Immunotherapy for cancer treatment

Abstract

The present invention relates to a kit-of-parts and a composition comprising a polyplex, which comprises 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. The present invention further relates to this composition or kit-of-parts for use in cancer treatment.

Description

Background Art

[0001] The present invention relates to the field of cancer treatment by immunotherapy. In particular, the present invention relates to polyplexes 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 kits of parts and compositions comprising 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. Further, the present invention relates to such compositions or kits of parts for use in cancer treatment.

[0002] Antibodies targeting tumor-associated antigens have become an important treatment for malignant tumors. Some monoclonal antibodies (mAbs) have been shown to be relatively well tolerated and effective in the treatment of many different malignant diseases. These antibodies are commonly used in clinics, but their effectiveness is generally moderate in most cases. mAbs need to overcome substantial obstacles to reach the antigen presented on the target cells and provide therapeutic value (Christiansen et al., Mol Cancer Ther, 2004, 3(11), 1493-1501). Furthermore, the efficiency of antibodies targeting tumor-associated antigens is reduced by the insufficient activation of the anti-tumor response of the immune system and the 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 management, and improved survival in some patients with advanced melanoma, non-small cell lung cancer (NSCLC), and other tumor types. Binding of PD-1 by either of its ligands, PD-L1 or PD-L2, induces a negative regulatory 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] Cytokines related to tumor necrosis factor (TNF) provide an essential communication network for linking multiple cell types to an effective host defense system against pathogens and malignant cells. Ligands (TNFSF) and receptors (TNFRSF) of the tumor necrosis factor superfamily provide important communication signals among various cell types during development. TNF receptors (TNFRs) share a conserved extracellular domain defined by a cysteine-rich signature. TNFRs with costimulatory properties are encoded by genes located within an immune response locus on chromosome region 1p36 and include GITR (glucocorticoid-induced TNF receptor), OX40, 4-1BB, and CD30 (Ward-Kavanagh, et al., The TNF Receptor Superfamily in Co-stimulating and Co-inhibitory Responses, Immunity 44, May 17, 2016).

[0005] Different approaches for treating malignant tumors are vaccine-based therapies. The molecular definition of tumor-associated antigens has led to the possibility of specific vaccines aimed at targeting tumor cells. Recombinant vaccines based on peptides or proteins derived from defined tumor-associated antigens (TAAs) are usually administered together with adjuvants or immunomodulators. These vaccines were able to induce antigen-specific T cell responses, but the clinical outcomes were unexpected (Guo et al., Adv Cancer Res, 2013, 119:421-475).

[0006] A further approach in cancer immunotherapy refers to the combination of antibodies and vaccines that activate anti-tumor immunity by blocking or inhibiting immune checkpoints. Immune checkpoints refer to a number of 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 utilize specific immune checkpoint pathways as a major mechanism of immune tolerance. Since many immune checkpoints are initiated by ligand-receptor interactions, they can be easily blocked or inhibited by antibodies or regulated by recombinant forms of ligands or receptors. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibodies were the first immunotherapeutic drugs of this class to obtain approval from the US Food and Drug Administration (FDA). However, the development of combination approaches of antibodies and vaccines remains a challenge. The combination strategy needs to be rationally designed and guided by mechanistic considerations and preclinical models (Pardoll, Nat Rev Cancer 2012, 12(4), 252-264).

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

[0008] Despite these initial promising results, the development of cancer immunotherapeutic agents continues to be a major challenge for tumor immunologists. Therefore, there is a high need for effective and tolerable immunotherapies for cancer treatment.

Summary of the Invention

[0009] The present invention provides a novel combinatorial immunotherapy approach for cancer treatment.

[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, wherein the polymer complex 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, and each of the one or more PEG moieties is linked to one of the one or more targeting moieties, each of the one or more targeting moieties is capable of binding to a cancer antigen, a polyplex, and b. At least one antibody 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 complex 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, and each of the one or more PEG moieties is linked to one of the one or more targeting moieties, each of the one or more targeting moieties is a polyplex that can bind to a cancer antigen, and b. at least one antibody that can modulate an immune checkpoint protein.

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

[0013] The inventors have unexpectedly found that combination therapy of a polyplex comprising double-stranded RNA (dsRNA) and a polymer complex with one or more anti-checkpoint antibodies according to the invention enhances the activity of the immune system and results in potent antitumor 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 the polyplex alone. In some individuals, the combination of the polyplex and the anti-checkpoint antibody eradicated the tumor completely and generated a persistent tumor defense and memory against cancer cells.

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

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

[0016] It has been found that treatment of tumors with immunomodulatory antibodies alone is not effective or shows only limited efficacy. With the anti-PD-1 antibody alone, the tumor size of RENCA tumors did not decrease (Figure 5). However, the combination of targeted and specifically delivered dsRNA and an immunomodulatory antibody showed an enhanced antitumor effect. The treatment of RENCA HER2 tumors with the HER2-targeted polyplex of the present invention, i.e., a triconjugate containing a PEI-PEG-HER2 affibody (PPHA) that forms a complex with poly IC (pIC / PPHA) and anti-PD-1 unexpectedly resulted in regression of tumor growth (Figure 6). Complete regression of the tumors was observed in the combination group with poly IC / PPHA + anti-PD-1. The cured mice showed complete protection from tumor rechallenge, indicating that an immune response against the tumors occurred.

[0017] Therefore, the combination of the targeted polyplex according to the present invention and an anti-checkpoint antibody can broaden the effectiveness of the antibody for patients who currently show no response. Utilizing the targeted delivery of dsRNA, preferably poly IC, in combination with an anti-checkpoint antibody shows significant effectiveness due to the ability of the compositions and kits of parts of the present invention to restore the immune system against tumors.

Brief Description of the Drawings

[0018]

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Mode for Carrying Out 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 following claims, unless the context requires otherwise, the word "comprise", "include", and variations such as "comprises" or "includes" and "comprising" or "including" are to be construed as including the recited integer, step, or group of integers or steps but not excluding any other integer, step, or group of integers or steps.

[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] When the term "about" or "approximately" is used in connection with a numerical value, it means a value within a range having a lower limit that is 0 to 10% less than the stated value and an upper limit that is 0 to 10% greater than the stated 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 deleted. All ranges of values disclosed herein refer to and should include all values within the 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, wherein the polymer complex comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties, the PEI is covalently bonded to one or more PEG moieties, and each of the one or more PEG moieties is linked to one of the one or more targeting moieties, each of the one or more targeting moieties is capable of binding to a cancer antigen, a polyplex, and b. At least one antibody 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, wherein the polymer complex comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties and one or more targeting moieties, wherein the PEI is covalently bound to the one or more PEG moieties, and each of the one or more PEG moieties is linked to one of the one or more targeting moieties, wherein each of the one or more targeting moieties is capable of binding to a cancer antigen, a composition, and b. At least one antibody 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] As used herein, the term "kit of parts" preferably refers to at least two separate parts, namely the combination of 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 kits of parts are known to those skilled in the art conventionally. In a particularly preferred embodiment, the kit of parts of the present invention or the parts of the kit of parts of the present invention, namely the polyplex and / or the one or more immunomodulatory antibodies, independently of each other, comprise at least one pharmaceutically acceptable diluent, excipient or carrier. In another preferred embodiment, the kit of parts of the present invention or the parts of the kit of parts of the present invention, namely the polyplex and / or the one or more immunomodulatory antibodies, independently of each other, comprise one or more adjuvants.

[0027] In certain embodiments, the compositions and kits of parts according to the present invention are formulated for administration by any known method. The compositions and kits of parts according to the present invention, namely the polyplex and / or the one or more immunomodulatory antibodies, and the pharmaceutical compositions can be formulated for any suitable route of administration including, but not limited to, intravenous, intracerebral (intracranial), 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 compositions and kits of parts according to the present invention, namely the polyplex and / or the one or more immunomodulatory antibodies, are formulated for intravenous, intraperitoneal or subcutaneous administration. More preferably, the compositions and kits of parts according to the present invention, namely the polyplex and / or the one or more immunomodulatory antibodies, are formulated as 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 present invention comprise the polyplexes of the present invention and one or more immune regulatory antibodies, and the polyplexes and the one or more immune regulatory antibodies are present in a therapeutically effective amount in the compositions and kits of parts.

[0030] The kit of parts of the present invention may comprise a container containing the polyplex and / or one or more antibodies, and / or a kit component, i.e., a device for administering 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 (NK) cells, and 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, and their soluble ligands or binding ligands and counter-receptors that can co-stimulate or co-inhibit the activity of the immune system. Preferably, the activity of the immune system is detected by measuring the T cell response as shown herein (e.g., Example 2).

[0032] Immune checkpoint proteins are important immune regulatory factors 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 the immune response. Immune homeostasis is regulated by a careful balance of activating and inhibitory immune checkpoint proteins. Under normal circumstances, immune checkpoints allow the immune system to respond to infections and malignancies while protecting tissues from any harm that may result from this action.

[0033] Tumor cells utilize these checkpoints and have developed several strategies to evade the host immune defense. The expression of immune checkpoint proteins can be dysregulated by tumors as an important immune tolerance mechanism. Therefore, inhibition or activation of checkpoint receptors and ligands has emerged as a potential strategy to limit 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 for activating therapeutic anti-tumor immunity are blocking the inhibition of immune checkpoint proteins or activating 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 the physiological immune response to maintain self-tolerance and minimize accompanying tissue damage. Tumors utilize specific immune checkpoint pathways as a major mechanism of immune tolerance, particularly to T cells specific for tumor antigens. T cells have been the main focus of efforts to therapeutically manipulate endogenous anti-tumor immunity for the following reasons: their ability to selectively recognize peptide-derived from proteins in all cell compartments; their ability to directly recognize and kill antigen-expressing cells (by CD8+ effector T cells; CTL); and their ability to organize diverse immune responses (by CD4+ helper T cells), including the ability to integrate adaptive and innate effector mechanisms. Thus, agonists of co-stimulatory receptors or antagonists of inhibitory signals, both of which result in amplification of antigen-specific T cell responses, are agents in current clinical trials. Since many immune checkpoints are initiated by ligand-receptor interactions, they can be readily 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 the immune checkpoint protein and thus promotes the activity of the immune system. Promotion of immune system activity includes the generation of an enhanced immune response to an antigen and / or a decrease in 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 related to functional stimulation or enhancement of co-stimulatory immune checkpoint proteins, and inhibition of co-inhibitory immune checkpoint proteins related to reduced activity and complete blockade of co-inhibitory immune checkpoint proteins. The designation "modulating an immune checkpoint protein" includes stimulation of T cells, including T helper cells, CTL natural killer T cells, and natural killer cells (NK). The stimulation (or activation) induced by modulation of an immune checkpoint protein is preferably detected by measuring increased levels of cytokines, particularly interferon, especially IFN-γ, produced or released, particularly by T cells and NK cells, as compared to a control not administered an immune checkpoint protein modulating antibody, as shown herein (e.g., Example 2).

[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, CD40 / TNFRSF5, CD40L / CD154 / TNFSF5, OX40 / CD134, OX-40L / TNFSF4 / CD252, GITR (glucocorticoid-induced 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, SIGLEC9 (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-1 / LGALS9, TIM-3 / HAVCR2, TIGIT / VSTM3, HVEM (herpesvirus 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 preferably selected from the group consisting of the foregoing, without limitation.

[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-related checkpoint inhibitor or a non-T cell-related checkpoint inhibitor.

[0040] In a preferred embodiment, the 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 (inducible T cell co-stimulator) / 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-9 / LGALS9, TIM-3 / HAVCR2, and TIGIT / VSTM3. In a more preferred embodiment, the 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, ICOS ligand / 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more 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 more preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, 4-1BB, and OX40.

[0044] In a more 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 more 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 more 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 more 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 more 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 more 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 more preferred embodiment, the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, and 4-1BB. In a more preferred embodiment, the immune checkpoint protein is PD-1 or 4-1BB. In a more 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 more preferred embodiment, the immune checkpoint protein is PD-1 or PD-L1. In a more preferred embodiment, the immune checkpoint protein is PD-1. In a more preferred embodiment, the immune checkpoint protein is PD-L1. In a more preferred embodiment, the immune checkpoint protein is PD-L2. In a more 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 an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that selectively binds to an antigen, which includes a hapten, epitope, receptor or ligand, or a portion thereof. Thus, the term "antibody" encompasses not only the entire antibody molecule, but also antibody fragments, as well as variants (including derivatives), antibody fragments, and fusion proteins. The term "antibody" also refers to various forms including an antibody composed of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as full-length antibodies and portions thereof, such as, for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-bonded Fv, scFv, single-domain antibodies (dAb), 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, a counter-receptor, or their binding ligands and soluble ligands.

[0047] As used herein, the term "anti-..." refers 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 mimic a monoclonal antibody and bind to a target protein or peptide with high affinity, and thus is a member of the antibody mimic family. Preferably, the affibody has a high-affinity binding domain derived from protein A. HER2 is the target of the HER2 affibody.

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

[0050] Preferably, the antibody capable of regulating the immune checkpoint protein is a monoclonal antibody, a chimeric 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 complex, wherein the polymer complex comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties, the PEI is covalently bound to the 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 further aspect, the present invention provides a composition comprising: (a) a polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, wherein the polymer complex comprises polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties, the PEI is covalently bound to the 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 selected from the group consisting of 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 co-stimulator) / 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 selected from the group consisting of 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-H2, 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 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-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 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-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 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-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 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-TIGIT, anti-LAG3, anti-TIM3, anti-GITR, anti-CD40, anti-OX40, and anti-ICOS. 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-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM3, anti-GITR, anti-CD40, anti-OX40, and anti-ICOS. 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-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, anti-CD40, anti-OX40, and anti-ICOS. 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-4-1BB, anti-TIGIT, anti-LAG3, anti-TIM-3, anti-GITR, and anti-ICOS. 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-4-1BB, anti-LAG3, anti-TIGIT, anti-TIM-3, anti-GITR, and anti-ICOS. In a more 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 more 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 more 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 selected from the group consisting of (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-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 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-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-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 of (i) and at least one antibody of (ii).

[0054] In a more preferred embodiment, the at least one antibody is selected from the group consisting of (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-9 / 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 more 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-9 / 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-9 / 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 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, 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, or (ii) anti-PD-1, anti-PD-L1, anti-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, 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 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-OX40 / CD134, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (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) 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-4-1BBL / CD137L, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, 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-4-1BBL / CD137L, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

[0056] In a preferred embodiment, the antibody capable of regulating 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 regulating an immune checkpoint protein is a lipocalin that binds to 4-1BB and HER2.

[0057] Preferred examples of the antibody that can regulate the immune checkpoint protein PD-1 (anti-PD-1) include pembrolizumab, nivolumab (also known as MDX-1106 or BMS-936558, disclosed in Topalian et al., 2012. N. Engl. J. Med. 366:2443-2454, U.S. Patent No. 8008449 B2), semiprilimab, IBI308, BCD-100, PDR001, tislelizumab, camrelizumab, pidilizumab (disclosed in Rosenblatt et al., 2011, J Immunother. 34:409-18), and ramucirumab (for example, disclosed as hPD109A and its humanized derivatives h409All, h409A16, and h409A17 in International Publication No. 2008 / 156712, Hamid et al., 2013, N. Engl. J. Med. 369:134-144), and a human antibody or humanized antibody selected from the group consisting of 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 the antibody that can regulate the immune checkpoint protein PD-1 (anti-PD-1) are pembrolizumab or nivolumab.

[0058] Preferred examples of the antibody that can regulate the immune checkpoint protein PD-L1 (anti-PD-L1) include durvalumab, avelumab, and atezolizumab, MEDI-4736 (for example, disclosed in International Publication No. 2011 / 066389Al), MPDL328 OA (for example, disclosed in U.S. Patent No. 8217149 B2), and MIH1 (Affymetrix). More preferred examples of the antibody that can regulate 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 that can regulate 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 that can regulate the immune checkpoint protein CD27 is CDX-1127, an agonistic anti-CD27 monoclonal antibody.

[0061] Preferred examples of such antibodies that can regulate the immune checkpoint protein OX40 are MEDI0562, a humanized OX40 agonist, MEDI6469, a mouse OX4 agonist, and MEDI6383, an OX40 agonist.

[0062] A preferred example of such an antibody that can regulate the immune checkpoint protein KIR is lirilumab, a monoclonal antibody against KIR.

[0063] A preferred example of such an antibody that can regulate the immune checkpoint protein 4-1BB is urelumab. A preferred example of such an antibody that can regulate the immune checkpoint protein LAG-3 is relatlimab.

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

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

[0066] In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of activating (stimulating) a co-stimulatory 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 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 activating a co-stimulatory immune checkpoint protein (checkpoint activator). The checkpoint activator activates a co-stimulatory immune checkpoint protein. The checkpoint activator delivers an activation signal to T cells, B cells or natural killer cells by directly activating the receptors of that cell type, inhibiting or blocking an inhibitory ligand, or activating the counter-receptor of an antigen-presenting cell (APC).

[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 derepresses, i.e., reduces or eliminates, the inhibitory function of an inhibitory immune checkpoint protein. The checkpoint inhibitor delivers an antagonistic signal to T cells, B cells or natural killer cells by directly antagonizing the receptors of that cell type, activating an inhibitory ligand, or antagonizing the counter-receptor of an antigen-presenting cell (APC).

[0068] In another preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein is an immune checkpoint activator or inhibitor. In another preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein 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 activating 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, CD122, HVEM / TNFRSF14, TNFSF14 / LIGHT / CD258, CD70 / CD27L / TNFSF7, CD28 / TP44, CD30, CD30 ligand, 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, 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, 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 activating 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, CD122, HVEM / TNFRSF14, TNFSF14 / LIGHT / CD258, CD70 / CD27L / TNFSF7, CD28 / TP44, CD30, CD30 ligand, 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, 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 / 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 activating 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, TL1A, DR3, LTβR, TNF, TNFR2, ICOS / AILIM / CD278, and ICOS ligand / B7-H2, 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, 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 (i) at least one antibody capable of acting on 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, TL1A, DR3, LTβR, TNF, TNFR2, ICOS / AILIM / CD278, and ICOS ligand / B7-H2, 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 / 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 more preferred embodiment, 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 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 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, 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, 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 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 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, 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, 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 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 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 / 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, 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 CD137 / 4-1BB, 4-1BBL / CD137L, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and ICOS ligand / B7-H2, or (ii) capable of antagonizing a coinhibitory immune checkpoint protein. At least one antibody capable of, 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, an antibody, or (iii) a mixture of both.

[0070] In a preferred embodiment of the composition or kit of parts of the present invention, at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of activating a co-stimulatory immune checkpoint protein, wherein the co-stimulatory 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), an antibody, 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, B7-1, B7-2, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3 and TIGIT, an antibody, 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, 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, 4-1BB ligand (4-1BBL), CD40, CD40 ligand (CD40L), OX40, OX-40 ligand (OX-40L), GITR, GITR ligand (GITRL), ICOS and ICOS ligand (ICOSL), an antibody, 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, an antibody, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). AVCR2, and TIGIT / VSTM3, or a mixture of both (iii). In a more preferred embodiment of the composition or kit of parts of the present invention, 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, GITR, and ICOS, an antibody, 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, an antibody, 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, 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, GITR, and ICOS, an antibody, 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, an antibody, 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, 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, an antibody, 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, an antibody, 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, at least one antibody capable of regulating 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, ICOS, and GITR, an antibody, 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, an antibody, 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 present invention, at least one antibody capable of regulating 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, an antibody, 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, an antibody, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a more preferred embodiment of the composition or kit of parts of the present invention, 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, OX40, and GITR, an antibody, 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, an antibody, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a more preferred embodiment of the composition or kit of parts of the present invention, 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 and OX40, an antibody, 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, an antibody, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

[0075] In a further preferred embodiment, 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 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 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, or (iii) a mixture of both. In a further preferred embodiment, 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, 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, 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 CD137 / 4-1BB, 4-1BBL / CD137L, 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, or (iii) a mixture of both. In a further preferred embodiment, 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 CD137 / 4-1BB, 4-1BBL / CD137L, 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, or (iii) a mixture of both.

[0076] In a further preferred embodiment, 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, and 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, 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, 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, and 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. In a further preferred embodiment, 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, and 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, 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, GITR, OX40, ICOS, and CD40. In a further preferred embodiment, 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 CD137 / 4-1BB, 4-1BBL / CD137L, GITR / TNFRSF18, GITR ligand / TNFSF18, ICOS / AILIM / CD278, and ICOS ligand / B7-H2. In a further preferred embodiment, 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 CD137 / 4-1BB or 4-1BBL / CD137L. In a further preferred embodiment, 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 CD137 / 4-1BB.

[0077] In a further preferred embodiment, 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, and 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, 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, and 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, 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, and the agonizing antibody is anti-CD137 / 4-1BB or anti-4-1BBL / CD137L. In a further preferred embodiment, 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, and the agonizing antibody is anti-CD137 / 4-1BB.

[0078] In a more preferred embodiment, 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, and the coinhibitory 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, 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 more preferred embodiment, 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, and the coinhibitory 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 more preferred embodiment, 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, and the coinhibitory 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 more preferred embodiment, 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, and the coinhibitory 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 more preferred embodiment, 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, and the coinhibitory 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.In a further preferred embodiment, 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, 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, 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, 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, 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, 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, at least one antibody capable of regulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, and 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, at least one antibody capable of regulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, and 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, at least one antibody capable of regulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a coinhibitory immune checkpoint protein, and the coinhibitory 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, 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, and the antagonistic antibody is selected from the group consisting of 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. In a further preferred embodiment, 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, and the antagonistic 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 present invention comprises two or more antibodies capable of modulating an immune checkpoint protein. In a preferred embodiment, the composition or kit of parts of the present invention comprises one, two or three antibodies capable of modulating an immune checkpoint protein. In a preferred embodiment, the composition or kit of parts of the present invention comprises two or three antibodies capable of modulating an immune checkpoint protein. In a preferred embodiment, the composition or kit of parts of the present invention comprises two antibodies capable of modulating an immune checkpoint protein. In a preferred embodiment, the composition or kit of parts of the present invention comprises three antibodies capable of modulating an immune checkpoint protein.

[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 the immune checkpoint protein comprised in the composition or kit of parts of the present invention is (i) An antibody capable of regulating the immune checkpoint protein CD27 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, (ii) An antibody capable of regulating the immune checkpoint protein CD40 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, (iii) An antibody capable of regulating the immune checkpoint protein GITR 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, (iv) An antibody capable of regulating the immune checkpoint protein OX40 and at least one antibody capable of regulating 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 regulating the immune checkpoint protein 4-1BB and at least one antibody capable of regulating 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 which is a mixture of.

[0084] In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised in the composition or kit of parts of the present invention is a mixture 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. In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised in the composition or kit of parts of the present invention is a mixture of (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. In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised in the composition or kit of parts of the present invention is a mixture of (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. In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised in the composition or kit of parts of the present invention is a mixture of (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. In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised in the composition or kit of parts of the present invention is a mixture of (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.In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised in the composition or kit of parts of the present invention is a mixture of (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.

[0085] In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised 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 modulating an immune checkpoint protein, said antibody being selected from the group consisting of anti-PD-1, anti-PD-L1 and anti-PD-L2. In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised 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 modulating an immune checkpoint protein, said antibody being selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2 and anti-CTLA-4. In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised 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 modulating an immune checkpoint protein, said antibody being selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2 and anti-CTLA-4. In a preferred embodiment, at least one antibody capable of modulating an immune checkpoint protein comprised 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 modulating an immune checkpoint protein, said antibody being 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, at least one antibody capable of modulating an immune checkpoint protein comprised 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 modulating an immune checkpoint protein, said antibody being 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, at least one antibody capable of regulating an immune checkpoint protein included in the composition or kit of parts of the present invention is a mixture of (vi) anti-ICOS and at least one antibody capable of regulating an immune checkpoint protein, and the antibody is selected from the group consisting of anti-PD-1, anti-PD-L1, and anti-PD-L2.

[0086] In a preferred embodiment, the polyplex of the present 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, the PEI being 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 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.

[0087] In a preferred embodiment, the polyplex of the present invention comprises double-stranded RNA (dsRNA) and a polymer complex consisting of polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, one or more linkers, and one or more targeting moieties, the PEI being 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 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.

[0088] In a preferred embodiment, the polyplex of the present invention consists of double-stranded RNA (dsRNA) and a polymer complex, and the polymer complex consists of polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, one or more linkers, and one or more target moieties. The PEI is covalently bonded to one or more PEG moieties, and each of the one or more PEG moieties is linked to one of the one or more target moieties via one of the one or more linkers. Each of the one or more target moieties can bind to a cancer antigen.

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

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

[0091] The polyIC of the polyplex for use according to the present invention may be composed of dsRNA, and each strand consists 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 a more preferred embodiment, each strand consists of 20 to 300 ribonucleotides.

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

[0093] The poly-IC is bound to the polymer complex via a non-covalent bond or a covalent bond, with the non-covalent bond being preferred. In a preferred embodiment, the poly-IC is non-covalently bound to PEI, preferably by an ionic bond.

[0094] The polyplex according to the present invention comprises a polymer complex, the polymer complex comprising polyethyleneimine (PEI), which is a polycation having the ability to condense with 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 linear polyethyleneimine (LPEI). In a preferred embodiment, the LPEI contains hydroxy groups located at one or both ends of the LPEI. Preferably, the hydroxy group is in place of the terminal -NH2 group of the LPEI.

[0096] In a preferred embodiment of the present invention, PEI or preferably LPEI has a molecular weight of about 10 - 30 kDa. In a preferred embodiment of the present invention, 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, PEI or preferably LPEI has a molecular weight of about 22 kDa (LPEI 22k ). In yet another preferred embodiment, PEI or preferably 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 synonymously with "LPEI 22k " herein. The expression "PEI has a molecular weight of about 22 kDa" is omitted and is omitted and used synonymously with "PEI 22k " herein.

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

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

[0099] The polyplexes for use according to the present invention comprise one or more polyethylene glycol (PEG) moieties. The 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 polyethylene glycol (PEG). The functional groups can react with either the PEI or preferably the LPEI or the target moiety.

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

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

[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) and the at least one PEG moiety has a molecular weight of about 2 kDa (PEG 2k ) is present. In a more preferred embodiment, the PEI is LPEI having a molecular weight of about 20 kDa (LPEI 20k ) and the at least one PEG moiety has a molecular weight of about 2 kDa (PEG 2k ).

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

[0104] As used herein, the terms "PEI covalently attached to one PEG moiety [...]" (used interchangeably herein as "PEI-PEG 1:1") or "LPEI covalently attached to one PEG moiety [...]" (used interchangeably herein as "LPEI-PEG 1:1") refer to the molar ratio of PEI to PEG or LPEI to PEG, and PEI-PEG 1:1 or LPEI-PEG 1:1 typically and preferably means that about 1 mole of PEG per mole of PEI or LPEI is included in the polymer complex. As used herein, the term "PEI covalently attached to three PEG moieties [...]" (used interchangeably herein as "PEI-PEG 1:3") or the term "LPEI covalently attached to three PEG moieties [...]" (used interchangeably herein as "LPEI-PEG 1:3") typically and preferably means that about 3 moles of PEG per mole of PEI or LPEI are included in the polymer complex. These values are preferably 1 determined by 1H-NMR analysis. The relative integral values of the hydrogen atoms of PEG (-CH2-CH2-O-) and the integral values of the hydrogen atoms of PEI or LPEI (-CH2-CH2-NH-) are preferably 1 used to determine the values by 1H-NMR. The term "about" 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 poly IC and the PEI is covalently attached to 1 to 3 PEG moieties. In a preferred embodiment, the dsRNA is poly IC and the PEI is covalently attached to 1, 2, or 3 PEG moieties. In a more preferred embodiment, the dsRNA is poly IC and the PEI is LPEI covalently attached to 1 to 3 PEG moieties. In a more preferred embodiment, the dsRNA is poly IC and the PEI is LPEI covalently attached to 1, 2, or 3 PEG moieties.

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

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

[0108] In a preferred embodiment, the dsRNA is poly I:C, and the PEI is covalently bound to three PEG moieties. In a more preferred embodiment, the dsRNA is poly I:C, and the PEI is LPEI covalently bound to three PEG moieties. In a more preferred embodiment, the dsRNA is poly I:C, and the PEI is LPEI covalently bound to three PEG moieties 22k moieties. In a more preferred embodiment, the dsRNA is poly I:C, and the PEI is three PEG 0.3-8k , preferably PEG 2k moieties. In a more preferred embodiment, the dsRNA is poly I:C, and the PEI is LPEI covalently bound to three PEG moieties 22k moieties. In a more preferred embodiment, the dsRNA is poly I:C, and the PEI is three PEG 0.3-8k , preferably PEG 2k moieties. In a more preferred embodiment, the dsRNA is poly I:C, and the PEI is LPEI covalently bound to three PEG 22k moieties. In a more preferred embodiment, the dsRNA is poly I:C, and the PEI is three PEG

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

[0110] In a preferred embodiment, the dsRNA is poly I:C, and the one or more PEG moieties of the polymer complex are PEG 0.3-8k , preferably PEG 2k moieties. In a preferred embodiment, the dsRNA is poly I:C, and the LPEI is covalently bound to one, two or three PEG 0.3-8k moieties, preferably PEG 2k moieties. In a preferred embodiment, the dsRNA is poly I:C, and the LPEI is one PEG0.3-8k a moiety, preferably PEG 2k is covalently attached to the moiety (LPEI-PEG 1:1). In a preferred embodiment, the dsRNA is poly IC and the LPEI is PEG 0.3-8k a moiety, preferably PEG 2k is covalently attached to the moiety (LPEI-PEG 1:3).

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

[0112] As used herein, the term "cancer antigen" (used interchangeably herein with tumor antigen or tumor marker) refers to an antigenic substance that is produced by tumor cells or presented on the surface of tumor cells that elicit an immune response in a 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 further 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 poly IC, the PEI is LPEI covalently bound to 1 to 3 PEG moieties, and the cancer antigen is EGFR or PSMA. In another preferred embodiment, the dsRNA is poly IC, the PEI is LPEI covalently bound to 1 to 3 PEG moieties, and the cancer antigen is EGFR. In another preferred embodiment, the dsRNA is poly IC, the PEI is LPEI covalently bound to 1 to 3 PEG moieties, and the cancer antigen is PSMA. In another preferred embodiment, the dsRNA is poly IC, the PEI is LPEI covalently bound to 1 to 3 PEG moieties, and the cancer antigen is HER2.

[0114] In another preferred embodiment, the dsRNA is poly IC, the PEI is LPEI covalently bound to 1 to 3 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 poly IC, the PEI is LPEI covalently bound to 1 to 3 PEG moieties, and the targeting moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is poly IC, the PEI is LPEI covalently bound to 1 to 3 PEG moieties, and the targeting moiety is a HER2 affibody or a HER2 antibody.

[0115] Treatment of EGFR (epidermal growth factor receptor) - overexpressing cells with PEI - PEG - EGF (epidermal growth factor) / poly IC polyplex 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, an increase in the secretion of cytokines (IFN - γ and TNFα) was observed in PBMC incubated with the supernatant from PEG - EGF / poly IC polyplex - treated cells overexpressing EGFR, which was not observed for the medium of EGFR - low - expressing cells (see Figure 12 and Example 9 herein). The combination of PEI - PEG - EGF / poly IC with nivolumab or anti - 41BB antibody resulted in a further increase in cytokine IFN - γ release in PBMC compared to treatment with PEI - PEG - EGF / poly IC, nivolumab or anti - 41BB antibody alone (see Figures 3, 4 and 10 and Examples 2 and 7 herein). Treatment of mice with RENCA EGFR tumors with PEI - PEG - EGF / poly IC in combination with anti - PD - 1 showed an increase in survival rate compared to anti - PD - 1 or PEI - PEG - EGF / poly IC, demonstrating the synergistic effect achieved by the composition and combination therapy of the present invention, respectively, as shown in Figures 5 and Example 3 herein.

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

[0117] It should be particularly noted that the vector poly IC / PEI-PEG-DUPA (pIC / PPD) containing PEI-PEG tethered to the PSMA ligand DUPA (2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid) induced apoptosis, cytokine secretion, and the recruitment of human peripheral blood mononuclear cells (PBMCs) and selectively delivered poly IC to prostate cancer cells overexpressing PSMA. In particular, pIC / PPD resulted in activation of PBMCs evident from the production of IFN-β, IP-10, and RANTES, chemotaxis, and strong expression of IL-2, and resulted in secretion of high levels of TNF-α and IFN-γ (Langut et al., PNAS, December 26, 2017, vol. 114, no. 52).

[0118] Accordingly, 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 an increase in the activation of the immune system and more potent antitumor activity. This is particularly the case for the 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, HER2 affibody, 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 poly IC and the PEI is LPEI covalently bound to 1 to 3 PEG moieties 15-25k , preferably LPEI 22k and the targeting moiety is EGF, preferably human EGF, HER2 affibody, HER2 antibody or DUPA. In another preferred embodiment, the dsRNA is poly IC and the PEI is LPEI covalently bound 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 poly IC and the PEI is LPEI covalently bound to 1 to 3 PEG moieties 15-25k , preferably LPEI 22k and the targeting moiety is HER2 affibody or HER2 antibody.

[0120] In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k moieties covalently bound LPEI, and the targeting moiety is EGF, preferably human EGF, HER2 affibody, HER2 antibody or DUPA. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k, preferably PEG 1-3k , more preferably PEG 2k is LPEI covalently attached to the target moiety, and the target moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1 to 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k is LPEI covalently attached to the target moiety, and the target moiety is a HER2 affibody or a HER2 antibody.

[0121] In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k is LPEI covalently attached to the target moiety 22k and the target moiety is EGF, preferably human EGF, a HER2 affibody, a HER2 antibody or DUPA. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k is LPEI covalently attached to the target moiety 15-25k , preferably LPEI 22k and the target moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1 to 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k is LPEI covalently attached to the target moiety 15-25k , preferably LPEI 22k and the target moiety is a HER2 affibody or a HER2 antibody.

[0122] In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bonded to 1, 2 or 3 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 bonded to 1, 2 or 3 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 polyIC, the PEI is LPEI covalently bonded to 1, 2 or 3 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 bonded to 1, 2 or 3 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 target portion 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 target portion 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 target portion 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 target portion 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 target portion 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 target portion 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 target portion 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 bonded to 1, 2 or 3 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 bonded to 1, 2 or 3 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 bonded to 1, 2 or 3 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 bonded to 1, 2 or 3 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 bound to 1, 2, or 3 PEG moieties, 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, 4-1BB, and 4-1BB ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, 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, and 4-1BB. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, 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, and 4-1BB. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, the target 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 bound to 1, 2, or 3 PEG moieties, the target 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 bound to 1, 2, or 3 PEG moieties, the target 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 bound to 1, 2, or 3 PEG moieties, the target 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is GITR or GITR ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently bound to 1, 2 or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is ICOS or ICOS ligand.In another preferred embodiment, the dsRNA is poly IC, the PEI is LPEI covalently bound to 1, 2 or 3 PEG moieties, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is OX40 or OX40 ligand.

[0127] In another preferred embodiment, the dsRNA is poly I:C, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the 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 poly I:C, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the 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 poly I:C, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the 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 polyIC, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the 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. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the 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 bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the 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 bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the 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 bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2 or 3 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 bound to 1, 2, or 3 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 bound to 1, 2, or 3 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 bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-GITR or anti-GITR ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-ICOS or ICOS ligand. In another preferred embodiment, the dsRNA is polyIC, the PEI is LPEI covalently bound to 1, 2, or 3 PEG moieties, the targeting moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-OX40 or anti-OX40 ligand.

[0128] In another preferred embodiment, the dsRNA is polyIC, and the PEI is 1, 2, or 3 PEG 0.3-8k , preferably PEG 2k moieties covalently bound to LPEI 22kand 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 (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 LPEI covalently bound to 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k moieties. 22k and 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 LPEI covalently bound to 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k moieties. 22k and 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, 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 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k moieties. 22kand the target portion 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 I:C and the PEI is LPEI covalently bound to 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k portions. 22k and the target portion 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 poly I:C and the PEI is LPEI covalently bound to 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k portions. 22k and the target portion 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 poly I:C and the PEI is LPEI covalently bound to 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k portions. 22k and the target portion 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 poly I:C and the PEI is LPEI covalently bound to 1, 2 or 3 PEG 0.3-8k , preferably PEG2k LPEI covalently attached 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, GITR, CD40, and OX40. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k LPEI covalently attached 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, GITR, and OX40. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k LPEI covalently attached 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 poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k LPEI covalently attached 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, 4-1BB and 4-1BB ligand. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 2k LPEI covalently attached to the moiety 22kand 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, and 4-1BB. In another preferred embodiment, the dsRNA is poly IC and the PEI is LPEI covalently bound to 1, 2, or 3 PEG 0.3-8k , preferably PEG 2k moieties. 22k and 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, and 4-1BB. In another preferred embodiment, the dsRNA is poly IC and the PEI is LPEI covalently bound to 1, 2, or 3 PEG 0.3-8k , preferably PEG 2k moieties. 22k and the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1 or 4-1BB. In another preferred embodiment, the dsRNA is poly IC and the PEI is LPEI covalently bound to 1, 2, or 3 PEG 0.3-8k , preferably PEG 2k moieties. 22k and the target 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 poly IC and the PEI is LPEI covalently bound to 1, 2, or 3 PEG 0.3-8k , preferably PEG 2k moieties. 22k and the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1. In another preferred embodiment, the dsRNA is poly IC and the PEI is LPEI covalently bound to 1, 2, or 3 PEG 0.3-8k , preferably PEG 2k moieties. 22k and the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L1. In another preferred embodiment, the dsRNA is poly IC and the PEI is LPEI covalently bound to 1, 2, or 3 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 PD-L2. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 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 4-1BB. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 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 CTLA-4. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22k wherein the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is LAG-3. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22k wherein the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIGIT. In another preferred embodiment, the dsRNA is poly IC and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k LPEI covalently bound to the moiety 15-25k , preferably LPEI 22kand the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIM3. In another preferred embodiment, the dsRNA is poly I:C, and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k -conjugated LPEI 15-25k , preferably LPEI 22k and the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is GITR or GITR ligand. In another preferred embodiment, the dsRNA is poly I:C, and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k -conjugated LPEI 15-25k , preferably LPEI 22k and the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is ICOS or ICOS ligand. In another preferred embodiment, the dsRNA is poly I:C, and the PEI is 1, 2 or 3 PEG 0.3-8k , preferably PEG 1-3k , more preferably PEG 2k -conjugated LPEI 15-25k , preferably LPEI 22k and the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is OX40 or OX40 ligand.

[0132] In another preferred embodiment, the dsRNA is poly I:C, the PEI is LPEI covalently bound to 1, 2 or 3 PEG moieties, the target moiety is EGF, preferably human EGF, and 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 regulating the immune checkpoint protein CD40 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, (iii) An antibody capable of regulating the immune checkpoint protein GITR 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, (iv) An antibody capable of regulating the immune checkpoint protein OX40 and at least one antibody capable of regulating 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 regulating the immune checkpoint protein 4-1BB and at least one antibody capable of regulating 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 is a mixture of.

[0133] In another preferred embodiment, the dsRNA is poly IC and the PEI is LPEI covalently bound to one, two, or three PEG 0.3-8k , preferably PEG 2k moieties, and the target moiety is EGF, preferably human EGF, and the at least one antibody capable of regulating an immune checkpoint protein is 22k (i) An antibody capable of regulating the immune checkpoint protein CD27 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, ​(ii) An antibody capable of regulating the immune checkpoint protein CD40 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, (iii) An antibody capable of regulating the immune checkpoint protein GITR 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, (iv) An antibody capable of regulating the immune checkpoint protein OX40 and at least one antibody capable of regulating 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 regulating the immune checkpoint protein 4-1BB and at least one antibody capable of regulating 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 is a mixture of.

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

[0135] In the polyplex of the present invention, the one or more PEG moieties are attached to one of the one or more target moieties. In a preferred embodiment, the one or more PEG moieties are directly linked to one of the one or more target moieties or are linked to one of the one or more target moieties 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 selected from (C1-C8) alkylene, (C2-C8) alkenylene, (C2-C8) alkynylene, (C6-C 10 ) arylene-diyl, or heteroarylene diyl, RX is absent or is S-, and R3 is absent or has the formula

Chemical formula

[0137] In a further preferred embodiment, the polymer complex is of the formulas (i)-(viii):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0138] In a more preferred embodiment, the polymer complex 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 target moiety HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA moiety). In a more preferred embodiment, the polymer complex 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 target moiety HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-(DUPA moiety).

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

[0140] In a more preferred embodiment, the target moiety is a HER2 affibody or a HER2 antibody, the polymer complex is of formula (i) or (v), and the HER2 affibody or HER2 antibody is linked via its mercapto group.

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

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

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

[0144] In another preferred embodiment, the dsRNA is poly I:C, the target 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 complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly I:C, the target 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 complex is of formula (i), (ii) or (vi).

[0145] In another preferred embodiment, the dsRNA is polyIC, the target 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 complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target 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 complex is of formula (i), (ii) or (vi).

[0146] In another preferred embodiment, the dsRNA is polyIC, the target 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 complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target 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 complex is of formula (i), (ii) or (vi).

[0147] In another preferred embodiment, the dsRNA is poly I:C, the target 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 complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly I:C, the target 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 complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly I:C, the target 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 complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly I:C, the target 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 complex is of formula (i), (ii) or (vi).

[0148] In another preferred embodiment, the dsRNA is polyIC, 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, 4-1BB and 4-1BB ligand. In another preferred embodiment, the dsRNA is polyIC, 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 and 4-1BB. In another preferred embodiment, the dsRNA is polyIC, 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, and 4-1BB, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1 or 4-1BB, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1, PD-L1 or PD-L2, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L1, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L2, and the polymer complex is of formula (i), (ii) or (vi).In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is 4-1BB. In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is CTLA-4, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is LAG-3, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIGIT. In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIM3, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is GITR or GITR ligand, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is ICOS or ICOS ligand, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the immune checkpoint protein is OX40 or OX40 ligand, and the polymer complex is of formula (i), (ii) or (vi).

[0149] In another preferred embodiment, the dsRNA is polyIC, the target portion is EGF, preferably human EGF, the antibody that modulates the 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 complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target portion is EGF, preferably human EGF, the antibody that modulates the 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 complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is polyIC, the target portion is EGF, preferably human EGF, the antibody that modulates the 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 complex is of formula (i), (ii) or (vi).In another preferred embodiment, the dsRNA is poly IC, the target portion is EGF, preferably human EGF, the antibody that regulates the 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 complex is of formula (i), (ii) or (vi).

[0150] In another preferred embodiment, the dsRNA is poly IC, the target portion is EGF, preferably human EGF, the antibody that regulates the 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 complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly IC, the target portion is EGF, preferably human EGF, the antibody that regulates the 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 complex is of formula (i), (ii) or (vi).

[0151] In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the antibody that modulates the 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 target moiety is EGF, preferably human EGF, and the antibody that modulates the 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 target moiety is EGF, preferably human EGF, and the antibody that modulates the 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 complex is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-PD-1 or anti-4-1BB, and the polymer complex is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the target 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, and the polymer complex is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is PD-1, and the polymer complex is of formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is polyIC, the target moiety is EGF, preferably human EGF, and the antibody that modulates the immune checkpoint protein is anti-PD-L1, and the polymer complex is of formula (i), (ii), or (vi).In another preferred embodiment, the dsRNA is poly I:C, the target portion is EGF, preferably human EGF, the antibody that regulates the immune checkpoint protein is anti-PD-L2, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly I:C, the target portion is EGF, preferably human EGF, and the antibody that regulates the immune checkpoint protein is anti-4-1BB. In another preferred embodiment, the dsRNA is poly I:C, the target portion is EGF, preferably human EGF, the antibody that regulates the immune checkpoint protein is anti-CTLA-4, and the polymer complex is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly I:C, the target portion is EGF, preferably human EGF, the antibody that regulates the immune checkpoint protein is anti-LAG-3, and the polymer complex is of formula (i), (ii) or (vi).

[0152] In another preferred embodiment, the dsRNA is poly I:C, the polymer complex is of formula (i), (ii) or (vi), the target portion is EGF, preferably human EGF, and at least one antibody capable of regulating an immune checkpoint protein is (i) at least one antibody capable of regulating the immune checkpoint protein CD27 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, (ii) at least one antibody capable of regulating the immune checkpoint protein CD40 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, (iii) at least one antibody capable of regulating the immune checkpoint protein GITR 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, (iv) an antibody capable of regulating the immune checkpoint protein OX40 and at least one antibody capable of regulating 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 regulating the immune checkpoint protein 4-1BB and at least one antibody capable of regulating 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 which is a mixture of.

[0153] In another preferred embodiment, the dsRNA is poly I:C, the polymer complex is of formula (i), (ii) or (vi), the target moiety is EGF, preferably human EGF, and at least one antibody capable of regulating an immune checkpoint protein is (i) at least one antibody selected from the group consisting of anti-CD27 and anti-PD-1, anti-PD-L1, and anti-PD-L2, (ii) anti-CD40 of an antibody capable of regulating an immune checkpoint protein and at least one antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-CTLA-4, anti (iii) anti-GITR of an antibody capable of regulating an immune checkpoint protein 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) anti-OX40 of an antibody capable of regulating an immune checkpoint protein 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 anti-4-1BB antibody of 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, anti-PD-L2, anti-OX40, anti-LAG-3, and anti-CTLA-4, or (vi) A mixture of an anti-ICOS antibody of 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 polyplex of the present invention contains one or more targeting moieties. The targeting moiety can be any one of a natural, natural or modified ligand or its paralog, or a non-natural ligand such as an antibody or a single-chain variable fragment (scFv), or an antibody mimetic such as an affibody or an aptamer against a cancer antigen.

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

[0156] In another preferred embodiment, one or more target moieties of the polyplexes of the present 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 target moieties of the polyplexes of the present 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 target moieties of the polyplexes of the present 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 target moieties of the polyplexes of the present 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 target moieties of the polyplexes of the present 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 target moieties of the polyplexes of the present 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 target moieties of the polyplex of the present 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 target moieties of the polyplex of the present 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 target moieties of the polyplex of the present 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 target moieties of the polyplex of the present 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 target moieties are EGF including mouse and human EGF. In a more preferred embodiment, the one or more target moieties are hEGF. In another preferred embodiment, the one or more target moieties of the polyplex of the present invention are hEGF of SEQ ID NO: 2.

[0160] In another preferred embodiment, the one or more targeting moieties of the polyplex of the present invention are HER2 affibodies or HER2 antibodies, preferably HER2 affibodies. In another preferred embodiment, the one or more targeting moieties of the polyplex of the present invention are HER2 affibodies, preferably the HER2 affibody is the one of SEQ ID NO: 1. In another preferred embodiment, the one or more targeting moieties of the polyplex of the present invention are EGF, preferably human EGF (hEGF) or 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 polyplex of the present invention are DUPA moiety (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-).

[0161] In a further aspect, the present invention relates to a composition or kit of parts according to the present 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 mammalian cancer treatment. Preferably, the mammal is a human.

[0163] In a preferred embodiment, the present invention relates to a method for treating cancer, which comprises 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, glioma, head and neck cancer, prostate cancer, penile cancer, testicular germ cell cancer, neuroendocrine tumor, and hepatocellular cancer. In a preferred embodiment, the polyplex comprised in the kit of parts according to the present invention is administered separately from one or more antibodies capable of modulating immune checkpoint proteins.

[0165] In one embodiment, the present invention provides the use of the pharmaceutical composition or kit of parts of the present invention for cancer treatment, 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 regulating an immune checkpoint protein.

[0166] In a preferred embodiment, the use of the kit of parts of the present invention in cancer treatment comprises separate dosing of the polyplex and at least one antibody capable of regulating an immune checkpoint protein. The polyplex and the at least one antibody capable of regulating an immune checkpoint protein can be administered simultaneously or sequentially (continuously), i.e., at different times. The polyplex and the at least one antibody capable of regulating an immune checkpoint protein can be combined prior to administration, administered together as a composition, or administered separately, as included in the kit of parts of the present invention. In a more preferred embodiment, the polyplex and the at least one antibody capable of regulating 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 can be administered by intravenous, intracerebral (intracranial), oral, intramuscular, subcutaneous, transdermal, intradermal, transmucosal, intranasal, sublingual, intraperitoneal or intraocular routes. 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 present invention is for systemic administration. More preferably, the kit of parts or composition according to the present invention is administered intravenously or intraperitoneally, and 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 continuously or simultaneously, preferably continuously. 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 continuously 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 present invention and the at least one antibody capable of modulating an immune checkpoint protein are administered continuously or simultaneously, preferably continuously, one compound (or part) of the kit of parts of the present 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 present invention and the at least one antibody capable of modulating an immune checkpoint protein are administered continuously or simultaneously, preferably continuously, 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 prior to at least one antibody capable of modulating an immune checkpoint protein. In another preferred embodiment, the polyplex and at least one antibody capable of modulating an immune checkpoint protein are administered sequentially, the polyplex is administered via intravenous injection, and at least one antibody capable of modulating an immune checkpoint protein is administered via intraperitoneal or intravenous injection, and the polyplex is administered prior to the antibody.

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

[0173] The kit of parts of the present invention can further be used as an additional treatment. As used herein, "additional treatment" means the combination of the polyplex and the at least one antibody for use in treatment, and the subject being treated starts a first treatment regimen using one or more different parts of the kit of parts before starting a second treatment regimen of one or more different parts of the kit of parts in addition to the first treatment regimen, such that not all of the reagents used in the treatment are started simultaneously. For example, one or more immune-modulating antibodies are administered to a patient who has already received the polyplex of the present invention, or vice versa.

[0174] Herein, the present invention will be illustrated by the following non-limiting examples.

[0175] Example Hereinafter, the present invention will be further described by way of example. The preferred polyplex of the present invention was used. The synthesis of the preferred polyplex was carried out as described in International Publication No. 2015 / 173824, particularly as described in Examples 2, 8, 10 to 13 of International Publication No. 2015 / 173824. A preferred polymer complex containing EGF as a target 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 more preferred polyplex of the present invention used herein contains HER2 as a target moiety in the polymer complex, particularly an anti-HER2 affibody and polyIC as double-stranded RNA. The preferred polyplex is abbreviated herein as "polyIC / PPHA".

[0176] Effect of Example 1 - 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 / polyIC for 5 hours.

[0177] The secretion of human IP-10 (CXCL10) was quantified by ELISA assay (ABTS ELISA Development Kit, Peprotech). The secretion of IP10 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. Then, A431 cells were stained with a PD-L1-PE labeled antibody (Biolegend, catalog number 393607) on ice in PBS containing 2% FCS for 40 minutes, washed, and analyzed with a FACS instrument. The expression of PD-L1 was significantly increased (MFI = 751) after treatment with PEI-PEG-EGF / polyIC compared to untreated control cells (MFI = 431). An isotype control was used as a negative control (MFI = 13).

[0179] The expression of PD-L1 was significantly higher in PEI-PEG-EGF / poly I:C-treated cells than in untreated cells (Figs. 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 / poly I:C. Furthermore, in MCF7 and U87, EGFR-low-expressing cell lines, IP10 levels were not affected. The increase in IP10 after stimulation with PEI-PEG-EGF / poly I:C provided a theoretical basis for addressing the immunostimulatory properties of the combination of the present invention.

[0181] Furthermore, the expression of PD-L1 was significantly higher after treatment with PEI-PEG-EGF / poly I:C (MFI = 751) compared to untreated cells (MFI = 431), providing a theoretical basis for developing a combination with nivolumab (Fig. 2).

[0182] Example 2 - In vitro effects of PEI-PEG-EGF / poly I:C in combination with the immunomodulatory therapy nivolumab and 4-1BB antibody Nivolumab: 40,000 A431 cells were treated with PEI-PEG-EGF / poly I:C at concentrations of 0.125, 0.25, 0.5, 1 μg / ml for 5 hours. Then, 200,000 PBMCs were stimulated with CD3 (5 μg / ml) and antigen-exposed for 48 hours in dilution medium (1:2) containing PEI-PEG-EGF / poly I:C alone (0.125 μg / ml) or in combination with nivolumab (20 μg / ml). After 48 hours, the medium was collected from the antigen-exposed PBMCs, and the production of INFγ was measured by ELISA assay (Fig. 3).

[0183] Combination of PEI-PEG-EGF / poly I:C with nivolumab significantly increased IFN-γ production by PBMCs (Fig. 3).

[0184] 4-1BB antibody: 40,000 A431 cells were treated with PEI-PEG-EGF / poly I:C at a concentration of 0.5 μg / ml for 5 hours. 200,000 PBMCs from healthy donors were either stimulated with CD3 (0.5 μg / ml) or left unstimulated and co-cultured with A431 cells treated with PEI-PEG-EGF / poly I:C 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 I:C and anti-4-1BB significantly increased IFN-γ production by PBMCs (Figure 4).

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

[0187] Therefore, the combination of PEI-PEG-EGF / poly I:C and immunotherapy results in increased activation of the immune system and more potent antitumor activity. From a clinical perspective, the potential induction of an antitumor immune response by combination therapy of PEI-PEG-EGF / poly I:C and immunological strategies is very attractive because such combinations should increase the proportion of patients responding to immunotherapy and decrease the incidence of resistance development.

[0188] Example 3 - In vivo efficacy of the combination of PEI-PEG-mEGF / poly I:C + anti-PD-1 The effect of PEI-PEG-EGF / poly I:C + anti-PD-1 on RencaEGFR lung metastases in immunocompetent mice was examined.

[0189] Materials and Methods: Cells: RencaEGFR; polyplex PEI-PEG-EGF / polyIC and LPEI / EGF Mice: 1 / 0.75); Dalton's polyIC; N / P: 8 polyplex-containing 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 lung, 250,000 RencaEGFR cells were intravenously injected into 40 Balb / c immunocompetent female mice (6 weeks old, body weight: 18 - 21 g). Ten days later, the animals were divided into four groups of 10 animals / group (untreated (UT), anti-PD-1, PEI-PEG-EGF / polyIC, and PEI-PEG-EGF / polyIC + aPD1 (anti-PD-1)).

[0191] Mice with RencaEGFR tumors were intravenously treated on days 0, 2, 4, 7, 10 with 250 μg / kg of PEI-PEG-EGF / polyIC alone, 6 injections / week for 2 weeks, or in combination with 10 mg / kg intraperitoneal anti-PD-1 (RPM1-14, rat IgG2a, Biox cell). Subsequently, survival rates were analyzed. Appearance of cancer clinical symptoms: A 10% weight loss between the last two weight measurements, or a 20% weight loss between the first and last weight measurements, slow / abnormal movement, hunchback, abnormal breathing justifies the sacrifice of the mouse. The presence of lung tumors was visually confirmed in the sacrificed mice. Mice were sacrificed according to scored parameters such as weight measurements and the general health status of the mice. After sacrificing the mice, organs were collected and analyzed by IHC to detect the presence of lung metastases.

[0192] Conclusion: Tumor uptake was 100% in both groups (untreated (UT), anti-PD-1), and the final mean survival periods were 41.5 days and 41.8 days. All mice developed tumors after cell injection, indicating the high reliability of the model. Anti-PD-1 alone did not inhibit tumor growth. On day 60, one animal in the PEI-PEG-EGF / polyIC (PPE / PIC) alone group was still alive. The preliminary increase in survival by PEI-PEG-EGF / polyIC was approximately 32%. Only 3 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 the combination of PEI-PEG-EGF / polyIC + anti-CTLA-4 As described in Example 3, to induce the formation of RencaEGFR tumors in the lungs, RencaEGFR cells are intravenously injected into 40 Balb / c immunocompetent female mice. Ten days later, the animals are divided into 4 groups: untreated control (UT), anti-CTLA-4, PEI-PEG-EGF / polyIC, and PEI-PEG-EGF / polyIC + anti-CTLA-4. Mice with EGFR tumors are treated on days 0, 2, 4, 7, and 10 with PEI-PEG-EGF / polyIC alone or in combination with anti-CTLA-4.

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

[0196] Reduction of cancer clinical symptoms, inhibition of tumor growth, and increase in survival rate are expected.

[0197] Example 5 - Combination Therapy with PolyIC / PPHA and Anti-PD-1 Antibody in Immunocompetent Mice with Tumors Overexpressing HER-2 Materials and Methods: RENCA-HER2 cells (renal cell carcinoma cells overexpressing HER2) (5×10 6 ) were subcutaneously (s.c.) injected into the right flank of immunocompetent BALB / c female mice (6 - 8 weeks old, Harlan Laboratories). Mice with subcutaneous RENCA HER2 tumors, with an average tumor volume of 235 mm 3 were randomly assigned to 4 groups of 7 - 8 animals / group (untreated, anti-PD-1, polyplex of polyIC / PEI-PEG-HER2 affibody (PPHA) and anti-PD-1 + polyIC / PPHA). PolyIC / PPHA is preferably synthesized as described in the examples of International Publication No. WO 2015 / 173824, more preferably Example 2 and pages 18 and onwards of International Publication No. WO 2015 / 173824. The HER2 affibody is preferably synthesized as described on pages 20 and onwards of International Publication No. WO 2015 / 173824.

[0198] The mice were treated intravenously (i.v.) with 0.25 mg / kg of 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 number BE0146), 200 mg / mouse were injected intraperitoneally (i.p.) every 4 - 6 days. Tumor xenografts were measured with calipers and tumor volume was determined using the formula: length × width 2 / 2 and plotted as mean ± SEM.

[0199] Conclusions: A 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 did not show further tumor growth for an additional 60 days. The cured mice showed complete protection from tumor rechallenge (5×10 6) It shows that an immune response to the tumor has occurred.

[0200] Example 6 - In vitro experiment, PD-L1 expression Materials and methods: Increased expression of PD-L1 was shown 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, the cells were trypsinized, washed, and incubated with the anti-PD-L1 antibody CD274 (PD-L1, B7-H1)[10F.9G2] Tonbo cat#50-1243-U025 or isotype control for 1 hour. The expression of PD-L1 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] Conclusion: RENCA HER-2 cells were treated with 1 microgram / ml of polyIC / PPHA for 24 hours, and the expression of PD-L1 was analyzed using flow cytometry. A significant increase in PD-L1 expression was observed after treatment with 1 microgram / ml of polyIC / PPHA compared to untreated cells, indicating that targeted delivery of polyIC induces upregulation of PD-L1 expression.

[0202]

Table 2

[0203] Example 7 - PBMCs are indirectly activated by the polyplexes of the present invention containing polyIC and the targeted polymer complex The following experiment demonstrates the advantages of the combination of the polyplexes of the present invention and nivolumab over monotherapy.

[0204] Materials and methods: IFNγ gamma release from PBMCs exposed to the supernatant of A431 cells treated with PEI-PEG-EGF / poly I:C 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 / poly I:C polyplexes were prepared in HBG (Hepes-buffered glucose). The PEI-PEG-EGF triconjugate was composed of PEGylated linear polyethyleneimine conjugated to hEGF (hEGF-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid) via an MCC linker and synthesized according to Example 10 of International Publication No. WO 2015 / 173824. Anti-PD-1 antibody (nivolumab) was utilized alone or in combination with PEI-PEG-EGF / poly I:C polyplexes. Anti-CD3 antibody (clone OKT3) was used to stimulate PBMCs after transferring the medium from treated A431 cells. Anti-PD-1 antibody (nivolumab) was used to test the combination of PEI-PEG-EGF / poly I:C polyplexes and checkpoint blockade. Secretion of human interferon γ was determined using an ELISA kit (BD Bioscience) according to the manufacturer's instructions.

[0206] A431 cells were seeded in a flat-bottom 96-well plate (40,000 cells / 90 μl of DMEM medium). In parallel, a plate containing only the medium (90 μl of medium without cells) was prepared as a negative control. The next day, A431 cells or the medium alone were treated with 0, 0.125, or 1 μg / ml of PEI-PEG-EGF / poly(I:C) polyplex (10 μl / well) at 37°C for 5 hours. Cryopreserved PBMCs were thawed and allowed to recover in a U-bottom 96-well plate (200,000 cells / 100 μl) in RPMI-1640 medium at 37°C for at least 5 hours. After 5 hours, the supernatant (SN) from A431 cells treated with PEI-PEG-EGF / poly(I:C) polyplex or the medium "treated" with PEI-PEG-EGF / poly(I:C) polyplex was transferred to the PBMCs. Subsequently, the PBMCs were stimulated with anti-CD3 antibody (OKT3, 500 ng / ml) and / or treated with nivolumab (20 μg / ml) and incubated overnight at 37°C. The SN of the stimulated PBMCs was collected and stored at -20°C until analyzed by ELISA.

[0207] To model the effect of the combination of PEI-PEG-EGF / poly I:C polyplex and nivolumab treatment on in vitro immune cell activation, the SN from A431 cells treated with PEI-PEG-EGF / poly I:C polyplex was transferred to human PBMC from healthy donors and treated in combination with nivolumab as follows. A431 cells were treated with the indicated concentration of PEI-PEG-EGF / poly I:C polyplex for 5 hours. As a control, cell-free medium was "treated" with PEI-PEG-EGF / poly I:C polyplex and incubated for 5 hours as well. After 5 hours, PBMCs stimulated or not stimulated with anti-CD3 antibody were treated with: nivolumab alone, SN from A431 cells treated with PEI-PEG-EGF / poly I:C polyplex alone, medium "treated" with PEI-PEG-EGF / poly I:C polyplex alone, SN from A431 cells treated with nivolumab + PEI-PEG-EGF / poly I:C polyplex, or medium "treated" with nivolumab + PEI-PEG-EGF / poly I:C polyplex. PBMCs were incubated overnight at 37°C. To evaluate PBMC activation, IFN-γ secreted from PBMCs was quantified by ELISA.

[0208] Results: As shown in Figure 10 and Table 3, CD3-stimulated PBMCs that received SN from A431 cells treated with PEI-PEG-EGF / poly I:C polyplex secreted significantly more IFN-γ (311.1 - 326.3 pg / ml) than PBMCs that received SN from untreated (UT) cells (12.7 pg / ml). The secretion of IFN-γ was further enhanced by the addition of nivolumab (502 - 523 pg / ml). PBMCs that received SN + nivolumab from untreated cells secreted low levels of IFN-γ (62.5 pg / ml). IFN-γ secretion was not induced by nivolumab treatment alone. The "treated" medium (without cells) alone with PEI-PEG-EGF / poly I:C polyplex or in combination with nivolumab did not result in an increase in IFN-γ secretion compared to UT medium. These results demonstrate that the combination of PEI-PEG-EGF / poly I:C polyplex and nivolumab results in an increase in PBMC activation, as seen by an increase in IFN-γ secretion, compared to PEI-PEG-EGF / poly I:C polyplex alone. Furthermore, PBMCs were activated by cytokines secreted from A431 cancer cells treated with PEI-PEG-EGF / poly I:C polyplex, rather than by PEI-PEG-EGF / poly I:C polyplex treatment alone.

[0209] Conclusion: The combination of polyplex containing poly I:C and the targeting moiety human EGF with nivolumab was repeated in a medium transfer experiment after treatment of A431 cells. IFN-γ levels were measured by ELISA, demonstrating that the combination of the two agents resulted in an increase in IFN-γ compared to either agent alone. As a control, the single agent (polyplex or nivolumab) or combination was incubated for the same length of time in cell-free medium. The medium was then transferred to PBMCs. IFN-γ levels were significantly lower in this group compared to PBMCs that received medium transferred from A431-treated cells. These results demonstrate that PBMC activation is not directly mediated by the poly I:C polyplex, but rather is mediated through the secretion of chemokines from cancer cells treated with the poly I:C polyplex.

[0210]

Table 3

[0211] Example 8 - Secretion of cytokines after treatment with PEI - PEG - EGF / Poly I:C polyplex and each component alone To evaluate whether the PEI - PEG - EGF / Poly I:C polyplex or its components induce the secretion of pro - inflammatory cytokines, three cell lines including two EGFR - overexpressing strains (MDA - MB - 468 and A431) and a low - EGFR strain (MCF7) were treated with PEI - PEG - EGF / Poly I:C polyplex, tricongugate 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 - overexpressing MDA - MB - 468 and A431) and low - EGFR (MCF7) were treated with the indicated concentrations of PEI - PEG - EGF / Poly I:C polyplex, PEI - PEG - EGF tricongugate or poly I:C for 5 hours. Then the medium was collected and analyzed for CCL5 (RANTES), IP10 and GROα. PEI - PEG - EGF was composed of a PEGylated linear polyethyleneimine that binds to hEGF via a linker MCC and was synthesized according to Example 10 of International Publication No. WO 2015 / 173824.

[0213] Tumor cell lines: MDA - MB - 468, A431 and MCF7 cells were provided by ATCC. The cell lines were passaged regularly once or twice a week and maintained in culture up to 20 passages. The cell lines were grown in RPMI - 1640 medium (25 mM HEPES containing 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 in a humidified atmosphere of 5% CO2 at 37°C.

[0214] Treatment: PEI-PEG-EGF / poly IC polyplex or pIC alone was incubated with HBG (HEPES-buffered glucose). The reagent was used at a concentration of 0-1 μg / ml of pIC. The triconjugate PEI-PEG-EGF alone was prepared in the same manner as the PEI-PEG-EGF / poly IC polyplex without pIC.

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

[0216] Results: Treatment with PEI-PEG-EGF / poly IC polyplex in MDA-MB-468 and A431, which are EGFR-high-expressing cells, induced the secretion of IP-10, GRO-α and CCL5, while cytokine expression was not observed in EGFR-low-expressing MCF7 cells (Figure 11A, B and C respectively). Up to 337 and 268 pg / ml of IP10, 496 and 757 pg / ml of GROα and 607 and 100 pg / ml of RANTES 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 / poly IC polyplex induces the secretion of pro-inflammatory cytokines, not its individual components.

[0217] Example 9 - Activation of PBMC by PEI-PEG-EGF / poly IC polyplex and pIC / PPHA Materials and Methods: Tumor cell lines: MDA-MB-468, A431, and MCF7 cells were provided by ATCC. The cell lines were passaged regularly once or twice a week and maintained in culture up to 20 passages. The cell lines were grown at 37°C in a humidified atmosphere of 5% CO2 in RPMI-1640 medium (25 mM HEPES containing 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.

[0218] Reagents: The PEI-PEG-EGF triconjugate was composed of PEGylated linear polyethyleneimine conjugated to hEGF (human epidermal growth factor) via an MCC linker and synthesized according to Example 10 of International Publication No. WO 2015 / 173824. PEI-PEG-EGF / poly(I:C) polyplexes and PEI-PEG-EGF / pLGA polyplexes (PEI-PEG-EGF and poly-L-glutamic acid (pLGA), Sigma, 50 - 100 kDa p4886) were prepared in HBG (HEPES-buffered glucose) using naked poly(I:C). The reagents were used at concentrations of 0 - 1 μg / ml of poly(I:C) or pLGA within the polyplexes.

[0219] PBMC isolation (buffy coat) from healthy donors: 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 the secretion of IFN-γ and TNF-α, 40,000 cells per well or medium alone were treated with the following compounds: PEI-PEG-EGF / poly(I:C) polyplexes with pIC concentrations of 0 to 1 μg / ml, pIC (0 to 1 μg / ml) alone, or PEI-PEG-EGF polyplexed with polyglutamic acid (PEI-PEG-EGF / pLGA polyplex) at 0 to 1 μg / ml. After 5 hours of incubation, only the supernatants and media were collected and transferred to 200,000 PBMCs stimulated with a-CD3 (0.5 μg / ml) and incubated for an additional 16 hours. In parallel, the supernatants from each cell line treated with each compound alone were incubated for 16 hours in the absence of PBMCs. After incubation, the media were collected and TNF-α, IFN-γ, and IL-2 were measured by ELISA assay (Peprotech and Invitrogen) and read using a Synergy H1 plate reader manufactured by Biotek.

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

[0222] In PBMCs incubated with supernatants from MDA-MB-468 and A431 treated with PEI-PEG-EGF / poly IC polyplexes, an increase in IFN-γ secretion was observed. In PBMCs derived from MDA-MB-468 and A431, IFN-γ of up to 3587 pg / ml and 2023 pg / ml was detected, respectively. PBMCs incubated with supernatants from MCF7 cells treated with PEI-PEG-EGF / poly IC polyplexes did not induce cytokine secretion. PBMCs treated with cell-free medium containing PEI-PEG-EGF / poly IC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes (PBMC alone) did not induce IFN-γ secretion in all cancer cell lines. No IFN-γ secretion was observed in the supernatants of any cancer cell lines treated with PEI-PEG-EGF / poly IC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes without PBMCs.

[0223] In PBMCs incubated with media from MDA-MB-468 and A431 cells treated with PEI-PEG-EGF / poly IC polyplexes, a significant increase in TNFα secretion was observed. In PBMCs derived from MDA-MB-468 and A431, TNFα of up to 2190 pg / ml and 3438 pg / ml was detected, respectively. PBMCs incubated with media from MCF7 cells treated with PEI-PEG-EGF / poly IC polyplexes did not induce cytokine secretion. Media from PBMCs treated with PEI-PEG-EGF / poly IC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes (PBMC alone) did not induce a significant increase in TNFα secretion in all cancer cell lines compared to UT cells. No secretion of IFN-γ and TNFα was observed in the supernatants from cancer cell lines treated with PEI-PEG-EGF / poly IC polyplexes, pIC, or PEI-PEG-EGF / pLGA polyplexes without PBMCs.

[0224] Example 10 - Efficacy and Selectivity of PEI-PEG-EGF / Poly IC Polyplex Compared with Naked pIC, PEI or Lipid-Based Transfection Reagents Complexed with pIC The purpose of the study was to measure the selectivity and efficacy of PEI-PEG-EGF / Poly IC polyplex against single components and non-targeted delivery systems and pIC analogs.

[0225] Materials and Methods: Cell Lines: EGFR-high expressing cell lines (BT20, MDA-MB-468, and HCC70), moderately EGFR-expressing cell line (U87MG), EGFR-low 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: The PEI-PEG-EGF triconjugate was composed of PEGylated linear polyethyleneimine conjugated to hEGF via an MCC linker and was synthesized according to Example 10 of International Publication No. 2015 / 173824. Using the PEI-PEG-EGF triconjugate and pIC, HBG (HEPES-buffered glucose) containing PEI-PEG-EGF / Poly IC polyplex was prepared. Using JetPEI (Polyplus) and pIC, jetPEI-pIC polyplex was prepared according to the manufacturer's instructions. Using Lipofectamine RNAiMax (Invitrogen, P / N 56531) and pIC, RNAiMax-pIC polyplex was prepared according to the manufacturer's instructions. Using poly-L-glutamic acid (pLGA, Sigma 50-100 kDa) and PEI-PEG-EGF, a PEI-PEG-EGF / pLGA polyplex having the same net charge as the PEI-PEG-EGF / Poly IC polyplex was prepared.

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

[0228] Results: The effects of PEI-PEG-EGF / poly(I:C) polyplexes on the survival rates of high- and low-EGFR-expressing cancer cell lines and non-cancerous cell lines were compared with those of naked pIC, jetPEI-pIC, RNAiMax-pIC (lipid-based transfection reagents), and PEI-PEG-EGF / pLGA polyplexes.

[0229] The jetPEI-pIC polyplex was significantly less effective than the PEI-PEG-EGF / poly(I:C) polyplex in all EGFR-overexpressing cells: the IC50 of the PEI-PEG-EGF / poly(I:C) polyplex was 1 / 10 to 1 / 20 of the IC50 of jetPEI-pIC. In cancer cell lines or non-cancerous cell lines with low EGFR, jetPEI-pIC was up to 2-fold more toxic than the PEI-PEG-EGF / poly(I:C) polyplex (Figures 13A - E). These results demonstrate an increase in the efficacy and selectivity of the PEI-PEG-EGF / poly(I:C) polyplex compared to jetPEI-pIC.

[0230] Compared to treatment with RNAiMax-pIC, an increase in the efficacy of the PEI-PEG-EGF / poly IC polyplex was observed in 3 out of 4 EGFR-overexpressing cell lines. Only in A431 cells, the IC50 of RNAiMax-pIC was lower (one-fourth) than that of the PEI-PEG-EGF / poly IC polyplex (Figure 13). In BT20, MDA-MB-468, and HCC70 cells, the IC50 of the PEI-PEG-EGF / poly IC polyplex was approximately one-tenth of the IC50 of RNAiMax-pIC. RNAiMax-pIC was found to be toxic in normal cells (WI-3 and MCF10A) as well as in cancer cells with low EGFR expression, while the PEI-PEG-EGF / poly IC polyplex was not toxic to these cells.

[0231] At the tested concentrations, naked pIC had no effect in all cell lines.

[0232] The PEI-PEG-EGF / pLGA polyplex was significantly less effective (less than one-thirtieth) than the PEI-PEG-EGF / poly IC polyplex against cancer cells overexpressing EGFR. The PEI-PEG-EGF / pLGA polyplex functions as a control for the PEI-PEG-EGF / poly IC polyplex. The results clearly demonstrate that the efficacy of the PEI-PEG-EGF / poly IC polyplex is mediated via targeted pIC therapy.

[0233] The PEG-EGF / poly IC polyplex showed high specificity and efficacy in cells overexpressing EGFR, while not inducing toxicity in normal cell lines. The efficacy of the PEI-PEG-EGF / poly IC polyplex was demonstrated to be mediated via pIC. In contrast to the PEI-PEG-EGF / poly IC polyplex, RNAiMax / pIC and jetPEI / pIC were less effective and selective than the PEI-PEG-EGF / poly IC polyplex. RNAiMax / pIC was also highly toxic in normal cells. The present disclosure includes the following preferred embodiments. (1) A kit of parts, comprising: a. A polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, wherein the polymer complex 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 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 the above polyplex; b. At least one antibody capable of modulating an immune checkpoint protein, and the above at least one antibody The kit of parts as described above. (2) A composition, comprising: a. A polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, wherein the polymer complex 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 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 the above polyplex; b. At least one antibody capable of modulating an immune checkpoint protein, and the above at least one antibody The composition as described above. (3) 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, 4-1BB ligand (4-1BBL), CD40, CD40 ligand (CD40L), OX40, OX-40 ligand (OX-40L), GITR, GITR ligand (GITRL), ICOS, and ICOS ligand (ICOSL), and the above at least one antibody, 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, said at least one antibody, or (iii) A mixture of at least one antibody of (i) and at least one antibody of (ii) The composition of (1) or the kit of parts according to (2). (4) 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, and 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. The composition or kit of parts according to any one of (1) to (3). (5) The immune checkpoint protein is 4-1BB or PD-1. The composition or kit of parts according to any one of (1) to (4). (6) At least one antibody capable of regulating an immune checkpoint protein is (i) An antibody capable of regulating the immune checkpoint protein CD27 and an antibody capable of regulating an immune checkpoint protein selected from the group consisting of PD-1, PD-L1, and PD-L2. (ii) An antibody capable of regulating the immune checkpoint protein CD40 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. (iii) An antibody capable of regulating the immune checkpoint protein GITR 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. (iv) An antibody capable of regulating the immune checkpoint protein OX40 and at least one antibody capable of regulating 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 regulating the immune checkpoint protein 4-1BB, and at least one antibody capable of regulating 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 A mixture according to any one of (1)-(5) of the composition or kit of parts. (7) The composition or kit of parts according to any one of (1)-(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 (1)-(7), wherein the polyethyleneimine (PEI) is linear polyethyleneimine (LPEI). (9) The composition or kit of parts according to any one of (1)-(8), wherein the dsRNA is polyinosine-polycytidylic acid double-stranded RNA (polyIC). (10) The composition or kit of parts according to any one of (1)-(9), wherein the cancer antigen is EGFR, HER2, or PSMA, preferably the cancer antigen is EGFR. (11) One or more target moieties are selected from the group consisting of EGF, HER2 affibody, HER2 antibody, and DUPA moiety (HOOC(CH 2 ) 2 -CH(COOH)-NH-CO-NH-CH(COOH)-(CH 2 ) 2 -CO-). The composition or kit of parts according to any one of (1)-(10). (12) The composition or kit of parts according to any one of (1)-(11), wherein one or more target moieties are EGF, preferably human EGF. (13) The composition or kit of parts according to any one of (1)-(12) for use in the treatment of mammalian, preferably human cancer. The composition or kit of parts for use according to (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, glioma, head and neck cancer, prostate cancer, penile cancer, testicular germ cell cancer, neuroendocrine tumor, and hepatocellular cancer. The kit of parts for use according to (13) or (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.

Sequence Listing 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, wherein the double-stranded RNA (dsRNA) is polyinosine-polycytidylic acid double-stranded RNA (poly I:C), the polymer complex 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, and 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 the above polyplex; b. At least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of activating a co-stimulatory immune checkpoint protein, (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii), and the immune checkpoint protein is 4-1BB or PD-1, and the above at least one antibody comprising the above kit of parts.

2. A composition, comprising: a. A polyplex comprising double-stranded RNA (dsRNA) and a polymer complex, wherein the double-stranded RNA (dsRNA) is polyinosine-polycytidylic acid double-stranded RNA (poly I:C), the polymer complex 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, and 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 the above polyplex; b. At least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of activating a co-stimulatory immune checkpoint protein, (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii), and the at least one antibody, wherein the immune checkpoint protein is 4-1BB or PD-1 comprising the composition.

3. The kit of parts according to claim 1, or the composition according to claim 2, wherein the PEI is covalently bound to one, two or three PEG moieties.

4. The kit of parts according to claim 1 or 3, or the composition according to claim 2 or 3, wherein the PEI is covalently bound to one or three PEG moieties.

5. The kit of parts according to any one of claims 1 and 3 to 4, or the composition according to any one of claims 2 to 4, wherein the polyethyleneimine (PEI) is linear polyethyleneimine (LPEI).

6. The kit of parts according to any one of claims 1 and 3 to 5, or the composition according to any one of claims 2 to 5, wherein the cancer antigen is EGFR, HER2 or PSMA.

7. The kit of parts according to any one of claims 1 and 3 to 6, or the composition according to any one of claims 2 to 6, wherein the cancer antigen is EGFR.

8. wherein the one or more target moieties are selected from the group consisting of EGF, HER2 affibody, HER2 antibody, and DUPA moiety (HOOC(CH ２ )) ２ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ２ )) ２ -CO-), the kit of parts according to any one of claims 1 and 3 to 7, or the composition according to any one of claims 2 to 7.

9. The kit of parts according to any one of claims 1 and 3 to 8, or the composition according to any one of claims 2 to 8, wherein the one or more target moieties are EGF.

10. The kit of parts according to any one of claims 1 and 3 to 9, or the composition according to any one of claims 2 to 9, wherein the one or more target moieties are human EGF.

11. The kit of parts according to any one of claims 1 and 3 to 10 for use in the treatment of mammalian cancer, or the composition according to any one of claims 2 to 10.

12. The composition or kit of parts according to claim 11, wherein the mammal is a human.

13. The composition or kit of parts according to claim 11 or 12, 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, glioma, head and neck cancer, prostate cancer, penile cancer, testicular germ cell cancer, neuroendocrine tumor, and hepatocellular cancer.

14. The kit of parts according to any one of claims 11 to 13, wherein the polyplex is administered to a mammal separately from at least one antibody capable of regulating an immune checkpoint protein.

15. The kit of parts according to claim 14, wherein the mammal is a human.

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