Immunotherapy for treatment of cancer

Abstract

To provide a novel combinatorial immunotherapeutic approach for the treatment of cancer.SOLUTION: The present invention relates to a kit-of-parts and a composition, comprising a polyplex comprising a double stranded RNA (dsRNA) and a polymeric conjugate comprising a polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties and one or more targeting moieties, and at least one antibody, wherein each of one or more targeting moieties is capable of binding to a cancer antigen, and at least one antibody is capable of modulating an immune checkpoint protein. Further, the invention relates to this composition or kit-of-parts for use in the treatment of cancer.SELECTED DRAWING: None

Description

[Background technology]

[0001] The present invention relates to the field of cancer treatment by immunotherapy. In particular, the present invention relates to a kit of parts and a composition comprising a polyplex comprising double-stranded RNA (dsRNA) and a polymer complex comprising polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties, and at least one antibody, wherein each of the one or more targeting moieties is capable of binding to a cancer antigen, and the at least one antibody is capable of modulating an immune checkpoint protein. Furthermore, the present invention relates to this composition or kit of parts for use in cancer treatment.

[0002] Antibodies targeting tumor-associated antigens have become an important therapeutic approach for malignant tumors. Several monoclonal antibodies (mAbs) have proven relatively well tolerated and effective in treating many different malignancies. Although these antibodies are commonly used in clinics, their efficacy is often modest. mAbs must overcome substantial obstacles to reach antigens displayed on target cells and provide therapeutic value (Christiansen et al., Mol Cancer Ther, 2004, 3(11), 1493-1501). Furthermore, the efficacy of antibodies targeting tumor-associated antigens is reduced by insufficient activation of the immune system's antitumor response and by inhibition of the immune response induced by the tumor itself.

[0003] Checkpoint blockade antibodies targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have demonstrated acceptable toxicity, promising clinical responses, durable disease control, and improved survival in some patients with advanced melanoma, non-small cell lung cancer (NSCLC), and other tumor types. Engagement of PD-1 by either its ligand, PD-L1 or PD-L2, induces a negative control signal, resulting in inhibition of T cell proliferation, cytokine production, and cytotoxic activity (Ma et al., Current status and perspectives in translational biomarker research for PD-1 / PD-L1 immune checkpoint blockade therapy, Journal of Hematology & Oncology (2016) 9:47).

[0004] Tumor necrosis factor (TNF)-related cytokines provide an essential communication network for coordinating multiple cell types into an effective host defense system against pathogens and malignant cells. Tumor necrosis factor superfamily ligands (TNFSFs) and receptors (TNFRs) provide important communication signals between various cell types during development. TNF receptors (TNFRs) share a conserved ectodomain defined by a cysteine-rich signature. TNFRs with costimulatory capabilities are encoded by genes located within the immune response locus on chromosome 1p36 and include GITR (glucocorticoid-induced tumor necrosis factor), OX40, 4-1BB, and CD30 (Ward-Kavanagh, et al., The TNF Receptor Superfamily in Co-stimulating and Co-inhibitory Responses, Immunity44, May 17, 2016).

[0005] A different approach to treating malignant tumors is vaccine-based therapy. The molecular definition of tumor-associated antigens has introduced the possibility of specific vaccines aimed at targeting tumor cells. Recombinant vaccines based on peptides or proteins derived from defined tumor-associated antigens (TAA) are usually administered together with adjuvants or immunomodulators. Although these vaccines were able to induce antigen-specific T cell responses, clinical outcomes have been disappointing (Guo et al., Adv Cancer Res, 2013, 119:421-475).

[0006] A further approach in cancer immunotherapy involves combining vaccines with antibodies that activate antitumor immunity by blocking or inhibiting immune checkpoints. Immune checkpoints refer to the numerous inhibitory pathways connected to the immune system that are important for maintaining self-tolerance and regulating the duration and amplitude of physiological immune responses. Tumors exploit specific immune checkpoint pathways as a primary mechanism of immune tolerance. Because many immune checkpoints are initiated by ligand-receptor interactions, they can be easily blocked or inhibited by antibodies or regulated by recombinant forms of the ligand or receptor. The cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibody was the first immunotherapeutic agent in this class to receive U.S. Food and Drug Administration (FDA) approval. However, developing a combination antibody-vaccine approach remains a challenge. Combination strategies need to be intelligently designed and guided by mechanistic considerations and preclinical models (Pardoll, Nat Rev Cancer 2012, 12(4), 252-264).

[0007] Several novel combinations of immunotherapy in oncology have been suggested (Morrisey et al. Clin. Transl. Sci 2016, 9, 89-104), including non-antigen-specific immunotherapy with naked polyIC and a blocking antibody targeting the programmed cell death-1 (PD-1) pathway, which was able to inhibit tumors in mouse models of B16 melanoma, Lewis lung carcinoma, and MC38 colon carcinoma (Nagato et al., OncoImmunology 2014, 3:e28440).

[0008] Despite initial promising results, the development of immunotherapeutic agents for cancer remains a major challenge for tumor immunologists. Therefore, there is a great need for effective and well-tolerated immunotherapies for cancer treatment. Summary of the Invention

[0009] The present invention provides a novel combinatorial immunotherapeutic approach for the treatment of cancer.

[0010] In a first aspect, the present invention provides a kit-of-parts comprising: a. A polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, the polymer conjugate comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties; the PEI is covalently bound to one or more PEG moieties, each of which is linked to one of the one or more targeting moieties; a polyplex, wherein each of the one or more targeting moieties is capable of binding to a cancer antigen; and b. At least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein.

[0011] In a further aspect, the present invention provides a composition comprising: a. A polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, the polymer conjugate comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties; the PEI is covalently bound to one or more PEG moieties, each of which is linked to one of the one or more targeting moieties; a polyplex, wherein each of the one or more targeting moieties is capable of binding to a cancer antigen; and b. At least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein.

[0012] In a further aspect, the present invention provides a composition or kit-of-parts according to the present invention for use in treating cancer in a mammal.

[0013] The present inventors unexpectedly found that combined treatment of a polyplex comprising double-stranded RNA (dsRNA) and a polymer complex with one or more anti-checkpoint antibodies according to the present invention enhanced immune system activity and resulted in potent anti-tumor activity. Tumor growth was more potently inhibited by the combination of a polyplex comprising dsRNA and a polymer complex with one or more anti-checkpoint antibodies than by polyplex alone. In some individuals, the combination of a polyplex with an anti-checkpoint antibody completely eradicated tumors and generated persistent tumor defense and memory against cancer cells.

[0014] Interferon secretion was increased by the combination of the polyplex of the present invention and an anti-checkpoint antibody compared to polyplex alone. When immune cells, preferably peripheral blood mononuclear cells (PBMCs), were exposed to medium from EGFR-overexpressing cells treated with the polyplex of the present invention or cultured in the presence of cells treated with the polyplex of the present invention, the PBMCs were induced to secrete interferon. Addition of an anti-checkpoint antibody thereto further increased interferon secretion (see Figures 3 and 4).

[0015] In immunocompetent mice bearing HER2-overexpressing tumors, the mice showed complete tumor regression after combined treatment with an anti-checkpoint antibody, preferably a monoclonal anti-PD-1 antibody, and the polyplexes of the present invention. Tumor rechallenge in cured mice did not induce tumor growth, indicating that an immune response against the tumor had been generated.

[0016] Treatment of tumors with immunomodulatory antibodies alone has been found to be ineffective or to show only limited efficacy. Anti-PD-1 antibodies alone did not reduce tumor size in RENCA tumors (Figure 5). However, the combination of targeted, specifically delivered dsRNA with immunomodulatory antibodies demonstrated enhanced antitumor efficacy. Surprisingly, treatment of RENCA HER2 tumors with the HER2-targeting polyplex of the present invention, i.e., a triconjugate comprising PEI-PEG-HER2 affibody (PPHA) complexed with polyIC (pIC / PPHA), and anti-PD-1, resulted in tumor growth regression (Figure 6). Complete tumor regression was observed in the polyIC / PPHA + anti-PD-1 combination group. Cured mice showed complete protection from tumor rechallenge, indicating the generation of an immune response against the tumor.

[0017] Thus, the combination of targeted polyplexes according to the present invention with anti-checkpoint antibodies can extend the effectiveness of antibodies to patients who currently do not show any response. Utilizing targeted delivery of dsRNA, preferably PolyIC, in combination with anti-checkpoint antibodies shows significant efficacy due to the ability of the compositions and kits of parts of the present invention to restore the immune system against tumors. [Brief explanation of the drawings]

[0018] [Figure 1]IP-10 secretion from cancer cells after treatment with PEI-PEG-EGF / PolyIC. A431, U87, and MCF7 cells (40,000 cells / well) were treated with various concentrations of PEI-PEG-EGF / PolyIC (0.125, 0.25, 0.5, and 1 μg / ml) for 5 hours. Human IP-10 (CXCL10) secretion was higher in A431 EGFR-expressing cells compared with U87 and MCF7 cells, which express very little EGFR. [Figure 2] PD-L1 (used interchangeably herein, synonymous with "PDL1") expression in A431 cells after treatment with PEI-PEG-EGF / PolyIC. A431 cells were treated with PEI-PEG-EGF / PolyIC at a concentration of 0.125 μg / ml for 5 hours. PD-L1 expression was significantly increased after PEI-PEG-EGF / PolyIC treatment (MFI=751, (C)) compared to untreated control cells (MFI=431, (B)). An isotype control was used as a negative control (MFI=13, (A)). [Figure 3] Combination therapy of PEI-PEG-EGF / PolyIC with nivolumab significantly increased IFN-γ production by PBMCs. Combining PEI-PEG-EGF / PolyIC with nivolumab significantly increased IFN-γ production by PBMCs exposed to antigen for 48 hours in dilution medium containing PEI-PEG-EGF / PolyIC alone (0.125 μg / ml) or in combination with nivolumab (20 μg / ml). [Figure 4] Combining PEI-PEG-EGF / PolyIC with 4-1BB antibody significantly increased IFN-γ production by PBMCs. PBMCs were stimulated with CD3 (0.5 μg / ml) and cocultured for 16 hours with A431 cells treated with PEI-PEG-EGF / PolyIC alone (0.5 μg / ml) or in combination with antibody against 4-1BB (10 μg / ml). [Figure 5]Antitumor activity of RMP1-14 in treating subcutaneous RENCA mouse kidney cancer model: No antitumor activity of anti-PD-1 (used interchangeably herein and synonymous with anti-PD1) antibody (RMP1-14) in treating subcutaneous tumors in the RENCA mouse kidney cancer xenograft model (Syngeneic Models for Developing Cancer Therapeutics Targeting Immune System, Lan Zhang et al., EORTC-NCI-AACR International Conference on Molecular Targets and Cancer Therapeutics, November 18-21, 2014, Barcelona, Spain, P013). [Figure 6] The effects of polyplexes consisting of PolyIC and the chemical vector polyethyleneimine-polyethylene glycol (PP) conjugated to a HER2 affibody (HA) (abbreviated herein as PolyIC / PPHA) and anti-PD-1 (PolyIC / PPHA+PD-1) on subcutaneous RENCA HER2 tumor growth in immunocompetent mice (subcutaneous RENCA HER2 xenograft model). Mice bearing subcutaneous RENCA HER2 tumors were randomly divided into four groups of 7-8 animals per group with a mean tumor volume of 235 mm3. Mice were treated intravenously with PolyIC / PPHA, 6.25 mg / mouse, N / P8, every 24 hours. At the indicated time points, anti-PD-1 was injected intraperitoneally at 200 mg / mouse. There appeared to be no significant difference between PolyIC / PPHA alone and the combination of PolyIC / PPHA+anti-PD-1, although two mice in the PolyIC / PPHA+anti-PD-1 group remained tumor-free 50 days after the start of treatment. [Figure 7] Individual tumor volumes at day 21 in immunocompetent mice (subcutaneous RENCA HER2 xenograft model). Complete regression of tumor growth was observed in 2 of 8 mice in PolyIC / PPHA + anti-PD-1 treated animals. These mice remained tumor-free following re-challenge. [Figure 8]Increased expression of PD-L1 in RENCA HER2 cells after treatment with PolyIC / PPHA. Median raw values calculated using the X-axis channel: PE-A [Figure 9] Effect of PEI-PEG-EGF / PolyIC+anti-PD-1 on Renca EGFR lung metastasis in immunocompetent mice. [Figure 10] The combination of PEI-PEG-EGF / PolyIC polyplexes with nivolumab increases PBMC activation, as demonstrated by IFN-γ (used interchangeably herein and synonymous with IFNγ) ELISA. A431 or medium alone was treated with the indicated concentrations of PEI-PEG-EGF / PolyIC polyplexes for 5 hours. PBMCs were stimulated or unstimulated with anti-CD3, treated with nivolumab, or treated without nivolumab. Supernatants from PEI-PEG-EGF / PolyIC polyplex-treated or untreated (UT) A431 cancer cells or medium containing PEI-PEG-EGF / PolyIC polyplexes or untreated medium were then transferred to PBMCs. After overnight incubation, IFN-γ ELISA was performed to quantify PBMC activation. [Figure 11] PEI-PEG-EGF / PolyIC polyplexes induce cytokine secretion. Three cell lines, namely, high-EGFR (epidermal growth factor receptor)-expressing cells (MDA-MB-468 and A431) and low-EGFR-expressing cells (MCF7), were treated with the indicated concentrations of PEI-PEG-EGF / PolyIC polyplexes, PEI-PEG-EGF triconjugate, or pIC for 5 hours. The medium was then collected and analyzed for (A) IP10, (B) GROα, and (C) CCL5 (RANTES) using ELISA assays. [Figure 12]PEI-PEG-EGF / PolyIC polyplexes induce PBMC activation as measured by IFN-γ and TNFα ELISA. Three cell lines, namely MDA-MB-468 (high EGFR), A431 (high EGFR), and MCF7 (low EGFR), or medium alone, were treated with PEI-PEG-EGF / PolyIC, pIC, and PEI-PEG-EGF / pLGA polyplexes and treated with the indicated concentrations of pIC or pLGA (poly-L-glutamic acid) within the polyplexes for 5 hours. Supernatants and medium were collected and transferred to PBMCs for 16 hours. Supernatants from treated cancer cells without PBMCs were used as controls and were similarly incubated for 16 hours. IFN-γ and TNFα were quantified using ELISA after treatment of MDA-MB-468 (A, D), A431 (B, E), and MCF7 cells (C, F), respectively. [Figure 13] PEI-PEG-EGF / PolyIC polyplex treatment induces cancer cell death in EGFR-overexpressing cells with high efficacy and selectivity compared to control treatment. Cancer cells with differential EGFR expression levels were treated with the following reagents: PEI-PEG-EGF / PolyIC, PIC alone (naked PIC), JETPEI-PIC, RNAIMAX-PIC, and PEI-PEG-EGF / PLGA polyplex for 72 hours. The concentrations shown reflect the concentration of PIC or PLGA. Cell viability was analyzed using CELTITER-GLO. For each compound, the percentage survival was normalized to that of untreated cells. (A, B) High-EGFR-expressing cell lines: BT20, MDA-MB-468, A431, and HCC70; (C) moderate-EGFR-expressing cell lines: U87MG; (D) low-EGFR-expressing cell lines: U138 and MCF7; and (E) non-cancer cell lines: WI-38 and MCF10A. DETAILED DESCRIPTION OF THE INVENTION

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0020] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" or "include", as well as variations such as "comprises / includes" and "comprising / including", should be understood to imply the inclusion of elements, stated integers, steps or groups thereof, but not the exclusion of any other elements, stated integers, steps or groups thereof.

[0021] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0022] The term "about" or "approximately," when used in connection with a numerical value, is meant to encompass numerical values within a range having a lower limit of 0-10% less than the stated numerical value and an upper limit of 0-10% greater than the stated numerical value. The term "about" or "approximately" preferably means ±10%, more preferably ±5%, even more preferably ±3%, or most preferably ±0% (each with respect to a given numerical value). In each embodiment of the present invention, "about" can be omitted. All ranges of values disclosed herein refer to and include all values within that range, including the values defining the range.

[0023] In one aspect, the present invention refers to a composition comprising: a. A polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, the polymer conjugate comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties; the PEI is covalently bound to one or more PEG moieties, each of which is linked to one of the one or more targeting moieties; a polyplex, wherein each of the one or more targeting moieties is capable of binding to a cancer antigen; and b. At least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein.

[0024] In a further aspect, the present invention refers to a kit-of-parts comprising: a. A composition comprising a polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, the polymer conjugate comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties; the PEI is covalently bound to one or more PEG moieties, each of which is linked to one of the one or more targeting moieties; a composition, wherein each of the one or more targeting moieties is capable of binding to a cancer antigen; and b. At least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein.

[0025] In a preferred embodiment, the composition of the present invention comprises at least one pharmaceutically acceptable diluent, excipient, or carrier. In a particularly preferred embodiment, the composition according to the present invention is a fixed-dose composition comprising a polyplex and one or more immunomodulatory antibodies in a single dosage form. In another preferred embodiment, the pharmaceutical composition comprises one or more adjuvants.

[0026] The term "kit of parts" as used herein preferably refers to a combination of at least two separate parts, i.e., the polyplex and the one or more immunomodulatory antibodies. In a preferred embodiment, the composition is a pharmaceutical composition. The arrangement and construction of such kit of parts are conventionally known to those skilled in the art. In a particularly preferred embodiment, the kit of parts of the invention or components of the kit of parts of the invention, i.e., the polyplex and / or one or more immunomodulatory antibodies, independently comprise at least one pharmaceutically acceptable diluent, excipient, or carrier. In another preferred embodiment, the kit of parts of the invention or components of the kit of parts of the invention, i.e., the polyplex and / or one or more immunomodulatory antibodies, independently comprise one or more adjuvants.

[0027] In certain embodiments, the compositions and kits of parts according to the invention are formulated for administration by any known method. The compositions and kits of parts according to the invention, i.e., polyplexes and / or one or more immunomodulatory antibodies, and pharmaceutical compositions, may be formulated for any suitable route of administration, including, but not limited to, intravenous, intracerebral (intracerebral), oral, intramuscular, subcutaneous, transdermal, intradermal, transmucosal, intranasal, sublingual, intraperitoneal, or intraocular administration.

[0028] In another more preferred embodiment, the composition or kit-of-parts according to the present invention is formulated for systemic administration. Even more preferably, the composition and kit-of-parts according to the present invention, i.e., the polyplex and / or one or more immunomodulatory antibodies, are formulated for intravenous, intraperitoneal, or subcutaneous administration. More preferably, the composition and kit-of-parts according to the present invention, i.e., the polyplex and / or one or more immunomodulatory antibodies, are formulated in one or more dosage forms suitable for injection, preferably as a solution, emulsion, or suspension suitable for injection.

[0029] In one embodiment, the compositions and kits of parts according to the invention comprise a polyplex of the invention and one or more immunomodulatory antibodies, wherein the polyplex and the one or more immunomodulatory antibodies are present in the compositions and kits of parts in therapeutically effective amounts.

[0030] The kit-of-parts of the present invention may comprise a container containing a polyplex and / or one or more antibodies, and / or a device for administering the kit components, i.e., the polyplex and / or one or more antibodies. In a preferred embodiment, the kit-of-parts of the present invention comprises at least one container containing an effective amount of the polyplex, at least one container containing an effective amount of the one or more antibodies, and optionally instructions for use.

[0031] The terms "immune checkpoint protein" or "immune checkpoint" are known and described in the art (e.g., Pardoll, 2012, Nature Rev Cancer 12:252-264; Darvin et al., 2018, Experimental & Molecular Medicine 50:165). As used herein, the term "immune checkpoint protein" refers to receptors on T cells, B cells, and natural killer cells (NK), as well as their soluble ligands or binding ligands and counter-receptors that can stimulate or inhibit the activity of the immune system. In a preferred embodiment, the immune checkpoint protein refers to receptors on T cells and natural killer cells, as well as their soluble ligands or binding ligands and counter-receptors that can costimulate or co-inhibit the activity of the immune system. Preferably, the activity of the immune system is detected by measuring T cell responses, as shown herein (e.g., Example 2).

[0032] Immune checkpoint proteins are important immunoregulators in maintaining immune homeostasis and preventing autoimmunity. They consist of both stimulatory and inhibitory receptors and ligands that are important for maintaining self-tolerance and regulating the type, magnitude, and duration of immune responses. Immune homeostasis is regulated by a careful balance of activating and inhibitory immune checkpoint proteins. Under normal circumstances, immune checkpoints enable the immune system to respond to infections and malignancies while protecting tissues from any harm that may result from this action.

[0033] Tumor cells have developed several strategies to exploit these checkpoints and evade host immune defenses. Expression of immune checkpoint proteins can be dysregulated by tumors as an important immune resistance mechanism. Therefore, inhibition or activation of checkpoint receptors and ligands has emerged as a potential strategy to restrict tumor-infiltrating lymphocytes (TILs) that inhibit signals from tumors and circulating monocytes, block negative signals and cytokines that inhibit T cell activity, and stimulate systemic immunity.

[0034] Among the most promising approaches to activating therapeutic antitumor immunity is the blockade of inhibitory or activation of stimulatory immune checkpoint proteins. Immune checkpoints refer to inhibitory and activating proteins of the immune system that are important for regulating the duration and amplitude of physiological immune responses to maintain self-tolerance and minimize collateral tissue damage. Tumors exploit specific immune checkpoint pathways as a primary mechanism of immune tolerance, particularly to tumor antigen-specific T cells. T cells have been the primary focus of efforts to therapeutically manipulate endogenous antitumor immunity due to their ability to selectively recognize peptides derived from proteins in all cellular compartments; their ability to directly recognize and kill antigen-expressing cells (by CD8+ effector T cells; CTLs); and their ability to orchestrate diverse immune responses (by CD4+ helper T cells), including the integration of adaptive and innate effector mechanisms. Therefore, agonists of costimulatory receptors or antagonists of inhibitory signals, both of which lead to the amplification of antigen-specific T cell responses, are currently in clinical trials. Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be easily modulated by antibodies.

[0035] Preferably, the immune checkpoint protein is a human immune checkpoint protein.

[0036] In the present invention, an antibody capable of modulating an immune checkpoint protein is any compound that modulates the function of an immune checkpoint protein and thus promotes immune system activity. Promotion of immune system activity includes the generation of an enhanced immune response to an antigen and / or the reduction of an immunosuppressive immune response to an antigen. Preferably, promotion of immune system activity results in immune-mediated elimination of tumor cells.

[0037] The term "modulate" (or "modulator") includes activation, with respect to functional stimulation or enhancement of costimulatory immune checkpoint proteins, and inhibition, with respect to reduced activity and complete blockage of costimulatory immune checkpoint proteins. The term "modulating an immune checkpoint protein" includes stimulation of T cells, including T helper cells, CTLs (natural killer T cells), and natural killer (NK) cells. Stimulation (or activation) induced by modulation of immune checkpoint proteins is preferably detected by measuring increased levels of cytokines, such as interferons, particularly IFN-γ, produced or released, particularly by T cells and NK cells, as shown herein (e.g., Example 2), compared to controls not administered the immune checkpoint protein-modulating antibody.

[0038] Examples of immune checkpoint proteins include PD-1 (programmed death 1, used interchangeably herein and synonymous with PD1), PD-L1, PD-L2, CTLA-4 / B7-1 / CD152 (cytotoxic T lymphocyte-associated protein 4), CD137 / 4-1BB, 4-1BBL / CD137L, TIM-3 (T-cell immunoglobulin domain and mucin domain 3), LAG3, By-He, H4, HAVCR2, ID01, C D40 / TNFRSF5, CD40L / CD154 / TNFSF5, OX40 / CD134, OX-40L / TNFSF4 / CD252, GITR (glucocorticoid-inducible TNFR family related gene) / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, ICOS ligand / B7-H2, CD122, CD155 / PVR, CD226 / DNAM-1, CD27, HVEM / TNFRSF14, TNFSF14 / LIGHT / CD258, CD70 / CD27L / TNFSF7, CD28 / TP44, CD80 / B7-1, CD86 / B7-2, A2AR, KIR (killer cell immunoglobulin-like receptor), NOX2 / nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2, SIGLEC7 (sialic acid-binding immunoglobulin-type lectin 7) / CD328, SIGL EC9 (sialic acid-binding immunoglobulin-type lectin 9) / CD329, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, VTCN1 / B7-H4 / B7S1 / 7x, VISTA (V-domain Ig suppressor of T-cell activation) / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, indoleamine 2,3-dioxygenase-dioxygenase / IDO, TDO / tryptophan 2,3-dioxygenase, galectin- / LGALS9, TIM-3 / HAVCR2, TIGIT / VSTM3, HVEM (herpes virus entry mediator) / TNFRSF14, BTLA (B and T lymphocyte attenuator) / CD272, CD160, CEACAM1 / CD66a, indoleamine, SIRP, alpha / CD172a, CD47, CD48 / SLAMF2, CD30, CD30L, TMIGD2, HHLA2, TL1A, DR3, LTβR, TNF, TNFR2, and 2B4 / CD244, and are preferably selected from the group consisting of the above.

[0039] In a preferred embodiment, the immune checkpoint protein is a protein of the B7-CD28 family or the TNFR family. In a preferred embodiment, the immune checkpoint protein is a T cell-associated checkpoint inhibitor or a non-T cell-associated checkpoint inhibitor.

[0040] In a preferred embodiment, the immune checkpoint protein is CD137 / 4-1BB, 4-1BBL / CD137L, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, OX40 / CD134, OX-40L / TNFSF4 / CD252, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS (inducible T cell costimulator) / AILIM / CD278, ICOS ligand / B7-H2, CD122, A2AR (adenosine A2A receptor) , KIR, NOX2, SIGLEC7 / CD328, SIGLEC9 / CD329, PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, 2,3-dioxygenase / IDO, galectin- / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3. In a further preferred embodiment, the immune checkpoint protein is CD137 / 4-1BB, 4-1BBL / CD137L, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, OX40 / CD134, OX-40L / TNFSF4 / CD252, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, ICOS ligand and B7-H2, PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, galectin-9 / LGALSAL9, TIM-3 / HAVCR2, and TIGIT / VSTM3. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, 4-1BB ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS, ICOS ligand, CD40, and CD40 ligand.In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, 4-1BB ligand, TIGIT, LAG3, TIM3, GITR, GITR ligand, CD40, CD40L, OX40, OX-40L, ICOS, and ICOS ligand. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM-3, GITR, CD40, OX40, and ICOS.

[0041] In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR, OX40, OX-40L, ICOS, and CD40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM3, GITR, CD40, OX40, and ICOS. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR, OX40, OX-40L, ICOS, and CD40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, TIGIT, LAG3, TIM3, GITR, CD40, OX40, and ICOS.

[0042] In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, LAG3, TIM3, GITR, CD40, OX40, and ICOS. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR, CD40, OX40, and ICOS. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, LAG3, TIM3, GITR, CD40, OX40, and ICOS. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, LAG3, TIM3, GITR, CD40, and OX40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, LAG3, TIM3, GITR, CD40 and OX40.

[0043] In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40, and OX40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR, CD40, and OX40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40, and OX40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, and OX40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, and OX40.

[0044] In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, LAG3, and TIM3. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, LAG3, and TIM3.

[0045] In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, TIGIT, LAG3, TIM-3, GITR, and ICOS. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, LAG3, TIGIT, TIM-3, and GITR. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, CTLA-4, 4-1BB, LAG3, TIGIT, TIM-3, GITR, and ICOS. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, and 4-1BB. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, and 4-1BB. In a further preferred embodiment, the immune checkpoint protein is PD-1 or 4-1BB. In a further preferred embodiment, the immune checkpoint protein is PD-1. In another preferred embodiment, the immune checkpoint protein is PD-1, PD-L1, or PD-L2. In a further preferred embodiment, the immune checkpoint protein is PD-1 or PD-L1. In a further preferred embodiment, the immune checkpoint protein is PD-1. In a further preferred embodiment, the immune checkpoint protein is PD-L1. In a further preferred embodiment, the immune checkpoint protein is PD-L2. In a further preferred embodiment, the immune checkpoint protein is 4-1BB. In another preferred embodiment, the immune checkpoint protein is CTLA-4.

[0046] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that selectively binds to an antigen, where the antigen includes a hapten, epitope, receptor, or ligand, or portions thereof. Thus, the term "antibody" encompasses not only whole antibody molecules, but also antibody fragments, as well as variants (including derivatives), antibody fragments, and fusion proteins. The term "antibody" also refers to various forms, including antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as full-length antibodies and portions thereof, such as immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab', F(ab'), Fv, disulfide-linked Fv, scFv, single-domain antibodies (dAbs), diabodies, naked antibodies, antibody-drug conjugates, and bispecific or trispecific antibodies, anti-idiotypic antibodies, anticalins, and functionally active epitope-binding fragments thereof. In particularly preferred embodiments, the antibody capable of modulating an immune checkpoint protein is a monoclonal antibody, a humanized antibody, or a fully human antibody. As used herein, the term "antibody capable of modulating an immune checkpoint protein" refers to an antibody that modulates the activity of a checkpoint receptor, counter-receptor, or their bound and soluble ligands.

[0047] As used herein, the term "anti-..." relates to an antibody that selectively binds to the target mentioned after the term "anti."

[0048] As used herein, the term "affibody" refers to a protein designed to bind to a target protein or peptide with high affinity, mimicking a monoclonal antibody, and thus is a member of the antibody mimetic family. Preferably, the affibody has a high-affinity binding domain derived from Protein A. HER2 is the target of HER2 affibodies.

[0049] In a preferred embodiment, the HER2 affibody comprises an affibody selected from the group consisting of ZHER2:2891, ABY-025, ZHER2:342, and ZHER2:2395, preferably ZHER2:2891.

[0050] Preferably, the antibody capable of modulating an immune checkpoint protein is a monoclonal antibody, a chimerized antibody, a humanized antibody, a human antibody, a fusion protein, or a combination thereof.

[0051] In a further aspect, the present invention provides a kit-of-parts comprising: (a) a polyplex comprising double-stranded RNA (dsRNA) and a polymer conjugate, the polymer conjugate comprising polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties, wherein the PEI is covalently bound to one or more PEG moieties, each of the one or more PEG moieties is linked to one of the one or more targeting moieties, and each of the one or more targeting moieties is capable of binding to a cancer antigen; and (b) at least one antibody. In yet a further aspect, the present invention provides a composition comprising: (a) a polyplex comprising double-stranded RNA (dsRNA) and a polymer conjugate, the polymer conjugate comprising polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties, wherein the PEI is covalently bound to one or more PEG moieties, each of the one or more PEG moieties is linked to one of the one or more targeting moieties, and each of the one or more targeting moieties is capable of binding to a cancer antigen; and (b) at least one antibody. In a preferred embodiment, the antibody is anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, CD40 / TNFRSF5, anti-CD40L / CD154 / TNFSF5, anti-OX40 / CD134, anti-OX-40L / TNFSF4 / CD252, anti-GITR / TNFRSF18, anti-GITR ligand / TNFSF18, anti-ICOS (inducible T cell costimulator) / AILIM / CD278, anti-ICOS ligand / B7-H2, anti-CD122, anti-A2AR (adenosine A2A receptor), anti-KIR, anti-NOX2 , anti-SIGLEC7 / CD328, anti-SIGLEC9 / CD329, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-CD80 / B7-1, anti-CD86 / B7-2, anti-B7-H3 / CD276, anti-B7-H4 / B7S1 / 7x, anti-VISTA / B7-H5 / GI24, anti-LAG3 / CD223 / lymphocyte activation gene 3, anti-2,3-dioxygenase / IDO, anti-galectin- / LGALS9, anti-TIM-3 / HAVCR2, and anti-TIGIT / VSTM3.In a further preferred embodiment, the antibody is anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, anti-CD40 / TNFRSF5, anti-CD40L / CD154 / TNFSF5, anti-OX40 / CD134, anti-OX-40L / TNFSF4 / CD252, anti-GITR / TNFRSF18, anti-GITR ligand / TNFSF18, anti-ICOS / AILIM / CD278, anti-ICOS ligand / B7-H 2, selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-CD80 / B7-1, anti-CD86 / B7-2, anti-B7-H3 / CD276, anti-B7-H4 / B7S1 / 7x, anti-VISTA / B7-H5 / GI24, anti-LAG3 / CD223 / lymphocyte activation gene 3, anti-galectin-9 / LGALS9, anti-TIM-3 / HAVCR2, and anti-TIGIT / VSTM3. In a more preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-4-1BB ligand (4-1BBL), anti-TIGIT, anti-LAG3, anti-TIM3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR4, anti-GITR ligand, anti-GITR, anti-OX40, anti-OX-40L, anti-ICOS, anti-ICOS ligand, anti-CD40, and anti-CD40 ligand. In a more preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-4-1BB ligand, anti-TIGIT, anti-LAG3, anti-TIM3, anti-GITR, anti-GITR ligand, anti-CD40, anti-CD40L, anti-OX40, anti-OX-40L, anti-ICOS, and anti-ICOS ligand.

[0052] In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR4, anti-GITR, anti-OX40, anti-ICOS, and anti-CD40. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM3, anti-GITR, anti-CD40, anti-OX40, and anti-ICOS. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM3, anti-GITR, anti-CD40, anti-OX40, and anti-ICOS. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, anti-CD40, anti-OX40, and anti-ICOS. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, and anti-ICOS. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1BB, anti-LAG3, anti-TIGIT, anti-TIM-3, anti-GITR, and anti-ICOS. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-CTLA-4, anti-4-1BB, anti-LAG3, anti-TIGIT, anti-TIM-3, anti-GITR, and anti-ICOS. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1BB, anti-GITR, anti-CD40, anti-ICOS, and anti-OX40. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1BB, anti-GITR, anti-CD40, and anti-OX40.In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1BB, anti-GITR, and anti-OX40. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1BB, and anti-OX40. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, and anti-4-1BB. In a further preferred embodiment, the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-4-1BB. In a further preferred embodiment, the antibody is anti-PD-1 or anti-4-1BB. In a further preferred embodiment, the antibody is anti-PD-1. In another preferred embodiment, the antibody is anti-PD-1, anti-PD-L1, or anti-PD-L2. In a further preferred embodiment, the antibody is anti-PD-1 or anti-PD-L1. In a further preferred embodiment, the antibody is anti-PD-1. In a further preferred embodiment, the antibody is anti-PD-L1. In a further preferred embodiment, the antibody is anti-PD-L2. In a further preferred embodiment, the antibody is anti-4-1BB. In another preferred embodiment, the antibody is anti-CTLA-4.

[0053] In a further preferred embodiment, the at least one antibody is (i) anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, anti-CD40 / TNFRSF5, anti-CD40L / CD154 / TNFSF5, anti-OX40 / CD134, anti-OX-40L / TNFSF4 / CD252, anti-GITR / TNFRSF18, anti-GITR ligand / TNFSF18, anti-ICOS / AILIM / CD278, and anti-ICOS ligand / B7-H2; or (ii) anti-PD-1, anti-PD anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-CD80 / B7-1, anti-CD86 / B7-2, anti-B7-H3 / CD276, anti-B7-H4 / B7S1 / 7x, anti-VISTA / B7-H5 / GI24, anti-LAG3 / CD223 / lymphocyte activation gene 3, anti-galectin- / LGALS9, anti-TIM-3 / HAVCR2, and anti-TIGIT / VSTM3; or (iii) a mixture of at least one antibody in (i) and at least one antibody in (ii). In a further preferred embodiment, the at least one antibody is (i) anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, anti-GITR / TNFRSF18, anti-GITR ligand / TNFSF18, anti-ICOS / AILIM / CD278, and anti-ICOS ligand / B7-H2; or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-CD80 / B7-1, anti-CD 86 / B7-2, anti-B7-H3 / CD276, anti-B7-H4 / B7S1 / 7x, anti-VISTA / B7-H5 / GI24, anti-LAG3 / CD223 / lymphocyte activation gene 3, anti-galectin- / LGALS9, anti-TIM-3 / HAVCR2, and anti-TIGIT / VSTM3; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

[0054] In a further preferred embodiment, the at least one antibody is (i) anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, anti-CD40 / TNFRSF5, anti-CD40L / CD154 / TNFSF5, anti-OX40 / CD134, anti-OX-40L / TNFSF4 / CD252, anti-GITR / TNFRSF18, anti-GITR ligand / TNFSF18, anti-ICOS / AILIM / CD278, and anti-ICOS ligand / B7-H2; or (ii) ), anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-H3 / CD276, anti-B7-H4 / B7S1 / 7x, anti-VISTA / B7-H5 / GI24, anti-LAG3 / CD223 / lymphocyte activation gene 3, anti-galectin- / LGALS9, anti-TIM-3 / HAVCR2, and anti-TIGIT / VSTM3; or (iii) a mixture of at least one antibody in (i) and at least one antibody in (ii). In a further preferred embodiment, the at least one antibody is selected from the group consisting of: (i) anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, anti-GITR / TNFRSF18, anti-GITR Ligand / TNFSF18, anti-ICOS / AILIM / CD278, and anti-ICOS Ligand / B7-H2; or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-H3 / CD276, anti-B7-H4 / B7S1 / 7x, anti-VISTA / B7-H5 / GI24, anti-LAG3 / CD223 / lymphocyte activation gene 3, anti-galectin- / LGALS9, anti-TIM-3 / HAVCR2, and anti-TIGIT / VSTM3; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).In a further preferred embodiment, the at least one antibody is selected from the group consisting of: (i) anti-CD137 / 4-1BB, anti-CD40 / TNFRSF5, anti-OX40 / CD134, anti-GITR / TNFRSF18, anti-ICOS / AILIM / CD278; or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-H3 / CD276, anti-B7-H4 / B7S1 / 7x, anti-VISTA / B7-H5 / GI24, anti-LAG3 / CD223 / lymphocyte activation gene 3, anti-galectin- / LGALS9, anti-TIM-3 / HAVCR2, and anti-TIGIT / VSTM3; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a further preferred embodiment, the at least one antibody is selected from the group consisting of (i) anti-CD137 / 4-1BB, anti-CD40 / TNFRSF5, anti-OX40 / CD134, anti-GITR / TNFRSF18, anti-ICOS / AILIM / CD278, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, or (iii) a mixture of at least one antibody in (i) and at least one antibody in (ii). In a further preferred embodiment, the at least one antibody is selected from the group consisting of (i) anti-CD137 / 4-1BB, anti-CD40 / TNFRSF5, anti-OX40 / CD134, anti-GITR / TNFRSF18, anti-ICOS / AILIM / CD278, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, or (iii) a mixture of at least one antibody in (i) and at least one antibody in (ii). In a further preferred embodiment, the at least one antibody is selected from the group consisting of (i) anti-CD137 / 4-1BB, anti-CD40 / TNFRSF5, anti-OX40 / CD134, anti-GITR / TNFRSF18, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, or (iii) a mixture of at least one antibody in (i) and at least one antibody in (ii). In a further preferred embodiment, the at least one antibody is selected from the group consisting of (i) anti-CD137 / 4-1BB, anti-CD40 / TNFRSF5, anti-OX40 / CD134, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, or (iii) a mixture of at least one antibody in (i) and at least one antibody in (ii).In a further preferred embodiment, the at least one antibody is selected from the group consisting of: (i) anti-CD137 / 4-1BB, anti-OX40 / CD134; or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2; or (iii) a mixture of at least one antibody in (i) and at least one antibody in (ii).

[0055] In a further preferred embodiment, the at least one antibody is selected from the group consisting of: (i) anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, anti-GITR / TNFRSF18, anti-GITR Ligand / TNFSF18, anti-ICOS / AILIM / CD278, and anti-ICOS Ligand / B7-H2; or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-LAG3 / CD223 / Lymphocyte Activation Gene 3, anti-TIM-3 / HAVCR2, and anti-TIGIT / VSTM3; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a further preferred embodiment, the at least one antibody is selected from the group consisting of (i) anti-4-1BB, anti-GITR, anti-OX40, anti-ICOS, and anti-CD40, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-TIGIT, anti-LAG3, anti-TIM3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR4, and anti-GITRL, or (iii) a mixture of at least one antibody of (i) with at least one antibody of (ii). In a further preferred embodiment, the at least one antibody is selected from the group consisting of (i) anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-CTLA-4, or (iii) a mixture of at least one antibody of (i) with at least one antibody of (ii). In a further preferred embodiment, the at least one antibody is selected from the group consisting of: (i) anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, or (iii) a mixture of at least one antibody in (i) and at least one antibody in (ii).

[0056] In a preferred embodiment, the antibody capable of modulating an immune checkpoint protein is a bispecific antibody capable of binding to a cancer antigen and an immune checkpoint protein. Preferably, the cancer antigen is EGFR, HER2, or PSMA. More preferably, the cancer antigen is EGFR or HER2, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, and 4-1BB. More preferably, the cancer antigen is EGFR or HER2, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, and 4-1BB. Even more preferably, the cancer antigen is EGFR or HER2, and the immune checkpoint protein is PD-1 or 4-1BB. Even more preferably, the cancer antigen is HER2 or EGFR, and the immune checkpoint protein is 4-1BB. In another preferred embodiment, the cancer antigen is HER2, and the immune checkpoint protein is 4-1BB. In another preferred embodiment, the cancer antigen is PSMA and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, and 4-1BB. In another preferred embodiment, the cancer antigen is EGFR and the immune checkpoint protein is 4-1BB. In a preferred embodiment, the antibody capable of modulating immune checkpoint proteins is a lipocalin that binds to 4-1BB and HER2.

[0057] Preferred examples of such antibodies capable of modulating the immune checkpoint protein PD-1 (anti-PD-1) are pembrolizumab, nivolumab (also known as MDX-1106 or BMS-936558, disclosed in Topalian et al., 2012. N. Eng. J. Med. 366:2443-2454, U.S. Pat. No. 8,008,449 B2), cemiplimab, IBI308, BCD-100, PDR001, tislelizumab, canrelizumab, pidilizumab (Rosenblatt et al., 2011, J. Immunol. 2011, 14:147-148, 2011). Immunother. 34:409-18), and lambrolizumab (e.g., disclosed as hPD109A and its humanized derivatives h409All, h409A16, and h409A17 in WO 2008 / 156712; Hamid et al., 2013, N. Engl. J. Med. 369:134-144), and soluble PD-1 ligands, including, but not limited to, PD-L2 Fc fusion protein (also known as B7-DC-Ig or AMP-244; disclosed in Mkrtichyan M, et al., 2012, J. Immunol. 189:2338-47). More preferred examples of such antibodies capable of modulating the immune checkpoint protein PD-1 (anti-PD-1) are pembrolizumab and nivolumab.

[0058] Preferred examples of the antibody capable of modulating the immune checkpoint protein PD-L1 (anti-PD-L1) are antibodies selected from the group consisting of durvalumab, avelumab, atezolizumab, MEDI-4736 (e.g., as disclosed in WO 2011 / 066389A1), MPDL328 OA (e.g., as disclosed in U.S. Pat. No. 8,217,149B2), and MIH1 (Affymetrix). More preferred examples of the antibody capable of modulating the immune checkpoint protein PD-L1 (anti-PD-L1) are antibodies selected from the group consisting of durvalumab, avelumab, and atezolizumab.

[0059] Preferred examples of such antibodies capable of modulating the immune checkpoint protein CTLA-4 are ipilimumab or tremelimumab, more preferably ipilimumab. Ipilimumab is a fully human CTLA-4 blocking antibody currently marketed under the name Yervoy (Bristol-Myers Squibb). A further CTLA-4 inhibitor is tremelimumab (see Ribas et al., 2013, J. Clin. Oncol. 31:616-22).

[0060] A preferred example of such an antibody capable of modulating the immune checkpoint protein CD27 is CDX-1127, an agonistic anti-CD27 monoclonal antibody.

[0061] Preferred examples of such antibodies capable of modulating the immune checkpoint protein OX40 are the humanized OX40 agonist MEDI0562, the murine OX4 agonist MEDI6469, and the OX40 agonist MEDI6383.

[0062] A preferred example of the antibody capable of regulating the immune checkpoint protein KIR is lirilumab, a monoclonal antibody against KIR.

[0063] A preferred example of the antibody capable of modulating the immune checkpoint protein 4-1BB is urelumab.A preferred example of the antibody capable of modulating the immune checkpoint protein LAG-3 is leratolimab.

[0064] A preferred example of such an antibody capable of modulating the immune checkpoint protein LAG3 is the monoclonal antibody BMS-986016.

[0065] In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, IBI308, BCD-100, PDR001, tislelizumab, camrelizumab, pidilizumab, lambrolizumab, h409All, h409A16, h409A17, soluble PD-1 ligands such as PD-L2 Fc fusion proteins, durvalumab, avelumab, atezolizumab, MEDI-4736, MPDL328 OA, CDX-1127, MIH1, MEDI0562, MEDI6469, MEDI6383, ipilimumab, tremelimumab, leratolimab, urelumab, anti-TIGIT antibodies, anti-TIM3 antibodies, anti-GITR antibodies, and anti-ICOS antibodies. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, IBI308, BMS-986016, BCD-100, PDR001, tislelizumab, camrelizumab, pidilizumab, lambrolizumab, h409All, h409A16, h409A17, soluble PD-1 ligands such as PD-L2 Fc fusion proteins, durvalumab, avelumab, atezolizumab, lirilumab, MEDI-4736, MPDL328 OA, MIH1, ipilimumab, tremelimumab, leratolimab, and urelumab. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, IBI308, BCD-100, PDR001, tislelizumab, camrelizumab, pidilizumab, lambrolizumab, h409All, h409A16, h409A17, soluble PD-1 ligands such as PD-L2 Fc fusion proteins, durvalumab, avelumab, atezolizumab, MEDI-4736, MPDL328 OA, MIH1, ipilimumab, tremelimumab, and urelumab.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, IBI308, BCD-100, PDR001, tislelizumab, camrelizumab, pidilizumab, lambrolizumab, h409All, h409A16, h409A17, soluble PD-1 ligands such as PD-L2 Fc fusion proteins, durvalumab, avelumab, atezolizumab, MEDI-4736, MPDL328 OA, MIH1, tremelimumab, and urelumab. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, IBI308, BCD-100, PDR001, tislelizumab, camrelizumab, pidilizumab, lambrolizumab, h409All, h409A16, h409A17, a soluble PD-1 ligand such as a PD-L2 Fc fusion protein, durvalumab, avelumab, atezolizumab, MEDI-4736, MPDL328 OA, and MIH1.

[0066] In preferred embodiments, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing (stimulating) a costimulatory immune checkpoint protein (referred to herein as a checkpoint activator), (ii) at least one antibody capable of antagonizing an inhibitory immune checkpoint protein (referred to herein as a checkpoint inhibitor), or (iii) a mixture of both (i) and (ii). In preferred embodiments, the at least one immune checkpoint modulating antibody capable of modulating an immune checkpoint protein is at least one antibody capable of agonizing a costimulatory immune checkpoint protein (checkpoint activator). The checkpoint activator activates the costimulatory immune checkpoint protein. Checkpoint activators deliver activation signals to T cells, B cells, or natural killer cells by agonizing directly on receptors on the cell types, by inhibiting or blocking inhibitory ligands, or by agonizing on counter-receptors on antigen-presenting cells (APCs).

[0067] In a preferred embodiment, the at least one immune checkpoint-modulating antibody capable of modulating an immune checkpoint protein is at least one antibody capable of antagonizing an inhibitory immune checkpoint protein (checkpoint inhibitor). The checkpoint inhibitor disinhibits, i.e., reduces or eliminates, the inhibitory function of the inhibitory immune checkpoint protein. Checkpoint inhibitors deliver an antagonistic signal to T cells, B cells, or natural killer cells by directly antagonizing receptors on the cell types, agonizing inhibitory ligands, or antagonizing counter-receptors on antigen-presenting cells (APCs).

[0068] In another preferred embodiment, the at least one antibody capable of modulating immune checkpoint proteins is an immune checkpoint activator or inhibitor. In another preferred embodiment, the at least one antibody capable of modulating immune checkpoint proteins is an immune checkpoint activator and inhibitor.

[0069] In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is CD155 / PVR, CD226 / DNAM-1, CD137 / 4-1BB, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, 4-1BBL / CD137L, OX40 / CD134, OX-40L / TNF SF4 / CD252, CD27, CD122, HVEM / TNFRSF14, TNFSF14 / LIGHT / CD258, CD70 / CD27L / TNFSF7, CD28 / TP44, CD30, CD30 ligand, TMIGD2, HHLA2, CD 80 / B7-1, CD86 / B7-2, GITR / TNFRSF18, GITR ligand / TNFSF18, DR3, TL1A, CD30, CD30L, TMIGD2, HHLA2, TL1A, DR3, LTβR, TNF, TNFR2, ICOS / AI or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, HVEM / TNFRSF14, BTLA, CD160. , LAG3 / CD223 / lymphocyte activation gene 3, CEACAM1 / CD66a, indoleamine, 2,3-dioxygenase / IDO, galectin- / LGALS9, TIM-3 / HAVCR2, 2B4 / CD244, SIRP, alpha / CD172a, CD47, CD48 / SLAMF2, TIGIT / VSTM3, A2AR, KIR, NOX2, SIGLEC7 / CD328, SIGLEC9 / CD329, and / or (iii) a mixture of both.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD155 / PVR, CD226 / DNAM-1, CD137 / 4-1BB, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, 4-1BBL / CD137L, OX40 / CD134, OX-40 L / TNFSF4 / CD252, CD27, CD122, HVEM / TNFRSF14, TNFSF14 / LIGHT / CD258, CD70 / CD27L / TNFSF7, CD28 / TP44, CD30, CD30 ligand, TMIGD2, H HLA2, CD80 / B7-1, CD86 / B7-2, GITR / TNFRSF18, GITR ligand / TNFSF18, DR3, TL1A, CD30, CD30L, TMIGD2, HHLA2, TL1A, DR3, LTβR, TNF, TNF R2, ICOS / AILIM / CD278, and ICOS ligand / B7-H2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, HVEM / TNFRSF14, BTLA, CD160, LAG3. / CD223 / lymphocyte activation gene 3, CEACAM1 / CD66a, indoleamine, 2,3-dioxygenase / IDO, galectin- / LGALS9, TIM-3 / HAVCR2, 2B4 / CD244, SIRP, alpha / CD172a, CD47, CD48 / SLAMF2, TIGIT / VSTM3, A2AR, KIR, NOX2, SIGLEC7 / CD328, SIGLEC9 / CD329, and / or (iii) a mixture of both.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD155 / PVR, CD226 / DNAM-1, CD137 / 4-1BB, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, 4-1BBL / CD137L, OX40 / CD134, OX-40L / TNFSF4 / CD252, CD27, HVEM / TNFRSF14, TNFSF14 / LIGHT / CD258, CD70 / CD27L / TNFSF7, C D28 / TP44, CD80 / B7-1, CD86 / B7-2, GITR / TNFRSF18, GITR ligand / TNFSF18, CD30, CD30L, TMIGD2, HHLA2, TL1A, DR3, LTβR, an antibody selected from the group consisting of TNF, TNFR2, ICOS / AILIM / CD278, and ICOS ligand / B7-H2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S. 1 / 7x, VISTA / B7-H5 / GI24, HVEM / TNFRSF14, BTLA, CD160, LAG3 / CD223 / lymphocyte activation gene 3, CEACAM1 / CD66a, indoleamine, galectin- / LGALS9, TIM-3 / HAVCR2, 2B4 / CD244, SIRP, alpha / CD172a, CD47, CD48 / SLAMF2, and TIGIT / VSTM3.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD155 / PVR, CD226 / DNAM-1, CD137 / 4-1BB, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, 4-1BBL / CD137L, OX40 / CD134, OX-40L / TNFSF4 / CD252, CD27, HVEM / TNFRSF14, TNFSF14 / LIGHT / CD258, CD70 / CD27L / TNFSF7, CD28 / TP44, CD80 / B7-1, CD86 / B7-2, GITR / TNFRSF18, GITR ligand / TNFSF18, CD30, CD30L, TMIGD2, HHLA2, and TL1A. , DR3, LTβR, TNF, TNFR2, ICOS / AILIM / CD278, and ICOS ligand / B7-H2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3 / CD276, B7-H4 / B7S1 / 7x, The antibody is selected from the group consisting of VISTA / B7-H5 / GI24, HVEM / TNFRSF14, BTLA, CD160, LAG3 / CD223 / lymphocyte activation gene 3, CEACAM1 / CD66a, indoleamine, galectin- / LGALS9, TIM-3 / HAVCR2, 2B4 / CD244, SIRP, alpha / CD172a, CD47, CD48 / SLAMF2, and TIGIT / VSTM3.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, OX40 / CD134, OX-40L / TNFSF4 / CD252, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and ICOS ligand / B7- H2, or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, galectin- / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3, or (iii) a mixture of both.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and ICOS ligand / B7-H2, or (ii) at least one antibody capable of agonizing a co-inhibitory immune checkpoint protein. (iii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, galectin- / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3; or (iv) a mixture of both.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, OX40 / CD134, OX-40L / TNFSF4 / CD252, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and IC or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, galectin- / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3, or (iii) a mixture of both. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and ICOS ligand / B7-H2, or (ii) an antibody capable of antagonizing a co-inhibitory immune checkpoint protein. wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, galectin- / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3, or (iii) a mixture of both.

[0070] In a preferred embodiment of the composition or kit-of-parts of the present invention, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is 4-1BB, 4-1BB ligand (4-1BBL), CD40, CD40 ligand (CD40L), OX40, OX-40 ligand (OX-40L), GITR, GITR ligand (GITRL), ICOS, and ICOS ligand. (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, B7-H3, B7-H4, VISTA, LAG-3, Galectin-9, TIM-3, and TIGIT; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

[0071] In a preferred embodiment of the composition or kit-of-parts of the present invention, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, 4-1BB ligand (4-1BBL), CD40, CD40 ligand (CD40L), OX40, OX-40 ligand (OX-40L), GITR, GITR ligand (GITRL), ICOS, and ICOS ligand (ICOSL), or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3, and TIGIT, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a further preferred embodiment of the composition or kit-of-parts of the present invention, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, CD40, OX40, GITR, and ICOS, or (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, wherein the coinhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3, and TIGIT, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).In a further preferred embodiment of the composition or kit-of-parts of the invention, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, CD40, OX40, GITR, and ICOS, or (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, wherein the coinhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3, and TIGIT, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

[0072] In a further preferred embodiment of the composition or kit-of-parts of the present invention, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, CD40, OX40, and GITR, or (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, wherein the coinhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3, and TIGIT, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

[0073] In a further preferred embodiment of the composition or kit-of-parts of the present invention, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, CD40, OX40, ICOS, and GITR, or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

[0074] In a further preferred embodiment of the composition or kit of parts of the invention, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of activating a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, CD40, OX40, and GITR; or (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, wherein the coinhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, and PD-L2, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a further preferred embodiment of the composition or kit-of-parts of the present invention, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, OX40, and GITR, or (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, wherein the coinhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, and PD-L2, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a further preferred embodiment of the composition or kit-of-parts of the present invention, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB and OX40, or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, and PD-L2, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

[0075] In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and ICOS ligand / B7-H2, or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, LAG3 / CD223 / lymphocyte activation gene 3, TIM-3 / HAVCR2, and TIGIT / VSTM3, or (iii) a mixture of both. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, GITR, OX40, ICOS, and CD40, or (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, wherein the coinhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, and GITRL, or (iii) a mixture of both.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, or (iii) a mixture of both. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, or (iii) a mixture of both.

[0076] In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of activating a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is CD155 / PVR, CD226 / DNAM-1, CD137 / 4-1BB, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, 4-1BBL / CD137L, OX40 / CD134, OX-40L / TNFSF4 / CD252, CD27, HVEM / T selected from the group consisting of NFRSF14, TNFSF14 / LIGHT / CD258, CD70 / CD27L / TNFSF7, CD28 / TP44, CD30, CD30 ligand (L), TMIGD2, HHLA2, CD80 / B7-1, CD86 / B7-2, GITR / TNFRSF18, GITR ligand / TNFSF18, DR3, TL1A, CD30, CD30L, TMIGD2, HHLA2, TL1A, DR3, LTβR, TNF, TNFR2, ICOS / AILIM / CD278, and ICOS ligand / B7-H2-1. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, OX40 / CD134, OX-40L / TNFSF4 / CD252, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and ICOS ligand ligand / B7-H2. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and ICOS ligand / B7-H2.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, GITR, OX40, ICOS, and CD40. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of CD137 / 4-1BB, 4-1BBL / CD137L, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and ICOS ligand / B7-H2. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is CD137 / 4-1BB or 4-1BBL / CD137L. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, and the costimulatory immune checkpoint protein is CD137 / 4-1BB.

[0077] In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the agonizing antibody is selected from the group consisting of anti-4-1BB, anti-GITR, anti-OX40, anti-ICOS, and anti-CD40. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the agonizing antibody is selected from the group consisting of anti-CD137 / 4-1BB, anti-4-1BBL / CD137L, anti-GITR / TNFRSF18, anti-GITR ligand / TNFSF18, anti-ICOS / AILIM / CD278, and anti-ICOS ligand / B7-H2. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the agonistic antibody is anti-CD137 / 4-1BB or anti-4-1BBL / CD137L. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the agonistic antibody is anti-CD137 / 4-1BB.

[0078] In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, or HVEM / TNFR. Selected from the group consisting of SF14, BTLA, CD160, LAG3 / CD223 / lymphocyte activation gene 3, CEACAM1 / CD66a, indoleamine, 2,3-dioxygenase / IDO, galectin- / LGALS9, TIM-3 / HAVCR2, 2B4 / CD244, SIRP, alpha / CD172a, CD47, CD48 / SLAMF2, TIGIT / VSTM3, A2AR, KIR, NOX2, SIGLEC7 / CD328, SIGLEC9 / CD329. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, HVEM / TNFRSF14, BTLA, CD160, LAG3 / CD223 / lymphocyte activation gene 3, CEACAM1 / CD66a, indoleamine, galectin- / LGALS9, TIM-3 / HAVCR2, 2B4 / CD244, SIRP, alpha / CD172a, CD47, CD48 / SLAMF2, and TIGIT / VSTM3.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, galectin- / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD80 / B7-1, CD86 / B7-2, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, galectin- / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, the co-inhibitory immune checkpoint protein being selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, HVEM / TNFRSF14, BTLA, Selected from the group consisting of CD160, LAG3 / CD223 / lymphocyte activation gene 3, CEACAM1 / CD66a, indoleamine, 2,3-dioxygenase / IDO, galectin- / LGALS9, TIM-3 / HAVCR2, 2B4 / CD244, SIRP, alpha / CD172a, CD47, CD48 / SLAMF2, TIGIT / VSTM3, A2AR, KIR, NOX2, SIGLEC7 / CD328, SIGLEC9 / CD329.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, HVEM / TNFRSF14, BTLA, CD160, LAG3 / CD223 / lymphocyte activation gene 3, CEACAM1 / CD66a, indoleamine, galectin- / LGALS9, TIM-3 / HAVCR2, 2B4 / CD244, SIRP, alpha / CD172a, CD47, CD48 / SLAMF2, and TIGIT / VSTM3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, galectin- / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3 / CD276, B7-H4 / B7S1 / 7x, VISTA / B7-H5 / GI24, LAG3 / CD223 / lymphocyte activation gene 3, galectin- / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3.In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, wherein the coinhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, and GITRL. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, wherein the coinhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, LAG3 / CD223 / lymphocyte-activation gene 3, TIM-3 / HAVCR2, and TIGIT / VSTM3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, and PD-L2.

[0079] In a further preferred embodiment of the composition or kit-of-parts of the present invention, the immune checkpoint protein is selected from the group consisting of 4-1BB, 4-1BB ligand, PD-1, PD-L1, PD-L2, and CTLA-4. More preferably, the immune checkpoint protein is selected from the group consisting of 4-1BB, 4-1BB ligand, PD-1, PD-L1, and PD-L2. In a further preferred embodiment of the composition or kit-of-parts of the present invention, the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, PD-L2, and CTLA-4. More preferably, the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, and PD-L2.

[0080] In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the antagonizing antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-anti-PD-L2, anti-CTLA-4, anti-LAG3 / CD223 / lymphocyte activation gene 3, anti-TIM-3 / HAVCR2, and anti-TIGIT / VSTM3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the antagonizing antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, and anti-PD-L2.

[0081] In a preferred embodiment, the composition or kit-of-parts of the invention comprises two or more antibodies capable of modulating immune checkpoint proteins. In a preferred embodiment, the composition or kit-of-parts of the invention comprises one, two or three antibodies capable of modulating immune checkpoint proteins. In a preferred embodiment, the composition or kit-of-parts of the invention comprises two or three antibodies capable of modulating immune checkpoint proteins. In a preferred embodiment, the composition or kit-of-parts of the invention comprises two antibodies capable of modulating immune checkpoint proteins. In a preferred embodiment, the composition or kit-of-parts of the invention comprises three antibodies capable of modulating immune checkpoint proteins.

[0082] In a preferred embodiment, the composition or kit-of-parts of the present invention further comprises a chemotherapeutic agent. In a preferred embodiment, the composition or kit-of-parts of the present invention is combined with radiotherapy.

[0083] In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein contained in the composition or kit of parts of the present invention is (i) an antibody capable of modulating the immune checkpoint protein CD27 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2; (ii) an antibody capable of modulating the immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iv) an antibody capable of modulating the immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and CTLA-4; (v) an antibody capable of modulating the immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or (vi) an antibody capable of regulating the immune checkpoint protein ICOS and at least one antibody capable of regulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4 It is a mixture of

[0084] In a preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein contained in the composition or kit of parts of the present invention is (i) a mixture of an antibody capable of modulating the immune checkpoint protein CD27 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2. In a preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein contained in the composition or kit of parts of the present invention is (ii) a mixture of an antibody capable of modulating the immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4. In a preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein contained in the composition or kit of parts of the present invention is (iii) a mixture of an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4. In a preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein contained in the composition or kit-of-parts of the present invention is (iv) a mixture of an antibody capable of modulating the immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and CTLA-4. In a preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein contained in the composition or kit-of-parts of the present invention is (v) a mixture of an antibody capable of modulating the immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4.In a preferred embodiment, the at least one antibody capable of regulating an immune checkpoint protein contained in the composition or kit-of-parts of the present invention is (vi) a mixture of an antibody capable of regulating the immune checkpoint protein ICOS and at least one antibody capable of regulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2.

[0085] In a preferred embodiment, the at least one antibody capable of regulating an immune checkpoint protein contained in the composition or kit of parts of the present invention is (i) a mixture of anti-CD27 and at least one antibody capable of regulating an immune checkpoint protein, wherein the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, and anti-PD-L2. In a preferred embodiment, the at least one antibody capable of regulating an immune checkpoint protein contained in the composition or kit of parts of the present invention is (ii) a mixture of anti-CD40 and at least one antibody capable of regulating an immune checkpoint protein, wherein the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-CTLA-4. In a preferred embodiment, the at least one antibody capable of regulating an immune checkpoint protein contained in the composition or kit of parts of the present invention is (iii) a mixture of anti-GITR and at least one antibody capable of regulating an immune checkpoint protein, wherein the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-CTLA-4. In a preferred embodiment, the at least one antibody capable of regulating an immune checkpoint protein contained in the composition or kit-of-parts of the present invention is (iv) a mixture of anti-OX40 and at least one antibody capable of regulating an immune checkpoint protein, wherein the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1BB, and anti-CTLA-4. In a preferred embodiment, the at least one antibody capable of regulating an immune checkpoint protein contained in the composition or kit-of-parts of the present invention is (v) a mixture of anti-4-1BB and at least one antibody capable of regulating an immune checkpoint protein, wherein the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-OX40, anti-LAG-3, and anti-CTLA-4.In a preferred embodiment, the at least one antibody capable of regulating an immune checkpoint protein contained in the composition or kit-of-parts of the present invention is (vi) a mixture of anti-ICOS and at least one antibody capable of regulating an immune checkpoint protein, wherein the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, and anti-PD-L2.

[0086] In a preferred embodiment, a polyplex of the invention comprises double-stranded RNA (dsRNA) and a polymer conjugate, the polymer conjugate comprising polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, one or more linkers, and one or more targeting moieties, wherein the PEI is covalently attached to one or more PEG moieties, each of the one or more PEG moieties is linked to one of the one or more targeting moieties via one of the one or more linkers, and each of the one or more targeting moieties is capable of binding to a cancer antigen.

[0087] In a preferred embodiment, a polyplex of the invention comprises double-stranded RNA (dsRNA) and a polymer conjugate, the polymer conjugate consisting of polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, one or more linkers, and one or more targeting moieties, wherein the PEI is covalently attached to one or more PEG moieties, each of the one or more PEG moieties is linked to one of the one or more targeting moieties via one of the one or more linkers, and each of the one or more targeting moieties is capable of binding to a cancer antigen.

[0088] In a preferred embodiment, a polyplex of the invention comprises double-stranded RNA (dsRNA) and a polymer complex, the polymer complex comprising polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, one or more linkers, and one or more targeting moieties, wherein the PEI is covalently attached to one or more PEG moieties, each of the one or more PEG moieties being linked to one of the one or more targeting moieties via one of the one or more linkers, and each of the one or more targeting moieties being capable of binding to a cancer antigen.

[0089] The polyplexes of the present invention include polyplexes containing double-stranded RNA (dsRNA). The term "dsRNA" typically and preferably refers to a double-stranded ribonucleotide polymer of any length, in which one or more ribonucleotides may be chemical analogs or modified derivatives of corresponding naturally occurring ribonucleotides. The term "dsRNA" typically and preferably also includes mismatched dsRNA.

[0090] In a preferred embodiment, the dsRNA is polyinosinic-polycytidylic acid double-stranded RNA (poly IC or pIC). Poly IC is double-stranded RNA, one strand of which is a polymer of inosinic acid and the other a polymer of cytidylic acid.

[0091] The polyIC of the polyplexes for use in accordance with the present invention may be composed of dsRNA, with each strand consisting of at least 22, preferably at least 45, ribonucleotides. In certain embodiments, each strand consists of 20 to 8000 ribonucleotides. In certain embodiments, each strand consists of 20 to 4000 ribonucleotides. In more preferred embodiments, each strand consists of 20 to 300 ribonucleotides.

[0092] As used herein, the term "molecular weight," particularly when referring to polymers such as polyIC, PEI, and PEG, refers to average molecular weight, preferably weight average molecular weight.

[0093] PolyIC is attached to the polymer complex via non-covalent or covalent bonds, with non-covalent bonds being preferred. In a preferred embodiment, the PolyIC is non-covalently attached to PEI, preferably by ionic bonds.

[0094] A polyplex according to the present invention comprises a polymer complex comprising polyethyleneimine (PEI), which is a polycation that has the ability to condense and non-covalently associate with nucleic acid molecules due to the polyanionic nature of the nucleic acid molecules.

[0095] In a preferred embodiment, the polyethyleneimine (PEI) is a linear polyethyleneimine (LPEI). In a preferred embodiment, the LPEI includes a hydroxy group located at one or both ends of the LPEI. Preferably, the hydroxy group is in place of a terminal -NH group of the LPEI.

[0096] In a preferred embodiment of the present invention, the PEI or preferably LPEI has a molecular weight of about 10-30 kDa. In a preferred embodiment of the present invention, the PEI or preferably LPEI has a molecular weight of about 15-25 kDa (PEI 15~25k / / LPEI 15~25k In an even more preferred embodiment, the PEI or preferably LPEI has a molecular weight of about 22 kDa (LPEI 22k In a still further preferred embodiment, the PEI or preferably the LPEI has a molecular weight of about 20 kDa. The expression "LPEI has a molecular weight of about 22 kDa" is omitted and is used herein as "LPEI". 22k The expression "PEI has a molecular weight of about 22 kDa" is omitted, and in this specification, "PEI 22k " is abbreviated and used synonymously.

[0097] In a preferred embodiment, the PEI or preferably LPEI has low dispersity. Preferably, the PEI or LPEI has a polydispersity index PDI of about 1.

[0098] In a preferred embodiment, one or more PEG moieties each independently form an -NH-CO- bond with the PEI or, preferably, LPEI.

[0099] Polyplexes for use according to the present invention comprise one or more polyethylene glycol (PEG) moieties. PEG moieties according to the present invention are also known as polyethylene oxide (PEO) moieties or polyoxyethylene (POE) moieties, depending on their molecular weight. As used herein, the term "polyethylene glycol moiety" (PEG moiety) typically and preferably refers to a PEG moiety containing two functional groups located at both ends of the polyethylene glycol (PEG). The functional groups can react with either the PEI or, preferably, the LPEI or the targeting moiety.

[0100] In a preferred embodiment, the PEG moiety is linear or branched. In another preferred embodiment, the PEG moiety is branched. In a further preferred embodiment, the PEG moiety is linear.

[0101] In one embodiment of the present invention, each of the at least one PEG moieties has a molecular weight of 1 kD or greater. In another embodiment, each of the at least one PEG moieties has a molecular weight of about 0.3-8 kDa, preferably about 0.5-5 kDa, and more preferably 1-3 kDa (PEG 1~3k ), most preferably 2 kDa (PEG 2k ) As indicated, the molecular weight corresponds to the average molecular weight. Therefore, the PEG preferably used 2k is 30-60 (some smaller, some larger) -(CH2CH2O) n It refers to a mixture of polyethylene glycols having an average value of n units and a molecular weight range of about 1300 to 2600 grams per mole.

[0102] In a preferred embodiment, the PEI has a molecular weight of about 10-30 kDa and the at least one PEG moiety has a molecular weight of about 0.3-8 kDa. In a more preferred embodiment, the PEI has a molecular weight of about 22 kDa (PEI 22k), wherein the at least one PEG moiety has a molecular weight of about 2 kDa (PEG 2k In a more preferred embodiment, the PEI has a molecular weight of about 20 kDa (LPEI 20k ), wherein the at least one PEG moiety has a molecular weight of about 2 kDa (PEG 2k )

[0103] In a preferred embodiment, PEI is covalently attached to 1 to 5 PEG moieties, preferably PEI is covalently attached to 1 to 3 PEG moieties. In a preferred embodiment, LPEI is covalently attached to 1 to 5 PEG moieties, preferably LPEI is covalently attached to 1 to 3 PEG moieties. In a preferred embodiment, PEI 15-25k , preferably PEI 22k 1 to 5 PEG 1-3k , preferably PEG 2k Covalently attached to the moiety, preferably PEI 15-25k , more preferably PEI 22k 1 to 3 PEG 1-3k , preferably PEG 2k In a preferred embodiment, the LPEI is covalently attached to the moiety. 15-25k , preferably LPEI 22k 1 to 5 PEG 1-3k , preferably PEG 2k Covalently attached to the moiety, preferably PEI 15-25k , more preferably PEI 22k 1 to 3 PEG 1-3k , preferably PEG 2k In a preferred embodiment, the PEI moiety is covalently attached to the 15-25k , preferably PEI 22k is one PEG 1-3k , preferably PEG 2k moiety or two or three PEG 1-3k , preferably PEG 2k In another preferred embodiment, the PEI is covalently attached to the moiety. 15-25k , preferably PEI 22k is one PEG 1-3k , preferably PEG 2kIn a further preferred embodiment, PEI is covalently bound to 15-25k , preferably PEI 22k is two PEG 1-3k , preferably PEG 2k In a further preferred embodiment, the PEI moiety is covalently attached to the 15-25k , preferably PEI 22k Three PEGs 1-3k , preferably PEG 2k In a preferred embodiment, the LPEI is covalently attached to the moiety. 15-25k , preferably LPEI 22k is one PEG 1-3k , preferably PEG 2k moiety or two or three PEG 1-3k , preferably PEG 2k In another preferred embodiment, the LPEI is covalently attached to the moiety. 15-25k , preferably LPEI 22k is one PEG 1-3k , preferably PEG 2k In a further preferred embodiment, the LPEI is covalently bound to 15-25k , preferably LPEI 22k is two PEG 1-3k , preferably PEG 2k In a further preferred embodiment, the LPEI is covalently attached to the moiety. 15-25k , preferably LPEI 22k Three PEGs 1-3k , preferably PEG 2k In another preferred embodiment, the PEI of the polyplex is covalently bound to one, two, or three PEG moieties. Preferably, the PEI of the polyplex is covalently bound to one or three PEG moieties. In another more preferred embodiment, the PEI of the polyplex is LPEI covalently bound to one, two, or three PEG moieties. More preferably, the PEI of the polyplex is LPEI covalently bound to one or three PEG moieties.

[0104] As used herein, the term "PEI[...] covalently attached to one PEG moiety" (used interchangeably herein with "PEI-PEG 1:1") or "LPEI[...] covalently attached to one PEG moiety" (used interchangeably herein with "LPEI-PEG 1:1") refers to the molar ratio of PEI to PEG or LPEI to PEG, with PEI-PEG 1:1 or LPEI-PEG 1:1 typically and preferably meaning that about 1 mole of PEG is included in the polymer conjugate per mole of PEI or LPEI. As used herein, the term "PEI[...] covalently attached to three PEG moieties" (used interchangeably herein with "PEI-PEG 1:3") or "LPEI[...] covalently attached to three PEG moieties" (used interchangeably herein with "LPEI-PEG 1:3") typically and preferably meaning that about 3 moles of PEG are included in the polymer conjugate per mole of PEI or LPEI. These values are preferably 1 The relative integrals of the hydrogen atoms of PEG (-CH-CH-O-) and PEI or LPEI (-CH-CH-NH-) are preferably determined by H-NMR analysis. 1 The value is determined by H-NMR. The term "approximately" as used herein preferably refers to a deviation of about 0% to 10%, more preferably about 0% to 5%, and even more preferably about 0% to 2%.

[0105] In a preferred embodiment, the dsRNA is polyIC and the PEI is covalently linked to one to three PEG moieties. In a preferred embodiment, the dsRNA is polyIC and the PEI is covalently linked to one, two, or three PEG moieties. In a more preferred embodiment, the dsRNA is polyIC and the PEI is LPEI covalently linked to one to three PEG moieties. In a more preferred embodiment, the dsRNA is polyIC and the PEI is LPEI covalently linked to one, two, or three PEG moieties.

[0106] In a preferred embodiment, the dsRNA is polyIC and the PEI is covalently linked to one, two, or three PEG moieties. In a more preferred embodiment, the dsRNA is polyIC and the PEI is LPEI covalently linked to one, two, or three PEG moieties. In a more preferred embodiment, the dsRNA is polyIC and the PEI is LPEI covalently linked to one, two, or three PEG moieties. 22k In a more preferred embodiment, the dsRNA is polyIC, and the PEI is 1, 2, or 3 PEG. 0.3-8k , preferably PEG 2k In a more preferred embodiment, the dsRNA is polyIC and the PEI is LPEI covalently linked to one, two, or three PEG moieties. 22k In a more preferred embodiment, the dsRNA is polyIC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k is.

[0107] In a preferred embodiment, the dsRNA is polyIC, and the PEI is covalently linked to one PEG moiety (PEI-PEG 1:1).In a more preferred embodiment, the dsRNA is polyIC, and the PEI is LPEI covalently linked to one PEG moiety (LPEI-PEG 1:1).In a more preferred embodiment, the dsRNA is polyIC, and the PEI is LPEI covalently linked to one PEG moiety. 22k In a more preferred embodiment, the dsRNA is polyIC and the PEI is one PEG 0.3-8k , preferably PEG 2k In a more preferred embodiment, the dsRNA is polyIC and the PEI is covalently linked to one PEG moiety (LPEI-PEG 1:1). 22k In a more preferred embodiment, the dsRNA is polyIC and the PEI is one PEG 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k(LPEI-PEG 1:1).

[0108] In a preferred embodiment, the dsRNA is polyIC, and the PEI is covalently linked to three PEG moieties. In a more preferred embodiment, the dsRNA is polyIC, and the PEI is LPEI covalently linked to three PEG moieties. In a more preferred embodiment, the dsRNA is polyIC, and the PEI is LPEI covalently linked to three PEG moieties. 22k In a more preferred embodiment, the dsRNA is polyIC, and the PEI is a PEG-1 PEG-2 PEG-3 PEG-4 PEG-4 PEG-5 PEG-6 PEG-6 PEG-7 PEG-8 PEG-9 PEG-10 PEG-11 PEG-12 PEG-13 PEG-14 PEG-15 PEG-16 PEG-17 PEG-18 PEG-19 PEG-19 PEG-19 PEG 0.3-8k , preferably PEG 2k In a more preferred embodiment, the dsRNA is polyIC and the PEI is LPEI covalently linked to three PEG moieties. 22k In a more preferred embodiment, the dsRNA is polyIC, and the PEI is a PEG-1 PEG-2 PEG-3 PEG-4 PEG-4 PEG-5 PEG-6 PEG-6 PEG-7 PEG-8 PEG-9 PEG-10 PEG-11 PEG-12 PEG-13 PEG-14 PEG-15 PEG-16 PEG-17 PEG-18 PEG-19 PEG-19 PEG-19 PEG 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k is.

[0109] In a preferred embodiment, the dsRNA of a polyplex is polyIC and the PEI of a polymer complex of a polyplex for use according to the invention is LPEI 22k In a preferred embodiment, the dsRNA of a polyplex is polyIC, and the PEI of a polymer complex of a polyplex for use according to the invention is PEI covalently linked to one, two, or three PEG moieties. 22k is.

[0110] In a preferred embodiment, the dsRNA is polyIC and the one or more PEG moieties of the polymer conjugate are PEG 0.3-8k , preferably PEG 2k In a preferred embodiment, the dsRNA is polyIC and the LPEI is one, two, or three PEGs. 0.3-8k moiety, preferably PEG 2k In a preferred embodiment, the dsRNA is polyIC and the LPEI is covalently linked to one PEG moiety.0.3-8k moiety, preferably PEG 2k In a preferred embodiment, the dsRNA is polyIC and the LPEI is covalently linked to a PEG moiety (LPEI-PEG 1:1). 0.3-8k , preferably PEG 2k It is covalently bound to the moiety (LPEI-PEG 1:3).

[0111] In a preferred embodiment, the dsRNA of the polyplex is polyIC and the LPEI of the polymer complex is LPEI 22k and the one or more PEG moieties are PEG 0.3-8k , preferably PEG 2k In a preferred embodiment, the dsRNA of the polyplex is polyIC and the LPEI is LPEI 22k and one PEG 0.3-8k , preferably PEG 2k moiety (LPEI-PEG 1:1) or three PEG 0.3-8k , preferably PEG 2k It is covalently bound to the moiety (LPEI-PEG 1:3).

[0112] As used herein, the term "cancer antigen" (used interchangeably herein with tumor antigen or tumor marker) refers to an antigenic substance produced by or presented on tumor cells that elicits an immune response in the host. In certain embodiments, the cancer antigen is selected from the group consisting of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), prostate surface membrane antigen (PSMA), insulin-like growth factor 1 receptor (IGF1R), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR). In a more preferred embodiment, the cancer antigen is selected from the group consisting of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and prostate surface membrane antigen (PSMA). In another preferred embodiment, the cancer antigen is EGFR. In another preferred embodiment, the cancer antigen is PSMA. In another preferred embodiment, the cancer antigen is HER2. In another preferred embodiment, the cancer antigen is EGFR or HER2. In another preferred embodiment, the cancer antigen is EGFR or PSMA.

[0113] In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to 1 to 3 PEG moieties, and the cancer antigen is EGFR or PSMA. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to 1 to 3 PEG moieties, and the cancer antigen is EGFR. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to 1 to 3 PEG moieties, and the cancer antigen is PSMA. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to 1 to 3 PEG moieties, and the cancer antigen is HER2.

[0114] In another preferred embodiment, the dsRNA is PolyIC, the PEI is LPEI covalently linked to one to three PEG moieties, and the targeting moiety is EGF, preferably human EGF, a HER2 affibody, a HER2 antibody, or DUPA. In another preferred embodiment, the dsRNA is PolyIC, the PEI is LPEI covalently linked to one to three PEG moieties, and the targeting moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is PolyIC, the PEI is LPEI covalently linked to one to three PEG moieties, and the targeting moiety is a HER2 affibody or a HER2 antibody.

[0115] Treatment of EGFR-high (epidermal growth factor receptor)-expressing cells with PEI-PEG-EGF (epidermal growth factor) / PolyIC polyplexes induced the secretion of pro-inflammatory cytokines (IP-10, GRO-α, and CCL5), which was not observed in EGFR-low expressing cells (see Figure 11 and Example 8 herein). Furthermore, increased secretion of cytokines (IFN-γ and TNFα) was observed in PBMCs incubated with supernatants from EGFR-high expressing PEG-EGF / PolyIC polyplex-treated cells, which was not observed in the medium from EGFR-low expressing cells (see Figure 12 and Example 9 herein). Combination of PEI-PEG-EGF / PolyIC with nivolumab or anti-41BB antibody resulted in a further increase in the cytokine IFN-γ release in PBMCs compared with treatment with PEI-PEG-EGF / PolyIC, nivolumab, or anti-41BB antibody alone (see Figures 3, 4, and 10 herein, Examples 2 and 7). Treatment of mice bearing RENCA EGFR tumors with PEI-PEG-EGF / PolyIC in combination with anti-PD-1 demonstrated increased survival compared to either anti-PD-1 or PEI-PEG-EGF / PolyIC, demonstrating the synergistic effects achieved with the compositions of the present invention and the combined treatment, respectively, as shown in Figure 5 and Example 3 herein.

[0116] Similarly, treatment with PolyIC / PEI-PEG conjugated to a HER2 affibody (pIC / PPHA) induced the expression of antiproliferative (e.g., IFN-γ, IFN-α) and immunostimulatory cytokines (e.g., IP-10, GRO-alpha, and RANTES), triggered immune cell recruitment and activation, and resulted in widespread cancer cell killing in vitro (Zigler et al., Cancer Immunol Res; 4(8) August 2016). As described in Example 5 and further shown in Figures 6-9 herein, treatment of RENCA HER2 tumors with a combination of HER2-targeted PolyIC polyplexes (PolyIC / PEI-PEG-HER2 affibody) plus anti-PD-1 according to the present invention resulted in complete regression of tumor growth and complete protection from tumor rechallenge.

[0117] Of particular note, the vector polyIC / PEI-PEG-DUPA (pIC / PPD), containing PEI-PEG tethered to the PSMA ligand DUPA (2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid), selectively delivered PolyIC to PSMA-overexpressing prostate cancer cells, inducing apoptosis, cytokine secretion, and recruitment of human peripheral blood mononuclear cells (PBMCs). Notably, pIC / PPD resulted in PBMC activation, as evidenced by the production of IFN-β, IP-10, and RANTES, chemotaxis, and strong expression of IL-2, leading to the secretion of high levels of TNF-α and IFN-γ (Langut et al., PNAS, December 26, 2017, Vol. 114, No. 52).

[0118] Therefore, the above clearly confirms and supports that the compositions and kits-of-parts of the present invention comprising a polyplex and at least one antibody capable of modulating an immune checkpoint protein can result in increased immune system activation and more potent anti-tumor activity. This is particularly the case for preferred compositions of the present invention comprising a polyplex, wherein the cancer antigen is EGFR, HER2, or PSMA, and / or the one or more targeting moieties are selected from EGF, a HER2 affibody, a HER2 antibody, and DUPA, and the antibody is capable of modulating an immune checkpoint protein, preferably an anti-4-1BB, anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody.

[0119] In another preferred embodiment, the dsRNA is polyIC and the PEI is PEI covalently linked to 1 to 3 PEG moieties. 15-25k , preferably LPEI 22k and the targeting moiety is EGF, preferably human EGF, a HER2 affibody, a HER2 antibody, or DUPA. In another preferred embodiment, the dsRNA is polyIC and the PEI is PEI covalently linked to 1 to 3 PEG moieties. 15-25k , preferably LPEI 22k and the targeting moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is polyIC and the PEI is PEI covalently linked to 1 to 3 PEG moieties. 15-25k , preferably LPEI 22k and the targeting moiety is a HER2 affibody or a HER2 antibody.

[0120] In another preferred embodiment, the dsRNA is polyIC and the PEI is substituted with 1, 2, or 3 PEGylation moieties. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k In another preferred embodiment, the dsRNA is polyIC and the PEI is covalently linked to one, two, or three PEG-1, PEG-2, or PEG-3 moieties, and the targeting moiety is EGF, preferably human EGF, a HER2 affibody, a HER2 antibody, or DUPA. 0.3-8k, preferably PEG 1-3k , more preferably PEG 2k In another preferred embodiment, the dsRNA is polyIC and the PEI is covalently bound to a moiety, and the targeting moiety is EGF, preferably human EGF. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k a LPEI covalently linked to a moiety, the targeting moiety being a HER2 affibody or a HER2 antibody.

[0121] In another preferred embodiment, the dsRNA is polyIC and the PEI is substituted with 1, 2, or 3 PEGylation moieties. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 22k and the targeting moiety is EGF, preferably human EGF, a HER2 affibody, a HER2 antibody, or DUPA. In another preferred embodiment, the dsRNA is polyIC and the PEI is 1, 2, or 3 PEG-1-binding domains. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22k and the targeting moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is polyIC and the PEI is 1 to 3 PEG- 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22k and the targeting moiety is a HER2 affibody or a HER2 antibody.

[0122] In another preferred embodiment, the dsRNA is poly IC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, 4-1BB ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS, ICOS ligand CD40, and CD40 ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, 4-1BB ligand, TIGIT, LAG3, TIM-3, GITR, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand, and ICOS.

[0123] In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, 4-1BB ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS, ICOS ligand CD40, and CD40 ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, 4-1BB ligand, TIGIT, LAG3, TIM-3, GITR, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand, and ICOS. In another preferred embodiment, the dsRNA is poly IC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR, OX40, ICOS, and CD40. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM-3, GITR, CD40, OX40, and ICOS.

[0124] In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, CTLA-4, GITR, CD40, OX40, and ICOS. In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40, OX40, and ICOS. In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40, and OX40. In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, CD40, and OX40. In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and OX40. In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, and 4-1BB. In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1 and 4-1BB.

[0125] In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40, OX40, and ICOS. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40, and OX40. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, and OX40. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and OX40.

[0126] In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, and 4-1BB ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, and 4-1BB. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, and 4-1BB. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1 or 4-1BB. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1, PD-L1, or PD-L2. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L1.In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L2. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is 4-1BB. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is CTLA-4. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is LAG-3. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIGIT. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIM3. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is GITR or a GITR ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is ICOS or an ICOS ligand.In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is OX40 or an OX40 ligand.

[0127] In another preferred embodiment, the dsRNA is poly IC, the PEI is PEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-4-1BB ligand (4-1BBL), anti-TIGIT, anti-LAG3, anti-TIM3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR4, anti-GITR ligand, anti-GITR, anti-OX40, anti-OX40L, anti-ICOS, anti-ICOS ligand anti-CD40, and anti-CD40 ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-4-1BB ligand, anti-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, anti-GITR ligand, anti-CD40, anti-CD40 ligand, anti-OX40, anti-OX40 ligand, anti-ICOS ligand and anti-ICOS. In another preferred embodiment, the dsRNA is polyIC, the PEI is PEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1BB, anti-4-1BB ligand, anti-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, anti-GITR ligand, anti-CD40, anti-CD40 ligand, anti-OX40, anti-OX40 ligand, anti-ICOS ligand, and anti-ICOS.In another preferred embodiment, the dsRNA is poly IC, the PEI is LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR4, anti-GITR, anti-OX40, anti-ICOS and anti-CD40. In another preferred embodiment, the dsRNA is PolyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, anti-CD40, anti-OX40, and anti-ICOS. In another preferred embodiment, the dsRNA is PolyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1BB, and anti-4-1BB ligand. In another preferred embodiment, the dsRNA is PolyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, and anti-4-1BB.In another preferred embodiment, the dsRNA is PolyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-4-1BB.In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-PD-1 or anti-4-1BB. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-PD-1, anti-PD-L1, or anti-PD-L2. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-PD-1. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-PD-L1. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-PD-L2. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-4-1BB. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-CTLA-4. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-LAG-3.In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-TIGIT. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-TIM3. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-GITR or an anti-GITR ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-ICOS or an ICOS ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two, or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-OX40 or an anti-OX40 ligand.

[0128] In another preferred embodiment, the dsRNA is polyIC and the PEI is substituted with 1, 2, or 3 PEGylation moieties. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22kwherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, 4-1BB ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS, ICOS ligand CD40, and CD40 ligand. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-31, PEG-32, PEG-33, PEG-34, PEG-35, PEG-35, PEG-35, PEG-45, PEG-46, PEG-47, PEG-48, PEG-49, PEG-51, PEG-52, PEG-53, PEG-54, PEG-55, PEG-55, PEG-55, PEG-55, PEG-56, PEG-57, PEG-58, PEG-59, PEG-59, PEG-59, PEG-59, PEG-51, PEG-51, PEG-52, PEG- 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, 4-1BB ligand, TIGIT, LAG3, TIM-3, GITR, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand, and ICOS.

[0129] In another preferred embodiment, the dsRNA is polyIC and the PEI is substituted with 1, 2, or 3 PEGylation moieties. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, 4-1BB ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS, ICOS ligand CD40, and CD40 ligand. In another preferred embodiment, the dsRNA is polyIC, and the PEI is one, two, or three PEGylation sites. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22kwherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, 4-1BB ligand, TIGIT, LAG3, TIM-3, GITR, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand, and ICOS. In another preferred embodiment, the dsRNA is polyIC, and the PEI is 1, 2, or 3 PEG-1, PEG-2, or PEG-3. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR, OX40, ICOS, and CD40. In another preferred embodiment, the dsRNA is polyIC and the PEI is 1, 2, or 3 PEGylation sites. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM-3, GITR, CD40, OX40, and ICOS.

[0130] In another preferred embodiment, the dsRNA is polyIC and the PEI is 1, 2, or 3 PEG 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR, CD40, OX40, and ICOS. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1, PEG-2, or PEG-3. 0.3-8k , preferably PEG2k LPEI covalently bound to the moiety 22k wherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40, and OX40. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1, PEG-2, or PEG-3. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, and OX40. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1, PEG-2, or PEG-3. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and OX40.

[0131] In another preferred embodiment, the dsRNA is polyIC and the PEI is substituted with 1, 2, or 3 PEGylation moieties. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, and 4-1BB ligand. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1, PEG-2, or PEG-3. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22kwherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, and 4-1BB. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1, PEG-2, or PEG-3. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, and 4-1BB. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1, PEG-2, or PEG-3. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1 or 4-1BB. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-4-1BB. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1, PD-L1, or PD-L2. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1, PEG-2, or PEG-3. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-2 or PEG-3 or PEG-4 or PEG-5 or PEG-6 or PEG-7 or PEG-8 or PEG-9 or PEG-11 or PEG-12 or PEG-13 or PEG-14 or PEG-15 or PEG-16 ... 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L1. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-2. 0.3-8k, preferably PEG 2k LPEI covalently bound to the moiety 22k In another preferred embodiment, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L2. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-2. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is 4-1BB. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-4-1BB. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is CTLA-4. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-2. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is LAG-3. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-2. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIGIT. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-2. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22kIn another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIM3. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-2. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is GITR or a GITR ligand. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-2. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22k In another preferred embodiment, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is ICOS or an ICOS ligand. In another preferred embodiment, the dsRNA is polyIC, and the PEI is PEG-1 or PEG-2. 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22k wherein the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is OX40 or an OX40 ligand.

[0132] In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of: (i) an antibody capable of modulating the immune checkpoint protein CD27 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2; (ii) an antibody capable of modulating the immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iv) an antibody capable of modulating the immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and CTLA-4; (v) an antibody capable of modulating the immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or (vi) an antibody capable of regulating the immune checkpoint protein ICOS and at least one antibody capable of regulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2; It is a mixture of

[0133] In another preferred embodiment, the dsRNA is poly(IC) and the PEI is one, two, or three PEGylation sites. 0.3-8k , preferably PEG 2k LPEI covalently bound to the moiety 22k wherein the targeting moiety is EGF, preferably human EGF, and the at least one antibody capable of modulating an immune checkpoint protein is (i) an antibody capable of modulating the immune checkpoint protein CD27 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2; (ii) an antibody capable of modulating the immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iv) an antibody capable of modulating the immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and CTLA-4; (v) an antibody capable of modulating the immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or (vi) an antibody capable of regulating the immune checkpoint protein ICOS and at least one antibody capable of regulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2; It is a mixture of

[0134] In a preferred embodiment, the composition or kit-of-parts of the present invention does not comprise, i.e. excludes, a cancer vaccine or a tumor-associated antigen. In a preferred embodiment, the composition or kit-of-parts of the present invention comprises a cancer vaccine or a tumor-associated antigen.

[0135] In the polyplexes of the present invention, the one or more PEG moieties are linked to one of the one or more targeting moieties. In a preferred embodiment, the one or more PEG moieties are linked to one of the one or more targeting moieties directly or via one of the one or more linkers.

[0136] In a further preferred embodiment, the linker is selected from -CO-R2-RX-R3 or a peptide moiety consisting of 3 to 7 amino acid residues, wherein R2 is (C1-C8) alkylene, (C2-C8) alkenylene, (C2-C8) alkynylene, (C6-C 10 ) arylene-diyl, or heteroarylene-diyl, wherein Rx is absent or is S-, and R3 is absent or is a group of the formula [ka] and In the formula, R4 is (C1-C8) alkylene, (C2-C8) alkenylene, (C2-C8) alkynylene (C1-C8) alkylene-(C3-C8) cycloalkylene, (C2-C8) alkenylene-(C3-C8) cycloalkylene, (C2-C8) alkynylene-(C3-C8) cycloalkylene, (C6-C 10 ) arylene-diyl, heteroarylene-diyl, (C1-C8) alkylene-(C6-C 10 ) arylene-diyl, or (C1-C8) alkylene-heteroarylenediyl, each of the (C1-C8) alkylene, (C2-C8) alkenylene, or (C2-C8) alkynylene is optionally selected from halogen, —COR5, —COOR5, —OCOOR5, —OCON(R5), —CN, —NO2, —SR5, —OR5, —N(R5), —CON(R5), —S02R5, —S03H, —S(═O)R5, (C6-C 10 )aryl, (C1-C4)alkylene-(C6-C 10 )aryl, heteroaryl, or (C1-C4)alkylene-heteroaryl, and optionally further substituted by one or more groups independently selected from S, O, or N, and / or NH—CO—, —CO—NH—, —N(R5)—, —N(C6-C 10 ) aryl-, (C6-C 10) arylene-diyl, or heteroarylene-diyl, and R5 is H or (C1-C8) alkyl.

[0137] In a further preferred embodiment, the polymer conjugate has formula (i)-(viii): [ka] (Wherein R6 is [ka] is) [ka] (Wherein R7 is [ka] is) [ka] (Wherein R6 is [ka] is) [ka] (Wherein R7 is [ka] is) and In the formula, T represents the targeting moiety, n corresponds to a molecular weight of 0.3 to 8 kD, preferably 0.5 to 5 kD, more preferably 1 to 3 kD, and most preferably 2 kD, and m corresponds to a molecular weight of 10 to 30 kD, preferably 15 to 25 kD, and even more preferably 22 kD.

[0138] In a further preferred embodiment, the polymer conjugate is of formula (ii) or (vii), wherein R6 is SEQ ID NO: 4 (-(NH-(CH2)7-CO)-Phe-Phe-(NH-CH2-CH(NH2)-CO)-Asp-Cys-), and wherein T represents the targeting moiety HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO- (DUPA moiety). In a further preferred embodiment, the polymer conjugate is of formula (iv) or (viii), wherein R7 is SEQ ID NO: 3 (-(NH-(CH2)7-CO)-Phe-Gly-Trp-Trp-Gly-Cys-), and wherein T represents the targeting moiety HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO- (DUPA moiety).

[0139] In a further preferred embodiment, the targeting moiety is a HER2 affibody or a HER2 antibody, and the polymer conjugate is of formula (i) or (v), and the HER2 affibody or HER2 antibody is linked via its sulfhydryl group; - the targeting moiety is hEGF (human epidermal growth factor; human EGF) and the polymer conjugate is of formula (i), (ii) or (vi), and the hEGF is linked via its amino group, or the targeting moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO- (DUPA moiety) and the polymer conjugate is of formula (iii), (iv), (vii) or (viii).

[0140] In a further preferred embodiment, the targeting moiety is a HER2 affibody or a HER2 antibody, the polymer conjugate is of formula (i) or (v), and the HER2 affibody or HER2 antibody is linked via its sulfhydryl group.

[0141] In a further preferred embodiment, the targeting moiety is hEGF and the polymer conjugate is of formula (i), (ii), or (vi). In a further preferred embodiment, the targeting moiety is hEGF and the polymer conjugate is of formula (i), (ii), or (vi), and the hEGF is linked via its amino group. In a further preferred embodiment, the targeting moiety is hEGF and the polymer conjugate is of formula (i), and the hEGF is linked via its amino group.

[0142] In a further preferred embodiment, the targeting moiety is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO- (DUPA moiety) and the polymer conjugate is of formula (iii), (iv), (vii) or (viii).

[0143] In a further preferred embodiment, the targeting moiety is a HER2 affibody or HER2 antibody, the polymer conjugate is of formula (i) or (v), and the HER2 affibody or HER2 antibody is linked via its sulfhydryl group. In a further preferred embodiment, the targeting moiety is hEGF, the polymer conjugate is of formula (i), (ii), or (vi), and the hEGF is linked via its amino group. In a further preferred embodiment, the targeting moiety is hEGF, the polymer conjugate is of formula (i), and the hEGF is linked via its amino group.

[0144] In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, 4-1BB ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS, ICOS ligand CD40, and CD40 ligand, and the polymer conjugate is according to formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is Poly IC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, 4-1BB ligand, TIGIT, LAG3, TIM-3, GITR, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand, and ICOS, and the polymer conjugate is of formula (i), (ii), or (vi).

[0145] In another preferred embodiment, the dsRNA is Poly IC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, 4-1BB ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS, ICOS ligand CD40, and CD40 ligand, and the polymer conjugate is according to formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is Poly IC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, 4-1BB ligand, TIGIT, LAG3, TIM-3, GITR, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand, and ICOS, and the polymer conjugate is of formula (i), (ii), or (vi).

[0146] In another preferred embodiment, the dsRNA is Poly IC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR, OX40, ICOS, and CD40, and the polymer conjugate is according to formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is Poly IC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM-3, GITR, CD40, OX40, and ICOS, and the polymer conjugate is of formula (i), (ii), or (vi).

[0147] In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR, CD40, OX40, and ICOS, and the polymer conjugate is according to formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40, and OX40, and the polymer conjugate is according to formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR, and OX40, and the polymer conjugate is according to formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, and OX40, and the polymer conjugate is according to formula (i), (ii), or (vi).

[0148] In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, and 4-1BB ligand. In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, and 4-1BB. In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, and 4-1BB, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is PD-1 or 4-1BB, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is PD-1, PD-L1, or PD-L2, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is PD-1, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is PD-L1, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is PD-L2, and the polymer conjugate is according to formula (i), (ii), or (vi).In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is 4-1BB. In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is CTLA-4, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is LAG-3, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIGIT. In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is TIM3, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is GITR or a GITR ligand, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is ICOS or an ICOS ligand, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the immune checkpoint protein is OX40 or an OX40 ligand, and the polymer conjugate is of formula (i), (ii), or (vi).

[0149] In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-4-1BB ligand (4-1BBL), anti-TIGIT, anti-LAG3, anti-TIM3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR4, anti-GITR ligand, anti-GITR, anti-OX40, anti-OX40L, anti-ICOS, anti-ICOS ligand anti-CD40, and anti-CD40 ligand, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is Poly IC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-4-1BB ligand, anti-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, anti-GITR ligand, anti-CD40, anti-CD40 ligand, anti-OX40, anti-OX40 ligand, anti-ICOS ligand, and anti-ICOS, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1BB, anti-4-1BB ligand (4-1BBL), anti-TIGIT, anti-LAG3, anti-TIM3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR4, anti-GITR ligand, anti-GITR, anti-OX40, anti-OX40L, anti-ICOS, anti-ICOS ligand, anti-CD40, and anti-CD40 ligand, and the polymer conjugate is of formula (i), (ii), or (vi).In another preferred embodiment, the dsRNA is Poly IC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1BB, anti-4-1BB ligand, anti-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, anti-GITR ligand, anti-CD40, anti-CD40 ligand, anti-OX40, anti-OX40 ligand, anti-ICOS ligand, and anti-ICOS, and the polymer conjugate is of formula (i), (ii), or (vi).

[0150] In another preferred embodiment, the dsRNA is Poly IC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR4, anti-GITR, anti-OX40, anti-ICOS, and anti-CD40, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is Poly IC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-1, anti-B7-2, anti-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, anti-CD40, anti-OX40, and anti-ICOS, and the polymer conjugate is of formula (i), (ii), or (vi).

[0151] In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1BB, and anti-4-1BB ligand. In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, and anti-4-1BB. In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates an immune checkpoint protein is selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-4-1BB, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates the immune checkpoint protein is anti-PD-1 or anti-4-1BB, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates the immune checkpoint protein is anti-PD-1, anti-PD-L1, or anti-PD-L2, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates the immune checkpoint protein is PD-1, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is PolyIC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates an immune checkpoint protein is anti-PD-L1, and the polymer conjugate is according to Formula (i), (ii), or (vi).In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates the immune checkpoint protein is anti-PD-L2, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-4-1BB. In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates the immune checkpoint protein is anti-CTLA-4, and the polymer conjugate is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the targeting moiety is EGF, preferably human EGF, the antibody that modulates the immune checkpoint protein is anti-LAG-3, and the polymer conjugate is of formula (i), (ii), or (vi).

[0152] In another preferred embodiment, the dsRNA is PolyIC, the polymer conjugate is of formula (i), (ii), or (vi), the targeting moiety is EGF, preferably human EGF, and the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of: (i) an antibody capable of modulating the immune checkpoint protein CD27 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2; (ii) an antibody capable of modulating the immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iv) an antibody capable of modulating the immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and CTLA-4; (v) an antibody capable of modulating the immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or (vi) an antibody capable of regulating the immune checkpoint protein ICOS and at least one antibody capable of regulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2; It is a mixture of

[0153] In another preferred embodiment, the dsRNA is PolyIC, the polymer conjugate is of formula (i), (ii), or (vi), the targeting moiety is EGF, preferably human EGF, and the at least one antibody capable of modulating an immune checkpoint protein is (i) anti-CD27, and at least one antibody selected from the group consisting of anti-PD-1, anti-PD-L1, and anti-PD-L2; (ii) an antibody capable of regulating immune checkpoint proteins, such as anti-CD40, and at least one antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-CTLA-4; (iii) an antibody capable of regulating immune checkpoint proteins, such as anti-GITR, and at least one antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-CTLA-4; (iv) an antibody capable of regulating immune checkpoint proteins, such as anti-OX40, and at least one antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1BB, and anti-CTLA-4; (v) an antibody capable of regulating an immune checkpoint protein, such as anti-4-1BB, and at least one antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, anti-OX40, anti-LAG-3, and anti-CTLA-4; or (vi) A mixture of anti-ICOS, an antibody capable of regulating immune checkpoint proteins, and at least one antibody selected from the group consisting of anti-PD-1, anti-PD-L1, and anti-PD-L2.

[0154] The polyplexes of the invention comprise one or more targeting moieties, which may be natural, natural, or modified ligands or paralogs thereof, or non-natural ligands such as antibodies, single-chain variable fragments (scFv), or antibody mimetics such as affibodies or aptamers directed against any one of the cancer antigens.

[0155] In preferred embodiments, the one or more targeting moieties of the polyplexes of the invention are selected from the group consisting of EGF, a HER2 affibody, a HER2 antibody, and a DUPA moiety (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In another preferred embodiment, the one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), a HER2 affibody, or a HER2 antibody. In another preferred embodiment, the one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), or a HER2 affibody.

[0156] In another preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), a HER2 antibody or a HER2 affibody, and the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, PD-L2, CTLA-4, ICOS, CD40, GITR, and OX40. In another preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), a HER2 antibody or a HER2 affibody, and the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, PD-L2, CTLA-4, CD40, GITR, and OX40. In another preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), a HER2 antibody or a HER2 affibody, and the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, PD-L2, GITR, and OX40. In another preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), a HER2 antibody or a HER2 affibody, and the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, PD-L2, and OX40. In another preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), a HER2 antibody or a HER2 affibody, and the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, PD-L2, and CTLA-4. In another preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), or a HER2 affibody, and the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, PD-L2, and CTLA-4.

[0157] In a more preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), a HER2 antibody or a HER2 affibody, and the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, and PD-L2. In a more preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), or a HER2 affibody, and the immune checkpoint protein is selected from the group consisting of 4-1BB, PD-1, PD-L1, and PD-L2.

[0158] In an even more preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), a HER2 antibody or a HER2 affibody, and the immune checkpoint protein is 4-1BB or PD-1. In an even more preferred embodiment, one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), or a HER2 affibody, and the immune checkpoint protein is 4-1BB or PD-1.

[0159] In a further preferred embodiment, the one or more targeting moieties are EGF, including mouse and human EGF. In a more preferred embodiment, the one or more targeting moieties are hEGF. In another preferred embodiment, the one or more targeting moieties of the polyplexes of the invention are hEGF of SEQ ID NO: 2.

[0160] In another preferred embodiment, the one or more targeting moieties of the polyplexes of the invention are a HER2 affibody or a HER2 antibody, preferably a HER2 affibody. In another preferred embodiment, the one or more targeting moieties of the polyplexes of the invention are a HER2 affibody, preferably the HER2 affibody is that of SEQ ID NO: 1. In another preferred embodiment, the one or more targeting moieties of the polyplexes of the invention are EGF, preferably human EGF (hEGF), or a DUPA moiety (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-). In another preferred embodiment, the one or more targeting moieties of the polyplexes of the invention are a DUPA moiety (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-).

[0161] In a further aspect, the present invention relates to a composition or kit-of-parts according to the invention for use in cancer treatment.

[0162] In a preferred embodiment, the present invention relates to a composition or kit-of-parts according to the present invention for use in the treatment of cancer in a mammal, preferably a human.

[0163] In a preferred embodiment, the present invention relates to a method for treating cancer, comprising administering the composition or the kit-of-parts according to the present invention to a patient in need thereof.

[0164] In a preferred embodiment, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, vulvar cancer, urothelial cancer, bladder cancer, kidney cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, glial tumors, head and neck cancer, prostate cancer, penile cancer, testicular embryonal carcinoma, neuroendocrine tumors, and hepatocellular carcinoma. In a preferred embodiment, the polyplex comprised in the kit-of-parts according to the present invention is administered separately from the one or more antibodies capable of modulating an immune checkpoint protein.

[0165] In one embodiment, the present invention provides the use of the pharmaceutical composition or kit-of-parts of the present invention for the treatment of cancer, wherein the polyplex is administered to a patient in a therapeutically effective amount in combination with a therapeutically effective amount of at least one antibody capable of modulating an immune checkpoint protein.

[0166] In a preferred embodiment, the use of the kit-of-parts of the present invention in cancer treatment involves the independent administration of a polyplex and at least one antibody capable of modulating an immune checkpoint protein. The polyplex and at least one antibody capable of modulating an immune checkpoint protein can be administered simultaneously or sequentially (continuously), i.e., at different times. The polyplex and at least one antibody capable of modulating an immune checkpoint protein, as included in the kit-of-parts of the present invention, can be combined prior to administration, administered together as a composition, or administered separately. In a more preferred embodiment, the polyplex and at least one antibody capable of modulating an immune checkpoint protein included in the kit-of-parts of the present invention are administered separately.

[0167] In certain embodiments, the kit of parts or composition for use according to the present invention is administered by any suitable route. The kit of parts or composition for use according to the present invention may be administered intravenously, intracerebrally (intracerebrally), orally, intramuscularly, subcutaneously, transdermally, intradermally, transmucosally, intranasally, sublingually, intraperitoneally, or intraocularly. In a preferred embodiment, the kit of parts or composition for use according to the present invention is administered systemically, i.e., enterally or parenterally. More preferably, the kit of parts or composition for use according to the present invention is administered intravenously, subcutaneously, or intraperitoneally.

[0168] In a more preferred embodiment, the kit-of-parts or composition according to the invention is for systemic administration. More preferably, the kit-of-parts or composition according to the invention is administered intravenously or intraperitoneally, even more preferably intravenously.

[0169] The polyplex of the kit of parts according to the present invention and the at least one antibody capable of modulating an immune checkpoint protein can be administered via the same route or, preferably, via different routes. More preferably, the polyplex of the kit of parts according to the present invention and the at least one antibody capable of modulating an immune checkpoint protein are administered via the same route. In a preferred embodiment, the polyplex of the kit of parts according to the present invention and the at least one antibody capable of modulating an immune checkpoint protein are administered sequentially or simultaneously, preferably sequentially. In a preferred embodiment, the polyplex of the kit of parts according to the present invention and the at least one antibody capable of modulating an immune checkpoint protein are administered sequentially via different routes. More preferably, the polyplex of the kit of parts according to the present invention and the at least one antibody capable of modulating an immune checkpoint protein are administered simultaneously via the same route.

[0170] In a preferred embodiment, the polyplex of the kit-of-parts according to the invention and the at least one antibody capable of modulating an immune checkpoint protein are administered sequentially or simultaneously, preferably sequentially, wherein one compound (or part) of the kit-of-parts according to the invention is administered by intraperitoneal injection and at least one other compound (or part) is administered by intravenous injection. In a preferred embodiment, the polyplex of the kit-of-parts according to the invention and the at least one antibody capable of modulating an immune checkpoint protein are administered sequentially or simultaneously, preferably sequentially, wherein the polyplex is administered by intravenous injection and the immunomodulatory antibody is administered by intraperitoneal or intravenous injection.

[0171] In another preferred embodiment, the polyplex is administered before the at least one antibody capable of modulating an immune checkpoint protein. In another preferred embodiment, the polyplex and the at least one antibody capable of modulating an immune checkpoint protein are administered sequentially, the polyplex is administered via intravenous injection, the at least one antibody capable of modulating an immune checkpoint protein is administered via intraperitoneal or intravenous injection, and the polyplex is administered before the antibody.

[0172] The ratio of the amount or concentration of polyplex to the amount or concentration of one or more antibodies to be administered in a kit-of-parts or composition of the invention can be varied, for example, to address the needs of a single patient or a subpopulation of patients to be treated, which may vary depending on the patient's age, sex, weight, condition, etc.

[0173] The kit-of-parts of the present invention can also be used as an add-on therapy. As used herein, "add-on therapy" refers to a collection of the polyplex and at least one antibody for use in a therapy, where a subject receiving treatment begins a first therapeutic regimen with one or more different parts of the kit-of-parts before beginning a second therapeutic regimen of one or more different parts of the kit-of-parts in addition to the first therapeutic regimen, such that not all of the reagents used in the therapy are initiated simultaneously. For example, one or more immunomodulatory antibodies are administered to a patient who has already received the polyplex of the present invention, or vice versa.

[0174] The invention will now be illustrated by the following non-limiting examples.

[0175] example The present invention is further illustrated by the following examples. A preferred polyplex of the present invention was used. The synthesis of the preferred polyplex was performed as described in WO 2015 / 173824, particularly as described in Examples 2, 8, 10-13 of WO 2015 / 173824. A preferred polymer conjugate containing EGF as a targeting moiety is abbreviated herein as "PEI-PEG-EGF," and the corresponding preferred polyplex is interchangeably abbreviated as "PEI-PEG-EGF / PolyIC" or "PPE / PIC." A further preferred polyplex of the present invention used herein comprises HER2, particularly a HER2 affibody, as a targeting moiety within the polymer conjugate, and PolyIC as a double-stranded RNA. The preferred polyplex is abbreviated herein as "PolyIC / PPHA."

[0176] Example 1: Effect of PEI-PEG-EGF / PolyIC alone A431, U87, and MCF7 cells (40,000 cells / well) were treated with various concentrations (0.125, 0.25, 0.5, 1 μg / ml) of PEI-PEG-EGF / Poly IC for 5 hours.

[0177] Human IP-10 (CXCL10) secretion was quantified by ELISA assay (ABTS ELISA Development Kit, Peprotech). IP10 secretion by A431 cells expressing high levels of EGFR was strongly increased after 5 hours of incubation with PEI-PEG-EGF / PolyIC (Figure 1).

[0178] A431 cells were treated with PEI-PEG-EGF / PolyIC at a concentration of 0.125 μg / ml for 5 hours. The cells were then stained with PD-L1 PE-labeled antibody (Biolegend, catalog no. 393607) in PBS containing 2% FCS for 40 minutes on ice, then washed and analyzed using a FACS instrument. PD-L1 expression was significantly increased (MFI=751) after PEI-PEG-EGF / PolyIC treatment compared to untreated control cells (MFI=431). An isotype control was used as a negative control (MFI=13).

[0179] PD-L1 expression was significantly higher after PEI-PEG-EGF / PolyIC treatment than in untreated cells ( Figures 2A–C ).

[0180] Conclusion: Secretion of IP10, a potent IFN-γ-inducible T cell chemokine, was strongly induced in the EGFR-overexpressing cell line A431 after stimulation with PEI-PEG-EGF / PolyIC. Furthermore, IP10 levels were unaffected in the EGFR-low expressing cell lines MCF7 and U87. The increase in IP10 after stimulation with PEI-PEG-EGF / PolyIC provided a rationale for addressing the immunostimulatory properties of the combination of the present invention.

[0181] Furthermore, PD-L1 expression was significantly higher after PEI-PEG-EGF / PolyIC treatment (MFI = 751) compared to untreated cells (MFI = 431), providing a rationale for developing a combination with nivolumab (Figure 2).

[0182] Example 2 - In vitro efficacy of PEI-PEG-EGF / Poly IC in combination with immunomodulatory therapy nivolumab and 4-1BB antibody Nivolumab: 40,000 A431 cells were treated with PEI-PEG-EGF / PolyIC at concentrations of 0.125, 0.25, 0.5, or 1 μg / ml for 5 hours. 200,000 PBMCs were then stimulated with CD3 (5 μg / ml) and challenged with PEI-PEG-EGF / PolyIC alone (0.125 μg / ml) diluted 1:2 in medium or in combination with nivolumab (20 μg / ml) for 48 hours. After 48 hours, medium was collected from the challenged PBMCs, and INF-γ production was measured by ELISA (Figure 3).

[0183] Combining PEI-PEG-EGF / PolyIC with nivolumab significantly increased IFN-γ production by PBMCs (Figure 3).

[0184] 4-1BB antibody: 40,000 A431 cells were treated with 0.5 μg / ml PEI-PEG-EGF / PolyIC for 5 hours. 200,000 PBMCs from healthy donors were stimulated with or without CD3 (0.5 μg / ml) and cocultured with A431 cells treated with PEI-PEG-EGF / PolyIC alone or in combination with an antibody against 4-1BB (Biolegend clone 4B4-1, 10 μg / ml) for 16 hours. After 16 hours, the medium was collected, and IFN-γ was measured by ELISA assay.

[0185] The combination of PEI-PEG-EGF / Poly IC and anti-4-1BB significantly increased IFN-γ production by PBMCs (FIG. 4).

[0186] Conclusions: Exposure of PBMCs to medium from EGFR-overexpressing A431 cells treated with PEI-PEG-EGF / PolyIC or culturing PBMCs in the presence of PEI-PEG-EGF / PolyIC-treated A431 cells induced PBMCs to secrete IFN-γ. Addition of either nivolumab or anti-41BB antibodies further increased IFN-γ.

[0187] Therefore, the combination of PEI-PEG-EGF / PolyIC with immunotherapy results in increased immune system activation and more potent antitumor activity. From a clinical perspective, the potential induction of antitumor immune responses by combined treatment with PEI-PEG-EGF / PolyIC and immunological strategies is highly attractive, as such combinations should increase the proportion of patients who respond to immunotherapy and reduce the incidence of resistance development.

[0188] Example 3 - In vivo efficacy of PEI-PEG-mEGF / PolyIC + anti-PD-1 combination The effects of PEI-PEG-EGF / PolyIC plus anti-PD-1 on RencaEGFR lung metastasis in immunocompetent mice were investigated.

[0189] Materials and Methods: Cells: Renca EGFR; Polyplexes PEI-PEG-EGF / PolyIC and LPEI / EGF mouse: 1 / 0.75; Dalton's PolyIC; N / P: 8 polyplexes in HBG (Hepes-buffered glucose). Anti-PD-1: Rat IgG2a, κ anti-mouse PD-1 antibody (Bioxcell clone RMP1-14).

[0190] To induce the formation of RencaEGFR tumors in the lungs, 250,000 RencaEGFR cells were intravenously injected into 40 Balb / c immunocompetent female mice (6 weeks old, body weight: 18-21 g). After 10 days, the animals were divided into four groups (10 animals per group): untreated (UT), anti-PD-1, PEI-PEG-EGF / PolyIC, and PEI-PEG-EGF / PolyIC + aPD-1 (anti-PD-1).

[0191] Mice bearing Renca EGFR tumors were treated intravenously on days 0, 2, 4, 7, and 10 with 250 μg / kg PEI-PEG-EGF / PolyIC alone, six injections per week for 2 weeks, or in combination with 10 mg / kg intraperitoneally anti-PD-1 (RPM1-14, rat IgG2a, Biox cells). Survival rates were then analyzed. The appearance of clinical signs of cancer: a 10% weight loss between the last two weighings or a 20% weight loss between the first and last weighing, slow / abnormal movement, hunched posture, or abnormal breathing warranted sacrifice of the mice. The presence of lung tumors was visually confirmed in sacrificed mice. Mice were sacrificed according to scored parameters, including weight measurements and the general health of the mice. After sacrifice, organs were collected and analyzed by IHC to detect the presence of lung metastases.

[0192] Conclusions: Tumor uptake was 100% in both groups (untreated (UT) and anti-PD-1), with final mean survival times of 41.5 and 41.8 days. All mice developed tumors after cell injection, demonstrating the high reliability of the model. Anti-PD-1 alone did not inhibit tumor growth. At day 60, one animal remained alive in the PEI-PEG-EGF / PolyIC (PPE / PIC) alone group. The preliminary increase in survival with PEI-PEG-EGF / PolyIC is approximately 32%. Only three animals died in the PEI-PEG-EGF / PolyIC + anti-PD-1 (PPE / PIC + aPD1) group. The preliminary increase in survival rate was approximately 63%, already suggesting a synergistic effect between PEI-PEG-EGF / PolyIC and anti-PD-1 (Figure 9).

[0193] [Table 1]

[0194] Example 4 - In vivo efficacy of PEI-PEG-EGF / PolyIC + anti-CTLA-4 combination RencaEGFR cells are intravenously injected into 40 Balb / c immunocompetent female mice to induce the formation of RencaEGFR tumors in the lungs, as described in Example 3. After 10 days, the animals are divided into four groups: untreated control (UT), anti-CTLA-4, PEI-PEG-EGF / PolyIC, and PEI-PEG-EGF / PolyIC + anti-CTLA-4. Mice bearing EGFR tumors are treated with PEI-PEG-EGF / PolyIC alone or in combination with anti-CTLA-4 on days 0, 2, 4, 7, and 10.

[0195] Mice are sacrificed according to the scoring parameters, such as body weight measurement and general health status of the mice. After the mice are sacrificed, organs are collected and analyzed by IHC to detect the presence of lung metastasis. Survival and the appearance of clinical symptoms of cancer are analyzed. The presence of lung tumors is visually confirmed in sacrificed mice.

[0196] It is expected to reduce the clinical symptoms of cancer, inhibit tumor growth, and increase survival rates.

[0197] Example 5 - Combination treatment with PolyIC / PPHA and anti-PD-1 antibody in immunocompetent mice bearing HER-2-overexpressing tumors Materials and Methods: RENCA-HER2 cells (HER2-overexpressing renal cell carcinoma cells) (5 × 10 6 ) was injected subcutaneously (sc) into the right flank of immunocompetent BALB / c female mice (6–8 weeks old, Harlan Laboratories). Mice bearing subcutaneous RENCA HER2 tumors grew to a mean tumor volume of 235 mm. 3 The subjects were randomly assigned to four groups (naive, anti-PD-1, PolyIC / PEI-PEG-HER2 affibody (PPHA) polyplex, and anti-PD-1 + PolyIC / PPHA) with 7-8 animals per group. PolyIC / PPHA is preferably synthesized as described in Examples, more preferably Example 2, of WO 2015 / 173824 and pages 18 et seq. of WO 2015 / 173824. The HER2 affibody is preferably synthesized as described in WO 2015 / 173824, pages 20 et seq.

[0198] Mice were treated intravenously (iv) with 0.25 mg / kg PolyIC / PPHA or 6.25 mg / mouse, N / P 8, every 24 hours for 10 days. Four doses of anti-PD-1 (BioXCell, InVivoMAb anti-mouse PD-1 (CD279), clone RMP1-14, catalog no. BE0146), 200 mg / mouse, were injected intraperitoneally (ip) every 4-6 days. Tumor xenografts were measured with calipers, and tumor volume was calculated using the formula: length x width. 2 / 2 and plotted as mean SEM.

[0199] Conclusions: Significant inhibition of tumor growth was observed in both the combination group and PolyIC / PPHA alone compared to anti-PD-1 antibody alone and compared to untreated tumors. Complete tumor regression was observed in 2 out of 8 mice in the combination group. These mice showed no tumor growth for an additional 60 days. Cured mice showed complete protection from tumor rechallenge (5 x 10 6), indicating that an immune response against the tumor has occurred.

[0200] Example 6 - In vitro experiments, PD-L1 expression Materials and Methods: Increased PD-L1 expression was demonstrated in RENCA HER2 cells after treatment with PolyIC / PPHA. RENCA HER2 cells were treated with PolyIC / PPHA at a concentration of 1 microgram / ml for 24 hours. Cells were analyzed using an anti-mouse PD-L1 antibody conjugated to phycoerythrin (PE). After treatment, cells were trypsinized, washed, and incubated with anti-PD-L1 antibody CD274 (PD-L1, B7-H1) [10F.9G2] Tonbo cat#50-1243-U025 or an isotype control for 1 hour. PD-L1 expression was analyzed using flow cytometry (BD FACS ARIAIII, BD Biosciences). Live cells were gated based on SSC and FSC. PE-positive cells were gated, and the mean PE was determined.

[0201] Conclusions: RENCA HER-2 cells were treated with 1 microgram / ml PolyIC / PPHA for 24 hours, and PD-L1 expression was analyzed using flow cytometry. Compared to untreated cells, a significant increase in PD-L1 expression was observed after treatment with 1 microgram / ml PolyIC / PPHA, indicating that targeted delivery of PolyIC induces upregulation of PD-L1 expression.

[0202] [Table 2]

[0203] Example 7 - PBMCs are indirectly activated by polyplexes of the invention comprising PolyIC and targeting polymer conjugates The following experiments demonstrate the advantages of combining a polyplex of the present invention with nivolumab over monotherapy.

[0204] Materials and Methods: IFNγ release from PBMCs exposed to the supernatant of A431 cells treated with PEI-PEG-EGF / PolyIC polyplexes was quantified. Cell lines: The cell types used were A431 cancer cells (ATCC) and PBMCs (healthy donor 147). A431 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin. Buffy coats were obtained from healthy donors. PBMCs were isolated from the buffy coats and cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin.

[0205] PEI-PEG-EGF / PolyIC polyplexes were prepared in Hepes-buffered glucose (HBG). PEI-PEG-EGF triconjugates consisted of PEGylated linear polyethyleneimine conjugated to hEGF (hEGF-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid) via an MCC linker and were synthesized according to Example 10 of WO 2015 / 173824. An anti-PD-1 antibody (nivolumab) was used alone or in combination with PEI-PEG-EGF / PolyIC polyplexes. An anti-CD3 antibody (clone OKT3) was used to stimulate PBMCs after removing the medium from treated A431 cells. The combination of PEI-PEG-EGF / PolyIC polyplexes with checkpoint blockade was tested using an anti-PD-1 antibody (nivolumab). Human interferon-γ secretion was determined using an ELISA kit (BD Bioscience) according to the manufacturer's instructions.

[0206] A431 cells were seeded in flat-bottom 96-well plates (40,000 cells / 90 μl of DMEM medium). In parallel, plates containing medium alone (90 μl of medium without cells) were prepared as negative controls. The next day, A431 cells or medium alone were treated with 0, 0.125, or 1 μg / ml of PEI-PEG-EGF / PolyIC polyplexes (10 μl / well) for 5 hours at 37°C. Frozen PBMCs were thawed and allowed to recover in RPMI-1640 medium in U-bottom 96-well plates (200,000 cells / 100 μl) for at least 5 hours at 37°C. After 5 hours, supernatants (SN) from PEI-PEG-EGF / PolyIC polyplex-treated A431 cells or PEI-PEG-EGF / PolyIC polyplex-treated cultures were transferred to the PBMCs. PBMCs were then stimulated with anti-CD3 antibody (OKT3, 500 ng / ml) and / or treated with nivolumab (20 μg / ml) and incubated overnight at 37°C. SNs of stimulated PBMCs were collected and stored at -20°C until analysis by ELISA.

[0207] To model the effect of combining PEI-PEG-EGF / PolyIC polyplexes with nivolumab treatment on immune cell activation in vitro, SNs from PEI-PEG-EGF / PolyIC polyplex-treated A431 cells were transferred to human PBMCs from healthy donors and treated in combination with nivolumab as follows: A431 cells were treated with the indicated concentrations of PEI-PEG-EGF / PolyIC polyplexes for 5 hours. As a control, cell-free medium was "treated" with PEI-PEG-EGF / PolyIC polyplexes and similarly incubated for 5 hours. After 5 hours, PBMCs stimulated with or without anti-CD3 antibody were treated with the following: nivolumab alone, SN from A431 cells treated with PEI-PEG-EGF / PolyIC polyplexes alone, PEI-PEG-EGF / PolyIC polyplex-treated medium alone, nivolumab + PEI-PEG-EGF / PolyIC polyplex-treated A431 cells, or nivolumab + PEI-PEG-EGF / PolyIC polyplex-treated medium. PBMCs were incubated overnight at 37°C. To assess PBMC activation, IFN-γ secreted from PBMCs was quantified by ELISA.

[0208] Results: As seen in Figure 10 and Table 3, CD3-stimulated PBMCs receiving SN from A431 cells treated with PEI-PEG-EGF / PolyIC polyplexes secreted significantly more IFN-γ (311.1–326.3 pg / ml) than PBMCs receiving SN from untreated (UT) cells (12.7 pg / ml). IFN-γ secretion was further enhanced by the addition of nivolumab (502–523 pg / ml). PBMCs receiving SN from untreated cells plus nivolumab secreted low levels of IFN-γ (62.5 pg / ml). Nivolumab treatment alone did not induce IFN-γ secretion. PEI-PEG-EGF / PolyIC polyplex "treated" medium (without cells) alone or in combination with nivolumab did not result in increased IFN-γ secretion compared to UT medium. These results demonstrate that the combination of PEI-PEG-EGF / PolyIC polyplexes with nivolumab results in increased activation of PBMCs, as seen by increased IFN-γ secretion, compared with PEI-PEG-EGF / PolyIC polyplexes alone. Furthermore, PBMCs are activated by cytokines secreted from A431 cancer cells treated with PEI-PEG-EGF / PolyIC polyplexes, but not with PEI-PEG-EGF / PolyIC polyplexes alone.

[0209] Conclusions: The combination of nivolumab with PolyIC and a polyplex containing the targeting moiety human EGF was repeated in a media transfer experiment after treatment of A431 cells. IFN-γ levels were measured by ELISA, demonstrating that the combination of both agents resulted in increased IFN-γ compared with either agent alone. As controls, single agents (polyplex or nivolumab) or the combination were incubated in cell-free media for the same time course. The media was then transferred to PBMCs. IFN-γ levels were significantly lower in this group compared with PBMCs receiving media transferred from A431-treated cells. These results demonstrate that PBMC activation is not directly mediated by PolyIC polyplexes, but rather via chemokine secretion from cancer cells treated with PolyIC polyplexes.

[0210] [Table 3]

[0211] Example 8 - Cytokine secretion after treatment with PEI-PEG-EGF / PolyIC polyplexes and each component alone To assess whether PEI-PEG-EGF / polyIC polyplexes or their components induce the secretion of proinflammatory cytokines, three cell lines, including two EGFR-high expressing lines (MDA-MB-468 and A431) and one EGFR-low expressing line (MCF7), were treated with PEI-PEG-EGF / polyIC polyplexes, triconjugated PEI-PEG-EGF, or pIC, and the secretion of IP-10, GRO-α, and CCL5 cytokines was measured by ELISA.

[0212] Materials and Methods: Three cell lines (EGFR-high expressing MDA-MB-468 and A431) and one EGFR-low expressing MCF7 were treated with the indicated concentrations of PEI-PEG-EGF / PolyIC polyplexes, PEI-PEG-EGF triconjugate, or PolyIC for 5 hours. The medium was then collected and analyzed for CCL5 (RANTES), IP10, and GROα. PEI-PEG-EGF was composed of PEGylated linear polyethyleneimine attached to hEGF via the linker MCC, synthesized according to Example 10 of WO 2015 / 173824.

[0213] Tumor cell lines: MDA-MB-468, A431, and MCF7 cells were provided by ATCC. Cell lines were routinely passaged once or twice a week and maintained in culture for up to 20 passages. Cell lines were grown in RPMI-1640 medium (25 mM HEPES with L-glutamine, #FG1385, Biochrom, Berlin, Germany) or DMEM supplemented with 10% (v / v) fetal bovine serum (Sigma, Taufkirchen, Germany), 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C in a humidified atmosphere of 5% CO2.

[0214] Treatment: PEI-PEG-EGF / PolyIC polyplexes or pIC alone were incubated in HBG (HEPES-buffered glucose). The reagents were used at concentrations of pIC ranging from 0 to 1 μg / ml. Triconjugated PEI-PEG-EGF alone was prepared in a similar manner to the PEI-PEG-EGF / PolyIC polyplexes without pIC.

[0215] Quantification of cytokine production by ELISA: To detect secretion of IP-10, Gro-α, and RANTES, 40,000 cells per well were treated with PEI-PEG-EGF / polyIC polyplexes and pIC concentrations of 0–1 μg / ml, or equivalent concentrations of PEI-PEG-EGF triconjugate or pIC for 5 h. Supernatants were collected, and cytokine secretion was quantified by IP-10, Gro-α, and RANTES ELISA (Peprotech) using a Synergy H1 plate reader (Biotek).

[0216] Results: Treatment of EGFR-highly expressing MDA-MB-468 and A431 cells with PEI-PEG-EGF / PolyIC polyplexes induced the secretion of IP-10, GRO-α, and CCL5, whereas no cytokine expression was observed in EGFR-low expressing MCF7 cells (Figures 11A, B, and C, respectively). Maximum IP-10 levels of 337 and 268 pg / ml, GRO-α levels of 496 and 757 pg / ml, and RANTES levels of 607 and 100 pg / ml were observed in A431 and MDA-MB-468, respectively. No cytokine secretion was observed in any cell line after treatment with PEI-PEG-EGF triconjugate or pIC alone. These results demonstrate that PEI-PEG-EGF / PolyIC polyplexes, but not their individual components, induce the secretion of pro-inflammatory cytokines.

[0217] Example 9 - Activation of PBMCs with PEI-PEG-EGF / polyIC polyplexes and pIC / PPHA Materials and Methods: Tumor cell lines: MDA-MB-468, A431, and MCF7 cells were provided by ATCC. Cell lines were routinely passaged once or twice a week and maintained in culture for up to 20 passages. Cell lines were grown in RPMI-1640 medium (25 mM HEPES with L-glutamine, #FG1385, Biochrom, Berlin, Germany) or DMEM supplemented with 10% (v / v) fetal bovine serum (Sigma, Taufkirchen, Germany), 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C in a humidified atmosphere of 5% CO2.

[0218] Reagents: PEI-PEG-EGF triconjugates consisted of PEGylated linear polyethyleneimine conjugated to hEGF (human epidermal growth factor) via an MCC linker and were synthesized according to Example 10 of WO 2015 / 173824. PEI-PEG-EGF / polyIC polyplexes and PEI-PEG-EGF / pLGA polyplexes (PEI-PEG-EGF and poly-L-glutamic acid (pLGA), Sigma, 50-100 kDa p4886) were prepared in HEPES-buffered glucose (HBG) and used naked pIC. Reagents were used at concentrations of 0-1 µg / ml of pIC or pLGA within the polyplexes.

[0219] PBMC isolation from healthy donors (buffy coat): Buffy coats from healthy donors were obtained from the University Hospital Basel Blood Bank. PBMCs were isolated from the buffy coats and cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin.

[0220] Quantification of cytokine production by ELISA: To quantify IFN-γ and TNF-α secretion, 40,000 cells per well or medium alone were treated with the following compounds: PEI-PEG-EGF / polyIC polyplexes at pIC concentrations of 0–1 μg / ml, pIC alone (0–1 μg / ml), or PEI-PEG-EGF polyplexed with polyglutamic acid (PEI-PEG-EGF / pLGA polyplexes) at 0–1 μg / ml. After 5 h of incubation, supernatants and medium alone were collected and transferred to 200,000 PBMCs stimulated with a-CD3 (0.5 μg / ml) for an additional 16 h. In parallel, supernatants from each cell line treated with each compound alone were incubated for 16 h in the absence of PBMCs. After incubation, the medium was collected and TNF-α, IFN-γ and IL-2 were measured by ELISA assays (Peprotech and Invitrogen) and read using a Biotek Synergy H1 plate reader.

[0221] Results: PBMC activation by cytokines secreted from cancer cells treated with PEI-PEG-EGF / polyIC polyplexes: Proinflammatory cytokines, such as IP-10, are known to induce lymphocyte recruitment and activation. To evaluate whether PEI-PEG-EGF / polyIC polyplexes or their drug substance pIC induce immune cell activation, three cell lines, one with high EGFR expression (MDA-MB-468 and A431) and one with low EGFR expression (MCF7), were treated with the indicated concentrations of PEI-PEG-EGF / polyIC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes (PEI-PEG-EGF polyplexed with polyglutamic acid (pLGA)). For PEI-PEG-EGF / pLGA polyplexes, the pIC in PEI-PEG-EGF / polyIC polyplexes was replaced with polyglutamic acid to demonstrate that the effect of PEI-PEG-EGF / polyIC polyplexes was pIC-mediated. After 5 hours of treatment, supernatants from the treated cancer cells were transferred to PBMCs from healthy donors (Figure 12; "PBMCs + SN" from each cell line). PBMCs were then incubated for 16 hours and analyzed by ELISA for cytokine secretion of IFN-γ and TNFα. In parallel, to demonstrate that PEI-PEG-EGF / PolyIC polyplexes did not directly induce PBMC activation, PEI-PEG-EGF / PolyIC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes were incubated in cell-free medium, this medium was transferred to PBMCs, and the PBMCs were incubated for 16 hours and tested by ELISA (Figure 12, "PBMCs alone"). As an additional control, supernatants from treated cancer cells incubated for the same time without PBMCs were also analyzed (Figure 12, "SN alone").

[0222] Increased IFN-γ secretion was observed in PBMCs incubated with supernatants from MDA-MB-468 and A431 cells treated with PEI-PEG-EGF / PolyIC polyplexes. IFN-γ levels of up to 3587 pg / ml and 2023 pg / ml were detected in PBMCs from MDA-MB-468 and A431 cells, respectively. PBMCs incubated with supernatants from MCF7 cells treated with PEI-PEG-EGF / PolyIC polyplexes did not induce cytokine secretion. PBMCs treated with cell-free medium containing PEI-PEG-EGF / PolyIC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes (PBMCs alone) did not induce IFN-γ secretion in any cancer cell line. No IFN-γ secretion was observed in the supernatants of any of the cancer cell lines treated with either PEI-PEG-EGF / polyIC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes without PBMCs.

[0223] A significant increase in TNFα secretion was observed in PBMCs incubated with medium from MDA-MB-468 and A431 cells treated with PEI-PEG-EGF / PolyIC polyplexes. Maximum TNFα levels of 2190 pg / ml and 3438 pg / ml were detected in PBMCs from MDA-MB-468 and A431 cells, respectively. PBMCs incubated with medium from MCF7 cells treated with PEI-PEG-EGF / PolyIC polyplexes did not induce cytokine secretion. Medium from PBMCs treated with PEI-PEG-EGF / PolyIC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes (PBMCs alone) did not induce a significant increase in TNFα secretion in any cancer cell line compared with UT cells. No secretion of IFN-γ or TNFα was observed in supernatants from cancer cell lines treated with PEI-PEG-EGF / polyIC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes without PBMCs.

[0224] Example 10 - Efficacy and selectivity of PEI-PEG-EGF / PolyIC polyplexes compared to naked pIC, PEI, or pIC complexed with lipid-based transfection reagents The objective of the study was to determine the selectivity and efficacy of PEI-PEG-EGF / PolyIC polyplexes versus single component and non-targeted delivery systems and pIC analogs.

[0225] Materials and Methods: Cell lines: Highly EGFR-expressing cell lines (BT20, MDA-MB-468, and HCC70), moderately EGFR-expressing cell lines (U87MG), lowly EGFR-expressing cell lines (MCF7 and U138), and non-cancer cell lines (WI-38 and MCF10A) were used in the experiments. Cells were cultured as described above.

[0226] Reagents: PEI-PEG-EGF triconjugates, consisting of PEGylated linear polyethyleneimine conjugated to hEGF via an MCC linker, were synthesized according to Example 10 of WO 2015 / 173824. PEI-PEG-EGF triconjugates and pIC were used to prepare PEI-PEG-EGF / PolyIC polyplexes in HEPES-buffered glucose (HBG). JetPEI (Polyplus) and pIC were used to prepare jetPEI-pIC polyplexes according to the manufacturer's instructions. Lipofectamine RNAiMax (Invitrogen, P / N 56531) and pIC were used to prepare RNAiMax-pIC polyplexes according to the manufacturer's instructions. Poly-L-glutamic acid (pLGA, Sigma 50–100 kDa) and PEI-PEG-EGF were used to prepare PEI-PEG-EGF / pLGA polyplexes with the same net charge as PEI-PEG-EGF / PolyIC polyplexes.

[0227] Treatment: 3000 cells / well were seeded in a 96-well plate. After 24 hours, cells were treated with the indicated concentrations of polyplexes (ranging from 2 μg / ml to 0.001 μg / ml). 72 hours after treatment, cell viability was assessed using CellTiter Glo (Promega), and luminescence was recorded using a synergy H1 cell plate reader (Biotek).

[0228] Results: The effect of PEI-PEG-EGF / polyIC polyplexes on the viability of high- and low-EGFR-expressing cancer cell lines and non-cancer cell lines was compared with that of naked pIC, jetPEI-pIC, RNAiMax-pIC (a lipid-based transfection reagent), and PEI-PEG-EGF / pLGA polyplexes.

[0229] JetPEI-pIC polyplexes were significantly less effective than PEI-PEG-EGF / PolyIC polyplexes in all EGFR-overexpressing cells: the IC50 of PEI-PEG-EGF / PolyIC polyplexes was 10-20 times lower than that of jetPEI-pIC. In cancer cell lines with low EGFR activity or non-cancer cell lines, jetPEI-pIC was up to two-fold more toxic than PEI-PEG-EGF / PolyIC polyplexes (Figure 13A-E). These results demonstrate the increased efficacy and selectivity of PEI-PEG-EGF / PolyIC polyplexes compared to jetPEI-pIC.

[0230] Increased efficacy of PEI-PEG-EGF / PolyIC polyplexes was observed in three of four EGFR-overexpressing cell lines compared with treatment with RNAiMax-pIC. Only in A431 cells was the IC50 of RNAiMax-pIC fourfold lower than that of PEI-PEG-EGF / PolyIC polyplexes (Figure 13). In BT20, MDA-MB-468, and HCC70 cells, the IC50 of PEI-PEG-EGF / PolyIC polyplexes was approximately tenfold lower than that of RNAiMax-pIC. RNAiMax-pIC was found to be toxic in normal cells (WI-3 and MCF10A) and cancer cells with low EGFR expression, whereas PEI-PEG-EGF / PolyIC polyplexes were not toxic to these cells.

[0231] At the concentrations tested, naked pIC was ineffective in all cell lines.

[0232] The PEI-PEG-EGF / pLGA polyplex was significantly less effective (less than 30-fold) than the PEI-PEG-EGF / PolyIC polyplex against EGFR-overexpressing cancer cells. The PEI-PEG-EGF / pLGA polyplex served as a control for the PEI-PEG-EGF / PolyIC polyplex. The results clearly demonstrate that the efficacy of the PEI-PEG-EGF / PolyIC polyplex is mediated through targeted pIC therapy.

[0233] PEG-EGF / PolyIC polyplexes showed high specificity and efficacy in EGFR-overexpressing cells while not inducing toxicity in normal cell lines. The efficacy of PEI-PEG-EGF / PolyIC polyplexes was demonstrated to be mediated through pIC. In contrast to PEI-PEG-EGF / PolyIC polyplexes, RNAiMax / pIC and jetPEI / pIC were less effective and selective than PEI-PEG-EGF / PolyIC polyplexes. RNAiMax / pIC was also highly toxic to normal cells. [Sequence List Free Text]

[0234] Sequence Listing 1 <223> synthesis Sequence Listing 3 <223> synthesis Sequence Listing 3 <223> X is 8-aminooctanoic acid. Sequence Listing 4 <223> synthesis Sequence Listing 4 <223> X is 8-aminooctanoic acid. Sequence Listing 4 <223> X is diaminopropionic acid.

Claims

1. A kit of parts comprising: a. A polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, the polymer conjugate comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties; PEI is covalently bound to one or more PEG moieties, each of the one or more PEG moieties being linked to one of the one or more targeting moieties; and the polyplex, wherein each of the one or more targeting moieties is capable of binding to a cancer antigen; b. at least one antibody capable of modulating an immune checkpoint protein; The above kit of parts including.

2. 1. A composition comprising: a. A polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, the polymer conjugate comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties; PEI is covalently bound to one or more PEG moieties, each of the one or more PEG moieties being linked to one of the one or more targeting moieties; and the polyplex, wherein each of the one or more targeting moieties is capable of binding to a cancer antigen; b. at least one antibody capable of modulating an immune checkpoint protein; The composition comprising:

3. At least one antibody capable of modulating an immune checkpoint protein, (i) at least one antibody capable of agonizing a costimulatory immune checkpoint protein, wherein the costimulatory immune checkpoint protein is selected from the group consisting of 4-1BB, 4-1BB ligand (4-1BBL), CD40, CD40 ligand (CD40L), OX40, OX-40 ligand (OX-40L), GITR, GITR ligand (GITRL), ICOS, and ICOS ligand (ICOSL); or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3, and TIGIT; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). The composition of claim 1 or the kit of parts of claim 2, wherein

4. The composition or kit-of-parts according to any one of claims 1 to 3, wherein the immune checkpoint protein is selected from the group consisting of 4-1BB, 4-1BB ligand, PD-1, PD-L1, PD-L2 and CTLA-4, more preferably the immune checkpoint protein is selected from the group consisting of 4-1BB, 4-1BB ligand, PD-1, PD-L1 and PD-L2.

5. The composition or kit-of-parts according to any one of claims 1 to 4, wherein the immune checkpoint protein is 4-1BB or PD-1.

6. At least one antibody capable of modulating an immune checkpoint protein, (i) an antibody capable of modulating the immune checkpoint protein CD27 and an antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2; (ii) an antibody capable of modulating the immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4; (iv) an antibody capable of modulating the immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and CTLA-4; (v) an antibody capable of modulating the immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or (vi) an antibody capable of modulating the immune checkpoint protein ICOS and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2. The composition or kit-of-parts according to any one of claims 1 to 5, which is a mixture of

7. Composition or kit-of-parts according to any one of claims 1 to 6, wherein the PEI is covalently bound to one, two or three PEG moieties, preferably one or three PEG moieties.

8. The composition or kit-of-parts according to any one of claims 1 to 7, wherein the polyethyleneimine (PEI) is linear polyethyleneimine (LPEI).

9. The composition or kit-of-parts according to any one of claims 1 to 8, wherein the dsRNA is polyinosinic-polycytidylic acid double-stranded RNA (poly IC).

10. The composition or kit-of-parts according to any one of claims 1 to 9, wherein the cancer antigen is EGFR, HER2 or PSMA, preferably the cancer antigen is EGFR.

11. The one or more targeting moieties may be EGF, a HER2 affibody, a HER2 antibody, and a DUPA moiety (HOOC(CH ２ ) ２ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ２ ) ２ The composition or kit-of-parts according to any one of claims 1 to 10, wherein the compound is selected from the group consisting of: -CO-.

12. Composition or kit-of-parts according to any one of claims 1 to 11, wherein the one or more target moieties is EGF, preferably human EGF.

13. A composition or kit-of-parts according to any one of claims 1 to 12 for use in the treatment of cancer in a mammal, preferably a human.

14. 14. The composition or kit-of-parts for use according to claim 13, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, vulvar cancer, urothelial cancer, bladder cancer, kidney cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, glial tumors, head and neck cancer, prostate cancer, penile cancer, testicular embryonal carcinoma, neuroendocrine tumors, and hepatocellular carcinoma.

15. 15. The kit-of-parts for use according to claim 13 or claim 14, wherein the polyplex is administered to a mammal, preferably a human, separately from at least one antibody capable of modulating an immune checkpoint protein.

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