Oncolytic virus therapy and immunotherapy

Abstract

To provide effective cancer therapies, including combinatorial cancer therapies.SOLUTION: Provided is a combination therapy for cancer that utilizes: (i) an oncolytic virus; (ii) a virus comprising a nucleic acid encoding an immunomodulatory factor; and (iii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen. In particular embodiments, the virus comprising a nucleic acid encoding an immunomodulatory factor comprises a nucleic acid encoding IL-12 and / or antagonist anti-PD-L1 antibody.SELECTED DRAWING: Figure 24D

Description

[Technical field]

[0001] The present disclosure relates to at least the fields of cell biology, molecular biology, immunology, virology, and medicine, such as cancer therapy. In certain embodiments, the disclosure relates to combination treatments involving the use of oncolytic virus therapy and immunotherapy. [Background technology]

[0002] Oncolytic virus therapy for head and neck squamous cell carcinoma (HNSCC) HNSCC is the sixth most common cancer worldwide. Treatment of locally advanced, recurrent and metastatic HNSCC is often limited by an unfavorable efficacy to toxicity ratio, and the median survival for patients with metastatic disease remains less than one year (Zandberg and Strome, Oral Oncology (2014) 50:627-632). HNSCC is a type of cancer that develops at or near the surface of the body. Because of the present locoregional disease, tumors are amenable to initial intratumoral injection of adenoviral vectors (Ad) to stimulate locoregional and even systemic antitumor immune responses (Liu et al., Nature Clinical Practice Oncology (2007) 4:101-117). Numerous clinical trials of conditionally replicating Ad (OncAd) or replication-defective Ad encoding therapeutic transgenes have demonstrated the safety and feasibility of Ad gene therapy for HNSCC, but failed to demonstrate improved overall survival, as intensive local treatment did not prevent metastasis to distant sites even when combined with chemo / radiotherapy (Liu et al., supra). OncAd is commonly administered intratumorally, but retargeting to metastatic tumors is poor (Koksi et al., Molecular Therapy: The Journal of the American Society of Gene Therapy (2015) 23:1641-1652).

[0003] OncAd carrying helper-dependent Ad (HDAd) expressing immunomodulatory molecules Adenovirus-based vectors (Ad) are attractive agents for cancer gene therapy because they can infect a variety of malignant cells and express high levels of soluble antigens and immunogenic transgenes (Cerullo et al., Advances in Cancer Research (2012) 115, 265-318). OncAd selectively replicates in cancer cells and is commonly used as an Ad-based vector in clinical trials for cancer gene therapy. However, OncAd has only limited transgene coding capacity (approximately 1.5 kb). Helper-dependent Ad (HDAd) lacks viral coding sequences and allows for a cargo capacity of up to 34 kb for the insertion of multiple transgenes in a single vector (Suzuki et al., Human Gene Therapy (2010) 21; 120-126). Because HDAd vector DNA encodes packaging signals, the OncAd replication machinery acts in trans to replicate and package both OncAd and HDAd in infected tumor cells, resulting in multiple cycles of production and release of both oncolytic virus and transgenes encoded by HDAd (combinatorial adenoviral vector: CAd-VEC; Farzad et al., Molecular Therapy - Oncolytics (2014) 1, 14008).

[0004] CAR T cell therapy In recent years, the use of T cells as cancer therapeutics has been facilitated by the expression of chimeric antigen receptors (CARs) directed against cancer cell antigens. (Reviewed in ). CAR-modified T cells have shown promise in the treatment of hematological malignancies (Garfall et al., The New England Journal of Medicine (2015) 373:1040). ~1047), but is less effective in treating solid tumors, which may be in part a result of the highly immunosuppressive nature of the solid tumor microenvironment (Quail et al., Nature Medicine (2013) 19:1423-1437). Due to immunosuppressive mechanisms at the tumor site, CAR T cells expand long-term despite expression of one or two costimulatory endodomains. Can't sustain it for very long. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Zandberg and Strome, Oral Oncology (2014) 50:627-632 [Non-Patent Document 2] Liu et al., Nature Clinical Practice Oncology (2007) 4:101–117 [Non-Patent Document 3] Molecular Therapy: The Journal of the American Society of Gene Therapy(2015)23:1641~1652 [Non-Patent Document 4] Cerullo et al., Advances in Cancer Research (2012) 115, 265–318 [Non-Patent Document 5] Suzuki et al., Human Gene Therapy (2010) 21;120-126 [Non-Patent Document 6] Farzad et al., Molecular Therapy - Oncolytics (2014)1, 14008 [Non-Patent Document 7] Kershaw et al., Nature (2013) 13:525-541 [Non-Patent Document 8] Garfall et al., The New England Journal of Medicine (2015) 373:1040-1047 [Non-Patent Document 9] Quail et al., Nature Medicine (2013) 19:1423-1437 Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure provides a solution to the long felt need for effective cancer therapies, including combinatorial cancer therapies. [Means for solving the problem]

[0007] A brief summary In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (i) Oncolytic viruses; (ii) a virus comprising a nucleic acid encoding an immunomodulator; and (iii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen; The present invention provides a method for treating cancer comprising administering to a subject

[0008] Also provided is a combination of (i) an oncolytic virus, (ii) a virus comprising a nucleic acid encoding an immune modulator, and (iii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen, for use in a method of treating cancer.

[0009] Also provided is the use of (i) an oncolytic virus, (ii) a virus comprising a nucleic acid encoding an immune modulator, and (iii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen in the manufacture of a medicament for use in a method of treating cancer.

[0010] (i) an oncolytic virus; and (ii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen; Also provided is a method of treating cancer comprising administering to a subject.

[0011] Also provided is a combination of (i) an oncolytic virus and (ii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen, for use in a method of treating cancer.

[0012] In the manufacture of a medicament for use in a method of treating cancer, the method comprises: (i) an oncolytic virus; and (ii) at least one compound comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen. Use of a single cell is also provided.

[0013] In some embodiments, the cells containing the CAR are specific for the oncolytic virus. (i) an oncolytic virus; and (ii) at least one immune cell specific for the oncolytic virus; Also provided is a method of treating cancer comprising administering to a subject.

[0014] Also provided is a combination of (i) an oncolytic virus and (ii) at least one immune cell specific for the oncolytic virus for use in a method of treating cancer. There is also provided the use of (i) an oncolytic virus, and (ii) at least one immune cell specific for the oncolytic virus, in the manufacture of a medicament for use in a method of treating cancer.

[0015] In some embodiments, the oncolytic virus is an oncolytic adenovirus (OncAd). In some embodiments, the oncolytic virus is derived from adenovirus 5 (Ad5). In some embodiments, the oncolytic virus encodes an E1A protein that exhibits reduced binding to Rb protein compared to the E1A protein encoded by Ad5. In some embodiments, the oncolytic virus encodes an E1A protein that lacks the amino acid sequence LTCHEACF (SEQ ID NO:52). In some embodiments, the oncolytic virus encodes an E1A protein that comprises, consists of, or consists essentially of the amino acid sequence SEQ ID NO:34. In some embodiments, the oncolytic virus comprises a nucleic acid that has one or more binding sites for one or more transcription factors. In some embodiments, the oncolytic virus comprises a nucleic acid that has one or more binding sites for STAT1.

[0016] In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator is a helper-dependent adenovirus (HDAd). In some embodiments, the immunomodulator is selected from an agonist of an effector immune response or an antagonist of an immunomodulatory response. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator comprises a nucleic acid encoding IL-12 and / or an antagonistic anti-PD-L1 antibody. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator comprises a nucleic acid encoding an enzyme capable of catalyzing the conversion of a non-toxic factor to a cytotoxic form. In some embodiments, the enzyme is selected from thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, horseradish peroxidase, and carboxylesterase. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator comprises a nucleic acid encoding a thymidine kinase.

[0017] In some embodiments, at least one cell comprising a CAR specific for a cancer cell antigen is a T cell. In some embodiments, the CAR comprises an antigen-binding domain capable of specifically binding to HER2. In some embodiments, the CAR comprises: LC-CRD1: SEQ ID NO: 10; LC-CRD2: SEQ ID NO:11; LC-CRD3: VL domain comprising SEQ ID NO: 12; and HC-CRD1: SEQ ID NO: 13; HC-CRD2: SEQ ID NO: 14; HC-CRD3: VH domain comprising SEQ ID NO: 15; or LC-CRD1: SEQ ID NO: 18; LC-CRD2: SEQ ID NO: 19; LC-CRD3: VL domain comprising SEQ ID NO: 20; and HC-CRD1: SEQ ID NO:21; HC-CRD2: SEQ ID NO:22; HC-CRD3: VH domain comprising SEQ ID NO:23; or LC-CRD1: SEQ ID NO:26; LC-CRD2: SEQ ID NO:27; LC-CRD3: VL domain comprising SEQ ID NO:28; and HC-CRD1: SEQ ID NO:29; HC-CRD2: SEQ ID NO:30; HC-CRD3: VH domain comprising SEQ ID NO:31; or LC-CRD1: SEQ ID NO:57; LC-CRD2: SEQ ID NO:58; LC-CRD3: VL domain comprising SEQ ID NO:59; and HC-CRD1: SEQ ID NO:60; HC-CRD2: SEQ ID NO:61; HC-CRD3: VH domain comprising SEQ ID NO:62 The antigen-binding domain comprises:

[0018] In some embodiments, the CAR is a VL comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO: 16, and a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO: 17; or a VL comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO:24, and a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO:25; or a VL comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO: 32, and a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO: 33; or A VL comprising or consisting of an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 63, and a VH comprising or consisting of an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 64. The antigen-binding domain comprises:

[0019] In some embodiments, the method further comprises: (a) isolating at least one cell from a subject, where in a specific embodiment, the cell is an immune cell; (b) modifying at least one cell to express or contain a cancer cell antigen-specific CAR or a nucleic acid encoding a cancer cell antigen-specific CAR; (c) optionally, expanding the at least one modified cell; and (d) administering at least one modified cell to a subject, wherein in a specific embodiment, the modified cell is provided to the subject at the time of administration along with one or more other agents for cancer therapy. Includes.

[0020] In some embodiments, the method of treating cancer comprises: (a) isolating at least one cell from a subject; (b) modifying at least one cell to express or contain a cancer cell antigen-specific CAR or a nucleic acid encoding a cancer cell antigen-specific CAR; (c) optionally, expanding the at least one modified cell; and (d) administering at least one modified cell to a subject. Includes.

[0021] In some embodiments, the method of treating cancer comprises: (a) isolating immune cells from a subject; (b) generating or expanding a population of immune cells specific for the oncolytic virus by a method comprising stimulating the immune cells by culture in the presence of antigen-presenting cells (APCs) that present a peptide of the oncolytic virus; and (c) administering to the subject at least one immune cell specific for the oncolytic virus.

[0022] In some embodiments, the cancer is selected from head and neck cancer, nasopharyngeal carcinoma (NPC), cervical cancer (CC), oropharyngeal carcinoma (OPC), gastric cancer (GC), hepatocellular carcinoma (HCC), and lung cancer.

[0023] The present disclosure also provides an oncolytic adenovirus (OncAd) encoding an E1A protein comprising, consisting of, or consisting essentially of the amino acid sequence SEQ ID NO:34.

[0024] The present disclosure also provides an oncolytic adenovirus (OncAd) comprising a nucleic acid having one or more binding sites for STAT1. In some embodiments, the OncAd comprises a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO:51, or an equivalent sequence as a result of codon degeneracy.

[0025] Also provided is an OncAd comprising a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO: 55, or an equivalent sequence as a result of codon degeneracy. In some embodiments, the OncAd encodes an E1A protein comprising, consisting of, or consisting essentially of the amino acid sequence SEQ ID NO: 34.

[0026] The present disclosure also provides a helper-dependent adenovirus (HDAd) comprising a nucleic acid encoding IL-12 and / or an antagonistic anti-PD-L1 antibody. In some embodiments, the HDAd additionally comprises a nucleic acid encoding an enzyme capable of catalyzing the conversion of a non-toxic factor into a cytotoxic form. In some embodiments, the enzyme is a thymidine kinase inhibitor. The enzyme is selected from kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, horseradish peroxidase and carboxylesterase.

[0027] In some embodiments, the HDAd additionally comprises a nucleic acid encoding a thymidine kinase. In cases where the HDAd nucleic acid encodes IL-12 and an anti-PD-L1 antibody, the respective expression sequences may or may not be regulated by the same regulatory sequence. In cases where the HDAd nucleic acid encodes both IL-12 and an anti-PD-L1 antibody, the location on the HDAd nucleic acid may be in any suitable configuration, for example, in the 5' to 3' direction, the nucleic acid region encoding IL-12 is either upstream or downstream of the nucleic acid region encoding the anti-PD-L1 antibody.

[0028] The present disclosure also provides: LC-CRD1: SEQ ID NO: 10; LC-CRD2: SEQ ID NO:11; LC-CRD3: VL domain comprising SEQ ID NO: 12; and HC-CRD1: SEQ ID NO: 13; HC-CRD2: SEQ ID NO: 14; HC-CRD3: VH domain comprising SEQ ID NO: 15; or LC-CRD1: SEQ ID NO: 18; LC-CRD2: SEQ ID NO: 19; LC-CRD3: VL domain comprising SEQ ID NO: 20; and HC-CRD1: SEQ ID NO:21; HC-CRD2: SEQ ID NO:22; HC-CRD3: VH domain comprising SEQ ID NO:23; or LC-CRD1: SEQ ID NO:26; LC-CRD2: SEQ ID NO:27; LC-CRD3: VL domain comprising SEQ ID NO:28; and HC-CRD1: SEQ ID NO:29; HC-CRD2: SEQ ID NO:30; HC-CRD3: VH domain comprising SEQ ID NO:31; or LC-CRD1: SEQ ID NO:57; LC-CRD2: SEQ ID NO:58; LC-CRD3: VL domain comprising SEQ ID NO:59; and HC-CRD1: SEQ ID NO:60; HC-CRD2: SEQ ID NO:61; HC-CRD3: VH domain comprising SEQ ID NO:62 Also provided is a chimeric antigen receptor (CAR) comprising an antigen binding domain comprising:

[0029] In some embodiments, the CAR is A VL comprising, consisting of, or consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or more sequence identity to SEQ ID NO: 16, and a VL having at least 75%, 80%, 85%, 90%, 95% or more sequence identity to SEQ ID NO: 17. a VH comprising, consisting of, or consisting essentially of an amino acid sequence with 5% or greater sequence identity; or a VL comprising, or consisting of, an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO:24, and a VH comprising, consisting of, or consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO:25; or a VL comprising, or consisting of, an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO: 32, and a VH comprising, consisting of, or consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% sequence identity to SEQ ID NO: 33; or a VL comprising, or consisting of, an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO:63, and a VH comprising, or consisting of, or consisting essentially of, an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% sequence identity to SEQ ID NO:64. The antigen-binding domain comprises:

[0030] The present disclosure also provides one or more nucleic acids, optionally isolated, encoding an oncolytic adenovirus (OncAd), a helper-dependent adenovirus (HDAd), or a chimeric antigen receptor (CAR) according to the present disclosure.

[0031] The present disclosure also provides an oncolytic adenovirus (OncAd), a helper-dependent adenovirus (HDAd), a chimeric antigen receptor (CAR), or a cell comprising one or more nucleic acids of the present disclosure.

[0032] The present disclosure also provides an oncolytic adenovirus (OncAd), a helper-dependent adenovirus (HDAd), a chimeric antigen receptor (CAR); a pharmaceutical composition comprising one or more nucleic acids or cells of the present disclosure, optionally with or in association with a pharma- ceutical acceptable carrier, diluent, excipient or adjuvant.

[0033] The present disclosure also provides methods of treating cancer comprising administering to a subject an oncolytic adenovirus (OncAd), a helper-dependent adenovirus (HDAd), a chimeric antigen receptor (CAR), one or more nucleic acids, cells or pharmaceutical compositions of the present disclosure.

[0034] The present disclosure also provides an oncolytic adenovirus (OncAd), a helper-dependent adenovirus (HDAd), a chimeric antigen receptor (CAR), one or more nucleic acids, cells or pharmaceutical compositions of the present disclosure for use in a method of treating cancer.

[0035] The present disclosure also provides the use of an oncolytic adenovirus (OncAd), a helper-dependent adenovirus (HDAd), a chimeric antigen receptor (CAR), one or more nucleic acids, cells or pharmaceutical compositions of the present disclosure in the manufacture of a medicament for treating cancer.

[0036] In some embodiments, according to various aspects of the present disclosure, the cancer is head and neck cancer, Selected from nasopharyngeal carcinoma (NPC), cervical carcinoma (CC), oropharyngeal carcinoma (OPC), gastric carcinoma (GC), hepatocellular carcinoma (HCC) and lung cancer.

[0037] The present disclosure also provides a kit of parts comprising a predetermined amount of an oncolytic adenovirus (OncAd), a helper-dependent adenovirus (HDAd), a chimeric antigen receptor (CAR), one or more nucleic acids, cells or pharmaceutical compositions according to the present disclosure.

[0038] Detailed Description The present disclosure relates to the combination of multiple therapeutic agents for the treatment of cancer. In particular, (i) an oncolytic virus, (ii) a virus that provides an immunomodulator, and (iii) an immune cell (e.g., T cell) with a CAR specific to a cancer cell antigen are used in combination as cancer therapy. The therapeutic agents are combined to provide an improved treatment effect compared to the effect seen when any one of the agents is used alone. In certain embodiments, at least two of the three therapeutic agents act additively to treat cancer, whereas in other embodiments, at least two of the three different therapeutic agents act synergistically to treat cancer.

[0039] Without wishing to be bound by any particular theory, it is believed that improved therapeutic effects are achieved by combining the advantageous features of oncolytic virus therapy (e.g., effective treatment of solid tumors) and CAR-T cell therapy (e.g., effective treatment of diffuse / metastatic cancers) while providing an immune environment favorable for CAR-T cell proliferation and activity.

[0040] Oncolytic viruses The present disclosure employs oncolytic viruses. Oncolytic viruses and their uses for treating cancer are reviewed, for example, in Chiocca and Rabkin Cancer Immunol Res (2014) 2(4):295-300, which is incorporated by reference in its entirety.

[0041] Oncolytic viruses replicate in cancer cells, causing the lysis of the cancer cells. Often, oncolytic viruses are selective for cancer cells over non-cancerous cells, e.g., oncolytic viruses generally replicate in dividing cells in preference to non-dividing cells. Thus, oncolytic viruses are useful for selectively killing cancer cells and destroying tumors, without substantially damaging normal non-cancerous cells / tissues.

[0042] Oncolytic virus therapy is associated with a number of advantageous features. Oncolytic viruses often target a number of oncogenic pathways and use multiple mechanisms of cytotoxicity, minimizing the chance of resistance. As mentioned above, oncolytic viruses selectively replicate in tumors and are non-pathogenic, thus exhibiting minimal toxicity. The viral dose in tumors also increases over time due to viral replication, and oncolytic viruses can also be genetically engineered to improve safety, for example by engineering susceptibility to drugs.

[0043] There are two main classes of oncolytic viruses: (i) Viruses that naturally replicate preferentially in cancer cells and are nonpathogenic in humans, often due to their high susceptibility to natural antiviral signaling or their dependency on oncogenic signaling pathways, such as autonomous parvoviruses, myxoma virus (MYXV; poxviruses), Newcastle disease virus (NDV; paramyxoviruses), reoviruses, and Seneca Valley virus (SVV; picornaviruses); (ii) A mutation in a gene, for example, that is required for replication in normal cells but not in cancer cells Viruses that have been genetically engineered with deletions, such as adenovirus (Ad), herpes simplex virus (HSV), vaccinia virus (VV), and vesicular stomatitis virus (VSV; rhabdovirus); or viruses that have been genetically engineered for use as vaccine vectors, such as measles virus (MV; paramyxovirus), poliovirus (PV; picornavirus), and VV (poxvirus).

[0044] Genetic engineering can include the insertion / alteration of functional sequences to provide enhanced selectivity, safety, and / or alter viral tropism against cancer cells.

[0045] For example, oncolytic viruses may be engineered to introduce tissue-specific internal ribosome entry sites (IRES) that only allow viral translation in target cells, and / or to introduce miRNAs / miRNA response elements (MREs), so that differential miRNA expression between healthy cells or specific tissues and tumor cells can prevent the virus from targeting healthy cells / tissues. Oncolytic viruses may also be engineered to cause transcription of the viral genome under the control of cell- or tissue-specific regulatory regions, such as promoters / enhancers (e.g., tumor cell-specific promoters). In some embodiments, the oncolytic viruses disclosed herein may include one or more modifications for such purposes.

[0046] Viruses may also be modified for targeting of transduction, for example, via modification of viral receptors / coat proteins to target tumor cells and / or detarget healthy cells / tissues.

[0047] Oncolytic viruses can be used to induce tumor growth in a subject, as described by Chiocca and Rabkin, supra. The oncolytic virus may be administered in such a way as to minimize anti-oncolytic virus responses (e.g., neutralization by anti-viral antibodies) and liver capture, and maximize tumor delivery. For example, the oncolytic virus may be administered to a cell carrier, such as in mesenchymal stromal cells, myeloid-derived suppressor cells (MDSCs), neural stem cells, T cells, cytokine-induced killer cells, or irradiated tumor cells, or coated with nanoparticles.

[0048] In some embodiments, the oncolytic virus of the present disclosure is or is derived from adenovirus (Ad), herpes simplex virus (HSV), vaccinia virus (VV), vesicular stomatitis virus (VSV); autonomous parvovirus, myxoma virus (MYXV), Newcastle disease virus (NDV), reovirus, Seneca Valley virus (SVV), measles virus, retrovirus, influenza virus, Sindbis virus (SINV) or poxvirus, for example.In some embodiments, the oncolytic virus is not vaccinia virus.In some embodiments, the oncolytic virus is not vaccinia virus JX-594.

[0049] As used herein, an oncolytic virus "derived from" a reference virus comprises the nucleic acid sequence or amino acid sequence that the reference virus has.In some embodiments, an oncolytic virus "derived from" a reference virus comprises one or more genes that the reference virus has.In some embodiments, an oncolytic virus "derived from" encodes one or more proteins that are encoded by the reference virus.

[0050] In some embodiments, an oncolytic virus derived from a reference virus may contain a nucleic acid sequence encoding one or more functional elements of the reference virus. "Functional element" may be, for example, a transcription regulator (e.g., promoter / enhancer), a regulator of post-transcriptional processing, a translation regulator, a regulator of post-transcriptional processing, a response element, a repeat sequence, or a viral protein. In some embodiments, the oncolytic virus derived from a reference virus may contain one or more genes of the reference virus or proteins encoded thereby.

[0051] In some embodiments, the oncolytic virus of the present disclosure is or is derived from an adenovirus (OncAd). OncAd is reviewed, for example, in Larson et al., Oncotarget. (2015) 6(24):19976-19989, the entire contents of which are incorporated by reference.

[0052] In some embodiments, the OncAd is or is derived from a species A, B, C, D, E, F, or G human adenovirus (i.e., HAdV-A, HAdV-B, HAdV-C, HAdV-D, HAdV-E, HAdV-F, or HAdV-G). In some embodiments, the OncAd is or is derived from a species C human adenovirus. In some embodiments, the OncAd is or is derived from Ad5, Ad2, Ad1, Ad6, or Ad57.

[0053] In some embodiments, the OncAd is a conditionally replicating adenovirus (or CRAd). In some embodiments, the OncAd has a reduced ability to infect, replicate in, and / or lyse non-cancerous cells (as opposed to infecting / replicating in comparable cancerous cells), e.g., as a result of genetic modification of the adenovirus from which the OncAd is derived. (compared to the ability to dissolve and / or dissolve it).

[0054] In some embodiments, the oncolytic virus comprises a modification to the sequence encoding one or more proteins. In some embodiments, the modification alters the production or activity of the encoded protein. In some embodiments, the modification is a truncation or deletion of the protein.

[0055] In some embodiments, the OncAd comprises a modification to an adenovirus early protein. In some embodiments, the modification is to a region encoding an E1A protein. In some embodiments, the OncAd encodes an E1A protein that has a reduced ability to bind to an Rb protein compared to a wild-type E1A protein (e.g., the E1A encoded by the adenovirus from which the OncAd is derived). In some embodiments, the OncAd encodes an E1A protein lacking the amino acid sequence LTCHEACF (SEQ ID NO:52). An example of an OncAd encoding an E1A protein lacking the amino acid sequence LTCHEACF (SEQ ID NO:52) is Onc5 / 3Ad2E1Δ24, as set forth in SEQ ID NO:55.

[0056] In some embodiments, the oncolytic virus encodes an E1A protein that comprises, consists of or essentially consists of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:34.

[0057] In some embodiments, the oncolytic virus comprises a nucleic acid sequence that provides one or more binding sites for one or more transcription factors. In some embodiments, the transcription factor is an activating transcription factor (i.e., a transcriptional activator). One or more binding sites for more transcription factors are preferably provided upstream (i.e., 5') to the nucleic acid sequence encoding one or more functional elements (e.g., viral proteins).

[0058] In some embodiments, the transcription factor is one that has increased expression or increased activity in cancerous cells compared to comparable non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type).

[0059] As used herein, "expression" may refer to gene expression or protein expression. Gene expression can be measured by various means known to those skilled in the art, such as by measuring the level of mRNA by quantitative real-time PCR (qRT-PCR) or by reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, such as by antibody-based methods, such as Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

[0060] An example of an OncAd that contains one or more binding sites for one or more transcription factors is ICOVIR15, described in Rojas et al., 2010 Mol Ther 18 1960-1971, which is incorporated by reference in its entirety. ICOVIR15 binds to the transcription factor E It contains eight binding sites for 2F.

[0061] In some embodiments, the oncolytic virus comprises one or more binding sites for a transcription factor whose gene or protein expression or activity in a cell is upregulated in response to a factor produced or expressed by an immune cell. In some embodiments, the factor produced or expressed by an immune cell is upregulated in response to an effector immune cell, such as CD8+ cytotoxic T lymphocytes (CTL), CD4+ T helper 1 (T H 1) It may be at least one cytokine / chemokine produced by, or a protein expressed on the cell surface of, a cell, a natural killer (NK) cell or a natural killer T (NKT) cell.

[0062] In some embodiments, the oncolytic virus of the present disclosure comprises one or more binding sites for STAT transcription factors.In some embodiments, the oncolytic virus comprises one or more binding sites for STAT1.The ICOSTAT OncAd described herein has eight binding sites for STAT1, and STAT1 is known to be upregulated by IFNγ.In certain embodiments, ICOSTAT is a particularly effective treatment for cancer, since the host's immune response to cancer cells is expected to promote the replication of oncolytic virus in situ.

[0063] In some embodiments, the oncolytic virus comprises more than one binding site for STAT1, for example at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 binding sites for STAT1. In some embodiments, the binding site for STAT1 may comprise, consist of, or essentially consist of the sequence TTCCGGGAA (SEQ ID NO: 53), or TTCTCGGAA (SEQ ID NO: 54). In some embodiments, the oncolytic virus of the present disclosure comprises one or more copies of the sequence TTCCGGGAA (SEQ ID NO: 53) or TTCTCGGAA (SEQ ID NO: 54).

[0064] In some embodiments, the oncolytic virus of the present disclosure has a sequence similar to SEQ ID NO:51 or an equivalent sequence as a result of codon degeneracy that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73% similar to SEQ ID NO:51 or an equivalent sequence as a result of codon degeneracy. , 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0065] In some embodiments, an oncolytic virus according to the present disclosure comprises, consists of, or consists essentially of a nucleic acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:55, or an equivalent sequence as a result of codon degeneracy.

[0066] In some embodiments, an oncolytic virus according to the present disclosure encodes the same protein as a protein encoded by an oncolytic virus comprising, consisting of, or consisting essentially of the nucleic acid set forth in SEQ ID NO: 55. In some embodiments, an oncolytic virus according to the present disclosure encodes the same protein as a protein encoded by an oncolytic virus comprising, consisting of, or consisting essentially of the nucleic acid set forth in SEQ ID NO: 51.

[0067] Viruses containing nucleic acids encoding immune modulators The present disclosure employs a virus that contains a nucleic acid that encodes an immunomodulator. The virus acts as a vector for delivering the immunomodulator. In certain embodiments, the virus contains a nucleic acid that encodes more than one immunomodulator.

[0068] Any virus can be used that can introduce nucleic acid encoding immunomodulator into cells (e.g., primary human immune cells).Suitable viruses include gamma retroviruses (e.g., murine leukemia virus (MLV)-derived vectors), lentiviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, and herpes viruses, such as those described in Maus et al., Annu Rev Immunol (2014) 32:189-225, or Morgan and Boyerinas, Biomedicines April 9, 2016, both of which are incorporated by reference in their entirety.In some embodiments, the virus that comprises nucleic acid encoding immunomodulator is or is derived from adenovirus, lentivirus, retrovirus, or herpes virus.

[0069] In some embodiments, the virus comprising nucleic acid encoding at least one immunomodulator is an oncolytic virus comprising nucleic acid encoding at least one immunomodulator.

[0070] The immunomodulatory agent encoded by the virus comprising a nucleic acid encoding an immunomodulatory agent according to the present disclosure is preferably selected to facilitate an immune response, particularly a cell-mediated immune response, against cancer in a subject. In one embodiment, the immunomodulatory agent is capable of promoting the induction of effector immune cells (e.g., CTL, T H The present invention provides conditions favorable for the activation, recruitment, proliferation, activity and / or survival of immune cells (immunoglobulin-1 cells, NK cells or NKT cells).

[0071] In some embodiments, the immunomodulatory factor is an agonist of an effector immune response, e.g., a cytokine or chemokine (e.g., IL-2, IL-7, IL-17, IL-12, IL-21, IL-15, MIP-1α or RANTES) that promotes activation, recruitment, proliferation, activity and / or survival of effector immune cells, an agonist antibody for a costimulatory receptor (e.g., 4-1BB, OX40, CD28, CD27, ICOS, CD30 or GITR), or a ligand for a costimulatory receptor (e.g., 4-1BBL, OX40L, CD80, CD86, CD70, ICOSL, CD30L, or GITRL). In some embodiments, the agonist of the effector immune response may be an antagonist of an immune checkpoint inhibitor, or an antagonist ligand of an immune checkpoint inhibitor, such as an antagonist antibody against PD-L1, PD-L2, PD-1, CTLA-4, LAG-3, TIM-3, Gal-9, TIGIT, VISTA, or BTLA, or an antagonist of a cytokine / chemokine that is an antagonist of the effector immune response, such as TGFβ (i.e., an antagonist anti-TGFβ antibody or a soluble / decoy TGFβ receptor). In some embodiments, the agonist of the effector immune response may be a molecule that binds to and utilizes bystander effector immune cells, such as T cells and NK cells.

[0072] In some embodiments, the immunomodulatory factor may be an antagonist of an immunomodulatory response, e.g., an antagonist of a cytokine / chemokine that promotes the activation, recruitment, proliferation, activity and / or survival of immunomodulatory cells, e.g., regulatory T cells (Tregs) and / or myeloid-derived suppressor cells (MDSCs), e.g., CCL9, CXCL10, CCL20, CCL22.

[0073] In some embodiments, the virus that comprises the nucleic acid that codes for the immunomodulator may also comprise the nucleic acid that codes for additional functional sequence.For example, the virus may comprise the nucleic acid that codes for the protein that reduces the growth / proliferation / survival of infected cells, or the protein that makes infected cells sensitive to the treatment with a given drug, or the protein that destroys tumor structure to facilitate immune cell infiltration (for example, the enzyme that digests tumor matrix).

[0074] In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator additionally comprises a nucleic acid encoding an enzyme capable of catalyzing the conversion of a non-toxic factor to its cytotoxic form. The enzyme can catalyze the conversion of a non-toxic prodrug to its active cytotoxic form.

[0075] Enzyme / prodrug systems are well known in the art, examples of which include those described in Malekshah et al., Curr Pharmacol Rep. (2016) 2(6):299-308, which is incorporated by reference in its entirety. Examples of non-toxic prodrugs, their active cytotoxic forms, and enzymes capable of catalyzing the conversion of the non-toxic prodrugs to their active cytotoxic forms are shown in Figure 2 of Malekshah et al.

[0076] In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator additionally comprises a nucleic acid encoding thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, horseradish peroxidase, and / or carboxylesterase.

[0077] For example, the virus may contain a nucleic acid encoding a thymidine kinase to render cells expressing the virus sensitive to treatment with ganciclovir (GCV), acyclovir (ACV) and / or valacyclovir. The virus may contain a nucleic acid encoding a cytosine deaminase to render cells expressing the virus sensitive to treatment with 5-fluorocytosine (5-FC), which is converted to 5-fluorouracil (5-FU) by cytosine deaminase. The virus may contain a nucleic acid encoding a nitroreductase to render cells expressing the virus sensitive to treatment with CB1954, nitro-CBI-DEI and / or PR-104A. The virus may contain a nucleic acid encoding a thymidine kinase to render cells expressing the virus sensitive to treatment with oxazaphosphorines (e.g., cyclophosphamide or The virus may contain a nucleic acid encoding a cytochrome P450 to render the virus sensitive to treatment with a ribozyme (e.g., ifosfamide). The virus may contain a nucleic acid encoding a carboxypeptidase G2 to render cells expressing the virus sensitive to treatment with a nitrogen mustard-based drug (e.g., CMDA or ZD2767P). The virus may contain a nucleic acid encoding a purine nucleoside phosphorylase to render cells expressing the virus sensitive to treatment with 6-methylpurine 2-deoxyriboside and / or fludarabine (e.g., 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo) or arabinofuranosyl-2-F-adenine monophosphate (F-araAMP). The virus may contain a nucleic acid encoding a horseradish peroxidase to render cells expressing the virus sensitive to treatment with indole-3-acetic acid (IAA). The virus may contain a nucleic acid encoding a carboxylesterase to render cells expressing the virus sensitive to treatment with irinotecan.

[0078] In some embodiments, the virus may contain a nucleic acid encoding an antagonist of a growth factor. In some embodiments, the virus may be a helper-dependent adenovirus (HDAd). HDAd are reviewed, for example, in Rosewell et al., J Genet Syndr Gene Ther (2011) Suppl 5:001, which is incorporated by reference in its entirety.

[0079] HDAd lack coding sequences for viral proteins and therefore have a large capacity (up to 37Kb) to transduce coding sequences of interest. HDAd are non-integrating, can efficiently transduce a wide variety of cell types independent of the cell cycle, and mediate long-term transgene expression without chronic toxicity.

[0080] HDAd contain only the cis-acting viral elements required for genome replication (inverted terminal repeats (ITRs)) and encapsidation (ψ) and are therefore helper virus dependent for propagation. When cells are infected with both helper virus and HDAd, the helper virus replication machinery acts in trans to replicate and package the HDAd.

[0081] In certain embodiments of the present disclosure, the oncolytic virus is an OncAd and the virus comprising a nucleic acid encoding an immune modulator is an HDAd, and the OncAd and HDAd can co-infect and replicate in cells of the cancer.

[0082] The dependence of HDAd on OncAd assistance provides highly localized expression of immune modulators: HDAd can only grow in cells co-infected with OncAd, meaning that OncAd is selective for replication in cancer cells, thus restricting expression of HDAd-encoded factors to cancer cells / tissues and minimizing side effects.

[0083] Furthermore, because OncAd and HDAd efficiently target and infect tumor cells, expression of immune modulators in those cells can alter the normally immunosuppressive tumor microenvironment to provide conditions that promote the activation, recruitment (i.e., tumor invasion / infiltration), proliferation, activity and / or survival of effector immune cells.

[0084] In particular, in the context of the present disclosure where the method of treatment employs the use of CAR-T cells, expression of the immune modulator encoded by the HDAd results in enhanced activation, recruitment, proliferation, activity and / or survival of the CAR-T cells.

[0085] In certain embodiments described herein, the virus comprising nucleic acid encoding an immunomodulatory factor is selected from the group consisting of IL-12p70, HSV-1 thymidine kinase and antagonist anti-PD-1. The HDAd comprises a nucleic acid encoding an L1 minibody.

[0086] In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator according to the present disclosure encodes IL-12. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator comprises a nucleic acid encoding an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:35.

[0087] In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator disclosed herein encodes an antagonist of PD-1 / PD-L1 signaling, hi some embodiments, the antagonist of PD-1 / PD-L1 signaling is an anti-PD-L1 antibody.

[0088] In some embodiments, the anti-PD-L1 antibody is LC-CRD1: SEQ ID NO:39; LC-CRD2: SEQ ID NO: 40; LC-CRD3: VL domain comprising SEQ ID NO: 41; and HC-CRD1: SEQ ID NO:42; HC-CRD2: SEQ ID NO:43; HC-CRD3: VH domain comprising SEQ ID NO: 44 The antigen-binding domain comprises:

[0089] In some embodiments, the anti-PD-L1 antibody comprises, consists of, or comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:45. and a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:46.

[0090] In some embodiments, a virus comprising a nucleic acid encoding an immune modulator comprises a nucleic acid encoding an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:38.

[0091] In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator according to the present disclosure comprises an amino acid sequence encoding a thymidine kinase. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator comprises a nucleic acid encoding an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:36.

[0092] In some embodiments, the virus comprising a nucleic acid encoding an immunomodulator according to the present disclosure has a sequence that is at least 60% similar to SEQ ID NO:50, or an equivalent sequence as a result of codon degeneracy. , 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0093] Chimeric antigen receptors (CARs) and CAR-expressing cells The present disclosure employs immune cells that contain a chimeric antigen receptor (CAR). 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), which is incorporated by reference in its entirety. CARs comprise an antigen-binding region and a signaling region linked to a cell membrane anchor region. An optional hinge region may separate the antigen-binding region from the cell membrane anchor region, which may act as a flexible linker.

[0094] The antigen-binding region of the CAR may be based on the antigen-binding region of an antibody specific for the antigen to which the CAR is targeted, or other agents capable of binding to the target. For example, the antigen-binding domain of the CAR may comprise the amino acid sequence for the complementarity determining region (CDR), or the entire light and heavy chain variable region amino acid sequence of an antibody that specifically binds to a target protein. The antigen-binding domain of the CAR may also target antigens based on other protein:protein interactions, such as ligand:receptor binding, e.g., IL-13Rα2-targeted CARs have been developed using antigen-binding domains based on IL-13 (see, e.g., Kahlon et al., 2004 Cancer Res 64(24):9160-9166).

[0095] The CAR of the present disclosure comprises an antigen-binding region specific to a cancer cell antigen. The antigen-binding region of the CAR may be provided in any suitable manner, for example as an scFv, Fab, etc. In some embodiments, the antigen-binding region of the CAR comprises or consists of a cancer cell antigen-binding scFv.

[0096] A cancer cell antigen is an antigen expressed by a cancer cell. A cancer cell antigen may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The expression of a cancer cell antigen may be associated with cancer. A cancer cell antigen may be aberrantly expressed by a cancer cell (e.g., a cancer cell antigen may be expressed with abnormal localization) or may be expressed in an abnormal structure by a cancer cell. A cancer cell antigen may be capable of eliciting an immune response.

[0097] In some embodiments, the antigen is expressed on the cell surface of the cancer cell (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen that binds to the bispecific antigen-binding polypeptide of the present disclosure is presented on the external surface of the cancer cell (i.e., is extracellular). In some embodiments, the antigen is anchored to the cell membrane, for example, via a transmembrane domain or other membrane anchor (e.g., a lipid anchor, e.g., a GPI anchor). In some embodiments, the cancer cell antigen is expressed on the cell surface of the cancer cell (i.e., is expressed in or at the cell membrane), but may also be expressed inside the cell (i.e., is expressed inside the comparable non-cancerous cell).

[0098] The cancer cell antigen may be a cancer-associated antigen. In some embodiments, the cancer cell antigen is an antigen whose expression is associated with the onset, progression and / or severity of symptoms of cancer. Cancer-associated antigens may be associated with the cause or pathology of cancer, or may be associated with the outcome of cancer. In some embodiments, the antigen is one whose expression is upregulated (e.g., at the RNA and / or protein level) by cancer cells, e.g., compared to expression levels by comparable non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type).

[0099] In some embodiments, cancer-associated antigens may be preferentially expressed by cancer cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type).In some embodiments, cancer-associated antigens may be the product of mutated oncogenes or mutated tumor suppressor genes.In some embodiments, cancer-associated antigens may be the product of overexpressed cellular proteins, cancer antigens produced by tumor viruses, oncofetal antigens, or cell surface glycolipids or glycoproteins.

[0100] Cancer cell antigens are Zarour HM, DeLeo A, Finn OJ et al., Categories of Tumor Antigens. In:Kufe DW, Pollock RE, Weichselbaum RR et al. eds., Holland-Frei Cancer Medicine. 6th ed. , Hamilton (ON): BC Decker; 2003. Cancer cell antigens include oncofetal antigen: CEA, immature laminin receptor, TAG-72; tumor viral antigens such as HPVE6 and E7; overexpressed proteins: BING-4, calcium activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1, surviving; cancer testis antigens. : BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; lineage control antigens: MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, prostate specific antigen; mutated antigens: β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII; antigens altered by post-translational modifications: MUC1, idiotypic antigens: Ig, TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, fetal-associated pancreatic protein (FAPP), alkaline phosphatase placenta-like 2 (ALPPL-2), Siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B.

[0101] In some embodiments, the cancer cell antigen is HER2. In some embodiments, the CAR of the present disclosure comprises an antigen binding domain capable of specific binding to HER2. In some embodiments, the CAR comprises an antigen binding domain comprising the CDR of an antibody capable of specific binding to HER2. In some embodiments, the CAR comprises an antigen binding domain comprising the VL and VH regions of an antibody capable of specific binding to HER2.

[0102] In certain embodiments, the CAR-expressing cell comprises two separate CARs, each of which targets a different cancer cell antigen, and in certain forms, at least one of the CARs targets HER2. In some cases, the CAR is bispecific to two different cancer cell antigens, and one of the two different cancer cell antigens can be HER2.

[0103] In some embodiments, the CAR is LC-CRD1: SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:26 or SEQ ID NO:57; LC-CRD2: SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:27 or SEQ ID NO:58; LC-CRD3: a VL domain comprising SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28 or SEQ ID NO: 59; and HC-CRD1: SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:29 or SEQ ID NO:60; HC-CRD2: SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:30 or SEQ ID NO:61; HC-CRD3: VH domain comprising SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31 or SEQ ID NO: 62 The antigen-binding domain comprises:

[0104] In some embodiments, the CAR is LC-CRD1: SEQ ID NO: 10; LC-CRD2: SEQ ID NO:11; LC-CRD3: VL domain comprising SEQ ID NO: 12; and HC-CRD1: SEQ ID NO: 13; HC-CRD2: SEQ ID NO: 14; HC-CRD3: VH domain comprising SEQ ID NO: 15 The antigen-binding domain comprises:

[0105] In some embodiments, the CAR comprises: LC-CRD1: SEQ ID NO: 18; LC-CRD2: SEQ ID NO: 19; LC-CRD3: VL domain comprising SEQ ID NO: 20; and HC-CRD1: SEQ ID NO:21; HC-CRD2: SEQ ID NO:22; HC-CRD3: VH domain comprising SEQ ID NO:23 The antigen-binding domain comprises:

[0106] In some embodiments, the CAR is LC-CRD1: SEQ ID NO:26; LC-CRD2: SEQ ID NO:27; LC-CRD3: VL domain comprising SEQ ID NO:28; and HC-CRD1: SEQ ID NO:29; HC-CRD2: SEQ ID NO:30; HC-CRD3: VH domain comprising SEQ ID NO:31 The antigen-binding domain comprises:

[0107] In some embodiments, the CAR comprises a VL domain: LC-CRD1: SEQ ID NO:57; LC-CRD2: SEQ ID NO:58; LC-CRD3: VL domain comprising SEQ ID NO:59; and HC-CRD1: SEQ ID NO:60; HC-CRD2: SEQ ID NO:61; HC-CRD3: VH domain comprising SEQ ID NO:62 The antigen-binding domain comprises:

[0108] In some embodiments, the CAR comprises, consists of, or consists essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16, 24, 32 or 63. ).

[0109] In some embodiments, the CAR comprises an antigen binding domain comprising a heavy chain variable region (VH) comprising, consisting of, or consisting essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17, 25, 33 or 64.

[0110] In some embodiments, the CAR comprises, consists of, or consists essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16; and an antigen-binding domain comprising a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:17. In some embodiments, the CAR comprises, consists of, or consists essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24; and an antigen-binding domain comprising a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:25.In some embodiments, the CAR comprises, consists of, or consists essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 32; and an antigen-binding domain comprising a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:33. In some embodiments, the CAR comprises, consists of, or consists essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 63; and an antigen-binding domain comprising a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:64.

[0111] In some embodiments, the CAR of the present disclosure comprises an antigen-binding region that comprises, consists of, or consists essentially of an antibody / antigen-binding fragment according to the present disclosure.

[0112] A cell membrane anchor region is provided between the antigen binding region and the signal transduction region of the CAR. The cell membrane anchor region is provided to fix the CAR to the cell membrane of the CAR-expressing cell with the antigen binding region in the extracellular space and the signal transduction region inside the cell. Suitable transmembrane domains include transmembrane regions from CD28, CD3-zeta, CD4 or CD8.

[0113] In some embodiments, the cell membrane anchor region comprises, consists of or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:4.

[0114] The signaling region of the CAR allows for the activation of T cells. The CAR signaling region may contain the amino acid sequence of the intracellular domain of CD3-ζ, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of T cells expressing the CAR. Signaling regions containing sequences of other ITAM-containing proteins have also been employed in CARs, for example, domains containing the ITAM-containing region of FcγRI (Haynes et al., 2001 J Immunol 166(1):182-187). From the intracellular domain of CD3-ζ CARs containing this signaling region are often referred to as first generation CARs.

[0115] In some embodiments, the cell membrane anchor region comprises, consists of or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6.

[0116] The signaling region of the CAR may also comprise a costimulatory sequence derived from the signaling region of a costimulatory molecule to facilitate the activation of the T cell expressing the CAR when it binds to a target protein.Suitable costimulatory molecules include at least CD28, OX40, 4-1BB, ICOS and CD27.CARs with a signaling region that includes additional costimulatory sequences are often referred to as second generation CARs.

[0117] In some cases, CARs are engineered to provide costimulation of different intracellular signaling pathways. For example, signaling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1BB-mediated signaling is via the TNF receptor-associated factor (TRAF) adaptor protein. Therefore, the signaling region of CARs sometimes contains costimulatory sequences from the signaling regions of more than one costimulatory molecule. CARs that contain signaling regions with multiple costimulatory sequences are often referred to as third generation CARs.

[0118] In some embodiments, the CAR of the present disclosure comprises one or more costimulatory sequences that comprise, consist of, or consist essentially of, or comprise an amino acid sequence derived from, the amino acid sequence of the intracellular domain of one or more of CD28, OX40, 4-1BB, ICOS, and CD27.

[0119] In some embodiments, the cell membrane anchor region comprises, consists of or consists essentially of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5.

[0120] The optional hinge region can provide a separation between the antigen-binding domain and the transmembrane domain and can act as a flexible linker. The hinge region can be a flexible domain that can orient the binding moieties in different directions. The hinge region can be from IgG1 or from the CH of an immunoglobulin. 2 CH 3 In some embodiments, the CAR of the present disclosure may be derived from the hinge region of IgG1 or the CH of an immunoglobulin. 2 CH 3 The invention also includes a hinge region which comprises, consists of, or consists essentially of, or is derived from, the amino acid sequence of the region.

[0121] In some embodiments, the cell membrane anchor region comprises, consists of or consists essentially of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:9.

[0122] In some embodiments, the CAR comprises, consists of or consists essentially of an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1, 2, 3 or 56.

[0123] The present disclosure also provides a cell comprising or expressing a CAR according to the present disclosure. A cell comprising or expressing a nucleic acid encoding a CAR according to the present disclosure is also provided. Engineering a CAR into a T cell can be performed in vitro for transduction and expansion in culture, for example during expansion of T cells for adoptive T cell therapy. Methods for 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, the entirety of which is incorporated by reference. "At least one cell" encompasses a plurality of cells, For example, it is understood to include a population of such cells.

[0124] The cell containing or expressing a CAR according to the present disclosure may be a eukaryotic cell, such as a mammalian cell. The mammal may be a human or a non-human mammal, such as a rabbit, guinea pig, rat, mouse or other rodent (including any animal in the rodent order), cat, dog, pig, sheep, goat, cow (including cattle, such as dairy cows, or any animal in the bovine genus), horse (including any animal in the equidae family), donkey, and non-human primate.

[0125] In some embodiments, the cells may be derived from or obtained from a human subject. When CAR-expressing cells are used to treat a subject, the cells may be derived from the subject to be treated with the CAR-expressing cells (i.e., the cells may be autologous) or the cells may be derived from a different subject (i.e., the cells may be xenogeneic).

[0126] 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., CD3γ, CD3ε, CD3ζ, or CD3δ), a TCR polypeptide (TCRα or TCRβ), CD27, CD28, CD4, or CD8.

[0127] In some embodiments, the cell is a T cell. In some embodiments, the T cell is a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)).

[0128] The use of CAR T cells is associated with the advantage that they can be administered systemically and become present in both primary tumors and metastatic tumors (Manzo et al., Human Molecular Genetics (2015)R67~73).

[0129] In some embodiments, the cell is antigen-specific T cell.In the embodiments described herein, "antigen-specific" T cell is a cell that shows the specific functional properties of T cell in response to the antigen that T cell is specific for or the cell that expresses said antigen.In some embodiments, the properties are the functional properties associated with effector T cell, such as cytotoxic T cell.

[0130] In some embodiments, antigen-specific T cells may exhibit one or more of the following characteristics: cytotoxicity, e.g., cytotoxicity against cells that contain / express the antigen for which T cells are specific; proliferation, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression, e.g., in response to the antigen for which T cells are specific or the cells that contain / express the antigen for which T cells are specific. Antigen-specific T cells contain a TCR that allows the cells to recognize peptides of the antigen that they are specific for when presented by appropriate MHC molecules. Antigen-specific T cells may be CD4+ T cells and / or CD8+ T cells.

[0131] In some embodiments, the antigen for which the T cells are specific may be a peptide or polypeptide of a virus, such as adenovirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).

[0132] T cells specific for viral antigens may be referred to herein as virus-specific T cells (VSTs). VSTs are CD4+ T cells (e.g., T HThe virus-specific T cells may be CD8+ T cells (e.g., CD4+ T cells) and / or CD8+ T cells (e.g., CTLs). T cells specific for a particular viral antigen may be described as specific for the relevant virus, e.g., T cells specific for an antigen of an adenovirus may be referred to as adenovirus-specific T cells, or "AdVSTs". The use of virus-specific T cells for CAR-T cell generation is associated with the advantage that while naive T cells may have limited long-term persistence after infusion, virus-specific T cells (VSTs) derived from memory compartments, and genetically engineered VSTs, have been shown to persist for up to 10 years after infusion in stem cell transplant recipients (Cruz et al., Cytotherapy (2010) 12:743-749). For example, VSTs expressing GD2.CAR have been shown to inhibit low tumor growth and tumor suppression. It has been shown to result in long-lasting, complete tumor responses following infusion in patients with high tumor burden (Sun et al., Journal for Immunotherapy of Cancer (2015) 3:5 and Pule et al., Nature Medicine (2008) 14:1264-1270).

[0133] In some embodiments, the cells comprising / expressing a CAR are virus-specific T cells (VSTs, e.g., virus-specific CD4+ T cells (e.g., T H cells) and / or virus-specific CD8+ T cells (e.g., CTLs). In some embodiments, the CAR-expressing cells are adenovirus-specific T cells (AdVST), cytomegalovirus-specific T cells (CMVST), Epstein-Barr virus-specific T cells (EBVST), influenza virus-specific T cells, measles virus-specific T cells, hepatitis B virus-specific T cells. (HBVST), Hepatitis C virus-specific T cells (HCVST), Human Immunodeficiency Virus-specific T cells (HIVST), Lymphocytic Choriomeningitis Virus-specific T cells (LCMVST), Herpes Simplex Virus-specific T cells (HSVST) or Human Papillomavirus (HPVST).

[0134] In some embodiments, the cell comprising / expressing a CAR is an oncolytic virus-specific immune cell (e.g., an oncolytic virus-specific T cell), e.g., as described herein.

[0135] Any cell of the present disclosure may be included in an isolated population of cells, and this population may be homogeneous or not.In a specific embodiment, the cell population has a majority of cells that are immune cells specific to oncolytic viruses and / or CAR-expressing cells.The cells in the cell population may contain one or more nucleic acids that code oncolytic adenovirus (OncAd), helper-dependent adenovirus (HDAd), chimeric antigen receptor (CAR), and / or one or more of OncAd, HDAd, and / or CAR.In a specific embodiment, the cell population has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the cells that contain one or more nucleic acids that code oncolytic adenovirus (OncAd), helper-dependent adenovirus (HDAd), chimeric antigen receptor (CAR), and / or one or more of OncAd, HDAd, and / or CAR.

[0136] Oncolytic virus-specific immune cells The disclosed aspects of the present invention provide an oncolytic virus-specific immune cell (also referred to herein as an immune cell specific for an oncolytic virus). The oncolytic virus-specific immune cell expresses / contains a receptor capable of recognizing an antigenic peptide of the oncolytic virus (e.g., when presented by an MHC molecule). The immune cell can express / contain such a receptor as a result of expression of an endogenous nucleic acid encoding such an antigenic receptor, or as a result of being engineered to express such a receptor.

[0137] In some embodiments, the oncolytic virus-specific immune cells may be cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. The lymphocytes may be, for example, T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or precursors thereof. The cells may express, for example, CD3 polypeptides (e.g., CD3γ, CD3ε, CD3ζ, or CD3δ), TCR polypeptides (TCRα or TCRβ), CD27, CD28, CD4, or CD8. In some embodiments, the oncolytic virus-specific immune cells are T cells, such as CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are T helper cells (T H In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)).

[0138] The oncolytic virus-specific immune cells (e.g., oncolytic virus-specific T cells) may be specific for an oncolytic virus described herein, i.e., the oncolytic virus-specific immune cells may be specific for one or more antigens of an oncolytic virus described herein.

[0139] Methods for generating / expanding populations of immune cells specific to an antigen of interest and / or a virus of interest are well known in the art and are described, for example, in Wang and Riviere Cancer Gene Ther. (2015) 22(2):85-94, which is incorporated by reference in its entirety.

[0140] Such methods may include contacting a heterogeneous population of immune cells (e.g. peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), tumor infiltrating lymphocytes (TILs)) with one or more peptides of an antigen of interest, or cells containing / expressing such antigens / peptides. The cells containing / expressing the antigen / peptide may be as a result of infection with a virus containing / encoding the antigen, uptake of the antigen / peptide by the cell, or expression of the antigen / peptide. Presentation is in the context of MHC molecules on the cell surface of the antigen presenting cells.

[0141] Cells containing / expressing antigens / peptides may be contacted with (or "pulsed" with) antigenic peptides according to methods well known to those skilled in the art. Antigenic peptides may be provided in the form of a library of peptide mixtures (corresponding to one or more antigens), which may also be referred to as a pepmix. The peptides of a pepmix may be overlapping peptides of 8-20 amino acids in length, covering all or part of the amino acid sequence of the relevant antigens.

[0142] Cells in a population of immune cells that contain a receptor specific for a peptide may be activated (stimulated to proliferate) after recognition of the peptide of an antigen presented by an antigen-presenting cell (APC) in the context of an appropriate co-stimulatory signal. It is understood that "immune cells specific for an oncolytic virus" encompasses a plurality of cells, for example, a population of such cells. Such populations can be generated / expanded in vitro and / or ex vivo.

[0143] In some embodiments, the oncolytic virus-specific immune cells are specific for oncolytic adenovirus (OncAd), such as the OncAd described herein. In some embodiments, the oncolytic virus-specific immune cells are specific for an antigen of OncAd. In some embodiments, the antigen is or is derived from an OncAd protein, such as a protein encoded by an early gene (e.g., E1 (e.g., E1A, E1B), E2 (e.g., E2A, E2B), E3 or E4), a protein encoded by a late gene (e.g., L1, L2, L3, L4 or L5), a protein encoded by IX, or a protein encoded by IVa2. In some embodiments, the antigen is or is derived from the hexon and / or penton of OncAd.

[0144] In some embodiments of the various aspects of the present disclosure, virus-specific immune cells can be generated / expanded (or may have been generated / expanded) by a method comprising stimulating a population of immune cells by culture in the presence of antigen-presenting cells (APCs) that present a peptide of the virus.

[0145] In some embodiments, immune cells specific for the oncolytic viruses disclosed herein are prepared by a method employing a pepmix comprising a mixture of overlapping peptides corresponding to human adenovirus 3 hexon and / or a pepmix comprising a mixture of overlapping peptides corresponding to human adenovirus 5 penton.

[0146] In some embodiments, the oncolytic virus-specific immune cells express / comprise a CAR, e.g., a CAR described herein. The oncolytic virus-specific immune cells may be engineered to express a CAR, e.g., by transfection / transduction of the oncolytic virus-specific immune cells with a nucleic acid encoding a CAR.

[0147] Combinations of the present disclosure Aspects of the invention include compositions and methods comprising / employing (i) an oncolytic virus; (ii) a virus comprising a nucleic acid encoding an immune modulator; and (iii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen.

[0148] Compositions and methods (i.e., without necessarily employing a virus that contains a nucleic acid encoding an immune modulator) that comprise / employ (i) an oncolytic virus; and (ii) at least one cell that comprises a chimeric antigen receptor (CAR) specific for a cancer cell antigen are also provided.

[0149] Also provided are compositions and methods comprising / recruiting (i) an oncolytic virus; and (ii) immune cells specific for the oncolytic virus. In some embodiments according to the various aspects described herein, the cell comprising / expressing the CAR is specific for the oncolytic virus employed (e.g., comprises an antigen receptor (e.g., TCR) specific for an antigen of the oncolytic virus). That is, in some embodiments, the specificity of the oncolytic virus and the cell comprising / expressing the CAR are matched. As an example, in some embodiments, the oncolytic virus is an adenovirus and the CAR-expressing cell comprising / expressing the CAR is an adenovirus-specific T cell.

[0150] Similarly, in various configurations described herein, the oncolytic virus is employed in combination with immune cells specific for the oncolytic virus (ie, the same oncolytic virus).

[0151] A "combination" as referred to herein encompasses products and compositions (e.g., pharmaceutical compositions) that include the components of the combination. A "combination" also encompasses therapeutic regimens that employ the components of the combination.

[0152] In some embodiments, the components of the combination are provided in separate compositions. In some embodiments, more than one component of the combination is provided in one composition. In some embodiments, the components of the combination are provided in one composition.

[0153] Similarly, in some embodiments, the components of the combination are administered separately. In some embodiments, the components of the combination are administered together with other components of the combination. In some embodiments, the components of the combination are administered together.

[0154] By way of illustration, in an example of a combination comprising an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, and at least one cell comprising a CAR specific for a cancer cell antigen, the oncolytic virus and the virus comprising a nucleic acid encoding an immunomodulator may be administered together, and the at least one cell comprising a CAR specific for a cancer cell antigen may be administered separately (e.g., subsequently).

[0155] When the components of the combination are administered together, administration may be simultaneous administration as described herein below. When the components of the combination are administered separately, administration may be simultaneous or sequential administration as described herein below. When the components of the combination are administered separately, administration of the separate components may or may not be via the same route of administration.

[0156] Functional properties Agents of the present disclosure can be defined by reference to one or more functional properties. Agents can be evaluated for functional properties, for example, by assays such as those described in the experimental examples. Similarly, combinations and methods of the present disclosure can be defined by reference to one or more functional properties and / or effects, for example, Such properties / effects can be assessed, for example, by assays such as those described in the experimental examples.

[0157] In some embodiments, an oncolytic virus according to the present disclosure may have one or more of the following features: · the ability to replicate in and / or cause cell death of cancer cells; a reduced ability to replicate in and / or cause cell killing of non-cancerous cells compared to its ability to replicate in and / or cause cell killing of cancer cells; · equivalent or improved ability to cause cell killing of cancer cells compared to the ability of one or more oncolytic viruses known in the art; · the ability to support the replication of helper-dependent adenovirus (HDAd); · Similar or improved ability to replicate in cancer cells compared to the ability of one or more oncolytic viruses known in the art.

[0158] In some embodiments, a cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen according to the present disclosure may have one or more of the following functions: the ability to bind to HER2; ·Ability to bind to HER2 expressing cells; · the ability to induce cell death in HER2 expressing cells; · A reduced ability to cause cell killing in cells that do not express HER2 compared with the ability to cause cell killing in cells that express HER2.

[0159] In some embodiments, the combination of an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, and at least one cell comprising a CAR specific for a cancer cell antigen may have one or more of the following functions: · improved ability to cause cell killing of cancer cells compared to the ability of any one of the components used alone or any two of the components used in combination to cause cell killing of cancer cells; The ability to cause cell killing of cancer cells that is synergistic (i.e., greater than additive) compared to the ability of each component used alone to cause cell killing of cancer cells.

[0160] In some embodiments, the combination of an oncolytic virus and at least one cell comprising a CAR specific for a cancer cell antigen may have one or more of the following functions: · improved ability to cause cell killing of cancer cells compared to the ability of either component used alone to cause cell killing of cancer cells; The ability to cause cell killing of cancer cells that is synergistic (i.e., greater than additive) compared to the ability of each component used alone to cause cell killing of cancer cells.

[0161] In some embodiments, the combination of oncolytic virus and oncolytic virus-specific immune cells may have one or more of the following functions: · improved ability to cause cell killing of cancer cells compared to the ability of either component used alone to cause cell killing of cancer cells; The ability to cause cell killing of cancer cells that is synergistic (i.e., greater than additive) compared to the ability of each component used alone to cause cell killing of cancer cells.

[0162] The analysis of the ability to cause cell death of cancer cells can be evaluated, for example, in vitro, by analysis of the number / viability of cancer cells. The analysis of the ability to cause cell death of cancer cells can also be evaluated, for example, in vivo, in a suitable model, by analysis of the number of cancer cells, size / viability of tumors. It can also be analyzed by analyzing the volume and / or some other correlation with cancer cell count (e.g., disease progression, severity of cancer symptoms, etc.).

[0163] therapeutic use Aspects of the present disclosure relate in particular to the use of an oncolytic virus, a virus comprising a nucleic acid encoding an immune modulator, and at least one T cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen in the treatment of cancer in a subject.

[0164] Thus, the present disclosure provides a method of treating cancer comprising administering to a subject an oncolytic virus; a virus comprising a nucleic acid encoding an immune modulator; and at least one T cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen.

[0165] The present disclosure also provides an oncolytic virus; a virus comprising a nucleic acid encoding an immunomodulator; and at least one T cell comprising a chimeric antigen receptor (CAR) specific to a cancer cell antigen for use in a method for treating cancer. Also provided is the use of an oncolytic virus; a virus comprising a nucleic acid encoding an immunomodulator; and at least one T cell comprising a chimeric antigen receptor (CAR) specific to a cancer cell antigen in the manufacture of a medicament for treating cancer.

[0166] The present disclosure also provides a method for treating cancer, comprising administering to a subject at least one cell comprising (i) an oncolytic virus; and (ii) a chimeric antigen receptor (CAR) specific to a cancer cell antigen. Also provided is at least one cell comprising (i) an oncolytic virus; and (ii) a chimeric antigen receptor (CAR) specific to a cancer cell antigen for use in the method for treating cancer. Also provided is the use of at least one cell comprising (i) an oncolytic virus; and (ii) a chimeric antigen receptor (CAR) specific to a cancer cell antigen in the manufacture of a medicament for use in the method for treating cancer.

[0167] The present disclosure also provides a method for treating cancer, comprising administering to a subject (i) an oncolytic virus; and (ii) an immune cell specific to the oncolytic virus. Also provided is an immune cell specific to the oncolytic virus (i) for use in the method for treating cancer. Also provided is the use of an immune cell specific to the oncolytic virus (i) for use in the method for treating cancer, in the manufacture of a medicament for use in the method for treating cancer.

[0168] Also provided is a method for treating cancer, comprising administering to a subject the OncAd, HDAd, CAR, one or more nucleic acids, cells and pharmaceutical compositions of the present disclosure.Also provided is the OncAd, HDAd, CAR, one or more nucleic acids, cells and pharmaceutical compositions of the present disclosure for use in a method for treating cancer.Also provided is the use of the OncAd, HDAd, CAR, one or more nucleic acids, cells and pharmaceutical compositions of the present disclosure in the manufacture of a medicament for treating cancer.

[0169] "Treatment" can be, for example, the reduction of cancer development or progression, the alleviation of cancer symptoms, or the reduction of cancer pathology.Treatment or alleviation of cancer can be effective in preventing the progression of cancer, for example, preventing the worsening of the condition, or slowing the rate of development of more severe pathology.In some embodiments, treatment or alleviation can result in the improvement of cancer, for example, the reduction of cancer symptoms, or the reduction of some other correlation of cancer severity / activity.Prevention of cancer can also refer to the prevention of the worsening of the condition or the prevention of cancer development, for example, preventing early cancer from developing into late stage.

[0170] In some embodiments, the treatment reduces the number of cancer cells or tissue containing cancer cells in the subject. In some embodiments, the treatment may be aimed at reducing the size and / or preventing the growth of at least one tumor in the subject.

[0171] In some embodiments, the treatment comprises administering to the subject an oncolytic virus according to the present disclosure. In some embodiments, the treatment may comprise administering to the subject a cell or a population of cells that comprises or encodes an oncolytic virus according to the present disclosure. In some embodiments, the treatment comprises administering to the subject an oncolytic virus and a virus that encodes an immunomodulator according to the present disclosure. In some embodiments, the treatment may comprise administering to the subject a cell or a population of cells that comprises or encodes an oncolytic virus and / or a virus that encodes an immunomodulator according to the present disclosure.

[0172] In some embodiments, the treatment may include modifying a cell or population of cells to contain / express a CAR according to the present disclosure. In some embodiments, the treatment may include administering to the subject a cell or population of cells that have been modified to contain / express a CAR according to the present disclosure. In some embodiments, the treatment is aimed at providing the subject with an immune cell or population of immune cells with specificity for a cancer cell antigen, for example by administering a CAR-expressing cell according to the present disclosure or generating a CAR-expressing cell according to the present disclosure.

[0173] In some embodiments, the treatment may comprise administering to the subject immune cells / populations of immune cells specific for the oncolytic virus of the present disclosure. In some embodiments, the treatment is aimed at providing to the subject immune cells / populations of immune cells with specificity for the oncolytic virus. In some embodiments, the treatment may comprise generating / expanding a population of immune cells specific for the oncolytic virus of the present disclosure.

[0174] In some embodiments, the treatment may include administering to the subject an immune cell / population of immune cells specific for the oncolytic virus disclosed herein that is modified to include / express a CAR disclosed herein. In some embodiments, the treatment is aimed at providing to the subject an immune cell / population of immune cells that has specificity for the oncolytic virus and also has specificity for a cancer cell antigen. In some embodiments, the treatment may include generating / expanding a population of immune cells specific for the oncolytic virus disclosed herein and modifying a cell or a population of cells to include / express a CAR disclosed herein.

[0175] The subject to be treated may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female, or may have any gender. The subject may be a patient. The subject may be a subject diagnosed with a cancer that requires treatment, a subject suspected of having such a cancer, or a subject at risk of developing such a cancer.

[0176] In some embodiments, the cancer to be treated comprises cells that express a cancer cell antigen, such as a cancer cell antigen described herein (e.g., HER2). In some embodiments, the cells express a cancer cell antigen (e.g., HER2) on the cell surface.

[0177] In some embodiments, the cancer to be treated comprises cells expressing a cancer cell antigen for which the CAR is specific. In some embodiments, the CAR comprises a cancer cell antigen binding domain. The cancer to be treated includes cells that express the cancer cell antigen, for example cells that express the cancer cell antigen on their cell surface.

[0178] In some embodiments, the cancer overexpresses a cancer cell antigen. Overexpression of a cancer cell antigen can be determined by detecting a level of expression of the cancer cell antigen that is greater than the level of expression by an equivalent non-cancerous cell / non-tumor tissue.

[0179] In some embodiments, cancer is HER2 expressing cancer, for example, HER2 expressing cancer on cell surface.In some embodiments, cancer overexpresses HER2.Overexpression of HER2 can be determined by detecting the expression level of HER2 that is greater than the expression level of HER2 by equivalent non-cancerous cell / non-tumor tissue.

[0180] In some embodiments, a subject to be treated according to the present disclosure is selected for treatment based on detection of expression / overexpression of a cancer cell antigen by cancer cells or tumors obtained from the subject.

[0181] The expression of a given cancer cell antigen can be determined by any suitable means. The expression can be gene expression or protein expression. Gene expression can be determined, for example, by detecting the mRNA encoding the cancer cell antigen, for example, by quantitative real-time PCR (qRT-PCR). Protein expression can be determined, for example, by detecting the cancer cell antigen, for example, by antibody-based methods, for example, by Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

[0182] The cancer to be treated / prevented according to the present disclosure may be any unwanted cell proliferation (or any disease in which unwanted cell proliferation appears), a neoplasm or tumor. The cancer may be benign or malignant, primary or secondary (metastatic). The cancer may be resistant (initially or after treatment) and / or the cancer may be recurrent. The neoplasm or tumor may be any abnormal growth or proliferation of cells and may be present in any tissue. The cancer may be of tissue / cells, e.g., from the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (with or without the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., kidney epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, palatine tonsils, trachea, uterus, vulva, white blood cells.

[0183] The cancer to be treated / prevented may be any type of cancer, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancer (e.g., Kaposi's sarcoma, AIDS-related lymphoma, primary central nervous system lymphoma), anal cancer, appendix cancer, astrocytoma, basal cell carcinoma of the skin, bile duct cancer (e.g., cholangiocarcinoma), bladder cancer, bone cancer (e.g., Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, cancer of unknown primary site, cardiac tumor, central nervous system cancer of the abdomen (e.g. atypical teratoid / rhabdoid tumor, embryonal tumor, germ cell tumor, primary central nervous system lymphoma), cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma (e.g. mycosis fungoides, Sézary syndrome), ductal carcinoma in situ (DCIS), endometrial cancer (uterine cancer), ependymoma, esophageal cancer, nasal neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (e.g. intraocular melanoma, retinoblastoma), fallopian tube cancer, malignant fibrous histiocytoma of bone tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), ovarian germ cell tumors, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, cardiac tumors, hepatocellular (liver) cancer, histiocytosis, Langerhans cells, Hodgkin's lymphoma, hypopharyngeal cancer, pancreatic islet cell tumors (pancreatic endocrine tumors), kidney (renal cell) cancer, laryngeal cancer, papillary Neoplastic syndromes, leukemia, oral cavity cancer, lung cancer (non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), lymphoma, male breast cancer, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, occult primary metastatic squamous cell carcinoma of the neck, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative Neoplasms, Myeloid Leukemia, Chronic Myeloid Leukemia, Acute Myeloid Leukemia (AML), Nasal and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Oral Cavity Cancer, Oral Cavity Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasmacytoma / Multiple Bone Marrow Tumors These include any one of the following: myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, vascular tumors, uterine sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma of the skin, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0184] In some embodiments, the cancer to be treated is one or more of nasopharyngeal carcinoma (NPC; e.g., Epstein-Barr virus (EBV)-positive NPC), cervical cancer (CC; e.g., human papillomavirus (HPV)-positive CC), oropharyngeal carcinoma (OPC; e.g., HPV-positive OPC), gastric cancer (GC; e.g., EBV-positive GC), hepatocellular carcinoma (HCC; e.g., hepatitis B virus (HBV)-positive HCC), lung cancer (e.g., non-small cell lung cancer (NSCLC)), and head and neck cancer (e.g., a cancer originating from tissues of the lip, mouth, nose, paranasal sinuses, pharynx, or larynx, e.g., head and neck squamous cell carcinoma (HNSCC)).

[0185] In some embodiments, the cancer is associated with or caused by a virus. In some embodiments, the cancer is an EBV positive cancer. In some embodiments, the cancer is an HPV positive cancer.

[0186] In some embodiments, the cancer is one of head and neck cancer, nasopharyngeal carcinoma (NPC), oropharyngeal cancer (OPC), cervical cancer (CC), gastric / stomach cancer, stomach cancer, or lung cancer.

[0187] Therapeutic methods may also include in vivo therapies, including those using autologous and / or heterologous cells or immortalized cell lines, ex vivo therapies, and adoptive immunotherapy.

[0188] Administration Administration is preferably in a "therapeutically effective amount", which is an amount sufficient to show benefit to an individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, such as determining dosing, is within the responsibility of a general practitioner or other physician, and typically takes into account the condition to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to physicians. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, published in 2000, Lippincott, Williams & Wilkins.

[0189] The viruses, CARs, nucleic acids, and cells of the present disclosure can be formulated as pharmaceutical compositions or medicaments for clinical use, and such pharmaceutical compositions or medicaments may include a pharma- ceutical acceptable carrier, diluent, excipient, or adjuvant. The pharmaceutical composition may be formulated for administration via local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes, such administration may include injection or infusion. Suitable formulations may include the virus, CAR, nucleic acid, or cells in a sterile or isotonic medium. The pharmaceutical composition may be formulated in a fluid, such as a gel form (foam). Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) to a selected area of ​​the human or animal body.

[0190] The virus comprising the nucleic acid encoding the oncolytic virus and / or immunomodulator may be formulated for intratumoral administration. In some embodiments, the method may include intratumoral administration of the virus comprising the nucleic acid encoding the oncolytic virus and / or immunomodulator.

[0191] The CAR-containing cells and / or oncolytic virus-specific immune cells may be formulated for intravenous administration. In some embodiments, the method may include intravenous administration of the CAR-containing cells and / or oncolytic virus-specific immune cells.

[0192] The administration of the components of the combinations disclosed herein (e.g., an oncolytic virus, a virus comprising a nucleic acid encoding an immune modulator; at least one T cell comprising a CAR specific for a cancer cell antigen; an immune cell specific for an oncolytic virus according to the disclosure) may be simultaneous or sequential. The disclosure also contemplates simultaneous or sequential administration of an OncAd, HDAd, CAR, one or more nucleic acids, cells, and pharmaceutical compositions of the disclosure.

[0193] Concurrent administration refers to administering the drugs together, for example as pharmaceutical compositions containing the drugs (i.e., combined preparations), or administering immediately after each other, optionally via the same administration route, for example, into the same artery, vein or other blood vessel.In certain embodiments, the oncolytic virus and the virus comprising the nucleic acid encoding the immunomodulator can be administered simultaneously in the form of combined preparations.In certain embodiments, when administered simultaneously, two or more drugs can be administered via different administration routes.In some embodiments, concurrent administration refers to administering at the same time, or within, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours or 48 hours.

[0194] Sequential administration refers to administering one or more of the drugs, followed by separate administration of the other drug after a given time interval.The two drugs do not necessarily have to be administered by the same route, but in some embodiments, they may be.The time interval may be any time interval, such as hours, days, weeks, months, or years.In some embodiments, sequential administration refers to administering 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 apart.

[0195] In some embodiments, the treatment may further include other therapeutic or preventive interventions, such as chemotherapy, immunotherapy, radiation therapy, surgery, vaccination, and / or hormonal therapy. Such other therapeutic or preventive interventions may be administered before, during, and / or after the therapy encompassed by the present disclosure, and the delivery of the other therapeutic or preventive interventions may be via a different administration route than the therapy encompassed by the present disclosure. Chemotherapy and radiation therapy refer to the treatment of cancer with drugs or with radiation therapy using ionizing radiation (e.g., X-rays or gamma rays), respectively. Drugs are chemical entities, such as small molecule pharmaceuticals, antibiotics, DNA intercalators, and the like. The drug may be a pharmacokinetic agent, a pharmacokinetic agent, a stimulator, a protein inhibitor (e.g., a kinase inhibitor), or a biological agent, such as an antibody, an antibody fragment, a nucleic acid or peptide aptamer, a nucleic acid (e.g., DNA, RNA), a peptide, a polypeptide, or a protein. The drug may be formulated as a pharmaceutical composition or medicament. The formulation may include one or more drugs (e.g., one or more active agents) together with one or more pharma- ceutically acceptable diluents, excipients, or carriers.

[0196] The chemotherapeutic agents may be administered by one or more routes of administration, such as parenteral, intravenous, oral, subcutaneous, intradermal or intratumoral routes. Chemotherapy can be administered according to a treatment plan, which may be a predetermined timetable, plan, scheme or schedule of chemotherapy administration, and such a plan may be prepared by a physician or medical technician and may be tailored to suit the patient in need of treatment.

[0197] A treatment plan may dictate one or more of the following: the type of chemotherapy to be administered to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of treatment holidays, if any, etc. In the case of combination therapy, a single treatment plan may be provided that dictates how each drug should be administered.

[0198] Chemotherapeutic and biological agents include alkylating agents, such as cisplatin, carboplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; purine or pyrimidine antimetabolites, such as azathioprine or mercaptopurine; alkaloids and terpenoids, such as vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine), podophyllotoxin, etoposide, teniposide, taxanes, such as paclitaxel (Taxo l(trademark), docetaxel; topoisomerase inhibitors, such as the type I topoisomerase inhibitors camptothecin, irinotecan, and topotecan, or the type II topoisomerase inhibitors amsacrine, etoposide, etoposide phosphate, teniposide; antitumor antibiotics (e.g., anthracycline antibiotics), such as dactinomycin, doxorubicin (Adriamycin(trademark), epirubicin, bleomycin, rapamycin); antibody-based anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-CTLA-4, anti-4-1BB, anti-GITR, anti-CD27, anti-BLTA, anti-OX43, anti-VEGF, anti-TNFα, anti-IL-2, anti-GpIIb / IIIa, anti-CD-52, anti-CD20, anti-RSV, anti-HER2 / neu (erbB2), anti-TNF receptor, anti-EGFR antibodies, monoclonal antibodies or antibody fragments, such as cetuximab, panitumumab, infliximab, basiliximab, bevacizumab ( Avastin®), abciximab, daclizumab, gemtuzumab, alemtuzumab, rituximab (Mabthera®), palivizumab, trastuzumab, etanercept, adalimumab, nimotuzumab; EGFR inhibitors, such as erlotinib, cetuximab and gefitinib; anti-angiogenic agents, such as bevacizumab (Avastin®); cancer vaccines, such as sipuleucel-T (Provenge®).

[0199] Additional chemotherapeutic agents include 13-cis-retinoic acid, 2-chlorodeoxyadenosine, 5-azacytidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, Abraxane, Accutane®, Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®, aldesleukin, alemtuzumab, ALIMTA, alitretinoin, Alkaban-AQ®, Alkeran®, all-trans retinoic acid, alpha interferon, altretamine, amethopterin, amifostine, aminoglutethimide, anagrelo, cyclosporine ... Lyde, Anandron®, Anastrozole, Arabinosylcytosine, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®, Arsenic Trioxide, Asparaginase, ATRA Avastin®, Azacitidine, BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, Leucovorin Calcium, Campath®, Camptosar®, Camptothecin-11, Capecitabine, Carac™, Carboplatin, Carmustine, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide, Cytadren®, CytarabineCytosar-U®, Cytoxan®, Dacogen, dactinomycin, darbepoetin alfa, dasatinib, daunomycin, daunorubicin, daunorubicin hydrochloride, daunorubicin liposomal, DaunoXome®, Decadron, decitabine, Delta-Cortef®, Deltasone®, denileukin, diftitox, DepoCyt™, dexamethasone, dexamethasone acetate, dexamethasone phosphate Sodium, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal, Droxia™, DTIC, DTIC-Dome®, Duralone®, Eligard®, Ellence®, Eloxatin™, Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux, El Lotinib, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos®, Etoposide, Etoposide phosphate, Eulexin®, Everolimus, Evista®, Exemestane, Faslodex®, Femara®, Filgrastim, Floxiridine, Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluoxymestane Lon, flutamide, folinic acid, FUDR®, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, Gleevec™, Gliadel® wafers, goserelin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Herceptin®, hexadrol, Hexalen®, hexamethylmelamine, HMM, Hycamtin®, Hydrea®, HydrocortAcetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate, Hydrocortisone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin®, Idarubicin, Ifex®, IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib Mesylate, Imidazole Carboxamide, Interferon Alpha, Interferon Alpha-2b (PEG Conjugate), Interleukin-2, Interleukin-11, Intron A® (Interferon Alpha-2b), Iressa®, Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, Kidrolase, Lanacort®, Lapatinib , L-Asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran, Leukine™, Leuprolide, Leulocristine, Leustatin™, Liposomal Ara-C, Liquid Pred™, Lomustine, L-PAM, L-Sarcolysin, Lupron™, Lupron Depot™, Matulane™, Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride, Medralone™, Medrol™, Me gace®, megestrol, megestrol acetate, melphalan, mercaptopurine, mesna, Mesnex™, methotrexate, methotrexate sodium, methylprednisolone, Meticorten®, mitomycin, mitomycin-C, mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, mustine, Mutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine, Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®, Nilutamide, Nipent®, nitrogen mustard, Novaldex®, Novantrone® mark), octreotide, octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxal™, Oprelvekin, Orapred®, Orasone®, oxaliplatin, paclitaxel, paclitaxel protein-bound, pamidronate, panitumumab, Panretin®, Paraplatin®, Pediapred®, PEG-interferon, pegaspargase, pegfilgrastim, PEG-INTRON™, PEG-L-asparaginase, pemetrexed, pentostatin, phenylalanine mustard, Platinol®, Platinol-AQ®, prednisolone, prednisone, Prelone®, procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with Carmustine ImplantPurinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®, Rituximab, Roferon-A® (Interferon alpha-2a), Rubex®, Rubidomycin hydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim, Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin, SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®, Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA, Thalidomide, Thalomid®, TheraCys®, Thioguanine Tabloid®, Thiophosphamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan, Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin, Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®, Vepesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate, Vincasar It may be selected from Pfs®, vincristine, vinorelbine, vinorelbine tartrate, VLB, VM-26, vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™, Zinecard®, Zoladex®, zoledronic acid, Zolinza, Zometa®.

[0200] In an embodiment of the present disclosure, a nucleic acid / virus encoding an enzyme capable of catalyzing the conversion of a non-toxic factor to a cytotoxic form is employed, and the method may further comprise administering with a prodrug substrate for the enzyme. The prodrug may be a nucleic acid / virus encoding an enzyme capable of catalyzing the conversion of a non-toxic factor to a cytotoxic form. It may be administered simultaneously with or sequentially to the administration of

[0201] In some embodiments, for example, when the nucleic acid / virus codes for thymidine kinase, the prodrug is selected from ganciclovir (GCV), acyclovir (ACV) and / or valacyclovir. In some embodiments, for example, when the nucleic acid / virus codes for cytosine deaminase, the prodrug is 5-fluorocytosine (5-FC). In some embodiments, for example, when the nucleic acid / virus codes for nitroreductase, the prodrug is selected from CB1954, nitro-CBI-DEI and / or PR-104A. In some embodiments, for example, when the nucleic acid / virus codes for cytochrome P450, the prodrug is an oxazaphosphorine (e.g., cyclophosphamide or ifosfamide). In some embodiments, for example, when the nucleic acid / virus codes for carboxypeptidase G2, the prodrug is a nitrogen mustard-based drug (e.g., CMDA or ZD2767P). In some embodiments, e.g., when the nucleic acid / virus encodes a purine nucleoside phosphorylase, the prodrug is 6-methylpurine 2-deoxyriboside and / or fludarabine (e.g., 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo), or arabinofuranosyl-2-F-adenine monophosphate (F-araAMP). In some embodiments, e.g., when the nucleic acid / virus encodes a horseradish peroxidase, the prodrug is indole-3-acetic acid (IAA). In some embodiments, e.g., when the nucleic acid / virus encodes a carboxylesterase, the prodrug is irinotecan.

[0202] Multiple doses of an agent of the present disclosure (e.g., a virus (OncAd, HdAd), a CAR, one or more nucleic acids, vectors, cells, compositions, combinations, prodrugs) can also be provided. One or more or each of the doses can be accompanied by simultaneous or sequential administration of another therapeutic agent.

[0203] The multiple doses may be separated by a predetermined time interval, which may be selected to be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or more hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. As an example, doses may be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0204] Adoption In an embodiment of the present disclosure, the method of treatment comprises adoptive transfer of immune cells. Adoptive cell transfer (ACT) generally refers to a process by obtaining cells (e.g., immune cells) from a subject, typically by taking a blood sample from which the cells are isolated. The cells are then typically treated or modified in some way, and then administered to either the same subject (adoptive transfer is autologous cell transfer) or a different subject (adoptive transfer is heterologous cell transfer). The purpose of this treatment is typically to provide the subject with a population of cells with certain desirable characteristics, or to increase the frequency of cells with such characteristics in the subject. In the present disclosure, adoptive transfer can be performed with the purpose of introducing a cell or a population of cells into a subject and / or increasing the frequency of a cell or a population of cells in a subject.

[0205] In some embodiments, the subject from which the cells are isolated is the subject to which the modified cells are administered. (i.e., the adoptive transfer is autologous cell transfer). In some embodiments, the subject from which the cells are isolated is a different subject than the subject to which the modified cells are administered (i.e., the adoptive transfer is xenogeneic cell transfer).

[0206] Adoptive transfer of T cells is described, for example, in Kalos and June 2013, Immunity 39(1):49-60, which is incorporated by reference in its entirety. Adoptive transfer of NK cells is described, for example, in Davis et al., 2015, Cancer J. 21(6):486-491, which is incorporated by reference in its entirety.

[0207] Such cells may be, for example, neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. The lymphocytes may be, for example, T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or precursors thereof. In some embodiments, the cells are T cells. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are T helper cells (T H In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)). In some embodiments, the T cell is a virus-specific T cell. In some embodiments, the T cell is specific for EBV, HPV, HBV, HCV, or sHIV.

[0208] In some embodiments, the cell is the immune cell specific to oncolytic virus as described herein.Therefore, in some embodiments, the method comprises administering to subject at least one immune cell specific to oncolytic virus.In some embodiments, the method of the present disclosure comprises generating / expanding a population of immune cells specific to oncolytic virus and administering to subject at least one immune cell specific to oncolytic virus.

[0209] In some embodiments, the method further comprises: (a) isolating immune cells from a subject; (b) generating or expanding a population of immune cells specific for the oncolytic virus by a method comprising stimulating the immune cells by culture in the presence of antigen-presenting cells (APCs) that present a peptide of the oncolytic virus; and (c) administering to the subject at least one immune cell specific for the oncolytic virus.

[0210] In some embodiments, the steps of the method for producing oncolytic virus-specific immune cells may include one or more of: taking a blood sample from a subject; isolating PBMCs from the blood sample; generating / expanding a population of oncolytic virus-specific immune cells (e.g., by culturing PBMCs in the presence of cells (e.g., APCs) that contain / express an antigen / peptide of the oncolytic virus); culturing the oncolytic virus-specific immune cells in in vitro or ex vivo cell culture; harvesting the oncolytic virus-specific immune cells; mixing the oncolytic virus-specific immune cells with an adjuvant, diluent or carrier; and administering the modified cells to a subject.

[0211] The present disclosure also provides a method for treating cancer in a subject, comprising administering at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen. In connection with the features of this disclosure, in some embodiments, the method additionally comprises a step for producing at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen. The CAR may be a first generation, second generation, or third generation CAR or a subsequent generation CAR. The CAR may be, for example, one, two, three, or more costimulatory domains.

[0212] In some embodiments, the method includes modifying at least one cell obtained from the subject to express or contain a CAR according to the disclosure, optionally expanding the modified at least one cell, and administering the modified at least one cell to the subject.

[0213] In some embodiments, the method further comprises: (a) isolating at least one cell from a subject; (b) modifying at least one cell to express or contain a CAR according to the present disclosure or a nucleic acid encoding a CAR according to the present disclosure; (c) optionally, expanding the at least one modified cell; and (d) administering at least one modified cell to a subject. Includes.

[0214] In some embodiments, the cell that comprises / expresses a CAR specific to a cancer cell antigen is an immune cell specific to an oncolytic virus as described herein.In some embodiments, the method includes modifying an immune cell specific to an oncolytic virus to express or comprise a CAR according to the present disclosure, optionally expanding the modified immune cell specific to the oncolytic virus, and administering the modified immune cell specific to the oncolytic virus to a subject.

[0215] In some embodiments, the method further comprises: (a) isolating immune cells from a subject; (b) generating or expanding a population of immune cells specific for the oncolytic virus by a method comprising stimulating the immune cells by culture in the presence of antigen-presenting cells (APCs) that present a peptide of the oncolytic virus; (c) modifying at least one immune cell specific for the oncolytic virus to express or contain a CAR according to the present disclosure or a nucleic acid encoding a CAR according to the present disclosure; (d) optionally, expanding at least one immune cell specific for the modified oncolytic virus; and (e) administering to the subject at least one immune cell specific for the modified oncolytic virus. Includes.

[0216] At least one cell modified according to the disclosure of the present invention may be modified to contain / express CAR according to methods known to those skilled in the art. Modification may include nucleic acid transfer for permanent or transient expression of the transferred nucleic acid. Any suitable genetic engineering platform can be used to modify cells according to the disclosure of the present invention. Suitable methods for modifying cells include the use of genetic engineering platforms such as gammaretroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, which is incorporated herein by reference above.

[0217] In some embodiments, the steps of the method for producing at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen include obtaining a blood sample from the subject; isolating and / or expanding at least one cell from the blood sample; The method may include one or more of culturing at least one cell in an in vitro or ex vivo cell culture; modifying at least one cell by introducing into the at least one cell a CAR described herein or a nucleic acid encoding a CAR described herein; expanding the at least one modified cell; harvesting the at least one modified cell; mixing the modified cell with an adjuvant, diluent or carrier; and administering the modified cell to a subject.

[0218] In some embodiments, the method may additionally include treating cells to induce / enhance expression of CAR or nucleic acid encoding CAR. For example, the nucleic acid may include a control element for inducibly upregulating expression of CAR from the nucleic acid in response to treatment with a particular agent. In some embodiments, the treatment may be performed in vivo by administering the agent to a subject to which the cell modified according to the present disclosure is administered. In some embodiments, the treatment may be performed ex vivo or in vitro by administering the agent to cells in ex vivo or in vitro culture.

[0219] One of skill in the art can determine appropriate reagents and procedures for adoptive transfer of cells according to the present disclosure, for example, by reference to Dai et al., 2016 J Nat Cancer Inst 108(7):djv439, which is incorporated by reference in its entirety.

[0220] In a related aspect, the present disclosure provides a method of preparing a modified cell, comprising modifying at least one cell by introducing into the cell a CAR according to the present disclosure or a nucleic acid encoding a CAR according to the present disclosure, the method being preferably carried out in vitro or ex vivo.

[0221] Compositions / Products / Kits The present disclosure also provides an oncolytic virus as described herein, optionally isolated. Nucleic acids encoding the optionally isolated oncolytic viruses are also provided. Cells comprising or including nucleic acids encoding the oncolytic viruses, optionally isolated, are also provided.

[0222] The present disclosure also provides a virus comprising an optionally isolated nucleic acid encoding an immunomodulatory factor described herein. An optionally isolated nucleic acid encoding the virus is also provided. An optionally isolated cell comprising the virus or comprising a nucleic acid encoding the virus is also provided.

[0223] The present disclosure also provides an optionally isolated chimeric antigen receptor (CAR) as described herein. An optionally isolated nucleic acid encoding the CAR is also provided. An optionally isolated cell comprising a CAR or comprising a nucleic acid encoding a CAR is also provided.

[0224] The present disclosure also provides compositions comprising an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, a chimeric antigen receptor, one or more nucleic acids, or a cell according to the present disclosure.

[0225] The oncolytic virus, the virus comprising a nucleic acid encoding an immunomodulator, the chimeric antigen receptor, one or more nucleic acids or cells according to the present disclosure may be formulated as a pharmaceutical composition for clinical use and may include a pharma- ceutically acceptable carrier, diluent, excipient or adjuvant. The combinations of the present disclosure may be provided in a single composition or as multiple compositions containing the components of the combination.

[0226] In accordance with the present disclosure, methods for the production of pharma- ceutical useful compositions are also provided, such production methods may comprise one or more steps selected from isolating an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, a chimeric antigen receptor, one or more nucleic acids or cells described herein; and / or mixing an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, a chimeric antigen receptor, one or more nucleic acids or cells described herein with a pharma- ceutical acceptable carrier, adjuvant, excipient or diluent.

[0227] For example, a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of cancer, comprising formulating a pharmaceutical composition or medicament by mixing an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, a chimeric antigen receptor, one or more nucleic acids or cells described herein with a pharma- ceutical acceptable carrier, adjuvant, excipient or diluent.

[0228] The present disclosure also provides a kit of parts comprising one or more of an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, a chimeric antigen receptor, a nucleic acid, a cell or a composition according to the present disclosure.

[0229] In some embodiments, the kit may have at least one container with a predetermined amount of an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, a chimeric antigen receptor, one or more nucleic acids, or a cell or composition according to the present disclosure. The kit may have containers containing individual components of the combinations disclosed herein, or may have containers containing combinations of components of the combinations disclosed herein.

[0230] The kit can provide an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, a CAR, a nucleic acid, a cell, or a composition with instructions for administration to a patient to treat a specified cancer. The oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulator, a CAR, one or more nucleic acids, cells, or compositions can be formulated to be suitable for injection or infusion into a tumor or blood.

[0231] In some embodiments, the kit may include materials for producing cells according to the present disclosure.For example, the kit may include materials for modifying cells to express or include a virus or its antigen / peptide, CAR or one or more nucleic acids according to the present disclosure, or materials for introducing a virus or its antigen / peptide or one or more nucleic acids according to the present disclosure into cells.The kit may also include materials for producing immune cells specific for oncolytic viruses, for example, the kit may include a pepmix of one or more antigens of oncolytic viruses.

[0232] In some embodiments, the kit may further comprise at least one container with a predetermined amount of another therapeutic agent (for example, anti-infective agent or chemotherapeutic agent).In such an embodiment, the kit may also comprise a second medicament or pharmaceutical composition, in which case the two medicaments or pharmaceutical compositions may be administered simultaneously or separately, so as to provide a combined treatment for cancer.The therapeutic agent may also be formulated to be suitable for injection or infusion into tumor or blood.

[0233] Sequence identity Pairwise and multiple sequence alignments for purposes of determining percent identity between two or more amino acid or nucleic acid sequences can be performed in a variety of ways known to those of skill in the art. For example, publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software can be used. When using such software, it is preferred to use the default parameters, e.g., with regard to gap penalties and extension penalties.

[0234] [Table 1-1]

[0235] [Table 1-2]

[0236] [Table 1-3]

[0237] [Table 1-4]

[0238] [Table 1-5]

[0239] [Table 1-6]

[0240]

Table 1-7

[0241]

Table 1-8

[0242]

Table 1-9

[0243]

Table 1-10

[0244]

Table 1-11

[0245]

Table 1-12

[0246]

Table 1-13

[0247]

Table 1-14

[0248]

Table 1-15

[0249]

Table 1-16

[0250]

Table 1-17

[0251]

Table 1-18

[0252]

Table 1-19

[0253]

Table 1-20

[0254]

Table 1-21

[0255]

Table 1-22

[0256]

Table 1-23

[0257]

Table 1-24

[0258]

Table 1-25

[0259]

Table 1-26

[0260]

Table 1-27

[0261]

Table 1-28

[0262]

Table 1-29

[0263]

Table 1-30

[0264]

Table 1-31

[0265]

Table 1-32

[0266]

Table 1-33

[0267]

Table 1-34

[0268]

Table 1-35

[0269]

Table 1-36

[0270]

Table 1-37

[0271] [Table 1-38]

[0272] [Table 1-39]

[0273] [Table 1-40]

[0274] [Table 1-41]

[0275] [Table 1-42]

[0276] [Table 1-43]

[0277] The present disclosure includes combinations of the described aspects and preferred features except where such combinations are clearly unacceptable or explicitly avoided. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0278] Disclosed forms and embodiments of the present invention will now be illustrated by way of example with reference to the accompanying drawings. Further forms and embodiments are expected to be apparent to those skilled in the art. All documents mentioned in this specification are incorporated herein by reference.

[0279] Throughout this specification, including the claims which follow, unless the context requires otherwise, the words "comprise", as well as "comprises" and "comprises" and "comprising" are used interchangeably. Variations such as "and" shall be understood to indicate the inclusion of a stated integer or step or group of integers or steps, but not to the exclusion of any other integer or step or group of integers or steps.

[0280] As used in this specification and the appended claims, the singular forms "a," "an," "an "An" and "the" are used interchangeably unless the context clearly indicates otherwise. It should be noted that the scope of the numerical referent is intended to encompass all such references. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0281] Where nucleic acid sequences are disclosed, the reverse complements thereof are also expressly contemplated. BRIEF DESCRIPTION OF THE DRAWINGS BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and studies illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings. [Brief description of the drawings]

[0282] [Figure 1A] FIG. 1A shows a schematic diagram of an example of a HER2-specific CAR construct. [Figure 1B] FIG. 1B shows a schematic diagram of an example protocol for transducing T cells to produce HER2-specific CAR-T. [Diagram 2]Graphs showing HER2-CAR, CCR7, CD45RO and PD-1 expression in T cells transduced with the indicated HER2-CAR constructs as determined by flow cytometry. [Diagram 3] Graph showing expression of HER2-CAR, CCR7, CD45RO, PD-1, LAG-3 and TIM-3 in CD4 and CD8 T cells after transduction with anti-HER2 clonal E4CAR construct as determined by flow cytometry. [Figure 4A] FIG. 4A is a bar graph showing cell killing of MDA cells (not expressing HER2 on the cell surface; negative control), MDA-HER2 cells (expressing HER2 on the cell surface; positive control), FaDu and SCC47 cells with anti-HER2 clone C5, and E4 and F1 CAR-T cells (or non-transduced (NT) cells) in vitro as determined by 51Cr release assay. [Figure 4B] FIG. 4B shows a graph depicting the expression of HER2 in MDA-HER2, FaDu and SCC47 cells, but not in MDA cells, as determined by flow cytometry. [Diagram 5] 1 is a bar graph showing cell killing of FaDu and SCC47 cells genetically modified to express firefly luciferase (ffLuc) by anti-HER2 clones C5, E4, and F1 CAR-T cells (or non-transduced (NT) cells) in vitro as determined by ffLuc activity assay. Data are presented as mean ± SD (n=4). *P<0.001. [Figure 6] FIG. 6 shows a schematic diagram of an example sequence of an ICOSTAT oncolytic adenoviral construct. [Figure 7A]Graphs depicting the ability of ICOSTAT oncolytic adenovirus to kill A549 (FIGS. 7A and 7F), FaDu (FIG. 7B), SCC47 (FIG. 7C), WI-38 (FIG. 7D) and ARPE-19 (FIG. 7E) cells after infection with the indicated concentrations of viral particles (Vp) as determined by MTS viability assay. Helper-dependent adenovirus (HDAd) is included as a control condition. [Figure 7B] Same as explanation for Figure 7A. [Figure 7C] Same as explanation for Figure 7A. [Figure 7D] Same as explanation for Figure 7A. [Figure 7E] Same as explanation for Figure 7A. [Figure 7F] Same as explanation for Figure 7A. [Figure 8A] 1 is a bar graph showing the ability of ICOSTAT oncolytic adenovirus to replicate and act as a helper for the replication of helper-dependent adenovirus (HDAd) as determined by copy number analysis by quantitative real-time PCR. The virus designated "Onc5 / 3AdicoSTAT" is ICOSTAT. "+HD" indicates coinfection with ICOSTAT and HDAd. [Figure 8B] Same as explanation for Figure 8A. [Figure 9A] FIG. 9A is a graph showing the replication of ICOSTAT oncolytic adenovirus in FaDu cells (FIG. 9A) in the presence or absence of 10 ng / ml IFNγ in cell culture medium. [Figure 9B] 9B is a graph showing the replication of ICOSTAT oncolytic adenovirus in SCC47 cells (FIG. 9B) in the presence or absence of 10 ng / ml IFNγ in cell culture medium. [Figure 10A] FIG. 10A is a schematic diagram of the HDAdIL-12_TK_PDL1 construct. [Figure 10B] FIG. 10B is a bar graph showing the production of IL-12p70 by cells transfected with the indicated helper-dependent adenoviral (HDAd) constructs. [Figure 10C] Figure 10C is a photograph of a western blot showing the production of anti-PD-L1 minibodies by cells transfected with HDAd constructs. [Figure 10D] FIG. 10D is a photograph of wells demonstrating HSV thymidine kinase production by cells transfected with HDAd constructs. [Figure 11] Figure 1 shows an ELISA analysis of PD-L1 minibody binding activity to recombinant human PD-L1 using serially diluted cell culture media from A549 cells transfected with a plasmid encoding GFP (pGFP; negative control), a plasmid encoding the anti-PD-L1 minibody described in Tanoue et al., supra (pPDL1miniTanoue) or a plasmid encoding the anti-PD-L1 minibody encoded by HDAdIL-12_TK_PD-L1 (pPDL1mini). Serially diluted anti-human PD-L1 antibody was used as a positive control (PDL1 IgG). [Figure 12A] 12A is a schematic diagram of an example sequence of an Onc5 / 2E1Δ24 oncolytic adenoviral construct (FIG. 12A). [Figure 12B] 12B is a schematic diagram of the sequence of the plasmid encoding the Onc5 / 2E1Δ24 oncolytic adenoviral construct. [Figure 13A] Graphs depicting the ability of Onc5 / 3Ad2E1A oncolytic adenovirus to kill FaDu (FIG. 13A), SCC47 (FIG. 13B), WI-38 (FIG. 13C) and ARPE-19 (FIG. 13D) cells after infection with the indicated concentrations of viral particles (Vp) as determined by MTS viability assay. Helper-dependent adenovirus (HDAd) is included as a control condition. [Figure 13B] Same as explanation for Figure 13A. [Figure 13C] Same as explanation for Figure 13A. [Figure 13D] Same as explanation for Figure 13A. [Figure 14]Graph showing the number of HER2-specific CAR T cells after the indicated days of cell culture following in vitro transduction with the indicated CAR constructs. [Figure 15A] Figures 15A-C are images and graphs showing the results of in vivo analysis of the anti-cancer activity of adoptively transferred luciferase-expressing T cells in an orthotopic FaDu cell-derived model of head and neck squamous cell carcinoma. Figures 15A and 15B show the number and location of cells expressing luciferase-expressing non-transduced T cells (NT) and luciferase-expressing T cells expressing C5, F1 or A3 HER2-specific CARs in mice on the indicated days after cell infusion. Figure 15C shows the percentage of surviving subjects in the different treatment groups on the indicated days after cell infusion. The negative control condition in which mice were not administered T cells is also shown as (-). [Figure 15B] Same as explanation for Figure 15A. [Figure 15C] Same as explanation for Figure 15A. [Figure 16A] Figures 16A-C are images and graphs showing the results of an analysis of adoptively transferred T cells in NSG mice in vivo. Figure 16A shows the number and location of cells expressing luciferase-expressing non-transduced T cells (NT), and luciferase-expressing T cells expressing C5, F1 or A3 HER2-specific CARs in the mice on the indicated days after cell infusion. [Figure 16B] FIG. 16B shows measurements of total flux (photons per second; p / sec) on the ventral surface per mouse in the different groups on the indicated days after cell infusion. [Figure 16C] FIG. 16C shows the body weights of mice in the different treatment groups on the indicated days after cell infusion, expressed as a percentage of day 0 body weight. [Figure 17A]Figures 17A-C are scatter plots and histograms showing the results of flow cytometric characterization of F1HER2-specific CAR T cells used in experiments to analyze the anti-cancer activity of CAdtrio and adoptively transferred T cells in combination in vivo. Figure 17A shows the percentage of CD4+ and CD8+ T cells in the F1.CAR-T population. [Figure 17B] Figure 17B shows the percentage of cells expressing HER2 CAR on the cell surface. [Figure 17C] Figure 17C shows the percentage of cells in the F1.CAR-T population that express CCR7 and / or CD45RO. [Figure 18A] Figures 18A-D are images and graphs showing the results of an in vivo analysis of the anti-cancer activity of the combination of CAdtrio and adoptively transferred T cells in an orthotopic FaDu cell-derived model of head and neck squamous cell carcinoma. Figure 18A shows the number and arrangement of cells expressing luciferase-expressing non-transduced T cells (NT), and luciferase-expressing T cells expressing an F1HER2-specific CAR in mice at the indicated days after cell infusion. The top right panel (Y-axis is labeled as total flux) is "days after CAR T cell injection." The bottom two panels are "days after CAdtrio injection." [Figure 18B] FIG. 18B shows measurements of total flux (photons per second; p / sec) on the ventral surface per mouse in the different groups on the indicated days after administration of CAdtrio. [Figure 18C] FIG. 18C shows the body weights of mice in the different treatment groups on the indicated days after administration of CAdtrio, expressed as a percentage of body weight on day 0. [Figure 18D] Figure 18D shows the percentage of surviving subjects in the different treatment groups on the indicated days after CAdtrio administration, as well as the negative control condition in which mice were not administered CAdtrio or T cells (-). [Figure 19A]Figures 19A-C are images and graphs showing the results of an in vivo analysis of the anti-cancer activity of different ratios of combinations of Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1 and adoptively transferred HER2-specific CAR T cells in an orthotopic FaDu cell-derived model of head and neck squamous cell carcinoma. Figure 19A shows the number and location of luciferase-expressing FaDu cells in mice on the indicated days after administration of CAdtrio. [Figure 19B] FIG. 19B shows measurements of total flux (photons per second; p / sec) on the ventral surface per mouse in the different groups on the indicated days after administration of CAdtrio. [Figure 19C] FIG. 19C shows the body weights of mice in the different treatment groups on the indicated days after administration of CAdtrio, expressed as a percentage of body weight on day 0. [Figure 20A] Figures 20A-D are bar graphs and graphs showing the results of in vivo analysis of the combination of Onc5 / 3Ad2E1Δ24 and HDAdIL-12_TK_PD-L1 and ganciclovir (GCV) in an ectopic FaDu cell-derived model of head and neck squamous cell carcinoma. Figures 20A and 20B show GAPDH-normalized copy numbers of Onc5 / 3Ad2E1Δ24 (Figure 20A) and HDAdIL-12_TK_PD-L1 (Figure 20B) in tumors of mice administered the combination of Onc5 / 3Ad2E1Δ24 and HDAdIL-12_TK_PD-L1 (CAdtrio) 22 days after infection with or without GCV treatment. [Figure 20B] Figures 20A and 20B show GAPDH-normalized copy numbers of Onc5 / 3Ad2E1Δ24 (Figure 20A) and HDAdIL-12_TK_PD-L1 (Figure 20B) in tumors from mice administered a combination of Onc5 / 3Ad2E1Δ24 and HDAdIL-12_TK_PD-L1 (CAdtrio) on day 22 post-infection with or without GCV treatment. [Figure 20C]FIG. 20C shows tumor volume in mm3 in mice administered a combination of Onc5 / 3Ad2E1Δ24 and HDAdIL-12_TK_PD-L1(CAdtrio) on the indicated days after CAdtrio injection, with or without GCV treatment. [Figure 20D] FIG. 20D shows IL-12 levels detected by ELISA analysis of blood samples obtained on the indicated days after CAdtrio injection, with or without GCV treatment. [Figure 21A] Figures 21A-C are bar graphs and images showing the results of an analysis of transgene expression in cancer cell lines infected with different HDAd viruses cultured in the presence or absence of ganciclovir (GCV). Figure 21A shows the levels of IL-12 in cell culture supernatants as determined by ELISA. [Figure 21B] Figure 21B shows anti-PD-L1 minibodies detected in cell culture supernatants by Western blot. [Figure 21C] FIG. 21C shows viable cells by crystal violet staining at the end of the experiment. [Figure 22A] Figures 22A and 22B are scatter plots showing the results of flow cytometric characterization of adenovirus-specific T cells (AdVST) used in the experiments of Example 9. Figure 22A shows the percentages of CD4+ and CD8+ T cells in the AdVST population. [Figure 22B] FIG. 22B shows the percentage of cells in the AdVST population that express CCR7 and / or CD45RO. [Figure 23A] Figures 23A-C are scatter plots and histograms showing the results of flow cytometric characterization of AdVST transduced with F1.CAR used in the experiments of Example 9. Figure 23A shows the percentage of CD4+ and CD8+ T cells in the transduced population. [Figure 23B] Figure 23B shows the percentage of cells expressing HER2 CAR on the cell surface. [Figure 23C]Figure 23C shows the percentage of cells in the F1.CAR-AdVST population that express CCR7 and / or CD45RO. [Figure 24A] Figures 24A-D are images and graphs showing the results of an in vivo analysis of the anti-cancer activity of adenovirus-specific T cells (AdVST), AdVST transduced with F1.CAR, a combination of AdVST transduced with F1.CAR and Onc5 / 3Ad2E1Δ24, and a combination of AdVST transduced with F1.CAR and Onc5 / 3Ad2E1Δ24+HDAdIL-12_TK_PD-L1 ("CAdtrio"). Figure 24A shows the number and location of luciferase-expressing FaDu cells in mice on the indicated days after administration of CAdtrio. [Figure 24B] Figure 24B shows measurements of total flux (photons per second; p / sec) on the ventral surface for different groups of mice on the indicated days after administration of CAdtrio. For Figure 24B, *P<0.04, **P<0.07, ***P<0.02. [Figure 24C] Figure 24C shows the body weights of mice in the different treatment groups on the indicated days after administration of CAdtrio, expressed as a percentage of body weight on day 0. For Figure 24C, *P<0.01, **P<0.04, ***P<0.02. [Figure 24D] Figure 24D shows the percentage of surviving subjects in the different treatment groups on the indicated days after administration of CAdtrio. For Figure 24D, *P=0.03, **P=0.02. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0283] Numbered Disclosure Description The following numbered paragraphs describe particular aspects and embodiments of the present disclosure: 1. A method of treating cancer comprising administering to a subject: (i) an oncolytic virus; (ii) a virus comprising a nucleic acid encoding an immune modulator; and (iii) at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen.

[0284] 2. The method of paragraph 1, wherein the oncolytic virus is an oncolytic adenovirus (OncAd). 3. The method of paragraph 1 or paragraph 2, wherein said oncolytic virus is derived from adenovirus 5 (Ad5).

[0285] 4. The method of any one of paragraphs 1 to 3, wherein the oncolytic virus encodes an E1A protein that exhibits reduced binding to Rb protein compared to the E1A protein encoded by Ad5.

[0286] 5. The method of any one of paragraphs 1 to 4, wherein the oncolytic virus encodes an E1A protein lacking the amino acid sequence LTCHEACF (SEQ ID NO:52). 6. The method of any one of paragraphs 1 to 5, wherein the oncolytic virus encodes an E1A protein comprising or consisting of the amino acid sequence SEQ ID NO:34.

[0287] 7. The method of any one of paragraphs 1 to 6, wherein the oncolytic virus comprises a nucleic acid having one or more binding sites for one or more transcription factors.

[0288] 8. The method of any one of paragraphs 1 to 7, wherein the oncolytic virus comprises a nucleic acid having one or more binding sites for STAT1. 9. The method of any one of paragraphs 1 to 8, wherein the virus comprising a nucleic acid encoding an immunomodulator is a helper-dependent adenovirus (HDAd).

[0289] 10. The method of any one of paragraphs 1 to 9, wherein the immunomodulatory factor is selected from an agonist of an effector immune response or an antagonist of an immunomodulatory response. 11. The method of any one of paragraphs 1 to 10, wherein the virus comprising nucleic acid encoding an immune modulator comprises nucleic acid encoding IL-12 and / or an antagonistic anti-PD-L1 antibody.

[0290] 12. The method of any one of paragraphs 1 to 11, wherein the virus comprising nucleic acid encoding an immunomodulator comprises nucleic acid encoding a thymidine kinase. 13. The method of any one of paragraphs 1 to 12, wherein at least one cell comprising a CAR specific for the cancer cell antigen is a T cell.

[0291] 14. The method of any one of paragraphs 1 to 13, wherein the CAR comprises an antigen-binding domain capable of specific binding to HER2. 15. The method according to any one of paragraphs 1 to 14, wherein the CAR is LC-CRD1: SEQ ID NO: 10; LC-CRD2: SEQ ID NO:11; LC-CRD3: VL domain comprising SEQ ID NO: 12; and HC-CRD1: SEQ ID NO: 13; HC-CRD2: SEQ ID NO: 14; HC-CRD3: VH domain comprising SEQ ID NO: 15; or LC-CRD1: SEQ ID NO: 18; LC-CRD2: SEQ ID NO: 19; LC-CRD3: VL domain comprising SEQ ID NO: 20; and HC-CRD1: SEQ ID NO:21; HC-CRD2: SEQ ID NO:22; HC-CRD3: VH domain comprising SEQ ID NO:23; or LC-CRD1: SEQ ID NO:26; LC-CRD2: SEQ ID NO:27; LC-CRD3: VL domain comprising SEQ ID NO:28; and HC-CRD1: SEQ ID NO:29; HC-CRD2: SEQ ID NO:30; HC-CRD3: VH domain comprising SEQ ID NO:31 The method comprises administering to a subject an antigen-binding domain comprising:

[0292] 16. The method according to any one of paragraphs 1 to 15, wherein the CAR is a VL comprising, consisting of or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 16, and a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 17; or a VL comprising, consisting of or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO:24, and a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO:25; or A VL comprising, consisting of or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 32, and a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 33. The method comprises administering to a subject an antigen-binding domain comprising:

[0293] 17. The method according to any one of paragraphs 1 to 17, wherein the method further comprises: (a) isolating at least one cell from a subject; (b) modifying at least one cell to express or contain a cancer cell antigen-specific CAR or a nucleic acid encoding a cancer cell antigen-specific CAR; (c) optionally, expanding the at least one modified cell; and (d) administering at least one modified cell to a subject. 17. The method of any one of paragraphs 1 to 16, comprising:

[0294] 18. The method, wherein said cancer is selected from head and neck cancer, nasopharyngeal carcinoma (NPC), cervical cancer (CC), oropharyngeal carcinoma (OPC), gastric cancer (GC), hepatocellular carcinoma (HCC) and lung cancer. 19. An oncolytic adenovirus (OncAd) encoding an E1A protein comprising or consisting of the amino acid sequence SEQ ID NO:34.

[0295] 20. An oncolytic adenovirus (OncAd) comprising a nucleic acid having one or more binding sites for STAT1. 21. The OncAd of paragraph 20, comprising a nucleic acid sequence having at least 60% sequence identity to SEQ ID NO:51, or an equivalent sequence as a result of codon degeneracy.

[0296] 22. A helper-dependent adenovirus (HDAd) comprising nucleic acid encoding IL-12 and / or an antagonistic anti-PD-L1 antibody. 23. The HDAd according to paragraph 22, wherein the HDAd additionally comprises a nucleic acid encoding a thymidine kinase.

[0297] 24. A chimeric antigen receptor (CAR), LC-CRD1: SEQ ID NO: 10; LC-CRD2: SEQ ID NO:11; LC-CRD3: VL domain comprising SEQ ID NO: 12; and HC-CRD1: SEQ ID NO: 13; HC-CRD2: SEQ ID NO: 14; HC-CRD3: VH domain comprising SEQ ID NO: 15; or LC-CRD1: SEQ ID NO: 18; LC-CRD2: SEQ ID NO: 19; LC-CRD3: VL domain comprising SEQ ID NO: 20; and HC-CRD1: SEQ ID NO:21; HC-CRD2: SEQ ID NO:22; HC-CRD3: VH domain comprising SEQ ID NO:23; or LC-CRD1: SEQ ID NO:26; LC-CRD2: SEQ ID NO:27; LC-CRD3: VL domain comprising SEQ ID NO:28; and HC-CRD1: SEQ ID NO:29; HC-CRD2: SEQ ID NO:30; HC-CRD3: VH domain comprising SEQ ID NO:31 A chimeric antigen receptor (CAR) comprising an antigen-binding domain comprising:

[0298] 25. The CAR: a VL comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 16, and a VH comprising, or consisting of, or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 17; or a VL comprising, consisting of or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO:24, and a VH comprising, consisting of or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO:25; or A VL comprising, or consisting of, an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 32, and a VH comprising, or consisting of, or consisting essentially of an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 33. 25. The CAR of paragraph 24, comprising an antigen binding domain comprising:

[0299] 26. A nucleic acid encoding an oncolytic adenovirus (OncAd) according to any one of paragraphs 19 to 21, a helper-dependent adenovirus (HDAd) according to paragraph 22 or paragraph 23, or a chimeric antigen receptor (CAR) according to paragraph 24 or paragraph 25, optionally an isolated or artificial nucleic acid.

[0300] 27. A cell comprising an oncolytic adenovirus (OncAd) according to any one of paragraphs 19 to 21, a helper-dependent adenovirus (HDAd) according to paragraph 22 or paragraph 23, a chimeric antigen receptor (CAR) according to paragraph 24 or paragraph 25, or a nucleic acid according to paragraph 26, optionally wherein the cell is artificially and is not found in nature.

[0301] 28. A pharmaceutical composition comprising an oncolytic adenovirus (OncAd) according to any one of paragraphs 19 to 21, a helper-dependent adenovirus (HDAd) according to paragraph 22 or paragraph 23, a chimeric antigen receptor (CAR) according to paragraph 24 or paragraph 25, a nucleic acid according to paragraph 26, or a cell according to paragraph 27, and a pharma- ceutical acceptable carrier, diluent, excipient or adjuvant.

[0302] 29. A method of treating cancer comprising administering to a subject an oncolytic adenovirus (OncAd) according to any one of paragraphs 19 to 21, a helper-dependent adenovirus (HDAd) according to paragraph 22 or paragraph 23, a chimeric antigen receptor (CAR) according to paragraph 24 or paragraph 25, a nucleic acid according to paragraph 26, a cell according to paragraph 27 or a pharmaceutical composition according to paragraph 28.

[0303] 30. An oncolytic adenovirus (OncAd) according to any one of paragraphs 19 to 21, a helper-dependent adenovirus (HDAd) according to paragraph 22 or paragraph 23, a chimeric antigen receptor (CAR) according to paragraph 24 or paragraph 25, a nucleic acid according to paragraph 26, a cell according to paragraph 27 or a pharmaceutical composition according to paragraph 28 for use in a method for treating cancer.

[0304] 31. Use of an oncolytic adenovirus (OncAd) according to any one of paragraphs 19 to 21, a helper-dependent adenovirus (HDAd) according to paragraph 22 or paragraph 23, a chimeric antigen receptor (CAR) according to paragraph 24 or paragraph 25, a nucleic acid according to paragraph 26, a cell according to paragraph 27 or a pharmaceutical composition according to paragraph 28 in the manufacture of a medicament for treating cancer.

[0305] 32. The method, use or use according to any one of paragraphs 29 to 31, wherein said cancer is selected from head and neck cancer, nasopharyngeal carcinoma (NPC), cervical cancer (CC), oropharyngeal carcinoma (OPC), gastric cancer (GC), hepatocellular carcinoma (HCC) and lung cancer.

[0306] 33. A kit of parts comprising a predetermined amount of an oncolytic adenovirus (OncAd) according to any one of paragraphs 19 to 21, a helper-dependent adenovirus (HDAd) according to paragraph 22 or paragraph 23, a chimeric antigen receptor (CAR) according to paragraph 24 or paragraph 25, a nucleic acid according to paragraph 26, a cell according to paragraph 27 or a pharmaceutical composition according to paragraph 28. EXAMPLES

[0307] In the following examples, the inventors describe the generation and functional characterization of novel HER-2-specific CAR and CAR-T cells, oncolytic adenoviruses and helper-dependent adenoviruses.

[0308] Example 1: HER2-specific CAR-T cells 1.1 Generation of HER2-specific CAR constructs and CAR-T cells HER2-binding CAR constructs were prepared. Briefly, DNA encoding scFv (i.e., VL and VH domains joined by a linker sequence) for anti-HER2 antibody clones C5, E4, F1, or A3 was cloned into a CAR construct backbone containing a 5' signal peptide (SP), and CD28 transmembrane (TM) and intracellular domain sequences together with a 3' CD3ζ intracellular domain sequence. Figure 1A shows a schematic of the three HER2-binding CAR constructs.

[0309] HER2-specific CAR-T cells were generated as shown diagrammatically in FIG. 1B. Briefly, human PBMCs were isolated from blood samples by Ficoll density gradient centrifugation. Cells were treated by stimulation with anti-CD3 (OKT3) / anti-CD28 in the presence of IL-2 to promote T cell activation and proliferation, and cells were transduced with a retrovirus encoding the HER2 CAR construct. T cells were expanded by culturing in the presence of 100 IU / mL recombinant human IL-2 and frozen 6 days after transduction. T cells transduced with the HER2-specific CAR construct were immediately expanded by culturing in vitro (see, e.g., FIG. 14).

[0310] T cells were thawed and expanded for 5 days in the presence of 100 IU / mL recombinant human IL-2 and used for in vitro / in vivo experiments and phenotypic analysis. 1.2 Characterization of HER2-specific CAR-T cells 1.2.1 Surface markers and HER2 CAR expression T cells transduced with HER2 CAR constructs encoding scFv of anti-HER2 antibody clone E4 were characterized by flow cytometry for the expression of different cell surface molecules. Expanded HER2-specific CAR T cells were stained with fluorescently labeled monoclonal antibodies for 30 min at 4°C. Live / dead cell discrimination was achieved by including 7AAD (BD Pharmingen) in the staining. Stained cells were analyzed by flow cytometry for 1 h at 4°C. Analysis was performed using an allios flow cytometer and Kaluza software (BD Bioscience) according to the manufacturer's instructions.

[0311] The results are shown in Figures 2 and 3. Strong surface expression of HER2-CAR was detected in the transduced cells (Figure 2). Figure 3 shows the results of characterization of T cells transduced with HER2(E4)-CAR. HER2(E4)-CAR expressing CD3+, CD4+ and CD8+ cells showed increased expression of PD-1, LAG-3 and TIM-3, and reduced levels of CCR7, compared to non-transduced cells (Figure 3).

[0312] 1.2.2 Cell-killing activity HER2-CAR-T cells were analyzed for their ability to kill HER2-expressing cancer cells in vitro in a cell killing assay.

[0313] In the first experiment, cells from a HER2-negative MDA cell line (negative control), MDA cells stably expressing HER2 (MDA-HER2; positive control), the pharyngeal squamous cell carcinoma cell line FaDu, or the head and neck squamous cell carcinoma cell line SCC47 were treated with Chromium-51 ( 51 The cells were labeled with Cr and co-cultured with non-transduced T cells (NT) or HER2-CAR-T cells expressing the indicated CARs at an effector:target cell ratio of 20:1 for 4 h. After centrifugation, the HER2-CAR-T cells in the cell culture medium were counted using a liquid scintillation counter. 51 Cr levels were counted. The results are shown in Figure 4A. HER2-CAR-T cells were shown to kill cancer cells expressing HER2. This experiment was performed using a 10:1 effector:target cell ratio with similar results.

[0314] The expression of HER2 in MDA-HER2, FaDu and SCC47 was confirmed by flow cytometry. Briefly, cells were stained with fluorescently labeled monoclonal anti-HER2 antibody or isotype control antibody for 30 min at 4°C. Live / dead cell discrimination was achieved by including 7AAD (BD Pharmingen) in the staining. Stained cells were analyzed using a Gallios flow cytometer and Kaluza software (BD Biosciences) according to the manufacturer's instructions. The results are shown in Figure 4B. It was confirmed that MDA cells do not express HER2, whereas MDA-HER2, FaDu and SCC47 express HER2.

[0315] In a separate experiment, FaDu and SCC47 cells genetically modified to express firefly luciferase (ffLuc) were seeded into wells of a 24-well plate and co-cultured with HER2(C5)-CAR-T cells, HER2(E4)-CAR-T cells, or HER2(F1)-CAR-T cells at an effector:target cell ratio of 1:5 for 3 days and analyzed using a plate reader (Life Technologies). fLuc activity was measured. The results are shown in Figure 5. HER2-CAR-T cells were shown to kill HER2-expressing cancer cells as evidenced by a reduction in ffLuc activity (relative light units, RLU). Experiments were performed using an effector:target cell ratio of 1:20 with similar results.

[0316] Example 2: OncAd constructs 2.1 Generation of OncAd constructs Use recombinant DNA technology to prepare a new construct that codes for oncolytic adenovirus.In a particular embodiment, OncAd is produced by modifying known viruses.For example, the region that codes for E1A protein from adenovirus 5, such as the sequence LTCHEACF (SEQ ID NO: 52) that is involved in binding with Rb protein, is replaced with the sequence that codes for E1A protein from adenovirus 2, which also has the sequence LTCHEACF (SEQ ID NO: 52) deleted.

[0317] ICOSTAT, shown in Figure 6, was produced from ICOVIR15, e.g., as disclosed in Rojas et al., 2010 Mol Ther 18 1960-1971. Briefly, the region of ICOVIR15 encoding eight copies of a binding site for the transcription factor E2F was replaced with a region encoding eight tandem copies of a binding site for the transcription factor STAT1. The sequence of ICOSTAT is shown in SEQ ID NO: 51.

[0318] Using recombinant DNA techniques, Onc5 / 3Ad2E1Δ24 (also referred to herein as "Onc5 / 2E1Δ24"), shown in SEQ ID NO:55 and depicted diagrammatically in FIG. 12, was also prepared. Onc5 / 3Ad2E1Δ24 is described, for example, in Fueyo et al., 2000 Oncogene 19:2-12 (incorporated by reference in its entirety; Onc5Δ24 is described in Fueyo et al., Onc5 / 3Ad2E1Δ24 has a similar structure to Onc5Δ24 disclosed in (also referred to as "Δ24"), but differs in that Onc5 / 3Ad2E1Δ24 encodes the E1A protein from adenovirus type 2 (Ad2) lacking the sequence LTCHEACF (SEQ ID NO: 52) rather than the E1A protein from adenovirus type 5 (Ad5) lacking the sequence LTCHEACF (SEQ ID NO: 52).

[0319] 2.2 Cell-killing activity The ability of the selected oncolytic adenovirus or ICOSTAT generated in Example 2.1 to kill cancer cells can be analyzed, for example, by MTS assay. Briefly, cells of human alveolar basal epithelial adenocarcinoma cell line A549 cells, FaDu cells, SCC47 cells, or non-cancerous WI-38 human lung fibroblasts or ARPE-19 human retinal pigment epithelial cells were seeded into wells of a 96-well plate and infected with different amounts of helper-dependent, non-replicating adenovirus (HDAd; as a negative control), selected oncolytic adenovirus (e.g. Onc5 / 3Ad2E1Δ24 as described in Example 2.1), or ICOSTAT as described in Example 2.1 above.

[0320] The cells may be cultured for, for example, 4 days, and then MTS reagent (Promega) may be added to each well and the cells may be incubated for 2 hours at 37° C. Live cells are The cells can be analyzed by measuring the absorbance at 490 nm using a light reader. The readings are calculated based on the readings for each type of untreated cells (i.e. untreated cells = 10 0% cell viability) may be used for normalization, with wells containing no cells being considered as 0%.

[0321] In certain embodiments, the selected oncolytic virus can kill cancer cells in a dose-dependent manner.In certain embodiments, the selected oncolytic virus also shows a lower level of cell killing of non-cancerous cells, such as WI-38 and ARPE-19 cells, compared to the level of virus-induced cell killing of cancerous cells.

[0322] Figures 7A-7F show that ICOSTAT can kill cancer cells (i.e., A549, FaDu and SCC47 cells) in a dose-dependent manner (Figures 7A-7C and 7F), and shows lower levels of cell killing of non-cancerous cells WI-38 and ARPE-19 cells compared to the levels of cell killing of cancerous cells (Figures 7D and 7E).

[0323] Figures 13A-13D show that Onc5 / 3Ad2E1Δ24 can kill cancer cells (i.e., FaDu and SCC47 cells) in a dose-dependent manner (Figures 13A and 13B), and shows lower levels of cell killing in non-cancerous WI-38 and ARPE-19 cells compared to the levels of cell killing in cancerous cells (Figures 13C and 13D).

[0324] 2.3 Ability to rescue helper-dependent adenovirus (HDAd) The ability of the selected oncolytic adenoviruses or ICOSTAT generated in Example 2.1 to support the replication of helper-dependent adenoviruses (HDAd) can be analyzed by co-infecting cancer cells with OncoAd and HDAd and determining the viral copy number. Briefly, FaDu or SCC47 cells were plated in 24-well plates and infected with HDAd alone or with OncAd+HDAd (at a ratio of OncAd:HDAd of 1:10) at 10 viral particles per HDAd cell. Cells were harvested 48 hours post-infection, DNA was extracted and assayed using a Bio-Rad iQ5 Real-Time PCR Detection System (Bio-Rad) and Applied Biosystems SYBR Green PCR Master Mix (Life Technologies). Both HDAd and Onc.Ad vector copies are analyzed by quantitative real-time PCR using a PCR probe (95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 30 s). Copy numbers detected for GAPDH are used to normalize copy numbers.

[0325] In certain embodiments, the selected oncolytic virus is capable of replicating itself and the HDAd sufficiently. Figures 8A and 8B show that ICOSTAT (designated "Onc5 / 3AdicoSTAT" in the figures) was found to be capable of replicating itself (Figure 8A) and HDAd (Figure 8B).

[0326] 2.4 Effect of IFNγ on ICOSTAT replication in cancer cells The effect of IFNγ treatment on ICOSTAT OncAd replication was analyzed. Briefly, FaDu and SCC47 cells were plated in 24-well plates, cells were infected with 10 vp / cell of selected oncolytic virus or icoSTAT, 3 h post-infection, cell culture medium was replaced with medium containing or not containing 10 ng / mL recombinant IFNγ, and cell culture medium was replaced with fresh medium, again containing or not containing 10 ng / mL recombinant IFNγ, at 24 and 48 h post-infection. Cells were harvested at 3, 24, 48 and 72 h post-infection, DNA was extracted from the cells, and viral copy numbers were analyzed by quantitative real-time PCR and normalized using the copy numbers detected for GAPDH.

[0327] Figures 9A and 9B show that ICOSTAT was able to replicate in FaDu and SCC47 cells in the presence or absence of IFNγ. Example 3: Helper-Dependent Ad (HDAd) Constructs 3.1 HDAd Construction and Production A novel construct encoding a helper-dependent adenovirus was prepared using recombinant DNA technology. Figure 10A shows a schematic of the coding sequence of the resulting construct, named HDAdIL-12_TK_PD-L1. HDAdIL-12_TK_PD-L1 contains sequences encoding (i) human IL-12p70 (alpha and beta chain coding sequences), (ii) HSV-1 thymidine kinase with an HA tag, and (iii) an expression cassette for anti-PD-L1 minibody (comprising the CDRs of anti-PD-L1 clone H12_gl, e.g., as described in WO2016111645A1). Each of the three coding sequences has its own polyA signal sequence.

[0328] The HDAd HDΔ28E4EGFP construct (HDAdeGFP) containing an EGFP transgene driven by the CMV promoter was produced as described in Farzad et al., Oncolytics 2014 1:14008.

[0329] The HDAd "HDIL12_PDL1" contains sequences encoding the human IL-12 p70 protein and an anti-PD-L1 minibody (atezolizumab) derived from YW243.55.S70. The anti-PD-L1 minibody of this construct consists of the scFv of YW243.55.S70 fused to the hinge, CH2 and CH3 regions of human IgG1 and a C-terminal HA tag (e.g., as described in Tanoue et al., Cancer Res. (2017) 77(8):2040-2051).

[0330] 3.2 Expression of the encoded protein Cancer cells transfected with plasmid HDAd vectors and media samples were collected to analyze IL-12p70 and anti-PD-L1 minibody levels in the cell culture medium of transfected cells 48 hours post-transfection.

[0331] IL-12p70 levels in the culture medium were measured using the BD Cytokine Multiplex Bead Array System (BD Biosciences) according to the manufacturer's instructions. The results are shown in Figure 10B. Cells transfected with the HDAdIL-12_TK_PD-L1 construct were found to produce higher levels of IL-12p70 than cells transfected with the HDIL-12_PD-L1 construct.

[0332] Secretion of anti-PD-L1 minibodies into cell culture medium was detected by Western blot analysis using an anti-HA antibody (to detect minibodies with an HA tag). Figure 10C shows that cells transfected with the HDAdIL-12_TK_PD-L1 construct secreted anti-PD-L1 minibodies into the cell culture medium.

[0333] In another experiment, the cell culture medium of cells transfected with different constructs was replaced with medium containing 10 ng / ml ganciclovir (GCV) 8 hours after transfection, then the cell culture medium was replaced with medium containing 10 ng / ml every 24 hours, and after 7 days, the wells were stained with crystal violet solution to reveal viable cells.

[0334] The results shown in Figure 10D confirm that cells transfected with the HDAdIL-12_TK_PD-L1 construct express thymidine kinase. In further experiments, A549, FaDu or SCC47 cells (n=4 wells per condition) were treated with HDAdIL-12_TK_PD-L1, HDAd_PD-L1 in vitro. (see, e.g., Tanoue et al., supra), or a control HDAd encoding eGFP (Farzad et al., supra). Cells were either cultured in the absence of ganciclovir for 48 h or the medium was changed 8 h postinfection and then every 24 h with medium containing 10 ng / ml ganciclovir.

[0335] Secretion of IL-12 into cell culture supernatants was analyzed by ELISA, and secretion of anti-PD-L1 minibodies was analyzed by Western blot using an anti-HA antibody (anti-PD-L1 minibodies contain a C-terminal HA tag). At the end of the experiment, wells were stained with crystal violet solution to reveal viable cells.

[0336] The results are shown in Figures 21A-21C. Expression of the transgene encoded by the HDAd in the different cancer cell lines analyzed was confirmed. 3.3 Confirmation of anti-PD-L1 minibody binding to PD-L1 The ability of the anti-PD-L1 minibody encoded by HDAdIL-12_TK_PD-L1 to bind to PD-L1 was analyzed by ELISA.

[0337] Briefly, Immulon 2 high binding 96-well plates (VWR) were coated with 500 ng / well of recombinant human PD-L1 (BioVision). After blocking with PBS-T containing 0.05% BSA, serially diluted cell culture medium from A549 cells transfected with a plasmid encoding GFP (pGFP; negative control), a plasmid encoding the anti-PD-L1 minibody described in Tanoue et al. above (pPDL1miniTanoue) or a plasmid encoding the anti-PD-L1 minibody encoded by HDAdIL-12_TK_PD-L1 (pPDL1mini) was added and incubated for 24 hours at 4°C. A serially diluted anti-human PD-L1 antibody (BioLegend) starting at 10 μg / well was used as a positive control (PDL1 IgG). After washing the plates with PBS-T, HRP-conjugated anti-human IgG (for PD-L1 mini and PDL1 mini Tanoue) or HRP-conjugated anti-mouse IgG (Bio-Rad; for PD-L1 IgG and Iso IgG) were added for detection and incubated at room temperature for 1 h. The plates were then developed and the absorbance was measured at 450 nm using a Tecan reader (TECAN).

[0338] The results are shown in Figure 11. An anti-PD-L1 minibody comprising the CDRs of the anti-PD-L1 antibody clone H12 was found to bind to human PD-L1 in a dose-dependent manner, with an avidity comparable to (or greater than) that of binding by the anti-PD-L1 minibody described in Tanoue et al., supra.

[0339] Example 4: Analysis of cancer treatment in vivo The anticancer effects of treatment with the combinations (1) selected oncolytic viruses + HDAdIL-12_TK_PD-L1 + HER2-CAR-T and (2) ICOSTAT + HDAdIL-12_TK_PD-L1 + HER2-CAR-T are demonstrated in vivo in mouse xenograft tumor models.

[0340] In the first experiment, 1 x 10 in PBS 6 FaDu cells are injected subcutaneously into NSG male mice. After 12 days, 1 × 10 8 Viral particles (1) oncolytic virus and HDAdIL-12_TK_PD-L1 or (2) ICOSTAT+HDAdIL-12_TK_PD-L1 are injected intratumorally at a ratio of 1:20 OncAd:HDAd.

[0341] In the second experiment, 0.5 × 10 6 Inject 1 × 10 FaDu cells orthotopic into NSG male mice. Six days later, 1 × 10 8 Virus particles (1) oncolytic virus and HDAdIL-12_TK_PD-L1 or (2) ICOSTAT+HDAdIL-12_TK_ PD-L1 will be injected intratumorally at a ratio of 1:20 OncAd:HDAd.

[0342] In both experiments, 3 days after administration of viral particles, 1 × 10 6 HER2-CAR T cells will be administered intravenously. In both experiments, the control conditions were as follows:

[0343] [Table 2]

[0344] Tumor size was monitored and tumor volume was calculated using the formula: width x y = 0.05; 2 Calculate using x length x 0.5. The use of the combination of oncolytic virus, HDAdIL-12_TK_PD-L1 and HER2 CAR-T (test condition 1) is found to have an improved antitumor effect compared to the use of either agent alone (conditions 8, 10 or 11) or compared to the use of two of the three agents (conditions 3, 4 and 6).

[0345] Similarly, the use of the combination of ICOSTAT, HDAdIL-12_TK_PD-L1 and HER2 CAR-T (test condition 2) is found to have an improved antitumor effect compared to the use of any of the single agents (conditions 9, 10 or 11) or compared to the use of two of the three agents (conditions 3, 5 and 7).

[0346] Similar results are observed when xenograft tumors are established using SCC47 and A549 cells. Example 5: Analysis of the anti-cancer activity of HER2-specific CAR-T cells in vivo The anti-cancer activity of HER2-specific CAR-T cells (see Example 1 above) was investigated in vivo in a FaDu cell-derived xenograft model of head and neck squamous cell carcinoma.

[0347] Simply put, 0.5 x 10 6 FaDu cells were injected orthotopic into NSG male mice. Nine days later, mice were 6 1 x 10 T cells genetically modified to express firefly luciferase transduced with HER2-CAR constructs 6 Firefly luciferase expressing T cells transduced with C5, F1 or A3CAR constructs were injected via the tail vein. Experiments in which mice were not injected with T cells on day 9 were included as control conditions.

[0348] The luciferase activity (and therefore the number and distribution of administered T cells), body weight, and survival of the mice were monitored over time. D-luciferin was injected intraperitoneally (1.5 mg per mouse) and imaged 10 min later using an IVIS imager (Xenogen). Luciferase activity was monitored by imaging the mice.

[0349] Figures 15A and 15B show the results of 0, 4, 7, 14, 28, 42, and 5 days after injection of luciferase-expressing T cells (i.e., non-transduced T cells or HER2-specific CAR-T cells). Images acquired at days 6 and 70 are shown (days refer to days after ffLuc T cell injection). It was shown that systemically injected T cells migrated to the orthotopic tumor site. T cells that were not modified to express a HER2-specific CAR were undetectable after 7 days. In contrast, HER2-specific CAR-T cells persisted and remained detectable throughout the experiment.

[0350] Figure 15C shows the survival percentage of mice subjected to different treatments over the course of the experiment. Administration of HER2-specific CAR-T cells was found to increase survival. In a separate experiment, NOD scid gamma (NSG) mice were injected with 1 × 10 6 HER2-CAR construct-transduced firefly luciferase-expressing T cells, or 1 × 10 6 Firefly luciferase-expressing T cells transduced with C5, F1 or A3CAR constructs were injected via the tail vein, and luciferase activity was monitored as described above, as well as mouse weight over time.

[0351] The results of the experiment are shown in Figures 16A-16C. C5 CAR-T cells were found to expand nonspecifically in NSG mice (Figure 16A). No significant weight loss was observed in NSG mice administered HER2-specific CAR-T cells (Figure 16C).

[0352] Example 6: Analysis of the anticancer activity of the combination of oncolytic virus, HDAd virus and HER2-specific CAR-T cells in vivo The anticancer activity of the combination of oncolytic virus, HdAd, and HER-specific CAR-T cell therapy was investigated in vivo in a FaDu cell-derived xenograft model of head and neck squamous cell carcinoma.

[0353] Simply put, 0.5 x 10 6FaDu cells were injected orthotopic into NSG male mice. Six days later, one group of mice was then injected intratumorally with a combination of Onc5 / 3Ad2E1Δ24 (described in Example 2.1) and HDAdIL-12_TK_PD-L1 (described in Example 3.1) (this combination of OncAd and HdAd is referred to herein as "CAdtrio"). A total of 1×10 7 Viral particles were administered at a ratio of 1:10 Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1.

[0354] Three days later, mice were inoculated with 1 × 10 6 A control group of mice that did not receive CAdtrio were injected with 1 × 10 T cells engineered to express firefly luciferase transduced with a HER2-specific CAR construct corresponding to clone F1 via the tail vein. 6 Firefly luciferase-expressing T cells transduced with the HER2-CAR construct were injected via the tail vein, and an additional control group of mice did not receive CAdtrio or were not injected with T cells. The luciferase activity, weight and survival of the mice were monitored over time.

[0355] Prior to use in experiments, F1.CART cells were characterized by flow cytometry. Results are shown in Figures 17A-17C. The cells were found to contain 72.5% CD4+ and CD8+ cells. 87% of the cells were determined to express HER2 CAR on the cell surface. 39% of the cells were CCDR7+CD45RO+ and 59.2% of the cells were CCR7-CD45RO+.

[0356] Figures 18A-18D show the results of experiments analyzing the therapeutic efficacy of a combination of oncolytic virus, HDAd virus and HER2-specific CAR-T cells to treat cancer in vivo. The combination of Onc5 / 3Ad2E1Δ24, HDAdIL-12_TK_PD-L1 and F1.CART improved survival over treatment with F1.CART cells alone. It was found that:

[0357] In further experiments, two different ratios of Onc5 / 3Ad2E1Δ24 to HDAdIL-12_TK_PD-L1 were investigated. Simply put, 0.5 x 10 6 FaDu cells modified to express firefly luciferase were injected orthotopic into NSG male mice. Six days later, mice were injected intratumorally with: (i) 1 × 10 Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1 at a 1:10 ratio 7 CAdtrio virus particles; (ii) 1 × 10 Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1 at a ratio of 1:20 7 CAdtrio virus particles; (iii) 1 × 10 Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1 at a 1:10 ratio 8 Cadtrio virus particles; or (iv) 1 × 10 of Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1 at a ratio of 1:20 8 CAdtrio virus particles.

[0358] Three days later, mice were inoculated with 1 × 10 6 F1 CAR construct-transduced T cells (which do not express firefly luciferase) were injected via the tail vein. Cancer was monitored over time by analysis of luciferase activity as described above, and mouse weight was also monitored.

[0359] The results of the experiment are shown in Figures 19A-19C. Mice treated with a 1:10 ratio of Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1 generally had fewer luciferase-expressing FaDu cells than mice treated with a 1:20 ratio of Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1, with 1x10 8 Mice administered with 1 x 10 CAdtrio virus particles typically received 1 x 107 Mice administered 1000 viral particles of CAdtrio had fewer FaDu cells expressing luciferase than mice administered 1000 viral particles of CAdtrio (Figure 19B).

[0360] Example 7: Analysis of the anticancer activity of a combination of oncolytic virus, HDAd virus and ganciclovir (GCV) in vivo The anticancer activity of oncolytic virus and combination of HdAd (encoding thymidine kinase) (i.e., CAdtrio) with ganciclovir (GCV) was investigated in vivo in a FaDu cell-derived xenograft model of head and neck squamous cell carcinoma.

[0361] Ectopic FaDu tumors were established by subcutaneous injection of FaDu cells into the flank of mice. Mice were then injected with 1 × 10 8 CAdtrio viral particles were injected intratumorally at a ratio of 1:10 Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1. One group of mice (n=5) was then injected intraperitoneally with 10mg / kg ganciclovir on days 2, 3, 4, 5, 7, 10, 14, 17, and 21 after CAdtrio injection.

[0362] Blood samples were collected from mice on days 2, 7, 14 and 21 and analyzed for IL-12 expression by ELISA. Tumor volume was monitored throughout the experiment. On day 22, copy numbers of Onc.Ad and HDAd vectors were determined in DNA extracted from tumors by quantitative real-time PCR analysis and normalized using the copy numbers detected for GAPDH.

[0363] The results of the experiment are shown in Figures 20A-20D. On day 22, ganciclovir (GCV) treatment did not significantly affect Onc.Ad vector copy number (Figure 20A), but GCV treatment was also found to improve tumor control (Fig. 20C), but did not significantly affect blood levels of IL-12 (Fig. 20D).

[0364] Example 8: Generation of oncolytic virus-specific T cells and oncolytic virus-specific T cells expressing a CAR specific for HER 8.1 Generation and characterization of oncolytic virus-specific T cells Adenovirus-specific T cells (AdVST) and activated T cells (ATC) were prepared as follows.

[0365] Anti-CD3 (clone OKT3) and anti-CD28 agonist antibodies were coated onto wells of tissue culture plates by adding 0.5 ml of a 1:1000 dilution of 1 mg / ml antibody and incubated at 37°C for 2-4 hours or at 4°C overnight.

[0366] PBMCs were isolated from blood samples obtained from healthy donors according to standard Ficoll-Paque methods. ATC: 1×10 6 PBMCs (in 2 ml cell culture medium) were stimulated by culturing on plates coated with anti-CD3 / CD28 agonist antibodies in CTL cell culture medium (containing 50% Advanced RPMI, 50% Click medium, 10% FBS, 1% Glutamax, 1% penicillin / streptomycin) supplemented with 10 ng / ml IL-7 and 5 ng / ml IL-15. Cells were incubated at 37°C, 5% CO 2 The following day, 1 ml of cell culture medium was replaced with fresh CTL medium containing 20 ng / ml IL-7 and 10 ng / ml IL-15.

[0367] ATCs were maintained in culture and then harvested and either used experimentally or cryopreserved between days 5 and 7. AdVST: 1×10 6 PBMCs (in 2 ml cell culture medium) were stimulated by culturing on plates coated with anti-CD3 / CD28 agonist antibodies in CTL cell culture medium supplemented with 10 ng / ml IL-7 and 100 ng / ml IL-15.

[0368] 20 μl of 1:200 diluted adenovirus-specific hexon pepmix (JPT Cat. No. PM-HAdV3) or penton pepmix (JPT Cat. No. PM-HAdV5) was added to the wells. The cells were incubated at 37 °C for 24 h in 5% CO 2 The cells were maintained in an atmosphere at 37° C. After 48 hours, the cells were fed with CTL medium and supplemented with IL-7 and IL-15 to a final concentration of 10 ng / ml IL-7 and 100 ng / ml IL-15.

[0369] 8.2 Generation of oncolytic virus-specific T cells expressing CAR On day 3, 0.125 × 10 6 AdVST was resuspended at a concentration of cells / ml.

[0370] Retronectin-coated plates were prepared by incubating Retronectin (Clontech) diluted 1:100 in PBS for 2–4 h at 37 °C or overnight at 4 °C. The wells were washed with CTL medium, 1 ml of retroviral supernatant of a HER2-specific CAR retrovirus was added to the wells, and the plates were centrifuged at 2000 g for 1.5 h. At the end of the centrifugation step, the retroviral supernatant was aspirated and the wells of the plate were filled with 2 ml of AdVST suspension (i.e., 0.25 × 10 6 The plate was centrifuged at 400 g for 5 min and incubated in 5% CO 2 Incubate at 37°C in a cool, dark place. Incubated.

[0371] After 48 hours (ie, day 6), the cell culture medium was aspirated and replaced with CTL cell culture medium containing 10 ng / ml IL-7 and 100 ng / ml IL-15. On day 9, cells were harvested and either used in experiments or cryopreserved, or subjected to a second stimulation to expand the AdVST expressing the CAR (see Example 8.3).

[0372] 8.3 Expansion of AdVST and CAR-AdVST AdVST and AdVST expressing CAR were expanded by further stimulation as required as follows.

[0373] Autologous ATCs pulsed with pepmix were used as APCs, and K562cs cells (see, e.g., Ngo et al., J Immunother. (2014) 37(4):193-203) were used as costimulatory cells. The final ratio of AdVST or CAR-AdVST:ATC:K562cs cells in stimulation cultures was 1:1:3–5.

[0374] AdVST or CAR-AdVST was administered at 0.2 × 10 6 The cells were resuspended to a concentration of 10 cells / ml. 1×10 6 ATCs were incubated with 10 μl of 200-fold diluted adenovirus-specific hexon pepmix (JPT Cat. No. PM-HAdV3) or penton pepmix (JPT Cat. No. PM-HAdV5) at 37°C for 30 min. The ATCs were then irradiated at 30 Gy and harvested. 6 K562cs cells were irradiated with 100 Gy.

[0375] ATC and K562cs cells were then mixed in a total volume of 5 ml of CTL medium, supplemented with 20 ng / ml IL-7 and 200 ng / ml IL-15, 1 ml of this mixture was added to a well of a 24-well plate, and 1 ml of AdVST suspension or CAR-AdVST suspension was added to the well.

[0376] Cells were incubated at 37 °C in 5% CO 2 The cells were maintained at 37° C. in an ambient atmosphere. After 3–4 days, cell culture medium was added as needed, and after 6–7 days, the expanded AdVST or CAR-AdVST cells were harvested for use in experiments.

[0377] Example 9: Analysis of the anticancer activity of combinations of oncolytic viruses, HDAds, oncolytic virus-specific T cells, and oncolytic virus-specific T cells expressing CAR in vivo The anticancer activity of different combinations of oncolytic viruses, HDAds, oncolytic virus-specific T cells, and oncolytic virus-specific T cells expressing a CAR was investigated in vivo in a FaDu cell-derived xenograft model of head and neck squamous cell carcinoma.

[0378] Simply put, 0.5 x 10 6 FaDu cells engineered to express firefly luciferase were injected orthotopic into NSG male mice. Six days later, groups of mice were injected intratumorally with: (i) 1 × 10 of Onc5 / 3Ad2E1Δ24:HDAdIL-12_TK_PD-L1 at a 1:10 ratio 7 Cadtrio virus particles; or (ii) 1 × 10 7 Virus particles Onc5 / 3Ad2E1Δ24.

[0379] Three days later, the mice were injected via the tail vein with: (a) 1×10 6 AdVSTs, or (b) 1×10 6 Anti-HER2 CAR clone F1 (prepared as described in Example 8) AdVST transduced with the vector (produced in vitro).

[0380] Prior to use in experiments, AdVST and F1.CAR-AdVST were characterized by flow cytometry. The results of the analysis are shown in Figures 22A and 22B and Figures 23A-23C.

[0381] Cancer was monitored over time by analysis of luciferase activity as described above, and mouse body weights were also monitored. The results of the experiment are shown in Figures 24A-24D. The greatest level of tumor control was observed in mice treated with the combination of CAdtrio+AdVST expressing a HER2-specific CAR (i.e., treatment group (i)(b)).

Claims

1. For use in the treatment of cancer, (i) oncolytic adenovirus (OncAd); (ii) a helper-dependent adenovirus (HDAd) comprising nucleic acids encoding IL-12 and an antagonistic anti-PD-L1 antibody; and (iii) at least one cell comprising a chimeric antigen receptor (CAR). A pharmaceutical comprising the combination of:

2. A pharmaceutical composition for use in the treatment of cancer, comprising an oncolytic adenovirus (OncAd), Treatment is further (i) a helper-dependent adenovirus (HDAd) comprising nucleic acids encoding IL-12 and an antagonistic anti-PD-L1 antibody; and (ii) at least one cell comprising a chimeric antigen receptor (CAR); The pharmaceutical composition comprising the administration of

3. A medicament for use in the treatment of cancer, comprising a helper-dependent adenovirus (HDAd) comprising a nucleic acid encoding IL-12 and an antagonistic anti-PD-L1 antibody, Treatment is further (i) oncolytic adenovirus (OncAd), and (ii) at least one cell comprising a chimeric antigen receptor (CAR); The pharmaceutical composition comprising the administration of

4. A medicament for use in the treatment of cancer, comprising at least one cell comprising a chimeric antigen receptor (CAR), Treatment is further (i) oncolytic adenovirus (OncAd), and (ii) Helper-dependent adenovirus (HDAd) comprising nucleic acids encoding IL-12 and an antagonistic anti-PD-L1 antibody. The pharmaceutical composition comprising the administration of

5. 1. A medicament for use in the treatment of cancer comprising (i) an oncolytic adenovirus (OncAd) and (ii) a helper-dependent adenovirus (HDAd) comprising nucleic acids encoding IL-12 and an antagonistic anti-PD-L1 antibody, wherein the treatment further comprises administering at least one cell comprising a chimeric antigen receptor (CAR).

6. The pharmaceutical according to any one of claims 1 to 5, wherein the helper-dependent adenovirus comprising a nucleic acid encoding IL-12 and an antagonistic anti-PD-L1 antibody further comprises a nucleic acid encoding thymidine kinase.

7. The pharmaceutical according to any one of claims 1 to 6, wherein the oncolytic virus is derived from adenovirus 5 (Ad5).

8. The pharmaceutical according to any one of claims 1 to 7, wherein the oncolytic virus encodes an E1A protein that exhibits reduced binding to Rb protein compared to the E1A protein encoded by Ad5.

9. The pharmaceutical according to any one of claims 1 to 8, wherein the oncolytic virus encodes an E1A protein lacking the amino acid sequence LTCHEACF (SEQ ID NO:52).

10. The method according to any one of claims 1 to 9, wherein the oncolytic virus encodes an E1A protein comprising or consisting of the amino acid sequence SEQ ID NO:

34.

11. The method of any one of claims 1 to 10, wherein the oncolytic virus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 55 or an equivalent sequence as a result of codon degeneracy.

12. The pharmaceutical according to any one of claims 1 to 11, wherein the at least one cell comprising a CAR specific to a cancer cell antigen is a T cell.

13. The pharmaceutical according to any one of claims 6 to 12, wherein the thymidine kinase is HSV-1 thymidine kinase.

14. The treatment of the cancer, (a) isolating at least one cell from a subject; (b) modifying the at least one cell to express or contain a cancer cell antigen-specific CAR, or a nucleic acid encoding a cancer cell antigen-specific CAR; (c) optionally expanding the at least one modified cell; and (d) administering the at least one modified cell to a subject. The pharmaceutical composition according to any one of claims 1 to 13, comprising:

15. The pharmaceutical agent according to any one of claims 1 to 14, wherein the cancer is selected from head and neck cancer, nasopharyngeal carcinoma (NPC), cervical cancer (CC), oropharyngeal carcinoma (OPC), gastric cancer (GC), hepatocellular carcinoma (HCC) and lung cancer.

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