Cancer therapy comprising car engineered t cells and parvovirus h-1

Abstract

Compositions, methods, uses and kits for treating a subject having cancer are provided.SOLUTION: Provided are compositions, methods, uses, and kits for immunotherapy, such as adaptive cell therapy, e.g., T cell therapy, and combination therapy comprising an oncolytic virus (particularly parvovirus H-1), for treating a subject having cancer. T cell therapies include cells expressing recombinant receptors, such as chimeric antigen receptors (CARs). In some embodiments, the cancer is a solid tumor or a hematologic malignancy.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions, methods, uses, and kits for combination therapy including immunotherapy, such as adaptive cell therapy, e.g., T cell therapy, and oncolytic viruses (particularly parvovirus H-1), for treating subjects with cancer. T cell therapy includes cells expressing a recombinant receptor, such as a chimeric antigen receptor (CAR). In some embodiments, the cancer is a solid tumor or a hematological malignancy. [Background technology]

[0002] For many years, cancer treatment has been based on surgery, chemotherapy, and radiation, and more recently, targeted therapy. While these approaches have contributed to improved outcomes, most malignant tumors still have a poor prognosis. Targeted anticancer approaches offer personalized treatments to combat the complexities of most malignancies and increase the odds of success. Currently, there is growing interest in immunotherapy, which harnesses the power of the patient's immune system to fight disease. One approach to cancer immunotherapy involves genetically engineering a patient's T cells to express chimeric antigen receptors (CARs) that recognize and attack tumor cells. CARs consist of a targeting ectodomain derived from an antibody or ligand fused to a hinge, transmembrane domain, and intracellular T cell signaling domain. When expressed by T cells, CARs confer antigen specificity determined by the targeting domain. In contrast to conventional T cell receptors (TCRs), which recognize antigens in a major histocompatibility complex (MHC)-dependent manner, CARs can potentially redirect T cell effector functions to any protein or non-protein target expressed on the cell surface. This strategy thereby circumvents the need for antigen processing and presentation by the target cell and is applicable to non-classical T cell targets. Avoiding human MHC restriction makes the CAR-T cell approach a universal treatment, broadening the potential applicability of adoptive T cell therapy.

[0003] Four generations of CARs are being investigated in preclinical and ongoing clinical studies (Mirzaei et al., Frontiers in Immunology 2017, Vol. 8, Art. 1850). The CAR "generation" typically refers to the intracellular signaling domain incorporated into the receptor molecule. First-generation CARs contain only CD3ζ as the intracellular signaling domain; second-generation CARs contain CD3ζ plus a single costimulatory domain, such as CD28, 4-1BB (CD137), CD27, or OX40; and third-generation CARs contain CD3ζ and two costimulatory domains, such as CD28, 4-1BB, or other costimulatory molecules (see Figure 3). CARs can be further engineered by the introduction of additional genes, including those encoding potent anti-tumor cytokines (e.g., IL-12 and IL-15) or costimulatory ligands (e.g., 4-1BBL), thus producing "armored" fourth-generation CAR T cells (Maus et al., Blood 2014, 123(1), 2625-2635; Pegram et al., Cancer J. 2014, 20(2):127).

[0004] Chimeric antigen receptors targeting the B-cell receptor-associated protein CD19, developed for the treatment of B-cell leukemia and lymphoma, have been the most clinically tested to date. Numerous advances in CD19-CAR T-cell therapy across multiple centers employing different therapeutic designs have led to the successful commercialization of this adoptive immunotherapy. Two CD19-targeted CAR-T cell products, Novartis' (East Hanover, NJ, USA) Kymriah® (Tisagenlecleucel) and Kite Pharma's (Santa Monica, CA, USA) Yescarta® (Axicabtagene ciloleucel), were approved by the US FDA in 2017 for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL), respectively. CAR-T cell therapy has achieved remarkable outcomes in children and young adults with relapsed and often refractory disease, with complete response (CR) rates of 70–90% (Cummins et al., Leuk. Lymphoma 2017, pp. 1–15). In lymphoma and other B-cell malignancies, CAR T-cell therapy is effective but shows lower CR rates (approximately 55%) (Cummins et al., Leuk. Lymphoma 2017, pp. 1–15). Both FDA-approved CARs specifically bind CD19, an antigen that works well as a target for hematologic malignancies because it is nearly uniformly expressed on malignant cells and appears on all B cells, both healthy and malignant. Therefore, although CD19-CAR-T cell treatment can cause B-cell aplasia, symptoms can be managed with intravenous immunoglobulin and close infection monitoring.

[0005] Despite advances in the treatment of hematological cancers with CAR-T cells, the treatment of solid tumors has proven more challenging. The limited success of CAR-T cell therapy for solid tumors can be attributed to a number of factors, including (i) the lack of unique tumor-associated antigens (TAA) in most cancers; (ii) the inability of ex vivo-expanded CAR-T cells to persist and proliferate after adoptive transfer; (iii) inefficient transport of CAR-T cells to tumor sites; (iv) heterogeneous expression of target antigen(s), leading to the growth of antigen-negative tumor variants; (v) the lack of survival and growth factors (e.g., IL-2); (vi) the presence of immunosuppressive molecules and cells; and (vii) a metabolically hostile tumor microenvironment (Zhang et al., Int. J. Biol. Sci. 2016, 12:718-729). Many strategies and approaches have been attempted to overcome these obstacles, including arming CAR-T cells with knockout of PD-1 expression or cytokine / chemokine secretion, and using CAR-T cells in combination with checkpoint inhibitors (Heczey et al., Mol. Ther. 2017, 25:2214-2224; Rupp et al., Sci. rep 2017, 7:737; Hedge et al., Cancer Immunol. Immunother. 2017, 66:1113-1121). Despite these efforts, no CAR-T cells have been clinically approved for the treatment of solid tumors to date, although several clinical studies are ongoing. Summary of the Invention

[0006] Therefore, improved strategies are needed to improve the efficacy of CAR-T cells for treating solid tumors. These improved strategies may include improving the persistence, activity, and / or proliferation of the cells upon administration to a subject. DETAILED DESCRIPTION OF THE INVENTION

[0007] According to the present invention, this is achieved by the subject matter defined in the claims.

[0008] The present inventors have successfully demonstrated that the combined use of (a) immune cells (particularly T cells) genetically modified to express a chimeric antigen receptor (CAR) [hereinafter "CAR cells"] and (b) an oncolytic virus (particularly parvovirus H-1) improves the efficacy of tumor treatment, particularly for treating solid tumors. Accordingly, the present invention relates to a pharmaceutical combination or pharmaceutical formulation comprising (a) T cells genetically modified to express a chimeric antigen receptor (CAR) and (b) parvovirus H-1.

[0009] The persistently poor outcomes of immunotherapy, including CAR-T technology for treating solid tumors, are caused by a barrier surrounding the solid tumor that makes T cell infiltration difficult or even impossible. This barrier, called the "tumor microenvironment" (TME), is the cellular environment in which the tumor resides, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix. The tumor and the surrounding microenvironment are closely related and constantly interact. While tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, immune cells in the microenvironment can influence the growth and evolution of cancerous cells. Clinical and preclinical studies have shown that reversing the immune inhibitory pathways induced in many cancers may require modification of CAR T cells, for example, by including costimulatory signaling or additional activators. In contrast to certain hematological cancers that have responded well to CAR T-cell therapy, solid tumors not only lack the traditional costimulatory molecules expressed on malignant and normal B-lymphocyte targets in hematological malignancies, but also have evolved mechanisms to actively suppress the immune system. Several immunosuppressive pathways may limit the full potential of adoptive CAR T-cell therapy. Inhibitory immune receptors are often expressed on T cells after persistent tumor antigen encounters, including T-cell membrane protein-3 (TIM-3), lymphocyte-activation protein-3 (LAG-3), T-cell Ig and ITIM domain (TIGIT), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), and programmed death-1 (PD-1). Upregulation of these receptors limits the persistence and activity of CAR T-cell antitumor responses. Therefore, tumors employ multiple strategies to evade or misdirect tumor-specific immune responses.

[0010] Therefore, the present inventors combined CAR-T cell technology with the oncolytic virus parvovirus H-1 to convert so-called "cold tumors" (i.e., tumors with low immune cell infiltration) into "hot tumors" (immunogenic tumors, i.e., tumors with moderate or high immune cell infiltration). The concepts of "cold" and "hot" tumors are well known to those skilled in the art. Cold tumors are typically rich in immunosuppressive cytokines and contain large numbers of Treg cells and myeloid-derived suppressor cells (MDSCs). Cold tumors typically contain small numbers of TH1 cells, NK cells, and CD8+ T cells, as well as small numbers of functional antigen-presenting cells (APCs) (e.g., dendritic cells (DCs)). In contrast, hot tumors are rich in TH1-type chemokines and contain large numbers of effector immune cells (TH1 cells, NK cells, and CD8+ T cells) and large numbers of DCs. The chemokines CXCL9, CXCL10 and CX3CL1 play important roles in attracting T cells in many cancer types.

[0011] The degree of immune cell infiltration can be measured, for example, by the so-called "immunoscore," which is used to predict the clinical outcome of cancer patients. The consensus immune score is a scoring system that summarizes the density of CD3+ and CD8+ T cells within a tumor and their infiltrative margin. For example, immune scores can be classified as low, medium, or high depending on the CD3+ / CD8+ T cell density, with a density of 0-25% being preferably scored as low, a density of 25-70% being preferably scored as medium, and a density of 70-100% being preferably scored as high (Pages F. et al. (2018) Lancet 391(10135):2128-2139). Cold tumors are defined as having a low degree of immune cell infiltration, i.e., preferably a low immune score. Hot tumors are defined as having a moderate or high degree of immune cell infiltration, i.e., preferably a moderate or high immune score.

[0012] Some tumor types belong to the hot tumor type even before treatment (e.g. melanoma). Nevertheless, the present invention allows for further enhancement of T cell activation and immune cell infiltration into tumors.

[0013] Cold tumors typically respond poorly to immunotherapy and cell-based therapy. The present invention is based on the discovery that oncolytic viruses can improve such responsiveness by increasing immune cell infiltration into tumors, thereby positively influencing the TME. Therefore, patients with cold tumors (e.g., colorectal carcinoma, ovarian cancer, lung cancer) will particularly benefit from treatment. As a result, tumors may show better response to cell-based therapy. Cold tumors can thereby be converted to hot tumors by application of parvovirus H-1, which acts as a "door opener" and renders tumors susceptible to T cell therapy.

[0014] Thus, provided herein is an adaptive cell therapy, e.g., immunotherapy such as T cell therapy, in combination with parvovirus H-1 for treating subjects with cancer, particularly solid tumors. T cell therapy includes cells expressing a recombinant receptor, such as a chimeric antigen receptor (CAR).

[0015] Chimeric antigen receptor and CAR cells "Chimeric antigen receptors" (CARs) are recombinant receptors that provide both antigen-binding and immune cell activation functions. CAR structure and engineering are reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1).

[0016] Thus, a chimeric antigen receptor (CAR) refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains in a CAR polypeptide construct are in the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the domains in a CAR polypeptide construct are not adjacent to each other, e.g., are in different polypeptide chains.

[0017] In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In some embodiments, the costimulatory molecule is selected from 41BB (i.e., CD137), CD27, ICOS, and / or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. In this regard, see Figure 3.

[0018] A CAR that comprises an antigen-binding domain (e.g., an scFv, single-domain antibody, or TCR (e.g., a TCR alpha-binding domain or a TCR beta-binding domain)) that targets a specific cancer cell antigen or tumor marker X, where X can be a cancer cell antigen described herein. For example, a CAR that comprises an antigen-binding domain that targets CEA is called a CEA-CAR. CARs can be expressed in any cell, such as an immune effector cell (e.g., a T cell or an NK cell) described below.

[0019] The present disclosure also provides a cell comprising or expressing a CAR according to the present disclosure. Also provided is a cell comprising or expressing a nucleic acid encoding a CAR according to the present disclosure.

[0020] The cell may be an immune cell. The cell may be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be, for example, a T cell, a B cell, a NK cell, a NKT cell, or an innate lymphoid cell (ILC), or a precursor thereof. The cell may express, for example, a CD3 polypeptide (e.g., CD3y CD3 or CD35), a TCR polypeptide (TCRa or TCR), CD27, CD28, CD4, or CD8.

[0021] In preferred embodiments, the cells are T cells. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD3+,CD8+ T cells. In some embodiments, the T cells are cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)).

[0022] The use of CAR T cells is associated with the advantage that they can be administered systemically and are applied to both primary and metastatic tumors.

[0023] In some embodiments, the cell is an antigen-specific T cell. In embodiments herein, an "antigen-specific" T cell is a cell that exhibits a particular functional property of a T cell in response to the antigen for which the T cell is specific, or a cell that expresses the antigen. In some embodiments, the property is a functional property associated with an effector T cell, e.g., a cytotoxic T cell.

[0024] In some embodiments, antigen-specific T cells may exhibit one or more of the following characteristics: cytotoxicity, e.g., cytotoxicity against cells containing / expressing the antigen for which the T cell is specific; proliferation, e.g., in response to the antigen for which the T cell is specific or to cells containing / expressing the antigen for which the T cell is specific, IFNy expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression. Antigen-specific T cells comprise a TCR that can recognize peptides of the antigen for which the T cell is specific when presented by the appropriate MHC molecule. Antigen-specific T cells may be CD4+ T cells and / or CD8+ T cells.

[0025] Engineering a CAR into a T cell can be performed in vitro during culture for transduction and expansion, such as occurs during T cell expansion for adoptive T cell therapy. Methods for collecting and engineering immune cells to express a CAR are known to those skilled in the art and are described, for example, in Wang and Riviere Mol Ther. Oncolytics. (2016) 3:16015. It will be understood that "at least one cell" encompasses a plurality of cells, e.g., a population of such cells.

[0026] Cells containing or expressing a CAR according to the present disclosure can be eukaryotic immune cells, e.g., mammalian immune cells, as defined above. The mammal can be a human or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse, or other rodent (including any animal of the order Rodents), cat, dog, pig, sheep, goat, cattle (including cattle, e.g., dairy cows, or any animal of the order Bovidae), horse (including any animal of the order Equines), donkey, and non-human primate). In some embodiments, the cells can be from a human subject or can be obtained from a human subject. When CAR-expressing cells are to be used in the treatment of a subject, the cells can be from the subject to be treated with the CAR-expressing cells (i.e., the cells can be autologous), or the cells can be from a different subject (i.e., the cells can be allogeneic).

[0027] Currently, most CAR-T clinical trials use autologous CAR-T, but the patient's own T cells usually have quality and quantity deficiencies; the manufacturing costs of autologous CAR-T are higher. Therefore, allogeneic CAR-T may also be useful. However, the antigen receptor TCR on allogeneic T cells may recognize alloantigens in the recipient, thereby causing graft-versus-host disease (GVHD). Furthermore, the expression of HLA on allogeneic T cells can rapidly trigger host immune cell rejection. Therefore, using gene editing tools such as ZFN, TALEN, and CRISPR / Cas9 to knock out TCR, MHC, and related signaling pathway genes on allogeneic T cells to prevent host rejection of allogeneic CAR-T is an important step toward realizing universal CAR-T.

[0028] In some cases, the subject treated with CAR-T cells has undergone lymphodepletion. Myeloablative lymphodepletion can be achieved by thymectomy and / or irradiation. Non-myeloablative lymphodepletion can be achieved by treatment with cyclophosphamide and fludarabine. The reason for optional lymphodepletion is to reduce the subject's lymphocyte pool before adoptive transfer of CAR T cells. This can increase the efficacy of treatment by eliminating regulatory T cells and competing elements of the subject's immune system ("cytokine sink").

[0029] The principle of CAR-T therapy is illustrated in Figure 2. Patient T cells are collected (e.g., by leukapheresis), expanded, and genetically engineered to express a chimeric antigen receptor (CAR) that recognizes a single tumor antigen. A large number of CAR-T cells are expanded in vitro and then infused back into the patient for cellular immunotherapy. CARs are genetically expressed chimeric proteins containing the antigen-binding domain of an antibody (e.g., a single-chain antibody scFv) linked to a T-cell signaling domain. A key advantage of CAR-T cell adoptive immunotherapy is its more precise nature. The CAR-T cell adoptive immunotherapy system uses genetic engineering of T cells to bypass MHC-restricted antigen presentation using the principles of antigen-antibody binding, thereby achieving precise targeting.

[0030] Currently, research and development of CAR-T therapy is primarily focused on the construction of CARs through various modifications to enhance the targeting, immune killing, durability, and safety of CAR-T cells.

[0031] In some embodiments, method steps for producing at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen can include one or more of: obtaining a blood or cancer biopsy sample from a subject; testing whether the sample expresses a particular cancer cell antigen, isolating and / or growing at least one cell from the sample; culturing at least one cell in in vitro or ex vivo cell culture; introducing a CAR described herein, or a nucleic acid encoding a CAR described herein, into at least one cell, thereby modifying at least one cell; growing at least one modified cell; harvesting at least one modified cell; mixing the modified cell with an adjuvant, diluent, or carrier; or administering the modified cell to a subject.

[0032] In some embodiments, the method may further include treating the cells to induce / enhance expression of the CAR or a nucleic acid encoding the CAR. For example, the nucleic acid may contain a regulatory element for inducible upregulation of expression of the CAR from the nucleic acid in response to treatment with a particular agent. In some embodiments, the treatment may be in vivo by administration of the agent to a subject to which modified cells according to the present disclosure have been administered. In some embodiments, the treatment may be ex vivo or in vitro by administration of the agent to cells in ex vivo or in vitro culture.

[0033] Those skilled in the art can determine appropriate reagents and procedures for adoptive transfer of cells according to the present disclosure, for example, by referring to Dai et al., 2016 J Nat Cancer Inst 108(7):439.

[0034] In the process of CAR protein expression on the surface of T cells, a viral vector is required to synthesize a DNA sequence capable of expressing the CAR protein in cells using DNA synthesis technology. Therefore, the CAR DNA sequence is loaded into a plasmid vector using molecular cloning technology. Preferably, the plasmid vector has multiple cloning sites that allow genes or DNA sequences to be expressed into intracellular proteins. After these CAR proteins are expressed, they are fixed on the surface of T cells. Alternatively, virus-mediated gene expression technologies such as lentivirus, retrovirus, adenovirus, and adeno-associated virus (AAV) can be used.

[0035] Cancer cell antigens (also called "tumor antigens") suitable for cancer treatment by CAR-T technology are reviewed in Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003, and reference is made to these antigens for the present invention, which are incorporated herein by reference. Examples of cancer cell antigens include, but are not limited to, CD19; CD123; CD22; CD30; CD70, CD97, CD171; CS-1; C-type lectin-like molecule-1, CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family members; B-cell maturation antigen; Tn antigen ((TnAg) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-fake tyrosine kinase 3 (FFT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); cancer antigen 125 (CA125), epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-1 (IL-1) 1 (IL-1); leukin-13 receptor subunit alpha-2; mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostatic acid phosphatase (PAP); elongation factor 2 mutated (EFF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (macropain) subunit, beta type 9 (LMP2); glycoprotein 100 (gplOO); oncogene polypeptide consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globo-H glycoceramide (GloboH); mammary differentiation antigen (NY-BR -1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-la); melanoma-associated antigen 1 (MAGE-A1); chromosome 12p ETS translocation mutant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; survivin; telomerase; prostate cancer tumor antigen-1, RAS family antigens or mutants (k-RAS, N-RAS), human telomerase reverse transcriptase (hTERT);Sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); prostate-specific antigen (PSA), androgen receptor; cyclin B1; v-myc avian myelopathy tomato sis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (FCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); cyclin-dependent kinase inhibitor p16; INK4a intestinal carboxylesterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); MSI frameshift mutants (e.g., those mentioned in WO 2014 / 090265), Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0036] In some embodiments, the antigen is mesothelin, EGFRvIII, GD2, Tn antigen, PSMA, PSA, CD70, CD97, TAG72, CD44v6, CEA, CA125, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-1Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, legumain, HPV E6 or E7, p16 INK4a , ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta-human chorionic gonadotropin, AFP, thyroglobulin, PRAC1, globoH, RAGE1, MN-CA IX, MSI frameshift mutant, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, or GFRa4.

[0037] Examples of CAR-T therapy and methods for producing and preparing CAR and T cells are described in Katz et al., Cancer Gene Therapy (2020), 27:341-355, Hege et al., Journal for ImmunoTherapy of Cancer (2017), 5:22, and Koneru et al., Journal of Translational Medicine (2015), 13:102. These methods are incorporated herein by reference.

[0038] Oncolytic viruses The term "oncolytic virus" refers to a virus from the Parvoviridae family, in particular "parvovirus", more particularly parvovirus H-1 or a related rodent parvovirus selected from LuIII, minute virus of mice (MMV), mouse parvovirus (MPV), minute virus of rat (RMV), rat parvovirus (RPV) or rat virus (RV).

[0039] As used herein, oncolytic viruses include wild-type or modified replication-competent derivatives thereof, as well as related viruses or vectors based on such viruses or derivatives. Suitable oncolytic viruses, derivatives, etc., as well as cells that can be used to actively produce said viruses and are useful for therapy, can be readily determined within the skill of the art based on the disclosure herein without undue empirical effort.

[0040] Parvovirus H-1 (H-1PV) belongs to the Parvoviridae family and is a small (approximately 25 nm in diameter), non-enveloped icosahedral particle containing a 5.1 kb-long, single-stranded DNA genome. The genome organization of H-1PV consists of two transcription units under the control of two promoters: the P4 early promoter and the P38 late promoter. P4 regulates the expression of the genes encoding the nonstructural (NS) proteins (NS1 and NS2) and P38, which encodes the capsid (VP) proteins (VP1, VP2, and VP3). The virus preferentially propagates in rapidly dividing cancer cells. This tumor selectivity is not due to better viral uptake by cancerous cells, but rather to the fact that cancer cells overexpress factors such as cyclin A, E2F, or CREB / ATF, which are required for viral DNA replication. Furthermore, cancer cells often lack the ability to mount an efficient antiviral immune response that favors viral propagation. Viruses are known to activate multiple cell death pathways. Depending on the cell type and growth conditions, H-1PV can induce apoptosis, necrosis, or cathepsin B-dependent cell death. The major nonstructural protein, NS1, is the master regulator of viral DNA replication, viral gene expression, and cytotoxicity. Expression of NS1 alone, as well as the whole virus, is sufficient to induce cell cycle arrest, apoptosis, and cell lysis through the accumulation of reactive oxygen species and DNA damage.

[0041] Treatment details Preferably, in the pharmaceutical formulations of the present invention, the oncolytic virus, i.e., parvovirus H-1, and immune cells (e.g., T cells) genetically modified to express a chimeric antigen receptor (CAR) are present in effective amounts and combined with a pharmaceutically acceptable carrier.

[0042] According to the present invention, the terms "pharmaceutical combination", "pharmaceutical composition" or "pharmaceutical formulation" are used interchangeably.

[0043] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or may not have a disease or disorder (e.g., cancer), but that may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient." The term "patient" refers to an individual or subject for treatment, particularly a diseased individual or subject.

[0044] In one embodiment of the present disclosure, the objective is to provide an immune response against diseased cells expressing an antigen, such as cancer cells expressing a tumor antigen, and to treat a disease, such as a cancer disease, in which cells expressing an antigen, such as a tumor antigen, are involved. An immune response against the antigen can be elicited that can be therapeutic or partially or completely protective. The pharmaceutical compositions described herein are applicable to inducing or enhancing an immune response. Thus, the pharmaceutical compositions described herein are useful in the prophylactic and / or therapeutic treatment of diseases in which the antigen is involved.

[0045] As used herein, "immune response" refers to the body's integrated response to an antigen or a cell expressing an antigen, and refers to a cellular immune response and / or a humoral immune response. Cellular immune responses include, but are not limited to, cellular responses to cells expressing an antigen. Such cells may be characterized by the expression of the antigen on their cell surface or by the presentation of the antigen by class I or class II MHC molecules. Cellular responses involve T lymphocytes, which can be classified as helper T cells (also called CD4+ T cells), which play a central role by regulating the immune response, or killer cells (also called cytotoxic T cells, CD8+ T cells, or CTLs), which induce apoptosis in infected or cancer cells. In one embodiment, administration of a pharmaceutical composition of the present disclosure includes stimulation of an anti-tumor CD8+ T cell response against cancer cells expressing one or more tumor antigens.

[0046] The present disclosure contemplates an immune response that may be protective, preventative, prophylactic, and / or therapeutic. As used herein, "induces an immune response" may indicate that there was no immune response to a particular antigen prior to induction, or that there was a basal level of immune response to a particular antigen prior to induction that was enhanced after induction. Thus, "induces an immune response" includes "enhances an immune response."

[0047] The term "immunotherapy" relates to the treatment of a disease or condition by inducing or enhancing an immune response.

[0048] The terms "vaccination" or "immunization" refer to the process of administering an antigen to an individual with the intent of inducing an immune response, for example, for therapeutic or prophylactic reasons.

[0049] As used herein, the term "drug" is understood to mean a substance that produces a desired effect in a tissue, system, animal, mammal, human, or other subject.

[0050] As used herein, the term "treat" and its derivatives refer to therapeutic therapy. In relation to a particular condition, treating means (1) ameliorating the condition or one or more of the biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that leads to or causes the condition, or (b) one or more of the biological manifestations of the condition, (3) alleviating one or more of the symptoms, effects, or side effects associated with the condition, or (4) slowing the progression of the condition or one or more of the biological manifestations of the condition.

[0051] The term "pharmacologically effective amount" or "therapeutically effective amount" refers to an amount that alone or together with further doses achieves a desired response or a desired effect. In the case of the treatment of a particular disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, particularly halting or reversing the progression of the disease. The desired response in the treatment of a disease can also be delaying or preventing the onset of the disease or symptoms. The effective amount of the compositions described herein depends on the condition being treated, the severity of the disease, individual patient parameters including age, physiological condition, size, and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Thus, the administered dose of the compositions described herein can depend on various such parameters. If the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) can be used. As used herein, "effective amount" refers to the amount of any of the components or constituents of a pharmaceutical formulation that elicits the biological or medical response of a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means any amount that results in improved treatment, cure, prevention, or amelioration of a disease, disorder, or side effect, compared to a corresponding subject who does not receive such amount. The term also includes within its scope amounts effective to enhance normal physiological function. "Effective doses" useful for treating and / or preventing these diseases or disorders can be determined using methods known to those skilled in the art.

[0052] Administration of a therapeutically effective amount of a combination of the present invention is advantageous over the individual component compounds in that the combination provides one or more of the following improved properties when compared to administration of a therapeutically effective amount of the component compounds individually: i) greater anti-cancer efficacy than the most active single agent; ii) synergistic or highly synergistic anti-cancer activity; iii) an administration protocol that provides enhanced anti-cancer activity with a reduced side effect profile; iv) reduced toxic effect profile; v) increased therapeutic window; or vi) increased bioavailability of one or both of the component compounds.

[0053] "Pharmaceutically acceptable" is meant to encompass any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the patient to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and administered to a subject in an effective dosage. Additional pharmaceutically compatible carriers can include gels, bioabsorbable matrix materials, implantable elements containing a therapeutic agent, or any other suitable vehicle, delivery or dispensing means or material(s).

[0054] Pharmaceutical compositions of the present invention may contain salts, buffering agents, preservatives, and optionally other therapeutic agents. In one embodiment, a pharmaceutical composition of the present disclosure comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0055] Suitable preservatives for use in pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0056] The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0057] The term "diluent" refers to a drug that is being diluted and / or thinned. Furthermore, the term "diluent" includes any one or more of a fluid, liquid or solid suspension and / or mixture medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0058] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which an active ingredient is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. As used herein, a carrier can be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's, lactated Ringer's, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0059] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0060] Pharmaceutical carriers, excipients, or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0061] As used herein, the term "cancer" refers to an abnormal growth of cells or tissues and is understood to include malignant neoplastic growth. The term "neoplastic" means or relates to a neoplasm. In some embodiments, the cancer is a solid tumor, particularly liver cancer (e.g., hepatocellular carcinoma), gastric cancer, ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer (e.g., (adeno)carcinoma of the cecum, appendix, ascending colon, descending colon, transverse colon, sigmoid colon, rectal carcinoma, or anal carcinoma), lung cancer (e.g., squamous cell lung carcinoma, non-small cell lung cancer (NSCLS), small cell lung cancer (SCLC)), soft tissue sarcoma, osteosarcoma, fibrosarcoma, skin cancer (e.g., malignant melanoma), testicular cancer, breast cancer, fibrosarcoma, neuroblastoma, brain cancer (e.g., glioma: ependymoma, astrocytoma, oligodendroglioma, brain stem glioma, oligoastrocytoma (e.g., glioblastoma multiforme, medulloblastoma)), bladder cancer, intestinal cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), pleural mesothelioma, head and neck squamous cell carcinoma (HNSCC), nasopharyngeal carcinoma (NPC) or oropharyngeal carcinoma (OPC). The term "cancer" also encompasses metastases of the mentioned tumors in various organs. In a further preferred embodiment, the tumor to be treated is a recurrent tumor. A particular advantage of the pharmaceutical preparation of the present invention is that even cancer-initiating stem cells can be successfully treated. This has a positive effect on avoiding tumor recurrence and metastasis formation.

[0062] In some embodiments, the cancer is a hematological cancer, particularly an acute or chronic leukemia or lymphoma. In some embodiments, the leukemia is selected from acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), or chronic lymphocytic leukemia (CLL). In some embodiments, the lymphoma is a non-Hodgkin's lymphoma. In some embodiments, the non-Hodgkin's lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt's lymphoma.

[0063] Administration of the compounds can be achieved through different systemic or local methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intratumoral, intranasal, or intradermal administration. The route of administration will, of course, depend on the type of treatment and the type of compound included in the pharmaceutical composition. The dosing regimen for the virus and CAR-T can be easily determined by the attending physician in the art based on patient data, findings, and other clinical factors (e.g., the patient's size, body surface area, age, sex, the specific virus, the specific inhibitor administered, etc., the time and route of administration, the type and characteristics of the tumor, the patient's overall health, and other drug therapies the patient is receiving). The selection of a dosing regimen (also referred to herein as an administration regimen) for the combination therapy of the present invention depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the availability of target cells, tissues, or organs in the individual being treated. Preferably, the dosing regimen maximizes the amount of each therapeutic agent delivered to the patient, consistent with an acceptable level of side effects. Thus, the dosage and frequency of administration of each therapeutic agent in the combination will depend, in part, on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance is available for selecting appropriate doses of antibodies, cytokines, and small molecules.For example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd. Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1193) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcek Dekker,New York,NY;Beart et al,(2003)New Engl.J.Med.348:601-608;Milgrom et al.(1999)New Engl.J.Med 341:1966-1973;Slamon et al.(2001)New Engl.J.Med.344:783-792;Beniaminovitz et al. al.(2000)New Engl.J.Med.342:613-619;Ghosh et al.(2003)New Engl.J.Med.348:24-32;Lipsky et al.(2000)New Engl.J.Med.343;1594-1602;Physicians`Desk Reference 2003(Physicians`Desk Reference,57. th ed);Medical Economics Company;ISBN;1563634457;57 th See, e.g., the American Journal of Clinical Oncology, Vol. 12, No. 1, 2002, pp. 1177-1182, 2002. Determination of an appropriate dosing regimen can be made by the clinician using, for example, parameters or factors known or suspected in the art to affect or predicted to affect treatment, and will depend, for example, on the patient's medical history (e.g., previous treatments), the type and stage of the cancer being treated, and biomarkers of response to one or more therapeutic agents in the combination therapy.

[0064] Since the virus combined with CAR cells according to the present invention contains infectious viral particles capable of penetrating the blood system, treatment can be carried out or at least initiated by intravenous injection of the virus.Since long-term intravenous treatment may be prone to inefficiency as a result of the formation of neutralizing antibodies against the virus, different administration modes can be adopted after the initial regimen of intravenous virus administration, or such different administration techniques, such as intratumoral virus administration, can be alternatively used throughout the entire course of virus treatment.However, in a preferred embodiment, administration is carried out throughout the entire course of treatment by intravenous administration.

[0065] In another specific administration technique, the virus (virus, vector, and / or cellular agent) can be administered to the patient from a source implanted in the patient. For example, a small subcutaneous reservoir (Lickham reservoir) placed in the patient during tumor removal or another procedure can be connected to a catheter, e.g., made of silicone or other biocompatible material, allowing the parvovirus composition to be injected locally at various times without further surgical intervention. The virus or derived vector can also be injected into the tumor using stereotactic or navigational targeting techniques.

[0066] Administration of the virus can also be achieved by continuous infusion of the virus particles or a fluid containing the virus particles at a low flow rate through an implanted catheter using a suitable pump system, such as a peristaltic infusion pump or a convection-enhanced delivery (CED) pump.

[0067] Another method of administering the virus-combined portion is through an implantable article constructed and designed to dispense the parvovirus into the desired cancer tissue. For example, a wafer impregnated with the virus, particularly parvovirus H-1, can be used, and the wafer is attached to the edge of the resection cavity at the end of surgical tumor removal. Multiple wafers can be used in such therapeutic interventions. Cells actively producing the virus or virus-based vector can be injected into the tumor or tumor cavity after tumor removal.

[0068] CAR-T cells and CAR-T cell compositions can be administered according to an appropriate dosing schedule. In certain embodiments, CAR T cells are administered once, with subsequent doses depending on clinical criteria. If an individual does not respond or only partially responds, the patient can have the CAR-T cell composition administered a second, third, or fourth time until a desired clinical response is observed. The dosage of CAR-T cells is generally at least 1x10 6 Contains 5x10 cells 8 The dose may contain no more than 100 cells. Cells may be administered based on the total amount of viable PBMCs of an individual transduced with the CAR construct. In certain embodiments, a single dose contains between 1 million and 100 million transduced PBMCs.

[0069] The parvovirus can be administered according to a suitable administration schedule. In certain embodiments, the virus is administered once, with subsequent doses depending on clinical criteria. If an individual does not respond or only partially responds, the patient can receive a second, third, or fourth dose until the desired clinical response is observed. The dosage of the parvovirus is generally at least 1 x 10 6 Contains pfu but 5x10 11 Contains no more than 1 x 10 pfu of cells. 7 pfu~5x10 10 A dose of pfu is administered.

[0070] It may also enable the clinical use of viruses and / or CAR cells at lower therapeutic doses, which improve safety and maintain or even enhance anti-cancer effects while reducing and / or avoiding side effects. Considering the strong synergistic effect between viruses and CAR cells, it is possible to predict a reduction in the therapeutic dose, for example, half or one-third of the previously used single component dose, while maintaining the desired therapeutic effect. Considering the lower dose, (severe) side effects may be reduced or even avoided.

[0071] In the case of parvoviruses, the infectious effect is to kill tumor cells but spare normal cells, and such infection can be achieved, for example, by intravenous or intratumoral use of a suitable parvovirus, such as parvovirus H-1, or related viruses or vectors based on such viruses, to provide tumor-specific therapy without adverse neurological or other side effects.

[0072] The combination therapy of the present invention is typically used to treat tumors large enough to be detected by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scan. In some preferred embodiments, the combination therapy of the present invention is used to treat tumors of at least about 200 mm 3 , 300mm 3 , 400mm 3 , 500mm 3 , 750mm 3 , or up to 1000mm 3 Advanced stage tumors having dimensions of 0.1 mm or less are treated.

[0073] The combination therapy of the present invention may be used before or after surgery to remove the tumor, and may be used before, during, or after chemotherapy or radiation therapy.

[0074] Thus, in some embodiments, treatment may further include other therapeutic or prophylactic interventions, such as chemotherapy, immunotherapy, radiation therapy, surgery, vaccination, and / or hormonal therapy. Such other therapeutic or prophylactic interventions may occur before, during, and / or after a treatment encompassed by the present disclosure, and delivery of the other therapeutic or prophylactic interventions may occur via a different route of administration than a treatment encompassed by the present disclosure. Chemotherapy and radiation therapy refer to the treatment of cancer with therapeutic agents or ionizing radiation (e.g., radiation therapy using X-rays or g-rays), respectively.

[0075] In some embodiments, at least one of the therapeutic agents in the combination therapy is administered using the same dosing regimen (dosage, frequency, and duration of treatment) as would typically be used when the agent is used as a monotherapy to treat the same cancer. In other embodiments, the patient receives a lower total amount of at least one therapeutic agent in the combination therapy than when the agent is used as a monotherapy, e.g., a lower dose, a less frequent dose, and / or a shorter duration of treatment.

[0076] The additional therapeutic agent can be, for example, a chemotherapeutic agent, a biotherapeutic agent (including, but not limited to, antibodies against VEGF, EGFR, Her2 / neu; growth factor receptors, CD20, CD40, CD40L, CTLA-4, OX-40 4-1BB, and ICOS; antibody fragments), a nucleic acid (DNA or RNA), an immunogenic agent (e.g., attenuated cancerous cells, tumor antigens, tumor-derived antigens, or antigen-presenting cells such as dendritic cells pulsed with nucleic acids), an immunostimulatory cytokine (e.g., IL-2, IFN-α, IFN-γ, GM-CSF), and cells transfected with a gene encoding an immunostimulatory cytokine (such as, but not limited to, GM-CSF), or a checkpoint inhibitor (such as, but not limited to, an anti-PD1 antibody or an anti-PD-L1 antibody, e.g., nivolizumab, pembrolizumab, ipilimumab, tremelimumab, avelumab).

[0077] Examples of chemotherapeutic agents include alkylating agents, such as cyclophosphamide, busulfan, camptothecin, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, antibiotics, bleomycin, caminomycin, dactinomycin, daunorubicin, idarubicin, 5-fluorouracil (5-FU), methotrexate, cytarabine, platinum analogs (such as cisplatin and carboplatin); vinblastine, platinum; etoposide (V P-16); ifosfamide, mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin, xeroda; ibandronate; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoids, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene or aromatase inhibitors.

[0078] The present invention also relates to the use of (a) an oncolytic virus (particularly parvovirus H-1) and (b) immune cells (particularly T cells) genetically modified to express a chimeric antigen receptor (CAR) for preparing (a) a therapeutic formulation(s) or pharmaceutical composition(s) or combination for treating cancer.

[0079] The administration of (a) and (b) can be simultaneous or sequential, with (a) and (b) being preferably administered sequentially or separately. This means that (a) and (b) can be provided in a single unit dosage form to be taken together or as separate entities (e.g., in separate containers) so that they are administered simultaneously or with a time lag. This time lag can be from 1 hour to 1 week, preferably from 12 hours to 3 days, and most preferably from 24 to 60 hours. It is also possible to administer the virus via a different administration method from the CAR cells. In this regard, it may be advantageous to administer either the virus or the CAR cells intratumorally and the other systemically. In a particularly preferred embodiment, the virus is administered intravenously and the CAR cells are administered intratumorally. Preferably, the virus and CAR cells are administered as separate compounds. Combination treatment using two agents is also possible.

[0080] Each therapeutic agent in the combination therapy of the invention can be administered alone or in a pharmaceutical preparation comprising the therapeutic agent and one or more pharmaceutically acceptable carriers, excipients, and diluents (also referred to herein as a "pharmaceutical composition" or "pharmaceutical formulation") in an amount in accordance with standard pharmaceutical practice as described above. Each therapeutic agent in the combination therapy of the invention can be administered simultaneously, concurrently, or sequentially in any order.

[0081] Simultaneous administration refers to the administration of agents together, e.g., as a pharmaceutical composition containing the agents (i.e., a combined preparation), or shortly after each other, optionally via the same route of administration, e.g., into the same artery, vein, or other blood vessel.

[0082] Sequential administration refers to the administration of one or more agents followed by separate administration of another agent after a given time interval. Sequential administration is particularly useful when the therapeutic agents in the combination therapy are in different dosage forms (solid / liquid) and / or administered on different dosing schedules, for example, when one is administered at least daily and the biologic therapeutic agent is administered less frequently, for example, once a week, once every two weeks, or once every three weeks. The time interval may be any time interval, including hours, days, weeks, or months. In some embodiments, sequential administration refers to administrations separated by a time interval of at least 10 minutes, 30 minutes, 1 hour, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months. In a preferred embodiment, the parvovirus is administered first, i.e., at a fixed time interval before the administration of CAR-T cells. This time difference may be at least 10 hours, but may be as long as 4 days, preferably 18 to 72 hours, and most preferably 24 to 60 hours. The two agents need not be administered by the same route, although in some embodiments they are.

[0083] The CAR cells and oncolytic viruses described herein can be provided as a kit comprising a first container, a second container, and a package insert. The first container contains at least one dose of CAR cells, and the second container contains at least one dose of the oncolytic virus, and the package insert or label includes instructions for treating a patient for cancer with the therapeutic preparation. The first and second containers can be composed of the same or different shapes (e.g., vials, syringes, and booles) and / or materials (e.g., plastic or glass). The kit can further include other materials that may be useful for administering the preparation, such as diluents, filters, IV bags and lines, needles, and syringes.

[0084] The present invention demonstrates for the first time that the combined use of the oncolytic virus H-1PV and CAR cells can be an effective approach against solid tumors.

[0085] Without wishing to be bound by theory, as previously mentioned, tumors hide from attack by the immune system. Many solid tumors have a microenvironment that makes it impossible for activated immune cells to invade the tumor, leading to the body's immune tolerance to the tumor. This tumor infiltration is currently possible through the use of oncolytic viruses, particularly parvoviruses, more specifically parvovirus H-1, which attack the tumor and alter its microenvironment. In other words, oncolytic viruses can "naked" the tumor through tumor lysis, allowing CAR-T cells to begin infiltrating the tumor. Oncolytic viruses can be seen as a door opener for a successful immune response due to their ability to stimulate pro-inflammatory and pro-migration cytokines and chemokines (Figure 23). Under this concept, because oncolytic virus therapy transforms non-immunogenic tumors into immunogenic ones, it should be possible to treat solid tumors for which CAR-T treatments have previously failed. Considering the general principle, this works with any oncolytic virus and any cell-based therapy as long as it alters the tumor microenvironment. This may have a long-term effect in preventing disease recurrence, potentially adding to the initial tumor lysis. The combined effect makes the tumor more susceptible to the immune system, especially after previous treatment with the virus.

[0086] The invasive margin of tumors has been found to be a highly specific region dominated by T cell-attracting chemokines and myeloid cell-associated factors, involving many different immune cell subtypes, including immunosuppressive cells and T cells (Halama et al., Cancer Cell 29:587-601 (2016); WO 2016 / 066634). Because such a distinct microenvironment is difficult to reproduce in animal models (Ellis and Fidler, Nat. Med. 16:974-975 (2010)), we established an ex vivo cell migration analysis model to study T cell infiltration and positioning in the original environment of tumor patients. The experimental design of the model is shown in Figure 1. The use of colorectal cancer liver metastasis (CRC-LM) should be understood as an example. The principle is the same for any tumor tissue.

[0087] Thus, in this application, a proof-of-concept of the combinatorial effect has been demonstrated in an explant model (Figure 1) that can recapitulate the distinct microenvironments of the original environment of a sample taken from a tumor patient, allowing for ex vivo cell migration analysis to study T cell infiltration. In this application, engaging CARs has been shown to activate and modulate the tumor environment, as evidenced by increased TH1 cytokines and more pro-migration chemokines. Furthermore, upregulation of tonic T cell-stimulating interleukins (prolonged activation and initiation of differentiation) has been observed. CARs bearing cancer cell antigens relevant to the tumor sample tested (e.g., CEA<->colorectal cancer; CA-125<->ovarian carcinoma) produce dramatically different cytokine environments compared to "mock-CARs" bearing cancer cell antigens not relevant to the tumor sample tested (see Figures 9, 10, 11, 12, 16, 17, 20, 21, and 22). Thus, distinct characteristics of CEA-CARs versus mock-CARs have been recognized, implying that specific CARs and their activation result in distinct anti-tumor environments. In this application, we used CEA-CARs as mock-CARs in an ovarian cancer model and CA-125CARs as mock-CARs in a CRC-LM cancer model. Comparing cytokine regulation between CARs with and without parvovirus H-1 addition clearly shows that parvovirus addition not only enhances T cell activation and migration, but also antigen presentation, including GM-CSF, IL-10, CXCL9, CXCL10 (IP-10), IL-5, and IL-6 (Figure 13). This implies a massive increase in TH1-type cytokines, massive upregulation of migration-promoting chemokines, and upregulation of tonic T cell-stimulating interleukins (prolonged activation and initiation of differentiation).

[0088] Furthermore, the presence of parvovirus H-1 has been shown to significantly improve deep tumor tissue infiltration (Figures 6, 7, 8, 14, 15, 18, and 19). Therefore, in summary, the addition of parvovirus H-1 significantly improves T cell infiltration. The infiltration of specific CAR-modified T cells is enhanced, replacing simple T cell infiltration. The infiltrating specific CAR-T cells are activated to produce TH1 cytokines and chemokines for further infiltration, which is most likely due to the CAR engaging the tumor. This effect is not observed when a mock-CAR, which does not engage the tumor, is used.

[0089] The term "about" means "approximately" or "nearly," and in the context of numerical values ​​or ranges set forth herein, means, in one embodiment, ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0090] As used in the context of describing the present disclosure (particularly in the context of the claims), the terms "a," "an," and "the" and similar references should be construed to encompass both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better explain the disclosure and does not limit the scope of the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0091] Unless otherwise indicated, the term "comprising" is used in the context of this specification to indicate that additional members may optionally be present in addition to the members of the list introduced by "comprising." However, for certain embodiments of the present disclosure, the term "comprising" is intended to encompass the possibility that additional members are not present, i.e., for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of."

[0092] Furthermore, the drawings, materials, methods, and examples are illustrative only and not intended to be limiting. Headings, subheadings, or numbered or lettered elements, e.g., (a), (b), (i), etc., are presented solely for ease of reading. The use of headings or numbered or lettered elements herein does not require that the steps or elements be performed in alphabetical order or that the steps or elements are necessarily separate from one another. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0093] In the figures and examples below, "Parvoryx" refers to an administrable formulation containing wild-type parvovirus H-1. [Brief explanation of the drawings]

[0094] [Figure 1] Human CRC liver metastasis ex vivo cell migration analysis model [Figure 2] Principles of CAR-T therapy [Figure 3] CAR cell generation [Figure 4] Immunohistochemistry to confirm CEA target structure positivity [Figure 5] CMFDA-labeled CAR-transduced patient T cells were placed in the explant medium, and the explants were harvested 24 hours later. Subsequent fluorescence imaging shows that the surface of the explant (colorectal cancer liver metastasis) has infiltrated to a depth of 200 μm (white line). [Figure 6] CMFDA-labeled CEA-CAR-transduced patient T cells after administration of ParvOryx (in culture medium) show enhanced massive infiltration exceeding 200 μm in depth (white line). Enlargement shows CMFDA-positive lymphocytes within colorectal cancer liver metastasis tissue. [Figure 7] Density of specific CEA-CAR transduced patient T cells compared with and without previous application of ParvOryx virus (Mann-Whitney non-parametric test). [Figure 8] Comparison of specific CEA-CAR vs. mock CAR transduced T cell infiltration demonstrating the clear ability of ParvOryx administration to enhance infiltration. [Figure 9] Negligible differences for specific CEA-CAR vs. mock-CAR transduced T cell infiltration (Mann-Whitney non-parametric test). ParvOryx administration enhances T cell infiltration independent of CAR specificity. [Figure 10] Cytokine level differences between specific CEA-CAR versus mock-CAR transduced T cell infiltrated tissues highlighting the specific TH1-like activation pattern only for specific CAR-transduced T cells. [Figure 11] Cytokine level differences between specific CEA-CAR versus mock-CAR transduced T cell infiltrated tissues highlighting the specific TH1-like activation pattern only for specific CAR-transduced T cells. [Figure 12] Cytokine level differences between specific CEA-CAR versus mock-CAR transduced T cell infiltrated tissues highlighting the specific TH1-like activation pattern only for specific CAR-transduced T cells. [Figure 13] Percentage differences in tissue cytokines comparing infiltrated specific CEA-CAR-transduced T cells with those infiltrated with concomitant ParvOryx administration. Asterisks indicate differential infiltration-dependent cytokine modulation and indicate specific T cell activation. [Figure 14]CMFDA-labeled CA125-CAR-transduced patient T cells after administration of ParvOryx (media) show enhanced massive infiltration in ovarian cancer to a depth of over 200 μm. Top: No parvovirus; bottom: + parvovirus. Enlargement shows CMFDA-positive lymphocytes within ovarian cancer tissue (white arrow at bottom). [Figure 15] Density of specific CA125-CAR transduced patient T cells compared with and without previous application of ParvOryx virus (Mann-Whitney non-parametric test). [Figure 16] Cytokine level differences between specific CA125-CAR versus mock-CAR transduced T cell infiltrated tissues highlighting the specific TH1-like activation pattern only for specific CAR-transduced T cells. [Figure 17] Differences in cytokine levels between specific CA125-CAR versus mock-CAR transduced T cell infiltrated tissues highlighting the specific TH1-like activation pattern only for specific CAR-transduced T cells and for the maintenance and differentiation of signals such as IL-7. [Figure 18] CMFDA-labeled CA125-CAR-transduced patient T cells after administration of ParvOryx (media) show enhanced massive infiltration exceeding 200 μm depth in ovarian cancer. [Figure 19] Density of specific CA125-CAR transduced patient T cells compared with and without previous application of ParvOryx virus (Mann-Whitney non-parametric test). [Figure 20] Cytokine level differences between specific CA125-CAR versus mock-CAR transduced T cell infiltrated tissues highlighting the specific TH1-like activation pattern only for specific CAR-transduced T cells. [Figure 21] Cytokine level differences between specific CA125-CAR versus mock-CAR transduced T cell infiltrated tissues highlighting the specific TH1-like activation pattern only for specific CAR-transduced T cells. [Figure 22]Differences in cytokine levels between specific CA125-CAR versus mock-CAR transduced T cell infiltrated tissues highlighting a specific TH1-like activation pattern for specific CAR-transduced T cells only, and for maintenance and differentiation of signals such as IL-5 and IL-7. [Figure 23] ParvOryx-induced changes in cytokine levels (mean values ​​shown for three ovarian cancer explants at 24 hours of treatment as a ratio to untreated controls) and CD8 T cell density (last column). The cytokines listed above are identical to those seen in other cancer entities (colorectal liver metastases and pancreatic cancer).

[0095] example patient: (1) Colorectal cancer with liver metastasis Patients who have had multiple lines of chemotherapy Resection of CRC liver metastases (CRCLM) CEA-positive tumor cells Successful peripheral blood sampling and T cell extraction Successful CAR transfection (mock and CEA-specific CAR) (2) Ovarian cancer (advanced stage) OvCa183 Patients receiving first-line chemotherapy Resection of abdominal tumor signs (OVCA) CA125 positive tumor cells according to pathology report Successful peripheral blood sampling and T cell extraction Successful CAR transfection (mock and CA125-specific CAR) (3) Ovarian cancer (advanced stage) OvCa184 Patients after first-line chemotherapy Resection of abdominal tumor signs (OVCA) CA125-positive tumor cells confirmed by pathology Successful peripheral blood sampling and T cell extraction Successful CAR transfection (mock and CA125-specific CAR)

[0096] Example 1: Off-factory model tissue culture Freshly resected tumor tissue (CRCLM or ovarian cancer) was transferred directly from the operating room to the laboratory in 0.9% saline solution (Sigma-Aldrich) on ice. Using forceps and a scalpel, each tissue was placed in a Petri dish and divided into small pieces containing equal proportions of invasive margins. One tissue piece was directly frozen and kept as a control. An additional piece of adjacent tissue (e.g., liver) was separated for autologous T cell isolation. The tissue pieces were placed in a 96-well plate and cultured under sterile conditions at 37°C and 5% carbon dioxide in MEM containing 2.9% 7.5% sodium bicarbonate solution and 1% 200 mM L-glutamine solution (Sigma-Aldrich). If necessary, the pH of the medium was adjusted to 7.4 using sodium hydroxide. After 24–72 h, the tissue was harvested, placed in a small plastic bowl (Sakura Finetek Germany GmbH) containing tissue embedding compound (VWR), and directly frozen in liquid nitrogen. The frozen tissue was stored at -80°C until further use.

[0097] Isolation and labeling of T cells For the isolation of autologous T cells, approximately 0.5 cm 2 Freshly excised human CRC-LM tissue fragments (adjacent liver, if available) were placed in a Petri dish and minced with a scalpel. The sheared tissue was gently placed on a cell strainer (40 μm mesh) with forceps and transferred to a 50 ml Falcon tube using RPMI (Sigma-Aldrich). The flow-through was passed through a new cell strainer for a second time, and the isolated cells were stained with 5 μM CellTracker™ Green CMFDA (Thermo Fisher Scientific) for 1 hour in a cell culture flask. Nonadherent T cells were isolated by negative bead-based isolation using the Dynabeads Untouched Human T Cells Kit (Thermo Fisher Scientific). Autologous CMFDA T cells were directly resuspended in tissue culture medium for ex vivo cell migration analysis.

[0098] Donor T cells were obtained from peripheral blood of healthy donors by density gradient centrifugation using Ficoll-Paque Plus (Sigma-Aldrich), followed by 1.5 hours of separation of nonadherent cells in cell culture flasks and negative bead-based T cell isolation. To stimulate proliferation, isolated donor T cells were cultured for 48 hours in X-vivo 15 medium (Biozym) with the addition of anti-human CD3 antibody (1:10,000, clone OKT3, BioLegend) and 300 units of IL-2 (PeproTech) daily. T cells were stained with 5 μM CMFDA for 1 hour and cryopreserved in FBS (Biochrom) containing 10% DMSO. Frozen CMFDA donor cells were stored at -80°C until use in ex vivo cell migration analysis.

[0099] Example 2: Creation of CAR-T cells To obtain primary T cells, 50 ml of blood was collected from three healthy donors into EDTA collection tubes. Fresh blood was thoroughly pipetted onto a layer of 10 ml Ficoll (Ficoll Paque™, density: 1.077; GE Healthcare) and centrifuged at 750 x g for 30 minutes without braking. Lymphocytes were removed and washed twice with PBS. Cell counts were determined, and CD3 + T cell isolation was performed using the Human Pan T Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer's instructions. The number of T cells was determined after sorting and was 1 x 10 6 / ml / cm 2The cells were seeded in activation medium (XVIVO20 (Lonza) containing 100 ng / ml anti-CD3 antibody (clone: ​​OKT3; Janssen-Cilag) and 300 U / ml interleukin-2 (ProLeukin®; Novartis)) for 24 h at 37°C. After activation, T cells were washed three times in PBS and further incubated in culture medium (XVIVO20; 300 U / ml IL-2). 48 h after T cell isolation, lentiviral CAR transduction was performed using the spinoculation method. Therefore, cells were plated in Retronectin®-coated 24-well plates (16 μg / ml Retronectin®; 1x10 6 CD3 + cells / ml / cm 2 ) were seeded onto the cells, and lentiviral particles encoding a CAR gene expression cassette (4H11scFv-IgG-28BBz or SCA431scFV-IgG-28BBz) were added at an MOI of 10. The plates were centrifuged at 2000g and 32°C for 1.5 hours, followed by incubation at 37°C for 24 hours. The medium containing the lentiviral particles was then removed and replaced with culture medium. 72 hours after the initial cell sorting, CAR-expressing CD3 + The percentage of cells was determined by flow cytometry. Detailed information for the different donors is shown in the table below. [Table 1]

[0100] Example 3: Processing in the off-factory model ParvOryx, a GMP-grade preparation of H-1 PV, was used. H-1 PV was grown in tissue culture at 1x10 7 pfu / ml, and after 60 minutes the above number of CAR cells was added and incubated for a further 60 minutes.

[0101] Explants treated with parvovirus H-1 and / or CAR-T cells or mock-CAR-T cells were then subjected to histological analysis (Figures 4, 5, 6, 8) and multiplex protein analysis (Figures 10, 11, 12, 16, 17).

Claims

1. A pharmaceutical combination comprising (a) an oncolytic virus and (b) an immune cell, particularly a T cell, that has been genetically modified to express a chimeric antigen receptor (CAR) specific for a cancer cell antigen.

2. 2. The pharmaceutical combination of claim 1, wherein the oncolytic virus is parvovirus H-1 or a related rodent parvovirus selected from LuIII, minute virus of mice (MMV), mouse parvovirus (MPV), minute virus of rat (RMV), rat parvovirus (RPV) or rat virus (RV).

3. The cancer cell antigen is selected from the group consisting of mesothelin, EGFRvIII, GD2, Tn antigen, PSMA, PSA, CD70, CD97, TAG72, CD44v6, CEA, CA125, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, and Ephrin. B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, legumain, HPV E6 or E7, p16 ＩＮＫ４ａ , ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, MSI frameshift mutant, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, or GFRα4.

4. The pharmaceutical combination according to claim 3, wherein the cancer cell antigen is CEA or CA125.

5. 5. The pharmaceutical combination of any one of claims 1 to 4, further comprising one or more additional therapeutic agents selected from a chemotherapeutic agent, a biotherapeutic agent, an immunogenic agent, an immunostimulatory cytokine, and a cell transfected with a gene encoding an immunostimulatory cytokine.

6. A pharmaceutical combination according to any one of claims 1 to 5 for use in a method for treating cancer.

7. 7. The pharmaceutical combination for use according to claim 6, wherein the oncolytic virus and the immune cells genetically modified to express a chimeric antigen receptor (CAR) specific for a cancer cell antigen are administered sequentially.

8. The pharmaceutical combination for use according to claim 6 or 7, wherein the immune cells are T cells.

9. The pharmaceutical combination for use according to any one of claims 6 to 8, wherein the use is for treating solid tumors, blood cancers and / or cancer-initiating stem cells.

10. 10. The pharmaceutical combination for use according to any one of claims 6 to 9, wherein the cancer is colon cancer, bladder cancer, liver cancer, breast cancer, kidney cancer, head and neck squamous cell carcinoma, lung cancer, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, brain cancer, cervical cancer, renal cell carcinoma, or gastric cancer.

11. The pharmaceutical composition for use according to any one of claims 6 to 10, wherein the oncolytic virus and / or the CAR immune cells are administered by intratumoral or intravenous administration.

12. 1. A kit comprising a first container, a second container, and a package insert, wherein the first container contains at least one dose of a pharmaceutical composition containing an oncolytic virus, the second container contains at least one dose of immune cells, preferably T cells, genetically modified to express a chimeric antigen receptor (CAR) specific for a cancer cell antigen, and the package insert comprises instructions for treating an individual with cancer.

13. 13. The kit of claim 12, wherein the cancer is colon cancer, bladder cancer, liver cancer, breast cancer, kidney cancer, head and neck squamous cell carcinoma, lung cancer, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, brain cancer, cervical cancer, renal cell carcinoma, or gastric cancer.

14. A method for inhibiting the proliferation and / or activity of tumor antigen-positive cells in an individual, comprising the step of providing to said individual a therapeutically effective amount of the pharmaceutical combination of any one of claims 1 to 5.

15. 15. The method of claim 14, wherein the components (a) and (b) of the pharmaceutical combination are provided separately by intratumoral or intravenous administration.

16. An oncolytic virus for use in a method of increasing TH1-type cytokines, upregulating pro-migration chemokines, and upregulating tonic T cell stimulating interleukins in the tumor microenvironment.

17. 17. The oncolytic virus for use according to claim 16, wherein the oncolytic virus is parvovirus H-1 or a related rodent parvovirus selected from LuIII, minute virus of mice (MMV), mouse parvovirus (MPV), minute virus of rat (RMV), rat parvovirus (RPV) or rat virus (RV).

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