阅读说明：本技术 用于治疗癌症的免疫疗法 (Immunotherapy for the treatment of cancer ) 是由 D·布洛卡 D·可莱克希亚 L·德’阿米科 E·基塔斯 A·列维茨基 E·波博-维勒 A 于 2020-04-03 设计创作，主要内容包括：本发明涉及一种成套试剂盒和一种组合物,所述成套试剂盒包括以下并且所述组合物包括以下：多聚复合物,所述多聚复合物包括双链RNA(dsRNA)和聚合缀合物,所述聚合缀合物包括聚乙烯亚胺(PEI)、一个或多个聚乙二醇(PEG)部分和一个或多个靶向部分,并且其中所述一个或多个靶向部分中的每个靶向部分能够与癌抗原结合；以及至少一种抗体,其中所述至少一种抗体能够调节免疫检查点蛋白。进一步地,本发明涉及用于治疗癌症的此组合物或成套试剂盒。(The present invention relates to a kit of parts and a composition, said kit of parts comprising the following and said composition comprising the following: a multimeric complex comprising double-stranded rna (dsrna) and a polymeric conjugate comprising Polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties, and wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen; and at least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein. Further, the present invention relates to such a composition or kit of parts for use in the treatment of cancer.)

1. A kit of parts comprising

a. A polyplex (polyplex) comprising double stranded RNA (dsRNA) and a polymeric conjugate,

wherein the polymeric conjugate comprises a Polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties,

wherein the PEI is covalently bound to one or more PEG moieties and each PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties, and

wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen; and

b. at least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein.

2. A composition comprising

a. A multimeric complex comprising double-stranded RNA (dsRNA) and a polymeric conjugate,

wherein the polymeric conjugate comprises a Polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, and one or more targeting moieties,

wherein the PEI is covalently bound to one or more PEG moieties and each PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties, and

wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen; and

b. at least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein.

3. The composition according to claim 1 or kit of parts according to claim 2, wherein said at least one antibody capable of modulating an immune checkpoint protein is

(i) At least one antibody capable of agonizing 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 ligand (GITRL), ICOS and ICOS ligand (ICOSL); or

(ii) At least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3 and TIGIT; or

(iii) A mixture of at least one antibody of (i) and at least one antibody of (ii).

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

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

6. The composition or kit of parts according to any one of the preceding claims, wherein said at least one antibody capable of modulating an immune checkpoint protein is a mixture of:

(i) an antibody capable of modulating an immune checkpoint protein CD27 and an antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1 and PD-L2;

(ii) an antibody capable of modulating an immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iv) an antibody capable of modulating an immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB and CTLA-4;

(v) an antibody capable of modulating immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or

(vi) An antibody capable of modulating the immune checkpoint protein ICOS and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1 and PD-L2.

7. The composition or kit of parts according to any one of the preceding claims, wherein the PEI is covalently bound to one, two or three PEG moieties, preferably to one or three PEG moieties.

8. The composition or kit of parts according to any one of the preceding claims, wherein the Polyethyleneimine (PEI) is Linear Polyethyleneimine (LPEI).

9. The composition or kit of parts according to any one of the preceding claims, wherein the dsRNA is polyinosine-polycytidylic acid double stranded rna (poly ic).

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

11. The composition or kit of parts according to any one of the preceding claims, wherein the one or more targeting 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)-(CH₂)₂-CO-)。

12. The composition or kit of parts according to any one of the preceding claims, wherein the one or more targeting moieties is EGF, preferably human EGF.

13. The composition or kit of parts according to any one of the preceding claims for use in the treatment of cancer in a mammal, preferably a human.

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

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

Background

Antibodies targeting tumor-associated antigens have become an important therapeutic modality for malignancies. Several monoclonal antibodies (mabs) have been shown to be relatively well tolerated and effective for treating a number of different malignant diseases. Although these antibodies are commonly used clinically, their efficacy is generally modest. mAbs must overcome a substantial barrier to render the antigen presented on target cells of therapeutic value (Christiansen et al, molecular Cancer Ther, 2004,3(11), 1493-1501). Furthermore, the efficiency of antibodies targeting tumor-associated antigens may be reduced by insufficient activation of the anti-tumor response of the immune system and inhibition of the immune response induced by the tumor itself.

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 response, durable disease control, and improved survival in some patients with advanced melanoma, non-small cell lung cancer (NSCLC), and other tumor types. PD-1 induces negative control signals by engagement of its ligand PD-L1 or PD-L2, resulting in inhibition of T cell proliferation, cytokine production and cytotoxic activity. (Ma et al, "Current status and perspectives in transformed biomarker research for PD-1/PD-L1 immune checkpoint blockade therapy for PD-1/PD-L1 immune checkpoint blockade therapy)", (Journal of Hematology and Oncology & Oncology), (2016)9: 47).

Tumor Necrosis Factor (TNF) -related cytokines provide the communication network necessary for an effective host defense system that coordinates multiple cell types into pathogens and malignant cells. Tumor necrosis factor ligand superfamily (TNFSF) and Tumor Necrosis Factor Receptor Superfamily (TNFRSF) provide key communication signals between various cell types during development. FTNF Receptors (TNFRs) share a conserved extracellular domain defined by cysteine-rich features. TNFR with Co-stimulatory reputation are encoded by genes residing within The immune response locus in chromosomal region 1p36 and comprise GITR (glucocorticoid-induced tumor necrosis factor), OX40, 4-1BB, and CD30(Ward-Kavanagh et al, "The TNF Receptor Superfamily in Co-stimulatory and Co-inhibitory Responses," immunization (Immunity) 44,2016, 5 months and 17 days).

A different approach for treating malignancies is vaccine-based therapy. The molecular definition of tumor-associated antigens opens the possibility to introduce specific vaccines targeting tumor cells. Recombinant vaccines based on peptides or proteins from defined Tumor Associated Antigens (TAAs) are usually administered together with adjuvants or immunomodulators. Although these vaccines are capable of inducing antigen-specific T cell responses, the clinical outcome is disappointing (Guo et al, Adv Cancer Res, 2013,119: 421-.

An additional approach to cancer immunotherapy refers to combining a vaccine with antibodies that activate anti-tumor immunity by blocking or inhibiting immune checkpoints. Immune checkpoints refer to a number of inhibitory pathways hardwired into the immune system that are important for maintaining self-tolerance and modulating the duration and magnitude of physiological immune responses. Tumors recruit certain immune checkpoint pathways as the primary mechanism of immune resistance. Because many immune checkpoints are triggered by ligand-receptor interactions, they may be susceptible to blockade or inhibition by antibodies, or modulation by recombinant forms of the ligand or receptor. Cytotoxic T lymphocyte-associated antigen 4(CTLA-4) antibodies are the first antibodies to obtain united states Food and Drug Administration (FDA) approval among such other immunotherapeutic agents. However, the development of combinatorial approaches to vaccines with antibodies remains a challenge. The combinatorial strategies must be intelligently designed and guided by mechanical considerations and preclinical models (pardol, nature Cancer reviews 2012,12(4), 252-264).

Several novel combinations of immunotherapy for oncology have been proposed (Morrisey et al, clin. trans. sci.) 2016,9,89-104), including non-antigen specific immunotherapy with naked poly ic and blocking antibodies targeting the programmed cell death-1 (PD-1) pathway, which are capable of inhibiting tumors in mouse models of cancer of B16 melanoma, Lewis lung cancer (Lewis lung carcinoma) and MC38 colon cancer (Nagato et al, oncoimmunological (OncoImmunology) 2014,3: e 28440).

Despite encouraging initial results, the development of cancer immunotherapy remains a major challenge for tumor immunologists. Therefore, effective and well-tolerated immunotherapy for the treatment of cancer is highly desirable.

Disclosure of Invention

The present invention provides a novel combination immunotherapeutic method for treating cancer.

In a first aspect, the present invention provides a kit of parts comprising

a. A multimeric complex comprising double-stranded rna (dsrna) and a polymeric conjugate, wherein the polymeric conjugate comprises Polyethylenimine (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 PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties, and wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen; and

b. at least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein.

In a further aspect, the present invention provides a composition comprising:

a. a multimeric complex comprising double-stranded rna (dsrna) and a polymeric conjugate, wherein the polymeric conjugate comprises Polyethylenimine (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 PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties, and wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen; and

b. at least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein.

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.

The inventors have surprisingly found that a combination treatment with a multimeric complex comprising double-stranded rna (dsrna) and a polymeric conjugate and one or more anti-checkpoint antibodies according to the invention enhances the activity of the immune system and leads to effective anti-tumor activity. Tumor growth is inhibited more effectively by combining a multimeric complex comprising a dsRNA and a polymeric conjugate and one or more anti-checkpoint antibodies than the multimeric complex alone. In some individuals, the combination of multimeric complexes and anti-checkpoint antibodies completely eradicates the tumor and results in sustained tumor defense and memory against cancer cells.

Interferon secretion is increased by combining a multimeric complex of the invention with an anti-checkpoint antibody compared to the multimeric complex alone. Exposing immune cells, preferably Peripheral Blood Mononuclear Cells (PBMCs), to culture medium from EGFR-overexpressing cells treated with a multimeric complex of the invention or culturing the immune cells in the presence of cells treated with a multimeric complex of the invention induces interferon secretion by the PBMCs. Addition of an anti-checkpoint antibody thereto resulted in a further increase in interferon secretion (see fig. 3 and 4).

In immunocompetent mice carrying HER2 overexpressing tumors, the mice show complete tumor regression after combined treatment with an anti-checkpoint antibody, preferably a monoclonal anti-PD-1 antibody, and a multimeric complex of the invention. Tumor re-challenge in cured mice did not induce tumor growth, indicating that an immune response to the tumor had developed.

It was found that treatment of tumors with immunomodulatory antibodies alone was ineffective or showed only limited efficacy. anti-PD-1 antibody alone did not reduce the tumor size of RENCA tumors (fig. 5). However, the combination of targeted and specifically delivered dsRNA and immunomodulatory antibodies showed an increased anti-tumor effect. Treatment of RENCA HER2 tumors with the HER 2-targeted multimeric complexes of the invention, i.e., the triple conjugate comprising PEI-PEG-HER2 affibody (PPHA) complexed with polyIC (pIC/PPHA) + anti-PD-1, surprisingly resulted in regression of tumor growth (FIG. 6). Complete tumor regression was observed in the combination group with polyIC/PPHA + anti-PD-1. The cured mice showed complete protection against tumor re-challenge, indicating that an immune response to the tumor has been generated.

The combination of targeted multimeric complexes and anti-checkpoint antibodies according to the invention thus enables a broadening of the efficacy of the antibodies for patients not showing any response at present. Targeted delivery using a combination of dsRNA, preferably poly ic, and an anti-checkpoint antibody shows significant efficacy due to the ability of the compositions and kits of the invention to restore the immune system against tumors.

Drawings

FIG. 1: IP-10 secretion from cancer cells following treatment with PEI-PEG-EGF/polyIC. A431, U87 and MCF7 cells (40,000 cells per well) were treated with various concentrations (0.125, 0.25, 0.5, 1. mu.g/ml) of PEI-PEG-EGF/polyIC for 5 hours. Human IP-10(CXCL10) secretion was higher in A431 EGFR expressing cells compared to U87 and MCF7 cells expressing very little EGFR.

FIG. 2: PD-L1 (used interchangeably herein with "PDL 1" and equivalent to "PDL 1") on a431 cells after treatment with PEI-PEG-EGF/poly ic. A431 cells were treated with PEI-PEG-EGF/polyIC at a concentration of 0.125. mu.g/ml for 5 hours. PD-L1 expression was significantly increased after PEI-PEG-EGF/poly ic treatment (MFI 751, (C)) compared to untreated control cells (MFI 431, (B)). Isotype control was used as a negative control (MFI ═ 13, (a)).

FIG. 3: combination of therapy with PEI-PEG-EGF/polyIC with Nivolumab (Nivolumab) significantly increased IFN-y production from PBMC. Combining PEI-PEG-EGF/polyIC with nivolumab significantly increased IFN-y production from PBMC challenged with diluted medium containing PEI-PEG-EGF/polyIC alone (0.125. mu.g/ml) or with nivolumab (20. mu.g/ml) for 48 hours.

FIG. 4: combining PEI-PEG-EGF/polyIC with 4-1BB antibody significantly increased IFN-y production by PBMC. PBMCs were stimulated with CD3 (0.5. mu.g/ml) and co-cultured for 16 hours with A431 cells treated with PEI-PEG-EGF/polyIC alone (0.5. mu.g/ml) or with a combination of antibodies to 4-1BB (10. mu.g/ml).

FIG. 5: anti-tumor activity of RMP1-14 in the treatment of s.c. renca murine renal cancer model: anti-PD-1 (used interchangeably with anti-PD 1 and equivalent to anti-PD 1) antibody (RMP1-14) had no anti-tumor activity in the treatment of subcutaneous tumors in the RENCA murine kidney Cancer xenograft model ("Syngeneic model for development of Cancer Therapeutics Targeting the Immune System", Lan Zhang et al, EORTC-NCI-AACR International Molecular Targets and Cancer Therapeutics (International Conference on Molecular Targets and Cancer Therapeutics), 11.18-21 days 2014, Barcelona (Barcelona, Spain), P013).

FIG. 6: multimeric complexes of poly ic and the chemical carrier polyethyleneimine-polyethylene glycol (PP) conjugated to HER2 affibody (HA), abbreviated herein as poly ic/PPHA plus the effect of anti-PD-1 (poly ic/PPHA + PD-1) on s.c. renca HER2 tumor growth (s.c. renca HER2 xenograft model) in immunocompetent mice. Mice bearing s.c. RENCA HER2 tumors were randomly divided into 4 groups, 7-8 animals/group, with an average tumor volume of 235mm³. Mice were treated intravenously every 24 hours with polyIC/PPHA, 6.25 mg/mouse, N/P8. anti-PD-1, 200 mg/mouse was injected intraperitoneally at the indicated time points. Although there appears to be no significant difference between polyIC/PPHA alone and the polyIC/PPHA + anti-PD-1 combination therapy, two mice in the polyIC/PPHA + anti-PD-1 group remained tumor-free 50 days after the start of treatment.

FIG. 7: effect of immunocompetent mice (s.c. RENCA HER2 xenograft model) at day 21. Complete regression of tumor growth was observed in 2 out of 8 mice in polyIC/PPHA + anti-PD-1 treated animals. These mice remained tumor-free after re-challenge.

FIG. 8: increased expression of PD-L1 in RENCA HER2 cells following treatment with polyIC/PPHA. Using an X-axis channel: PE-a calculates the original value of the median.

FIG. 9: effect of PEI-PEG-EGF/polyIC + anti-PD-1 on Lunca EGFR metastasis in immunocompetent mice.

FIG. 10: the combination of PEI-PEG-EGF/poly ic polyplex and nivolumab increased PBMC activation as demonstrated by IFN- γ (used interchangeably herein and is equivalent to IFN γ) ELISA. A431 alone or the medium was treated with the indicated concentrations of PEI-PEG-EGF/polyIC polyplex for 5 hours. PBMCs were stimulated or unstimulated with anti-CD 3 and treated with or without nivolumab. Supernatants from PEI-PEG-EGF/polyIC polyplex-treated or Untreated (UT) A431 cancer cells or PEI-PEG-EGF/polyIC polyplex in culture medium or untreated medium were then transferred to PBMCs. After overnight incubation, IFN- γ ELISA was performed to quantify PBMC activation.

FIG. 11: the PEI-PEG-EGF/polyIC polyplex induces cytokine secretion. Three cell lines, high EGFR (epidermal growth factor receptor) expressing cells (MDA-MB-468 and A431) and low EGFR expressing cells (MCF7), were treated with indicated concentrations of PEI-PEG-EGF/polyIC polyplexes, PEI-PEG-EGF tricojugates or pIC for 5 hours. The media was then harvested and analyzed for (a) IP10, (B) GRO α, and (C) CCL5(RANTES) using an ELISA assay.

FIG. 12: PEI-PEG-EGF/polyIC polyplex induced PBMC activation as measured by IFN-. gamma.and TNF. alpha. ELISA. Three cell lines MDA-MB-468 (high EGFR), A431 (high EGFR) and MCF7 (low EGFR) or medium alone were treated with indicated concentrations of PEI-PEG-EGF/polyIC, pIC and PEI-PEG-EGF/pLGA polyplexes of pIC or pLGA (poly-L-glutamic acid) for 5 hours in polyplexes. Supernatants and media were collected and transferred to PBMCs for 16 hours. Supernatants from treated cancer cells without PBMC were used as controls and incubated for 16 hours as well. In the case of MDA-MB-468(A, D); a431(B, E); and MCF7 cells (C, F), IFN- γ and TNF α were quantified using ELISA.

FIG. 13: treatment with PEI-PEG-EGF/poly ic polyplex induced cancer cell death of EGFR overexpressing cells with high efficacy and selectivity as compared to control treatment. Cancer cells with different EGFR expression levels were treated with the following reagents for 72 hours: PEI-PEG-EGF/polyIC, pIC alone (naked pIC), jetPEI-pIC, RNAiMax-pIC and PEI-PEG-EGF/pLGA polyplex. The concentrations shown reflect the concentrations of pIC or pLGA. Cell viability was analyzed using Celtiter-Glo. For each compound, the percent survival was normalized to the percent survival of untreated cells. (A, B) high EGFR expressing cell lines BT20, MDA-MB-468, A431 and HCC70, (C) medium EGFR expressing cell line U87MG, (D) low EGFR expressing cell lines U138 and MCF7, and (E) non-cancer cell lines WI-38 and MCF 10A.

Detailed Description

Unless defined otherwise, 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.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer, step or group of elements, integers, steps or groups thereof but not the exclusion of any other element, integer, step or group of elements, integers, steps or groups thereof.

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

The term "about" or "approximately" when used in conjunction with a numerical value is intended to encompass values having a lower limit in the range of 0-10% less than the indicated value and an upper limit in the range of 0-10% greater than the indicated value. The term "about" or "approximately" means preferably ± 10%, more preferably ± 5%, still more preferably ± 3% or most preferably ± 0% (with reference to the given numerical values, respectively). In each of the embodiments of the present invention, "about" may be deleted. All ranges of values disclosed herein are intended to mean and include any and all values that fall within the recited ranges, including the values that define the recited ranges.

In one aspect, the present invention relates to a composition comprising

a. A multimeric complex comprising double-stranded RNA (dsRNA) and a polymeric conjugate,

wherein the polymeric conjugate comprises a 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 PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties, and wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen; and

b. at least one antibody, wherein the at least one antibody is capable of modulating an immune checkpoint protein.

In a further aspect, the invention relates to a kit of parts comprising

a. A composition, wherein the composition comprises a multimeric complex comprising double-stranded rna (dsrna) and a polymeric conjugate, wherein the polymeric conjugate comprises Polyethylenimine (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 PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties, and wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen; and

b. at least one antibody, wherein said antibody is capable of modulating an immune checkpoint protein.

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

The term "kit of parts" as used herein preferably refers to at least two separate parts, i.e. the combination of the multimeric complex 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 is conventionally known to the person skilled in the art. In a particularly preferred embodiment, said kit of parts of the invention or said parts of the kit of parts of the invention, i.e. the multimeric complex and/or the one or more immunomodulatory antibodies, comprise, independently of each other, at least one pharmaceutically acceptable diluent, excipient or carrier. In another preferred embodiment, said kit of parts of the invention or said parts of the kit of parts of the invention, i.e. the multimeric complex and/or the one or more immunomodulatory antibodies, comprise, independently of each other, one or more adjuvants.

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

In another more preferred embodiment, the composition or kit of parts according to the invention is formulated for systemic administration. Still more preferably, the composition and kit of parts according to the invention, i.e. the multimeric complex and/or the one or more immunomodulatory antibodies, are formulated for intravenous, intramembranous or subcutaneous administration. More preferably, the composition and kit of parts according to the invention, i.e. the multimeric complex 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.

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

The kit of parts of the invention may comprise a container containing the polyplex and/or the one or more antibodies and/or a device for administering the parts of the kit, i.e. the polyplex and/or the one or more antibodies. In a preferred embodiment, said kit of parts of the invention comprises at least one container comprising an effective dose of said multimeric complexes and at least one container comprising an effective dose of said one or more antibodies and optionally an instruction insert.

The term "immune checkpoint protein" or "immune checkpoint" is known and described in the art (see, e.g., pardol, 2012, "natural Cancer review (Nature Rev Cancer) 12: 252-. The term "immune checkpoint protein" as used herein refers to receptors of T cells, B cells and natural killer cells (NK) and soluble or bound ligands and counter-receptors thereof that can stimulate or inhibit the activity of the immune system. In a preferred embodiment, the immune checkpoint protein refers to receptors for T cells and natural killer cells, as well as soluble or bound ligands and counter-receptors thereof that can co-stimulate or co-inhibit the activity of the immune system. Preferably, said activity of the immune system is detected by measuring T cell responses as shown herein (e.g. example 2).

Immune checkpoint proteins are important immune modulators for maintaining immune homeostasis and preventing autoimmunity. These immune modulators consist of stimulatory and inhibitory receptors and ligands that are important for maintaining self-tolerance and modulating the type, magnitude and duration of immune responses. Immune homeostasis is carefully balanced and regulated by activating and suppressing immune checkpoint proteins. Under normal circumstances, immune checkpoints allow the immune system to respond to infections and malignancies while protecting tissues from any damage that may result from this action.

Several strategies have been developed for tumor cells that exploit these checkpoints and bypass host immune defenses. The expression of immune checkpoint proteins may be deregulated by tumors as an important immune resistance mechanism. Therefore, inhibition or activation of checkpoint receptors and ligands has become a potential strategy to limit Tumor Infiltrating Lymphocytes (TILs) to inhibit signals from tumors and circulating monocytes, block negative signals and cytokines that inhibit T cell activity, and stimulate systemic immunity.

One of the most promising approaches to activating therapeutic anti-tumor immunity is to block inhibition of immune checkpoint proteins or to activate stimulatory immune checkpoint proteins. Immune checkpoints refer to inhibitory and activating proteins of the immune system that are critical to maintaining self-tolerance and modulating the duration and magnitude of the physiological immune response to minimize collateral tissue damage. Tumor recruitment selects certain immune checkpoint pathways as the primary mechanism of immune resistance of T cells that are specific for tumor antigens in particular. T cells have been the main focus of therapeutic manipulation of endogenous anti-tumor immunity for the following reasons: the ability of T cells to selectively recognize peptides derived from proteins in all cell compartments; the ability of T cells to directly recognize and kill antigen expressing cells (via CD8+ effector T cells, CTLs); and the ability of T cells to orchestrate the integrative adaptation and innate effector mechanisms of various immune responses (via CD4+ helper T cells). Thus, agonists of co-stimulatory receptors or antagonists of inhibitory signals are agents in current clinical testing, both of which result in amplification of antigen-specific T cell responses. Because many immune checkpoints are triggered by ligand-receptor interactions, they may be readily modulated by antibodies.

Preferably, the immune checkpoint protein is a human immune checkpoint protein.

Within the present invention, an antibody capable of modulating an immune checkpoint protein is any compound that modulates the function of an immune checkpoint protein and thereby promotes the activity of the immune system. Promotion of immune system activity comprises causing an enhanced immune response to an antigen and/or a reduced immunosuppressive immune response to an antigen. Preferably, the promotion of immune system activity results in immune-mediated tumor cell elimination.

The term "modulation" (or "modulator") encompasses activation associated with stimulation or enhancement of the function of a costimulatory immune checkpoint protein, and inhibition of a suppressive co-inhibitory immune checkpoint protein associated with reduction of activity and complete blockade of the co-inhibitory immune checkpoint protein. The designation "modulating an immune checkpoint protein" encompasses stimulating T cells comprising T helper cells, CTL natural killer T cells, and natural killer cells (NK). Stimulation (or activation) induced by modulation of the immune checkpoint protein is preferably detected by measuring an increase in the level of a cytokine such as an interferon, in particular IFN- γ produced or released by T and NK cells, in particular, as compared to a control not administered an immune checkpoint protein modulating antibody as described herein (e.g., example 2).

Examples of immune checkpoint proteins include, but are not limited to and preferably are selected from the group consisting of: PD-1 (programmed death 1, used interchangeably herein with PD1 and corresponding to 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 and mucin domains 3), LAG3, By-He, H4, HACR 2, ID01, CD40/TNFRSF5, CD40L/CD 154/TNFRSF 5, OX40/CD134, OX-40L/TNFRSF 4/CD252, GITR (glucocorticoid-induced TNFR family-related gene)/TNFRSF 18, GITR ligand/TNFRSF 18, ICOS/AILIM/CD278, ICOS ligand/B7-H2, CD122, CD 155/CD 226, DNARFRSF 226, HVRSF 27/14, TNFRSF 3626/CD 3, PVR-III, TNFSF14/LIGHT/CD258, CD70/CD27L/TNFSF7, CD28/TP44, CD80/B7-1, CD86/B7-2, A2AR, KIR (killer cell immunoglobulin-like receptor), NOX 2/NADPH oxidase isomer 2 of nicotinamide adenine dinucleotide phosphate, SIGLEC7 (sialic acid-binding immunoglobulin lectin 7)/CD328, SIGLEC9 (sialic acid-binding immunoglobulin lectin 9)/CD329, CD80/B7-1, CD86/B7-2, B7-H3/CD276, CN1/B7-H4/B7S1/7x, VISTA (T cell activation V domain Ig inhibitor)/B7-H5/GI 24, LAG3/CD 3/lymphocyte activation gene 3, indole-2/dioxygenase 3/dioxygenase/bis-O-3/bis-O-dioxygenase/2, TDO/tryptophan 2, 3-dioxygenase, galectin-/LGALS 9, TIM-3/HAVCR2, TIGIT/VSTM3, HVEM (herpes virus invasion mediator)/TNFRSF 14, BTLA (B and T lymphocyte attenuator)/CD 272, CD160, CEACAM1/CD66a, indoleamine, SIRP, alpha/CD 172a, CD47, CD48/SLAMF2, CD30, CD30L, TMIGD2, HHLA2, TL1A, DR3, LT beta R, TNF, TNFR2 and 2B4/CD 244.

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

In a preferred embodiment, the immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, CD40/TNFRSF5, CD40L/CD 154/TNFRSF L, OX L/CD 134, OX-40L/TNFRSF L/CD 252, GITR/TNFRSF L, GITR ligand/TNFRSF L, ICOS (inducible T cell costimulatory molecule)/AILIM/CD 278, ICOS ligand/B L-H L, CD122, A2L (adenosine A2L receptor), KIR, NOX L, SIGLEC L/CD 328, SIGLEC L/CD 329, PD-1, PD-L L, CTLA-4, CD L/B L-1, CD L/B L-2, B L-H L/CD, B L-36276-H36276/B7S L/7, CTLA-X-72, CTLA-B L/B L, GAL L/B L, LGA-72/LGA 3/LGA L-L, LGA L/L L, LAG 3/LAG 3, and LGA 3/L L-3-activating genes, TIM-3/HAVCR2 and TIGIT/VSTM 3. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, CD40/TNFRSF5, CD40L/CD 154/TNFRSF 5, OX 2/CD 134, OX-40L/TNFRSF 4/CD252, GITR/TNFRSF18, GITR ligand/TNFRSF 18, ICOS/AILIM/CD278, ICOS ligand/B7-H2, PD-1, PD-L1, PD-L2, CTLA-4, CD 2/B2-1, CD 2/B2-2, B2-H2/CD 276, B2-H2/B7S 2/7 x, VISTA/B2-H2/GI 2, LAG 2/CD 223/lymphocyte activating gene 3, galectin-9/LGALS 2, VSIT-3/TIGSF 2 and TIGSTIM-TIGSF 69572. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS ligand CD40 and CD40 ligand. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB ligand, TIGIT, LAG3, TIM3, GITR ligand, CD40, CD40L, OX40, OX-40L, ICOS and ICOS ligand. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, 4-1BB, TIGIT, LAG3, TIM-3, GITR, CD40, OX40 and ICOS.

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

In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, LAG3, TIM3, GITR, CD40, OX40, and ICOS. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR, CD40, OX40 and ICOS. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L24-1BB, LAG3, TIM3, GITR, CD40, OX40, and ICOS. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, LAG3, TIM3, GITR, CD40 and OX 40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, LAG3, TIM3, GITR, CD40 and OX 40.

In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40 and OX 40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR, CD40 and OX 40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40 and OX 40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, GITR and OX 40. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB and OX 40.

In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, LAG3 and TIM 3. In a further preferred embodiment, the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, LAG3 and TIM 3.

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

As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that selectively binds to an antigen, including haptens, epitopes, receptors or ligands or portions thereof. As such, the term "antibody" encompasses not only intact antibody molecules, but also antibody fragments as well as variants (including derivatives), antibody fragments and fusion proteins thereof. The term "antibody" also refers to antibodies comprising two immunoglobulin heavy chains and two immunoglobulin light chains, as well as forms comprising full-length antibodies and portions thereof; comprising, for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab ', F (ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAb), diabodies, naked antibodies, antibody-drug conjugates and bi-or tri-specific antibodies, anti-idiotypic antibodies, anticalins, and functionally active epitope-binding fragments thereof. In a particularly preferred embodiment, said antibody capable of modulating an immune checkpoint protein is a monoclonal antibody, a humanized antibody or a fully human antibody. The term "antibody capable of modulating an immune checkpoint protein" as used herein refers to an antibody that modulates the activity of a checkpoint receptor, counter receptor or binding and soluble ligand thereof.

The term "anti", as used herein, relates to an antibody that selectively binds to a target mentioned after the term "anti".

As used herein, the term "affinity antibody" refers to a protein that is engineered to bind with high affinity to a target protein or peptide that mimics a monoclonal antibody, and is therefore a member of the family of antibody mimetics. Preferably, the affibody has a high affinity binding domain derived from protein a. HER2 is a target for HER2 affibody.

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

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

In a further aspect, the present invention provides a kit of parts comprising: (a) a multimeric complex comprising double-stranded rna (dsrna) and a polymeric conjugate, wherein the polymeric conjugate comprises Polyethylenimine (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 PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties, and wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen; and (b) at least one antibody. In yet a further aspect, the present invention provides a composition comprising: (a) a multimeric complex comprising double-stranded rna (dsrna) and a polymeric conjugate, wherein the polymeric conjugate comprises Polyethylenimine (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 PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties, and wherein each targeting moiety 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-CD 137/4-1BB, anti-4-1 BBL/CD137L, CD40/TNFRSF5, anti-CD 40L/CD 154/TNFRSF 5, anti-OX 40/CD134, anti-OX-40L/TNFRSF 4/CD252, anti-GITR/TNFRSF 4, anti-GITR ligand/TNFRSF 4, anti-ICOS (inducible T cell costimulatory molecule)/AILIM/CD 278, anti-ICOS ligand/B4-H4, anti-CD 122, anti-A2 4 (adenosine A2 4 receptor), anti-KIR, anti-NOX 4, anti-SIGLEC 4/CD 328, anti-GLLEC 4/CD 329, anti-PD-1, anti-PD-L4, anti-CTLA-4, anti-CD 4/B4-1, anti-CD 4/B4-H-4, anti-CD 4/5-CD 4/CD 4, anti-CTLA-3/4, anti-CD 4/4-activating gene, anti-GI/4/GI 3/4, Anti-2, 3-dioxygenase/IDO, anti-galectin-/LGALS 9, anti-TIM-3/HAVCR 2 and anti-TIGIT/VSTM 3. In further preferred embodiments, the antibody is selected from the group consisting of: anti-CD 137/4-1BB, anti-4-1 BBL/CD137L, anti-CD 40/TNFRSF5, anti-CD 40L/CD 154/TNFRSF 5, anti-OX 40/CD134, anti-OX-40L/TNFRSF 4/CD252, anti-GITR/TNFRSF 18, anti-GITR ligand/TNFRSF 18, anti-ICOS/AILIM/CD 278, anti-ICOS ligand/B7-H2, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-CD 2/B2-1, anti-CD 2/B2-2, anti-B2-H2/CD 276, anti-B2-H2/B7S 2/7 x, anti-VISTA/B2-H2/2, anti-LAG 2/TIG-3, anti-GALT-TIGG-3/TIGG-TIGSF-3, anti-TIGG-TIGSF-4, anti-TIGSF-TIGG-2, anti-TIGG-4, anti-TIGG-2, anti-TIGG-4, anti-TIGG-2, anti-TIGG-3, anti-TIGG-2, anti-TIGG-4, anti-2, anti-TIGG-4, anti-TIGG-2, anti-TIGG-2, anti-2, and TIGG-2, anti-TIGG-TIGGAGIRE-TIGGAGLID-2, anti-TIGGIRE-TIGGAGE-TIGGIRE-2, anti-TIGGIRE-4, anti-TIGGIRE-2, and TIGGIRE-4, and TIGGIRE-4, and TIGGIRE-2, anti-4-TIGGIRE-4, and TIGGIRE-TIGG. In further preferred embodiments, 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-4-1 BB ligand (4-1BBL), anti-TIGIT, anti-LAG 3, anti-TIM 3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR 4, anti-GITR ligand, anti-GITR, anti-OX 40, anti-OX-40L, anti-ICOS ligand, anti-CD 40, and anti-CD 40 ligand. In further preferred embodiments, 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-4-1 BB ligand, anti-TIGIT, anti-LAG 3, anti-TIM 3, anti-GITR ligand, anti-CD 40, anti-CD 40L, anti-OX 40, anti-OX-40L, anti-ICOS and anti-ICOS ligands.

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-4-1 BB, anti-TIGIT, anti-LAG 3, anti-TIM 3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR 4, anti-GITR, anti-OX 40, anti-ICOS and anti-CD 40. In further preferred embodiments, 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-4-1 BB, anti-TIGIIT, anti-LAG 3, anti-TIM 3, anti-GITR, anti-CD 40, anti-OX 40, and anti-ICOS. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1 BB, anti-TIGIT, anti-LAG 3, anti-TIM 3, anti-GITR, anti-CD 40, anti-OX 40, and anti-ICOS. In further preferred embodiments, 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-4-1 BB, anti-TIGIIT, anti-LAG 3, anti-TIM-3, anti-GITR, anti-CD 40, anti-OX 40, and anti-ICOS. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1 BB, anti-TIGIT, anti-LAG 3, anti-TIM-3, anti-GITR and anti-ICOS. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1 BB, anti-LAG 3, anti-TIGIT, anti-TIM-3, anti-GITR and anti-ICOS. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-CTLA-4, anti-4-1 BB, anti-LAG 3, anti-TIGIT, anti-TIM-3, anti-GITR and anti-ICOS. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1 BB, anti-GITR, anti-CD 40, anti-ICOS and anti-OX 40. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1 BB, anti-GITR, anti-CD 40, and anti-OX 40. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1 BB, anti-GITR, and anti-OX 40. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1 BB, and anti-OX 40. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, and anti-4-1 BB. In further preferred embodiments, the antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-4-1 BB. In a further preferred embodiment, the antibody is anti-PD-1 or anti-4-1 BB. 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-4-1 BB. In another preferred embodiment, the antibody is anti-CTLA-4.

In further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-4-1 BBL/CD137L, anti-CD 40/TNFRSF5, anti-CD 40L/CD 154/TNFRSF 5, anti-OX 40/CD134, anti-OX-40L/TNFRSF 4/CD252, anti-GITR/TNFRSF 18, anti-GITR ligand/TNFRSF 18, anti-ICOS/AILIM/CD 278 and anti-ICOS ligand/B7-H2, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-CD 2/B2-1, anti-CD 2/B2-2, anti-B2-H2/CD 276, anti-B2-H2/B7S 2/7 x, anti-VISTA/B2-H2/2, anti-CD 2/LAG 3/TIGS activating genes, anti-GI-TIGSF/TIGSF 223, anti-TIGSF-2, anti-TIGS-TIGSF-4, anti-TIGSD-TIGSF-4, anti-TIGSF-TIGG-1, anti-TIGG-1, anti-TIGG-2, anti-TIGG-4, anti-1, anti-TIGG-2, anti-TIGG-4, anti-4-1, anti-4-TIGG-4-1, anti-4-TIGG-1, anti-TIGG-2, anti-TIGG-4-TIGG-4, anti-4-1, anti-2, anti-4-TIGG-4-2, anti-4-1-4-1, anti-4-1, anti-4-1, anti-TIGG-4-1, anti-4-; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-4-1 BBL/CD137L, anti-GITR/TNFRSF 18, anti-GITR ligand/TNFSF 18, anti-ICOS/AILIM/CD 278, and anti-ICOS ligand/B7-H2, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-CD 80/B7-1, anti-CD 86/B7-2, anti-B7-H3/CD 276, anti-B7-H4/B7S 1/7x, anti-VISTA/B7-H5/GI 24, anti-LAG 3/CD 223/lymphocyte activation gene 3, anti-galectin-/LGALS 9, anti-3/HAVCCR 2, and anti-TIGIT/VSTM 3; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

In further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-4-1 BBL/CD137L, anti-CD 40/TNFRSF5, anti-CD 40L/CD 154/TNFRSF 5, anti-OX 40/CD134, anti-OX-40L/TNFRSF 4/CD252, anti-GITR/TNFRSF 18, anti-GITR ligand/TNFRSF 18, anti-ICOS/AILIM/CD 278, and anti-ICOS ligand/B7-H2, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-H3/CD 276, anti-B7-H4/B7S 1/7x, anti-VISTA/B7-H5/GI 24, anti-LAG 3/CD 223/lymphocyte activator gene 3, anti-agglutinin-/LS 9, anti-VSCR-TIGTM 2/TIGTM 3; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-4-1 BBL/CD137L, anti-GITR/TNFRSF 18, anti-GITR ligand/TNFSF 18, anti-ICOS/AILIM/CD 278, and anti-ICOS ligand/B7-H2, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-H3/CD 276, anti-B7-H4/B7S 1/7x, anti-VISTA/B7-H5/GI 24, anti-LAG 3/CD 223/lymphocyte activation gene 3, anti-galectin-/LS 9, anti-3/HAVCR 2, and anti-TIGIT/VSTM 3; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-CD 40/TNFRSF5, anti-OX 40/CD134, anti-GITR/TNFRSF 18, anti-ICOS/AILIM/CD 278, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-B7-H3/CD 276, anti-B7-H4/B7S 1/7x, anti-VISTA/B7-H5/GI 24, anti-LAG 3/CD 223/lymphocyte activation gene 3, anti-galectin-/LGALS 9, anti-3/HAVCR 2, and anti-TIG/VSTM 3; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-CD 40/TNFRSF5, anti-OX 40/CD134, anti-GITR/TNFRSF 18, anti-ICOS/AILIM/CD 278, 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 further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-CD 40/TNFRSF5, anti-OX 40/CD134, anti-GITR/TNFRSF 18, anti-ICOS/AILIM/CD 278, 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 further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-CD 40/TNFRSF5, anti-OX 40/CD134, anti-GITR/TNFRSF 18, 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 further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-CD 40/TNFRSF5, anti-OX 40/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 further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-OX 40/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 further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-4-1 BBL/CD137L, anti-GITR/TNFRSF 18, anti-GITR ligand/TNFSF 18, anti-ICOS/AILIM/CD 278, and anti-ICOS ligand/B7-H2, or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-LAG 3/CD 223/lymphocyte activation gene 3, anti-TIM-3/HAVCR 2, and anti-TIGIT/vs 3; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-4-1 BB, anti-GITR, anti-OX 40, anti-ICOS and anti-CD 40; or (ii) anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-TIGIT, anti-LAG 3, anti-TIM 3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR 4, and anti-GITRL; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-4-1 BBL/CD 137L; 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 further preferred embodiments, the at least one antibody is selected from the group consisting of: (i) anti-CD 137/4-1BB, anti-4-1 BBL/CD 137L; 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 preferred embodiment, the antibody capable of modulating an immune checkpoint protein is a bispecific antibody capable of binding to a cancer antigen and an immune checkpoint protein. Preferably, the cancer antigen is EGFR, HER2 or PSMA, more preferably, the cancer antigen is EGFR or HER2, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4 and 4-1 BB. 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 4-1 BB. More preferably, the cancer antigen is EGFR or HER2 and the immune checkpoint protein is PD-1 or 4-1 BB. Still more preferably, the cancer antigen is HER2 or EGFR and the immune checkpoint protein is 4-1 BB. In another preferred embodiment, the cancer antigen is HER2 and the immune checkpoint protein is 4-1 BB. 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 4-1 BB. In another preferred embodiment, the cancer antigen is EGFR and the immune checkpoint protein is 4-1 BB. In a preferred embodiment, said antibody capable of modulating an immune checkpoint protein is a lipocalin binding to 4-1BB and HER 2.

Preferred examples of said antibodies capable of modulating the immune checkpoint protein PD-1 (anti-PD-1) are human or humanized antibodies selected from the group consisting of: pembrolizumab (pembrolizumab), nivolumab (also known as MDX-1106 or BMS-936558, Topalian et al, 2012, New England journal of medicine (N.Eng.J.Med.), (366: 2443-2454, disclosed in US 8008449B 2), cimiraprizumab (cemipimab), IBI308, BCD-100, PDR001, tirelezumab (tiselizumab), carprilizumab (camelizumab), pidilizumab (pidelizumab) (disclosed in Rosenblatt et al, J Immunotherapy (J Immunotherapeutic.) -34: 409-18), and palbrizumab (labrolizumab) (disclosed in the following, for example, in hPD A as well as humanized derivatives thereof, Alphantih A.201132, and Alphan.20132; published in Habrolizumab, also known as MDX-1106, Michel et al, Michel.3, Michelizumab et al, published in US 800134, Michelizumab (Cemifolimab), Mirabizumab (pidelimab (pidiun) (disclosed in Rosenh et al, 389.),34; 2011.), 2012, J Immunol 189: 2338-47). A more preferred example of such an antibody capable of modulating the immune checkpoint protein PD-1 (anti-PD-1) is pembrolizumab or nivolumab.

Preferred examples of said antibodies capable of modulating the immune checkpoint protein PD-L1 (anti-PD-L1) are antibodies selected from the group consisting of: dolavizumab (durvalumab), avizumab (avelumab) and atezumab (atezolizumab), MEDI-4736 (disclosed in, e.g., WO 2011/066389 Al), MPDL328OA (disclosed in, e.g., US 8217149B 2) and MIH1 (Affymetrix). A more preferred example of said antibody capable of modulating the immune checkpoint protein PD-L1 (anti-PD-L1) is an antibody selected from the group consisting of: dolvacizumab, avilumumab and astuzumab.

Preferred examples of said antibodies capable of modulating the immune checkpoint protein CTLA-4 are ipilimumab (ipilimumab) or tremelimumab (tremelimumab), more preferably ipilimumab. Ipilimumab is a fully human CTLA-4 blocking antibody currently marketed under the name Yervoy (Bristol-Myers Squibb). An additional CTLA-4 inhibitor is tremelimumab (see Ribas et al, 2013, journal of clinical oncology (j.clin.oncol.) 31: 616-22).

A preferred example of such an antibody capable of modulating the immune checkpoint protein CD27 is CDX-1127, an agonistic anti-CD 27 monoclonal antibody.

Preferred examples of said antibodies capable of modulating the immune checkpoint protein OX40 are MEDI0562, a humanized OX40 agonist; MEDI6469, a murine OX4 agonist; and MEDI6383, an OX40 agonist.

A preferred example of such an antibody capable of modulating an immune checkpoint protein KIR is liriluzumab (Lirilumab), a monoclonal antibody directed against KIR.

A preferred example of such an antibody capable of modulating immune checkpoint protein 4-1BB is umeluzumab (ureluab). A preferred example of such an antibody capable of modulating the immune checkpoint protein LAG-3 is riltilimab (relatlimab).

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

In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of: pembrolizumab, nivolumab, cimeprinizumab, IBI308, BCD-100, PDR001, tirezumab, carprilizumab, pidilizumab, palbocilizumab, h409All, h409a16, h409a17, soluble PD-1 ligand such as PD-L2Fc fusion protein, dolacruzumab, avillumab, astuzumab, MEDI-4736, MPDL328OA, CDX-1127, MIH1, MEDI0562, MEDI6469, MEDI6383, ipilimumab, tremelimumab, rillizumab, reulumumab, anti-TIGIT antibody, anti-TIGIT 3 antibody, anti-GITR antibody, and anti-ICOS antibody. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of: pembrolizumab, nivolumab, cimirapril mab, IBI308, BMS-986016, BCD-100, PDR001, tiragluzumab, carprilizumab, pidilizumab, pabolilizumab, h409All, h409a16, h409a17, soluble PD-1 ligands such as PD-L2Fc fusion protein, daclizumab, avilumumab, alemtuzumab, liriluzumab, MEDI-4736, MPDL328OA, MIH1, ipilimumab, tremelimumab, riluzumab, and Urumumab. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of: pembrolizumab, nivolumab, cimeprinizumab, IBI308, BCD-100, PDR001, tirezumab, carprilizumab, pidilizumab, palboclizumab, h409All, h409a16, h409a17, soluble PD-1 ligands such as PD-L2Fc fusion protein, duvaluzumab, aviluzumab, atuzumab, MEDI-4736, MPDL328OA, MIH1, ipilimumab, tremelimumab, and Urreulizumab. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of: pembrolizumab, nivolumab, cimeprinizumab, IBI308, BCD-100, PDR001, tirezumab, carprilizumab, pidilizumab, palboclizumab, h409All, h409a16, h409a17, soluble PD-1 ligands such as PD-L2Fc fusion protein, bevacizumab, avilumab, astuzumab, MEDI-4736, MPDL328OA, MIH1, tremelimumab, and urelumab. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is selected from the group consisting of: pembrolizumab, nivolumab, cimeprinizumab, IBI308, BCD-100, PDR001, tirezumab, carprilizumab, pidilizumab, palboclizumab, h409All, h409a16, h409a17, soluble PD-1 ligands such as PD-L2Fc fusion protein, bevacizumab, avilumab, atelizumab, MEDI-4736, MPDL328OA, and MIH 1.

In a preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody (referred to herein as a checkpoint activator) capable of agonizing a costimulatory immune checkpoint protein; (ii) at least one antibody capable of antagonizing inhibition of an immune checkpoint protein (referred to herein as 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 (checkpoint activator) capable of agonizing a co-stimulatory immune checkpoint protein. The checkpoint activator activates a costimulatory immune checkpoint protein. Checkpoint activators deliver activation signals directly to T cells, B cells, or natural killer cells by agonizing receptors of the cell type, inhibiting or blocking inhibitory ligands, or agonizing counter receptors on Antigen Presenting Cells (APCs).

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 the inhibition of an immune checkpoint protein (checkpoint inhibitor). The checkpoint inhibitor derepresses, i.e. reduces or removes, the inhibitory function of the inhibitory immune checkpoint protein. Checkpoint inhibitors deliver an antagonist signal directly to T cells, B cells, or natural killer cells by antagonizing receptors of the cell type, agonizing inhibitory ligands, or antagonizing counterreceptors on Antigen Presenting Cells (APCs).

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

In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD155/PVR, CD226/DNAM-1, CD137/4-1BB, CD40/TNFRSF5, CD40L/CD 154/TNFRSF 5, 4-1BBL/CD137L, OX40/CD134, OX-40L/TNFRSF 4/CD252, CD27, CD122, HVEM/TNFRSF14, TNFRSF 14/LIGHT/CD258, CD70/CD 27L/TNFRSF 7, CD28/TP44, CD30, CD30 ligand, TMIGD2, HHLA2, CD80/B7-1, CD86/B7-2, GITR/TNFRSF18, GITR ligand/TNFRSF 18, DR3, TL1A, CD30, CD30 LIFLX 30, TMIGD 30, HHLA 30, TNFRS 30, CD 30-TICS 30, CD30, TNF 30, CD30, and CD 36LT/LT 30/LT; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD80/B7-1, CD86/B7-2, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, HVEM/TNFRSF14, BTLA, CD160, LAG3/CD 223/lymphocyte activating gene 3, CEACAM1/CD66a, indoleamine, 2, 3-dioxygenase/IDO, galectin-/LGALS 9, TIM-3/HAVCR2, 2B4/CD244, SIRP, alpha/CD 172a, CD47, CD 48/AMF 2, TIGIT/VSTM3, A2AR, KIR, NOX2, CD 7/GLEC 328, GLEC7/CD 9, GLEC 328, GLEC9, SIEC 329, or mixtures thereof (III). In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD155/PVR, CD226/DNAM-1, CD137/4-1BB, CD40/TNFRSF5, CD40L/CD 154/TNFRSF 5, 4-1BBL/CD137L, OX40/CD134, OX-40L/TNFRSF 4/CD252, CD27, CD122, HVEM/TNFRSF14, TNFRSF 14/LIGHT/CD258, CD70/CD 27L/TNFRSF 7, CD28/TP44, CD30, CD30 ligand, TMIGD2, HHLA2, CD80/B7-1, CD86/B7-2, GITR/TNFRSF18, GITR ligand/TNFRSF 18, DR3, TL1A, CD30, CD30 LIFLX 30, TMIGD 30, HHLA 30, TNFRS 30, CD 30-TICS 30, CD30, TNF 30, CD30, and CD 36LT/LT 30/LT; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, HVEM/TNFRSF14, BTLA, CD160, LAG3/CD 223/lymphocyte activating gene 3, CEACAM1/CD66a, indoleamine, 2, 3-dioxygenase/IDO, galectin-/LGALS 9, TIM-3/HAVCR2, 2B4/CD244, SIRP, α/CD172a, CD47, CD48/SLAMF2, TIG/VSTM 3, A2AR, KIR, NOX2, SIGLEC7/CD328, SIGLEC9/CD329, and (iii) mixtures of the two. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD155/PVR, CD226/DNAM-1, CD137/4-1BB, CD40/TNFRSF5, CD40L/CD 154/TNFRSF 5, 4-1BBL/CD137L, OX40/CD134, OX-40L/TNFRSF 4/CD252, CD27, HVEM/TNFRSF14, TNFRSF 14/LIGHT/CD258, CD70/CD 27L/TNFRSF 7, CD28/TP44, CD80/B7-1, CD86/B7-2, GITR/TNFRSF18, GITR ligand/TNFRSF 18, CD30, CD30L, TMIGD2, HHLA2, TL1A, DR3, LT beta R, TNF, ICTNFR 2, ICOS/AILIM/CD278 and ICOS ligand 7/B2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD80/B7-1, CD86/B7-2, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, HVEM/TNFRSF14, BTLA, CD160, LAG3/CD 223/lymphocyte activating gene 3, CEACAM1/CD66a, indoleamine, galectin-/LGALS 9, TIM-3/HAVCR2, 2B4/CD244, SIRP, alpha/CD 172a, CD47, CD48/SLAMF2 and TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD155/PVR, CD226/DNAM-1, CD137/4-1BB, CD40/TNFRSF5, CD40L/CD 154/TNFRSF 5, 4-1BBL/CD137L, OX40/CD134, OX-40L/TNFRSF 4/CD252, CD27, HVEM/TNFRSF14, TNFRSF 14/LIGHT/CD258, CD70/CD 27L/TNFRSF 7, CD28/TP44, CD80/B7-1, CD86/B7-2, GITR/TNFRSF18, GITR ligand/TNFRSF 18, CD30, CD30L, TMIGD2, HHLA2, TL1A, DR3, LT beta R, TNF, ICTNFR 2, ICOS/AILIM/CD278 and ICOS ligand 7/B2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, HVEM/TNFRSF14, BTLA, CD160, LAG3/CD 223/lymphocyte activating gene 3, CEACAM1/CD66a, indoleamine, galectin-/LGALS 9, TIM-3/HAVCCR 2, 2B4/CD244, SIRP, alpha/CD 172a, CD47, CD48/SLAMF2 and TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, CD40/TNFRSF5, CD40L/CD 154/TNFRSF 5, OX40/CD134, OX-40L/TNFRSF 4/CD252, GITR/TNFRSF18, GITR ligand/TNFRSF 18, ICOS/AILIM/CD278, and ICOS ligand/B7-H2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD80/B7-1, CD86/B7-2, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, LAG3/CD 223/lymphocyte activation gene 3, galectin-/LGALS 9, TIM-3/HAVCR2 and TIGIT/VSTM 3; or (iii) a mixture of both. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, GITR/TNFRSF18, GITR ligand/TNFSF 18, ICOS/AILIM/CD278, and ICOS ligand/B7-H2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD80/B7-1, CD86/B7-2, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, LAG3/CD 223/lymphocyte activation gene 3, galectin-/LGALS 9, TIM-3/HAVCR2 and TIGIT/VSTM 3; or (iii) a mixture of both. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, CD40/TNFRSF5, CD40L/CD 154/TNFRSF 5, OX40/CD134, OX-40L/TNFRSF 4/CD252, GITR/TNFRSF18, GITR ligand/TNFRSF 18, ICOS/AILIM/CD278, and ICOS ligand/B7-H2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, LAG3/CD 223/lymphocyte activation gene 3, galectin-/LGALS 9, TIM-3/HAVCR2 and TIGIT/VSTM 3; or (iii) a mixture of both. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, GITR/TNFRSF18, GITR ligand/TNFSF 18, ICOS/AILIM/CD278, and ICOS ligand/B7-H2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, LAG3/CD 223/lymphocyte activation gene 3, galectin-/LGALS 9, TIM-3/HAVCR2 and TIGIT/VSTM 3; or (iii) a mixture of both.

In a preferred embodiment of the composition or kit of parts of the invention, said at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing 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 ligand (GITRL), ICOS and ICOS ligand (ICOSL); or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3 and TIGIT; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

In a preferred embodiment of the composition or kit of parts of the invention, said at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing 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 ligand (GITRL), ICOS and ICOS ligand (ICOSL); or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3 and TIGIT; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). AVCR2 and TIGIT/VSTM 3; or (iii) a mixture of both. In a further preferred embodiment of the composition or kit of parts of the invention, said at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: 4-1BB, CD40, OX40, GITR and ICOS; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3 and TIGIT; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a further preferred embodiment of the composition or kit of parts of the invention, said at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: 4-1BB, CD40, OX40, GITR and ICOS; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, B7-H4, VISTA, LAG-3, galectin-9, TIM-3 and TIGIT; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

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

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

In a further preferred embodiment of the composition or kit of parts of the invention, said at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: 4-1BB, CD40, OX40 and GITR; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a further preferred embodiment of the composition or kit of parts of the invention, said at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: 4-1BB, OX40, and GITR; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii). In a further preferred embodiment of the composition or kit of parts of the invention, said at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: 4-1BB and OX 40; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2; or (iii) a mixture of at least one antibody of (i) and at least one antibody of (ii).

In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, GITR/TNFRSF18, GITR ligand/TNFSF 18, ICOS/AILIM/CD278, and ICOS ligand/B7-H2; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, LAG3/CD 223/lymphocyte activation gene 3, TIM-3/HAVCR2 and TIGIT/VSTM 3; or (iii) a mixture of both. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: 4-1BB, GITR, OX40, ICOS and CD 40; 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, TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4 and GITRL; or (iii) a mixture of both. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD 137L; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4; or (iii) a mixture of both. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD 137L; or (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2; or (iii) a mixture of both.

In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD155/PVR, CD226/DNAM-1, CD137/4-1BB, CD/TNFRSF, CD 40/CD 154/TNFRSF, 4-1BBL/CD137, OX/CD 134, OX-40L/TNFRSF/CD 252, CD, HVEM/TNFRSF, TNFRSF/LIGHT/CD 258, CD/CD 27/TNFRSF, CD/TP, CD ligand (L), TMIGD, HHLA, CD/B-1, CD/B-2, GITR/TNFRSF, GITR ligand/TNFRSF, DR, TL1, CD30, TMIGD, HHLA, TL1, DR, LT beta R, TNF, TNFR, ICOS/AILIM/CD278, and ICOS ligand/B-H. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is: (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, CD40/TNFRSF5, CD40L/CD 154/TNFRSF 5, OX40/CD134, OX-40L/TNFRSF 4/CD252, GITR/TNFRSF18, GITR ligand/TNFRSF 18, ICOS/AILIM/CD278, and ICOS ligand/B7-H2. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, GITR/TNFRSF18, GITR ligand/TNFSF 18, ICOS/AILIM/CD278, and ICOS ligand/B7-H2. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: 4-1BB, GITR, OX40, ICOS and CD 40. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is selected from the group consisting of: CD137/4-1BB, 4-1BBL/CD137L, GITR/TNFRSF18, GITR ligand/TNFSF 18, ICOS/AILIM/CD278, and ICOS ligand/B7-H2. In a further preferred embodiment, said at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein said co-stimulatory immune checkpoint protein is CD137/4-1BB or 4-1BBL/CD 137L. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the co-stimulatory immune checkpoint protein is CD137/4-1 BB.

In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the agonistic antibody is selected from the group consisting of: anti-4-1 BB, anti-GITR, anti-OX 40, anti-ICOS and anti-CD 40. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein the agonistic antibody is selected from the group consisting of: anti-CD 137/4-1BB, anti-4-1 BBL/CD137L, anti-GITR/TNFRSF 18, anti-GITR ligand/TNFSF 18, anti-ICOS/AILIM/CD 278, and anti-ICOS ligand/B7-H2. In a further preferred embodiment, said at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein said agonistic antibody is anti-CD 137/4-1BB or anti-4-1 BBL/CD 137L. In a further preferred embodiment, said at least one antibody capable of modulating an immune checkpoint protein is (i) at least one antibody capable of agonizing a co-stimulatory immune checkpoint protein, wherein said agonistic antibody is anti-CD 137/4-1 BB.

In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD80/B7-1, CD86/B7-2, B7-H3/CD276, B7-H4/B7S 4/7 x, VISTA/B4-H4/GI 4, HVEM/TNFRSF 4, BTLA, CD160, LAG 4/CD 223/lymphocyte activating gene 3, CEACAM 4/CD 3666, indoleamine, 2, 3-bis-oxygenase/IDO, galectin-/LGALS 4, TIM-3/HAVCCR 4, 2B4/CD244, SIRP, alpha/CD 172 4, CD SL3672, CD 4/AMF 4, TIGIT/VSTM 4, A2 4, KIR, NOX 4, CD 4/GLEC 328, SIEC 4/SIEC 329. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD80/B7-1, CD86/B7-2, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, HVEM/TNFRSF14, BTLA, CD160, LAG3/CD 223/lymphocyte activating gene 3, CEACAM1/CD66a, indoleamine, galectin-/LGALS 9, TIM-3/HAVCR2, 2B4/CD244, SIRP, alpha/CD 172a, CD47, CD48/SLAMF2 and TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD80/B7-1, CD86/B7-2, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, LAG3/CD 223/lymphocyte activation gene 3, galectin-/LGALS 9, TIM-3/HAVCR2 and TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD80/B7-1, CD86/B7-2, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, LAG3/CD 223/lymphocyte activation gene 3, galectin-/LGALS 9, TIM-3/HAVCR2 and TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, HVEM/TNFRSF14, BTLA, CD160, LAG3/CD 223/lymphocyte activating gene 3, CEACAM1/CD66a, indoleamine, 2, 3-dioxygenase/IDO, galectin-/LGALS 9, TIM-3/HAVCR2, 2B4/CD244, SIRP, alpha/CD 172a, CD47, CD48/SLAMF2, TIG/VSTM 3, A2AR, KIR, NOX2, SIGLEC7/CD328, SIGLEC9/CD 329. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, HVEM/TNFRSF14, BTLA, CD160, LAG3/CD 223/lymphocyte activating gene 3, CEACAM1/CD66a, indoleamine, galectin-/LGALS 9, TIM-3/HAVCCR 2, 2B4/CD244, SIRP, alpha/CD 172a, CD47, CD48/SLAMF2 and TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, LAG3/CD 223/lymphocyte activation gene 3, galectin-/LGALS 9, TIM-3/HAVCR2 and TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, B7-H3/CD276, B7-H4/B7S1/7x, VISTA/B7-H5/GI24, LAG3/CD 223/lymphocyte activation gene 3, galectin-/LGALS 9, TIM-3/HAVCR2 and TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4 and GITRL. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, LAG3/CD 223/lymphocyte activation gene 3, TIM-3/HAVCR2 and TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the co-inhibitory immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1 and PD-L2.

In a further preferred embodiment of the composition or kit of parts of the 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 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.

In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the antagonistic antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-LAG 3/CD 223/lymphocyte activation gene 3, anti-TIM-3/HAVCR 2 and anti-TIGIT/VSTM 3. In a further preferred embodiment, the at least one antibody capable of modulating an immune checkpoint protein is (ii) at least one antibody capable of antagonizing a co-inhibitory immune checkpoint protein, wherein the antagonistic antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, and anti-PD-L2.

In a preferred embodiment, the composition or kit of parts of the invention comprises more than one antibody capable of modulating an immune checkpoint protein. In a preferred embodiment, the composition or kit of parts of the invention comprises 1, 2 or 3 antibodies capable of modulating an immune checkpoint protein. In a preferred embodiment, the composition or kit of parts of the invention comprises 2 or 3 antibodies capable of modulating an immune checkpoint protein. In a preferred embodiment, the composition or kit of parts of the invention comprises 2 antibodies capable of modulating an immune checkpoint protein. In a preferred embodiment, the composition or kit of parts of the invention comprises 3 antibodies capable of modulating an immune checkpoint protein.

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

In a preferred embodiment, said at least one antibody comprised in said composition or kit of parts of the invention capable of modulating an immune checkpoint protein is a mixture of:

(i) an antibody capable of modulating an immune checkpoint protein CD27 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1 and PD-L2;

(ii) an antibody capable of modulating an immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iv) an antibody capable of modulating an immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB and CTLA-4;

(v) an antibody capable of modulating immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or

(vi) An antibody capable of modulating the immune checkpoint protein ICOS and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4.

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

In a preferred embodiment, the at least one antibody comprised in the composition or kit of parts of the invention capable of modulating an immune checkpoint protein is a mixture of: (i) an anti-CD 27 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, the at least one antibody comprised in the composition or kit of parts of the invention capable of modulating an immune checkpoint protein is a mixture of: (ii) an anti-CD 40 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, the at least one antibody comprised in the composition or kit of parts of the invention capable of modulating an immune checkpoint protein is a mixture of: (iii) 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, the at least one antibody comprised in the composition or kit of parts of the invention capable of modulating an immune checkpoint protein is a mixture of: (iv) anti-OX 40 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-4-1 BB and anti-CTLA-4. In a preferred embodiment, the at least one antibody comprised in the composition or kit of parts of the invention capable of modulating an immune checkpoint protein is a mixture of: (v) an anti-4-1 BB 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-OX 40, anti-LAG-3, and anti-CTLA-4. In a preferred embodiment, the at least one antibody comprised in the composition or kit of parts of the invention capable of modulating an immune checkpoint protein is a mixture of: (vi) at least one antibody against ICOS 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, the multimeric complex of the invention comprises double-stranded rna (dsrna) and a polymeric conjugate, wherein the polymeric conjugate comprises Polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, one or more linkers, and one or more targeting moieties; wherein the PEI is covalently bound to one or more PEG moieties and each PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties through one of the one or more linkers, and wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen.

In a preferred embodiment, the multimeric complex of the invention comprises double-stranded rna (dsrna) and a polymeric conjugate, wherein the polymeric conjugate consists of Polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, one or more linkers, and one or more targeting moieties; wherein the PEI is covalently bound to one or more PEG moieties and each PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties through one of the one or more linkers, and wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen.

In a preferred embodiment, the multimeric complex of the invention consists of double-stranded rna (dsrna) and a polymeric conjugate, wherein the polymeric conjugate consists of Polyethyleneimine (PEI), one or more polyethylene glycol (PEG) moieties, one or more linkers, and one or more targeting moieties; wherein the PEI is covalently bound to one or more PEG moieties and each PEG moiety of the one or more PEG moieties is linked to one of the one or more targeting moieties through one of the one or more linkers, and wherein each targeting moiety of the one or more targeting moieties is capable of binding to a cancer antigen.

The multimeric complex of the present invention includes a multimeric complex comprising double-stranded rna (dsrna). The term "dsRNA" typically and preferably refers to a double-stranded polymer of ribonucleotides of any length, wherein one or more of the ribonucleotides may be a chemical analogue or modified derivative of the corresponding naturally occurring ribonucleotide. The term "dsRNA" typically and preferably also comprises mismatched dsRNA.

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

The poly ic for multimeric complexes used according to the invention may consist of dsRNA, wherein each strand consists of at least 22, preferably at least 45 ribonucleotides. In a certain embodiment, each strand consists of 20 to 8000 ribonucleotides. In a certain embodiment, each strand consists of 20 to 4000 ribonucleotides. In a more preferred embodiment, each strand consists of 20 to 300 ribonucleotides.

As used herein and particularly when referring to polymers such as poly ic, PEI and PEG, the term "molecular weight" refers to an average molecular weight, preferably a weight average molecular weight.

PolyIC is bound to the polymeric conjugate by non-covalent or covalent bonds, with non-covalent binding being preferred. In a preferred embodiment, the poly ic is non-covalently bound to the PEI, preferably by ionic bonding.

Polyplexes according to the present invention include polymeric conjugates, wherein the polymeric conjugates include Polyethylenimine (PEI), which is a polycation capable of condensing and associating with nucleic acid molecules in a non-covalent manner due to the polycationic nature.

In a preferred embodiment, the Polyethyleneimine (PEI) is Linear Polyethyleneimine (LPEI). In a preferred embodiment, the LPEI comprises hydroxyl groups located at one or either end of the LPEI. Preferably, the hydroxyl group replaces the terminal-NH of the LPEI₂A group.

In a preferred embodiment of the invention, the molecular weight of the PEI or preferably the LPEI is about 10-30 kDa. In a preferred embodiment of the invention, the molecular weight of the PEI or preferably the LPEI is about 15-25kDa (PEI)_15-25k//LPEI_15-25k). In a further more preferred embodiment, the molecular weight of the PEI or preferably the LPEI is about 22kDa (LPEI)_22k). In yet another preferred embodiment, the molecular weight of the PEI or preferably the LPEI is about 20 kDa. The expression "molecular weight of LPEI about 22 kDa" is abbreviated as "LPEI_22k"and is used synonymously herein with. The expression "molecular weight of PEI is about 22 kDa" is abbreviated "PEI_22k"and is used synonymously herein with.

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

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

Multimeric complexes for use according to the invention comprise one or more polyethylene glycol (PEG) moieties. The PEG moiety according to the invention is also referred to as polyethylene oxide (PEO) or Polyoxyethylene (POE) moiety, depending on its molecular weight. As used herein, the term "polyethylene glycol moiety" (PEG moiety) typically and preferably refers to a PEG moiety comprising two functional groups located on either end of the polyethylene glycol (PEG). The functional group is capable of reacting with the PEI or preferably the LPEI or the targeting moiety.

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

In one embodiment of the invention, each PEG moiety of the at least one PEG moiety has a molecular weight of 1kD or greater. In another embodiment, each PEG moiety of the at least one PEG moiety has a molecular weight of about 0.3-8kDa, preferably about 0.5-5Da, more preferably 1-3kDa (PEG)_1-3k) Most preferably 2kDa (PEG)_2k). As indicated, the molecular weights correspond to the average molecular weight, so that the preferred and used PEG_2kIs referred to as- (CH)₂CH₂O)_nA mixture of polyethylene glycols having an average value n of units between 30 and 60 (some of which are even smaller and some of which are larger) and a molecular weight in the range of about 1300 to 2600 grams per mole.

In a preferred embodiment, the molecular weight of the PEI is between about 10 and 30kDa, and the molecular weight of the at least one PEG moiety is between about 0.3 and 8 kDa. In a more preferred embodiment, the molecular weight of PEI is about 22kDa (PEI)_22k) And the at least one PEG moiety has a molecular weight of about 2kDa (PEG)_2k). In a more preferred embodiment, the PEI is a molecular weight of about 20kDa (LPEI)_20k) And the at least one PEG moiety has a molecular weight of about 2kDa (PEG)_2k)。

In a preferred embodiment, the PEI is covalently attached to from one to five PEG moieties, preferably PEI is covalently attached to from one to three PEG moieties. In a preferred embodiment, the LPEI is covalently attached to one to five PEG moieties, wherein preferably the LPEI is covalently attached to one to three PEG moieties. In a preferred embodiment, PEI_15-25kPreferably PEI_22kWith one to five PEGs_1-3kPreferably PEG_2kThe moieties being covalently linked, wherein preferably PEI_15-25kFurther preferably PEI_22kWith one to three PEGs_1-3kPreferably PEG_2kThe moieties are covalently linked. In a preferred embodiment, the LPEI_15-25kPreferably LPEI_22kWith one to five PEGs_1-3kPreferably PEG_2kThe moieties being covalently linked, wherein preferably PEI_15-25kFurther preferably PEI_22kWith one to three PEGs_1-3kPreferably PEG_2kThe moieties are covalently linked. In a preferred embodiment, PEI_15-25kPreferably PEI_22kWith a PEG_1-3kPreferably PEG_2kPartial or two or three PEGs_1-3kPreferably PEG_2kThe moieties are covalently linked. In another preferred embodiment, PEI_15-25kPreferably PEI_22kWith one or more PEGs_1-3kPreferably PEG_2kAnd (3) covalent linkage. In a further preferred embodiment, PEI_15-25kPreferably PEI_22kWith two PEGs_1-3kPreferably PEG_2kThe moieties are covalently linked. In a further preferred embodiment, PEI_15-25kPreferably PEI_22kWith three PEGs_1-3kPreferably PEG_2kThe moieties are covalently linked. In a preferred embodiment, the LPEI_15-25kPreferably LPEI_22kWith a PEG_1-3kPreferably PEG_2kPartial or two or three PEGs_1-3kPreferably PEG_2kThe moieties are covalently linked. In another preferred embodiment, the LPEI_15-25kPreferably LPEI_22kWith a PEG_1-3kPreferably PEG_2kAnd (3) covalent linkage. In a further preferred embodiment, the LPEI_15-25kPreferably LPEI_22kWith two PEGs_1-3kPreferably PEG_2kThe moieties are covalently linked. In a further preferred embodiment, the LPEI_15-25kPreferably LPEI_22kWith three PEGs_1-3kPreferably PEG_2kThe moieties are covalently linked. In another preferred embodiment, said PEI of said polyplex is associated with one or both ofOne or three PEG moieties are covalently attached. Preferably, the PEI of the polyplex is covalently bound to one or three PEG moieties. In another more preferred embodiment, the PEI of the polyplex is an LPEI covalently bound to one, two, or three PEG moieties. More preferably, the PEI of the polyplex is an LPEI covalently bound to one or three PEG moieties.

As used herein, the term "PEI covalently attached to a PEG moiety.]"(used interchangeably herein with" PEI-PEG1: 1 ") or" an LPEI covalently attached to a PEG moiety.]"used interchangeably herein with" LPEI-PEG1: 1 ") refers to the molar ratio of PEI to PEG or LPEI to PEG, where PEI-PEG1: 1 or LPEI-PEG1: 1 typically and preferably means about one mole of PEG is included per mole of PEI or LPEI in the polymeric conjugate. As used herein, the term "PEI covalently attached to three PEG moieties.]"(used interchangeably herein with" PEI-PEG1:3 ") or the term" LPEI [. to which three PEG moieties are covalently attached.]"(used interchangeably herein with" LPEI-PEG1:3 ") typically and preferably means that the polymeric conjugate comprises about three moles of PEG per mole of PEI or LPEI. Said value is preferably determined by¹H-NMR analysis. PEG (-CH)₂-CH₂The relative integer values of the hydrogen atoms on-O-) and PEI or LPEI (-CH)₂-CH₂The integer value of the hydrogen atom on-NH-) is preferably used for the reaction by¹H-NMR confirmed values. The term "about" preferably means herein a deviation of about 0% -10%, more preferably about 0% -5%, still more preferably about 0% -2%.

In a preferred embodiment, the dsRNA is poly ic and the PEI is covalently linked to one to three PEG moieties. In a preferred embodiment, the dsRNA is poly ic and the PEI is covalently linked to one, two or three PEG moieties. In a more preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one to three PEG moieties. In a more preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one, two or three PEG moieties.

In a preferred embodimentIn embodiments, the dsRNA is poly ic and the PEI is covalently linked to one, two, or three PEG moieties. In a more preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one, two or three PEG moieties. In a more preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one, two or three PEG moieties_22k. In a more preferred embodiment, the dsRNA is poly ic and the PEI is with one, two or three PEGs_0.3-8kPreferably PEG2_kA portion of covalently attached LPEI. In a more preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one, two or three PEG moieties_22k. In a more preferred embodiment, the dsRNA is poly ic and the PEI is with one, two or three PEGs_0.3-8kPreferably PEG2_kPartially covalently linked LPEI_22k。

In a preferred embodiment, the dsRNA is poly ic and the PEI is covalently linked to a PEG moiety (PEI-PEG 1: 1). In a more preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to a PEG moiety (LPEI-PEG1: 1). In a more preferred embodiment, the dsRNA is polyIC and the PEI is an LPEI covalently linked to a PEG moiety_22k. In a more preferred embodiment, the dsRNA is polyIC and the PEI is conjugated to a PEG_0.3-8kPreferably PEG_2kPartially covalently attached LPEI (PEI-PEG 1: 1). In a more preferred embodiment, the dsRNA is polyIC and the PEI is an LPEI covalently linked to a PEG moiety_22k. In a more preferred embodiment, the dsRNA is polyIC and the PEI is conjugated to a PEG_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22k(PEI-PEG 1:1)。

In a preferred embodiment, the dsRNA is poly ic and the PEI is covalently linked to three PEG moieties. In a more preferred embodiment, the dsRNA is polyIC and the PEI is conjugated to three PEG moietiesCovalently attached LPEI. In a more preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to three PEG moieties_22k. In a more preferred embodiment, the dsRNA is polyIC and the PEI is conjugated to three PEGs_0.3-8kPreferably PEG2_kA portion of covalently attached LPEI. In a more preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to three PEG moieties_22k. In a more preferred embodiment, the dsRNA is polyIC and the PEI is conjugated to three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22k。

In a preferred embodiment, the dsRNA of the polyplex is poly ic and the PEI for the polymeric conjugate of the polyplex for use according to the invention is LPEI_22k. In a preferred embodiment, the dsRNA of the polyplex is poly ic and the PEI for the polymeric conjugate of the polyplex for use according to the invention is a LPEI covalently linked to one, two or three PEG moieties_22k。

In a preferred embodiment, the dsRNA is poly ic and the one or more PEG moieties of the polymeric conjugate are PEG_0.3-8kPreferably PEG_2k. In a preferred embodiment, the dsRNA is poly ic, and the LPEI is conjugated to one, two or three PEGs_0.3-8kPreferably PEG_2kThe moieties are covalently linked. In a preferred embodiment, the dsRNA is polyIC and the LPEI is conjugated to a PEG_0.3-8kPreferably PEG_2kMoieties (LPEI-PEG1: 1) are covalently attached. In a preferred embodiment, the dsRNA is poly ic, and the LPEI and PEG are_0.3-8kPreferably PEG_2kMoieties (LPEI-PEG1:3) are covalently attached.

In a preferred embodiment, the dsRNA of the polyplex is poly ic and the LPEI of the polymeric conjugate is LPEI_22kAnd said one or more PEG moieties are PEG_0.3-8kPreferably PEG_2kAnd (4) partial. In a preferred embodimentIn one embodiment, the dsRNA of the polyplex is polyIC and the LPEI is LPEI_22kAnd with a PEG_0.3-8kPreferably PEG_2kPartial (LPEI-PEG1: 1) or three PEGs_0.3-8kPreferably PEG_2kMoieties (LPEI-PEG1:3) are covalently attached.

As used herein, the term "cancer antigen" (used herein synonymously with tumor antigen or tumor marker) refers to an antigenic substance produced in or presented on tumor cells that triggers 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 further preferred embodiments, 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 HER 2. In another preferred embodiment, the cancer antigen is EGFR or HER 2. In another preferred embodiment, the cancer antigen is EGFR or PSMA.

In another preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one to three PEG moieties and the cancer antigen is EGFR or PSMA. In another preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one to three PEG moieties and the cancer antigen is EGFR. In another preferred embodiment, the dsRNA is poly ic, and the PEI is an LPEI covalently linked to one to three PEG moieties, and the cancer antigen is PSMA. In another preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one to three PEG moieties and the cancer antigen is HER 2.

In another preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one to three PEG moieties 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 linked to one to three PEG moieties and the targeting moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is poly ic and the PEI is an LPEI covalently linked to one to three PEG moieties and the targeting moiety is HER2 affibody or HER2 antibody.

Treatment of PEI-PEG-EGF (epidermal growth factor)/poly ic polyplex on high EGFR (epidermal growth factor receptor) expressing cells induced proinflammatory cytokine (IP-10, GRO- α and CCL5) secretion that was not observed in low EGFR expressing cells (see figure 11 herein, example 8). Furthermore, increased secretion of cytokines (IFN- γ and TNF α) was observed in PBMCs incubated with supernatants from cells treated with PEG-EGF/poly ic polyplex with high EGFR expression, which was not observed in media from low EGFR expressing cells (see fig. 12 herein, example 9). The combination of PEI-PEG-EGF/poly ic with nivolumab or anti-41 BB antibody resulted in a further increase in cytokine IFN-y release in PBMCs compared to treatment with PEI-PEG-EGF/poly ic, nivolumab or anti-41 BB antibody alone (see figures 3, 4 and 10, examples 2 and 7 herein). Treatment of RENCA EGFR tumor-bearing mice with a combination of PEI-PEG-EGF/polyIC and anti-PD-1 showed increased survival compared to anti-PD-1 or PEI-PEG-EGF/polyIC, demonstrating the synergy achieved with the compositions and combinations of the present invention, respectively, as shown in FIG. 5 and example 3.

Similarly, treatment with polyIC/PEI-PEG conjugated to HER2 affibody (pIC/PPHA) induced expression of antiproliferative cytokines (e.g., IFN-. gamma., IFN-. alpha.) and immunostimulatory cytokines (e.g., IP-10, GRO-. alpha., and RANTES), triggering immune cell recruitment and activation and resulting in extensive Cancer cell killing in vitro (Zigler et al, Cancer immunology Res; 4(8)2016 8). As described in example 5 and further shown in fig. 6-9, treatment of RENCA HER2 tumors with HER 2-targeted poly ic polyplex (poly ic/PEI-PEG-HER2 affibody) plus anti-PD-1 combinations according to the invention resulted in complete regression of tumor growth and complete protection against tumor re-challenge.

Of particular note here is the carrier polyIC/PEI-PEG-DUPA (pIC/PPD) comprising PEI-PEG tethered to PSMA ligand DUPA (2- [3- (1, 3-dicarboxypropyl) ureido ] glutarate) that selectively delivers polyIC to PSMA overexpressing prostate cancer cells, thereby inducing apoptosis, cytokine secretion and recruitment of human Peripheral Blood Mononuclear Cells (PBMCs). Specifically, pIC/PPD leads to the production of IFN- β, IP-10 and RANTES, to chemotaxis and to activation of PBMCs apparent from strong expression of IL-2, and to high levels of secretion of TNF- α and IFN- γ (Langut et al, proceedings of the national academy of sciences (PNAS), 12/26/2017, vol 114, 52).

Thus, it is clearly demonstrated and supported above that the compositions and kits of the invention comprising a multimeric complex and at least one antibody capable of modulating an immune checkpoint protein are capable of leading to an increased activation of the immune system and a more effective antitumor activity. This is especially the case for preferred compositions of the invention comprising a multimeric complex, 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 wherein the antibody is capable of modulating an immune checkpoint protein, preferably an anti-4-1 BB, anti-PD-1, anti-PD-L1 or anti-PD-L2 antibody.

In another preferred embodiment, the dsRNA is polyIC and the PEI is LPEI_15-25kPreferably an LPEI covalently linked to one to three PEG moieties_22kAnd the targeting moiety is EGF, preferably human EGF, HER2 affibody, HER2 antibody or DUPA. In another preferred embodiment, the dsRNA is polyIC and the PEI is LPEI_15-25kPreferably an LPEI covalently linked to one to three PEG moieties_22kAnd the targeting moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is polyIC and the PEI is LPEI_15-25kPreferably covalently linked to one to three PEG moietiesLPEI of_22kAnd the targeting moiety is HER2 affibody or a HER2 antibody.

In another preferred embodiment, the dsRNA is poly ic and the PEI is with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kThe moiety is a covalently attached 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 with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kThe moiety is a covalently attached LPEI and the targeting moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one to three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kA moiety covalently attached to the LPEI, and the targeting moiety is a HER2 affibody or a HER2 antibody.

In another preferred embodiment, the dsRNA is poly ic and the PEI is with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kPartially covalently linked LPEI_22kAnd 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 with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kPartially covalently linked LPEI_15-25kPreferably LPEI_22kAnd the targeting moiety is EGF, preferably human EGF. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one to three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kPartially covalently linked LPEI_15-25kPreferably LPEI_22kAnd the targeting moiety is HER2 affibody or a HER2 antibody.

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

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

In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, CTLA-4, GITR, CD40, OX40 and ICOS. In another preferred embodiment, the dsRNA is poly ic, 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 poly ic, 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 OX 40. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, CD40 and OX 40. In another preferred embodiment, the dsRNA is poly ic, 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 OX 40. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2 and 4-1 BB. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1 and 4-1 BB.

In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40, OX40 and ICOS. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, GITR, CD40 and OX 40. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB, GITR and OX 40. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB and OX 40.

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

In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating 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-4-1 BB ligand (4-1BBL), anti-TIGIT, anti-LAG 3, anti-TIM 3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR 4, anti-GITR ligand, anti-GITR, anti-OX 40, anti-OX-40L, anti-ICOS ligand, anti-CD 40, and anti-CD 40 ligand. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating 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-4-1 BB ligand, anti-TIGIT, anti-LAG 3, anti-TIM-3, anti-GITR ligand, anti-CD 40, anti-CD 40 ligand, anti-OX 40, anti-OX 40 ligand, anti-ICOS ligand, and anti-ICOS. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1 BB ligand, anti-TIGIT, anti-LAG 3, anti-TIM-3, anti-GITR ligand, anti-CD 40, anti-CD 40 ligand, anti-OX 40, anti-OX 40 ligand, anti-ICOS ligand, and anti-ICOS. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating 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-4-1 BB, anti-TIGIT, anti-LAG 3, anti-TIM 3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR 4, anti-GITR, anti-OX 40, anti-ICOS and anti-CD 40. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating 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-4-1 BB, anti-TIGIIT, anti-LAG 3, anti-TIM-3, anti-GITR, anti-CD 40, anti-OX 40, and anti-ICOS. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1 BB, and anti-4-1 BB ligands. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, and anti-4-1 BB. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-4-1 BB. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-PD-1 or anti-4-1 BB. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-PD-1, anti-PD-L1 or anti-PD-L2. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-PD-1. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-PD-L1. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-PD-L2. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-4-1 BB. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-CTLA-4. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-LAG-3. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-TIGIT. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-TIM 3. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-GITR or an anti-GITR ligand. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-ICOS or an ICOS ligand. In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-OX 40 or anti-OX 40 ligand.

In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe 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 ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS ligand CD40 and CD40 ligand. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe 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 ligand, TIGIT, LAG3, TIM-3, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand and ICOS.

In another preferred embodiment, the dsRNA is polyIC, the dsRNA is conjugated to a second strand of a third strand of a second strand of a third strand of a second strand of a third strand of a fourth strand of a third strand of a fourth strands of a fourth strands of a fourth of a strands of a fourth strands of a fourth strands of aThe PEI is with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe 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 ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS ligand CD40, and CD40 ligand. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe 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 ligand, TIGIT, LAG3, TIM-3, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand, and ICOS. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe 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 CD 40. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe 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.

In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR, CD40, OX40 and ICOS. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe 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 OX 40. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe 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 OX 40. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe 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 OX 40.

In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB and 4-1BB ligands. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4 and 4-1 BB. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartial covalent attachment of LPEI_22kThe targeting moiety is EGF, preferably human EGF, and the immunizing agentThe disease checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2 and 4-1 BB. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1 or 4-1 BB. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1, PD-L1 or PD-L2. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kThe targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L1. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L2. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is 4-1 BB. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immunizingThe checkpoint protein is CTLA-4. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kPartially covalently linked LPEI_15-25kPreferably LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is LAG-3. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kPartial covalent attachment of LPEI_15-25kPreferably LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIGIT. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kPartially covalently linked LPEI_15-25kPreferably LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIM 3. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kPartially covalently linked LPEI_15-25kPreferably LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is GITR or a GITR ligand. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kPartially covalently linked LPEI_15-25kPreferably LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is ICOS or an ICOS ligand. In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_1-3kFurther preferably PEG_2kPartially covalently linked LPEI_15-25kPreferably LPEI_22kAnd the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is OX40 or OX40 ligand.

In another preferred embodiment, the dsRNA is poly ic, the PEI is an LPEI covalently linked to one, two or three PEG moieties, the targeting moiety is EGF, preferably human EGF, and the at least one antibody capable of modulating an immune checkpoint protein is a mixture of: (i) an antibody capable of modulating an immune checkpoint protein CD27 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1 and PD-L2;

(ii) an antibody capable of modulating an immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iv) an antibody capable of modulating an immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB and CTLA-4;

(v) an antibody capable of modulating immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or

(vi) An antibody capable of modulating the immune checkpoint protein ICOS and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1 and PD-L2.

In another preferred embodiment, the dsRNA is polyIC and the PEI is conjugated with one, two or three PEGs_0.3-8kPreferably PEG_2kPartially covalently linked LPEI_22kSaid targeting moiety is EGF, preferably human EGF, and said at least one antibody capable of modulating an immune checkpoint protein is a mixture of: (i) can regulate immunity examinationAn antibody to the dot protein CD27 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1 and PD-L2;

(ii) an antibody capable of modulating an immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iv) an antibody capable of modulating an immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB and CTLA-4;

(v) an antibody capable of modulating immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or

(vi) An antibody capable of modulating the immune checkpoint protein ICOS and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1 and PD-L2.

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

In a multimeric complex of the invention, the one or more PEG moieties are linked to one of the one or more targeting moieties. In a preferred embodiment, the one or more PEG moieties are linked to one of the one or more targeting moieties directly or via one of the one or more linkers.

In a further preferred embodiment, the linker is selected from the group consisting of-CO-R2-RX-R3 or a peptide moiety consisting of 3 to 7 amino acid residues, wherein R2 is selected from (C)₁-C₈) Alkylene group,(C₂-C₈) Alkenylene, (C)₂-C₈) Alkynylene group, (C)₆-C₁₀) Arylene-diyl or heteroarylene-diyl; RX is absent or-S-; r3 is absent or has the formula

Wherein R4 is selected from (C)₁-C₈) Alkylene, (C)₂-C₈) Alkenylene, (C)₂-C₈) Alkynylene (C)₁-C₈) Alkylene- (C)₃-C₈) Cycloalkylene, (C)₂-C₈) Alkenylene- (C)₃-C₈) Cycloalkylene, (C)₂-C₈) Alkynylene- (C)₃-C₈) Cycloalkylene, (C)₆-C₁₀) Arylene-diyl, heteroarylene-diyl, (C)₁-C₈) Alkylene- (C)₆-C₁₀) Arylene-diyl or (C)₁-C₈) Alkylene-heteroarylene diyl; wherein said (C)₁-C₈) Alkylene, (C)₂-C₈) Alkenylene or (C)₂-C₈) Each of the alkynylene groups is optionally independently selected from the group consisting of halo, -COR5, -COOR5, -OCOOR5, -OCON (R5)₂、-CN、-NO₂、-SR5、-OR5、-N(R5)₂、-CON(R5)₂、-SO₂R5、-SO₃H、-S(＝O)R5、(C₆-C₁₀) Aryl group, (C)₁-C₄) Alkylene- (C)₆-C₁₀) Aryl, heteroaryl or (C)₁-C₄) Alkylene-heteroaryl, and further substituted with one or more identical or different heteroatoms selected from S, O or N and/or each independently selected from-NH-CO-, -CO-NH-, -N (R5) -, -N (C)₆-C₁₀) Aryl-, (C)₆-C₁₀) At least one radical interruption of the arylene-diyl or heteroarylene-diyl group; and R5 is H or (C)₁-C₈) An alkyl group.

In a further preferred embodiment, the polymeric conjugate has the formulae (i) - (viii):

wherein R6 is

Wherein R7 is

Wherein R6 is

Wherein R7 is

Wherein said T represents said targeting moiety and n corresponds to a molecular weight of 0.3-8kD, preferably 0.5-5kD, more preferably 1-3kD, most preferably 2 kD; and m corresponds to a molecular weight of 10-30kD, preferably 15-25kD, further preferably 22 kD.

In a further preferred embodiment, the polymeric conjugate has the formula (ii) or (vii), wherein R6 is SEQ ID No.4(- (NH- (CH) s)₂)₇-CO)-Phe-Phe-(NH-CH₂-CH(NH₂) -CO) -Asp-Cys-), and wherein T represents a targeting moiety HOOC (CH)₂)₂-CH(COOH)-NH-CO-NH-CH(COOH)-(CH₂)₂-CO- (DUPA part). In addition toIn a preferred embodiment, the polymeric conjugate has the formula (iv) or (viii), wherein R7 is SEQ ID NO.3(- (NH- (CH) s)₂)₇-CO) -Phe-Gly-Trp-Gly-Cys-), and wherein T represents the targeting moiety HOOC (CH)₂)₂-CH(COOH)-NH-CO-NH-CH(COOH)-(CH₂)₂-CO- (DUPA part).

In a further preferred embodiment of the present invention,

-the targeting moiety is HER2 affibody or HER2 antibody and the polymeric conjugate has formula (i) or (v) and the HER2 affibody or HER2 antibody is linked through its thiol group;

-the targeting moiety is hEGF (human epidermal growth factor; human EGF), and the polymeric conjugate has formula (i), (ii) or (vi), wherein the hEGF is linked through its amino group; or

-the targeting moiety is HOOC (CH)₂)₂-CH(COOH)-NH-CO-NH-CH(COOH)-(CH₂)₂-CO- (DUPA moiety), and the polymeric conjugate has formula (iii), (iv), (vii), or (viii).

In a further preferred embodiment, the targeting moiety is HER2 affibody or a HER2 antibody, and the polymeric conjugate has formula (i) or (v), and the HER2 affibody or the HER2 antibody is linked through its thiol group.

In a further preferred embodiment, the targeting moiety is hEGF and the polymeric conjugate is of formula (i), (ii) or (vi). In a further preferred embodiment, the targeting moiety is hEGF, and the polymeric conjugate has formula (i), (ii), or (vi), wherein the hEGF is linked through its amino group. In a further preferred embodiment, the targeting moiety is hEGF, and the polymeric conjugate is of formula (i), wherein the hEGF is linked through its amino group.

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

In a further preferred embodiment, the targeting moiety is HER2 affibody or a HER2 antibody, and the polymeric conjugate has formula (i) or (v), and the HER2 affibody or the HER2 antibody is linked through its thiol group. In a further preferred embodiment, the targeting moiety is hEGF, and the polymeric conjugate has formula (i), (ii), or (vi), wherein the hEGF is linked through its amino group. In a further preferred embodiment, the targeting moiety is hEGF, and the polymeric conjugate is of formula (i), wherein the hEGF is linked through its amino group.

In another preferred embodiment, the dsRNA is poly ic, 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 ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS ligand CD40, and CD40 ligand, and wherein the polymeric conjugate has formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is poly ic, 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 ligand, TIGIT, LAG3, TIM-3, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand, and ICOS, and wherein the polymeric conjugate has formula (i), (ii), or (vi).

In another preferred embodiment, the dsRNA is poly ic, 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 ligand (4-1BBL), TIGIT, LAG3, TIM3, B7-H3, B7-H4, VISTA, CCR4, GITR ligand, GITR, OX40, OX-40L, ICOS ligand CD40, and CD40 ligand, and wherein the polymeric conjugate has formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is poly ic, 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 ligand, TIGIT, LAG3, TIM-3, GITR ligand, CD40, CD40 ligand, OX40, OX40 ligand, ICOS ligand, and ICOS, and wherein the polymeric conjugate has formula (i), (ii), or (vi).

In another preferred embodiment, the dsRNA is poly ic, 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, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, 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, and wherein the polymeric conjugate has formula (i), (ii), or (vi).

In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR, CD40, OX40, and ICOS, and wherein the polymeric conjugate has formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is poly ic, 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, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, GITR and OX40, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB and OX40, and wherein the polymeric conjugate has formula (i), (ii) or (vi).

In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB and 4-1BB ligands. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4 and 4-1 BB. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is selected from the group consisting of: PD-1, PD-L1, PD-L2, and 4-1BB, and wherein the polymeric conjugate has formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1 or 4-1BB, and wherein the polymeric conjugate is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1, PD-L1 or PD-L2, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-1, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L1, and wherein the polymeric conjugate is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is PD-L2, and wherein the polymeric conjugate is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is 4-1 BB. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is CTLA-4, and wherein the polymeric conjugate is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is LAG-3, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIGIT. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is TIM3, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is a polyIC, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is GITR or a GITR ligand, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic and the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is ICOS or ICOS ligand, and wherein the polymeric conjugate is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is a poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein is OX40 or OX40 ligand, and wherein the polymeric conjugate is of formula (i), (ii) or (vi).

In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating 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-4-1 BB ligand (4-1BBL), anti-TIGIIT, anti-LAG 3, anti-TIM 3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR 4, anti-GITR ligand, anti-GITR, anti-OX 40, anti-OX-40L, anti-ICOS ligand, anti-CD 40, and anti-CD 40 ligand, and wherein the polymeric conjugate has formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating 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-4-1 BB ligand, anti-TIGIT, anti-LAG 3, anti-TIM-3, anti-GITR ligand, anti-CD 40, anti-CD 40 ligand, anti-OX 40, anti-OX 40 ligand, anti-ICOS ligand, and anti-ICOS, and wherein the polymeric conjugate has formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1 BB ligand (4-1BBL), anti-TIGIT, anti-LAG 3, anti-TIM 3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR 4, anti-GITR ligand, anti-GITR, anti-OX 40, anti-OX-40L, anti-ICOS ligand, anti-CD 40, and anti-CD 40 ligand, and wherein the polymeric conjugate has formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1 BB ligand, anti-TIGIT, anti-LAG 3, anti-TIM-3, anti-GITR ligand, anti-CD 40, anti-CD 40 ligand, anti-OX 40, anti-OX 40 ligand, anti-ICOS ligand, and anti-ICOS, and wherein the polymeric conjugate has formula (i), (ii), or (vi).

In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating 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-4-1 BB, anti-TIGIT, anti-LAG 3, anti-TIM 3, anti-B7-H3, anti-B7-H4, anti-VISTA, anti-CCR 4, anti-GITR, anti-OX 40, anti-ICOS, and anti-CD 40, and wherein the polymeric conjugate has formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating 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-4-1 BB, anti-TIGIIT, anti-LAG 3, anti-TIM-3, anti-GITR, anti-CD 40, anti-OX 40, and anti-ICOS, and wherein the polymeric conjugate has formula (i), (ii), or (vi).

In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-4-1 BB, and anti-4-1 BB ligands. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, and anti-4-1 BB. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-4-1 BB, and wherein the polymeric conjugate has formula (i), (ii), or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-PD-1 or anti-4-1 BB, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-PD-1, anti-PD-L1 or anti-PD-L2, and wherein the polymeric conjugate is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is PD-1, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-PD-L1, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-PD-L2, and wherein the polymeric conjugate has formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-4-1 BB. In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-CTLA-4, and wherein the polymeric conjugate is of formula (i), (ii) or (vi). In another preferred embodiment, the dsRNA is poly ic, the targeting moiety is EGF, preferably human EGF, and the immune checkpoint protein modulating antibody is anti-LAG-3, and wherein the polymeric conjugate has formula (i), (ii) or (vi).

In another preferred embodiment, the dsRNA is poly ic, the polymeric conjugate has formula (i), (ii) or (vi), the targeting moiety is EGF, preferably human EGF, and the at least one antibody capable of modulating an immune checkpoint protein is a mixture of: (i) an antibody capable of modulating an immune checkpoint protein CD27 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1 and PD-L2;

(ii) an antibody capable of modulating an immune checkpoint protein CD40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iii) an antibody capable of modulating the immune checkpoint protein GITR and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2 and CTLA-4;

(iv) an antibody capable of modulating an immune checkpoint protein OX40 and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, 4-1BB and CTLA-4;

(v) an antibody capable of modulating immune checkpoint protein 4-1BB and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1, PD-L2, OX40, LAG-3, and CTLA-4; or

(vi) An antibody capable of modulating the immune checkpoint protein ICOS and at least one antibody capable of modulating an immune checkpoint protein selected from the group consisting of: PD-1, PD-L1 and PD-L2.

In another preferred embodiment, the dsRNA is poly ic, the polymeric conjugate has formula (i), (ii) or (vi), the targeting moiety is EGF, preferably human EGF, and the at least one antibody capable of modulating an immune checkpoint protein is a mixture of: (i) anti-CD 27 and at least one antibody selected from the group consisting of: anti-PD-1, anti-PD-L1, and anti-PD-L2;

(ii) an antibody capable of modulating an immune checkpoint protein anti-CD 40 and at least one antibody selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-CTLA-4;

(iii) an antibody capable of modulating an immune checkpoint protein anti-GITR and at least one antibody selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, and anti-CTLA-4;

(iv) an antibody capable of modulating an immune checkpoint protein anti-OX 40 and at least one antibody selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-4-1 BB, and anti-CTLA-4;

(v) an antibody capable of modulating an immune checkpoint protein anti-4-1 BB and at least one antibody selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-PD-L2, anti-OX 40, anti-LAG-3, and anti-CTLA-4; or

(vi) An antibody capable of modulating an immune checkpoint protein against ICOS and at least one antibody selected from the group consisting of: anti-PD-1, anti-PD-L1, and anti-PD-L2.

Multimeric complexes of the invention comprise one or more targeting moieties. The targeting moiety may be a natural (native), natural (native) or modified ligand or paralogs thereof, or a non-natural ligand such as an antibody, a single chain variable fragment (scFv) or an antibody mimetic such as an affibody or aptamer of any of the cancer antigens.

In a preferred embodiment, the one or more targeting moieties of a multimeric complex of the invention 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)-(CH₂)₂-CO-). In another preferred embodiment, said one of the multimeric complexes of the inventionThe one or more targeting moieties are EGF, preferably human EGF (hegf), HER2 affibody or HER2 antibody. In another preferred embodiment, the one or more targeting moieties of multimeric complexes of the invention is EGF, preferably human EGF (hegf) or HER2 affibody.

In another preferred embodiment, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf), HER2 antibody or 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 OX 40. In another preferred embodiment, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf), HER2 antibody or 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 OX 40. In another preferred embodiment, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf), HER2 antibody or HER2 affibody, and the immune checkpoint protein is selected from the group consisting of: 4-1BB, PD-1, PD-L1, PD-L2, GITR and OX 40. In another preferred embodiment, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf), HER2 antibody or HER2 affibody, and the immune checkpoint protein is selected from the group consisting of: 4-1BB, PD-1, PD-L1, PD-L2 and OX 40. In another preferred embodiment, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf), HER2 antibody or 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, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf) or 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 a more preferred embodiment, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf), HER2 antibody or 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, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf) or HER2 affibody, and the immune checkpoint protein is selected from the group consisting of: 4-1BB, PD-1, PD-L1, and PD-L2.

In yet a more preferred embodiment, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf), HER2 antibody or HER2 affibody, and the immune checkpoint protein is 4-1BB or PD-1. In yet a more preferred embodiment, the one or more targeting moieties of the multimeric complex of the invention is EGF, preferably human EGF (hegf) or HER2 affibody, and the immune checkpoint protein is 4-1BB or PD-1.

In further preferred embodiments, the one or more targeting moieties is EGF, including mouse and human EGF. In a more preferred embodiment, the one or more targeting moieties is hEGF. In another preferred embodiment, the one or more targeting moieties of the multimeric complex of the invention is hEGF of SEQ ID NO 2.

In another preferred embodiment, the one or more targeting moieties of a multimeric complex of the invention is HER2 affibody or a HER2 antibody, preferably HER2 affibody. In another preferred embodiment, the one or more targeting moieties of a multimeric complex of the invention is HER2 affibody, preferably the HER2 affibody has SEQ ID NO: 1. In another preferred embodiment, the targeting moiety or moieties of multimeric complexes of the invention are EGF, preferably human EGF (hegf) or DUPA moieties (HOOC (CH)₂)₂-CH(COOH)-NH-CO-NH-CH(COOH)-(CH₂)₂-CO-). In another preferred embodiment, the one or more targeting moieties of a multimeric complex of the invention are DUPA moieties (HOOC (CH)₂)₂-CH(COOH)-NH-CO-NH-CH(COOH)-(CH₂)₂-CO-)。

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

In a preferred embodiment, the invention relates to a composition or kit of parts for use in the treatment of cancer in a mammal according to the invention. Preferably, the mammal is a human.

In a preferred embodiment, the present invention relates to a method for the treatment of cancer, wherein said composition or said kit of parts according to the present invention is administered to a patient in need thereof.

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, renal cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, glioma, head and neck cancer, prostate cancer, penile cancer, testicular-embryonic cancer, neuroendocrine tumors, and hepatocellular carcinoma. In a preferred embodiment, said multimeric complexes comprised in the kit of parts according to the invention are administered separately from one or more antibodies capable of modulating an immune checkpoint protein.

In one embodiment, the invention provides the use of a pharmaceutical composition or kit of parts for the treatment of cancer according to the invention, wherein said multimeric complex is administered to a patient in a therapeutically effective amount in combination with a therapeutically effective amount of said at least one antibody capable of modulating an immune checkpoint protein.

In a preferred embodiment, the kit of parts of the invention for use in the treatment of cancer comprises independently administering a multimeric complex and said at least one antibody capable of modulating an immune checkpoint protein. The multimeric complex and the at least one antibody capable of modulating an immune checkpoint protein may be administered simultaneously or sequentially (consecutively), i.e. chronologically staggered. The multimeric complex and the at least one antibody capable of modulating an immune checkpoint protein as comprised in the kit of parts of the invention may be combined prior to administration and may be administered together in the form of a composition or may be administered separately. In a more preferred embodiment, said multimeric complex as comprised in the kit of parts of the invention and said at least one antibody capable of modulating an immune checkpoint protein are administered separately.

In certain embodiments, the kit of parts or composition for use according to the invention is administered by any suitable route. The kit of parts or the composition for use according to the invention may be administered by intravenous, intracerebral (intracerebral), oral, intramuscular, subcutaneous, transdermal, intradermal, transmucosal, intranasal, sublingual, intraperitoneal or intraocular routes. In a preferred embodiment, the kit of parts or the composition for use according to the invention is administered systemically, i.e. enterally or parenterally. More preferably, the kit of parts or the composition for use according to the invention is administered intravenously, subcutaneously or intraperitoneally.

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

Said multimeric complex and said at least one antibody capable of modulating an immune checkpoint protein of a kit of parts according to the invention may be administered by the same route or preferably by different routes. More preferably, said multimeric complex and said at least one antibody capable of modulating an immune checkpoint protein of a kit of parts according to the invention are administered by the same route. In a preferred embodiment, said multimeric complex and said at least one antibody capable of modulating an immune checkpoint protein of a kit of parts according to the invention are administered sequentially or simultaneously, preferably sequentially. In a preferred embodiment, said multimeric complex and said at least one antibody capable of modulating an immune checkpoint protein of a kit of parts according to the invention are administered sequentially by different routes. More preferably, said multimeric complex and said at least one antibody capable of modulating an immune checkpoint protein of a kit of parts according to the invention are administered simultaneously by the same route.

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

In another preferred embodiment, said multimeric complex is administered before said at least one antibody capable of modulating an immune checkpoint protein. In another preferred embodiment, the multimeric complex and the at least one antibody capable of modulating an immune checkpoint protein are administered sequentially, wherein the multimeric complex is administered by intravenous injection and the at least one antibody capable of modulating an immune checkpoint protein is administered intraperitoneally or intravenously, and wherein the multimeric complex is administered prior to the antibody.

The ratio of the amount or concentration of multimeric complexes to the amount or concentration of one or more antibodies to be administered in a kit of parts or composition of the invention may be varied, for example to address the needs of the individual patient or patient subpopulation to be treated, which needs may differ depending on the age, sex, weight, condition, etc. of the patients.

The kit of parts of the invention may further be used as an add-on therapy. As used herein, "add-on therapy" means a combination of the multimeric complex and the at least one antibody for use in therapy, wherein a subject receiving therapy begins a first treatment regimen with one or more different parts of the kit of parts followed by a second treatment regimen with one or more different parts of the kit of parts other than the first treatment regimen, such that not all agents used in therapy begin at the same time. For example, one or more immunomodulatory antibodies are administered to a patient who has received a multimeric complex of the invention or vice versa.

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

Examples of the invention

In the following, the invention is further illustrated by means of examples. The preferred multimeric complexes of the present invention have been used to this point. The synthesis of the preferred multimeric complexes has been performed as described in WO 2015/173824, and in particular as described in examples 2, 8, 10 to 13 of WO 2015/173824. Preferred polymeric conjugates that include EGF as the targeting moiety are abbreviated herein as "PEI-PEG-EGF" and corresponding preferred multimeric complexes are interchangeably abbreviated as "PEI-PEG-EGF/poly ic" or "PPE/PIC". Further preferred multimeric complexes for use in the invention and herein include HER2, in particular HER2 affibody as targeting moiety within a polymeric conjugate and poly ic as a double stranded RNA. The preferred polyplexes are abbreviated herein as "PolyIC/PPHA".

EXAMPLE 1 Effect of PEI-PEG-EGF/PolyIC alone

A431, U87 and MCF7 cells (40,000 cells per well) were treated with various concentrations (0.125, 0.25, 0.5, 1. mu.g/ml) of PEI-PEG-EGF/polyIC for 5 hours.

Human IP-10(CXCL10) secretion was quantified by ELISA assay (ABTS ELISA development kit by Peprotech, Pappertak). IP10 secretion was strongly increased in a431 cells expressing high EGFR levels after 5 hours incubation with PEI-PEG-EGF/poly ic (figure 1).

A431 cells were treated with PEI-PEG-EGF/polyIC at a concentration of 0.125. mu.g/ml for 5 hours. A431 cells were then stained with PD-L1-PE labeled antibody (Biolegend, Cat. 393607) in PBS with 2% FCS on ice for 40 minutes, then washed and analyzed by FACS instruments. PD-L1 expression was significantly increased after PEI-PEG-EGF/poly ic treatment (MFI 751) compared to untreated control cells (MFI 431). Isotype control was used as negative control (MFI ═ 13).

PD-L1 expression was significantly higher after PEI-PEG-EGF/polyIC treatment than untreated cells (FIGS. 2A-C).

And (4) conclusion: potent IFN-y-induced secretion of T cell chemokine-IP 10 was strongly induced in the EGFR overexpressing cell line A431 following PEI-PEG-EGF/polyIC stimulation. Furthermore, IP10 levels were not affected in low EGFR expressing cell lines, MCF7 and U87. The increase in IP10 following PEI-PEG-EGF/polyIC stimulation provides a rationale for addressing the immunostimulatory properties of the combinations of the invention.

In addition, PD-L1 expression was significantly higher after PEI-PEG-EGF/poly ic treatment (MFI 751) compared to untreated cells (MFI 431), which provides the principle for the development of combinations with nivolumab (fig. 2).

Example 2 in vitro Effect of PEI-PEG-EGF/polyIC in combination with immunomodulatory therapies nivolumab and 4-1BB antibody

Nivolumab: 40.000A431 cells were treated with PEI-PEG-EGF/polyIC at concentrations of 0.125, 0.25, 0.5, 1. mu.g/ml for 5 hours. 200.000 PBMCs were then stimulated with CD3 (5. mu.g/ml) and challenged with diluted medium (1:2) containing PEI-PEG-EGF/polyIC alone (0.125. mu.g/ml) or in combination with nivolumab (20. mu.g/ml) for 48 hours. After 48 hours, media from the challenged PBMCs were collected and INFy production was measured by ELISA assay (fig. 3).

Combining PEI-PEG-EGF/polyIC with nivolumab significantly increased IFN-y production from PBMC (FIG. 3).

4-1BB antibody: 40.000A431 cells were treated with PEI-PEG-EGF/polyIC at a concentration of 0.5. mu.g/ml for 5 hours. 200,000 PBMCs from healthy donors were stimulated or unstimulated with CD3 (0.5. mu.g/ml) and co-cultured with A431 cells treated with PEI-PEG-EGF/polyIC alone or in combination with an antibody to 4-1BB (Biolegend clone, 4B 4-1; 10. mu.g/ml) for 16 hours. After 16 hours the medium was collected and IFN-y was measured by ELISA assay.

Combining PEI-PEG-EGF/polyIC with anti-4-1 BB significantly increased IFN-y production by PBMC (FIG. 4).

And (4) conclusion: exposure of PBMCs to media from EGFR-overexpressing A431 cells treated with PEI-PEG-EGF/polyIC or culturing PBMCs in the presence of A431 cells treated with PEI-PEG-EGF/polyIC induces IFN-y secretion by PBMCs. Addition of nivolumab or anti-41 BB antibody resulted in a further increase of IFN-y.

Thus, the combination of PEI-PEG-EGF/poly ic with immunotherapy results in an increased activation of the immune system and more effective anti-tumor activity. From a clinical point of view, the potential induction of anti-tumor immune responses when PEI-PEG-EGF/poly ic is treated in combination with an immunization strategy is very attractive, as such combinations should increase the percentage of patients that respond to immunotherapy and reduce the incidence of developing drug resistance.

Example 3 in vivo efficacy of the combination PEI-PEG-mEGF/polyIC + anti-PD-1

The effect of PEI-PEG-EGF/polyIC + anti-PD-1 on RencaEGFR lung metastasis in immunocompetent mice was examined.

Materials and methods: cell: RencaEGFR; multimeric complexes PEI-PEG-EGF/poly ic and-LPEI/EGF mice: 1/0.75); poly IC from Dalton (Dalton); multimeric complex-containing HBG (Hepes buffered glucose), N/P: 8. anti-PD-1: rat IgG2a, a kappa anti-mouse PD-1 antibody (Bioxcell clone RMP 1-14).

250,000RencaEGFR cells were injected intravenously into 40Balb/c immunocompetent female mice (6 weeks old, weight: 18-21gr) to induce the formation of RencaEGFR tumors in the lungs. After 10 days, animals were divided into 4 groups of 10 animals/group (untreated (UT), anti-PD-1, PEI-PEG-EGF/polyIC and PEI-PEG-EGF/polyIC + aPD1 (anti-PD-1).

RencaEGFR tumor bearing mice were treated intravenously with PEI-PEG-EGF/polyIC alone at 250 μ g/kg, 6 injections/week for 2 weeks or intraperitoneally at 10mg/kg on days 0, 2, 4, 7, 10 in combination with anti-PD-1 (RPM1-14, rat IgG2a, Biox cells). Survival was then analyzed. The appearance of clinical symptoms of cancer: a 10% weight reduction between 2 final weighings or a 20% weight reduction between 1 st weighings and final weighings; slow/abnormal movement; bending the back; abnormal breathing confirmed the rationality for mouse sacrifice. The presence of tumors in the lungs was visually confirmed in the sacrificed mice. Mice were sacrificed according to scoring parameters such as mouse weight measurements, general health, etc. After the mice are sacrificed, organs are collected and IHC will be analyzed to detect the presence of lung metastases.

And (4) conclusion: the uptake rate of tumors was 100% for both groups (untreated (UT), anti-PD-1), with final average survival of 41.5 and 41.8 days. All mice had tumors after cell injection, which shows the high reliability of the model. anti-PD-1 alone failed to inhibit tumor growth. One animal remained alive in the PEI-PEG-EGF/polyIC (PPE/PIC) group alone on day 60. The survival rate was initially increased by about 32% using PEI-PEG-EGF/polyIC. In the PEI-PEG-EGF/polyIC + anti-PD-1 (PPE/PIC + aPD1) group, only 3 animals died. The survival rate was initially increased by about 63%, which has shown synergy between PEI-PEG-EGF/polyIC and anti-PD-1 (FIG. 9).

TABLE 1

	UT	aPD1	PPE/PIC	PPE/PIC+aPD1
					Average survival rate (Tian)	41.5	41.8	54.8	67.8
(%) survival improvement		0.7	32.0	63.4

Example 4 in vivo efficacy of the combination PEI-PEG-EGF/polyIC + anti-CTLA-4

RencaEGFR cells were injected intravenously into 40Balb/c immunocompetent female mice to induce the formation of RencaEGFR tumors in the lungs as described in example 3. After 10 days, animals were divided into 4 groups, untreated control (UT), anti-CTLA-4, PEI-PEG-EGF/polyIC and PEI-PEG-EGF/polyIC + anti-CTLA-4. Mice bearing EGFR tumors were treated with PEI-PEG-EGF/poly ic alone or in combination with anti-CTLA-4 on day 0, day 2, day 4, day 7, day 10.

Mice were sacrificed according to scoring parameters such as mouse weight measurements, general health, etc. After the mice are sacrificed, organs will be collected and analyzed for IHC to detect the presence of pulmonary metastases. Survival was analyzed as well as the appearance of clinical symptoms of cancer. The presence of tumors in the lungs was visually confirmed in the sacrificed mice.

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

Example 5-combination therapy with polyIC/PPHA and anti-PD-1 antibody in immunocompetent mice bearing HER-2 overexpressing tumors

Materials and methods: RENCA-HER2 cells (renal cell carcinoma cells overexpressing HER2) (5X 10⁶) Subcutaneously (s.c.) injected into immunocompetent BALB/c female mice (6-8 weeks old; right flank of Harlan Laboratories, Harlan Laboratories). Mice bearing s.c. RENCA HER2 tumors were randomly divided into 4 groups (untreated, multimeric complexes of anti-PD-1, polyIC/PEI-PEG-HER2 affibody (PPHA) and anti-PD-1 + polyIC/PPHA), 7-8 animals/group, with an average tumor volume of 235mm³. Preferably the PolyIC/PPHA is synthesized as described in the examples of WO 2015/173824, more preferably example 2 and page 18ff of WO 2015/173824. Preferably a synthetic HER as described in WO 2015/173824, page 20ff2 affibody.

Mice were treated intravenously (i.v.) every 24 hours for 10 days with polyIC/PPHA 0.25mg/kg or 6.25 mg/mouse, N/P8. 4 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 x width²And/2 and plotted as mean SEM.

And (4) conclusion: significant inhibition of tumor growth was observed in both the combination panel and the poly ic/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 any tumor growth for an additional 60 days. Cured mice showed complete protection against tumor re-challenge (5 × 10)⁶) This indicates that an immune response to the tumor has been generated.

Example 6 in vitro experiment, PD-L1 expression

Materials and methods: shows increased expression of PD-L1 in RENCA HER2 cells following 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 Phycoerythrin (PE) -conjugated anti-mouse PD-L1 antibody. After treatment, the cells were trypsinized, washed and incubated with anti-PD-L1 antibody CD274(PD-L1, B7-H1) [10F.9G2] Tonbo catalog No. 50-1243-U025 or isotype control for 1 hour. PD-L1 expression was analyzed using flow cytometry (BD FACS ARIAIII; Bidi Biosciences, BD Biosciences). Live cells were gated on SSC and FSC. PE positivity was gated and the mean PE determined.

And (4) conclusion: RENCA HER-2 cells were treated with 1 microgram/ml polyIC/PPHA for 24 hours and analyzed for PD-L1 expression using flow cytometry. A significant increase in PD-L1 expression was observed after treatment with 1 microgram/ml polyIC/PPHA compared to untreated cells, indicating that targeted delivery of polyIC induces upregulation of PD-L1 expression.

TABLE 2

Example 7-direct activation of PBMCs by multimeric complexes of the invention comprising polyIC and targeted polymeric conjugates

The following experiments demonstrate the benefit of the combination of multimeric complexes of the invention with nivolumab over single agent therapy.

Materials and methods: the IFN γ gamma release from PBMCs exposed to supernatants of a431 cells treated with PEI-PEG-EGF/poly ic polyplex was quantified. Cell line: the cell types used were a431 cancer cells (ATCC) and PBMCs (healthy donor 147). A431 cells were cultured in DMEM medium with 10% fetal bovine serum, 100 units/ml penicillin (penicillin), 100. mu.g/ml streptomycin (streptomycin). Buffy coat was obtained from healthy donors. PBMCs were isolated from buffy coats and cultured in RPMI-1640 medium with 10% fetal bovine serum, 100 units/ml penicillin, 100. mu.g/ml streptomycin.

PEI-PEG-EGF/polyIC polyplex was prepared in HBG (Hepes buffered glucose). The PEI-PEG-EGF triconjugate consisted of a pegylated linear polyethyleneimine conjugated to hEGF (hEGF-4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid) via an MCC linker and synthesized according to WO 2015/173824, example 10. anti-PD-1 antibody (nivolumab) alone or in combination with PEI-PEG-EGF/polyIC polyplex was used. After transfer of media from treated a431 cells, PBMCs were stimulated with anti-CD 3 antibody (clone OKT 3). The combination of PEI-PEG-EGF/polyIC polyplex and checkpoint blockade was tested using an anti-PD-1 antibody (nivolumab). Human interferon gamma secretion was measured using an ELISA kit (Bidy biosciences) according to the manufacturer's instructions.

A431 cells were seeded in flat bottom 96-well plates (40,000 cells/90. mu.l DMEM medium). In parallel, plates with medium alone (90 μ l of medium without cells) were prepared as negative controls. The next day, A431 cells alone or the culture medium were treated with 0, 0.125 or 1. mu.g/ml PEI-PEG-EGF/polyIC polyplex (10. mu.l/well) for 5 hours at 37 ℃. Frozen PBMCs were thawed and allowed to recover in RPMI-1640 medium at 37 ℃ for at least 5 hours (200,000 cells/100 μ l) in U-bottom 96-well plates. After 5 hours, Supernatants (SN) from A431 cells treated with PEI-PEG-EGF/polyIC polyplex or from media "treated" with PEI-PEG-EGF/polyIC polyplex were transferred to PBMCs. Thereafter, PBMCs were stimulated with anti-CD 3 antibody (OKT3, 500ng/ml) or/and treated with nivolumab (20. mu.g/ml) and incubated o/n at 37 ℃. SN of stimulated PBMC was collected and stored at-20 ℃ until analysis by ELISA.

To model the effect of combining PEI-PEG-EGF/poly ic polyplexes with nivolumab treatment on immune cell activation in vitro, SN from a431 cells treated with PEI-PEG-EGF/poly ic polyplexes was transferred into human PBMCs from healthy donors and treated in combination with nivolumab as follows. A431 cells were treated with indicated concentrations of PEI-PEG-EGF/polyIC polyplex for 5 hours. As a control, medium without cells was "treated" with PEI-PEG-EGF/polyIC polyplex and further incubated for 5 hours. After 5 hours, PBMCs stimulated or unstimulated with anti-CD 3 antibody were treated with: nivolumab alone; SN from a431 cells treated with PEI-PEG-EGF/poly ic polyplex alone; separate culture media "treated" with PEI-PEG-EGF/poly ic polyplex; nivolumab plus SN from a431 cells treated with PEI-PEG-EGF/poly ic polyplex; or nivolumab plus medium "treated" with PEI-PEG-EGF/polyIC polyplex. PBMC were incubated o/n at 37 ℃. To assess PBMC activation, IFN- γ secreted from PBMCs was quantified by ELISA.

As a result: as can be seen in figure 10 and table 3, CD3 stimulated PBMC that received SN from a431 cells treated with PEI-PEG-EGF/poly ic polyplex secreted much more IFN- γ (311.1-326.3pg/ml) than PBMC that received SN from Untreated (UT) cells (12.7 pg/ml). IFN- γ secretion was further enhanced by the addition of nivolumab (502-523 pg/ml). PBMCs receiving SN from untreated cells plus nivolumab secreted low levels of IFN- γ (62.5 pg/ml). Nivolumab treatment alone did not induce IFN- γ secretion. Medium "treated" with PEI-PEG-EGF/poly ic polyplex (no cells) alone or in combination with nivolumab did not result in increased IFN- γ secretion compared to UT medium. These results demonstrate that the combination of PEI-PEG-EGF/poly ic polyplex with nivolumab results in increased activation of PBMCs compared to PEI-PEG-EGF/poly ic polyplex, as seen by increased IFN- γ secretion. In addition, PBMC were activated by cytokines secreted from A431 cancer cells treated with PEI-PEG-EGF/polyIC polyplex, rather than by PEI-PEG-EGF/polyIC polyplex alone.

And (4) conclusion: after a431 cell treatment, the combination of multimeric complex comprising poly ic and targeting moiety human EGF and nivolumab was repeated in medium transfer experiments. IFN- γ levels were measured by ELISA and demonstrated that the combination of the two agents resulted in an increase in IFN- γ compared to the individual agents alone. As a control, single agents (polyplexes or nivolumab) or combinations were incubated in medium without cells for the same time course. The medium was then transferred to PBMCs. IFN- γ levels were significantly lower in this group compared to PBMCs receiving media transferred from a 431-treated cells. These results demonstrate that activation of PBMCs is not directly mediated by poly ic polyplexes, but rather by secretion of chemokines from cancer cells treated with poly ic polyplexes.

Table 3: the combination of PEI-PEG-EGF/polyIC polyplex and nivolumab increased PBMC activation as demonstrated by IFN-. gamma.ELISA.

Example 8-cytokine secretion after treatment with PEI-PEG-EGF/polyIC polyplex alone and Each component

To assess whether PEI-PEG-EGF/polyIC polyplex or a component thereof induces pro-inflammatory cytokine secretion, three cell lines comprising two high EGFR expression lines (MDA-MB-468 and A431) and a low EGFR line (MCF7) were treated with PEI-PEG-EGF/polyIC polyplex, the triconjugate PEI-PEG-EGF or pIC and IP-10 GRO-alpha and measured for CCL5 cytokine secretion by ELISA.

Materials and methods: three cell lines (high EGFR expressing MDA-MB-468 and A431) and low EGFR (MCF7) were treated with the indicated concentrations of PEI-PEG-EGF/polyIC polyplex, PEI-PEG-EGF triconjugate or polyIC for 5 hours. The media was then harvested and analyzed for CCL5(RANTES), IP10, and GRO α. PEI-PEG-EGF consists of pegylated linear polyethyleneimine synthesized according to WO 2015/173824, example 10 conjugated to hEGF via linker MCC.

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

And (3) treatment: PEI-PEG-EGF/poly IC polyplex alone or pIC was incubated in HBG (HEPES buffered glucose). Reagents with pIC concentrations between 0-1. mu.g/ml were used. The individual tri-conjugates PEI-PEG-EGF were prepared in a similar manner to the PEI-PEG-EGF/polyIC polyplex without pIC.

Cytokine production was quantified by ELISA: to detect IP-10, Gro-alpha and RANTES secretion, 40,000 cells per well were treated with PEI-PEG-EGF/polyIC polyplex and a pIC concentration between 0-1. mu.g/ml, or the equivalent concentration of PEI-PEG-EGF triconjugate or pIC, for 5 hours. Supernatants were collected and cytokine secretion was quantified by IP-10, GRO α and RANTES ELISA (Peprotech) using a Synergy H1 plate reader (Biotek).

As a result: PEI-PEG-EGF/polyIC polyplex treatment of high EGFR expressing cells MDA-MB-468 and A431 induced secretion of IP-10 GRO-alpha and CCL5, while no cytokine expression was observed in low EGFR expressing MCF7 cells (FIGS. 11A, B and C, respectively). Up to 337 and 268pg/ml IP10, 496 and 757pg/ml GRO α and 607 and 100pg/ml RANTES were observed in A431 and MDA-MB-468, respectively. Cytokine secretion was not observed in all cell lines after treatment with PEI-PEG-EGF triconjugate or pIC alone. These results demonstrate that PEI-PEG-EGF/polyIC polyplex, but not its individual components, induces proinflammatory cytokine secretion.

Example 9-activation of PBMC by PEI-PEG-EGF/polyIC polyplex and pIC/PPHA

Materials and methods: tumor cell lines: MDA-MB-468, A431 and MCF7 cells were supplied by ATCC. Cell lines were routinely passaged once or twice a week and maintained in culture for up to 20 passages. The cell lines were grown in RPMI-1640 medium (25 mM HEPES with L-glutamine, # FG1385 from Bessen GmbH, Berlin, Germany) or DMEM supplemented with 10% (v/v) fetal bovine serum (Sigma, Doffy, Germany), 100 units/ml penicillin, 100. mu.g/ml streptomycin, with 5% CO2 in a humidified atmosphere at 37 ℃.

Reagent: the PEI-PEG-EGF triconjugate consists of a pegylated linear polyethyleneimine conjugated to hEGF (human epidermal growth factor) via an MCC linker and synthesized according to WO 2015/173824, example 10. PEI-PEG-EGF/polyIC polyplex and PEI-PEG-EGF/pLGA polyplex (PEI-PEG-EGF and poly-L-glutamic acid (pLGA) from Sigma, 50-100kDa p4886) were prepared in HBG (HEPES buffered glucose) and utilized naked pIC. Reagents with pIC or pLGA concentrations of 0 to 1. mu.g/ml were used in multimeric complexes.

PBMC isolation from healthy donors (buffy coat): buffy coats from healthy donors were obtained from the University Hospital Blood Bank (University Hospital base Blood Bank). PBMCs were isolated from buffy coats and cultured in RPMI-1640 medium with 10% fetal bovine serum, 100 units/ml penicillin, 100. mu.g/ml streptomycin.

Cytokine production was quantified by ELISA: to quantify IFN- γ and TNF- α secretion, 40,000 cells per well or individual media were treated with the following compounds: PEI-PEG-EGF/polyIC polyplex with pIC concentration between 0-1. mu.g/ml, pIC alone (0-1. mu.g/ml) or PEI-PEG-EGF (PEI-PEG-EGF/pLGA polyplex) polyplex with polyglutamic acid polyplex of 0-1. mu.g/ml. After 5 hours of incubation, the supernatant and individual media were collected and transferred to 200,000 PBMCs stimulated with a-CD3 (0.5. mu.g/ml) and incubated for another 16 hours. In parallel, supernatants from each cell line treated with each compound alone were incubated for 16 hours in the absence of PBMCs. After incubation, the media was collected and TNF- α, IFN- γ and IL-2 were measured by ELISA assay (dept and Invitrogen) and read using the Synergy H1 plate reader from beton.

As a result: PBMC activation by cytokines secreted from cancer cells treated with PEI-PEG-EGF/poly ic polyplex: proinflammatory cytokines such as IP-10 are known to induce recruitment and activation of lymphocytes. To assess whether PEI-PEG-EGF/polyIC polyplex or its drug substance pIC induced immune cell activation, three cell lines were treated with indicated concentrations of PEI-PEG-EGF/polyIC polyplex, pIC or PEI-PEG-EGF/pLGA polyplex (PEI-PEG-EGF complexed with polyglutamic acid (pLGA)) for high EGFR expression (MDA-MB-468 and A431) and low EGFR (MCF 7). In the PEI-PEG-EGF/pLGA polyplex, pIC of the PEI-PEG-EGF/polyIC polyplex was replaced by polyglutamic acid to demonstrate that the effect of the PEI-PEG-EGF/polyIC polyplex is pIC mediated. After 5 hours of treatment, supernatants from treated cancer cells were transferred into PBMCs from healthy donors (FIG. 12; PBMC + SN from each cell line). PBMCs were then incubated for 16 hours and analyzed by ELISA for secretion of IFN- γ and TNF α cytokines. Meanwhile, in order to prove that the PEI-PEG-EGF/polyIC polyplex does not directly induce the activation of PBMC, PEI-PEG-EGF/polyIC polyplex, pIC or PEI-PEG-EGF/pLGA polyplex was incubated in a medium without cells, and this medium was transferred to PBMC, followed by incubation for 16 hours and testing by ELISA (FIG. 12; "PBMC alone"). As an additional control, supernatants from treated cancer cells incubated for the same time period, but without PBMCs, were also analyzed (FIG. 12; "SN alone").

Increased secretion of IFN-. gamma.was observed in PBMCs incubated with supernatants from MDA-MB-468 and A431 treated with PEI-PEG-EGF/polyIC polyplex. Up to 3587pg/ml and 2023pg/ml of IFN- γ were detected in PBMCs from MDA-MB-468 and A431, respectively. PBMCs incubated with supernatants from MCF7 cells treated with PEI-PEG-EGF/poly ic polyplex did not induce any cytokine secretion. PBMCs treated with medium containing PEI-PEG-EGF/polyIC polyplex, pIC or PEI-PEG-EGF/pLGA polyplex (PBMC alone) without cells did not induce IFN- γ secretion in all cancer cell lines. No IFN- γ secretion was observed in PBMC-free supernatants from any cancer cell lines treated with PEI-PEG-EGF/polyIC polyplex, pIC or PEI-PEG-EGF/pLGA polyplex.

A significant increase in TNF α secretion was observed in PBMCs incubated with media from MDA-MB-468 and A431 cells treated with PEI-PEG-EGF/polyIC polyplex. TNF α was detected in PBMCs from MDA-MB-468 and A431 at most 2190pg/ml and 3438pg/ml, respectively. PBMCs incubated with media from MCF7 cells treated with PEI-PEG-EGF/polyIC polyplex did not induce cytokine secretion. Culture media from PBMCs treated with PEI-PEG-EGF/poly ic polyplex, pIC or PEI-PEG-EGF/pLGA polyplex (PBMC alone) did not induce a significant increase in TNF α secretion in all cancer cell lines compared to UT cells. No IFN-. gamma.and TNF. alpha.secretion was observed in PBMC-free supernatants from cancer cell lines treated with PEI-PEG-EGF/polyIC polyplex, pIC or PEI-PEG-EGF/pLGA polyplex.

Example 10 efficacy and selectivity of PEI-PEG-EGF/polyIC polyplex compared to naked pIC, pIC complexed with PEI or lipids based on transfection reagents

The aim of the study was to measure the selectivity and potency of PEI-PEG-EGF/poly ic polyplexes with single components and non-targeted delivery systems as well as pIC analogs.

Materials and methods: cell line: high EGFR expressing cell lines (BT20, MDA-MB-468, and HCC70), medium EGFR expressing cell lines (U87MG), low EGFR expressing cell lines (MCF7 and U138), and non-cancer cell lines (WI-38 and MCF10A) were used for experiments. Cells were cultured as above.

Reagent: the PEI-PEG-EGF triconjugate consists of a pegylated linear polyethyleneimine synthesized according to WO 2015/173824, example 10 conjugated to hEGF by an MCC linker. PEI-PEG-EGF triconjugate and pIC were used to prepare PEI-PEG-EGF/polyIC polyplexes in HBG (HEPES buffered glucose). JetPEI (Polyplus) and pIC were used to prepare a JetPEI-pIC polyplex according to the manufacturer's instructions. Lipofectamin RNAiMax (Invitrogen, P/N56531) and pIC were used to prepare RNAiMax-pIC polyplexes according to the manufacturer's instructions. Poly-L-glutamic acid (pLGA; sigma 50-100kDa) and PEI-PEG-EGF were used to prepare a PEI-PEG-EGF/pLGA polyplex having the same net charge as the PEI-PEG-EGF/polyIC polyplex.

And (3) treatment: 3000 cells/well were seeded in 96-well plates. After 24 hours, the cells were treated with the indicated concentration of multimeric complexes (ranging from 2. mu.g/ml to 0.001. mu.g/ml). 72 hours after treatment, cell viability was assessed by CellTiter Glo (Promega) and luminescence recorded using a synergy H1 cell plate reader (Bolten).

As a result: the effect of the activity of PEI-PEG-EGF/polyIC polyplex on high and low EGFR expressing cancer cell lines as well as on non-cancerous lines was compared to the effect of naked pIC, jetPEI-pIC, RNAiMax-pIC (lipid based transfection reagent) and PEI-PEG-EGF/pLGA polyplex.

In all EGFR overexpressing cells, JetPEI-pIC polyplexes were significantly less efficient than PEI-PEG-EGF/poly ic polyplexes: the IC50 of the PEI-PEG-EGF/polyIC polyplex is 1/20-1/10 of the IC50 of jetPEI-pIC. In cancer or non-cancer cell lines with low EGFR, jetPEI-pIC was up to 2-fold more toxic than PEI-PEG-EGF/polyIC polyplex (FIGS. 13A-E). These results demonstrate the increased efficacy and selectivity of PEI-PEG-EGF/polyIC polyplex compared to jetPEI-pIC.

Increased efficacy of the PEI-PEG-EGF/polyIC polyplex was observed in 3 of 4 EGFR overexpressing cell lines compared to treatment with RNAiMax-pIC. In A431 cells only, the IC50 of RNAiMax-pIC was lower than the IC50 of PEI-PEG-EGF/polyIC polyplex (IC 50 of RNAiMax-pIC was 1/4 of the IC50 of PEI-PEG-EGF/polyIC polyplex) (FIG. 13). In BT20, MDA-MB-468, and HCC70 cells, the IC50 of the PEI-PEG-EGF/polyIC polyplex is about 1/10 of the IC50 of the RNAiMax-pIC. RNAiMax-pIC was found to be toxic in normal cells (WI-3 and MCF10A) as well as cancer cells with low EGFR expression, whereas PEI-PEG-EGF/polyIC polyplex was not toxic to these cells.

At the concentrations tested, naked pIC was not effective in all cell lines.

The PEI-PEG-EGF/pLGA polyplex was significantly less effective (more than 30 fold) on EGFR overexpressing cancer cells than the PEI-PEG-EGF/polyIC polyplex. The PEI-PEG-EGF/pLGA polyplex served as a control for the PEI-PEG-EGF/polyIC polyplex. The results clearly demonstrate that the efficacy of the PEI-PEG-EGF/polyIC polyplex is mediated by targeted pIC treatment.

The PEG-EGF/poly ic polyplex showed high specificity and efficacy in EGFR-overexpressing cells, while it did not induce toxicity in normal cell lines. It was demonstrated that the efficacy of PEI-PEG-EGF/polyIC polyplex was mediated by pIC. Compared to PEI-PEG-EGF/polyIC polyplexes, RNAiMax/pIC and jetPEI/pIC are less effective and less selective than PEI-PEG-EGF/polyIC polyplexes. RNAiMax/pIC is also highly toxic in normal cells.