TW202011977A - Oncolytic virotherapy and immunotherapy - Google Patents

Abstract

The present disclosure concerns combination therapy for cancer that utilizes (i) an oncolytic virus; (ii) a virus comprising 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 comprises nucleic acid encoding an immunomodulatory factor comprises nucleic acid encoding IL-12 and/or antagonist anti-PD-L1 antibody.

Description

Oncolytic virus therapy and immunotherapy

This application claims the priority of US 62/660305 filed on April 20, 2018. For all purposes, the contents and elements of which are incorporated herein by reference. Technical field

This disclosure is at least related to the fields of cell biology, molecular biology, immunology, virology and medicine, including cancer therapy. In certain embodiments, the present disclosure relates to the use of a combination therapy of oncolytic virus therapy and immunotherapy.

BACKGROUND OF THE INVENTION Oncolytic virus therapy for head and neck squamous cell carcinoma (HNSCC) , HNSCC, is the sixth most prevalent cancer in the world. The treatment of locally advanced, recurrent, and metastatic HNSCC is often limited by the unfavorable drug-to-toxicity ratio, and the median survival of patients with metastatic disease is still less than 1 year (Zandberg and Strome, Oral Oncology (2014) 50: 627-632). Because HNSCC is a localized disease that appears on or near the body, it is suitable for injecting adenovirus vector (Ad) into the original tumor in order to arouse localized and even systemic antitumor immune responses (Liu et al., Nature Clinical Practice Oncology (2007) 4: 101-117). Many clinical trials of conditional replication Ad (OncAd) or replication-defective Ad encoding therapeutic transgenes have proven to be safe and feasible Ad Gene Therapy for HNSCC, but failed to show an improvement in overall survival rate due to intensive local treatment , Even if combined with chemotherapy/radiotherapy, it cannot prevent metastasis to the distal site (Liu et al. , ibid.). OncAd is generally administered intratumorally and it is difficult to re-target metastatic tumors (Koksi et al., Molecular Therapy: The Journal of the American Society of Gene Therapy (2015) 23:1641-1652). OncAd and ancillary Ad (HDAd) expressing immunomodulatory molecules

Adenovirus-based vectors (Ad) can infect a range of malignant cells and express high-level soluble antigens and immunogenic transgenes, making them attractive as cancer gene therapy reagents (Cerullo et al., Advances in Cancer Research (2012) 115, 265-318). OncAd selectively replicates in cancer cells and is an Ad-based vector commonly used in cancer gene therapy in clinical trials. However, OncAd has limited coding capacity for transgenes (~1.5 kb). Helper-dependent Ad (HDAd) has no viral coding sequence, allows multiple transgenes to be inserted in a single vector, and has a loading capacity of up to 34 kb (Suzuki et al., Human Gene Therapy (2010) 21; 120-126). Because the HDAd vector DNA encodes packaging information, the OncAd replication mechanism in turn replicates and packages OncAd and HDAd in infected tumor cells, causing multiple cycles of production and release of both oncolytic viruses and HDAd-encoded transgenes (combined adenovirus Carrier: CAd-VEC; Farzad et al. , Molecular Therapy-Oncolytics (2014) 1, 14008). CAR T cell therapy

Recently, the expression of cancer cell antigen-directed chimeric antigen receptors (CARs; in the review of Kershaw et al., Nature (2013) 13: 525-541) has promoted the use of T cells as cancer therapeutics. CAR-modified T cells have shown promise for the treatment of hematological malignancies (Garfall et al., The New England Journal of Medicine (2015) 373:1040-1047), but they are less effective in treating solid tumors, part of which It may be the result of the highly immunosuppressive nature of the solid tumor microenvironment (Quail et al., Nature Medicine (2013) 19:1423-1437). Due to the immunosuppressive mechanism at the tumor site, despite expressing one or two co-stimulated internal domains (endodomain), CAR T cells cannot expand and sustain for a long time.

The present disclosure provides solutions that address the long-standing need for effective cancer therapies, including combination cancer therapies.

Summary of the invention In one aspect, the present disclosure provides a method of treating cancer, which includes administering to a body: (i) an oncolytic virus; (ii) a virus containing nucleic acids encoding immunomodulatory factors; and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is a combination of methods for treating cancer, comprising (i) an oncolytic virus, (ii) a virus comprising nucleic acid encoding an immunomodulatory factor; and (iii) at least one comprising specificity for cancer cell antigens Cells of the chimeric antigen receptor (CAR).

Also provided is the use of a medicament for manufacturing a method for treating cancer, which uses (i) an oncolytic virus, (ii) a virus containing a nucleic acid encoding an immunomodulatory factor; and (iii) at least one containing a cancer cell antigen Cells with specific chimeric antigen receptors (CAR).

A method of treating cancer is also provided, which includes administering to a body: (i) an oncolytic virus; and (ii) At least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is a combination of methods for treating cancer, comprising (i) an oncolytic virus, and (ii) at least one cell comprising a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is the use of a medicament for the treatment of cancer, which uses (i) an oncolytic virus, and (ii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens .

Also provided is an oncolytic virus for a method of treating cancer, the method comprising administering to an individual individually or simultaneously: (i) an oncolytic virus; and (ii) At least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is a method for treating cancer of at least one cell comprising a chimeric antigen receptor (CAR) specific for cancer cell antigens, the method comprising administering to an individual individually or simultaneously: (i) an oncolytic virus; and (ii) At least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is the use of oncolytic viruses in the manufacture of a medicament, which can be used to include the following methods for administering an individual individually or simultaneously: (i) an oncolytic virus; and (ii) At least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is the use of at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens for the manufacture of a medicament, which can be used to include the following methods of administering an individual individually or simultaneously to the following: (i) an oncolytic virus; and (ii) At least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

In some embodiments, the CAR-containing cells are specific to the oncolytic virus.

Also provided is a method of treating cancer, which includes administering to a body: (i) an oncolytic virus; and (ii) at least one immune cell specific for the oncolytic virus.

Also provided is a combination of methods for treating cancer comprising (i) an oncolytic virus; and (ii) at least one immune cell specific for the oncolytic virus.

Also provided is the use of a medicament for the method of treating cancer, which uses (i) an oncolytic virus; and (ii) at least one immune cell specific for the oncolytic virus.

Also provided is an oncolytic virus for a method of treating cancer, the method comprising administering an individual individually or simultaneously: (i) an oncolytic virus; and (ii) at least one immune cell with specificity for the oncolytic virus.

Also provided is a method for treating cancer of at least one immune cell specific for oncolytic viruses, the method comprising administering to an individual individually or simultaneously: (i) an oncolytic virus; and (ii) at least one immune cell specific for the oncolytic virus.

Also provided is the use of oncolytic viruses in the manufacture of a medicament, which comprises the following methods of administering the following individually or simultaneously to an individual: (i) an oncolytic virus; and (ii) at least one immune cell specific for the oncolytic virus.

Also provided is the use of at least one immune cell specific for oncolytic viruses in the manufacture of a medicament, which comprises the following methods of administering an individual individually or simultaneously to an individual: (i) an oncolytic virus; and (ii) at least one immune cell specific for the oncolytic virus.

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 the E1A protein, which exhibits less binding to the Rb protein than the E1A protein encoded by Ad5. In some embodiments, the oncolytic virus encodes an E1A protein that lacks the amino acid sequence LTCHEACF (sequence identification number 52). In some embodiments, the oncolytic virus code comprises the following, or consists of, or consists essentially of the E1A protein: SEQ ID NO: 34 amino acid sequence. In some embodiments, the oncolytic virus comprises a nucleic acid having one or more binding sites for one or more transcription factors. In some embodiments, the oncolytic virus comprises a nucleic acid having one or more binding sites for STAT1.

In some embodiments, the virus containing nucleic acid encoding an immunomodulatory factor is a helper-dependent adenovirus (HDAd). In some embodiments, the immunomodulatory factor 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 immunomodulatory factor comprises a nucleic acid encoding IL-12 and/or antagonist anti-PD-L1 antibody. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulatory factor comprises a nucleic acid encoding an enzyme capable of catalytically converting a non-toxic factor into a cytotoxic form. In some embodiments, the enzyme is selected from the group consisting of: thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, horseradish peroxidase, and carboxyl Esterase. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulatory factor comprises a nucleic acid encoding a thymidine kinase.

In some embodiments, the at least one cell comprising a CAR specific for cancer cell antigens is a T cell. In some embodiments, the CAR contains an antigen binding domain capable of specifically binding HER2. In some embodiments, the CAR comprises an antigen-binding domain, which contains: A VL domain, which contains: LC-CRD1: sequence identification number 10; LC-CRD2: sequence identification number 11; LC-CRD3: sequence identification number 12; And a VH domain, which includes: HC-CRD1: sequence identification number 13; HC-CRD2: sequence identification number 14; HC-CRD3: sequence identification number 15; or A VL domain, which contains: LC-CRD1: sequence identification number 18; LC-CRD2: sequence identification number 19; LC-CRD3: sequence identification number 20; And a VH domain, which includes: HC-CRD1: sequence identification number 21; HC-CRD2: sequence identification number 22; HC-CRD3: sequence identification number 23; or A VL domain, which contains: LC-CRD1: sequence identification number 26; LC-CRD2: sequence identification number 27; LC-CRD3: Sequence identification number 28; And a VH domain, which includes: HC-CRD1: sequence identification number 29; HC-CRD2: sequence identification number 30; HC-CRD3: sequence identification number 31; or A VL domain, which contains: LC-CRD1: Sequence identification number 57; LC-CRD2: Sequence identification number 58; LC-CRD3: Sequence identification number 59; And a VH domain, which includes: HC-CRD1: sequence identification number 60; HC-CRD2: sequence identification number 61; HC-CRD3: Sequence identification number 62.

In some embodiments, the CAR includes an antigen-binding domain, which includes: A VL, which contains the following, or consists of, or consists essentially of: an amino acid having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 16 Sequence; and a VH, which includes the following, or consists of, or consists essentially of: having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 17 Amino acid sequence; or A VL, which contains the following, or consists of, or consists essentially of: an amino acid having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 24 Sequence; and a VH, which includes the following, or consists of, or consists essentially of: having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 25 Amino acid sequence; or A VL, which contains, consists of, or consists essentially of: an amino acid having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 32 Sequence; and a VH, which includes the following, or consists of, or consists essentially of: having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 33 Amino acid sequence; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 63; and a VH comprising or consisting of: having at least 75% sequence identification number 64 Amino acid sequence of sequence identity.

In some embodiments, the method additionally includes: (a) at least one cell is isolated from a body, and in a specific embodiment, the cell is an immune cell; (b) modifying the at least one cell to express or contain a CAR specific for cancer cell antigens, or a nucleic acid encoding a CAR specific for cancer cell antigens, (c) optionally expand the modified at least one cell, and; (d) administering the modified at least one cell to an individual; in a specific embodiment, upon administration, the modified cell is provided to the individual along with one or more other cancer therapeutic agents.

In some embodiments, the method of treating cancer comprises: (a) Separate at least one cell from a body; (b) modifying the at least one cell to express or contain a CAR specific for cancer cell antigens, or a nucleic acid encoding a CAR specific for cancer cell antigens, (c) optionally expanding the modified at least one cell, and; (d) administering the modified at least one cell to a body.

In some embodiments, the method of treating cancer comprises: (a) Separate at least one immune cell from a body; (b) generating or expanding an immune cell population specific for oncolytic viruses using a method comprising: stimulating the immune cells by culturing in the presence of antigen-presenting cells (APCs) presenting the oncolytic virus peptides, and; (c) administering at least one immune cell specific to the oncolytic virus to a body.

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

The present disclosure also provides an oncolytic adenovirus (OncAd), which encodes an E1A protein comprising, consisting of, or consisting essentially of: the amino acid sequence of sequence identification number 34.

The present disclosure also provides an oncolytic adenovirus (OncAd), which includes 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 more sequence identity with sequence identification number 51, Or the equivalent sequence caused by codon degeneracy.

Also provided is an OncAd, which contains a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 55, Or the equivalent sequence caused by codon degeneracy. In some embodiments, the OncAd code comprises the following, or consists of, or consists essentially of the E1A protein: amino acid sequence of SEQ ID NO:34.

The present disclosure also provides a help-dependent adenovirus (HDAd), which comprises a nucleic acid encoding IL-12 and/or antagonist anti-PD-L1 antibody. In some embodiments, the HDAd additionally contains a nucleic acid encoding an enzyme capable of catalytically converting non-toxic factors to cytotoxic forms. In some embodiments, the enzyme is selected from the group consisting of: thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, horseradish peroxidase, and carboxyl Esterase.

In some embodiments, the HDAd additionally comprises a nucleic acid encoding thymidine kinase. In the case where the HDAd nucleic acid encodes IL-12 and anti-PD-L1 antibody, the respective expression sequences may or may not be regulated by the same regulatory sequence. In the case where the HDAd nucleic acid encodes both IL-12 and anti-PD-L1 antibody, the positioning on the HDAd nucleic acid may have any suitable configuration, such as encoding IL-12 in the 5'to 3'direction The nucleic acid region is upstream or downstream of the nucleic acid region encoding the anti-PD-L1 antibody.

The disclosure also provides a chimeric antigen receptor (CAR), which includes an antigen binding domain, which includes: A VL domain, which contains: LC-CRD1: sequence identification number 10; LC-CRD2: sequence identification number 11; LC-CRD3: sequence identification number 12; And a VH domain, which includes: HC-CRD1: sequence identification number 13; HC-CRD2: sequence identification number 14; HC-CRD3: sequence identification number 15; or A VL domain, which contains: LC-CRD1: sequence identification number 18; LC-CRD2: sequence identification number 19; LC-CRD3: sequence identification number 20; And a VH domain, which includes: HC-CRD1: sequence identification number 21; HC-CRD2: sequence identification number 22; HC-CRD3: sequence identification number 23; or A VL domain, which contains: LC-CRD1: sequence identification number 26; LC-CRD2: sequence identification number 27; LC-CRD3: Sequence identification number 28; And a VH domain, which includes: HC-CRD1: sequence identification number 29; HC-CRD2: sequence identification number 30; HC-CRD3: sequence identification number 31; or A VL domain, which contains: LC-CRD1: Sequence identification number 57; LC-CRD2: Sequence identification number 58; LC-CRD3: Sequence identification number 59; And a VH domain, which includes: HC-CRD1: sequence identification number 60; HC-CRD2: sequence identification number 61; HC-CRD3: Sequence identification number 62.

In some embodiments, the CAR includes an antigen-binding domain, which includes: A VL, which contains the following, or consists of, or consists essentially of: an amino acid having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 16 Sequence, and a VH, which includes the following, or consists of, or consists essentially of: having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 17 Amino acid sequence; or A VL comprising or consisting of: an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 24, and a VH comprising The following, or consist of, or consist essentially of: an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 25; or A VL comprising or consisting of the following: an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 32, and a VH comprising The following, or consist of, or consist essentially of: an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% sequence identity with sequence identification number 33; or A VL comprising or consisting of: an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or more sequence identity with sequence identification number 63, and a VH comprising The following, or consist of, or consist essentially of: an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% sequence identity with sequence identification number 64.

The present disclosure also provides an optionally isolated nucleic acid or multiple nucleic acids encoding oncolytic adenovirus (OncAd), helper dependent adenovirus (HDAd) and/or chimeric antigen receptor (CAR) according to the present disclosure .

The present disclosure also provides a cell comprising oncolytic adenovirus (OncAd), helper dependent adenovirus (HDAd), chimeric antigen receptor (CAR), and/or nucleic acid or multiple nucleic acids according to the present disclosure.

The present disclosure also provides an optionally isolated nucleic acid or multiple nucleic acids encoding oncolytic adenovirus (OncAd) and/or chimeric antigen receptor (CAR) according to the present disclosure. The present disclosure also provides a cell comprising an oncolytic adenovirus (OncAd), a chimeric antigen receptor (CAR), and/or a nucleic acid or a plurality of nucleic acids according to the present disclosure. Also provided is a cell comprising oncolytic adenovirus (OncAd) and chimeric antigen receptor (CAR) according to the present disclosure.

The present disclosure also provides a pharmaceutical composition comprising oncolytic adenovirus (OncAd), helper-dependent adenovirus (HDAd), chimeric antigen receptor (CAR); nucleic acid or multiple nucleic acids or cells according to the present disclosure, and It can be combined with or contained in a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

The present disclosure also provides a method of treating cancer, which comprises administering an oncolytic adenovirus (OncAd), helper dependent adenovirus (HDAd), chimeric antigen receptor (CAR), nucleic acid or multiple Nucleic acid, cell or pharmaceutical composition.

The present disclosure also provides an oncolytic adenovirus (OncAd), helper dependent adenovirus (HDAd), chimeric antigen receptor (CAR), nucleic acid or multiple nucleic acids, cells or Pharmaceutical composition.

The present disclosure also provides a use for manufacturing a medicament for treating cancer using oncolytic adenovirus (OncAd), helper dependent adenovirus (HDAd), chimeric antigen receptor (CAR), nucleic acid or more Nucleic acid, cell or pharmaceutical composition.

In certain embodiments according to various aspects of the present disclosure, the cancer is selected from the group consisting of head and neck cancer, nasopharyngeal cancer (NPC), cervical cancer (CC), oropharyngeal cancer (OPC), gastric cancer (GC), liver Cellular carcinoma (HCC) and lung cancer.

The present disclosure also provides a modular kit comprising a predetermined amount of oncolytic adenovirus (OncAd), helper-dependent adenovirus (HDAd), chimeric antigen receptor (CAR), nucleic acid or multiple Nucleic acid, cell and/or pharmaceutical composition.

Detailed description This disclosure relates to the combined use of multiple therapeutic agents to treat cancer. In particular, a combination of (i) oncolytic viruses, (ii) viruses that provide immunomodulatory factors, and (iii) CAR-carrying immune cells (such as T cells) specific for cancer cell antigens is used as a cancer therapy. The combination of the therapeutic agents provides improved therapeutic effects compared to the effects seen when any one of the agents is used alone. In some embodiments, at least two of the three therapeutic agents treat cancer in an additive manner, while in other embodiments, at least two of the three different therapeutic agents treat in a synergistic manner cancer.

Without wishing to be bound by any particular theory, but the improvement of therapeutic effect is believed to be due to the combination of oncolytic virus therapy (eg, effective treatment of solid tumors) and CAR-T cell therapy (eg, effective treatment of diffuse/metastatic cancer) Advantageous features are achieved by combining and providing an immune environment favorable to the proliferation and activity of CAR-T cells. Oncolytic virus

This disclosure uses oncolytic viruses. Oncolytic viruses and their uses in the treatment of cancer are described in, for example, Chiocca and Rabkin Cancer Immunol Res (2014) 2(4): 295-300, which is hereby incorporated by reference in its entirety.

Oncolytic viruses replicate in cancer cells and cause cell lysis. Usually they will choose cancer cells rather than non-cancer cells. For example, oncolytic viruses generally prefer to replicate in dividing cells rather than non-dividing cells. Therefore, oncolytic viruses can selectively kill cancer cells and destroy tumors without causing substantial damage to normal and non-cancer cells/tissues.

Oncolytic virus therapy has many advantages. Oncolytic viruses usually target many carcinogenic pathways and use multiple cytotoxic mechanisms to minimize the chance of resistance. As described above, because oncolytic viruses selectively replicate in tumors and are non-pathogenic, they exhibit minimal toxicity. And due to the replication of the virus, the dose of virus in the tumor will increase over time, and the safety of the oncolytic virus can also be genetically improved, such as the sensitivity to drugs by genetic engineering modification.

There are two main types of oncolytic viruses: (i) Viruses that replicate naturally in cancer cells preferentially, and often due to increased sensitivity to innate antiviral signals or dependence on oncogenic signaling pathways, are non-pathogenic in humans, including spontaneously small Viruses, myxoma virus (MYXV; pox virus), Newcastle disease virus (NDV; paramyxovirus), Rio virus and Seneca valley virus (SVV); picornavirus); and (ii) Viruses that perform genetic manipulations such as mutations/deletions on genes required for replication in normal, but not cancer cells, including adenovirus (Ad), herpes simplex virus (HSV), acne virus (VV), and vesicular Stomatitis virus (VSV; baculovirus); or viruses genetically manipulated as vaccine vectors, including measles virus (MV; paramyxovirus), poliovirus (PV; picornavirus), and VV (pox virus) ).

Genetic manipulation may include insertion/alteration of functional sequences to provide enhanced cancer cell selectivity, safety, and/or modification of viral tropism.

For example, oncolytic viruses can be genetically engineered to introduce tissue-specific internal ribosome entry sites (IRESs) that only allow the virus to be translated in target cells, and/or to introduce miRNAs/miRNA response elements (MREs); healthy cells or certain Different miRNA expression between these tissues and tumor cells prevents the virus from being targeted by healthy cells/tissues. Oncolytic viruses can also be engineered to place transcription of the viral genome under the control of cell or tissue-specific regulatory regions, such as promoters/enhancers (eg, tumor cell-specific promoters). In some embodiments, oncolytic viruses according to the present disclosure may contain one or more modifications for this purpose.

Viruses can also be modified for transduction targeting, such as by modifying viral receptors/coating proteins in order to target tumor cells and/or not target healthy cells/tissues.

Oncolytic viruses can be administered in the manner described above in Chiocca and Rabkin, ie causing the smallest anti-oncolytic virus response in the individual (eg, neutralization with antiviral antibodies) and then sealed in the liver, and maximizing tumor delivery Change. For example, oncolytic viruses can be administered in a cell carrier, such as mesenchymal stromal cells, myelogenous inhibitory cells (MDSCs), neural stem cells, T cells, cytokine-induced killer cells or irradiated tumor cells, or can be coated On nanoparticles.

In some embodiments, the oncolytic virus of the present disclosure is or is derived from adenovirus (Ad), herpes simplex virus (HSV), acne virus (VV), vesicular stomatitis virus (VSV); spontaneous parvovirus , Myxoma virus (MYXV), Newcastle disease virus (NDV), Rio virus, Seneca valley virus (SVV), measles virus, retrovirus, influenza virus, Sindbis virus (SINV) or pox virus. In some embodiments, the oncolytic virus is not an acne virus. In some embodiments, the oncolytic virus is not acne virus JX-594.

The oncolytic virus "derived from" a reference virus as used herein includes the nucleic acid sequence or amino acid sequence possessed by the reference virus. In some embodiments, an oncolytic virus "derived from" a reference virus includes one or more genes owned by the reference virus. In some embodiments, an oncolytic virus "derived from" a reference virus encodes one or more proteins encoded by the reference virus.

In some embodiments, an oncolytic virus derived from a reference virus may include a nucleic acid sequence encoding one or more functional elements of the reference virus. "Functional elements" can be, for example, transcriptional regulators (eg, promoters/enhancers), post-transcriptional processing regulators, translational regulators, post-transcriptional processing regulators, response elements, repetitive sequences, or viral proteins. In some embodiments, an oncolytic virus derived from a reference virus may include one or more genes of the reference virus or its encoded protein.

In some embodiments, the oncolytic virus of the present disclosure is or is derived from adenovirus (OncAd). OncAds is reviewed in, for example, Larson et al. , Oncotarget. (2015) 6(24): 19976–19989, which is hereby incorporated by reference in its entirety.

In some embodiments, the OncAd is or derived from species A, B, C, D, E, F, or G human adenovirus (eg, 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 species C human adenovirus. In some embodiments, the OncAd is or is derived from Ad5, Ad2, Ad1, Ad6, or Ad57.

In some embodiments, the OncAd is a conditionally replicating adenovirus (or CRAd).

In some embodiments, the OncAd has a reduced ability to infect non-cancer cells, replicate in non-cancer cells, and/or lyse non-cancer cells (compared to infection of comparable cancer cells/replication and/or replication in comparable cancer cells) The ability to dissolve comparable cancer cells), for example, as a result of genetic modification of the OncAd-derived adenovirus.

In some embodiments, the oncolytic virus contains modifications to one or more protein coding sequences. In some embodiments, the modification changes the yield or activity of the encoded protein. In some embodiments, the modification is truncation or deletion of the protein.

In some embodiments, the OncAd contains modifications to adenovirus early proteins. In some embodiments, the modification is to the region encoding the E1A protein. In some embodiments, the OncAd encodes an E1A protein, which has less ability to bind to the Rb protein than the wild-type E1A protein (eg, E1A encoded by the adenovirus derived from the OncAd). In some embodiments, the OncAd encodes the E1A protein lacking the amino acid sequence LTCHEACF (sequence identification code 52). An example of OncAd that contains the E1A protein encoding the amino acid sequence LTCHEACF (sequence identification number 52) is Onc5/3Ad2E1Δ24 shown in sequence identification number 55.

In some embodiments, the oncolytic virus encodes an E1A protein, which comprises, consists of, or consists essentially of: having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or have 100 % Amino acid sequence with sequence identity.

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 activated transcription factor (ie, transcription-activating protein). The one or more binding sites provided for one or more transcription factors are preferably provided upstream (i.e., 5' to) of the nucleic acid sequence encoding one or more functional elements (e.g., viral proteins).

In some embodiments, the transcription factor is a transcription factor that has increased expression or increased activity in cancer cells compared to comparable non-cancer cells (eg, non-cancer cells derived from the same tissue/cell type).

"Expression" herein may mean gene expression or protein expression. Gene expression can be measured by various methods known to those skilled in the art, such as quantitative real-time PCR (qRT-PCR) to measure mRNA levels or by reporter gene-based methods. Similarly, protein expression can be measured by various methods well known to those skilled in the art, such as by antibody-based methods, such as by Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT or reporter gene-based methods.

An example of OncAd containing 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 fully incorporated herein by reference . ICOVIR15 contains 8 binding sites for the transcription factor E2F.

In some embodiments, the oncolytic virus contains one or more binding sites for transcription factors, and the expression or activity of genes or proteins in cells is up-regulated in response to factors produced or expressed by immune cells. In some embodiments, the factors produced or expressed by immune cells may be effector immune cells, such as CD8+ cytotoxic T lymphocytes (CTL), CD4+ T helper 1 (TH1) cells, natural killer (NK ) At least one cytokine/chemokine produced by cells or natural killer T (NKT) cells, or a protein expressed on the cell surface thereof.

In some embodiments, the oncolytic virus of the present disclosure includes one or more binding sites for STAT transcription factors. In some embodiments, the oncolytic virus contains one or more binding sites for STAT1. The ICOSTAT OncAd described herein has 8 binding sites for STAT1, and STAT1 is known to be up-regulated by IFNγ. In a specific embodiment, ICOSTAT is particularly effective for cancer treatment because the host's immune response to cancer cells promotes the on-site replication of the oncolytic virus.

In some embodiments, the oncolytic virus contains more than one binding site for STAT1, such as 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 include or consist of or consist essentially of the sequence: sequence TTCCGGGAA (sequence identification number 53) or TTCTCGGAA (sequence identification number 54). In some embodiments, the oncolytic virus of the present disclosure includes one or more copies of the sequence TTCCGGGAA (sequence identification number 53) or TTCTCGGAA (sequence identification number 54).

In some embodiments, the oncolytic virus according to the present disclosure comprises or consists of or consists essentially of: having at least 60%, 61%, 62%, 63%, 64%, 65 with sequence identification number 51 %, 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 a nucleic acid sequence with 100% sequence identity, or an equivalent sequence caused by codon degeneration.

In some embodiments, the oncolytic virus according to the present disclosure comprises or consists of or consists essentially of: having at least 60%, 61%, 62%, 63%, 64%, 65% with sequence identification number 55 , 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%, Nucleic acid sequence with 99% or 100% sequence identity, or equivalent sequence caused by codon degeneracy.

In some embodiments, the oncolytic virus according to the present disclosure encodes the same protein as the protein encoded by the oncolytic virus comprising, consisting of, or consisting essentially of: the nucleic acid represented by SEQ ID NO:55. In some embodiments, the oncolytic virus according to the present disclosure encodes the same protein as the protein encoded by the oncolytic virus comprising, consisting of, or consisting essentially of: a nucleic acid represented by sequence identification number 51. Viruses containing nucleic acids encoding immunomodulatory factors

The present disclosure uses viruses containing nucleic acids encoding immunomodulatory factors. The virus acts as a carrier to deliver the immunomodulatory factor. In certain embodiments, the virus contains nucleic acids encoding more than one immunomodulatory factor.

Any virus capable of introducing nucleic acids encoding immunomodulatory factors into cells (eg, human primary immune cells) can be used. Suitable viruses include gamma retroviruses (eg, vectors derived from mouse leukemia virus (MLV)), lentiviruses, adenoviruses, adeno-associated viruses, acne viruses, and herpes viruses, such as Maus et al. , Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 2016 4, 9, which are fully incorporated in this case for reference. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulatory factor is or is derived from adenovirus, lentivirus, retrovirus, or herpes virus.

In some embodiments, the virus comprising a nucleic acid encoding at least one immunomodulatory factor is an oncolytic virus comprising a nucleic acid encoding at least one immunomodulatory factor.

The immunomodulatory factor encoded by the virus according to the present invention containing a nucleic acid encoding an immunomodulatory factor is preferably selected to promote an immune response to an individual's cancer, especially a cell-mediated immune response. In one embodiment, the immunomodulatory factor to provide beneficial effects of immune cells (e.g., CTLs, T _H 1 cells, NK cells or NKT cells) of the activation, recruitment, proliferation, and activity conditions / or survival.

In some embodiments, the immunomodulatory factor may be an agonist of an effector immune response, such as a cytokine or chemokine (e.g., IL-2) that promotes the activation, recruitment, proliferation, activity, and/or survival of effector immune cells , IL-7, IL-17, IL-12, IL-21, IL-15, MIP-1α or RANTES), costimulatory receptors (eg, 4-1BB, OX40, CD28, CD27, ICOS, CD30 or GITR ) Agonist antibody, or co-stimulating receptor (eg, 4-1BBL, OX40L, CD80, CD86, CD70, ICOSL, CD30L or GITRL) ligand. In some embodiments, the agonist of the effector immune response may be an antagonist of an immune checkpoint inhibitor, or an antagonist of a ligand of an immune checkpoint inhibitor, such as for PD-L1, PD-L2, PD- 1. Antagonistic antibodies to CTLA-4, LAG-3, TIM-3, Gal-9, TIGIT, VISTA, or BTLA, or antagonists of cytokines/chemokines (antagonists of effector immune responses; such as TGFβ) ( That is, antagonistic anti-TGFβ antibody or soluble/bait TGFβ receptor). In some embodiments, the agonist of the effector immune response may be a molecule for attracting and collecting bystander effector immune cells such as T cells and NK cells.

In some embodiments, the immunomodulatory factor may be an antagonist of an immunomodulatory response, such as promoting the activation, recruitment, proliferation, and proliferation of immunomodulatory cells, such as regulatory T cells (Tregs) and/or myeloid-derived suppressor cells (MDSCs), Antagonists of active and/or surviving cytokines/chemokines, such as CCL9, CXCL10, CCL20, CCL22.

In some embodiments, the virus containing nucleic acids encoding immunomodulatory factors may additionally contain nucleic acids encoding additional functional sequences. For example, the virus may contain a protein encoding for reducing the growth/proliferation/survival of infected cells, or a protein for sensitizing infected cells to a specified therapeutic agent, or a protein for destroying tumor structures to promote immune cell infiltration (such as , Enzymes that digest tumor matrix) nucleic acids.

In some embodiments, the virus comprising a nucleic acid encoding an immunomodulatory factor additionally comprises a nucleic acid encoding an enzyme capable of catalytically converting a non-toxic factor into a cytotoxic form. The enzyme converts non-toxic prodrugs into their active cytotoxic form.

Enzyme/prodrug systems are well known in the industry, including those described in Malekshah et al. Curr Pharmacol Rep. (2016) 2(6): 299–308, which is hereby incorporated by reference in its entirety. Examples of non-toxic prodrugs, their active cytotoxic forms and the ability to convert the non-toxic prodrugs into their active cytotoxic forms are shown in Figure 2 of Malekshah et al.

In some embodiments, the virus comprising a nucleic acid encoding an immunomodulatory factor additionally comprises thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylation Nucleic acids of enzymes, horseradish peroxidase and/or carboxylesterase.

For example, the virus may contain a nucleic acid encoding thymidine kinase, which is used to direct cells expressing the virus to ganciclovir (GCV), aciclovir (ACV), and/or valaciclovir )'S treatment is sensitive. The virus may contain a nucleic acid encoding cytosine deaminase, which is used to convert cells expressing the virus to 5-fluorocytosine (5-FC), which is converted to 5-fluorouracil by cytosine deaminase (5 -FU), the treatment is sensitive. The virus may contain a nucleic acid encoding a nitroreductase for sensitizing cells expressing the virus to CB1954, nitro-CBI-DEI and/or PR-104A treatment. The virus may contain a nucleic acid encoding cytochrome P450, which is used to sensitize cells expressing the virus to the treatment of oxynitrazides (eg, cyclophosphamide or ifosfamide). The virus may contain a nucleic acid encoding carboxypeptidase G2, which is used to sensitize cells expressing the virus to the treatment of nitrogen mustard drugs (eg, CMDA or ZD2767P). The virus may contain a nucleic acid encoding a purine nucleoside phosphorylase for the cells expressing the virus to 6-methylpurine 2-deoxyriboside and/or fludarabine (eg, 6-methyl Purine-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 horseradish peroxidase, which is used to sensitize cells expressing the virus to indole-3-acetic acid (IAA) treatment. The virus may include a carboxylesterase-encoding enzyme The nucleic acid is used to sensitize cells expressing the virus to irinotecan.

In some embodiments, the virus may include nucleic acids encoding antagonists of growth factors.

In some embodiments, the virus may be helper-dependent adenovirus (HDAd). HDAds is reviewed in, for example, Rosewell et al. , J Genet Syndr Gene Ther (2011) Suppl 5:001, which is hereby incorporated by reference in its entirety.

HDAds lack a viral protein coding sequence and therefore have a large capacity (up to 37Kb) for transduction of the coding sequence of interest. HDAds are non-embedded, can effectively transduce multiple cell types independently of the cell cycle, and mediate long-term transgene expression without chronic toxicity.

HDAds contain only cis-acting viral elements required for genome replication (inverted terminal repeats (ITRs)) and capsidization (ψ), and therefore rely on helper viruses for proliferation. When cells are infected with both helper viruses and HDAds, the helper virus replication mechanism will copy and package HDAds in a trans manner.

In a specific embodiment of the present disclosure, the oncolytic virus is OncAd, and the virus containing nucleic acid encoding an immunomodulatory factor is HDAd, and the OncAd and HDAd can co-infect and replicate in cancer cells.

HDAd's dependence on OncAd's assistance provides highly localized expression of immunomodulatory factors. That is, because HDAd can only proliferate in cells co-infected with OncAd, and then because OncAd is selective for replication in cancer cells, the expression of factors encoded by HDAd is limited to cancer cells/tissues, minimizing side effects.

In addition, because OncAd and HDAd can effectively target and infect tumor cells, the expression of immunomodulatory factors in these cells will change the normal immunosuppressive tumor microenvironment, and provide for the activation and recruitment of effector immune cells (ie, Tumor penetration/infiltration), hyperplasia, activity and/or survival conditions.

In particular, in the case where the treatment method of the present disclosure uses CAR-T cells, the expression of the immunomodulatory factor encoded by the HDAd provides enhanced activation, recruitment, proliferation, activity, and/or survival of the CAR-T cells.

In a specific embodiment herein, the virus comprising a nucleic acid encoding an immunomodulatory factor is HDAd comprising a nucleic acid encoding IL-12p70, HSV-1 thymidine kinase, and antagonist anti-PD-L1 minibody.

In some embodiments, a virus comprising a nucleic acid encoding an immunomodulatory factor according to the present disclosure encodes IL-12. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulatory factor, comprising coding and sequence identification number 35 has 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 a nucleic acid with an amino acid sequence that is 100% sequence identical.

In some embodiments, according to the viruses of the present disclosure that include nucleic acids encoding immunomodulatory factors, antagonists of messages encoding PD-1/PD-L1. In some embodiments, the antagonist of the PD-1/PD-L1 message is an anti-PD-L1 antibody.

In some embodiments, the anti-PD-L1 antibody comprises an antigen binding domain, which comprises a VL domain, which comprises: LC-CRD1: Sequence identification number 39; LC-CRD2: sequence identification number 40; LC-CRD3: Sequence identification number 41; And a VH domain, which includes: HC-CRD1: sequence identification number 42; HC-CRD2: sequence identification number 43; HC-CRD3: Sequence identification number 44.

In some embodiments, the anti-PD-L1 antibody comprises a VL comprising or consisting of or consisting essentially of: having at least 75%, 76%, 77%, 78%, 79 with sequence identification number 45 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or an amino acid sequence with 100% sequence identity; and a VH which contains or consists of or consists essentially of: having at least 75 with the sequence identification number 46 %, 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 an amino acid sequence with 100% sequence identity.

In some embodiments, the virus comprising a nucleic acid encoding an immunomodulatory factor, comprising coding and sequence identification number 38 has 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 a nucleic acid with an amino acid sequence that is 100% sequence identical.

In some embodiments, a virus comprising a nucleic acid encoding an immunomodulatory factor according to the present disclosure includes an amino acid sequence encoding thymidine kinase. In some embodiments, the virus comprising a nucleic acid encoding an immunomodulatory factor, comprising coding and sequence identification number 36 has 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 a nucleic acid with an amino acid sequence that is 100% sequence identical.

In some embodiments, a virus comprising a nucleic acid encoding an immunomodulatory factor according to the present disclosure comprises, consists of, or consists essentially of: having a sequence identification number 50 of 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 a nucleic acid sequence with 100% sequence identity, or an equivalent sequence caused by codon degeneration. Chimeric antigen receptors (CARs) and CAR expressing cells

The present disclosure uses immune cells comprising chimeric antigen receptors (CAR).

Chimeric antigen receptors (CARs) are recombinant receptors that provide both antigen binding and immune cell activation functions. The CAR structure and engineering are summarized in, for example, Dotti et al. , Immunol Rev (2014) 257(1), which is fully incorporated herein by reference. CARs include an antigen binding region and a signal transduction region connected to the anchoring region of the cell membrane. The optional hinge region can provide a separation between the antigen binding region and the cell membrane anchoring interval, and can serve as a flexible linker.

The antigen-binding region of CAR can be based on the antigen-binding region of an antibody (which is specific for the antigen targeted by CAR), or other agents that can bind to the target. For example, the antigen-binding domain of the CAR may include amino acid sequences that will specifically bind to the complementarity determining regions (CDRs) of the antibody of the target protein or the entire light chain and heavy chain variable region amino acid sequences. The antigen-binding domain of CARs can be based on other proteins: protein interactions to target antigens, such as ligands: receptor binding; for example, using an IL-13-based antigen-binding domain to develop a CAR targeting IL-13Rα2 (see Kahlon et al . 2004 Cancer Res 64(24): 9160-9166).

The CAR of the present disclosure contains an antigen-binding region specific for cancer cell antigens. The antigen-binding region of the CAR can be provided in any suitable format, such as scFv, Fab, etc. In some embodiments, the antigen-binding region of the CAR comprises or consists of cancer cell antigen-binding scFv.

Cancer cell antigen is an antigen expressed by cancer cells. The cancer cell antigen can be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Cancer cell antigen expression may be related to cancer. Cancer cell antigens may be abnormal expression of cancer cells (eg, cancer cell antigens may be expressed at abnormal locations) or may be abnormal structures expressed by cancer cells. Cancer cell antigens may be able to trigger an immune response.

In some embodiments, the antigen is expressed on the cell surface of cancer cells (ie, the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen bound by the bispecific antigen-binding polypeptide of the present disclosure appears on the outer surface of the cancer cell (ie, is extracellular). In some embodiments, the antigen is anchored to the cell membrane through, for example, a transmembrane domain or other membrane anchor (eg, lipid anchor, such as a GPI anchor). In some embodiments, the cancer cell antigen is expressed on the cell surface of the cancer cell (ie, expressed in or on the cell membrane), but may be expressed inside the cell (ie, expressed in a comparable non-cancer cell).

The cancer cell antigen may be a cancer-associated antigen. In some embodiments, the cancer cell antigen is an antigen whose expression correlates with the development, progression, and/or severity of symptoms of the cancer. The cancer-associated antigen may be related to the cause or pathology of cancer, or may be abnormally expressed due to cancer. In some embodiments, the antigen is an expression level (eg, RNA and/or protein level) compared to an equivalent non-cancer cell (eg, non-cancer cell derived from the same tissue/cell type) ) Antigens upregulated by cancer cells.

In some embodiments, the cancer-associated antigen may be preferentially expressed by cancer cells rather than by comparable non-cancer cells (eg, non-cancer cells derived from the same tissue/cell type). In some embodiments, the cancer-associated antigen may be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of overexpressed cell proteins, cancer antigens produced by oncogenic viruses, cancer embryo antigens, or cell surface glycolipids or glycoproteins.

Cancer cell antigens are reviewed in Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al. , editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton ( ON): BC Decker; 2003. Cancer cell antigens include cancer embryo antigens: CEA, immature laminin receptor, TAG-72; oncogenic viral antigens such as HPV E6 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-testicle antigens: BAGE, CAGE, GAGE, MAGE, SAGE , XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; lineage-restricted antigens: MART1, Gp100, tyrosinase, TRP-1/2, MC1R, prostate specific antigen; mutant antigen: β- Catenin, RCA1/2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII; antigens changed after translation: MUC1, idiotype antigens: Ig, TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose regulatory protein 78 (GRP78), vimentin (vimentin), nucleolin, fetal-acinus pancreatic protein (FAPP), alkali Sex phosphatase placenta-like type 2 (ALPPL-2), sialin 5 (siglec-5), pressure-induced phosphorylation protein 1 (STIP1), protein tyrosine kinase 7 (PTK7) and cyclophilin B.

In some embodiments, the cancer cell antigen is HER2. In some embodiments, the CAR of the present disclosure includes an antigen binding domain capable of specifically binding HER2. In some embodiments, the CAR includes an antigen-binding domain that includes CDRs of antibodies that specifically bind HER2. In some embodiments, the CAR includes an antigen-binding domain that includes the VL and VH regions of an antibody that specifically binds HER2.

In a specific embodiment, the CAR-expressing cell contains two separate CARs, each targeting a different cancer cell antigen, and in a specific aspect, at least one of the CARs targets HER2. In some cases, the CAR has dual specificity for two different cancer cell antigens, one of which may be HER2.

In some embodiments, the CAR comprises an antigen binding domain, which comprises a VL domain, which comprises: LC-CRD1: sequence identification number 10, sequence identification number 18, sequence identification number 26 or sequence identification number 57; LC-CRD2: sequence identification number 11, sequence identification number 19, sequence identification number 27 or sequence identification number 58; LC-CRD3: sequence identification number 12, sequence identification number 20, sequence identification number 28 or sequence identification number 59; And a VH domain, which includes: HC-CRD1: sequence identification number 13, sequence identification number 21, sequence identification number 29 or sequence identification number 60; HC-CRD2: sequence identification number 14, sequence identification number 22, sequence identification number 30 or sequence identification number 61; HC-CRD3: sequence identification number 15, sequence identification number 23, sequence identification number 31 or sequence identification number 62.

In some embodiments, the CAR comprises an antigen binding domain, which comprises a VL domain, which comprises: LC-CRD1: sequence identification number 10; LC-CRD2: sequence identification number 11; LC-CRD3: sequence identification number 12; And a VH domain, which includes: HC-CRD1: sequence identification number 13; HC-CRD2: sequence identification number 14; HC-CRD3: Sequence identification number 15.

In some embodiments, the CAR comprises an antigen binding domain, which comprises a VL domain, which comprises: LC-CRD1: sequence identification number 18; LC-CRD2: sequence identification number 19; LC-CRD3: sequence identification number 20; And a VH domain, which includes: HC-CRD1: sequence identification number 21; HC-CRD2: sequence identification number 22; HC-CRD3: Sequence identification number 23.

In some embodiments, the CAR comprises an antigen binding domain, which comprises a VL domain, which comprises: LC-CRD1: sequence identification number 26; LC-CRD2: sequence identification number 27; LC-CRD3: Sequence identification number 28; And a VH domain, which includes: HC-CRD1: sequence identification number 29; HC-CRD2: sequence identification number 30; HC-CRD3: Sequence identification number 31.

In some embodiments, the CAR comprises an antigen binding domain, which comprises a VL domain, which comprises: LC-CRD1: Sequence identification number 57; LC-CRD2: Sequence identification number 58; LC-CRD3: Sequence identification number 59; And a VH domain, which includes: HC-CRD1: sequence identification number 60; HC-CRD2: sequence identification number 61; HC-CRD3: Sequence identification number 62.

In some embodiments, the CAR includes an antigen-binding domain that includes a light chain variable region (VL) that includes or consists of or consists essentially of: and sequence identification numbers 16, 24, 32 Or 63 has 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 amino acid sequences with 100% sequence identity.

In some embodiments, the CAR comprises an antigen binding domain comprising a heavy chain variable region (VH), the region comprising or consisting of or consisting essentially of: and sequence identification number 17, 25, 33 or 64 has 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 an amino acid sequence with 100% sequence identity.

In some embodiments, the CAR includes an antigen-binding domain that includes a VL that includes or consists of or consists essentially of: having at least 75%, 76%, 77%, 78 with sequence identification number 16 %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Amino acid sequence of 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and a VH, which contains or consists of or consists essentially of the following: and sequence identification number 17 Have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, Amino acid sequences of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the CAR comprises an antigen-binding domain comprising a VL comprising or consisting of or consisting essentially of: having at least 75%, 76%, 77%, 78 with the sequence identification number 24 %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or an amino acid sequence with 100% sequence identity, and a VH, which contains or consists of or consists essentially of the following: and sequence identification number 25 Have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, Amino acid sequences of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the CAR comprises an antigen-binding domain comprising a VL comprising or consisting of or consisting essentially of: having at least 75%, 76%, 77%, 78 with sequence identification number 32 %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Amino acid sequence of 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and a VH, which contains or consists of or consists essentially of the following: and sequence identification number 33 Have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, Amino acid sequences of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the CAR comprises an antigen-binding domain comprising a VL comprising or consisting of or consisting essentially of: having at least 75%, 76%, 77%, 78 with sequence identification number 63 %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Amino acid sequence of 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and a VH, which contains or consists of or consists essentially of the following: and sequence identification number 64 Have 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 an amino acid sequence with 100% sequence identity.

In some embodiments, the CAR of the present disclosure includes an antigen-binding region that includes or consists of or consists essentially of the following: an antibody/antigen-binding fragment according to the present disclosure.

The cell membrane anchor region is provided between the antigen binding region and the signal transduction region of the CAR. The cell membrane anchor region provides the CAR anchor 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 those derived from CD28, CD3-ζ, CD4 or CD8.

In some embodiments, the cell membrane anchor region comprises or consists of or consists essentially of: having at least 80%, 85%, 90%, 91%, 92%, 93%, 94% with sequence identification number 4 , 95%, 96%, 97%, 98%, 99% or 100% amino acid sequences with sequence identity.

The signaling region of CAR allows activation of T cells. The CAR signaling region may include the amino acid sequence of the CD3-ζ intracellular domain, which provides immunoreceptor tyrosine activation motifs (ITAMs) for phosphorylation and activation of CAR expressing T cells. In CARs, a signal transduction region containing other ITAM protein-containing sequences is also used, such as an ITAM region-containing domain containing FcyRI (Haynes et al. , 2001 J Immunol 166(1):182-187). CARs that contain signal transduction regions derived from the CD3-ζ intracellular domain are generally referred to as first-generation CARs.

In some embodiments, the cell membrane anchor region comprises or consists of or consists essentially of: having at least 80%, 85%, 90%, 91%, 92%, 93%, 94% with sequence identification number 6 , 95%, 96%, 97%, 98%, 99% or 100% amino acid sequences with sequence identity.

The signal transduction region of CARs may also contain a co-stimulation sequence derived from the signal transduction region of the co-stimulatory molecule, so as to promote the activation of CAR expressing T cells when bound to the target protein. Suitable costimulatory molecules include at least CD28, OX40, 4-1BB, ICOS, and CD27. CARs with signal transduction regions that include additional co-stimulatory sequences are generally called second-generation CARs.

In some cases, CARs are designed to provide co-stimulation of different intracellular signaling pathways. For example, the signal transduction associated with CD28 co-stimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, while 4-1BB-mediated signal transduction is via the TNF receptor-related factor (TRAF) adaptor protein. Therefore, the message-passing region of CARs sometimes contains costimulatory sequences derived from the message-passing region of more than one costimulatory molecule. CARs that contain multiple common stimulation sequences in the messaging area are generally called third-generation CARs.

In some embodiments, the CAR of the present disclosure includes one or more costimulatory sequences that include or consist of or consist essentially of an amino acid sequence, the amino acid sequence Amino acid sequences comprising or consisting of or consisting essentially of or derived from the following: intracellular domains of one or more of CD28, OX40, 4-1BB, ICOS, and CD27.

In some embodiments, the cell membrane anchor region comprises or consists of or consists essentially of: having at least 80%, 85%, 90%, 91%, 92%, 93%, 94% with sequence identification number 5 , 95%, 96%, 97%, 98%, 99% or 100% amino acid sequences with sequence identity.

The optional hinge region can provide separation between the antigen binding domain and the transmembrane domain, and can serve as a flexible linker. The hinge region may be a flexible domain that allows the binding motif to be oriented in different directions. The hinge region can be derived from the CH ₂ CH ₃ region of IgG1 or immunoglobulin. In some embodiments, the CAR of the present disclosure includes a hinge region that includes or consists of an amino acid sequence or consists essentially of an amino acid sequence, the amino acid sequence includes the following Consists or consists essentially of or derived from the following: the hinge region of IgG1 or the amino acid sequence of CH ₂ CH ₃ of immunoglobulins.

In some embodiments, the cell membrane anchor region comprises or consists of or consists essentially of: having at least 85%, 90%, 91%, 92%, 93%, 94%, 95% with the sequence identification number 9 , 96%, 97%, 98%, 99% or 100% sequence identity of amino acid sequences.

In some embodiments, the CAR comprises or consists of or consists essentially of: having at least 70%, 71%, 72%, 73%, 74%, 75 with sequence identification number 1, 2, 3, or 56 %, 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 an amino acid sequence with 100% sequence identity.

The present disclosure also provides a cell containing or expressing the CAR according to the present disclosure. Also provided is a cell containing or expressing a nucleic acid encoding a CAR according to the present disclosure. The engineering of bringing CAR into T cells can be carried out during the cultivation for transduction in test tubes, and the expansion takes place during the expansion of T cells such as adoptive T cell therapy. Methods for constructing immune cells to express CARs are known to those skilled in the art, and are described in, for example, Wang and Rivière Mol Ther Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety. It can be understood that "at least one cell" includes a plurality of cells, such as a cell population.

The cell containing or expressing the CAR according to the present invention may be a eukaryotic cell, such as a mammalian cell. The mammal can be a human or non-human mammal (eg, rabbit, guinea pig, rat, mouse, or other rodent (including any rodent)), cat, dog, pig, sheep, goat, cow (including cow , Such as milk cows or any bovine animal), horses (including any equine animals), donkeys and non-human primates).

In some embodiments, the cell can be from, or can be obtained from a human individual. When a CAR-expressing cell is used to treat an individual, the cell may be from an individual to be treated with the CAR-expressing cell (ie, the cell may be autologous) or the cell may be from a different individual (ie, the cell Can be foreign).

The cell may be an immune cell. The cells may be cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or mononuclear cells. The lymphocytes can be, for example, T cells, B cells, NK cells, NKT cells, or congenital lymphocytes (ILC) or their precursors. The cell may express, for example, a CD3 polypeptide (eg, CD3γ, CD3ε, CD3ζ, or CD3δ), a TCR polypeptide (TCRα or TCRβ), CD27, CD28, CD4, or CD8.

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 cells are CD3+, CD8+ T cells. In some embodiments, the T cell is a cytotoxic T cell (eg, cytotoxic T lymphocyte (CTL)).

The use of CAR T cells has the advantage of being systemically administered and directed to both primary and metastatic tumors (Manzo et al. , Human Molecular Genetics (2015) R67-73).

In some embodiments, the cell is an antigen-specific T cell. In the embodiments herein, an "antigen-specific" T cell is a cell that exhibits certain functional characteristics of T cells in response to an antigen specific to T cells or a cell expressing the antigen. In some embodiments, the characteristic is a functional characteristic associated with effector T cells, such as cytotoxic T cells.

In some embodiments, antigen-specific T cells may exhibit one or more of the following characteristics: cytotoxicity against cells such as those containing/expressing antigens specific for T cells; in response to antigens specific for T cells or containing/ Cell proliferation expressing antigens specific for T cells, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression and/or FAS ligand (FASL) expression. Antigen-specific T cells contain a TCR that can recognize peptides specific to T cells when appropriate MHC molecules are presented. The antigen-specific T cells may be CD4+ T cells and/or CD8+ T cells.

In some embodiments, the antigen specific for T cells may be a virus, such as adenovirus, cytomegalovirus (CMV), EB virus (EBV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus Virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV) or herpes simplex virus (HSV), peptides or polypeptides.

T cells specific for viral antigens may be referred to herein as virus-specific T cells (VST). VSTs can be CD4+ T cells (eg, T _H cells) and/or CD8+ T cells (eg, CTLs). T cells specific for specific virus antigens can be described as related virus specific T cells; for example, T cells specific for adenovirus antigens can be referred to as adenovirus specific T cells, or "AdVST". The use of virus-specific T cells to generate CAR-T cells has the following advantages: Although primitive T cells have limited long-term durability after infusion, virus-specific T cells (VSTs) derived from memory intervals and genetically modified VSTs have been It has been shown that it persists for more than 10 years after infusion into a stem cell transplant recipient (Cruz et al. , Cytotherapy (2010) 12:743-749). For example, VST expressing GD2.CARs has shown long-term persistence after infusion and has produced a complete tumor response in patients with low tumor burden (Sun et al., Journal for Immunotherapy of Cancer (2015) 3:5 and Pule et al. , Nature Medicine (2008) 14: 1264-1270).

In some embodiments, the CAR-containing/expressing cells are virus-specific T cells (VST, such as virus-specific CD4+ T cells (eg, T _H cells) and/or virus-specific CD8+ T cells (eg, CTL). In some embodiments, the CAR expressing cells are adenovirus-specific T cells (AdVST), cytomegalovirus-specific T cells (CMVST), EB 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).

In some embodiments, the CAR-containing/expressing cells are oncolytic virus-specific immune cells (eg, oncolytic virus-specific T cells), as described herein.

Any cell of the present disclosure may be included in a population of isolated cells that may or may not be homogeneous. In a specific embodiment, most of the cells in the cell population are immune cells specific for oncolytic viruses and/or CAR-expressing. The cells in the cell population may comprise oncolytic adenovirus (OncAd), helper-dependent adenovirus (HDAd), chimeric antigen receptor (CAR) and/or encode one or more of the OncAd, HDAd and/or CAR Nucleic acid or multiple nucleic acids. In particular embodiments, the cell population has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of cells containing oncolytic adenovirus (OncAd), Helper dependent adenovirus (HDAd), chimeric antigen receptor (CAR) and/or nucleic acid or nucleic acids encoding one or more of the OncAd, HDAd and/or CAR. Oncolytic virus-specific immune cells

The aspect of the present disclosure provides oncolytic virus-specific immune cells (also referred to herein as oncolytic virus-specific immune cells). Oncolytic virus-specific cells express/contain receptors capable of recognizing oncolytic virus antigen peptides (eg, when presented by MHC molecules). The immune cell can express/contain this receptor because of the expression of the endogenous nucleic acid encoding the antigen receptor or because it has been engineered to express this receptor.

In some embodiments, the oncolytic virus-specific immune cells may be cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or mononuclear spheres. The lymphocytes can be, for example, T cells, B cells, NK cells, NKT cells, or congenital lymphocytes (ILC) or their precursors. The cell may express, for example, a CD3 polypeptide (eg, CD3γ, CD3ε, CD3ζ, or CD3δ), a TCR polypeptide (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 cell is a T helper cells (T _H cells)). In some embodiments, the T cell is a cytotoxic T cell (eg, cytotoxic T lymphocyte (CTL)).

As described herein, the oncolytic virus-specific immune cells (eg, oncolytic virus-specific T cells) may be specific for oncolytic viruses. That is, the oncolytic virus-specific immune cells can be specific to one or more oncolytic virus antigens as described herein.

Methods for generating/expanding immune cell populations specific for the antigen of interest and/or virus of interest are known in the industry and described in Wang and Rivière Cancer Gene Ther. (2015) 22(2) :85-94, which is hereby incorporated by reference in its entirety.

This method may involve combining a heterogeneous immune cell population (eg, peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), tumor infiltrating lymphocytes (TILs)) with one or more polypeptides of the antigen of interest or containing / Contact with cells expressing the antigen/peptide. The cell containing/expressing the antigen/peptide may be the result of infection with a virus containing/encoding the antigen, the cell absorbing its antigen/peptide or expressing its antigen/peptide. This presentation is usually in the context of MHC molecules at the cell surface of the antigen presenting cells.

Cells containing/expressing the antigen/peptide can be contacted with the peptide of the antigen ("shock") by methods well known to those skilled in the art. Antigen peptides can be provided in a peptide mixture library (corresponding to one or more antigens), which can be called a peptide mixture. The peptide in the peptide mixture may be, for example, overlapping peptides of 8-20 amino acids in length, and may cover all or part of the amino acid sequence of the relevant antigen.

After identifying peptides of antigens presented by antigen-presenting cells (APCs) in the context of appropriate co-stimulation messages, cells within the immune cell population containing receptors specific for the peptide can be activated (and stimulate hyperplasia). It should be understood that "immune cells specific for oncolytic viruses" include multiple cells, such a group of cells. This population can be generated/expanded in vitro and/or ex vivo.

In some embodiments, immune cells specific for oncolytic viruses are specific for oncolytic adenoviruses (OncAd), such as OncAd as described herein. In some embodiments, immune cells specific for oncolytic viruses are specific for OncAd antigens. In some embodiments, the antigen is, or is derived from, an OncAd protein, such as a protein encoded by an early gene (eg, E1 (eg, E1A, E1B), E2 (eg, E2A, E2B), E3, or E4), encoded by a late gene Protein (eg, L1, L2, L3, L4, or L5), protein encoded by IX, or protein encoded by IVa2. In some embodiments, the antigen is, or is derived from OncAd hexon and/or penton.

In some embodiments, according to various aspects of the present disclosure, a method specific to the virus-specific immune cells may be produced/expanded (or already produced/expanded) by: Cultured in the presence of (APCs) to stimulate immune cell populations.

In some embodiments, immune cells specific for oncolytic viruses according to the present disclosure are obtained by using PepMix containing a mixture of overlapping peptides corresponding to 3 hexon of human adenovirus and/or containing an overlap corresponding to 5 penton of human adenovirus Prepared by PepMix peptide mixture.

In some embodiments, the oncolytic virus-specific immune cells express/contain CAR, as described herein. By transfecting/transducing the oncolytic virus-specific immune cells with a nucleic acid encoding CAR, the oncolytic virus-specific immune cells can be engineered to express CAR. The combination of this disclosure

Aspects of the present invention include compositions and methods that include/use (i) an oncolytic virus; (ii) a virus that includes nucleic acids encoding immunomodulatory factors; and (iii) at least one that contains specificity for cancer cell antigens Chimeric antigen receptor (CAR) cells.

Also provided is an oncolytic virus for use in a method of treating cancer, the method comprising administering to a body separately, simultaneously or sequentially: (i) an oncolytic virus; (ii) a virus containing nucleic acids encoding immunomodulatory factors; and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is a virus containing a nucleic acid encoding an immunomodulatory factor for a method of treating cancer, the method comprising administering to an individual separately, simultaneously or sequentially: (i) an oncolytic virus; (ii) a virus containing nucleic acids encoding immunomodulatory factors; and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is a method for treating cancer of at least one cell comprising a chimeric antigen receptor (CAR) specific for cancer cell antigens, the method comprising administering to an individual separately, simultaneously or sequentially: (i) an oncolytic virus; (ii) a virus containing nucleic acids encoding immunomodulatory factors; and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is the use of an oncolytic virus in the manufacture of a medicament, which comprises the following methods of administering an individual separately, simultaneously or sequentially: (i) an oncolytic virus; (ii) a virus containing nucleic acids encoding immunomodulatory factors; and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is the use of a virus containing a nucleic acid encoding an immunomodulatory factor in the manufacture of a medicament for the method of administering the following separately, simultaneously, or sequentially to an individual: (i) an oncolytic virus; (ii) a virus containing nucleic acids encoding immunomodulatory factors; and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided is the use of at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens for the manufacture of a medicament for the administration of a separate, simultaneous or sequential administration of the following method: (i) an oncolytic virus; (ii) a virus containing nucleic acids encoding immunomodulatory factors; and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

Also provided are compositions and methods which comprise/express (i) an oncolytic virus; and (ii) at least one cell comprising a chimeric antigen receptor (CAR) specific for cancer cell antigens (ie, no It is also necessary to use a virus containing nucleic acids encoding immunomodulatory factors).

Also provided are compositions and methods comprising/expressing: (i) an oncolytic virus; and (ii) a pair of immune cells specific for the oncolytic virus. In some embodiments, the compositions and methods can include/use a virus that includes nucleic acids encoding immunomodulatory factors, as described herein.

In some embodiments, the compositions and methods provided herein do not contain a virus (or the step of administering the virus to an individual) comprising a nucleic acid encoding an immunomodulatory factor, as described herein. For example, the composition and method include/use (i) an oncolytic virus; and (ii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigen or the oncolytic virus Specific immune cells; does not contain a virus containing nucleic acids encoding immunomodulators (or the step of administering the virus to a body).

In some embodiments according to various aspects described herein, the CAR-containing/expressing cell is specific for the oncolytic virus used (eg, contains an antigen that is specific for the antigen of the oncolytic virus Body (eg, TCR)). That is, in some embodiments, the specificity of the oncolytic virus and the CAR-containing/expressing cell is consistent. For example, in some embodiments, the oncolytic virus is an adenovirus, and the CAR-expressing cells containing/expressing CAR are adenovirus-specific T cells.

Similarly, in the various aspects herein, oncolytic viruses are used in combination with immune cells specific for the oncolytic virus (ie, the same oncolytic virus).

As used herein, "combination" encompasses products and compositions (eg, pharmaceutical compositions) that include the components of the combination. "Combination" also encompasses treatment regimens that use the components of the combination.

In some embodiments, the components of the combination are provided as individual compositions. In some embodiments, more than one component of the combination is provided as a composition. In some embodiments, the components of the combination are provided as one composition.

Similarly, in some embodiments, the components of the combination are administered individually. In some embodiments, a component of the combination is administered together with another component of the combination. In some embodiments, the components of the combination are administered together.

For example, in a combination example comprising an oncolytic virus, a virus comprising a nucleic acid encoding an immunomodulatory factor, and at least one cell comprising a CAR specific for cancer cell antigens, the oncolytic virus and the The virus of the factor nucleic acid can be administered together, and the at least one cell containing a CAR specific for cancer cell antigens is administered individually (eg, later).

When the components of the combination are administered together, the administration can be simultaneous administration, as described below. When the components in the combination are administered individually, the administration can be simultaneous administration or sequential administration, as described below. In the case where the components in the combination are administered individually, the administration of the individual components may or may not be administered through the same administration route. Features

The agents of the present disclosure can be defined by reference to one or more functional characteristics. The function of this agent can be evaluated by, for example, the analytical methods described in the experimental examples. Similarly, the combinations and methods of the present disclosure can be defined by reference to one or more functional characteristics and/or effects, and this characteristic/effect can be evaluated by the analysis method as described in the experimental example.

In some embodiments, oncolytic viruses according to the present disclosure may have one or more of the following functional characteristics: • The ability to replicate and/or cause cells to kill cancer cells in cancer cells; • Compared with the ability to replicate and/or cause cells to kill cancer cells in cancer cells, it has the ability to replicate and/or cause cells to kill non-cancer cells in non-cancer cells; •Compared with the ability of one or more oncolytic viruses known in the industry, it has comparable or improved ability to cause cells to kill cancer cells; • Ability to assist in the replication of dependent adenovirus (HDAd); •Compared with the ability of one or more oncolytic viruses known in the industry, it has comparable or improved ability to replicate in cancer cells.

In some embodiments, cells containing a chimeric antigen receptor (CAR) specific for cancer cell antigens according to the present disclosure may have one or more of the following functional characteristics: • The ability to combine HER2; • Ability to bind HER2 expressing cells; • The ability to cause cells to kill HER2 expressing cells; • Compared with the ability to cause cells to kill HER2 expressing cells, it has the ability to cause cells to kill cells that do not express HER2.

In some embodiments, a combination of an oncolytic virus, a virus containing nucleic acids encoding immunomodulators, and at least one cell containing a CAR specific for cancer cell antigens may have one or more of the following functional characteristics: • Compared with the ability of using any one component alone or combining any two components to cause cells to kill cancer cells, it has the ability to improve the cells to kill cancer cells. •Compared with the ability of using components alone to cause cells to kill cancer cells, it has a synergistic (ie, superadditive) ability to cause cells to kill cancer cells.

In some embodiments, a combination of an oncolytic virus and at least one cell comprising a CAR specific for cancer cell antigens may have one or more of the following functional characteristics: • Compared with the ability of using each component alone to cause cells to kill cancer cells, it has an improved ability to cause cells to kill cancer cells. •Compared with the ability of using components alone to cause cells to kill cancer cells, it has a synergistic (ie, superadditive) ability to cause cells to kill cancer cells.

In some embodiments, a combination of an oncolytic virus and a pair of specific immune cells of the oncolytic virus may have one or more of the following functional characteristics: • Compared with the ability of using each component alone to cause cells to kill cancer cells, it has an improved ability to cause cells to kill cancer cells. •Compared with the ability of using components alone to cause cells to kill cancer cells, it has a synergistic (ie, superadditive) ability to cause cells to kill cancer cells.

The analysis of the ability of cells to kill cancer cells can be evaluated by analyzing the number/survival rate of cancer cells in vitro. Analysis of the ability of the cell to kill cancer cells can also be analyzed in vivo in an appropriate model, such as by analyzing the number of cancer cells, tumor size/volume, and/or some other correlations of cancer cell numbers (eg, disease progression , The severity of cancer symptoms, etc.). Therapeutic application

The aspect of the present disclosure is particularly relevant to the use of an oncolytic virus, a virus containing a nucleic acid encoding an immunomodulatory factor, and at least one chimeric antigen receptor (CAR) specific for cancer cell antigens in the treatment of cancer in one body ) Of T cells.

Accordingly, the present disclosure provides a method of treating cancer, which comprises administering to an individual: an oncolytic virus; a virus containing a nucleic acid encoding an immunomodulatory factor; and at least one containing a chimera specific for cancer cell antigens T cell of antigen receptor (CAR).

The present disclosure also provides an oncolytic virus as a method for treating cancer; a virus containing a nucleic acid encoding an immunomodulatory factor; and at least one T containing a chimeric antigen receptor (CAR) specific for cancer cell antigens cell. Also provided is a use for manufacturing a medicament for treating cancer, which uses an oncolytic virus; a virus containing a nucleic acid encoding an immunomodulatory factor; and at least one containing a chimeric antigen receptor specific for cancer cell antigen (CAR ) Of T cells.

The present disclosure also provides a method of treating cancer, which comprises administering to an individual: (i) an oncolytic virus; and (ii) at least one comprising a chimeric antigen receptor (CAR) specific for cancer cell antigens cell. Also provided are (i) an oncolytic virus for use in a method of treating cancer; and (ii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens. Also provided is the use of a medicament for manufacturing a method for treating cancer, which uses (i) an oncolytic virus; and (ii) at least one chimeric antigen receptor (CAR) containing specificity for cancer cell antigens Of cells.

The present disclosure also provides a method of treating cancer, which comprises administering to one body: (i) an oncolytic virus; and (ii) a pair of immune cells specific for the oncolytic virus. Also provided are (i) an oncolytic virus for use in a method of treating cancer; and (ii) a pair of immune cells specific for the oncolytic virus. Also provided is a use for manufacturing a medicament for a method of treating cancer, which uses (i) an oncolytic virus; and (ii) a pair of immune cells specific for the oncolytic virus.

Also provided is a method for treating cancer, which includes administering OncAds, HDAds, CARs, nucleic acid/multiple nucleic acids, cells, and pharmaceutical compositions of the present disclosure to an individual. Also provided are OncAds, HDAds, CARs, nucleic acid/multiple nucleic acids, cells, and pharmaceutical compositions of the present disclosure for use in methods of treating cancer. Also provided is the use of manufacturing an agent for treating cancer, which uses OncAds, HDAds, CARs, nucleic acid/multiple nucleic acids, cells, and pharmaceutical compositions of the present disclosure.

'Treatment' can be, for example, reducing the development or progression of cancer, alleviating the symptoms of cancer, or reducing the pathology of cancer. The treatment or alleviation of cancer can effectively prevent the progression of cancer, such as preventing the deterioration of the condition or slowing the rate of progression to a more serious disease state. In some embodiments, treatment or mitigation may lead to improvement of cancer, such as reducing the symptoms of cancer or reducing some other factors related to the severity/activity of cancer. The prevention of cancer means preventing the deterioration of the condition of cancer or the development of cancer, such as preventing the early stage cancer from developing into the advanced stage.

In some embodiments, the treatment may focus on reducing the number of cancer cells or the amount of tissue containing cancer cells in the individual. In some embodiments, the treatment may focus on reducing the size and/or preventing the growth of at least one tumor in the individual.

In some embodiments, the treatment comprises administering an oncolytic virus according to the present disclosure to the individual. In some embodiments, the treatment may include administering to a body a cell or cell population that contains or encodes an oncolytic virus according to the present disclosure. In some embodiments, the treatment includes administering to the individual an oncolytic virus and a virus encoding an immunomodulatory factor according to the present disclosure. In some embodiments, the treatment may include administering to a body a cell or cell population comprising or encoding an oncolytic virus according to the present disclosure and/or a virus encoding an immunomodulatory factor.

In some embodiments, the treatment may include modifying a cell or cell population to include/express the CAR according to the present disclosure. In some embodiments, the treatment may include administering to a body a cell or cell population modified to contain/express the CAR of the present disclosure. In some embodiments, the treatment focuses on providing the individual with an immune cell or immune cell population specific for a pair of cancer cell antigens, such as by administering CAR-expressing cells according to the present disclosure, or generating CAR according to the present disclosure Expressive cells.

In some embodiments, the treatment may include administering to an individual an immune cell/immune cell population specific for oncolytic viruses according to the present disclosure. In some embodiments, the treatment focuses on providing the individual with an immune cell/immune cell population specific for the oncolytic virus. In some embodiments, the treatment may include generating/expanding a population of immune cells specific for oncolytic viruses according to the present disclosure.

In some embodiments, the treatment may include administering to an individual an immune cell/immune cell population that is specific to oncolytic viruses according to the present disclosure, modified to include/express the CAR according to the present disclosure. In some embodiments, the treatment focuses on providing the individual with an immune cell/immune cell population that is specific for a pair of oncolytic viruses and for cancer cell antigens. In some embodiments, the treatment may include generating/expanding a population of immune cells specific for oncolytic viruses according to the present disclosure, and modifying the cells or cells of the population to include/express the CAR according to the present disclosure.

The individual receiving treatment can be any animal or human. The individual is preferably a mammal, more preferably a human. The individual may be a non-human mammal, but more preferably a human. The individual may be male or female or any sex. The individual may be a patient. The individual may have been diagnosed with cancer in need of treatment, may be suspected of having this cancer, or may be at risk of developing this cancer.

In some embodiments, the cancer to be treated comprises cells expressing cancer cell antigens, such as cancer cell antigens (eg, HER2) as described herein. In some embodiments, the cell expresses cancer cell antigen (eg, HER2) on the cell surface.

In some embodiments, the cancer to be treated comprises cells expressing CAR-specific cancer cell antigens. In some embodiments, the CAR comprises a cancer cell antigen binding domain, and the cancer to be treated comprises cells expressing the cancer cell antigen, such as cells expressing the cancer cell antigen on the cell surface.

In some embodiments, the cancer overexpresses the cancer cell antigen. By detecting that the expression level of the cancer cell antigen is greater than that of a comparable non-cancer/non-tumor tissue, it can be determined that the cancer cell antigen is overexpressed.

In some embodiments, the cancer is a cancer that expresses HER2, such as a cancer that expresses HER2 on the cell surface. In some embodiments, the cancer overexpresses HER2. Overexpression of HER2 can be determined by detecting that the expression level of HER2 is greater than that of comparable non-cancer/non-tumor tissues.

In some embodiments, the individual to be treated according to the present disclosure is selected based on the expression/overexpression of cancer cell antigens detected on cancer cells or tumors obtained from the individual.

The expression of designated cancer cell antigens can be determined by any suitable method. Expression can be gene expression or protein expression. Gene expression can be determined by, for example, detecting mRNA encoding the cancer cell antigen, for example, using quantitative real-time PCR (qRT-PCR). Protein expression can be determined by, for example, detecting the cellular antigen, for example, using antibody-based methods, such as Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

The cancer to be treated/prevented according to the present invention may be any harmful cell proliferation (or any disease itself manifested by harmful cell proliferation), neoplasm or tumor. The cancer may be benign or malignant, and may be primary or secondary (metastatic). The cancer may be resistant (primitive or post-treatment) and/or the cancer may be relapsed. The neoplasm or tumor can be any abnormal cell growth or proliferation, and can be located in any tissue. The cancer may be derived from, for example, the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, Endometrium, epithelial cells (eg, renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, and mediastinum , Mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, Tissues/cells of tongue, tonsils, trachea, uterus, vulva, white blood cells.

The cancer to be treated/prevented can be any type of cancer, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), adrenal cortical cancer, AIDS-related cancer (ie, Kaposi's sarcoma, AIDS-related lymphoma Tumor, primary CNS lymphoma), anal cancer, appendix cancer, astrocytoma, basal cell carcinoma of the skin, cholangiocarcinoma (eg, cholangiocarcinoma), bladder cancer, bone cancer (eg, Ewing’s sarcoma, osteosarcoma, Malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor, Birch's lymphoma, carcinoid tumor, cancer of unknown primary site, heart tumor, central nervous system cancer (eg, atypical teratoma/striated muscle) Cell tumor, embryonic cell tumor, germ cell tumor, primary CNS lymphoma), cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative tumor, colorectal Cancer, craniopharyngioma, cutaneous T-cell lymphoma (eg, mycosis fungoides, Sézary syndrome), carcinoma in situ of the duct (DCIS), endometrial cancer (uterine cancer), ependymoma, esophageal cancer , Olfactory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (eg, intraocular melanoma, retinoblastoma), fallopian tube cancer, bone malignant fibrous histiocytoma, gallbladder cancer, stomach ( Stomach) cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), ovarian germ cell tumor, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumor, liver cell (liver) cancer, tissue cells Hyperplasia, Langerhans' cell, Hodgkin's lymphoma, hypopharyngeal carcinoma, islet cell tumor (pancreatic neuroendocrine tumor), kidney (renal cell) cancer, laryngeal cancer, papillomatosis, leukemia, lip and oral cancer, Lung cancer (non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), lymphoma, male breast cancer, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, occult primary metastatic squamous cell neck cancer, Involved in NUT gene among line cancer, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasma cell tumor, mycosis granuloma, myelodysplastic syndrome, myelodysplastic abnormality/myeloproliferative tumor, myeloid leukemia, chronic Myeloid leukemia, acute myeloid leukemia (AML), nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, lip and oral cancer, oropharyngeal cancer, osteosarcoma, Ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus cancer, nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell tumor/multiple bone marrow Tumor, pleural lung blastoma, pregnancy breast cancer, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, hemangioma, uterine sarcoma, skin cancer, small intestine cancer , Skin squamous cell carcinoma, T-cell lymphoma, laryngeal carcinoma, thymoma, thymic carcinoma, thyroid cancer, renal pelvis and ureteral transitional cell carcinoma, urethral cancer, vaginal cancer, vulvar cancer, or Wilms Any one of the tumors.

In some embodiments, the cancer to be treated is nasopharyngeal cancer (NPC; such as EB virus (EBV) positive NPC), cervical cancer (CC; such as human papilloma virus (HPV) positive CC), oropharyngeal cancer (OPC ; Such as HPV positive OPC), gastric cancer (GC; such as EBV positive GC), hepatocellular carcinoma (HCC; such as hepatitis B virus (HBV) positive HCC), lung cancer (such as non-small cell lung cancer (NSCLC)) and head and neck cancer (Eg, one or more of cancers originating from the tissues of the lips, mouth, nose, sinuses, pharynx, or larynx, such as head and neck squamous cell carcinoma (HNSCC)).

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

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

Medical methods also involve in vivo, ex vivo, and adoptive immunotherapy, including those using autologous and/or heterologous cells or immortal cell lines. Drug administration

It is best to administer the drug in a "therapeutically effective amount", which is sufficient to show the effect on the individual. The actual amount administered and the rate and duration of administration depend on the nature and severity of the disease to be treated. Treatment prescriptions, such as dosage decisions, are within the responsibility of general practitioners and other doctors, and usually take into account the condition to be treated, the condition of the individual patient, the location of delivery, the method of administration, and other factors known to practitioners. Examples of the technologies and plans mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Viruses, CARs, nucleic acids, and cells according to the present disclosure can be formulated into pharmaceutical compositions or agents for clinical use, and can contain a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. The composition can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or oral administration Transdermal route of administration, which may include injection or infusion. Suitable formulations can include viruses, CARs, nucleic acids, or cells formulated in sterile or isotonic media. Medicaments and pharmaceutical compositions can be formulated in fluid form, including gels. The fluid formulation can be formulated for injection or infusion administration (eg, through a catheter) to selected areas of the human or animal body.

The oncolytic viruses and/or viruses containing nucleic acids encoding immunomodulatory factors can be formulated for intratumoral administration. In some embodiments, the method may comprise intratumoral administration of the oncolytic virus and/or virus comprising a nucleic acid encoding an immunomodulatory factor.

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

Components of the combination of the present disclosure (eg, oncolytic viruses according to the present disclosure, viruses containing nucleic acids encoding immunomodulatory factors; at least one T cell of CAR specific for cancer cell antigens; specific for oncolytic viruses Sexual immune cells) can be administered simultaneously or sequentially. The present disclosure also contemplates administering the OncAds, HDAds, CARs, nucleic acid/multiple nucleic acids, cells, and pharmaceutical compositions of the present disclosure simultaneously or sequentially.

Simultaneous administration also means that the agents are administered together, for example, in the form of a pharmaceutical composition containing the agents (ie, a combined preparation) or next to each other and optionally administered through the same route to the same artery or vein Or other blood vessels. In certain embodiments, the oncolytic virus and the virus containing nucleic acids encoding immunomodulatory factors can be administered simultaneously in a combined preparation. In some embodiments, when administered simultaneously, two or more of these agents may be administered through different administration routes. In some embodiments, simultaneous administration means at the same time or at, 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 Inward investment.

Sequential administration means administration of one or more of these agents, followed by administration of the other of the agents individually after a specified time interval. The two agents are not necessarily administered in the same way, although in the case of some embodiments. The time interval can be any time interval, including hours, days, weeks, months, or years. In some embodiments, sequential administration means 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, One of 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months or 6 months is administered individually.

In some embodiments, the treatment may further include other treatments or preventive interventions, such as chemotherapy, immunotherapy, radiation therapy, surgery, vaccines, and/or hormone therapy. This other therapeutic or prophylactic intervention can be performed before, during, and/or after the therapies included in this disclosure, and the delivery of other therapeutic or prophylactic interventions can be through a different route of administration than the therapies of this disclosure. Chemotherapy and radiation therapy mean the treatment of cancer with drugs or ionizing radiation (eg, radiotherapy using X-rays or gamma rays). The drug may be a chemical entity, such as small molecule pharmacy, antibiotics, DNA intercalator, protein inhibitor (eg, kinase inhibitor) or biological agent, such as antibody, antibody fragment, nucleic acid or peptide aptamer, nucleic acid (eg , DNA, RNA), peptides, peptides or proteins. The medicine can be formulated as a pharmaceutical composition or medicament. The formulation may contain one or more drugs (eg, one or more active agents) and one or more pharmaceutically acceptable diluents, excipients, or carriers.

Chemotherapy can be administered via one or more routes of administration, such as parenteral, intravenous, oral, subcutaneous, intradermal, or intratumoral.

Chemotherapy can be administered according to a treatment plan. The treatment plan may be a scheduled schedule, plan, plan or schedule of chemotherapy administration made by a clinician or medical practitioner, and may be customized to suit the patient in need of treatment.

The treatment plan may mean one or more of the following: the type of chemotherapy administered to the patient; the dose of each drug or radiation; the time interval between administrations; if any, the number of any treatment holidays and Nature and so on. In terms of co-therapy, a single treatment plan can be provided, which indicates how to administer each drug.

Chemotherapeutic drugs and biological agents can be selected from: alkalizing agents such as cisplatin, carboplatin, methyldi(chloroethyl)amine, cyclophosphamide, chlorambucil, ifosfamide; purine or Pyrimidine antimetabolites, such as azathioprine or mercaptopurine; alkaloids and terpenes, such as vinca alkaloids (eg, vincristine, vinblastine, vinorelbine, vindesine), Podophyllotoxin, etoposide, teniposide, paclitaxel, such as paclitaxel (TaxolTM), paclitaxel; topoisomerase inhibitors, such as type I topoisomer Enzyme inhibitors camptothecins irinotecan and topotecan, or type II topoisomerase inhibitors amsacrine, etoposide, etoposide phosphate, Teniposide; antitumor antibiotics (eg, anthracycline antibiotics), such as radiocin, doxorubicin (Adriamycin ^TM ), epirubicin (epirubicin), bleomycin, rapamycin (rapamycin); antibody bases, such as 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 antibody, monoclonal antibody or antibody fragment, Examples include: cetuximab (cetuximab), panitumumab (panitumumab), infliximab (infliximab), baliximab (basiliximab), bevacizumab (bevacizumab; Avastin®), A Abciximab, daclizumab, gemtuzumab, alemtuzumab, rituximab (rituximab; Mabthera®), palivizumab ( palivizumab), trastuzumab, etanercept, adalimumab, nimotuzumab; EGFR inhibitors such as erlotinib, Cetuximab and gefitinib (gefitinib); anti-angiogenesis agents, such as betelimab (Avastin®); cancer vaccines, such as Sipuleucel-T (Provenge®).

Additional chemotherapeutic drugs can be selected from: 13-cis-vitamin A acid, 2-chlorodeoxyadenosine, 5-Azacitidine, 5-fluorouracil, 6-mercaptopurine, 6 -Thioguanine, albumin-bound paclitaxel (Abraxane), Accutane®, radiocin-D Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®, Aldesleukin, alemtuzumab, ALIMTA, allevi A acid (Alitretinoin), Alkaban-AQ®, Alkeran®, all-trans retinoid acid, alpha interferon, hexamethylmelamine, methotrexate, amifostine, amine gluten, anagrelide ( Anagrelide), Anandron®, Anastrozole, cytosine arabinoside, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®, arsenic trioxide, asparaginase, ATRA Avastin®, azacitidine , BCG, BCNU, Bendamustine (Bendamustine), Betimab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU, Blenoxane®, bleomycin, bortezomib (Bortezomib), Butacaine Sulfate, Busulfex®, Calcium Tetrahydrofolate, Campath®, Camptosar®, Camptothecin-11, Capecitabine, Carac™, Carboplatin, Carmustine (Carmustine), Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, chlorambucil, cisplatin, aldoxalic acid, cladribine ), cortisone, Cosmegen®, CPT-11, cyclophosphamide, Cytadren®, cytarabine Cytosar-U®, Cytoxan®, Dacogen, radiocin, Darbepoetin Alfa, Dasatinib, Daunomycin (Daunorubicin), Daunomycin hydrochloride, Daunomycin liposomes, DaunoXome®, Decadron, Decitabine , Delta-Cortef®, Deltasone®, Denileukin (Denileukin), Diphtheria Toxin (Diftitox), DepoCyt™, Dexamethasone, Dexamethasone Acetate, Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Paclitaxel, Doxil®, Doxorubicin, Doxorubicin Liposome, Droxia™, DTIC, DTIC-Dome®, Duralone ®, Eligard™, Elllence™, Eloxatin™, Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, Erwinia L-Asparaginase (Erwinia L-asparaginase), Estramustine, Ethyol, Etopophos®, Etoposide, Etoposide, Eulexin®, Everolimus (Everolimus), Evista®, Exemestane, Faslodex®, Femara®, filgrastim (Filgrastim), Fluxuridine, Fludara®, fludarabine, Fluoroplex®, fluorouracil, Fluoxymethylene testosterone, Flutamide, Aldehyde Folic Acid, FUDR®, Fulvestrant, Gefitinib, Gemcitabine, Getuzumab ozogamicin ( Gemtuzumab ozogamicin), Gleevec™, Gliadel® Wafer, Goserelin (Goserelin), granular leukocyte colony stimulating factor, granular leukocyte macrophage colony stimulating factor, Herceptin®, Hexadrol, Hexalen®, hexamethylmelamine, HMM, Hycamtin ®, Hydrea®, Hydrocort Acetate®, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone phosphate, hydroxyurea, timobizumab (Ibritumomab; Ibritumomab Tiuxetan ), Idamycin®, Idarubicin, Ifex®, IFN-α, Ifosfamide, IL-11, IL-2, Imatinib mesylate, imidazole carboxylate Acetamide, interferon alpha, interferon alpha-2b (PEG conjugate), interleukin-2, interleukin-11, Intron A® (interferon alpha-2b), Iressa®, irinotecan, isotretinoin ( Isotretinoin), Ixabepilone, Ixempra ™, Kidrolase, Lanacort®, Lapatinib, L-Asparaginase, LCR, Lenalidomide, Letrozole, Folinic acid ( Leucovorin), Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™, liposomal cytarabine, Pred® solution, Lomustine, L-PAM, L L-Sarcolysin, Lupron®, Lupron Depot®, Matulane®, Maxidex, methyl di(chloroethyl)amine, Mechlorethamine Hydrochloride, Medralone®, Medrol®, Megace®, Megestrol, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate, Methotrexate sodium, Methylprednisolone Methylprednisolone, Meticorten®, mitomycin, mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, nitrogen mustard, Mutamycin®, Myleran® , Mylocel™, Mylotarg®, Navelbine®, Nelarabine, Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®, Nilutamide, Nipent®, Nitrogen (Nitrogen) Mustard), Novaldex®, Novantrone®, Octreotide, Octreotide, Octreotide Acetate, Oncospar®, Oncovin®, Ontak®, Onxal™, Oprevelkin, Orapred®, Orasone®, Oxaliplatin , Paclitaxel, Albumin-bound Paclitaxel, Pamidronate, Panitumumab, Panretin®, Paraplatin®, Pediapred®, PEG interferon, Pegaspargase, Pefegrastim ( Pegfilgrastim), PEG-INTRON™, PEG-L-asparaginase, PEMETREXED, penastatin (Pentosta tin), phenylalanine nitrogen mustard, Platinol®, Platinol-AQ®, Prednisolone, Prednisolone, Prednisone, Prelone®, Procarbazine, PROCRIT®, Proleukin®, with Prolifeprospan 20 of Carmustine implants, Purinethol®, 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, streptozotocin, SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®, Taxotere®, Temodar®, Temozolomide, Temsirolimus, teniposide, TESPA, thalidomide, Thalomid®, TheraCys®, thioguanine, thioguanine Tabloid®, thiophosphoramide, Thioplex®, thiophene Thiotepa (Thiotepa), TICE®, Toposar®, Toprolk, Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin, Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®, VePesid®, Vesanoid®, Viadur™, Vidaza®, vinblastine, vinblastine sulfate, Vincasar Pfs®, vincristine, vinblastine Vinorelbine, vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™, Zinecard®, Zoladex®, zoledron Acid (Zoledronic acid), Zolinza, Zometa®.

In the disclosed embodiment in which a nucleic acid/virus encoding an enzyme capable of catalytically converting a non-toxic factor into a cytotoxic form is used, the method may further include administering a prodrug matrix of the enzyme. The prodrug can be administered simultaneously or sequentially with the nucleic acid/virus encoding the enzyme that can catalyze the conversion of non-toxic factors into cytotoxic form.

In some embodiments, the prodrug is selected from ganciclovir (GCV), acyclovir (ACV), and/or deciclovir, wherein the nucleic acid/virus encodes thymidine kinase. In some embodiments, the prodrug is 5-fluorocytosine (5-FC), such as where the nucleic acid/virus encodes cytosine deaminase. In some embodiments, the prodrug is selected from CB1954, nitro-CBI-DEI, and/or PR-104A, such as where the nucleic acid/virus encodes a nitroreductase. In some embodiments, the prodrug is oxazinophos (eg, cyclophosphamide or ifosfamide), such as where the nucleic acid/virus encodes cytochrome P450. In some embodiments, the prodrug is a nitrogen mustard (eg, CMDA or ZD2767P), such as where the nucleic acid/virus encodes carboxypeptidase G2. In some embodiments, the prodrug is 6-methylpurine 2-deoxyriboside and/or fludarabine (eg, 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo) or arabinofuranosyl-2-F-adenine monophosphate (F-araAMP), as in which the nucleic acid/virus encodes purine nucleoside phosphorylase In some embodiments, the prodrug is indole-3-acetic acid (IAA), such as where the nucleic acid/virus encodes horseradish peroxidase. In some embodiments, the prodrug is irinotecan, such as The nucleic acid/virus encodes a carboxylesterase.

Multiple doses of the disclosed agents (eg, viruses (OncAds, HdAds), CARs, nucleic acid/multiple nucleic acids, vectors, cells, compositions, compositions, prodrugs) can be provided. One or more or each of these doses may be administered simultaneously or sequentially with another therapeutic agent.

Multiple doses can be separated at a predetermined time interval, which can be selected as 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 one of 1, 2, 3, 4, 5 or 6 months. For example, a dose can be administered every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day). Adoptive transfer

In an embodiment of the present disclosure, the method of treatment includes adoptive immune cell transfer. Adoptive cell transfer (ACT) generally refers to the process of obtaining cells (eg, immune cells) from an individual, usually by drawing a blood sample from which cells are isolated. After that, the cells are usually treated or modified by some methods, and then administered to the same individual (adoptive transfer is autologous cells) or different individuals (adoptive transfer is heterologous cells). The purpose of this treatment is usually to provide a cell with certain desired characteristics, or to increase the frequency of cells with this characteristic in the individual. In the present disclosure, the purpose of adoptive transfer is to introduce cells or cell populations into a body, and/or to increase the frequency of cells or cell populations in a body.

In some embodiments, the individual isolated from the cell is the individual administered the modified cell (ie, adoptive transfer is an autologous cell). In some embodiments, the individual isolated from the cell and the individual administered the modified cell are different individuals (ie, adoptive transfer is a heterologous cell).

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

The cells may be, for example, neutrophils, eosinophilic spheres, basophilic spheres, dendritic cells, lymphocytes or mononuclear spheres. The lymphocytes can be, for example, T cells, B cells, NK cells, NKT cells, or congenital lymphocytes (ILC) or their precursors. 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 cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cell is a T helper cells (T _H cells). In some embodiments, the T cell is a cytotoxic T cell (eg, 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.

In some embodiments, the cell is an immune cell specific for oncolytic viruses, as described herein. Accordingly, in some embodiments, the method includes administering to a body at least one immune cell specific for oncolytic viruses. In some embodiments, the methods of the present disclosure include generating/expanding a population of immune cells specific for oncolytic viruses, and administering at least one immune cell specific for oncolytic viruses to a body.

In some embodiments, the method includes: (a) Isolate immune cells from a body; (b) generating or expanding an immune cell population specific for oncolytic viruses using a method comprising: stimulating the immune cells by culturing in the presence of antigen-presenting cells (APCs) presenting the oncolytic virus peptides, and ; (c) administering at least one immune cell specific to the oncolytic virus to a body.

In some embodiments, the method steps for generating immune cells specific for the oncolytic virus may include one or more of the following: taking a blood sample from a body; and isolating PBMCs from the blood sample; Generate/expand immune cell populations specific for oncolytic viruses (eg, by culturing PBMCs in the presence of cells containing/expressing oncolytic virus antigens/peptides (eg, APCs)); in vitro or in vitro cell culture Culture immune cells specific for oncolytic viruses; collect immune cells specific for oncolytic viruses; mix immune cells specific for oncolytic viruses with adjuvants, diluents, or carriers; The modified cells are administered.

The present disclosure also provides a method of treating cancer in a body, which comprises administering at least one cell comprising a chimeric antigen receptor (CAR) specific for a cancer cell antigen. In conjunction with this feature of the present disclosure, in some embodiments, the method additionally includes the step of generating the at least one cell comprising a chimeric antigen receptor (CAR) specific for cancer cell antigens. The CAR may be the CAR of the first generation, the second generation, or the third generation or the subsequent generation. The CAR may contain, for example, one, two, three or more costimulatory domains.

In some embodiments, the method includes modifying at least one cell from a body to express or include a CAR according to the present disclosure, optionally expanding the modified at least one cell, and administering the modified to a body At least one cell.

In some embodiments, the method includes: (a) Separate at least one cell from a body; (b) modifying the at least one cell to express or contain the CAR according to the present disclosure, or a nucleic acid encoding the CAR according to the present disclosure, (c) optionally expand the modified at least one cell, and; (d) administering the modified at least one cell to a body.

In some embodiments, the cell comprising/expressing a CAR specific for cancer cell antigens is an immune cell specific for oncolytic viruses, as described herein. In some embodiments, the method includes modifying immune cells specific for oncolytic viruses to express or include CARs according to the present disclosure, optionally expanding the modified immune cells specific for oncolytic viruses, and for A body administered the modified immune cell specific for the oncolytic virus.

In some embodiments, the method includes: (a) Isolate immune cells from a body; (b) Use methods that include the following methods to generate or expand a population of immune cells specific for oncolytic viruses: stimulate immune cells by the presence of antigen-presenting cells (APCs) presenting peptides of the oncolytic virus to cultivate; (c) modifying at least one immune cell specific for oncolytic viruses to express or contain the CAR according to the present disclosure, or a nucleic acid encoding the CAR according to the present disclosure, (d) optionally expand the modified at least one immune cell specific for oncolytic viruses, and; (e) administering the modified at least one immune cell specific for oncolytic viruses to one body.

The at least one cell modified according to the present disclosure can be modified to include/express CAR according to methods well known to those skilled in the art. The modification may include nucleic acid transfer for permanent or temporary expression of the transferred nucleic acid. Any suitable genetic engineering platform can be used to modify the cells according to the present disclosure. Suitable methods for modifying cells include the use of genetic engineering platforms such as gamma retroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, such as Maus et al ., As described in Annu Rev Immunol (2014) 32:189-225, this case is hereby incorporated by reference.

In some embodiments, the method steps for generating the at least one cell comprising a chimeric antigen receptor (CAR) specific for cancer cell antigens may include one or more of the following: taking blood from a body A sample; at least one cell is isolated and/or expanded from the blood sample; the at least one cell is cultured in vitro or in vitro cell culture; the CAR described herein is introduced into the at least one cell, or the code described herein is encoded The nucleic acid of CAR, thereby modifying the at least one cell; expanding the at least one modified cell; collecting the at least one modified cell; mixing the modified cell with an adjuvant, diluent, or carrier; administering the agent to a body Modified cells.

In some embodiments, the method may additionally include treating the cell to cause/increase expression of the CAR or CAR-encoding nucleic acid. For example, the nucleic acid may include a control element for inducing up-regulation of the CAR expression from the nucleic acid in response to treatment with a specific agent. In some embodiments, the treatment can be performed in vivo by administering the agent to an individual who has administered the modified cell according to the present disclosure. In some embodiments, the treatment can be performed ex vivo or in vitro by administering the agent to cells cultured ex vivo or in vitro.

Those familiar with this technique can determine the appropriate reagents and procedures for adoptive cell transfer according to this disclosure, for example, refer to Dai et al. , 2016 J Nat Cancer Inst 108(7): djv439, which is fully incorporated herein This case is for reference.

In a related aspect, the present disclosure provides a method of preparing a modified cell, the method comprising introducing a CAR according to the present disclosure or a nucleic acid encoding the CAR according to the present disclosure into a cell, thereby modifying the at least one cell. The method is preferably performed in vitro or ex vivo. Composition/ Product/ Set

The present disclosure also provides an oncolytic virus (optionally isolated) as described herein. Also provided is a nucleic acid encoding the oncolytic virus (optionally isolated). Also provided are cells containing the oncolytic virus, or nucleic acids encoding the oncolytic virus (optionally isolated).

The present disclosure also provides a virus (optionally isolated) comprising a nucleic acid encoding an immunomodulatory factor as described herein. Also provided is a nucleic acid encoding the virus (optionally isolated). Also provided is a cell (optionally isolated) containing the virus, or containing a nucleic acid encoding the virus.

The present disclosure also provides a chimeric antigen receptor (CAR) as described herein (optionally isolated). Also provided is a nucleic acid encoding the CAR　 (optionally isolated). Also provided is a cell (optionally isolated) containing the CAR, or a nucleic acid encoding the CAR.

The present disclosure also provides a composition comprising an oncolytic virus according to the present disclosure, a virus containing a nucleic acid encoding an immunomodulatory factor, a chimeric antigen receptor, a nucleic acid/multiple nucleic acids, or cells.

Oncolytic viruses, viruses containing nucleic acids encoding immunomodulatory factors, chimeric antigen receptors, nucleic acids/multiple nucleic acids or cells according to the present disclosure can be formulated into pharmaceutical compositions for clinical use and can contain a pharmaceutical Acceptable carriers, diluents, excipients or adjuvants. The combination of the present disclosure may be provided in the form of a single component, or in the form of multiple components including the components of the combination.

According to the present invention, there is also provided a method for producing a pharmaceutically useful composition, which production method may comprise one or more steps selected from the following: isolated from oncolytic viruses described herein, comprising nucleic acids encoding immunomodulatory factors Viruses, chimeric antigen receptors, nucleic acids/multiple nucleic acids or cells; and/or the oncolytic viruses described herein, viruses containing nucleic acids encoding immunomodulatory factors, chimeric antigen receptors, nucleic acids/multiple nucleic acids Or the cells are mixed with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

For example, a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for treating cancer, the method comprising by mixing an oncolytic virus described herein, a virus encoding a nucleic acid encoding an immunomodulatory factor, and embedding Combine the antigen receptor, nucleic acid/multiple nucleic acids or cells and a pharmaceutically acceptable carrier, adjuvant, excipient or diluent to prepare a pharmaceutical composition or medicament.

The present disclosure also provides a modular kit comprising one or more of oncolytic viruses according to the present disclosure, viruses encoding nucleic acids encoding immunomodulatory factors, chimeric antigen receptors, nucleic acids, cells or components.

In some embodiments, the kit may have at least one container having a predetermined amount of oncolytic virus according to the present disclosure, a virus encoding a nucleic acid encoding an immunomodulatory factor, a chimeric antigen receptor, a nucleic acid/nucleic acids, Or a cell, or a composition according to this disclosure. The kit may have a container containing the individual components of the disclosed combination, or may have a container containing the combination of the components of the disclosed combination.

The kit can provide the oncolytic virus, virus containing nucleic acids encoding immunomodulatory factors, CAR, nucleic acids, cells or compositions, and instructions for administration to patients for the treatment of specific cancers. The oncolytic virus, virus containing nucleic acids encoding immunomodulatory factors, CARs, nucleic acids/nucleic acids, cells, or compositions can be formulated to be suitable for injection or infusion into tumors or blood.

In some embodiments, the kit may include materials for producing cells according to the present disclosure. For example, the kit may contain materials for modifying cells to express or contain the virus or its antigen/peptide, CAR or nucleic acid/nucleic acid according to the present disclosure, or for the use of the virus or its antigen according to the present disclosure/ Peptide or nucleic acid/materials into which cells are introduced. The kit may contain materials for the production of immune cells specific for oncolytic viruses; for example, the kit may contain a peptide mixture of one or more oncolytic virus antigens.

In some embodiments, the kit may further include at least one container having a predetermined amount of another therapeutic agent (eg, anti-infective agent or chemotherapeutic agent). In this embodiment, the kit may also include a second agent or pharmaceutical composition, so that the two agents or pharmaceutical compositions can be administered simultaneously or individually, so that they provide combined treatment of the cancer. The therapeutic agent can also be formulated to be suitable for injection or infusion into tumor or blood. Sequence identity

In order to determine the percent identity between two or more amino acid or nucleic acid sequences, paired and multiple sequence alignments can be achieved in various ways known to those skilled in the art, for example, using public computer software such as Clustal Omega (Söding, 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. When using this software, it is better to use the default parameters, such as space penalty and extension penalty. Sequence ***

This disclosure includes combinations of the described aspects and preferred features unless such combinations are clearly not allowed or explicitly avoided.

The chapter titles used in this article are for organizational purposes only and should not be interpreted as limitations of the subject matter.

The aspects and embodiments of the present disclosure will now be exemplified with reference to the drawings. Further aspects and embodiments will be apparent to those familiar with this skill. All documents mentioned in this article are hereby incorporated into this case for reference.

Throughout the specification, including the scope of subsequent patent applications, unless the context requires otherwise, the word "comprising" and its variants will be understood to include the integers or steps, or groups of integers or steps, but does not exclude any other Integer or step, or integer or step group.

It should be noted that the singular forms "a", "an", "an" and "the" used in this specification and the appended patent applications include plural referents unless the context clearly dictates otherwise. The range herein may be expressed as from "about" one specific value and/or to "about" another specific value. When expressed in this range, another embodiment includes from the one specific value and/or to the other specific value. Similarly, when the value is expressed as an approximate value, by using the leading "about", it can be understood that the specific value forms another embodiment.

The reverse complementary sequence of the disclosed nucleic acid sequence is also explicitly considered. Description of numbers in this disclosure

The following numbered paras illustrate specific aspects and embodiments of the present disclosure:

1. A method of treating cancer comprising administering to a body: (i) an oncolytic virus; (ii) a virus containing nucleic acids encoding immunomodulatory factors; and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

2. The method according to item 1, wherein the oncolytic virus is an oncolytic adenovirus (OncAd).

3. The method of item 1 or item 2, wherein the oncolytic virus is derived from adenovirus 5 (Ad5).

4. The method of any one of items 1 to 3, wherein the oncolytic virus encodes an E1A protein, which shows less binding to the Rb protein than the E1A protein encoded by Ad5.

5. The method according to any one of items 1 to 4, wherein the oncolytic virus encodes an E1A protein lacking the amino acid sequence LTCHEACF (sequence identification number 52).

6. The method according to any one of items 1 to 5, wherein the oncolytic virus encodes an E1A protein comprising or consisting of: the amino acid sequence of SEQ ID NO: 34.

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

8. The method of any one of items 1 to 7, wherein the oncolytic virus comprises a nucleic acid having one or more binding sites for STAT1.

9. The method according to any one of items 1 to 8, wherein the virus comprising a nucleic acid encoding an immunomodulatory factor is a helper-dependent adenovirus (HDAd).

10. The method according to any one of items 1 to 9, wherein the immunomodulatory factor is selected from: an agonist that effects an immune response or an antagonist that is an immune regulatory response.

11. The method according to any one of items 1 to 10, wherein the virus comprising a nucleic acid encoding an immunomodulatory factor comprises a nucleic acid encoding IL-12 and/or antagonist anti-PD-L1 antibody.

12. The method according to any one of items 1 to 11, wherein the virus comprising a nucleic acid encoding an immunomodulatory factor comprises a nucleic acid encoding a thymidine kinase.

13. The method according to any one of items 1 to 12, wherein the at least one cell comprising a CAR specific for cancer cell antigen is a T cell.

14. The method of any one of items 1 to 13, wherein the CAR comprises an antigen binding domain capable of specifically binding HER2.

15. The method of any one of items 1 to 14, wherein the CAR comprises an antigen binding domain, which contains: A VL domain, which contains: LC-CRD1: sequence identification number 10; LC-CRD2: sequence identification number 11; LC-CRD3: sequence identification number 12; And a VH domain, which includes: HC-CRD1: sequence identification number 13; HC-CRD2: sequence identification number 14; HC-CRD3: sequence identification number 15; or A VL domain, which contains: LC-CRD1: sequence identification number 18; LC-CRD2: sequence identification number 19; LC-CRD3: sequence identification number 20; And a VH domain, which includes: HC-CRD1: sequence identification number 21; HC-CRD2: sequence identification number 22; HC-CRD3: sequence identification number 23; or A VL domain, which contains: LC-CRD1: sequence identification number 26; LC-CRD2: sequence identification number 27; LC-CRD3: Sequence identification number 28; And a VH domain, which includes: HC-CRD1: sequence identification number 29; HC-CRD2: sequence identification number 30; HC-CRD3: Sequence identification number 31.

16. The method according to any one of items 1 to 15, wherein the CAR comprises an antigen-binding domain, which comprises: A VL comprising the following, or consisting of, or consisting essentially of: an amino acid sequence having at least 75% sequence identity with sequence identification number 16; and a VH, comprising the following, or consisting of the following, Or basically consist of the following: an amino acid sequence having at least 75% sequence identity with sequence identification number 17; or A VL comprising the following, or consisting of, or consisting essentially of: an amino acid sequence having at least 75% sequence identity with sequence identification number 24; and a VH, comprising the following, or consisting of, Or basically consist of the following: an amino acid sequence having at least 75% multiple sequence identity with sequence identification number 25; or A VL comprising the following, or consisting of, or consisting essentially of: an amino acid sequence having at least 75% sequence identity with the sequence identification number 32; and a VH, comprising the following or consisting of, Or it basically consists of an amino acid sequence having at least 75% sequence identity with sequence identification number 33.

17. If the method of any one of items 1 to 16, the method additionally includes: (a) Separate at least one cell from a body; (b) modifying the at least one cell to express or contain a CAR specific for cancer cell antigens, or a nucleic acid encoding a CAR specific for cancer cell antigens, (c) optionally expanding the modified at least one cell, and; (d) administering the modified at least one cell to a body.

18. The method according to any one of items 1 to 17, wherein the cancer is selected from head and neck cancer, nasopharyngeal cancer (NPC), cervical cancer (CC), oropharyngeal cancer (OPC), gastric cancer (GC), liver Cellular carcinoma (HCC) and lung cancer.

19. An oncolytic adenovirus (OncAd), which encodes an E1A protein comprising, consisting of, or consisting essentially of: the amino acid sequence of sequence identification number 34.

20. An oncolytic adenovirus (OncAd) comprising a nucleic acid having one or more binding sites for STAT1.

21. The OncAd of item 20, wherein the OncAd contains a nucleic acid sequence having a sequence identity of at least 60% with the sequence identification number 51, or an equivalent sequence caused by codon degeneration.

22. A helper dependent adenovirus (HDAd) comprising a nucleic acid encoding IL-12 and/or antagonist anti-PD-L1 antibody.

23. The HDAd of item 22, wherein the HDAd additionally comprises a nucleic acid encoding thymidine kinase.

24. A chimeric antigen receptor (CAR), comprising an antigen binding domain, comprising: A VL domain, which contains: LC-CRD1: sequence identification number 10; LC-CRD2: sequence identification number 11; LC-CRD3: sequence identification number 12; And a VH domain, which includes: HC-CRD1: sequence identification number 13; HC-CRD2: sequence identification number 14; HC-CRD3: sequence identification number 15; or A VL domain, which contains: LC-CRD1: sequence identification number 18; LC-CRD2: sequence identification number 19; LC-CRD3: sequence identification number 20; And a VH domain, which includes: HC-CRD1: sequence identification number 21; HC-CRD2: sequence identification number 22; HC-CRD3: sequence identification number 23; or A VL domain, which contains: LC-CRD1: sequence identification number 26; LC-CRD2: sequence identification number 27; LC-CRD3: Sequence identification number 28; And a VH domain, which includes: HC-CRD1: sequence identification number 29; HC-CRD2: sequence identification number 30; HC-CRD3: Sequence identification number 31.

25. The CAR of item 24, wherein the CAR comprises an antigen binding domain, which comprises: A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 16 and a VH comprising or consisting of, or consisting essentially of: and Sequence identification number 17 has an amino acid sequence with at least 75% sequence identity; or A VL comprising the following, or consisting of, or consisting essentially of: an amino acid sequence having at least 75% sequence identity with sequence identification number 24, and a VH comprising the following, or consisting of, Or basically consist of the following: an amino acid sequence having at least 75% sequence identity with sequence identification number 25; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 32, and a VH comprising or consisting of, or consisting essentially of: and The sequence identification number 33 has an amino acid sequence of at least 75% identity.

26. An optionally isolated or artificial nucleic acid encoding oncolytic adenovirus (OncAd) according to any one of items 19 to 21, helper-dependent adenovirus (HDAd) such as item 22 or item 23 ), or chimeric antigen receptor (CAR) such as item 24 or item 25.

27. A cell comprising an oncolytic adenovirus (OncAd) according to any one of items 19 to 21, a help-dependent adenovirus (HDAd) such as item 22 or item 23, such as item 24 or item The chimeric antigen receptor (CAR) of sub 25, or the nucleic acid of item 26, optionally wherein the cell is artificial and cannot be found in nature.

28. A pharmaceutical composition comprising an oncolytic adenovirus (OncAd) according to any one of items 19 to 21, a help-dependent adenovirus (HDAd) according to item 22 or item 23, such as item 24 Or the chimeric antigen receptor (CAR) of item 25, the nucleic acid of item 26, or the cell of item 27, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

29. A method of treating cancer, comprising administering an oncolytic adenovirus (OncAd) according to any one of items 19 to 21, a help-dependent adenovirus (HDAd) such as item 22 or item 23 to a body ), the chimeric antigen receptor (CAR) of item 24 or item 25, the nucleic acid of item 26, the cell of item 27, or the pharmaceutical composition of item 28.

30. Oncolytic adenovirus (OncAd) according to any of items 19 to 21, helper-dependent adenovirus (HDAd) such as item 22 or item 23, chimerism such as item 24 or item 25 An antigen receptor (CAR), a nucleic acid such as item 26, a cell such as item 27, or a pharmaceutical composition as described in item 28, are used in a method of treating cancer.

31. An oncolytic adenovirus (OncAd) according to any one of items 19 to 21, a help-dependent adenovirus (HDAd) such as item 22 or item 23, embedding such as item 24 or item 25 The use of an antigen receptor (CAR), a nucleic acid such as item 26, a cell such as item 27, or a pharmaceutical composition as described in item 28 for the preparation of a medicament for the treatment of cancer.

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

33. A modular kit comprising a predetermined amount of oncolytic adenovirus (OncAd) according to any one of items 19 to 21, helper-dependent adenovirus (HDAd) such as item 22 or item 23 , Such as the chimeric antigen receptor (CAR) of item 24 or item 25, the nucleic acid of item 26, the cell of item 27, or the pharmaceutical composition of item 28.

1A. A method of treating cancer comprising administering to a body: (i) an oncolytic virus; (ii) a virus containing nucleic acids encoding immunomodulatory factors; and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

2A. A combination of methods for treating cancer having (i) an oncolytic virus; (ii) a virus containing a nucleic acid encoding an immunomodulatory factor; and (iii) at least one containing a specificity for cancer cell antigens Cells of the chimeric antigen receptor (CAR).

3A. One (i) an oncolytic virus (ii) a virus containing a nucleic acid encoding an immunomodulatory factor and (iii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens is in preparation The use of an agent, which is used in a method of treating cancer.

4A. A method of treating cancer comprising administering to a body: (i) an oncolytic virus; and (ii) At least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens.

5A. A combination of methods for treating cancer having (i) an oncolytic virus; and (ii) at least one cell comprising a chimeric antigen receptor (CAR) specific for cancer cell antigens.

6A. Use of (i) an oncolytic virus and (ii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens for the preparation of a medicament for the treatment of cancer Method.

7A. The method of item 1A or item 4A, the combination of item 2A or item 5A, or the use of item 3A or item 6A, in which the CAR-containing cells are specific to the oncolytic virus .

8A. A method of treating cancer comprising administering to a body: (i) an oncolytic virus; and (ii) at least one immune cell specific for the oncolytic virus.

9A. A combination of methods for treating cancer having (i) an oncolytic virus, and (ii) at least one immune cell specific for the oncolytic virus.

10A. A use of (i) an oncolytic virus and (ii) at least one immune cell specific for the oncolytic virus in the preparation of a medicament for use in a method of treating cancer.

11A. The method, combination or use of any of items 1A to 10A, wherein the oncolytic virus is an oncolytic adenovirus (OncAd).

12A. The method, combination or use of any of items 1A to 11A, wherein the oncolytic virus is derived from adenovirus 5 (Ad5).

13A. The method, combination, or use of any one of items 1A to 12A, wherein the oncolytic virus encodes an E1A protein that exhibits less binding to the Rb protein than the E1A protein encoded by Ad5.

14A. The method, combination or use of any of items 1A to 13A, wherein the oncolytic virus encodes an E1A protein lacking the amino acid sequence LTCHEACF (sequence identification number 52).

15A. The method, combination or use of any of items 1A to 14A, wherein the oncolytic virus encodes an E1A protein comprising or consisting of: the amino acid sequence of sequence identification number 34.

16A. The method, combination or use of any of items 1A to 15A, wherein the oncolytic virus comprises a nucleic acid having one or more binding sites for one or more transcription factors.

17A. The method, combination or use of any of items 1A to 16A, wherein the oncolytic virus comprises a nucleic acid having one or more binding sites for STAT1.

18A. The method, combination or use of any of items 1A to 7A or 11A to 17A, wherein the at least one cell comprising a CAR specific for cancer cell antigens is a T cell.

19A. The method, combination or use of any of items 1A to 7A or 11A to 18A, wherein the CAR comprises an antigen binding domain capable of specifically binding HER2.

20A. The method, combination or use of any of items 1A to 7A or 11A to 19A, wherein the CAR comprises an antigen binding domain comprising: A VL domain, which contains: LC-CRD1: sequence identification number 10; LC-CRD2: sequence identification number 11; LC-CRD3: sequence identification number 12; And a VH domain, which includes: HC-CRD1: sequence identification number 13; HC-CRD2: sequence identification number 14; HC-CRD3: sequence identification number 15; or A VL domain, which contains: LC-CRD1: sequence identification number 18; LC-CRD2: sequence identification number 19; LC-CRD3: sequence identification number 20; And a VH domain, which includes: HC-CRD1: sequence identification number 21; HC-CRD2: sequence identification number 22; HC-CRD3: sequence identification number 23; or A VL domain, which contains: LC-CRD1: sequence identification number 26; LC-CRD2: sequence identification number 27; LC-CRD3: Sequence identification number 28; And a VH domain, which includes: HC-CRD1: sequence identification number 29; HC-CRD2: sequence identification number 30; HC-CRD3: sequence identification number 31; or A VL domain, which contains: LC-CRD1: Sequence identification number 57; LC-CRD2: Sequence identification number 58; LC-CRD3: Sequence identification number 59; And a VH domain, which includes: HC-CRD1: sequence identification number 60; HC-CRD2: sequence identification number 61; HC-CRD3: Sequence identification number 62.

21A. The method, combination or use of any of items 1A to 7A or 11A to 20A, wherein the CAR comprises an antigen binding domain comprising: A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 16; and a VH comprising or consisting of: having at least 75% sequence identification number 17 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 24; and a VH comprising or consisting of: having at least 75% sequence identification number 25 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 32; and a VH comprising or consisting of: having at least 75% sequence identification number 33 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 63; and a VH comprising or consisting of: having at least 75% sequence identification number 64 Amino acid sequence of sequence identity.

22A. The method, combination or use of any of items 1A to 3A or 11A to 21A, wherein the virus comprising a nucleic acid encoding an immunomodulatory factor is a helper dependent adenovirus (HDAd).

23A. The method, combination or use of any one of items 1A to 3A or 11A to 22A, wherein the immunomodulatory factor is selected from: an agonist of an effect immune response or an antagonist of an immunomodulatory response.

24A. The method, combination or use of any of items 1A to 3A or 11A to 23A, wherein the virus comprising a nucleic acid encoding an immunomodulatory factor comprises an IL-12 and/or antagonist anti-PD-L1 antibody Nucleic acid.

25A. The method, combination or use of any of items 1A to 3A or 11A to 24A, wherein the virus comprising a nucleic acid encoding an immunomodulatory factor comprises an enzyme encoding an enzyme capable of catalytically converting a non-toxic factor into a cytotoxic form Nucleic acid.

26A. The method, combination or use of item 25A, wherein the enzyme is selected from the group consisting of: thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase , Horseradish peroxidase and carboxylesterase.

27A. The method, combination or use of any of items 1A to 26A, wherein the method of treating cancer comprises: (a) Separate at least one cell from a body; (b) modifying the at least one cell to express or contain a CAR specific for cancer cell antigens, or a nucleic acid encoding a CAR specific for cancer cell antigens, (c) optionally expanding the modified at least one cell, and; (d) administering the modified at least one cell to a body.

28A. The method, combination or use of any of items 1A to 27A, wherein the method of treating cancer comprises: (a) Isolate immune cells from a body; (b) Use a method including the following to generate or expand an immune cell population specific for oncolytic viruses: stimulate the immune cells by the presence of antigen-presenting cells (APCs) presenting peptides of the oncolytic virus Cultivate, and; (c) administering at least one immune cell specific to the oncolytic virus to a body.

29A. The method, combination or use of any one of items 1A to 28A, wherein the cancer is selected from head and neck cancer, nasopharyngeal cancer (NPC), cervical cancer (CC), oropharyngeal cancer (OPC), gastric cancer (GC), hepatocellular carcinoma (HCC) and lung cancer.

30A. An oncolytic adenovirus (OncAd), which encodes an E1A protein comprising or consisting of: the amino acid sequence of SEQ ID NO:34.

31A. An oncolytic adenovirus (OncAd) comprising a nucleic acid having one or more binding sites for STAT1.

32A. An oncolytic adenovirus (OncAd) comprising a nucleic acid sequence having at least 60% sequence identity with sequence identification number 51, or an equivalent sequence resulting from codon degeneration.

33A. An oncolytic adenovirus (OncAd) comprising a nucleic acid sequence having at least 60% sequence identity with sequence identification number 55, or an equivalent sequence resulting from codon degeneration.

34A. A helper dependent adenovirus (HDAd) comprising a nucleic acid encoding IL-12 and/or antagonist anti-PD-L1 antibody.

35A. The HDAd of item 34A, wherein the HDAd additionally contains a nucleic acid encoding an enzyme capable of catalytically converting a non-toxic factor into a cytotoxic form.

36A. The HDAd of item 34A or item 35A, wherein the enzyme is selected from: thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase , Horseradish peroxidase and carboxylesterase.

37A. A chimeric antigen receptor (CAR) comprising an antigen binding domain, comprising: A VL domain, which contains: LC-CRD1: sequence identification number 10; LC-CRD2: sequence identification number 11; LC-CRD3: sequence identification number 12; And a VH domain, which includes: HC-CRD1: sequence identification number 13; HC-CRD2: sequence identification number 14; HC-CRD3: sequence identification number 15; or A VL domain, which contains: LC-CRD1: sequence identification number 18; LC-CRD2: sequence identification number 19; LC-CRD3: sequence identification number 20; And a VH domain, which includes: HC-CRD1: sequence identification number 21; HC-CRD2: sequence identification number 22; HC-CRD3: sequence identification number 23; or A VL domain, which contains: LC-CRD1: sequence identification number 26; LC-CRD2: sequence identification number 27; LC-CRD3: Sequence identification number 28; And a VH domain, which includes: HC-CRD1: sequence identification number 29; HC-CRD2: sequence identification number 30; HC-CRD3: sequence identification number 31; or A VL domain, which contains: LC-CRD1: Sequence identification number 57; LC-CRD2: Sequence identification number 58; LC-CRD3: Sequence identification number 59; And a VH domain, which includes: HC-CRD1: sequence identification number 60; HC-CRD2: sequence identification number 61; HC-CRD3: Sequence identification number 62.

38A. The CAR of item 37A, wherein the CAR comprises an antigen binding domain, which comprises: A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 16; and a VH comprising or consisting of: having at least 75% sequence identification number 17 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 24; and a VH comprising or consisting of: having at least 75% sequence identification number 25 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 32; and a VH comprising or consisting of: having at least 75% sequence identification number 33 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 63; and a VH comprising or consisting of: having at least 75% sequence identification number 64 Amino acid sequence of sequence identity.

39A. An optionally isolated or artificial nucleic acid or multiple nucleic acids encoding oncolytic adenovirus (OncAd) as in any of items 30A to 33A, as aided in as in any of items 34A to 36A Dependent adenovirus (HDAd), or chimeric antigen receptor (CAR) such as item 37A or item 38A.

40A. A cell comprising the oncolytic adenovirus (OncAd) according to any one of items 30A to 33A, the help-dependent adenovirus (HDAd) according to any one of items 34A to 36A, such as item The chimeric antigen receptor (CAR) of item 37A or item 38A, or the nucleic acid or nucleic acids such as item 39A.

41A. A pharmaceutical composition comprising the oncolytic adenovirus (OncAd) according to any one of items 30A to 33A, the help-dependent adenovirus (HDAd) according to any one of items 34A to 36A, Such as item 37A or item 38A chimeric antigen receptor (CAR), item 39A nucleic acid or nucleic acids, or item 40A cells, and a pharmaceutically acceptable carrier, diluent, Excipient or adjuvant.

42A. A method of treating cancer comprising administering an oncolytic adenovirus (OncAd) such as any one of items 30A to 33A, an auxiliary dependent type such as any one of items 34A to 36A to a body Adenovirus (HDAd), chimeric antigen receptor (CAR) such as item 37A or item 38A, nucleic acid or multiple nucleic acids such as item 39A, cells such as item 40A, or pharmaceutical composition such as item 41A Thing.

43A. Oncolytic adenovirus (OncAd) according to any one of items 30A to 33A, Help-dependent adenovirus (HDAd) according to any one of items 34A to 36A, such as item 37A or items A chimeric antigen receptor (CAR) of 38A, a nucleic acid or a plurality of nucleic acids such as item 39A, a cell such as item 40A, or a pharmaceutical composition such as item 41A are used in a method of treating cancer.

44A. An oncolytic adenovirus (OncAd) according to any of items 30A to 33A, a help-dependent adenovirus (HDAd) according to any of items 34A to 36A, such as item 37A or item The use of a chimeric antigen receptor (CAR) of sub-38A, nucleic acid or multiple nucleic acids such as item 39A, cells such as item 40A or pharmaceutical compositions such as item 41A for the preparation of a medicament, which is for use Used to treat cancer.

45A. The method according to item 42A, the OncAd, HDAd, CAR, nucleic acid or nucleic acids, cells or pharmaceutical compositions used in item 43, or the use according to item 44A, wherein the cancer is selected from head and neck cancer , Nasopharyngeal cancer (NPC), cervical cancer (CC), oropharyngeal cancer (OPC), gastric cancer (GC), hepatocellular carcinoma (HCC) and lung cancer.

46A. A modular kit having a predetermined amount of oncolytic adenovirus (OncAd) as in any of items 30A to 33A, and an auxiliary dependent type as in any of items 34A to 36A Adenovirus (HDAd), chimeric antigen receptor (CAR) such as item 37A or item 38A, nucleic acid or multiple nucleic acids such as item 39A, cells such as item 40A or pharmaceutical composition such as item 41A Thing. example

In the following examples, the present inventors illustrate the functional characteristics of the novel HER-2 specific CARs and CAR-T cells, oncolytic adenovirus, and helper-dependent adenovirus. Example 1 : HER2 specific CAR-T cells 1.1 Generation of HER2 specific CAR constructs and CAR-T cells

Preparation of HER2 binding CAR construct. Briefly, DNA encoding scFv (ie, the VL and VH domains joined by a linker sequence) of the anti-HER2 antibody strains C5, E4, F1, or A3 is cloned into the 5'message peptide (SP) And CD28 transmembrane (TM) and intracellular domain sequences, and the 3'CD3ζ intracellular domain sequence in the CAR construct backbone. Three HER2 binding CAR constructs are shown schematically in Figure 1A.

The HER2 specific CAR-T cells are generated as shown schematically in Figure 1B. Briefly, Ficoll density gradient centrifugation was used to separate PBMC from blood samples. In the presence of IL-2, cells were treated with anti-CD3 (OKT3)/anti-CD28 stimulation to promote T cell activation and proliferation, and then transduced with retrovirus encoding the HER2 CAR construct. T cells were expanded by culturing in the presence of 100 IU/mL recombinant human IL-2, and then frozen 6 days after transduction. T cells transduced with this HER2 specific CAR construct are easily expanded by in vitro culture (see Figure 14).

The T cells were thawed and expanded for 5 days in the presence of 100 IU/mL of recombinant human IL-2, and then used for in vitro/in vivo experiments and phenotype analysis. 1.2 Characterization of HER2 specific CAR-T cells 1.2.1 Surface markers and HER2 CAR expression

Flow cytometry was used to characterize the expression of different cell surface molecules of T cells transduced with the HER2 CAR construct encoding the scFv of the anti-HER2 antibody strain E4. Fluorescently labeled monoclonal antibodies were used to stain HER2 specific CAR T cells for 30 minutes at 4ºC. By including 7AAD (BD Pharmingen) at the time of staining, the distinction between living and dead cells is achieved. The stained cells were analyzed using Gallios flow cytometer and Kaluza software (BD Bioscience) according to the manufacturer's instructions.

The results are shown in 2 and 3. A strong surface expression of HER2-CARs was detected on the transduced cells (Figure 2).

Figure 3 shows the characterization results of T cells transduced with HER2(E4)-CAR. Compared to non-transduced cells, CD3+ cells, CD4+ cells and CD8+ cells expressing HER2(E4)-CAR showed increased expression of PD-1, LAG-3 and TIM-3, while the expression level of CCR7 decreased (image 3). 1.2.2 Cell killing activity

In the cell killing analysis, the ability of HER2-CAR-T cells to kill HER2-expressing cancer cells in vitro was analyzed.

In the first experiment, chromium-51 ( ⁵¹ Cr) was used to identify HER2-negative MDA cell lines (negative control group), MDA cells stably expressing HER2 (MDA-HER2; positive control group), pharyngeal squamous cell carcinoma cell lines FaDu or head and neck squamous cell carcinoma cell line SCC47 cells, and the ratio of non-transduced T cells (NT) or HER2-CAR-T cells expressing designated CARs in effector cells: target cells is 20:1 A total of 4 hours of cultivation under the conditions. After centrifugation, use a liquid scintillation counter to calculate the level of ⁵¹ Cr in the cell culture medium. The results are shown in Figure 4A; it is shown that HER2-CAR-T cells kill HER2-expressing cancer cells. Similar results were obtained when the experiment was conducted using the effect cell:target cell ratio of 10:1.

Flow cytometry was used to confirm the expression of HER2 on MDA-HER2, FaDu and SCC47. Briefly, fluorescently labeled single anti-HER2 antibody or isotype control antibody was used to stain cells at 4ºC for 30 minutes. By including 7AAD (BD Pharmingen) at the time of staining, the distinction between living and dead cells is achieved. The stained cells were analyzed using Gallios flow cytometer and Kaluza software (BD Bioscience) according to the manufacturer's instructions. The results are shown in Figure 4B; it was confirmed that MDA cells did not express HER2, while MDA-HER2, FaDu, and SCC47 did express HER2.

In another experiment, FaDu and SCC47 cells that were genetically modified to express firefly luciferase (ffLuc) were inoculated into the wells of a 24-well disk and were combined with HER2(C5)-CAR-T cells and HER2(E4) -CAR-T cells or HER2(F1)-CAR-T cells were co-cultured for 3 days under the condition of effector: target cell ratio of 1:5, and then ffLuc activity was measured using a disk reader (Life Technologies). The results are shown in Figure 5; as evidenced by the decrease in ffLuc activity (relative light units, RLU), it is shown that HER2-CAR-T cells kill HER2-expressing cancer cells. Similar results were obtained when the experiment was conducted using the effect cell:target cell ratio of 1:20. Example 2 : OncAd Construct 2.1 OncAd Construct

Using recombinant DNA technology to prepare novel constructs encoding oncolytic adenoviruses.

In specific embodiments, OncAd is produced by modifying known viruses. For example, a region encoding the E1A protein from adenovirus 5, such as a region lacking the sequence LTCHEACF (sequence identification number 52) involved in the binding of the Rb protein, to encode the E1A protein from adenovirus 2 also lacks the sequence LTCHEACF (sequence identification) No. 52).

The ICOSTAT shown in Fig. 6 was produced by ICOVIR15 as disclosed in Rojas et al. 2010 Mol Ther 18 1960-1971. Briefly, the region encoding 8 copies of the binding site for transcription factor E2F in ICOVIR15 was replaced with the region encoding 8 serial copies of the binding site for transcription factor STAT1. The sequence of ICOSTAT is shown in the serial number 51.

Recombinant DNA technology was also used to prepare Onc5/3Ad2E1Δ24 (also referred to herein as “Onc5/2E1Δ24”) shown in sequence identification number 55 and illustrated in FIG. 12. Onc5/3Ad2E1Δ24 has the same Onc5Δ24 as disclosed in Fueyo et al. 2000 Oncogene 19:2-12 (which is hereby incorporated by reference in its entirety; in Fueyo et al., Onc5Δ24 is also called “Δ24”) Similar structure, but the difference is that Onc5/3Ad2E1Δ24 encodes the E1A protein from the missing sequence LTCHEACF (sequence identification number 52) of adenovirus type 2 (Ad2) instead of the missing sequence from adenovirus type 5 (Ad5) E1A protein of LTCHEACF (Sequence ID No. 52). 2.2 Cell killing activity

For example, MTS can be used to analyze the ability of the preferred oncolytic adenovirus or ICOSTAT produced in Example 2.1 to kill cancer cells. Briefly, 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 in 96-well disks Wells, then use different numbers of helper-dependent, non-replicating adenoviruses (HDAd; negative control group), preferred oncolytic adenoviruses (eg, Onc5/3Ad2E1Δ24 as described in Example 2.1) or as described in Example 2.1 above ICOSTAT infection.

The cells are cultured for 4 days, for example, MTS reagent (Promega) is added to each well, and the cells are incubated at 37°C for 2 hours. Living cells were analyzed by measuring the absorbance at 490nm using a disk reader. The readings of various types of untreated cells (ie, untreated cells = 100% cell survival rate) can be used to normalize the readings, and the wells without cells are regarded as 0%.

In certain embodiments, the preferred oncolytic virus is capable of killing cancer cells in a dose-dependent manner. In a specific embodiment, the preferred oncolytic virus also exhibits a lower level of killing non-cancer cells such as WI-38 and ARPE-19 cells than the virus killing cancer cells.

Figures 7A to 7F show that ICOSTAT can kill cancer cells (ie, A549, FaDu, and SCC47 cells) in a dose-dependent manner (Figures 7A to 7C and 7F), and compared to the level of killing cancer cells, killing non-cancer cells The cells WI-38 and ARPE-19 cells showed lower levels (Figures 7D and 7E).

Figures 13A to 13D show that Onc5/3Ad2E1Δ24 can kill cancer cells (ie, FaDu and SCC47 cells) in a dose-dependent manner (Figures 13A and 13B), and compared to the level of killing cancer cells, killing non-cancer cells WI- 38 and ARPE-19 cells showed lower levels (Figures 13C and 13D). 2.3 The ability to help assist dependent adenovirus (HDAd)

The ability of the preferred oncolytic adenovirus or the ICOSTAT produced in Example 2.1 to assist in the replication of dependent adenovirus (HDAd) can be analyzed by co-infecting cancer cells with OncoAd and HDAd, and then determining the number of viral replicas. Briefly, FaDu or SCC47 cells were inoculated into 24-well plates and infected with HDAd of 10 virions/cell alone, or OncAd+HDAd (OncAd:HDAd ratio 1:10). Collect cells 48 hours after infection, extract DNA and analyze HDAd and Onc.Ad vectors by quantitative real-time PCR using the Bio-Rad iQ5 real-time PCR detection system (Bio-Rad) and Applied Biosystems SYBR green PCR master mix (Life Technologies) Number of copies of both (10 minutes at 95°C, then 45 cycles of 10 seconds at 95°C, 15 seconds at 60°C and 30 seconds at 72°C). Normalize the number of replicas measured against GAPDH.

In certain embodiments, the preferred oncolytic virus can fully replicate itself with HDAd.

Figures 8A and 8B show that ICOSTAT (named "Onc5/3AdicoSTAT" in the figure) is able to copy itself (Figure 8A) and HDAd (Figure 8B). 2.4 The effect of IFNγ on the replication of ICOSTAT in cancer cells

The effect of IFNγ treatment on ICOSTAT OncAd replication was analyzed. In short, inoculate FaDu and SCC47 cells into a 24-well plate and infect the cells with the preferred oncolytic virus or icoSTAT of 10vp/cell, 3 hours after infection, with or without 10ng/mL recombinant IFNγ The medium replaces the cell culture medium 3 hours after infection, and then 24 and 48 hours after infection, the cell culture medium is replaced again with a new medium with/without 10 ng/mL recombinant IFNγ. Cells were collected 3, 24, 48 and 72 hours after infection, DNA was extracted from the cells, the number of virus replicas was analyzed by quantitative real-time PCR and the number of replicas measured with GAPDH was normalized.

Figures 9A and 9B show that ICOSTAT can replicate in FaDu cells and SCC47 cells with or without IFNγ. Example 3 : Auxiliary dependent Ad (HDAd) construct 3.1 HDAd construct and generation

Using recombinant DNA technology to prepare novel constructs encoding helper-dependent adenoviruses. The resulting coding sequence of the construct named HDAd IL-12_TK_PD-L1 is shown in FIG. 10A. HDAdIL-12_TK_PD-L1 contains (i) human IL-12p70 (sequences encoding α and β chains), (ii) HSV-1 thymidine kinase and (iii) anti-PD-L1 microsomes (including as in WO 2016111645 A1 The CDRs of the anti-PD-L1 strain H12_gl) include the sequence of the HA tag expression cassette. Each of the three coding sequences has its own polyadenylation (polyA) message sequence.

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

The HDAd "HDIL12_PDL1" contains a sequence encoding human IL-12p70 protein and an anti-PD-L1 microsome (atezolizumab) derived from YW243.55.S70. The anti-PD-L1 microsomes in this construct include the hinge-fused scFv of YW243.55.S70, the CH2 and CH3 regions of human IgG1, and the C-terminal HA tag (e.g. Tanoue et al. Cancer Res. (2017) 77( 8): described in 2040-2051). 3.2 Expression of encoded protein

Cancer cells were transfected with plastid HDAd vector, and culture medium samples 48 hours after transfection were collected to analyze IL-12p70 and anti-PD-L1 microbody levels in the cell culture medium of the transfected cells.

Using the BD cytokine multiple bead array system (BD Biosciences), according to the manufacturer's instructions, the level of IL-12p70 in the culture medium was measured. The results are shown in Figure 10B. It was found that cells transfected with the HDAd IL-12_TK_PD-L1 construct produced higher levels of IL-12p70 than cells transfected with the HDIL-12_PD-L1 construct.

The Western blot method was used to detect anti-PD-L1 microsomes secreted into the cell culture medium using anti-HA antibodies (microsomes for detecting HA tags). Figure 10C shows that cells transfected with the HDAdI L-12_TK_PD-L1 construct secrete anti-PD-L1 microsomes into the cell culture medium.

In another embodiment, cells are transfected with different constructs, and the cell culture medium is replaced with a medium containing 10 ng/ml ganciclovir (GCV) 8 hours after transfection. After that, the cell culture medium was replaced with a medium containing 10 ng/ml every 24 hours, and after 7 days, the wells were stained with crystal violet solution to show live cells.

The results are shown in FIG. 10D, and it was confirmed that cells transfected with the HDAd IL-12_TK_PD-L1 construct expressed cytidine kinase.

In another embodiment, A549, FaDu or SCC47 cells are infected in vitro with HDAd IL-12_TK_PD-L1 , HDAd_PD-L1 (see above Tanoue et al .) or control HDAd encoding eGFP (see Farzad et al. above) (n=4 holes/condition). The cells were cultured without ganciclovir for 48 hours, or 8 hours after infection and every 24 hours thereafter, the medium was replaced with a medium containing 10 ng/ml ganciclovir.

The secretion of IL-12 secreted into the cell culture supernatant was analyzed by ELISA, and the secretion of anti-PD-L1 microsomes was analyzed by Western blotting using an anti-HA antibody (anti-PD-L1 microsomes containing a C-terminal HA tag). At the end of the experiment, the wells were stained with crystal violet to show live cells.

The results are shown in FIGS. 21A to 21C, and the expression of the transgene encoded by HDAd in the different cancer cell lines analyzed was confirmed. 3.3 Confirmation of anti-PD-L microsome binding to PD-L1

The ability of anti-PD-L1 microsomes encoded by HDAd IL-12_TK_PD-L1 to bind to PD-L1 was analyzed by ELISA.

Briefly, Immulon 2 high-binding 96-well disk (VWR) was coated with 500 ng/well of recombinant human PD-L1 (BioVision). After closing the disk with PBS-T containing 3% BSA, add plastids that have been encoded with GFP (pGFP; negative control group) and plastids encoding anti-PD-L1 microsomes (pPDL1) as described above by Tanoue et al. mini Tanoue) or a serially diluted cell culture medium of A549 cells transfected with plastids (pPDL1 mini) encoding anti-pPDL1 microsomes (encoded by HDAd IL-12_TK_PD-L1 ) and incubated at 4ºC for 24 hours. As a positive control group (PDL1 IgG), anti-human PD-L1 antibody (BioLegend) serially diluted from 10 µg/well was used. After washing the dishes with PBS-T, add HRP-labeled anti-human IgG (for PD-L1 mini and PDL1 mini Tanoue) or HRP-labeled anti-mouse IgG (BioRad; for PD-L1 IgG and Iso IgG) for For detection, and incubated for 1 hour at room temperature. After that, the disk was developed, and the absorbance at 450 nm was measured using a Tecan reader (TECAN).

The results are shown in Figure 11. Anti-PD-L1 antibody containing CDRs of anti-PD-L1 antibody strain H12 was found to bind to human PD-L1 in a dose-dependent manner, binding to the anti-PD-L1 microsome described in Tanoue et al. Compared with the force, it has a comparable (or higher) binding force. Example 4 : Analysis of cancer treatment in vivo

It was proved in the mouse xenograft tumor model that (1) the preferred oncolytic virus + HDAd IL-12_TK_PD-L1 + HER2-CAR-T and (2) ICOSTAT + HDAd IL-12_TK_PD-L1 + HER2-CAR-T Anti-cancer effect of combination therapy in vivo.

In the first experiment, 1×10 ⁶ FaDu cells formulated in PBS were injected subcutaneously into NSG male mice. 12 days later, the tumor was injected into 1x10 ⁸ viral particles (1) with an oncolytic virus HDAd IL-12_TK_PD-L1 (2 ) ICOSTAT HDAd IL-12_TK_PD-L1, OncAd or +: HDAd ratio of 1:20.

In the second experiment, 0.5×10 ⁶ FaDu cells were injected into NSG male mice in situ. After 6 days, intratumoral injection of 1x10 ⁸ viral particles (1) with an oncolytic virus HDAdIL-12_TK_PD-L1 (2) ICOSTAT HDAd IL-12_TK_PD-L1, OncAd or +: HDAd ratio of 1:20.

In two experiments, 3 days after the administration of virions, 1×10 ⁶ HER2-CAR T cells were administered intravenously.

In the two experiments, the control conditions included the following:

The tumor size was monitored, and the tumor volume was calculated using the equation: width ² x length x 0.5.

It was found that the oncolytic virus, HDAdIL-12_TK_PD-L1 and HER2 were used compared to the use of either agent alone (Condition 8, 10 or 11), or compared to the use of two of the three agents (Condition 3, 4 and 6) The combination of CAR-T (Test Condition 1) has an improved anti-tumor effect.

Similarly, it was found that ICOSTAT, HDAdIL-12_TK_PD-L1 and ICOSTAT were used in comparison with either agent alone (Condition 9, 10 or 11) or compared to the use of two of the three agents (Condition 3, 5 and 7). The combination of HER2 CAR-T (Test Condition 2) has an improved anti-tumor effect.

Similar results were observed when using SCC47 cells and A549 cells to establish xenograft tumors. Example 5 : Analysis of the anticancer activity of HER2 specific CAR-T cells in vivo

In the xenograft model of squamous cell head and neck cancer-derived FaDu cells, the anti-cancer activity of HER2 specific CAR-T cells (see Example 1 above) in vivo was studied.

Briefly, 0.5x10 ⁶ cells were injected orthotopically into FaDu th NSG male mice. After 9 days, mice were injected with 1x10 ⁶ T cells expressing firefly luciferase gene modified by HER2-CAR construct or 1x10 ⁶ constructed with C5, F1 or A3 CAR via tail vein Transduced firefly luciferase expressing T cells. This experiment included control conditions in which mice were not injected with T cells on day 9.

Monitor changes in luciferase activity (hence the number and distribution of administered T cells), body weight, and mouse survival rate over time. The luciferase activity was monitored by intraperitoneal injection of D-luciferin (1.5 mg/mouse), and 10 minutes later the mice were imaged using an IVIS imager (Xenogen).

15A and 15B show the day 0, day 4, day 7, day 14, day 14 after injection of luciferase expressing T cells (ie, non-transduced T cells or HER2 specific CAR-T cells) Images obtained on day 28, day 42, day 56, and day 70 (days means the number of days after ffLuc T cell injection). Systemically infused T cells showed movement to the tumor site in situ. After 7 days, no T cells were detected that were not modified to express HER2 specific CAR. On the contrary, HER2-specific CAR-T cells were continuously detected throughout the experiment.

Figure 15C shows the percentage survival rate of mice receiving different treatments after the entire experiment. It was found that the survival rate of HER2 specific CAR-T cells administered was increased.

In another experiment, NOD scid γ (NSG) mice were injected with 1x10 ⁶ firefly luciferase expressing T cells that were not transduced with the HER2-CAR construct through the tail vein, or 1x10 ⁶ had been C5 , F1 or A3 CAR construct transduced firefly luciferase expressing T cells. The luciferase activity was monitored as described above, and the body weight of mice was also monitored over time.

The experimental results are shown in FIGS. 16A to 16C. C5 CAR-T cells were found to expand non-specifically in NSG mice (Figure 16A). No significant weight loss was observed in mice administered HER2-specific CAR-T cells (Figure 16C). Example 6 : Analysis of the anticancer activity in vivo of a combination of oncolytic virus, HDAd virus and HER2 specific CAR-T cells

In the xenotransplantation model of squamous cell head and neck cancer-derived FaDu cells, the anti-cancer activity of the combination therapy of oncolytic virus, HDAd and HER-specific CAR-T cells in vivo was studied.

Briefly, 0.5x10 ⁶ cells were injected orthotopically into the FaDu th NSG male mice. After 6 days, a combination of Onc5/3Ad2E1Δ24 (described in Example 2.1) and HDAd IL-12_TK_PD-L1 described in Example 3.1 (the combination of OncAd and HdAd is referred to herein as "CAd" trio ”). Using Onc5/3Ad2E1Δ24:HDAd IL-12_TK_PD-L1 as a ratio of 1:10, all 1×10 ⁷ virus particles were administered.

Three days later, 1×10 ⁶ T cells that had been transduced with HER2 specific CAR construct (corresponding to strain F1) and genetically engineered to express firefly luciferase were administered to mice through the tail vein. In the control mouse population not administered with CAd trio , 1x106 ⁶ firefly luciferase expressing T cells that were not transduced by the HER2-CAR construct were injected through the tail vein, but not in another control mouse population T cells were not injected when CAd trio was administered. Monitor the luciferase activity, body weight and survival rate of mice over time.

Prior to their use in experiments, F1.CART cells were characterized by flow cytometry, and the results are shown in FIGS. 17A to 17C. The cells were found to contain 72.5% of CD4+ cells and CD8+ cells. It was determined that 87% of cells expressed HER2 CAR on the cell surface. 39% of the cells are CCDR7+CD45RO+, and 59.2% of the cells are CCR7-CD45RO+.

The experimental results of analyzing the therapeutic effect of the combination of oncolytic virus, HDAd virus and HER2 specific CAR-T cells in vivo to treat cancer are shown in FIGS. 18A to 18D. It was found that the combination of Onc5/3Ad2E1Δ24, HDAd IL-12_TK_PD-L1 and F1.CART can improve the survival rate of treatment with F1.CART cells only.

In another experiment, two different ratios of Onc5/3Ad2E1Δ24 to HDAd IL-12_TK_PD-L1 were studied .

Briefly, 0.5x10 ⁶ FaDu cells modified to express firefly luciferase were injected into NSG male mice in situ. After 6 days, mice were injected into the tumor: (i) 1x10 ⁷ CAd trio virus particles, the ratio of Onc5/3Ad2E1Δ24: HDAd IL-12_TK_PD-L1 was 1:10; (ii) 1x10 ⁷ CAd trio virus particles , Onc5 / 3Ad2E1Δ24: HDAd IL 12_TK_PD L1-- ratio of 1:20; (iii) 1x10 ⁸ th CAd trio virions, Onc5 / 3Ad2E1Δ24: HDAd IL 12_TK_PD -L1- ratio of 1:10; or (iv) 1x10 ⁸ th CAd trio virions, Onc5 / 3Ad2E1Δ24: ratio HDAd IL-12_TK_PD-L1 of 1:20.

Three days after injection of 1x10 ⁶ Construction singled form past F1 CAR transduced T cells through the tail vein (not expressing firefly luciferase). By analyzing the luciferase activity as described above, the cancer is monitored over time, and the body weight of the mice is also monitored.

The results of the experiment are shown in FIGS. 19A to 19C. Mice administered with Onc5/3Ad2E1Δ24:HDAd IL-12_TK_PD-L1 ratio of 1:10 usually have less fluorescence than mice administered with Onc5/3Ad2E1Δ24:HDAd IL-12_TK_PD-L1 ratio of 1:20 luciferase expressing FaDu cells, and administered with 1x10 ⁸ th CAd trio of mice virions than generally administered with 1x10 ⁷ th CAd trio of mice with viral particles less luciferase expressing FaDu cells (FIG. 19B). Example 7 : Analysis of anticancer activity in vivo of the combination of oncolytic virus, HDAd virus and ganciclovir (GCV)

In a xenograft model of squamous cell head and neck cancer-derived FaDu cells, the in vivo anticancer activity of a combination of oncolytic virus and HdAd (encoding thymidine kinase) (ie, CAd trio ) and ganciclovir (GCV) was studied.

Ectopic FaDu tumors were established by subcutaneous injection of FaDu cells in the flank of mice. After the tumor was injected into the mice 1x10 ⁸ th CAd trio virions, Onc5 / 3Ad2E1Δ24: ratio HDAd IL-12_TK_PD-L1 of 1:10. On the 2nd, 3rd, 4th, 5th, 7th, 10th, 14th, 17th and 21st days after CAd trio injection, in a group of mice (n=5) Intraperitoneal injection of 10mg/kg ganciclovir.

Blood samples were collected from mice on Day 2, Day 7, Day 14 and Day 21, and then the expression of IL-12 was analyzed by ELISA. The tumor volume was monitored throughout the experiment. On the 22nd day, quantitative real-time PCR analysis was used to determine the number of Onc.Ad and HDAd vectors in the DNA extracted from the tumor, and the number of copies detected for GAPDH was used for normalization.

The experimental results are shown in Figs. 20A to 20D. Ganciclovir (GCV) treatment did not significantly affect the number of Onc. Ad vector copies on day 22 (Figure 20A), but significantly reduced the number of HDAd vector copies (Figure 20B). It was also found that GCV treatment improved tumor control (Figure 20C), but did not significantly affect the level of IL-12 in the blood (Figure 20D). Example 8 : Generation of oncolytic virus-specific T cells and HER- specific CAR- expressing oncolytic virus-specific T cells 8.1 Generation and characterization of oncolytic virus-specific T cells

Oncolytic virus-specific T cells (AdVST) and activated T cells (ATCs) are prepared as follows.

By adding 0.5ml of 1mg/ml antibody diluted 1:1000, and then incubating at 37°C for 2-4 hours or overnight at 4°C, the anti-CD3 (strain OKT3) and anti-CD28 The agonistic antibody is coated on the well of the tissue culture plate.

According to the standard Ficoll-Paque method, PBMCs are isolated from blood samples obtained from healthy donors. ATCs :

By coating the anti-CD3/CD28 agonist antibody coated discs in CTL cell culture medium (containing 50% Advanced RPMI, 50% Click medium, 10%) supplemented with 10ng/ml IL-7 and 5ng/ml IL-15 FBS, 1% GlutaMax, 1% penicillin / streptomycin) culture, 1x10 ⁶ th stimulation of PBMCs (2ml of cell culture medium). Keep the cells in an environment of 37°C and 5% CO ₂ . The next day, 1 ml of the cell culture medium was replaced with a new CTL medium containing 20 ng/ml IL-7 and 10 ng/ml IL-15.

Keep culturing ATCs, and then collect between 5-7 days and use for experiment or cryopreservation. AdVSTs :

Stimulate 1x10 ⁶ PBMCs (in 2ml of cell culture medium) by cultivating on CTL cell culture medium supplemented with 10ng/ml IL-7 and 100ng/ml IL-15 on a plate coated with anti-CD3/CD28 agonist antibody in).

Add 20 μl of 200-fold diluted adenovirus-specific Hexon Pepmix (JPT Cat# PM-HAdV3) or Penton PepMix (JPT Cat# PM-HAdV5) to the wells. The cells were kept at 37°C, 5% CO ₂ environment. After 48 hours, the cells were fed with CTL medium, and IL-7 and IL-15 were added to a final concentration of 10 ng/ml IL-7 and 100 ng/ml IL-15. 8.2 Generation of CAR- expressing oncolytic virus-specific T cells

On day 3, the AdVSTs at a concentration 0.125 x 10 ⁶ cells / ml and suspended in medium containing 10ng / ml CTL IL-7 and 100ng / ml IL-15 in the cells.

The Retronectin coated disks were prepared by incubating RetroNectin (Clontech) diluted 1:100 in PBS at 37°C for 2-4 hours, or 4°C overnight. The wells were washed with CTL medium, 1 ml of HER2 specific CAR retrovirus retroviral supernatant was added to the wells, and then the plate was centrifuged at 2000 g for 1.5 hours. At the end of the centrifugation step, aspirate the retrovirus supernatant, and then add 2 ml of AdVST suspension (ie, 0.25× ^{10 6} cells) to the wells of the dish. The disk was centrifuged at 400g for 5 minutes, and then incubated at 37°C in a 5% CO ₂ environment.

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 collected for experimental use or cryopreservation, or a second stimulation was performed to expand CAR-expressing AdVSTs (see Example 8.3). 8.3 AdVSTs of amplification and CAR-AdVSTs

As follows, further stimulation and expansion of AdVSTs and CAR-expressing AdVSTs as needed.

Pepmix-impacted autologous ATCs were used as APCs, and K562c cells (see, eg, Ngo et al., J Immunother. (2014) 37(4):193-203) were used as costimulatory cells. The final ratio of AdVSTs or CAR-AdVSTs: ATCs: K562cs in the stimulating medium is 1:1:3-5.

AdVSTs or CAR-AdVSTs were resuspended in CTL medium to a concentration of 0.2× ^{10 6} cells/ml.

At 37 ° C for at 10μl 200 dilution of adenovirus specific Hexon Pepmix (JPT Cat # PM- HAdV3) or Penton PepMix (JPT Cat # PM- HAdV5) cultivating 1x10 ⁶ th ATCs 30 minutes. Afterwards, the ATCs were irradiated with 30 Gy and collected. 3-5x10 ⁶ K562cs cells were irradiated with 100Gy.

Then mix ATCs and K562cs cells in a total volume of 5ml of CTL medium, add 20ng/ml IL-7 and 200ng/ml IL-15, add 1ml of this mixture to the wells of the 24-well plate, and Add 1ml of AdVST suspension or CAR-AdVST suspension to the well.

Maintain the cells in a 37°C, 5% CO ₂ environment. After 3-4 days, the cell culture medium is added as needed, and after 6-7 days, the expanded AdVSTs or CAR-AdVSTs cells are collected for the experiment. Example 9 : Analysis of anticancer activity in vivo of a combination of oncolytic virus, HDAd , oncolytic virus-specific T cells and CAR- expressing oncolytic virus-specific T cells

In the xenograft model of squamous cell head and neck cancer FaDu cells, the in vivo anticancer activity of different combinations of oncolytic virus, HDAd, oncolytic virus-specific T cells and CAR-expressing oncolytic virus-specific T cells was studied.

Briefly, 0.5x10 ⁶ FaDu cells processed to express firefly luciferase were injected into NSG male mice in situ. After 6 days, inject into the tumor of the mouse group: (i) 1x10 ⁷ CAd trio virus particles, the ratio of Onc5/3Ad2E1Δ24:HDAd IL-12_TK_PD-L1 is 1:10; or (ii) 1x10 ⁷ Onc5/ 3Ad2E1Δ24 virions. Three days later, the mice were injected via the tail vein: (a) 1x10 ⁶ th AdVSTs, or (b) 1x10 ⁶ with resistance to HER2 CAR F1 clones are transduced AdVSTs (prepared as described in Example 8).

Prior to their use in experiments, flow cytometry was used to characterize AdVSTs and F1.CAR-AdVSTs. The analysis results are shown in FIGS. 22A and 22B and FIGS. 23A to 23C.

By analyzing the luciferase activity as described above, the cancer was monitored over time, and the body weight of the mouse was also monitored.

The experimental results are shown in FIGS. 24A to 24D. In mice treated with a combination of CAd trio + HER2 specific CAR expressing AdVSTs (ie, treatment group (i)(b)), the highest level of tumor control and survival rate were observed.

Embodiments and studies describing the principles of the present disclosure will now be discussed with reference to the drawings.

Figures 1A and 1B . Figure 1A shows a schematic diagram of an example of a HER2 specific CAR construct. FIG. 1B shows a schematic diagram of an example of a plan for transducing T cells to produce HER2-specific CAR-T.

Figure 2 shows the expression graph of HER2-CARs, CCR7, CD45RO and PD-1 on T cells transduced with the designated HER2-CAR construct as determined by flow cytometry.

Figure 3 shows HER2-CAR, CCR7, CD45RO, PD-1, LAG-3 and TIM on CD4 and CD8 T cells transduced with the anti-HER2 strain E4 CAR construct as determined by flow cytometry -3 expression chart.

4A and 4B, and 4A is a by ⁵¹ Cr release assay analysis, the anti-HER2 clones are C5, ((NT) or non-transduced cells) and E4 F1 CAR-T cells in vitro cell killing MDA cells (which does not A histogram of HER2 will be expressed on the cell surface; negative control group), MDA-HER2 cells (which will express HER2 on the cell surface; positive control group), FaDu and SCC47 cells. Figure 4B shows a graph determined by flow cytometry, indicating that HER2 is expressed on MDA-HER2 cells, FaDu, and SCC47 cells, but not on MDA cells.

Figure 5 shows that the anti-HER2 strain C5, E4, and F1 CAR-T cells (or non-transduced (NT) cells) were modified in vitro to express the firefly luciferase (ffLuc) as determined by ffLuc activity analysis A graph of FaDu and SCC47 cells. The data is expressed as mean ± SD (n=4). *P> 0.001.

Figure 6 . Figure 6 shows a schematic diagram of an exemplary sequence of the ICOSTAT oncolytic adenovirus construct.

Figures 7A to 7F . It was determined by MTS survival analysis that after infection with the indicated concentration of virions (Vp), ICOSTAT oncolytic adenovirus killed A549 cells (Figures 7A and 7F), FaDu cells (Figure 7B), SCC47 cells (Figure 7C), Capability chart of WI-38 cells (Figure 7D) and ARPE-19 cells (Figure 7E). Helper-dependent adenovirus (HDAd) is included as a control condition.

Figures 8A and 8B . A bar graph showing the ability of ICOSTAT oncolytic adenovirus replication and assisted replication of helper-dependent adenovirus (HDAd) by quantitative analysis of quantitative real-time PCR. The virus named "Onc5/3AdicoSTAT" is ICOSTAT. "+HD" refers to the co-infection of ICOSTAT and HDAd.

Figures 9A and 9B . A graph showing the replication of ICOSTAT oncolytic adenovirus in FaDu cells (Figure 9A) and SCC47 cells (Figure 9B) in the presence or absence of 10 ng/ml IFNγ in the cell culture medium.

Figures 10A to 10D . FIG. 10A is a schematic diagram of the HDAd IL-12_TK_PDL1 construct. Figure 10B is a bar graph showing IL-12p70 produced by cells transfected with a designated helper-dependent adenovirus (HDAd) construct. FIG. 10C is a photograph of Western blotting methods showing anti-PD-L1 microsomes produced by cells transfected with the HDAd construct. Figure 10D is a photograph showing the pores of HSV thymidine kinase produced by cells transfected with the HDAd construct.

Figure 11 . ELISA analysis chart showing the binding of PD-L1 microsomes to recombinant human PD-L1, using plastids encoding GFP (pGFP; negative control group), encoding Tanoue et al. Serially diluted cell culture medium of A549 cells transfected with plastid (pPDL1 mini Tanoue) or plastid (pPDL1 mini) encoding anti-pPDL1 microsome (encoded by HDAd IL-12_TK_PD-L1 ). Serially diluted anti-human PD-L1 antibody was used as a positive control group (PDL1 IgG).

Figures 12A and 12B . Example sequence diagram of the Onc5/2E1Δ24 oncolytic adenovirus construct (Figure 12A) and the plastid encoding the Onc5/2E1Δ24 oncolytic adenovirus construct (Figure 12B).

Figures 13A to 13D . Shown by MTS survival analysis, after infection with the indicated concentration of virions (Vp), Onc5/3Ad2E1A oncolytic adenovirus kills FaDu cells (Figure 13A), SCC47 cells (Figure 13B), WI-38 cells (Figure 13C ) And ARPE-19 cells (Figure 13D) ability chart. Helper-dependent adenovirus (HDAd) is included as a control condition.

Figure 14 shows a graph of the number of HER2 specific CAR T cells after transduction with a designated CAR construct and after a specified number of days of cell culture in vitro.

Figures 15A to 15C . Images and graphs showing the results of in vivo anticancer activity analysis of adoptively transferred luciferase expressing T cells in a squamous cell head and neck cancer in situ FaDu cell-derived model. Figures 15A and 15B show luciferase expressing non-transduced T cells (NT) in mice and luciferase expressing T expressing C5, F1 or A3 HER2 specific CARs at a specified number of days after cell infusion The number and location of cells. Figure 15C shows the percentage of surviving individuals in different treatment groups at the specified number of days after infusion of the cells. The negative control condition (-) in which T cells were not administered was also shown.

Figures 16A to 16C . Images and graphs showing the results of in vivo analysis of adoptively transferred T cells in NSG mice. Figure 16A shows luciferase expressing non-transduced T cells (NT) and luciferase expressing T cells expressing C5, F1 or A3 HER2 specific CARs in mice at the specified number of days after infusion of the cells Number and location. Figure 16B shows the measured values of total ventral flux (photons/sec; p/s) of mice of different groups at the specified number of days after infusion of the cells. Figure 16C shows the weight of mice in different treatment groups, expressed as a percentage of body mass on day 0, at the specified number of days after infusion of the cells.

Figures 17A to 17C . Scatterplot and histogram showing the results of F1 HER2 specific CAR T cells used in an in vivo anticancer activity analysis experiment using flow cytometry to characterize the combination of CAd trio and adoptively transferred T cells. Figure 17A shows the percentage of CD4+ T cells and CD8+ T in F1. CAR-T population. Figure 17B shows the percentage of cells expressing HER2 CAR on the cell surface. Figure 17C shows the percentage of cells expressing CCR7 and/or CD45RO in the F1. CAR-T population.

Figures 18A to 18D . Images and graphs showing the results of in vivo anticancer activity analysis of the combination of CAd trio and adoptively transferred T cells in a squamous cell head and neck cancer in situ FaDu cell-derived model. Figure 18A shows the number and location of luciferase-expressing non-transduced T cells (NT) and luciferase-expressing T cells expressing F1 HER2 specific CAR in mice at the specified number of days after infusion of the cells . The upper right graph (the Y axis is labeled as total flux) is the "days after CAR T cell injection". The bottom two graphs are "Days after CAd trio injection. Figure 18B shows the measured values of total flux (photons/sec; p/s) on the ventral surface of different groups of mice at the specified number of days after CAd trio administration. Figure 18C displaying the specified number of days after administration CAd trio, the different treatment groups in weight of mice, expressed as a percentage of day 0 of the weight. FIG. 18D is displayed when the specified number of days after administration CAd trio, the different treatment groups in the percentage of survival of individuals of It also shows that there is no negative control condition (-) for administration of CAd trio or T cells.

Figures 19A to 19C . Showing the ratio of Onc5/3Ad2E1Δ24: combination of HDAd IL-12_TK_PD-L1 and adoptively transferred HER2 specific CAR T cells, in vivo anti-cancer activity analysis results in FaDu cell-derived model of squamous cell head and neck cancer in situ Images and charts. Figure 19A shows the number and location of luciferase expressing FaDu cells in mice at the specified number of days after administration of CAd trio . Figure 19B shows the measured values of total flux on the ventral surface of mice of different groups (photons/sec; p/s) at the specified number of days after administration of CAd trio . Figure 19C shows the weight of mice in different treatment groups, expressed as a percentage of body weight on day 0, at the specified number of days after CAd trio administration.

Figures 20A to 20D . Bar charts and graphs showing the results of in vivo analysis of the combination of Onc5/3Ad2E1Δ24 and HDAd IL-12_TK_PD-L1 with ganciclovir (GCV) in an ectopic FaDu cell-derived model of squamous cell head and neck cancer. Figures 20A and 20B show Onc5/3Ad2E1Δ24 (Figure 20A) in mouse tumors administered with a combination of Onc5/3Ad2E1Δ24 and HDAd IL-12_TK_PD-L1 (CAd trio ) with or without GCV on day 22 after infection ) And the number of GAPDH normalized copies of HDAd IL-12_TK_PD-L1 (Figure 20B). FIG. 20C shows the tumor volume (mm ³ ) of mice administered the combination of Onc5/3Ad2E1Δ24 and HDAd IL-12_TK_PD-L1 (CAd trio ) with or without GCV at the specified number of days after CAd trio injection. FIG. 20D shows the IL-12 levels measured by ELISA analysis of blood samples taken with or without GCV at the specified number of days after CAd trio injection.

Figures 21A to 21C . Bar graphs and images showing the results of expression analysis of transgenes of cancer cell lines cultured with or without ganciclovir (GCV) infected with different HDAd viruses. Figure 21A shows the level of IL-12 in the cell culture supernatant determined by ELISA. Figure 21B shows anti-PD-L1 microsomes detected in cell culture supernatant by Western blotting. Figure 21C shows live cells detected using crystal violet staining at the end of the experiment.

Figures 22A and 22B . Scatterplot showing the results of characterizing adenovirus-specific T cells (AdVSTs) used in the Example 9 experiment by flow cytometry. Figure 22A shows the percentage of CD4+ T cells and CD8+ T cells in the AdVST population. Figure 22B shows the percentage of cells expressing CCR7 and/or CD45RO within the AdVST population.

Figures 23A to 23C . Scatterplot and histogram showing the results of characterizing F1.CAR-transduced AdVSTs used in the Example 9 experiment by flow cytometry. Figure 23A shows the percentage of CD4+ T cells and CD8+ T cells in the transduced population. Figure 23B shows the percentage of cells expressing HER2 CAR on the cell surface. Figure 23C shows the percentage of cells expressing CCR7 and/or CD45RO in the F1.CAR-AdVST population.

Figures 24A to 24D . Shows adenovirus-specific T cells (AdVSTs), F1.CAR transduced AdVSTs, F1.CAR transduced AdVSTs combined with Onc5/3Ad2E1Δ24, F1.CAR transduced AdVSTs and Onc5/3Ad2E1Δ24 + HDAd IL-12_TK_PD- The combination of L1 ("CAd trio "), images and graphs of the results of anti-cancer activity analysis in vivo. Figure 24A shows the number and location of luciferase expressing FaDu cells in mice at the specified number of days after administration of CAd trio . Figure 24B shows the measured values of the total ventral flux of mice of different groups (photons/sec; p/s) at the specified number of days after administration of CAd trio . Figure 24C shows the weight of mice in different treatment groups, expressed as a percentage of body mass on day 0, at the specified number of days after administration of CAd trio . Figure 24D shows the percentage of surviving individuals in different treatment groups at the specified number of days after administration of CAd trio .

(no)

Claims (44)

A method of treating cancer, which includes administering to a body: (i) an oncolytic virus; and (ii) At least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens. A combination of methods for treating cancer having (i) an oncolytic virus; and (ii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens. Use of (i) an oncolytic virus and (ii) at least one cell containing a chimeric antigen receptor (CAR) specific for cancer cell antigens for the preparation of a medicament for use in a method of treating cancer . For example, the method of claim 1, the combination of claim 2, or the use of claim 3, wherein the CAR-containing cell is specific to the oncolytic virus. A method of treating cancer, which includes administering to a body: (i) an oncolytic virus; and (ii) at least one immune cell specific for the oncolytic virus. A combination of methods for treating cancer having (i) an oncolytic virus, and (ii) at least one immune cell specific for the oncolytic virus. A use of (i) an oncolytic virus and (ii) at least one immune cell specific for the oncolytic virus in the preparation of a medicament for use in a method of treating cancer. The method, combination or use of any one of claims 1 to 7, wherein the oncolytic virus is an oncolytic adenovirus (OncAd). The method, combination or use of any one of claims 1 to 8, wherein the oncolytic virus is derived from adenovirus 5 (Ad5). The method, combination or use of any one of claims 1 to 9, wherein the oncolytic virus encodes the E1A protein, which exhibits less binding to the Rb protein than the E1A protein encoded by Ad5. The method, combination or use of any one of claims 1 to 10, wherein the oncolytic virus encodes an E1A protein lacking the amino acid sequence LTCHEACF (sequence identification number 52). The method, combination or use according to any one of claims 1 to 11, wherein the oncolytic virus encodes an E1A protein comprising or consisting of: the amino acid sequence of sequence identification number 34. The method, combination or use of any one of claims 1 to 12, wherein the oncolytic virus comprises a nucleic acid having one or more binding sites for one or more transcription factors. The method, combination or use of any one of claims 1 to 13, wherein the oncolytic virus comprises a nucleic acid having one or more binding sites for STAT1. The method, combination or use of any one of claims 1 to 4 or 8 to 14, wherein the at least one cell comprising a CAR specific for cancer cell antigen is a T cell. The method, combination or use of any one of claims 1 to 4 or 8 to 15, wherein the CAR comprises an antigen binding domain capable of specifically binding HER2. The method, combination or use of any one of claims 1 to 4 or 8 to 16, wherein the CAR comprises an antigen binding domain, which comprises: A VL domain, which contains: LC-CRD1: sequence identification number 10; LC-CRD2: sequence identification number 11; LC-CRD3: sequence identification number 12; And a VH domain, which includes: HC-CRD1: sequence identification number 13; HC-CRD2: sequence identification number 14; HC-CRD3: sequence identification number 15; or A VL domain, which contains: LC-CRD1: sequence identification number 18; LC-CRD2: sequence identification number 19; LC-CRD3: sequence identification number 20; And a VH domain, which includes: HC-CRD1: sequence identification number 21; HC-CRD2: sequence identification number 22; HC-CRD3: sequence identification number 23; or A VL domain, which contains: LC-CRD1: sequence identification number 26; LC-CRD2: sequence identification number 27; LC-CRD3: Sequence identification number 28; And a VH domain, which includes: HC-CRD1: sequence identification number 29; HC-CRD2: sequence identification number 30; HC-CRD3: sequence identification number 31; or A VL domain, which contains: LC-CRD1: Sequence identification number 57; LC-CRD2: Sequence identification number 58; LC-CRD3: Sequence identification number 59; And a VH domain, which includes: HC-CRD1: sequence identification number 60; HC-CRD2: sequence identification number 61; HC-CRD3: Sequence identification number 62. The method, combination or use of any one of claims 1 to 4 or 8 to 17, wherein the CAR comprises an antigen binding domain, which comprises: A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 16; and a VH comprising or consisting of: having at least 75% sequence identification number 17 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 24; and a VH comprising or consisting of: having at least 75% sequence identification number 25 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 32; and a VH comprising or consisting of: having at least 75% sequence identification number 33 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 63; and a VH comprising or consisting of: having at least 75% sequence identification number 64 Amino acid sequence of sequence identity. The method, combination or use of any one of claims 1 to 18, wherein the method, combination or use further comprises a virus comprising a nucleic acid encoding an immunomodulatory factor. The method, combination, or use of claim 19, wherein the virus containing nucleic acid encoding an immunomodulatory factor is a helper-dependent adenovirus (HDAd). The method, combination or use of claim 19 or 20, wherein the immunomodulatory factor is selected from: an agonist of an effector immune response or an antagonist of an immunomodulatory response. The method, combination or use of any one of claims 19 to 21, wherein the virus comprising a nucleic acid encoding an immunomodulatory factor comprises an IL-12 and/or antagonist anti-PD-L1 antibody (antagonist anti-PD-L1 antibody). The method, combination or use of any one of claims 19 to 22, wherein the virus comprising a nucleic acid encoding an immunomodulatory factor comprises a nucleic acid encoding an enzyme capable of catalytically converting a non-toxic factor into a cytotoxic form. The method, combination or use of claim 23, wherein the enzyme is selected from the group consisting of: thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, spicy Root peroxidase and carboxylesterase. The method, combination or use of any one of claims 1 to 24, wherein the method of treating cancer comprises: (a) Separate at least one cell from a body; (b) modifying the at least one cell to express or contain a CAR specific for cancer cell antigens, or a nucleic acid encoding a CAR specific for cancer cell antigens, (c) optionally expanding the modified at least one cell, and; (d) administering the modified at least one cell to a body. The method, combination or use of any one of claims 1 to 25, wherein the method of treating cancer comprises: (a) Isolate immune cells from a body; (b) Use a method including the following to generate or expand a population of immune cells specific for oncolytic viruses: stimulate the immune cells by the presence of antigen-presenting cells (APCs) presenting peptides of the oncolytic virus Come down to cultivate, and; (c) administering at least one immune cell specific to the oncolytic virus to a body. The method, combination or use of any one of claims 1 to 26, wherein the cancer is selected from head and neck cancer, nasopharyngeal cancer (NPC), cervical cancer (CC), oropharyngeal cancer (OPC), gastric cancer (GC ), hepatocellular carcinoma (HCC) and lung cancer. An oncolytic adenovirus (OncAd), which encodes an E1A protein comprising or consisting of: the amino acid sequence of SEQ ID NO:34. An oncolytic adenovirus (OncAd), which contains a nucleic acid having one or more binding sites for STAT1. An oncolytic adenovirus (OncAd), which contains a nucleic acid sequence having at least 60% sequence identity with sequence identification number 51 or an equivalent sequence caused by codon degeneracy. An oncolytic adenovirus (OncAd), which contains an equivalent sequence resulting from the degeneracy of a nucleic acid sequence or codon with at least 60% sequence identity with sequence identification number 55. A helper-dependent adenovirus (HDAd) comprising nucleic acids encoding IL-12 and/or antagonist anti-PD-L1 antibodies. The HDAd of claim 32, wherein the HDAd additionally contains a nucleic acid encoding an enzyme capable of catalytically converting a non-toxic factor into a cytotoxic form. The HDAd according to claim 32 or claim 33, wherein the enzyme is selected from: thymidine kinase, cytosine deaminase, nitroreductase, cytochrome P450, carboxypeptidase G2, purine nucleoside phosphorylase, spicy Root peroxidase and carboxylesterase. A chimeric antigen receptor (CAR), which comprises an antigen binding domain, which comprises: A VL domain, which contains: LC-CRD1: sequence identification number 10; LC-CRD2: sequence identification number 11; LC-CRD3: sequence identification number 12; And a VH domain, which includes: HC-CRD1: sequence identification number 13; HC-CRD2: sequence identification number 14; HC-CRD3: sequence identification number 15; or A VL domain, which contains: LC-CRD1: sequence identification number 18; LC-CRD2: sequence identification number 19; LC-CRD3: sequence identification number 20; And a VH domain, which includes: HC-CRD1: sequence identification number 21; HC-CRD2: sequence identification number 22; HC-CRD3: sequence identification number 23; or A VL domain, which contains: LC-CRD1: sequence identification number 26; LC-CRD2: sequence identification number 27; LC-CRD3: Sequence identification number 28; And a VH domain, which includes: HC-CRD1: sequence identification number 29; HC-CRD2: sequence identification number 30; HC-CRD3: sequence identification number 31; or A VL domain, which contains: LC-CRD1: Sequence identification number 57; LC-CRD2: Sequence identification number 58; LC-CRD3: Sequence identification number 59; And a VH domain, which includes: HC-CRD1: sequence identification number 60; HC-CRD2: sequence identification number 61; HC-CRD3: Sequence identification number 62. The CAR of claim 35, wherein the CAR includes an antigen binding domain, which includes: A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 16; and a VH comprising or consisting of: having at least 75% sequence identification number 17 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 24; and a VH comprising or consisting of: having at least 75% sequence identification number 25 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 32; and a VH comprising or consisting of: having at least 75% sequence identification number 33 Amino acid sequence of sequence identity; or A VL comprising or consisting of: an amino acid sequence having at least 75% sequence identity with sequence identification number 63; and a VH comprising or consisting of: having at least 75% sequence identification number 64 Amino acid sequence of sequence identity. An optionally isolated or artificial nucleic acid or multiple nucleic acids encoding oncolytic adenovirus (OncAd) according to any one of claims 28 to 31, and an auxiliary dependent type according to any one of claims 32 to 24 Adenovirus (HDAd), or chimeric antigen receptor (CAR) as in claim 35 or claim 36. A cell comprising an oncolytic adenovirus (OncAd) according to any one of claims 28 to 31, a help-dependent adenovirus (HDAd) according to any one of claims 32 to 34, or 35 or a request The chimeric antigen receptor (CAR) of item 36, or the nucleic acid or nucleic acids of claim 37. A pharmaceutical composition comprising the oncolytic adenovirus (OncAd) according to any one of claims 28 to 31, the help-dependent adenovirus (HDAd) according to any one of claims 32 to 34, such as claim 35 Or the chimeric antigen receptor (CAR) of claim 36, the nucleic acid or nucleic acids as in claim 37, or the cells as in claim 38, and a pharmaceutically acceptable carrier, diluent, excipient or Adjuvant. A method of treating cancer comprising administering an oncolytic adenovirus (OncAd) according to any one of claims 28 to 31, and a help-dependent adenovirus (HDAd) according to any one of claims 32 to 34 to a body ), the chimeric antigen receptor (CAR) as in claim 35 or claim 36, the nucleic acid or nucleic acids as in claim 37, the cells as in claim 38, or the pharmaceutical composition as in claim 39. Oncolytic adenovirus (OncAd) according to any one of claims 28 to 31, helper-dependent adenovirus (HDAd) according to any one of claims 32 to 34, chimerism as in request 35 or 36 An antigen receptor (CAR), the nucleic acid or nucleic acids as in claim 37, the cells as in claim 38, or the pharmaceutical composition as in claim 39, which are used in a method of treating cancer. An oncolytic adenovirus (OncAd) as in any one of claims 28 to 31, a help-dependent adenovirus (HDAd) as in any one of claims 32 to 34, as embedded in claim 35 or 36 Antigen receptor (CAR), the nucleic acid or nucleic acids as in claim 37, the cells as in claim 38, or the pharmaceutical composition as in claim 39 for the preparation of a medicament for the treatment of cancer. The method according to claim 40, the OncAd, HDAd, CAR, nucleic acid or nucleic acids, cells or pharmaceutical compositions provided in claim 41, or the use according to claim 42, wherein the cancer is selected from head and neck cancer, nasal Pharyngeal cancer (NPC), cervical cancer (CC), oropharyngeal cancer (OPC), gastric cancer (GC), hepatocellular carcinoma (HCC) and lung cancer. A modular kit comprising a predetermined amount of oncolytic adenovirus (OncAd) according to any one of claims 28 to 31, and help-dependent adenovirus (HDAd) according to any one of claims 32 to 34 , The chimeric antigen receptor (CAR) of claim 35 or claim 36, the nucleic acid or nucleic acids of claim 37, the cell of claim 38, or the pharmaceutical composition of claim 39.

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