Antigen-specific T cell receptors and T cell epitopes

Abstract

This provides clinically relevant T cell receptors and immune effector cells useful for immunotherapy. [Solution] A T cell receptor is provided, wherein the T cell receptor specifically binds to an epitope sequence consisting of a specific sequence, or a fragment of a sequence having at least 70%, at least 80%, or at least 90% of the amino acid residues from the specific sequence, and the T cell receptor comprises a T cell receptor α chain containing all three CDR sequences of the T cell receptor α chain consisting of a specific sequence; and a T cell receptor β chain containing all three CDR sequences of the T cell receptor β chain of Sequence ID No. 30.

Description

[Technical Field]

[0001] The present invention relates to providing clinically relevant T cell receptors and T cell epitopes useful for immunotherapy. The present invention also relates to therapies comprising immune effector cells, such as T cells, that express the T cell receptors disclosed herein and / or are specific to the T cell epitopes disclosed herein. In one embodiment, immune effector cells are engineered to express T cell receptors, for example, by being genetically modified to express T cell receptors. Such genetic modification may be performed ex vivo or in vitro, after which the immune effector cells may be administered to a subject in need of treatment, or may be performed in vivo in a subject in need of treatment. These methods are particularly useful for the treatment of cancers characterized by disease cells expressing antigens to which T cell receptors are directed. T cell receptor engineered immune effector cells may be provided to a subject by administration of T cell receptor engineered immune effector cells, or by generating T cell receptor engineered immune effector cells in the subject. Furthermore, target antigens for T cell receptors may be provided to a subject by administration of antigens targeted by T cell receptors, polynucleotides encoding antigens, or cells expressing antigens to the subject. The antigens targeted by the T cell receptor may include naturally occurring antigens or variants thereof, or fragments of naturally occurring antigens or variants thereof. In one particularly preferred embodiment, the polynucleotide encoding the antigen is RNA. The methods and agents described herein are particularly useful for treating diseases characterized by disease cells expressing antigens to which T cell receptors or T cell receptor-modified immune effector cells are directed. [Background technology]

[0002] The immune system plays a crucial role not only in pathogen-associated diseases but also in cancer, autoimmunity, and allergies. T cells and NK cells are important mediators of the anti-tumor immune response. CD8 + T cells and NK cells can directly lyse tumor cells. On the other hand, CD4 + T cells are CD8+ CD4 can mediate the influx of various immune subsets, including T cells and NK cells, into tumors. + T cells are dendritic cells (DCs) that have antitumor CD8 + It can allow priming of the T cell response and directly act on tumor cells through IFNγ-mediated upregulation and proliferation inhibition of MHC. CD8 + and CD4 + Tumor-specific T cell responses can be induced by vaccination or adoptive transfer of T cells.

[0003] The recognition and binding of specific antigens by T cells is mediated by T cell receptors (TCRs) expressed on the surface of T cells. T cell receptors on T cells can bind to major histocompatibility complex (MHC) molecules and interact with immunogenic peptides (epitopes) presented on the surface of target cells. Specific binding of TCRs triggers a signaling cascade within the T cell, leading to proliferation and differentiation into mature effector T cells.

[0004] MHC and antigen binding are mediated by complementarity-determining regions 1, 2, and 3 (CDR1, CDR2, and CDR3) of the TCR. CDR3 of the β-chain is most important for antigen recognition.

[0005] Active immunity tends to induce and expand antigen-specific T cells in patients that can specifically recognize and kill disease cells. In contrast, passive immunity can be achieved through adoptive transfer of T cells that have been expanded in vitro and optionally genetically modified (adoptive T cell therapy).

[0006] Active immunization (vaccination) can utilize various antigen forms, including whole cancer cells, proteins, peptides, or immune vectors such as RNA, DNA, or viral vectors, which can be directly applied in vivo or in vitro by pulsing the dendritic cells (DCs) after transfer to the patient.

[0007] Adoptive cell transfer (ACT) immunotherapy can be broadly defined as a form of passive immunity using previously sensitized T cells that are expanded ex vivo from a low precursor frequency to a clinically appropriate cell count before being transferred to a non-immune recipient or autohost. The cell types used in ACT experiments have included lymphokine-activated killer (LAK) cells (Mule, J.Jet al. (1984) Science 225, 1487-1489; Rosenberg, SA et al. (1985) N.Engl.J.Med.313, 1485-1492), tumor-infiltrating lymphocytes (TILs) (Rosenberg, SA et al. (1994) J.Natl.Cancer Inst.86, 1159-1166), donor lymphocytes after hematopoietic stem cell transplantation (HSCT), and tumor-specific T cell lines or clones (Dudley, ME et al. (2001) J.Immunother.24, 363-373; Yee, C. et al. (2002) Proc.Natl.Acad.Sci.USA 99, 16168-16173). An alternative approach involves adoptive transfer of autologous T cells reprogrammed to express tumor-reactive immune receptors of defined specificity during short-term ex vivo culture, followed by reinfusion into the patient (Kershaw MH et al. (2013) Nature Reviews Cancer 13(8):525-41). This strategy makes ACT applicable to a variety of common malignancies, even when tumor-reactive T cells are not present in the patient.

[0008] Shared non-mutated tumor-associated antigens (TAAs), such as cancer / germline genes or lineage-specific differentiation markers, are frequently expressed across human oncologies and are attractive immunotherapeutic targets (Coulie, PG, et al., Nat. Rev. Cancer 14, 135-146 (2014)). TAAs are thought to be susceptible to central T cell tolerance (Kyewski, B. & Derbinski, J., Nat. Rev. Immunol. 4, 688-98 (2004)), which may contribute to the generally weak, clinically ineffective T cell response observed in the majority of vaccine trials to date (Melero, I. et al., Nat. Rev. Clin. Oncol. 11, 509-524 (2014); Romero, P. et al., Sci. Transl. Med. 8, 334 ps9 (2016)). The inventors recently introduced a systemically administered nanoparticle liposomal RNA vaccine class (RNA-LPX) for whole-body targeting of dendritic cells (DCs) in the lymphoid compartment. When spatiotemporally aligned to deliver vaccine antigens, RNA-LPX mediates potent co-stimulation through type I interferon-driven antiviral immune mechanisms, resulting in a significant expansion of antitumor effector T cells, even against autoantigens (Kranz, L M et al., Nature 534, 396-401 (2016); De Vries, J. & Figdor, C., Nature 534, 329-31 (2016)). For the first human trials of this approach, the inventors initiated a Phase I dose-escalation study in patients with advanced melanoma who had radiologically evaluable metastatic disease at baseline or who had undergone resection of unevaluable disease (Lipo-MERIT, NCT02410733).

[0009] The vaccine (referred to as melanoma FixVac) consists of four lipid complex RNAs encoding non-mutant TAA NY-ESO-1, MAGE-A3, tyrosinase, and TPTE, each known for its restricted expression in normal tissues, high immunogenicity, and high prevalence in human melanoma (Simon, P. et al., Cancer Immunol. Res. 2, 1230-44 (2014); Cheever, MA et al., Clin. Cancer Res. 15, 5323-37 (2009)). The single-stranded 5' cap vaccine messenger RNA (Figure 1A) contains functional sequence elements that improve the efficiency of its translation, particularly in immature DCs (Holtkamp, ​​S. et al., Blood 108, 4009-17 (2006); Orlandini von Niessen, AGet al., Mol. Ther. 27, 824-836 (2018)). The open reading frame of each TAA is in-frame fused to a tetanus toxoid helper epitope and endosomal targeting domain at the C-terminus, as well as to an N-terminal secretory signal that enhances the processing and presentation of tumor antigen-derived epitopes on individual HLA-class I and II molecules of the patient (Kreiter, S. et al., J.Immunol. 180, 309-318 (2008)).

[0010] Patients expressing at least one TAA confirmed by qRT-PCR analysis were eligible for this trial. Melanoma FixVac was administered according to a prime / repeat boost protocol, followed optionally by monthly treatment (Figure 1B). In the dose escalation portion, target dose levels ranging from 7.2 μg and 400 μg of total RNA were tested. Patients in the dose escalation cohort were escalated to the target dose by intra-individual dose escalation at weekly intervals. The expansion portion included three dose cohorts using melanoma FixVac alone, and patients treated with melanoma FixVac in combination with an anti-PD1 or BRAF / MEK inhibitor.

[0011] Melanoma FixVac mediated durable objective responses in heavily pretreated metastatic and progressive tumors as a single agent and in combination with anti-PD1 therapy. Clinical responses correlated with the induction of HLA class I and II restricted TAA-specific T cell responses. Characterization of the corresponding HLA class I restricted TCRs confirmed their ability to mediate the recognition and lysis of tumor cell lines that endogenously express TAAs after transfer into CD8 + T cells from healthy donors. Similarly, HLA class II restricted TCRs were functional after transfer into CD4 + T cells from healthy donors.

[0012] Most vaccine-induced or vaccine-amplified CD4 + or CD8 + T cell responses were detected against NY-ESO-1 and MAGE-A3, both of which are known for their restricted expression in normal tissues, high immunogenicity, and high morbidity in human melanoma (Simon, P. et al., Cancer Immunol. Res. 2, 1230-44 (2014); Cheever, M. A. et al., Clin. Cancer Res. 15, 5323-37 (2009)). The corresponding TCRs are expected to have therapeutic value in the context of TCR gene therapy. Furthermore, a novel HLA-B*4001 restricted epitope of NY-ESO-1 124-133 was discovered and confirmed to be processed and presented by melanoma cells that endogenously express NY-ESO-1.

Prior Art Documents

Non-Patent Documents

[0013]

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Non-licensed literature 9

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[0014] Immunotherapy strategies are a promising option for treating cancer. Precise definition of peptide epitopes derived from tumor antigens can contribute to improving the specificity and efficiency of vaccination strategies. Furthermore, adoptive transfer of T cells engineered to express defined antigen-specific T cell receptors (TCRs) can specifically target tumor-associated antigens, thereby leading to the selective destruction of malignant cells. [Means for solving the problem]

[0015] The present invention relates in particular to T cell receptors specific to tumor-associated antigens NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively, when presented on the surface of cells such as disease cells or antigen-presenting cells, as well as peptides comprising epitopes recognized by these T cell receptors.

[0016] In one embodiment, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-44, 101, and 102, or a variant of said amino acid sequence.

[0017] In one embodiment, the peptide has a length of 200 amino acids or less, 150 amino acids or less, 100 amino acids or less, 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 20 amino acids or less, or 15 amino acids or less.

[0018] In one embodiment, the peptide may be an MHC class I or class II presenting peptide, preferably an MHC class I presenting peptide, or may be processed to produce a processing product which, if present in a cell, is an MHC class I or class II presenting peptide, preferably an MHC class I presenting peptide. Preferably, the MHC class I or class II presenting peptide has a sequence substantially corresponding to a given amino acid sequence, i.e., an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-44, 101, and 102, or a variant of said amino acid sequence. Preferably, the peptide according to the present invention can stimulate a cellular response to a disease involving cells characterized by the presentation of antigens from which the peptide originates, i.e., NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively, by class I MHC.

[0019] In a further aspect, the present invention relates to nucleic acids encoding the peptide of the present invention.

[0020] Such nucleic acids can be present in plasmids or expression vectors and can be functionally linked to a promoter. In one embodiment, the nucleic acid is RNA.

[0021] In a further embodiment, the present invention relates to cells genetically modified to express the peptide of the present invention.

[0022] The cells may be recombinant cells that can secrete encoded peptides or their processing products, express them on their surface, and may further express MHC molecules that preferably bind to the peptides or their processing products, and preferably present the peptides or their processing products on the cell surface. In one embodiment, the cells endogenously express MHC molecules. In a further embodiment, the cells recombinantly express MHC molecules and / or peptides. The cells are preferably nonproliferative. In a preferred embodiment, the cells are antigen-presenting cells, particularly dendritic cells, monocytes, or macrophages.

[0023] In one embodiment, the cell contains nucleic acid encoding a peptide.

[0024] In one embodiment, a cell presents a peptide or a processing product thereof. The processing product may be a peptide having a given amino acid sequence, i.e., an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-44, 101, and 102, or a variant of said amino acid sequence.

[0025] In a further embodiment, the present invention relates to cells that present the peptide or its processing product.

[0026] The processing product may be a peptide having a given amino acid sequence, i.e., an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-44, 101, and 102, or a variant of said amino acid sequence. Cells may present the peptide or its processing product by MHC molecules on their surface. In one embodiment, cells endogenously express MHC molecules. In a further embodiment, cells recombinantly express MHC molecules. In one embodiment, the MHC molecules of a cell are loaded with a peptide (pulsed with a peptide) by adding the peptide to the cell. Cells may recombinantly express the peptide and present the peptide or its processing product on their cell surface. Cells are preferably non-proliferative. In a preferred embodiment, the cells are antigen-presenting cells such as dendritic cells, monocytes, or macrophages.

[0027] In a further embodiment, the present invention relates to immune effector cells that are reactive with the peptide of the present invention.

[0028] In one embodiment, immune effector cells are reactive with the peptide of the present invention when presented on the cell surface. Immune effector cells may be cells sensitized in vitro to recognize the peptide. Immune effector cells may be T cells, preferably cytotoxic T cells. Preferably, immune effector cells bind to a sequence in the peptide substantially corresponding to a given amino acid sequence, i.e., an amino acid sequence selected from the group consisting of SEQ ID NOs. 39-44, 101, and 102, or a variant of said amino acid sequence.

[0029] In a further embodiment, the present invention relates to a T cell receptor that is reactive with the peptide of the present invention, or a polypeptide chain of the T cell receptor.

[0030] In a further embodiment, the present invention relates to a T cell receptor polypeptide or a T cell receptor comprising the T cell receptor polypeptide, The aforementioned T cell receptor polypeptide is (i) A T cell receptor polypeptide comprising at least one, preferably two, more preferably all three, CDR sequences of a T cell receptor α chain or a variant thereof, selected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99, as well as (ii) T cell receptor polypeptide comprising a T cell receptor α chain sequence or a variant thereof selected from SEQ ID NOs. 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99 Selected from the group consisting of .

[0031] In one embodiment, the T cell receptor polypeptide is the T cell receptor α chain.

[0032] In one embodiment, the present invention relates to a T cell receptor α chain or a T cell receptor comprising the T cell receptor α chain, The aforementioned T cell receptor α chain is (i) A T cell receptor α chain comprising at least one, preferably two, more preferably all three, CDR sequences of a T cell receptor α chain or a variant thereof, selected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99, and (ii) T cell receptor α chain containing a T cell receptor α chain sequence or a variant thereof selected from SEQ ID NOs. 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99. Selected from the group consisting of .

[0033] The CDR sequence is underlined in the sequence of the T cell receptor α chain shown herein.

[0034] In a further embodiment, the present invention relates to a T cell receptor polypeptide or a T cell receptor comprising the T cell receptor polypeptide, The aforementioned T cell receptor polypeptide is (i) A T cell receptor polypeptide comprising at least one, preferably two, more preferably all three, CDR sequences of a T cell receptor β chain or a variant thereof, selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 92, 94, 96, 98 and 100, and (ii) A T cell receptor polypeptide comprising a T cell receptor β chain sequence or a variant thereof selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 92, 94, 96, 98, and 100. Selected from the group consisting of .

[0035] In one embodiment, the T cell receptor polypeptide is the T cell receptor β chain.

[0036] In one embodiment, the present invention relates to a T cell receptor β chain or a T cell receptor comprising the T cell receptor β chain, The aforementioned T cell receptor β chain is (i) A T cell receptor β chain comprising at least one, preferably two, more preferably all three, CDR sequences of a T cell receptor β chain or a variant thereof selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 92, 94, 96, 98 and 100, and (ii) T cell receptor β chain containing a T cell receptor β chain sequence or a variant thereof selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 92, 94, 96, 98 and 100 Selected from the group consisting of .

[0037] The CDR sequence is underlined in the T cell receptor β chain sequence shown herein.

[0038] In a further embodiment, the present invention is (I) T cell receptor, (i) at least one, preferably two, more preferably all three, of the CDR sequences of the T cell receptor α chain or its variant of sequence number x, and (ii) at least one, preferably two, more preferably all three, of the CDR sequences of the T cell receptor β chain of sequence number x+1 or its variant; Here, x is selected from 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99. T cell receptors including; Furthermore (II) T cell receptor, (i) the T cell receptor α chain sequence of sequence number x or a variant thereof, and (ii) The T cell receptor β chain sequence of sequence number x+1 or a variant thereof; Here, x is selected from 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99. T cell receptors including This relates to T cell receptors selected from a group consisting of the following.

[0039] In one embodiment, the present invention is (I) T cell receptor, (i) A T cell receptor α chain comprising at least one, preferably two, more preferably all three, CDR sequences of the T cell receptor α chain of sequence number x or a variant thereof, and (ii) A T cell receptor β chain comprising at least one, preferably two, more preferably all three, of the CDR sequences of the T cell receptor β chain of sequence number x+1 or a variant thereof; Here, x is selected from 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99. T cell receptors including; Furthermore (II) T cell receptor, (i) A T cell receptor α chain containing the T cell receptor α chain sequence of sequence number x or a variant thereof, and (ii) A T cell receptor β chain containing the T cell receptor β chain sequence of sequence number x+1 or a variant thereof; Here, x is selected from 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99. T cell receptors including; This relates to T cell receptors selected from a group consisting of the following.

[0040] The above-mentioned T cell receptors are preferably specific to tumor-associated antigens NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively, when presented on the surface of cells such as disease cells or antigen-presenting cells.

[0041] In a further embodiment, the present invention relates to nucleic acids encoding a T cell receptor chain or T cell receptor.

[0042] In a further embodiment, the present invention relates to cells genetically modified to express the T cell receptor chain or T cell receptor of the present invention.

[0043] In one embodiment, the cell contains a T cell receptor chain or a nucleic acid encoding a T cell receptor.

[0044] The cells may be effector cells or stem cells, preferably immune effector cells. In one embodiment, the cells are immune effector cells. The immune effector cells may be T cells, preferably cytotoxic T cells. Preferably, the immune effector cells are reactive to tumor-associated antigens NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively, when presented on the surface of cells such as disease cells or antigen-presenting cells, and are specifically reactive to the peptides of the present invention, and preferably bind to a sequence in the peptide substantially corresponding to a given amino acid sequence, i.e., an amino acid sequence selected from the group consisting of SEQ ID NOs. 39-44, 101, and 102, or a variant of said amino acid sequence.

[0045] In a further embodiment, the present invention relates to a method for preparing immunoeffector cells genetically modified to express the T cell receptor of the present invention, comprising delivering a nucleic acid encoding the T cell receptor to the immunoeffector cells.

[0046] In one embodiment, the method involves contacting immune effector cells with particles containing nucleic acids. In one embodiment, the particles further include targeting molecules for targeting immune effector cells. In one embodiment, the nucleic acids are delivered to the immune effector cells by contacting them with the particles.

[0047] In one embodiment, the genetically modified immune effector cells are present in vivo or in vitro.

[0048] In one embodiment, the genetically modified immunoeffector cells are present in vivo in the subject, and the method involves administering particles to the subject.

[0049] Furthermore, the present invention generally encompasses the treatment of diseases by targeting disease cells expressing the antigens NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively. The treatments described herein may be therapeutic or prophylactic measures for malignant diseases.

[0050] This method provides selective eradication of cells presenting the tumor antigens NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively, thereby minimizing adverse effects on normal cells that do not present these antigens. Therefore, preferred diseases for treatment are malignant diseases, particularly cancers as described herein, in which at least one of the antigens described herein is expressed and presented. Target cells may express antigens in relation to MHC for recognition by T cell receptors (TCRs).

[0051] In one embodiment, immune effector cells genetically modified to express a T cell receptor (TCR) that targets cells via binding to an antigen (or its processing product) may be provided to a subject by administration of the genetically modified immune effector cells to the subject or by generating the genetically modified immune effector cells in the subject. Genetic modification may be achieved using particles containing a nucleic acid encoding a T cell receptor for genetic modification and optionally a targeting molecule for targeting the immune effector cells. The particles may deliver the nucleic acid to cells in vitro / ex vivo and in vivo. A vaccine antigen, which may be a disease-associated antigen or a variant thereof (e.g., an epitope of a disease-associated antigen, particularly an epitope recognized by a TCR, e.g., a given amino acid sequence, i.e., an amino acid sequence selected from the group consisting of SEQ ID NOs. 39-44, 101 and 102, or a peptide or protein containing a variant of said amino acid sequence), a nucleic acid encoding it, or a cell expressing the antigen may be administered (optionally after nucleic acid expression by appropriate target cells) to provide the antigen for stimulation, priming, and / or expansion of immune effector cells genetically modified to express an antigen receptor, and the immune effector cells target the antigen or its processing product. In one embodiment, the polynucleotide encoding the vaccine antigen is RNA. In one embodiment, the RNA encoding the vaccine antigen targets a secondary lymphoid organ. Immune effector cells, such as stimulated, primed, and / or expanded T cells in a patient, can recognize cells expressing the antigen that result in the eradication of disease cells. In one embodiment, the immune effector cells are CD8 + T cells. In one embodiment, the targeting molecule described herein is CD8 + It binds to the CD8 receptor on T cells. In one embodiment, immune effector cells bind to CD4 + T cells. In one embodiment, the targeting molecule described herein is CD4 + It binds to the CD4 receptor on T cells. In one embodiment, immune effector cells bind to CD4 + T cells and / or CD8 +The cells are T cells. In one embodiment, the targeting molecule described herein binds to CD3 on T cells. In one embodiment, the immune effector cells are against tumors or cancer. In one embodiment, the target cell population or target tissue is tumor cells or tumor tissue, particularly of solid tumors. In one embodiment, the target antigen is a tumor antigen.

[0052] The methods and agents described herein are particularly useful for treating diseases characterized by disease cells expressing antigens directed at immune effector cells, namely the tumor antigens NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively. Preferably, the cells are genetically modified to stably express a T cell receptor on their surface. In one embodiment, immune effector cells from either the target to be treated or a different target are administered to the target to be treated. The administered immune effector cells may be genetically modified ex vivo before administration or in vivo in the target after administration to express the T cell receptor described herein. In one embodiment, the immune effector cells are endogenous in the target to be treated (and therefore not administered to the target to be treated) and are genetically modified in vivo in the target to express the T cell receptor described herein. Thus, immune effector cells may be genetically modified ex vivo or in vivo to express a T cell receptor. Therefore, such genetic modification using T cell receptors can be performed in vitro, after which immune effector cells can be administered to subjects requiring treatment, or it can be performed in vivo in subjects requiring treatment.

[0053] In a further embodiment, the present invention is (i) The peptide of the present invention; (ii) Nucleic acids of the present invention; (iii) Cells of the present invention; and (iv) Immunoeffector cells of the present invention This relates to a pharmaceutical composition containing one or more of the following.

[0054] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier and may optionally contain one or more adjuvants, stabilizers, etc. The pharmaceutical composition may be in the form of a therapeutic or prophylactic vaccine. In one embodiment, the pharmaceutical composition is for use in treating or preventing malignant diseases as described herein.

[0055] Administration of the above pharmaceutical composition may provide an MHC class II presenting epitope capable of inducing a CD4+ helper T cell response and / or a CD8+ T cell response to the antigen described herein. Alternatively, administration of the above pharmaceutical composition may further provide an MHC class I presenting epitope capable of inducing a CD8+ T cell response to the antigen described herein.

[0056] In one embodiment, the relevant antigens are NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively, and the pharmaceutical composition of the present invention is useful for the treatment and / or prevention of malignant diseases.

[0057] In a further embodiment, the present invention relates to a method for treating a subject, comprising administering the pharmaceutical composition of the present invention to the subject.

[0058] In a further embodiment, the present invention relates to a method for treating a subject, comprising providing an immune effector cell genetically modified to express the T cell receptor of the present invention.

[0059] In one embodiment, the method of the above embodiment is a method for inducing an immune response in the subject. In one embodiment, the immune response is a T cell-mediated immune response. In one embodiment, the immune response is an immune response to a target cell population or target tissue expressing an antigen. In one embodiment, the target cell population or target tissue is cancer cells or cancer tissue. In one embodiment, the cancer cells or cancer tissue is a solid tumor.

[0060] In a further embodiment, the present invention relates to a method for treating a subject having a disease, disorder or condition associated with the expression or upregulation of an antigen, comprising providing a subject with genetically modified immune effector cells expressing the T cell receptor of the present invention, wherein the T cell receptor targets a disease, disorder or condition-related antigen or a cell expressing a disease, disorder or condition-related antigen.

[0061] In one embodiment, the disease, disorder, or condition is cancer, and the antigen associated with the disease, disorder, or condition is a tumor antigen. In one embodiment, the associated antigens are NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively, and the method of the present invention is useful for the treatment and / or prevention of malignant diseases.

[0062] In one embodiment, the disease, disorder, or condition is a solid tumor.

[0063] In one embodiment of the method described above, immune effector cells genetically modified to express T cell receptors are provided to a subject by administering the immune effector cells genetically modified to express T cell receptors, or by generating the immune effector cells genetically modified to express T cell receptors in the subject.

[0064] In one embodiment of the method described above, immune effector cells genetically modified to express T cell receptors are prepared by a method comprising delivering nucleic acids encoding T cell receptors to the immune effector cells.

[0065] In one embodiment of the method described above, immune effector cells genetically modified to express a T cell receptor are prepared by a method comprising contacting the immune effector cells with particles containing nucleic acids encoding the T cell receptor. In one embodiment, the particles further comprise a targeting molecule for targeting the immune effector cells. In one embodiment, the nucleic acid is delivered to the immune effector cells by contacting the immune effector cells with the particles.

[0066] In one embodiment of the method described above, the genetically modified immune effector cells are present in vivo or in vitro.

[0067] In one embodiment of the method described above, the genetically modified immunoeffector cells are present in vivo in the subject, and the method includes administering particles to the subject.

[0068] In one embodiment of the method described above, the method is a method for treating or preventing cancer in a subject. In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is associated with the expression or upregulation of tumor antigens targeted by T cell receptors. In one embodiment, the antigens in question are NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively, and the method of the present invention is useful for treating and / or preventing malignant diseases.

[0069] In one embodiment of the method described above, the method further comprises administering an antigen targeted by a T cell receptor, a polynucleotide encoding the antigen, or a host cell genetically modified to express the antigen. In one embodiment, the polynucleotide encoding the antigen is RNA. In one embodiment, the host cell genetically modified to express the antigen contains the polynucleotide encoding the antigen.

[0070] In one embodiment of the method described above, the immune effector cells genetically modified to express the T cell receptor contain a polynucleotide encoding the T cell receptor. In one embodiment, the nucleic acid is RNA. In another embodiment, the nucleic acid is DNA.

[0071] In one embodiment of the method described above, the gene modification is transient or stable.

[0072] In one embodiment of the method described above, gene modification is performed by a virus-based method, a transposon-based method, or a gene-editing-based method. In one embodiment, the gene-editing-based method includes CRISPR-based gene editing.

[0073] In one embodiment of the method described above, the particles are non-viral particles.

[0074] In one embodiment of the method described above, the particles are lipid-based and / or polymer-based particles.

[0075] In one embodiment of the method described above, the particles are nanoparticles.

[0076] In one embodiment of the method described above, the particles are functionalized with a targeting molecule on their surface.

[0077] In one embodiment of the method described above, the particles are functionalized with a targeting molecule by linking the targeting molecule to at least one particle-forming component.

[0078] In one embodiment of the method described above, the targeting molecule targets CD8, CD4, or CD3.

[0079] In one embodiment of the method described above, the immune effector cell is a T cell.

[0080] In one embodiment of the method described above, the immune effector cells are CD4+ or CD8+ T cells.

[0081] The compositions and agents described herein can preferably induce or promote cellular responses to diseases characterized by presentation of antigens described herein by class I MHC, such as malignant diseases, preferably cytotoxic T cell activity.

[0082] In one embodiment, the present invention provides agents and compositions described herein for use in the therapeutic methods described herein.

[0083] The treatments for malignant diseases described herein may be combined with surgical resection and / or radiation and / or conventional chemotherapy.

[0084] Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0085] [Figure 1] Study design and melanoma FixVac-mediated immune activation. Figure 1A: Molecular structures of four vaccine RNAs. The 5' cap, 5' and 3' untranslated regions (UTRs), and poly(A) tail are optimized for RNA stability and translation efficiency in human dendritic cells. Signal peptides (SPs), tetanus toxoid CD4+ epitopes P2 and P16, and MHC class I transport domains (MITDs) are fused to each TAA coding sequence to enhance presentation and immunogenicity on HLA class I and II molecules. Figure 1B: High-level clinical trial design. [Figure 2] TAA-specific T cell immunity and clinical responses induced by melanoma FixVac. Figure 2A: Ex vivo CD8+ T cell response of patient A2-009 measured after pulsed PBMCs with individual TAA PepMixes. PBMCs incubated with culture medium were used as a control. Figure 2B: (B) Dynamics of vaccine-associated antigen-specific T cells determined by multimer analysis of T cells stimulated with single peptides or PepMixes. Figure 2C: (C) Dynamics of vaccine-associated antigen-specific T cells determined by intracellular cytokine staining (ICS) of T cells stimulated with single peptides or PepMixes. [Figure 3]Discovery and characterization of NY-ESO-1 specific TCRs from post-treatment PBMCs of patient A2-009. Figure 3A: PBMCs were stimulated with NY-ESO-1 PepMix, and single IFNγ-positive CD8+ T cells were sorted by flow cytometry for TCR cloning (control; HIV-gag OLP pool). Figure 3B: HLA-restrictiveness and epitope specificity of NY-ESO-1-TCRs were analyzed after co-culture of TCR-transfected CD8+ T cells with HLA-transfected K562 cells using IFNγ ELISPOT. Figure 3C: Specific lysis of NY-ESO-1-positive (SK-MEL-37) and NY-ESO-1-negative (SK-MEL-28) melanoma cell lines by NY-ESO-1-TCR-transfected T cells was evaluated using xCELLigence cell index (CI) impedance measurement. Specific lysis of HLA-transfected melanoma cell lines was measured after 12 hours of co-culture (E:T=20:1). Bars represent mean specific lysis percentage + sd. Figure 3D: TRB chain frequency of NY-ESO-1 specific TCRs. [Figure 4A] Figure 4: TAA-specific T-cell immunization in patient 53-02 with partial response to melanoma FixVac monotherapy. Figure 4A: CT scans of the right lower and middle lobes of the lung before (a) and after (b) initiation of melanoma FixVac treatment. [Figure 4B] Figure 4B: Size dynamics of multiple melanoma lesions in treated patients as evaluated by CT. Lesions that decreased to less than a quantifiable size are plotted at a diameter of 0.1 mm. T; target lesion, NT; non-target lesion, according to irRECISTv1.1. [Figure 4C] Figure 4C: Dynamics of NY-ESO-196-104-specific Cw*0304-restricted CD8+ T cells analyzed by HLA multimer staining. [Figure 4D] Figure 4D: Dynamics of the ex vivo frequency of NY-ESO-1 specific cytokine-secreting CD8+ T cells, measured by intracellular cytokine staining (ICS) after stimulation with NY-ESO-196-104 peptide. [Figure 4E]Figure 4E: (Upper panel) Killing of melanoma cell lines by vaccine-induced CD8+ T cells from post-treatment PBMCs. Specific killing of HLA-transfected melanoma cell lines by CD8+ T cells from short-term IVS cultures (E:T=20:1) was analyzed after 63 hours of co-culture. Bars represent the mean lysis percentage + sd of three technical replicas. (Lower panel) Frequency of NY-ESO-196-104 multimer-specific CD8+ T cells after IVS of PBMCs from three different time points under treatment (-1 day is baseline, 22 days after 3 vaccinations, 64 days after 7 vaccinations). [Figure 5] Discovery and characterization of NY-ESO-196-104-specific HLA-Cw*0304-restricted TCRs. Figure 5A: Sorting gate for multimer-positive CD8+ T cells for TCR cloning. Control, fluorescence minus 1 (FMO) samples. Figure 5B: Recognition of peptide-pulsed HLA-Cw*0304-transfected K562 cells by NY-ESO-1-TCR-transfected CD8+ T cells in IFNγ ELISpot. Control; HIV-gag OLP pool, NY-ESO-1; NY-ESO-1 PepMix. Figure 5C: Cytotoxicity of NY-ESO-1-TCR-transfected CD8+ T cells after 24-hour co-culture (E:T=50:1) with HLA-transfected melanoma cell lines. Bars represent mean specific lysis %+sd of three technical replicas. Figure 5D: Dynamics of the frequency of NY-ESO-1 specific TCR chronotypes in TCR repertoire data obtained from PBMCs before and after vaccination. [Figure 6A] Figure 6: Discovery and characterization of two NY-ESO-1124-133-specific HLA-B*4001-restricted TCRs. Figure 6A: PBMCs were stimulated with NY-ESO-1 PepMix, and a single IFNγ-positive CD8+ T cell was sorted by flow cytometry for TCR cloning (control; HIV-gag OLP pool). [Figure 6B] Figure 6B: HLA-restrictiveness and epitope specificity of NY-ESO-1-TCR were analyzed after co-culture of TCR-transfected CD8+ T cells with HLA-transfected K562 cells using IFNγ ELISpot. [Figure 6C] Figure 6C: HLA-restrictiveness and epitope specificity of NY-ESO-1-TCR were analyzed after co-culture of TCR-transfected CD8+ T cells with HLA-transfected K562 cells using IFNγ ELISpot. [Figure 6D] Figure 6D: Cytotoxicity of NY-ESO-1 specific TCRs identified in patient post-vaccination samples. TCR-transfected CD8+ T cells were stimulated for 12 hours with an HLA-transfected melanoma cell line at an effector-to-target ratio of 20:1. Bars represent the mean specific lysis percentage + sd of three technical replicas. [Figure 6E] Figure 6E: Dynamics of the frequency of NY-ESO-1 specific TCR chronotypes in TCR repertoire data obtained from PBMCs before and after vaccination. [Figure 7] Correlation between MAGE-A3-specific T cell immunity and TAA expression and tumor mutational burden in patients C2-28 with partial response under melanoma FixVac in combination with anti-PD1. Figure 7A: Clinical response as % change in total target lesion size. Figure 7B: De novo-induced MAGE-A3-specific CD8+ T cells measured by flow cytometry (lower panel: exemplary plot) in patients C2-28. [Figure 8]Discovery and characterization of MAGE-A3168-176-specific HLA-A*0101-restricted TCRs. Figure 8A: Post-treatment PBMCs were stained with HLA-A*0101 / MAGE-A3168-176 multimers, and single multimer-positive CD8+ T cells were sorted by flow cytometry for TCR cloning. Control, fluorescence minus 1 (FMO) samples. Figure 8B: Recognition of MAGE-A3-negative SK-MEL-29(B) melanoma cells by MAGE-A3168-176-specific TCRs obtained from post-treatment PBMCs of C2-28 using a bioluminescence-based T cell activation assay. Figure 8C: Recognition of MAGE-A3-positive SK-MEL-28(C) melanoma cells by MAGE-A3168-176-specific TCRs obtained from post-treatment PBMCs of C2-28 using a bioluminescence-based T cell activation assay. Bars represent the mean luminescence value + sd of two technical replicas. [Figure 9] MAGE-A3-specific T cell immunization in patients C1-40 with partial response under melanoma FixVac in combination with anti-PD1. Figure 9A: CT scans of the right middle lobe and left lower lobe of the lung in patients C1-40 before (pre) and after (post) initiation of melanoma FixVac treatment. Figure 9B: Dynamics of ex vivo frequency of MAGE-A3168-176-specific HLA-A*0101-restricted T cells analyzed by multimer staining. Figure 9C: (Upper panel) Killing of melanoma cell lines by vaccine-induced CD8+ T cells from post-treatment PBMCs. Specific killing of HLA-transfected melanoma cell lines by CD8+ T cells from short-term IVS cultures (E:T=8.5:1) of PBMCs collected at different time points under treatment was analyzed after 8 hours of co-culture. Bars represent the mean lysis percentage + sd of three technical replicas (only two replicas for day 36). (Bottom panel) Frequency of MAGE-A3168-176 multimer-specific CD8+ T cells after IVS. [Figure 10]Discovery and characterization of MAGE-A3168-176-specific HLA-A*0101-restricted TCRs from patient C1-40. Figure 10A: CD8+ IVS cells from post-treatment PBMCs (after 8 vaccinations) were stimulated with MAGE-A3168-176, and single IFNγ-positive CD8+ T cells were sorted by flow cytometry for TCR cloning. Control, SSX241-49. Figure 10B: Recognition of HLA-A*0101-transfected K562 cells by MAGE-A3168-176-specific TCRs obtained from post-treatment PBMCs of C1-40 using a bioluminescence-based T cell activation assay. Bars represent the mean luminescence value + sd of two technical replicas. [Figure 11] TAA-specific T cell immunity in patients A2-10 with partial response. Figure 11A: CT scans of inguinal lymph node metastases in patients A2-10 obtained before and after the initiation of vaccination. Figure 11B: CD4+ T cell response of patients after in vitro stimulation with TAA-coding RNA before and after 8 vaccinations. Cells were restimulated in the IFNγ-ELISpot assay by co-culture with auto-DCs transfected with RNA (TAA or luciferase as a control), auto-DCs pulsed with TAA (PepMix), or unpulsed DCs (no peptide). Bars represent the median + range of spot counts from three replicates. [Figure 12A] Figure 12: Characterization of the HLA class II-restricted TAA-specific T cell response in patients A2-10 with partial response. Figure 12A: CD4+ T cells from IVS cultures were restimulated in PepMix-pulsed DCs and sorted by flow cytometry for TCR cloning (control, HIV-gag OLP pool). [Figure 12B] Figure 12B: Determination of HLA-restrictiveness (B) using TCR-transfected CD4+ T cells and HLA-transfected K562 cells via IFNγ ELISpot. [Figure 12C] Figure 12C: Determination of epitope specificity (C) using TCR-transfected CD4+ T cells and HLA-transfected K562 cells with IFNγ ELISpot. [Figure 12D] Figure 12D: Dynamics of TCR chronotype frequencies in peripheral blood by ex vivo TCR repertory analysis. [Figure 13A] Figure 13: Discovery and characterization of HLA-A*02:01-restricted NY-ESO-1 specific TCRs from checkpoint inhibitor-treated (CIT) NSCLC patients. Figure 13A: CD8+ T cells obtained from NSCLC patient EL28 after CIT treatment were stimulated with NY-ESO-1 IVT-RNA transfected iDCs, and single T cells were sorted for TCR cloning based on the expression of the activation marker CD137 and / or IFNγ via flow cytometry. Cells were gated with single viable CD8+ T lymphocytes. Control, iDCs transfected with RNA encoding an unrelated antigen (eGFP). [Figure 13B] Figure 13B: HLA-restrictiveness and target specificity of NY-ESO-1-TCR were analyzed after co-culturing TRCCD8-EL28-NYE#2 transfected CD8+ T cells with HLA-transfected and peptide-pulsed K562 cells using IFNγ ELISPOT. Control, CD8+ T cells transfected without RNA; effector only (Eff); positive control, Staphylococcus enterotoxin B (SEB). [Figure 13C] Figure 13C: Epitope specificity of TCR-transfected Jurkat cells and peptide-pulsed HLA-transfected K562 target cells was analyzed using a bioluminescence-based T cell activation assay. Bars represent the average luminescence value + sd of three technical replicas. [Figure 13D] Figure 13D: Recognition of endogenously expressed NY-ESO-1 was determined using IFNγ ELISPOT in TRCD8-EL28-NYE#2 transfected CD8+ T cells and NSCLC cell line LCLC-103H. Positive control: phytohemagglutinin-L (PHA-L). [Figure 13E]Figure 13E: Specific lysis of endogenous NY-ESO-1 expressing NSCLC cells by TCRCD8-EL28-NYE#2 expressing CD8+ T cells. TCR-transfected T cells were co-cultured with HLA-A*0201-transfected LCLC-103H cells for 24 hours in the presence (w peptide) or absence (w / o peptide) of NY-ESO-1157-165 (E:T=30:1). Control and T cells transfected without RNA are also shown. Bars represent the mean specific lysis %+sd of three technical replicas. [Figure 13F] Figure 13F: Lysis of endogenous NY-ESO-1 expressing melanoma cell lines mediated by TCRCD8-EL28-NYE#2. TCR-transfected CD8+ T cells were co-cultured for 24 hours with NY-ESO-1 positive (SK-Mel-37) and negative (SK-Mel-28) melanoma cell lines (E:T=20:1). Bars represent the mean specific lysis %+sd of three technical replicas. [Figure 13G] Figure 13G: TRB chain frequency of NY-ESO-1 specific chronotype TCRCD8-EL28-NYE#2 in PBMCs of patients before and after CIT treatment. [Figure 14A] Figure 14: Discovery and characterization of four HLA-A*01:01-restricted KRAS-Q61-H specific TCRs from primary NSCLC patients. Figure 14A: CD8+ T cells were stimulated in iDCs transfected with Multitope#7 IVT-RNA encoding six neoepitopes, including KRAS-Q61H48-74. Single T cells were sorted by flow cytometry based on the expression of the activation marker CD137 and / or IFNγ for TCR cloning. Control, iDCs transfected with IVT-RNA encoding the unrelated antigen eGFP. [Figure 14B] Figure 14B: Using IFNγ ELISPOT, the HLA-restrictiveness and specificity of Multitope#7 (including KRAS-Q61H48-74) were determined after co-culturing TCR-transfected CD8+ T cells with K562 cells transfected with HLA-A*0101 and Multitope#7 RNA. [Figure 14C]Figure 14C: Epitope specificity of KRAS-Q61H55-64 TCR. TCR-transfected CD8+ T cells were stimulated with HLA-A*0101-transfected K562 target cells, peptide-pulsed using IFNγ ELISPOT. Control, unrelated peptide from HIV-gag; effector only (Eff); nonspecific stimulation with Staphylococcus enterotoxin B (SEB). [Figure 14D] Figure 14D: Functional avidity of HLA-A*01:01-restricted KRAS-Q61H55-64-specific TCRs. TCR-transfected CD8+ T cells co-cultured with HLA-A*0101-transfected K562 cells pulsed with a titration of KRAS-Q61H55-64 peptide were analyzed by IFNγ ELISPOT. [Figure 14E] Figure 14E: Specific lysis of endogenous KRAS-Q61H-expressing NSCLC cells mediated by TCRCD8-EL8-LM7#1. TCR-transfected CD8+ T cells were co-cultured with HLA-A*0101-transfected NSCLC cell line NCI-H-460 for 24 hours in the presence (w peptide) or absence (w / o peptide) of KRAS-Q61H55-64 (E:T=30:1). T cells transfected without control and RNA; bars represent mean specific lysis %+sd of three technical replicas. [Modes for carrying out the invention]

[0086] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, and that these may vary. Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention, and that the scope of the present invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0087] Preferably, the terms used herein are defined as those found in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G. W. Heuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0088] Unless otherwise indicated, the implementation of this disclosure will utilize conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0089] The elements of this disclosure are described below. These elements are listed along with specific embodiments, but it should be understood that they may be combined in any way and in any number to create further embodiments. The various examples and embodiments described should not be construed as limiting this disclosure to only the embodiments expressly described. This description should be understood as disclosing and encompassing embodiments that combine the expressly described embodiments with any number of disclosed elements. Furthermore, any rearrangement and combination of all described elements should be considered disclosed by this description unless specifically indicated in the context.

[0090] The term "about" means approximately or nearly, and in the context of the numbers or ranges described herein, in one embodiment, means ±20%, ±10%, ±5%, or ±3% of the listed or claimed numbers or ranges.

[0091] In the context describing this disclosure (particularly in the context of the claims), the terms “one” and “it,” and similar references, should be interpreted as encompassing both singular and plural, unless otherwise specifically indicated herein or unless the contextual context clearly contradicts this interpretation. Enumerations of value ranges herein are intended simply as a way of concisely referring to each separate value belonging to that range individually. Unless otherwise specifically indicated herein, each individual value is incorporated herein as if it were individually listed herein. All methods described herein may be performed in any suitable order unless otherwise specifically indicated herein or unless the contextual context clearly contradicts this interpretation. The use of any examples or illustrative language provided herein (e.g., “etc.”) is intended solely to better illustrate this disclosure and does not impose limitations on the claims. No language herein should be interpreted as referring to any unclaimed element essential to the practice of this disclosure.

[0092] Unless otherwise specified, the term “including” is used in the context of this Document to indicate that there may be additional members in addition to the members of the list introduced by “including”. However, the term “including” is intended to encompass the possibility that there may be no additional members, i.e., for the purposes of this embodiment, “including” should be understood to mean “consisting of”.

[0093] Throughout this specification, several sources are referenced. Each source referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety, either above or below. Nothing in this specification should be construed as an acknowledgment that this disclosure had no prior rights to such disclosure.

[0094] The following definitions are provided, applicable to all aspects of this disclosure. Unless otherwise indicated, the following terms have the meanings set forth below. Terms not defined have the meanings widely recognized in their respective art.

[0095] definition References to sequence numbers 39 through 44 should be understood as referring to sequence numbers 39, 40, 41, 42, 43, and 44 individually.

[0096] As used herein, terms such as “reduce,” “decrease,” “inhibit,” or “impair” relate to an overall reduction or ability to produce an overall reduction of a level, for example, a binding level, preferably by 5% or more, 10% or more, 20% or more, more preferably by 50% or more, and most preferably by 75% or more.

[0097] Terms such as “increase,” “boost,” or “exceed” preferably relate to an increase or boost of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, most preferably at least 100%, at least 200%, at least 500%, or even more.

[0098] The term "multiple" in relation to objects refers to a specific group of a certain number of those objects. In certain embodiments, this term means 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 10 19 , 1020 , 10 21 , 10 22 , or 10 23 This refers to the group described above.

[0099] According to this disclosure, the term “peptide” includes oligopeptides and polypeptides and refers to substances containing a sequence of amino acids linked together by peptide bonds, numbering approximately 2 or more, approximately 3 or more, approximately 4 or more, approximately 6 or more, approximately 8 or more, approximately 10 or more, approximately 13 or more, approximately 16 or more, approximately 20 or more, and up to approximately 50, approximately 100 or approximately 150. The terms “protein” or “polypeptide” refer to larger peptides, particularly peptides having at least approximately 151 amino acids, but the terms “peptide,” “protein,” and “polypeptide” are generally used as synonyms herein.

[0100] A “therapeutic protein,” when administered to a subject in a therapeutically effective amount, exerts a positive or beneficial effect on the subject’s condition or pathology. In one embodiment, a therapeutic protein may have curative or palliative properties and may be administered to improve, alleviate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein may also have prophylactic properties and may be used to delay the onset of a disease or to reduce the severity of such a disease or pathological condition. The term “therapeutic protein” may include whole proteins or peptides, or may refer to therapeutically active fragments thereof. It may also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, antigens and cytokines for vaccination.

[0101] With respect to an amino acid sequence (peptide or protein), a “fragment” refers to a sequence representing a portion of the amino acid sequence, i.e., a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A N-terminal shortened fragment (C-terminal fragment) can also be obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, insofar as it contains a start codon that acts to initiate translation of the truncated open reading frame. An amino acid sequence fragment contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. Preferably, an amino acid sequence fragment contains at least 6, particularly 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 the amino acid sequence.

[0102] In this specification, “variant,” “variant protein,” or “variant polypeptide” means a protein that differs from the wild-type protein by at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or a modified wild-type polypeptide. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, for example, 1 to about 20 amino acid modifications compared to the parent polypeptide, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0103] As used herein, "parent polypeptide," "parent protein," "precursor polypeptide," or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to produce a variant. The parent polypeptide may be a wild-type polypeptide, or a variant or modified form of a wild-type polypeptide.

[0104] In this specification, “wild-type,” “WT,” or “natural” means the amino acid sequence found in nature, including allelic mutations. Wild-type proteins or polypeptides have an amino acid sequence that is not intentionally modified.

[0105] For the purposes of this disclosure, the term "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all splice variants, post-translational modification variants, conformational variants, isoform variants, and species homologs, in particular those naturally expressed by cells. The term "variant" also includes, in particular, fragments of amino acid sequences.

[0106] Amino acid insertion variants involve the insertion of one or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, but random insertions are also possible with appropriate screening of the resulting product. Amino acid addition variants involve amino-terminal and / or carboxyl-terminal fusions of one or more amino acids, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from a sequence, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position in the protein. Amino acid deletion variants containing a deletion at the N-terminus and / or C-terminus of a protein are also called N-terminal and / or C-terminal cleavage variants. Amino acid substitution variants are characterized by the removal of at least one residue in a sequence and the insertion of another residue in its place. It is preferable to modify amino acid sequences at non-conserved positions between homologous proteins or peptides, and / or to substitute amino acids with other amino acids of similar properties. Preferably, amino acid changes in peptides and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of the families of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: Glycine, alanine; Valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; Lysine, arginine; and Phenylanine, tyrosine.

[0107] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 65%, 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 preferably given for an amino acid region that 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 total 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 preferably given 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, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, for example, using Align, with a standard setting, preferably EMBOSS::Needle, Matrix:Blosum62, Gap Open 10.0, Gap Extension 0.5.

[0108] "Sequence similarity" indicates the proportion of amino acids that are identical or represent a conserved amino acid substitution. "Sequence identity" between two amino acid sequences indicates the proportion of amino acids that are identical between those sequences.

[0109] The term "identity percentage" is intended to indicate the percentage of amino acid residues that are identical between two sequences being compared, obtained after best alignment. This percentage is purely statistical, and the differences between the two sequences are randomly distributed across their entire length. Sequence comparisons between two amino acid sequences are typically performed by comparing them after optimal alignment, and the comparison is done segment by segment or "comparison window" by segment to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be achieved manually, by local homology algorithms (Smith and Waterman, 1981, Ads App.Math.2, 482; Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443; Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 85, 2444; or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0110] The identity percentage is calculated by determining the number of identical positions in the two sequences being compared, dividing this number by the total number of positions being compared, and multiplying the result by 100 to obtain the identity percentage between these two sequences.

[0111] Homologous amino acid sequences, according to this disclosure, exhibit identity of at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98%, or at least 99% of the amino acid residues.

[0112] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. Manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions, or deletions is described in detail, for example, Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, such as solid-phase synthesis and similar methods.

[0113] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a “functional fragment” or “functional variant.” The terms “functional fragment” or “functional variant” of an amino acid sequence refer to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., functionally equivalent. With respect to an antigen or antigen peptide, one particular function is one or more immunostimulatory activities indicated by the amino acid sequence from which the fragment or variant is derived, and / or binding to molecules such as MHC and receptors (one or more) to which the amino acid sequence from which the fragment or variant is derived. With respect to an antigen receptor such as a T cell receptor, one particular function is one or more immunostimulatory activities indicated by the amino acid sequence from which the fragment or variant is derived, and / or binding to molecules such as MHC and epitopes to which the amino acid sequence from which the fragment or variant is derived. As used herein, the terms “functional fragment” or “functional variant” refer, in particular, to a variant molecule or sequence that includes 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 still performs one or more functions of the parent molecule or sequence, for example, and can bind to a target molecule. In one embodiment, the alteration of the amino acid sequence of the parent molecule or sequence does not significantly affect or alter the binding properties of the molecule or sequence. In a different embodiment, the binding of the functional fragment or functional variant may be reduced but still significantly present, for example, the binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the binding of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0114] An amino acid sequence (peptide, protein, or polypeptide) "derived" from a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the original amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to that particular sequence or fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant or fragment of that particular sequence. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be modified to have a sequence different from the naturally occurring sequence from which they are derived, while retaining the desired activity of the natural sequence.

[0115] Where used herein, “Instructional Materials” or “Instructions” includes publications, records, figures, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The kit's instructional materials may, for example, be affixed to the container containing the compositions of the present invention, or shipped together with the container containing the compositions. Alternatively, the instructional materials may be shipped separately from the container, with the intention that the instructional materials and the compositions be used in conjunction by the recipient.

[0116] "Isolated" means modified or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not "isolated," but the same nucleic acids or peptides that have been partially or completely separated from their naturally occurring coexisting substances are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0117] In the context of this invention, the term "recombinant" means "produced through genetic manipulation." Preferably, "recombinant products," such as recombinant cells, in the context of this invention do not exist in nature.

[0118] As used herein, the term “naturally occurring” refers to the fact that a substance can be found in nature. For example, peptides or nucleic acids that are present in living organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified by humans in a laboratory are considered naturally occurring.

[0119] As used herein, the term “specifically binding” means molecules such as TCRs that recognize a specific target, such as an antigen or antigenic peptide, but substantially do not recognize or bind to other molecules in the sample or subject. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such species cross-reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different alleles of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the term “specifically binding” or “specifically binding” can be used with respect to the interaction between an antibody, protein, or peptide and a second chemical species to mean that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, an antibody generally recognizes and binds to a specific protein structure rather than the protein itself. If an antibody is specific to epitope “A”, in a reaction involving labeled “A” and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) reduces the amount of labeled A that binds to the antibody.

[0120] The term “genetic modification” includes the transfection of cells with nucleic acids. The term “transfection” relates to the introduction of nucleic acids, particularly RNA, into cells. For the purposes of this invention, the term “transfection” also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, and the cells may be present in a subject, e.g., a patient. Accordingly, according to this invention, the cells for nucleic acid transfection described herein may be present in vitro or in vivo, and for example, the cells may form an organ, tissue, and / or part of an organism of a patient. According to this invention, transfection may be transient or stable. In some applications of transfection, it is sufficient that the transfected genetic material is expressed only transiently. RNA can be transfected into cells to transiently express the protein it encodes. Since nucleic acids introduced in the process of transfection are not usually incorporated into the nuclear genome, the foreign nucleic acids are diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desirable that the transfected nucleic acids actually remain in the genome of the cells and their daughter cells, stable transfection must occur. Such stable transfection can occur when the nucleic acid introduced during the transfection process is integrated into the nuclear genome, and can be achieved, for example, by using a virus-based or transposon-based system for transfection. Generally, cells genetically modified to express antigen receptors, such as T cell receptors, are stably transfected with nucleic acids encoding the antigen receptor, whereas generally, nucleic acids encoding the antigen are transiently transfected into cells.

[0121] Immune effector cells Cells used in connection with the present invention, into which nucleic acids (DNA or RNA) encoding antigen receptors, particularly T cell receptors, may be introduced, include in particular lytic cells, especially immune effector cells such as lymphoid cells, preferably T cells, and more preferably cytotoxic lymphocytes selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. When activated, these cytotoxic lymphocytes each cause the destruction of target cells. For example, cytotoxic T cells cause the destruction of target cells by one or both of the following means: First, when activated, T cells release cytotoxic substances such as perforin, granzyme, and granulysin. Perforin and granulysin create pores in the target cell, and granzyme enters the cell, triggering a cytoplasmic caspase cascade that induces apoptosis (programmed cell death) of the cell. Second, apoptosis may be induced via Fas-Fas ligand interaction between the T cell and the target cell. The cells used in connection with the present invention are preferably autologous cells, but heterologous cells or allogeneic cells can also be used.

[0122] In the context of the present invention, the term "effector function" includes any function mediated by components of the immune system that result in inhibition of tumor growth and / or inhibition of tumor development, including, for example, the death of diseased cells such as tumor cells, or the suppression of tumor dissemination and metastasis. Preferably, the effector function in the context of the present invention is a T cell-mediated effector function. Such a function is a helper T cell (CD4 + In the case of T cells, cytokine release and / or CD8 + This includes activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, elimination of cells, i.e., cells characterized by antigen expression, via apoptosis or perforin-mediated cytolysis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic death of target cells expressing the antigen.

[0123] In the context of the present invention, the terms “immune effector cell” or “immunoreactive cell” refer to cells that exert effector function during an immune response. In one embodiment, “immune effector cells” can bind to antigens, such as antigens presented in relation to MHC on the cell, and mediate an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, “immune effector cells” refer to T cells, preferably CD4 + and / or CD8 + T cells, most preferably CD8 + These are T cells. According to the present invention, the term “immune effector cells” also includes cells that can mature into immune cells (e.g., T cells, particularly T helper cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells are CD34 + This includes hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells is analogous to clonal selection in the immune system when exposed to antigens.

[0124] Preferably, “immune effector cells” recognize antigens with some specificity, particularly when presented in relation to MHC on the surface of disease cells such as cancer cells. Preferably, such recognition allows the antigen-recognizing cells to be responsive or reactive. + If it is a T cell, such responsiveness or reactivity is due to the release of cytokines and / or CD8 +This may include activation of lymphocytes (CTLs) and / or B cells. If the cells are CTLs, such responsiveness or reactivity may include the elimination of cells, i.e., cells characterized by antigen expression, for example, through apoptosis or perforin-mediated cytolysis. According to the present invention, CTL responsiveness may include sustained calcium flow, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic death of target cells expressing the antigen. CTL responsiveness may also be determined using artificial reporters that accurately demonstrate CTL responsiveness. Such CTLs that recognize an antigen and are responsive or reactive are also referred to herein as “antigen-responsive CTLs.”

[0125] In one embodiment, the genetically modified immune effector cells are immune effector cells that express a TCR. The immune effector cells used in accordance with the present invention may express endogenous antigen receptors such as T cell receptors or B cell receptors, or they may not express endogenous antigen receptors.

[0126] "Lymphoid cells" are cells or precursor cells of such cells that, after optional and appropriate modification, for example, after the transfer of an antigen receptor such as a TCR, can generate an immune response, such as a cellular immune response, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells may be immune effector cells as described herein. Preferred lymphoid cells are T cells that can be modified to express an antigen receptor on their cell surface. In one embodiment, lymphoid cells lack endogenous expression of the T cell receptor.

[0127] The terms "T cell" and "T lymphocyte" are used interchangeably herein, and T helper cell (CD4) + Cytotoxic T cells (CTL, CD8 +This includes T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes a target antigen and, preferably, exhibits effector function. A T cell is considered antigen-specific if it kills target cells that express the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, in a chromium-releasing assay or proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-gamma) can be measured.

[0128] T cells belong to the group of white blood cells known as lymphocytes and play a central role in cellular immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence of special receptors on their cell surface called T cell receptors (TCRs). The thymus is the main organ involved in the maturation of T cells. Several different subsets of T cells have been discovered, each with distinct functions.

[0129] Among its many functions, T helper cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface, hence the name CD4. + They are also known as T cells. Helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist the active immune response.

[0130] Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface, therefore CD8 + They are also known as T cells. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0131] Regulatory T cells, or Tregs, are a subpopulation of T cells that modulate the immune system, maintain tolerance to autoantigens, and prevent autoimmune diseases. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0132] As used herein, the term “naive T cell” refers to a mature T cell that has never encountered its congener antigens in the periphery, unlike activated T cells or memory T cells. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), absence of the activation markers CD25, CD44, or CD69, and absence of the memory CD45RO isoform.

[0133] As used herein, the term “memory T cells” refers to a subgroup or subpopulation of T cells that have previously encountered and responded to their congeneral antigens. Upon a second encounter with the antigen, memory T cells may be regenerated to initiate a faster and stronger immune response than the first time the immune system responded to the antigen. Memory T cells are CD4 + or CD8 + It can be one of the following, and usually expresses CD45RO.

[0134] According to the present invention, the term "T cell" also includes cells that can mature into T cells upon appropriate stimulation.

[0135] Most T cells possess a T cell receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor is produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and consists of two distinct peptide chains called α- and β-TCR chains. γδ T cells (gamma delta T cells) are a small subset of T cells that have a different T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is far rarer than αβ T cells (2% of all T cells).

[0136] The structure of the T cell receptor is very similar to that of an immunoglobulin Fab fragment, which is a region defined as a combination of the light and heavy chains of an antibody arm. Each chain of the TCR is a member of the immunoglobulin superfamily and has one N-terminal immunoglobulin (Ig) variable (V) domain, one Ig constant (C) domain, a transmembrane / transcellular transmembrane region, and a short cytoplasmic tail at the C-terminus.

[0137] The TCRs described herein may have naturally occurring, non-naturally occurring, or manipulated TCR formats. The TCRs described herein may preferably be alpha-beta heterodimers having an alpha-chain constant domain sequence and a beta-chain constant domain sequence. The alpha-chain and beta-chain constant domain sequences may be modified by cleavage or substitution, for example, to remove a naturally occurring disulfide bond. In one embodiment, the TCRs described herein may be single-chain formats of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, where Vα and Vβ are the TCR α and β variable regions, respectively, Cα and Cβ are the TCR α and β constant regions, respectively, and L is a linker sequence. The TCRs described herein may associate with or ligate with other parts, such as a detectable label, therapeutic agent, or PK modification moiety. The TCRs described herein may be TCR anti-CD3 fusions comprising the TCR and an anti-CD3 antibody or antibody fragment. Anti-CD3 antibodies or antibody fragments can be covalently bound to the C-terminus or N-terminus of the alpha or beta chain of the TCR.

[0138] According to the present invention, the term “variable region of T cell receptor” refers to the variable domain of the TCR chain. With respect to the T cell receptor sequences described herein (e.g., SEQ ID NOs. 5-38 and 91-100), the term “variant” as used herein refers, in one embodiment, to a fragment of a T cell receptor sequence that contains only the variable region or domain, i.e., a fragment of a T cell receptor sequence that does not contain the constant region sequence portion.

[0139] Both the α and β chains of the TCR have three hypervariable regions or complementarity-determining regions (CDRs), but the β chain's variable region has an additional hypervariable region (HV4) that does not normally come into contact with the antigen and is therefore not considered a CDR. CDR3 is the primary CDR involved in the recognition of processed antigens, while CDR1 of the α chain has been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC. HV4 of the β chain is not thought to be involved in antigen recognition but has been shown to interact with superantigens.

[0140] The phrase "T cell receptor chain containing the CDR sequence of the T cell receptor chain" refers to a T cell receptor chain that contains the CDR of the other T cell receptor chain as its respective CDR.

[0141] According to the present invention, the term “at least one CDR sequence” refers in particular to one or more complementarity-determining regions (CDRs) of the α and / or β chains of the T cell receptor, preferably at least one CDR3 region. Preferably, the term “at least one CDR sequence” means at least one CDR3 sequence. In one embodiment, the term “CDR sequence of the T cell receptor chain” preferably relates to CDR1, CDR2 and CDR3 of the α and / or β chains of the T cell receptor. In one embodiment, the one or more complementarity-determining regions (CDRs) are selected from the set of complementarity-determining regions CDR1, CDR2 and CDR3. In a particularly preferred embodiment, the term “at least one CDR sequence” refers to the complementarity-determining regions CDR1, CDR2 and CDR3 of the α and / or β chains of the T cell receptor.

[0142] In one embodiment, a variable domain of a TCR comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein includes the CDRs together with their intervening framework regions.

[0143] In one embodiment, a TCR α chain, which is part of a TCR and optionally further includes a TCR β chain, comprises a variable domain containing at least one, preferably two, more preferably all three, of the CDR sequences of the TCR α chain variable domain described herein.

[0144] In one embodiment, a TCR β chain, which is part of a TCR optionally further comprising a TCR α chain, comprises a variable domain containing at least one, preferably two, more preferably all three, of the CDR sequences of the TCR β chain variable domain described herein.

[0145] The constant domain of the TCR domain consists of short ligature sequences in which cysteine ​​residues form disulfide bonds, creating a link between the two chains.

[0146] Nucleic acids, such as RNA encoding T cell receptor (TCR) chains, can be introduced into immune effector cells, such as T cells or other lytic cells. In appropriate embodiments, TCR α and β chains are cloned from antigen-specific T cells or T cell lines and used in adoptive T cell therapy. The present invention provides T cell receptors specific to antigens or antigenic peptides (epitopes) disclosed herein. Generally, this aspect of the present invention relates to T cell receptors that recognize or bind antigenic peptides presented in relation to MHC. The T cell receptor, for example, the nucleic acids encoding the α and β chains of the T cell receptor provided according to the present invention, may be contained in separate nucleic acid molecules, such as expression vectors, or in a single nucleic acid molecule. Thus, the term “nucleic acid encoding a T cell receptor” refers to nucleic acid molecules encoding T cell receptor chains on the same or preferably different nucleic acid molecules.

[0147] The term "peptide-reactive immune effector cells" refers to immune effector cells that exhibit the effector function of the immune effector cells described herein when they recognize peptides, particularly when they are presented in association with MHC molecules on the surface of disease cells such as antigen-presenting cells or malignant cells.

[0148] The term “peptide-reactive T cell receptor” refers to a T cell receptor that, when present on immune effector cells, particularly when presented in association with MHC molecules on the surface of disease cells such as antigen-presenting cells or malignant cells, recognizes peptides so that immune effector cells can exert the effector function of the immune effector cells described herein.

[0149] The term "antigen-reactive cell" or similar terms refers to cells that, when presented in association with MHC molecules on the surface of antigen-presenting cells or disease cells such as malignant cells, recognize antigens and exert the effector function of the immune effector cells described herein.

[0150] The term “antigen-specific cell” or similar terms relating to cells that recognize an antigen and, when presented in association with an MHC molecule on the surface of disease cells such as antigen-presenting cells or malignant cells, particularly when an antigen-specific T cell receptor is provided, and which preferably exert the effector function of an immune effector cell as described herein. T cells and other lymphoid cells are considered antigen-specific if the cell kills a target cell that expresses an antigen and / or presents an antigenic peptide after binding to the target cell via the antigen expressed by such target cell or the antigenic peptide presented by such target cell, or secretes cytokines.

[0151] T cells can generally be prepared in vitro or ex vivo using standard procedures. For example, T cells can be isolated from bone marrow, peripheral blood, or fractions of bone marrow or peripheral blood of a mammal, such as a patient, using a commercially available cell isolation system. Alternatively, T cells may originate from related or unrelated human, non-human animal, cell line, or culture. A sample containing T cells may be, for example, peripheral blood mononuclear cells (PBMCs).

[0152] As used herein, the terms “NK cells” or “natural killer cells” refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of T cell receptors. As provided herein, NK cells can also be differentiated from stem cells or progenitor cells.

[0153] Genetic modification to express antigen receptors Cells described herein, such as immune effector cells, can be genetically modified ex vivo / in vitro or in vivo in a subject being treated to express antigen receptors or their processing products, such as T cell receptors (TCRs) that bind to antigens, particularly when presented by target cells, e.g., antigen-presenting cells or disease cells. Cells may naturally express antigen receptors or may be modified to express antigen receptors (e.g., ex vivo / in vitro or in vivo in a subject being treated). In one embodiment, the modification to express antigen receptors is performed ex vivo / in vitro. The modified cells may then be administered to the patient. In one embodiment, the modification to express antigen receptors is performed in vivo. The cells may be endogenous cells of the patient or may be administered to the patient.

[0154] Various methods can be used to introduce antigen receptors, such as TCR constructs, into cells, including T cells, to produce genetically modified cells that express the antigen receptor. Such methods include non-viral-based DNA transfection, non-viral-based RNA transfection (e.g., mRNA transfection), transposon-based systems, and virus-based systems. Non-viral-based DNA transfection carries a low risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids that do not contain the integration element. Virus-based systems include the use of gamma-retroviruses and lentiviral vectors. Gamma-retroviruses are relatively easy to use to produce T cells and efficiently and persistently transduce them, and have been preliminaryly proven to be safe in terms of integration into primary human T cells. Lentiviral vectors also efficiently and persistently transduce T cells, but are more expensive to manufacture. They are also potentially safer than retrovirus-based systems.

[0155] Immune effector cells, especially CD8 + The particles described herein, which can be functionalized with the targeting moieties described herein for the specific targeting of T cells, may be used ex vivo / in vitro or in vivo to deliver nucleic acids encoding antigen receptors, such as T cell receptors, to immune effector cells, such as T cells, to produce genetically modified cells that express antigen receptors.

[0156] In one embodiment of all aspects of the present invention, T cells or T cell precursors are transfected with nucleic acids encoding an antigen receptor either ex vivo or in vivo. In one embodiment, a combination of ex vivo and in vivo transfection may be used. In one embodiment of all aspects of the present invention, the T cells or T cell precursors are derived from the subject being treated. In one embodiment of all aspects of the present invention, the T cells or T cell precursors are derived from a subject different from the subject being treated.

[0157] In one aspect of the present invention, genetically modified T cells can be produced in vivo and thus almost instantaneously using particles such as nanoparticles described herein that target T cells. For example, lipid and / or polymer-based nanoparticles can be coupled to CD3, CD8, or CD4-specific targeting moieties to bind to CD3, CD8, or CD4 on T cells or T cell subpopulations, respectively. Upon binding to T cells, these nanoparticles are endocytized. Their contents, e.g., nucleic acids encoding antigen receptors, e.g., plasmid DNA encoding the antitumor antigen TCR, can be directed to the T cell nucleus because they contain, for example, peptides containing microtubule-associated sequences (MTAS) and nuclear localization signals (NLS). By including nucleic acids encoding antigen receptors, e.g., transposons adjacent to the TCR gene expression cassette, and separate nucleic acids encoding highly active transposases, e.g., plasmids, efficient integration of nucleic acids encoding antigen receptors, e.g., TCR vectors, into chromosomes can be enabled.

[0158] Another possibility is to intentionally position antigen receptor coding sequences, such as TCR coding sequences, at specific loci using CRISPR / Cas9 methods, such as prime editing as described in Anzalone et al. (2019) Nature 576(7785):149-157. For example, an antigen receptor could be knocked in, and an existing T cell receptor (TCR) could be knocked out while the antigen receptor is under the dynamic regulatory control of an endogenous promoter that would otherwise suppress the expression of the endogenous TCR.

[0159] Therefore, in addition to nucleic acids encoding antigen receptors, the particles described herein may also deliver gene editing tools such as CRISPR / Cas9 (or related) or transposon systems such as Sleeping Beauty or Piggyback as cargo. Such tools for genome integration / editing (e.g., transposases, gene editing tools such as CRISPR / Cas9) may be delivered as proteins or coding nucleic acids (DNA or RNA). Nevertheless, delivery of mRNA or auto-amplified RNA is also an option for inducing transient expression of antigen receptors such as T cell receptors (TCRs).

[0160] In one embodiment of all aspects of the present invention, cells genetically modified to express an antigen receptor are stably or transiently transfected with a nucleic acid encoding the antigen receptor. Thus, the nucleic acid encoding the antigen receptor is either integrated into or not integrated into the cell's genome.

[0161] In one embodiment of all aspects of the present invention, cells genetically modified to express an antigen receptor are inactivated for the expression of endogenous T cell receptor and / or endogenous HLA.

[0162] In one embodiment of all aspects of the present invention, the cells described herein may be autologous, allogeneic, or syngeneic to the target being treated. In one embodiment, the disclosure assumes the extraction of cells from a patient and subsequent re-delivery of the cells to the patient. In one embodiment, the disclosure does not assume the extraction of cells from a patient. In the latter case, all steps of genetic modification of the cells are performed in vivo.

[0163] The term "autologous" is used to describe something that originates from the same source. For example, "autotransplantation" refers to the transplantation of tissue or organs from the same source. Such a procedure is advantageous because it overcomes immunological barriers that would otherwise lead to rejection.

[0164] The term "homogenetic" is used to describe things that originate from different individuals of the same species. Two or more individuals are said to be homogeneous if they do not have identical genes at one or more gene loci.

[0165] The term "related" is used to describe individuals or tissues that have the same genotype, i.e., identical twins or animals of the same inbred lineage, or tissues derived from them.

[0166] The term "xenotransplant" is used to describe something consisting of multiple different elements. For example, transferring bone marrow from one individual to another constitutes xenotransplantation. Xenogenes are genes that originate from a source other than the target organism.

[0167] Nucleic acid containing particles In the context of this disclosure, the term “particle” refers to a structured entity formed by a molecule or molecular complex. In one embodiment, the term “particle” refers to a micro-sized or nano-sized structure, such as a micro-sized or nano-sized dense structure dispersed in a medium. In one embodiment, the particles are nucleic acid-containing particles, such as particles containing DNA, RNA, or mixtures thereof.

[0168] Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acids are involved in particle formation. This leads to complex formation and the spontaneous formation of nucleic acid particles. In one embodiment, the nucleic acid particles are nanoparticles.

[0169] As used in this disclosure, “nanoparticles” refers to particles having an average diameter suitable for parenteral administration.

[0170] "Nucleic acid particles" can be used to deliver nucleic acids to target sites of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles may be formed from at least one cationic or ionizable lipid or lipid-like substance such as DOTAP, at least one cationic polymer such as protamine, or a mixture thereof, and nucleic acids. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0171] While not intended to be bound by any particular theory, it is thought that cationic or ionizable lipids or lipid-like substances and cationic polymers, together with nucleic acids, form aggregates, and these aggregates result in colloidally stable particles.

[0172] In one embodiment, the particles described herein further comprise at least one lipid or lipid-like substance other than a cationic or cationically ionizable lipid or lipid-like substance, at least one polymer other than a cationic polymer, or a mixture thereof.

[0173] In some embodiments, nucleic acid particles comprise multiple types of nucleic acid molecules, and the molecular parameters of the nucleic acid molecules may be similar or different from one another, such as molar mass or basic structural elements such as molecular structure, capping, coding region, or other features.

[0174] In one embodiment, the nucleic acid particles described herein may have an average diameter in the range of about 30 nm to about 1000 nm, about 50 nm to about 800 nm, about 70 nm to about 600 nm, about 90 nm to about 400 nm, or about 100 nm to about 300 nm.

[0175] For example, nucleic acid particles produced by the process described herein exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or less than or equal to about 0.2. As an example, nucleic acid particles may exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

[0176] The nucleic acid particles described herein can be prepared using a wide range of methods, which may include obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain nucleic acid particles.

[0177] As used herein, the term “colloid” refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are microscopic, with particle sizes ranging from 1 to 1000 nanometers. The mixture may be referred to as a colloid or colloidal suspension. The term “colloid” may sometimes refer only to the particles in the mixture and not to the entire suspension.

[0178] For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, conventionally used and appropriately adapted methods for preparing liposome vesicles are applicable herein. The most commonly used methods for preparing liposome vesicles share the following basic steps: (i) dissolution of lipids in an organic solvent, (ii) drying of the resulting solution, and (iii) hydration of the dried lipids (using various aqueous media).

[0179] In the membrane hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of the flask. The obtained lipid film is hydrated using a suitable aqueous medium to obtain a liposome dispersion. Further miniaturization steps may also be included.

[0180] Reverse-phase evaporation is an alternative to membrane hydration for preparing liposome vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Brief sonication of this mixture is necessary for homogenization of the system. Removal of the organic phase under reduced pressure yields a milky gel, which subsequently becomes a liposome suspension.

[0181] Other methods having the property of not containing organic solvents may also be used in accordance with this disclosure to prepare colloids.

[0182] LNPs typically consist of four components: ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. Each component plays a role in payload protection, enabling effective intracellular delivery. LNPs can be prepared by rapidly mixing lipids dissolved in ethanol with nucleic acids in an aqueous buffer.

[0183] The term "average diameter" refers to the average hydrodynamic diameter of a particle measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, resulting in the so-called Z, which has the dimension of length. 平均 , and provides a dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "average diameter", "diameter", or "size" of the particle is given by this Z. 平均 It is used synonymously with the value.

[0184] The "polydispersion index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis, as mentioned in the definition of "average diameter." Under certain preconditions, this can be considered a measure of the size distribution of the nanoparticle aggregate.

[0185] It has been previously described that various types of nucleic acid-containing particles are suitable for the delivery of nucleic acids in particulate form (e.g., Kaczmarek, JCet al., 2017, Genome Medicine 9, 60). In the case of nonviral nucleic acid delivery vehicles, encapsulation of nucleic acids in nanoparticles can physically protect the nucleic acids from degradation and, depending on their specific chemical properties, can aid in cellular uptake and endosomal extrusion.

[0186] This disclosure describes particles comprising nucleic acids, at least one cationic or ionizable lipid or lipid-like substance, and / or at least one cationic polymer that associates with nucleic acids to form nucleic acid particles, and compositions comprising such particles. Nucleic acid particles may comprise nucleic acids complexed with the particles in various forms by non-covalent interactions. The particles described herein are not viral particles, in particular infectious viral particles; that is, they cannot virally infect cells.

[0187] Appropriate cationic or ionizable lipids or lipid-like substances and cationic polymers that form nucleic acid particles are included in the term “particle-forming components” or “particle-forming agents.” The term “particle-forming components” or “particle-forming agents” refers to any component that associates with nucleic acids to form nucleic acid particles. Such components include any component that may be part of a nucleic acid particle.

[0188] Cationic polymers Polymers are commonly used materials for nanoparticle-based delivery due to their high degree of chemical flexibility. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups often consist of amines that change their protonation state in the pH range of 5.5–7.5, which is thought to lead to ionic imbalances resulting in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamines, protamines, and polyethyleneimines, as well as naturally occurring polymers such as chitosan, are all applied to nucleic acid delivery and are suitable as cationic polymers herein. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-aminoesters) are widely used in nucleic acid delivery, particularly due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0189] As used herein, "polymer" is given its ordinary meaning, i.e., a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeating units can all be the same, or in some cases, there can be multiple types of repeating units present in the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties as described herein, can also be present in the polymer.

[0190] When multiple types of repeating units are present in a polymer, that polymer is said to be a "copolymer". It should be understood that the polymers used herein can be copolymers. The repeating units forming the copolymer can be arranged in any manner. For example, the repeating units can be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., a copolymer comprising one or more regions each containing a first repeating unit (e.g., a first block) and one or more regions each containing a second repeating unit (e.g., a second block), etc. Block copolymers can have two (diblock copolymers), three (triblock copolymers), or more separate blocks.

[0191] In certain embodiments, the polymer is biocompatible. A biocompatible polymer is typically a polymer that does not cause significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer can be chemically and / or biologically degraded in a physiological environment such as in the body.

[0192] In certain embodiments, the polymer can be protamine or a polyalkyleneimine, particularly protamine.

[0193] The term "protamine" refers to any of several relatively low molecular weight strongly basic proteins that are rich in arginine and found in the sperm cells of various animals (such as fish), often associating with DNA in place of somatic histones. Specifically, the term "protamine" refers to proteins found in fish sperm that are strongly basic, water-soluble, do not coagulate with heat, and primarily produce arginine upon hydrolysis. In purified form, they are used in long-acting insulin formulations to neutralize the anticoagulant effect of heparin.

[0194] As used herein, the term “protamine” is intended to include any protamine amino acid sequence and fragments thereof obtained from or derived from natural or biological sources, as well as polymeric forms of such amino acid sequence or fragments, and artificial, specifically designed for a particular purpose, unisolated (synthesized) polypeptides from natural or biological sources.

[0195] In one embodiment, the polyalkyleneimine includes polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. The preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75 × 10⁻⁶. 2 ~10 7 Da, preferably 1000-10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0196] According to this disclosure, linear polyalkyleneimines such as linear polyethyleneimine (PEI) are preferred.

[0197] The cationic polymers intended for use herein (including polycationic polymers) include any cationic polymers that can electrostatically bind to nucleic acids. In one embodiment, the cationic polymers intended for use herein include any cationic polymer to which nucleic acids can associate, for example, by forming a complex with nucleic acids or by forming vesicles in which nucleic acids are encapsulated or enclosed.

[0198] The particles described herein may also include polymers other than cationic polymers, namely non-cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0199] Lipids and lipid-like substances The terms “lipid” and “lipid-like substance” are broadly defined herein as molecules containing one or more hydrophobic moieties or groups, and optionally one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are also often referred to as amphiphilic substances. Lipids are typically poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecule to self-assemble into organized structures and various phases. One of these phases consists of lipid bilayers, such as vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the presence of long-chain saturated and unsaturated aliphatic hydrocarbon groups, as well as nonpolar groups, including but not limited to those substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups may include polar and / or charged groups, and may include carbohydrates, phosphate groups, carboxylic acid groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0200] As used herein, the term “amphiphilic” refers to a molecule having both a polar and a nonpolar moiety. Often, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water, while the nonpolar moiety is insoluble in water. Furthermore, the polar moiety may have either a formal positive charge or a formal negative charge. Alternatively, the polar moiety may have both a formal positive and a formal negative charge and may be a zwitterion or an internal salt. For the purposes of this disclosure, amphiphilic compounds may be, but are not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0201] The terms “lipid-like substance,” “lipid-like compound,” or “lipid-like molecule” refer to substances that are structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense. For example, this term includes compounds that can form amphiphilic layers, such as vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment, and includes surfactants or synthetic compounds that have both hydrophilic and hydrophobic parts. Generally speaking, this term refers to molecules that have hydrophilic and hydrophobic parts with different structural arrangements, which may or may not be similar to the structure of lipids. Where used herein, the term “lipid” should be interpreted as encompassing both lipids and lipid-like substances unless otherwise specifically indicated herein or unless the context clearly contradicts it.

[0202] Specific examples of amphiphilic compounds that may be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0203] In certain embodiments, amphiphilic compounds are lipids. The term “lipid” refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenolipids (derived from the condensation of isoprene subunits). The term “lipid” is sometimes used as a synonym for fat, but fat is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0204] Fatty acids, or fatty acid residues, are a diverse group of molecules made up of hydrocarbon chains ending in a carboxylic acid group; this arrangement confers a polar, hydrophilic end and a water-insoluble, nonpolar, hydrophobic end to the molecule. Typically 4–24 carbon chains, they can be saturated or unsaturated and can bond to functional groups including oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, it can be either cis or trans geometric isomerized, which significantly affects the molecule's stereochemistry. A cis double bond, when combined with more double bonds in the fatty acid chain, results in the bending of the fatty acid chain. Other major lipid classes in the fatty acid category are fatty acid esters and fatty acid amides.

[0205] Glycerolipids are composed of monosubstituted, disubstituted, and trisubstituted glycerols, the most well known being fatty acid triesters of glycerol called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is esterified, typically by a different fatty acid. A further subclass of glycerolipids is represented by glycosylglycerols, characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

[0206] Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) that have a glycerol core attached to two fatty acid-derived "tails" by ester bonds and to a single "head" group by a phosphate ester bond. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid), include phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0207] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The major sphingoid bases in mammals are generally referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives that have amide-linked fatty acids. These fatty acids are typically saturated or monounsaturated, with chain lengths of 16–26 carbon atoms. The major sphingophospholipid in mammals is sphingomyelin (ceramidephosphocholine), while insects primarily contain ceramidephosphoethanolamine, and fungi have phytoceramidephosphoinositol and mannose-containing head groups. Sphingoglycolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples of these include simple and complex sphingoglycolipids such as cerebrosides and gangliosides.

[0208] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelin.

[0209] Glycolipids represent compounds in which fatty acids are directly linked to a sugar backbone, forming a structure compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The most well-known glycolipid is the acylated glucosamine precursor of the lipid A component of lipopolysaccharides of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with seven fatty acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli (E. coli) is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0210] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by classical enzymes, as well as iterative and multimodular enzymes that share mechanistic features with fatty acid synthases. They include a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources and have great structural diversity. Many polyketides are cyclic molecules whose skeletons are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0211] According to the present disclosure, lipids and lipid-like substances can be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH.

[0212] Cationic or cation-ionizable lipids or lipid-like substances The nucleic acid particles described herein may comprise at least one cationic or cationically ionizable lipid or lipid-like substance as a particle-forming agent. The cationic or cationically ionizable lipid or lipid-like substance intended for use herein comprises any cationic or cationically ionizable lipid or lipid-like substance that can electrostatically bind to nucleic acids. In one embodiment, the cationic or cationically ionizable lipid or lipid-like substance intended for use herein can associate with nucleic acids, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is encapsulated or enclosed.

[0213] As used herein, “cationic lipid” or “cationic lipid-like substance” refers to a lipid or lipid-like substance that has a net positive charge. Cationic lipids or lipid-like substances bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have lipophilic moieties such as sterols, acyl chains, diacyl chains or more, and the lipid head groups typically carry a positive charge.

[0214] In certain embodiments, cationic lipids or lipid-like substances have a net positive charge only at specific pH levels, particularly acidic pH levels, but at different, preferably higher, pH levels such as physiological pH, they preferably have no net positive charge, and preferably are chargeless, i.e., neutral. This ionizable behavior is thought to enhance efficacy by facilitating endosomal escape and reducing toxicity compared to particles that remain cationic at physiological pH levels.

[0215] For the purposes of this disclosure, such “cationically ionizable” lipids or lipid-like substances are included in the term “cationic lipids or lipid-like substances” unless otherwise inconsistent with the context.

[0216] In one embodiment, a cationic or cationically ionizable lipid or lipid-like substance comprises a head group containing at least one positively charged or protonable nitrogen atom (N).

[0217] Examples of cationic lipids include 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); 1,2-diacyloxy-3-di Methylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleil Oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3- Beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadieneoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadieneoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3] -Dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminonium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminonium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium (DOBAQ), 2 -({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine(octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammoniumpropane(DMDAP), 1,2-dipalmitoyl-3-dimethylammoniumpropane(DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-Di[oleyloxy]-benzamide (MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-Bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amonium bromide (DMORIE), Di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate (ATX), N,N-dimethyl- 2,3-Bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N, 12 -5) includes, but is not limited to, 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazine-1-yl]ethyl]amino]dodecane-2-ol (lipidoid C12-200). DOTAP, DODMA, DOTMA, DODAC, and DOSPA are preferred. In certain embodiments, at least one cationic lipid is DOTAP.

[0218] In some embodiments, cationic lipids may constitute about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the particles.

[0219] Further lipids or lipid-like substances The particles described herein may also contain lipids or lipid-like substances other than cationic or cationically ionizable lipids or lipid-like substances, i.e., non-cationic lipids or lipid-like substances (including non-cationically ionizable lipids or lipid-like substances). Collectively, anionic and neutral lipids or lipid-like substances are referred to herein as non-cationic lipids or lipid-like substances. By optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties such as cholesterol and lipids in addition to ionizable / cationic lipids or lipid-like substances, particle stability and the effectiveness of nucleic acid delivery can be enhanced.

[0220] Further lipids or lipid-like substances may be incorporated, which may or may not affect the overall charge of the nucleic acid particles. In certain embodiments, the further lipids or lipid-like substances are noncationic lipids or lipid-like substances. Noncationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, “neutral lipids” refers to any of a number of lipid species that exist in uncharged or neutral zwitterionic forms at a selected pH. In preferred embodiments, the further lipids include one of the following neutral lipid components: (1) phospholipids, (2) cholesterol or its derivatives, or (3) mixtures of phospholipids and cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0221] Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids include, in particular, diacylphosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), and dibehenoylphosphatidylcholine (DB PC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 This includes Lyso PC and phosphatidylethanolamines, particularly diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphytanoylphosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids having various hydrophobic chains.

[0222] In certain preferred embodiments, the further lipids are DSPC, or DSPC and cholesterol.

[0223] In certain embodiments, the nucleic acid particles include both a cationic lipid and further lipids. In exemplary embodiments, the cationic lipid is DOTAP, and the further lipids are DSPC or DSPC and cholesterol.

[0224] While we do not wish to be bound by theory, the amount of at least one cationic lipid compared to the amount of at least one further lipid may affect important nucleic acid particle properties such as charge, particle size, stability, tissue selectivity, and the biological activity of nucleic acids. Therefore, in some embodiments, the molar ratio of at least one cationic lipid to at least one further lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

[0225] In some embodiments, noncationic lipids, particularly neutral lipids (e.g., one or more phospholipids and / or cholesterol), may constitute about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 0 mol% to about 70 mol%, about 0 mol% to about 60 mol%, or about 0 mol% to about 50 mol% of the total lipids present in the particles.

[0226] target molecule One or more of the particle-forming components described herein, such as polymers, lipids and / or lipid-like substances, can cause particles to affect immune effector cells, particularly CD8 + The targeting molecule may contain, or be functionalized with, one or more targeting molecules directed toward T cells, such as T cells. The targeting molecule may be conjugated to any particle-forming component, such as lipids, lipid-like substances, or polymers, and may be covalently, noncovalently, or ligated. When fused to nucleic acid particle components such as lipids or proteins, the targeting molecule may bind specifically to CD8 and may show increased transfection of CD8+ T cells in vitro and in vivo compared to particles not functionalized with the targeting molecule. The targeting molecule includes, but is not limited to, antibodies, antibody fragments, and ankyrin repeat proteins.

[0227] CD8 is the primary marker of the cytotoxic subset of T lymphocytes. CD8 is a type I single-pass transmembrane protein expressed on the surface of immune cells as a disulfide-bonded homodimer or heterodimer molecule. The CD8 heterodimer consists of CD8α and CD8β chains and is expressed only on the surface of immature CD4+CD8+ double-positive thymocytes and mature peripheral cytotoxic αβ T cells. The homodimer consists of two CD8α chains and is expressed on a much broader range of immune cells. In addition to classical cytotoxic αβ T cells and thymocytes, it is found on natural killer T (NKT) cells, a subset of dendritic cells (DCs), and a subpopulation of natural killer (NK) cells. Both CD8αβ and CD8αα can mediate MHC-I binding; however, the heterodimer form is more dominant on the surface of MHC-I-restricted cytotoxic T cells. Notably, the CD8ββ homodimer does not exist in nature.

[0228] The term "antibody" includes immunoglobulins, which comprise at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), with more conserved regions called framework regions (FRs) interposed between them. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies bind to antigens, preferably specifically. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or they can be the immunoreactive portion or fragment of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in this invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0229] The term "antibody fragment" refers to a portion of an intact antibody, typically containing the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0230] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in an antibody molecule in its naturally occurring conformation.

[0231] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in naturally occurring conformations, and κ-light chain and λ-light chain refer to the two main antibody light chain isotypes.

[0232] nucleic acid As used herein, the terms “polynucleotide” or “nucleic acid” are intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated.

[0233] Nucleic acids may be included in vectors. As used herein, the term “vector” includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phages, viral vectors such as retroviruses, adenoviruses or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of an operablely linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment and may lack the functional sequences necessary for the expression of the desired DNA fragment.

[0234] In one embodiment of all aspects of the present invention, a nucleic acid, such as a nucleic acid encoding an antigen receptor or a nucleic acid encoding a vaccine antigen, is expressed in a target cell to be treated in order to provide the antigen receptor or vaccine antigen. In one embodiment of all aspects of the present invention, the nucleic acid is transiently expressed in the target cell. Therefore, in one embodiment, the nucleic acid is not integrated into the cell's genome. In one embodiment of all aspects of the present invention, the nucleic acid is RNA, preferably in vitro transcription RNA. In one embodiment of all aspects of the present invention, antigen expression occurs on the cell surface. In one embodiment of all aspects of the present invention, the vaccine antigen is expressed and presented in relation to the MHC.

[0235] In one embodiment of all aspects of the present invention, a nucleic acid encoding a vaccine antigen is expressed in cells such as target antigen-presenting cells to be treated, which provide the vaccine antigen for binding by immune effector cells expressing an antigen receptor, and such binding results in stimulation, priming, and / or expansion of the immune effector cells expressing the antigen receptor.

[0236] The nucleic acids described herein may be recombinant and / or isolated molecules.

[0237] In this disclosure, the term “RNA” refers to nucleic acid molecules containing ribonucleotide residues. In preferred embodiments, RNA comprises all or most of the ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. RNA includes, but is not limited to, isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and 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 the addition of non-nucleotide material to internal RNA nucleotides or to the ends (one or both) of RNA. In this disclosure, nucleotides in RNA are also construed to be non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. In this disclosure, these modified RNAs are considered analogues of naturally occurring RNA.

[0238] In certain embodiments of this disclosure, RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As is established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide-coding region, and a 3' untranslated region (3'-UTR). In some embodiments, RNA is produced by in vitro transcription or chemosynthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid comprising deoxyribonucleotides.

[0239] In one embodiment, the RNA is in vitro transcription RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for regulating transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0240] In certain embodiments of this disclosure, RNA is replicon RNA or simply “replicon,” in particular self-replicating RNA. In one particularly preferred embodiment, the replicon or self-replicating RNA is derived from or contains elements derived from ssRNA viruses, particularly positive-strand ssRNA viruses such as alphaviruses. Alphaviruses are typical representative examples of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837–856 for a review of the alphavirus life cycle). The total genome length of many alphaviruses is typically in the range of 11,000–12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in the transcription, modification, and replication of viral RNA, as well as protein modification) and structural proteins (forming the viral particle). Typically, there are two open reading frames (ORFs) within the genome. The four non-structural proteins (nsP1-nsP4) are typically encoded together by a first-order reading frame (ORF) beginning near the 5' end of the genome, while the alphaviral structural proteins are found downstream of the first ORF and are encoded together by a second ORF extending near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding non-structural proteins are translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol.87, pp.111-124). After infection, i.e., in the early stages of the viral life cycle, the (+) strand genomic RNA acts directly like messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or target organisms.A simple approach involves replacing the open reading frame encoding the alphaviral structural protein with the open reading frame encoding the protein of interest. Alphaviral-based trans replication systems rely on alphaviral nucleotide sequence elements on two separate nucleic acid molecules: one encoding a viral replicase, and the other being replicatable in trans by the replicase (hence the name trans replication system). Trans replication requires the presence of both of these nucleic acid molecules within a given host cell. The nucleic acid molecule that can be replicatable in trans by the replicase must contain specific alphaviral sequence elements that enable recognition and RNA synthesis by the alphaviral replicase.

[0241] In one embodiment, RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine, pseudouridine, and / or 1-methylpsoiduridine.

[0242] In some embodiments, the RNA according to this disclosure includes a 5' cap. In one embodiment, the RNA according to this disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified by a 5' cap analogue. The term “5' cap” refers to a structure found at the 5' end of an mRNA molecule, and generally consists of a guanosine nucleotide linked to the mRNA by a 5'-5' triphosphate bond. In one embodiment, this guanosine is methylated at position 7. Providing a 5' cap or a 5' cap analogue to RNA can be achieved by in vitro transcription in which the 5' cap is co-transcribed onto the RNA strand, or by post-transcriptional binding to the RNA using a capping enzyme.

[0243] In some embodiments, the building block cap for RNA is m2 7,3'-O Gppp(m1 2'-O )ApG(sometimes m2 7,3'O G(5')ppp(5')m2'-O It is also called ApG, and it has the following structure: [ka]

[0244] The following are RNA and m2 7,3'O G(5')ppp(5')m 2'-O This is an example of cap 1 RNA containing ApG: [ka]

[0245] The following is another example of capped 1 RNA (without cap analogues): [ka]

[0246] In some embodiments, RNA is structured as follows: [ka] Anti-reverse direction cap (ARCA cap (m2)) of a cap analogue having 7,3'O It is modified with a "cap 0" structure using G(5')ppp(5')G).

[0247] The following are RNA and m2 7,3'O This is an example of cap 0 RNA containing G(5')ppp(5')G: [ka]

[0248] In some embodiments, the "cap 0" structure is structure: [ka] Cap analog β-S-ARCA(m2 7,2'OIt is generated using G(5')ppSp(5')G).

[0249] The following is β-S-ARCA(m2 7,2'O This is an example of capped RNA containing G(5')ppSp(5')G) and RNA: [ka]

[0250] A particularly preferred cap is the 5' cap m2 7,2'O Includes G(5')ppSp(5')G.

[0251] In some embodiments, the RNA according to this disclosure includes a 5'-UTR and / or a 3'-UTR. The terms “untranslated region” or “UTR” refer to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region within an RNA molecule such as an mRNA molecule. Untranslated regions (UTRs) may be located on the 5' side (upstream) (5'-UTR) and / or the 3' side (downstream) (3'-UTR) of the open reading frame. The 5'-UTR, if present, is located at the 5' end upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5' cap (if present) and, for example, directly adjacent to the 5' cap. The 3'-UTR, if present, is located at the 3' end downstream of the stop codon of the protein-coding region, although the term “3'-UTR” preferably does not include a poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present) and, for example, directly adjacent to the poly(A) sequence.

[0252] In some embodiments, the RNA according to this disclosure includes a 3'-poly(A) sequence.

[0253] As used in the present invention, the terms “polyA tail” or “polyA sequence” typically refer to a continuous or discontinuous sequence of adenylate residues located at the 3' end of an RNA molecule. PolyA tails or polyA sequences are known to those skilled in the art and may follow the 3'-UTR of the RNA described herein. A continuous polyA tail is characterized by a sequence of adenylate residues. In nature, continuous polyA tails are typical. The RNA disclosed herein may have a polyA tail that is bound to the free 3' end of the RNA by template-independent RNA polymerase after transcription, or a polyA tail encoded by DNA and transcribed by template-dependent RNA polymerase.

[0254] The polyA tail, consisting of approximately 120 A nucleotides, has been shown to have beneficial effects on RNA levels in transfected eukaryotic cells, as well as on the levels of proteins translated from the open reading frame located upstream (5' end) of the polyA tail (Holtkamp et al., 2006, Blood, vol.108, pp.4009-4017).

[0255] The polyA tail can be of any length. In some embodiments, the polyA tail contains, essentially consists of, or comprises 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, particularly about 120 A nucleotides. In this context, “essentially consists of” means that most of the nucleotides in the polyA 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% of the nucleotide number in the polyA tail, are A nucleotides, but the remaining nucleotides may be non-A nucleotides such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consists of" means that all nucleotides in the polyA tail, i.e., 100% of the nucleotides in the polyA tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0256] In some embodiments, the poly(A) tail is bound during RNA transcription, for example, during the preparation of in vitro transcription RNA, based on a DNA template containing repeating dT nucleotides (deoxythymidylate) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is referred to as the poly(A) cassette.

[0257] In some embodiments, poly(A) cassettes present in the coding strand of DNA are essentially composed of dA nucleotides but interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5–50, 10–30, or 10–20 nucleotides in length. Such cassettes are disclosed in International Publication 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication 2016 / 005324 A1 may be used in the present invention. Poly(A) cassettes, essentially composed of dA nucleotides but with four nucleotides (dA, dC, dG, and dT) evenly distributed and interrupted by a random sequence having, for example, a length of 5–50 nucleotides, are still associated with beneficial properties relating to the sustained proliferation of plasmid DNA in E. coli at the DNA level and, at the RNA level, to the support of RNA stability and translation efficiency. As a result, in some embodiments, the poly-A tail contained in the RNA molecules described herein is essentially composed of A nucleotides but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such a random sequence may be 5–50, 10–30, or 10–20 nucleotides in length.

[0258] In some embodiments, nucleotides other than A nucleotides are not adjacent to the polyA tail at their 3' end, i.e., the polyA tail is not masked, or it is not followed by a nucleotide other than A at its 3' end.

[0259] In some embodiments, the polyA tail may contain at least 20, at least 30, at least 40, at least 80, or at least 100 nucleotides, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the polyA tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 nucleotides, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the polyA tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 nucleotides, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the polyA tail contains at least 100 nucleotides. In some embodiments, the polyA tail contains approximately 150 nucleotides. In some embodiments, the polyA tail contains approximately 120 nucleotides.

[0260] According to this disclosure, the vaccine antigen is preferably administered as a single-stranded 5'-capped mRNA that is translated into the respective protein upon entering an antigen-presenting cell (APC). Preferably, the RNA contains structural elements (5' cap, 5'-UTR, 3'-UTR, poly(A) tail) optimized for the maximum efficacy of the RNA in terms of stability and translational efficiency.

[0261] In one embodiment, β-S-ARCA(D1) is used as a specific capping structure for the 5' end of the RNA. In one embodiment, the 5'-UTR sequence is derived from human α-globin mRNA. In one embodiment, two repeating 3'-UTRs derived from human β-globin mRNA are placed between the coding sequence and the poly(A) tail to ensure higher maximal protein levels and long-term mRNA persistence. Alternatively, the 3'-UTR may be a combination of two sequence elements (FI elements) derived from “split amino-terminal enhancer” (AES) mRNA (referred to as F) and mitochondrial-encoded 12S ribosomal RNA (referred to as I). These were identified by an ex vivo selection process of sequences that confer RNA stability and enhance total protein expression (see International Publication 2017 / 060314, incorporated herein by reference). In one embodiment, a poly(A) tail, measured to be 110 nucleotides long, is used, consisting of a stretch of 30 adenosine residues followed by a sequence of 10 nucleotide linkers and another 70 adenosine residues. This poly(A) tail sequence is designed to enhance RNA stability and translation efficiency in dendritic cells.

[0262] The RNA is preferably administered as lipoplex particles, preferably containing DOTMA and DOPE, as further described below. Such particles are preferably administered systemically, particularly intravenously.

[0263] In the context of this disclosure, the term “transcription” refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into peptides or proteins.

[0264] In relation to RNA, the terms "expression" or "translation" refer to the process in the ribosomes of a cell in which a strand of mRNA directs the assembly of amino acid sequences to make a peptide or protein.

[0265] "Code" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties that arise therefrom, which have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and act as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, if the transcription and translation of mRNA corresponding to a gene produce a protein in a cell or other biological system, that gene codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0266] According to this disclosure, the term “RNA-encoded” means that RNA, when present in a suitable environment such as within the cells of a target tissue, can direct the assembly of amino acids to produce the peptide or protein it encodes during the translation process. In one embodiment, RNA can interact with cellular translation mechanisms that enable the translation of peptides or proteins. Cells may produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide or protein, or express it on their surface.

[0267] As used herein, “endogenous” means any substance produced from or within an organism, cell, tissue, or system.

[0268] As used herein, the term “exogenous” means any substance introduced from or produced outside of an organism, cell, tissue, or system.

[0269] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence. Expression can be transient or stable. According to the present invention, the term “expression” also includes “ectopic expression” or “abnormal expression.”

[0270] As used herein, the terms “linked,” “fused,” and “fused” are interchangeable. These terms refer to the combination of two or more elements, components, or domains.

[0271] The term “cell” includes any living cell, i.e., a viable cell capable of performing its normal metabolic functions. In one embodiment, the term refers to any cell that can be transformed or transfected with exogenous nucleic acids. According to the present invention, the term “cell” includes prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells). Mammalian cells, such as cells from humans, mice, hamsters, pigs, goats, and primates, are particularly preferred. Cells may originate from a number of tissue types and may include primary cells and cell lines. In certain embodiments, cells are antigen-presenting cells, particularly dendritic cells, monocytes or macrophages, or immune effector cells, particularly T cells such as cytotoxic T cells. Cells may be recombinant cells and may contain nucleic acids, particularly nucleic acids encoding peptides or proteins, which can be delivered to the cell, for example, by transfection. Cells can secrete encoded peptides or proteins, express them on their surface, and preferably further express MHC molecules that bind to the peptides or proteins or their processing products. In one embodiment, cells endogenously express MHC molecules. In further embodiments, cells recombinantly express MHC molecules and / or peptides or proteins or their processing products. Cells are preferably non-proliferative.

[0272] Cytokine The methods described herein may include, for example, providing one or more cytokines to a subject by administering to the subject one or more cytokines, polynucleotides encoding one or more cytokines, or host cells expressing one or more cytokines.

[0273] As used herein, the term "cytokine" includes naturally occurring cytokines and their functional variants (including fragments of naturally occurring cytokines and their variants). One particularly preferred cytokine is IL2.

[0274] Cytokines are a category of small proteins (approximately 5–20 kDa) that are crucial for cellular signaling. The release of cytokines influences the behavior of surrounding cells. Cytokines are involved in autocrine, parasecrine, and endocrine signaling as immunomodulators. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor, but generally do not include hormones or growth factors (despite some overlap in terminology). Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by multiple types of cells. Cytokines act via receptors and are particularly important in the immune system; they regulate the balance between humoral and cellular immune responses and modulate the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the actions of other cytokines in complex ways.

[0275] IL2 Interleukin 2 (IL2) is a cytokine that induces the proliferation of antigen-activated T cells and stimulates natural killer (NK) cells. The biological activity of IL2 is mediated through the multi-subunit IL2 receptor complex (IL2R), which consists of three polypeptide subunits spanning the cell membrane: p55 (IL2Rα, alpha subunit, also known as CD25 in humans), p75 (IL2Rβ, beta subunit, also known as CD122 in humans), and p64 ​​(IL2Rγ, gamma subunit, also known as CD132 in humans). The T cell response to IL2 depends on a variety of factors, including (1) the concentration of IL2; (2) the number of IL2R molecules on the cell surface; and (3) the number of IL2Rs occupied by IL2 (i.e., the affinity of the binding interaction between IL2 and IL2R) (Smith, "Cell Growth Signal Transduction is Quantal," In Receptor Activation by Antigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995). The IL2:IL2R complex is internalized upon ligand binding, and the different components undergo different sorting. When administered as an intravenous (iv) bolus, IL2 has rapid systemic clearance (an initial clearance phase with a half-life of 12.9 minutes, followed by a slower clearance phase with a half-life of 85 minutes) (Konrad et al., Cancer Res. 50:2009-2017, 1990).

[0276] In eukaryotic cells, human IL-2 is synthesized as a 153-amino acid precursor polypeptide, from which 20 amino acids are removed to produce mature secreted IL-2. Recombinant human IL-2 is produced in E. coli, insect cells, and mammalian COS cells.

[0277] According to this disclosure, IL2 (optionally as part of extended PK IL2) may be naturally occurring IL2 or a fragment or variant thereof. IL2 may be human IL2 and may originate from any vertebrate, in particular any mammal.

[0278] extended PK group The cytokine polypeptides described herein can be prepared as fusion polypeptides or chimeric polypeptides comprising a cytokine moiety and a heterogeneous polypeptide (i.e., a polypeptide that is not a cytokine or a variant thereof). The resulting molecules, hereinafter referred to as "extended pharmacokinetic (PK) cytokines," have an extended circulating half-life compared to free cytokines. The extended circulating half-life of extended PK cytokines allows in vivo serum cytokine concentrations to be maintained within the therapeutic range, potentially leading to enhanced activation of many types of immune cells, including T cells. Due to their favorable pharmacokinetic profile, extended PK cytokines can be administered at lower frequencies and for longer periods compared to unmodified cytokines.

[0279] As used herein, “half-life” refers to the time required for the serum or plasma concentration of a compound, such as a peptide or protein, to decrease by 50% in vivo, for example, due to degradation and / or clearance or sequestration by natural mechanisms. Extended PK cytokines suitable for use herein, such as extended PK interleukins (ILs), are stabilized in vivo, and their half-lives are increased, for example, by fusion with serum albumin (e.g., HSA or MSA) that resists degradation and / or clearance or sequestration. Half-life can be determined by any method known in itself, such as pharmacokinetic analysis. Appropriate techniques will be apparent to those skilled in the art and may include, for example, the steps of appropriately administering an appropriate dose of an amino acid sequence or compound to a subject; collecting blood samples or other samples from the subject at periodic intervals; determining the level or concentration of the amino acid sequence or compound in the blood samples; and calculating, from the data (plots of) thus obtained, the time required for the level or concentration of the amino acid sequence or compound to decrease by 50% compared to the initial level at administration. Further details are provided in standard handbooks such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists, and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0280] Cytokines can be fused to extended PK groups that increase their circulating half-life. Non-limiting examples of extended PK groups are described below. It should be understood that other PK groups that increase the circulating half-life of cytokines or their variants are also applicable to this disclosure. In certain embodiments, the extended PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0281] As used herein, the term “PK” is an acronym for “pharmacokinetics” and encompasses, for example, the properties of a compound including absorption, distribution, metabolism, and excretion by a subject. As used herein, “extended PK group” refers to a protein, peptide, or parity that, when fused to or administered together with a biologically active molecule, increases the circulating half-life of the biologically active molecule. Examples of extended PK groups include serum albumin (e.g., HSA), immunoglobulin Fc or Fc fragments and their variants, transferrin and its variants, and human serum albumin (HSA) conjugates (disclosed in U.S. Patent Applications Publications 2005 / 0287153 and 2007 / 0003549). Other exemplary extended PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, which is incorporated herein by reference in whole. As used herein, “extended PK cytokine” refers to a cytokine parity combined with an extended PK group. In one embodiment, an extended PK cytokine is a fusion protein in which a cytokine portion is linked to or fused to an extended PK group. As used herein, "extended PK IL" refers to an interleukin (IL) portion (including an IL variant portion) combined with an extended PK group. In one embodiment, an extended PK IL is a fusion protein in which an IL portion is linked to or fused to an extended PK group. An exemplary fusion protein is an HSA / IL2 fusion in which an IL2 portion is fused with an HSA.

[0282] In certain embodiments, the serum half-life of the extended PK cytokine is increased compared to the cytokine alone (i.e., the cytokine not fused to the extended PK group). In certain embodiments, the serum half-life of the extended PK cytokine is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer than the serum half-life of the cytokine alone. In certain embodiments, the serum half-life of the extended PK cytokine is at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 10 times, 12 times, 13 times, 15 times, 17 times, 20 times, 22 times, 25 times, 27 times, 30 times, 35 times, 40 times, or 50 times longer than the serum half-life of the cytokine alone. In certain embodiments, the serum half-life of the prolonged PK cytokine is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0283] In certain embodiments, the extended PK group comprises serum albumin or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which, for the purposes of this disclosure, are included in the term “albumin”). The polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form albumin fusion proteins. Such albumin fusion proteins are described in U.S. Patent Application Publication No. 20070048282.

[0284] As used herein, “albumin fusion protein” refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) with at least one molecule of a therapeutic protein, particularly IL2 (or a variant thereof). Albumin fusion proteins can be produced by the translation of a nucleic acid in which a polynucleotide encoding the therapeutic protein is in-frame bound to a polynucleotide encoding albumin. Once the therapeutic protein and albumin become part of the albumin fusion protein, they may be referred to as “part,” “region,” or “fragment” of the albumin fusion protein (e.g., “therapeutic protein portion” or “albumin protein portion”). In a very preferred embodiment, the albumin fusion protein comprises at least one molecule of therapeutic protein (including, but not limited to, a mature form of the therapeutic protein) and at least one molecule of albumin (including, but not limited to, a mature form of albumin). In one embodiment, the albumin fusion protein is processed by host cells, such as hepatocytes, of the target organ of the administered RNA and secreted into circulation. Processing of the nascent albumin fusion protein occurring in the secretory pathway of the host cell used for RNA expression may include, but are not limited to, signal peptide cleavage; disulfide bond formation; proper folding; carbohydrate addition and processing (e.g., N- and O-linked glycosylation); specific proteolytic cleavage; and / or assembly into a multimeric protein. The albumin fusion protein is preferably encoded by an unprocessed form of RNA having a signal peptide, particularly at its N-terminus, and after secretion by the cell, preferably exists in a processed form, particularly with the signal peptide cleaved. In the most preferred embodiment, “processed form of albumin fusion protein” refers to the albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as “mature albumin fusion protein.”

[0285] In preferred embodiments, albumin-fused proteins containing a therapeutic protein have higher plasma stability compared to the plasma stability of the same therapeutic protein when it is not fused to albumin. Plasma stability typically refers to the period from when the therapeutic protein is administered in vivo and transported into the bloodstream until it is broken down, removed from the bloodstream by organs such as the kidneys or liver, and ultimately removed from the body. Plasma stability is calculated with respect to the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

[0286] As used herein, “albumin” collectively refers to an albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activity) of albumin. In particular, “albumin” refers to human albumin or its fragments or variants, in particular the mature form of human albumin, or albumin or its fragments derived from other vertebrates, or variants of these molecules. Albumin may be derived from any vertebrate, in particular any mammal, such as humans, mice, cattle, sheep, or pigs. Non-mammalian albumins include, but are not limited to, hens and salmon. The albumin portion of an albumin fusion protein may be derived from a different animal than the therapeutic protein portion.

[0287] In certain embodiments, albumin is human serum albumin (HSA), or a fragment or variant thereof, as disclosed in, for example, U.S. Patent No. 5,876,969, International Publication No. 2011 / 124718, International Publication No. 2013 / 075066, and International Publication No. 2011 / 0514789.

[0288] The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and include human serum albumin (and its fragments and variants) as well as albumin (and its fragments and variants) from other species.

[0289] As used herein, an albumin fragment sufficient to prolong the therapeutic activity or plasma stability of a therapeutic protein refers to an albumin fragment of sufficient length or structure to stabilize or extend the therapeutic activity or plasma stability of a protein, such that the plasma stability of the therapeutic protein portion of an albumin fusion protein is prolonged or enhanced compared to its plasma stability in the unfused state.

[0290] The albumin portion of the albumin fusion protein may contain the full length of the albumin sequence, or one or more fragments thereof that can stabilize or extend therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids long, or may contain about 15, 20, 25, 30, 50 or more consecutive amino acids from the albumin sequence, or may contain part or all of a specific domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is a mature form of HSA.

[0291] Generally speaking, albumin fragments or variants are at least 100 amino acids long, preferably at least 150 amino acids long.

[0292] According to this disclosure, albumin may be naturally occurring albumin or a fragment or variant thereof. Albumin may be human albumin and may originate from any vertebrate, in particular any mammal.

[0293] Preferably, the albumin fusion protein contains albumin as the N-terminal portion and a therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein containing albumin as the C-terminal portion and a therapeutic protein as the N-terminal portion may also be used.

[0294] In one embodiment, a therapeutic protein(s) is bound to albumin via a peptide linker(s). The linker peptides between the fusion regions provide greater physical separation between the regions and thus can maximize the accessibility of the therapeutic protein region for binding, for example, to its homologous receptor. The linker peptides may be composed of amino acids so that they are flexible or more rigid. The linker sequence may be cleavable by proteases or chemically.

[0295] As used herein, the term “Fc region” refers to a portion of innate immunoglobulin formed by the Fc domains (or Fc portions) of each of the two heavy chains of innate immunoglobulin. As used herein, the term “Fc domain” refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain in which the Fc domain does not contain an Fv domain. In certain embodiments, the Fc domain begins in a hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain includes at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc domain includes at least one of the hinge (e.g., upper, middle, and / or lower hinge regions), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, the Fc domain includes a complete Fc domain (i.e., the hinge domain, the CH2 domain, and the CH3 domain). In certain embodiments, the Fc domain includes a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, the Fc domain includes a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain consists of a CH3 domain or a portion thereof. In certain embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In certain embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As used herein, an Fc domain generally refers to a polypeptide that includes all or part of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides that include the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides that include only, for example, the hinge, CH2, and CH3 domains.The Fc domain may originate from any species and / or any subtype of immunoglobulin, including but not limited to human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The Fc domain encompasses native Fc and Fc variant molecules. As described herein, it will be understood by those skilled in the art that any Fc domain may be modified so that its amino acid sequence differs from the native Fc domain of naturally occurring immunoglobulin molecules. In certain embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

[0296] The Fc domains of polypeptides described herein may be derived from different immunoglobulin molecules. For example, the Fc domain of a polypeptide may include CH2 and / or CH3 domains derived from the IgG1 molecule, as well as a hinge region derived from the IgG3 molecule. In another example, the Fc domain may include a chimeric hinge region that is partly derived from the IgG1 molecule and partly from the IgG3 molecule. In yet another example, the Fc domain may include a chimeric hinge that is partly derived from the IgG1 molecule and partly from the IgG4 molecule.

[0297] In certain embodiments, the extended PK group comprises an Fc domain or a fragment thereof, or a variant of an Fc domain or a fragment thereof (all of which are included in the term “Fc domain” for the purposes of this disclosure). The Fc domain does not contain a variable region that binds to an antigen. Fc domains suitable for use in this disclosure can be obtained from several different sources. In certain embodiments, the Fc domain is derived from human immunoglobulin. In certain embodiments, the Fc domain is derived from the human IgG1 constant region. However, it is understood that the Fc domain may be derived from immunoglobulins of other mammalian species, including, for example, rodent species (e.g., mouse, rat, rabbit, guinea pig) or non-human primate species (e.g., chimpanzee, macaque).

[0298] Furthermore, the Fc domain (or a fragment or variant thereof) may originate from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, as well as any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

[0299] Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains containing Fc domain sequences lacking specific effector function and / or possessing specific modifications that reduce immunogenicity can be selected. Many sequences of antibodies and antibody-coding genes are publicly available, and suitable Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using techniques widely accepted in the art.

[0300] In certain embodiments, the extended PK group is a serum albumin-binding protein, such as those described in U.S. Patent Application No. 2005 / 0287153, U.S. Patent Application No. 2007 / 0003549, U.S. Patent Application No. 2007 / 0178082, U.S. Patent Application No. 2007 / 0269422, U.S. Patent Application No. 2010 / 0113339, International Publication No. 2009 / 083804, and International Publication No. 2009 / 133208, which are incorporated herein by reference in their entirety. In certain embodiments, the extended PK group is transferrin, as disclosed in U.S. Patent No. 7,176,278 and U.S. Patent No. 8,158,579, which are incorporated herein by reference in their entirety. In certain embodiments, the extended PK group is a serum immunoglobulin-binding protein, such as those disclosed in U.S. Patent Application No. 2007 / 0178082, U.S. Patent Application No. 2014 / 0220017, and U.S. Patent Application No. 2017 / 0145062, which are incorporated herein by reference in their entirety. In certain embodiments, the extended PK group is a serum albumin-binding fibronectin (Fn)-based scaffold domain protein, such as those disclosed in U.S. Patent Application No. 2012 / 0094909, which are incorporated herein by reference in their entirety. A method for producing a fibronectin-based scaffold domain protein is also disclosed in U.S. Patent Application No. 2012 / 0094909. A non-limiting example of an Fn3-based extended PK group is Fn3(HSA), i.e., a human serum albumin-binding Fn3 protein.

[0301] In certain embodiments, an extended PK cytokine suitable for use according to this disclosure may use one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that links two or more domains in the linear amino acid sequence of a polypeptide chain (e.g., an extended PK portion and an IL portion such as IL2). For example, a peptide linker may be used to link a cytokine portion to an HSA domain.

[0302] For example, linkers suitable for fusing an extended PK group to IL2 are well known in the art. Exemplary linkers include glycine-serine polypeptide linkers, glycine-proline polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a glycine-serine polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0303] In addition to, or instead of, the heterologous polypeptides described herein may include sequences encoding a “marker” or “reporter.” Examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin B phosphotransferase (HPH), thymidine kinase (TK), β-galactosidase, and xanthine guanine phosphoribosyltransferase (XGPRT).

[0304] antigen The subject matter disclosed herein may include immune effector cells that can be generated in a subject by active immunization, for example, by vaccination using peptides described herein, or by passive immunization, for example, by administering immune effector cells having specific specificity, which can be genetically modified as described herein. Immune effector cells, in particular immune effector cells expressing antigen receptors, for example, immune effector cells genetically engineered to express antigen receptors, may be brought into contact with a congenital antigen molecule (also referred herein as “antigen targeted by antigen receptors,” “vaccine antigen,” or simply “antigen”) in a subject to be treated, the antigen molecule or its processing product, for example, a fragment thereof, which binds to an antigen receptor such as a TCR carried by the immune effector cell, in particular when presented by MHC. In one embodiment, the congenital antigen molecule includes an antigen or fragment thereof expressed by a target cell targeted by the immune effector cell, or a variant of the antigen or fragment.

[0305] Accordingly, the methods described herein include the step of administering a homogeneous antigen molecule, a nucleic acid encoding it, or cells expressing the homogeneous antigen molecule to a target. In one embodiment, the nucleic acid encoding the homogeneous antigen molecule is expressed in the target cells to provide the homogeneous antigen molecule. In one embodiment, the nucleic acid encoding the homogeneous antigen molecule is transiently expressed in the target cells. In one embodiment, the nucleic acid encoding the homogeneous antigen molecule is RNA. In one embodiment, the homogeneous antigen molecule or the nucleic acid encoding it is administered systemically. In one embodiment, after systemic administration of the nucleic acid encoding the homogeneous antigen molecule, expression of the nucleic acid encoding the homogeneous antigen molecule occurs in the spleen. In one embodiment, after systemic administration of the nucleic acid encoding the homogeneous antigen molecule, expression of the nucleic acid encoding the homogeneous antigen molecule occurs in antigen-presenting cells, preferably professional antigen-presenting cells. In one embodiment, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In one embodiment, after systemic administration of the nucleic acid encoding the homogeneous antigen molecule, expression of the nucleic acid encoding the homogeneous antigen molecule in the lungs and / or liver does not occur, or does not occur essentially. In one embodiment, after systemic administration of a nucleic acid encoding a congenital antigen molecule, the expression of the nucleic acid encoding the congenital antigen molecule in the spleen is at least five times higher than the expression level in the lungs. In one embodiment, the congenital antigen molecule is processed, and the processing product is presented in relation to the MHC for TCR recognition.

[0306] Peptides and protein antigens provided to a subject according to the present invention (by administering either peptides and protein antigens, or nucleic acids encoding peptides and protein antigens, particularly RNA, or cells expressing peptides and protein antigens), i.e., vaccine antigens, preferably result in stimulation, priming, and / or expansion of immune effector cells in the administered subject. The stimulated, primed, and / or expanded immune effector cells are preferably directed toward a target antigen, particularly a target antigen expressed by disease cells, tissues, and / or organs, i.e., disease-related antigens. Thus, vaccine antigens may comprise disease-related antigens, or fragments or variants thereof. In one embodiment, such fragments or variants are immunologically equivalent to disease-related antigens. In the context of this disclosure, the terms “antigen fragment” or “antigen variant” mean an agent that results in stimulation, priming, and / or expansion of immune effector cells, and the stimulated, primed, and / or expanded immune effector cells target the antigen, i.e., disease-related antigen, particularly when presented by disease cells, tissues, and / or organs. Therefore, a vaccine antigen may correspond to or contain a disease-related antigen, correspond to or contain a fragment of a disease-related antigen, or correspond to or contain an antigen homologous to a disease-related antigen or a fragment thereof. If a vaccine antigen contains a fragment of a disease-related antigen or an amino acid sequence homologous to a fragment of a disease-related antigen, the fragment or amino acid sequence may contain an epitope of the disease-related antigen targeted by the antigen receptor of an immune effector cell or a sequence homologous to an epitope of a disease-related antigen. Therefore, according to this disclosure, a vaccine antigen may contain an immunogenic fragment of a disease-related antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-related antigen. The “immunogenic fragment of an antigen” as used in this disclosure preferably relates to a fragment of an antigen that can stimulate, prime, and / or expand immune effector cells or cells expressing an antigen that carry an antigen receptor that binds to the antigen.The vaccine antigen (similar to a disease-related antigen) preferably provides an associated epitope for binding by antigen receptors present on immune effector cells. In one embodiment, the vaccine antigen (similar to a disease-related antigen) is expressed by cells such as antigen-presenting cells in relation to MHC and expressed on the cell surface in order to provide an associated epitope for binding by immune effector cells. The vaccine antigen may be a recombinant antigen.

[0307] In one embodiment of all aspects of the present invention, a nucleic acid encoding a vaccine antigen is expressed in a target cell to provide an antigen or a processing product thereof for binding by an antigen receptor expressed by an immune effector cell, the binding resulting in stimulation, priming, and / or expansion of the immune effector cell.

[0308] The term “immunologically equivalent” means that immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, exhibit the same or essentially the same immunological properties and / or exert the same or essentially the same immunological effects, for example, with respect to the type of immunological effect. In the context of this disclosure, the term “immunologically equivalent” is preferably used with respect to the immunological effects or properties of an antigen or antigen variant used for immunization. For example, if an amino acid sequence induces an immune response that has specificity to react with a reference amino acid sequence when exposed to a target immune system, such as T cells that bind to a reference amino acid sequence or cells that express a reference amino acid sequence, then the amino acid sequence is immunologically equivalent to the reference amino acid sequence. Thus, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties and / or exerts the same or essentially the same effects with respect to the stimulation, priming, and / or expansion of T cells with respect to the antigen targeted by the T cell.

[0309] As used herein, “activation” or “stimulation” may refer to a state of immune effector cells, such as T cells, that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with the initiation of signaling pathways, induction of cytokine production, and detectable effector function. The term “activated immune effector cells” refers, among other things, to immune effector cells undergoing cell division.

[0310] The term "priming" refers to the process by which immune effector cells, such as T cells, first come into contact with their specific antigens, triggering differentiation into effector cells, such as effector T cells.

[0311] The terms “clonal expansion” or “expansion” refer to the process by which a particular entity increases. In the context of this disclosure, the terms are preferably used in relation to an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and specific lymphocytes that recognize the antigen are amplified. Preferably, clonal expansion results in the differentiation of lymphocytes.

[0312] The term "antigen" refers to an active substance containing an epitope that can elicit an immune response. The term "antigen" particularly includes proteins and peptides. In one embodiment, an antigen is presented on the surface of an immune system cell, such as an antigen-presenting cell like a dendritic cell or macrophage. An antigen or its processing product, such as a T cell epitope, is bound by an antigen receptor in one embodiment. Thus, an antigen or its processing product can react specifically with immune effector cells, such as T lymphocytes (T cells). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen.

[0313] 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 containing an epitope that stimulates the host's immune system to produce a cellular antigen-specific immune response and / or humoral antibody response to the disease. Therefore, disease-associated antigens or their epitopes may be used for therapeutic purposes. Disease-associated antigens may be associated with microorganisms, typically infections caused by microbial antigens, or with cancer, typically tumors.

[0314] The term “tumor antigen” or “tumor-associated antigen” refers to components of cancer cells that may originate from the cytoplasm, cell surface, and cell nucleus. In particular, the term refers to antigens produced intracellularly or as surface antigens on tumor cells. Tumor antigens are typically selectively expressed by cancer cells (e.g., expressed at higher levels in cancer cells than in non-cancerous cells), and in some cases, expressed only by cancer cells. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, claudin family cell surface proteins such as claudin 6, claudin 18.2, and claudin 12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, and Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan A, MC1R, Myosin / m, MUC This includes 1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, Proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, Survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, and WT-1.

[0315] In preferred embodiments, the antigens are tumor-associated antigens such as NY-ESO-1, MAGE-A3, tyrosinase, and KRAS, respectively, and the present invention comprises stimulating an antitumor CTL response against malignant cells that express such tumor-associated antigens, preferably presenting such tumor-associated antigens together with class I MHC.

[0316] NY-ESO-1 is a cancer / testicular antigen that is expressed only in normal adult testicular germ cells in normal adult tissues and in various cancers. It induces specific humoral and cellular immunity in patients with NY-ESO-1 expressing cancers.

[0317] The term "NY-ESO-1" preferably refers to human NY-ESO-1, and more particularly to a protein comprising the amino acid sequence described in Sequence ID No. 1 of the sequence listing or a variant of said amino acid sequence.

[0318] In various aspects of the present invention, whenever NY-ESO-1, particularly SEQ ID NO: 1, the epitope sequence of NY-ESO-1, particularly SEQ ID NOs: 39, 40, 41, 42, and 101, respectively, or T cell receptor sequences specific to NY-ESO-1, particularly SEQ ID NOs: 5-22, 91, and 92, are involved, the objective is preferably to induce an immune response against malignant cells expressing NY-ESO-1, preferably characterized by the presentation of NY-ESO-1, and / or to treat or prevent malignant diseases involving cells expressing NY-ESO-1. Preferably, the immune response includes stimulating an anti-NY-ESO-1 CTL response against malignant cells expressing NY-ESO-1, preferably presenting NY-ESO-1 together with class I MHC.

[0319] MAGE-A3 is melanoma-associated antigen 3 (MAGE-A3). MAGE-A3 is a tumor-specific protein and has been identified in many tumors, particularly melanoma, non-small cell lung cancer, and hematological malignancies.

[0320] The term "MAGE-A3" preferably refers to human MAGE-A3, and more particularly to a protein comprising the amino acid sequence described in Sequence ID No. 2 of the sequence listing or a variant of said amino acid sequence.

[0321] In various aspects of the present invention, whenever MAGE-A3, particularly SEQ ID NO: 2, the epitope sequence of MAGE-A3, particularly SEQ ID NOs: 43 and 44, respectively, or MAGE-A3-specific T cell receptor sequences, particularly SEQ ID NOs: 23-34, are involved, the objective is preferably to induce an immune response against malignant cells expressing MAGE-A3, preferably characterized by presentation of MAGE-A3, and / or to treat or prevent malignant diseases involving cells expressing MAGE-A3. Preferably, the immune response comprises stimulating an anti-MAGE-A3 CTL response against malignant cells expressing MAGE-A3, preferably presenting MAGE-A3 together with class I MHCs.

[0322] Tyrosinase is an oxidase, the rate-limiting enzyme that controls melanin production. Normally, tyrosinase is produced in small amounts, but its levels are significantly elevated in melanoma cells.

[0323] The term "tyrosinase" preferably refers to human tyrosinase, and more particularly to a protein comprising the amino acid sequence described in Sequence ID No. 3 of the sequence listing or a variant thereof.

[0324] According to various aspects of the present invention, whenever tyrosinase, particularly SEQ ID NO: 3, a tyrosinase epitope sequence, or a tyrosinase-specific T cell receptor sequence, particularly SEQ ID NOs: 35-38, is involved, the objective is preferably to induce an immune response against malignant cells expressing tyrosinase, preferably characterized by tyrosinase presentation, and / or to treat or prevent malignant diseases involving tyrosinase-expressing cells. Preferably, the immune response comprises stimulating an anti-tyrosinase CTL response against malignant cells expressing tyrosinase, preferably presenting tyrosinase together with class I MHC.

[0325] The term "TPTE" refers to a "tensin homology transmembrane phosphatase." The term "TPTE" preferably refers to human TPTE, in particular a protein comprising the amino acid sequence described in Sequence ID No. 4 of the sequence listing or a variant thereof.

[0326] In contrast to TPTE expression in healthy tissues, which is limited to the testes and where transcript levels are below the detection limit in all other normal tissue specimens, TPTE expression is found across a variety of cancer types, including malignant melanoma, breast cancer, lung cancer, prostate cancer, ovarian cancer, renal cell carcinoma, and cervical cancer.

[0327] TPTE transcription is initiated during malignant transformation mediated by cancer-associated DNA hypomethylation. Furthermore, TPTE promotes cancer progression and the metastatic spread of cancer cells. In particular, TPTE is essential for efficient chemotaxis, a process involved in multiple aspects of cancer progression, including cancer invasion and metastasis, which affect the homing and metastatic sites of cancer cells. TPTE expression in primary tumors is associated with a significantly higher rate of metastatic disease.

[0328] In various aspects of the present invention, whenever TPTE, particularly SEQ ID NO: 4, the epitope sequence of TPTE, or a T cell receptor sequence specific to TPTE is involved, the objective is preferably to induce an immune response against malignant cells expressing TPTE, preferably characterized by TPTE presentation, and / or to treat or prevent malignant diseases involving cells expressing TPTE. Preferably, the immune response includes stimulating an anti-TPTE CTL response against malignant cells expressing TPTE, preferably presenting TPTE together with class I MHC.

[0329] KRAS is a protein that is part of the RAS / MAPK pathway. This protein sends signals from outside the cell to the cell nucleus, directing the cell to proliferate or differentiate. The KRAS protein is a GTPase and was first identified as an oncogene in Kirsten rat sarcoma virus. The KRAS gene is a proto-oncogene.

[0330] The term "KRAS" preferably refers to human KRAS, particularly the amino acid sequence described in Sequence ID No. 90 of the sequence listing, or a variant of said amino acid sequence, in particular a variant of said amino acid sequence in which the glutamine at position 61 is replaced by histidine (Q61H).

[0331] In various aspects of the present invention, whenever KRAS, particularly SEQ ID NO: 90 or SEQ ID NO: 90(Q61H), an epitope sequence of KRAS, particularly SEQ ID NO: 102, or a T cell receptor sequence specific to KRAS, particularly SEQ ID NOs: 93-100, is involved, the objective is preferably to induce an immune response against malignant cells expressing KRAS, preferably characterized by KRAS presentation, and / or to treat or prevent malignant diseases involving KRAS-expressing cells. Preferably, the immune response includes stimulating an anti-KRAS CTL response against malignant cells expressing KRAS, preferably presenting KRAS together with class I MHC.

[0332] The antigen sequences described above include any variants of the sequences, particularly mutants, splice variants, conformational variants, isoform variants, allele variants, species variants, and species homologs, especially those occurring in nature. Allele variants are associated with normal sequence changes in a gene, and their significance is often unclear. Complete gene sequencing often identifies numerous allele variants for a given gene. Species homologs are nucleic acids or amino acid sequences originating from a different species than that of a given nucleic acid or amino acid sequence. The terms “NY-ESO-1,” “MAGE-A3,” “tyrosinase,” “TPTE,” and “KRAS” shall encompass (i) splice variants, (ii) post-translational modification variants, including variants with different glycosylation, particularly N-glycosylation states, (iii) conformational variants, and (iv) disease-related and non-disease-related variants. Preferably, "NY-ESO-1", "MAGE-A3", "tyrosinase", "TPTE", or "KRAS" exist in their native conformation.

[0333] "Target cells" include cells that are targets of immune responses, such as cellular immune responses. Target cells include cells that present antigens or antigen epitopes, i.e., peptide fragments derived from antigens, and include undesirable cells such as malignant cells as described herein. In preferred embodiments, target cells are cells that express the antigens described herein, and preferably present the antigens together with class I MHC.

[0334] The terms “expressed on the cell surface” or “associated with the cell surface” mean that a molecule, such as a receptor, is positioned in association with the cell’s plasma membrane, with at least a portion of the molecule facing the extracellular space of the cell and accessible from the outside of the cell, for example, by an antibody located outside the cell. In this context, the portion is preferably at least four, preferably at least eight, preferably at least twelve, and more preferably at least twenty amino acids. Association can be direct or indirect. For example, association may be by one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, saccharide, or other structure that may be found on the outer layer of the cell’s plasma membrane. For example, a molecule associating with the cell surface may be a transmembrane protein having an extracellular portion, or a protein that associates with the cell surface by interacting with another protein that is a transmembrane protein.

[0335] "Cell surface" or "surface of a cell" is used according to its common meaning in the art and therefore includes the outside of the cell accessible by binding by proteins and other molecules. An antigen is expressed on the surface of a cell if it is located on the cell surface and accessible by binding by, for example, an antigen-specific antibody applied to the cell. In one embodiment, an antigen receptor expressed on the surface of a cell is an intrinsic membrane protein having an extracellular portion that recognizes the antigen or its processing product.

[0336] In the context of the present invention, the terms “exodomain” refer to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is accessible from the outside of the cell, preferably by binding to a molecule such as an antibody located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0337] The term “epitope” refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope may be recognized by T cells, B cells, or antibodies. An antigen epitope may consist of a continuous or discontinuous portion of the antigen and may be about 5 to about 100, for example, about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length. For example, an epitope may preferably be 8, 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, the epitope is about 10 to about 25 amino acids in length. The term “epitope” includes T cell epitopes.

[0338] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in association with an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or disease cells in immune responses, and MHC proteins or molecules bind to peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the bound peptide is typically about 8 to 10 amino acids long, but longer or shorter peptides may also be effective. For class II MHC / peptide complexes, the bound peptide is typically about 10 to 25 amino acids long, particularly about 13 to 18 amino acids long, but longer and shorter peptides may also be effective.

[0339] Preferably, antigen peptides disclosed herein, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-44, 101, and 102, or a variant of said amino acid sequence, can stimulate an immune response, preferably a cellular response, to cells characterized by the expression of an antigen from which they originate, and preferably by the presentation of an antigen. Preferably, antigen peptides can stimulate a cellular response to cells characterized by presenting an antigen together with class I MHC, and preferably can stimulate antigen-responsive CTLs. Preferably, the antigen peptides according to the present invention are MHC class I and / or class II presenting peptides, or can be processed to produce MHC class I and / or class II presenting peptides. Preferably, the sequence that binds to an MHC molecule is selected from SEQ ID NOs: 39-44, 101, and 102.

[0340] When the antigen peptide is presented directly, i.e., without processing and especially without cleavage, the antigen peptide has a length suitable for binding to MHC molecules, particularly class I MHC molecules, preferably 7 to 20 amino acids long, more preferably 7 to 12 amino acids long, more preferably 8 to 11 amino acids long, and especially 8, 9, or 10 amino acids long. Preferably, the sequence of the directly presented antigen peptide substantially corresponds to, and preferably is exactly identical to, a sequence selected from SEQ ID NOs: 39 to 44, 101, and 102.

[0341] When the antigen peptide is presented after processing, particularly after cleavage, the peptide produced by processing has a length suitable for binding to MHC molecules, particularly class I MHC molecules, preferably 7 to 20 amino acids long, more preferably 7 to 12 amino acids long, more preferably 8 to 11 amino acids long, particularly 8, 9, or 10 amino acids long. Preferably, the sequence of the peptide presented after processing substantially corresponds to, and preferably is completely identical to, a sequence selected from SEQ ID NOs: 39 to 44, 101, and 102. Thus, in one embodiment, the antigen peptide according to the present invention comprises a sequence selected from SEQ ID NOs: 39 to 44, 101, and 102, and after processing of the antigen peptide, a sequence selected from SEQ ID NOs: 39 to 44, 101, and 102 is formed.

[0342] Peptides having an amino acid sequence substantially corresponding to the sequence of a peptide presented by an MHC molecule may differ in one or more residues that are not essential for TCR recognition of the peptide presented by the MHC or for peptide binding to the MHC. Such substantially corresponding peptides can also preferably stimulate antigen-specific cellular responses, such as antigen-specific CTLs. Peptides having an amino acid sequence different from the presented peptide in which residues do not affect TCR recognition but improve the stability of binding to MHC can improve the immunogenicity of the antigen peptide and may be referred to herein as “optimized peptides.” A reasonable approach to designing substantially corresponding peptides can be employed using existing knowledge of which of these residues is more likely to affect binding to either MHC or TCR. The resulting functional peptide is intended as an antigen peptide. The sequences described above are encompassed by the term “variant” as used herein.

[0343] Cells such as antigen-presenting cells can be loaded with MHC class I-presenting peptides by exposing the cells to the peptide, i.e., by pulsing them with the peptide, or by transduction into cells with a nucleic acid, preferably RNA, that encodes a peptide or protein containing the peptide to be presented, such as a nucleic acid encoding an antigen.

[0344] In some embodiments, the present invention includes antigen-presenting cells loaded with an antigen peptide. In this regard, protocols may be based on in vitro culture / differentiation of dendritic cells engineered to artificially present an antigen peptide. The preparation of genetically engineered dendritic cells may involve introducing nucleic acids encoding an antigen or antigen peptide into dendritic cells. Transfection of dendritic cells with mRNA is a promising antigen loading technique that stimulates potent antitumor immunity. Such transfection can be performed ex vivo, and a pharmaceutical composition containing such transfected cells can then be used for therapeutic purposes. Alternatively, a gene delivery vehicle targeting dendritic cells or other antigen-presenting cells may be administered to a patient to result in transfection occurring in vivo. In vivo and ex vivo transfection of dendritic cells can generally be carried out using any method known in the art, such as described in, for example, International Publication No. 97 / 24447, or using the gene gun approach described in Mahvi et al., Immunology and Cell Biology 75:456-460, 1997. Antigen loading of dendritic cells can be achieved by incubating dendritic cells or progenitor cells with an antigen, DNA (naked or in a plasmid vector), or RNA, or with recombinant bacteria or viruses expressing the antigen (e.g., vaccinia virus, fowlpox virus, adenovirus, or lentiviral vector).

[0345] Peptides and protein antigens may be 2 to 100 amino acids long, for example, containing 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the peptide may contain more than 50 amino acids. In some embodiments, the peptide may contain more than 100 amino acids.

[0346] According to the present invention, the vaccine antigen must be recognizable by immune effector cells. Preferably, when the antigen is recognized by immune effector cells, in the presence of appropriate co-stimulatory signals, it can induce stimulation, priming, and / or expansion of immune effector cells carrying antigen receptors that recognize the antigen. In relation to embodiments of the present invention, the antigen is preferably presented on the surface of a cell, preferably an antigen-presenting cell. Recognition of the antigen on the surface of diseased cells can result in an immune response against the antigen (or the cell expressing the antigen).

[0347] chemotherapy In certain embodiments, further treatments may be administered to the patient in combination with the treatments described herein. Such further treatments include classic cancer treatments, such as radiotherapy, surgery, hyperthermia, and / or chemotherapy.

[0348] Chemotherapy is a type of cancer treatment that typically uses one or more anticancer drugs (chemotherapeutic agents) as part of a standardized chemotherapy regimen. The term chemotherapy has come to imply the nonspecific use of intracellular toxins to inhibit mitosis. This implied exclusion of more selective drugs that block extracellular signals (signaling). The development of therapies with specific molecular or gene targets that inhibit growth-promoting signals from classical endocrine hormones (primarily estrogen for breast cancer and androgens for prostate cancer) is now called hormone therapy. In contrast, other inhibitions of growth signals, such as those related to receptor tyrosine kinases, are referred to as targeted therapies.

[0349] Importantly, the use of drugs (whether chemotherapy, hormone therapy, or targeted therapy) constitutes systemic therapy for cancer in that they are introduced into the bloodstream and, in principle, can address cancer at any anatomical location within the body. Systemic therapy is often used in combination with other modalities that constitute local therapy for cancer (i.e., treatments whose effectiveness is limited to the anatomical area to which it is applied), such as radiotherapy, surgery, or hyperthermia.

[0350] Traditional chemotherapy agents are cytotoxic by interfering with cell division (mitosis), but cancer cells vary greatly in their sensitivity to these drugs. For the most part, chemotherapy can be thought of as a way to damage or stress cells, and if apoptosis is initiated, it can lead to cell death.

[0351] Chemotherapy agents include alkylating agents, antimetabolites, antimicrotubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.

[0352] Alkylating agents have the ability to alkylate many molecules, including proteins, RNA, and DNA. Subtypes of alkylating agents include nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatins and their derivatives, as well as non-classical alkylating agents. Nitrogen mustards include mechloretamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, and busulfan. Nitrosoureas include N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin. Tetrazines include dacarbazine, mitozolomide, and temozolomide. Aziridines include thiotepa, mitomycin, and diazicone (AZQ). Cisplatins and their derivatives include cisplatin, carboplatin, and oxaliplatin. These impair cellular function by forming covalent bonds with amino groups, carboxyl groups, sulfhydryl groups, and phosphate groups in biologically important molecules. Non-classical alkylating agents include procarbazine and hexamethylmelamine. In one particularly preferred embodiment, the alkylating agent is cyclophosphamide.

[0353] Antimetabolites are a group of molecules that interfere with the synthesis of DNA and RNA. Many of them have structures similar to the building blocks of DNA and RNA. Antimetabolites are similar to either nucleic acid bases or nucleosides, but have altered chemical groups. These drugs exert their effects by blocking enzymes necessary for DNA synthesis or by being incorporated into DNA or RNA. Subtypes of antimetabolites include folate antagonists, fluoropyrimidines, deoxynucleoside analogs, and thiopurines. Folate antagonists include methotrexate and pemetrexed. Fluoropyrimidines include fluorouracil and capecitabine. Deoxynucleoside analogs include cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nerarabine, cladribine, clofarabine, and pentostatin. Thiopurines include thioguanine and mercaptopurine.

[0354] Antimicrotubule agents inhibit cell division by interfering with microtubule function. Vinca alkaloids inhibit microtubule formation, while taxanes inhibit microtubule degradation. Vinca alkaloids include vinorelbine, vindesine, and vinflunine. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0355] Topoisomerase inhibitors are drugs that affect the activity of two enzymes: topoisomerase I and topoisomerase II, and include irinotecan, topotecan, camptothecin, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, melbaron, and acralubicin.

[0356] Cytotoxic antibiotics are a diverse group of drugs with various mechanisms of action. A common theme they share in their chemotropic applications is the disruption of cell division. The most important subgroups are anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, and acralubicin) as well as bleomycin; other notable examples include mitomycin C, mitoxantrone, and actinomycin.

[0357] In one embodiment, lymphocyte depletion therapy may be applied by administering, for example, cyclophosphamide and fludarabine before administering immune effector cells. Such therapy may increase cell persistence as well as the incidence and duration of clinical responses.

[0358] Immune checkpoint inhibitors In certain embodiments, immune checkpoint inhibitors are used in combination with other therapeutic agents described herein.

[0359] As used herein, “immune checkpoint” refers to co-stimulatory and inhibitory signals that modulate the magnitude and quality of antigen recognition by T cell receptors. 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. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 that replaces CD28 binding. In certain embodiments, the inhibitory signal is the interaction between LAG3 and MHC class II molecules. In certain embodiments, the inhibitory signal is the interaction between TIM3 and galectin 9.

[0360] As used herein, “immune checkpoint inhibitor” refers to a molecule that completely or partially reduces, inhibits, interferes with or modulates one or more checkpoint proteins. In certain embodiments, an immune checkpoint inhibitor prevents an inhibitory signal associated with an immune checkpoint. In certain embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that interferes with an inhibitory signaling signal associated with an immune checkpoint. In certain embodiments, an immune checkpoint inhibitor is a small molecule that interferes with an inhibitory signaling signal. In certain embodiments, an immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that prevents interactions between checkpoint blocking proteins, for example, an antibody or fragment thereof that interferes with the interaction between PD-1 and PD-L1. In certain embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that interferes with the interaction between CTLA-4 and CD80 or CD86. In certain embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that interferes with the interaction between LAG3 and its ligand, or between TIM-3 and its ligand. A checkpoint inhibitor may also be in the form of a soluble form of the molecule (or its variant) itself, for example, a soluble PD-L1 or PD-L1 fusion.

[0361] The “programmed death 1 (PD-1)” receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term “PD-1” includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs having at least one common epitope with hPD-1.

[0362] "Programmed death ligand 1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2) that, upon binding to PD-1, downregulate T cell activation and cytokine secretion. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs having at least one common epitope with hPD-L1.

[0363] Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a T cell surface molecule and a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 and CD86. As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs having at least one common epitope with hCTLA-4.

[0364] Lymphocyte Activator Gene 3 (LAG3) is an inhibitory receptor associated with inhibiting lymphocyte activity by binding to MHC class II molecules. This receptor enhances the function of Treg cells and CD8 + It inhibits the function of effector T cells. As used herein, the term "LAG3" includes human LAG3 (hLAG3), variants, isoforms, and species homologs of hLAG3, as well as analogs having at least one common epitope.

[0365] T cell membrane protein 3 (TIM3) is an inhibitory receptor involved in inhibiting lymphocyte activity by inhibiting the TH1 cell response. Its ligand is galectin 9, which is upregulated in various types of cancer. As used herein, the term "TIM3" includes human TIM3 (hTIM3), variants, isoforms, and species homologs of hTIM3, as well as analogs having at least one common epitope.

[0366] The "B7 family" refers to undefined receptor inhibitory ligands. The B7 family includes B7-H3 and B7-H4, both of which are upregulated in tumor cells and tumor-infiltrating cells.

[0367] In certain embodiments, immune checkpoint inhibitors suitable for use in the methods disclosed herein are antibodies targeting inhibitory signal antagonists, such as PD-1, PD-L1, CTLA-4, LAG3, B7-H3, B7-H4, or TIM3. These ligands and receptors are reviewed in Pardoll, D., Nature. 12:252-264, 2012.

[0368] In certain embodiments, an immune checkpoint inhibitor is an antibody or its antigen-binding moiety that interferes with or inhibits signaling from inhibitory immunomodulators. In certain embodiments, an immune checkpoint inhibitor is a small molecule that interferes with or inhibits signaling from inhibitory immunomodulators.

[0369] In certain embodiments, inhibitory immunomodulators are components of the PD-1 / PD-L1 signaling pathway. Therefore, certain embodiments of the present disclosure provide targeted administration of an antibody or its antigen-binding moiety that interferes with the interaction between the PD-1 receptor and its ligand, PD-L1. Antibodies that bind to PD-1 and interfere with the interaction between PD-1 and its ligand, PD-L1, are known in the art. In certain embodiments, the antibody or its antigen-binding moiety specifically binds to PD-1. In certain embodiments, the antibody or its antigen-binding moiety specifically binds to PD-L1, inhibiting its interaction with PD-1 and thereby increasing immune activity.

[0370] In certain embodiments, inhibitory immunomodulators are components of the CTLA4 signaling pathway. Accordingly, certain embodiments of the present disclosure provide target administration of antibodies or their antigen-binding moieties that target CTLA4 and interfere with its interaction with CD80 and CD86.

[0371] In certain embodiments, inhibitory immunomodulators are components of the LAG3 (lymphocyte activator gene 3) signaling pathway. Accordingly, certain embodiments of the present disclosure provide targeted administration of antibodies or their antigen-binding moieties that target LAG3 and interfere with its interaction with MHC class II molecules.

[0372] In certain embodiments, inhibitory immunomodulators are components of the B7 family signaling pathway. In certain embodiments, the B7 family members are B7-H3 and B7-H4. Accordingly, certain embodiments of this disclosure provide targeted administration of antibodies or their antigen-binding moieties that target B7-H3 or B7-H4. Although the B7 family does not have defined receptors, these ligands are upregulated in tumor cells or tumor-infiltrating cells. Preclinical mouse models have shown that blocking these ligands can enhance anti-tumor immunity.

[0373] In certain embodiments, inhibitory immunomodulators are components of the TIM3 (T cell membrane protein 3) signaling pathway. Accordingly, certain embodiments of the present disclosure provide targeted administration of an antibody or its antigen-binding moiety that targets TIM3 and interferes with its interaction with galectin 9.

[0374] It will be understood by those skilled in the art that other immune checkpoint targets can also be targeted by antagonists or antibodies, provided that the targeting results in the stimulation of an immune response, such as an antitumor immune response, which may be reflected in increased T cell proliferation, enhanced T cell activation, and / or increased cytokine production (e.g., IFN-γ, IL-2).

[0375] RNA targeting According to the present invention, the peptides, proteins, or polypeptides described herein, particularly the vaccine antigens, are especially preferably administered in the form of RNA encoding the peptides, proteins, or polypeptides described herein. In one embodiment, the different peptides, proteins, or polypeptides described herein are encoded by different RNA molecules.

[0376] In one embodiment, RNA is formulated into a delivery vehicle. In one embodiment, the delivery vehicle contains particles. In one embodiment, the delivery vehicle contains at least one lipid. In one embodiment, the at least one lipid contains at least one cationic lipid. In one embodiment, the lipid forms a complex with the RNA and / or encapsulates the RNA. In one embodiment, the lipid is contained in a vesicle that encapsulates the RNA. In one embodiment, the RNA is formulated into liposomes.

[0377] According to this disclosure, after administration of the RNA described herein, at least a portion of the RNA is delivered to target cells. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cells. In one embodiment, the RNA is translated by the target cells to produce an encoded peptide or protein.

[0378] Some aspects of this disclosure include targeted delivery of RNA disclosed herein (e.g., RNA encoding a vaccine antigen).

[0379] In one embodiment, the disclosure includes targeting the lymphatic system, particularly secondary lymphoid organs, and more specifically the spleen. Targeting the lymphatic system, particularly secondary lymphoid organs, and more specifically the spleen is particularly preferred when the administered RNA is RNA encoding a vaccine antigen.

[0380] In one embodiment, the target cells are spleen cells. In one embodiment, the target cells are antigen-presenting cells such as professional antigen-presenting cells in the spleen. In one embodiment, the target cells are dendritic cells in the spleen.

[0381] The lymphatic system is part of the circulatory system and is an important part of the immune system, including a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphoid organs, a network of lymphatic vessel conductions, and circulating lymph. Primary or central lymphoid organs produce lymphocytes from immature progenitor cells. The thymus and bone marrow constitute primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphocytes and initiate adaptive immune responses.

[0382] RNA can be delivered to the spleen by so-called lipoplex formulations, in which RNA is bound to liposomes containing cationic lipids and optionally further lipids or helper lipids to form an injectable nanoparticle formulation. Liposomes can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. RNA lipoplex particles targeting the spleen are described in International Publication No. 2013 / 143683, incorporated herein by reference. It has been found that RNA lipoplex particles having a net negative charge can be used to selectively target splenic tissue or splenic cells, such as antigen-presenting cells, particularly dendritic cells. Thus, RNA accumulation and / or RNA expression occur in the spleen after administration of RNA lipoplex particles. Therefore, the RNA lipoplex particles of this disclosure can be used to express RNA in the spleen. In one embodiment, RNA accumulation and / or RNA expression in the lungs and / or liver does not occur or is essentially absent after administration of RNA lipoplex particles. In one embodiment, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen, after administration of RNA lipoplex particles. Thus, the RNA lipoplex particles of this disclosure can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0383] In the context of this disclosure, the term “RNA lipoplex particles” refers to particles comprising lipids, particularly cationic lipids, and RNA. Such cationic lipids are described above. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in complexation and the spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using cationic lipids such as DOTMA and further lipids such as DOPE. In one embodiment, the RNA lipoplex particles are nanoparticles.

[0384] Further lipids may be incorporated to adjust the overall positive-to-negative charge ratio and physical stability of the RNA lipoplex particles. Such further lipids are described above. In certain embodiments, the further lipids are neutral lipids. As used herein, “neutral lipids” refers to lipids having a net charge of zero. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalin, cholesterol, and cerebrosides. In certain embodiments, the further lipids are DOPE, cholesterol, and / or DOPC.

[0385] In certain embodiments, the RNA lipoplex particles contain both cationic lipids and further lipids. In exemplary embodiments, the cationic lipid is DOTMA and the further lipid is DOPE.

[0386] In some embodiments, the molar ratio of at least one cationic lipid to at least one further lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In certain embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one further lipid is about 2:1.

[0387] In one embodiment, the RNA lipoplex particles described herein have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, RNA lipoplex particles have an average diameter of approximately 200 nm, approximately 225 nm, approximately 250 nm, approximately 275 nm, approximately 300 nm, approximately 325 nm, approximately 350 nm, approximately 375 nm, approximately 400 nm, approximately 425 nm, approximately 450 nm, approximately 475 nm, approximately 500 nm, approximately 525 nm, approximately 550 nm, approximately 575 nm, approximately 600 nm, approximately 625 nm, approximately 650 nm, approximately 700 nm, approximately 725 nm, approximately 750 nm, approximately 775 nm, approximately 800 nm, approximately 825 nm, approximately 850 nm, approximately 875 nm, approximately 900 nm, approximately 925 nm, approximately 950 nm, approximately 975 nm, or approximately 1000 nm. In one embodiment, RNA lipoplex particles have an average diameter in the range of approximately 250 nm to approximately 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter in the range of about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0388] The charge of the RNA lipoplex particles of this disclosure is the sum of the charges present in at least one cationic lipid and the charges present in the RNA. The charge ratio is the ratio of the positive charges present in at least one cationic lipid to the negative charges present in the RNA. The charge ratio of the positive charges present in at least one cationic lipid to the negative charges present in the RNA is calculated by the following formula: Charge ratio = [(concentration of cationic lipid (mol)) * (total number of positive charges in cationic lipid)] / [(concentration of RNA (mol)) * (total number of negative charges in RNA)].

[0389] The spleen-targeting RNA lipoplex particles described herein at physiological pH preferably have a net negative charge, such as a positive-to-negative charge ratio of about 1.9:2 to about 1:2. In certain embodiments, the positive-to-negative charge ratio in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

[0390] RNA delivery systems have a unique selectivity for the liver. This is related to lipid-based particles, cationic and neutral nanoparticles, and especially lipid nanoparticles such as liposomes, nanomicelles, and lipophilic ligands in bioconjugates. Hepatic accumulation is caused by the discontinuous nature of the hepatic vascular system or lipid metabolism (liposomes and lipid or cholesterol conjugates).

[0391] In one embodiment of targeted delivery of cytokines such as IL2, the target organ is the liver, and the target tissue is liver tissue. Delivery to such target tissue is particularly preferred when the presence of cytokines in this organ or tissue is desirable, and / or when the expression of large amounts of cytokines is desirable, and / or when the systemic presence of cytokines, especially in significant amounts, is desirable or required.

[0392] In one embodiment, the RNA encoding the cytokine is administered in a formulation for targeting the liver. Such formulations are described herein above.

[0393] For in vivo delivery of RNA to the liver, drug delivery systems can be used to transport RNA to the liver by preventing its degradation. For example, polyplex nanomicelles, consisting of a poly(ethylene glycol) (PEG) coated surface and an mRNA-containing core, are a useful system because the nanomicelles provide excellent in vivo stability of RNA under physiological conditions. Furthermore, the stealth properties provided by the polyplex nanomicelle surface, composed of dense PEG palisades, effectively evade the host's immune defenses.

[0394] Pharmaceutical composition Nucleic acids, nucleic acid particles, peptides, proteins, polypeptides, RNA, RNA particles, immune effector cells, and further agents, such as immune checkpoint inhibitors, described herein may be administered in the form of any suitable pharmaceutical composition, which may include a pharmaceutically acceptable carrier and optionally include one or more adjuvants, stabilizers, etc. In one embodiment, the pharmaceutical composition is for use in the treatment or prevention of diseases involving the antigens described herein, such as cancerous diseases, for example, as described herein.

[0395] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably together with a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations. In connection with this disclosure, pharmaceutical compositions include nucleic acids, nucleic acid particles, peptides, proteins, polypeptides, RNA, RNA particles, immune effector cells, and / or further agents as described herein.

[0396] The pharmaceutical compositions of this disclosure may comprise one or more adjuvants, or may be administered together with one or more adjuvants. The term “adjuvant” relates to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a group of heterogeneous compounds such as oil emulsions (e.g., Freund’s adjuvants), inorganic compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune stimuli complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, and chemokines. Cytokines may include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL15, IFNα, IFNγ, GM-CSF, and LT-a. Further known adjuvants include aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA51. Other suitable adjuvants for use in this disclosure include lipopeptides such as Pam3Cys.

[0397] The pharmaceutical compositions described herein are generally applied in terms of "pharmaceutically effective amounts" and "pharmaceutically acceptable formulations."

[0398] The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interact with the action of the active ingredient in a pharmaceutical composition.

[0399] The terms “pharmaceutical effective dose” or “therapeutic effective dose” refer to the amount obtained alone or in combination with additional doses to achieve the desired response or effect. In the case of treating a particular disease, the desired response preferably relates to inhibiting the course of the disease. This includes slowing the progression of the disease, in particular interrupting or reversing its progression. The desired response in the treatment of a disease may also be the delay or prevention of the onset of the disease or condition. The effective dose of the compositions described herein depends on the individual parameters of the patient, including the condition being treated, the severity of the disease, age, physiological state, size and weight, the duration of treatment, the type of accompanying treatment (if any), the specific route of administration, and similar factors. Therefore, the dose administered of the compositions described herein may depend on such various parameters. If the patient’s response is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0400] The pharmaceutical compositions of this disclosure may comprise salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical composition of this disclosure comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0401] Suitable preservatives for use in the pharmaceutical compositions of this disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0402] As used herein, the term “excipient” refers to a substance that may be present in the pharmaceutical compositions of this disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

[0403] The term “diluent” refers to a substance used to dilute and / or reduce an agent. Furthermore, the term “diluent” includes one or more fluids, liquid or solid suspensions, and / or mixed media. Examples of suitable diluents include ethanol, glycerol, and water.

[0404] The term "carrier" refers to a component that may be natural, synthetic, organic, or inorganic, to which the active ingredient is combined to facilitate, enhance, or enable the administration of the pharmaceutical composition. As used herein, carriers may be one or more suitable solid or liquid fillers, diluents, or encapsulants suitable for administration to a target. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, Ringer's lactate solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, hydrogenated naphthalene, and in particular biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of this disclosure contains isotonic saline.

[0405] Pharmacopoeia-acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0406] The pharmaceutical carrier, excipient, or diluent may be selected in relation to the intended route of administration and standard pharmaceutical practices.

[0407] In one embodiment, the pharmaceutical composition described herein may be administered intravenously, intra-arterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration may include enteral administration, including absorption through the gastrointestinal tract, or parenteral administration. As used herein, “parenteral administration” refers to administration by any method other than through the gastrointestinal tract, such as intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous administration. In one embodiment of all aspects of the present invention, RNA encoding an antigen is administered systemically.

[0408] As used herein, the term “concurrent administration” means the process of administering different compounds or compositions (e.g., immune effector cells [which may be “administered” by in vivo generation in a subject] and an antigen, a polynucleotide encoding an antigen, or a host cell genetically modified to express an antigen) to the same patient. Different compounds or compositions may be administered simultaneously, essentially simultaneously, or sequentially. In one embodiment, the antigen, a polynucleotide encoding an antigen, or a host cell genetically modified to express an antigen is administered after the administration or generation of immune effector cells, for example, at least one day after the administration or generation of immune effector cells, for example, 1 to 10 days or 1 to 5 days later. Antigens, polynucleotides encoding antigens, or genetically modified host cells expressing antigens may be administered several times over time at regular or different time intervals, for example, at time intervals of 10 to 40 days after the administration or generation of immune effector cells expressing antigen receptors, and the first administration of antigens, polynucleotides encoding antigens, or genetically modified host cells expressing antigens may be at least 1 day, for example, 1 to 10 days or 1 to 5 days after the administration or generation of immune effector cells.

[0409] treatment Therefore, the agents, compositions, and methods described herein can be used to treat subjects having diseases, for example, diseases characterized by the presence of disease cells expressing the antigens described herein. Particularly preferred diseases are cancers. Thus, the agents, compositions, and methods may be useful in treating cancers in which cancer cells express the tumor antigens described herein.

[0410] Immunotherapy may be carried out using one of a variety of techniques in which the drugs provided herein function to remove antigen-expressing cells from a patient. Such removal may occur as a result of enhancing or inducing an immune response in the patient that is specific to the antigen or the cells expressing the antigen.

[0411] In certain embodiments, immunotherapy may be active immunotherapy, and the treatment relies on in vivo stimulation of the endogenous host immune system to respond to diseased cells by administration of an immune response modulator (such as peptides and nucleic acids provided herein).

[0412] In other embodiments, the immunotherapy may be passive immunotherapy, and the treatment involves the delivery of a drug (such as effector cells) that has an established tumor immunoreactivity that can directly or indirectly mediate an antitumor effect and does not necessarily depend on an intact host immune system. Examples of effector cells include T lymphocytes (such as CD8+ cytotoxic T lymphocytes and CD4+ T helper lymphocytes), as well as antigen-presenting cells (such as dendritic cells and macrophages). T cell receptors specific to the peptides listed herein may be cloned, expressed, and transferred to other effector cells for adoptive immunotherapy.

[0413] As described above, the immunoreactive peptides provided herein can be used to rapidly expand antigen-specific T cell cultures to generate a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells such as dendritic cells, macrophages, monocytes, fibroblasts, and / or B cells can be pulsed with immunoreactive peptides or transfected with one or more nucleic acids using standard techniques well known in the art. Cultured effector cells for therapeutic use must be able to grow in vivo, be widely distributed, and survive for extended periods. Studies have shown that cultured effector cells can be induced to grow in vivo and survive for extended periods in considerable numbers by repeated stimulation with IL2-supplemented antigens (see, e.g., Cheever et al. (1997), Immunological Reviews 157, 177).

[0414] Alternatively, nucleic acids expressing the peptides described herein may be introduced into antigen-presenting cells taken from a patient and clonally augmented ex vivo for transplantation back into the same patient.

[0415] The transfected cells can be reintroduced into the patient in a sterile form, preferably by intravenous, intracavitary, intraperitoneal, or intratumoral administration, using any means known in the art.

[0416] The methods disclosed herein may include the administration of autologous T cells activated in response to a peptide or antigen-presenting cells expressing the peptide. Such T cells may be CD4+ and / or CD8+ and may be proliferated as described above. The T cells may be administered to a subject in a dose effective in inhibiting the development of disease.

[0417] The term "disease" refers to an abnormal condition that affects an individual's body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. Diseases can be caused by factors originating from external sources, such as infections, or by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often more broadly used to refer to a condition that causes pain, impairment, suffering, social problems, or death in the affected individual, or similar problems in those in contact with the individual. In this broader sense, disease sometimes includes injury, helplessness, disability, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Many diseases, and living with them, can alter one's worldview and personality, so diseases usually affect individuals not only physically but also emotionally.

[0418] In this context, the terms “treatment,” “to treat,” or “therapeutic intervention” refer to the management and care of an individual aimed at combating a condition such as a disease or disorder. The term is intended to encompass all forms of treatment for a given condition in which an individual is afflicted, including the administration of therapeutically effective compounds to alleviate symptoms or complications, to slow the progression of a disease, disorder or condition, to alleviate or reduce symptoms and complications, and / or to cure or eliminate a disease, disorder or condition, and to prevent the condition. Prevention should be understood as the management and care of an individual aimed at combating a disease, condition or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0419] The term “therapeutic treatment” refers to any treatment that improves the health of an individual and / or extends (increases) their lifespan. Such treatment may eliminate disease in an individual, stop or delay the onset of disease in an individual, inhibit or delay the onset of disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual that currently has or has previously had the disease.

[0420] The terms “preventive measures” or “preventive measures” refer to any treatment intended to prevent the development of disease in an individual. The terms “preventive measures” and “preventive measures” are used interchangeably herein.

[0421] The terms “individual” and “subject” are used interchangeably herein. They refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that is susceptible to, but may or may not have, a disease or disorder (e.g., cancer). In many embodiments, the individual is a human. Unless otherwise specified, the terms “individual” and “subject” do not indicate a specific age and therefore encompass adults, the elderly, children, and newborns. In embodiments of this disclosure, the “individual” or “subject” is a “patient.”

[0422] The term "patient" means an individual or subject for treatment, in particular an individual or subject that is ill.

[0423] In one embodiment of this disclosure, the objective is to provide an immune response against disease cells that express antigens, such as cancer cells that express tumor antigens, and to treat diseases such as cancer that involve cells that express antigens such as tumor antigens.

[0424] An immune response to the antigen may be induced, which may be therapeutic, partially protective, or completely protective. Therefore, the pharmaceutical compositions described herein are applicable to induce or enhance immune responses. Accordingly, the pharmaceutical compositions described herein are useful for the prophylactic and / or therapeutic treatment of diseases involving the antigen.

[0425] As used herein, “immune response” refers to the integrated bodily response to an antigen or an antigen-expressing cell, and includes cellular and / or humoral immune responses.

[0426] The terms "cell-mediated immunity," "cellular immunity," "cellular immune response," or similar terms are intended to include cellular responses directed towards cells characterized by antigen expression, particularly those characterized by antigen presentation by class I or class II MHC. The cellular response relates to cells called T cells or T lymphocytes that act as either "helper" or "killer" cells. Helper T cells (CD4+ T cells (also called CD8 cells) play a central role in regulating the immune response, and killer cells (cytotoxic T cells, cytolytic T cells, CD8 cells) play a central role in this process. + T cells (also known as CTLs) kill disease cells such as cancer cells and prevent the production of further disease cells.

[0427] The terms "cells characterized by antigen presentation," "cells that present antigens," "antigens presented by cells," "presented antigens," or similar expressions refer to cells that present disease cells, such as malignant cells, or antigens expressed by such cells or fragments derived from such antigens, in relation to MHC molecules, particularly MHC class I molecules, for example, by antigen processing. Similarly, the term "disease characterized by antigen presentation" refers to diseases involving cells characterized by antigen presentation, particularly using class I MHC. Antigen presentation by cells can be achieved by transfecting cells with nucleic acids, such as RNA, that encode antigens.

[0428] This disclosure envisions an immune response that may be protective, preventive, prophylactic, and / or therapeutic. As used herein, “inducing (or inducing) an immune response” may indicate that there was no immune response to a particular antigen prior to induction, or that there was a baseline level of immune response to a particular antigen prior to induction, which was enhanced after induction. Thus, “inducing (or inducing) an immune response” includes “enhancing (or enhancing) an immune response.”

[0429] The term "immunotherapy" refers to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

[0430] The terms "immunization" or "vaccination" refer to the process of administering an antigen to an individual for the purpose of inducing an immune response, for example, for therapeutic or preventive reasons.

[0431] The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf foreign pathogens within the macrophage and kill them through hydrolytic and oxidative attacks that lead to the degradation of the pathogens. Peptides from the degraded proteins are displayed on the surface of macrophage cells, which can be recognized by T cells and directly interact with antibodies on the surface of B cells, leading to the activation of T and B cells and further stimulation of the immune response. Macrophages belong to the class of antigen-presenting cells. In one embodiment, the macrophage is a splenic macrophage.

[0432] The term “dendritic cell” (DC) refers to another subtype of phagocytic cell belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells originate from hematopoietic bone marrow progenitor cells. These progenitor cells first differentiate into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T-cell activating ability. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. When they come into contact with presentable antigens, they are activated and become mature dendritic cells, and begin to migrate to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, breaking down their proteins into small fragments, and upon maturation, they use MHC molecules to present these fragments on their cell surface. At the same time, they upregulate cell surface receptors such as CD80, CD86, and CD40, which act as co-receptors in T-cell activation, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to the lymph nodes. Here, they act as antigen-presenting cells, activating helper T cells, killer T cells, and B cells by presenting antigens along with non-antigen-specific costimulatory signals. Therefore, dendritic cells can actively induce immune responses related to T cells or B cells. In one embodiment, the dendritic cells are splenic dendritic cells.

[0433] The term "antigen-presenting cell" (APC) refers to one of the various types of cells that can display, acquire, and / or present at least one antigen or antigen fragment on (or on) its cell surface. Antigen-presenting cells can be distinguished into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0434] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules necessary for interaction with naive T cells. When T cells interact with the MHC class II molecular complex on the membrane of antigen-presenting cells, the antigen-presenting cells produce costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0435] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but express them in response to 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.

[0436] "Antigen processing" refers to the breakdown of an antigen into processing products, which are fragments of the antigen (e.g., breakdown of proteins into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by a cell such as an antigen-presenting cell.

[0437] The terms “antigen-related disease,” “antigen-expressing cell-related disease,” or similar terms refer to any disease related to an antigen, such as a disease characterized by the presence of an antigen. An antigen-related disease may be a cancerous disease or simply cancer. As described above, the antigen may be a disease-related antigen, such as a tumor-related antigen. In one embodiment, an antigen-related disease is a disease in which antigen-expressing cells are involved, preferably a disease in which antigen-presenting cells are involved.

[0438] Malignant lesions are medical conditions, particularly tumors, that tend to progress progressively and potentially lead to death. They are characterized by anaplastic, invasive, and metastatic properties. Malignant is a corresponding adjectival medical term used to describe a disease that is severe and progressively worsening. As used herein, the term “malignant disease” preferably refers to cancer or neoplastic disease. Similarly, as used herein, the term “malignant cell” preferably refers to cancer cells or tumor cells. Malignant tumors can be contrasted with non-cancerous benign tumors in that malignant lesions are not self-limiting in their growth, can invade adjacent tissues, and may have the ability to spread (metastasize) to distant tissues, whereas benign tumors do not possess any of these characteristics. Malignant tumors are essentially synonymous with cancer. Malignant tumors, malignant neoplasms, and malignant tumors are essentially synonymous with cancer.

[0439] According to the present invention, the terms “tumor” or “tumor disease” refer to a swelling or lesion formed by the abnormal growth of cells (called neoplastic cells or tumor cells). “Tumor cells” means abnormal cells that grow by rapid, uncontrolled proliferation and continue to grow after the stimulus that initiated new growth has ceased. Tumors exhibit a partial or complete lack of structural organization and functional coordination with normal tissue, and typically form a distinct tissue mass that can be benign, premalignant, or malignant.

[0440] A benign tumor is a tumor that lacks all three malignant characteristics of cancer. Therefore, by definition, a benign tumor does not grow aggressively indefinitely, does not invade surrounding tissues, and does not spread (metastasize) to non-adjacent tissues. Common examples of benign tumors include nevi and uterine fibroids.

[0441] The term "benign" refers to mild, non-progressive diseases, and indeed, many types of benign tumors are harmless to health. However, some neoplasms defined as "benign tumors" because they lack the invasive characteristics of cancer can still have adverse effects on health. Examples include tumors that cause "mass effects" (compression of vital organs essential for life, such as blood vessels) or "functional" tumors of endocrine tissue that can overproduce certain hormones (examples include thyroid adenomas, adrenocortical adenomas, and pituitary adenomas).

[0442] Benign tumors are typically surrounded by an outer surface that inhibits their ability to behave malignantly. In some cases, certain “benign” tumors may later develop into malignant cancers, which arises from further genetic changes in a subpopulation of neoplastic cells in the tumor. A prominent example of this phenomenon is the tubular adenoma, a common type of colon polyp that is a significant precursor to colon cancer. The cells of a tubular adenoma, like most tumors that often progress to cancer, exhibit specific abnormalities in cellular maturation and appearance known collectively as dysplasia. These cellular abnormalities are not seen in benign tumors that rarely or never become cancerous, but are seen in other precancerous tissue abnormalities that do not form individual masses, such as precancerous lesions of the cervix. Some authorities prefer to refer to dysplastic tumors as “premalignant” and reserve the term “benign” for tumors that rarely or never develop into cancer.

[0443] A neoplasm is an abnormal tissue mass resulting from neoplasia. Neoplasia (from the Greek word for new growth) is the abnormal proliferation of cells. Cell growth outpaces the growth of the surrounding normal tissue and does not coordinate with normal tissue growth. The growth persists in the same excessive manner even after the cessation of stimulation. This usually results in a lump or tumor. Neoplasms can be benign, premalignant, or malignant.

[0444] The term "tumor growth" or "tumor development" according to the present invention relates to the tendency of a tumor to increase in size and / or the tendency of tumor cells to proliferate.

[0445] Preferably, the "malignant disease" according to the present invention is a cancerous disease or a tumor disease, and the malignant cells are cancer cells or tumor cells. Preferably, the "malignant disease" is characterized by cells expressing tumor-associated antigens such as NY-ESO-1, MAGE-A3, tyrosinase, TPTE, and KRAS, respectively.

[0446] Cancer (medical term: malignant neoplasm) is a class of diseases characterized by uncontrolled growth (division beyond normal limits), invasion (invasion and destruction of adjacent tissues), and sometimes metastasis (spread to other parts of the body via the lymphatic system or bloodstream). These three malignant characteristics of cancer are self-limiting and distinguish cancer from benign tumors that do not invade or metastasize. Most cancers form tumors, but some cancers, such as leukemia, do not.

[0447] Cancers are classified by the type of cells that resemble the tumor, and therefore by the tissue from which the tumor is presumed to originate. These are histology and location, respectively.

[0448] Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specifically, examples of such cancers include bone cancers, hematological cancers, lung cancers, liver cancers, pancreatic cancers, skin cancers, head and neck cancers, cutaneous or intraocular melanomas, uterine cancers, ovarian cancers, rectal cancers, anal cancers, gastric cancers, colon cancers, breast cancers, prostate cancers, uterine cancers, cancers of the genitals and reproductive organs, Hodgkin's disease, esophageal cancers, small intestine cancers, cancers of the endocrine system, thyroid cancers, parathyroid cancers, adrenal cancers, soft tissue sarcomas, bladder cancers, kidney cancers, renal cell carcinomas, renal pelvis cancers, neoplasms of the central nervous system (CNS), neuroectodermal carcinomas, spinal axial tumors, gliomas, meningiomas, and pituitary adenomas. The term “cancer” as used in this disclosure also includes cancer metastases. In one embodiment, cancer is melanoma, in particular malignant melanoma. In one embodiment, cancer is NSCLC.

[0449] The term "melanoma" or "malignant melanoma" refers to a type of cancer that arises from pigment-containing cells known as melanocytes. Melanoma typically occurs in the skin, but can rarely occur in the mouth, intestines, or eyes (uveal melanoma).

[0450] The term "NSCLC," or "non-small cell lung cancer," refers to a type of epithelial lung cancer other than small cell lung cancer (SCLC). NSCLC accounts for approximately 85% of all lung cancers. The most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma, but several other types occur less frequently. Some of the less common types are pleomorphic tumors, carcinoid tumors, salivary gland carcinomas, and unclassified carcinomas.

[0451] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process, depending on the separation of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and subsequent transport by the blood before invading the target organ. Finally, the growth of a new tumor at the target site, i.e., a secondary or metastatic tumor, depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, as tumor cells or components may remain and exhibit metastatic potential. In one embodiment, the term "metastasis" according to this invention refers to "distant metastasis," which is a metastasis away from the primary tumor and the regional lymph node system.

[0452] Recurrence or relapse occurs when a person becomes ill again with a condition they have previously suffered from. For example, if a patient has suffered from a tumor, has been successfully treated for the disease, and then develops the disease again, the newly developed disease may be considered a recurrence or relapse. However, according to the present invention, recurrence or relapse of a tumor may occur at the site of the original tumor, but not necessarily. For example, if a patient has suffered from an ovarian tumor and has been successfully treated for it, recurrence or relapse could be the development of an ovarian tumor or a tumor at a site other than the ovary. Tumor recurrence or relapse also includes situations in which the tumor develops at a site different from the original tumor site and situations in which it develops at the original tumor site. Preferably, the original tumor that the patient was treated for is a primary tumor, and the tumor at a site different from the original tumor site is a secondary or metastatic tumor.

[0453] As used herein, the terms “solid tumor” or “solid carcinoma” refer to the occurrence of a cancerous mass, as is well known in the art, for example, in Harrison’s Principles of Internal Medicine, 14th edition. Preferably, the terms refer to cancer or carcinoma of body tissue other than blood, preferably other than blood, bone marrow, and lymphatic tissue. For example, but not limited to, solid tumors include cancers of the prostate, lung, colorectal tissue, bladder, oropharyngeal / laryngeal tissue, kidney, breast, endometrium, ovary, cervix, stomach, pancreas, brain, and central nervous system.

[0454] Combination strategies in cancer treatment can be desirable due to the resulting synergistic effects, which can be considerably more potent than the effects of monotherapy approaches. In one embodiment, a pharmaceutical composition is administered together with an immunotherapy agent. As used herein, “immunotherapy agent” refers to any agent that may be involved in the activation of a specific immune response and / or immune effector function(s). This disclosure intends to use antibodies as immunotherapy agents. While not wishing to be bound by theory, antibodies can achieve therapeutic effects against cancer cells through a variety of mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting tumor cell proliferation. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies may induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or, by binding to complement proteins, may result in direct cytotoxicity known as complement-dependent cell-mediated cytotoxicity (CDC). Non-limiting examples of anticancer antibodies and potential antibody targets (in parentheses) that may be used in combination with this disclosure include: avagovomab (CA-125), absiximab (CD41), adecatumumab (EpCAM), aftuzumab (CD20), aracizumab pegol (VEGFR2), artumomab penteate (CEA), amatsuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), alsitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), vectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), vibatuzumab meltansine (CD44) v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), cantuzumab meltansine (mucin CanAg), cantuzumab lavtansine (MUC1), capromab pendetide (prostate cancer cells), carrumab (CNT0888), catumakisomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab vogatox (EpCAM), thixumumab (IGF-1 receptor), clodiximab (claudin), cribatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), darotuzumab (insulin) (Lymphoma-like growth factor I receptor), denosumab (RANKL), detumomab (B lymphoma cells), droditumab (DR5), eclomeximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enabatuzumab (PDL192), encituximab (NPC-1C), epratuzumab (CD22), ertzumaxomab (HER2 / neu, CD3), etalacizumab (integrin ανβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficratuzumab (SCH900105), Figitumumab (IGF-1 receptor), Frambotumab (glycoprotein 75), Fresolimmab (TGF-β), Galiximab (CD80), Ganitumab (IGF-I), Gemtuzumab Ozogamicin (CD33), Gevokizumab (ILIβ), Gilentuximab (carbonic anhydrase 9 (CA-IX)), Grembatumumab Vedotin ( GPNMB), ibritumomab tiuxetan (CD20), iculcumab (VEGFR-1), igoboma (CA-125), indatuximab blutancin (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), rabetsuzumab (CEA), lexatumumab Br (TRAIL-R2), ribivirumab (hepatitis B surface antigen), lintuzumab (CD33), rorbotuzumab meltansine (CD56), lucatumumab (CD40), lumiliximab (CD23), mapatumumab (TRAIL-R1), matsuzumab (EGFR), mepolizumab (IL5), milatuzumab (CD74), mitumomab (GD3 ganglion Oside), mogamulizumab (CCR4), moxetumomab pasdotox (CD22), nacolomab butafenatox (C242 antigen), naptumomab estafenatox (5T4), namatumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-R)a) Onartuzumab (human scattering factor receptor kinase), oportuzumab monatox (EpCAM), olegobomab (CA-125), oxerumab (OX-40), panitumumab (EGFR), patritumumab (HER3), pemtumoma (MUC1), pertuzumab (HER2 / neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), lacosumomab (N-glycolylneuraminic acid), radretumumab (fibronectin extradomain B), rafibirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor), samarizumab (CD200), ci Brotuzumab (FAP), siltuximab (IL6), tavarmab (BAFF), tacutuzumab tetraxetan (α-fetoprotein), tapritumomab paptox (CD19), tenatumomab (tenascin C), teprotumumab (CD221), tisilimmumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucozouzumab cermoloykin (EpCAM), ubrituximab (MS4A1), urerumab (4-1BB), boroximab (integrin α5β1), botumumab (tumor antigen CTAA) 16.88), saltumumab (EGFR), and zanorimumab (CD4).

[0455] The references to documents and tests made herein are not intended to constitute an acknowledgment that any of the foregoing constitutes relevant prior art. All statements relating to the contents of these documents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the contents of these documents.

[0456] The following description is provided to enable those skilled in the art to create and use various embodiments. Specific descriptions of apparatus, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the examples described and shown herein, but should be given a scope consistent with the claims. [Examples]

[0457] The techniques and methods used herein are either described herein or known by themselves, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd The procedure shall be carried out as described in the Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. All procedures, including the use of kits and reagents, shall be carried out according to the manufacturer's instructions unless otherwise specified.

[0458] Example 1: Materials and Method patient material PBMCs for immunomonitoring were isolated from peripheral blood or leukocyte apheresis samples by density gradient centrifugation using Ficoll-Hypaque (Amersham Biosciences). Immature DCs (S. Holtkamp et al., Blood. 108, 4009-17 (2006)) or ultra-short-term cultured mature DCs (M. Dauer et al., J. Immunol. 170, 4069-76 (2003)) were generated as described above.

[0459] cell line The K562, LCLC-103H, NCI-H-460, and SK-Mel-28 cell lines were obtained from ATCC. SK-Mel-29 was obtained from Memorial Sloan Kettering Cancer Center, NY-York. The SK-Mel-37 cell line is described by Carey TE et al., Proc Natl Acad Sci, 1976. Jurkat T cell lines expressing a luciferase reporter driven by an NFAT response element are produced by Promega.

[0460] In vitro stimulation (IVS) of PBMCs CD4 + and CD8 + T cells were isolated from cryopreserved PBMCs using microbeads (Miltenyi Biotec). IVS cultures were set up with RNA or peptides. For RNA-based IVS, CD4 or CD8 depleted PBMCs were allowed to stand overnight, then treated with vaccine antigen, eGFP, influenza matrix protein 1 (M1), or tetanus p2 / p16 sequence (each corresponding to CD4). + and CD8 + CD4 cells were electroporated with RNA encoding a positive control for T cells, left to stand at 37°C for 3 hours, and irradiated with 15 Gy. Subsequently, the cells were left to stand overnight. + / CD8 + T cells and electroporated and irradiated antigen-presenting cells were combined in a 2:1 effector-to-target ratio. In the case of peptide IVS, CD4 was used in the presence of ultra-short-term cultured mature DCs pulsed with OLP encoding either MAGE-A3, tyrosinase, TPTE, or NY-ESO-1. + T cells were enlarged (E:T = 10:1). CD8 + To expand T cells, CD4-depleted PBMCs are purified in the presence of IL-4 and GM-CSF (1000 U / mL each) and their respective peptides. +CD8 cells were co-cultured with T cells (E:T = 1:10). One day after the start of IVS, fresh culture medium containing 10 U / ml IL-2 (Proleukin S, Novartis) and 5 ng / mL IL-15 (Peprotech) was added. IL-4 and GM-CSF (1000 U / mL each) were further added to the peptide-stimulated CD8 IVS cultures. For tumor cell lysis experiments, peptide-pulsed bulk PBMCs were also used in IVS and harvested after 6-8 days of culture. For longer cultures, IL-2 was supplemented 7 days after setting up the IVS cultures. Eleven days after stimulation, cells were analyzed by flow cytometry and used in the ELISpot assay.

[0461] IFNγ ELISpot Multiscreen filter plates (Merck Millipore) pre-coated with an IFNγ-specific antibody (Mabtech) were washed with PBS and blocked with X-VIVO 15 (Lonza) containing 2% human serum albumin (CSL-Behring) for 1–5 hours. (0.5–3 × 10⁻⁶) 5Each effector cell / well was stimulated for 16–20 hours with either a peptide (ex vivo setting), autogenous DCs electroporated with RNA or loaded with a peptide (post-IVS), or HLA class I or II transfected K562 cell peptide (TCR validation). For analysis of ex vivo T cell responses, cryopreserved PBMCs were subjected to ELISpot after a 2–5 hour rest period at 37°C. Alternatively, CD4 or CD8 depleted PBMCs were used as CD8 or CD4 effectors. All tests were performed two or three times in duplicate and included positive controls (Staphylococcus enterotoxin B (Sigma Aldrich), anti-CD3 (Mabtech)) and reference donor cells with known reactivity. Spots were visualized with biotin-conjugated anti-IFNγ antibody (Mabtech) and then incubated with ExtrAvidin-Alkaline Phosphatase (Sigma-Aldrich) and BCIP / NBT substrate (Sigma-Aldrich). Alternatively, a secondary antibody directly conjugated with ALP was used (ELISpotPro kit, Mabtech). Plates were scanned using CTL's ImmunoSpot® Series S five Versa ELISpot Analyzer (S5Versa-02-9038) or AID Classic Robot ELISPOT Reader and analyzed with ImmunoCapture V6.3 or AID ELISPOT 7.0 software. Spot counts were summarized as the median for 3 or 2 overlapping runs, respectively. T cell responses stimulated by vaccine RNA or peptide were compared to target cells electroporated with control RNA (luciferase) or unloaded target cells, respectively. Responses were measured at 1x10⁶ in the ex vivo setting. 5 A minimum of 5 spots per cell, or 5x10 in the post-IVS setting. 4 A cell was defined as positive if it had at least 25 spots, and more than twice the number of spots compared to the control.

[0462] Flow cytometry Antigen specific CD8+ T cells were identified using fluorophore-conjugated HLA multimers (Immudex). Cells were first stained with the multimers and subsequently stained with cell surface markers (antibody clones in parentheses) CD28 (CD28.8), CD197 (150503), CD45RA (HI100); CD3 (UCHT1 or SK7), CD16 (3G8), CD14 (MφP9), CD19 (SJ25C1), CD27 (L128), CD279 (EH12), CD134 (ACT35), CD8 (RPA-T8), all purchased from BD; CD19 (HIB19), CD4 (OKT4) from Biolegend, and viability stained using DAPI (BD) or Fixable Viability Dye eFluor™ 780 (eBioscience). Singlet, live, multimer-positive events were identified within CD3 (or CD8)-positive, CD4 / CD14 / CD16 / CD19-negative, or CD3 (or CD8)-positive / CD4-negative events. For detection of antigen-specific T cells after IVS, singlet, live, CD3 + , CD8 + / multimer + lymphocytes were gated. To define the multimer-positive population, gates were set relative to fluorescence minus one (FMO) controls.

[0463] For intracellular cytokine staining, auto-DCs electroporated with RNA encoding a single neoepitope were added in a 10:1 E:T ratio and cultured at 37°C for approximately 16 hours in the presence of brefeldin A and monensin. Cells were stained with survival dyes (Fixable Viability Dye eFluor® 506 or Viability Dye eFluor® 780, eBioscience) and surface markers CD8 (RPA-T8 or SK1), CD4 (SK3), CD16 (3G8), CD14 (MφP9), all purchased from BD; and CD19 (HIB19) and CD4 (OKT4) from Biolegend. After permeabilization, intracellular cytokine staining was performed using antibodies against IFNγ (B27, BD) and TNF (Mab11, BD, or Biolegend) for intracellular cytokine staining. Singlet, live, IFNγ, and TNF-positive events were identified within CD8 and CD4-positive, and CD14 / CD16 / CD19-negative (when stained according to the marker panel used) events.

[0464] Cell surface expression of transfected TCR genes was analyzed using anti-TCR antibodies (Beckman Coulter) and CD8 or CD4-specific antibodies (SK-1, BD) against the appropriate variable region family or constant region of the TCR-β chain. HLA antigens of antigen-presenting cells used to evaluate the function of TCR-transfected T cells were detected by staining with HLA class II-specific antibodies (9-49, Beckman Coulter and REA623, Miltenyi Biotec) and HLA class I-specific antibodies (DX17, BD Biosciences).

[0465] Data was acquired using LSR Fortessa SORP, FACSCelesta, or FACSCanto II (BD) and analyzed using FlowJo software (Tree Star).

[0466] HLA antigen cloning HLA antigens were synthesized by Eurofins Genomics Germany GmbH according to their respective high-resolution HLA typing results. HLA-DQA sequences were amplified from donor-specific cDNA using 2.5 U Pfu polymerase with DQA1_s (PHO-GCC ACC ATG ATC CTA AAC AAA GCT CTG MTG C) and DQA1_as (TAT GCG ATC GCT CAC AAK GGC CCY TGG TGT CTG) primers. HLA antigens were cloned into appropriately digested IVT vectors (Simon, P. et al. Cancer Immunol. Res. 2, 1230-44 (2014)).

[0467] RNA introduction into cells RNA was added to cells suspended in X-VIVO 15 medium (Lonza) in a pre-cooled, 4mm gap sterile electroporation cuvette (Bio-Rad). Electroporation was performed using a BTX ECM 830 square wave electroporation system (T cells: 500V / 3ms / 1 pulse; iDC: 300V / 12ms / 1 pulse; bulk PBMC: 400V / 6ms / 1 pulse; MZ-GaBa-018: 225V / 3ms / 2 pulses; K562: 200V / 8ms / 3 pulses).

[0468] peptide NY-ESO-1, tyrosinase, MAGE-A3 and TPTE (full length), or short (8-11-mer) epitopes derived from these antigens, KRAS-Q61H 55-64 A pool of duplicate peptides (PepMix®) encoding epitopes and control antigens (HIV-gag, SSX2) was used. All synthetic peptides were purchased from JPT Peptide Technologies GmbH and dissolved to a final concentration of 3 mM in water containing 10% DMSO (short peptides) or in 100% DMSO (PepMix®).

[0469] Single cell sorting Sorting of single antigen-specific T cells was performed using either ex vivo PBMC or IVS cultures, based on stimulated IFNγ secretion or multimer binding. For stimulation, PBMC were pulsed with overlapping peptides covering the relevant antigen or control antigen, and expanded T cells after IVS were cultured with DC pulsed with self-peptides. After 4 hours, cells were harvested and treated with fluorescent dye-conjugated antibodies against CD3, CD8, CD4, CD137 (4B4-1) (BD) and IFNγ using an IFNγ secretion assay kit (Miltenyi Biotec). Alternatively, PBMC were stained with respective multimers. Sorting of single neoantigen-specific T cells was performed on a FACSAria™ or FACSMelody™ flow cytometer (both from BD Biosciences) using BD FACSDiva™ and BD FACSChorus™ software, respectively. Antigen-specific T cells were identified with respect to control samples stimulated with control antigen or stained without multimer. One T cell (CD3 + CD8 + single, live CD3 within gate + and CD8 + / IFNγ + CD4 + / IFNγ + or CD8 + / multimer + lymphocytes or CD137 + / IFNγ + (gated) was recovered into a 96-well V-bottom plate (Greiner Bio-One) containing 6 μL of mild hypotonic cell lysis buffer per well consisting of 0.2% Triton X-100 in RNase-free water, 0.2 μL of RiboLock RNase inhibitor (Thermo Scientific), 5 ng of poly(A) carrier RNA (Qiagen) and 1 μL of dNTP mix (10 mM, Biozym). The plate was sealed, centrifuged and stored at -65 °C to -85 °C immediately after sorting.

[0470] Cloning of antigen-specific TCR The TCR gene was cloned from single T cells with the following modifications as previously described (M. Dauer, et al., J. Immunol. 170, 4069-76 (2003)). Plates containing the selected cells were thawed, and template-switched cDNA synthesis was performed using RevertAid H-reverse transcriptase (Thermo Fisher) with TCR alpha and beta constant gene-specific primers (TRAC 5'-catcacaggaactttctgggctg-3', TRBC1 5'-gctggtaggacaccgaggtaaagc-3', and TRBC2 5'-gctggtaagactcggaggtgaagc-3'), followed by pre-amplification using PfuUltra Hotstart DNA Polymerase (Agilent). After both cDNA synthesis and PCR, residual primers were removed by treatment with 5U exonuclease I (NEB). Aliquots of cDNA were used for Vα / Vβ gene-specific multiplex PCR. The products were analyzed using a capillary electrophoresis system (Qiagen). Samples with bands of 430–470 bp were size-fractionated on agarose gels, the bands were excised, and purified using the Gel Extraction Kit (Qiagen). The purified fragments were sequenced using the IMGT / V-Quest tool. 30 Each V(D)J junction was analyzed using [method name]. The DNA of the corresponding novel and productively reconstituted TCR strands was digested with NotI and cloned into a pST1 vector containing a suitable constant region for complete TCR-α / β strand in vitro transcription (Simon, P. et al. Cancer Immunol. Res. 2, 1230-44 (2014)).

[0471] Single-cell TCR (scTCR) sequencing For selected patients, TCRs were obtained from sorted single cells using an NGS-based scTCRseq workflow. Template switch cDNA synthesis was performed using TCR alpha and beta constant gene-specific primers (TRAC 5'-catcacaggaactttctgggctg-3' and TRBC 5'-cacgtggtcggggwagaagc-3'), followed by treatment with 5U exonuclease I. Each cDNA was amplified by PCR, and then mixed with 2.5U PfuUltra Hotstart DNA Polymerase (Agilent), 1× PCR buffer, 0.2 mM dNTPs, 0.2 μM of one of eight tagged forward primers Tag130-RBCx-TS 5'-cgatccagactagacgctcaggaagxxxxxaagcagtggtatcaacgcagagt-3', and 0.1 μM of each tagged nested TCR alpha and beta constant gene-specific primer Tag146-TRAC 5'-caatatgtgaccgccgagtcccaggttagagtctctcagctggtacacggcag-3' and Tag146-TRBC Barcodes were generated column by column using 5'-caatatgtgaccgccgagtcccaggggctcaaacacagcgacctcgggtg-3' (2 min at 95°C; 5 cycles of 30 sec at 94°C, 30 sec at 61°C, and 1 min at 72°C; 5 cycles of 30 sec at 94°C, 30 sec at 64°C, and 1 min at 72°C; 8 cycles of 30 sec at 94°C, and 2 min at 72°C; 6 min at 72°C). Samples from each column were pooled and purified twice using AMPure XP beads (Agencourt) treated with exonuclease I in between.For each pool, one-third of the purified TCR cDNA was further amplified by PCR using 1 μl of PfuUltra II Fusion Hotstart DNA Polymerase (Agilent), 1× reaction buffer, 0.2 mM dNTPs, forward primer Tag-130 5'-(n)nnnncgatccagactagacgctcaggaag-3', and one of 12 Tag-146 reverse oligo 5'-xxxxxcaatatgtgaccgccgagtcccagg-3' containing a different barcode for each column (1 min at 95°C; 20 seconds at 94°C, 20 seconds at 64°C, and 30 seconds at 72°C for 24 cycles; 3 min at 72°C). After pooling the PCR products and purifying them with AMPure XP beads and exonuclease I, TCR sequencing libraries were constructed using the TruSeq DNA Nano kit (Illumina). The scTCR library was sequenced using paired-end 300-base pair sequencing on an Illumina MiSeq with a sequencing depth of 10,000 reads per well. The sequencing data were demultiplexed to the single-cell level using bcl2fastq software (Illumina), followed by an in-house Python script. TCR sequences were then obtained using MiXCR-2.1.5 (Bolotin, et al., Nat. Methods 12, 380-1 (2015)). Selected pairs of alpha and beta VDJ fragments were synthesized (Eurofins Genomics) and cloned as described above for subsequent in vitro transcription.

[0472] Bulk TCR sequencing Total RNA was collected at multiple time points during vaccination using the RNeasy Mini Kit (QIAGEN) at a rate of 1 × 10⁶ 6 The TCRs were isolated from rapid-frozen PBMCs. Libraries were prepared using the SMARTer Human TCR a / b Profiling Kit (Clontech) and sequenced using the Illumina MiSeq system. The total number of TCR reads per sample was 1 x 10⁶. 6 ~4x10 6The range was as follows. The data was analyzed using VDJtools (Shugay, M. et al., PLoS Comput. Biol. 11, e1004503 (2015)) and MiXCR.

[0473] Characterization of functional TCRs TCR-transfected CD4 from healthy donors + or CD8 + T cells were co-cultured with peptide-pulsed HLA class I or II transfected K562 cells and tested using the IFNγ-ELISpot assay. Alternatively, Jurkat cells using the T Cell Activation Bioassay (NFAT, Promega) were transfected with RNA encoding CD8α and TCRα / β and tested against target cells. T cell activation was analyzed by luminescence measurement (Infinite F200 PRO, TECAN) after the addition of Bio-Glo reagent (Promega).

[0474] TCR-mediated cytotoxicity was evaluated by cell index (CI) impedance measurement using the xCELLigence MP system (OMNI Life Science) according to the supplier's instructions. Target cells were placed in 96-well PET E plates (ACEA Biosciences Inc.) at a rate of 2 × 10⁶ cells per well. 4 Cells were seeded at the specified concentrations. After 24 hours, TCR-transfected T cells were added using different E:T ratios and monitored every 30 minutes for up to 48 hours using the xCELLigence system. Specific lysis was calculated after the instructed co-culture time.

[0475] Example 2: Discovery and characterization of HLA class I-restricted NY-ESO-1 specific TCRs in LipoMERIT patients A2-09 and 53-02 A systemically administered nanoparticle liposomal RNA vaccine (RNA-LPX) was tested in a Phase I dose-escalation study in patients with advanced melanoma (Lipo-MERIT, NCT02410733).

[0476] The vaccine (referred to as melanoma FixVac) consists of four lipid complex RNAs encoding non-mutant TAA NY-ESO-1, MAGE-A3, tyrosinase, and TPTE, each known for its restricted expression in normal tissues, high immunogenicity, and high prevalence in human melanoma (Simon, P. et al., Cancer Immunol. Res. 2, 1230-44 (2014); Cheever, MA et al., Clin. Cancer Res. 15, 5323-37 (2009)). The single-stranded 5' cap vaccine messenger RNA (Figure 1A) contains functional sequence elements that improve the efficiency of its translation, particularly in immature DCs (Holtkamp, ​​S. et al., Blood 108, 4009-17 (2006); Orlandini von Niessen, AGet al., Mol.Ther. (2018). doi:10.1016 / j.ymthe.2018.12.011). The open reading frame of each TAA is in-frame fused to a secretory signal at the N-terminus and a tetanus toxoid helper epitope and endosomal targeting domain at the C-terminus, enhancing the processing and presentation of tumor antigen-derived epitopes on individual HLA-class I and II molecules of the patient (Kreiter, S. et al., J.Immunol. 180, 309-318 (2008)).

[0477] Patients expressing at least one TAA confirmed by qRT-PCR analysis were eligible for this trial. Melanoma FixVac was administered according to the prime / repeat boost protocol, followed by optional monthly treatment (Figure 1B).

[0478] To determine the immunogenicity of TAA encoded by four RNAs, T cell responses in pre- and post-vaccination blood samples from patient A2-009 were analyzed by IFNγ-ELISPOT, multimer staining, and ICS (Figure 2A-C).

[0479] All three orthogonal assays were performed on NY-ESO-1 specific CD8 after FixVac treatment. +We demonstrated T cell induction and expansion. NY-ESO-1 specific T cells were barely detectable at baseline, but within the first 4–8 weeks, circulating CD8 + This showed a rapid increase of up to nearly 1% in T cells (Figure 2B).

[0480] To analyze the vaccine-induced NY-ESO-1 specific T cell response at the molecular level, the TCR-α / β chain was cloned from a single NY-ESO-1 specific T cell. For this purpose, post-treatment PBMCs were stimulated with NY-ESO-1 encoding a duplicated 15-mer peptide (OLP), and a single NY-ESO-1 specific T cell was selected by flow cytometry based on IFNγ activation-induced secretion (Figure 3A). For functional characterization, the cloned TCR-α / β chain in vitro transcription (IVT) RNA was co-transfected into healthy donor T cells. Specificity and HLA class I restriction were assessed by co-culturing TCR-transfected CD8 cells with K562 cells pulsed with a NY-ESO-1 peptide pool expressing individual patient HLA class I alleles. + T cells were used and evaluated by IFNγ-ELISPOT. In particular, four NY-ESO-1 specific TCRs were identified and shown to be constrained to B*3503 (Figure 3B, Table 1). CD8 + To evaluate whether NY-ESO-1-specific TCRs isolated from T cells can mediate cell lysis effector function, we used pre-activated TCR transfected CD8 + Specific death of NY-ESO-1-positive and negative melanoma cell lines by T cells was analyzed using an impedance-based cytotoxicity assay (Figure 3C). Indeed, all TCRs mediated antigen-specific lysis of NY-ESO-1-positive SK-MEL-37 cells, but not of NY-ESO-1-negative SK-MEL-28 cells. Tracking of the NY-ESO-1-TCR-β sequence in TCR deep sequencing data revealed that its frequency was undetectable or extremely low in pre-vaccinated PBMCs, while a high chronotype frequency was detected in post-vaccinated PBMCs (Figure 3D).

[0481] To examine the contribution of melanoma FixVac to treatment efficacy, we analyzed the immune responses of selected response patients for whom blood samples were available. Patient 53-02, who joined the trial after disease progression under pembrolizumab treatment, experienced a partial response (PR) of approximately 8 months with melanoma FixVac, accompanied by regression of multiple lung and subcutaneous metastases (Figure 4A, B). The patient initiated a robust de novo immune response to NY-ESO-1 and MAGE-A3 detectable by ex vivo ELISpot (data not shown). NY-ESO-1 96-104 Vaccine-inducing HLA-Cw*0304 restrictive CD8 for epitopes + The T cell response (Jackson, H. et al., J. Immunol. 176, 5908-17 (2006)) was identified by HLA multimer staining, which is related to peripheral blood CD8 + T cells rapidly increased to over 10% and remained high under continuous vaccination (Figure 4C). Analysis of peripheral blood T cells by ICS as an orthogonal assay showed that whole peripheral blood CD8 + NY-ESO-1-reactive IFNγ constitutes up to 15% of the T cell population. + The figure shows an expansion of T cells (Figure 4D). Vaccine-induced T cells are NY-ESO-1 96-104 The short-term IVS cultures from vaccinated PBMCs with expanded epitopes exhibited strong antigen-specific cytotoxic activity, as demonstrated by the death of endogenous NY-ESO-1 expressing melanoma cells (Figure 4E).

[0482] To further characterize this population, single NY-ESO-1 specific T cells were isolated from post-vaccination blood samples based on their binding to HLA multimers (Figure 5A). Single-cell TCR cloning revealed an HLA-Cw*0304-restricted TCR capable of mediating the lysis of NY-ESO-1-expressing SK-MEL-37 melanoma cells. CD8We identified -53-02-NY#107 (Figure 5B, Table 1) (Figure 5C). TCR beta-chronotyping analysis of blood samples before and after vaccination showed that the Cw*0304-restricted NY-ESO-1-TCR was undetectable at baseline and expanded with repeated vaccination (Figure 5D).

[0483] To identify NY-ESO-1-TCRs that recognize other HLA-presenting NY-ESO-1 epitopes, post-treatment PBMCs were stimulated with NY-ESO-1 encoding OLP, and CD8 was identified based on antigen-specific IFNγ release. + T cells were selected (Figure 6A). Two NY-ESO-1 TCRs and TCRs were shown to recognize the B*4001-restricted NY-ESO-1 epitope. CD8 -53-02-NY#109 and #110 were identified (Figure 6B, Table 1). Testing of different NY-ESO-1 peptides revealed that both TCRs are NY-ESO-1, an epitope not yet described in combination with HLA-B*4001. 124-133 We demonstrated that it recognizes (Figure 6C). Both NY-ESO-1-TCRs mediated the efficient death of endogenously NY-ESO-1 expressing melanoma cells SK-MEL-37, confirming that the identified epitopes are endogenously processed and presented by melanoma cells (Figure 6D). TCR beta-chronotype analysis of blood samples before and after vaccination showed that the B*4001-restricted NY-ESO-1-TCR was undetectable at baseline and expanded with repeated vaccination (Figure 6E).

[0484] These data indicate that the objective tumor response in this patient is related to a vaccine-induced polyepitopetic T-cell response against NY-ESO-1.

[0485] Example 3: Discovery and characterization of HLA class I-restricted MAGE-A3 specific TCRs in LipoMERIT patients C2-28 and C1-40 Patient C2-28 had melanoma with multiple liver and subcutaneous metastases and participated in the trial after radiologically confirmed progression on ipilimumab / nivolumab combination therapy. The patient was switched to melanoma FixVac in combination with nivolumab and experienced a partial response (PR) (Figure 7A). After 8 doses of vaccination, immune responses to NY-ESO-1 and MAGE-A3 were detected by IVS ELISpot (data not shown). HLA multimer staining was detectable ex vivo 2 months after initiation of melanoma FixVac, and peripheral blood CD8 over a time course of several months. + Previously described HLA-A*0101-restricted MAGE-A3 leads to a continuous increase of up to 2% of T cells. 168-176 We clarified the vaccine-induced T cell response to the epitope (Hanagiri, T., et al., Cancer Immunol. Immunother. 55, 178-84 (2006)) (Figure 7B).

[0486] Three MAGE-A3-specific TCRs were found to be MAGE-A3 after vaccination. 168-176 It was discovered in multimer-specific T cells (Figure 8A, Table 1). All three TCRs are RNA encoding MAGE-A3 or MAGE-A3 168-176 After being pulsed with the peptide, MAGE-A3-negative target cells were recognized (Figure 8B), and two of these cells showed specific recognition of MAGE-A3 endogenously expressed on melanoma cells (Figure 8C).

[0487] Patient C1-40 had a history of pembrolizumab-responsive metastatic melanoma and experienced disease progression with multiple rapidly progressing lung lesions 7 months after discontinuation of anti-PD1 therapy. Treatment with nivolumab was initiated, and 8 weeks later, melanoma FixVac was added to this anti-PD1 therapy. The patient experienced a partial response with shrinkage of lung metastases (Figure 9A). HLA multimer staining showed HLA-A*0101-restricted MAGE-A3. 168-176We revealed a strong vaccine-induced T cell response to the epitope (Figure 9B). Notably, short-term cultures of lymphocytes obtained from blood samples after vaccination showed highly efficient death of MAGE-A3-positive melanoma cells, demonstrating the functionality of these vaccine-induced MAGE-A3-specific T cells (Figure 9C).

[0488] Using IVS cultures from post-treatment PBMCs, MAGE-A3 168-176 CD8 of patient C1-40 secreting IFNγ after restimulation with peptide-pulsed iDCs. + MAGE-A3 from T cells 168-176 We discovered a specific HLA A*0101-restricted TCR (Figure 10A, Table 1). CD8 -C1-40-MA3#1 was shown to mediate the recognition of HLA-A*0101 transfected target cells pulsed with MAGE-A3 peptide or transfected with MAGE-A3 RNA (Figure 10B).

[0489] Example 4: Discovery and characterization of HLA class II-restricted MAGE-A3, NY-ESO-1, and tyrosinase-specific TCRs in LipoMERIT patient A2-010 Patient A2-10 had a history of checkpoint inhibitor-resistant melanoma with rapidly progressing, multi-metastatic disease on ipilimumab and nivolumab. With melanoma FixVac monotherapy, the patient experienced a 6-month partial response with regression of multiple lymph node and lung metastases (Figure 11A). Post-treatment PBMCs showed IFNγ against MAGE-A3 and NY-ESO-1. + / CD4 + A T cell response was detected (Figure 11B).

[0490] CD4 derived from IVS culture + Using T cells, we discovered HLA class II-restricted TAA-specific T cells after restimulation with auto-iDCs pulsed with TAA encoding OLP (Figure 12A). We identified several TCR chronotypes for NY-ESO-1, tyrosinase, and MAGE-A3 with different HLA class II restriction, and CD4+ The MAGE-A3 TCRs were recovered by single-cell cloning from T cells (Figure 12B, Table 1). It has been previously reported that the two MAGE-A3-TCRs are presented immunodominantly and indiscriminately on various HLA-DRB1 alleles (Hu, Y. et al., Cancer Immunol. Immunother. 63, 779-86 (2014)). 281-295 Epitope recognition was achieved (Figure 12C). Tracking of identified chronotypes in TCR profiling data revealed that most of them were below the detection threshold frequency before vaccination but were amplified by the vaccine (Figure 12D).

[0491] Example 5: Discovery and characterization of HLA-A*0201-restricted NY-ESO-1 specific TCRs derived from NSCLC patients treated with checkpoint inhibitors. To discover functional transcatheter receptors (TCRs) that specifically recognize antigens expres...

Claims

1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-44, 101, and 102, or a variant of said amino acid sequence.

2. A nucleic acid encoding the peptide described in claim 1.

3. Cells genetically modified to express the peptide described in claim 1.

4. The cell according to claim 3, comprising a nucleic acid encoding the peptide.

5. The cell according to claim 3 or 4, which presents the peptide or its processing product.

6. A cell that presents the peptide or its processing product according to claim 1.

7. A cell according to any one of claims 3 to 6, which is an antigen-presenting cell.

8. An immune effector cell that is reactive with the peptide described in claim 1.

9. A T cell receptor that is reactive with the peptide described in claim 1, or a polypeptide chain of the T cell receptor.

10. A T cell receptor polypeptide or a T cell receptor comprising the T cell receptor polypeptide, The T cell receptor polypeptide described above (i) A T cell receptor polypeptide comprising at least one, preferably two, more preferably all three, CDR sequences of a T cell receptor α chain or a variant thereof, selected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99, and (ii) T cell receptor polypeptide comprising a T cell receptor α chain sequence or a variant thereof selected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99 Selected from the group consisting of, A T cell receptor polypeptide or a T cell receptor comprising the T cell receptor polypeptide.

11. A T cell receptor polypeptide or a T cell receptor comprising the T cell receptor polypeptide, The T cell receptor polypeptide described above (i) A T cell receptor polypeptide comprising at least one, preferably two, more preferably all three, CDR sequences of a T cell receptor β chain or a variant selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 92, 94, 96, 98 and 100, and (ii) T cell receptor polypeptide comprising a T cell receptor β chain sequence or a variant thereof selected from SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 92, 94, 96, 98 and 100 Selected from the group consisting of, A T cell receptor polypeptide or a T cell receptor comprising the T cell receptor polypeptide.

12. It is a T cell receptor, (I)(i) at least one, preferably two, more preferably all three, of the CDR sequences of the T cell receptor α chain or a variant of sequence number x, and (ii) At least one, preferably two, more preferably all three, of the CDR sequences of the T cell receptor β chain of sequence number x+1 or its variant; Here, x is selected from 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99. T cell receptors including; Furthermore (II) (i) The T cell receptor α chain sequence of sequence number x or a variant thereof, and (ii) The T cell receptor β chain sequence of sequence number x+1 or a variant thereof; Here, x is selected from 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 91, 93, 95, 97, and 99. T cell receptors including A T cell receptor selected from the group consisting of the following.

13. A nucleic acid encoding a T cell receptor chain or T cell receptor according to any one of claims 9 to 12.

14. Cells genetically modified to express the T cell receptor chain or T cell receptor described in any one of claims 9 to 12.

15. The cell according to claim 14, comprising the T cell receptor chain or nucleic acid encoding a T cell receptor.

16. The cells according to claim 14 or 15, which are immune effector cells.

17. A method for preparing immunoeffector cells genetically modified to express a T cell receptor according to any one of claims 9 to 12, comprising delivering a nucleic acid encoding the T cell receptor to the immunoeffector cells.

18. The method according to claim 17, comprising bringing the immune effector cells into contact with particles containing the nucleic acid.

19. The method according to claim 18, wherein the particles further comprise a targeting molecule for targeting the immune effector cells.

20. The method according to claim 18 or 19, wherein contacting the immune effector cells with the particles delivers the nucleic acid to the immune effector cells.

21. The method according to any one of claims 17 to 20, wherein the genetically modified immune effector cells are present in vivo or in vitro.

22. The method according to any one of claims 18 to 21, wherein the genetically modified immune effector cells are present in vivo in a subject, and the method comprises administering the particles to the subject.

23. A pharmaceutical composition, (i) The peptide according to claim 1; (ii) The nucleic acid according to claim 2 or 13; (iii) The cells according to any one of claims 3 to 7 and 14 to 16; and (iv) The immune effector cell according to claim 8 A pharmaceutical composition containing one or more of the following.

24. A method for treating a subject, comprising administering the pharmaceutical composition described in claim 23 to the subject.

25. A method for treating a subject, comprising providing the subject with immune effector cells genetically modified to express a T cell receptor according to any one of claims 9 to 12.

26. The method according to claim 24 or 25, which is a method for inducing an immune response in the subject.

27. The method according to claim 26, wherein the immune response is a T cell-mediated immune response.

28. The method according to claim 26 or 27, wherein the immune response is an immune response against a population of target cells or a target tissue that expresses an antigen.

29. The method according to claim 28, wherein the target cell population or target tissue is cancer cells or cancer tissue.

30. The method according to claim 29, wherein the cancer cells or cancer tissue are solid tumors.

31. A method for treating a subject having a disease, disorder or condition associated with the expression or increased expression of an antigen, comprising providing the subject with genetically modified immune effector cells expressing a T cell receptor according to any one of claims 9 to 12, wherein the T cell receptor targets an antigen associated with the disease, disorder or condition or cells expressing an antigen associated with the disease, disorder or condition.

32. The method according to claim 31, wherein the disease, disorder or condition is cancer, and the antigen associated with the disease, disorder or condition is a tumor antigen.

33. The method according to claim 31 or 32, wherein the disease, disorder, or condition is a solid tumor.

34. The method according to any one of claims 25 to 33, wherein the immune effector cells genetically modified to express the T cell receptor are provided to the subject by administering the immune effector cells genetically modified to express the T cell receptor, or by generating the immune effector cells genetically modified to express the T cell receptor in the subject.

35. The method according to any one of claims 25 to 34, wherein the immune effector cells, which have been genetically modified to express the T cell receptor, are prepared by a method comprising delivering a nucleic acid encoding the T cell receptor to the immune effector cells.

36. The method according to any one of claims 25 to 35, wherein the immune effector cells, which have been genetically modified to express the T cell receptor, are prepared by a method comprising contacting the immune effector cells with particles containing nucleic acids encoding the T cell receptor.

37. The method according to claim 36, wherein the particles further comprise a targeting molecule for targeting the immune effector cells.

38. The method according to claim 36 or 37, wherein contacting the immune effector cells with the particles delivers the nucleic acid to the immune effector cells.

39. The method according to any one of claims 25 to 38, wherein the genetically modified immune effector cells are present in vivo or in vitro.

40. The method according to any one of claims 36 to 39, wherein the genetically modified immune effector cells are present in vivo in a subject, and the method comprises administering the particles to the subject.

41. The method according to any one of claims 24 to 40, which is a method for treating or preventing cancer in a subject.

42. The method according to claim 41, wherein the cancer is a solid tumor.

43. The method according to claim 41 or 42, wherein the cancer is related to the expression or increased expression of a tumor antigen targeted by the T cell receptor.

44. The method according to any one of claims 24 to 43, further comprising administering to the subject an antigen targeted by the T cell receptor, a polynucleotide encoding the antigen, or a host cell genetically modified to express the antigen.

45. The method according to claim 44, wherein the polynucleotide encoding the antigen is RNA.

46. The method according to claim 44, wherein a host cell genetically modified to express the antigen comprises a polynucleotide encoding the antigen.

47. The method according to any one of claims 17 to 22 and 25 to 46, wherein the immune effector cells genetically modified to express the T cell receptor include a polynucleotide encoding the T cell receptor.

48. The method according to claim 47, wherein the nucleic acid is RNA.

49. The method according to claim 47, wherein the nucleic acid is DNA.

50. The method according to any one of claims 17 to 22 and 25 to 49, wherein the gene modification is transient or stable.

51. The method according to any one of claims 17 to 22 and 25 to 50, wherein the gene modification is carried out by a virus-based method, a transposon-based method, or a gene editing-based method.

52. The method according to claim 51, wherein the gene editing-based method includes gene editing based on CRISPR.

53. The method according to any one of claims 18 to 22 and 36 to 52, wherein the particles are non-viral particles.

54. The method according to any one of claims 18 to 22 and 36 to 53, wherein the particles are lipid-based and / or polymer-based particles.

55. The method according to any one of claims 18 to 22 and 36 to 54, wherein the particles are nanoparticles.

56. The method according to any one of claims 18 to 22 and 36 to 55, wherein the particles are functionalized with a targeted molecule on their surface.

57. The method according to any one of claims 18 to 22 and 36 to 56, wherein the particles are functionalized with the targeted molecule by linking the targeted molecule to at least one particle-forming component.

58. The method according to any one of claims 19 to 22 and 37 to 57, wherein the targeted molecule targets CD8, CD4, or CD3.

59. The method according to any one of claims 17 to 22 and 25 to 58, wherein the immune effector cell is a T cell.

60. The method according to any one of claims 17 to 22 and 25 to 59, wherein the immune effector cells are CD4+ or CD8+ T cells.

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