Combination therapy comprising an RNA cancer vaccine and a multispecific binding agent targeting PD-1 / PD-l1 and VEGF / vegfr for cancer treatment

Abstract

The present invention relates to combination therapy using an RNA anti-cancer vaccine in combination with a binding agent that binds to (i) PD-1, PD-L1, or both, and (ii) VEGF, VEGF receptor (VEGFR), or both to reduce or prevent progression of cancer or to treat cancer.

Description

[0001] COMBINATION THERAPY COMPRISING AN RNA CANCER VACCINE AND A MULTISPECIFIC BINDING AGENT TARGETING PD-1 / PD-L1 AND VEGF / VEGFR FOR CANCER TREATMENT

[0002] Technical Field

[0003] The present invention relates to combination therapy using an RNA anti -cancer vaccine in combination with a binding agent that binds to (i) PD-1, PD-L1, or both, and (ii) VEGF, VEGF receptor (VEGFR), or both to reduce or prevent progression of cancer or to treat cancer.

[0004] Background

[0005] The immune system of humans and other mammals provides protection against infection and disease through mechanisms of innate and adaptive immunity. The evolutionary ancient innate immune system provides a rapid, z.e., within minutes, but non-specific immune response which relies on invariant receptors that recognize common molecular patterns associated with pathogens (antigens). In contrast, the immune response of the adaptive immune system is considerably slower, z.e., taking days to weeks, but involves highly specific antigen receptors on B cells (B lymphocytes) and T cells (T lymphocytes) for pathogen recognition. Like the innate immune system, the adaptive immune system also comprises a humoral immune response and a cell-mediated immune response. The humoral immune response is primarily driven by antibodies produced by B cells, which are able to recognize and neutralize foreign target antigens. In contrast, the cell-mediated immune response involves the activation of macrophages, neutrophils, natural killer (NK) cells, and antigen-specific cytotoxic T cells, and the release of various cytokines in response to the recognition of an antigen.

[0006] Besides the protection against pathogens, the immune system plays a pivotal role in cancer prevention, development, and defense (Gonzalez et al., Genes Dev. 2018, 32(19-20): 1267-1284). Therefore, the field of cancer immunotherapy has attracted great attention from both the scientific and clinical communities over the past two decades. The overarching concept of cancer immunotherapy is to activate, induce and / or enhance a specific immune response in patients to control and / or eliminate the cancer disease. Cancer immunotherapies can generally be categorized into active and passive immunization strategies, depending on their ability to (re-)activate the immune system against cancer cells. Passive forms of cancer immunotherapy include tumor-targeting monoclonal antibodies (mAbs) and adoptively transferred T cells, while anti-cancer vaccines and immunomodulatory antibodies (such as immune checkpoint inhibitors and co-stimulatory antibodies) are considered active forms of cancer immunotherapy (Galluzi et al., Oncotarget 2014, 5(24): 12472-12508).

[0007] Anti-cancer vaccines aim to elicit a tumor-specific immune response by active immunization, e.g., by inducing and expanding cancer antigen-specific T cells in patients, which are able to specificallyrecognize and kill malignant cells. The identification of a growing number of tumor-associated antigens (TAA) has led to a broad collection of suitable targets for immunotherapy using anti -cancer vaccination strategies. Specific TAAs can be delivered to the patients using different vaccination strategies, including proteins, peptides or immunizing vectors such as RNA, DNA or viral vectors that can be applied either directly in vivo or in vitro by pulsing of dendritic cells (DCs) following transfer into the patient. In particular, the field of RNA-based anti -cancer vaccines has developed rapidly in recent years (Liu etal., ACS Nano 2023, 17, 20: 19550-19580; Sahin etal., Nature 2020, 585: 107-112).

[0008] Therapeutic anti-cancer vaccines that stimulate the immune system against specific TAAs have shown promising results, but their efficacy has so far fallen short of expectations. Tumor heterogeneity, immunosuppressive tumor microenvironments, optimal TAA candidate identification, and immune response evaluation are still major challenges of anti -cancer vaccine development and application, resulting in their overall low efficacy in clinical trials (Fan et al., Sig. Transduct. Target Ther. 2023, 8:450; Wang et al., Front. Immunol. 2023, 14:1246682). Curing established tumors is a particular challenge for RNA-based anti-cancer vaccines as the immune response elicited by the RNA cancer vaccines can be insufficient to induce the complete rejection of large tumor masses.

[0009] PD-1 and PD-L1 are inhibitory checkpoint molecules regulating the immune system and enabling selftolerance. At the same time inhibitory checkpoint molecules are ideal targets for cancer immunotherapy. In tumor-draining lymph nodes and within the tumor microenvironment, a subset of CD4+ and CD8+ T cells are characterized by the co-expression of multiple TCR-inducible molecules including high levels of programmed cell death 1 (PD-1) (Gros et al., J. Clin Invest 2014;124(5):2246-59; Seifert et al., Cancers (Basel) 12; Simoni etal., Nature 557: 575-579). Upregulation of PD-1 on T cells can contribute to T-cell exhaustion and reduce T-cell activation upon binding to its ligand programmed cell death 1 ligand 1 (PD-L1) (Yu et al., Eur J Pharmacol 881: 173240). PD-L1 expression is often upregulated by tumor cells, particularly in inflamed tumors (Teng, et al., Cancer Res 75: 2139-2145). Thereby, the tumor cells provide an inhibitory signal to the activated T cells through which they can evade T-cell mediated cytotoxicity. Antibodies that block the PD-1 / PD-L1 inhibitory axis can restore T-cell function (Boussiotis et al., N Engl J Med 375: 1767-1778; Chen et al., Nature 541: 321-330).

[0010] Vascular endothelial growth factor (VEGF) is a member of the platelet-derived growth factor family of cystine-knot growth factors, i.e., a group of signal proteins involved in vasculogenesis and angiogenesis. Upon binding to tyrosine kinase receptors (VEGF receptors (VEGFRs)), the VEGFR dimerizes and becomes activated through transphosphorylation. Overexpression of VEGF can cause vascular diseases. Drugs which target VEGF (e.g., aflibercept, bevacizumab, ranibizumab, and pegaptanib) can inhibit VEGF and, thus, slow such diseases.Despite these promising improvements, response to immunotherapy treatment is not guaranteed in all cancer subjects and clinical efforts evaluating PD-1 / PD-L1 and VEGF / VEGFR antagonists as monotherapy have found limited success.

[0011] There is therefore an unmet need for improved methods for cancer treatment.

[0012] Summary

[0013] The present inventors have surprisingly found that the effectiveness of an RNA anti-cancer vaccine can be increased, e.g. synergistically, by co-administration of a bispecific antibody which antagonizes the PD-1 / PD-L1 interaction and the VEGF / VEGFR interaction. Thus, the present disclosure generally embraces the immunotherapeutic treatment of cancer in a subject comprising (1) the administration of RNA, z.e., RNA anti-cancer vaccine, encoding one or more amino acid sequences, z.e., at least one amino acid sequence (vaccine antigen), said at least one amino acid sequence comprising a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof, z.e., an antigenic peptide or protein. Thus, the vaccine antigen comprises an epitope of a tumor antigen for inducing an immune response against the tumor antigen in the subject. RNA encoding vaccine antigen is administered to provide (following expression of the polynucleotide by appropriate target cells) antigen for induction, z.e., stimulation, priming and / or expansion, of an immune response which is targeted to target antigen (tumor antigen) or a procession product thereof. The vaccine RNA treatment described herein is combined with (2) the administration of a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction, thereby blocking the PD-1 / PD-L1 inhibitory axis and inhibiting the VEGF / VEGFR pathway in cancer cells.

[0014] Thus, in a first aspect, the present disclosure provides a combination for use as a medicament, wherein the combination comprises: (a) an anti -cancer vaccine comprising at least one RNA, wherein the at least one RNA encodes at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and (b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction (this binding agent is also referred to herein as "anti-PD-l / PD-LlxVEGF / VEGFR binding agent").

[0015] In a second aspect, the present disclosure provides a method of treating cancer in a subject comprising administering to the subject: (a) an anti-cancer vaccine, wherein the anti-cancer vaccine comprises atleast one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and (b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction.

[0016] In a related aspect, the present disclosure provides an anti -cancer vaccine comprising at least one RNA, wherein the at least one RNA encodes at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof, for use in a method of treating cancer in a subject, the method comprising administering to the subject (a) the anti-cancer vaccine and (b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction.

[0017] In a further related aspect, the present disclosure provides a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction, for use in a method of treating cancer in a subject, the method comprising administering to the subject (a) the binding agent and (b) an anti-cancer vaccine comprising at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof

[0018] In a further related aspect, the present disclosure provides a binding agent and an anti-cancer vaccine for use in a method of treating cancer in a subject, the method comprising administering to the subject the binding agent and the anti -cancer vaccine, wherein (a) the binding agent comprises (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction; and (b) the anti -cancer vaccine comprises at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof.

[0019] In a third aspect, the present disclosure provides a medical preparation, combination, kit, or composition comprising: (a) an anti-cancer vaccine comprising at least one RNA, wherein the at least one RNAencodes at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and (b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction").

[0020] In a related aspect, the present disclosure provides a combination comprising: (a) an anti -cancer vaccine comprising at least one RNA, wherein the at least one RNA encodes at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and (b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction (this binding agent is also referred to herein as "anti-PD-1 / PD-LlxVEGF / VEGFR binding agent").

[0021] In a fourth aspect, the present disclosure provides the medical preparation, combination, kit, or composition of the third aspect for use as a medicament, e.g., for pharmaceutical use.

[0022] In aspects which are related to the second aspect, the present disclosure also provides:

[0023] (A) the use of an anti -cancer vaccine for the preparation of a medicament for treating cancer in a subject, wherein the anti -cancer vaccine comprises at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof, and wherein the medicament is to be administered in a combination therapy with (b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction;

[0024] (B) the use of a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction, for the preparation of a medicament for treating cancer in a subject, wherein the medicament is to be administered in a combination therapy with an anti-cancer vaccine, wherein the anti-cancer vaccine comprises at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; or(C) the use of (a) an anti-cancer vaccine and (b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction, for the preparation of a medicament for treating cancer in a subject, wherein the anti -cancer vaccine comprises at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof.

[0025] In a further aspect, the present disclosure provides a kit comprising (i) an anti-cancer vaccine, wherein the anti-cancer vaccine comprises at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof, and (ii) a binding agent comprising (i) a first binding region which binds to PD-1, PD-Ll, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction.

[0026] In a related aspect, the present disclosure provides a kit comprising (1) a composition comprising an anti-cancer vaccine, wherein the anti-cancer vaccine comprises at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and (2) a composition comprising a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction.Brief description of the Figures

[0027] Fig. 1 depicts (A) the expression of CLDN6 as measured by flow cytometry and (B) the expression of all six human PRAME, MAGE-A3, MAGE-CI, MAGE-A4, KK-LC-1 and CLDN6 antigens, normalized to the expression of endogenous Hprt, as measured by digital droplet PCR in the selected TC-1 clone expressing all six human TAAs CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI antigens.

[0028] Fig. 2 depicts the mean tumor growth curves of mice treated with an RNA cancer vaccine (Vaccine 1) comprising six different RNA constructs encoding the amino acid sequences of six human TAAs (CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI) in combination with an anti-PD-LlxVEGF antibody (PD-LlxVEGF; human IgGl). C57BL / 6 mice were inoculated subcutaneously (s.c.) with TC-1 tumor cells expressing all six human TAAs (CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI) on day 0 and were vaccinated intravenously (i.v.) once a week for two times (day 9 and 16; indicated by dotted lines) with Vaccine 1. Mice were treated in addition with PD-LlxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 9, 12 and 16; indicated by dotted lines). Monotherapy groups received either Vaccine 1 or PD-LlxVEGF. The control group was treated with vehicle. Last observation carried forward, if mice sacrificed due to tumor size: n>5 mice. Statistical analysis was performed using a Mixed-effects analysis followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 1 + PD-LlxVEGF.

[0029] Fig. 3 depicts the individual tumor growth curves of mice treated with Vaccine 1 comprising six different RNA constructs encoding the amino acid sequences of six human TAAs (CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI) in combination with an anti -PD-LlxVEGF antibody (PD-LlxVEGF). C57BL / 6 mice were inoculated s.c. with TC-1 tumor cells expressing all six human TAAs (CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI) on day 0 and were vaccinated i.v. once a week for two times (day 9 and 16; indicated by dotted lines) with Vaccine 1. Mice were treated in addition with PD-LlxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 9, 12, and 16; indicated by dotted lines). Monotherapy groups received either Vaccine 1 or PD-LlxVEGF. The control group was treated with vehicle. The number of complete responses (CR) is indicated for each group in the respective graphs.

[0030] Fig. 4 depicts the survival of mice treated with an RNA cancer vaccine (Vaccine 1) comprising six different RNA constructs encoding the amino acid sequences of six human TAAs (CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI) in combination with an anti -PD-LlxVEGF antibody (PD-LlxVEGF; human IgGl). C57BL / 6 mice were inoculated s.c. with TC-1 tumor cells expressing all six human TAAs CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI on day 0 and were vaccinated i.v. once a week for two times with Vaccine 1. Mice were treated in addition with PD-LlxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 9, 12, and 16). Monotherapy groups received either Vaccine 1 or PD-LlxVEGF. The control group was treated with vehicle. Statistical analysis was performed using a Log rank (Mantel-Cox) test. Significance is shown compared to Vaccine 1 + PD-LlxVEGF.

[0031] Fig. 5 depicts the number of CD8+ T cells in the blood of mice treated with an RNA cancer vaccine (Vaccine 1) comprising four different RNA constructs encoding the amino acid sequences of four human TAAs (CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI) in combination with an anti-PD-LlxVEGF antibody (PD-LlxVEGF; human IgGl) by flow cytometry. C57BL / 6 mice were inoculated s.c. with TC-1 tumor cells expressing all six human TAAs CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-C 1 on day 0 and were vaccinated i.v. once a week for two times (day 9 and 16) with Vaccine 1. Mice were treated in addition with PD-LlxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 9, 12, and 16). Monotherapy groups received either Vaccine 1 or PD-LlxVEGF. The control group was treated with vehicle. Blood was taken on Day 23. Statistical analysis was performed using One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 1 + PD-LlxVEGF.

[0032] Fig. 6 depicts the expression of the MAGE-A3 antigen, normalized to the expression of endogenous Hprt, as measured by digital droplet PCR in the selected B 16F 10 clone expressing one of the four human TAAs.

[0033] Fig. 7 depicts the mean tumor growth curves of mice treated with an RNA cancer vaccine (Vaccine 2) comprising four different RNA constructs encoding the amino acid sequences of four human TAAs (MAGE-A3, NY-ESO-1, tyrosinase, TPTE) in combination with an anti -PD-LlxVEGF antibody (PD-LlxVEGF; mouse IgG2a). C57BL / 6 mice are inoculated subcutaneously (s.c.) with B 16F10 tumor cells expressing human MAGE-A3 antigen on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13; indicated by dotted lines) with Vaccine 2. Mice were treated in addition with PD-LlxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9, and 13; indicated by dotted lines). Monotherapy groups received either Vaccine 2 or PD-LlxVEGF. The control group was treated with vehicle. Last observation carried forward, if mice sacrificed due to tumor size: n>5 mice. Statistical analysis was performed using a Mixed-effects analysis followed by Dunnett’ s test for multiple comparisons. Significance is shown compared to Vaccine 2 + PD-LlxVEGF

[0034] Fig. 8 depicts the survival of mice treated with an RNA cancer vaccine (Vaccine 2) comprising four different RNA constructs encoding the amino acid sequences of four human TAAs (MAGE-A3, NY-ESO-1, tyrosinase, and TPTE) in combination with an anti-PD-LlxVEGF antibody (PD-LlxVEGF; mouse IgG2a). C57BL / 6 mice were inoculated subcutaneously (s.c.) with B16F10 tumor cellsexpressing human MAGE-A3 antigen on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13) with Vaccine 2. Mice were treated in addition with PD-LlxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9 and 13). Monotherapy groups received either Vaccine 2 or PD-LlxVEGF. The control group was treated with vehicle. Statistical analysis was performed using a Log-rank (Mantel-Cox) test. Significance is shown compared to Vaccine 2 + PD-LlxVEGF.

[0035] Fig. 9 depicts the number of CD8+ T cells (A) and the fractions of interleukin- 18 receptor a (IL-18Ra) + (B), granzyme B (gzmB) + (C) and killer cell lectin like receptor G1 (KLRG1) + (D) CD8+ T cells in the blood of mice treated with an RNA cancer vaccine (Vaccine 2) comprising four different RNA constructs encoding the amino acid sequences of four human TAAs (MAGE-A3, NY-ESO-1, tyrosinase, and TPTE) in combination with an anti -PD-LlxVEGF antibody (PD-LlxVEGF; mouse IgG2a) by flow cytometry. C57BL / 6 mice were inoculated subcutaneously (s.c.) with B16F10 tumor cells expressing human MAGE-A3 antigen on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13) with Vaccine 2. Mice were treated in addition with PD-LlxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9 and 13). Monotherapy groups receive either Vaccine 2 or PD-LlxVEGF. The control group was treated with vehicle. Blood was taken on Day 20. Statistical analysis was performed using One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 2 + PD-LlxVEGF.

[0036] Fig. 10 depicts the expression of the HPV16 E6 and E7 antigens, normalized to the expression of endogenous Hprt, as measured by digital droplet PCR in the selected B16F10 clone expressing the two TAAs.

[0037] Fig. 11 depicts the survival of mice treated with an RNA cancer vaccine (Vaccine 3) comprising two different RNA constructs encoding the amino acid sequences of two tumor-specific antigens (HPV16 E6 and E7) in combination with an anti-PD-LlxVEGF antibody (PD-LlxVEGF; mouse IgG2a). C57BL / 6 mice were inoculated subcutaneously (s.c.) with B16F10 tumor cells expressing HPV16 E6 and E7 antigen on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13) with Vaccine 3. Mice were treated in addition with PD-LlxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9 and 13). Monotherapy groups received either Vaccine 3 or PD-LlxVEGF. The control group was treated with vehicle. Statistical analysis was performed using a Logrank (Mantel-Cox) test. Significance is shown compared to Vaccine 3 + PD-LlxVEGF.

[0038] Fig. 12 depicts the number of E7-specific CD8+ T cell in blood of mice treated with an RNA cancer vaccine (Vaccine 3) comprising two different RNA constructs encoding the amino acid sequences of two tumor-specific antigens (HPV16 E6 and E7) in combination with an anti-PD-LlxVEGF antibody (PD-LlxVEGF; mouse IgG2a) by flow cytometry. C57BL / 6 mice were inoculated subcutaneously (s.c.)with Bl 6F 10 tumor cells expressing HPV16 E6 and E7 on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13) with Vaccine 3. Mice are treated in addition with PD-LlxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9 and 13). Monotherapy groups receive either Vaccine 3 or PD-LlxVEGF. The control group is treated with vehicle. Blood was taken on Day 20. Statistical analysis was performed using One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 3 + PD-LlxVEGF.

[0039] Fig. 13 depicts the mean tumor growth curves of mice treated with an RNA cancer vaccine (Vaccine 1) comprising six different RNA constructs encoding the amino acid sequences of six human TAAs (CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI) in combination with an anti-PD-lxVEGF antibody (PD-lxVEGF). C57BL / 6 mice were inoculated s.c. with TC-1 tumor cells expressing all six human TAAs CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI on day 0 and were vaccinated i.v. once a week for two times (day 9 and 16; indicated by dotted lines) with Vaccine 1. Mice were treated in addition with PD-lxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 9, 12, and 16; indicated by dotted lines). Monotherapy groups received either Vaccine 1 or PD-lxVEGF. The control group was treated with vehicle. Last observation carried forward, if mice sacrificed due to tumor size: n>5 mice. Statistical analysis was performed using a Mixed-effects analysis followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 1 + PD-lxVEGF.

[0040] Fig. 14 depicts the individual tumor growth curves of mice treated with an RNA cancer vaccine (Vaccine 1) comprising six different RNA constructs encoding the amino acid sequences of six human TAAs (CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI) in combination with an anti-PD-lxVEGF antibody (PD-lxVEGF). C57BL / 6 mice were inoculated s.c. with TC-1 tumor cells expressing all six human TAAs CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI on day 0 and were vaccinated i.v. once a week for two times (day 9 and 16; indicated by dotted lines) with Vaccine 1. Mice were treated in addition with PD-lxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 9, 12, and 16; indicated by dotted lines). Monotherapy groups received either Vaccine 1 or PD-lxVEGF. The control group was treated with vehicle. The number of complete responses (CR) is indicated for each group in the respective graphs.

[0041] Fig. 15 depicts the number of CD8+ T cells (A) and the fractions of interleukin- 18 receptor a (IL-18Ra) + CD8+ T cells (B) in blood of mice treated with an RNA cancer vaccine (Vaccine 1) comprising six different RNA constructs encoding the amino acid sequences of six human TAAs (CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI) in combination with an anti-PD-lxVEGF antibody (PD-lxVEGF) by flow cytometry. C57BL / 6 mice were inoculated s.c. with TC-1 tumor cells expressing all six human TAAs CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and MAGE-CI on day 0and were vaccinated i.v. once a week for two times (day 9 and 16) with Vaccine 1. Mice were treated in addition with PD-lxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 9, 12, and 16). Monotherapy groups received either Vaccine 1 or PD-lxVEGF. The control group was treated with vehicle. Blood was taken on Day 23. Statistical analysis was performed using One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 1 + PD-lxVEGF.

[0042] Fig. 16 depicts the number of CD8+ T cells in blood of mice treated with an RNA cancer vaccine (Vaccine 2) comprising four different RNA constructs encoding the amino acid sequences of four human TAAs (MAGE-A3, NY-ESO-1, tyrosinase, and TPTE) in combination with an anti-PD-lxVEGF antibody (PD-lxVEGF) by flow cytometry. C57BL / 6 mice were inoculated subcutaneously (s.c.) with B16F10 tumor cells expressing human MAGE-A3 antigen on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13) with Vaccine 2. Mice were treated in addition with PD-lxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9 and 13). Monotherapy groups receive either Vaccine 2 or PD-lxVEGF. The control group was treated with vehicle. Blood was taken on Day 20. Statistical analysis was performed using One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 2 + PD-lxVEGF.

[0043] Fig. 17 depicts the mean tumor growth curves of mice treated with an RNA cancer vaccine (Vaccine 3) comprising two different RNA constructs encoding the amino acid sequences of two tumor-specific antigens (HPV16 E6 and E7) in combination with an anti -PD-lxVEGF antibody (PD-lxVEGF). C57BL / 6 mice were inoculated subcutaneously (s.c.) with B16F10 tumor cells expressing HPV16 E6 and E7 antigen on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13; indicated by dotted lines) with Vaccine 3. Mice were treated in addition with PD-lxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9 and 13; indicated by dotted lines). Monotherapy groups received either Vaccine 3 or PD-lxVEGF. The control group was treated with vehicle. Last observation carried forward, if mice sacrificed due to tumor size: n>5 mice. Statistical analysis was performed using a Mixed-effects analysis followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 3 + PD-lxVEGF.

[0044] Fig. 18 depicts the individual tumor sizes of mice treated with an RNA cancer vaccine (Vaccine 3) comprising two different RNA constructs encoding the amino acid sequences of two tumor-specific antigens (HPV16 E6 and E7) in combination with an anti-PD- IxVEGF antibody (PD-lxVEGF) at the time when mice were euthanized. C57BL / 6 mice were inoculated subcutaneously (s.c.) with B16F10 tumor cells expressing HPV16 E6 and E7 antigen on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13) with Vaccine 3. Mice were treated in addition with PD-lxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9 and 13). Monotherapy groupsreceived either Vaccine 3 or PD-lxVEGF. The control group was treated with vehicle. Statistical analysis was performed using a Mixed-effects analysis followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 3 + PD-lxVEGF.

[0045] Fig. 19 depicts the individual tumor growth curves of mice treated with an RNA cancer vaccine (Vaccine 3) comprising two different RNA constructs encoding the amino acid sequences of two tumor-specific antigens (HPV16 E6 and E7) in combination with an anti -PD-lxVEGF antibody (PD-lxVEGF). C57BL / 6 mice were inoculated subcutaneously (s.c.) with B16F10 tumor cells expressing HPV16 E6 and E7 antigen on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13; indicated by dotted lines) with Vaccine 3. Mice were treated in addition with PD-lxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9 and 13; indicated by dotted lines). Monotherapy groups received either Vaccine 3 or PD-lxVEGF. The control group was treated with vehicle. The number of complete responses (CR) is indicated for each group in the respective graphs.

[0046] Fig. 20 depicts the number of E7-specific CD8+ T cell (A) and the fractions of interleukin- 18 receptor a (IL-18Ra) + E7-specific CD8+ T cells (B) in blood of mice treated with an RNA cancer vaccine (Vaccine 3) comprising two different RNA constructs encoding the amino acid sequences of two tumorspecific antigens (HPV16 E6 and E7) in combination with an anti-PD-lxVEGF antibody (PD-lxVEGF) by flow cytometry. C57BL / 6 mice were inoculated subcutaneously (s.c.) with B16F10 tumor cells expressing HPV16 E6 and E7 on day 0 and vaccinated intravenously (i.v.) two times (day 6 and 13) with Vaccine 3. Mice are treated in addition with PD-lxVEGF three times intraperitoneally (i.p.), starting on the same day as the vaccine (day 6, 9 and 13). Monotherapy groups receive either Vaccine 3 or PD-lxVEGF. The control group is treated with vehicle. Blood was taken on Day 20. Statistical analysis was performed using One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to Vaccine 3 + PD-lxVEGF.

[0047] Fig. 21 depicts the cytotoxicity of PD-lxVEGF in combination with MAGE-A3 TCR-CD8+ T cells against Sk-Mel-5 tumor cells assessed as the change in the cell index (ACI, A) or as specific lysis [%] of Sk-Mel-5 tumor cells (B) measured using an impedance -based xCELLigence system. Specific lysis is displayed for the time point (40.2 h, Donor 1; 54.7 h Donor 2) of the highest difference in per cent specific lysis between MAGE-A3 TCR-CD8+ T cells and MAGE-A3 TCR-CD8+ T cells in combination with PD-lxVEGF. Treatment with PD-lxVEGF and addition of CD8+ T cells was initiated one day after seeding the tumor cells at E:T ratios 16:1 (Donor 1) or 8:1 (Donor 2). The final concentration of PD-lxVEGF was 80 nM. Triplicates per donor were measured. One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to MAGE-A3 TCR-CD8+ T cells + PD-lxVEGF.Fig. 22 depicts the cytotoxicity of PD-lxVEGF in combination with MAGE-A3 TCR-CD8+ T cells against Sk-Mel-5 tumor cells assessed as the change in the cell index (ACI, A) or as specific lysis [%] of Sk-Mel-5 tumor cells (B) measured using an impedance -based xCELLigence system. Specific lysis is displayed for the time point (53.7 h, Donor 1; 48.7 h, Donor 2) of the highest difference in per cent specific lysis between MAGE-A3 TCR-CD8+ T cells and MAGE-A3 TCR-CD8+ T cells in combination with PD-lxVEGF. Treatment with PD-lxVEGF and addition of CD8+ T cells was initiated one day after seeding the tumor cells at E:T ratios 8: 1 (Donor 1) or 4: 1 (Donor 2). The final concentration of PD-lxVEGF was 80 nM. Triplicates per donor were measured. One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to MAGE-A3 TCR-CD8+ T cells + PD-lxVEGF.

[0048] Fig. 23 depicts the cytotoxicity of PD-lxVEGF in combination with HPV16 E7 CD8+ TCR-T cells against MDA-MB-231 tumor cells assessed as the change in the cell index (ACI, A) or as specific lysis [%] of MDA-MB-231 tumor cells (B) measured using an impedance -based xCELLigence system. Specific lysis is displayed for the time point (138.8 h, Donor 1; 119.3 h, Donor 2; 119.3 h, Donor 3) of the highest difference in per cent specific lysis between HPV16 E7 TCR-CD8+ T cells and HPV16 E7 TCR-CD8+ T cells in combination with PD-lxVEGF. Treatment with PD-lxVEGF and addition of CD8+ T cells was initiated one day after seeding the tumor cells at E:T ratios 2.5:1 (Donor 1), 1:1 (Donor 2) or 2.5:1 (Donor 3). The final concentration of PD-lxVEGF was 80 nM. Duplicates (E7 TCR-CD8+ T cells, staurosporine, PD-lxVEGF + E7 TCR-CD8+ T cells) or Quadruplicates (vehicle, PD-lxVEGF) per donor were measured. One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to HPV16 E7 TCR-CD8+ T cells + PD-lxVEGF.

[0049] Fig. 24 depicts the cytotoxicity of PD-lxVEGF in combination with HPV16 E7 TCR-CD8+ T cells against MDA-MB-231 tumor cells assessed as the change in the cell index (ACI, A) or as specific lysis [%] of MDA-MB-231 tumor cells (B) measured using an impedance -based xCELLigence system. Specific lysis is displayed for the time point (138.8 h, Donor 1; 138.3 h, Donor 2; 133.8, Donor 3) of the highest difference in per cent specific lysis between HPV16 E7 TCR-CD8+ T cells and HPV16 E7 TCR-CD8+ T cells in combination with PD-lxVEGF. Treatment with PD-lxVEGF and addition of CD8+ T cells was initiated one day after seeding the tumor cells at E:T ratios 1 : 1 (Donor 1), 0.4: 1 (Donor 2) or 1:1 (Donor 3). The final concentration of PD-lxVEGF was 80 nM. Duplicates (E7 TCR-CD8+ T cells, staurosporine, PD-lxVEGF + E7 TCR-CD8+ T cells) or Quadruplicates (vehicle, PD-lxVEGF) per donor were measured. One-Way ANOVA followed by Dunnett’s test for multiple comparisons. Significance is shown compared to HPV16 E7 TCR-CD8+ T cells + PD-lxVEGF.The following table provides a listing of certain sequences referenced herein.

[0050] Table 1: Description of the sequences

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Detailed Description

[0089] Although the present disclosure is further described in more detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0090] In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. For example, if in a preferred embodiment of the binding agent used herein the binding agent comprises one or two anti-PD-Ll single domain antibodies (e.g., one or two anti-PD-Ll VHHs) and in another preferred embodiment of the binding agent used herein the binding agent comprises a second binding region which binds to VEGF-A, then in a further preferred embodiment of the binding agent used herein the binding agent comprises one or two anti-PD-Ll single domain antibodies (e.g., one or two anti-PD-Ll VHHs) and a second binding region which binds to VEGF-A.

[0091] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0092] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (c , e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rbmpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0093] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0094] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0095] Citation of documents and studies referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.

[0096] Definitions

[0097] In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0098] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The term "consisting essentially of' means excluding other members, integers or steps of any essential significance. The term "comprising" encompasses the term "consisting essentially of' which, in turn, encompasses the term "consisting of'. Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of' or "consisting of'. Likewise, at each occurrence in the present application, the term "consisting essentially of' may be replaced with the term "consisting of'.The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0099] Where used herein, "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

[0100] In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

[0101] The term "binding agent" in the context of the present disclosure refers to any agent capable of binding to desired antigens. In certain embodiments, the binding agent is an antibody, antibody fragment, or construct thereof. The binding agent may also comprise synthetic, modified or non-naturally occurring moieties, in particular non-peptide moieties. Such moieties may, for example, link desired antigenbinding functionalities or regions such as antibodies or antibody fragments. In one embodiment, the binding agent is a synthetic construct comprising antigen-binding CDRs or variable regions.

[0102] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably to antigen receptors such as antibodies or the B cell receptor (BCR). The immunoglobulins are characterized by a structural domain, z.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane bound immunoglobulins as well as soluble immunoglobulins. Membrane bound immunoglobulins are also termed surface immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally termed antibodies.

[0103] The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch.

[0104] 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)). Briefly, immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains or regions, such as the VL or VL (variable light chain) domain / region, CL or CL (constant light chain) domain / region, VH or VH(variable heavy chain) domain / region, and the CH or CH (constant heavy chain) domains / regions CHI (CHI), CH2 (CH2), CH3 (CH3), and CH4 (CH4). The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The hinge region is the region between the CHI and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a VL and a CL. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (LRs). Each VH and VL is typically composed of three CDRs and four ERs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to Kabat or IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also internet http address www.imgt.org. Unless otherwise stated or contradicted by context, reference to amino acid positions in the constant regions in the present disclosure is according to the EU-numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May;63(l):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

[0105] There are five types of mammalian immunoglobulin heavy chains, i.e., a, 5. a, y, and p which account for the different classes of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM. As opposed to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminus. In mammals there are two types of light chains, i.e., lambda and kappa. The immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different isotypes of the immunoglobulins, wherein the variable part is highly divers and accounts for antigen recognition.

[0106] The term "amino acid" and "amino acid residue" may herein be used interchangeably, and are not to be understood limiting. Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each amino acid. In the context of the present disclosure, amino acids may be classified based on structure and chemical characteristics. Thus, classes of amino acids may be reflected in one or both of the following tables:Table 2: Main classification based on structure and general chemical characterization of R group

[0107]

[0108] Table 3: Alternative Physical and Functional Classifications of Amino Acid Residues

[0109]

[0110] Unless indicated to the contrary, an "amino acid" or "amino acid residue" as used herein preferably refers to one of the 22 natural amino acids, 20 of which are listed in Table 2 (and are encoded by the universal genetic code) and the remaining 2 of which are selenocysteine and pyrrolysine (these 2 amino acids are incorporated into proteins by unique synthesis mechanisms). These 22 amino acids are also called "proteinogenic amino acids" or "natural amino acids". The term "unnatural amino acid" as used herein refers to any amino acid which is not a natural amino acid (e.g., citrulline and homocysteine are examples of unnatural amino acids).

[0111] For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide, protein or polypeptide) comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence.

[0112] Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.Amino acid addition variants comprise amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0113] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C-terminal truncation variants.

[0114] Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Substitution of one amino acid for another may be classified as a conservative or non-conservative substitution. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and / or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, z.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. In the context of the present disclosure, a "conservative substitution" is a substitution of one amino acid with another amino acid having similar structural and / or chemical characteristics, such substitution of one amino acid residue for another amino acid residue of the same class as defined in any of the two tables above: for example, leucine may be substituted with isoleucine as they are both aliphatic, branched hydrophobes. Similarly, aspartic acid may be substituted with glutamic acid since they are both small, negatively charged residues. Naturally occurring amino acids may also be generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups:

[0115] - glycine, alanine;

[0116] - valine, isoleucine, leucine;

[0117] - aspartic acid, glutamic acid;

[0118] - asparagine, glutamine;

[0119] - serine, threonine;

[0120] - lysine, arginine; and

[0121] - phenylalanine, tyrosine.

[0122] The term "antibody" (Ab) in the context of the present disclosure refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability tospecifically bind to an antigen (in particular an epitope on an antigen) under typical physiological conditions, preferably with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally defined period (such as a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or time sufficient for the antibody to recruit an effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the foregoing. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of a VH are termed HCDR1, HCDR2 and HCDR3 (or CDR-H1, CDR-H2 and CDR-H3), the CDRs of a VL are termed LCDR1, LCDR2 and LCDR3 (or CDR-L1, CDR-L2 and CDR-L3). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further subdivided into constant domain CHI, a hinge region, and constant domains CH2 and CH3 (arranged from amino-terminus to carboxy-terminus in the following order: CHI, CH2, CH3). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as Clq. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.

[0123] The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The terms "binding region" and "antigen-binding region" are used herein interchangeably and refer to the region which interacts with the antigen and comprises at least a single variable domain, such as a heavy chain variable (VH) region or both a VH region and a VL region. An antibody as used herein comprises not only monospecific antibodies, but also multispecificantibodies which comprise multiple, such as two or more, e.g., three or more, different antigen-binding regions.

[0124] As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, z.e., retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) a Fab’ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in WO 2007 / 059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature 341. 544-546 (1989)), which consists essentially ofaVH domain and also called domain antibodies (Holt eta / ; Trends Biotechnol. 2003 Nov;21(ll):484-90) or single domain antibodies; (vi) camelid or Nanobody molecules (Revets et al Expert Opin Biol Ther. 2005 Jan;5(l): 111-24); and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242. 423-426 (1988) and Huston et al., PNAS USA 85. 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0125] A "single domain antibody" (sdAb) is an antibody composed of a single variable domain (e.g., heavy chain variable region) composed of antibody fragments. Typically, a single domain antibody, domain antibody or nanobody consists of 4 framework regions (FR1-FR4) and 3 complementarity determining regions (CDR1-CDR3). In some embodiments, the single domain antibody has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. These antibodies do not require light chain variable regions to bindantigens with high affinity and specificity. Examples of single domain antibodies include, but are not limited to, VHH fragments, and VNAR fragments. Compared with antibodies composed of heavy chain and light chain, single domain antibodies have high solubility, high stability to heat, pH, protease and other deforming agents, and only need single chain expression to facilitate large scale production.

[0126] A "VNAR" as used herein refers to a variable new Antigen receptor which is the smallest single domain antibody derived from the variable domain of shark immunoglobulin new antigen receptor (IgNAR) of cartilaginous fishes.

[0127] An antibody as generated can possess any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (for instance IgG (such as IgGl, IgG2, IgG3, IgG4), IgD, IgA (such as IgAl, IgA2), IgE, IgM, or IgY) that is encoded by heavy chain constant region genes. When a particular isotype, e.g., IgGl, is mentioned herein, the term is not limited to a specific isotype sequence, e.g., a particular IgGl sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g., IgGl, than to other isotypes. Thus, e.g., an IgGl antibody disclosed herein may be a sequence variant of a naturally occurring IgGl antibody, including variations in the constant regions.

[0128] IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any of which are suitable for use in some of the embodiments herein. Common allotypic variants in human populations are those designated by the letters a, f, n, z or combinations thereof. In any of the embodiments herein, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In further embodiments, the human IgG Fc region comprises a human IgGl.

[0129] The term "multispecific antibody" in the context of the present disclosure refers to an antibody having at least two different antigen-binding regions defined by different antibody sequences. In some embodiments, said different antigen-binding regions bind different epitopes on the same antigen. However, in preferred embodiments, said different antigen-binding regions bind different target antigens. In one embodiment, the multispecific antibody is a "bispecific antibody" or "bs". A multispecific antibody, such as a bispecific antibody, can be of any format, including any of the bispecific or multispecific antibody formats described herein below.

[0130] The term "full-length" when used in the context of an antibody indicates that the antibody is not a fragment, but contains all of the domains of the particular isotype normally found for that isotype in nature, e.g., the VH, CHI, CH2, CH3, hinge, VL and CL domains for an IgGl antibody.The term "human antibody", as used herein, is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and a human immunoglobulin constant domain. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, have been grafted onto human framework sequences.

[0131] The term "chimeric antibody" as used herein refers to an antibody wherein the variable region is derived from a non-human species (e.g., derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by antibody engineering. "Antibody engineering" is a term used generically for different kinds of modifications of antibodies, and processes for antibody engineering are well-known for the skilled person. In particular, a chimeric antibody may be generated by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, the chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody may be performed by other methods than those described herein. Chimeric monoclonal antibodies for therapeutic applications in humans are developed to reduce anticipated antibody immunogenicity of non-human antibodies, e.g., rodent antibodies. They may typically contain non-human (e.g., murine or rabbit) variable regions, which are specific for the antigen of interest, and human constant antibody heavy and light chain domains. The terms "variable region" or "variable domain" as used in the context of chimeric antibodies, refer to a region which comprises the CDRs and framework regions of both the heavy and light chains of an immunoglobulin, as described below.

[0132] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO 92 / 22653 and EP 0 629240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.

[0133] As used herein, a protein which is "derived from" another protein, e.g., a parent protein, means that one or more amino acid sequences of the protein are identical or similar to one or more amino acid sequences in the other or parent protein. For example, in an antibody, binding arm, antigen-binding region, constant region, or the like which is derived from another or a parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical or similar to those of the other or parent antibody, binding arm, antigen-binding region, or constant region. Examples of such one or more amino acid sequences include, but are not limited to, those of the VH and VL CDRs and / or one or more or all of the framework regions, VH, VL, CL, hinge, or CH regions. For example, a humanized antibody can be described herein as "derived from" a non-human parent antibody, meaning that at least the VL and VH CDR sequences are identical or similar to the VH and VL CDR sequences of said non-human parent antibody. A chimeric antibody can be described herein as being "derived from" a non-human parent antibody, meaning that typically the VH and VL sequences may be identical or similar to those of the non-human parent antibody. Another example is a binding arm or an antigen-binding region which may be described herein as being "derived from" a particular parent antibody, meaning that said binding arm or antigen-binding region typically comprises identical or similar VH and / or VL CDRs, or VH and / or VL sequences to the binding arm or antigen-binding region of said parent antibody. As described elsewhere herein, however, amino acid modifications such as mutations can be made in the CDRs, constant regions or elsewhere in the antibody, binding arm, antigen-binding region or the like, to introduce desired characteristics. When used in the context of one or more sequences derived from a first or parent protein, a "similar" amino acid sequence preferably has a sequence identity of at least about 50%, such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 97%, 98% or 99%.

[0134] Non-human antibodies can be generated in a number of different species, such as mouse, rabbit, chicken, guinea pig, llama and goat.

[0135] Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of antibody genes, and such methods are well known to a person skilled in the art.Hybridoma production in such non-human species is a very well established procedure. Immunization protocols and techniques for isolation of splenocytes of immunized animals / non-human species for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0136] When used herein, unless contradicted by context, the term "Fab-arm" or "arm" refers to one heavy chain-light chain pair and is used interchangeably with "half molecules" herein.

[0137] The term "binding arm comprising an antigen-binding region" means an antibody molecule or fragment that comprises an antigen-binding region. Thus, a binding arm can comprise, e.g., the six VH and VL CDR sequences, the VH and VL sequences, a Fab or Fab' fragment, or a Fab-arm.

[0138] When used herein, unless contradicted by context, the term "Fc region" refers to an antibody region consisting of the two Fc sequences of the heavy chains of an immunoglobulin, wherein said Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain. In one embodiment, the term "Fc region", as used herein, refers to a region comprising, in the direction from the N- to C-terminal end of the antibody, at least a hinge region, a CH2 region and a CH3 region. An Fc region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.

[0139] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgGl antibody corresponds to amino acids 216-230 according to the EU numbering as set forth in Kabat (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp 662,680,689 (1991). However, the hinge region may also be any of the other subtypes as described herein.

[0140] The term "CHI region" or "CHI domain" as used herein refers to the CHI region of an immunoglobulin heavy chain. Thus, for example, the CHI region of a human IgGl antibody corresponds to amino acids 118-215 according to the EU numbering as set forth in Kabat (ibid). However, the CHI region may also be any of the other subtypes as described herein.

[0141] The term "CH2 region" or "CH2 domain" as used herein refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgGl antibody corresponds to amino acids 231-340 according to the EU numbering as set forth in Kabat (ibid). However, the CH2 region may also be any of the other subtypes as described herein.The term "CH3 region" or "CH3 domain" as used herein refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgGl antibody corresponds to amino acids 341-447 according to the EU numbering as set forth in Kabat (ibid). However, the CH3 region may also be any of the other subtypes as described herein.

[0142] The term "monovalent antibody" means in the context of the present disclosure that an antibody molecule is capable of binding a single molecule of the antigen, and thus is not capable of antigen crosslinking.

[0143] A "PD-1 antibody" or "anti-PD-1 antibody" is an antibody as described above, which is directed against the antigen PD-1 and which preferably binds specifically to the antigen PD-1.

[0144] A "PD-L1 antibody" or "anti-PD-Ll antibody" is an antibody as described above, which is directed against the antigen PD-L1 and which preferably binds specifically to the antigen PD-L1.

[0145] A "VEGF antibody" or "anti-VEGF antibody" is an antibody as described above, which is directed against the antigen VEGF and which preferably binds specifically to the antigen VEGF.

[0146] A "VEGFR antibody" or "anti-VEGFR antibody" is an antibody as described above, which is directed against the antigen VEGFR and which preferably binds specifically to the antigen VEGFR.

[0147] The term "VEGFR2" refers to Vascular Endothelial Growth Factor Receptor 2 (also known as KDR or FLK1) and variants thereof that retain at least part of the biological activity of VEGFR2. As used herein, VEGFR2 may include all species of native sequence VEGFR2, including human, rat, mouse and chicken. The term "VEGFR2" may be used to include variants, isoforms and species homologs of human VEGFR2. In some embodiments, "VEGFR2" refers to the wild-type human form of VEGFR2. Antibodies used according to the invention may cross-react with VEGFR2 from species other than human, in particular VEGFR2 from rhesus monkey (Macaca mulatta). Examples of human and rhesus VEGFR2 amino acid sequences are provided in Table 1. In certain embodiments, the antibodies may be completely specific for human VEGFR2 and may not exhibit non-human cross-reactivity. In one embodiment, VEGFR2 is human VEGFR, in particular human VEGFR comprising the amino acid sequence of SEQ ID NO: 220. In some embodiments, VEGFR2 is monkey VEGFR2, in particular Macaca VEGFR2 comprising the amino acid sequence of SEQ ID NO: 221.

[0148] As used herein, the terms "binding" or "capable of binding" in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less, about IO10M or less, orabout 1011M or even less, when determined, for instance, using Bio-Layer Interferometry (BLI) or using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. The antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is higher is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the degree to which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold.

[0149] The term "kd" (sec1), as used herein, refers to the dissociation rate constant of a particular antibodyantigen interaction. Said value is also referred to as the kOff value.

[0150] The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0151] Two antibodies have the "same specificity" if they bind to the same antigen and to the same epitope. Whether an antibody to be tested recognizes the same epitope as a certain antigen-binding antibody, i. e. , the antibodies bind to the same epitope, may be tested by different methods well known to a person skilled in the art.

[0152] The competition between the antibodies can be detected by a cross-blocking assay. For example, a competitive ELISA assay may be used as a cross-blocking assay. E.g., target antigen may be coated on the wells of a microtiter plate and antigen-binding antibody and candidate competing test antibody may be added. The amount of the antigen-binding antibody bound to the antigen in the well indirectly correlates with the binding ability of the candidate competing test antibody that competes therewith for binding to the same epitope. Specifically, the larger the affinity of the candidate competing test antibody is for the same epitope, the smaller the amount of the antigen-binding antibody bound to the antigen-coated well. The amount of the antigen-binding antibody bound to the well can be measured by labeling the antibody with detectable or measurable labeling substances.

[0153] An antibody competing for binding to an antigen with another antibody, e.g., an antibody comprising heavy and light chain variable regions as described herein, or an antibody having the specificity for an antigen of another antibody, e.g., an antibody comprising heavy and light chain variable regions as described herein, may be an antibody comprising variants of said heavy and / or light chain variableregions as described herein, e.g. modifications in the CDRs and / or a certain degree of identity as described herein.

[0154] A binding region as defined herein (such as an antibody or a fragment thereof as defined herein) "antagonizes the PD-1 / PD-L1 interaction" means that the binding region is antagonistic towards the PD-1 / PD-L1 signaling pathway. In particular, such binding region which antagonizes the PD-1 / PD-L1 interaction totally or partially inhibits, reduces, interferes with or negatively modulates the PD-1 / PD-L1 signaling pathway by binding to PD-1, PD-L1, or both. Preferably, the binding region which antagonizes the PD-1 / PD-L1 interaction mediates its antagonistic activity in a manner that totally inhibits (z.e., blocks) or partially reduces the binding of PD-L1 to PD-1.

[0155] A binding region as defined herein (such as an antibody or a fragment thereof as defined herein) "antagonizes the VEGF / VEGFR interaction" means that the binding region is antagonistic towards the VEGF / VEGFR signaling pathway. In particular, such binding region which antagonizes the VEGF / VEGFR interaction totally or partially inhibits, reduces, interferes with or negatively modulates the VEGF / VEGFR signaling pathway by binding to VEGF, VEGFR, or both. Preferably, the binding region which antagonizes the VEGF / VEGFR interaction mediates its antagonistic activity in a manner that totally inhibits (z.e., blocks) or partially reduces the binding of VEGF to VEGFR.

[0156] An "isolated multispecific antibody" as used herein is intended to refer to a multispecific antibody which is substantially free of other antibodies having different antigenic specificities (for instance an isolated bispecific antibody that specifically binds to PD-L1 and VEGF is substantially free of monospecific antibodies that specifically bind to PD-L1 or VEGF).

[0157] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0158] The present disclosure also describes antibodies, such as monoclonal antibodies, comprising functional variants of the VL regions, VH regions, or one or more CDRs of the antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of a antibody still allows the antigen-binding region of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or the specificity / selectivity of the parent antibody and in some cases such an antibody may be associated with greater affinity, selectivity and / or specificity than the parent antibody.Such functional variants typically retain significant sequence identity to the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (z.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g., be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.

[0159] In the context of the present disclosure, unless otherwise indicated, the following notations are used to describe a mutation: i) substitution of an amino acid in a given position is written as, e.g., L234A (with the numbering according to the EU-index) which means a substitution of a leucine in position 234 of the protein with an alanine; and ii) for specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, the substitution of leucine with alanine in position 234 is designated as: L234A (with the numbering according to the EU-index), and the substitution of leucine with any amino acid residue in position 234 is designated as L234X. In case of deletion of leucine in position 234 it is indicated by L234*. As noted, amino acids are numbered according to the EU numbering (EU-index) as set forth in Kabat (Kabat, E.A.; National Institutes of Health (U.S.) Office of the Director. Sequences of Proteins of Immunological Interest, 5th ed.; DIANE Publishing: Collingdale, PA, USA, 1991).

[0160] Exemplary variants include those which differ from the VH and / or VL and / or CDRs of the parent sequences mainly by conservative substitutions; for example, 12, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.

[0161] In the context of the present disclosure, conservative substitutions may be defined by substitutions within the classes of amino acids as defined in tables 2 and 3.

[0162] As used herein, "immune checkpoint" refers to regulators of the immune system, and, in particular, costimulatory and inhibitory signals that regulate the amplitude and quality of T cell receptor recognition of an antigen. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1 and / or PD-L2. The terms "checkpoint inhibitor" (CPI) and "immune checkpoint (ICP) inhibitor" are used herein synonymously. The terms refer to molecules, such as binding agents, which totally or partially reduce, inhibit, interfere with or negatively modulate one or more checkpoint proteins or that totally or partially reduce, inhibit,interfere with or negatively modulate expression of one or more checkpoint proteins, like molecules, such as binding agents, which inhibit an immune checkpoint, in particular, which inhibit the inhibitory signal of an immune checkpoint. In one embodiment, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to one or more molecules regulating checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to precursors of one or more checkpoint proteins e.g., on DNA- or RNA-level. Any agent that functions as a checkpoint inhibitor according to the present disclosure can be used. The term "partially" as used herein means at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% in the level, e.g., in the level of inhibition of a checkpoint protein.

[0163] In one embodiment, the checkpoint inhibitor can be any compound, such as any binding agent, which inhibits the inhibitory signal of an immune checkpoint. In one embodiment, the inhibitory signal is the interaction between PD-1 and PD-L1 and / or PD-L2 (such a checkpoint inhibitor which inhibits the interaction between PD-1 and PD-L1 and / or PD-L2 is also called a PD-1 / PD-L1 checkpoint inhibitor herein). In one embodiment, the checkpoint inhibitor is at least one selected from the group consisting of PD- 1 inhibitors, PD-L 1 inhibitors, and PD-L2 inhibitors. In one embodiment, the checkpoint inhibitor may be a blocking antibody, such as a PD-1 blocking antibody, a PD-L1 blocking antibody, or a PD-L2 blocking antibody. Examples of a PD-1 blocking antibody include pembrolizumab, nivolumab, cemiplimab, and spartalizumab. Examples of a PD-L1 blocking antibody include atezolizumab, durvalumab, avelumab, sotiburafusp alpha, and palverafusp.

[0164] In one embodiment, the immune checkpoint inhibitor suitable for use in the aspects of the present disclosure is an antibody which targets PD-1 or PD-L1. In a preferred embodiment, the immune checkpoint inhibitor suitable for use in the aspects of the present disclosure is pembrolizumab.

[0165] The "Programmed Death-1 (PD-1)" receptor refers to an immuno-inhibitory receptor belonging to the CD28 family. PD-1 (also known as CD279) is expressed predominantly on previously activated T cells in vivo, and binds to two ligands, PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273). The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. "Programmed Death Ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD- 1. The term "PD-L1" as used herein includes human PD-L1 (hPD-Ll), variants, isoforms, and species homologs of hPD-Ll, such as macaque (cynomolgus monkey), African elephant, wild boar and mouse PD-L1 (cf„ e.g., Genbank accession no. NP_054862.1, XP_005581836, XP_003413533, XP_005665023 and NP_068693, respectively), and analogs having at least one common epitope withhPD-Ll. The term "PD-L2" as used herein includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, and analogs having at least one common epitope with hPD-L2. The ligands of PD-1 (PD-L1 and PD-L2) are expressed on the surface of antigen-presenting cells, such as dendritic cells or macrophages, and other immune cells. Binding of PD-1 to PD-L1 or PD-L2 results in downregulation of T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 are able to switch off T cells expressing PD-1 what results in suppression of the anticancer immune response. The interaction between PD-1 and its ligands results in a decrease in tumor infdtrating lymphocytes, a decrease in T cell receptor mediated proliferation, and immune evasion by the cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well.

[0166] Many of the immune checkpoints are regulated by interactions between specific receptor and ligand pairs, such as those described above. Thus, immune checkpoint proteins mediate immune checkpoint signaling. For example, checkpoint proteins directly or indirectly regulate T cell activation, T cell proliferation and / or T cell function. Cancer cells often exploit these checkpoint pathways to protect themselves from being attacked by the immune system. Hence, the function of checkpoint proteins is typically the regulation of T cell activation, T cell proliferation and / or T cell function. Immune checkpoint proteins thus regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Many of the immune checkpoint proteins belong to the B7:CD28 family or to the tumor necrosis factor receptor (TNFR) super family and, by binding to specific ligands, activate signaling molecules that are recruited to the cytoplasmic domain (Suzuki etal., 2016, Jap J Clin One, 46:191-203).

[0167] The term "VEGF" as used herein refers to vascular endothelial growth factor (VEGF) which is a member of the platelet-derived growth factor family of cystine-knot growth factors, i. e., a group of signal proteins involved in vasculogenesis and angiogenesis. Upon binding to tyrosine kinase receptors (VEGF receptors (VEGFRs)), the VEGFR dimerizes and becomes activated through transphosphorylation. Overexpression of VEGF can cause vascular diseases. Drugs which target VEGF (e.g., aflibercept, bevacizumab, ranibizumab, and pegaptanib) can inhibit VEGF and, thus, slow such diseases. In one embodiment, VEGF is human VEGF, in particular human VEGF comprising the amino acid sequence of SEQ ID NO: 176. In some embodiments, VEGF is human VEGF-A, in particular human VEGF-A comprising the amino acid sequence of SEQ ID NO: 177.

[0168] The term " VEGFR" as used herein refers to a VEGF receptor, which is tyrosine kinase receptor. Upon binding of a VEGF to the VEGFR, the VEGFR dimerizes and becomes activated through transphosphorylation."Enhancing T cell function" means to induce, cause or stimulate a T cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T cells. Examples of enhancing T cell function include increased secretion of y-interferon from CD8+ T cells, increased proliferation, increased antigen responsiveness (e.g., tumor clearance) relative to such levels before the intervention. In one embodiment, the level of enhancement is as least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, or more. Manners of measuring this enhancement are known to one of ordinary skill in the art.

[0169] The term "inhibitory nucleic acid" or "inhibitory nucleic acid molecule" as used herein refers to a nucleic acid molecule, e.g., DNA or RNA, that totally or partially reduces, inhibits, interferes with or negatively modulates one or more checkpoint proteins. Inhibitory nucleic acid molecules include, without limitation, oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers).

[0170] The term "oligonucleotide" as used herein refers to a nucleic acid molecule that is able to decrease protein expression, in particular expression of a checkpoint protein, such as the checkpoint proteins described herein. Oligonucleotides are short DNA or RNA molecules, typically comprising from 2 to 50 nucleotides. Oligonucleotides may be single -stranded or double-stranded. A checkpoint inhibitor oligonucleotide may be an antisense-oligonucleotide.

[0171] Antisense-oligonucleotides are single -stranded DNA or RNA molecules that are complementary to a given sequence, in particular to a sequence of the nucleic acid sequence (or a fragment thereof) of a checkpoint protein. Antisense RNA is typically used to prevent protein translation of mRNA, e.g., of mRNA encoding a checkpoint protein, by binding to said mRNA. Antisense DNA is typically used to target a specific, complementary (coding or non-coding) RNA. If binding takes place, such a DNA / RNA hybrid can be degraded by the enzyme RNase H. Moreover, morpholino antisense oligonucleotides can be used for gene knockdowns in vertebrates. For example, Kryczek et al., 2006 (J Exp Med, 203:871-81) designed B7-H4-specific morpholines that specifically blocked B7-H4 expression in macrophages, resulting in increased T cell proliferation and reduced tumor volumes in mice with tumor associated antigen (TAA)-specific T cells.

[0172] The terms "siRNA" or "small interfering RNA" or "small inhibitory RNA" are used interchangeably herein and refer to a double-stranded RNA molecule with a typical length of 20-25 base pairs that interferes with expression of a specific gene, such as a gene coding for a checkpoint protein, with a complementary nucleotide sequence. In one embodiment, siRNA interferes with mRNA therefore blocking translation, e.g., translation of an immune checkpoint protein. Transfection of exogenoussiRNA may be used for gene knockdown, however, the effect may be only transient, especially in rapidly dividing cells. Stable transfection may be achieved, e.g., by RNA modification or by using an expression vector. Useful modifications and vectors for stable transfection of cells with siRNA are known in the art. siRNA sequences may also be modified to introduce a short loop between the two strands resulting in a "small hairpin RNA" or "shRNA". shRNA can be processed into a functional siRNA by Dicer. shRNA has a relatively low rate of degradation and turnover. Accordingly, the immune checkpoint inhibitor may be a shRNA.

[0173] The term "aptamer" as used herein refers to a single-stranded nucleic acid molecule, such as DNA or RNA, typically in a length of 25-70 nucleotides that is capable of binding to a target molecule, such as a polypeptide. In one embodiment, the aptamer binds to an immune checkpoint protein such as the immune checkpoint proteins described herein. For example, an aptamer according to the disclosure can specifically bind to an immune checkpoint protein or polypeptide, or to a molecule in a signaling pathway that modulates the expression of an immune checkpoint protein or polypeptide. The generation and therapeutic use of aptamers is well known in the art (see, e.g., US 5,475,096).

[0174] The terms "small molecule inhibitor" or "small molecule" are used interchangeably herein and refer to a low molecular weight organic compound, usually up to 1000 daltons, that totally or partially reduces, inhibits, interferes with, or negatively modulates one or more checkpoint proteins as described above. Such small molecular inhibitors are usually synthesized by organic chemistry, but may also be isolated from natural sources, such as plants, fungi, and microbes. The small molecular weight allows a small molecule inhibitor to rapidly diffuse across cell membranes. For example, various A2AR antagonists known in the art are organic compounds having a molecular weight below 500 daltons.

[0175] The term "cell based therapy" refers to the transplantation of cells (e.g., T lymphocytes, dendritic cells, or stem cells) expressing an immune checkpoint inhibitor into a subject for the purpose of treating a disease or disorder (e.g., a cancer disease).

[0176] The term "oncolytic virus" as used herein refers to a virus capable of selectively replicating in and slowing the growth or inducing the death of a cancerous or hyperproliferative cell, either in vitro or in vivo, while having no or minimal effect on normal cells. An oncolytic virus for the delivery of an immune checkpoint inhibitor comprises an expression cassette that may encode an immune checkpoint inhibitor that is an inhibitory nucleic acid molecule, such as a siRNA, shRNA, an oligonucleotide, antisense DNA or RNA, an aptamer, an antibody or a fragment thereof or a soluble immune checkpoint protein or fusion. The oncolytic virus preferably is replication competent and the expression cassette is under the control of a viral promoter, e.g., synthetic early / late poxvirus promoter. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdoviruses (e.g., picomaviruses such as SenecaValley virus; SVV-001), coxsackievirus, parvovirus, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retroviruses (e.g., influenza viruses), measles virus, reovirus, Sinbis virus, vaccinia virus, as exemplarily described in WO 2017 / 209053 (including Copenhagen, Western Reserve, Wyeth strains), and adenovirus (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAdl, H101, AD5 / 3-D24-GMCSF). Generation of recombinant oncolytic viruses comprising a soluble form of an immune checkpoint inhibitor and methods for their use are disclosed in WO 2018 / 022831, herein incorporated by reference in its entirety. Oncolytic viruses can be used as attenuated viruses.

[0177] "Treatment cycle" is herein defined as the time period, within the effects of separate dosages of the therapeutic agent (e.g., the anti-PD-1 / PD-LlxVEGF / VEGFR binding agent or anti -cancer vaccine) cease to exist due to its pharmacodynamics, or in other words the time period after the subject's body is essentially cleared from the administrated therapeutic agent. Multiple small doses in a small time window, e.g. within 2-24 few hours, such as 2-12 hours or on the same day, might be equal to a larger single dose.

[0178] In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and / or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications. In one embodiment, "treatment" refers to the administration of an effective amount of a therapeutically active agent, such as of an anti-PD- 1 / PD-L IxVEGF / VEGFR binding agent and / or of an anti -cancer vaccine as disclosed herein, with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.

[0179] The resistance to, failure to respond to and / or relapse from treatment with a therapeutic agent (e.g., the anti-PD- 1 / PD-L IxVEGF / VEGFR binding agent or anti-cancer vaccine) of the present disclosure may be determined according to the Response Evaluation Criteria in Solid Tumors; version 1.1 (RECIST Criteria vl.l). The RECIST Criteria are set forth in the table below (LD: longest dimension).Table 4: Definition of Response (RECIST Criteria vl.l)

[0180] <

[0181] >

[0182] >

[0183] <

[0184]

[0185] The "best overall response" is the best response recorded from the start of the treatment until disease progression / recurrence (the smallest measurements recorded since the treatment started will be used as the reference for PD). Subjects with CR or PR are considered to be objective response. Subjects with CR, PR or SD are considered to be in disease control. Subjects with NE are counted as non-responders. The best overall response is the best response recorded from the start of the treatment until disease progression / recurrence (the smallest measurements recorded since the treatment started will be used as the reference for PD). Subjects with CR, PR or SD are considered to be in disease control. Subjects with NE are counted as non-responders.

[0186] The objective response rate (ORR) is the percentage of all subjects in a study or treatment group who have either a partial or complete response to the treatment. The ORR can be calculated by adding the numbers of subjects having CR and the numbers of subjects having PR and dividing the resulting sum by the total number of subjects in the treatment group. The ORRevai, z.e., the ORR of all evaluable subjects in a study or treatment group, is the percentage of all evaluable subjects in a study or treatment group who have either a partial or complete response to the treatment.

[0187] The disease control rate (DCR) is the percentage of all subjects in a study or treatment group who have either a complete response, a partial response, or a stable disease to the treatment (CR, PR or SD). The DCR can be calculated by adding the numbers of subjects having CR, the numbers of subjects having PR, and the numbers of subjects having SD and dividing the resulting sum by the total number of subjects in the treatment group. The DCRevai, z.e., the DCR of all evaluable subjects in a study ortreatment group, is the percentage of all evaluable subjects in a study or treatment group who have either a complete response, a partial response, or a stable disease to the treatment (CR, PR or SD).

[0188] "Duration of response (DOR)" only applies to subjects whose confirmed best overall response is CR or PR and is defined as the time from the first documentation of objective tumor response (CR or PR) to the date of first PD or death due to underlying cancer.

[0189] "Progression-free survival (PFS)" is defined as the number of days from Day 1 in Cycle 1 to the first documented progression or death due to any cause.

[0190] "Overall survival (OS)" is defined as the number of days from Day 1 in Cycle 1 to death due to any cause. If a subject is not known to have died, then OS will be censored at the latest date the subject was known to be alive (on or before the cut-off date).

[0191] In the context of the present disclosure, the term "treatment regimen" refers to a structured treatment plan designed to improve and maintain health.

[0192] The term "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a therapeutic agent, such as an antibody, or anti -cancer vaccine, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used. In case that unwanted side effects occur in a patient with a dose, lower doses (or effectively lower doses achieved by a different, more localized route of administration) may be used.

[0193] The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B-cell surface, resulting in T- and B-cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In one embodiment, the macrophages are splenic macrophages.The term "dendritic cell" (DC) refers to another subtype of phagocytic cells belonging to the class of antigen presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T-cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T-cell activation such as CD80, CD86, and CD40 greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node. Here they act as antigen-presenting cells and activate helper T cells and killer T cells as well as B cells by presenting them antigens, alongside non-antigen specific co-stimulatory signals. Thus, dendritic cells can actively induce a T-cell- or B-cell-related immune response. In one embodiment, the dendritic cells are splenic dendritic cells.

[0194] The term "antigen presenting cell" (APC) is a cell of a variety of cells capable of displaying, acquiring, and / or presenting at least one antigen or antigenic fragment on (or at) its cell surface. Antigen-presenting cells can be distinguished in professional antigen presenting cells and non-professional antigen presenting cells.

[0195] The term "professional antigen presenting cells" relates to antigen presenting cells which constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen presenting cell, the antigen presenting cell produces a co-stimulatory molecule inducing activation of the T cell. Professional antigen presenting cells comprise dendritic cells and macrophages.

[0196] The term "non-professional antigen presenting cells" relates to antigen presenting cells which do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines such as interferon-gamma. Exemplary, non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or vascular endothelial cells.

[0197] The term "disease involving an antigen" or "disease involving an epitope" refers to any disease which implicates an antigen or epitope, e.g., a disease which is characterized by the presence of an antigen orepitope. The disease involving an antigen or epitope can be a cancer disease or simply cancer. As mentioned above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen and the epitope may be derived from such antigen.

[0198] As used herein, the term "cancer disease" or "cancer" refers to or describes the physiological condition in an individual that is typically characterized by unregulated cell growth and typically a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and / or migration. By "cancer cell" is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancers include bone cancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skin cancer (e.g., melanoma, such as mucosal, cutaneous or intraocular melanoma), cancer of the head or neck, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer (e.g., triple negative breast cancer), prostate cancer, uterine cancer, carcinoma of the sexual and reproductive organs, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma. Particular forms of cancer that can be treated by the compositions and methods described herein include lung cancer (in particular, non-small cell lung cancer (NSCLC), e.g., advanced unresectable Stage III or metastatic Stage IV NSCLC), skin cancer (in particular, melanoma, e.g., cutaneous melanoma), and HPV-positive cancer (in particular, head and neck squamous cell carcinoma (HNSCC) or cervical cancer).

[0199] The term "cancer" according to the present disclosure also comprises cancer metastases. By "metastasis" is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor, i. e. a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the present disclosure relates to "distant metastasis" which relates to a metastasis which is remote from the primary tumor and the regional lymph node system.The term "triple negative breast cancer" or TNBC as used herein refers to a breast cancer type in which there is neither expression of the estrogen and progesterone receptor nor an overexpression or gene amplification of the human epidermal growth factor receptor 2 (HER2). Typically, TNBC is HER2 negative and has <1% expression of estrogen receptors and progesterone receptors by immunostaining. I.e., this breast cancer type is not affected by the hormones estrogen or progesterone or by the HER2 gene.

[0200] Terms such as "reduce", "inhibit", "interfere", and "negatively modulate" as used herein means an overall decrease or the ability to cause an overall decrease, for example, of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, or about 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, i.e. a reduction to zero or essentially to zero.

[0201] Terms such as "increase" or "enhance" in one embodiment relate to an increase or enhancement by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0202] "Physiological pH" as used herein refers to a pH of about 7.5.

[0203] The term "ionic strength" refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges. Thus, ionic strength I is represented mathematically by the formula

[0204]

[0205] in which c is the molar concentration of a particular ionic species and z the absolute value of its charge. The sum E is taken over all the different kinds of ions (i) in solution. According to the disclosure, the term "ionic strength" in one embodiment relates to the presence of monovalent ions. Regarding the presence of divalent ions, in particular divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is in one embodiment sufficiently low so as to prevent degradation of RNA. In one embodiment, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of the phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent ions is 20 pM or less. In one embodiment, there are no or essentially no free divalent ions.As used in the present disclosure, "% by weight" refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).

[0206] The term "freezing" relates to the solidification of a liquid, usually with the removal of heat.

[0207] The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used herein interchangeably.

[0208] The term "spray-drying" refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.

[0209] The term "cryoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the freezing stages.

[0210] The term "lyoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.

[0211] The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.

[0212] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes. In one embodiment, the term "particle" relates to a micro- or nanosized structure, such as a micro- or nano-sized compact structure.

[0213] In particulate formulation, it is possible that each RNA species (e.g. RNA encoding the different vaccine antigens) is separately formulated as an individual particulate formulation. In that case, each individual particulate formulation will comprise one RNA species. The individual particulate formulations may be present as separate entities, e.g. in separate containers. Such formulations are obtainable by providing each RNA species separately (typically each in the form of an RNA-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific RNA species that is being provided when the particles are formed (individual particulate formulations). In one embodiment, a composition such as a pharmaceutical composition comprises more than one individual particle formulation. Respective pharmaceutical compositions arereferred to as mixed particulate formulations. Mixed particulate formulations according to the disclosure are obtainable by forming, separately, individual particulate formulations, as described above, followed by a step of mixing of the individual particulate formulations. By the step of mixing, a formulation comprising a mixed population of RNA-containing particles is obtainable (for illustration: e.g. a first population of particles may contain RNA encoding a vaccine antigen, and a second formulation of particles may contain RNA encoding a different vaccine antigen). Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations. Alternatively, it is possible that different RNA species of the pharmaceutical composition (e.g. RNA encoding a vaccine antigen and RNA encoding a different vaccine antigen) are formulated together as a combined particulate formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of different RNA species together with a particle -forming agent, thereby allowing the formation of particles. As opposed to a mixed particulate formulation, a combined particulate formulation will typically comprise particles which comprise more than one RNA species. In a combined particulate composition different RNA species are typically present together in a single particle.

[0214] As used in the present disclosure, "nanoparticle" refers to a particle comprising RNA and at least one cationic lipid and having an average diameter suitable for intravenous administration.

[0215] The term "average diameter" refers to the mean hydrodynamic diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called ZaVerage with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here "average diameter", "diameter" or "size" for particles is used synonymously with this value of the ZaVerage.

[0216] The term "polydispersity index" is used herein as a measure of the size distribution of an ensemble of particles, e.g., nanoparticles. The polydispersity index is calculated based on dynamic light scattering measurements by the so-called cumulant analysis.

[0217] The term "ethanol injection technique" refers to a process, in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example lipid vesicle formation such as liposome formation. Generally, the RNA lipoplex particles described herein are obtainable by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in one embodiment, formed as follows: an ethanol solution comprising lipids, such as cationic lipids like DOTMA and additional lipids, is injected into an aqueous solution under stirring. In one embodiment, the RNA lipoplex particles described herein are obtainable without a step of extrusion.The term "extruding" or "extrusion" refers to the creation of particles having a fixed, cross-sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores.

[0218] As used herein, an "instructional material" or "instructions" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods described herein. The instructional material of the kit of the disclosure may, for example, be affixed to a container which contains the compositions of the disclosure or be shipped together with a container which contains the compositions. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.

[0219] As used herein, the term "vaccine" refers to a composition that induces an immune response upon inoculation into a subject. In some embodiments, the induced immune response provides therapeutic immunity.

[0220] The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In one embodiment, a "recombinant object" in the context of the present disclosure is not occurring naturally.

[0221] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but that may be discovered and / or isolated in the future from a natural source.

[0222] According to the present disclosure, the term "peptide" comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "protein" refers to large peptides, in particular peptides having at least about 151 amino acids, but the terms "peptide" and "protein" are used herein usually as synonyms. A preferred example of a peptide is an antigen, such as tumor-associated antigen.The term "linker peptide" according to the disclosure relates to a peptide added between two peptide domains such as epitopes or vaccine sequences to connect said peptide domains. There is no particular limitation regarding the linker sequence. However, it is preferred that the linker sequence reduces steric hindrance between the two peptide domains, is well translated, and supports or allows processing of the epitopes. Furthermore, the linker peptide should have no or only little immunogenic sequence elements. Linker peptides preferably should not create non-endogenous epitopes like those generated from the junction suture between adjacent epitopes, which might generate unwanted immune reactions. Therefore, the vaccine antigens should preferably contain linker sequences which are able to reduce the number of unwanted MHC binding junction epitopes. Hoyt et al. (EMBO J. 25(8), 1720-9, 2006) and Zhang et al. (J. Biol. Chem., 279(10), 8635-41, 2004) have shown that glycine-rich sequences impair proteasomal processing and thus the use of glycine rich linker sequences act to minimize the number of linker-contained peptides that can be processed by the proteasome. Furthermore, glycine was observed to inhibit a strong binding in MHC binding groove positions (Abastado etal., J. Immunol. 151(7), 3569- 75, 1993). Schlessinger et al. (Proteins, 61(1), 115-26, 2005) had found that amino acids glycine and serine included in an amino acid sequence result in a more flexible protein that is more efficiently translated and processed by the proteasome, enabling better access to the encoded epitopes. The linker peptide each may comprise 3 or more, 6 or more, 9 or more, 10 or more, 15 or more, 20 or more and preferably up to 50, up to 45, up to 40, up to 35 or up to 30 amino acids. Preferably the linker peptide is enriched in glycine and / or serine amino acids. Preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the amino acids of the linker peptide are glycine and / or serine. In one preferred embodiment, a linker peptide is substantially composed of the amino acids glycine and serine. In one embodiment, the linker peptide comprises the amino acid sequence (GGS)a(GSS)b(GGG)c(SSG)d(GSG)e, wherein a, b, c, d and e is independently a number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and wherein a + b + c + d + e are different from 0 and preferably are 2 or more, 3 or more, 4 or more or 5 or more. In one embodiment, the linker peptide comprises the sequence GGSGGGGSG. In one embodiment, the linker peptide comprises the sequence GGSGGGGSGG. In one embodiment, the linker peptide comprises the sequence GSSGGGGSPGGGSS.

[0223] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term "portion" thereof may designate a continuous or a discontinuous fraction of said structure.

[0224] The terms "part" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure. When used in context of a composition, the term "part" means a portion of thecomposition. For example, a part of a composition may any portion from 0.1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.

[0225] "Fragment", with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3 '-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5'-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.

[0226] According to the present disclosure, a part or fragment of a peptide or protein preferably has at least one functional property of the peptide or protein from which it has been derived. Such functional properties comprise a pharmacological activity, the interaction with other peptides or proteins, an enzymatic activity, the interaction with antibodies, and the selective binding of nucleic acids. E.g.. a pharmacological active fragment of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the fragment has been derived. A part or fragment of a peptide or protein preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50, consecutive amino acids of the peptide or protein. A part or fragment of a peptide or protein preferably comprises a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the peptide or protein.

[0227] By "variant", with reference to an amino acid sequence (peptide or protein), herein is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid modifications, and preferably from 1 to about 10 or from 1 to about 5 (such a 1, 2, 3, 4, or 5) amino acid modifications compared to the parent.

[0228] By "wild type" or "WT" or "native" herein is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.Preferably the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: meedle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0229] The disclosure describes nucleic acid sequences and amino acid sequences having a certain degree of similarity or identity to a given nucleic acid sequence or amino acid sequence, respectively (a reference sequence). "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0230] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

[0231] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0232] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference amino acid sequence consists of 200 amino acid residues, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acid residues, in some embodiments continuous amino acid residues. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0233] Nucleic acid sequences or amino acid sequences having a particular degree of similarity or identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of said given sequence, e.g., and in some instances, are functionally equivalent to said given sequence. One important property includes an immunogenic property, in particular when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a particular degree of similarity or identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0234] Homologous amino acid sequences exhibit according to the present disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0235] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparingpeptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

[0236] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, z.e., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response. In one embodiment, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0237] An amino acid sequence (peptide, protein or polypeptide) "derived from" a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

[0238] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as,for example, a host cell. In a preferred embodiment, the anti-cancer vaccine used in the present disclosure is in substantially purified form.

[0239] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid. The term "transfection" relates to the introduction of nucleic acids, in particular RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and / or an organism of a patient. According to the present disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection. Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.

[0240] According to the present disclosure, an analog of a peptide or protein is a modified form of said peptide or protein from which it has been derived and has at least one functional property of said peptide or protein. E.g., a pharmacological active analog of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the analog has been derived. Such modifications include any chemical modification and comprise single or multiple substitutions, deletions and / or additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids and / or proteins or peptides. In one embodiment, "analogs" of proteins or peptides include those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective / blocking groups, proteolytic cleavage or binding to an antibody or to another cellular ligand. The term "analog" also extends to all functional chemical equivalents of said proteins and peptides.

[0241] "Activation" or "stimulation", as used herein, refers to the state of an immune effector cell such as a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" (such as "activated T cells") refers to, among other things, immune effector cells (such as T cells) that are undergoing cell division.The term "priming" refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.

[0242] The term "clonal expansion" or "expansion" refers to a process wherein a specific entity is multiplied. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which immune effector cells (such as lymphocytes) are stimulated by an antigen, proliferate, and the specific immune effector cell (such as the specific lymphocyte) recognizing said antigen is amplified. Preferably, clonal expansion leads to differentiation of the immune effector cells (such as lymphocytes).

[0243] An "antigen" according to the present disclosure covers any substance that will elicit an immune response and / or any substance against which an immune response or an immune mechanism such as a cellular response is directed. This also includes situations wherein the antigen is processed into antigen peptides and an immune response or an immune mechanism is directed against one or more antigen peptides, in particular if presented in the context of MHC molecules. In particular, an "antigen" relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T-cells). According to the present disclosure, the term "antigen" comprises any molecule which comprises at least one epitope, such as a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule which, optionally after processing, induces an immune reaction, which is preferably specific for the antigen (including cells expressing the antigen). In one embodiment, an antigen is a disease-associated antigen, such as a tumor antigen, or an epitope derived from such antigen.

[0244] According to the present disclosure, any suitable antigen may be used, which is a candidate for an immune response, wherein the immune response may be both a humoral as well as a cellular immune response. In the context of some embodiments of the present disclosure, the antigen is preferably presented by a cell, preferably by an antigen presenting cell, in the context of MHC molecules, which results in an immune response against the antigen. An antigen is preferably a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may be a tumor antigen.

[0245] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule which contains epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include antigens associated with cancer, typically tumors, such as tumor antigens.In a preferred embodiment, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular those which primarily occur intracellularly or as surface antigens of tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, z.e., an antigen derived from a pathogen, e.g., from a virus, bacterium, unicellular organism, or parasite, for example a viral antigen such as viral ribonucleoprotein or coat protein. In particular, the antigen should be presented by MHC molecules which results in modulation, in particular activation of cells of the immune system, preferably CD4+and CD8+lymphocytes, in particular via the modulation of the activity of a T-cell receptor.

[0246] The terms "tumor antigen" and "tumor-associated antigen" are used herein synonymously and refer to a constituent of cancer cells which may be derived from the cytoplasm, the cell surface or the cell nucleus. In particular, these terms refer to those antigens which are produced intracellularly or as surface antigens on tumor cells. According to the present disclosure, a tumor antigen preferably comprises any antigen which is characteristic for tumors or cancers as well as for tumor or cancer cells with respect to type and / or expression level.

[0247] A tumor antigen disclosed herein may be CLDN6 (SEQ ID NO: 1), HPV E6 (e.g., HPV 16 E6 (SEQ ID NO: 37), HPV 18 E6 (SEQ ID NO: 45), HPV 31 E6 (SEQ ID NO: 53), HPV 33 E6 (SEQ ID NO: 61), HPV 45 E6 (SEQ ID NO: 69), HPV 58 E6 (SEQ ID NO: 77)), HPV E7 (e.g., HPV 16 E7 (SEQ ID NO: 41), HPV 18 E7 (SEQ ID NO: 49), HPV 31 E7 (SEQ ID NO: 57), HPV 33 E7 (SEQ ID NO: 65), HPV 45 E7 (SEQ ID NO: 73), HPV 58 E7 (SEQ ID NO: 81)), KK-LC-1 (SEQ ID NO: 5), MAGE-A3 (SEQ ID NO: 9), MAGE-A4 (SEQ ID NO: 13), MAGE-CI (SEQ ID NO: 21), NY-ESO-1 (SEQ ID NO: 25), PRAME (SEQ ID NO: 17), TPTE (SEQ ID NO: 29), or tyrosinase (SEQ ID NO: 33).

[0248] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies, T cells or B cells, in particular when presented in the context of MHC molecules. In one embodiment, "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked or covered by the specifically antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen-binding peptide).An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably between about 5 and about 100, preferably between about 5 and about 50, more preferably between about 8 and about 0, most preferably between about 10 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that the epitope in the context of the present disclosure is a T cell epitope.

[0249] Terms such as "epitope", "fragment of an antigen", "immunogenic peptide" and "antigen peptide" are used interchangeably herein and preferably relate to an incomplete representation of an antigen which is preferably capable of eliciting an immune response against the antigen or a cell expressing or comprising and preferably presenting the antigen. Preferably, the terms relate to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (z.e., specifically bound) by a T cell receptor, in particular if presented in the context of MHC molecules. Certain preferred immunogenic portions bind to an MHC class I or class II molecule. The term "epitope" refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system. For example, the epitope may be recognized by T cells, B cells or antibodies. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is between about 10 and about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0250] The term "T cell epitope" refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. The proteins encoded by the MHC are expressed on the surface of cells, and display both selfantigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell. In the case of class I MHC / peptide complexes, the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.The term "immunologically equivalent" means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of antigens or antigen variants. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence, if said amino acid sequence when exposed to T cells binding to the reference amino acid sequence or cells expressing the reference amino acid sequence induces an immune reaction having a specificity of reacting with the reference amino acid sequence, in particular stimulation, priming and / or expansion of T cells. Thus, a molecule which is immunologically equivalent to an antigen exhibits the same or essentially the same properties and / or exerts the same or essentially the same effects regarding the stimulation, priming and / or expansion of T cells as the antigen to which the T cells are targeted.

[0251] In certain embodiments of the present disclosure, the RNA (such as mRNA) encodes at least one epitope. In certain embodiments, the epitope is derived from a tumor antigen as described herein.

[0252] The peptide and protein antigen can be 2 to 100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

[0253] The peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibodies and T cell responses to the peptide or protein.

[0254] In one embodiment, vaccine antigen, z.e., an antigen whose inoculation into a subject induces an immune response, is recognized by an immune effector cell. Preferably, the vaccine antigen if recognized by an immune effector cell is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and / or expansion of the immune effector cell carrying an antigen receptor recognizing the vaccine antigen. In the context of the embodiments of the present disclosure, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen presenting cell. In one embodiment, an antigen is presented by a diseased cell (such as tumor cell or an infected cell). In one embodiment, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In one embodiment, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and / or expansion of said T cells. In one embodiment, binding of a TCR when expressed by T cells and / orpresent on T cells to an antigen presented on diseased cells results in cytolysis and / or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.

[0255] In one embodiment, an antigen receptor is an antibody or B cell receptor which binds to an epitope in an antigen. In one embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

[0256] The term "expressed on the cell surface" or "associated with the cell surface" means that a molecule such as an antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by antibodies located outside the cell. In this context, a part is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0257] "Cell surface" or "surface of a cell" is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by, e.g., antigen-specific antibodies added to the cells.

[0258] The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or a fragment thereof.

[0259] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+T cells) and cytotoxic T cells (CTLs, CD8+T cells) which comprise cytolytic T cells. The term "antigenspecific T cell" or similar terms relate to a T cell which recognizes the antigen to which the T cell is targeted, in particular when presented on the surface of antigen presenting cells or diseased cells such as cancer cells in the context of MHC molecules and preferably exerts effector functions of T cells. T cells are considered to be specific for antigen if the cells kill target cells expressing an antigen. T cell specificity may be evaluated using any of a variety of standard techniques, for example, within a chromium release assay or proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-y) can be measured. In certain embodiments of the present disclosure, the RNA (in particular mRNA) encodes at least one epitope.The term "target" shall mean an agent such as a cell or tissue which is a target for an immune response such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, z.e., a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell expressing an antigen and preferably presenting said antigen with class I MHC.

[0260] "Antigen processing" refers to the degradation of an antigen into processing products which are fragments of said antigen (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, preferably antigen-presenting cells to specific T-cells.

[0261] By "antigen-responsive CTL" is meant a CD8+T-cell that is responsive to an antigen or a peptide derived from said antigen, which is presented with class I MHC on the surface of antigen presenting cells.

[0262] According to the present disclosure, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFNy and TNFa, up-regulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen expressing target cells. CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness.

[0263] The terms "immune response" and "immune reaction" are used herein interchangeably in their conventional meaning and refer to an integrated bodily response to an antigen or a cell expressing an antigen and preferably refers to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the term "immune response to" or "immune response against" with respect to an agent such as an antigen, cell or tissue, relates to an immune response such as a cellular response directed against the agent. An immune response may comprise one or more reactions selected from the group consisting of developing antibodies against one or more antigens and expansion of antigen-specific T-lymphocytes, preferably CD4+and CD8+T-lymphocytes, more preferably CD8+T-lymphocytes, which may be detected in various proliferation or cytokine production tests in vitro.

[0264] The terms "inducing an immune response" and "eliciting an immune response" and similar terms in the context of the present disclosure refer to the induction of an immune response, preferably the induction of a cellular immune response, a humoral immune response, or both. The immune response may be protective / preventive / prophylactic and / or therapeutic. The immune response may be directed against any immunogen or antigen or antigen peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (such as influenza virus (A, B, or C), CMV or RSV)). "Inducing" in this context may mean that there was no immune response against a particularantigen or pathogen before induction, but it may also mean that there was a certain level of immune response (e.g., a basal level of immune response) against a particular antigen or pathogen before induction and after induction said immune response is enhanced. Thus, "inducing the immune response" in this context also includes "enhancing the immune response". Preferably, after inducing an immune response in an individual, said individual is protected from developing a disease such as an infectious disease or a cancerous disease or the disease condition is ameliorated by inducing an immune response.

[0265] The term "cellular immune response" includes, without limitation, a cellular response directed to cells expressing an antigen and being characterized by presentation of an antigen with class I or class II MHC molecule. The cellular response relates to T lymphocytes, which may be classified as helper T cells (also termed CD4+ T cells) that play a central role by regulating the immune response or killer cells (also termed cytotoxic T cells, CD8+ T cells, or CTLs) that induce apoptosis in infected cells or cancer cells. In one embodiment, administering a pharmaceutical composition as described herein involves stimulation of an anti-tumor CD8+ T-cell response against cancer cells expressing one or more tumor antigens. In a specific embodiment, the tumor antigens are presented with class I MHC molecule. The term "humoral immune response" refers to a process in living organisms wherein antibodies are produced in response to agents and organisms, which they ultimately neutralize and / or eliminate. The specificity of the antibody response is mediated by T and / or B cells through membrane-associated receptors that bind antigen of a single specificity. Following binding of an appropriate antigen and receipt of various other activating signals, B lymphocytes divide, which produces memory B cells as well as antibody secreting plasma cell clones, each producing antibodies that recognize the identical antigenic epitope as was recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and the resulting escalation in antibody response when re-exposed to a specific antigen.

[0266] The term "immunotherapy" relates to the treatment of a disease or condition by inducing, or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

[0267] The terms "vaccination" and "immunization" describe the process of administering one or more immunogen(s) or antigen(s) or derivatives thereof, in particular in the form of RNA (especially mRNA) coding therefor, as described herein to an individual and inducing an immune response against said one or more immunogen(s) or antigen(s) or cells characterized by presentation of said one or more immunogen(s) or antigen(s). These terms may also relate to the process of treating an individual for therapeutic or prophylactic reasons, e.g., for treating cancer.

[0268] The term "co-administered" or "co-administration" or the like as used herein refers to administration of two or more agents concurrently, simultaneously, or essentially at the same time, either as part of asingle formulation or as multiple formulations that are administered by the same or different routes. "Essentially at the same time" as used herein means within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 6 hours period of each other.

[0269] By "cell characterized by presentation of an antigen" or "cell presenting an antigen" or "MHC molecules which present an antigen on the surface of an antigen presenting cell" or similar expressions is meant a cell such as a diseased cell, in particular a tumor cell or an infected cell, or an antigen presenting cell presenting the antigen or an antigen peptide, either directly or following processing, in the context of MHC molecules, preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules.

[0270] In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA (especially mRNA) may be translated into peptide or protein. According to the present disclosure, the term "transcription" comprises "in vitro transcription" .

[0271] The term "in vitro transcription" or "IVT" as used herein means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)). The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Preferably, cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present disclosure encompassed by the term "vector". According to the present disclosure, the RNA used herein preferably is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription according to the disclosure is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™). For providing modified RNA (such as mRNA), correspondingly modified nucleotides, such as modified naturally occurringnucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription.

[0272] The term "uracil," as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is:

[0273]

[0274] The term "uridine," as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is:

[0275]

[0276] UTP (uridine 5 ’-triphosphate) has the following structure:

[0277]

[0278] Pseudo-UTP (pseudouridine 5 ’-triphosphate) has the following structure:

[0279]

[0280] "Pseudouridine" is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0281] Another exemplary modified nucleoside is N1 -methyl -pseudouridine (m IT). which has the structure:

[0282]

[0283] N1 -methyl -pseudo-UTP has the following structure:

[0284]

[0285] Another exemplary modified nucleoside is 5 -methyl -uridine (m5U), which has the structure:

[0286]

[0287] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence. With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0288] The term "optional" or "optionally" as used herein means that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0289] As used herein, the terms "linked", "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.

[0290] The term "disease" (also referred to as "disorder" herein) refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality.

[0291] The term "therapeutic treatment" or "treatment" or "treating" relates to any treatment which improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and / or decrease the recurrence in an individual who currently has or who previously has had a disease.

[0292] The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably. Similarly, the term "method for preventing" in the context of progression of a disease, such as progression of a tumor or cancer, relates to any method that is intended to prevent the disease from progressing in an individual.

[0293] The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other nonmammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer) but may or may not have the disease or disorder, or may have a need for prophylactic intervention such as vaccination, or may have a need for interventions such as by protein replacement. In many embodiments, the individual is a human being. Unlessotherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient".

[0294] The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.

[0295] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 24 (such as 1 to 12 or 1 to 10) carbon atoms, z.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms, abbreviated as C1.24 alkyl, (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, abbreviated as CMO alkyl), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1 -methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl -propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2 -ethyl -hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. A "substituted alkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent, as specified herein. Examples of a substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0296] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene comprises from 1 to 24 (such as 1 to 12 or 1 to 10) carbon atoms, z.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (z.e., 1,1 -ethylene, 1,2-ethylene), propylene (z.e., 1,1 -propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3-propylene), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1,1 -iso-butylene, 1,2-iso-butylene, and 1,3 -iso-butylene), the pentylene isomers (e.g., 1,1 -pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-iso-pentylene, 1,1 -sec-pentyl, 1,1 -neo-pentyl), the hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1.3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1 -isohexylene), the heptylene isomers (e.g., 1,1 -heptylene, 1,2-heptylene, 1,3-heptylene, 1,4-heptylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1 -isoheptylene), the octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1.4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1 -isooctylene), and the like. The straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also bedesignated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene). Generally, instead of using the ending "ylene" for alkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2-butylene can also be called butan-l,2-diyl). A "substituted alkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent, as specified herein.

[0297] The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), z.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 24 (such as 2 to 12 or 2 to 10) carbon atoms, z.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 12, abbreviated as C2-12 alkenyl, (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carboncarbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (z.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3 -hexenyl, 4-hexenyl, 5-hexenyl, 1 -heptenyl, 2-heptenyl, 3 -heptenyl, 4-heptenyl, 5 -heptenyl, 6-heptenyl, 1 -octenyl, 2-octenyl, 3 -octenyl, 4-octenyl, 5 -octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. A "substituted alkenyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom isreplaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0298] The term "alkenylene" refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (such as 1 to 4), z.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 24 (such as 2 to 12 or 2 to 10) carbon atoms, z.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethen- 1,2-diyl, vinylidene (also called ethenylidene), 1 -propen- 1,2-diyl, 1 -propen- 1,3 -diyl, 1 -propen-2, 3 -diyl, allylidene, 1-buten- 1,2-diyl, l-buten-l,3-diyl, l-buten-l,4-diyl, l-buten-2,3-diyl, l-buten-2,4-diyl, 1-buten-3,4-diyl, 2-buten- 1,2-diyl, 2-buten-l,3-diyl, 2-buten-l,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4-diyl, 2-buten-3,4-diyl, and the like. If an alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. A "substituted alkenylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0299] The term "alkynyl" refers to a a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. Alkynyl groups can optionally have one or more carbon carbon double bonds. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon triple bonds. Preferably, the alkynyl group comprises from 2 to 24 (such as 2 to 12 or 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkynyl group comprises from 2 to 12, abbreviated as C2-12 alkynyl, (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon triple bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. A "substituted alkynyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0300] The terms "carbocycle" and "carbocyclic ring" are used herein synonymously and represent cyclic nonaromatic versions of "alkyl" and "alkenyl" with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, z.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary carbocycle groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl. The carbocycle group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). A "substituted carbocycle" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a carbocycle group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the carbocycle group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0301] The term "carbocyclene" means a diradical of a carbocycle and, thus, refers to a diradical of a saturated or unsaturated, non-aromatic cyclic hydrocarbon with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, z.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary carbocyclene groups include cyclopropylene, cyclopropenylene, cyclobutylene, cyclobutenylene, cyclopentylene, cyclopentenylene, cyclohexylene, cyclohexenylene, cycloheptylene, cycloheptenylene, cyclooctylene, cyclooctenylene, cyclononylene, cyclononenylene, cylcodecylene, cylcodecenylene, and adamantylene. The carbocyclene group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). A "substituted carbocyclene" means that one or more (such as 1 to the maximum numberof hydrogen atoms bound to a carbocyclene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the carbocyclene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0302] The term "cycloalkyl" represents cyclic non-aromatic versions of "alkyl" with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, z.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary carbocycle groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, and adamantyl. The cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). A "substituted cycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0303] The term "cycloalkylene" represents cyclic non-aromatic versions of "alkylene" and is ageminal, vicinal or isolated diradical. In certain embodiments, the cycloalkylene (i) is monocyclic or polycyclic (such as bi- or tricyclic) and / or (ii) is 3- to 14-membered (z.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 3- to 12-membered or 3- to 10-membered). In one embodiment the cycloalkylene is a mono-, bi- or tricyclic 3- to 14-membered (z.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 3- to 12-membered or 3- to 10-membered) cycloalkylene. Generally, instead of using the ending "ylene" for cycloalkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2-cyclopropylene can also be called cyclopropan-l,2-diyl). Exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantanylene (e.g.. tricyclo|3.3. 1. l3 7|dccan-2.2-diyl). A "substituted cycloalkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0304] The term "cycloalkenylene" represents cyclic non-aromatic versions of "alkenylene" and is a geminal, vicinal or isolated diradical. Generally, the maximal number of carbon-carbon double bonds in thecycloalkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the cycloalkenylene group by 2 and, if the number of carbon atoms in the cycloalkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an cycloalkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the cycloalkenylene group has 1 to 6 (such as 1 to 4), z.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. In certain embodiments, the cycloalkenylene (i) is monocyclic or polycyclic (such as bi-or tricyclic) and / or (ii) is 3- to 14-membered (z.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 3- to 12-membered or 3- to 10-membered). In one embodiment the cycloalkenylene is a mono-, bi- or tricyclic 3- to 14-membered (z.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 3- to 12-membered or 3- to 10-membered) cycloalkenylene. Exemplary cycloalkenylene groups include cyclohexenylene, cycloheptenylene, cyclopropenylene, cyclobutenylene, cyclopentenylene, and cyclooctenylene. A "substituted cycloalkenylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0305] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes. A "substituted aryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the aryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein. Examples of a substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (z.e., 2-, 3-, or 4-methoxyphenyl).

[0306] The term "arylene" refers to a diradical of an aryl group and, thus, refers to a diradical of an aromatic cyclic hydrocarbon with preferably 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenylene) or two or more condensed rings (e.g., naphthylene). Arylene does not encompass fullerenes. A "substituted arylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an arylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or upto 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the arylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0307] The term "heteroaryl" or "heteroaromatic ring" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Alternatively, it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include fiiranyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, IH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. A "substituted heteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0308] The term "heteroarylene" refers to a diradical of an heteroaryl group and, thus, refers to an arylene group as defined above in which one or more carbon atoms in the arylene group are replaced by heteroatoms of O, S, or N. Preferably, heteroarylene refers to diradical of a five or six-membered aromatic monocyclic ring, wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Alternatively, it means a diradical of an aromatic bicyclic or tricyclic ring system, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroarylene group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroarylene groupsinclude furanylene, thienylene, oxazolylene, isoxazolylene, oxadiazolylene, pyrrolylene, imidazolylene, pyrazolylene, triazolylene, tetrazolylene, thiazolylene, isothiazolylene, thiadiazolylene, pyridylene, pyrimidinylene, pyrazinylene, triazinylene, benzofuranylene, indolylene, isoindolylene, benzothienylene, IH-indazolylene, benzimidazolylene, benzoxazolylene, indoxazinylene, benzisoxazolylene, benzothiazolylene, benzisothiazolylene, benzotriazolylene, quinolinylene, isoquinolinylene, benzodiazinylene, quinoxalinylene, quinazolinylene, benzotriazinylene, pyridazinylene, phenoxazinylene, thiazolopyridinylene, pyrrolothiazolylene, phenothiazinylene, isobenzofuranylene, chromenylene, xanthenylene, pyrrolizinylene, indolizinylene, indazolylene, purinylene, quinolizinylene, phthalazinylene, naphthyridinylene, cinnolinylene, pteridinylene, carbazolylene, phenanthridinylene, acridinylene, perimidinylene, phenanthrolinylene, and phenazinylene. Exemplary 5- or 6-memered heteroarylene groups include furanylene, thienylene, oxazolylene, isoxazolylene, oxadiazolylene, pyrrolylene, imidazolylene (e.g., 2-imidazolylene), pyrazolylene, triazolylene, tetrazolylene, thiazolylene, isothiazolylene, thiadiazolylene, pyridylene (e.g., 4-pyridylene), pyrimidinylene, pyrazinylene, triazinylene, and pyridazinylene. A "substituted heteroarylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroarylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroarylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0309] The terms "heterocycle", "heterocyclyl", and "heterocyclic ring" are used herein synonymously and mean a carbocyclic group as defined above in which one or more (such as 1, 2, 3, or 4) carbon atoms in the carbocyclic group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorous, or sulfur, preferably O, S, or N. A heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of the heterocyclyl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The terms "heterocycle", "heterocyclyl", and "heterocyclic ring" are also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. A "substituted heterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.The term "heterocyclylene" refers to a diradical of a heterocycle group and, thus, refers to a carbocyclene group as defined above in which in which one or more (such as 1, 2, 3, or 4) carbon atoms in the carbocyclene group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorous, or sulfur, preferably O, S, or N. A heterocyclylene group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of the heterocyclylene group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclene" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroarylene groups. Exemplary heterocyclylene groups include morpholinylene, pyrrolidinylene, imidazolidinylene, pyrazolidinylene, piperidinylene (also called piperidylene), piperazinylene, di- and tetrahydrofuranylene, di- and tetrahydrothienylene, di- and tetrahydropyranylene, and urotropinylene. A "substituted heterocyclylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0310] The term "heterocycloalkyl" means a cycloalkyl group as defined above in which one or more (such as 1, 2, 3, or 4) carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorous, or sulfur, preferably O, S, orN. A heterocycloalkyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of the heterocycloalkyl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocycloalkyl" is also meant to encompass completely hydrogenated forms of the above-mentioned heteroaryl groups. Exemplary heterocycloalkyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. A "substituted heterocycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocycloalkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0311] The term "heterocycloalkylene" as used herein means a heterocyclyl group as defined above which contains at least one ring heteroatom (such as those selected from the group consisting of O, S, N, B, Si, and P) and in which one hydrogen atom has been removed resulting in a geminal, vicinal or isolateddiradical. In some embodiments, the heteroatoms of the heterocycloalkylene group are selected from the group consisting of O, S, andN. For example, the heterocycloalkylene may be O / S-heterocycloalkylene, such as O-heterocycloalkylene. In some embodiments, in each ring of the heterocycloalkylene group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The heterocycloalkylene may be monocyclic or polycyclic (such as bi- or tricyclic). In some embodiments, the heterocycloalkylene is a mono-, bi- or tricyclic 4- to 14-membered (z.e., 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 4- to 12-membered or 4- to 10-membered) heterocycloalkylene. A "substituted heterocycloalkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocycloalkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1stlevel substituent as specified herein.

[0312] The expression "partially hydrogenated form" of an unsaturated compound or group as used herein means that part of the unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group without removing all unsaturated moieties. The phrase "completely hydrogenated form" of an unsaturated compound or group is used herein interchangeably with the term "perhydro" and means that all unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group. For example, partially hydrogenated forms of a 5-membered heteroaryl group (containing 2 double bonds in the ring, such as furan) include dihydro forms of said 5-membered heteroaryl group (such as 2, 3 -dihydrofuran or 2,5-dihydrofuran), whereas the tetrahydro form of said 5-membered heteroaryl group (e.g., tetrahydrofuran, z.e., THF) is a completely hydrogenated (or perhydro) form of said 5 -membered heteroaryl group. Likewise, for a 6-membered heteroaryl group having 3 double bonds in the ring (such as pyridyl), partially hydrogenated forms include di- and tetrahydro forms (such as di- and tetrahydropyridyl), whereas the hexahydro form (such as piperidinyl in case of the heteroaryl pyridyl) is the completely hydrogenated (or perhydro) derivative of said 6-membered heteroaryl group. Consequently, a hexahydro form of an aryl or heteroaryl can only be considered a partially hydrogenated form according to the present disclosure if the aryl or heteroaryl contains at least 4 unsaturated moieties consisting of double and triple bonds between ring atoms.

[0313] The term "aromatic" as used in the context of hydrocarbons means that the whole molecule has to be aromatic. For example, if a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as carbocycle for the purposes of the present disclosure. Likewise, if a bi- or polycyclic aryl (such as naphthyl) is hydrogenated the resulting hydrogenated bi-or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as carbocycle for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic). A similardistinction is made within the present application between heteroaryl and heterocyclyl. For example, indolinyl, i.e., a dihydro variant of indolyl, is classified as heterocyclyl for the purposes of the present disclosure, since only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.

[0314] The term "hydrocarbon" includes non-cyclic (e.g., linear (straight) or branched) as well as cyclic organic molecules all of which are primarily composed of carbon and hydrogen atoms. It should be appreciated that one or more of the hydrogen atoms in the hydrocarbon may be substituted with other atoms, e.g., halogen, oxygen or sulfur. In some embodiments, the hydrocarbon is non-cyclic, but can also include a cyclic (z.e., carbocyclic, such as cycloalkyl or cycloalkenyl) group or an aryl group, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0315] The term "hydrocarbyl" as used herein relates to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, hydrocarbyl groups are non-cyclic, e.g., linear (straight) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, carbocyclic, carbocyclene, aryl groups, and combinations thereof (such as arylalkyl (aralkyl), etc.). Particular examples of hydrocarbyl groups are C1-30 alkyl (such as Ce-so alkyl, Cs-24 alkyl, or C10-20 alkyl), C2-30 alkenyl (such as Ce-3o alkenyl, Cs-24 alkenyl, or C10-20 alkenyl) having 1, 2, or 3 double bonds, aryl, and aryl(Ci-e alkyl). In some embodiments, the hydrocarbyl group is optionally substituted (e.g., with one or more 1stlevel substituents as defined herein), provided that the overall polarity of the hydrocarbon remains relatively nonpolar. In some embodiments, the hydrocarbyl group is the hydrocarbyl chain of naturally occurring fatty acids and may have at least 8 carbon atoms, e.g., the hydrocarbyl group is a (preferably linear) Cs-2o alkyl chain, e.g., a (preferably linear) Cio-is alkyl chain).

[0316] The term "aliphatic group" as used herein relates to a non-aromatic hydrocarbon / hydrocarbyl group as defined herein. An aliphatic group may be saturated or unsaturated and / or may be straight, branched, cyclic, or any combination thereof. Typical examples of aliphatic groups include alkyl, alkenyl, alkynyl, carbocyclic, carbocyclene groups, and combinations thereof (such as cycloalkylalkyl, etc.). Particular examples of aliphatic groups are C1.30 alkyl (such as Ce-3o alkyl, Cs-24 alkyl, or C10-20 alkyl), C2-30 alkenyl (such as Ce-3o alkenyl, Cs-24 alkenyl, or C10-20 alkenyl) having 1, 2, or 3 double bonds, and C3-10 cycloalkyl. In some embodiments, the aliphatic group is optionally substituted (e.g., with one or more 1stlevel substituents as defined herein), provided that the overall polarity of the aliphatic group remains relatively nonpolar. In some embodiments, the aliphatic group is the hydrocarbyl chain of naturally occurring fatty acids and may have at least 8 carbon atoms, e.g., the hydrocarbyl group is a (preferably linear) Cs-2o alkyl chain, e.g., a (preferably linear) Cio-is alkyl chain).

[0317] The term "halogen" as used herein means fluoro, chloro, bromo, or iodo.The term "cyano" as used herein relates to the group -CN.

[0318] The term "azido" as used herein relates to the group -Ns.

[0319] The term "nitro" as used herein relates to the group -NO2.

[0320] The term "hydroxyl" or "hydroxy" as used herein relates to the group -OH.

[0321] The term "alkoxyl" as used herein relates to the group -O-alkyl, wherein alkyl is as defined herein.

[0322] The term "aryloxy" as used herein relates to the group -O-aryl, wherein aryl is as defined herein.

[0323] The term "arylalkyl" as used herein relates to an alkyl group as defined herein which is substituted by an aryl group as defined herein.

[0324] The term "alkylaryl" as used herein relates to an aryl group as defined herein which is substituted by an alkyl group as defined herein.

[0325] The term "heteroarylalkyl" as used herein relates to an alkyl group as defined herein which is substituted by a heteroaryl group as defined herein.

[0326] The term "formidamidine" as used herein is synonym to the term "formamidine" and refers to the group HN=CH-NH2.

[0327] The term "benzyl" as used herein relates to the group phenylmethylene (C6H5-CH2-).

[0328] The term "amino" or "amine" as used herein includes unsubstituted amino (z.e., the group -NH2) and substituted amino (z. e. , mono- or disubstituted amino, wherein one or two of the hydrogen atoms have been replaced with a group other than hydrogen). An amino group may be monovalent (e.g., -NRR, wherein each R is independently H or an organic group, such as R72or R73as defined below) or divalent (e.g., -NR-, wherein R is H or an organic group, such as R72as defined below). In some embodiments, the term "amino" means the group -N(R72)(R73), wherein R72and R73are, in each case, independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, carbocycle, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R72and R73may join together with the nitrogen atom to which they are attached to form the group -N=CR75R76, wherein each of the alkyl, alkenyl, alkynyl, carbocycle, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more (such as 1 to the maximumnumber of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, carbocycle, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70; R75and R76are independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, carbocycle, cycloalkyl, aryl, heteroaryl, heterocyclyl, and -NHyR802-y, or R75and R76may join together with the atom to which they are attached to form a ring which is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the ring, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70, wherein each of the alkyl, alkenyl, alkynyl, carbocycle, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, carbocycle, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70; y is an integer from 0 to 2; R80is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocycle, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, carbocycle, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the alkyl, alkenyl, alkynyl, carbocycle, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70; and R70is other than H, preferably a 1stlevel substituent as disclosed herein. In some embodiments, each of R72and R73is independently H or a hydrocarbyl group, such as selected from the group consisting of H, Ci-6 alkyl, aryl, and aryl(Ci.e alkyl), wherein each of the hydrocarbyl groups (such as each of the Ci-6 alkyl, aryl, and aryl(Ci.e alkyl) groups) is optionally substituted with one or more (such as 1 to the maximum number of hydrogen atoms bound to the hydrocarbyl group (such as Ci-6 alkyl, aryl, or aryl(Ci-6 alkyl) group)), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R70.

[0329] The term "amide" or "amido" as used herein relates to a group comprising the structure -C(O)NH-(including its isomerically arranged structure -NHC(O)-, unless it is specified to the contrary). Preferably, each of both ends of the amide structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of a linker) (if both ends are linked to the same organic group the amide moiety is also referred to as lactam). An amide group may be monovalent (e.g., -C(O)NRR or -NRC(O)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(O)NR- or -NRC(O)-, wherein R is H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").The term "thioamide" as used herein relates to a group comprising the structure -C(S)NR- (including its isomerically arranged structure -N(R)C(S)-, unless it is specified to the contrary), wherein one of both ends of the thioamide structure is covalently linked to a C atom of an organic group (e.g., an alkylene group as further component of a linker) and the other end is covalently linked to H or to a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino"). A thioamide group may be monovalent (e.g., -C(S)NRR or -N(R)C(S)R, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -C(S)NR- or -N(R)C(S)-, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino").

[0330] The term "ester" as used herein relates to a group comprising the structure -C(O)O- (including its isomerically arranged structure -OC(O)-, unless it is specified to the contrary). Preferably, each of both ends of the ester structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of a linker) (if both ends are linked to the same organic group the ester moiety is also referred to as lactone). An ester group may be monovalent (e.g., -C(O)OR or -OC(O)R, wherein R is independently an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e g., -C(O)O- or -OC(O)-)).

[0331] The term "thioester" (also called "thioate") as used herein relates to a group comprising the structure -SC(O)- (including its isomerically arranged structures -C(O)S-, -OC(S), and -C(S)O-, unless it is specified to the contrary), wherein each of both ends of the thioate structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of a linker). A thioate group may be monovalent (e.g., -SC(O)R or -C(O)SR or -OC(S)R or -C(S)OR, wherein each R is independently H or an organic group, such as one of the organic groups specified in the definition of R72indicated above in the definition of the term "amino") or divalent (e.g., -SC(O)- or -C(O)S- or -OC(S)- or -C(S)O-).

[0332] The term "optionally substituted" indicates that one or more (such as 1 to the maximum number of hydrogen atoms bound to a group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atom(s) may be replaced with a group (z.e., a 1stlevel substituent) different from hydrogen, and / or any two 1stlevel substituents which are bound to the same carbon atom of a carbocycle or heterocyclyl group may join together to form =0, =S, =NH, or =N(CI-3 alkyl).Typical 1stlevel substituents are preferably selected from the group consisting of C1-3 alkyl, phenyl, halogen (e.g., F, Cl, or Br), -CF3, -OH, -OCH3, -SCH3, -NH2 z(CH3)z, -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1, or 2 and C1-3 alkyl is methyl, ethyl, propyl or isopropyl.

[0333] In the present specification, a structural formula of a compound may represent a certain isomer of said compound. It is to be understood, however, that the present disclosure includes all isomers such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, mesomers, and the like which occur structurally and isomer mixtures and is not limited to the description of the formula. Furthermore, in the present specification, a structural formula of a compound may represent a specific salt and / or solvate of said compound. It is to be understood, however, that the present disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates) and is not limited to the description of the specific salt and / or solvate.

[0334] "Isomers" are compounds having the same molecular formula but differ in structure ("structural isomers") or in the geometrical (spatial) positioning of the functional groups and / or atoms ("stereoisomers").

[0335] "Enantiomers" are a pair of stereoisomers which are non-superimposable mirror-images of each other. A "racemic mixture" or "racemate" contains a pair of enantiomers in equal amounts and is denoted by the prefix (±). "Diastereomers" are stereoisomers which are non-superimposable and which are not mirror-images of each other.

[0336] "Tautomers" are structural isomers of the same chemical substance that spontaneously and reversibly interconvert into each other, even when pure, due to the migration of individual atoms or groups of atoms; z.e., the tautomers are in a dynamic chemical equilibrium with each other. An example of tautomers are the isomers of the keto-enol-tautomerism.

[0337] "Mesomers" of a molecule have the same chemical structure but differ in the way electrons are formally assigned to atoms in the Lewis structure depictions of the molecule. Examples of mesomers are the contributing structures of the thiocyanate anion (S=C=N-and S-C=N) and the contributing structures

[0338] and hybrid structure of benzene:

[0339]

[0340] "Conformers" are stereoisomers that can be interconverted just by rotations about formally single bonds, and include - in particular - those leading to different 3-dimentional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane.The term "solvate" as used herein refers to an addition complex of a dissolved material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal. The amount of solvent contained in the addition complex may be stoichiometric or non-stoichiometric. A "hydrate" is a solvate wherein the solvent is water.

[0341] In isotopically labeled compounds one or more atoms are replaced by a corresponding atom having the same number of protons but differing in the number of neutrons. For example, a hydrogen atom may be replaced by a deuterium or tritium atom. Exemplary isotopes which can be used in the present disclosure include deuterium, tritium,nC,13C,14C,15N,18F,32P,32S,35S,36C1, and125I.

[0342] RNA

[0343] According to the present disclosure, the term "RNA" means a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered / modified nucleotides or nucleosides (z.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (z.e., naturally occurring) nucleotide residues or analogs thereof).

[0344] In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. Said RNA may express the encoded peptide, polypeptide, or protein. For example, said RNA may be RNA encoding an antigen. In some embodiments, RNA is able to interactwith the cellular translation machinery allowing translation of the peptide, polypeptide or protein. A cell may produce the encoded peptide, polypeptide or protein intracellularly (e.g. in the cytoplasm), may secrete the encoded peptide, polypeptide or protein, or may produce it on the surface.

[0345] In some embodiments of all aspects of the disclosure, the RNA is mRNA. According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" . Generally, mRNA encodes a peptide, polypeptide or protein.

[0346] As established in the art, the RNA (such as mRNA) generally contains a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. In one embodiment, the RNA (such as mRNA) is produced by in vitro transcription using a DNA template (where DNA refers to a nucleic acid that contains deoxyribonucleotides). The in vitro transcription methodology is known to the skilled person. Furthermore, a variety of in vitro transcription kits is commercially available. For providing modified RNA (such as mRNA), correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription.

[0347] In one embodiment, RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA, such as IVT-mRNA), and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0348] In some embodiments, the RNA (such as mRNA) which preferably encodes a peptide, polypeptide or protein has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0349] In some embodiments of the present disclosure, the RNA (such as mRNA) is "replicon RNA" (such as "replicon mRNA") or simply a "replicon", in particular "self-replicating RNA" (such as "self-replicating mRNA") or "self-amplifying RNA" (or "self-amplifying mRNA"). In certain embodiments, the repliconor self-replicating RNA (such as self-replicating mRNA) is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp.

[0350] 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5 ’-cap, and a 3’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould etal., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234). Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

[0351] In some embodiments of the present disclosure, the RNA (such as mRNA) described herein (e.g., contained in the compositions of the present disclosure and / or used in the methods of the present disclosure) contains one or more modifications, e.g., in order to increase its stability and / or increase translation efficiency and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in order to increase expression of the RNA (such as mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, preferably without altering the sequence of the expressed peptide or protein. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include the following: a 5'-cap structure; an extension or truncation ofthe naturally occurring poly(A) tail; an alteration of the 5'- and / or 3 '-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the G / C content of the RNA). The term "modification" in the context of modified RNA (such as mRNA) according to the present disclosure preferably relates to any modification of an RNA (such as mRNA) which is not naturally present in said RNA (mRNA).

[0352] In some embodiments, the RNA (such as mRNA) described herein comprises a 5 '-cap structure. In some embodiments, the mRNA does not have uncapped 5'-triphosphates. In some embodiments, the RNA (such as mRNA) described herein may comprise a conventional 5'-cap and / or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine 5 '-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the mRNA (z.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the mRNA). The guanosine may be methylated at position N7(resulting in the cap structure m7Gppp). The term "5'-cap analog" refers to a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2'- or 3 '-position of the m7guanosine structure in order to avoid an integration of the 5 '-cap analog in the reverse orientation (such 5 '-cap analogs are also called antireverse cap analogs (ARCAs)). Particularly preferred 5 '-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5'-cap analogs at the P-phosphate (such as m27’2 OG(5')ppsp(5')G (referred to as beta-S-ARCA or -S-ARCA)), as described in PCT / EP2019 / 056502. Providing an RNA (such as mRNA) with a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5 '-cap compound, wherein said 5 '-cap structure is co-transcriptionally incorporated into the generated RNA (such as mRNA) strand, or the RNA (such as mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the mRNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

[0353] In some embodiments, the RNA (such as mRNA) comprises a 5'-cap structure selected from the group consisting of m27’2 OG(5’)ppsp(5')G (in particular its DI diastereomer), m27’3 OG(5')ppp(5')G, and m27’3 OGppp(mi2 O)ApG. In some embodiments, RNA encoding a peptide, polypeptide or protein comprising an antigen or epitope comprises m27’2OG(5’)ppsp(5')G (in particular its DI diastereomer) as 5 '-cap structure.

[0354] In some embodiments, the RNA (such as mRNA) comprises a capO, capl, or cap2, preferably capl or cap2. According to the present disclosure, the term "capO" means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'. According to the present disclosure, the term "capl" means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCHs moiety atposition 2'. According to the present disclosure, the term "cap2" means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.

[0355] The 5 '-cap analog beta-S-ARCA (P-S-ARCA) has the following structure:

[0356]

[0357] The "DI diastereomer of beta-S-ARCA" or "beta-S-ARCA(Dl)" is the diastereomer of beta-S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time. The HPLC preferably is an analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column, preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml / min can be applied. In one embodiment, a gradient of methanol in ammonium acetate, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH = 5.9, within 15 min is used. UV-detection (VWD) can be performed at 260 nm and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0358] The 5'-cap analog m27’3 OGppp(mi2 O)ApG (also referred to as m27’3 OG(5')ppp(5')m2 OApG) which is a building block of a capl has the following structure:

[0359]

[0360] An exemplary capO mRNA comprising P-S-ARCA and mRNA has the following structure:

[0361]

[0362] An exemplary capO mRNA comprising m27’3 OG(5')ppp(5')G and mRNA has the following structure:

[0363]

[0364] An exemplary cap 1 mRNA comprising m27’3°Gppp(mi2°)ApG and mRNA has the following structure:

[0365]

[0366] A particularly preferred cap comprises the 5’-cap m27’2°G(5’)ppsp(5’)G. In some embodiments, at least one RNA described herein comprises the 5 ’-cap m272°G(5’)ppsp(5’)G. In some embodiments, each RNA described herein comprises the 5’-cap m27’2°G(5’)ppsp(5’)G.

[0367] In some embodiments, the RNA (such as mRNA) comprises a 3’-poly(A) sequence. As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylateresidues which is typically located at the 3 '-end of an RNA (such as mRNA) molecule. Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs (such as mRNAs) described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. RNAs (such as mRNAs) disclosed herein can have a poly-A tail attached to the free 3 '-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template -dependent RNA polymerase.

[0368] It has been demonstrated that a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of mRNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0369] The poly-A tail may be of any length. In some embodiments, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0370] In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.

[0371] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 Al may be used in the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficialproperties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A tail contained in an RNA (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0372] In some embodiments, no nucleotides other than A nucleotides flank a poly-A tail at its 3'-end, z.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A.

[0373] In some embodiments, a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides. In some embodiments, the poly-A tail comprises or consists of the nucleotide sequence of SEQ ID NO: 93. In some embodiments, the poly-A sequence has a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 93.

[0374] In some embodiments, at least one RNA comprises a poly-A tail. In some embodiments, each RNA comprises a poly-A tail. In some embodiments, the poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, upto 400, upto 300, upto 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may comprise the poly-A tail shown in SEQ ID NO: 93. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides.

[0375] In some embodiments, at least one RNA comprises a poly-A tail comprising the nucleotide sequence of SEQ ID NO: 93, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 93. In some embodiments, each RNA comprises a poly-A tail comprising the nucleotide sequence of SEQ ID NO: 93, or a nucleotide sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 93.

[0376] In some embodiments, RNA (such as mRNA) used in present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5 '-UTR) and / or 3' (downstream) of an open reading frame (3 '-UTR). A 5 '-UTR, if present, is located at the 5'-end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3 '-end, downstream of the termination codon of a protein-encoding region, but the term "3 '-UTR" does preferably not include the poly-A sequence. Thus, the 3 '-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3'-UTR into the 3'-non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'-UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs may be autologous or heterologous to the RNA (preferably mRNA) into which they are introduced. In one particular embodiment the 3'-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2 -globin, alpha 1 -globin, or beta-globin, preferably betaglobin, more preferably human beta-globin. For example, the RNA (preferably mRNA) may be modified by the replacement of the existing 3 '-UTR with or the insertion of one or more, preferably two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alpha 1 -globin, beta-globin, preferably beta-globin, more preferably human beta-globin.

[0377] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 91. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 92.

[0378] In some embodiments, at least one RNA (such as mRNA) used in present disclosure comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 91, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 91. In some embodiments, each RNA (such as mRNA) used in present disclosure comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 91, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 91.

[0379] In some embodiments, at least one RNA (such as mRNA) used in present disclosure comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 92, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 92.In some embodiments, each RNA (such as mRNA) used in present disclosure comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 92, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 92.

[0380] In one embodiment (in particular in order to increase the stability of the RNA and / or decrease the immunogenicity of the RNA and / or decrease the cytotoxicity of the RNA), the RNA may have one or more modified nucleosides and / or one or more modified nucleotides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine. For example, in some embodiments, uridine in the RNA (such as mRNA) described herein is replaced (partially (z.e., at least one uridine I the RNA (such as mRNA) described herein is replaced) or completely, preferably completely) by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0381] In some embodiments, the modified uridine replacing uridine is pseudouridine (y), Nl-methyl-pseudouridine (ml\| / ), or 5 -methyl -uridine (m5U).

[0382] An RNA (such as mRNA) which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) is referred to herein as "T-modified", whereas the term "ml'P-modified" means that the RNA (such as mRNA) contains N(l)-methylpseudouridine (replacing partially or completely, preferably completely, uridine). Furthermore, the term "m5U-modified" means that the RNA (such as mRNA) contains 5 -methyluridine (replacing partially or completely, preferably completely, uridine). Such T- or m I - or m5U-modified RNAs usually exhibit decreased immunogenicity compared to their unmodified forms and, thus, are preferred in applications where the induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(l)-methylpseudouridine replacing completely uridine.

[0383] In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine in the RNA (such as mRNA) may be any one or more of 3 -methyl -uridine (m3U), 5 -methoxy-uridine (mo5U), 5 -aza-uridine, 6-aza-uridine, 2-thio-5 -aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5 -bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5 -carboxymethyl -uridine (cm5U), 1-carboxymethyl-pseudouridine, 5 -carboxyhydroxymethyl -uridine (chm5U), 5 -carboxyhydroxymethyl -uridine methyl ester (mchm5U), 5 -methoxy carbonylmethyl -uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5 -aminomethyl -2 -thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1 -ethyl -pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5 -methylaminomethyl -2-seleno-uridine (mnm5se2U), 5 -carbamoylmethyl -uridine(ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl -pseudouridine, 5-taurinomethyl-uridine (rm5U).

[0384] 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), l-taurinomethyl-4-thio-pseudouridine), 5 -methyl -2 -thio-uridine (m5s2U), 1 -methyl -4-thio-pseudouridine (m 1 s4\| / )_ 4-thio-l-methyl-pseudouridine, 3 -methyl -pseudouridine (m3\| / ). 2-thio-l -methyl -pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l -methyl- 1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1 -methyl -pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 \| / ), 5- (isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (\| / m), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-0H-ara-uridine, 5-(2 -carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.

[0385] In some embodiments, at least one RNA (such as mRNA) comprises a modified nucleoside in place of at least one uridine. In some embodiments, at least one RNA (such as mRNA) comprises a modified nucleoside in place of each uridine. In some embodiments, each RNA (such as mRNA) comprises a modified nucleoside in place of at least one uridine. In some embodiments, each RNA (such as mRNA) comprises a modified nucleoside in place of each uridine. In some embodiments, the modified uridine replacing at least one (e.g., all) uridine is selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (ml\| / ), 5 -methyl -uridine (m5U), and combinations thereof. In some embodiments, the modified nucleoside comprises pseudouridine (v)- Insome embodiments, the modified nucleoside comprises N1 -methyl -pseudouridine (ml\| / ). In some embodiments, the modified nucleoside comprises 5 -methyl -uridine (m5U).

[0386] In some embodiments, at least one RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (y), N I -methyl -pseudouridine (ml\| / ), and 5-methyl-uridine (m5U). In some embodiments, each RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (y), N1 -methyl -pseudouridine (ml\| / ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (v) and N1 -methyl -pseudouridine (ml\| / ). In some embodiments, the modified nucleosides comprise pseudouridine (v) and 5 -methyl -uridine (m5U). In some embodiments, the modified nucleosides comprise N1 -methyl -pseudouridine (m h| / ) and 5 -methyl -uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (y), Nl-methyl-pseudouridine (m h| / ). and 5 -methyl -uridine (m5U).

[0387] In some embodiments, the RNA (such as mRNA) comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in one embodiment, in the RNA (such as mRNA) 5 -methylcytidine is substituted partially or completely, preferably completely, for cytidine. In one embodiment, the RNA (such as mRNA) comprises 5 -methylcytidine and one or more selected from pseudouridine (y), N 1 -methyl -pseudouridine (m h| / ). and 5-methyl-uridine (m5U). In one embodiment, the RNA (such as mRNA) comprises 5 -methylcytidine and N1 -methyl -pseudouridine (m h| / ). In some embodiments, the RNA (such as mRNA) comprises 5 -methylcytidine in place of each cytidine and N1 -methyl -pseudouridine (m H| / ) in place of each uridine.

[0388] In some embodiments, at least one RNA (such as mRNA) comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. In some embodiments, each RNA (such as mRNA) comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in said RNA (such as mRNA) 5 -methylcytidine may be substituted partially or completely, preferably completely, for cytidine. In some embodiments, at least one RNA (such as mRNA) comprises 5 -methylcytidine and one or more selected from pseudouridine (y), Nl-methyl-pseudouridine (ml\| / ), and 5-methyl-uridine (m5U). In some embodiments, each RNA (such as mRNA) comprises 5 -methylcytidine and one or more selected from pseudouridine (y), Nl-methyl-pseudouridine (ml\| / ), and 5-methyl-uridine (m5U). In one embodiment, at least one RNA (such as mRNA) comprises 5 -methylcytidine and Nl-methyl-pseudouridine (ml\| / ). In one embodiment, each RNA (such as mRNA) comprises 5 -methylcytidine and Nl-methyl-pseudouridine (ml\| / ). In some embodiments, at least one RNA (such as mRNA) comprises 5 -methylcytidine in place of each cytidine and Nl-methyl-pseudouridine (m h| / ) in place of each uridine. In some embodiments, each RNA (such as mRNA) comprises 5 -methylcytidine in place of each cytidine and Nl-methyl-pseudouridine (m h| / ) in place of each uridine.

[0389] The codons of the RNA (preferably mRNA) described in the present disclosure may further be optimized, e.g., to increase the G / C content of the RNA and / or to replace codons which are rare in the cell (or subject) in which the peptide or protein of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject) (so called codon-optimization). In some embodiments, the amino acid sequence encoded by the RNA described in the present disclosure (in particular, the amino acid sequence comprising a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof described herein) is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increasedcompared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and / or increased in the G / C content compared to the corresponding sequence regions of the wild type coding sequence. In one embodiment, the codonoptimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0390] The term "codon-optimized" refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present invention, coding regions are preferably codon-optimized for optimal expression in a subject to be treated using the RNA (preferably mRNA) described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA (preferably mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".

[0391] In some embodiments of the disclosure, the guanosine / cytosine (G / C) content of the coding region of the RNA described herein (in particular, of at least one RNA (preferably mRNA) encoding at least one amino acid sequence, such as each RNA (preferably mRNA) encoding at least one amino acid sequence) is increased compared to the G / C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences having an increased G (guanosine)ZC (cytosine) content are more stable than sequences having an increased A (adenosine ) / U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and / or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or U or contain a lower content of A and / or U nucleotides.

[0392] In various embodiments, the G / C content of the coding region of the RNA (in particular, mRNA) described herein (in particular, of at least one RNA (preferably mRNA) encoding at least one amino acid sequence, such as each RNA (preferably mRNA) encoding at least one amino acid sequence) is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild type RNA.A combination of the above described modifications, i.e., incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleosides / nucleotides with synthetic nucleosides / nucleotides (e.g., 5 -methylcytidine for cytidine and / or pseudouridine ( ) or N(l)-methylpseudouridine (m I ) or 5 -methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency. Thus, in some embodiments, the RNA (preferably mRNA) described in the present disclosure (in particular, at least one RNA (preferably mRNA) encoding at least one amino acid sequence, such as each RNA (preferably mRNA) encoding at least one amino acid sequence), contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, z.e., (i) incorporation of a 5'-cap structure; (ii) incorporation of a poly-A sequence, unmasking of a poly-A sequence; (iii) alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5 -methylcytidine for cytidine and / or pseudouridine ( ) or N(l)-methylpseudouridine (m IT) or 5-methyluridine (m5U) for uridine); and (v) codon optimization. In some embodiments, the RNA (preferably mRNA) described in the present disclosure (in particular, at least one RNA (preferably mRNA) encoding at least one amino acid sequence, such as each RNA (preferably mRNA) encoding at least one amino acid sequence) comprises a capl or cap2, preferably a capl structure. In some embodiments, the poly-A sequence comprises at least 100 nucleotides. In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 93. In some embodiments, the a 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 91, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 91. In some embodiments, the 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 92, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 92.

[0393] In one embodiment, after administration of the RNA (in particular, mRNA) described herein, e.g., formulated as RNA lipoplex particles, at least a portion of the RNA is delivered to a target cell. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to produce the peptide(s) or protein(s) it encodes. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell or macrophage. RNA lipoplex particles (in particular, mRNA lipoplex particles) described herein may be used for delivering RNA to such target cell. Accordingly, the present disclosure also relates to a method for delivering RNA (in particular, mRNA) to a target cell in a subject comprising the administration of the RNA lipoplex particles (in particular, mRNA lipoplex particles) described herein to the subject. In one embodiment, the RNA (in particular, mRNA) is delivered to the cytosol ofthe target cell. In one embodiment, the RNA (in particular, mRNA) is translated by the target cell to produce the peptide(s) or protein(s) encoded by the RNA.

[0394] According to the disclosure, the term "RNA encodes" means that the RNA, if present in the appropriate environment, such as within cells of a target tissue, can direct the assembly of amino acids to produce the peptide or protein it encodes during the process of translation. In one embodiment, RNA is able to interact with the cellular translation machinery allowing translation of the peptide or protein. A cell may produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or in the nucleus), may secrete the encoded peptide or protein, or may produce it on the surface.

[0395] Human papillomavirus infection (HPV infection) is an infection caused by the human papillomavirus (HPV), a DNA virus from the papillomavirus family. HPV is the most frequently sexually transmitted viral infection. HPV infection has been established as a causative agent for a variety of cancers in the genital and head and neck region. All cervical cancers are HPV-positive, 88% of anal, 78% of vaginal and to a lesser extent vulva and penile cancers. Of the head and neck region, HPV-attributed cancers account for 30.8% of oropharyngeal cancers, whereas oral cavity and the larynx are affected to a much lesser extent. The incidence of HPV-negative oropharyngeal squamous cell carcinomas (OPSCC) decreased by 50% from 1988 to 2004, whereas HPV-positive oropharyngeal carcinomas increased more than two-fold. Over 100 different HPV types exist which can be classified as either "low" or "high" risk depending on their ability to mediate malignant transformation. High risk HPV 16 and HPV 18 are the most frequent oncogenic HPV types, with HPV 16 accounting for 90% of all HPV-positive head and neck squamous cell carcinomas (HNSCC) and 66% of all cervical cancers.

[0396] High risk HPV viruses are non-enveloped double-stranded DNA viruses that owe their transforming capacity to their oncogenes E6 and E7. HPV infects the mucosal and cutaneous squamous epithelium and has an intra-epithelial infection cycle. HPV expresses six early (El, E2, E4, E5, E6 and E7) and two late (LI and L2) proteins. El and E2 are needed for the initiation of viral replication, whereas E2 also regulates the expression of E6 and E7 as a transcriptional repressor. During rolling circle genome integration, the E2 gene is disrupted, leading to higher expression of E6 and E7. LI and L2 are viral capsid proteins that are crucial for virus assembly and, via the help ofE4, release of viral particles. After an acute phase of viral infection, HPV gene products may remain within cells of the mucosal and cutaneous squamous epithelium with persistent expression of E6 and E7 that may drive malignant transformation. The HPV proteins E6 and E7 are able to deregulate the cell cycle, inhibit apoptosis and thus drive cancer progression. E6 possesses the ability to abrogate the function of the tumor suppressor protein p53, which governs cell cycle progression and apoptosis. The oncogenic potential of E7 is determined by its ability to regulate several cellular factors such as the retinoblastoma pocket protein(pRb), which is involved in G1 to S phase transition. E7 targets pRb family members to proteosomal degradation, resulting in impaired DNA repair, cell cycle checkpoints and loss of genomic integrity.

[0397] HPV-driven cancers are marked by the persistent expression of the viral oncogenes E6 and E7, which are key oncogenic drivers.

[0398] HNSCC is an aggressive and difficult to treat cancer type, associated with a reduction of life quality and significant morbidity. Although multiple vaccine formats have entered clinical testing, no HPV vaccine has been approved for therapeutic use in HPV-positive cervical or HNSCC cancer patients yet.

[0399] According to the disclosure, the term "HPV-positive cancer" includes cancers which are caused by HPV infection and / or wherein HPV or certain components, in particular HPV E6 and / or HPV E7, are detectable. Examples of such cancers include, but are not limited to, anogenital, cervical and penile cancers and cancer in the head and neck region such as cancer in the genital region or in the head and neck region. In one embodiment, an HPV-positive cancer is head and neck squamous cell carcinoma (HNSCC) or cervical cancer.

[0400] Aspects and embodiments of the present disclosure

[0401] In a one aspect, the present disclosure provides a medical preparation, combination, kit, or composition comprising: (a) an anti-cancer vaccine comprising at least one RNA, wherein the at least one RNA encodes at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and (b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction.

[0402] As demonstrated in the present disclosure, a combined treatment with an RNA-based cancer vaccine and a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction further enhances tumor antigen-specific immune responses, thereby potentiating antitumor activity.

[0403] Anti-cancer vaccine

[0404] One component of the medical preparation, combination, kit, or composition of the third aspect or of the combination used according to the first aspect is an anti-cancer vaccine in the form of at least one RNA. The at least one RNA encodes at least one amino acid sequence (z.e., a set of one or more vaccineantigens), said at least one amino acid sequence comprising a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof, z.e., an antigenic peptide or protein. Thus, the vaccine antigen comprises an epitope for at least one tumor antigen for inducing an immune response against the tumor antigens in the subject. The at least one RNA (encoding vaccine antigens) is administered to provide (following expression of the polynucleotide by appropriate target cells) antigens for induction, z.e., stimulation, priming and / or expansion, of an immune response which is targeted to target antigens (tumor antigens) or a procession product thereof.

[0405] A peptide and protein antigen described herein (e.g., CLDN6 protein, HPV E6 protein, HPV E7 protein, KK-LC- 1 protein, MAGE-A3 protein, MAGE-A4 protein, MAGE-C 1 protein, PRAME protein, TPTE protein, and tyrosinase protein) when provided to a subject by administration of RNA encoding the antigen, z.e., a vaccine antigen, preferably results in stimulation, priming and / or expansion of immune effector cells (such as T cells) in the subject. Said stimulated, primed and / or expanded immune effector cells (such as T cells) are preferably directed against the target antigen, in particular the target antigen expressed by diseased cells, tissues and / or organs, z.e., the disease-associated antigen, in particular tumor-associated antigen. Thus, a vaccine antigen may comprise the tumor-associated antigen, or a fragment or variant thereof. In one embodiment, such fragment or variant is immunologically equivalent to the tumor-associated antigen. In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" means an agent which results in stimulation, priming and / or expansion of immune effector cells (such as T cells) which stimulated, primed and / or expanded immune effector cells (such as T cells) target the disease-associated antigen, in particular the tumor-associated antigen, particularly when expressed on the surface of diseased cells, tissues and / or organs. Thus, the vaccine antigen administered according to the disclosure may correspond to or may comprise the tumor-associated antigen, may correspond to or may comprise a fragment of the tumor-associated antigen or may correspond to or may comprise an antigen which is homologous to the tumor-associated antigen or a fragment thereof. If the vaccine antigen administered according to the disclosure comprises a fragment of the tumor-associated antigen or an amino acid sequence which is homologous to a fragment of the tumor-associated antigen said fragment or amino acid sequence may comprise an epitope of the tumor-associated antigen or a sequence which is homologous to an epitope of the tumor-associated antigen, wherein the T cells bind to said epitope. Thus, according to the disclosure, an antigen may comprise an immunogenic fragment of the tumor-associated antigen or an amino acid sequence being homologous to an immunogenic fragment of the tumor-associated antigen. An "immunogenic fragment of an antigen" according to the disclosure preferably relates to a fragment of an antigen which is capable of stimulating, priming and / or expanding immune effector cells (such as T cells). It is preferred that the vaccine antigen (similar to the tumor-associated antigen) provides the relevant epitope for binding by immune effector cells (such as T cells). It is also preferred that the vaccine antigen (similar to the tumor-associatedantigen) is expressed on the surface of a cell such as an antigen-presenting cell so as to provide the relevant epitope for binding by the immune effector cells (such as T cells). The vaccine antigen according to the disclosure may be a recombinant antigen.

[0406] In some embodiments, the at least one RNA encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequences, wherein each of said 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid sequences comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof. In some embodiments, the at least one RNA encodes 2 or more amino acid sequences, wherein each of said 2 or more amino acid sequences comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof. In some embodiments, the at least one RNA encodes 3 or more amino acid sequences, wherein each of said 3 or more amino acid sequences comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof. In some embodiments, the at least one RNA encodes 4 or more amino acid sequences, wherein each of said 4 or more amino acid sequences comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof. In some embodiments, the at least one RNA encodes 5 or more amino acid sequences, wherein each of said 5 or more amino acid sequences comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof. In some embodiments, the at least one RNA encodes 6 or more amino acid sequences, wherein each of said 6 or more amino acid sequences comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof.

[0407] In some embodiments, each of the at least one amino acid sequence is encoded by a separate RNA. Thus, in some embodiments where the set of one or more vaccine antigens comprises two vaccine antigens, the anti -cancer vaccine comprises two RNA entities: one RNA entity encoding one vaccine antigen, and the other RNA entity encoding the other vaccine antigen. Likewise, in some embodiments where the set of one or more vaccine antigens comprises three vaccine antigens, the anti-cancer vaccine comprises three RNA entities: one RNA entity encoding vaccine antigen 1, one RNA entity encoding vaccine antigen 2, and one RNA entity encoding vaccine antigen 3. In some embodiments, the anticancer vaccine comprises up to six different RNAs each encoding a different vaccine antigen (e.g., selected from human tumor-associated antigens expressed by the majority of non-small cell lung cancers (NSCLCs)).

[0408] In an alternative embodiment, the at least one amino acid sequence is encoded by a single RNA. Thus, in some embodiments where the set of one or more vaccine antigens comprises two vaccine antigens, the anti-cancer vaccine comprises one RNA entity encoding both vaccine antigens. Likewise, in someembodiments where the set of one or more vaccine antigens comprises three vaccine antigens, the anticancer vaccine comprises one RNA entity encoding vaccine antigens 1 to 3.

[0409] In some embodiments, a tumor antigen is selected from a set of tumor antigens which are characteristic for a certain tissue (e.g., lung cancer, skin cancer, breast cancer, ovarian cancer, or HPV -positive cancer).

[0410] In some embodiments, a tumor antigen comprises any antigen which is characteristic for one of the following cancer tissues: lung cancer; skin cancer; and HPV-positive cancer. In some embodiments, each tumor antigen for a set of two or more vaccine antigens is selected from tumor antigens which are characteristic for one of the following cancer tissues: lung cancer; skin cancer; and HPV-positive cancer.

[0411] In some preferred embodiments, a tumor antigen comprises a lung cancer antigen (e.g., an NSCLC antigen). For example, a tumor antigen may be CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, PRAME, and / or MAGE-CI . In some embodiments, each tumor antigen for a set of two or more vaccine antigens is selected from antigens characteristic for lung cancer (e.g., NSCLC). For example, each tumor antigen for a set of two or more vaccine antigens is selected from the group consisting of CLDN6, KK-LC-1, MAGE-A3, MAGE-A4, MAGE-CI, and PRAME. In some embodiments, compositions, kits, combinations, or medical preparations described herein comprise RNA encoding a CLDN6 vaccine antigen, RNA encoding a KK-LC- 1 vaccine antigen, RNA encoding a MAGE-A3 vaccine antigen, RNA encoding a MAGE-A4 vaccine antigen, RNA encoding a MAGE-CI vaccine antigen, and RNA encoding a PRAME vaccine antigen. Likewise, methods described herein comprise administration of RNA encoding a CLDN6 vaccine antigen, RNA encoding a KK-LC-1 vaccine antigen, RNA encoding a MAGE-A3 vaccine antigen, RNA encoding a MAGE-A4 vaccine antigen, RNA encoding a MAGECI vaccine antigen, and RNA encoding a PRAME vaccine antigen. In some embodiments, the at least one RNA encodes at least the following amino acid sequences (1) to (6): (1) an amino acid sequence comprising CLDN6, an immunogenic variant thereof, or an immunogenic fragment of the CLDN6 or the immunogenic variant thereof; (2) an amino acid sequence comprising KK-LC-1, an immunogenic variant thereof, or an immunogenic fragment of the KK-LC-1 or the immunogenic variant thereof; (3) an amino acid sequence comprising MAGE-A3, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A3 or the immunogenic variant thereof; (4) an amino acid sequence comprising MAGE-A4, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A4 or the immunogenic variant thereof; (5) an amino acid sequence comprising PRAME, an immunogenic variant thereof, or an immunogenic fragment of the PRAME or the immunogenic variant thereof; (6) an amino acid sequence comprising MAGE-C 1 , an immunogenic variant thereof, or an immunogenic fragment of the MAGE-C 1 or the immunogenic variant thereof.Thus, in some embodiments, the medical preparation, combination, kit, or composition of the third aspect comprises the following RNAs ( l)-(6): (1) RNA encoding an amino acid sequence comprising CLDN6, an immunogenic variant thereof, or an immunogenic fragment of the CLDN6 or the immunogenic variant thereof; (2) RNA encoding an amino acid sequence comprising KK-LC-1, an immunogenic variant thereof, or an immunogenic fragment of the KK-LC-1 or the immunogenic variant thereof; (3) RNA encoding an amino acid sequence comprising MAGE-A3, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A3 or the immunogenic variant thereof; (4) RNA encoding an amino acid sequence comprising MAGE-A4, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A4 or the immunogenic variant thereof; (5) RNA encoding an amino acid sequence comprising PRAME, an immunogenic variant thereof, or an immunogenic fragment of the PRAME or the immunogenic variant thereof; (6) RNA encoding an amino acid sequence comprising MAGE-CI, an immunogenic variant thereof, or an immunogenic fragment of the MAGECI or the immunogenic variant thereof.

[0412] In some preferred embodiments, a tumor antigen comprises a skin cancer antigen (e.g., a melanoma antigen). For example, a tumor antigen may be MAGE-A3, NY-ESO-1, TPTE, and / or tyrosinase. In some embodiments, each tumor antigen for a set of two or more vaccine antigens is selected from antigens characteristic for skin cancer (e.g., melanoma). For example, each tumor antigen for a set of two or more vaccine antigens is selected from the group consisting of MAGE-A3, NY-ESO-1, TPTE, and tyrosinase. In some embodiments, compositions, kits, combinations, or medical preparations described herein comprise RNA encoding a MAGE -A3 vaccine antigen, RNA encoding a NY-ESO-1 vaccine antigen, RNA encoding a TPTE vaccine antigen, and RNA encoding a tyrosinase vaccine antigen. Likewise, methods described herein comprise administration of RNA encoding a MAGE-A3 vaccine antigen, RNA encoding a NY-ESO-1 vaccine antigen, RNA encoding a TPTE vaccine antigen, and RNA encoding a tyrosinase vaccine antigen. In some embodiments, the at least one RNA encodes at least the following amino acid sequences (1) to (4): (1) an amino acid sequence comprising MAGE-A3, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A3 or the immunogenic variant thereof; (2) an amino acid sequence comprising NY-ESO-1, an immunogenic variant thereof, or an immunogenic fragment of the NY -ESO- 1 or the immunogenic variant thereof; (3) an amino acid sequence comprising TPTE, an immunogenic variant thereof, or an immunogenic fragment of the TPTE or the immunogenic variant thereof; (4) an amino acid sequence comprising tyrosinase, an immunogenic variant thereof, or an immunogenic fragment of the tyrosinase or the immunogenic variant thereof.

[0413] Thus, in some embodiments, the medical preparation, combination, kit, or composition of the third aspect comprises the following RNAs ( l)-(4): (1) RNA encoding an amino acid sequence comprising MAGE-A3, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A3 or theimmunogenic variant thereof; (2) RNA encoding an amino acid sequence comprising NY-ESO-1, an immunogenic variant thereof, or an immunogenic fragment of the NY-ESO-1 or the immunogenic variant thereof; (3) RNA encoding an amino acid sequence comprising TPTE, an immunogenic variant thereof, or an immunogenic fragment of the TPTE or the immunogenic variant thereof; and (4) RNA encoding an amino acid sequence comprising tyrosinase, an immunogenic variant thereof, or an immunogenic fragment of the tyrosinase or the immunogenic variant thereof.

[0414] In some preferred embodiments, a tumor antigen comprises an HPV-positive cancer antigen (such as head and neck squamous cell carcinoma antigen (HNSCC) or cervical cancer). For example, a tumor antigen may be HPV E6 and / or HPV E7. In some embodiments, each tumor antigen for a set of two or more vaccine antigens is selected from antigens characteristic for HPV-positive cancer (e.g., HNSCC or cervical cancer). For example, each tumor antigen for a set of two or more vaccine antigens is selected from the group consisting of HPV E6 and HPV E7. In some embodiments, compositions, kits, combinations, or medical preparations described herein comprise RNA encoding an HPV E6 vaccine antigen, and RNA encoding an HPV E7 vaccine antigen. Likewise, methods described herein comprise administration of RNA encoding an HPV E6 vaccine antigen, and RNA encoding an HPV E7 vaccine antigen. In some embodiments, the at least one RNA encodes at least the following amino acid sequences (1) and (2): (1) an amino acid sequence comprising HPV E6, an immunogenic variant thereof, or an immunogenic fragment of the HPV E6 or the immunogenic variant thereof; (2) an amino acid sequence comprising HPV E7, an immunogenic variant thereof, or an immunogenic fragment of the HPV E7 or the immunogenic variant thereof.

[0415] Thus, in some embodiments, the medical preparation, combination, kit or composition of the third aspect comprises the following RNAs (1) and (2): (1) RNA encoding an amino acid sequence comprising HPV E6, an immunogenic variant thereof, or an immunogenic fragment of the HPV E6 or the immunogenic variant thereof; (2) RNA encoding an amino acid sequence comprising HPV E7, an immunogenic variant thereof, or an immunogenic fragment of the HPV E7 or the immunogenic variant thereof.

[0416] In some embodiments, at least one RNA of the anti -cancer vaccine is modified as specified herein.

[0417] For example, in some embodiments of the anti -cancer vaccine, (1) at least one amino acid sequence comprises an amino acid sequence which breaks immunological tolerance; (2) at least one RNA is coadministered with RNA encoding an amino acid sequence which breaks immunological tolerance; (3) at least one amino acid sequence is encoded by a coding sequence which is codon-optimized; (4) the G / C content of at least one amino acid sequence is increased compared to wild type coding sequence; (5) at least one RNA comprises a 5'-cap structure, preferably m27’2 0Gppsp(5')G; (6) at least one RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 91, or a nucleotide sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 91; (7) at least one amino acid sequence comprises an amino acid sequence enhancing antigen processing and / or presentation; (8) at least one RNA comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 92, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 92; (9) at least one RNA comprises a poly-A sequence; and / or (10) at least one RNA has one or more modified nucleosides and / or one or more modified nucleotides.

[0418] Thus, in some embodiments of the anti-cancer vaccine, at least one amino acid sequence (e.g., each of the at least one amino acid sequence) comprises an amino acid sequence which breaks immunological tolerance.

[0419] In some embodiments of the anti -cancer vaccine, at least one RNA (e.g., each RNA) is co-administered with RNA encoding an amino acid sequence which breaks immunological tolerance.

[0420] In some embodiments of the anti -cancer vaccine, the amino acid sequence which breaks immunological tolerance comprises helper epitopes, preferably tetanus toxoid-derived helper epitopes. In some embodiments, (i) the RNA encoding the amino acid sequence which breaks immunological tolerance comprises the nucleotide sequence of SEQ ID NO: 90, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 90; and / or (ii) the amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 89.

[0421] In some embodiments of the anti-cancer vaccine, at least one amino acid sequence (e.g., each of the at least one amino acid sequence) is encoded by a coding sequence which is codon-optimized, wherein the codon-optimization preferably does not change the sequence of the encoded amino acid sequence.

[0422] In some embodiments of the anti-cancer vaccine, the G / C content of at least one amino acid sequence (e.g., of each of the at least one amino acid sequence) is increased compared to wild type coding sequence, wherein the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0423] In some embodiments of the anti-cancer vaccine at least one amino acid sequence (e.g., each of the at least one amino acid sequence) is encoded by a coding sequence which is codon-optimized and the G / Ccontent of which is increased compared to wild type coding sequence, wherein the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0424] In some embodiments of the anti-cancer vaccine, at least one RNA (e.g., each RNA) comprises a 5'-cap structure, preferably m2’’2 0Gppsp(5')G.

[0425] In some embodiments of the anti-cancer vaccine, at least one RNA (e.g., each RNA) comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 91, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 91.

[0426] In some embodiments of the anti-cancer vaccine, at least one amino acid sequence (e.g., each of the at least one amino acid sequence) comprises an amino acid sequence enhancing antigen processing and / or presentation. In some embodiments, the amino acid sequence enhancing antigen processing and / or presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domain of an MHC molecule, preferably an MHC class I molecule. In some embodiments, (i) the RNA encoding the amino acid sequence enhancing antigen processing and / or presentation comprises the nucleotide sequence of SEQ ID NO: 88, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 88; and / or (ii) the amino acid sequence enhancing antigen processing and / or presentation comprises the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 87.

[0427] In some embodiments of the anti-cancer vaccine, at least one RNA (e.g., each RNA) comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 92, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 92.

[0428] In some embodiments of the anti-cancer vaccine, at least one RNA (e.g., each RNA) comprises a poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 nucleotides. In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 93.

[0429] In some embodiments of the anti-cancer vaccine, (a) at least one amino acid sequence (e.g., each of the at least one amino acid sequence) comprises an amino acid sequence which breaks immunologicaltolerance, wherein (i) the RNA encoding the amino acid sequence which breaks immunological tolerance comprises the nucleotide sequence of SEQ ID NO: 90, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 90; and / or (ii) the amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 89; (b) at least one amino acid sequence (e.g., each of the at least one amino acid sequence) is encoded by a coding sequence which is codon-optimized and the G / C content of which is increased compared to wild type coding sequence, wherein the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence; (c) at least one RNA (e.g., each RNA) comprises a 5'-cap structure, preferably m27’2°Gppsp(5')G; (d) at least one amino acid sequence (e.g., each of the at least one amino acid sequence) comprises an amino acid sequence enhancing antigen processing and / or presentation, wherein (i) the RNA encoding the amino acid sequence enhancing antigen processing and / or presentation comprises the nucleotide sequence of SEQ ID NO: 88, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 88; and / or (ii) the amino acid sequence enhancing antigen processing and / or presentation comprises the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 87; (e) at least one RNA (e.g., each RNA) comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 92, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 92; and (f) at least one RNA (e.g., each RNA) comprises a poly-A sequence. In some embodiments, the poly-A sequence comprises at least 100 nucleotides. In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 93.

[0430] In some embodiments, the anti-cancer vaccine is formulated with liposomes to yield RNA-lipoplexes.

[0431] Molecular Structures and Functions of vaccine antigens

[0432] CLDN6

[0433] The human claudin 6 gene (CLDN6) is localized on chromosome 16 and contains two isoforms which encode a protein of 220 amino acids. CLDN6 is highly conserved among species, and belongs to the group of claudins which consists of at least 27 members. In general, claudins, including CLDN6, are important for epithelial barrier regulation and belong to the group of tight junction molecules. CLDN6 contains four transmembrane domains, two extracellular loops, intracellular N- and C-termini, and a PDZ -binding domain, and has been shown to play a role in maintaining permeability barriers and trans-epithelial resistance in epidermal cells. Additionally, CLDN6 appears to be required for normal blastocyst formation.

[0434] In one embodiment, CLDN6 has the amino acid sequence according to SEQ ID NO: 1.

[0435] A CLDN6 vaccine antigen comprises an amino acid sequence comprising CLDN6, an immunogenic variant thereof, or an immunogenic fragment of the CLDN6 or the immunogenic variant thereof. In one embodiment, a CLDN6 vaccine antigen comprises (1) the amino acid sequence of SEQ ID NO: 1 or 2, (2) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 2, or (3) an immunogenic fragment of (1) or (2). Additionally, the CLDN6 vaccine antigen comprising the amino acid sequence of SEQ ID NO: 1 may further comprise one or more (preferably all) of the following (ii) and (iii), whereas the CLDN6 vaccine antigen comprising the amino acid sequence of SEQ ID NO: 2 may further comprise the following (iii): (ii) an amino acid sequence breaking immunological tolerance (preferably, as described herein, e.g., comprising the amino acid sequence of SEQ ID NO: 89); (iii) an amino acid sequence enhancing antigen processing and / or presentation (preferably, as described herein, e.g., comprising the amino acid sequence of SEQ ID NO: 87). In one embodiment, a CLDN6 vaccine antigen comprises the amino acid sequence of SEQ ID NO: 2.

[0436] RNA encoding a CLDN6 vaccine antigen may encode an amino acid sequence comprising (1) the amino acid sequence of SEQ ID NO: 1 or 2, (2) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 2, or (3) an immunogenic fragment of (1) or (2). Additionally, the RNA encoding an CLDN6 vaccine antigen comprising the amino acid sequence of SEQ ID NO: 1 may further comprise one or more (preferably all) of the following nucleotide sequences (i) and (iii) to (vi), whereas the RNA encoding an CLDN6 vaccine antigen comprising the amino acid sequence of SEQ ID NO: 2 may further comprise one or more (preferably all) of the following nucleotide sequences (i), and (iv) to (vi): (i) a 5'-UTR (preferably, as described herein, e.g., a Kozak sequence comprising the sequence of SEQ ID NO: 91); (iii) a nucleotide sequence encoding an amino acid sequence breaking immunological tolerance (preferably, as described herein, e.g., a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 90); (iv) a nucleotide sequence encoding an amino acid sequence enhancing antigen processing and / or presentation (preferably, as described herein, e.g., a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 88); (v) a 3'-UTR (preferably, as described herein, e.g., an FI element comprising the sequence of SEQ ID NO: 92); (vi) a poly-A tail (preferably, as described herein, e.g., A30L70 comprising the nucleotide sequence of SEQ ID NO: 93). In one embodiment, RNA encoding a CLDN6 vaccine antigen may comprise (I1) the nucleotide sequence of SEQ ID NO: 3 or 4, (2') a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3 or 4, or (3') a fragment of (I1) or (2'). In one embodiment, RNA encoding a CLDN6 vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 4; and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 2.KK-LC-1

[0437] Kita-kyushu lung cancer antigen 1 (KK-LC-1), also cancer / testis antigen 83, CT83, CXorf61, is aprotein and tumor antigen from the group of cancer / testis antigens. KK-LC-1 has a length of 113 amino acids. KK-LC-1 is rarely found as a tumor antigen in healthy cells (except in immune privileged spermatocytes), but is often expressed in various tumors, e.g. non-small cell lung cancer.

[0438] In one embodiment, KK-LC-1 has the amino acid sequence according to SEQ ID NO: 5.

[0439] A Kita-kyushu lung cancer antigen 1 (KK-LC-1) vaccine antigen comprises an amino acid sequence comprising KK-LC-1, an immunogenic variant thereof, or an immunogenic fragment of the KK-LC-1 or the immunogenic variant thereof. In one embodiment, a KK-LC-1 vaccine antigen comprises (1) the amino acid sequence of SEQ ID NO: 5 or 6, (2) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5 or 6, or (3) an immunogenic fragment of (1) or (2). Additionally, the KK-LC-1 vaccine antigen comprising the amino acid sequence of SEQ ID NO: 5 may further comprise one or both (preferably both) of the following (ii) and (iii), whereas the KK-LC-1 vaccine antigen comprising the amino acid sequence of SEQ ID NO: 6 may further comprise the following (iii): (ii) an amino acid sequence breaking immunological tolerance (preferably, as described herein, e.g., comprising the amino acid sequence of SEQ ID NO: 89); (iii) an amino acid sequence enhancing antigen processing and / or presentation (preferably, as described herein, e.g., comprising the amino acid sequence of SEQ ID NO: 87). In one embodiment, a KK-LC-1 vaccine antigen comprises the amino acid sequence of SEQ ID NO: 6.

[0440] RNA encoding a KK-LC-1 vaccine antigen may encode an amino acid sequence comprising (1) the amino acid sequence of SEQ ID NO: 5 or 6, (2) an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5 or 6, or (3) an immunogenic fragment of (1) or (2). Additionally, the RNA encoding a KK-LC-1 vaccine antigen comprising the amino acid sequence of SEQ ID NO: 5 may further comprise one or more (preferab...

Claims

Claims1. A combination for use as a medicament, wherein the combination comprises:(a) an anti-cancer vaccine comprising at least one RNA, wherein the at least one RNA encodes at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and(b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction.

2. A method of treating cancer in a subject comprising administering to the subject:(a) an anti-cancer vaccine, wherein the anti-cancer vaccine comprises at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and(b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction.

3. An anti-cancer vaccine comprising at least one RNA, wherein the at least one RNA encodes at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof, for use in a method of treating cancer in a subject, the method comprising administering to the subject (a) the anti-cancer vaccine and (b) a binding agent comprising (i) a first binding region which binds to PD- 1 , PD-L 1 , or both and which antagonizes the PD-1 / PD-L 1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction.

4. A binding agent comprising (i) a first binding region which binds to PD-1 , PD-L1 , or both and which antagonizes the PD-1 / PD-L 1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction, for use in a method of treating cancer in a subject, the method comprising administering to the subject (a) the binding agent and (b) an anti-cancer vaccine comprising at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein saidat least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof.

5. A binding agent and an anti-cancer vaccine for use in a method of treating cancer in a subject, the method comprising administering to the subject the binding agent and the anti-cancer vaccine, wherein (a) the binding agent comprises (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction; and (b) the anti-cancer vaccine comprises at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof.

6. A medical preparation, combination, kit, or composition comprising:(a) an anti-cancer vaccine comprising at least one RNA, wherein the at least one RNA encodes at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and(b) a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction; ora kit comprising(1) a composition comprising an anti-cancer vaccine, wherein the anti-cancer vaccine comprises at least one RNA, the at least one RNA encoding at least one amino acid sequence, wherein said at least one amino acid sequence comprises a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof; and (2) a composition comprising a binding agent comprising (i) a first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction; and (ii) a second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction.

7. The combination for use of claim 1, the method of claim 2, the anti-cancer vaccine for use of claim 3, the binding agent for use of claim 4, the binding agent and anti-cancer vaccine for use of claim 5, or the medical preparation, combination, kit, or composition of claim 6, wherein the tumor antigen is selected from a set of tumor antigens comprising CLDN6, humanpapillomavirus (HPV) E6, HPV E7, KK-LC-1, MAGE-A3, MAGE-A4, MAGE-CI, NY-ESO- 1 , PRAME, TPTE, and tyrosinase.

8. The combination for use of claim 1 or 7, the method of claim 2 or 7, the anti-cancer vaccine for use of claim 3 or 7, the binding agent for use of claim 4 or 7, the binding agent and anti-cancer vaccine for use of claim 5 or 7, or the medical preparation, combination, kit, or composition of claim 6 or 7, wherein the tumor antigen is selected from a set of tumor antigens consisting of CLDN6, HPV E6, HPV E7, KK-LC-1, MAGE-A3, MAGE-A4, MAGE-CI, NY-ESO-1, PRAME, TPTE, and tyrosinase.

9. The combination for use of any one of claims 1 , 7, and 8, the method of any one of claims 2, 7, and 8, the anti-cancer vaccine for use of any one of claims 3, 7, and 8, the binding agent for use of any one of claims 4, 7, and 8, the binding agent and anti-cancer vaccine for use of claim 5, 7, or 8, or the medical preparation, combination, kit, or composition of any one of claims 6 to 8, wherein the at least one RNA encodes one or more amino acid sequences, two or more amino acid sequences, three or more amino acid sequences, four or more amino acid sequences, five or more amino acid sequences, or six or more amino acid sequences, each of said amino acid sequences comprising a tumor antigen, an immunogenic variant thereof, or an immunogenic fragment of the tumor antigen or the immunogenic variant thereof.

10. The combination for use of any one of claims 1 and 7 to 9, the method of any one of claims 2 and 7 to 9, the anti-cancer vaccine for use of any one of claims 3 and 7 to 9, the binding agent for use of any one of claims 4 and 7 to 9, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 9, or the medical preparation, combination, kit, or composition of any one of claims 6 to 9, wherein the at least one RNA encodes at least the following amino acid sequences ( 1 )-(6):(1) an amino acid sequence comprising CLDN6, an immunogenic variant thereof, or an immunogenic fragment of the CLDN6 or the immunogenic variant thereof;(2) an amino acid sequence comprising KK-LC-1, an immunogenic variant thereof, or an immunogenic fragment of the KK-LC-1 or the immunogenic variant thereof;(3) an amino acid sequence comprising MAGE-A3, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A3 or the immunogenic variant thereof;(4) an amino acid sequence comprising MAGE-A4, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A4 or the immunogenic variant thereof;(5) an amino acid sequence comprising PRAME, an immunogenic variant thereof, or an immunogenic fragment of the PRAME or the immunogenic variant thereof;(6) an amino acid sequence comprising MAGE-CI, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-CI or the immunogenic variant thereof.

11. The combination for use of any one of claims 1 and 7 to 10, the method of any one of claims 2 and 7 to 10, the anti-cancer vaccine for use of any one of claims 3 and 7 to 10, the binding agent for use of any one of claims 4 and 7 to 10, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 10, or the medical preparation, combination, kit, or composition of any one of claims 6 to 10, which comprises the following RNAs (l)-(6) or which comprises administering the following RNAs (1 )-(6):(1) RNA encoding an amino acid sequence comprising CLDN6, an immunogenic variant thereof, or an immunogenic fragment of the CLDN6 or the immunogenic variant thereof; (2) RNA encoding an amino acid sequence comprising KK-LC-1, an immunogenic variant thereof, or an immunogenic fragment of the KK-LC-1 or the immunogenic variant thereof; (3) RNA encoding an amino acid sequence comprising MAGE-A3, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A3 or the immunogenic variant thereof; (4) RNA encoding an amino acid sequence comprising MAGE-A4, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-A4 or the immunogenic variant thereof; (5) RNA encoding an amino acid sequence comprising PRAME, an immunogenic variant thereof, or an immunogenic fragment of the PRAME or the immunogenic variant thereof; (6) RNA encoding an amino acid sequence comprising MAGE-CI, an immunogenic variant thereof, or an immunogenic fragment of the MAGE-CI or the immunogenic variant thereof.

12. The combination for use of any one of claims 1 and 7 to 9, the method of any one of claims 2 and 7 to 9, the anti-cancer vaccine for use of any one of claims 3 and 7 to 9, the binding agent for use of any one of claims 4 and 7 to 9, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 9, or the medical preparation, combination, kit, or composition of any one of claims 6 to 9, wherein the at least one RNA encodes at least the following amino acid sequences (1 )-(4):(1) an amino acid sequence comprising MAGE- A3, an immunogenic variant thereof, or an immunogenic fragment of the MAGE- A3 or the immunogenic variant thereof;(2) an amino acid sequence comprising NY-ESO-1, an immunogenic variant thereof, or an immunogenic fragment of the NY-ESO-1 or the immunogenic variant thereof;(3) an amino acid sequence comprising TPTE, an immunogenic variant thereof, or an immunogenic fragment of the TPTE or the immunogenic variant thereof; and(4) an amino acid sequence comprising tyrosinase, an immunogenic variant thereof, or an immunogenic fragment of the tyrosinase or the immunogenic variant thereof.

13. The combination for use of any one of claims 1 , 7 to 9, and 12, the method of any one of claims 2, 7 to 9, and 12, the anti-cancer vaccine for use of any one of claims 3, 7 to 9, and 12, the binding agent for use of any one of claims 4, 7 to 9, and 12, the binding agent and anti-cancer vaccine for use of any one of claims 5, 7 to 9, and 12, or the medical preparation, combination, kit, or composition of any one of claims 6 to 9 and 12, which comprises the following RNAs (1 )-(4) or which comprises administering the following RNAs (l)-(4):(1) RNA encoding an amino acid sequence comprising MAGE- A3, an immunogenic variant thereof, or an immunogenic fragment of the MAGE- A3 or the immunogenic variant thereof; (2) RNA encoding an amino acid sequence comprising NY-ESO-1, an immunogenic variant thereof, or an immunogenic fragment of the NY-ESO-1 or the immunogenic variant thereof; (3) RNA encoding an amino acid sequence comprising TPTE, an immunogenic variant thereof, or an immunogenic fragment of the TPTE or the immunogenic variant thereof;(4) RNA encoding an amino acid sequence comprising tyrosinase, an immunogenic variant thereof, or an immunogenic fragment of the tyrosinase or the immunogenic variant thereof.

14. The combination for use of any one of claims 1 and 7 to 9, the method of any one of claims 2 and 7 to 9, the anti-cancer vaccine for use of any one of claims 3 and 7 to 9, the binding agent for use of any one of claims 4 and 7 to 9, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 9, or the medical preparation, combination, kit, or composition of any one of claims 6 to 9, wherein the at least one RNA encodes at least the following amino acid sequences (1) and (2):(1) an amino acid sequence comprising HPV E6, an immunogenic variant thereof, or an immunogenic fragment of the HPV E6 or the immunogenic variant thereof;(2) an amino acid sequence comprising HPV E7, an immunogenic variant thereof, or an immunogenic fragment of the HPV E7 or the immunogenic variant thereof.

15. The combination for use of any one of claims 1, 7 to 9, and 14, the method of any one of claims 2, 7 to 9, and 14, the anti-cancer vaccine for use of any one of claims 3, 7 to 9, and 14, the binding agent for use of any one of claims 4, 7 to 9, and 14, the binding agent and an anti-cancer vaccine for use of any one of claims 5, 7 to 9, and 14, or the medical preparation, combination, kit, or composition of any one of claims 6 to 9 and 14, which comprises the following RNAs (1) and (2) or which comprises administering the following RNAs (1) and (2):(1) RNA encoding an amino acid sequence comprising HPV E6, an immunogenic variant thereof, or an immunogenic fragment of the HPV E6 or the immunogenic variant thereof; (2) RNA encoding an amino acid sequence comprising HPV E7, an immunogenic variant thereof, or an immunogenic fragment of the HPV E7 or the immunogenic variant thereof.

16. The combination for use of any one of claims 1 and 7 to 15, the method of any one of claims 2 and 7 to 15, the anti-cancer vaccine for use of any one of claims 3 and 7 to 15, the binding agent for use of any one of claims 4 and 7 to 15, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 15, or the medical preparation, combination, kit, or composition of any one of claims 6 to 15, wherein each of the at least one amino acid sequence is encoded by a separate RNA.

17. The combination for use of any one of claims 1 and 7 to 16, the method of any one of claims 2 and 7 to 16, the anti-cancer vaccine for use of any one of claims 3 and 7 to 16, the binding agent for use of any one of claims 4 and 7 to 16, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 16, or the medical preparation, combination, kit, or composition of any one of claims 6 to 16, wherein at least one amino acid sequence comprises an amino acid sequence which breaks immunological tolerance and / or at least one RNA is co-administered with RNA encoding an amino acid sequence which breaks immunological tolerance.

18. The combination for use of any one of claims 1 and 7 to 17, the method of any one of claims 2 and 7 to 17, the anti-cancer vaccine for use of any one of claims 3 and 7 to 17, the binding agent for use of any one of claims 4 and 7 to 17, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 17, or the medical preparation, combination, kit, or composition of any one of claims 6 to 17, wherein each of the at least one amino acid sequence comprises an amino acid sequence which breaks immunological tolerance and / or each RNA is coadministered with RNA encoding an amino acid sequence which breaks immunological tolerance.

19. The combination for use of claim 17 or 18, the method of claim 17 or 18, the anti-cancer vaccine for use of claim 17 or 18, the binding agent for use of claim 17 or 18, the binding agent and anticancer vaccine for use of claim 17 to 18, or the medical preparation, combination, kit, or composition of claim 17 or 18, wherein the amino acid sequence which breaks immunological tolerance comprises helper epitopes, preferably tetanus toxoid-derived helper epitopes.

20. The combination for use of any one of claims 17 to 19, the method of any one of claims 17 to 19, the anti-cancer vaccine for use of any one of claims 17 to 19, the binding agent for use of any one of claims 17 to 19, the binding agent and anti-cancer vaccine for use of any one of claims 17 to 19, or the medical preparation, combination, kit, or composition of any one of claims 17 to 19, wherein(i) the RNA encoding the amino acid sequence which breaks immunological tolerance comprises the nucleotide sequence of SEQ ID NO: 90, or a nucleotide sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 90; and / or(ii) the amino acid sequence which breaks immunological tolerance comprises the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 89.

21. The combination for use of any one of claims 1 and 7 to 20, the method of any one of claims 2 and 7 to 20, the anti-cancer vaccine for use of any one of claims 3 and 7 to 20, the binding agent for use of any one of claims 4 and 7 to 20, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 20, or the medical preparation, combination, kit, or composition of any one of claims 6 to 20, wherein at least one amino acid sequence is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence, wherein the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

22. The combination for use of any one of claims 1 and 7 to 21, the method of any one of claims 2 and 7 to 21 , the anti-cancer vaccine for use of any one of claims 3 and 7 to 21 , the binding agent for use of any one of claims 4 and 7 to 21 , the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 21, or the medical preparation, combination, kit, or composition of any one of claims 6 to 21, wherein each of the at least one amino acid sequence is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence, wherein the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

23. The combination for use of any one of claims 1 and 7 to 22, the method of any one of claims 2 and 7 to 22, the anti-cancer vaccine for use of any one of claims 3 and 7 to 22, the binding agent for use of any one of claims 4 and 7 to 22, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 22, or the medical preparation, combination, kit, or composition of any one of claims 6 to 22, wherein at least one RNA comprises a 5'-cap structure, preferably m27-2°Gppsp(5’)G.

24. The combination for use of any one of claims 1 and 7 to 23, the method of any one of claims 2 and 7 to 23, the anti-cancer vaccine for use of any one of claims 3 and 7 to 23, the binding agent for use of any one of claims 4 and 7 to 23, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 23, or the medical preparation, combination, kit, or composition ofany one of claims 6 to 23, wherein each RNA comprises a 5 '-cap structure, preferably m7,2’’ °Gppsp(5')G.

25. The combination for use of any one of claims 1 and 7 to 24, the method of any one of claims 2 and 7 to 24, the anti-cancer vaccine for use of any one of claims 3 and 7 to 24, the binding agent for use of any one of claims 4 and 7 to 24, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 24, or the medical preparation, combination, kit, or composition of any one of claims 6 to 24, wherein at least one RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 91, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 91.

26. The combination for use of any one of claims 1 and 7 to 25, the method of any one of claims 2 and 7 to 25, the anti-cancer vaccine for use of any one of claims 3 and 7 to 25, the binding agent for use of any one of claims 4 and 7 to 25, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 25, or the medical preparation, combination, kit, or composition of any one of claims 6 to 25, wherein each RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 91 , or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%>, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 91.

27. The combination for use of any one of claims 1 and 7 to 26, the method of any one of claims 2 and 7 to 26, the anti-cancer vaccine for use of any one of claims 3 and 7 to 26, the binding agent for use of any one of claims 4 and 7 to 26, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 26, or the medical preparation, combination, kit, or composition of any one of claims 6 to 26, wherein at least one amino acid sequence comprises an amino acid sequence enhancing antigen processing and / or presentation.

28. The combination for use of any one of claims 1 and 7 to 27, the method of any one of claims 2 and 7 to 27, the anti-cancer vaccine for use of any one of claims 3 and 7 to 27, the binding agent for use of any one of claims 4 and 7 to 27, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 27, or the medical preparation, combination, kit, or composition of any one of claims 6 to 27, wherein each of the at least one amino acid sequence comprises an amino acid sequence enhancing antigen processing and / or presentation.

29. The combination for use of claim 27 or 28, the method of claim 27 or 28, the anti-cancer vaccine for use of claim 27 or 28, the binding agent for use of claim 27 or 28, the binding agent and anti-cancer vaccine for use of claim 27 or 28, or the medical preparation, combination, kit, or composition of claim 27 or 28, wherein the amino acid sequence enhancing antigen processing and / or presentation comprises an amino acid sequence corresponding to the transmembrane and cytoplasmic domain of an MHC molecule, preferably an MHC class I molecule.

30. The combination for use of any one of claims 27 to 29, the method of any one of claims 27 to 29, the anti-cancer vaccine for use of any one of claims 27 to 29, the binding agent for use of any one of claims 27 to 29, the binding agent and anti-cancer vaccine for use of any one of claims 27 to 29, or the medical preparation, combination, kit, or composition of any one of claims 27 to 29, wherein(i) the RNA encoding the amino acid sequence enhancing antigen processing and / or presentation comprises the nucleotide sequence of SEQ ID NO: 88, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 88; and / or(ii) the amino acid sequence enhancing antigen processing and / or presentation comprises the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 87.

31. The combination for use of any one of claims 1 and 7 to 30, the method of any one of claims 2 and 7 to 30, the anti-cancer vaccine for use of any one of claims 3 and 7 to 30, the binding agent for use of any one of claims 4 and 7 to 30, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 30, or the medical preparation, combination, kit, or composition of any one of claims 6 to 30, wherein at least one RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 92, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 92.

32. The combination for use of any one of claims 1 and 7 to 31, the method of any one of claims 2 and 7 to 31 , the anti-cancer vaccine for use of any one of claims 3 and 7 to 31 , the binding agent for use of any one of claims 4 and 7 to 31 , the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 31 , or the medical preparation, combination, kit, or composition of any one of claims 6 to 31, wherein each RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 92, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 92.

33. The combination for use of any one of claims 1 and 7 to 32, the method of any one of claims 2 and 7 to 32, the anti-cancer vaccine for use of any one of claims 3 and 7 to 32, the binding agent for use of any one of claims 4 and 7 to 32, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 32, or the medical preparation, combination, kit, or composition of any one of claims 6 to 32, wherein at least one RNA comprises a poly-A sequence.

34. The combination for use of any one of claims 1 and 7 to 33, the method of any one of claims 2 and 7 to 33, the anti-cancer vaccine for use of any one of claims 3 and 7 to 33, the binding agent for use of any one of claims 4 and 7 to 33, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 33, or the medical preparation, combination, kit, or composition of any one of claims 6 to 33, wherein each RNA comprises a poly-A sequence.

35. The combination for use of claim 33 or 34, the method of claim 33 or 34, the anti-cancer vaccine for use of claim 33 or 34, the binding agent for use of claim 33 or 34, the binding agent and anticancer vaccine for use of claim 33 or 34, or the medical preparation, combination, kit, or composition of claim 33 or 34, wherein the poly-A sequence comprises at least 100 nucleotides.

36. The combination for use of any one of claims 33 to 35, the method of any one of 33 to 35, the anti-cancer vaccine for use of any one of claims 33 to 35, the binding agent for use of any one of claims 33 to 35, the binding agent and anti-cancer vaccine for use of any one of claims 33 to 35, or the medical preparation, combination, kit, or composition of any one of claims 33 to 35, wherein the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 93.

37. The combination for use of any one of claims 1 and 7 to 36, the method of any one of claims 2 and 7 to 36, the anti-cancer vaccine for use of any one of claims 3 and 7 to 36, the binding agent for use of any one of claims 4 and 7 to 36, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 36, or the medical preparation, combination, kit, or composition of any one of claims 6 to 36, wherein the first binding region which binds to PD-1 , PD-L1 , or both and which antagonizes the PD-1 / PD-L1 interaction comprises an anti-PD-1 antibody or a PD-1 binding fragment thereof.

38. The combination for use of claim 37, the method claim 37, the anti-cancer vaccine for use of claim 37, the binding agent for use of claim 37, the binding agent and anti-cancer vaccine for use of claim 37, or the medical preparation, combination, kit, or composition of claim 37, wherein the anti-PD-1 antibody is selected from the group consisting of Pembrolizumab,Nivolumab, Cemiplimab, Dostarlimab, Toripalimab, Retifanlimab, Jankistomig, and PD-1 binding fragments thereof.

39. The combination for use of any one of claims 1 and 7 to 38, the method of any one of claims 2 and 7 to 38, the anti-cancer vaccine for use of any one of claims 3 and 7 to 38, the binding agent for use of any one of claims 4 and 7 to 38, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 38, or the medical preparation, combination, kit, or composition of any one of claims 6 to 38 , wherein the first binding region which binds to PD- 1 , PD-L 1 , or both and which antagonizes the PD-1 / PD-L 1 interaction comprises:(i-la) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 98, 99, and 100, respectively, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 101, 102, and 103, respectively;(i-lb) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 104, 105, and 106, respectively, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 107, 108, and 103, respectively;(i-lc) the amino acid sequence of SEQ ID NO: 109 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 109;(i-ld) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 109, and comprising the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 98, 99, 100, 101, 102, and 103, respectively; or(i-le) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 109, and comprising the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 104, 105, 106, 107, 108, and 103, respectively.

40. The combination for use of any one of claims 1 and 7 to 38, the method of any one of claims 2 and 7 to 38, the anti-cancer vaccine for use of any one of claims 3 and 7 to 38, the binding agent for use of any one of claims 4 and 7 to 38, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 38, or the medical preparation, combination, kit, or composition of any one of claims 6 to 38, wherein the first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L 1 interaction comprises:(i-2a) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, HI, and 112, respectively, or the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 113, respectively (e.g., the first binding region comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 112, respectively, or a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 113, respectively);(i-2b) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 116, respectively, or the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 117, respectively (e.g., the first binding region comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 116, respectively, or a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 117, respectively);(i-2c) the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118 preferably comprises the HCDR1 , HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 112, respectively, or comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 116, respectively; or(i-2d) the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119 preferably comprises the HCDR1 , HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 113, respectively, or comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 117, respectively.

41. The combination for use of any one of claims 1 and 7 to 38, the method of any one of claims 2 and 7 to 38, the anti-cancer vaccine for use of any one of claims 3 and 7 to 38, the binding agent for use of any one of claims 4 and 7 to 38, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 38, or the medical preparation, combination, kit, or composition of any one of claims 6 to 38, wherein the first binding region which binds to PD-1 , PD-L1 , or both and which antagonizes the PD-1 / PD-L1 interaction comprises:(i-5a) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 190, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 191;(i-5b) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 184, 185, and 186, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 187, 188, and 189, respectively;(i-5c) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 191 ;(i-5d) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 191, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 184, 185, and 186, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 187, 188, and 189, respectively; or(i-5e) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ED NO: 190, and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 191.

42. The combination for use of any one of claims 1 and 7 to 38, the method of any one of claims 2 and 7 to 38, the anti-cancer vaccine for use of any one of claims 3 and 7 to 38, the binding agent for use of any one of claims 4 and 7 to 38, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 38, or the medical preparation, combination, kit, or composition of any one of claims 6 to 38, wherein the first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction comprises:(i-6a) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 190, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 193;(i-6b) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 184, 185, and 186, respectively, and a light chain variableregion (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 187, 192, and 189, respectively;(i-6c) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 193;(i-6d) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ of ID NO: 193, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 184, 185, and 186, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 187, 192, and 189, respectively; or(i-6e) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 193.

43. The combination for use of any one of claims 1 and 7 to 38, the method of any one of claims 2 and 7 to 38, the anti-cancer vaccine for use of any one of claims 3 and 7 to 38, the binding agent for use of any one of claims 4 and 7 to 38, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 38, or the medical preparation, combination, kit, or composition of any one of claims 6 to 38, wherein the first binding region which binds to PD-1 , PD-L1 , or both and which antagonizes the PD-1 / PD-L1 interaction comprises:(i-7a) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 190, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 195;(i-7b) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 184, 185, and 186, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 187, 194, and 189, respectively;(i-7c) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 190, and a lightchain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 195;(i-7d) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 195, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 184, 185, and 186, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 187, 194, and 189, respectively; or(i-7e) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 195.

44. The combination for use of any one of claims 1 and 7 to 38, the method of any one of claims 2 and 7 to 38, the anti-cancer vaccine for use of any one of claims 3 and 7 to 38, the binding agent for use of any one of claims 4 and 7 to 38, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 38, or the medical preparation, combination, kit, or composition of any one of claims 6 to 38, wherein the first binding region which binds to PD-1 , PD-L1 , or both and which antagonizes the PD-1 / PD-L1 interaction comprises:(i-8a) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 190, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 197;(i-8b) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 184, 185, and 186, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 187, 196, and 189, respectively;(i-8c) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 197;(i-8d) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 197, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 184, 185, and 186, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 187, 196, and 189, respectively; or(i-8e) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 190, and a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 197.

45. The combination for use of any one of claims 1 and 7 to 38, the method of any one of claims 2 and 7 to 38, the anti-cancer vaccine for use of any one of claims 3 and 7 to 38, the binding agent for use of any one of claims 4 and 7 to 38, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 38, or the medical preparation, combination, kit, or composition of any one of claims 6 to 38, wherein the first binding region which binds to PD-1 , PD-L1 , or both and which antagonizes the PD-1 / PD-L1 interaction comprises:(i-lOa) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 229, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 230;(i-lOb) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 222, 223, and 224, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively;(i-lOc) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 226, 227, and 228, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively;(i-lOd) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 229, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 230;(i-lOe) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 229, and a lightchain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 230, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 222, 223, and 224, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively; or(i-101) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 229, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 230, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 226, 227, and 228, respectively, and the VL comprises the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively.

46. The combination for use of any one of claims 1 and 7 to 36, the method of any one of claims 2 and 7 to 36, the anti-cancer vaccine for use of any one of claims 3 and 7 to 36, the binding agent for use of any one of claims 4 and 7 to 36, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 36, or the medical preparation, combination, kit, or composition of any one of claims 6 to 36, wherein the first binding region which binds to PD-1 , PD-L1 , or both and which antagonizes the PD-1 / PD-L1 interaction comprises an anti-PD-Ll antibody or a PD- L1 binding fragment thereof.

47. The combination for use of claim 46, the method of claim 46, the anti-cancer vaccine for use of claim 46, the binding agent for use of claim 46, the binding agent and an anti-cancer vaccine for use of claim 46, or the medical preparation, combination, kit, or composition of claim 46, wherein the anti-PD-Ll antibody is selected from the group consisting of Atezolizumab, Durvalumab, Avelumab, Sotiburafusp alpha, Palverafiisp, and PD-L1 binding fragments thereof.

48. The combination for use of any one of claims 1, 7 to 36, 46, and 47, the method of any one of claims 2, 7 to 36, 46, and 47, the anti-cancer vaccine for use of any one of claims 3, 7 to 36, 46, and 47, the binding agent for use of any one of claims 4, 7 to 36, 46, and 47, the binding agent and anti-cancer vaccine for use of any one of claims 5, 7 to 36, 46, and 47, or the medical preparation, combination, kit, or composition of any one of claims 6 to 36, 46, and 47, whereinthe first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD- 1 / PD-L1 interaction comprises:(i-3a) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 120, 121, and 122, respectively;(i-3b) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 123, 124, and 125, respectively; or(i-3c) the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 126, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 126 preferably comprises the HCDR1 , HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 120, 121, and 122, respectively, or comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 123, 124, and 125, respectively.

49. The combination for use of any one of claims 37 to 48, the method of any one of claims 37 to 48, the anti-cancer vaccine for use of any one of claims 37 to 48, the binding agent for use of any one of claims 37 to 48, the binding agent and anti-cancer vaccine for use of any one of claims 37 to 48, or the medical preparation, combination, kit, or composition of any one of claims 37 to 48, wherein the first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD-1 / PD-L1 interaction comprises a single-chain fragment (e.g., scFv) or a VHH.

50. The combination for use of any one of claims 1, 7 to 36, 46, and 47, the method of any one of claims 2, 7 to 36, 46, and 47, the anti-cancer vaccine for use of any one of claims 3, 7 to 36, 46, and 47, the binding agent for use of any one of claims 4, 7 to 36, 46, and 47, the binding agent and anti-cancer vaccine for use of any one of claims 5, 7 to 36, 46, and 47, or the medical preparation, combination, kit, or composition of any one of claims 6 to 36, 46, and 47, wherein the first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD- 1 / PD-L1 interaction comprises:(i-4a) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 133, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 134;(i-4b) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 127, 128, and 129, respectively, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 130, 131, and 132, respectively;(i-4c) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 133, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 134; or(i-4d) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 133, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 134, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 127, 128, and 129, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 130, 131, and 132, respectively.

51. The combination for use of any one of claims 1, 7 to 36, 46, and 47, the method of any one of claims 2, 7 to 36, 46, and 47, the anti-cancer vaccine for use of any one of claims 3, 7 to 36, 46, and 47, the binding agent for use of any one of claims 4, 7 to 36, 46, and 47, the binding agent and anti-cancer vaccine for use of any one of claims 5, 7 to 36, 46, and 47, or the medical preparation, combination, kit, or composition of any one of claims 6 to 36, 46, and 47, wherein the first binding region which binds to PD-1, PD-L1, or both and which antagonizes the PD- 1 / PD-L1 interaction comprises:(i-9a) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 209, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 210;(i-9b) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 198, 199, and 200, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 201, 202, and 203, respectively;(i-9c) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 204, 205, and 206, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 207, 208, and 203, respectively;(i-9d) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 209, and a lightchain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 210;(i-9e) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 209, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 210, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 198, 199, and 200, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 201, 202, and 203, respectively; or(i-9f) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 209, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 210, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 204, 205, and 206, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 207, 208, and 203, respectively.

52. The combination for use of any one of claims 1 and 7 to 51, the method of any one of claims 2 and 7 to 51 , the anti-cancer vaccine for use of any one of claims 3 and 7 to 51 , the binding agent for use of any one of claims 4 and 7 to 51 , the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 51 , or the medical preparation, combination, kit, or composition of any one of claims 6 to 51, wherein the second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction comprises an anti- VEGF antibody, preferably an anti-VEGF-A antibody, or a VEGF binding fragment thereof.

53. The combination for use of any one of claims 1 and 7 to 52, the method of any one of claims 2 and 7 to 52, the anti-cancer vaccine for use of any one of claims 3 and 7 to 52, the binding agent for use of any one of claims 4 and 7 to 52, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 52, or the medical preparation, combination, kit, or composition of any one of claims 6 to 52, wherein the anti-VEGF antibody is selected from Bevacizumab, Ranibizumab, and VEGF binding fragments thereof.

4. The combination for use of any one of claims 1 and 7 to 53, the method of any one of claims 2 and 7 to 53, the anti-cancer vaccine for use of any one of claims 3 and 7 to 53, the binding agent for use of any one of claims 4 and 7 to 53, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 53, or the medical preparation, combination, kit, or composition of any one of claims 6 to 53, wherein the second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction comprises: (ii-la) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 146, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 147;(ii-lb) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and a light chain variable region (VL) comprising the CDR1 , CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively;(ii-lc) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively;(ii-ld) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147;(ii-le) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively;(ii-lf) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively;(ii-lg) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151 and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152;(ii-lh) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151 and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively; or(ii-li) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151 and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

55. The combination for use of any one of claims 1 and 7 to 53, the method of any one of claims 2 and 7 to 53, the anti-cancer vaccine for use of any one of claims 3 and 7 to 53, the binding agent for use of any one of claims 4 and 7 to 53, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 53, or the medical preparation, combination, kit, or composition of any one of claims 6 to 53, wherein the second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction comprises:(ii-2a) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 235, and a light chain variable region (VL) comprising the LCDR1 , LCDR2, and LCDR3 sequences of SEQ ID NO: 230;(ii-2b) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 231, 136, and 232, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively;(ii-2c) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 233, 142, and 234, respectively, and a light chain variable region (VL) comprising the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively;(ii-2d) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 235, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 230;(ii-2e) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 235, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 230, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 231, 136, and 232, respectively, and the VL comprises the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively; or(ii-2f) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 235, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 230, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 233, 142, and 234, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively.

6. The combination for use of any one of claims 1 and 7 to 53, the method of any one of claims 2 and 7 to 53, the anti-cancer vaccine for use of any one of claims 3 and 7 to 53, the binding agent for use of any one of claims 4 and 7 to 53, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 53, or the medical preparation, combination, kit, or composition of any one of claims 6 to 53, wherein the second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction comprises: (ii-2’a) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 146, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 230;(ii-2’b) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively;(ii-2’c) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively;(ii-2’d) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 230;(ii-2’e) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 230, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively; or(ii-2’f) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 230, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively.

57. The combination for use of any one of claims 1 and 7 to 56, the method of any one of claims 2 and 7 to 56, the anti-cancer vaccine for use of any one of claims 3 and 7 to 56, the binding agent for use of any one of claims 4 and 7 to 56, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 56, or the medical preparation, combination, kit, or composition of any one of claims 6 to 56, wherein the second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction is in the format of a full-length antibody or a VEGF binding antibody fragment thereof.

58. The combination for use of any one of claims 1 and 7 to 51 , the method of any one of claims 2 and 7 to 51 , the anti-cancer vaccine for use of any one of claims 3 and 7 to 51 , the binding agent for use of any one of claims 4 and 7 to 51 , the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 51, or the medical preparation, combination, kit, or composition of any one of claims 6 to 51, wherein the second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction comprises a VEGFR domain, such as a VEGFR- 1 domain comprising the amino acid sequence of SEQ ID NO: 153, a VEGFR-2 domain comprising the amino acid sequence of SEQ ID NO: 154, a VEGFR-3a domain comprising the amino acid sequence of SEQ ID NO: 155, a VEGFR-3b domain comprising the amino acid sequence of SEQ ID NO: 215, or a combination thereof.

59. The combination for use of any one of claims 1 , 7 to 51 , and 58, the method of any one of claims 2, 7 to 51, and 58, the anti-cancer vaccine for use of any one of claims 3, 7 to 51, and 58, the binding agent for use of any one of claims 4, 7 to 51, and 58, the binding agent and anti-cancer vaccine for use of any one of claims 5, 7 to 51 , and 58, or the medical preparation, combination, kit, or composition of any one of claims 6 to 51 and 58, wherein the second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction comprises at least one VEGFR- 1 domain, or at least one VEGFR-2 domain, or at least one VEGFR-3a domain, or at least one VEGFR-1 domain and at least one VEGFR-2 domain, such as two VEGFR-1 domains, or two VEGFR-2 domains, or two VEGFR- 1 domains and two VEGFR-2 domains, or two VEGFR-3a domains.

60. The combination for use of any one of claims 1, 7 to 51, 58, and 59, the method of any one of claims 2, 7 to 51 , 58, and 59, the anti-cancer vaccine for use of any one of claims 3, 7 to 51 , 58,and 59, the binding agent for use of any one of claims 4, 7 to 51, 58, and 59, the binding agent and anti-cancer vaccine for use of any one of claims 5, 7 to 51, 58, and 59, or the medical preparation, combination, kit, or composition of any one of claims 6 to 51, 58, and 59, wherein the second binding region which binds to VEGF, VEGF receptor (VEGFR), or both and which antagonizes the VEGF / VEGFR interaction comprises:(ii-3a) the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 153;(ii-3b) the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SE Q ID NO: 154;(ii-3c) the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 155 (such as the amino acid sequence of SEQ ID NO: 215);(ii-3d) the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 156;(ii-3e) the amino acid sequence of SEQ ID NO: 215 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 215; or(ii-3f) the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 157.

61. The combination for use of any one of claims 1 and 7 to 60, the method of any one of claims 2 and 7 to 60, the anti-cancer vaccine for use of any one of claims 3 and 7 to 60, the binding agent for use of any one of claims 4 and 7 to 60, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 60, or the medical preparation, combination, kit, or composition of any one of claims 6 to 60, wherein the binding agent is a multispecific antibody, such as a bispecific antibody.

62. The combination for use of claim 61 , the method of claim 61 , the anti-cancer vaccine for use of claim 61, the binding agent for use of claim 61, the binding agent and anti-cancer vaccine for use of claim 61, or the medical preparation, combination, kit, or composition of claim 61, wherein the first binding region of the multispecific antibody binds to PD-L1 and the second binding region binds to VEGF, preferably to VEGF- A.

63. The combination for use of claim 61 or 62, the method of claim 61 or 62, the anti-cancer vaccine for use of claim 61 or 62, the binding agent for use of claim 61 or 62, the binding agent and anticancer vaccine for use of claim 61 or 62, or the medical preparation, combination, kit, or composition of claim 61 or 62, wherein the multispecific antibody comprises an anti-VEGF antibody and at least one anti-PD-Ll single domain antibody, preferably an anti-VEGF antibody and two anti-PD-Ll single domain antibodies, more preferably an anti-VEGF antibody and two anti-PD-Ll VHHs.

64. The combination for use of claim 63, the method of claim 63, the anti-cancer vaccine for use of claim 63, the binding agent for use of claim 63, the binding agent and anti-cancer vaccine for use of claim 63, or the medical preparation, combination, kit, or composition of claim 63, wherein each heavy chain of the anti-VEGF antibody is terminally fused to an anti-PD-Ll single domain antibody, preferably via a linker.

65. The combination for use of any one of claims 1 and 7 to 64, the method of any one of claims 2 and 7 to 64, the anti-cancer vaccine for use of any one of claims 3 and 7 to 64, the binding agent for use of any one of claims 4 and 7 to 64, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 64, or the medical preparation, combination, kit, or composition of any one of claims 6 to 64, wherein the binding agent comprises:(A) (1) at least one anti-PD-Ll single domain antibody comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 120, 121, and 122, respectively, or comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 123, 124, and 125, respectively; and / or (2) an anti-VEGF antibody comprising (i) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and a light chain variable region (VL) comprising the CDR1 , CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively; or (ii) a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively; or(B) (1) at least one anti-PD-Ll single domain antibody comprising the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 126, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 126 preferably comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 120, 121, and122, respectively, or the HCDR1, HCDR2, and HCDR3 sequences set forth in: 123, 124, and 125, respectively; and / or (2) an anti-VEGF antibody comprising (i) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 1 5, 136, and 137, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively; or (ii) a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

66. The combination for use of claim 65, the method of claim 65, the anti-cancer vaccine for use of claim 65, the binding agent for use of claim 65, the binding agent and anti-cancer vaccine for use of claim 65, or the medical preparation, combination, kit, or composition of claim 65, wherein the anti-VEGF antibody comprises:(ii-ld) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147;(ii-le) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively;(ii-lf) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and the VL comprises the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively;(ii-lg) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152;(ii-lh) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively; or(ii-li) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

67. The combination for use of any one of claims 1 and 7 to 66, the method of any one of claims 2 and 7 to 66, the anti-cancer vaccine for use of any one of claims 3 and 7 to 66, the binding agent for use of any one of claims 4 and 7 to 66, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 66, or the medical preparation, combination, kit, or composition of any one of claims 6 to 66, wherein the binding agent comprises (A) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 158, and (B) a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein (I) the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and (ii) the HCDR1, HCDR2, andHCDR3 sequences set forth in: SEQ ID NO: 120, 121, and 122, respectively, or the HC comprises (i*) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and (ii*) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 123, 124, and 125, respectively, and (II) the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively, or the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

68. The combination for use of any one of claims 1 and 7 to 67, the method of any one of claims 2 and 7 to 67, the anti-cancer vaccine for use of any one of claims 3 and 7 to 67, the binding agent for use of any one of claims 4 and 7 to 67, the binding agent and anti-cancer vaccine for use of any one of claims 5 and 7 to 67, or the medical preparation, combination, kit, or composition of any one of claims 6 to 67, wherein the binding agent comprises (A) two heavy chains (HCs) each comprising the amino acid sequence of SEQ ID NO: 158, and (B) two light chains (LCs) each comprising the amino acid sequence of SEQ ID NO: 152.

69. The combination for use of claim 61 , the method of claim 61 , the anti-cancer vaccine for use of claim 61, the binding agent for use of claim 61, the binding agent and anti-cancer vaccine for use of claim 61, or the medical preparation, combination, kit, or composition of claim 61, wherein the first binding region of the multispecific antibody binds to PD-1 and the second binding region binds to VEGF, preferably to VEGF-A.

70. The combination for use of claim 69, the method of claim 69, the anti-cancer vaccine for use of claim 69, the binding agent for use of claim 69, the binding agent and anti-cancer vaccine for use of claim 69, or the medical preparation, combination, kit, or composition of claim 69, wherein the multispecific antibody comprises an anti- VEGF antibody and at least one anti-PD- 1 scFv antibody, preferably an anti-VEGF antibody and two anti-PD-1 scFv antibodies.

71. The combination for use of claim 70, the method of claim 70, the anti-cancer vaccine for use of claim 70, the binding agent for use of claim 70, the binding agent and anti-cancer vaccine for use of claim 70, or the medical preparation, combination, kit, or composition of claim 70, wherein each heavy chain of the anti-VEGF antibody is terminally fused to an anti-PD-1 scFv antibody, preferably via a linker.

72. The combination for use of any one of claims 69 to 71, the method of any one of claims 69 to 71, the anti-cancer vaccine for use of any one of claims 69 to 71, the binding agent for use of any one of claims 69 to 71, the binding agent and anti-cancer vaccine for use of any one ofclaims 69 to 71, or the medical preparation, combination, kit, or composition of any one of claims 69 to 71 , wherein the binding agent comprises:(A) (1) at least one anti-PD-1 scFv antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 98, 99, and 100, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 101, 102, and 103, respectively, and (2) an anti-VEGF antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively;(B) (1) at least one anti-PD-1 scFv antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 104, 105, and 106, respectively, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 107, 108, and 103, respectively, and (2) an anti-VEGF antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively;(C) (1) at least one anti-PD-1 scFv antibody comprising the amino acid sequence of SEQ ID NO: 109 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 109, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 109 preferably comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 98, 99, 100, 101, 102, and 103, respectively, and (2) an anti-VEGF antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively; or(D) (1) at least one anti-PD-1 scFv antibody comprising the amino acid sequence of SEQ ID NO: 109 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 109, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 109 preferably comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ IDNO: 104, 105, 106, 107, 108, and 103, respectively, and (2) an anti-VEGF antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively.

73. The combination for use of any one of claims 69 to 72, the method of any one of claims 69 to 72, the anti-cancer vaccine for use of any one of claims 69 to 72, the binding agent for use of any one of claims 69 to 72, the binding agent and anti-cancer vaccine for use of any one of claims 69 to 72, or the medical preparation, combination, kit, or composition of any one of claims 69 to 72, wherein the anti-VEGF antibody comprises:(ii-ld) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147;(ii-le) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively;(ii-lf) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ED NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively;(ii-lg) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 151, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152;(ii-lh) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and the LC comprises the LCDRl, LCDR2, and LCDR3 sequences set forth in: SEQ LD NO: 138, 139, and 140, respectively; or(ii-li) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and the LC comprises the LCDRl, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

74. The combination for use of any one of claims 69 to 73, the method of any one of claims 69 to 73, the anti-cancer vaccine for use of any one of claims 69 to 73, the binding agent for use of any one of claims 69 to 73, the binding agent and anti-cancer vaccine for use of any one of claims 69 to 73, or the medical preparation, combination, kit, or composition of any one of claims 69 to 73, wherein the binding agent comprises (A) at least one heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 159, and (B) at least one light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein (I) the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and (ii) the HCDR1, HCDR2, HCDR3, LCDRl, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 104, 105, 106, 107, 108, and 103, respectively, or the HC comprises (i’) the HCDR1, HCDR2, and HCDR3 sequences setforth in: SEQ ID NO: 141, 142, and 143, respectively, and (ii') the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 98, 99, 100, 101, 102, and 103, respectively, and (II) the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively, or the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

75. The combination for use of any one of claims 69 to 74, the method of any one of claims 69 to 74, the anti-cancer vaccine for use of any one of claims 69 to 74, the binding agent for use of any one of claims 69 to 74, the binding agent and anti-cancer vaccine for use of any one of claims 69 to 74, or the medical preparation, combination, kit, or composition of any one of claims 69 to 74, wherein the binding agent comprises (A) two heavy chains (HCs) each comprising the amino acid sequence of SEQ ID NO: 159, and (B) two light chains (LCs) each comprising the amino acid sequence of SEQ ID NO: 152.

76. The combination for use of claim 69, the method of claim 69, the anti-cancer vaccine for use of claim 69, the binding agent for use of claim 69, the binding agent and anti-cancer vaccine for use of claim 69, or the medical preparation, combination, kit, or composition of claim 69, wherein the multispecific antibody comprises an anti-VEGF antibody and at least one anti-PD- 1 single domain antibody, preferably an anti-VEGF antibody and at least two anti-PD-1 single domain antibodies, more preferably an anti-VEGF antibody and at least two anti-PD-1 VHHs.

77. The combination for use of claim 76, the method of claim 76, the anti-cancer vaccine for use of claim 76, the binding agent for use of claim 76, the binding agent and anti-cancer vaccine for use of claim 76, or the medical preparation, combination, kit, or composition of claim 76, wherein each heavy chain of the anti-VEGF antibody is terminally fused to an anti-PD-1 single domain antibody, preferably via a linker.

78. The combination for use of any one of claims 69, 76, and 77, the method of any one of claims 69, 76, and 77, the anti-cancer vaccine for use of any one of claims 69, 76, and 77, the binding agent for use of any one of claims 69, 76, and 77, the binding agent and anti-cancer vaccine for use of any one of claims 69, 76, and 77, or the medical preparation, combination, kit, or composition of any one of claims 69, 76, and 77, wherein the binding agent comprises:(A) (1) at least one anti-PD-1 single domain antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 112, respectively, or a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 113, respectively; and (2) an anti-VEGF antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively;(B) (1) at least one anti-PD-1 single domain antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 116, respectively, or VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 117, respectively; and (2) an anti-VEGF antibody comprising a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and a light chain variable region (VL) comprising the CDR1 , CDR2, and CDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively;(C) (1) at least one anti-PD-1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118 preferably comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 112, respectively, or comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 116, respectively, and (2) an anti-VEGF antibody comprising (i) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively, or (ii) a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and a VL comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively; or(D) (1) at least one anti-PD-1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119 preferably comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 113, respectively, or the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 114, 115, and 117, respectively, and (2) an anti-VEGF antibody comprising (i) a heavy chain variable region (VH) comprising the HCDR1 , HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and a light chain variable region (VL) comprising theCDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively, or (ii) a VH comprising the HCDR1 , HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and a VL comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

79. The combination for use of any one of claims 69 and 76 to 78, the method of any one of claims 69 and 76 to 78, the anti-cancer vaccine for use of any one of claims 69 and 76 to 78, the binding agent for use of any one of claims 69 and 76 to 78, the binding agent and anti-cancer vaccine for use of any one of claims 69 and 76 to 78, or the medical preparation, combination, kit, or composition of any one of claims 69 and 76 to 78, wherein the anti-VEGF antibody comprises: (ii-ld) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147;(ii-le) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively;(ii-lf) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 147, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively;(ii-lg) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151, and a light chain (LC)comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152;(ii-lh) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively; or(ii-li) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 151, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and the LC comprises the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

80. The combination for use of any one of claims 69 and 76 to 79, the method of any one of claims 69 and 76 to 79, the anti-cancer vaccine for use of any one of claims 69 and 76 to 79, the binding agent for use of any one of claims 69 and 76 to 79, the binding agent and anti-cancer vaccine for use of any one of claims 69 and 76 to 79, or the medical preparation, combination, kit, or composition of any one of claims 69 and 76 to 79, wherein the binding agent comprises (A) at least one heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 160, and (B) at least one light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein (I) the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and (ii) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 112, respectively, or the HC comprises (i') the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and (ii') the HCDR1, HCDR2, and HCDR3,sequences set forth in: SEQ ID NO: 114, 115, and 116, respectively, and (II) the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively, or the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

81. The combination for use of any one of claims 69 and 76 to 79, the method of any one of claims 69 and 76 to 79, the anti-cancer vaccine for use of any one of claims 69 and 76 to 79, the binding agent for use of any one of claims 69 and 76 to 79, the binding agent and anti-cancer vaccine for use of any one of claims 69 and 76 to 79, or the medical preparation, combination, kit, or composition of any one of claims 69 and 76 to 79, wherein the binding agent comprises (A) at least one heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 161, and (B) at least one light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein (I) the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and (ii) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 112, respectively, or the HC comprises (i') the HCDR1 , HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and (ii') the HCDR1, HCDR2, and HCDR3, sequences set forth in: SEQ ID NO : 114, 115, and 116, respectively, and (II) the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively, or the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

82. The combination for use of any one of claims 69 and 76 to 79, the method of any one of claims 69 and 76 to 79, the anti-cancer vaccine for use of any one of claims 69 and 76 to 79, the binding agent for use of any one of claims 69 and 76 to 79, the binding agent and anti-cancer vaccine for use of any one of claims 69 and 76 to 79, or the medical preparation, combination, kit, or composition of any one of claims 69 and 76 to 79, wherein the binding agent comprises (A) at least one heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 162, and (B) at least one light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 152, wherein (I) the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and(ii) the HCDR1 , HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 110, 111, and 113, respectively, or the HC comprises (!') the HCDR1 , HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and (ii') the HCDR1, HCDR2, and HCDR3, sequences set forth in: SEQ ID NO: 114, 115, and 117, respectively, and (II) the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 140, respectively, or the LCDR1, LCDR2, andLCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 140, respectively.

83. The combination for use of any one of claims 69 and 76 to 82, the method of any one of claims 69 and 76 to 82, the anti-cancer vaccine for use of any one of claims 69 and 76 to 82, the binding agent for use of any one of claims 69 and 76 to 82, the binding agent and anti-cancer vaccine for use of any one of claims 69 and 76 to 82, or the medical preparation, combination, kit, or composition of any one of claims 69 and 76 to 82, wherein the binding agent comprises (A) two heavy chains (HCs) each comprising the amino acid sequence of SEQ ID NO: 160, the amino acid sequence of SEQ ID NO: 161, or the amino acid sequence of SEQ ID NO: 162, and (B) two light chains (LCs) each comprising the amino acid sequence of SEQ ID NO: 152.

84. The combination for use of claim 69, the method of claim 69, the anti-cancer vaccine for use of claim 69, the binding agent for use of claim 69, the binding agent and anti-cancer vaccine for use of claim 69, or the medical preparation, combination, kit, or composition of claim 69, wherein the multispecific antibody comprises at least one Fab fragment of an anti-VEGF antibody and at least one Fab fragment of an anti-PD-1 antibody, preferably at least two Fab fragments of an anti-VEGF antibody and at least two Fab fragments of an anti-PD-1 antibody, more preferably two Fab fragments of an anti-VEGF antibody and two Fab fragments of an anti- PD-1 antibody.

85. The combination for use of claim 84, the method of claim 84, the anti-cancer vaccine for use of claim 84, the binding agent for use of claim 84, the binding agent and anti-cancer vaccine for use of claim 84, or the medical preparation, combination, kit, or composition of claim 84, wherein the two Fab fragments of the anti-VEGF antibody are the same; and / or the two Fab fragments of the anti-PD-1 antibody are the same.

86. The combination for use of claim 84 or 85, the method of claim 84 or 85, the anti-cancer vaccine for use of claim 84 or 85, the binding agent for use of claim 84 or 85, the binding agent and anticancer vaccine for use of claim 84 or 85, or the medical preparation, combination, kit, or composition of claim 84 or 85, wherein the at least one Fab fragment of the anti-PD-1 antibodyis terminally fused to the at least one Fab fragment of the anti-VEGF antibody, preferably via a linker.

87. The combination for use of any one of claims 84 to 86, the method of any one of claims 84 to 86, the anti-cancer vaccine for use of any one of claims 84 to 86, the binding agent for use of any one of claims 84 to 86, the binding agent and anti-cancer vaccine for use of any one of claims 84 to 86, or the medical preparation, combination, kit, or composition of any one of claims 84 to 86, wherein the multispecific antibody comprises a heavy chain comprising at least one heavy chain Fd fragment of the anti-PD-1 antibody and at least one heavy chain Fd fragment of the anti-VEGF antibody, preferably of the anti-VEGF antibody 2.

88. The combination for use of claim 87, the method of claim 87, the anti-cancer vaccine for use of claim 87, the binding agent for use of claim 87, the binding agent and anti-cancer vaccine for use of claim 87, or the medical preparation, combination, kit, or composition of claim 87, wherein the heavy chain comprises from N to C terminal direction (i) a heavy chain Fd fragment of an anti-PD-1 antibody followed by a heavy chain Fd fragment of the anti-VEGF antibody, preferably of the anti-VEGF antibody 2; or (ii) a heavy chain Fd fragment of an anti-VEGF antibody, preferably of the anti-VEGF antibody 2, followed by a heavy chain Fd fragment of the anti-PD-1 antibody89. The combination for use of claim 87 or 88, the method of claim 87 or 88, the anti-cancer vaccine for use of claim 87 or 88, the binding agent for use of claim 87 or 88, the binding agent and anticancer vaccine for use of claim 87 or 88, or the medical preparation, combination, kit, or composition of claim 87 or 88, wherein the anti-PD-1 xVEGF antibody comprises (i) two heavy chains (HCs) each comprising (a) a Fab fragment of the anti-PD-1 antibody, preferably the Fab fragment of the anti-PD-1 antibody A10, and (b) a Fab fragment of the anti-VEGF antibody, preferably of the anti-VEGF antibody 2; and (ii) four light chains (LCs), wherein each Fab fragment of the anti-VEGF antibody is fused to the Fab fragment of the anti-PD-1 antibody through a linker comprising the amino acid sequence of SEQ ID NO: 219 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 219.

90. The combination for use of any one of claims 84 to 89, the method of any one of claims 84 to 89, the anti-cancer vaccine for use of any one of claims 84 to 89, the binding agent for use of any one of claims 84 to 89, the binding agent and anti-cancer vaccine for use of any one of claims 84 to 89, or the medical preparation, combination, kit, or composition of any one of claims 84 to 89, wherein the the multispecific antibody comprises two Fab fragments of the anti-PD-1 antibody and two Fab fragments of the anti-VEGF antibody, preferably wherein (i) the two Fab fragments of the anti-PD-1 antibody are the same; (ii) the two Fab fragments of the anti-VEGF antibody are the same; and / or (iii) the two Fab fragments of the anti-PD-1 antibody are each fused to an Fc region, preferably to an Fc region of IgG4 or an Fc region derived from IgG4.

91. The combination for use of any one of claims 84 to 90, the method of any one of claims 84 to 90, the anti-cancer vaccine for use of any one of claims 84 to 90, the binding agent for use of any one of claims 84 to 90, the binding agent and anti-cancer vaccine for use of any one of claims 84 to 90, or the medical preparation, combination, kit, or composition of any one of claims 84 to 90, wherein the multispecific antibody comprises (i) a CHI domain, preferably comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 236; (ii) a CL domain, preferably comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 150; and / or (iii) an IgG4 Fc region comprising a S228P mutation, preferably an IgG4 Fc region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 237.

92. The combination for use of any one of claims 84 to 91, the method of any one of claims 84 to 91, the anti-cancer vaccine for use of any one of claims 84 to 91, the binding agent for use of any one of claims 84 to 91, the binding agent and anti-cancer vaccine for use of any one of claims 84 to 91, or the medical preparation, combination, kit, or composition of any one of claims 84 to 91, wherein the multispecific antibody comprises:(A) (1) at least one anti-PD-1 VH comprising the amino acid sequence of SEQ ID NO: 229 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 229, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 229 preferably comprises the HCDR1 , HCDR2, HCDR3 sequences set forth in: SEQ ID NO: 222, 223, and 224, respectively, or the HCDR1, HCDR2, HCDR3 sequences set forth in SEQ ID NO: 226, 227, and 228, respectively; and / or (2) at least one anti-VEGF VH comprising the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 146, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 146, preferably comprising (i) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, or the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively; or (ii) a VL comprising the comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively;(B) (1) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 238 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 238, and / or one or more amino acid additions, deletions and / or substitutions in a framework region thereof; and (2) a light chain sequence comprising the amino acid sequence of SEQ ID NO: 239 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 239, and / or one or more amino acid additions, deletions and / or substitutions in a framework region thereof;(C) (1) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 238, and (2) a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 239, wherein the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and (ii) the HCDR1 , HCDR2, HCDR3 sequences set forth in: SEQ ID NO: 222, 223, and 224, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively;(C’) (l)a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 238, and (2) a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 239, wherein the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and (ii) the HCDR1, HCDR2, HCDR3 sequences set forth in: SEQ ID NO: 226, 227, and 228, respectively,and the LC comprises the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively;(D) (1) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 238 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 238; and (2) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 239 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 239;(E) (1) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 238; and (2) a light chain sequence comprising the amino acid sequence of SEQ ID NO: 239; or (F) (1) two heavy chains each comprising the amino acid sequence of SEQ ID NO: 238; and (2) four light chains each comprising the amino acid sequence of SEQ ID NO: 239.

93. The combination for use of any one of claims 84 to 92, the method of any one of claims 84 to 92, the anti-cancer vaccine for use of any one of claims 84 to 92, the binding agent for use of any one of claims 84 to 92, the binding agent and anti-cancer vaccine for use of any one of claims 84 to 92, or the medical preparation, combination, kit, or composition of any one of claims 84 to 92, wherein the multispecific antibody comprises:(A) (1) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 240 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 240, and / or one or more amino acid additions, deletions and / or substitutions in a framework region thereof; and (2) a light chain sequence comprising the amino acid sequence of SEQ ID NO: 239 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 239, and / or one or more amino acid additions, deletions and / or substitutions in a framework region thereof;(B) (1) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 240, and (2) a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 239, wherein the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 135, 136, and 137, respectively, and (ii) the HCDR1, HCDR2, HCDR3 sequences set forth in: SEQ ID NO: 222, 223, and 224, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively;(B’) (1) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 240, and (B') a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 239, wherein the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 141, 142, and 143, respectively, and (ii) the HCDR1, HCDR2, HCDR3 sequences set forth in: SEQ ID NO: 226, 227, and 228, respectively, and the LC comprises the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively;(C) (1) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 240 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 240; and (2) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 239 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 239;(D) (I) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 240; and (2) a light chain sequence comprising the amino acid sequence of SEQ ID NO: 239; or (E) (1 ) two heavy chains each comprising the amino acid sequence of SEQ ID NO: 240; and (2) four light chains each comprising the amino acid sequence of SEQ ID NO: 239.

94. The combination for use of any one of claims 84 to 92, the method of any one of claims 84 to 92, the anti-cancer vaccine for use of any one of claims 84 to 92, the binding agent for use of any one of claims 84 to 92, the binding agent and anti-cancer vaccine for use of any one of claims 84 to 92, or the medical preparation, combination, kit, or composition of any one of claims 84 to 92, wherein the multispecific antibody comprises:(A) (1) at least one anti-PD-1 VH comprising the amino acid sequence of SEQ ID NO: 229 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 229, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 229 preferably comprises the HCDR1, HCDR2, HCDR3 sequences set forth in: SEQ ID NO: 222, 223, and 224, respectively, or the HCDR1, HCDR2, HCDR3 sequences set forth in SEQ ID NO: 226, 227, and 228, respectively; and / or (2) an anti-VEGF VH comprising the amino acid sequence of SEQ ID NO: 235 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 235, wherein the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 235 preferably comprises (i) a heavy chain variable region (VH) comprising the HCDR1 , HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 231, 136, and 232, respectively, or the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 233, 142, and 234; and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively; or (ii) a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively;(B) (1) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 241 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 241, and / or one or more amino acid additions, deletions and / or substitutions in a framework region thereof; and (2) a light chain sequence comprising the amino acid sequence of SEQ ID NO: 239 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 239, and / or one or more amino acid additions, deletions and / or substitutions in a framework region thereof;(C) (1) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 241, and (2) a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 239, wherein the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 231, 136, and 232, respectively, and (ii) the HCDR1, HCDR2, HCDR3 sequences set forth in: SEQ ID NO: 222, 223, and 224, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 138, 139, and 225, respectively;(C’) (1) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 241, and (2) a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 239, wherein the HC comprises (i) the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 233, 142, and 234, respectively, and (ii) the HCDR1 , HCDR2, HCDR3 sequences set forth in: SEQ ID NO: 226, 227, and 228, respectively, and the LC comprises the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 144, 145, and 225, respectively;(D) (1) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 241 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 241; and (2) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 239 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 239;(E) (1) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 241; and (2) a light chain sequence comprising the amino acid sequence of SEQ ID NO: 239; or (F) (1) two heavy chains each comprising the amino acid sequence of SEQ ID NO: 241 ; and (ii) four light chains each comprising the amino acid sequence of SEQ ID NO: 239.

95. The combination for use of claim 61 , the method of claim 61 , the anti-cancer vaccine for use of claim 61, the binding agent for use of claim 61, the binding agent and anti-cancer vaccine for use of claim 61, or the medical preparation, combination, kit, or composition of claim 61, wherein the first binding region of the multispecific antibody binds to PD-L1 and the second binding region comprises at least one VEGFR domain.

96. The combination for use of claim 95, the method of claim 95, the anti-cancer vaccine for use of claim 95, the binding agent for use of claim 95, the binding agent and anti-cancer vaccine for use of claim 95, or the medical preparation, combination, kit, or composition of claim 95, wherein the multispecific antibody comprises a fusion protein comprising an anti-PD-Ll antibody and the at least one VEGFR domain.

97. The combination for use of claim 96, the method of claim 96, the anti-cancer vaccine for use of claim 96, the binding agent for use of claim 96, the binding agent and anti-cancer vaccine for use of claim 96, or the medical preparation, combination, kit, or composition of claim 96, wherein:(i) the at least one VEGR domain is terminally fused to the heavy chain of the anti-PD-Ll antibody, preferably via a linker;(ii) the anti-PD-Ll antibody comprises a full-length anti-PD-Ll antibody; and / or(iii) the anti-PD-Ll antibody is a humanized antibody.

98. The combination for use of any one of claims 95 to 97, the method of any one of claims 95 to 97, the anti-cancer vaccine for use of any one of claims 95 to 97, the binding agent for use of any one of claims 95 to 97, the binding agent and anti-cancer vaccine for use of any one of claims 95 to 97, or the medical preparation, combination, kit, or composition of any one of claims 95 to 97, wherein the binding agent comprises:(A) (1) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 133, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 134; and (2) at least one VEGFR domain comprising the amino acid sequence of SEQ ID NO: 155;(B) (1) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 127, 128, and 129, respectively, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 130, 131, and 132, respectively; and (2) at least one VEGFR domain comprising the amino acid sequence of SEQ ID NO: 155; or(C) (1) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 133, and a light chain variable region (VL) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 134, wherein preferably the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 127, 128, and 129, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 130, 131, and 132, respectively; and (2) at least one VEGFR domain comprising the amino acid sequence of SEQ ID NO: 155.

99. The combination for use of any one of claims 95 to 98, the method of any one of claims 95 to 98, the anti-cancer vaccine for use of any one of claims 95 to 98, the binding agent for use of any one of claims 95 to 98, the binding agent and anti-cancer vaccine for use of any one of claims 95 to 98, or the medical preparation, combination, kit, or composition of any one of claims 95 to 98, wherein the binding agent comprises (A) at least one heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 163, and (B) at least one light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 164, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 127, 128, and 129, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 130, 131, and 132, respectively.

100. The combination for use of any one of claims 95 to 98, the method of any one of claims 95 to 98, the anti-cancer vaccine for use of any one of claims 95 to 98, the binding agent for use of any one of claims 95 to 98, the binding agent and anti-cancer vaccine for use of any one ofclaims 95 to 98, or the medical preparation, combination, kit, or composition of any one of claims 95 to 98, wherein the binding agent comprises (A) at least one heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 165, and (B) at least one light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 164, wherein the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 127, 128, and 129, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 130, 131, and 132, respectively.

101. The combination for use of any one of claims 95 to 100, the method of any one of claims 95 to 100, the anti-cancer vaccine for use of any one of claims 95 to 100, the binding agent for use of any one of claims 95 to 100, the binding agent and anti-cancer vaccine for use of any one of claims 95 to 100, or the medical preparation, combination, kit, or composition of any one of claims 95 to 100, wherein the binding agent comprises (A) two heavy chains (HCs) each comprising the amino acid sequence of SEQ ID NO: 163 or the amino acid sequence of SEQ ID NO: 165, and (B) two light chains (LCs) each comprising the amino acid sequence of SEQ ID NO: 164.

102. The combination for use of any one of claims 95 to 97, the method of any one of claims 95 to 97, the anti-cancer vaccine for use of any one of claims 95 to 97, the binding agent for use of any one of claims 95 to 97, the binding agent and anti-cancer vaccine for use of any one of claims 95 to 97, or the medical preparation, combination, kit, or composition of any one of claims 95 to 97, wherein the binding agent comprises:(A) (1) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 209, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 210; and (2) at least one VEGFR domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 215, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 215;(B) (1) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 198, 199, and 200, respectively, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 201, 202, and 203, respectively; and (2) at least one VEGFR domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 215, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 215; or(C) (1) a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 204, 205, and 206, respectively, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 207, 208, and 203, respectively; and (2) at least one VEGFR domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 215, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 215.

103. The combination for use of any one of claims 95 to 97 and 102, the method of any one of claims 95 to 97 and 102, the anti -cancer vaccine for use of any one of claims 95 to 97 and 102, the binding agent for use of any one of claims 95 to 97 and 102, the binding agent and anti-cancer vaccine for use of any one of claims 95 to 97 and 102, or the medical preparation, combination, kit, or composition of any one of claims 95 to 97 and 102, wherein the binding agent comprises: (A) (1 ) a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 216 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 216, and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof; and (2) a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 217 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 217, and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof;(B) (1) a heavy chain (HC) comprising or consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 216, and (2) a light chain (LC) comprising or consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 217, wherein (i) theHC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 198, 199, and 200, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 201, 202, and 203, respectively, or (ii) the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 204, 205, and 206, respectively, and the LC comprises the LCDR1 , LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 207, 208, and 203, respectively; or(C) (1) a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 216 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 216; and (2) a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 217 or an aminoacid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 217.

104. The combination for use of any one of claims 95 to 97 and 102, the method of any one of claims 95 to 97 and 102, the anti-cancer vaccine for use of any one of claims 95 to 97 and 102, the binding agent for use of any one of claims 95 to 97 and 102, the binding agent and anti-cancer vaccine for use of any one of claims 95 to 97 and 102, or the medical preparation, combination, kit, or composition of any one of claims 95 to 97 and 102, wherein the binding agent comprises: (A) (1) a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 218 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 218, and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof; and (2) a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 217 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 217, and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof;(B) (1) a heavy chain (HC) comprising or consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 218, and (2) a light chain (LC) comprising or consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 217, wherein (i) the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 198, 199, and 200, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 201, 202, and 203, respectively, or (ii) the HC comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in: SEQ ID NO: 204, 205, and 206, respectively, and the LC comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in: SEQ ID NO: 207, 208, and 203, respectively; or(C) (1) a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 218 or an amino acid sequence comprising or consisting of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 218; and (2) a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 217 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 217.

105. The combination for use of any one of claims 95 to 97 and 102 to 104, the method of any one of claims 95 to 97 and 102 to 104, the anti-cancer vaccine for use of any one of claims 95 to 97 and 102 to 104, the binding agent for use of any one of claims 95 to 97 and 102 to 104, the binding agent and anti-cancer vaccine for use of any one of claims 95 to 97 and 102 to 104, or the medical preparation, combination, kit, or composition of any one of 95 to 97 and 102 to 104, wherein the binding agent comprises (1) two heavy chains each comprising or consisting of the amino acid sequence of SEQ ID NO: 216 or the amino acid sequence of SEQ 218; and (2) two light chains each comprising or consisting of the amino acid sequence of SEQ ID NO: 217.

106. The combination for use of claim 61 , the method of claim 61 , the anti-cancer vaccine for use of claim 61, the binding agent for use of claim 61, the binding agent and anti-cancer vaccine for use of claim 61, or the medical preparation, combination, kit, or composition of claim 61, wherein the first binding region of the multispecific antibody binds to PD-1 and the second binding region binds to VEGFR, preferably VEGFR2, preferably wherein the first binding region and the second binding region are separate, or overlap, partially or completely.

107. The combination for use of claim 106, the method of claim 106, the anti-cancer vaccine for use of claim 106, the binding agent for use of claim 106, the binding agent and anti -cancer vaccine for use of claim 106, or the medical preparation, combination, kit, or composition of claim 106, wherein the multispecific antibody comprises an IgGl, lgG2, lgG3, lgG4, IgAl or lgA2 immunoglobulin constant region, preferably wherein the multispecific antibody comprises an immunologically inert immunoglobulin constant region.

108. The combination for use of claim 106 or 107, the method of claim 106 or 107, the anti-cancer vaccine for use of claim 106 or 107, the binding agent for use of claim 106 or 107, the binding agent and anti-cancer vaccine for use of claim 106 or 107, or the medical preparation, combination, kit, or composition of claim 106 or 107, wherein the multispecific antibody comprises (1) a VH comprising: (i) a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 184; (ii) a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 185; and (iii) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 186, and / or (II) a VL comprising: (i) a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 187; (ii) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 188, (ii’) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 192, (ii”) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 194, or (ii’”) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 196; and (iii) a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 189.

109. The combination for use of any one of claims 106 to 108, the method of any one of claims 106 to 108, the anti-cancer vaccine for use any one of claims 106 to 108, the binding agent for use any one of claims 106 to 108, the binding agent and anti-cancer vaccine for use of one of claims 106 to 108, or the medical preparation, combination, kit, or composition any one of claims 106 to 108, wherein the multispecific antibody comprises a VH comprising or consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 190; and / or a VL comprising or consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 191, 193, 195, or 197.

110. The combination for use of any one of claims 106 to 109, the method of any one of claims 106 to 109, the anti-cancer vaccine for use any one of claims 106 to 109, the binding agent for use any one of claims 106 to 109, the binding agent and anti-cancer vaccine for use of one of claims 106 to 109, or the medical preparation, combination, kit, or composition any one of claims 106 to 109, wherein the multispecific antibody comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 190; and / or a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 191, 193, 195, or 197.

111. The combination for use of any one of claims 1 and 7 to 110, the method of any one of claims 2 and 7 to 110, the anti-cancer vaccine for use of any one of claims 3 and 7 to 110, the binding agent for use of any one of claims 4 and 7 to 110, the binding agent and anti-cancer vaccine for use of one of claims 5 and 7 to 110, or the medical preparation, combination, kit, or composition of any one of claims 6 to 110, wherein the anti-cancer vaccine is formulated as a liquid, a solid, or a combination thereof.

112. The combination for use of any one of claims 1 and 7 to 111, the method of any one of claims 2 and 7 to 111 , the anti-cancer vaccine for use of any one of claims 3 and 7 to 111 , the binding agent for use of any one of claims 4 and 7 to 111, the binding agent and anti-cancer vaccine for use of one of claims 5 and 7 to 111 , or the medical preparation, combination, kit, or composition of any one of claims 6 to 111, wherein the anti-cancer vaccine is formulated for injection or infusion.

113. The combination for use of any one of claims 1 and 7 to 112, the method of any one of claims 2 and 7 to 112, the anti-cancer vaccine for use of any one of claims 3 and 7 to 112, the bindingagent for use of any one of claims 4 and 7 to 112, the binding agent and anti-cancer vaccine for use of one of claims 5 and 7 to 112, or the medical preparation, combination, kit, or composition of any one of claims 6 to 112, wherein the anti-cancer vaccine is formulated for intravenous administration.

114. The combination for use of any one of claims 1 and 7 to 113, the method of any one of claims 2 and 7 to 113, the anti-cancer vaccine for use of any one of claims 3 and 7 to 113, the binding agent for use of any one of claims 4 and 7 to 113, the binding agent and anti-cancer vaccine for use of one of claims 5 and 7 to 113, or the medical preparation, combination, kit, or composition of any one of claims 6 to 113, wherein the at least one RNA is formulated or is to be formulated as lipoplex particles.

115. The combination for use of claim 114, the method of claim 114, the anti-cancer vaccine for use of claim 114, the binding agent for use of claim 114, the binding agent and anti -cancer vaccine for use of claim 114, or the medical preparation, combination, kit, or composition of claim 114, wherein the RNA lipoplex particles are obtainable or obtained by mixing the at least one RNA with liposomes.

116. The combination for use of any one of claims 1 and 7 to 115, the method of any one of claims 2 and 7 to 115, the anti-cancer vaccine for use of any one of claims 3 and 7 to 115, the binding agent for use of any one of claims 4 and 7 to 115, the binding agent and anti-cancer vaccine for use of one of claims 5 and 7 to 115, or the medical preparation, combination, kit, or composition of any one of claims 6 to 115, wherein the binding agent is formulated as a liquid, a solid, or a combination thereof, preferably as a liquid.

117. The combination for use of any one of claims 1 and 7 to 116, the method of any one of claims 2 and 7 to 116, the anti-cancer vaccine for use of any one of claims 3 and 7 to 116, the binding agent for use of any one of claims 4 and 7 to 116, the binding agent and anti -cancer vaccine for use of one of claims 5 and 7 to 116, or the medical preparation, combination, kit, or composition of any one of claims 6 to 116, wherein the binding agent is formulated for injection or infusion.

118. The combination for use of any one of claims 1 and 7 to 117, the method of any one of claims 2 and 7 to 117, the anti-cancer vaccine for use of any one of claims 3 and 7 to 117, the binding agent for use of any one of claims 4 and 7 to 117, the binding agent and anti-cancer vaccine for use of one of claims 5 and 7 to 117, or the medical preparation, combination, kit, or composition of any one of claims 6 to 117, wherein one or both of the anti-cancer vaccine and the binding agent is / are administered systemically, preferably intravenously.

119. The combination for use of any one of claims 1 and 7 to 118, the method of any one of claims 2 and 7 to 118, the anti-cancer vaccine for use of any one of claims 3 and 7 to 118, the binding agent for use of any one of claims 4 and 7 to 118, the binding agent and anti-cancer vaccine for use of one of claims 5 and 7 to 118, or the medical preparation, combination, kit, or composition of any one of claims 6 to 118, wherein the binding agent is to be administered or is administered prior to, simultaneously with, or after administration of the anti-cancer vaccine.

120. The combination for use of any one of claims 1 and 7 to 119 or the medical preparation, combination, kit, or composition of any one of claims 6 to 119, further comprising a further therapeutic agent.

121. The medical preparation, combination, kit, or composition of any one of claims 6 to 120, which comprises a pharmaceutical composition.

122. The medical preparation, combination, kit, or composition of claim 121, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

123. The medical preparation, combination, kit, or composition of any one of claims 6 to 122, which is in the form of a kit of parts.

124. The medical preparation, combination, kit, or composition of any one of claims 6 to 123, wherein the anti-cancer vaccine and the binding agent are in separate vials, preferably each RNA and the binding agent are in separate vials.

125. The medical preparation, combination, kit, or composition of any one of claims 121 to 124, further comprising instructions for use of the medical preparation or composition for treating or preventing cancer.

126. The medical preparation, combination, kit, or composition of any one of claims 6 to 125 for use as a medicament.

127. The combination for use of any one of claims 1 and 7 to 120 or the medical preparation, combination, kit, or composition for use of claim 126, wherein the medicament is for administration to a subject.

128. The combination for use of any one of claims 1 , 7 to 120, and 127, or the medical preparation, combination, kit, or composition for use of claim 126 or 127, wherein the use as a medicament comprises a therapeutic or prophylactic treatment of a disease or disorder.

129. The combination for use of claim 128 or the medical preparation, combination, kit, or composition for use of claim 128, wherein the therapeutic or prophylactic treatment of a disease or disorder comprises treating, ameliorating or preventing cancer.

130. The combination for use of claim 129 or the medical preparation, combination, kit, or composition for use of claim 129, wherein the therapeutic or prophylactic treatment of a disease or disorder further comprises administering radiotherapy, preferably local radiotherapy.

131. The method of any one of claims 2 and 7 to 119, the anti-cancer vaccine for use of any one of claims 3 and 7 to 119, the binding agent for use of any one of claims 4 and 7 to 119, or the binding agent and anti-cancer vaccine for use of one of claims 5 and 7 to 119, wherein the method further comprises administering radiotherapy, preferably local radiotherapy, and / or administering a further therapeutic agent.

132. The combination for use of claim 129 or 130, the method of any one of claims 2, 7 to 119, and 131 , the anti-cancer vaccine for use of any one of claims 3, 7 to 119, and 131 , the binding agent for use of any one of claims 4, 7 to 119, and 131, the binding agent and anti-cancer vaccine for use of one of claims 5, 7 to 119, and 131, or the medical preparation, combination, kit, or composition for use of claim 129 or 130, wherein the cancer is selected from the group consisting of lung cancer (such as non-small cell lung cancer (NSCLC)), skin cancer (such as melanoma), breast cancer (such as triple-negative breast cancer (TNBC)), ovarian cancer, and HPV-positive cancer (such as head and neck squamous cell carcinoma (HNSCC) or cervical cancer), preferably from the group consisting of lung cancer (such as non-small cell lung cancer (NSCLC)), skin cancer (such as melanoma), and HPV-positive cancer (such as head and neck squamous cell carcinoma (HNSCC)).

133. The combination for use of any one of claims 127 to 130 and 132, the method of any one of claims 2, 7 to 119, 131, and 132, the anti-cancer vaccine for use of any one of claims 3, 7 to 119, 131, and 132, the binding agent for use of any one of claims 4, 7 to 119, 131, and 132, the binding agent and anti-cancer vaccine for use of one of claims 5, 7 to 119, 131, and 132, or the medical preparation, combination, kit, or composition for use of any one of claims 127 to 130 and 132, wherein the subject is a human.

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