CN114262691A - Isolated oncolytic adenoviruses capable of expressing foreign genes, vectors, therapeutic agents and uses thereof - Google Patents

Abstract

The present invention provides isolated oncolytic adenoviruses, vectors, therapeutics and uses thereof that can be used to express foreign genes. The oncolytic adenovirus is a selective replication type recombinant oncolytic adenovirus obtained by carrying out gene modification on adenovirus, and the genome of the oncolytic adenovirus has the following characteristics: 1) comprises an E1B gene regulatory element; 2) the coding region of the E1B gene is deleted, and a foreign gene is inserted at the site, and the foreign gene is positioned behind the E1B promoter and controlled by the regulatory elements of the E1B gene; 3) the upstream of the foreign gene comprises cDNA of E1A transcribing E1A 13s mRNA, which is wild-type or Rb protein binding region deleted. The oncolytic adenovirus can more effectively introduce nucleic acid with a marker polypeptide coding sequence into tumor and/or cancer cells, more efficiently express exogenous marker polypeptide in the tumor and/or cancer cells, enable the exogenous marker polypeptide to enter an MHC I antigen presentation pathway, and enhance the recognition sensitivity of T cell receptor modified immune cells to the tumor and/or cancer cells.

Description

Isolated oncolytic adenoviruses capable of expressing foreign genes, vectors, therapeutic agents and uses thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an oncolytic adenovirus, a vector, a therapeutic agent and application thereof.

Background

With the remarkable antitumor efficacy of immune checkpoint inhibitors in clinical trials (see "Cancer Cell 27, 450-461 (2015)"), and the long-term effective therapeutic effect of adoptive T Cell therapy represented by CAR-T (CAR) T cells on hematological tumors, tumor immunotherapy has become one of the most promising areas. Adoptive Cell Therapy (ACT), including adoptive transfer of Tumour Infiltrating Lymphocytes (TILs), T cells expressing foreign tumour specific T cell receptors (TCR-T), or CAR-T cells, has proven to be the most promising immunotherapy for one type of cancer (see the literature "N Engl J Med 2017; 377: 2545-.

Although CAR-T therapy targeting CD19 or BCMA has shown significant clinical efficacy against B cell type hematological tumors, CAR-T has not shown significant clinical benefit to solid tumor patients (see document "J immunolther.2019; 42: 126-. While adoptive transfer of TIL or transfer of TCR-T cells modified with a tumor antigen-specific TCR gene showed clinical efficacy in patients with solid tumors (see references "Adv Immunol.2016; 130: 279-94"). At present, clinical trials of TCR-T for the treatment of various solid tumors are in various stages (see the literature "Technol Cancer Res treat. 2019; 1-13"). Although adoptive T cell therapy is a promising approach to the treatment of solid tumors, there are several obstacles to further improving their efficacy. One such problem is tumor tissue heterogeneity, which is manifested by heterogeneous expression of tumor antigens specifically recognized by T cells in tumor cells (see "Int J cancer. 2001Jun 15; 92 (6): 856-60"). The lack or insufficient expression of the target antigen in some tumor cells may allow them to escape recognition and killing by the adoptive T cells. During tumorigenesis and development, the antigen processing and presentation mechanisms in tumor cells are often abnormal, and are manifested by HLA class I molecule mutation or reduced expression, and reduced expression of beta 2-microglobulin, TAP, tapasin enzyme, LMP complex, etc., so that tumor antigens cannot be effectively presented on the cell surface to escape recognition and elimination by tumor antigen-specific T cells (see document J Natl Cancer Inst.2013; 105 (16): 1172-87). Furthermore, tumor-specific T cells recognize epitope peptides (epitope peptides) presented by major histocompatibility complex (MHC, human MHC is an HLA molecule), and the function of T cells to recognize antigens is restricted by MHC, and only tumor epitope peptides presented by specific MHC molecules can be recognized. Tumor cells of tumor patients express specific tumor antigens and have specific HLA alleles so as to become suitable populations for adoptive T cell therapy, and therefore the application range of adoptive T cell therapy is obviously limited by the restriction of MHC. Another factor that affects the anti-tumor efficacy of adoptive T cells is the immunosuppressive properties of the Tumor Microenvironment (TME), which affects proliferation, differentiation, cytotoxicity and homing of adoptive T cells (see the literature "Curr Opin immunol. 2016Apr; 39: 1-6").

One solution to the above-mentioned problems encountered with adoptive T cell therapies is to use oncolytic viruses as vectors to label tumor cells to express target antigens that are specifically recognized by adoptive T cells, and to use the immunogenicity of the virus itself to improve the immunosuppressive properties of the tumor microenvironment. The oncolytic virus can selectively replicate in tumor cells after infecting the tumor cells, and can crack the tumor cells to achieve the effect of specifically killing the tumor cells through the massive proliferation of the subviral. The released daughter viruses, in turn, can selectively infect and lyse other tumor cells to maximize the clearance of tumor tissue (see literature "Nat Biotechnol.2012Jul 10; 30(7): 658-70"). Aberrant signaling pathways in tumor cells, such as RAS, TP53, RB1, PTEN, WNT, affect the antiviral mechanisms of the cells themselves, making viruses more readily replicated in tumor cells, a major cause of tumor selectivity (see "Nat Rev cancer.201711; 17(11): 633). In addition to its own oncolytic effect, oncolytic viruses can also alter the microenvironment of tumor tissue, primarily by inducing secreted cytokines, attracting natural immune cells, releasing tumor antigens, providing immune risk signals, etc., thereby enhancing anti-tumor immune responses locally to the tumor (see "J.Clin. invest.2018; 128,1258-1260"). Adenovirus as a vector was an earlier developed oncolytic virus, and Ad5 type adenovirus H101 based on E1B55K Gene deficiency was the first approved oncolytic virus product (see literature "Hum Gene ther.2018Feb; 29(2): 151-. The molecular structure and biological characteristics of adenovirus are studied more deeply, so that adenovirus is easier to be transformed into oncolytic virus through genetic engineering. The genome of oncolytic viruses is often modified to increase the selectivity of oncolytic viral tumor cells. In addition, tumor-selectivity may also be provided by using tumor-specific gene promoters to drive genes necessary for adenovirus replication.

However, modifications to the oncolytic viral genome will tend to affect the transcriptional replication of the virus within the cell and the ability to lyse the cell, such that tumor cells infected with the virus cannot be lysed. In addition, the completion of the replication cycle by oncolytic viruses requires the involvement of a large number of cellular components dependent on the host tumor cells and is regulated by a unique molecular mechanism (see the literature "J Virol.2008 Aug; 82(15): 7252-63"), while the gene regulation and protein expression of each tumor cell are different, and after tumor cells with different growth states and properties are infected by oncolytic viruses, some tumor cells cannot complete the replication cycle and lyse the cells (see the literature "Nat Rev cancer. 2002; 2(12):938 and 950"). In addition, neutralizing antibodies produced by the host against the oncolytic virus and tumor tissue limitations on viral particle spread may reduce the oncolytic effect of the oncolytic virus.

Therefore, there is still a strong need to improve the antitumor efficacy of adoptive transfer T cells and the antitumor efficacy of oncolytic viruses.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides that adoptive T cells are combined by using oncolytic viruses to enhance the tumor killing function and the application range of the adoptive T cells, and tumor cells which can not complete the virus replication cycle are eliminated by the T cells. To this end, the present invention further provides oncolytic viruses that are capable of efficiently expressing target antigens recognized by specific T cells in tumor cells, and that can be used in combination with adoptive TCR-T cell therapy to further enhance the efficacy of solid tumor immunotherapy.

In particular, to solve the problems of the prior art as described above, the present invention provides an isolated oncolytic adenovirus for expressing a foreign gene, a vector, a therapeutic agent and uses thereof.

Specifically, the present invention provides:

(1) an isolated oncolytic adenovirus for expressing a foreign gene, wherein the oncolytic adenovirus is a selective replication-competent recombinant oncolytic adenovirus obtained by genetically modifying adenovirus, and the genome of the recombinant oncolytic adenovirus has the following characteristics:

1) contains E1B gene regulatory elements, which include E1B promoter and E1B and pIX shared polyadenylation addition signal sequence;

2) the coding region of the E1B gene is deleted, and when the foreign gene needs to be inserted, the foreign gene is inserted at the site of the coding region of the E1B gene, is positioned behind the E1B promoter and is controlled by the regulatory elements of the E1B gene;

3) upstream of the foreign gene, a cDNA sequence of E1A transcribing the E1A 13s mRNA is contained, and the cDNA is wild type or Rb protein binding region deleted, the Rb protein binding region deleted is the wild type cDNA from which the nucleotide sequence shown in SEQ ID NO.7 is removed, or the Rb protein binding region deleted encodes a mutated E1A protein, the mutated E1A protein is shown in SEQ ID NO. 6.

(2) The oncolytic adenovirus according to (1), wherein the nucleotide sequence of the E1B promoter is shown in SEQ ID NO.1, and the polyadenylation addition signal sequence shared by E1B and pIX is shown in aataaa.

(3) The oncolytic adenovirus of (1), wherein the E1B gene comprises E1B-55K gene and E1B-19K gene.

(4) The oncolytic adenovirus of (1), wherein the nucleotide sequence of the coding region of the E1B gene is shown as SEQ ID NO. 3.

(5) The oncolytic adenovirus of (1), wherein the initiation site of the exogenous gene comprises a Kozak sequence, preferably, the Kozak sequence is shown as SEQ ID NO. 4.

(6) The oncolytic adenovirus of (1), wherein the nucleotide sequence of the wild-type E1A cDNA is shown as SEQ ID NO. 5.

(7) The oncolytic adenovirus of (1), wherein the cDNA for E1A that transcribes the E1A 13s mRNA is located upstream of the E1B promoter and partially coincides with the nucleotide sequence of the E1B promoter.

(8) The oncolytic adenovirus of (1), wherein the cDNA sequence of E1A transcribing the E1A 13s mRNA is under the control of an endogenous E1A promoter/enhancer, or under the control of an exogenous promoter; preferably, the nucleotide sequence of the endogenous E1A promoter/enhancer is shown in SEQ ID NO. 8.

(9) The oncolytic adenovirus of (1), wherein the cDNA sequence of E1A transcribing the E1A 13s mRNA is under the control of a foreign promoter, the nucleotide sequence shown as SEQ ID NO.9 is removed from the genome of the recombinant oncolytic adenovirus, and the foreign promoter nucleotide sequence is inserted at the removed site.

(10) The oncolytic adenovirus of (8) or (9), wherein the exogenous promoter comprises an EF-1 alpha promoter, a CMV promoter, a PKG promoter, an E2F promoter, an AFP promoter and a TERT promoter.

(11) The oncolytic adenovirus of (1), wherein the exogenous gene comprises: HLA protein coding sequence, marker polypeptide coding sequence, HLA protein coding sequence and beta 2-microglobulin coding sequence, or HLA protein coding sequence, beta 2-microglobulin coding sequence and marker polypeptide coding sequence.

(12) The oncolytic adenovirus of (11), wherein said HLA protein comprises HLA class I molecules comprising HLA-A, HLA-B and HLA-C.

(13) The oncolytic adenovirus of (12), wherein the HLA-C comprises a wild-type molecule, or at least one of the following mutations: 1) arginine at position 2 is mutated to alanine; 2) the 4 th nucleotide of the nucleotide sequence for coding the HLA-C protein is mutated from C to G, and the 5 th nucleotide is mutated from G to C; 3) isoleucine at position 362 mutated to threonine; 4) glutamic acid 359 th was mutated to valine.

(14) The oncolytic adenovirus of (11), wherein the marker polypeptide comprises the following amino acid sequences in operable linkage, in serial order: an amino acid sequence of an N-terminal signal peptide, an amino acid sequence of one or more epitope polypeptides, and optionally an amino acid sequence of a C-terminal endoplasmic reticulum retention signal, wherein when said tag polypeptide comprises a plurality of amino acid sequences of said epitope polypeptides, the amino acid sequences of each two adjacent said epitope polypeptides are linked by an amino acid sequence of a cleavable linker polypeptide; preferably, the cleavable linker polypeptide is a furin cleavage recognition polypeptide.

(15) The oncolytic adenovirus of (14), wherein the amino acid sequence of the epitope polypeptide is derived from the amino acid sequence of a naturally occurring protein or is an artificially synthesized amino acid sequence not occurring in nature; preferably, the naturally occurring protein includes a protein of human origin and proteins of other species than human.

(16) The oncolytic adenovirus of (14), wherein the amino acid sequence of the epitope polypeptide is derived from the amino acid sequence of a tumor-associated antigen or a tumor-specific antigen.

(17) The oncolytic adenovirus according to (16), wherein the tumor-associated antigen is selected from the group consisting of NY-ESO-1157-165, NY-ESO-11-11, NY-ESO-153-62, NY-ESO-118-27, Her2/neu 369-377, SSX-241-49, MAGE-A4230-239, MAGE-A10254-262, MAGE-C2336-344, MAGE-C2191-200, MAGE-C2307-315, MAGE-C242-50, MAGE-A1120-129, MAGE-A1230-238, MAGE-A1-169, KK-LC-176-84, p 5399-107, PRAME 301-309, alpha-fetoprotein 158-166, HPV 16-E62938, HPV 16-E-19, EBV-P151-59, and E-19, EBV-LMP 1125-133, KRAS: G12D 10-18, KRAS: G12D 8-16, KRAS: G12D 7-16, KRAS: G12C 8-16, KRAS: G12A 8-16, KRAS: G12S 8-16, KRAS: G12R 8-16, KRAS: G12V 8-16, KRAS: G12V 7-16, KRAS: G12V 5-14, KRAS: G12V 11-19, KRAS: G12V 5-14, KRAS: Q61H 55-64, KRAS: Q61L 55-64, KRAS: Q61R55-64, KRAS: G12D 5-14, KRAS: G13D 5-14, KRAS: G12A 5-14, KRAS: G12C 5-14, KRAS: G12S 5-14, KRAS: G12R 5-14, KRAS: G12D 10-19, TP 53: V157G 156 164, TP 53: R248Q 240-249, TP 53: R248W 240-249, TP 53: G245S 240-249, TP 53: V157F 156 164, TP 53: V157F 149-158, TP 53: Y163C 156-164, TP 53: R248Q 247-255 and TP 53: R248Q 245 254, TP 53: R248W 245 254, TP 53: G245S 245-254, TP 53: G249S 245-254, TP 53: Y220C 217-225, TP 53: R175H 168-176, TP 53: R248W 240-249, TP 53: K132N-134, CDC 73: Q254E 248-256, CYP2A 6: N438Y 436-444, CTNNB 1: T41A 41-49, CTNNB 1: S45P 41-49, CTNNB 1: T41A 34-43, CTNNB 1: S37Y 30-39, CTNNB 1: S33C 30-39, CTNNB 1: S45P 40-49, EGFR: L858R 852-: T790M 790 799, PIK3 CA: E542K 533-: H1047R1046-1055, GNAS: R201H 197-205, CDK 4: R24C 23-32, H3.3: K27M26-35, BRAF: V600E 591-: k73Rfs 141-148, NRAS: Q61R55-64, IDH 1: R132H 126-135, TVP 23C: C51Y 51-59, TVP 23C: C51Y42-51 and TVP 23C: C51Y 45-53.

(18) The oncolytic adenovirus according to (14), wherein the epitope polypeptide is NY-ESO-1157-165 as shown in SEQ ID NO.10 or KRAS as shown in SEQ ID NO. 11: G12D 10-18.

(19) A vector for preparing the oncolytic adenovirus of any one of (1) - (18), wherein the vector comprises the E1B gene regulatory element, lacks the E1B gene coding region, and comprises the cDNA sequence of E1A transcribing E1A 13s mRNA upstream of the foreign gene.

(20) A therapeutic agent for treating a tumor and/or cancer, comprising:

(a) a first composition, wherein the first composition comprises a first active ingredient comprising or comprising an oncolytic adenovirus according to any one of (1) - (18) for introduction into a tumor cell and/or a cancer cell in a first pharmaceutically acceptable carrier; and

(b) a second composition, wherein the second composition comprises a second active ingredient comprising a T cell receptor modified immune cell in a second pharmaceutically acceptable carrier.

(21) The therapeutic agent of (20), wherein the first composition and the second composition are each independently present in the therapeutic agent without intermixing.

(22) The therapeutic agent according to (20), wherein the immune cell comprises a primitive T cell or a precursor cell thereof, an NKT cell, or a T cell line.

(23) The therapeutic agent of (20), wherein the first composition comprises a therapeutically effective amount of the oncolytic adenovirus.

(24) The therapeutic agent according to (20), wherein the second composition comprises a therapeutically effective amount of the T cell receptor-modified immune cell.

(25) The therapeutic agent according to (20), wherein the oncolytic adenovirus is formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid intracavity administration, or intravenous administration.

(26) The therapeutic agent according to (20), wherein the immune cell is formulated to be administered intra-arterially, intravenously, subcutaneously, intradermally, intratumorally, intralymphatically, subarachnoid cavity, intramedullally, intramuscularly or intraperitoneally.

(27) Use of an oncolytic adenovirus according to any one of (1) - (18) for the manufacture of a medicament for the treatment of a tumor and/or cancer.

(28) The use of (27), wherein the tumor and/or cancer comprises: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

(29) Use of a therapeutic agent according to any one of (20) - (26) in the manufacture of a medicament for the treatment of a tumor and/or cancer.

(30) The use of (29), wherein the tumor and/or cancer comprises: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

(31) A method for treating a tumor and/or cancer, comprising administering an oncolytic adenovirus according to any one of (1) - (18) to a tumor and/or cancer patient.

(32) The method of (31), wherein the tumor and/or cancer comprises: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

(33) A method of treating a tumor and/or cancer, comprising:

administering a first composition of the therapeutic agents according to any one of (20) - (26) to a tumor and/or cancer patient; and

administering to the tumor and/or cancer patient a second composition of the therapeutic agents according to any one of (20) - (26).

(34) The method according to (33), comprising the steps of, in order:

1) administering the first composition to the tumor and/or cancer patient; and

2) administering a second composition of said therapeutic agents to said tumor and/or cancer patient after administering said first composition.

(35) The method of (34), wherein the tumor and/or cancer comprises: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

Compared with the prior art, the invention has the following advantages and positive effects:

the oncolytic adenovirus for expressing the exogenous gene constructed by the invention can be selectively replicated in tumor cells and/or cancer cells, and can express the exogenous gene more efficiently. Specifically, the invention utilizes the self E1B gene regulatory element in the genome of the oncolytic virus to regulate the expression of the exogenous gene, and avoids the possible interference of the inserted exogenous gene regulatory element on the expression of the genome of the virus to influence the effective replication of the virus and the expression of downstream genes. In addition, the length of the inserted foreign gene fragment can also be increased, so that the oncolytic viral vector can carry more foreign gene loads. In another aspect, the oncolytic adenovirus constructed in accordance with the present invention lacks the coding region of the E1B gene. The E1B-19K protein can inhibit apoptosis induced by tumor necrosis factor and FAS pathway, so that infected cells are resistant to killing by T cells. Removal of the E1B-19K gene increases the sensitivity of infected tumor cells to killer T cells. Removal of the E1B-55K gene increases the tumor cell oncolytic selectivity of oncolytic adenoviruses. In addition, the present inventors have found that, in the genome of oncolytic adenovirus, by designing the cDNA sequence of E1A transcribing E1A-13S mRNA upstream of the foreign gene instead of the E1A genomic gene, the present invention can transcribe only E1A-13S while avoiding the transcription of E1A-12S, thereby increasing the expression of the foreign gene and enhancing the replication of the viral genome.

Compared with similar oncolytic adenoviruses for expressing exogenous genes in the prior art, the oncolytic adenovirus for expressing exogenous genes constructed by the invention can more effectively introduce nucleic acid with a marked polypeptide coding sequence into tumor cells and/or cancer cells through ingenious design, and more efficiently express exogenous marked polypeptides in the tumor cells and/or the cancer cells, particularly epitope polypeptides derived from genetically mutated nascent antigens (neo-antigens), so that the epitope polypeptides enter an MHC I antigen presentation pathway, the expression quantity of HLA/antigen epitope polypeptide complexes on the surface of the tumor cells is remarkably increased, and the recognition sensitivity of immune cells modified by T cell receptors to the tumor cells and/or the cancer cells is further enhanced. In addition, the tumor cells are marked by a xenogenic HLA class I molecule (allo-HLA class I) carried by the fusogenic virus vector, so that the application range of the adoptive T cell therapy is greatly increased.

The oncolytic adenovirus for expressing the exogenous gene constructed by the invention can more efficiently express exogenous epitope peptide, beta 2-microglobulin and/or exogenous MHC class I molecules in tumor cells so as to obviously enhance the number of epitope peptide/MHC class I molecule complexes on the surface of the tumor cells, and can adopt immune cells specifically aiming at TCR modification of the epitope peptide to carry out combined treatment. When the oncolytic adenovirus is used as a vector to mediate the expression of exogenous antigen epitope peptide, beta 2-microglobulin and/or exogenous MHC I molecules in tumor cells, the killing of the oncolytic virus to the tumor and the killing of TCR modified immune cells to the tumor can generate a synergistic treatment effect.

The constructed oncolytic adenovirus guides the labeled polypeptide, the beta 2-microglobulin and/or HLA protein coding nucleic acid into the tumor cells and/or cancer cells, plays a role of killing the tumor cells and/or cancer cells by the oncolytic adenovirus, and further enhances the synergistic treatment effect achieved by the combination of the presentation of the exogenous antigen epitope peptide on the surface of the tumor cells and the immune cells modified by T cell receptors. In addition, the oncolytic virus can relieve the immunosuppression state of a tumor microenvironment while killing tumors, and improve the homing of T cell receptor modified immune cells; in addition, the immune cells modified by the T cell receptor can effectively eliminate tumor cells which cannot complete replication cycle and generate enough quantity of subviral to be cracked after being infected by the oncolytic virus; thereby achieving a further synergistic effect. In addition, the antigen released by the tumor cells lysed by the oncolytic virus can further activate the anti-tumor immunity of the organism, so that a better tumor killing effect can be realized than that of the immune cells modified by the oncolytic virus or a T cell receptor alone, and a synergistic treatment effect is realized.

Definition of

In the present invention, the words "tumor", "cancer", "tumor cell", "cancer cell" encompass meanings commonly recognized in the art.

The phrase "oncolytic virus" as used herein refers to a virus that is capable of selectively replicating and lysing tumor cells in a tumor cell.

The phrase "therapeutically effective amount" as used herein refers to an amount of a functional agent or pharmaceutical composition that is capable of exhibiting a detectable therapeutic or inhibitory effect, or that exerts an anti-tumor effect. The effect can be detected by any assay known in the art.

The words "administration" or "administering" as used herein refer to providing a compound, complex or composition (including viruses and cells) to a subject.

The word "patient" as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. In some embodiments, the patient has a tumor. In some instances, the patient is concurrently suffering from one or more types of cancer.

The phrase "synergistic effect" as used herein refers to an effect that two or more agents together exert, the effect being greater than the sum of the individual effects of each of the agents.

According to the present invention, "abnormal expression" or "abnormal expression" means that expression is altered, preferably increased, in comparison to non-tumorigenic normal cells or healthy individuals (i.e., individuals without disease) in connection with abnormal or abnormal expression of certain proteins, such as tumor-associated antigens. An increase in expression means an increase of at least 10%, in particular at least 20%, at least 50% or at least 100% or more. In one embodiment, expression is found only in diseased tissue, while expression is inhibited in healthy tissue.

As used herein, the term "about" refers to a value or range of values within 20%, or in some cases within 10%, or in some cases within 5%, or in some cases within 1%, or in some cases within 0.1% because such variations are suitable for performing the disclosed methods or for the intended purpose of the disclosed compositions.

The term "active ingredient" refers to an ingredient in a pharmaceutical product composition that is biologically active or has the intended pharmaceutical effect.

The term "adoptive cell transfer" or "adoptive cell therapy" or "ACT" refers to an immunotherapy in which the subject or patient's own immune cells (e.g., autologous T cells) or immune cells from a healthy donor (e.g., allogeneic T cells) are collected to treat their cancer. TCR-T cell therapy is one of the ACT.

As used herein, the term "and/or" includes "and", "or" and "all or any other combination of elements connected by the term" are meant.

The term "anti-tumor" refers to a biological effect that can be manifested in a variety of ways, including, but not limited to, reduction in tumor volume, reduction in the number of tumor cells, reduction in the number of tumor cell metastases, increase in life expectancy, reduction in tumor cell proliferation, reduction in tumor cell survival, or improvement in various physiological symptoms associated with a cancer condition, for example. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to first prevent tumorigenesis.

As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins and may be derived from natural or recombinant sources. The antibody may be a tetramer of immunoglobulin molecules.

As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. Such an immune response may involve the production of antibodies, or the activation of specific immunologically-competent cells, or both. The skilled person will appreciate that any macromolecule, including virtually all proteins or peptides, may be used as an antigen. Furthermore, the antigen may be derived from recombinant DNA or genomic DNA. It will be understood by those skilled in the art that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein which elicits an immune response encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will appreciate that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It is apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Furthermore, the skilled person will understand that an antigen need not be encoded by a "gene" at all. It will be apparent that the antigen may be synthetically produced or may be derived from a biological sample, or may be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or fluids with other biological components.

The term "antigen presentation mechanism" (or "antigen presentation mechanism") refers to an immune molecule or cell that processes and prepares an antigen for presentation to a T lymphocyte. The antigen presentation mechanism involves two different pathways for the processing of antigens from the organism's own (self) proteins or intracellular pathogens (e.g. viruses) or phagocytic pathogens (e.g. bacteria); subsequent presentation of these antigens on class I or class I Major Histocompatibility Complex (MHC) molecules depends on the route used. Both MHC class I and II must bind antigen before they can be stably expressed on the cell surface. MHC I antigen presentation generally involves the endogenous pathway of antigen processing, while MHC II antigen presentation involves the exogenous pathway of antigen processing.

The term "autologous" refers to any material that is derived from the same individual and subsequently reintroduced into the individual.

The term "beta 2-microglobulin" or "beta₂Microglobulin "is a component of MHC class I molecules with α 1, α 2 and α 3 proteins present on all nucleated cells (excluding erythrocytes). In humans, β 2 microglobulin is encoded by the B2M gene.

The term "C-terminus" (also referred to as C-terminus, carboxy-terminus, C-terminal tail, C-terminus or COOH-terminus) refers to the terminus of an amino acid chain (protein or polypeptide) that terminates with a free carboxy group (-COOH). When proteins are translated from messenger RNA, they are produced from the N-terminus to the C-terminus. The common practice for writing peptide sequences is to insert the C-at the right end into the sequence from the N-terminus to the C-terminus.

As used herein, the term "combination" refers to any arrangement of the various components (e.g., an oncolytic virus and one or more substances effective in anti-cancer therapy) that is possible. Such an arrangement includes mixtures of the components as well as individual combinations for simultaneous or sequential administration. The present invention includes combinations having different effective dosages. It will be appreciated that the optimum dosage of each component of the combination may be determined by one skilled in the art.

As used herein, the term "composition" or "pharmaceutical composition" refers to a chemical and/or biological composition suitable for administration to a subject or patient with an intended pharmaceutical effect (e.g., prophylactic and therapeutic effect). Examples of compositions suitable for such therapeutic applications include formulations for parenteral, subcutaneous, transdermal, intradermal, intramuscular, intracoronary, intramyocardial, intracerebral, intratumoral, intraperitoneal, intravenous (e.g., injectable) or intratracheal administration, such as sterile suspensions, emulsions and aerosols. Intratracheal administration may involve contacting or exposing lung tissue (e.g., alveoli) to a therapeutic agent comprising a therapeutically effective amount of nucleic acid and/or immune cells (e.g., T cells (e.g., TCR-modified T cells)) in a pharmaceutical carrier. In some cases, a pharmaceutical composition suitable for therapeutic use may be mixed with one or more pharmaceutically acceptable excipients, diluents, or carriers (e.g., sterile water, saline, dextrose, etc.).

As used herein, the terms "comprising," "having," "including," or "containing" when used to define products, compositions, and methods are open-ended and do not exclude additional unrecited elements or method steps. Thus, a polypeptide "comprises" an amino acid sequence when that amino acid sequence may be part of the final amino acid sequence of the polypeptide. Such polypeptides may have up to several hundred additional amino acid residues. "consisting essentially of means excluding any other component or step of significance. Thus, a composition consisting essentially of the components will not exclude trace contaminants and pharmaceutically acceptable carriers. When such an amino acid sequence is present, the polypeptide "consists essentially of" the amino acid sequence, eventually having only a few additional amino acid residues. "consisting of" means a trace element or step that excludes other components. For example, a polypeptide "consists of" an amino acid sequence when the polypeptide does not contain any other amino acids than the recited amino acid sequence.

The term "conditionally replication competent virus" or "conditionally replicating virus" or "CRV" or "selectively replicating virus" refers to a virus that is designed to be able to selectively replicate in tumor cells, causing their destruction, while retaining normal cells.

The term "constitutive promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, results in the production of the gene product in a cell under most or all of the physiological conditions of the cell.

The term "death receptor" refers to a member of the tumor necrosis factor receptor superfamily, characterized by a cytoplasmic region called the "death domain" that enables the receptor to initiate cytotoxic signals when bound to a cognate ligand.

The terms "obtained from", "derived from" or "derived from" are used to identify the original source of a component (e.g., a polypeptide, nucleic acid molecule, amino acid sequence), but are not intended to limit the method by which the component is prepared, which may be, for example, by chemical synthesis or recombinant means.

The term "pharmaceutically acceptable" or "pharmaceutically acceptable" means suitable for administration to a patient or subject to achieve the intended drug or drug effect without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio, for example.

As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable vehicle" or "pharmaceutically acceptable carrier" refers to a vehicle or carrier for administration of a therapeutic agent that is suitable for humans and/or mammals without undue adverse side effects ((e.g., toxicity, irritation, and allergic) and at a reasonable benefit/risk ratio.

The term "encoding" when applied to a polynucleotide refers to a polynucleotide that, if manipulated in its native state or by methods well known to those skilled in the art, can "encode" a polypeptide, which can be transcribed and/or translated to produce mRNA for the polypeptide and/or fragments thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom. A nucleic acid or nucleic acid sequence "encoding" a peptide refers to a nucleic acid comprising the coding sequence for the peptide. An amino acid sequence "encoding" a peptide refers to an amino acid sequence that contains a peptide sequence.

The term "endoplasmic reticulum retention signal sequence" ("endoplasmic reticulum retention signal sequence") refers to a signal sequence that retains a protein in the endoplasmic reticulum or ER after folding into an ER retention protein. The classical ER retention signal is the C-terminal KDEL (Lys-Asp-Glu-Leu) sequence.

The term "antigenic epitope", "epitope" or "antigenic determinant" refers to the part of an antigen that is recognized and bound by the immune system (in particular antibodies, B cells or T cells). The term "epitope peptide" or "antigenic epitope peptide" refers to an epitope or antigenic epitope in the form of a peptide.

As used herein, the term "excipient" or "additive" is intended to mean all substances in a pharmaceutical formulation that are not active ingredients, such as carriers (e.g., vector DNA, plasmids, vector viruses), binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents or coloring agents.

The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system. For example, the term "exogenous HLA protein" as used herein refers to an HLA protein from outside of a subject or patient, and the "exogenous HLA protein" may or may not be produced by a cell or tissue of the subject or patient.

The term "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue or system.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and/or the process by which transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may comprise splicing of the mRNA in a eukaryotic cell.

As used herein, "gene expression" refers to the process of making a functional gene product (e.g., a protein or RNA) from the genetic information, the sequence of DNA base pairs, in a gene. The basic process is the transcription of DNA into RNA, followed by translation of RNA into protein.

The term "Human Leukocyte Antigen (HLA)" refers to a complex or system of genes encoding human Major Histocompatibility Complex (MHC) proteins, also referred to as "HLA proteins". MHC proteins are cell surface proteins responsible for the regulation of the human immune system. The HLA gene complexes are located on a 3Mbp fragment on chromosome 6p21 and are highly polymorphic, meaning that they have many different alleles that can fine-tune the adaptive immune system. HLA corresponds to MHC class I (a, B and C), presents foreign antigens (e.g., viral antigens) from within the cell to the T lymphocytes, and the HLA class I/antigen peptide complex stimulates cytotoxic T cells (also known as CTLs), which in turn kill the target cell.

The term "HLA class I protein" refers to human MHC class I proteins or molecules. MHC class I molecules are transmembrane proteins, composed of a single alpha chain, and associated with β 2-microglobulin, which can be correctly folded and transported to the cell surface.

The term "immune cell" refers to a cell of the immune system, which can be classified as a lymphocyte (T cell, B cell and NK cell), neutrophil, and monocyte/macrophage. These are all types of leukocytes.

The term "immune danger signal" means that when tissue cells become stranded due to injury, infection, etc., they begin to secrete or express molecules on their surface that represent a "danger", or that components of the invading organism (e.g., viral DNA or RNA) are also sensitized by the immune system as danger signals.

The term "immunogen" refers to a specific type of antigen that is capable of eliciting an immune response.

The term "Tumor Microenvironment (TME)" or "immunosuppressive tumor microenvironment" refers to the environment surrounding a tumor, including surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and extracellular matrix (ECM). The tumor is closely associated with the surrounding microenvironment and constantly interacts. Tumors can suppress immune responses by releasing extracellular signals, promote tumor angiogenesis and induce peripheral immune tolerance, affecting the microenvironment, while immune cells in the microenvironment can influence the growth and evolution of cancer cells.

Terms such as "increase" or "enhancing" preferably relate to increasing or enhancing by about at least 5%, preferably at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, still more preferably at least 100%. These terms may also relate to the case where there is no detectable signal of a certain compound or condition at zero time and there is a detectable signal of a certain compound or condition at a particular point in time later than zero.

The term "inducible promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, results in the production of the gene product in a cell substantially only when an inducing agent corresponding to the promoter is present in the cell.

As used herein, the term "isolated" refers to a cell, protein, polypeptide, peptide, polynucleotide, vector, or the like that is removed from its natural environment (i.e., separated from at least one other component with which it is naturally associated or found in nature).

A lentiviral vector is a retrovirus that infects both dividing and non-dividing cells because their pre-integration complex (the viral "shell") can cross the entire membrane of the target nucleus. Lentiviral vectors are derived from human immunodeficiency virus.

As used herein, "linker peptide" or "linker sequence" refers to an amino acid sequence that links two other amino acid sequences. For example, a portion of an HLA class I protein may be linked to a portion of a tumor associated antigen sequence, e.g., an epitope sequence, via a linker sequence.

The term "loss of heterozygosity" or "LOH" refers to the loss of contribution of a parent to a cell, possibly due to direct deletion, due to unbalanced rearrangement, gene transformation, mitotic recombination alpha, or chromosome loss (monochromosomes).

The term "major histocompatibility complex" or "MHC" refers to a group of genes that encode cell surface proteins essential for the resulting immune system to recognize foreign molecules in vertebrates, which in turn determines histocompatibility. MHC molecules, which mediate the interaction of White Blood Cells (WBCs) with other white blood cells, bind to antigens produced by pathogens and are displayed on the cell surface for recognition by appropriate T cells. MHC determines the compatibility of donor organ transplants, as well as the sensitivity of the immunity to autoimmune diseases by cross-reactivity, and human MHC is also known as HLA (human leukocyte antigen) complex (often abbreviated as HLA). MHC is a tissue antigen that allows the immune system, more specifically T cells, to bind, recognize and tolerate itself (auto-recognition). MHC is also a chaperone for intracellular peptides that bind to MHC as potential foreign antigens and are presented to the T Cell Receptor (TCR). MHC interacts with TCR and its co-receptor to optimize the binding conditions for TCR-antigen interaction from the point of view of antigen binding affinity and specificity and efficiency of signal transduction.

The term "MHC class I protein" or "MHC class I molecule" refers to one of two major classes of Major Histocompatibility Complex (MHC) molecules or glycoproteins or proteins (the other class being MHC class II), which are present on the cell surface of all nucleated cells in a vertebrate. MHC I proteins form functional receptors on most nucleated cells in the body. The Major Histocompatibility Complex (MHC) class I molecules are responsible for presenting peptide epitopes to cytotoxic T cells. In humans, the Human Leukocyte Antigen (HLA) system is the locus of genes encoding class I and MHC class MHC molecules. HLA-A, -B and-C genes encode MHC class I (MHCI) proteins. Peptides, typically 8-11 amino acids in length, will bind to MHC I molecules by interacting with the groove formed by the two alpha helices positioned over the antiparallel beta sheet. Processing and presentation of peptide-MHC class I (pMHCI) molecules involves a series of sequential stages, including: a) protease-mediated protein digestion; b) peptide transport into the Endoplasmic Reticulum (ER) mediated by a transporter associated with antigen processing (TAP); c) forming pMHCI using newly synthesized MHC I molecules; d) pMHCI was transported to the cell surface. On the cell surface, pMHCI will interact with cytotoxic T cells via T Cell Receptors (TCRs). Following a complex pMHCI-TCR interaction, the identification of non-self antigens may lead to cytotoxic T cell activation through a series of biochemical events mediated by associated enzymes, co-receptors, adaptor molecules and transcription factors. The activated cytotoxic T cells will proliferate to generate a large number of effector T cells that express TCRs specific for the identified immunogenic peptide epitopes. Expansion of T cells with TCR specificity for an identified non-self epitope leads to immune-mediated apoptosis, revealing an activated non-self epitope.

The term "MHC protein" refers to a protein encoded by an MHC gene.

Immunosuppressive Tumor Microenvironment (TME): the ability of tumors to promote tolerant microenvironments and the activation of multiple immunosuppressive mechanisms that may work together to counteract potent immune responses, such as impaired antigen presentation by tumors, activation of negative costimulatory signals, and complicating immunosuppressive factors.

The term "mutant" or "mutant type" refers to a strain, gene or characteristic produced or produced by a mutational instance, which is typically a change in the DNA sequence of the genome or chromosome of an organism.

The term "N-terminal signal peptide" refers to the signal peptide (typically 16-30 amino acids in length) present at the N-terminus of most newly synthesized proteins that are intended to be directed toward the secretory pathway. These proteins include proteins that reside in certain organelles (endoplasmic reticulum, golgi or endosomes), either secreted from the cell or inserted into most cell membranes. The function of the signal peptide is to cause the cell to translocate the protein, usually to the cell membrane.

The term "neoantigen" refers to a newly formed antigen that has not been previously recognized by the immune system. For example, the neoantigen may be derived from an altered tumor protein resulting from a change in a tumor gene, including point mutations, insertions/deletions, amplifications/fusions, post-translational modifications, or from a viral protein.

As used herein, "nucleic acid" is interchangeable with the term "polynucleotide" and generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA or any combination thereof. "nucleic acid" includes but is not limited to single-stranded and double-stranded nucleic acids. The term "nucleic acid" as used herein also includes DNA or RNA as described above containing one or more modified bases. Thus, a DNA or RNA having a backbone modified for stability or other reasons is a "nucleic acid". The term "nucleic acid" as used herein includes such chemically, enzymatically or metabolically modified forms of nucleic acids, as well as chemical forms of DNA and RNA that are characteristic of viruses and cells, including, for example, simple and complex cells. "nucleic acid" or "nucleic acid sequence" may also include regions of single-or double-stranded RNA or DNA, or any combination.

As used herein, the terms "coding nucleic acid", "coding nucleic acid molecule", "DNA sequence code" and "DNA code" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the amino acid sequence along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

As used herein, the term "oncolytic virus" refers to a virus that is capable of selectively replicating in tumor cells (e.g., proliferative cells such as cancer cells) to slow the growth and/or lysis of the dividing cells, in vitro or in vivo, while replicating little or no in normal cells. Typically, an oncolytic virus comprises a viral genome packaged into a viral particle (or virion) and is infectious (i.e., capable of infecting and entering a host cell or subject). As used herein, the term encompasses DNA or RNA vectors (depending on the virus in question) and viral particles produced therefrom.

The term "one or more" refers to one or more than one number (e.g., 2, 3, 4, 5, etc.).

The phrases "operably linked" or "operably linked" refer to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Generally, it refers to the functional relationship of transcriptional regulatory sequences to transcriptional sequences. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system. Typically, promoter transcriptional regulatory sequences operably linked to a transcribed sequence are physically contiguous with the transcribed sequence, i.e., they are cis-acting. However, certain transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in the vicinity of the coding sequence whose transcription is enhanced.

The terms "polypeptide", "peptide" and "protein" refer to a polymer of amino acid residues comprising a plurality of amino acids joined by peptide bonds. The polymer may be linear, branched or cyclic, and may comprise naturally occurring and/or amino acid analogs, and may be interrupted by non-amino acids.

The term "plasmid" refers to an "additional" self-replicating genetic element found in a cell. Plasmids are used in genetic engineering to produce recombinant DNA and as a mechanism for transferring genes between organisms.

Point mutations or substitutions are genetic mutations in which a single nucleotide base is altered, inserted or deleted from a DNA or RNA sequence.

The term "polyadenylation signal" or "polyadenylation signal sequence" or "polyadenylation signal sequence" refers to a sequence motif recognized by RNA cleavage complexes that varies between groups of eukaryotes. Most human polyadenylation signals contain an AAUAAA sequence.

As used herein, the term "prevention" refers to prophylactic or preventative measures, which are intended to inhibit the development of an undesirable physiological change or disorder or condition. Preventing a disease or condition can include beginning administration of T cells obtained according to the methods provided herein at a time prior to the occurrence or presence of the disease or condition (or symptoms thereof) such that the disease or condition, or a pathological feature, consequence, or adverse effect thereof, does not occur.

The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery that is required to initiate specific transcription of a polynucleotide sequence.

As used herein, the term "receptor" refers to a molecule expressed on the surface of a cell, wherein the molecule is capable of binding a cellular ligand. Receptor-ligand binding, as used herein, is preferably capable of initiating or inhibiting a biochemical pathway and/or signaling cascade when a suitable ligand binds to the receptor.

The term "recombinant" refers to an organism, cell, protein, genetic material, DNA or RNA that is related to or is formed using recombinant techniques.

The term "recombinant DNA" refers to DNA or rDNA made by combining DNA from two or more sources. DNA fragments are excised from the normal position of a chromosome using restriction enzymes (also known as restriction endonucleases) and then inserted into other chromosomes or DNA molecules using enzymes known as ligases.

The term "recombinant TCR" refers to a TCR prepared by recombinant techniques.

As used herein, the term "replication-competent" or "replication-competent virus" refers to a virus that is replication-competent or whose replication is dependent on an agent (e.g., an upregulating agent) in a cancer cell.

In the context of the present specification, the term "replication competent virus" refers to a virus having all the necessary mechanisms of replication in cells in vitro and in vivo, i.e. without the aid of a packaging cell line. In this context, a viral vector capable of replication in a complementing packaging cell line (e.g. a viral vector deleted at least in the E1A region) is not a replication-competent virus.

In the context of the present specification, the term "replication deficient virus" refers to a virus that requires replication of a packaging cell line (comprising a transgene).

The term "retroviral vector" refers to a proviral sequence that can accommodate a gene of interest to allow for the incorporation of both into a target cell. The vector may also contain viral and cellular gene promoters, such as the CMV promoter, to enhance expression of the gene of interest in the target cell.

The term "self-cleaving linker peptide" refers to a short peptide (e.g., a 18-22 amino acid long peptide) present in a protein (e.g., a recombinant protein) that can trigger self-cleavage of the protein in a cell. Cleavage begins after protein translation. The exact cleavage mechanism for self-cleaving linker peptides remains uncertain. However, it is believed to involve ribosomal "hopping" of glycylprolyl peptide bond formation rather than true proteolytic cleavage.

Self-inactivating lentiviral vectors have been used to introduce genes into mature T cells to generate immunity to cancer by delivering Chimeric Antigen Receptors (CARs) or cloned T cell receptors.

The term "self protein" refers to a protein that is normally produced by a particular organism. The immune system of a particular organism should be tolerant to self-proteins. If not, there is autoimmunity.

As used herein, the term "silencer" refers to a DNA sequence capable of binding a transcriptional regulator, referred to as a repressor. The DNA comprises genes that provide a template for the production of messenger rna (mrna). The mRNA is then translated into protein. When a repressor protein binds to a silencer region of DNA, RNA polymerase is prevented from transcribing the RNA sequence into RNA. Due to the block of transcription, RNA cannot be translated into protein. Thus, silencers prevent the expression of a gene as a protein.

The term "solid tumor" refers to an abnormal mass of tissue or tumor that generally does not contain cysts or fluid areas. Solid tumors may be benign (non-cancerous) or malignant (cancerous).

As used herein, the terms "subject" or "patient" are used interchangeably and can encompass any vertebrate, including but not limited to humans, mammals, reptiles, amphibians, and fish. Advantageously, however, the subject or patient is a mammal, such as a human, or a mammal, such as a domesticated mammal (e.g., dog, cat, horse, etc.) or livestock (e.g., cow, sheep, pig, etc.). In an exemplary embodiment, the subject is a human. As used herein, the phrase "in need of" refers to a condition of a subject in which a therapeutic or prophylactic measure is needed. Such a state may include, but is not limited to, a subject suffering from a disease or condition such as cancer.

The term "suicide gene" refers to a gene encoding a protein capable of converting a prodrug into a cytotoxic compound. Suicide genes include, but are not limited to, genes encoding proteins having cytosine deaminase activity, thymidine kinase activity, uracil phosphoribosyltransferase activity, purine nucleoside phosphorylase activity, and thymidylate kinase activity. Examples of suicide genes and corresponding precursors of drugs comprising one nucleobase moiety are disclosed in the following table.

The term "surface expression" refers to the fusion of a protein of interest with a native surface protein of a host cell. This results in the recombinant protein being transported to the host surface and subsequently displayed on the host surface.

The term "T cell" refers to a lymphocyte (hence the name) that develops in the thymus and plays a key role in the immune response. T cells can be distinguished from other lymphocytes by the presence of T cell receptors on the cell surface. "cytotoxic T cells or CD8+ T cells or killer cells are able to directly kill virus infected cells as well as cancer cells. CD8+ T cells are also able to recruit other cells upon immunization using small signaling proteins called cytokines. Helper T cells or CD4+ T cells function by indirectly killing cells that are recognized as foreign: they determine whether and how other parts of the immune system respond to a particular perceived threat.

The phrase "in tandem" refers to a spatial relationship between two or more entities, such as a polynucleotide (e.g., DNA) and a polypeptide, arranged in such a way that they are placed one after the other.

As used herein, the term "target" refers to a molecule (e.g., a protein or peptide), cell or tissue or organism against which an immune response is directed.

The term "target antigen" refers to any substance against which an immune response is desired, but typically, the target antigen is a protein or peptide. The target antigen may comprise a full-length protein or fragment thereof (i.e., an immunogenic fragment) that induces an immune response. The target antigen or fragment thereof may be modified, for example, to reduce one or more biological activities of the target antigen or to enhance its immunogenicity.

The term "target cell" refers to any cell to which an immune response is desired or which can be specifically recognized by an immune cell, such as a (T cell).

As used herein, the term "T cell receptor" or "TCR" refers to a molecule found on the surface of a T cell or T lymphocyte that is responsible for recognizing an antigen fragment as a peptide that binds to a Major Histocompatibility Complex (MHC) molecule.

Unless otherwise indicated, the technical terms herein are used according to conventional usage. Definitions of terms commonly used in molecular biology can be found in the following documents: gene VII of Rie Ming, published by Oxford university Press, 1999; edited by Kendrew et al, encyclopedia of molecular biology, Blackwell Science Ltd, published 1994; and Robert meyers, editions, molecular biology and biotechnology: integrated reference, published by VCH Publishers, inc. 1995; and other similar references.

The term "therapeutic" as used herein refers to treatment. Therapeutic effects can be obtained by reducing, inhibiting, alleviating, preventing or eradicating the disease state.

As used herein, the term "therapeutically effective dose" or "therapeutically effective amount" or "effective dose" means that the desired response, alone or in combination with other doses, results in the desired response or, in the case of treatment of a particular disease or a particular condition, the desired response is associated with inhibition of disease progression, which may include slowing of disease progression, particularly disruption of disease progression. The desired response to treat a disease or condition can also be to delay the onset of the disease or condition or to inhibit the onset of the disease or condition. The effective amount of the composition of the present invention depends on the condition or disease, the severity of the disease, various parameters of the patient including age, physiological condition, height and weight, duration of treatment, the type of concomitant therapy that may be selected, the particular route of administration and the like. If the patient does not respond adequately to the initial dose, multiple or higher doses (or higher effective doses, which may be achieved by more limited routes of administration) may be employed.

The term "therapy" (and any form of therapy, such as "treatment") refers to both therapeutic and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or pathological condition. Treating cancer may include, but is not limited to, alleviation of one or more clinical indications, diminishment of tumor growth or tumor cell proliferation, diminishment of severity of clinical indications of one or more cancer conditions, diminishment of extent of disorders, stabilization of the disease state (i.e., not worsening) of the subject, delay or slowing, halting or reversing cancer progression, and achieving partial or complete remission. Treating cancer also includes extending survival by days, weeks, months or years if compared to prognosis of treatment according to standard medical practice (without combining T cells obtained according to the methods provided herein). Subjects in need of treatment can include subjects who have or have been diagnosed with cancer, as well as subjects who are susceptible to, may develop, or are suspected of having cancer (e.g., lymphoma or multiple myeloma) or infection.

The term "tumor antigen" refers to an antigenic substance produced in a tumor cell, i.e., it triggers an immune response in a host. Tumor antigens are useful tumor markers for identifying tumor cells by diagnostic tests and are potential candidates for cancer therapy.

The term "tumor-associated antigen" refers to an antigen that is present on some tumor cells as well as on some normal cells.

"Tumor Infiltrating Lymphocytes (TILs)" refers to the subject's own natural T cells that have infiltrated the subject's tumor. It can be harvested, activated, expanded and reintroduced (e.g., re-infused) into a subject, where it can seek and destroy tumors as part of cancer or tumor therapy.

The term "tumor-specific antigen" refers to an antigen that is present on a tumor cell and not on any other cell.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The term "expression vector" includes any vector (e.g., a plasmid, cosmid, or phage chromosome) that comprises a genetic construct in a form suitable for expression by a cell (e.g., linked to a promoter). In the present specification, "plasmid" and "vector" are used interchangeably, as the plasmid is the usual form of vector. Moreover, the invention is intended to include other vectors having equivalent functions.

The term "viral vector" is defined as a virus or viral particle comprising a polynucleotide to be delivered to a host cell in vivo, ex vivo or in vitro. Examples of the viral vector include a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an alphaviral vector and the like. Alphavirus vectors, such as semliki forest virus-based vectors and Sindbis virus-based vectors, have also been developed for gene therapy and immunotherapy. See literature: "Schlesinger and Dubensky (1999) Cur. Opin. Biotechnol.5: 434-; and "Ying, et al (1999) nat. Med.5(7): 823-.

The term "vp/day" refers to daily viral particles.

The term "wild" or "wild-type" refers to a line, gene or characteristic that is ubiquitous in nature, as opposed to atypical mutant types.

Drawings

FIG. 1A shows a schematic representation of a recombinant oncolytic adenoviral construct described herein. pAdEasy-NY-A2 (i.e., pAdEasy-EF1 alpha-NY-A2) represents a replication-defective adenovirus vector expressing the marker polypeptide comprising NY-ESO-1157-165 epitope peptide and HLA-A2 protein. The backbone of this construct is derived from adenovirus type 5 genomic DNA with a deletion of the E1 region (E1 del) and a deletion of the E3 region (E3 del). In the region of E1A, an expression unit is incorporated. The expression unit comprises a marker polypeptide coding sequence and HLA-A2 protein, wherein the marker polypeptide coding sequence comprises INSL5 signal peptide (INSL5 SP), three NY-ESO-1157-165 epitope peptides (NY epitope x3) connected through Furin protease cleavage sites, a linker peptide (Furin-F2A), and an EF1 alpha promoter (EF1 alpha pro) and an SV40 poly (A) signal sequence (SV40 pA) are arranged on two sides of the expression unit. LITR and RITR represent the left Inverted Terminal Repeat (ITR) and the right inverted terminal repeat, respectively. pAd-EF1 alpha-E1A-A2-F2A-NY (i.e., pAd-EF1 alpha-E1 Ad24-A2-F2A-NY) and pAd-EF1 alpha-E1A-A2-F2A-BM (i.e., pAd-EF1 alpha-E1 Ad24-A2-F2A-BM) represent adenovirus vectors with conditional replication ability, respectively, and comprise HLA-A2 gene and a marker polypeptide encoding the epitope peptide containing NY-ESO-1157-165 or gene carrying human beta 2-microglobulin. The backbone of these constructs was genomic DNA from adenovirus type 5 with deletions in the E1 and E3 regions. The mutant E1A gene (E1A del24) with 122-129 deletions was incorporated in the E1A region and flanked by the EF-1. alpha. promoter and the endogenous E1A polyA signal (E1A pA), respectively. The expression unit encoding HLA-A2 and a marker polypeptide with an ER retention signal (KDEL) is flanked by a5 'endogenous E1B promoter (E1B pro) and a 3' pIX gene region, including the endogenous E1B/IX poly A signal sequence (E1B/IX pA). On the other hand, the E1B gene region was deleted.

Figure 1B shows a schematic of a lentiviral vector for expressing a TCR. pCDH-EF1 alpha-TCR-NY represents an HIV-based lentiviral vector expressing a TCR specific for the NY-ESO-1157-165 epitope peptide. Deletion of the U3 region enhancer (U3 del) ensured self-inactivation of the lentiviral construct. The TCR gene encodes a β chain with variable (TCR- β V) and murine constant (murine TCR- β C) sequences and an α chain with variable (TCR- α V) and murine constant (murine TCR- α C) sequences, flanked by the EF1 α promoter and the lentiviral WPRE region.

FIG. 1C shows HLA-A2 expression from 293T cells after transfection with pShuttle vector expressing the marker polypeptide comprising NY-ESO-1157-165 epitope peptide and HLA-A2 protein. 293T cells were stained with FITC-labeled anti-HLA-A2 antibody and analyzed by flow cytometry. The dark bold lines show the expression of HLA-A2 on 293T cells transduced with a vector encoding the HLA-A2 protein. The light grey line is control 293T cells transduced with empty vector. Mean fluorescence intensity (Geom mean) of the gated population (gated population) is shown on the flow cytometer plot. The left panel shows HLA-A2 expression from 293T cells transduced with pShuttle-EF1 alpha-NY-A2, the middle panel shows HLA-A2 expression from 293T cells transduced with pShuttle-EF1 alpha-E1 Ad24-A2-F2A-NY, and the right panel shows HLA-A2 expression from 293T cells transduced with pShuttle-EF1 alpha-E1 Ad 24-A2-F2A-BM.

FIG. 1D shows that JRT cells expressed NY-ESO-1 specific TCRs after lentiviral transfection, which expressed different TCRs specific for NY-ESO-1157-165 epitope peptide in the context of HLA-A2. JRT cells were transfected with recombinant lentiviruses and analyzed within 7-10 days. JRT cells were stained with APC-labeled anti-CD 8 antibody and PE-labeled NY-ESO-1157-165/HLA-A2 tetramer and analyzed by flow cytometry. The percentage of tetramer positive cells in the gated JRT cell population is shown on the flow cytometry plots. The control group shown from left to right in figure 1 is JRT cells not transduced with lentiviruses. The "TCR-NY-LY" set shown in FIG. 2 from the left is a JRT cell transfected with a lentivirus produced from pCDH-EF1 α -TCR-NY-LY. The "TCR-NY-AE" group shown in FIG. 3 from the left is JRT cells transfected with lentivirus generated from pCDH-EF1 α -TCR-NY-AE. The "TCR-NY-LI" group, shown from left to right in FIG. 4, is a JRT cell transfected with lentivirus produced from pCDH-EF1 α -TCR-NY-LI.

FIG. 2A shows that NY-ESO-1 specific TCRs can recognize the NY-ESO-1157-165 epitope peptide presented by HLA-A2 on T2 cells. Recombinant lentivirus transduced JRT cells generated from pCDH-EF1 alpha-TCR-NY-LY (JRT-TCR-NY-LY), pCDH-EF1 alpha-TCR-NY-AE (JRT-TCR-NY-AE) or pCDH-EF1 alpha-TCR-NY-LI (JRT-TCR-NY-LI) were cultured with T2 cells, which T2 cells were treated with NY-ESO-1157-165 epitope peptide series diluted 10-fold from 1. mu.g/ml in concentration for 16 hours. Cells were harvested and stained with anti-CD 69 antibody to analyze expression of CD69 by flow cytometry. The X-axis is T2 target cells loaded with a range of concentrations of NY-ESO-1157-165 peptide, and the Y-axis is CD69 in gated JRT cells⁺Percentage of cells.

FIG. 2B shows that 293T cells expressing a tagged polypeptide comprising the NY-ESO-1157-165 epitope peptide and HLA-A2 can activate the NY-ESO-1 specific TCR on JRT cells. In duplicate wells, JRT cells transduced with recombinant lentivirus to express different TCRs specific for NY-ESO-1 were incubated with 293T cells transduced with pShuttle-EF1a-NY-A2 or pShuttle-EF1a-E1Ad24-A2-NY for 16 hours, the cells were harvested and stained with anti-CD 69 antibody to analyze the expression of CD69 by flow cytometry. The X axis shows that JRT cell expression specificity is directed to different TCRs of NY-ESO-1, including JRT-TCR-NY-LY, JRT-TCR-NY-AE and JRT-TCR-NY-LI. Y-axis is CD69 in gated JRT cells⁺Percentage of cells (mean. + -. SD; n ═ 2). "control" represents 293T target cells transduced with the empty pShuttle vector; "pShuttle-EF 1 a-NY-A2" represents 293T target cells transduced with pShuttle-EF1a-NY-A2 vector; "pShuttle-EF 1a-E1Ad 24-A2-NY" represents 293T target cells transduced with pShuttle-EF1a-E1Ad24-A2-NY vector. Data were analyzed by Student's t assay, representing p<0.01, represents p<0.05。

FIG. 2C shows that exogenous HLA-A2 can present NY-ESO-1157-165 epitope peptide derived from NY-ESO-1 protein to activate NY-ESO-1 specific TCR on JRT cells. In duplicate wells, co-transfected with pCDNA3.3 vector expressing full-length NY-ESO-1 protein (pCDNA3.3-NY) and pShuttle vector expressing exogenous HLA-A2 protein (pShuttle-EF1a-E1Ad24-A2-F2A-BM)293T cells were used as target cells to stimulate JRT cells transduced with NY-ESO-1 specific TCRs. 293T cells transduced with pCDNA3.3-NY or pShuttle-EF1a-E1Ad24-A2-F2A-BM alone were negative controls. As a positive control, 293T cells transduced with pShuttle-EF1a-E1Ad24-A2-F2A-BM alone and loaded with NY-ESO-1157-165 epitope peptide concentration at 1. mu.g/ml were used. Cells were harvested within 16 hours, stained with anti-CD 69 antibody and analyzed by flow cytometry. The X-axis shows that JRT cells express different TCRs specific to NY-ESO-1, including JRT-TCR-NY-LY, JRT-TCR-NY-AE and JRT-TCR-NY-LI. Y-axis is CD69 in gated JRT cells⁺Percentage of cells (mean. + -. SD; n ═ 2). "pCDNA3.3-NY" represents 293T target cells transduced with pCDNA3.3-NY only; "pShuttle-EF 1a-E1Ad 24-A2-F2A-BM" represents 293T target cells transduced only by pShuttle-EF1a-E1Ad 24-A2-F2A-BM; "pShuttle-EF 1a-E1Ad24-A2-F2A-BM + pCDNA3.3-NY" represents 293T target cells co-transduced with pShuttle-EF1a-E1Ad24-A2-F2A-BM and pCDNA3.3-NY; "pShuttle-EF 1a-E1Ad24-A2-F2A-BM + NY-ESO-1 polypeptide" represents target 293T cells transduced with pShuttle-EF1a-E1Ad24-A2-F2A-BM and loaded with NY-ESO-1157-165 peptide concentration. Data were analyzed by Student's t assay, representing p<0.01, represents p<0.05。

FIG. 2D shows that nucleic acids encoding foreign peptides and proteins from recombinant oncolytic adenoviral DNA can transduce 293T cells to express tagged polypeptides comprising NY-ESO-1157-165 epitope peptide and foreign HLA-A2. 293T cells were transduced with recombinant oncolytic adenoviral vectors pAd-EF1a-E1Ad24-A2-NY and pAd-EF1a-E1Ad24-A2-BM and used as target cells to stimulate JRT cells expressing NY-ESO-1 specific TCR. In duplicate wells, the mixed cultured cells were incubated for 16 hours and harvested for analysis of CD69 expression by flow cytometry. The X axis shows that JRT cells express different TCRs specific for NY-ESO-1, including JRT-TCR-NY-LY and JRT-TCR-NY-AE. Y-axis is CD69 in gated JRT cells⁺Percentage of cells (mean. + -. SD; n ═ 2). "control" is 293T cells that were not transfected; "pAd-EF 1a-E1Ad 24-A2-F2A-NY" and "pAd-EF 1a-E1Ad 24-A2-F2A-BM" represent 293T target cells transduced with pAd-EF1a-E1Ad24-A2-F2A-NY or pAd-EF1a-E1Ad24-A2-F2A-BM, respectively. "pAd-EF 1a-E1Ad24-A2-F2A-BM + pCDNA3.3-NY" representspAd-EF1a-E1Ad24-A2-F2A-BM and pCDNA3.3-NY co-transduced 293T target cells; "pAd-EF 1a-E1Ad24-A2-F2A-BM + NY-ESO-1 polypeptide" represents 293T target cells transduced with pAd-EF1a-E1A d24-A2-F2A-BM and loaded with NY-ESO-1157-165 peptide concentration. Data were analyzed by Student's t assay, representing p<0.01, represents p<0.05。

FIG. 3 shows that tumor cells can be sensitized by NY-ESO-1 specific TCR on JRT cells recognizing a labeled polypeptide comprising an epitope peptide of NY-ESO-1 and exogenous HLA-A2. Tumor cell lines A375, SKOV3 and SKOV3-NY (SKOV 3 cells transduced with pCDNA3.3-NY and stably expressing the NY-ESO-1 protein) were transduced with pShuttle-A2-F2A-NY or pShuttle-A2-F2A-BM and used as target cells for the stimulation of JRT cells expressing NY-ESO-1 specific TCRs. In duplicate wells, tumor cells and JRT cells were cultured in mixed culture for 16 hours, and cells were harvested for analysis of CD69 expression by flow cytometry. The X-axis shows that JRT cells express different TCRs specific for NY-ESO-1, including JRT-TCR-NY-LY and JRT-TCR-NY-AE. Y-axis is CD69 in gated JRT cells⁺Percentage of cells (mean. + -. SD; n ═ 2). "A375", "SKOV 3" and "SKOV 3-NY" represent untransduced target cells; "A375-pShuttle-A2-F2A-BM", "SKOV 3-pShuttle-A2-F2A-BM" and "SKOV 3-NY-pShuttle-A2-F2A-BM" represent tumor target cells transduced with pShuttle-A2-F2A-BM; "A375-pShuttle-A2-F2A-NY", "SKOV 3-pShuttle-A2-F2A-NY" and "SKOV 3-NY-pShuttle-A2-F2A-NY" represent tumor target cells transduced with pShuttle-A2-F2A-NY. Data were analyzed by Student's t assay, representing p<0.01, represents p<0.05。

FIG. 4A shows that PBMCs transfected with recombinant lentiviruses encoding NY-ESO-1-specific TCRs can express TCRs specific for the NY-ESO-1157-165 peptide in the context of HLA-A2. PBMC cells were infected with freshly prepared lentiviruses encoding different NY-ESO-1 specific TCRs, including TCR-NY-LY (left panel), TCR-NY-AE (middle panel) and TCR-NY-LI (right panel). Cells were harvested within 7-10 days, stained with APC-labeled anti-CD 8 antibody and PE-labeled NY-ESO-1157-165/HLA-A2 tetramer, and analyzed by flow cytometry. Flow cytometry plots showing CD8 in gated lymphocyte populations based on forward scatter and side scatter⁺Tetramers⁺Cells and CD8^-And tetramer⁺Percentage of cells.

FIG. 4B shows that tumor cells can be sensitized by a tagging polypeptide comprising an NY-ESO-1 epitope peptide and/or exogenous HLA-A2 to be recognized by an NY-ESO-1 specific TCR on primary T cells. Tumor cell lines A375, SKOV3 and SKOV3-NY were transduced with pShuttle-A2-F2A-NY or pShuttle-A2-F2A-BM and used as target cells to stimulate HLA-A2-PBMC cells expressing NY-ESO-1 specific TCR. In duplicate wells, tumor cells and PBMC cells were cultured in mixed culture for 24 hours at an E: T ratio of 10: 1. After incubation, supernatants were collected to assess IFN-. gamma.secretion by T cells. X-axis shows PBMC cells expressing different TCRs against NY-ESO-1, including PBMC-TCR-NY-LY, PBMC-TCR-NY-AE and PBMC-TCR-NY-LI. The Y-axis shows the concentration of IFN- γ produced by specific T cells (mean ± SD; n ═ 2). "A375", "SKOV 3" and "SKOV 3-NY" represent untransduced target cells; "A375 + A2 BM", "SKOV 3+ A2 BM" and "SKOV 3-NY + A2 BM" represent tumor target cells transduced with pShuttle-A2-F2A-BM; "A375 + A2 NY", "SKOV 3+ A2 NY" and "SKOV 3-NY + A2 NY" represent tumor target cells transduced with pShuttle-A2-F2A-NY; "A375 + A2BM + NY polypeptide", "SKOV 3+ A2BM + NY peptide" and "SKOV 3-NY + A2BM + NY polypeptide" represent tumor target cells transduced with pShuttle-A2-F2A-BM and loaded with a concentration of NY-ESO-1157-165 peptide of 1. mu.g/ml. Data were analyzed by Student's t assay, representing p <0.01 and p < 0.05.

FIG. 4C shows that more tumor cell lines can be sensitized by a marker polypeptide containing NY-ESO-1 epitope peptide and exogenous HLA-A2 and recognized by NY-ESO-1 specific TCR on primary T cells. Tumor cell lines A549, H1299 and HOS-C1 ("A549-NY", "H1299-NY" and "HOS-NY", respectively) were transduced with pShuttle-NY-A2 and used as target cells to stimulate PBMC cells transduced with the NY-ESO-1 specific TCR gene. Tumor cells and PBMC cells were cultured in triplicate wells in a mixed culture for 24 hours at an E: T ratio of 5: 1. After incubation, supernatants were collected to assess IFN-. gamma.secretion by T cells. The X-axis shows the combination of effector and target cells and the Y-axis shows the concentration of IFN- γ produced by a particular T cell (mean ± SD; n ═ 3). Mock effector cells were PBMC cells transduced with empty lentiviruses. Other effector cells are PBMC transduced with the TCR-NY-LY gene and PBMC transduced with the TCR-NY-LI. Data were analyzed by Student's t assay, representing p <0.01 and p < 0.05.

FIG. 5 shows the results of cell surface HLA-A2 expression detection by flow cytometry 48 hours after SKOV3 cells were infected with recombinant oncolytic adenovirus OAd-NY/A2 of different MOI in example 5 of the present invention. The horizontal axis represents the MOI of the recombinant oncolytic adenovirus OAd-NY/A2, and the vertical axis represents the expression percentage of HLA-A2.

FIG. 6 shows the results of IFN-. gamma.release from Mock-T or TCR-T targeting NY-ESO-1 co-cultured with SKOV3 or SKOV3 infected with recombinant oncolytic adenovirus OAd-NY/A2 in example 6 of the present invention. FIG. 6A shows the results of human melanoma cell line A375(NY-ESO-1 positive and HLA-A2 positive), FIG. 6B shows the results of human lung cancer cell line H1299(NY-ESO-1 positive and HLA-A2 negative), FIG. 6C shows the results of human ovarian cancer cell line SKOV3(NY-ESO-1 negative and HLA-A2 negative), and FIG. 6D shows the results of human sarcomatosis cell line HOSC1(NY-ESO-1 weak positive and HLA-A2 positive). In the figure, "Mock-T" represents a group of T cells expressing GFP (control group), "TCR-T" represents a group of T cells expressing a TCR targeting NY-ESO-1. The abscissa of the graph represents the different experimental groups and the ordinate represents the IFN-. gamma.concentration (pg/ml).

FIG. 7 shows the results of in vitro combined killing of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1 against human ovarian cancer cell line SKOV3 in example 7 of the invention. FIG. 7A shows the results of real-time killing, with time after tumor cell plating (i.e., after the start of the experiment) on the abscissa, and normalized cell index on the ordinate in hours (h), and the vertical downward arrows in the figure indicate the time points of addition of OAd-NY/A2 and Mock-T or TCR-T, respectively. FIG. 7B shows the calculated tumor growth inhibition rate of the groups analyzed for the cell index at the end of the experiment (90.8 hours) of FIG. 7A, with the abscissa representing the different experimental groups and the ordinate representing the tumor growth inhibition rate IR (%).

FIG. 8 shows the results of in vitro combined killing of human lung cancer cell line H1299 by recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1 in example 8 of the invention. FIG. 8A is the results of real-time killing, with time after tumor cell plating (i.e., after the start of the experiment) on the abscissa, and cell indices normalized on the ordinate in hours (h), and the vertical downward arrows in the figure indicate the time points of addition of OAd-NY/A2 and Mock-T or TCR-T, respectively. Fig. 8B is a graph showing the calculated tumor growth inhibition ratios of the 61.14 th group of fig. 8A in which the abscissa represents the different experimental groups and the ordinate represents the tumor growth inhibition ratio IR (%).

FIG. 9 shows the results of in vitro combined killing of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1 against human osteosarcoma cell line HOS C1 in example 9 of the present invention. FIG. 9A shows the results of real-time killing, with time after tumor cell plating (i.e., after the start of the experiment) on the abscissa, and normalized cell indices in hours (h) on the ordinate, and the time points for addition of OAd-NY/A2 and Mock-T or TCR-T, respectively, as indicated by the arrows pointing vertically downward in the figure. Fig. 9B shows the calculated tumor growth inhibition ratios of the 64.44 th group of fig. 9A by analyzing the cell indices, the abscissa of the abscissa is the different experimental groups, and the ordinate is the tumor growth inhibition ratio IR (%).

Detailed Description

The present invention is further described in the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, and those skilled in the art may make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.

The invention provides a separated oncolytic adenovirus for expressing exogenous genes, wherein the oncolytic adenovirus is a selective replication type recombinant oncolytic adenovirus obtained by carrying out gene modification on adenovirus, and the genome of the recombinant oncolytic adenovirus has the following characteristics:

1) contains E1B gene regulatory elements, which include E1B promoter and E1B and pIX shared polyadenylation addition signal sequence;

2) the coding region of the E1B gene is deleted, and when the foreign gene needs to be inserted, the foreign gene is inserted at the site of the coding region of the E1B gene and is positioned behind the E1B promoter and controlled by the regulatory elements of the E1B gene;

3) upstream of the foreign gene, a cDNA sequence of E1A transcribing E1A 13s mRNA is contained, and the cDNA is wild type or Rb protein binding region deleted, the Rb protein binding region deleted is the wild type cDNA from which the nucleotide sequence shown as SEQ ID NO.7 (i.e., AC _000008.1nt 923-nt 946) is removed (the AC _000008.1 is the GenBank accession number of NCBI (i.e., national center for Biotechnology information, website: https:// www.ncbi.nlm.nih.gov)), or the Rb protein binding region deleted encodes a mutant E1A protein, the mutant E1A protein is shown as SEQ ID NO. 6.

It was found that the partial sequence of intron 2 of the E1A gene of adenovirus type 5 coincided with the downstream E1B promoter, whereas read-through transcription of the E1A mRNA was required for activation of the E1B promoter early in viral infection. If the read-through transcription of the E1A gene is terminated, the cis (in cis) expression of the downstream E1b gene is greatly reduced. Therefore, the invention utilizes the self E1B gene regulatory element in the genome of the oncolytic virus to regulate the expression of the exogenous gene, and avoids the possible interference of the inserted exogenous gene regulatory element on the expression of the viral genome to influence the effective replication of the virus. In addition, the length of the inserted foreign gene fragment can also be increased, so that the oncolytic viral vector can carry more foreign gene loads. In another aspect, the oncolytic adenovirus constructed in accordance with the present invention lacks the coding region of the E1B gene. The E1B-19K protein can inhibit apoptosis induced by tumor necrosis factor and FAS pathway, so that infected cells are resistant to killing by T cells. Removal of the E1B-19K gene increases the sensitivity of infected tumor cells to killer T cells. Removal of the E1B-55K gene increases the tumor cell oncolytic selectivity of oncolytic adenoviruses. On the other hand, in the early stages of adenoviral infection, differential splicing of the mRNA transcript of the E1A gene occurs into two major E1A proteins, E1A-12S (243R) and E1A-13S (289R). The E1A-13S (289R) protein has a unique 46 amino acids in the conserved region (CR3), although E1A-12S (243R) and E1A-13S (289R) are very similar, but show significant differences in biological activity. E1A-13S is the major viral protein involved in activating viral gene expression through its CR3 interaction with various transcription factors. Viruses expressing E1a289R are able to drive viral gene and protein expression more efficiently and replicate their genomes faster and more than adenoviruses expressing E1A-12S. In addition, co-expression of E1A-12S (lacking CR3) inhibited the transcriptional activation function of E1A-13S, whereas inhibition of transcription by E1A-12S was at its N-terminus and correlated with the ability to bind p 300/CBP. The invention discovers that in the genome of the oncolytic adenovirus, by designing the cDNA sequence of E1A for transcribing E1A-13S mRNA instead of E1A genome gene at the upstream of the exogenous gene, the invention can transcribe only E1A-13S and avoid transcribing E1A-12S, thereby increasing the expression of the exogenous gene and enhancing the replication of the viral genome.

The core site of binding of the adenoviral E1A protein to the Rb protein is Leu-122-X-Cys-X-Glu (X denotes any amino acid residue). The E1A protein lacking this amino acid sequence is unable to bind to the Rb protein, resulting in the oncolytic adenovirus selectively replicating and lysing tumor cells in tumor cells deficient in the Rb/E2F1 pathway. Thus, in the genome of the recombinant oncolytic adenovirus of the present invention, when the cDNA sequence of E1A transcribing the E1A 13s mRNA is Rb protein binding region deleted, in one embodiment, the Rb protein binding region deleted is the wild type cDNA with the nucleotide sequence shown as SEQ ID No.7 (i.e., AC _000008.1nt 923-nt 946) removed. Accordingly, the deletion of the amino acid sequence of E1A protein encoded by the Rb protein binding region-deleted cDNA was L-T-C-H-E-A-G-F (Leu-Thr-Cys-His-Glu-Ala-Gly-Phe). In another embodiment, the Rb protein binding region deleted encodes a mutant E1A protein, said mutant E1A protein being represented by SEQ ID No.6, wherein the mutation sites are L122V, C124S and E126D.

Preferably, the adenovirus is a human adenovirus type C, including human adenovirus type 2 and human adenovirus type 5.

In a preferred embodiment of the invention, the nucleotide sequence of the E1B promoter is shown in SEQ ID NO.1 (i.e., AC _000008.1nt 1336-nt 1702), and the polyadenylation addition signal sequence shared by the E1B and pIX (i.e., E1B/pIX polyadenylation addition signal sequence) is shown in SEQ ID NO.2 (i.e., AC _000008.1nt 4038-nt 4043).

In the present invention, it is preferred that the deletion of the coding region of the E1B gene includes deletion of the coding regions of the E1B-55K gene and the E1B-19K gene.

More preferably, the nucleotide sequence of the coding region of the E1B gene is shown in SEQ ID NO.3 (i.e., AC _000008.1nt 1714-nt 3509).

Preferably, the start site of the foreign gene comprises a Kozak sequence, preferably the Kozak sequence is as set forth in SEQ ID NO.4 (i.e., GCCRCC)ATGG and R are purine (A or G)).

Preferably, the nucleotide sequence of the wild-type cDNA is shown in SEQ ID NO.5 (i.e., AC _000008.1nt 560-nt 1545 minus nt 1113-nt 1228).

In a preferred embodiment of the invention, the cDNA of E1A transcribing E1A 13s mRNA is located upstream of the E1B promoter, and since the added pA signal sequence (AC _000008.1nt 1611-nt1616) and pA addition site (AC _000008.1nt 1632) of the endogenous E1A gene, as well as part of the E1A gene sequence (nt 1336-nt 1552), are contained within the E1B promoter, the cDNA of E1A transcribing E1A 13s mRNA partially coincides with the nucleotide sequence of the E1B promoter. In one embodiment, the genome of the recombinant oncolytic adenovirus comprises the nucleotide sequence shown as SEQ ID No. 2:

atgagacatattatctgccacggaggtgttattaccgaagaaatggccgccagtcttttggaccagct gatcgaagaggtactggctgataatcttccacctcctagccattttgaaccacctacccttcacgaactgtatgat ttagacgtgacggcccccgaagatcccaacgaggaggcggtttcgcagatttttcccgactctgtaatgttggcgg tgcaggaagggattgacttactcacttttccgccggcgcccggttctccggagccgcctcacctttcccggcagcc cgagcagccggagcagagagccttgggtccggtttctatgccaaaccttgtaccggaggtgatcgatccacccagt gacgacgaggatgaagagggtgaggagtttgtgttagattatgtggagcaccccgggcacggttgcaggtcttgtc attatcaccggaggaatacgggggacccagatattatgtgttcgctttgctatatgaggacctgtggcatgtttgt ctacagtcctgtgtctgaacctgagcctgagcccgagccagaaccggagcctgcaagacctacccgccgtcctaaa atggcgcctgctatcctgagacgcccgacatcacctgtgtctagagaatgcaatagtagtacggatagctgtgact ccggtccttctaacacacctcctgagatacacccggtggtcccgctgtgccccattaaaccagttgccgtgagagt tggtgggcgtcgccaggctgtggaatgtatcgaggacttgcttaacgagcctgggcaacctttggacttgagctgt aaacgccccaggccataaggtgtaaacctgtgattgcgtgtgtggttaacgcctttgtttgctgaatgagttgatgtaagtttaataaagggtgagataatgtttaacttgcatggcgtgttaaatggggcggggcttaaagggtatataatgcgccgtgggctaatcttggttacatctgacctc(SEQ ID NO.2)。

wherein, the nucleotide sequence shown as SEQ ID NO.2 comprises: the E1A cDNA transcribed from E1A 13s mRNA (underlined) with deletion of the Rb protein binding region, and the E1B promoter (italicized). And the nucleotide sequence shown as SEQ ID NO.2 is positioned between the site of an endogenous E1A promoter/enhancer or a foreign promoter and the initiation site of the foreign gene.

In some embodiments of the invention, the cDNA sequence of E1A transcribing the E1A 13s mRNA is under the control of an endogenous E1A promoter/enhancer, or under the control of an exogenous promoter; preferably, the nucleotide sequence of the endogenous E1A promoter/enhancer is shown in SEQ ID NO.8 (i.e., AC _000008.1nt 1-nt 499).

In other embodiments of the invention, the cDNA sequence of E1A transcribing the E1A 13s mRNA is under the control of an exogenous promoter. In a specific embodiment, since the starting position of the oncolytic adenoviral genome (e.g., AC _000008.1nt 1-nt 375) comprises the viral ITRs, Ad Ψ and packaging elements, a foreign promoter is inserted after these positions. In an embodiment of the human adenovirus type 5, the nucleotide sequence shown as SEQ ID No.9 (i.e., AC _000008.1nt 376-nt 559) is removed from the genome of the recombinant oncolytic adenovirus, and the exogenous promoter is inserted into the genome from the removed site.

Preferably, the exogenous promoters include the EF-1 alpha promoter, the CMV promoter, the PKG promoter, the E2F promoter, the AFP promoter, and the TERT promoter.

In a preferred embodiment of the present invention, the exogenous gene includes: HLA protein coding sequence, marker polypeptide coding sequence, HLA protein coding sequence and beta 2-microglobulin coding sequence, or HLA protein coding sequence, beta 2-microglobulin coding sequence and marker polypeptide coding sequence.

The invention refers to the situation that an HLA protein coding sequence and a marker polypeptide coding sequence, an HLA protein coding sequence and a beta 2-microglobulin coding sequence and an HLA protein coding sequence, a beta 2-microglobulin coding sequence and a marker polypeptide coding sequence are inserted into the genome of the oncolytic adenovirus at the same time, or the HLA protein coding sequence and the beta 2-microglobulin coding sequence are inserted into the genome of the oncolytic adenovirus at the same time, or the HLA protein coding sequence, the beta 2-microglobulin coding sequence and the marker polypeptide coding sequence are inserted into the genome of the oncolytic adenovirus at the same time.

Preferably, the HLA protein comprises HLA class I molecules including HLA-A, HLA-B and HLA-C.

Preferably, the HLA protein is HLA-a 02: 01, and the amino acid sequence (containing a signal peptide sequence) is shown as SEQ ID NO. 12.

HLA-C expression on the cell surface is low, and at least one of the following mutations is performed on the inserted exogenous HLA-C in order to increase the expression of HLA-C: 1) arginine at position 2 was mutated to alanine. Thus, after removal of the initiator methionine, Ala at the N-terminus is acetylated, rendering the newly synthesized HLA-C more stable against degradation. 2) The nucleotide sequence encoding the HLA-C protein has a mutation from C to G at the 4 th nucleotide and from G to C at the 5 th nucleotide, thus forming a strong Kozak sequence (GCCRCC)ATGG, R are purines (A or G) to enhance translation and expression of the protein. 3) Isoleucine at position 362 mutated to threonine; 4) glutamic acid 359 th was mutated to valine. The C-terminus of HLA-C protein contains the double hydrophobic (LI) internalization and lysosomal targeting signal (dxsil), and isoleucine at position 362 is a key amino acid affecting the activity of this motif. Thus, the modification of isoleucine at position 362 in the HLA-C tail to threonine in HLA-A and B tails (I362T), and/or the mutation of glutamic acid at position 359 to valine, can improve the HLA-C profileAnd (5) performing surface expression. Since these mutations are not located in the region of the antigen polypeptide presentation, they do not affect the presentation of the antigen polypeptide and the recognition of the TCR.

Preferably, the HLA protein is HLA-C having a point mutation of I362T. More preferably, the HLA protein is HLA-C08: 02 protein, the amino acid sequence of which comprises R2A point mutation, I362T point mutation and E359V point mutation, and the amino acid sequence of which is shown as SEQ ID NO. 13.

Other HLA proteins include, but are not limited to, HLA-a 01: 01 protein, HLA-A02: 03 protein, HLA-a 02: 06 protein, HLA-a 03: 01 protein, HLA-A11: 01 protein, HLA-A24: 02 protein, HLA-a 30: 01 protein, HLA-A68: 01 protein, HLA-B08: 01 protein, HLA-B14: 02 protein, HLA-B1501, HLA-B58: 01, HLA-C07: 01 protein, HLA-C01: 02 protein.

The coding sequence for the HLA protein is expressed under the control of optional foreign gene expression regulatory elements including promoters, enhancers, silencers and polyadenylation signals, or gene expression regulatory elements of the pharmaceutically acceptable vector itself.

Generally, the antigen protein expressed in the cytoplasm can enter into MHC I antigen presenting path, after a series of protease hydrolysis, the formed short peptide (containing antigen epitope polypeptide) is transduced into endoplasmic reticulum through TAP molecule on endoplasmic reticulum membrane and is connected with HLA protein and beta therein₂The microglobulin is presented on the cell surface after formation of trimers (wherein HLA protein and beta)₂Microglobulin pairs to form MHC class I molecules) and is thus recognized by immune cells. Due to the loss of function of the MHC class I antigen presenting pathway which often occurs in tumor cells, the tumor antigen expressed in the cytoplasm cannot effectively form epitope polypeptide or enter endoplasmic reticulum and combine with HLA and beta 2-microglobulin to form a complex.

In the invention, by introducing the nucleic acid with the coding sequence of the labeled polypeptide into tumor cells and/or cancer cells, the exogenous labeled polypeptide expressed in the tumor cells and/or the cancer cells can enter an MHC class I antigen presenting pathway, so that the expression level of HLA/epitope polypeptide complexes on the surface of the tumor cells is increased, and the recognition sensitivity of the T cell receptor modified immune cells to the tumor cells and/or the cancer cells is enhanced.

Preferably, the tagged polypeptide comprises the following amino acid sequences in sequence and in tandem, operably linked: an amino acid sequence of an N-terminal signal peptide, an amino acid sequence of one or more epitope polypeptides, and optionally an amino acid sequence of a C-terminal endoplasmic reticulum retention signal, wherein when said tag polypeptide comprises a plurality of amino acid sequences of said epitope polypeptides, the amino acid sequences of each two adjacent said epitope polypeptides are linked by an amino acid sequence of a cleavable linker polypeptide; the amino acid sequence of the epitope polypeptide and optionally the amino acid sequence of the C-terminal endoplasmic reticulum retention signal may be linked by an amino acid sequence of a cleavable linker polypeptide. Preferably, the labeled polypeptide includes an amino acid sequence of the C-terminal endoplasmic reticulum retention signal. Preferably, the cleavable linker polypeptide is a furin cleavage recognition polypeptide.

The amino acid sequence of the epitope polypeptide can be derived from the amino acid sequence of a protein existing in the nature, or is an artificially synthesized amino acid sequence which does not exist in the nature. Preferably, the naturally occurring protein includes a protein of human origin and proteins of other species than human. More preferably, the amino acid sequence of the epitope polypeptide is derived from the amino acid sequences of tumor-associated antigen, tumor-specific antigen, and tumor neoantigen (neo-antigen) containing mutation points.

"tumor-associated antigen" generally refers to a normal protein derived from itself, but overexpressed or abnormally expressed in tumor cells, and includes carcinoembryonic antigen, tumor-testis antigen (CT antigen), and the like.

"tumor-specific antigen" generally refers to a mutein derived from itself, or a foreign viral protein involved in tumor development and progression.

In the present invention, "tumor-associated antigen" and "tumor-specific antigen" are sometimes collectively referred to as "tumor antigen".

The tumor antigen may be a tumor antigen as described in a Cancer Antigenic Peptide Database (Cancer Antigenic Peptide Database) (website https:// captured. icp. ucl. ac. be). Preferably, the tumor antigen may be a tumor antigen as described in table 1 below.

The epitope polypeptide can be a peptide segment with 8-11 amino acids capable of being presented by MHC class I molecules. The epitope polypeptide can be an epitope polypeptide as described in Cancer Antigenic Peptide Database (website https:// captured. icp. ucl. ac. be). Preferably, the epitope polypeptide may be an epitope polypeptide as described in table 1 below. In other embodiments, the epitope polypeptide is an epitope polypeptide having 4-9 consecutive identical amino acids (e.g., 4, 5, 6, 7, 8, or 9 consecutive identical amino acids) to the epitope polypeptide described in table 1 below, and these polypeptides are 8-11 amino acids in length.

TABLE 1 preferred tumor antigens and epitope polypeptides

In certain embodiments, each of the epitope polypeptides has a flexible linker at both ends that serves as a cleavage site for a proteolytic enzyme in the cytoplasm to release the epitope polypeptide. The flexible connecting fragment comprises GSGSR, AGSGSR and AGSGS.

In certain embodiments, the tagging polypeptide has a signal peptide at the amino terminus of the amino acid sequence of the one or more epitope polypeptides that can introduce the tagging polypeptide into the endoplasmic reticulum. The core of the signal peptide contains a long stretch of hydrophobic amino acids, forming a single alpha-helix. The amino terminus of the signal peptide often begins with a short positively charged amino acid sequence, and the terminus of the signal peptide usually has a cleavage site for an amino acid that is recognized and cleaved by a signal peptidase. After the connected exogenous polypeptide enters the endoplasmic reticulum, the signal peptide is identified and cut by the signal peptidase, and the exogenous polypeptide is released in the endoplasmic reticulum. Therefore, the labeled polypeptide carrying the signal peptide can directly enter the endoplasmic reticulum without being hydrolyzed by protease in the MHC class I antigen presentation pathway and transported by TAP molecules. The signal peptide can be a signal peptide (SEQ ID NO.14) consisting of amino acids 1-22 of the amino terminus of insulin-like protein (INSL 5).

In the case where the tagged polypeptide has a plurality of such epitope polypeptides, each two of such epitope polypeptides may be linked by a cleavable linker polypeptide. Cleavable linker polypeptides include Furin cleavage recognition polypeptides having a standard four amino acid motif that can be cleaved by Furin enzyme, i.e., R-X- [ KR]-R amino acid sequence (see literature "Molecular Therapy 2007; vol.15no.6, 1153-1159"). Preferably, the amino acid sequence of the cleavable linker polypeptide is R-R-K-R. The labeled polypeptide is introduced into endoplasmic reticulum by the signal peptide and then is expressed by R-X- [ KR]The epitope polypeptide connected with the R amino acid sequence is cut and hydrolyzed by furin enzyme in endoplasmic reticulum to release the epitope polypeptide and HLA and beta in endoplasmic reticulum₂-microglobulin forms an antigenic complex. Aminopeptidases and carboxypeptidases within the endoplasmic reticulum may also be involved in the enzymatic digestion and release of epitope polypeptides (see document "J Immunol.2009 November 1; 183(9): 5526-.

In order to allow retention of the tagged polypeptide introduced into the endoplasmic reticulum by the signal peptide within the lumen of the endoplasmic reticulum to facilitate release of the epitope polypeptide and binding of the HLA and β 2-microglobulin to form an antigenic complex, in certain embodiments, the tagged polypeptide has an endoplasmic reticulum retention signal peptide at the carboxy terminus of the amino acid sequence of the one or more epitope polypeptides. The amino acid sequence of the ER retention signal of soluble polypeptides (i.e.non-transmembrane proteins) is KDEL and the ER retention signal of ER membrane proteins is KKXX (see "Molecular Biology of the cell.2003; 14(3): 889-. In the present invention, the labeled polypeptide is a soluble polypeptide. Therefore, it is preferable that the endoplasmic reticulum retention signal peptide is a K-D-E-L fragment consisting of lysine-aspartic acid-glutamic acid-leucine residues.

More preferably, the tumor associated antigen is selected from the group consisting of NY-ESO-1157-165, NY-ESO-11-11, NY-ESO-153-62, NY-ESO-118-27, Her2/neu 369-377, SSX-241-49, MAGE-A4230-239, MAGE-A10254-262, MAGE-C2336-344, MAGE-C2191-200, MAGE-C2307-315, MAGE-C242-50, MAGE-A1120-129, MAGE-A1230-238, MAGE-A1161-169, KK-LC-176-84, p 5399-107, PRAME 301-309, alpha-fetoprotein 158-166, HPV 16-E629-38, HPV 16-E711-19, EBV-LMP 151-59, EBV-LMP-1125-133, HPV 16-E-1125-133, KRAS: G12D 10-18, KRAS: G12D 8-16, KRAS: G12D 7-16, KRAS: G12C 8-16, KRAS: G12A 8-16, KRAS: G12S 8-16, KRAS: G12R 8-16, KRAS: G12V 8-16, KRAS: G12V 7-16, KRAS: G12V 5-14, KRAS: G12V 11-19, KRAS: G12V 5-14, KRAS: Q61H 55-64, KRAS: Q61L 55-64, KRAS: Q61R55-64, KRAS: G12D 5-14, KRAS: G13D 5-14, KRAS: G12A 5-14, KRAS: G12C 5-14, KRAS: G12S 5-14, KRAS: G12R 5-14, KRAS: G12D 10-19, TP 53: V157G 156 164, TP 53: R248Q 240-249, TP 53: R248W 240-249, TP 53: G245S 240-249, TP 53: V157F 156 164, TP 53: V157F 149-158, TP 53: Y163C 156-164, TP 53: R248Q 247-255 and TP 53: R248Q 245 254, TP 53: R248W 245 254, TP 53: G245S 245-254, TP 53: G249S 245-254, TP 53: Y220C 217-225, TP 53: R175H 168-176, TP 53: R248W 240-249, TP 53: K132N-134, CDC 73: Q254E 248-256, CYP2A 6: N438Y 436-444, CTNNB 1: T41A 41-49, CTNNB 1: S45P 41-49, CTNNB 1: T41A 34-43, CTNNB 1: S37Y 30-39, CTNNB 1: S33C 30-39, CTNNB 1: S45P 40-49, EGFR: L858R 852-: T790M 790 799, PIK3 CA: E542K 533-: H1047R1046-1055, GNAS: R201H 197-205, CDK 4: R24C 23-32, H3.3: K27M26-35, BRAF: V600E 591-: k73Rfs 141-148, NRAS: Q61R55-64, IDH 1: R132H 126-135, TVP 23C: C51Y 51-59, TVP 23C: C51Y42-51 and TVP 23C: C51Y: 45-53.

More preferably, the epitope polypeptide is NY-ESO-1157-165 shown in SEQ ID NO.10 or KRAS shown in SEQ ID NO. 11: G12D 10-18.

Preferably, the tagged polypeptide comprises n of the epitope polypeptides linked by a cleavable linker polypeptide R-K-R, wherein n is an integer greater than or equal to 2, e.g. n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 …. Preferably, n is an integer between 2 and 20; it is also preferred that n is an integer between 2 and 10; it is also preferred that n-3-8. For example, the tagged polypeptides include 3-8 NY-ESO-1157-165 linked by a cleavable linker polypeptide or 3-8 KRAS linked by a cleavable linker polypeptide: G12D 10-18.

Considering that HLA proteins in tumor cells and/or cancer cells are also frequently deficient in expression, including complete deletion, haplotype deletion or allele deletion, and that the deletions of different tumor types range from 65% to 90% (see the literature "immunological today. 1997; 18: 89-95"), in some embodiments, the foreign gene comprises the HLA protein coding sequence and the marker polypeptide coding sequence, the HLA protein coding sequence and the marker polypeptide coding sequence are under the control of respective promoters, or the HLA protein coding sequence and the marker polypeptide coding sequence are under the control of the same promoter and the HLA protein coding sequence is operably linked to the marker polypeptide coding sequence by a cleavable linker polypeptide coding sequence.

Preferably, the HLA protein has a phenotype that is consistent with the phenotype of the HLA protein to which the marker polypeptide is capable of binding. The promoter may be a eukaryotic promoter, including persistently expressing promoters and inducibly expressing promoters, including (for example): PGK1 promoter, EF-1 alpha promoter, CMV promoter, SV40 promoter, Ubc promoter, CAG promoter, TRE promoter, CaMKIIa promoter, human beta actin (human beta actin) promoter.

Examples of said cleavable linker polypeptide linking said HLA protein to said marker polypeptide are known in the art, such as 2A polypeptides, 2A polypeptides including but not limited to F2A polypeptides from picornaviruses, and similar class 2A polypeptides from other viruses; it may also be a Furin-F2A junction fragment.

In some embodiments where the foreign gene comprises the HLA protein coding sequence and marker polypeptide coding sequence, preferably, the combination of HLA protein and marker polypeptide is as follows: HLA-A-02: 01 protein and a labeled polypeptide containing an epitope peptide NY-ESO-1157-165, which is shown as SEQ ID NO. 15; HLA-C08: 02 protein and an epitope-containing peptide KRAS: a labeled polypeptide of G12D 10-18, shown as SEQ ID NO. 16; HLA-A-01: 01 protein and a polypeptide comprising epitope peptide KRAS: Q61H 55-64, KRAS: Q61L 55-64 or KRAS: a tagged polypeptide of Q61R 55-64; HLA-A-02: 01. HLA-A-02: 03 or HLA-A-02: 06 protein and the antigen epitope peptide containing NY-ESO-1157-165, Her2/neu 369-377, NY-ESO-11-11, NY-ESO-153-62, NY-ESO-118-27, SSX-241-49, MAGE-A4230-239, MAGE-A10254-262, MAGE-C2336-344, MAGE-C2191-200, MAGE-C2307-315, MAGE-C242-50, MAGE-A1120-129, MAGE-A1230-238, MAGE-A1161-169, KK-LC-176-84, p 5399-107, PRAME 301-309-166, alpha fetoprotein 158-166, HPV 16-E629-38, HPV 16-E711-19, EBV-LMP 151-59, EBV-1125-133, KRAS: G12V 5-14, KRAS: G12D 5-14, KRAS: G13D 5-14, KRAS: G12A 5-14, KRAS: G12C 5-14, KRAS: G12S 5-14, KRAS: G12R 5-14, TP 53: R248Q 247-255 and TP 53: R248Q 245 254, TP 53: R248W 245 254, TP 53: G245S 245-254, TP 53: G249S 245-254, TP 53: Y240C 217-225, TP 53: R175H 168-176, CTNNB 1: T41A 34-43, CTNNB 1: S37Y 30-39, CTNNB 1: S33C 30-39, EGFR: T790M 790 799, GNAS: R201H 197-205, CDK 4: R24C 23-32, H3.3: K28M 27-36, TVP 23C: C51Y 51-59 or CDC 73: a tagged polypeptide of Q254E; HLA-A-03: 01 protein and the antibody comprising the epitope peptide KRAS: G12V 8-16, KRAS: G12V 7-16, CTNNB 1: S45P 41-49, CTNNB 1: S45P 40-49, BRAF: a marker polypeptide of V600E 591-601 or TP53-V157G 156-164; HLA-A11: 01 protein and the antibody comprising the epitope peptide KRAS: G12D 8-16, KRAS: G12D 7-16, KRAS: G12C 8-16, KRAS: G12A 8-16, KRAS: G12S 8-16, KRAS: G12R 8-16, KRAS: G12V 8-16, KRAS: G12V 7-16, TP 53: R248Q 240-249, TP 53: R248W 240-249, TP 53: G245S 240-249, TP 53: V157F 156 164, TP 53: V157F 149-158, TP 53: Y163C 156-164, CTNNB 1: T41A 41-49, CTNNB 1: S45P 41-49, EGFR: L858R 852-860 or PIK3 CA: E542K 533-542 labeled polypeptide; HLA-A24: 02 and epitope peptide TP 53: a tagged polypeptide of K132N 125-134; HLA-A68: 01 and a polypeptide comprising epitope peptide TP 53: R248W 240-249; HLA-B-08: 01 protein and a labeled polypeptide containing an epitope peptide CHD 4K 73Rfs 141-148; HLA-B-15: 01 and the peptide TVP 23C: C51Y42-51 or IDH 1: R132H 126-135; HLA-B58: 01 and the peptide TVP 23C: a C51Y 45-53 labeled polypeptide; HLA-C-01 comprising I362T and E359V mutations: 02 protein and an epitope-containing peptide KRAS: a labeled polypeptide of G12V 11-19; HLA-C-07 comprising I362T and E359V mutations: 01 and a polypeptide comprising epitope peptide PIK3 CA: a labeled polypeptide of H1047R 1046-1055; HLA-C-08 comprising the I362T and E359V mutations: 02 protein and an epitope-containing peptide KRAS: G12D 10-19 or KRAS: G12D 10-18.

When the foreign gene comprises the HLA protein coding sequence and the beta 2-microglobulin coding sequence, the HLA protein coding sequence and the beta 2-microglobulin coding sequence are respectively under the control of respective promoters, or the HLA protein coding sequence and the beta 2-microglobulin coding sequence are under the control of the same promoter, and the HLA protein coding sequence is operably connected with the beta 2-microglobulin coding sequence through a cleavable connecting polypeptide coding sequence.

This embodiment is applicable to tumors with a loss of β 2-microglobulin expression.

The promoter may be a eukaryotic promoter, including persistently expressing promoters and inducibly expressing promoters, including (for example): PGK1 promoter, EF-1 alpha promoter, CMV promoter, SV40 promoter, Ubc promoter, CAG promoter, TRE promoter, CaMKIIa promoter, human beta actin (human beta actin) promoter.

Examples of said cleavable linker polypeptide linking said HLA protein to said marker polypeptide are known in the art, such as 2A polypeptides, 2A polypeptides including but not limited to F2A polypeptides from picornaviruses, and similar class 2A polypeptides from other viruses; it may also be a Furin-F2A junction fragment.

Preferably, the β 2-microglobulin is a human protein or a murine protein.

Also preferably, the amino acid sequence of the beta 2-microglobulin is shown in SEQ ID NO. 17.

The present invention also provides a vector for preparing the oncolytic adenovirus, wherein the vector comprises the regulatory element of the E1B gene, lacks the coding region of the E1B gene, and comprises the cDNA sequence of E1A transcribing the E1A 13s mRNA upstream of the foreign gene. In one embodiment, the vector may have pShuttle as a backbone. The pShuttle plasmid is derived from the pBR322 plasmid and contains a kanamycin resistance gene derived from pZero 2.1 and a multiple cloning site into which a foreign gene is inserted. The two ends of the multiple cloning site are homologous recombination sequences of 5-type adenovirus genome. The left homologous recombination sequence is adenovirus type 5 nucleic acid sequence 34,931 and 35, 935. The right homologous recombination sequence is adenovirus type 5 nucleic acid sequence 3, 534-5, 790. In one embodiment, upstream of the E1A 13s sequence is the exogenous promoter.

The oncolytic adenovirus constructed by the invention can be used alone as a tumor vaccine and can also be used in combination with immune cells modified by a T cell receptor.

When the oncolytic adenovirus is used alone as a tumor vaccine, preferably, the exogenous gene of the oncolytic adenovirus is selected from a marker polypeptide coding sequence, an HLA protein coding sequence and a marker polypeptide coding sequence.

The present invention also provides a therapeutic agent for treating a tumor and/or cancer, comprising:

(a) a first composition, wherein the first composition comprises a first active ingredient comprising or comprising an oncolytic adenovirus according to the invention for introduction into a tumor cell and/or a cancer cell in a first pharmaceutically acceptable carrier; and

(b) a second composition, wherein the second composition comprises a second active ingredient comprising a T cell receptor modified immune cell in a second pharmaceutically acceptable carrier.

Preferably, in the case where the oncolytic adenovirus expresses an HLA protein, and expresses an HLA protein and a β 2-microglobulin, a tumor and/or cancer suitable for use in the therapeutic agent of the present invention should express an endogenous tumor antigen that provides an epitope polypeptide, with or without expression of an endogenous HLA protein capable of presenting the epitope polypeptide.

Preferably, in the case where the oncolytic adenovirus expresses a marker polypeptide, a tumor and/or cancer suitable for use in the therapeutic agent of the present invention should express an endogenous HLA protein capable of presenting the epitope polypeptide in the marker polypeptide, with or without expression of an endogenous tumor antigen capable of providing the epitope polypeptide.

Preferably, in the case where the oncolytic adenovirus expresses HLA protein and a marker polypeptide, and in the case where the oncolytic adenovirus expresses HLA protein, β 2-microglobulin and a marker polypeptide, the tumor and/or cancer suitable for the therapeutic agent of the present invention may or may not express an endogenous tumor antigen that provides an epitope polypeptide, and may or may not express an endogenous HLA protein that can present the epitope polypeptide.

In some preferred embodiments, the foreign gene of the oncolytic adenoviral vector is selected from the group consisting of a marker polypeptide coding sequence and an HLA protein coding sequence and a marker polypeptide coding sequence, the marker polypeptide having an amino acid sequence of one or more epitope polypeptides capable of being presented on the surface of the tumor and/or cancer cell by MHC class I molecules; and the T cell receptor modified immune cells are capable of specifically recognizing and binding the epitope polypeptide presented by the MHC class I molecule.

Preferably, the first composition and the second composition are each independently present in the therapeutic agent without intermixing.

In the therapeutic agent of the present invention, the immune cell modified with a T cell receptor includes a primitive T cell or a precursor cell thereof, an NKT cell, or a T cell line.

The T cell receptor includes at least one of an alpha chain and a beta chain, both of which comprise variable and constant regions, capable of specifically recognizing the epitope polypeptide on the surface of a tumor cell and/or cancer cell.

The variable regions of the TCR alpha and beta chains, used to bind the antigen polypeptide/major histocompatibility complex (MHC I), each include three hypervariable regions or Complementarity Determining Regions (CDRs), namely CDR1, CDR2, CDR 3. Wherein the CDR3 region is important for specific recognition of an antigen polypeptide presented by an MHC molecule. The TCR alpha chain is formed by recombining different V and J gene segments, and the beta chain is formed by recombining different V, D and J gene segments. The specificity of TCR for antigen polypeptide recognition is established by The corresponding CDR3 region formed by recombinant binding of a particular gene fragment, as well as by palindrome of The binding region and randomly inserted nucleotides (see "immunology: The immune system in health and disease.5)^thedition, Chapter 4, The generation of The lymphoma antigen receptors "). The MHC class I molecules include human HLA. The HLA includes: HLA-A, B, C.

Exogenous TCR alpha and beta chains expressed by T cells may be mismatched with the alpha and beta chains of the T cell's own TCR, which not only dilutes the expression of the correctly paired exogenous TCR, but also makes the antigenic specificity of the mismatched TCR unclear, thus potentially risking recognition of the autoantigen, and therefore the constant regions of the TCR alpha and beta chains are preferably modified to reduce or avoid the mismatch.

In one embodiment, the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the present inventors have found that the constant region of the alpha chain can be replaced, in whole or in part, by homologous sequences from other species, and/or the constant region of the beta chain can be replaced, in whole or in part, by homologous sequences from other species. More preferably, the other species is mouse.

Such substitutions may increase the amount of TCR expression in the cell and may further improve the specificity of the Her2/neu antigen for cells modified by the TCR.

The constant region of the alpha chain may be modified with one or more disulfide bonds, and/or the constant region of the beta chain may be modified with one or more disulfide bonds, for example 1 or 2.

In a specific embodiment, TCRs modified in two different ways, one by adding a disulfide bond to the TCR constant region by point mutation, are prepared in the literature "Cancer res.2007apr 15; 67(8) 3898-903 ", which is incorporated herein by reference in its entirety. The method of replacing the corresponding human TCR constant region sequence with the mouse TCR constant region sequence is described in the literature "Eur.J.Immunol.200636: 3052-3059", which is incorporated herein by reference in its entirety.

Preferably, said first composition comprises a therapeutically effective amount of said oncolytic adenovirus. Preferably, the oncolytic adenovirus is administered at a dose of 5 × 10⁷-5×10¹²vp/day, 1-2 times per day for 1-7 consecutive days.

Also preferably, said second composition comprises a therapeutically effective amount of said T cell receptor modified immune cells. Preferably, the total dosage range of 1 × 10 per treatment course is included³-1×10⁹One cell per Kg body weight of the T cell receptor modified immune cells.

The oncolytic adenovirus may be formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid intracavity administration, or intravenous administration.

The immune cells may be formulated for administration by arterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, subarachnoid, intramedullary, intramuscular, or intraperitoneal administration.

Preferably, the therapeutic agent consists of the first composition and the second composition.

It will be understood by those skilled in the art that the therapeutic agents of the present invention may also comprise suitable pharmaceutically acceptable adjuvants including pharmaceutically or physiologically acceptable carriers, excipients, diluents (including saline, PBS solution), and various additives including sugars, lipids, polypeptides, amino acids, antioxidants, adjuvants, preservatives, and the like.

The present invention also provides a kit of synergistic combinations for the treatment of tumors and/or cancers comprising:

a first container containing a first composition of therapeutic agents according to the present invention;

a second container containing a second composition in a therapeutic agent according to the present invention, wherein the first container and the second container are independent; and

instructions for timing and mode of administration are specified.

The invention also provides the application of the oncolytic adenovirus in preparing a medicament for treating tumors and/or cancers.

In some embodiments, the exogenous gene of the oncolytic adenovirus is selected from the group consisting of an HLA protein coding sequence, a marker polypeptide coding sequence, an HLA protein coding sequence and a β 2-microglobulin coding sequence, or an HLA protein coding sequence, a β 2-microglobulin coding sequence and a marker polypeptide coding sequence.

The tumors and/or cancers include: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

The invention also provides the use of the therapeutic agent of the invention in the manufacture of a medicament for the treatment of a tumour and/or cancer.

The tumors and/or cancers include: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

The invention also provides the use of a kit according to the invention for the manufacture of a medicament for the treatment of a tumour and/or cancer.

The tumors and/or cancers include: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

The present invention also provides a method of treating a tumor and/or cancer comprising administering to a patient having a tumor and/or cancer an oncolytic adenovirus according to the present invention.

Preferably, the foreign gene of the oncolytic adenovirus is selected from an HLA protein coding sequence, a marker polypeptide coding sequence, an HLA protein coding sequence and a β 2-microglobulin coding sequence, or an HLA protein coding sequence, a β 2-microglobulin coding sequence and a marker polypeptide coding sequence.

The tumors and/or cancers include: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

Preferably, the tumor and/or cancer expresses an endogenous tumor antigen that provides an epitope polypeptide, and also expresses an endogenous HLA-class I molecule that presents the epitope polypeptide.

The oncolytic adenovirus is administered in a therapeutically effective amount. Preferably, the oncolytic adenovirus is administered at a dose of 5 × 10⁷-5×10¹²vp/day, 1-2 times per day for 1-7 consecutive days.

The oncolytic adenovirus may be formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid intracavity administration, or intravenous administration.

The present invention also provides a method of treating a tumor and/or cancer comprising:

administering a first composition of therapeutic agents according to the present invention to a tumor and/or cancer patient; and

administering a second composition of the therapeutic agents according to the invention to the tumor and/or cancer patient.

The first and second compositions of the therapeutic agent can be administered simultaneously (e.g., intratumorally as a mixture, simultaneously), separately but simultaneously (e.g., administered intratumorally and intravenously, respectively), or sequentially (e.g., first the first composition and then the second composition; or first the second composition and then the first composition).

Preferably, the method comprises the following steps carried out in sequence:

1) first administering the first composition to the tumor and/or cancer patient; and

2) administering a second composition of said therapeutic agents to said tumor and/or cancer patient after administering said first composition.

Preferably, the second composition of said therapeutic agents is administered to said tumor and/or cancer patient on days 1-30 after the first composition is administered.

By "administering a second composition of said therapeutic agents to said tumor and/or cancer patient from day 1 to day 30 after the first administration of said first composition" is meant that the time interval between the administration of the first second composition and the administration of the first composition is from 1 to 30 days (e.g., 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 days), or the time interval between the administration of the first second composition and the administration of the first composition immediately preceding it is from 1 to 30 days (e.g., 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 days). Preferably, the time interval between the administration of the first second composition and the administration of the first composition immediately preceding it is 3-14 days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days).

In a preferred embodiment of the invention, the first composition comprises said oncolytic adenovirus, which is administered at a dose of 5 x 10⁷-5×10¹²vp/day, 1-2 times per day, for 1-7 consecutive days, or any value in between the ranges above.

In a preferred embodiment of the invention, the T cell receptor modified immune cells are administered in a total dose range of 1X 10 per treatment course³-1×10⁹One cell/Kg body weight. Preferably, administration is 1-3 times for 1 day, and 1-7 consecutive days.

In certain embodiments, the method of treating a tumor and/or cancer further comprises administering to the patient an additional agent useful for treating a tumor and/or cancer, and/or an agent useful for modulating the immune system of the patient, to enhance the number and function of the T cell receptor-modified immune cells in vivo. Such other drugs for treating tumors and/or cancers include, but are not limited to: chemotherapeutic agents, such as cyclophosphamide, fludarabine (fludarabine); a radiotherapeutic agent; immunosuppressants such as cyclosporin, azathioprine, methotrexate, mycophenolate mofetil (mycophenolate), FK 50; antibodies, for example, against CD3, IL-2, IL-6, IL-17, TNF α.

In certain embodiments, the method of treating a tumor and/or cancer further comprises administering to the patient an additional agent useful for treating a tumor and/or cancer, and/or an agent useful for modulating the immune system of the patient, for eliminating the number and function of the T cell receptor modified immune cells carrying a suicide gene in vivo when the T cell receptor modified immune cells produce severe toxic side effects. Such other drugs for treating tumors and/or cancers include, but are not limited to: chemically Induced Dimerization (CID) drugs, AP1903, phosphorylated ganciclovir (ganciclovir), anti-Cd 20 antibodies, anti-CMYC antibodies, anti-EGFR antibodies.

The oncolytic adenovirus may be formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid intracavity administration, or intravenous administration.

The T cell receptor-modified immune cell may be formulated for administration by arterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, subarachnoid, intramedullary, intramuscular, or intraperitoneal administration.

The tumors and/or cancers include: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

Preferably, in the case where the oncolytic adenovirus expresses an HLA protein, and expresses an HLA protein and a β 2-microglobulin, a tumor and/or cancer suitable for use in the therapeutic agent of the present invention should express an endogenous tumor antigen that provides an epitope polypeptide, with or without expression of an endogenous HLA protein capable of presenting the epitope polypeptide.

Preferably, in the case where the oncolytic adenovirus expresses a marker polypeptide, a tumor and/or cancer suitable for use in the therapeutic agent of the present invention should express an endogenous HLA protein capable of presenting the epitope polypeptide in the marker polypeptide, with or without expression of an endogenous tumor antigen capable of providing the epitope polypeptide.

Preferably, in the case where the oncolytic adenovirus expresses HLA protein and a marker polypeptide, and in the case where the oncolytic adenovirus expresses HLA protein, β 2-microglobulin and a marker polypeptide, the tumor and/or cancer suitable for the therapeutic agent of the present invention may or may not express an endogenous tumor antigen that provides an epitope polypeptide, and may or may not express an endogenous HLA protein that can present the epitope polypeptide.

The method within the scope of the present invention may be provided to a patient based on the patient's own circumstances of the tumor and/or cancer patient.

In order to select patients for treatment with the above-described oncolytic adenoviruses, therapeutic agents and kits, HLA-I typing and tumor antigen expression assays may be performed on the patients prior to use of the above-described treatments.

The typing of HLA class I molecules can be detected by using the existing techniques, for example, serological typing, PCR-SSP (sequence specific priming PCR), PCR-SSOP (sequence specific oligonucleotide probe), PCR-RFLP (restriction fragment length polymorphism), PCR-SBT (sequencing based typing), PCR-RSCA (reference strand mediated conformation analysis), and HLA typing based on Next Generation Sequencing (NGS).

The tumor antigen expression can be detected by the prior art, such as qPCR and FISH (fluorescence in situ hybridization) for detecting the gene overexpression of the specific tumor antigen, immunohistochemical, flow cytometry for detecting the protein overexpression of the specific tumor antigen, and the like. Detection of whether a tumor harbors a neoantigen with a particular point mutation is mainly performed by obtaining a somatic variant by comparing the sequences of tumor tissues and corresponding normal tissues based on Next Generation Sequencing (NGS) technology, including whole genome sequencing and/or RNA sequencing, to determine whether a tumor harbors and expresses a neoantigen with a particular point mutation (Frnt. Immunol 2019, (24); 10: 1392).

The present invention will be further explained or illustrated below by way of examples, which should not be construed as limiting the scope of the invention.

Examples of the present invention

Unless otherwise indicated, the experimental procedures used in the following examples were performed using conventional experimental protocols, procedures, materials and conditions in the field of biotechnology. For example, recombinant plasmids and viral vectors, or polypeptides and proteins, can be produced recombinantly using nucleic acids described herein using standard recombinant methods (Green and Sambrook, molecular cloning: A laboratory Manual, 4 th edition, Cold spring harbor Press, Cold spring harbor, NY, 2012).

Hereinafter, unless otherwise specified, the percentage concentration (%) of each agent refers to the volume percentage concentration (% (v/v)) of the agent.

The materials and methods used for examples 1-4 are as follows:

1. cell line:

the cell line used for the preparation of lentiviral particles or for use as target cells was 293T cells (ATCC CRL-3216). The presenting cell line used for presenting antigenic peptides is T2 cells (174xCEM. T2, ATCC CRL-1992). The cell line used for TCR gene expression and function analysis was JRT cells (j.rt3-T3.5,

TIB-153). Tumor cell lines used as target cells include: human melanoma cell line A375(ATCC CRL-1619), human fibrosarcoma cell line HT1080(ATCC CCL-121), human ovarian carcinoma cell line SKOV3(ATCC HTB-77), human lung cancer cell line H1299(ATCC CRL-5803), human osteosarcoma cell line HOS-C1(ATCC CRL-1547), and human lung cancer cell line A549(ATCC CCL-185). The plasmid pCDNA3.3-NY coding the NY-ESO-1 protein or the plasmid coding KRAS is constructed by a conventional method from the pCDNA3.3 plasmid (Thermo Fisher K830001)The plasmid pCDNA3.3-KRAS/G12d of G12D protein, which was transfected into SKOV3 cells respectively, and selected with 500. mu.g/mL geneticin (Thermo Fisher 10131027), can generate SKOV3 cell line expressing NY-ESO-1 protein or KRAS mutant protein with G12D point mutation. NY-ESO-1 (or CTAG-1B) is a well-known cancer-testis antigen (CTA) that is overexpressed in many types of cancer. The following describes a method for preparing pCDNA3.3-NY.

2. Cell culture medium:

293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine high glucose (VWR Cat. No. VWRL 0101-0500). Other cell lines were cultured in RPMI-1640 complete medium (Lonza, catalog No. 12-115F) supplemented with fetal bovine serum (ATCC 30-2020), 2mmol/L L-glutamic acid, 1 Xessential amino acids 50X (Invitrogen 11130-.

3. Peripheral blood:

human peripheral blood products from healthy donors were from the stanford blood center. Peripheral Blood Mononuclear Cells (PBMC) were generated from the residual leukocytes by extraction (LRS chamber, product code A1012) using Ficoll-Paque PLUS density gradient medium (GE Healthcare17144002) according to the manufacturer's instructions.

4. Preparation of target cells expressing foreign proteins:

293T cells or tumor cells were transfected with Lipofectamine 3000(Thermo Fisher L3000015) according to the manufacturer's instructions. Expression plasmids include plasmids encoding the marker polypeptides, exogenous HLA proteins or amino acid sequences described herein. The method for preparing the plasmid is described below. If a plasmid containing the adenoviral genome is used to transfect 293T cells, the plasmid is pre-digested with PacI enzyme (New England Biolabs, R0547S) to release the adenoviral genome. Cells transiently expressing the foreign protein can be used as target cells 48-72 hours after transfection. To generate a stable SKOV3 cell line expressing the NY-ESO-1 protein or a mutant KRAS protein having a G12D point mutation, SKOV3 cells were cultured with medium supplemented with 500. mu.g/mL of geneticin 72 hours after transfection with a plasmid encoding the corresponding protein.

5. Preparation of T cells expressing recombinant TCR:

to generate activated human T cells expressing TCR, PBMC cells in 24-well plates were cultured with RPMI-1640 complete medium supplemented with 2 μ g/ml anti-human CD3 antibody (Biolegend 317303) and 2 μ g/ml anti-human CD28 antibody (Biolegend 302914) for 24 hours, or PBMC were treated with human T-CD3/CD8 magnetic beads (Thermo Fisher 11131D) according to the manufacturer's instructions. After 24 hours, cells were cultured in RPMI-1640 complete medium supplemented with IL-2100 IU/ml, IL-75 ng/ml, IL-155 ng/ml. To generate a T cell line for expressing the TCR, β chain deficient mutant JRT (J.RT3-T3.5) cells from the Jurkat cell line were cultured with RPMI-1640 complete medium. To infect T cells with lentivirus encoding the TCR, activated PBMC or JRT cells were resuspended in 24-well plates with 1ml of freshly prepared lentivirus supernatant and Polybrene (Santa Cruz Biotechnology sc-134220) was added at 25 ℃. The final concentration was 5-8. mu.g/ml. The cells were centrifuged at 1000g and 32 ℃ for 2 hours. After 6 hours, the medium was changed to RPMI-1640 complete medium supplemented with IL-2100 IU/ml, IL-75 ng/ml, IL-155 ng/ml. Cells can also be transfected using a retroNectin dish (retroNectin precoat dish, 35 mm. phi.) (Takara T110A) according to the manufacturer's instructions.

6. Cell phenotype analysis by flow cytometry:

to analyze the expression of TCR in PBMC or JRT cells transfected with lentivirus encoding exogenous TCR, cells were resuspended in DPBS buffer (2.7mM KCl, 1.5mM KH) containing 1% FBS₂PO₄、136.9mM NaCl，8.9mM Na₂HPO₄·7H₂O, pH 7.4), and anti-human CD8 antibody labeled with APC (Biolegend 300912) and iTAg tetramer/PE-HLA-a 02: 01NY-ESO-1(SLLMWITQC) (MBL International TB-M011-1) staining. The flow cytometer was MACSQuran Analyzer 10(Miltenyi Biotec Corporation), and the results were analyzed by Flowjo software (Flowjo Corporation). To analyze HLA-A2 expression in 293T cells transfected with plasmids encoding HLA-A2 protein, the cells were treated with FITC anti-human HLA-A2 antibody(Biolegend 343303) were stained and analyzed by flow cytometry.

T cell functional assay:

to assess the specificity and function of the TCRs expressed by JRT cells, CD69 expression following antigen stimulation was assessed by flow cytometry according to methods in the art (see literature "cytometry. 1996; 26 (4): 305-10"). Briefly, TCR gene modified JRT cells were co-cultured with target cells for 16 hours in duplicate wells of a 96-well plate, e.g., in mixed culture with T2 cells loaded with treatment at different concentrations of antigenic peptide, 293T cells transduced with nucleic acids described herein, or tumors. Cells were stained with anti-CD 69 antibody (Biolegend310905) and analyzed by flow cytometry for CD69 according to the manufacturer's instructions⁺Frequency of JRT cells. To assess the specificity and function of TCRs transduced into PBMCs, IFN- γ secretion by specific T cells following antigen stimulation was measured by IFN- γ ELISA (enzyme linked immunosorbent assay). Briefly, TCR gene modified PBMCs were co-cultured with the above-described target cells in duplicate or triplicate wells of a 96-well plate. Cell supernatants were collected over 18-24 hours using IFN-gamma ELISA Read-Set-Go kit (eBioscience 88-7316) or human IFN-gamma DuoSet ELISA kit (R)&D Systems DY285B) IFN- γ ELISA assays were performed according to the manufacturer's instructions.

8. Preparation of recombinant TCR lentiviral vectors:

the TCR gene was cloned into a replication deficient lentiviral vector pCDH-EF1 alpha-MCS-PGK-GFP (System Biosciences CD 811A-1). The vector pCDH-EF 1. alpha. -MCS without GFP was generated by removing the PGK promoter and GFP gene from the vector pCDH-EF 1. alpha. -MCS-PGK-GFP. According to the methods described in patent application publications US8143376B2, WO2018099402a1 or reference "J Immunol 2010; 184(9),4936-46 "were determined to be within HLA-a 02: 01 against the background of the sequences of the TCR-. alpha. -V-D-J region and the TCR-. beta. -V-D-J region which are specific for the NY-ESO-1157-165-epitope peptide (TCR-. beta. -V-D-J region for US8143376B2, TCR-. beta. -LI for WO2018099402A1 and TCR-. beta. -NY-LI for reference J Immunol 2010; 184(9),4936-46 for TCR-. beta. -AE). In HLA-C08: 02, for KRAS: the sequences of the TCR-. alpha. -V-D-J region and TCR-. beta. -V-D-J region specific for the G12D 10-18 peptide were determined according to patent application publication WO2018026691 (denoted as TCR-RAS G12D). The sequences of the mouse TCR-. alpha.constant chain and the mouse TCR-. beta.constant chain were determined from the reference sequences (GeneBank KU254562 and EF154514.1, respectively). The nucleic acid sequences for TCR-NY-LY, TCR-NY-AE and TCR-NY-LI of NY-ESO-1157-165, the nucleic acid sequence for TCR-RAS-G12D of KRAS: G12D 10-18 (comprising the TCR beta chain with the mouse TCR-beta constant chain, the TCR alpha chain with the mouse TCR-alpha constant chain, the linker nucleic acid encoding the furin protease cleavage peptide, and the F2A peptide between the TCR alpha and beta chains, respectively) are shown in SEQ ID NO.18, 19, 20 or 21, respectively, and are synthesized by Integrated DNA Technologies or LifesSct LLC. The synthesized nucleic acid was cloned into the lentiviral vector pCDH-EF1 alpha-MCS-PGK-GFP or pCDH-EF1 alpha-MCS (without GFP) at a multiple cloning site downstream of the EF-1 alpha promoter according to the manufacturer's instructions. The lentiviral vector expressing the TCR against the NY-ESO-1157-165-epitope was designated pCDH-EF1 alpha-TCR-NY. The inserted nucleic acid was sequenced and no errors or mutations were found. Lentiviral vector plasmids were transformed into the competent bacterium stellar (Takara Bio, 636763) to prepare plasmid stocks for the preparation of lentiviral particles.

9. Preparation of recombinant TCR lentiviral particles:

TCR lentiviral particles were produced by 293T or 293FT cells (Thermo Fisher R70007) transfected with lentiviral vector plasmids containing the TCR genes. Briefly, 293T or 293FT cells grown in 6-well plates were co-transfected with TCR lentiviral vector plasmid and pPACKH 1-terminator packaging kit (System Biosciences LV500A-1) using Lipofectaine 3000 transfection reagent (invitrogen, 11668019) according to the manufacturer's instructions. After 48 hours of incubation, the supernatant was collected and filtered through a 0.4 μm filter. According to the manufacturer's instructions, with Lenti-X^TMThe concentrator (Takara, 631231) concentrates the virus supernatant. Freshly prepared TCR-lentiviruses were used to infect JRT cells or activated PBMCs.

10. Preparation of expression vectors encoding NY-ESO-1 protein or mutant KRAS G12D protein:

PureLink will be used according to the manufacturer's instructions^TMRNA Total RNA purified from HT1080 cells (NY-ESO-1+, KRAS wild-type) using Mini kit (Thermo Fisher 12183020) was used as a sample for PrimeScript^TMThe RT-PCR kit (Takara RR014A) generated a template for the RT-PCR product. The NY-ESO-1 full-length gene was generated by the following PCR primer pair: 5'-TATATAAGAGCAGAGCTGCCACCATGCAGGCCGAAGGCCGGGGCA-3' (SEQ ID NO.22) and 5'-TGATTGTCGACGCCCTTAGCGCCTCTGCCCTGAGGGAGGCTG-3' (SEQ ID NO. 23). The KRAS G12D full-length gene was generated by the following PCR primer pair: 5'-ATGACTGAATATAAACTTGTGGTAGTTGGAGCTGACGGCGTAGGCAAGAGTGCCTTG-3' (SEQ ID NO.24) and 5'-TGATTGTCGACGCCCTTACATAATTACACACTTTGTCTTTGACTTC-3' (SEQ ID NO. 25). The resulting gene was cloned into the TOPO cloning site of the pCDNA3.3 vector (Thermo Fisher K830001) according to the manufacturer's instructions. According to manufacturer's instructions, use

High-Fidelity 2X PCR Master Mix (New England Biolabs M0541L) the PCR described in this application was performed.

11. Preparing a replication-defective recombinant adenovirus plasmid for encoding the marker polypeptide and/or the exogenous HLA class I molecule:

the replication-defective recombinant adenovirus system described in the present application is based on the AdEasy system (Nature Protocols 2007; 2: 1236-1247). The nucleic acid for the EF1 alpha promoter was cloned from the pCDH-EF1 alpha-MCS-PGK-GFP plasmid (System Biosciences, CD811A-1) by the following PCR primer pairs: 5'-CTCATAGCGCGTAATGGCTCCGGTGCCCGTCAGTGGGCAG-3' (SEQ ID NO.26) and 5'-GAATTCGCTAGCTCTAGATCACGACACCTGAAATGGAAG-3' (SEQ ID NO.27) and was integrated into the pShuttle-CMV vector (Agilent Technologies, Cat. No. 24007) in place of the CMV promoter to produce the pShuttle-EF 1. alpha. vector. Encodes full-length HLA-a 02: 01 (as shown in SEQ ID NO. 28) and the cDNA encoding human β 2-microglobulin were produced by T2 cells and cloned into pcDNA3.3-TOPO vector according to the manufacturer's instructions. The nucleic acid shown in SEQ ID NO.29 encoding a marker polypeptide comprising the 3 epitope peptides NY-ESO-1157-165 and the nucleic acid shown in SEQ ID NO.30 encoding a linker peptide were synthesized by Integrated DNA Technologies. To prepare a nucleic acid as shown in SEQ ID No.31 encoding a tagged polypeptide linking HLA-a 201 protein to a linker peptide, the following steps were performed: PCR was performed using the synthesized nucleic acid encoding the tagged polypeptide as a template, using primer pairs 5'-AGAGCTAGCGAATTCAACATGAAAGGTTCCATCTTCAC-3' (SEQ ID NO.32) and 5'-ACACTGTGTAATCCACATCAATAGCGATCTCTTTC-3' (SEQ ID NO.33) to generate a nucleic acid fragment (denoted NY) encoding the tagged polypeptide with NY-ESO-1157-165; PCR was performed using the synthesized nucleic acid encoding the linker peptide as template with the following primer pairs: 5'-TGGATTACACAGTGTCGTCGTAAGCGATCCGGAAGCGCG-3' (SEQ ID NO.34) and 5'-CGCCATGACGGCCATGGGCCCAGGGTTGGACTCGACGTC-3' (SEQ ID NO.35) to produce a nucleic acid encoding a linker peptide; the full length HLA-a 02: the HLA-A201 gene (named A2) was generated by PCR of 5'-ATGGCCGTCATGGCGCCCCGA-3' (SEQ ID NO.36) and 5'-TCACACTTTACAAGCTGTGAGAGACAC-3' (SEQ ID NO.37) primers in cDNA pair of 01. The PCR products purified above were mixed as templates to perform PCR using the following primer pairs: 5'-ATGAAAGGTTCCATCTTCACATTGTTTTTGTTC-3' (SEQ ID NO.38) and 5'-CGCCATGACGGCCATGGGCCCAGGGTTGGACTCGACGTC-3' (SEQ ID NO.39) to generate a nucleic acid encoding a tagged polypeptide linking the HLA-A201 protein to a linker peptide. The resulting nucleic acid was cloned into the multiple cloning site of the pShuttle-EF1 alpha vector using conventional gene cloning techniques in the art to generate pShuttle-EF1 alpha-NY-A2.

12. Preparation of recombinant adenovirus plasmid

The pShuttle vector was linearized with PmeI (NEB Biolabs, R0560 s). After purification, the vector was transferred into the electroactive BJ5183-AD-1 bacterial strain (Agilent Technologies, 200157) by loading at 2500V, 200. omega. and 25. mu.F delivery concentrations in a Bio-Rad Gene concentration loading generator electroporator according to the manufacturer's instructions. Potential adenovirus recombinants were screened by restriction digestion with PacI (NEB Biolabs R0547S). A correct recombinant will usually produce one larger fragment (about 30kb), and one smaller fragment of 3.0 or 4.5 kb.

13. Preparation of a conditionally replication competent adenovirus plasmid encoding said tagged polypeptide and/or exogenous HLA class I molecule:

to generate a pShuttle vector comprising the adenovirus E1A gene driven by a foreign promoter and a nucleic acid encoding an HLA molecule binding to the marker polypeptide or an HLA molecule binding to β 2-microglobulin driven by the native E1B promoter, the following procedure was performed: the sequence shown in SEQ ID NO.2, which comprises the native E1B promoter sequence and a nucleic acid sequence encoding an E1A-13s protein containing a24 base pair deletion (E1A122-129 deletion) (native E1A polyA signal sequence with an E1A polyA addition site), was synthesized by Integrated DNA Technologies. PCR was performed using the synthesized gene sequence as a template, using the following primer pairs: 5'-ATGAGACATATTATCTGCCACGGAG-3' (SEQ ID NO.46) and 5'-CATGGTGGCGAGGTCAGATGTAAC-3' (SEQ ID NO.47), a gene fragment was obtained comprising the E1A-13s nucleic acid sequence with a24 base pair deletion and the native E1B promoter sequence (denoted E1Ad 24). Use of a plasmid encoding full length HLA-a 02: 01 or a cDNA encoding human β -2 microglobulin as a template for PCR (at this time, Kozak sequence was introduced), and HLA-a 02: the 01 gene fragment (denoted A2) and the beta-2 microglobulin gene fragment (denoted BM). Synthetic mutant HLA-C08: the 02 gene is shown as SEQ ID NO.49 (shown as C08), and the synthesized nucleic acid encodes a tagged polypeptide comprising 3 NY-ESO-1157-165 epitope peptides, as shown as SEQ ID NO.29 (shown as NY), or encodes a tagged polypeptide comprising 3 KRAS: a labeled polypeptide of the epitope peptide of G12D 10-18, as shown in SEQ ID NO.50 (denoted as RAS), or a synthetic nucleic acid encoding a linker peptide, as shown in SEQ ID NO.30 (denoted as F2A), was used as a template to perform PCR to obtain a corresponding gene fragment (Kozak sequence was introduced when PCR was performed using the mutant HLA-C08: 02 gene as a template). Combinations of nucleic acid fragments comprising E1Ad24-A2-F2A-NY, E1Ad24-C08-F2A-RAS, E1Ad24-A2-F2A-BM and E1Ad24-C08-F2A-BM were generated using the In-Fusion HD Cloning Plus kit (Takara 638909) according to the manufacturer's instructions. The resulting gene combinations were cloned downstream of the EF1 α promoter in the pShuttle-EF1 α vector to generate pShuttle-EF1 α -E1Ad24-A2-F2A-NY, pShuttle-EF1 α -E1Ad24-C08-F2A-RAS, pShuttle-EF1 α -E1Ad24-C08-F2A-BM and pShuttle-EF1 α -E1Ad 24-A2-F2A-BM. To generate the recombinant adenovirus plasmid, the pShuttle vector was linearized with PmeI and transformed into electrically active BJ5183-AD-1 by loading at a frequency delivery concentration of 2500V, 200 Ω and 25 μ F in a Bio-Rad Gene concentration Loading electroporator, as described above. The resulting recombinant oncolytic adenovirus plasmids were designated pAd-EF1 α -E1Ad24-A2-F2A-NY, pAd-EF1 α -E1Ad24-C08-F2A-RAS, pAd-EF1 α -E1Ad24-C08-F2A-BM, and pAd-EF1 α -E1Ad 24-A2-F2A-BM.

14. Preparation of recombinant adenovirus encoding the marker polypeptide and/or exogenous HLA class I molecule:

the replication-defective recombinant adenovirus plasmid and the recombinant oncolytic adenovirus plasmid with conditional replication ability obtained by the method are prepared to obtain the corresponding recombinant adenovirus by the method.

The recombinant adenovirus plasmid was digested with Pac I (NEB Biolabs R0547S) to release adenovirus genomic DNA. The linearized plasmid was purified by phenol/chloroform extraction and ADENO-X293 cells (Takara 632271) were transfected with Lipofectaine 3000 transfection reagent (Thermo Fisher L3000001) according to the manufacturer's instructions. Transfected cells were at 37 ℃ 5% CO₂For 14-20 days until a cytopathic effect (CPE) is observed. Four freeze-thaw vortex cycles were performed to release adenovirus from the cells and obtain viral particles. Two to four rounds of amplification are typically required to generate high titer viruses for large scale preparation. The procedure for the preparation of large-scale adenoviruses followed the method described in the reference (Nat Protoc 2007; 2(5), 1236-47). Adenovirus titers were determined by the Adeno-X GoStix kit (Takara 632270) according to the manufacturer's instructions. To infect target cells with recombinant adenovirus, the number of viral titers and the number of target cells are determined based on the determined MOI (multiplicity of infection, referring to the number of viral particles infected per cell). In general, expression of foreign genes can be detected 3 to 4 days after infection.

The materials and methods used for examples 5-9 and preparation 1 were as follows:

human ovarian cancer cell line SKOV3, human lung cancer cell line H1299 and human osteosarcoma cell line HOS C1 are purchased from ATCC;

human melanoma cell line a375 was purchased from the cell bank of the chinese academy of sciences;

human IFN-gamma ELISA kits were purchased from R & D;

an xcelligene RTCA S16 real-time unlabelled cell function analyzer was purchased from ACEA Bio;

APC-bound anti-human HLA-a2 antibody was purchased from BD, PE-bound anti-human CD3 antibody was purchased from BD;

Dynabeads^TMhuman T-Activator CD3/CD28 was purchased from Thermofish;

hrIL-2 was obtained from the hypericin company, hrIL-7 and hrIL-15 from the near shore protein company;

flow cytometer Novocyte was purchased from ACEA Bio.

Example 1

This example demonstrates that a foreign gene can be efficiently expressed by a genetic construct described herein, including an adenoviral vector comprising a nucleic acid encoding the marker polypeptide and/or a foreign HLA class I molecule; and lentiviral vectors comprising nucleic acids encoding T cell receptors. Figure 1A shows a schematic of the constructs described in the present application. pAdEasy-EF1 alpha-NY-A2 is a replication-defective adenovirus vector, expresses the labeled polypeptide shown as SEQ ID NO.51, and comprises NY-ESO-1157-165 epitope peptide connected with 3 Furin protein enzyme cutting sites and HLA-A2 protein shown as SEQ ID NO. 12. The labeled polypeptide and the HLA-A2 gene are connected by an F2A sequence. The expression unit of the marker polypeptide is flanked by an exogenous EF-1 alpha promoter and an SV40 poly (A) signal sequence. pAd-EF1 alpha-E1A-A2-F2A-NY is a replication-competent adenovirus vector comprising a nucleic acid encoding the HLA-A2 gene and the said marker polypeptide of 3 NY-ESO-1157-165 epitope peptides. pAd-EF1 alpha-E1A-A2-F2A-BM is a replication-competent adenovirus vector comprising nucleic acid encoding the HLA-A2 gene and human beta 2-microglobulin. The constructs pAd-EF1 α -E1Ad24-A2-F2A-NY and pAd-EF1 α -E1Ad24-A2-F2A-BM each comprised a nucleic acid encoding an E1-13S mutein (i.e., the cDNA sequence of E1A transcribing E1A 13S mRNA) containing a deletion of 24 residues of the E1A protein as shown in SEQ ID No. 43. The E1A-13s gene is flanked by an exogenous EF-1 alpha promoter and a natural E1A poly (A) signal sequence. In both replication competent constructs, the region of the E1B gene was deleted and the nucleic acids encoding the marker polypeptide and other foreign genes were inserted into this region and the expression of the foreign gene was driven using native E1B regulatory elements, including the native E1B promoter and the E1B/IX poly (A) signal. FIG. 1B is a schematic representation of lentiviral vectors pCDH-EF1 α -TCR-NY-LY, pCDH-EF1 α -TCR-NY-AE and pCDH-EF1 α -TCR-NY-LI comprising nucleic acids encoding respective NY-ESO-1 specific TCR β chain and α chain polypeptides (said polypeptides are shown in SEQ ID NO.52, 53, 54, respectively). The constant regions of both the β and α TCR chains were replaced by murine TCR constant region sequences. The beta chain and alpha chain of TCR are connected by polypeptide Furin protease cut point and F2A connection sequence, and the two ends are EF-1 alpha promoter and lentivirus post-transcriptional regulatory element (WPRE).

To assess whether the recombinant constructs could express foreign genes, HLA-A2 negative 293T cells were transfected with pShuttle-EF1 α -NY-A2, pShuttle-EF1 α -E1Ad24-A2-F2A-NY or pShuttle-EF1 α -E1Ad24-A2-F2A-BM (each flanked by genomic sequences homologous to adenovirus type 5). Cells were stained with anti-HLA-A2 antibody and expression of HLA-A2 was assessed by flow cytometry. FIG. 1C shows that 293T cells transfected with all three constructs containing the HLA-A2 gene can express HLA-A2, indicating that the regulatory elements in the constructs have the function of driving expression of foreign proteins and that HLA-A2 protein can be isolated and expressed on the cell surface by the Furin protease and F2A linker. The fluorescence intensity of HLA-A2 expression in 293T cells transduced with pShuttle-EF1 alpha-NY-A2 is lower than that of 293T cells transduced with pShuttle-EF1 alpha-E1 Ad24-A2-F2A-NY and pShuttle-EF1 alpha-E1 Ad24-A2-F2A-BM, indicating that the natural E1B promoter and E1B poly (A) signal may drive the expression of foreign genes more effectively in the context of adenovirus genome.

To assess whether the constructed recombinant lentiviral vectors expressed TCR, lentiviral particles expressing NY-ESO-1 specific TCR were prepared from 293T cells transfected with pCDH-EF1 α -TCR-NY vector and used to infect J.RT3-T3.5 cells (JRT cells, ATCC TIB 153). Infected JRT cells were stained with anti-CD 8 antibody and the NY-ESO-1157-165/HLA-A2 tetramer which specifically binds to the TCR which specifically recognizes the NY-ESO-1157-165 epitope polypeptide presented by HLA-A2. FIG. 1D shows that JRT cells transfected with lentivirus express different TCRs capable of binding the NY-ESO-1157-165/HLA-A2 tetramer, indicating that recombinant lentivirus expresses a specific TCR consisting of TCR α and β chains. The α and β chains pair through the contained murine constant regions to form the TCR/CD3 complex and are expressed on the surface of JRT cells.

Example 2

This example demonstrates that, once the construct vector expresses a tagged polypeptide and a foreign HLA class I protein, the epitope peptide can be released from the tagged polypeptide and presented by the foreign HLA class I molecule, forming an antigenic peptide/HLA class I complex that can be recognized by a specific TCR.

To assess the ability of the specific TCR expressed by JRT cells to recognize epitope peptides presented by foreign HLA class I molecules, JRT cells were first transfected with recombinant lentiviruses produced by 293T cells co-transfected with pCDH-EF1 α -TCR-NY-LY, pCDH-EF1 α -TCR-NY-AE or pCDH-EF1 α -TCR-NY-LI and the packaging vectors described above. JRT cells expressing specific TCR were co-cultured with HLA-A2 positive T2 cells and NY-ESO-1157-165 polypeptide in a 10-fold dilution series starting at 1. mu.g/ml was added to the culture wells. The percentage of CD69 positive JRT cells was analyzed by flow cytometry 16-24 hours after antigen stimulation. FIG. 2A shows that JRT cells expressing specific TCR expressed CD69 after stimulation with antigenic peptide, indicating that the activity of JRT cells activated by NY-ESO-1157-165 peptide presented by HLA-A2 showed an antigen dose-dependent relationship. In addition, it was shown that the difference in the sensitivity of different TCRs to epitope recognition by NY-ESO-1157-165-antigen was similar in the activity of TCR-NY-LY and TCR-NY-AE, but the sensitivity of TCR-NY-LI to epitope polypeptide NY-ESO-1157-165-antigen was about 10 times lower.

JRT cells transfected with NY-ESO-1157-165 antigen polypeptide-specific TCR were used as effector cells, pShuttle-EF1a-NY-A2 or pShuttle-EF1a-E1Ad24-A2-NY transfected HLA-A2-negative and NY-ESO-1-negative 293T cells were used as target cells, and 293T cells transduced with empty pShuttle vectors were used as control target cells to evaluate whether target cells transfected with vectors expressing marker polypeptides and exogenous HLA-A2 expressed epitope polypeptides recognized by specific T cells on the surface of the target cells. FIG. 2B shows that both pShuttle-EF1a-NY-A2 and pShuttle-EF1a-E1Ad24-A2-NY transfected 293T cells activated JRT cells expressing different NY-ESO-1157-165 polypeptide specific TCRs. The percentage of CD69+ JRT cells was significantly increased for 293T target cells expressing the above-described marker polypeptide and HLA-A2, compared to control target 293T transduced with the empty pShuttle vector (student's T-test, p < 0.01). The results showed that the marker polypeptide and the exogenous HLA-A2 were expressed separately in 293T cells and formed the HLA-A2/NY-ESO-1157-165 polypeptide complex recognized by specific TCR. In the group in which JRT cells expressing TCR-NY-LY and TCR-NY-AE were effector cells, target cells transfected with pShuttle-EF1 α -E1Ad24-A2-F2A-NY were able to induce more CD69+ JRT cells than target cells transfected with pShuttle-EF1 α -NY-A2 (student's t-test, p < 0.05). The result is consistent with the high expression of HLA-A2 after 293T cells are transfected by pShuttle-EF1 alpha-E1 Ad24-A2-F2A-NY, which shows that in adenovirus genome DNA, the natural E1B promoter and E1B poly (A) signals can effectively drive the expression of exogenous genes.

To evaluate the function of the foreign HLA class I molecules expressed by the constructs to present epitope peptides from endogenous antigenic proteins, 293T cells were co-transfected with pCDNA3.3-NY and pShuttle-EF1a-E1A d24-A2-F2A-BM and used as target cells. pCDNA3.3-NY encodes full-length NY-ESO-1 protein, and 293T cell expresses NY-ESO-1 protein as endogenous tumor antigen, thereby generating NY-ESO-1157-165 epitope peptide. If the NY-ESO-1 protein can provide NY-ESO-1157-165 epitope polypeptide through HLA class I antigen processing pathway and is presented by exogenous HLA-A2, the target cell can be recognized by JRT cell expressing specific TCR. 293T cells transfected only with pCDNA3.3-NY or pShuttle-EF1a-E1Ad24-A2-F2A-BM were used as negative controls. FIG. 2C shows that 293T cells transduced with pShuttle-EF1a-E1A 24-A2-F2A-BM alone were unable to activate JRT cells expressing a NY-ESO-1 specific TCR. However, when target cells were loaded with NY-ESO-1157-165 epitope polypeptide at a concentration of 1. mu.g/ml, CD69 expression was induced in a large number of TCR-expressing JRT cells, indicating that exogenous HLA-A2 was expressed in the target cells and can present antigenic polypeptide to activate T cells. In addition, when the target cells express both NY-ESO-1 protein and HLA-A2 molecules, mixed culture of JRT cells expressing NY-ESO-1 specific TCR can induce more CD69+ JRT cells (student's t test, p <0.05) than negative control target cells. The results show that the NY-ESO-1157-165 epitope polypeptide can be generated through an endogenous HLA class I processing mechanism and is recognized by a specific TCR after being presented by exogenous HLA-A2. Therefore, it is feasible to introduce exogenous HLA class I molecules as allo-HLA (allo-HLA) molecules into tumor cells to present epitope peptides from endogenous proteins. The endogenous protein producing epitope polypeptide may be an overexpressed tumor-associated antigen or a tumor neoantigen (neo-antigen) produced by a mutein.

To further assess whether nucleic acids encoding a tagged polypeptide and/or HLA class I molecule can express foreign polypeptides and proteins within the recombinant adenovirus genomic DNA framework, the adenovirus vector pAd-EF1a-E1Ad24-A2-NY was digested with PacI to obtain recombinant adenovirus genomic DNA comprising foreign nucleic acids encoding HLA-A2 protein and a tagged polypeptide having an NY-ESO-1157-165 epitope polypeptide. pAd-EF1a-E1A d24-A2-BM was also digested with PacI to obtain adenoviral genomic DNA expressing exogenous HLA-A2 and β 2-microglobulin. 293T cells were transfected with adenoviral DNA and used as target cells 48 hours later to stimulate JRT cells expressing NY-ESO-1 specific TCR. FIG. 2D shows that pAd-EF1a-E1Ad24-A2-BM express foreign HLA-A2 in 293T cells and are capable of presenting not only the NY-ESO-1157-165 epitope polypeptide loaded at a concentration of 1. mu.g/ml, but also the NY-ESO-1157-165 epitope polypeptide from the NY-ESO-1 protein. The percentage of CD69+ JRT cells was significantly increased compared to control target cells transfected with pAd-EF1a-E1Ad24-A2-BM alone (student's t-test, p < 0.01). 293T cells transfected with pAd-EF1a-E1Ad24-A2-NY adenovirus DNA express a marker polypeptide with NY-ESO-1157-165 epitope and exogenous HLA-A2 at the same time, and can also activate JRT cells expressing NY-ESO-1 specific TCR. Compared with the target cell transfected with pCDNA3.3-NY/pAd-EF1a-E1Ad24-A2-BM at the same time, the target cell transfected with pAd-EF1a-E1Ad24-A2-NY has similar capability of activating JRT-TCR-NY-LY cells. Although more JRT-TCR-NY-AE cells were activated by pAd-EF1a-E1Ad24-A2-NY target cells, there was no significant difference between the two groups. These results indicate that if the mechanism and function of HLA class I antigen processing and presentation in target cells is intact, epitope peptides can be produced from endogenous proteins, efficiently presented by exogenous HLA class I molecules introduced into target cells by the vector, and recognized by specific T cells. The foreign HLA class I molecule can be autologous HLA (auto-HLA) or allogeneic HLA (allo-HLA)

Example 3

This example demonstrates that when tumor cells are transduced with a genetic construct containing nucleic acids encoding the marker polypeptides and HLA class I proteins, epitope peptides can be presented by exogenous HLA class I molecules in the tumor cells. Whether or not the tumor cell expresses a specific tumor antigen or has an HLA class I allele presenting a specific epitope polypeptide, expression of the marker polypeptide and the exogenous HLA class I molecule can make the tumor cell a target cell recognized by a specific T cell. In addition, expression of exogenous HLA class I proteins can not only increase the immunogenicity of tumor cells, but also present epitope peptides derived from endogenous tumor antigens and activate HLA-restricted TCR specific for the epitope peptides.

A375 is a human melanoma cell line, representing HLA-A2⁺And NY-ESO-1⁺A tumor cell. SKOV3 is a human ovarian cancer cell line, representing HLA-A2^-And NY-ESO-1^-Double negative tumor cells. SKOV3 cells transduced pCDNA3.3-NY and stably expressing NY-ESO-1 protein represent HLA-A2 negative and NY-ESO-1 positive tumor cells. These tumor cells were transfected with pShuttle-EF1a-E1Ad24-A2-NY or pShuttle-EF1a-E1Ad24-A2-BM and used as target cells, co-cultured with JRT cells expressing NY-ESO-1 specific TCR.

FIG. 3 shows that both JRT-TCR-NY-LY and JRT-TCR-NY-AE can be activated by A375 cells, which indicates that NY-ESO-1157-165 epitope peptide can be produced from endogenous NY-ESO-1 protein and presented by its own HLA-A2 molecule. Although the A375 cells transfected with pShuttle-EF1a-E1Ad24-A2-BM expressed more HLA-A2, the sensitivity of the cells to be recognized by specific TCR was not significantly improved, which indicates that the NY-ESO-1157-165 peptide/HLA-A2 complex is not limited by the amount of HLA-A2 on the A375 cells, but is limited by the amount of NY-ESO-1157-165 polypeptide produced by HLA class I antigen processing mechanism. A375 cells transduced with pShuttle-EF1a-E1A d24-A2-NY significantly increased CD compared to control A375 cells69⁺Percentage of JRT-TCR-NY-LY cells (student's t-test, p<0.05), indicating that the labeled polypeptide can evade transporter proteins associated with antigen processing (TAP), deliver antigenic peptides into the lumen of the Endoplasmic Reticulum (ER), and be bound to HLA class I molecules. NY-ESO-1^-/HLA-A2^-Double negative SKOV3 cells and NY-ESO-1 transduced with pShuttle-EF1a-E1Ad24-A2-BM^-/HLA-A2⁺SKOV3 cells were unable to activate JRT-TCR-NY-LY and JRT-TCR-NY-AE cells. pShuttle-EF1a-E1Ad24-A2-NY can make SKOV3 cell be recognized by JRT cell expressing NY-ESO-1 specific TCR (student's t test, p test)<0.01), indicating that the epitope peptide expressed after the oncolytic viral vector transfects tumor cells can be combined with HLA-A2 molecule and recognized by specific TCR, regardless of the expression state of endogenous antigen or matched HLA class I allele. SKOV3-NY cells transduced by pShuttle-EF1a-E1Ad24-A2-BM can activate JRT cells of NY-ESO-1 specific TCR, which shows that exogenous HLA class I molecules introduced into tumor cells can also enable the tumor cells to be recognized by the specific TCR, but the tumor cells need to express tumor antigens to be used as sources for generating antigen peptides. In addition, there is a need for an intact antigen processing and presentation mechanism in tumor cells in order to process tumor antigen proteins to produce epitope peptides and to present them by foreign HLA class I molecules. Target cells transfected with pShuttle-A2-F2A-NY activated more JRT-TCR-NY-LY cells than SKOV3-NY transfected with pShuttle-A2-F2A-BM (student's t test, p test)<0.05), indicating that the tagged polypeptide produces more NY-ESO-1157-165 epitope polypeptide than the endogenous antigen protein.

Example 4

This example shows that tumor cells transfected with the nucleic acids encoding the marker polypeptides and/or exogenous HLA class I molecules can be recognized by primary T cells (primary T cells) expressing specific TCRs.

HLA-A2 negative PBMC are transfected with recombinant lentiviruses expressing NY-ESO-1 specific TCRs (including TCR-NY-LY, TCR-NY-AE or TCR-NY-LI). 7-10 days after transfection, PBMCs were stained with anti-CD 8-APC and NY-ESO-1157-165 tetramer-PE. FIG. 4A shows that T cells transduced with all three TCR genes can express the NY-ESO-1157-165 tetramerLabeled NY-ESO-1157-165-specific TCRs. In CD8⁺And CD8^-NY-ESO-1157-165 tetramer positive cells were observed in the cell population. Albeit CD8⁺The cells are NY-ESO-1 specific killer T Cells (CTL), CD8^-The cells may be CD4 expressing a NY-ESO-1 specific TCR⁺T helper cells. The results indicate that TCR-NY-LY, TCR-NY-AE and TCR-NY-LI have high binding affinity with the NY-ESO-1157-165/HLA-A2 complex and are independent of the helper function of CD 8. PBMC expressing NY-ESO-1 specific TCR were used as effector cells to assess the recognition sensitivity of tumor cells expressing the marker polypeptide and exogenous HLA class I molecules to specific T cells.

A375 cell line, SKOV3 cell line and SKV3-NY cell expressing full-length NY-ESO-1 protein respectively represent target cells with NY-ESO-1 and HLA-A2 double positive, NY-ESO-1 and HLA-A2 double negative and NY-ESO-1 single positive. These target cells were transfected with pShuttle-EF1a-E1Ad24-A2-NY or pShuttle-EF1a-E1A d24-A2-BM and used as target cells to evaluate the recognition function of specific TCRs expressed on primary T cells expressing the marker polypeptide and exogenous HLA-A2. Recombinant lentivirus-transfected PBMC were incubated with target cells for 16-24 hours at an E: T ratio of 10: 1. IFN- γ secretion in the supernatant was examined to assess the ability of the target cells to stimulate T. FIG. 4B shows that all target cells transfected with pShuttle-EF1a-E1A d24-A2-BM efficiently presented NY-ESO-1156-165 polypeptides and activated T cells expressing specific TCR, indicating that the exogenous HLA-A2 protein expressed by pShuttle-EF1a-E1A d24-A2-BM vector combined with the exogenous NY-ESO-1156-165 polypeptide formed the NY-ESO-1156-165/HLA-A2 complex and was recognized by primary T cells expressing NY-ESO-1 TCR specificity. Both A375 and SKOV3-NY cells transfected with pShuttle-EF1a-E1A d24-A2-NY and pShuttle-EF1a-E1A d24-A2-BM were recognized by T cells expressing specific TCRs. While SKOV3 cells were recognized by T cells expressing specific TCR only by transfecting pShuttle-EF1a-E1Ad24-A2-NY which simultaneously expresses HLA-A2 molecule and NY-ESO-1157-165 epitope peptide. The results demonstrate that the oncolytic virus vector expressing the labeled polypeptide containing the epitope peptide and the exogenous HLA class I molecule can not only make tumor cells recognized by JRT cells expressing specific TCR, but also can be recognized by primary T cells expressing specific TCR.

To assess whether other types of tumor cells can be transduced by nucleic acids encoding the marker polypeptides and/or exogenous HLA class I molecules, the human lung cancer cell line H1299 (NY-ESO-1)⁺/HLA-A2^-) Human osteosarcoma cell line HOS-C1(NY-ESO-1 low/HLA-A2)⁺) And human lung cancer cell line A549 (NY-ESO-1)^-/HLA-A2^-) The vector pShuttle-NY-A2 was transduced and used as target cells. PBMC transfected with recombinant lentiviruses expressing TCR-NY-LY or TCR-NY-LI were used as effector cells. Blank control effector cells were PBMCs transfected with empty lentiviruses. Effector or placebo cells were incubated with target cells at a ratio of E: T ═ 5:1 for 24 hours. The secretion of IFN-. gamma.in the supernatant was examined by ELISA. As shown in FIG. 4C, specific T cells expressing TCR-NY-LY or TCR-NY-LI produced large amounts of IFN- γ as compared to the control with placebo T cells (student's T-test, p<0.01), indicating that the tested tumor cells can be marked by the marking polypeptide expressing the NY-ESO-1157-165 antigen epitope peptide and the exogenous HLA-A2 protein and can be recognized by T cells expressing the NY-ESO-1 specific TCR.

Example 5: expression of HLA-A2 after recombinant oncolytic adenovirus OAd-NY/A2 infects SKOV3 cells

The recombinant oncolytic adenovirus "OAd-NY/A2" described below is a recombinant oncolytic adenovirus packaged from plasmid pAd-EF1 alpha-E1 Ad24-A2-F2A-NY according to the methods described above.

This example demonstrates that HLA-A2 can be expressed on the cell surface after the recombinant oncolytic adenovirus OAd-NY/A2 infects tumor cells. On day 0, according to 5X 10⁴One SKOV3 cell/well was seeded in 24-well plates. On day 1, recombinant oncolytic adenovirus OAd-NY/A2 was added to SKOV3 cells at 5, 10, 20, 50, 100MOI, respectively. After further culturing for 48 hours, the cells were harvested. The cells were stained with APC-conjugated anti-human HLA-A2 antibody (BD Co.) (dilution 1: 50) and the expression of HLA-A2 on the cell surface was detected using a flow cytometer Novocyte (purchased from ACEA Bio Inc.).

As a result, as shown in FIG. 5, the expression of HLA-A2 was successfully detected, and as the MOI increased, the expression ratio of HLA-A2 also increased.

Preparation example 1: preparation of Mock-T and NY-ESO-1 targeted TCR-T cells

The T cell culture medium was AIM V + 5% FBS +100IU/ml hrIL-2+10ng/ml hrIL-7+10ng/ml hrIL-15. On day 0, human PBMC (available from Hangzhou Kangwanda pharmaceutical science and technology Co., Ltd.) were revived at 5X 10⁵Cells/ml PBMCs were seeded into one well of a 24-well plate, 1 ml/well, and dynabeads were added as T: beads ═ 1: 1. After 6 hours of activation, either GFP lentivirus or TCR-NY-AE lentivirus was added at MOI ═ 5 for preparation of Mock-T and NY-ESO-1 targeted TCR-T cells in the examples below, respectively. On day 3, the whole medium was changed at 5X 10⁵-1×10⁶Cell density was adjusted per cell/ml. Then, every 1-2 days, make up the liquid according to 5X 10⁵-1×10⁶Cell density was adjusted per cell/ml. By day 10, all cells were harvested, counted, and the proportion of GFP expression in Mock-T and TCR-T cells was determined by flow cytometry, and CD3 expression in Mock-T and TCR-T cells was determined using PE-conjugated anti-human CD3 antibody.

Example 6: secretion of cytokine after co-culture of TCR-T of targeted NY-ESO-1 and tumor cells of recombinant oncolytic adenovirus OAd-NY/A2 infection marker

In this example, the secretion of IFN gamma was detected by ELISA after overnight coculture of TCR-T cells targeting NY-ESO-1 and tumor cells infected with recombinant oncolytic adenovirus OAd-NY/A2. The tumor cell strains detected comprise a human melanoma cell strain A375, a human lung cancer cell strain H1299, a human ovarian cancer cell strain SKOV3 and a human osteosarcoma cell strain HOSC 1. Day 0, according to 1X 10⁵Each tumor cell/well was seeded in 12-well plates. On day 1, 80. mu.l of recombinant oncolytic adenovirus OAd-NY/A2 was added to the corresponding tumor cells. On day 2, tumor cells and tumor cells marked for infection with recombinant oncolytic adenovirus OAd-NY/A2 were counted by trypsinization at 1X 10⁵Individual tumor cells/well were seeded in 96-well plates. TCR-T cells or Mock-T cells (expressing GFP as a negative control group of TCR-T) targeting NY-ESO-1 are treated according to the standard of 3 x 10⁵The number of effector cells and target cells is (are) inoculated into corresponding wells of a 96-well plateE: T) is 3:1, each group has 3 multiple holes. After co-cultivation overnight, human IFN-. gamma.DuoSet ELISA (from R) was used&Company D) the content of IFN γ in the culture supernatants was examined.

As shown in FIG. 6A, since A375 cells were NY-ESO-1 positive and HLA-A2 positive, IFN γ was secreted from TCR-T cells targeting NY-ESO-1 (shown as A375+ TCR-T) after stimulation with A375, compared to Mock-T cells of the negative control group (shown as A375+ Mock-T). Infection of A375 with recombinant oncolytic adenovirus OAd-NY/A2 did not result in secretion of IFN γ. If the T cells are co-cultured with A375 cells infected by recombinant oncolytic adenovirus OAd-NY/A2, after being stimulated by A375 marked by OAd-NY/A2 infection, the TCR-T cells (shown as A375+ TCR-T + OAd-NY/A2) targeting NY-ESO-1 can secrete more IFN gamma compared with the Mock-T (shown as A375+ Mock-T + OAd-NY/A2) of a negative control group. As shown in FIG. 6B, since H1299 cells were NY-ESO-1 positive and HLA-A2 negative, TCR-T cells targeting NY-ESO-1 (shown as H1299+ TCR-T) did not secrete IFN γ upon stimulation with H1299, compared to Mock-T cells of the negative control group (shown as H1299+ Mock-T). Infection of H1299 with recombinant oncolytic adenovirus OAd-NY/A2 also did not result in secretion of IFN γ. If T cells are co-cultured with H1299 cells infected with recombinant oncolytic adenovirus OAd-NY/A2, the TCR-T cells targeting NY-ESO-1 (shown as H1299+ TCR-T + OAd-NY/A2) can secrete more IFN gamma after being stimulated by H1299 marked by OAd-NY/A2 infection compared with Mock-T (shown as H1299+ Mock-T + OAd-NY/A2) in a negative control group. As shown in FIG. 6C, since SKOV3 cells were NY-ESO-1 negative and HLA-A2 negative, TCR-T cells targeting NY-ESO-1 (shown as SKOV3+ TCR-T) did not secrete IFN γ after stimulation with SKOV3, compared to Mock-T cells of the negative control group (shown as SKOV3+ Mock-T). The recombinant oncolytic adenovirus OAd-NY/A2 infected SKOV3 can not cause secretion of IFN gamma. If T cells and recombinant oncolytic adenovirus OAd-NY/A2 infected SKOV3 cells are co-cultured, after being stimulated by SKOV3 marked by OAd-NY/A2 infection, the TCR-T cells (shown as SKOV3+ TCR-T + OAd-NY/A2) targeting NY-ESO-1 can secrete more IFN gamma compared with the Mock-T (shown as SKOV3+ Mock-T + OAd-NY/A2) of a negative control group. As shown in FIG. 6D, since HOSC1 cells were weakly positive for NY-ESO-1 and positive for HLA-A2, TCR-T cells targeting NY-ESO-1 (shown as HOSC1+ TCR-T) secreted a small amount of IFN γ after stimulation by HOSC1, compared to Mock-T cells of the negative control group (shown as HOSC1+ Mock-T). Infection of HOSC1 with recombinant oncolytic adenovirus OAd-NY/A2 also did not cause secretion of IFN gamma. If T cells are co-cultured with HOSC1 cells infected by recombinant oncolytic adenovirus OAd-NY/A2 and stimulated by HOSC1 marked by OAd-NY/A2 infection, compared with Mock-T (shown as HOSC1+ Mock-T + OAd-NY/A2) of a negative control group, TCR-T cells targeting NY-ESO-1 (shown as HOSC1+ TCR-T + OAd-NY/A2) can secrete more IFN gamma.

The TCR targeting NY-ESO-1 recognizes HLA-A2 on the surface of the target cell and the NY-ESO-1 short peptide molecule presented by the same. If the tumor cells are positive by HLA-A2 and positive by NY-ESO-1, the recognition of TCR-T targeting NY-ESO-1 to target cells can be improved to a certain extent after the recombinant oncolytic adenovirus OAd-NY/A2 is infected with a marker. If the tumor cell is HLA-A2 negative or NY-ESO-1 negative or double negative, the original TCR-T can not recognize the target cell and can recognize and attack the tumor cell after the recombinant oncolytic adenovirus OAd-NY/A2 infects and marks the tumor cell.

Example 7: in-vitro combined killing of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1 on SKOV3

In the embodiment, the in-vitro combined killing effect of the recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T cells targeting NY-ESO-1 on human ovarian cancer cell lines SKOV3 is detected by an RTCA real-time killing monitoring method. On day 0, according to 5X 10³Individual tumor cells/well were seeded into 16 well E-plates. On day 1, recombinant oncolytic adenovirus OAd-NY/A2 was added to the corresponding wells at an MOI of 10. On day 2, 5X 10 of the solution was added to the corresponding wells³TCR-T cells or Mock-T cells (expressing GFP as a negative control group for TCR-T) targeting NY-ESO-1 were added to each T cell/well, 2 wells per group. The growth of SKOV3 cells was monitored by an xcelligene RTCA S16 real-time unlabeled cell function analyzer.

As shown in FIG. 7A, the growth of SKOV3 cells after inoculation (shown as SKOV3), the growth of SKOV3 cells was not affected by the addition of recombinant oncolytic adenovirus OAd-NY/A2 alone (shown as SKOV3+ OAd-NY/A2), and the growth of SKOV3 cells was weakly affected by the addition of TCR-T cells or Mock-T cells targeting NY-ESO-1 alone (shown as SKOV3+ Mock-T or SKOV3+ TCR-T). The combined action of the recombinant oncolytic adenovirus OAd-NY/A2 and Mock-T (shown as SKOV3+ OAd-NY/A2+ Mock-T in the figure) can slow down the growth of SKOV3 cells, while the combined action of the recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T cells targeting NY-ESO-1 (shown as SKOV3+ OAd-NY/A2+ TCR-T in the figure) has a very obvious killing effect on SKOV3 cells, so that the number of the SKOV3 cells is obviously reduced. The cell growth index at 90.8 hours at the end of the experiment was analyzed and the tumor growth inhibition rate (IR%) was calculated as: 100% × (experimental cellular index-SKOV 3 cellular index)/SKOV 3 cellular index (Cell index) obtained by RTCA instrument). The result is shown in fig. 7B, the recombinant oncolytic adenovirus, Mock-T or TCR-T act alone, only produces weak inhibition effect on the growth of SKOV3 cells, the combined action of the recombinant oncolytic adenovirus and Mock-T produces greater inhibition effect on the growth of SKOV3 cells, and the combined action group of the recombinant oncolytic adenovirus and TCR-T can significantly reduce the number of SKOV3 cells, and the killing effect is greater than the superposition of the effects of the individual actions of the recombinant oncolytic adenovirus and TCR-T, thereby producing synergistic killing effect.

Example 8: in vitro combined killing of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1 on H1299

In the embodiment, the in-vitro combined killing effect of the recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T cells targeting NY-ESO-1 on human lung cancer cell strains H1299 is detected by an RTCA real-time killing monitoring method. On day 0, according to 5X 10³Individual tumor cells/well were seeded into 16 well E-plates. On day 1, recombinant oncolytic adenovirus OAd-NY/A2 was added to the corresponding wells at an MOI of 10. On day 2, 1X 10 of the solution was added to the corresponding wells⁴TCR-T cells or Mock-T cells (expressing GFP as a negative control group for TCR-T) targeting NY-ESO-1 were added to each T cell/well, 2 wells per group. Growth of H1299 cells was monitored by an xCELLigence RTCA S16 real-time unlabeled cell function analyzer.

As shown in FIG. 8A, the growth of H1299 cells (shown as H1299) continued after inoculation, the growth of H1299 cells was hardly affected by the addition of recombinant oncolytic adenovirus OAd-NY/A2 alone (shown as H1299+ OAd-NY/A2), and the growth of H1299 cells was significantly affected by the addition of TCR-T cells or Mock-T cells targeting NY-ESO-1 alone (shown as H1299+ Mock-T or H1299+ TCR-T). The combined action of the recombinant oncolytic adenovirus OAd-NY/A2 and Mock-T (shown as H1299+ OAd-NY/A2+ Mock-T) can effectively kill H1299 cells, while the combined action of the recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T cells targeting NY-ESO-1 (shown as H1299+ OAd-NY/A2+ TCR-T) shows a very remarkable killing effect on the H1299 cells, so that the number of the H1299 cells is reduced more remarkably. The cell growth index at 61.14 hours at the experimental node was analyzed to calculate the tumor growth inhibition rate (IR%) as 100% × (experimental group cell index-H1299 cell index)/H1299 cell index. The result is shown in fig. 8B, the recombinant oncolytic adenovirus, Mock-T or TCR-T act alone and only have a certain inhibitory effect on the growth of H1299 cells, the combined action of the recombinant oncolytic adenovirus and Mock-T can reduce the number of H1299 cells, and the combined action group of the recombinant oncolytic adenovirus and TCR-T can significantly reduce the number of H1299 cells, and the killing effect is greater than the superposition of the effects of the individual actions of the recombinant oncolytic adenovirus and TCR-T, thereby generating the synergistic killing effect.

Example 9: in vitro combined killing of HOSC1 by recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1

In the embodiment, the in-vitro combined killing effect of the recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T cells targeting NY-ESO-1 on human osteosarcoma cell strain HOS C1 is detected by an RTCA real-time killing monitoring method. On day 0, according to 5X 10³Individual tumor cells/well were seeded into 16 well E-plates. On day 1, recombinant oncolytic adenovirus OAd-NY/A2 was added to the corresponding wells at an MOI of 30. On day 2, 1.5X 10 of the solution was added to the corresponding wells⁴TCR-T cells or Mock-T cells (expressing GFP as a negative control group for TCR-T) targeting NY-ESO-1 were added to each T cell/well, 2 wells per group. The growth of HOS C1 cells was monitored by an xCELLigence RTCA S16 real-time unlabeled cell function analyzer.

As shown in FIG. 9A, HOS C1 cells continued to grow after inoculation (shown as HOS C1), only the addition of recombinant oncolytic adenovirus OAd-NY/A2 weakly affected the growth of HOS C1 cells (shown as HOS C1+ OAd-NY/A2), and only TCR-T cells or Mock-T cells targeting NY-ESO-1 also inhibited the growth of HOS C1 cells to some extent (shown as HOS C1+ Mock-T or HOS C1+ TCR-T). The combined action of the recombinant oncolytic adenovirus OAd-NY/A2 and Mock-T (shown as HOS C1+ OAd-NY/A2+ Mock-T in the figure) can reduce the cell number of HOS C1, while the combined action of the recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T cells targeting NY-ESO-1 (shown as HOS C1+ OAd-NY/A2+ TCR-T in the figure) shows a very obvious killing effect on HOS C1 cells, so that the cell number of HOS C1 is obviously reduced. Analyzing the cell growth index at 61.14 hours at the end point of the experiment, and calculating the tumor growth inhibition rate, wherein the formula is as follows: 100% × (experimental group cell index-HOS C1 cell index)/HOS C1 cell index (IR%). As shown in FIG. 9B, the recombinant oncolytic adenovirus, Mock-T or TCR-T acting alone only slightly inhibited the growth of HOS C1 cells, the combined action of the recombinant oncolytic adenovirus and Mock-T slightly reduced the number of HOS C1 cells, and the combined action group of the recombinant oncolytic adenovirus and TCR-T significantly reduced the number of HOS C1 cells, and the killing effect was greater than the sum of the effects of the recombinant oncolytic adenovirus and TCR-T acting alone, thereby producing a synergistic killing effect.

Conclusion

Once the nucleic acid encoding the exogenous epitope peptide or HLA class I protein is delivered into the tumor cell via a vector (which may be a plasmid vector, a recombinant virus, a nanoparticle, or naked DNA or RNA), the labeled polypeptide comprising the epitope peptide and/or the exogenous HLA class I molecule allows the tumor cell to be recognized by T cells expressing a specific TCR. As suggested in the present application, combination therapy for the treatment of solid tumors may extend the scope of application of adoptive T cell therapy, which may not normally be included in patients tested, e.g. patients whose HLA types are mismatched (despite their tumor cells expressing a particular tumor antigen) may also benefit from treatment. In theory, regardless of the match of the HLA class I of tumor cells and the level of antigen expression in tumor cells, delivering nucleic acids encoding a labeled polypeptide comprising an epitope and an HLA class I molecule that can present the epitope peptide into tumor cells and allowing them to be recognized by specific T cells following delivery can be a versatile method of treating a variety of solid tumors. However, if normal cells are also labeled with a labeling nucleic acid and become targets for adoptive transfer of T cells, off-target toxic side effects may result. This risk can be avoided or controlled if the delivery vector can selectively express a marker epitope polypeptide or an exogenous HLA molecule in tumor cells, e.g. using an oncolytic virus having tumor cell selective replication capacity as suggested in the present application. In addition, the oncolytic virus vector enables tumor cells to directly express the labeled polypeptide of the HLAI molecules containing the antigenic epitopes and presenting the antigenic epitope peptides aiming at the defects of the antigen processing and presenting mechanisms which are frequently generated in the tumor cells, and enables the tumor cells to be targets for the adoptive transfer of T cells. In addition, as suggested by the present invention, allogeneic HLA class I molecules are delivered to tumor cells carrying the most common mutations of the tumor driver genes (e.g., KRAS or p53 mutations) so that they present tumor antigen neo-epitopes that cannot be presented by endogenous HLA class I molecules, and are recognized and cleared by subsequently transfused specific T cells. This combination therapy is a promising treatment to benefit more cancer patients.

Sequence listing

<110> synthetic immune products of Co., Ltd (Synimmune, Inc.)

HANGZHOU CONVERD Co.,Ltd.

<120> isolated oncolytic adenovirus capable of expressing foreign gene, vector, therapeutic agent and use thereof

<130> FI-204260-59:52/C

<160> 54

<170> SIPOSequenceListing 1.0

<210> 1

<211> 367

<212> DNA

<213> Adenovirus Type 5 (Type 5 Adenovir)

<400> 1

gtgtctagag aatgcaatag tagtacggat agctgtgact ccggtccttc taacacacct 60

cctgagatac acccggtggt cccgctgtgc cccattaaac cagttgccgt gagagttggt 120

gggcgtcgcc aggctgtgga atgtatcgag gacttgctta acgagcctgg gcaacctttg 180

gacttgagct gtaaacgccc caggccataa ggtgtaaacc tgtgattgcg tgtgtggtta 240

acgcctttgt ttgctgaatg agttgatgta agtttaataa agggtgagat aatgtttaac 300

ttgcatggcg tgttaaatgg ggcggggctt aaagggtata taatgcgccg tgggctaatc 360

ttggtta 367

<210> 2

<211> 1014

<212> DNA

<213> Adenovirus Type 5 (Type 5 Adenovir)

<400> 2

atgagacata ttatctgcca cggaggtgtt attaccgaag aaatggccgc cagtcttttg 60

gaccagctga tcgaagaggt actggctgat aatcttccac ctcctagcca ttttgaacca 120

cctacccttc acgaactgta tgatttagac gtgacggccc ccgaagatcc caacgaggag 180

gcggtttcgc agatttttcc cgactctgta atgttggcgg tgcaggaagg gattgactta 240

ctcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg gcagcccgag 300

cagccggagc agagagcctt gggtccggtt tctatgccaa accttgtacc ggaggtgatc 360

gatccaccca gtgacgacga ggatgaagag ggtgaggagt ttgtgttaga ttatgtggag 420

caccccgggc acggttgcag gtcttgtcat tatcaccgga ggaatacggg ggacccagat 480

attatgtgtt cgctttgcta tatgaggacc tgtggcatgt ttgtctacag tcctgtgtct 540

gaacctgagc ctgagcccga gccagaaccg gagcctgcaa gacctacccg ccgtcctaaa 600

atggcgcctg ctatcctgag acgcccgaca tcacctgtgt ctagagaatg caatagtagt 660

acggatagct gtgactccgg tccttctaac acacctcctg agatacaccc ggtggtcccg 720

ctgtgcccca ttaaaccagt tgccgtgaga gttggtgggc gtcgccaggc tgtggaatgt 780

atcgaggact tgcttaacga gcctgggcaa cctttggact tgagctgtaa acgccccagg 840

ccataaggtg taaacctgtg attgcgtgtg tggttaacgc ctttgtttgc tgaatgagtt 900

gatgtaagtt taataaaggg tgagataatg tttaacttgc atggcgtgtt aaatggggcg 960

gggcttaaag ggtatataat gcgccgtggg ctaatcttgg ttacatctga cctc 1014

<210> 3

<211> 1796

<212> DNA

<213> Adenovirus Type 5 (Type 5 Adenovir)

<400> 3

atggaggctt gggagtgttt ggaagatttt tctgctgtgc gtaacttgct ggaacagagc 60

tctaacagta cctcttggtt ttggaggttt ctgtggggct catcccaggc aaagttagtc 120

tgcagaatta aggaggatta caagtgggaa tttgaagagc ttttgaaatc ctgtggtgag 180

ctgtttgatt ctttgaatct gggtcaccag gcgcttttcc aagagaaggt catcaagact 240

ttggattttt ccacaccggg gcgcgctgcg gctgctgttg cttttttgag ttttataaag 300

gataaatgga gcgaagaaac ccatctgagc ggggggtacc tgctggattt tctggccatg 360

catctgtgga gagcggttgt gagacacaag aatcgcctgc tactgttgtc ttccgtccgc 420

ccggcgataa taccgacgga ggagcagcag cagcagcagg aggaagccag gcggcggcgg 480

caggagcaga gcccatggaa cccgagagcc ggcctggacc ctcgggaatg aatgttgtac 540

aggtggctga actgtatcca gaactgagac gcattttgac aattacagag gatgggcagg 600

ggctaaaggg ggtaaagagg gagcgggggg cttgtgaggc tacagaggag gctaggaatc 660

tagcttttag cttaatgacc agacaccgtc ctgagtgtat tacttttcaa cagatcaagg 720

ataattgcgc taatgagctt gatctgctgg cgcagaagta ttccatagag cagctgacca 780

cttactggct gcagccaggg gatgattttg aggaggctat tagggtatat gcaaaggtgg 840

cacttaggcc agattgcaag tacaagatca gcaaacttgt aaatatcagg aattgttgct 900

acatttctgg gaacggggcc gaggtggaga tagatacgga ggatagggtg gcctttagat 960

gtagcatgat aaatatgtgg ccgggggtgc ttggcatgga cggggtggtt attatgaatg 1020

taaggtttac tggccccaat tttagcggta cggttttcct ggccaatacc aaccttatcc 1080

tacacggtgt aagcttctat gggtttaaca atacctgtgt ggaagcctgg accgatgtaa 1140

gggttcgggg ctgtgccttt tactgctgct ggaagggggt ggtgtgtcgc cccaaaagca 1200

gggcttcaat taagaaatgc ctctttgaaa ggtgtacctt gggtatcctg tctgagggta 1260

actccagggt gcgccacaat gtggcctccg actgtggttg cttcatgcta gtgaaaagcg 1320

tggctgtgat taagcataac atggtatgtg gcaactgcga ggacagggcc tctcagatgc 1380

tgacctgctc ggacggcaac tgtcacctgc tgaagaccat tcacgtagcc agccactctc 1440

gcaaggcctg gccagtgttt gagcataaca tactgacccg ctgttccttg catttgggta 1500

acaggagggg ggtgttccta ccttaccaat gcaatttgag tcacactaag atattgcttg 1560

agcccgagag catgtccaag gtgaacctga acggggtgtt tgacatgacc atgaagatct 1620

ggaaggtgct gaggtacgat gagacccgca ccaggtgcag accctgcgag tgtggcggta 1680

aacatattag gaaccagcct gtgatgctgg atgtgaccga ggagctgagg cccgatcact 1740

tggtgctggc ctgcacccgc gctgagtttg gctctagcga tgaagataca gattga 1796

<210> 4

<211> 10

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<221> misc_feature

<222> (4)

<223> r is a or g

<400> 4

gccrccatgg 10

<210> 5

<211> 870

<212> DNA

<213> Adenovirus Type 5 (Type 5 Adenovir)

<400> 5

atgagacata ttatctgcca cggaggtgtt attaccgaag aaatggccgc cagtcttttg 60

gaccagctga tcgaagaggt actggctgat aatcttccac ctcctagcca ttttgaacca 120

cctacccttc acgaactgta tgatttagac gtgacggccc ccgaagatcc caacgaggag 180

gcggtttcgc agatttttcc cgactctgta atgttggcgg tgcaggaagg gattgactta 240

ctcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg gcagcccgag 300

cagccggagc agagagcctt gggtccggtt tctatgccaa accttgtacc ggaggtgatc 360

gatcttacct gccacgaggc tggctttcca cccagtgacg acgaggatga agagggtgag 420

gagtttgtgt tagattatgt ggagcacccc gggcacggtt gcaggtcttg tcattatcac 480

cggaggaata cgggggaccc agatattatg tgttcgcttt gctatatgag gacctgtggc 540

atgtttgtct acagtcctgt gtctgaacct gagcctgagc ccgagccaga accggagcct 600

gcaagaccta cccgccgtcc taaaatggcg cctgctatcc tgagacgccc gacatcacct 660

gtgtctagag aatgcaatag tagtacggat agctgtgact ccggtccttc taacacacct 720

cctgagatac acccggtggt cccgctgtgc cccattaaac cagttgccgt gagagttggt 780

gggcgtcgcc aggctgtgga atgtatcgag gacttgctta acgagcctgg gcaacctttg 840

gacttgagct gtaaacgccc caggccataa 870

<210> 6

<211> 289

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 6

Met Arg His Ile Ile Cys His Gly Gly Val Ile Thr Glu Glu Met Ala

1 5 10 15

Ala Ser Leu Leu Asp Gln Leu Ile Glu Glu Val Leu Ala Asp Asn Leu

20 25 30

Pro Pro Pro Ser His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp

35 40 45

Leu Asp Val Thr Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln

50 55 60

Ile Phe Pro Asp Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu

65 70 75 80

Leu Thr Phe Pro Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser

85 90 95

Arg Gln Pro Glu Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Ser Met

100 105 110

Pro Asn Leu Val Pro Glu Val Ile Asp Val Thr Ser His Asp Ala Gly

115 120 125

Phe Pro Pro Ser Asp Asp Glu Asp Glu Glu Gly Glu Glu Phe Val Leu

130 135 140

Asp Tyr Val Glu His Pro Gly His Gly Cys Arg Ser Cys His Tyr His

145 150 155 160

Arg Arg Asn Thr Gly Asp Pro Asp Ile Met Cys Ser Leu Cys Tyr Met

165 170 175

Arg Thr Cys Gly Met Phe Val Tyr Ser Pro Val Ser Glu Pro Glu Pro

180 185 190

Glu Pro Glu Pro Glu Pro Glu Pro Ala Arg Pro Thr Arg Arg Pro Lys

195 200 205

Met Ala Pro Ala Ile Leu Arg Arg Pro Thr Ser Pro Val Ser Arg Glu

210 215 220

Cys Asn Ser Ser Thr Asp Ser Cys Asp Ser Gly Pro Ser Asn Thr Pro

225 230 235 240

Pro Glu Ile His Pro Val Val Pro Leu Cys Pro Ile Lys Pro Val Ala

245 250 255

Val Arg Val Gly Gly Arg Arg Gln Ala Val Glu Cys Ile Glu Asp Leu

260 265 270

Leu Asn Glu Pro Gly Gln Pro Leu Asp Leu Ser Cys Lys Arg Pro Arg

275 280 285

Pro

<210> 7

<211> 24

<212> DNA

<213> Adenovirus Type 5 (Type 5 Adenovir)

<400> 7

cttacctgcc acgaggctgg cttt 24

<210> 8

<211> 499

<212> DNA

<213> Adenovirus Type 5 (Type 5 Adenovir)

<400> 8

catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60

ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120

gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180

gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240

taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300

agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360

gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420

cgggtcaaag ttggcgtttt attattatag tcagctgacg tgtagtgtat ttatacccgg 480

tgagttcctc aagaggcca 499

<210> 9

<211> 184

<212> DNA

<213> Adenovirus Type 5 (Type 5 Adenovir)

<400> 9

cgtggagact cgcccaggtg tttttctcag gtgttttccg cgttccgggt caaagttggc 60

gttttattat tatagtcagc tgacgtgtag tgtatttata cccggtgagt tcctcaagag 120

gccactcttg agtgccagcg agtagagttt tctcctccga gccgctccga caccgggact 180

gaaa 184

<210> 10

<211> 9

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 10

Ser Leu Leu Met Trp Ile Thr Gln Cys

1 5

<210> 11

<211> 9

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 11

Gly Ala Asp Gly Val Gly Lys Ser Ala

1 5

<210> 12

<211> 365

<212> PRT

<213> human (Homo sapiens)

<400> 12

Met Ala Val Met Ala Pro Arg Thr Leu Val Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Gln Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe

20 25 30

Phe Thr Ser Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala

50 55 60

Ala Ser Gln Arg Met Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly

65 70 75 80

Pro Glu Tyr Trp Asp Gly Glu Thr Arg Lys Val Lys Ala His Ser Gln

85 90 95

Thr His Arg Val Asp Leu Gly Thr Leu Arg Gly Tyr Tyr Asn Gln Ser

100 105 110

Glu Ala Gly Ser His Thr Val Gln Arg Met Tyr Gly Cys Asp Val Gly

115 120 125

Ser Asp Trp Arg Phe Leu Arg Gly Tyr His Gln Tyr Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Ile Ala Leu Lys Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Met Ala Ala Gln Thr Thr Lys His Lys Trp Glu Ala Ala His Val

165 170 175

Ala Glu Gln Leu Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu

180 185 190

Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Thr Asp Ala

195 200 205

Pro Lys Thr His Met Thr His His Ala Val Ser Asp His Glu Ala Thr

210 215 220

Leu Arg Cys Trp Ala Leu Ser Phe Tyr Pro Ala Glu Ile Thr Leu Thr

225 230 235 240

Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu Leu Val Glu

245 250 255

Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val

260 265 270

Val Pro Ser Gly Gln Glu Gln Arg Tyr Thr Cys His Val Gln His Glu

275 280 285

Gly Leu Pro Lys Pro Leu Thr Leu Arg Trp Glu Pro Ser Ser Gln Pro

290 295 300

Thr Ile Pro Ile Val Gly Ile Ile Ala Gly Leu Val Leu Phe Gly Ala

305 310 315 320

Val Ile Thr Gly Ala Val Val Ala Ala Val Met Trp Arg Arg Lys Ser

325 330 335

Ser Asp Arg Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser

340 345 350

Ala Gln Gly Ser Asp Val Ser Leu Thr Ala Cys Lys Val

355 360 365

<210> 13

<211> 366

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 13

Met Ala Val Met Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Glu Thr Trp Ala Cys Ser His Ser Met Arg Tyr Phe

20 25 30

Tyr Thr Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Val Gly Tyr Val Asp Asp Thr Gln Phe Val Gln Phe Asp Ser Asp Ala

50 55 60

Ala Ser Pro Arg Gly Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly

65 70 75 80

Pro Glu Tyr Trp Asp Arg Glu Thr Gln Lys Tyr Lys Arg Gln Ala Gln

85 90 95

Thr Asp Arg Val Ser Leu Arg Asn Leu Arg Gly Tyr Tyr Asn Gln Ser

100 105 110

Glu Ala Gly Ser His Thr Leu Gln Arg Met Tyr Gly Cys Asp Leu Gly

115 120 125

Pro Asp Gly Arg Leu Leu Arg Gly Tyr Asn Gln Phe Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Ile Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Lys Ala Ala Gln Ile Thr Gln Arg Lys Trp Glu Ala Ala Arg Glu

165 170 175

Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu

180 185 190

Arg Arg Tyr Leu Glu Asn Gly Lys Lys Thr Leu Gln Arg Ala Glu His

195 200 205

Pro Lys Thr His Val Thr His His Pro Val Ser Asp His Glu Ala Thr

210 215 220

Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Thr Leu Thr

225 230 235 240

Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu Leu Val Glu

245 250 255

Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val

260 265 270

Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu

275 280 285

Gly Leu Pro Glu Pro Leu Thr Leu Arg Trp Gly Pro Ser Ser Gln Pro

290 295 300

Thr Ile Pro Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val

305 310 315 320

Leu Ala Val Leu Gly Ala Val Met Ala Val Val Met Cys Arg Arg Lys

325 330 335

Ser Ser Gly Gly Lys Gly Gly Ser Cys Ser Gln Ala Ala Ser Ser Asn

340 345 350

Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala Cys Lys Ala

355 360 365

<210> 14

<211> 22

<212> PRT

<213> human (Homo sapiens)

<400> 14

Met Lys Gly Ser Ile Phe Thr Leu Phe Leu Phe Ser Val Leu Phe Ala

1 5 10 15

Ile Ser Glu Val Arg Ser

20

<210> 15

<211> 462

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 15

Met Ala Val Met Ala Pro Arg Thr Leu Val Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Gln Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe

20 25 30

Phe Thr Ser Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala

50 55 60

Ala Ser Gln Arg Met Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly

65 70 75 80

Pro Glu Tyr Trp Asp Gly Glu Thr Arg Lys Val Lys Ala His Ser Gln

85 90 95

Thr His Arg Val Asp Leu Gly Thr Leu Arg Gly Tyr Tyr Asn Gln Ser

100 105 110

Glu Ala Gly Ser His Thr Val Gln Arg Met Tyr Gly Cys Asp Val Gly

115 120 125

Ser Asp Trp Arg Phe Leu Arg Gly Tyr His Gln Tyr Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Ile Ala Leu Lys Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Met Ala Ala Gln Thr Thr Lys His Lys Trp Glu Ala Ala His Val

165 170 175

Ala Glu Gln Leu Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu

180 185 190

Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Thr Asp Ala

195 200 205

Pro Lys Thr His Met Thr His His Ala Val Ser Asp His Glu Ala Thr

210 215 220

Leu Arg Cys Trp Ala Leu Ser Phe Tyr Pro Ala Glu Ile Thr Leu Thr

225 230 235 240

Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu Leu Val Glu

245 250 255

Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val

260 265 270

Val Pro Ser Gly Gln Glu Gln Arg Tyr Thr Cys His Val Gln His Glu

275 280 285

Gly Leu Pro Lys Pro Leu Thr Leu Arg Trp Glu Pro Ser Ser Gln Pro

290 295 300

Thr Ile Pro Ile Val Gly Ile Ile Ala Gly Leu Val Leu Phe Gly Ala

305 310 315 320

Val Ile Thr Gly Ala Val Val Ala Ala Val Met Trp Arg Arg Lys Ser

325 330 335

Ser Asp Arg Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser

340 345 350

Ala Gln Gly Ser Asp Val Ser Leu Thr Ala Cys Lys Val Arg Ala Lys

355 360 365

Arg Ser Gly Ser Gly Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu

370 375 380

Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Lys Gly

385 390 395 400

Ser Ile Phe Thr Leu Phe Leu Phe Ser Val Leu Phe Ala Ile Ser Glu

405 410 415

Val Arg Ser Ser Leu Leu Met Trp Ile Thr Gln Cys Arg Arg Lys Arg

420 425 430

Ser Leu Leu Met Trp Ile Thr Gln Cys Arg Arg Lys Arg Ser Leu Leu

435 440 445

Met Trp Ile Thr Gln Cys Arg Arg Arg Arg Lys Asp Glu Leu

450 455 460

<210> 16

<211> 467

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 16

Met Ala Val Met Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Glu Thr Trp Ala Cys Ser His Ser Met Arg Tyr Phe

20 25 30

Tyr Thr Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Val Gly Tyr Val Asp Asp Thr Gln Phe Val Gln Phe Asp Ser Asp Ala

50 55 60

Ala Ser Pro Arg Gly Glu Pro Arg Ala Pro Trp Val Glu Gln Glu Gly

65 70 75 80

Pro Glu Tyr Trp Asp Arg Glu Thr Gln Lys Tyr Lys Arg Gln Ala Gln

85 90 95

Thr Asp Arg Val Ser Leu Arg Asn Leu Arg Gly Tyr Tyr Asn Gln Ser

100 105 110

Glu Ala Gly Ser His Thr Leu Gln Arg Met Tyr Gly Cys Asp Leu Gly

115 120 125

Pro Asp Gly Arg Leu Leu Arg Gly Tyr Asn Gln Phe Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Ile Ala Leu Asn Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Lys Ala Ala Gln Ile Thr Gln Arg Lys Trp Glu Ala Ala Arg Glu

165 170 175

Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu

180 185 190

Arg Arg Tyr Leu Glu Asn Gly Lys Lys Thr Leu Gln Arg Ala Glu His

195 200 205

Pro Lys Thr His Val Thr His His Pro Val Ser Asp His Glu Ala Thr

210 215 220

Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile Thr Leu Thr

225 230 235 240

Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Thr Glu Leu Val Glu

245 250 255

Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ala Val Val

260 265 270

Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val Gln His Glu

275 280 285

Gly Leu Pro Glu Pro Leu Thr Leu Arg Trp Gly Pro Ser Ser Gln Pro

290 295 300

Thr Ile Pro Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val

305 310 315 320

Leu Ala Val Leu Gly Ala Val Met Ala Val Val Met Cys Arg Arg Lys

325 330 335

Ser Ser Gly Gly Lys Gly Gly Ser Cys Ser Gln Ala Ala Ser Ser Asn

340 345 350

Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala Cys Lys Ala Arg Ala

355 360 365

Lys Arg Ser Gly Ser Gly Ala Pro Val Lys Gln Thr Leu Asn Phe Asp

370 375 380

Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Lys

385 390 395 400

Gly Ser Ile Phe Thr Leu Phe Leu Phe Ser Val Leu Phe Ala Ile Ser

405 410 415

Glu Val Arg Ser Gly Ala Asp Gly Val Gly Lys Ser Ala Arg Arg Lys

420 425 430

Arg Arg Arg Lys Arg Gly Ala Asp Gly Val Gly Lys Ser Ala Arg Arg

435 440 445

Lys Arg Gly Ala Asp Gly Val Gly Lys Ser Ala Arg Arg Lys Arg Lys

450 455 460

Asp Glu Leu

465

<210> 17

<211> 119

<212> PRT

<213> human (Homo sapiens)

<400> 17

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg

20 25 30

His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser

35 40 45

Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu

50 55 60

Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp

65 70 75 80

Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp

85 90 95

Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile

100 105 110

Val Lys Trp Asp Arg Asp Met

115

<210> 18

<211> 1824

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 18

atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 60

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 180

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 240

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 300

gctccctccc agacatctgt gtacttctgt gccagcagtt acgtcgggaa caccggggag 360

ctgttttttg gagaaggctc taggctgacc gtactggagg atctgagaaa tgtgactcca 420

cccaaggtct ccttgtttga gccatcaaaa gcagagattg caaacaaaca aaaggctacc 480

ctcgtgtgct tggccagggg cttcttccct gaccacgtgg agctgagctg gtgggtgaat 540

ggcaaggagg tccacagtgg ggtcagcacg gaccctcagg cctacaagga gagcaattat 600

agctactgcc tgagcagccg cctgagggtc tctgctacct tctggcacaa tcctcgcaac 660

cacttccgct gccaagtgca gttccatggg ctttcagagg aggacaagtg gccagagggc 720

tcacccaaac ctgtcacaca gaacatcagt gcagaggcct ggggccgagc agactgtggg 780

attacctcag catcctatca acaaggggtc ttgtctgcca ccatcctcta tgagatcctg 840

ctagggaaag ccaccctgta tgctgtgctt gtcagtacac tggtggtgat ggctatggtc 900

aaaagaaaga attcacgtgc caagcgatcc ggaagcggag cccctgtaaa gcagactttg 960

aattttgacc ttctcaagtt ggcgggagac gtcgagtcca accctgggcc catggagacc 1020

ctcttgggcc tgcttatcct ttggctgcag ctgcaatggg tgagcagcaa acaggaggtg 1080

acacagattc ctgcagctct gagtgtccca gaaggagaaa acttggttct caactgcagt 1140

ttcactgata gcgctattta caacctccag tggtttaggc aggaccctgg gaaaggtctc 1200

acatctctgt tgcttattca gtcaagtcag agagagcaaa caagtggaag acttaatgcc 1260

tcgctggata aatcatcagg acgtagtact ttatacattg cagcttctca gcctggtgac 1320

tcagccacct acctctgtgc tgtgaggccc ctgtacggag gaagctacat acctacattt 1380

ggaagaggaa ccagccttat tgttcatccg tatatccaga acccagaacc tgctgtgtac 1440

cagttaaaag atcctcggtc tcaggacagc accctctgcc tgttcaccga ctttgactcc 1500

caaatcaatg tgccgaaaac catggaatct ggaacgttca tcactgacaa aactgtgctg 1560

gacatgaaag ctatggattc caagagcaat ggggccattg cctggagcaa ccagacaagc 1620

ttcacctgcc aagatatctt caaagagacc aacgccacct accccagttc agacgttccc 1680

tgtgatgcca cgttgaccga gaaaagcttt gaaacagata tgaacctaaa ctttcaaaac 1740

ctgtcagtta tgggactccg aatcctcctg ctgaaagtag cgggatttaa cctgctcatg 1800

acgctgaggc tgtggtccag ttga 1824

<210> 19

<211> 1824

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 19

atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 60

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 180

gggctgaggc tgattcatta ctcagttgct gaaggtatca ctgaccaagg agaagtcccc 240

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 300

gctccctccc agacatctgt gtacttctgt gccagcagtt acgtcgggaa caccggggag 360

ctgttttttg gagaaggctc taggctgacc gtactggagg atctgagaaa tgtgactcca 420

cccaaggtct ccttgtttga gccatcaaaa gcagagattg caaacaaaca aaaggctacc 480

ctcgtgtgct tggccagggg cttcttccct gaccacgtgg agctgagctg gtgggtgaat 540

ggcaaggagg tccacagtgg ggtcagcacg gaccctcagg cctacaagga gagcaattat 600

agctactgcc tgagcagccg cctgagggtc tctgctacct tctggcacaa tcctcgcaac 660

cacttccgct gccaagtgca gttccatggg ctttcagagg aggacaagtg gccagagggc 720

tcacccaaac ctgtcacaca gaacatcagt gcagaggcct ggggccgagc agactgtggg 780

attacctcag catcctatca acaaggggtc ttgtctgcca ccatcctcta tgagatcctg 840

ctagggaaag ccaccctgta tgctgtgctt gtcagtacac tggtggtgat ggctatggtc 900

aaaagaaaga attcacgtgc caagcgatcc ggaagcggag cccctgtaaa gcagactttg 960

aattttgacc ttctcaagtt ggcgggagac gtcgagtcca accctgggcc catggagacc 1020

ctcttgggcc tgcttatcct ttggctgcag ctgcaatggg tgagcagcaa acaggaggtg 1080

acacagattc ctgcagctct gagtgtccca gaaggagaaa acttggttct caactgcagt 1140

ttcactgata gcgctattta caacctccag tggtttaggc aggaccctgg gaaaggtctc 1200

acatctctgt tgcttattca gtcaagtcag agagagcaaa caagtggaag acttaatgcc 1260

tcgctggata aatcatcagg acgtagtact ttatacattg cagcttctca gcctggtgac 1320

tcagccacct acctctgtgc tgtgaggccc acatcaggag gaagctacat acctacattt 1380

ggaagaggaa ccagccttat tgttcatccg tatatccaga acccagaacc tgctgtgtac 1440

cagttaaaag atcctcggtc tcaggacagc accctctgcc tgttcaccga ctttgactcc 1500

caaatcaatg tgccgaaaac catggaatct ggaacgttca tcactgacaa aactgtgctg 1560

gacatgaaag ctatggattc caagagcaat ggggccattg cctggagcaa ccagacaagc 1620

ttcacctgcc aagatatctt caaagagacc aacgccacct accccagttc agacgttccc 1680

tgtgatgcca cgttgaccga gaaaagcttt gaaacagata tgaacctaaa ctttcaaaac 1740

ctgtcagtta tgggactccg aatcctcctg ctgaaagtag cgggatttaa cctgctcatg 1800

acgctgaggc tgtggtccag ttga 1824

<210> 20

<211> 1809

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 20

atgggctcct ggaccctctg ctgtgtgtcc ctttgcatcc tggtagcaaa gcacacagat 60

gctggagtta tccagtcacc ccggcacgag gtgacagaga tgggacaaga agtgactctg 120

agatgtaaac caatttcagg acacgactac cttttctggt acagacagac catgatgcgg 180

ggactggagt tgctcattta ctttaacaac aacgttccga tagatgattc agggatgccc 240

gaggatcgat tctcagctaa gatgcctaat gcatcattct ccactctgaa gatccagccc 300

tcagaaccca gggactcagc tgtgtacttc tgtgccagca gtttaggctc caacgagcag 360

tacttcgggc cgggcaccag gctcacggtc acagaggatc tgagaaatgt gactccaccc 420

aaggtctcct tgtttgagcc atcaaaagca gagattgcaa acaaacaaaa ggctaccctc 480

gtgtgcttgg ccaggggctt cttccctgac cacgtggagc tgagctggtg ggtgaatggc 540

aaggaggtcc acagtggggt cagcacggac cctcaggcct acaaggagag caattatagc 600

tactgcctga gcagccgcct gagggtctct gctaccttct ggcacaatcc tcgcaaccac 660

ttccgctgcc aagtgcagtt ccatgggctt tcagaggagg acaagtggcc agagggctca 720

cccaaacctg tcacacagaa catcagtgca gaggcctggg gccgagcaga ctgtgggatt 780

acctcagcat cctatcaaca aggggtcttg tctgccacca tcctctatga gatcctgcta 840

gggaaagcca ccctgtatgc tgtgcttgtc agtacactgg tggtgatggc tatggtcaaa 900

agaaagaatt cacgtgccaa gcgatccgga agcggagccc ctgtaaagca gactttgaat 960

tttgaccttc tcaagttggc gggagacgtc gagtccaacc ctgggcccat ggaaactctc 1020

ctgggagtgt ctttggtgat tctatggctt caactggcta gggtgaacag tcaacaggga 1080

gaagaggatc ctcaggcctt gagcatccag gagggtgaaa atgccaccat gaactgcagt 1140

tacaaaacta gtataaacaa tttacagtgg tatagacaaa attcaggtag aggccttgtc 1200

cacctaattt taatacgttc aaatgaaaga gagaaacaca gtggaagatt aagagtcacg 1260

cttgacactt ccaagaaaag cagttccttg ttgatcacgg cttcccgggc agcagacact 1320

gcttcttact tctgtatgta cgaccagaac ggcaagatca tctttggaaa agggacacga 1380

cttcatattc tccccaatat ccagaaccca gaacctgctg tgtaccagtt aaaagatcct 1440

cggtctcagg acagcaccct ctgcctgttc accgactttg actcccaaat caatgtgccg 1500

aaaaccatgg aatctggaac gttcatcact gacaaaactg tgctggacat gaaagctatg 1560

gattccaaga gcaatggggc cattgcctgg agcaaccaga caagcttcac ctgccaagat 1620

atcttcaaag agaccaacgc cacctacccc agttcagacg ttccctgtga tgccacgttg 1680

accgagaaaa gctttgaaac agatatgaac ctaaactttc aaaacctgtc agttatggga 1740

ctccgaatcc tcctgctgaa agtagcggga tttaacctgc tcatgacgct gaggctgtgg 1800

tccagttga 1809

<210> 21

<211> 1812

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 21

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggtgaggg aagagtggac 360

ggctacacct tcggttcggg gaccaggtta accgttgtag aggatctgag aaatgtgact 420

ccacccaagg tctccttgtt tgagccatca aaagcagaga ttgcaaacaa acaaaaggct 480

accctcgtgt gcttggccag gggcttcttc cctgaccacg tggagctgag ctggtgggtg 540

aatggcaagg aggtccacag tggggtcagc acggaccctc aggcctacaa ggagagcaat 600

tatagctact gcctgagcag ccgcctgagg gtctctgcta ccttctggca caatcctcgc 660

aaccacttcc gctgccaagt gcagttccat gggctttcag aggaggacaa gtggccagag 720

ggctcaccca aacctgtcac acagaacatc agtgcagagg cctggggccg agcagactgt 780

gggattacct cagcatccta tcaacaaggg gtcttgtctg ccaccatcct ctatgagatc 840

ctgctaggga aagccaccct gtatgctgtg cttgtcagta cactggtggt gatggctatg 900

gtcaaaagaa agaattcacg tgccaagcga tccggaagcg gagcccctgt aaagcagact 960

ttgaattttg accttctcaa gttggcggga gacgtcgagt ccaaccctgg gcccatgagg 1020

caagtggcga gagtgatcgt gttcctgacc ctgagtactt tgagccttgc taagaccacc 1080

cagcccatct ccatggactc atatgaagga caagaagtga acataacctg tagccacaac 1140

aacattgcta caaatgatta tatcacgtgg taccaacagt ttcccagcca aggaccacga 1200

tttattattc aaggatacaa gacaaaagtt acaaacgaag tggcctccct gtttatccct 1260

gccgacagaa agtccagcac tctgagcctg ccccgggttt ccctgagcga cactgctgtg 1320

tactactgcc tcgtgggtga catggatcag gcaggaactg ctctgatctt tgggaaggga 1380

accaccttat cagtgagttc catccagaac ccagaacctg ctgtgtacca gttaaaagat 1440

cctcggtctc aggacagcac cctctgcctg ttcaccgact ttgactccca aatcaatgtg 1500

ccgaaaacca tggaatctgg aacgttcatc actgacaaaa ctgtgctgga catgaaagct 1560

atggattcca agagcaatgg ggccattgcc tggagcaacc agacaagctt cacctgccaa 1620

gatatcttca aagagaccaa cgccacctac cccagttcag acgttccctg tgatgccacg 1680

ttgaccgaga aaagctttga aacagatatg aacctaaact ttcaaaacct gtcagttatg 1740

ggactccgaa tcctcctgct gaaagtagcg ggatttaacc tgctcatgac gctgaggctg 1800

tggtccagtt ga 1812

<210> 22

<211> 45

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 22

tatataagag cagagctgcc accatgcagg ccgaaggccg gggca 45

<210> 23

<211> 42

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 23

tgattgtcga cgcccttagc gcctctgccc tgagggaggc tg 42

<210> 24

<211> 57

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 24

atgactgaat ataaacttgt ggtagttgga gctgacggcg taggcaagag tgccttg 57

<210> 25

<211> 46

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 25

tgattgtcga cgcccttaca taattacaca ctttgtcttt gacttc 46

<210> 26

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 26

ctcatagcgc gtaatggctc cggtgcccgt cagtgggcag 40

<210> 27

<211> 39

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 27

gaattcgcta gctctagatc acgacacctg aaatggaag 39

<210> 28

<211> 1098

<212> DNA

<213> human (Homo sapiens)

<400> 28

atggccgtca tggcgccccg aaccctcgtc ctgctactct cgggggctct ggccctgacc 60

cagacctggg cgggctctca ctccatgagg tatttcttca catccgtgtc ccggcccggc 120

cgcggggagc cccgcttcat cgcagtgggc tacgtggacg acacgcagtt cgtgcggttc 180

gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggt 240

ccggagtatt gggacgggga gacacggaaa gtgaaggccc actcacagac tcaccgagtg 300

gacctgggga ccctgcgcgg ctactacaac cagagcgagg ccggttctca caccgtccag 360

aggatgtatg gctgcgacgt ggggtcggac tggcgcttcc tccgcgggta ccaccagtac 420

gcctacgacg gcaaggatta catcgccctg aaagaggacc tgcgctcttg gaccgcggcg 480

gacatggcag ctcagaccac caagcacaag tgggaggcgg cccatgtggc ggagcagttg 540

agagcctacc tggagggcac gtgcgtggag tggctccgca gatacctgga gaacgggaag 600

gagacgctgc agcgcacgga cgcccccaaa acgcatatga ctcaccacgc tgtctctgac 660

catgaagcca ccctgaggtg ctgggccctg agcttctacc ctgcggagat cacactgacc 720

tggcagcggg atggggagga ccagacccag gacacggagc tcgtggagac caggcctgca 780

ggggatggaa ccttccagaa gtgggcggct gtggtggtgc cttctggaca ggagcagaga 840

tacacctgcc atgtgcagca tgagggtttg cccaagcccc tcaccctgag atgggagccg 900

tcttcccagc ccaccatccc catcgtgggc atcattgctg gcctggttct ctttggagct 960

gtgatcactg gagctgtggt cgctgctgtg atgtggagga ggaagagctc agatagaaaa 1020

ggagggagct actctcaggc tgcaagcagt gacagtgccc agggctctga tgtgtctctc 1080

acagcttgta aagtgtga 1098

<210> 29

<211> 198

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 29

atgaaaggtt ccatcttcac attgtttttg ttctccgtat tgttcgcaat cagcgaagtc 60

cgatcatccc tgttgatgtg gatcacgcag tgccgcagaa agaggtcact cttaatgtgg 120

ataacccaat gtaggcgaaa gagatcgcta ttgatgtgga ttacacagtg taggcgaagg 180

cggaaagacg agctttaa 198

<210> 30

<211> 96

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 30

cgtgccaagc gatccggaag cggagcccct gtaaagcaga ctttgaattt tgaccttctc 60

aagttggcgg gagacgtcga gtccaaccct gggccc 96

<210> 31

<211> 1365

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 31

atgaaaggtt ccatcttcac attgtttttg ttctccgtat tgttcgcaat cagcgaagtc 60

cgatcatccc tgttgatgtg gatcacgcag tgccgcagaa agaggtcact cttaatgtgg 120

ataacccaat gtaggcgaaa gagatcgcta ttgatgtgga ttacacagtg tcgtcgtaag 180

cgatccggaa gcggagcccc tgtaaagcag actttgaatt ttgaccttct caagttggcg 240

ggagacgtcg agtccaaccc tgggcccatg gccgtcatgg cgccccgaac cctcgtcctg 300

ctactctcgg gggctctggc cctgacccag acctgggcgg gctctcactc catgaggtat 360

ttcttcacat ccgtgtcccg gcccggccgc ggggagcccc gcttcatcgc agtgggctac 420

gtggacgaca cgcagttcgt gcggttcgac agcgacgccg cgagccagag gatggagccg 480

cgggcgccgt ggatagagca ggagggtccg gagtattggg acggggagac acggaaagtg 540

aaggcccact cacagactca ccgagtggac ctggggaccc tgcgcggcta ctacaaccag 600

agcgaggccg gttctcacac cgtccagagg atgtatggct gcgacgtggg gtcggactgg 660

cgcttcctcc gcgggtacca ccagtacgcc tacgacggca aggattacat cgccctgaaa 720

gaggacctgc gctcttggac cgcggcggac atggcagctc agaccaccaa gcacaagtgg 780

gaggcggccc atgtggcgga gcagttgaga gcctacctgg agggcacgtg cgtggagtgg 840

ctccgcagat acctggagaa cgggaaggag acgctgcagc gcacggacgc ccccaaaacg 900

catatgactc accacgctgt ctctgaccat gaagccaccc tgaggtgctg ggccctgagc 960

ttctaccctg cggagatcac actgacctgg cagcgggatg gggaggacca gacccaggac 1020

acggagctcg tggagaccag gcctgcaggg gatggaacct tccagaagtg ggcggctgtg 1080

gtggtgcctt ctggacagga gcagagatac acctgccatg tgcagcatga gggtttgccc 1140

aagcccctca ccctgagatg ggagccgtct tcccagccca ccatccccat cgtgggcatc 1200

attgctggcc tggttctctt tggagctgtg atcactggag ctgtggtcgc tgctgtgatg 1260

tggaggagga agagctcaga tagaaaagga gggagctact ctcaggctgc aagcagtgac 1320

agtgcccagg gctctgatgt gtctctcaca gcttgtaaag tgtga 1365

<210> 32

<211> 38

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 32

agagctagcg aattcaacat gaaaggttcc atcttcac 38

<210> 33

<211> 35

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 33

acactgtgta atccacatca atagcgatct ctttc 35

<210> 34

<211> 39

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 34

tggattacac agtgtcgtcg taagcgatcc ggaagcgcg 39

<210> 35

<211> 39

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 35

cgccatgacg gccatgggcc cagggttgga ctcgacgtc 39

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 36

atggccgtca tggcgccccg a 21

<210> 37

<211> 27

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 37

tcacacttta caagctgtga gagacac 27

<210> 38

<211> 33

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 38

atgaaaggtt ccatcttcac attgtttttg ttc 33

<210> 39

<211> 39

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 39

cgccatgacg gccatgggcc cagggttgga ctcgacgtc 39

<210> 40

<211> 28

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 40

atgagacata ttatctgcca cggaggtg 28

<210> 41

<211> 25

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 41

ttatggcctg gggcgtttac agctc 25

<210> 42

<211> 984

<212> DNA

<213> Adenovirus Type 5 (Type 5 Adenovir)

<400> 42

atgagacata ttatctgcca cggaggtgtt attaccgaag aaatggccgc cagtcttttg 60

gaccagctga tcgaagaggt actggctgat aatcttccac ctcctagcca ttttgaacca 120

cctacccttc acgaactgta tgatttagac gtgacggccc ccgaagatcc caacgaggag 180

gcggtttcgc agatttttcc cgagtctgta atgttggcgg tgcaggaagg gattgactta 240

ttcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg gcagcccgag 300

cagccggagc agagagcctt gggtccggtt tctatgccaa accttgtgcc ggaggtgatc 360

gatcttacct gccacgaggc tggctttcca cccagtgacg acgaggatga agagggtgag 420

gagtttgtgt tagattatgt ggagcacccc gggcacggtt gcaggtcttg tcattatcac 480

cggaggaata cgggggaccc agatattatg tgttcgcttt gctatatgag gacctgtggc 540

atgtttgtct acagtaagtg aaaattatgg gcagtcggtg atagagtggt gggtttggtg 600

tggtaatttt tttttaattt ttacagtttt gtggtttaaa gaattttgta ttgtgatttt 660

ttaaaaggtc ctgtgtctga acctgagcct gagcccgagc cagaaccgga gcctgcaaga 720

cctacccggc gtcctaaatt ggtgcctgct atcctgagac gcccgacatc acctgtgtct 780

agagaatgca atagtagtac ggatagctgt gactccggtc cttctaacac acctcctgag 840

atacacccgg tggtcccgct gtgccccatt aaaccagttg ccgtgagagt tggtgggcgt 900

cgccaggctg tggaatgtat cgaggacttg cttaacgagt ctgggcaacc tttggacttg 960

agctgtaaac gccccaggcc ataa 984

<210> 43

<211> 281

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

Met Arg His Ile Ile Cys His Gly Gly Val Ile Thr Glu Glu Met Ala

1 5 10 15

Ala Ser Leu Leu Asp Gln Leu Ile Glu Glu Val Leu Ala Asp Asn Leu

20 25 30

Pro Pro Pro Ser His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp

35 40 45

Leu Asp Val Thr Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln

50 55 60

Ile Phe Pro Asp Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu

65 70 75 80

Leu Thr Phe Pro Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser

85 90 95

Arg Gln Pro Glu Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Ser Met

100 105 110

Pro Asn Leu Val Pro Glu Val Ile Asp Pro Pro Ser Asp Asp Glu Asp

115 120 125

Glu Glu Gly Glu Glu Phe Val Leu Asp Tyr Val Glu His Pro Gly His

130 135 140

Gly Cys Arg Ser Cys His Tyr His Arg Arg Asn Thr Gly Asp Pro Asp

145 150 155 160

Ile Met Cys Ser Leu Cys Tyr Met Arg Thr Cys Gly Met Phe Val Tyr

165 170 175

Ser Pro Val Ser Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro

180 185 190

Ala Arg Pro Thr Arg Arg Pro Lys Met Ala Pro Ala Ile Leu Arg Arg

195 200 205

Pro Thr Ser Pro Val Ser Arg Glu Cys Asn Ser Ser Thr Asp Ser Cys

210 215 220

Asp Ser Gly Pro Ser Asn Thr Pro Pro Glu Ile His Pro Val Val Pro

225 230 235 240

Leu Cys Pro Ile Lys Pro Val Ala Val Arg Val Gly Gly Arg Arg Gln

245 250 255

Ala Val Glu Cys Ile Glu Asp Leu Leu Asn Glu Pro Gly Gln Pro Leu

260 265 270

Asp Leu Ser Cys Lys Arg Pro Arg Pro

275 280

<210> 44

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

cgcgtcgact actgtaatag taatcaatta cgg 33

<210> 45

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

gacgtcgact aagatacatt gatgagtttg gac 33

<210> 46

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

atgagacata ttatctgcca cggag 25

<210> 47

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

catggtggcg aggtcagatg taac 24

<210> 48

<211> 388

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

gtgtctagag aatgcaatag tagtacggat agctgtgact ccggtccttc taacacacct 60

cctgagatac acccggtggt cccgctgtgc cccattaaac cagttgccgt gagagttggt 120

gggcgtcgcc aggctgtgga atgtatcgag gacttgctta acgagcctgg gcaacctttg 180

gacttgagct gtaaacgccc caggccataa ggtgtaaacc tgtgattgcg tgtgtggtta 240

acgcctttgt ttgctgaatg agttgatgta agtttaataa agggtgagat aatgtttaac 300

ttgcatggcg tgttaaatgg ggcggggctt aaagggtata taatgcgccg tgggctaatc 360

ttggttacat ctgacctcgc caccatgg 388

<210> 49

<211> 1101

<212> DNA

<213> human (Homo sapiens)

<400> 49

atggcggtca tggcgccccg aaccctcatc ctgctgctct cgggagccct ggccctgacc 60

gagacctggg cctgctccca ctccatgagg tatttctaca ccgccgtgtc ccggcccggc 120

cgcggagagc cccgcttcat cgcagtgggc tacgtggacg acacgcagtt cgtgcagttc 180

gacagcgacg ccgcgagtcc aagaggggag ccgcgggcgc cgtgggtgga gcaggagggg 240

ccggagtatt gggaccggga gacacagaag tacaagcgcc aggcacagac tgaccgagtg 300

agcctgcgga acctgcgcgg ctactacaac cagagcgagg ccgggtctca caccctccag 360

aggatgtatg gctgcgacct ggggcccgac gggcgcctcc tccgcgggta taaccagttc 420

gcctacgacg gcaaggatta catcgccctg aatgaggacc tgcgctcctg gaccgccgcg 480

gacaaggcgg ctcagatcac ccagcgcaag tgggaggcgg cccgtgaggc ggagcagcgg 540

agagcctacc tggagggcac gtgcgtggag tggctccgca gatacctgga gaacgggaag 600

aagacgctgc agcgcgcgga acacccaaag acacacgtga cccaccatcc cgtctctgac 660

catgaggcca ccctgaggtg ctgggccctg ggcttctacc ctgcggagat cacactgacc 720

tggcagcggg atggcgagga ccaaactcag gacaccgagc ttgtggagac caggccagca 780

ggagatggaa ccttccagaa gtgggcagct gtggtggtgc cttctggaga agagcagaga 840

tacacgtgcc atgtgcagca cgaggggctg ccagagcccc tcaccctgag atgggggcca 900

tcttcccagc ccaccatccc catcgtgggc atcgttgctg gcctggctgt cctggctgtc 960

ctagctgtcc taggagctgt gatggctgtt gtgatgtgta ggaggaagag ctcaggtgga 1020

aaaggaggga gctgctctca ggctgcgtcc agcaacagtg cccagggctc tgatgtgtct 1080

ctcacagctt gtaaagccta a 1101

<210> 50

<211> 210

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

atgaaaggtt ccatcttcac attgtttttg ttctccgtat tgttcgcaat cagcgaagtc 60

cgatcaggag ctgatggcgt aggcaagagt gcccgcagaa agaggcgcag aaagaggggg 120

gccgatggtg ttggaaagag cgctaggcgg aagaggggcg ccgatggtgt cggaaaaagc 180

gcgcggcgga aacgaaaaga cgagctttaa 210

<210> 51

<211> 65

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

Met Lys Gly Ser Ile Phe Thr Leu Phe Leu Phe Ser Val Leu Phe Ala

1 5 10 15

Ile Ser Glu Val Arg Ser Ser Leu Leu Met Trp Ile Thr Gln Cys Arg

20 25 30

Arg Lys Arg Ser Leu Leu Met Trp Ile Thr Gln Cys Arg Arg Lys Arg

35 40 45

Ser Leu Leu Met Trp Ile Thr Gln Cys Arg Arg Arg Arg Lys Asp Glu

50 55 60

Leu

65

<210> 52

<211> 607

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala

1 5 10 15

Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu

20 25 30

Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His

35 40 45

Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu

50 55 60

Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro

65 70 75 80

Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg

85 90 95

Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser

100 105 110

Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg

115 120 125

Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser

130 135 140

Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

180 185 190

Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu

195 200 205

Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys

210 215 220

Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly

225 230 235 240

Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg

245 250 255

Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser

260 265 270

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

275 280 285

Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn

290 295 300

Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Pro Val Lys Gln Thr Leu

305 310 315 320

Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly

325 330 335

Pro Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln

340 345 350

Trp Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser

355 360 365

Val Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser

370 375 380

Ala Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu

385 390 395 400

Thr Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly

405 410 415

Arg Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr

420 425 430

Ile Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val

435 440 445

Arg Pro Leu Tyr Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr

450 455 460

Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro Glu Pro Ala Val Tyr

465 470 475 480

Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr

485 490 495

Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr

500 505 510

Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys

515 520 525

Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln

530 535 540

Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro

545 550 555 560

Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu

565 570 575

Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys

580 585 590

Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

595 600 605

<210> 53

<211> 607

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 53

Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala

1 5 10 15

Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu

20 25 30

Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His

35 40 45

Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu

50 55 60

Ile His Tyr Ser Val Ala Glu Gly Ile Thr Asp Gln Gly Glu Val Pro

65 70 75 80

Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg

85 90 95

Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser

100 105 110

Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg

115 120 125

Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser

130 135 140

Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

180 185 190

Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu

195 200 205

Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys

210 215 220

Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly

225 230 235 240

Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg

245 250 255

Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser

260 265 270

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

275 280 285

Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn

290 295 300

Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Pro Val Lys Gln Thr Leu

305 310 315 320

Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly

325 330 335

Pro Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln

340 345 350

Trp Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser

355 360 365

Val Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser

370 375 380

Ala Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu

385 390 395 400

Thr Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly

405 410 415

Arg Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr

420 425 430

Ile Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val

435 440 445

Arg Pro Thr Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr

450 455 460

Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro Glu Pro Ala Val Tyr

465 470 475 480

Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr

485 490 495

Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr

500 505 510

Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys

515 520 525

Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln

530 535 540

Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro

545 550 555 560

Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu

565 570 575

Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys

580 585 590

Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

595 600 605

<210> 54

<211> 602

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala

1 5 10 15

Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr

20 25 30

Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His

35 40 45

Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu

50 55 60

Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro

65 70 75 80

Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu

85 90 95

Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala

100 105 110

Ser Ser Leu Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu

115 120 125

Thr Val Thr Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu

130 135 140

Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu

145 150 155 160

Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp

165 170 175

Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln

180 185 190

Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg

195 200 205

Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln

210 215 220

Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser

225 230 235 240

Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala

245 250 255

Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala

260 265 270

Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val

275 280 285

Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser

290 295 300

Arg Ala Lys Arg Ser Gly Ser Gly Ala Pro Val Lys Gln Thr Leu Asn

305 310 315 320

Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro

325 330 335

Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu

340 345 350

Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser

355 360 365

Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser

370 375 380

Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val

385 390 395 400

His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg

405 410 415

Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile

420 425 430

Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Met Tyr Asp

435 440 445

Gln Asn Gly Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu

450 455 460

Pro Asn Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro

465 470 475 480

Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln

485 490 495

Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys

500 505 510

Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile

515 520 525

Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu

530 535 540

Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu

545 550 555 560

Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu

565 570 575

Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn

580 585 590

Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

595 600

Claims (35)

1. An isolated oncolytic adenovirus for expressing a foreign gene, wherein the oncolytic adenovirus is a selective replication-competent recombinant oncolytic adenovirus obtained by genetically modifying adenovirus, and the genome of the recombinant oncolytic adenovirus has the following characteristics:

1) contains E1B gene regulatory elements, which include E1B promoter and E1B and pIX shared polyadenylation addition signal sequence;

2) the coding region of the E1B gene is deleted, and when the foreign gene needs to be inserted, the foreign gene is inserted at the site of the coding region of the E1B gene, is positioned behind the E1B promoter and is controlled by the regulatory elements of the E1B gene;

3) upstream of the foreign gene, a cDNA sequence of E1A transcribing the E1A 13s mRNA is contained, and the cDNA is wild type or Rb protein binding region deleted, the Rb protein binding region deleted is the wild type cDNA from which the nucleotide sequence shown in SEQ ID NO.7 is removed, or the Rb protein binding region deleted encodes a mutated E1A protein, the mutated E1A protein is shown in SEQ ID NO. 6.

2. The oncolytic adenovirus of claim 1, wherein the nucleotide sequence of the E1B promoter is shown in SEQ ID NO.1, and the polyadenylation addition signal sequence shared by E1B and pIX is shown in aataaa.

3. The oncolytic adenovirus of claim 1, wherein the E1B gene comprises an E1B-55K gene and an E1B-19K gene.

4. The oncolytic adenovirus of claim 1, wherein the nucleotide sequence of the coding region of the E1B gene is represented by SEQ ID No. 3.

5. The oncolytic adenovirus of claim 1, wherein the start site of the exogenous gene comprises a Kozak sequence, preferably the Kozak sequence is shown in SEQ ID No. 4.

6. The oncolytic adenovirus of claim 1, wherein the nucleotide sequence of the wild-type E1A cDNA is set forth in SEQ ID No. 5.

7. The oncolytic adenovirus of claim 1, wherein the cDNA of E1A transcribing the E1A 13s mRNA is located upstream of the E1B promoter and partially coincides with the nucleotide sequence of the E1B promoter.

8. The oncolytic adenovirus of claim 1, wherein the cDNA sequence of E1A transcribing E1A 13s mRNA is under the control of an endogenous E1A promoter/enhancer, or under the control of an exogenous promoter; preferably, the nucleotide sequence of the endogenous E1A promoter/enhancer is shown in SEQ ID NO. 8.

9. The oncolytic adenovirus of claim 1, wherein the cDNA sequence of E1A transcribing E1A 13s mRNA is under the control of a foreign promoter, the nucleotide sequence shown as SEQ ID No.9 is removed from the genome of the recombinant oncolytic adenovirus, and the foreign promoter nucleotide sequence is inserted at the site of the removal.

10. The oncolytic adenovirus of claim 8 or 9, wherein the exogenous promoter comprises an EF-1 a promoter, a CMV promoter, a PKG promoter, an E2F promoter, an AFP promoter, and a TERT promoter.

11. The oncolytic adenovirus of claim 1, wherein the exogenous gene comprises: HLA protein coding sequence, marker polypeptide coding sequence, HLA protein coding sequence and beta 2-microglobulin coding sequence, or HLA protein coding sequence, beta 2-microglobulin coding sequence and marker polypeptide coding sequence.

12. The oncolytic adenovirus of claim 11, wherein said HLA proteins comprise HLA class I molecules comprising HLA-A, HLA-B and HLA-C.

13. The oncolytic adenovirus of claim 12, wherein the HLA-C comprises a wild-type molecule, or at least one of the following mutations: 1) arginine at position 2 is mutated to alanine; 2) the 4 th nucleotide of the nucleotide sequence for coding the HLA-C protein is mutated from C to G, and the 5 th nucleotide is mutated from G to C; 3) isoleucine at position 362 mutated to threonine; 4) glutamic acid 359 th was mutated to valine.

14. The oncolytic adenovirus of claim 11, wherein the marker polypeptide comprises the following amino acid sequences in operable linkage, in sequential tandem: an amino acid sequence of an N-terminal signal peptide, an amino acid sequence of one or more epitope polypeptides, and optionally an amino acid sequence of a C-terminal endoplasmic reticulum retention signal, wherein when said tag polypeptide comprises a plurality of amino acid sequences of said epitope polypeptides, the amino acid sequences of each two adjacent said epitope polypeptides are linked by an amino acid sequence of a cleavable linker polypeptide; preferably, the cleavable linker polypeptide is a furin cleavage recognition polypeptide.

15. The oncolytic adenovirus of claim 14, wherein the amino acid sequence of said epitope polypeptide is derived from the amino acid sequence of a naturally occurring protein, or is an artificially synthesized amino acid sequence not occurring in nature; preferably, the naturally occurring protein includes a protein of human origin and proteins of other species than human.

16. The oncolytic adenovirus of claim 14, wherein the amino acid sequence of said epitope polypeptide is derived from the amino acid sequence of a tumor-associated antigen or a tumor-specific antigen.

17. The oncolytic adenovirus of claim 16, wherein the tumor-associated antigen is selected from the group consisting of NY-ESO-1157-165, NY-ESO-11-11, NY-ESO-153-62, NY-ESO-118-27, Her2/neu 369-377, SSX-241-49, MAGE-A4230-239, MAGE-A10254-262, MAGE-C2336-344, MAGE-C2191-200, MAGE-C2307-315, MAGE-C242-50, MAGE-A1120-129, MAGE-A1230-238, MAGE-A1-169, KK-LC-176-84, p 5399-107, PRAME 301-309, alpha-fetoprotein 158-166, HPV 16-E62938, HPV 16-E-19, EBV-P151-59, and E-19, EBV-LMP 1125-133, KRAS: G12D 10-18, KRAS: G12D 8-16, KRAS: G12D 7-16, KRAS: G12C 8-16, KRAS: G12A 8-16, KRAS: G12S 8-16, KRAS: G12R 8-16, KRAS: G12V 8-16, KRAS: G12V 7-16, KRAS: G12V 5-14, KRAS: G12V 11-19, KRAS: G12V 5-14, KRAS: Q61H 55-64, KRAS: Q61L 55-64, KRAS: Q61R55-64, KRAS: G12D 5-14, KRAS: G13D 5-14, KRAS: G12A 5-14, KRAS: G12C 5-14, KRAS: G12S 5-14, KRAS: G12R 5-14, KRAS: G12D 10-19, TP 53: V157G 156 164, TP 53: R248Q 240-249, TP 53: R248W 240-249, TP 53: G245S 240-249, TP 53: V157F 156 164, TP 53: V157F 149-158, TP 53: Y163C 156-164, TP 53: R248Q 247-255 and TP 53: R248Q 245 254, TP 53: R248W 245 254, TP 53: G245S 245-254, TP 53: G249S 245-254, TP 53: Y220C 217-225, TP 53: R175H 168-176, TP 53: R248W 240-249, TP 53: K132N-134, CDC 73: Q254E 248-256, CYP2A 6: N438Y 436-444, CTNNB 1: T41A 41-49, CTNNB 1: S45P 41-49, CTNNB 1: T41A 34-43, CTNNB 1: S37Y 30-39, CTNNB 1: S33C 30-39, CTNNB 1: S45P 40-49, EGFR: L858R 852-: T790M 790 799, PIK3 CA: E542K 533-: H1047R1046-1055, GNAS: R201H 197-205, CDK 4: R24C 23-32, H3.3: K27M26-35, BRAF: V600E 591-: k73Rfs 141-148, NRAS: Q61R55-64, IDH 1: R132H 126-135, TVP 23C: C51Y 51-59, TVP 23C: C51Y42-51 and TVP 23C: C51Y 45-53.

18. The oncolytic adenovirus of claim 14, wherein the epitope polypeptide is NY-ESO-1157-165 as shown in SEQ ID No.10 or KRAS as shown in SEQ ID No. 11: G12D 10-18.

19. A vector for preparing the oncolytic adenovirus of any one of claims 1-18, wherein the vector comprises the E1B gene regulatory element, lacks the E1B gene coding region, and comprises the cDNA sequence of E1A transcribing E1A 13s mRNA upstream of the foreign gene.

20. A therapeutic agent for treating a tumor and/or cancer, comprising:

(a) a first composition, wherein the first composition comprises a first active ingredient comprising or comprising an oncolytic adenovirus according to any one of claims 1-18 for introduction into a tumor cell and/or a cancer cell in a first pharmaceutically acceptable carrier; and

(b) a second composition, wherein the second composition comprises a second active ingredient comprising a T cell receptor modified immune cell in a second pharmaceutically acceptable carrier.

21. The therapeutic agent of claim 20, wherein the first composition and the second composition are each independently present in the therapeutic agent without intermixing.

22. The therapeutic agent of claim 20, wherein the immune cells comprise naive T cells or precursor cells thereof, NKT cells, or a T cell strain.

23. The therapeutic agent of claim 20, wherein the first composition comprises a therapeutically effective amount of the oncolytic adenovirus.

24. The therapeutic agent of claim 20, wherein the second composition comprises a therapeutically effective amount of the T cell receptor-modified immune cell.

25. The therapeutic agent according to claim 20, wherein the oncolytic adenovirus is formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid intracavity administration, or intravenous administration.

26. The therapeutic agent according to claim 20, wherein the immune cell is formulated for administration by arterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, subarachnoid, intramedullary, intramuscular, or intraperitoneal administration.

27. Use of an oncolytic adenovirus according to any one of claims 1-18 in the manufacture of a medicament for the treatment of a tumor and/or cancer.

28. The use of claim 27, wherein the tumor and/or cancer comprises: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

29. Use of a therapeutic agent according to any one of claims 20-26 in the manufacture of a medicament for the treatment of a tumour and/or cancer.

30. The use of claim 29, wherein the tumor and/or cancer comprises: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

31. A method of treating a tumor and/or cancer comprising administering to a tumor and/or cancer patient an oncolytic adenovirus according to any one of claims 1-18.

32. The method of claim 31, wherein the tumor and/or cancer comprises: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

33. A method of treating a tumor and/or cancer, comprising:

administering a first composition of the therapeutic agent of any one of claims 20-26 to a tumor and/or cancer patient; and

administering to the tumor and/or cancer patient a second composition of the therapeutic agent of any one of claims 20-26.

34. The method of claim 33, comprising the following sequential steps:

1) administering the first composition to the tumor and/or cancer patient; and

2) administering a second composition of said therapeutic agents to said tumor and/or cancer patient after administering said first composition.

35. The method of claim 34, wherein the tumor and/or cancer comprises: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

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