WO2022057725A1 - 分离的能够用于表达外源基因的溶瘤腺病毒、载体、治疗剂及其用途 - Google Patents

分离的能够用于表达外源基因的溶瘤腺病毒、载体、治疗剂及其用途 Download PDF

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WO2022057725A1
WO2022057725A1 PCT/CN2021/117571 CN2021117571W WO2022057725A1 WO 2022057725 A1 WO2022057725 A1 WO 2022057725A1 CN 2021117571 W CN2021117571 W CN 2021117571W WO 2022057725 A1 WO2022057725 A1 WO 2022057725A1
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cancer
cells
tumor
hla
oncolytic adenovirus
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French (fr)
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侯亚非
胡放
绳纪坡
谭贤魁
陈璨
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杭州康万达医药科技有限公司
侯亚非
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464488—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/861—Adenoviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses

Definitions

  • the present invention belongs to the field of biotechnology, and in particular, relates to an isolated oncolytic adenovirus for expressing foreign genes, a vector, a therapeutic agent and the use thereof.
  • ACT adoptive transfer of tumor-infiltrating lymphocytes (TILs), T cells expressing exogenous tumor-specific T-cell receptors (TCR-Ts), or CAR-T cells, has been shown to be the most Promising immunotherapy for one type of cancer (see “N Engl J Med 2017;377:2545-2554”).
  • tumor tissue heterogeneity which is manifested by heterogeneous expression of tumor antigens specifically recognized by T cells in tumor cells (see “Int J Cancer. 2001 Jun 15;92(6):856-60” ).
  • Loss or insufficiency of target antigen expression in some tumor cells allows them to escape recognition and killing by adoptively transfused T cells.
  • the mechanism of antigen processing and presentation in tumor cells is often abnormal, manifested as HLA class I molecule mutation or decreased expression, decreased expression of ⁇ 2-microglobulin, TAP, tapasin enzyme, LMP complex, etc.
  • tumor antigen-specific T cells recognize epitope peptides (epitope peptides) presented by the major histocompatibility complex (MHC, human MHC is HLA molecule), and the function of T cells to recognize antigens is affected by MHC. Restricted, it can only recognize tumor epitope peptides presented by specific MHC molecules.
  • MHC major histocompatibility complex
  • the tumor cells of tumor patients express specific tumor antigens and have specific HLA alleles in order to become a suitable population for adoptive T cell therapy.
  • TME immunosuppressive tumor microenvironment
  • oncolytic virus as a carrier to label tumor cells so that they express target antigens that can be specifically recognized by adoptive T cells, and use the virus's own immunogenicity to label tumor cells.
  • the oncolytic virus infects the tumor cells, it selectively replicates in the tumor cells, and through the massive proliferation of the daughter virus, the tumor cells are lysed to achieve the effect of specifically killing the tumor cells.
  • the released daughter virus can in turn selectively infect and lyse other tumor cells to maximize tumor clearance (see document "Nat Biotechnol. 2012 Jul 10;30(7):658-70").
  • Adenovirus as a carrier is an oncolytic virus developed earlier.
  • Ad5-type adenovirus H101 based on E1B55K gene deficiency is the first oncolytic virus product approved for marketing (see the document "Hum Gene Ther. 2018 Feb; 29(2) :151-159”).
  • the molecular structure and biological characteristics of adenovirus have been studied in depth, making it easier for adenovirus to become an oncolytic virus through genetic engineering.
  • the genomes of oncolytic viruses are often modified to increase the tumor cell selectivity of oncolytic viruses.
  • the use of tumor-specific gene promoters to drive genes necessary for adenovirus replication can also provide tumor selectivity.
  • the modification of the oncolytic virus genome is bound to affect the transcription and replication of the virus in cells and its ability to lyse cells, so that virus-infected tumor cells cannot be lysed.
  • the completion of the replication cycle of oncolytic viruses requires the participation of a large number of cellular components of host tumor cells and is regulated by unique molecular mechanisms (see the document "J Virol. 2008 Aug; 82(15):7252-63"), and each The gene regulation and protein expression of individual tumor cells are different, which determines that after tumor cells with different growth states and properties are infected with oncolytic viruses, some tumor cells cannot complete the replication cycle and lyse cells (see the literature "Nat Rev Cancer. 2002; 2 (12):938-950”).
  • host-produced neutralizing antibodies against oncolytic viruses and tumor tissue restriction of viral particle spread can reduce the oncolytic effect of oncolytic viruses.
  • the present invention proposes to use oncolytic virus in combination with adoptive T cells to enhance the tumoricidal function and applicable scope of adoptive T cells, and to eliminate the inability to complete the virus replication cycle through T cells. of tumor cells.
  • the present invention further provides an oncolytic virus that can effectively express target antigens recognized by specific T cells in tumor cells, and can be combined with adoptive TCR-T cell therapy to further enhance the efficacy of solid tumor immunotherapy.
  • the present invention provides an isolated oncolytic adenovirus for expressing foreign genes, a vector, a therapeutic agent and the use thereof.
  • the present invention provides:
  • An isolated oncolytic adenovirus for expressing a foreign gene wherein the oncolytic adenovirus is a selective replication type recombinant oncolytic adenovirus obtained by genetically modifying an adenovirus, and the recombinant oncolytic adenovirus is a
  • the genome of a virus has the following characteristics:
  • the E1B gene coding region is deleted, and when it is necessary to insert the foreign gene, the foreign gene is inserted at the E1B gene coding region site, and the foreign gene is located after the E1B promoter, and is subject to the control of the E1B gene regulatory element;
  • the cDNA sequence of E1A that transcribes the E1A 13s mRNA is included, and the cDNA is a wild-type or Rb protein binding region deletion type, and the Rb protein binding region deletion type is the wild-type cDNA removed
  • the nucleotide sequence shown in SEQ ID NO.7, or the Rb protein binding region deletion type encodes a mutated E1A protein, and the mutated E1A protein is shown in SEQ ID NO.6.
  • exogenous promoter includes EF-1 ⁇ promoter, CMV promoter, PKG promoter, E2F promoter, AFP promoter and TERT Promoter.
  • HLA protein comprises HLA class I molecules including HLA-A, HLA-B and HLA-C.
  • the marker polypeptide comprises the following amino acid sequences operably linked in series: the amino acid sequence of the N-terminal signal peptide, one or more antigenic epitopes
  • the amino acid sequence of the polypeptide the amino acid sequence of the optional C-terminal endoplasmic reticulum retention signal, wherein when the marker polypeptide includes the amino acid sequence of a plurality of the antigenic epitope polypeptides, every two adjacent said antigens
  • the amino acid sequences of epitope polypeptides are connected by amino acid sequences of cleavable linking polypeptides; preferably, the cleavable linking polypeptides are furin cleavage recognition polypeptides.
  • a therapeutic agent for treating tumor and/or cancer comprising:
  • a first composition wherein the first composition comprises a first active ingredient in a first pharmaceutically acceptable carrier, the first active ingredient comprising or containing a basis for introduction into tumor cells and/or cancer cells (The oncolytic adenovirus of any one of 1)-(18); and
  • composition comprising a second active ingredient in a second pharmaceutically acceptable carrier, the second active ingredient comprising T cell receptor-modified immune cells.
  • the tumor and/or cancer comprises: breast cancer, head and neck tumor, 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder Carcinoma, ureteral carcinoma, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervix cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squam
  • the tumor and/or cancer comprises: breast cancer, head and neck tumor, 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder Carcinoma, ureteral carcinoma, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervix cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squam
  • (31) A method of treating tumor and/or cancer, comprising administering the oncolytic adenovirus according to any one of (1)-(18) to a tumor and/or cancer patient.
  • the tumor and/or cancer comprises: breast cancer, head and neck tumor, 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder Carcinoma, ureteral carcinoma, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervix cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squam
  • a method of treating tumor and/or cancer comprising:
  • the second composition of the therapeutic agent according to any one of (20)-(26) is administered to the tumor and/or cancer patient.
  • the tumor and/or cancer comprises: breast cancer, head and neck tumor, 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder Carcinoma, ureteral carcinoma, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervix cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squam
  • the present invention has the following advantages and positive effects:
  • the oncolytic adenovirus constructed in the present invention for expressing exogenous genes can selectively replicate in tumor cells and/or cancer cells, and express exogenous genes more efficiently.
  • the present invention utilizes its own E1B gene regulatory element in the oncolytic virus genome to regulate the expression of exogenous genes, avoiding possible interference caused by the inserted exogenous gene regulatory element to the expression of the viral genome, thereby affecting the effective replication of the virus, and expression of downstream genes.
  • the length of the inserted foreign gene fragment can also be increased, so that the oncolytic virus vector can carry more foreign gene load.
  • the oncolytic adenovirus constructed in the present invention lacks the coding region of the E1B gene.
  • E1B-19K protein can inhibit apoptosis induced by tumor necrosis factor and FAS pathways, making infected cells resistant to T cell killing. Deletion of the E1B-19K gene increases the sensitivity of infected tumor cells to killer T cells. Deletion of the E1B-55K gene increases the tumor cell oncolytic selectivity of oncolytic adenoviruses.
  • the present invention found that, in the genome of the oncolytic adenovirus, by designing the cDNA sequence of E1A that transcribes E1A-13S mRNA upstream of the exogenous gene instead of the E1A genome gene, the present invention can only transcribe E1A-13S and avoid transcription E1A-12S, thereby increasing the expression of foreign genes and enhancing the replication of the viral genome.
  • the oncolytic adenovirus for expressing exogenous genes constructed by the present invention can be more effectively realized by ingenious design than the similar oncolytic adenoviruses used for expressing exogenous genes in the prior art.
  • the nucleic acid is introduced into tumor cells and/or cancer cells, and exogenous marker polypeptides, especially epitope polypeptides derived from mutated neo-antigens (neo-antigen), are more efficiently expressed in tumor cells and/or cancer cells. It enters the MHC class I antigen presentation pathway, and the expression of HLA/antigen epitope polypeptide complexes on the tumor cell surface is significantly increased, thereby further enhancing the immune cells modified by the T cell receptor. The recognition sensitivity of cancer cells.
  • the labeling of tumor cells by heterologous HLA class I molecules (allo-HLA class I) carried by the cytotoxic virus vector also greatly increases the application scope of adoptive T cell therapy.
  • the oncolytic adenovirus constructed by the present invention for expressing exogenous genes can more efficiently express exogenous antigenic epitope peptides, ⁇ 2-microglobulin and/or exogenous MHC class I molecules in tumor cells, so as to significantly enhance the The number of epitope peptide/MHC class I molecule complexes on the surface of tumor cells, and the combination therapy can be carried out with TCR-modified immune cells specific for the epitope peptide.
  • the killing of the tumor by the oncolytic virus is related to TCR
  • the killing of tumors by modified immune cells can also produce synergistic therapeutic effects.
  • marker polypeptide, ⁇ 2-microglobulin and/or HLA protein-encoding nucleic acid is introduced into tumor cells and/or cancer cells through the constructed oncolytic adenovirus, and the oncolytic virus can kill tumor cells and/or cancer cells.
  • the above-mentioned synergistic therapeutic effect achieved by significantly enhancing the presentation of exogenous epitope peptides on the surface of tumor cells and T cell receptor-modified immune cells is further enhanced.
  • oncolytic viruses can relieve the immunosuppressive state of the tumor microenvironment and improve the homing of T cell receptor-modified immune cells while killing tumors.
  • tumor cells that are lysed cannot complete the replication cycle and produce a sufficient number of daughter viruses; thus, further synergy is achieved.
  • the antigens released by the tumor cells lysed by the oncolytic virus can further activate the body's own anti-tumor immunity, which can achieve better tumor killing effect than the use of oncolytic virus or T cell receptor-modified immune cells alone. synergistic treatment effect.
  • oncolytic virus refers to a virus capable of selectively replicating in and lysing tumor cells.
  • the phrase "therapeutically effective amount” refers to an amount of a functional agent or pharmaceutical composition capable of exhibiting a detectable therapeutic or inhibitory effect, or an amount that exerts an anti-tumor effect.
  • the effect can be detected by any assay known in the art.
  • administering refers to providing a compound, complex or composition (including viruses and cells) to a subject.
  • patient refers to a human or non-human organism. Accordingly, the methods and compositions described herein are applicable to human disease and veterinary disease.
  • the patient has a tumor. In some instances, the patient has one or more types of cancer at the same time.
  • the phrase "synergistic effect” refers to an effect of two or more agents together that is greater than the sum of the individual effects of each agent therein.
  • abnormal expression or “abnormal expression” means that the expression is altered, preferably increased, associated with certain proteins, such as tumors, compared to non-tumorigenic normal cells or healthy individuals (ie, individuals without disease).
  • Abnormal or abnormal expression of antigens 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, whereas expression is suppressed in healthy tissue.
  • the term "about” means within 20%, or in some cases within 10%, or in some cases within 5%, or in some cases within 1% % of a given value or range within, or in some cases within 0.1, as such variation is suitable for carrying out the disclosed methods or for the intended purpose of the disclosed compositions.
  • active ingredient refers to an ingredient in a pharmaceutical product composition that is biologically active or has an intended pharmaceutical effect.
  • ACT adoptive cell transfer
  • a subject or patient's own immune cells eg, autologous T cells
  • immune cells from a healthy donor eg, allogeneic T cells
  • TCR-T cell therapy is a type of ACT.
  • anti-tumor refers to a biological effect that can manifest in a variety of ways, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of tumor cell metastases, an increase in life expectancy, tumor cell proliferation reduction in tumor cell survival, or improvement in various physiological symptoms associated with cancer conditions.
  • Anti-tumor effect can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to prevent tumorigenesis in the first place.
  • antibody refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen.
  • Antibodies can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and can be derived from natural or recombinant sources.
  • Antibodies can be tetramers of immunoglobulin molecules.
  • the term "antigen" or "Ag” is defined as a molecule that elicits an immune response. This immune response may involve the production of antibodies, or the activation of specific immunologically immune cells, or both.
  • antigens can be derived from recombinant DNA or genomic DNA. Those of skill in the art will understand that any DNA comprising a nucleotide sequence or part of a nucleotide sequence encoding a protein that elicits an immune response encodes the term "antigen" as used herein.
  • the antigen need not be encoded solely by the full-length nucleotide sequence of the gene.
  • the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit a desired immune response.
  • the antigen need not be encoded by a "gene” at all.
  • antigens can be produced synthetically or can be derived from biological samples, or can be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or fluids with other biological components.
  • antigen presentation machinery refers to immune molecules or cells that process and prepare antigens for presentation to T lymphocytes.
  • the antigen presentation mechanism involves two distinct pathways for processing antigens from the organism's own (self) proteins or from intracellular pathogens (eg viruses) or phagocytic pathogens (eg bacteria); these antigens are in either class I or class I
  • MHC major histocompatibility complex
  • Both MHC classes I and II must bind antigen before they can be stably expressed on the cell surface.
  • MHC I antigen presentation typically involves the endogenous pathway of antigen processing, while MHC II antigen presentation involves the exogenous pathway of antigen processing.
  • autologous refers to any material derived from the same individual and subsequently reintroduced into that individual.
  • ⁇ 2-microglobulin or " ⁇ 2 - microglobulin” is a component of MHC class I molecules, which have ⁇ 1, ⁇ 2 and ⁇ 3 proteins present on all nucleated cells (excluding erythrocytes) . In humans, ⁇ 2-microglobulin is encoded by the B2M gene.
  • C-terminal refers to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).
  • a protein is translated from messenger RNA, it is produced from the N-terminus to the C-terminus.
  • the convention for writing peptide sequences is to insert the C-terminus on the right into the sequence from the N-terminus to the C-terminus.
  • the term "combination” refers to any possible arrangement of the various components (eg, an oncolytic virus and one or more substances effective in anticancer therapy). Such arrangements include mixtures of the components as well as separate combinations for simultaneous or sequential administration.
  • the present invention includes combinations having different effective doses. It will be appreciated that the optimal dosage of each component of the combination can be determined by one skilled in the art.
  • composition refers to a chemical and/or biological composition suitable for administration to a subject or patient with the intended pharmaceutical effect (eg, prophylactic and therapeutic effects).
  • compositions suitable for such therapeutic applications include parenteral, subcutaneous, transdermal, intradermal, intramuscular, intracoronary, intramyocardial, intracerebral, intratumoral, intraperitoneal, intravenous (eg, injectable) Or formulations for intratracheal administration, such as sterile suspensions, emulsions and aerosols.
  • Intratracheal administration may involve contacting or exposing lung tissue (eg, alveoli) to a therapeutic agent comprising a therapeutically effective amount of nucleic acid and/or immune cells (eg, T cells (eg, TCR-modified T cells) in a pharmaceutical carrier. cell)).
  • a therapeutic agent comprising a therapeutically effective amount of nucleic acid and/or immune cells (eg, T cells (eg, TCR-modified T cells) in a pharmaceutical carrier. cell)).
  • T cells eg, TCR-modified T cells
  • pharmaceutical compositions suitable for therapeutic use may be admixed with one or more pharmaceutically acceptable excipients, diluents or carriers (eg, sterile water, physiological saline, dextrose, etc.).
  • a polypeptide when used to define products, compositions and methods, the terms “comprising”, “having”, “comprising” or “containing” are open ended and do not exclude other unrecited elements or method steps.
  • a polypeptide “comprises” an amino acid sequence when it may be part of the final amino acid sequence of the polypeptide. Such polypeptides can have up to hundreds of additional amino acid residues.
  • Consisting essentially of means excluding any significant other components or steps.
  • a composition consisting essentially of the described components will not exclude trace contaminants and pharmaceutically acceptable carriers.
  • the polypeptide When such an amino acid sequence is present, the polypeptide "consists essentially of” the amino acid sequence, ultimately having only a few additional amino acid residues.
  • Consisting of means excluding trace elements or steps of other components.
  • a polypeptide consists of” an amino acid sequence when it does not contain any other amino acids other than the recited amino acid sequence.
  • conditional replication competent virus or "conditional replication virus” or “CRV” or “replicative selective virus” refers to a virus designed to be able to replicate selectively in tumor cells, resulting in their destruction, while sparing normal cells .
  • constitutive promoter refers to a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in a cell under most or all physiological conditions of the cell .
  • 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 cytotoxicity when bound to a cognate ligand Signal.
  • the terms “obtained from,” “originated from,” or “derived from” are used to identify the original source of a component (eg, 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 Methods of synthetic or recombinant means.
  • a component eg, polypeptide, nucleic acid molecule, amino acid sequence
  • 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 allergy), For example, with a reasonable reward/risk ratio.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable vehicle” or “pharmaceutically acceptable carrier” refers to a vehicle or carrier for administering a therapeutic agent suitable for human and and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), and with a reasonable benefit/risk ratio.
  • a “pharmaceutically acceptable carrier” includes nucleic acids that can be used to deliver and/or express of any vehicle or vector, including nanoparticles, lipids, plasmids, viruses or cells.
  • encoding when applied to a polynucleotide refers to a polynucleotide that "encodes" a polypeptide if manipulated in its native state or by methods well known to those skilled in the art, which can be transcribed and/or translated to produce mRNA for polypeptides and/or fragments thereof.
  • the antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.
  • 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 that "encodes" a peptide refers to an amino acid sequence that contains the peptide 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 resident protein.
  • the canonical ER retention signal is the C-terminal KDEL (Lys-Asp-Glu-Leu) sequence.
  • epitope refers to the portion of an antigen that is recognized and bound by the immune system, particularly antibodies, B cells or T cells.
  • epitope refers to an epitope or antigenic epitope in the form of a peptide.
  • excipient or “additive” is intended to mean all substances in a pharmaceutical formulation that are not active ingredients, such as carriers (eg, vector DNA, plasmids, vector viruses), binding agents, lubricants, enhancers Thickening agents, surfactants, preservatives, emulsifiers, buffers, flavoring or coloring agents.
  • carriers eg, vector DNA, plasmids, vector viruses
  • binding agents e.g, lubricants, enhancers Thickening agents, surfactants, preservatives, emulsifiers, buffers, flavoring or coloring agents.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • exogenous HLA protein refers to HLA protein from outside the subject or patient, and “exogenous HLA protein” may or may not be produced by cells or tissues of the subject or patient.
  • endogenous refers to any material derived from or produced within an organism, cell, tissue or system.
  • expression refers to the process by which a polynucleotide is transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells.
  • gene expression refers to the process of converting the genetic information in a gene, the sequence of DNA base pairs, into a functional gene product (eg, protein or RNA). The basic process is to transcribe DNA into RNA and then translate the RNA into protein.
  • human leukocyte antigen refers to a gene complex or system encoding human major histocompatibility complex (MHC) proteins, also referred to as "HLA proteins".
  • MHC proteins are cell surface proteins responsible for regulating the body's immune system.
  • the HLA gene complex is located on a 3-Mbp segment on chromosome 6p21 and is highly polymorphic, meaning they have many different alleles, allowing fine-tuning of the adaptive immune system.
  • HLA corresponds to MHC class I (A, B and C) and presents foreign antigens (such as viral antigens) from the interior of cells to T lymphocytes.
  • HLA class I/antigen peptide complexes can stimulate cytotoxic T cells (also known as CTL), which in turn kills target cells.
  • HLA class I protein refers to a human MHC class I protein or molecule.
  • MHC class I molecules are transmembrane proteins consisting of a single alpha chain, associated with beta2-microglobulin, for proper folding and transport to the cell surface.
  • immune cells refers to cells of the immune system, which can be classified into lymphocytes (T cells, B cells and NK cells), neutrophils and monocytes/macrophages. These are all types of white blood cells.
  • immune danger signal refers to when tissue cells are in distress due to injury, infection, etc., they begin to secrete or express "dangerous" molecules on their surface, or components of invading organisms (eg, viral DNA or RNA) Also sensitized by the immune system as a danger signal.
  • immunogen refers to a specific type of antigen capable of eliciting an immune response.
  • tumor microenvironment or “immunosuppressive tumor microenvironment” refers to the environment surrounding a tumor, including surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix (ECM). Tumors are closely related and constantly interacting with the surrounding microenvironment. Tumors can influence the microenvironment by releasing extracellular signals to suppress immune responses, promote tumor angiogenesis and induce peripheral immune tolerance, and immune cells in the microenvironment can influence the growth and evolution of cancer cells.
  • TME tumor microenvironment
  • ECM extracellular matrix
  • Terms such as “increase” or “enhance” preferably relate to an increase or enhancement of 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%, also preferably at least 100%. These terms may also relate to situations where there is no detectable signal for a certain compound or condition at time zero, but there is a detectable signal for a certain compound or condition at a particular point in time later than time zero.
  • inducible promoter refers to a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, results in substantially only when an inducer corresponding to the promoter is present in a cell The gene product is produced in the cell.
  • isolated refers to a cell, protein, polypeptide, peptide, polynucleotide, vector, etc. removed from its natural environment (ie, separated from at least one other component with which it are naturally associated or found in nature).
  • Lentiviral vectors are retroviruses that can infect both dividing and non-dividing cells because their preintegrated complexes (viral "capsids") can penetrate the intact membrane of the target cell nucleus.
  • Lentiviral vectors are derived from human immunodeficiency virus.
  • linker peptide or “linker sequence” refers to an amino acid sequence that joins two other amino acid sequences.
  • a portion of an HLA class I protein can be linked to a portion of a tumor-associated antigen sequence, such as an epitope sequence, via a linker sequence.
  • LHO loss of heterozygosity
  • LOH refers to the loss of one parent's contribution to a cell, possibly due to a direct deletion, due to unbalanced rearrangement, gene transformation, mitotic recombination alpha or chromosomal loss (single chromosome) .
  • MHC major histocompatibility complex
  • WBCs white blood cells
  • MHC tissue antigens that allow the immune system (more specifically T cells) to bind, recognize and tolerate itself (auto-recognition). MHC is also a chaperone molecule for intracellular peptides that bind to MHC and present to the T cell receptor (TCR) as a potential exogenous antigen. MHC interacts with TCR and its co-receptors to optimize binding conditions for TCR-antigen interaction in terms of antigen-binding affinity and specificity as well as signal transduction efficiency.
  • TCR T cell receptor
  • MHC class I protein or "MHC class I molecule” refers to major histocompatibility complex (MHC) molecules or glycoproteins or one of two major classes of proteins (the other being MHC class II), Found on the cell surface of all nucleated cells in vertebrates. MHC I proteins form functional receptors on most nucleated cells in the body. Major histocompatibility complex (MHC) class I molecules are responsible for the presentation of peptide epitopes to cytotoxic T cells. In humans, the human leukocyte antigen (HLA) system is the locus of genes encoding MHC class I and MHC class MHC molecules. HLA-A, -B and -C genes encode MHC class I (MHCI) proteins.
  • HLA human leukocyte antigen
  • Peptides typically 8-11 amino acids in length, will bind MHC I molecules by interacting with the groove formed by the two alpha helices located above the antiparallel beta sheets.
  • the 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 endosomes mediated by transporters associated with antigen processing (TAP). Plasma reticulum (ER); c) formation of pMHCI using newly synthesized MHC I molecules; d) transport of pMHCI to the cell surface. On the cell surface, pMHCI will interact with cytotoxic T cells through the T cell receptor (TCR).
  • TCP T cell receptor
  • cytotoxic T cell activation through a series of biochemical events mediated by related enzymes, co-receptors, adaptor molecules and transcription factors. Activated cytotoxic T cells will proliferate to generate large numbers of effector T cells expressing TCRs specific for the identified immunogenic peptide epitopes. Expansion of T cells with TCR specificity for the identified non-self epitopes results in immune-mediated apoptosis, revealing activated non-self epitopes.
  • MHC protein refers to a protein encoded by an MHC gene.
  • Immunosuppressive tumor microenvironment The ability of the tumor to promote a tolerance-promoting microenvironment and the activation of multiple immunosuppressive mechanisms that may work together to counteract an effective immune response, such as tumor-induced impaired antigen presentation, negative Activation of sexual co-stimulatory signaling and complicating immunosuppressive factors.
  • mutant refers to a strain, gene or characteristic produced or produced by an instance of a mutation, which is usually a change in the DNA sequence of an organism's genome or chromosome.
  • N-terminal signal peptide refers to the signal peptide (usually 16-30 amino acids in length) present at the N-terminus of most newly synthesized proteins destined for the secretory pathway. These proteins include those that reside in certain organelles (endoplasmic reticulum, Golgi or endosome), are secreted from cells, or are inserted into most cell membranes. The function of signal peptides is to prompt cells to translocate proteins, usually to the cell membrane.
  • neoantigen refers to a newly formed antigen not previously recognized by the immune system.
  • neoantigens can be derived from altered tumor proteins resulting from alterations in tumor genes, including point mutations, insertions/deletions, amplifications/fusions, post-translational modifications, or from viral proteins.
  • 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 of them.
  • Nucleic acid includes, but is not limited to, single- and double-stranded nucleic acids.
  • nucleic acid also includes DNA or RNA as described above containing one or more modified bases. Thus, DNA or RNA having a backbone modified for stability or other reasons is a "nucleic acid”.
  • 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.
  • a "nucleic acid” or “nucleic acid sequence” may also include regions of single- or double-stranded RNA or DNA or any combination.
  • encoding nucleic acid refers to the sequence or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The sequence of these deoxyribonucleotides determines the amino acid sequence along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.
  • an oncolytic virus refers to a virus capable of selectively replicating in tumor cells (eg, proliferative cells such as cancer cells) in vitro or in vivo to slow the growth and/or lysis of the dividing cells virus, while in normal cells there is little or no replication.
  • an oncolytic virus comprises a viral genome packaged into a virion (or virion) and is infectious (ie, capable of infecting and entering a host cell or subject).
  • the term encompasses DNA or RNA vectors (depending on the virus in question) as well as viral particles produced therefrom.
  • one or more refers to one or more numbers (eg, 2, 3, 4, 5, etc.).
  • operably linked refers to a functional relationship between two or more polynucleotide (eg, DNA) segments. Generally, it refers to the functional relationship of transcriptional regulatory sequences to transcribed sequences.
  • a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates transcription of the coding sequence in a suitable host cell or other expression system.
  • promoter transcriptional regulatory sequences operably linked to a transcribed sequence are physically contiguous with the transcribed sequence, ie, they are cis-acting.
  • certain transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in the vicinity of the coding sequence in which they enhance transcription.
  • polypeptide refers to a polymer of amino acid residues comprising a plurality of amino acids joined by peptide bonds.
  • the polymers may be linear, branched or cyclic, and may contain naturally occurring and/or amino acid analogs, and may be interrupted by non-amino acids.
  • Plasmid refers to an "extra" self-replicating genetic element found in a cell. Plasmids are used in genetic engineering to generate 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 changed, inserted or deleted from a DNA or RNA sequence.
  • polyadenylation signal or “polyadenylation signal sequence” or “polyadenylation signal” or “polyadenylation signal sequence” or “polyadenylation signal” or “Polyadenylation signal sequence” refers to sequence motifs recognized by RNA cleavage complexes that vary among eukaryotic groups. Most human polyadenylation signals contain the AAUAAA sequence.
  • prevention refers to prophylactic or preventive measures aimed at inhibiting undesired physiological changes or the development of a disorder or condition.
  • Preventing a disease or condition can include starting administration of T cells obtained according to the methods provided herein at a time before the disease or condition (or symptoms thereof) appears or exists, such that the disease or condition, or its pathological features, consequences, or adverse effects does not occur .
  • promoter refers to a DNA sequence recognized by a cell's synthetic machinery or introduced synthetic machinery and required to initiate specific transcription of a polynucleotide sequence.
  • 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 preferably enables the initiation or inhibition of biochemical pathways and/or signaling cascades when a suitable ligand binds to the receptor.
  • recombinant refers to or refers to an organism, cell, protein, genetic material, DNA or RNA formed using recombinant techniques.
  • recombinant DNA refers to DNA or rDNA made by combining DNA from two or more sources. DNA fragments are cleaved from their normal locations on chromosomes using restriction enzymes (also called restriction endonucleases), and then inserted into other chromosomes or DNA molecules using enzymes called ligases.
  • restriction enzymes also called restriction endonucleases
  • recombinant TCR refers to a TCR prepared by recombinant techniques.
  • replication-competent or “replication-competent virus” refers to a replication-competent virus or a virus whose replication depends on factors (eg, up-regulators) in cancer cells.
  • replication-competent virus refers to a virus that has all the necessary mechanisms for replication in cells in vitro and in vivo, ie without the aid of a packaging cell line.
  • a viral vector capable of replicating in complementary packaging cell lines eg a viral vector deleted at least in the E1A region
  • replication-deficient virus refers to a virus that requires a packaging cell line (comprising a transgene) for replication.
  • the term "retroviral vector” refers to a proviral sequence that can accommodate a gene of interest to allow incorporation of both into target cells.
  • the vector may also contain viral and cellular gene promoters, such as the CMV promoter, to enhance expression of the gene of interest in target cells.
  • self-cleaving linker peptide refers to a short peptide (eg, 18-22 amino acid long peptide) present in a protein (eg, a recombinant protein) that can trigger self-cleavage of the protein in a cell. Cleavage begins after protein translation. The exact molecular mechanism of the self-cleaving linker peptide remains uncertain. However, it is believed to involve ribosomal "jumping" 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 against cancer by delivering chimeric antigen receptors (CARs) or cloned T cell receptors.
  • CARs chimeric antigen receptors
  • cloned T cell receptors cloned T cell receptors
  • self protein refers to a protein normally produced by a particular organism.
  • the immune system of a given organism should be able to tolerate its own proteins. If not, there is autoimmunity.
  • the term "silencer” refers to a DNA sequence capable of binding transcriptional regulators, called repressors.
  • DNA contains genes that provide the template for the production of messenger RNA (mRNA). This mRNA is then translated into protein.
  • mRNA messenger RNA
  • the repressor protein binds to silencer regions of DNA, it prevents RNA polymerase from transcribing RNA sequences into RNA. Since transcription is blocked, RNA cannot be translated into protein. Thus, silencers prevent genes from being expressed as proteins.
  • solid tumor refers to an abnormal mass of tissue or tumor that typically does not contain cysts or areas of fluid. Solid tumors may be benign (noncancerous) or malignant (cancerous).
  • the terms "subject” or “patient” are used interchangeably and can encompass any vertebrate including, but not limited to, humans, mammals, reptiles, amphibians and fish.
  • the subject or patient is a mammal such as a human, or a mammal such as a domesticated mammal (eg dog, cat, horse, etc.) or domestic animal (eg cow, sheep, pig, etc.).
  • the subject is a human.
  • the phrase "in need” refers to a condition of a subject in which treatment or preventive measures are required. Such states may include, but are not limited to, subjects suffering from diseases or conditions such as cancer.
  • 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 a nucleobase moiety are disclosed in the table below.
  • surface expression refers to a fusion of the protein of interest to the native surface protein of the host cell. This results in the transport of the recombinant protein to the host surface and subsequent display on the host surface.
  • T cell refers to a type of 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 can 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. Help Sexual T cells, or CD4+ T cells, function by indirectly killing cells that are recognized as foreign: they determine whether and how the rest of the immune system responds to a particular perceived threat.
  • tandem refers to the spatial relationship between two or more entities (eg, a polynucleotide (eg, DNA) and a polypeptide) arranged in such a way that they are placed one after the other.
  • entities eg, a polynucleotide (eg, DNA) and a polypeptide
  • target refers to a molecule (eg, protein or peptide), cell or tissue or organism against which an immune response is directed.
  • 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 a fragment thereof (ie, an immunogenic fragment) that induces an immune response.
  • the target antigen or fragment thereof can be modified, eg, to reduce one or more biological activities of the target antigen or to enhance its immunogenicity.
  • target cell refers to any cell to which an immune response is desired or that can be specifically recognized by immune cells such as (T cells).
  • T cell receptor refers to a molecule found on the surface of T cells or T lymphocytes that is responsible for recognizing antigenic fragments as bound to major histocompatibility complex (MHC) molecules of peptides.
  • MHC major histocompatibility complex
  • therapeutic refers to treatment. Therapeutic effect can be obtained by reducing, inhibiting, alleviating, alleviating, preventing or eradicating a disease state.
  • the term "therapeutically effective dose” or “therapeutically effective amount” or “effective amount” or “effective dose” means, alone or in combination with other doses, that results in a desired response or in the case of treating a particular disease or condition,
  • the desired response is associated with inhibition of disease progression, which may include a slowdown in disease progression, particularly disruption of disease progression.
  • a desired response to treat a disease or disorder may also be to delay the onset of the disease or disorder or inhibit the onset of the disease or disorder.
  • the effective amount of the composition of the present invention will depend on the condition or disease, the severity of the disease, various parameters of the patient, including age, physical condition, height and weight, duration of treatment, type of optional concomitant therapy, and particular route of administration. and similar factors. If the patient does not respond adequately to the initial dose, multiple doses or higher doses (or higher effective doses, which can be achieved by a more restricted route of administration) may be employed.
  • Treating cancer may include, but is not limited to, alleviating one or more clinical indications, reducing tumor growth or tumor cell proliferation, reducing the severity of one or more clinical indications for a cancer condition, reducing the extent of the condition, stabilizing the subject disease state (ie not worsening), delay or slow, stop or reverse cancer progression, and achieve partial or complete remission.
  • Treating cancer also includes prolonging survival by days, weeks, months, or years if compared to the prognosis of treatment according to standard medical practice (without incorporating T cells obtained according to the methods provided herein).
  • Subjects in need of treatment can include subjects who have had or been diagnosed with cancer, as well as subjects prone, likely to develop, or suspected of having cancer (eg, lymphoma or multiple myeloma) or infection.
  • tumor antigen refers to an antigenic substance produced in tumor cells, ie it triggers an immune response in the host. Tumor antigens are useful tumor markers for identifying tumor cells by diagnostic tests and are potential candidates for cancer therapy.
  • tumor-associated antigen refers to an antigen that is present on some tumor cells as well as some normal cells.
  • TILs Tumor-infiltrating lymphocytes
  • tumor-specific antigen refers to an antigen that is present on tumor cells and not on any other cell.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • expression vector includes any vector (eg, plasmid, cosmid, or phage chromosome) that contains a genetic construct in a form suitable for cellular expression (eg, linked to a promoter).
  • plasmid and "vector” are used interchangeably, as a plasmid is a common form of vector.
  • the present invention is intended to include other vectors having equivalent functions.
  • 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.
  • viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphavirus vectors, 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 references: “Schlesinger and Dubensky (1999) Cur. Opin. Biotechnol. 5:434-439"; and "Ying, et al. (1999) Nat. Med. 5(7):823-827".
  • vp/day refers to viral particles per day.
  • wild or wild-type refers to a strain, gene or trait that is ubiquitous under natural conditions as opposed to atypical mutant types.
  • FIG. 1A shows a schematic representation of the recombinant oncolytic adenovirus constructs described herein.
  • pAdEasy-NY-A2 i.e. pAdEasy-EF1 ⁇ -NY-A2
  • the backbone of this construct is derived from adenovirus type 5 genomic DNA with deletion of the El region (E1del) and deletion of the E3 region (E3del). In the E1A region, expression units are incorporated.
  • the expression unit comprises a marker polypeptide coding sequence including the INSL5 signal peptide (INSL5 SP), three NY-ESO-1 157-165 epitopes linked by a Furin protease cleavage site and an HLA-A2 protein
  • the peptide (NY epitope x3), the linker peptide (Furin-F2A), the expression unit is flanked by the EF1 ⁇ promoter (EF1 ⁇ pro) and the SV40 poly(A) signal sequence (SV40pA).
  • LITR and RITR stand for left inverted terminal repeat (ITR) and right inverted terminal repeat, respectively.
  • pAd-EF1 ⁇ -E1A-A2-F2A-NY i.e.
  • pAd-EF1 ⁇ -E1Ad24-A2-F2A-NY and pAd-EF1 ⁇ -E1A-A2-F2A-BM (i.e. pAd-EF1 ⁇ -E1Ad24-A2-F2A) -BM) represents a conditional replication-competent adenoviral vector, containing the HLA-A2 gene and the marker polypeptide encoding the NY-ESO-1 157-165 epitope peptide, or with the human ⁇ 2-microglobulin gene, respectively.
  • the backbone of these constructs is the genomic DNA of adenovirus type 5 with deletions of the El and E3 regions.
  • E1A del24 A mutant E1A gene with a 122-129 deletion (E1A del24) was incorporated in the E1A region and flanked by the EF-1 ⁇ promoter and 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 the 5' endogenous E1B promoter (E1B pro) and the 3' pIX gene region, including the endogenous E1B /IX poly A signal sequence (E1B/IX pA).
  • E1B gene region was deleted.
  • FIG. 1B shows a schematic diagram of the lentiviral vector used to express TCR.
  • pCDH-EF1 ⁇ -TCR-NY represents an HIV-based lentiviral vector expressing a TCR specific for the NY-ESO-1 157-165 epitope peptide. Deletion of the U3 region enhancer (U3del) ensures self-inactivation of the lentiviral construct.
  • the TCR gene encodes a beta chain with a variable sequence (TCR- ⁇ V) and a murine constant sequence (murine TCR- ⁇ C) and an ⁇ chain with a variable sequence (TCR- ⁇ V) and a murine constant sequence (murine TCR- ⁇ C), It is flanked by the EF1 ⁇ promoter and the lentiviral WPRE region.
  • Figure 1C shows HLA-A2 expression in 293T cells after transfection with pShuttle vector expressing the marker polypeptide comprising the NY-ESO-1 157-165 epitope peptide and HLA-A2 protein.
  • 293T cells were stained with FITC-labeled anti-HLA-A2 antibody and analyzed by flow cytometry.
  • the dark thick line shows the expression of HLA-A2 on 293T cells transduced with a vector encoding the HLA-A2 protein.
  • Light grey line is control 293T cells transduced with empty vector.
  • the mean fluorescence intensity (Geom mean) of the gated population is shown on the flow cytometry plot.
  • the left panel shows HLA-A2 expression in 293T cells transduced with pShuttle-EF1 ⁇ -NY-A2
  • the middle panel shows HLA-A2 expression in 293T cells transduced with pShuttle-EF1 ⁇ -E1Ad24-A2-F2A-NY
  • the right panel shows Expression of HLA-A2 in 293T cells transduced with pShuttle-EF1 ⁇ -E1Ad24-A2-F2A-BM.
  • FIG. 1D shows that JRT cells express a NY-ESO-1 specific TCR following lentivirus transfection that expresses a peptide specific for the NY-ESO-1157-165 epitope in the context of HLA-A2 sexually different TCRs.
  • JRT cells were transfected with recombinant lentivirus and analyzed within 7-10 days.
  • JRT cells were stained with APC-labeled anti-CD8 antibody and PE-labeled NY-ESO-1 157-165/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 plot.
  • the control group shown in Figure 1 from left to right is JRT cells not transduced with lentivirus.
  • the "TCR-NY-LY” group shown in Figure 2 from left to right is JRT cells transfected with lentivirus produced from pCDH-EF1 ⁇ -TCR-NY-LY.
  • the "TCR-NY-AE” panel shown in Figure 3 from left to right is JRT cells transfected with lentivirus produced from pCDH-EF1 ⁇ -TCR-NY-AE.
  • the "TCR-NY-LI” group shown in Figure 4 from left to right is JRT cells transfected with lentivirus produced from pCDH-EF1 ⁇ -TCR-NY-LI.
  • Figure 2A shows that NY-ESO-1-specific TCR can recognize the NY-ESO-1 157-165 epitope peptide presented by HLA-A2 on T2 cells.
  • pCDH-EF1 ⁇ -TCR-NY-LY JRT-TCR-NY-LY
  • pCDH-EF1 ⁇ -TCR-NY-AE JRT-TCR-NY-AE
  • pCDH-EF1 ⁇ -TCR-NY-LI JRT - TCR-NY-LI
  • the X-axis is T2 target cells loaded with a range of concentrations of NY-ESO-1 157-165 peptide concentration, and the Y-axis is the percentage of CD69 + cells in gated JRT cells.
  • FIG. 2B shows that 293T cells expressing a marker polypeptide containing the NY-ESO-1 157-165 epitope peptide and HLA-A2 can activate NY-ESO-1 specific TCR on JRT cells.
  • JRT cells transduced with recombinant lentiviruses to express different TCRs specific for NY-ESO-1 were transduced with pShuttle-EF1a-NY-A2 or pShuttle-EF1a-E1Ad24-A2-NY 293T cells were incubated for 16 hours, cells were harvested and stained with anti-CD69 antibody to analyze 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, JRT-TCR-NY-AE and JRT-TCR-NY-LI.
  • Control represents 293T target cells transduced with empty pShuttle vector;
  • pShuttle-EF1a-NY-A2 represents 293T target cells transduced with pShuttle-EF1a-NY-A2 vector;
  • pShuttle-EF1a-E1Ad24-A2- NY represents 293T target cells transduced with pShuttle-EF1a-E1Ad24-A2-NY vector.
  • Data were analyzed with Student's t-test, ** represents p ⁇ 0.01, * represents p ⁇ 0.05.
  • Figure 2C shows that exogenous HLA-A2 can present the NY-ESO-1 157-165 epitope peptide derived from NY-ESO-1 protein to activate NY-ESO-1-specific TCR on JRT cells.
  • the pCDNA3.3 vector expressing the full-length NY-ESO-1 protein pCDNA3.3-NY
  • the pshuttle vector expressing the exogenous HLA-A2 protein pShuttle-EF1a-E1Ad24-A2-F2A
  • -BM Co-transfected 293T cells were used as target cells to stimulate JRT cells transduced with NY-ESO-1 specific TCR.
  • 293T cells transduced with pCDNA3.3-NY or pShuttle-EF1a-E1Ad24-A2-F2A-BM alone were used as negative controls.
  • 293T cells transduced with pShuttle-EF1a-E1Ad24-A2-F2A-BM alone and treated with NY-ESO-1 157-165 epitope peptide loading at 1 ⁇ g/ml were used as positive controls.
  • Cells were harvested within 16 hours, stained with anti-CD69 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.
  • "pCDNA3.3-NY” represents 293T target cells transduced with pCDNA3.3-NY only;
  • pShuttle-EF1a-E1Ad24-A2-F2A-BM represents only transduction with pShuttle-EF1a-E1Ad24-A2-F2A-BM 293T target cells transduced;
  • pShuttle-EF1a-E1Ad24-A2-F2A-BM+pCDNA3.3-NY” represents the 293T target co-transduced with pShuttle-EF1a-E1Ad24-A2-F2A-BM and pCDNA3.3-NY Cells;
  • "pShuttle-EF1a-E1Ad24-A2-F2A-BM+NY-ESO-1 polypeptide” represents transduced with pShuttle-EF1a-E1A
  • Figure 2D shows that nucleic acids encoding exogenous peptides and proteins in recombinant oncolytic adenoviral DNA can transduce 293T cells to express tagged polypeptides comprising the NY-ESO-1 157-165 epitope peptide and exogenous 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 expressing NY-ESO-1-specific TCR cell.
  • 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.
  • Control is 293T cells that were not transfected; "pAd-EF1a-E1Ad24-A2-F2A-NY” and "pAd-EF1a-E1Ad24-A2-F2A-BM” represent pAd-EF1a-E1Ad24-A2-F2A, respectively - 293T target cells transduced with NY or pAd-EF1a-E1Ad24-A2-F2A-BM.
  • pAd-EF1a-E1Ad24-A2-F2A-BM+pCDNA3.3-NY represents 293T target cells co-transduced with pAd-EF1a-E1Ad24-A2-F2A-BM and pCDNA3.3-NY;
  • pAd-EF1a -E1Ad24-A2-F2A-BM+NY-ESO-1 polypeptide represents 293T target cells transduced with pAd-EF1a-E1A d 24-A2-F2A-BM and treated with NY-ESO-1 157-165 peptide concentration loading .
  • Data were analyzed with Student's t-test, ** represents 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 marker polypeptide comprising a NY-ESO-1 epitope peptide and exogenous HLA-A2.
  • Tumor cell lines A375, SKOV3 and SKOV3-NY were transduced with pShuttle-A2-F2A-NY or pShuttle-A2-F2A-BM (SKOV3 cells transduced with pCDNA3.3-NY and stably expressing NY-ESO-1 protein) , and used as target cells to stimulate JRT cells expressing NY-ESO-1 specific TCR.
  • tumor cells and JRT cells were mixed 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.
  • 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.
  • Cells were harvested within 7-10 days, stained with APC-labeled anti-CD8 antibody and PE-labeled NY-ESO-1 157-165/HLA-A2 tetramer, and analyzed by flow cytometry.
  • Flow cytometry plots show percentages of CD8 + , tetramer + cells and CD8- and tetramer + cells in gated lymphocyte populations based on forward scatter and side scatter.
  • FIG. 4B shows that tumor cells can be sensitized by marker polypeptides comprising NY-ESO-1 epitope peptides and/or exogenous HLA-A2 for recognition by NY-ESO-1 specific TCRs 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 expressing NY-ESO-1 specific TCR -A2-PBMC cells.
  • tumor cells and PBMC cells were mixed for 24 hours at an E:T ratio of 10:1. After incubation, supernatants were collected to assess T cell secretion of IFN- ⁇ .
  • the 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.
  • Figure 4C shows that more tumor cell lines can be sensitized with marker peptides containing NY-ESO-1 epitope peptides and exogenous HLA-A2, and specific for NY-ESO-1 on primary T cells TCR identification.
  • Tumor cell lines A549, H1299, and HOS-C1 resulting in "A549-NY”, “H1299-NY” and “HOS-NY", respectively) were transduced with pShuttle-NY-A2 and used as target cells for stimulation with PBMC cells transduced with NY-ESO-1 specific TCR gene.
  • tumor cells and PBMC cells were mixed and cultured for 24 hours at an E:T ratio of 5:1.
  • Mock effector cells were PBMC cells transduced with empty lentivirus.
  • Other effector cells were PBMCs transduced with TCR-NY-LY gene and PBMCs transduced with TCR-NY-LI.
  • Data were analyzed with Student's t-test, ** represents p ⁇ 0.01, * represents p ⁇ 0.05.
  • Figure 5 shows the results of detecting the expression of HLA-A2 on the cell surface by flow cytometry after infecting SKOV3 cells with recombinant oncolytic adenovirus OAd-NY/A2 with different MOIs for 48 hours in Example 5 of the present invention.
  • the abscissa is the MOI of the recombinant oncolytic adenovirus OAd-NY/A2, and the ordinate is the expression percentage of HLA-A2.
  • Figure 6 shows the IFN release after co-culture of Mock-T or TCR-T targeting NY-ESO-1 with SKOV3 or recombinant oncolytic adenovirus OAd-NY/A2 infected SKOV3 in Example 6 of the present invention - Result of gamma.
  • Figure 6A shows the results of human melanoma cell line A375 (NY-ESO-1 positive and HLA-A2 positive)
  • Figure 6B shows the results of human lung cancer cell line H1299 (NY-ESO-1 positive and HLA-A2 negative)
  • Figure 6C Figure 6D is the result of human ovarian cancer cell line SKOV3 (NY-ESO-1 negative and HLA-A2 negative)
  • Figure 6D is the result of human osteosarcoma cell line HOSC1 (NY-ESO-1 weakly positive and HLA-A2 positive).
  • “Mock-T” indicates a T cell group expressing GFP (control group)
  • TCR-T indicates a T cell group expressing TCR targeting NY-ESO-1.
  • the abscissa in the figure represents different experimental groups, and the ordinate represents the concentration of IFN- ⁇ (pg/ml).
  • FIG. 7 shows the combined killing results of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1 on human ovarian cancer cell line SKOV3 in vitro in Example 7 of the present invention.
  • Figure 7A shows the results of real-time killing.
  • the abscissa is the time after tumor cells are plated (that is, after the experiment starts), in hours (h), and the ordinate is the normalized cell index. is the time point of adding OAd-NY/A2 and Mock-T or TCR-T.
  • Figure 7B is the analysis of the cell index of each group at the end of the experiment in Figure 7A (90.8 hours), the calculated tumor growth inhibition rate, the abscissa is the different experimental groups, the ordinate is the tumor growth inhibition rate IR (%).
  • Figure 8 shows the combined killing results of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1 in vitro on human lung cancer cell line H1299 in Example 8 of the present invention.
  • Figure 8A shows the results of real-time killing.
  • the abscissa is the time after tumor cells are plated (that is, after the experiment starts), in hours (h), and the ordinate is the normalized cell index.
  • the vertical downward arrows in the figure point to respectively is the time point of adding OAd-NY/A2 and Mock-T or TCR-T.
  • Figure 8B is the analysis of the cell index of each group at the 61.14th hour in Figure 8A, the calculated tumor growth inhibition rate, the abscissa is the different experimental groups, the ordinate is the tumor growth inhibition rate IR (%).
  • Figure 9 shows the combined killing results of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1 in vitro on human osteosarcoma cell line HOS C1 in Example 9 of the present invention.
  • Figure 9A is the result of real-time killing, the abscissa is the time after the tumor cells are plated (that is, after the experiment starts), in hours (h), the ordinate is the normalized cell index, the vertical downward arrows in the figure point to respectively is the time point of adding OAd-NY/A2 and Mock-T or TCR-T.
  • Figure 9B is the analysis of the cell index of each group at 64.44 hours in Figure 9A, and the calculated tumor growth inhibition rate, the abscissa is the different experimental groups, and the ordinate is the tumor growth inhibition rate IR (%).
  • the present invention provides an isolated oncolytic adenovirus for expressing foreign genes, wherein the oncolytic adenovirus is a selective replication type recombinant oncolytic adenovirus obtained by genetically modifying the adenovirus, and the recombinant oncolytic adenovirus is a
  • the genome of adenovirus has the following characteristics:
  • E1B gene regulatory element comprising an E1B promoter and a polyadenylation addition signal sequence shared by E1B and pIX;
  • the E1B gene coding region is deleted, and, when the exogenous gene needs to be inserted, the exogenous gene is inserted at the E1B gene coding region site, and the exogenous gene is located after the E1B promoter, and is subject to the the control of the E1B gene regulatory element;
  • the cDNA sequence of E1A that transcribes the E1A 13s mRNA is included, and the cDNA is a wild-type or Rb protein binding region deletion type, and the Rb protein binding region deletion type is the wild-type cDNA removed
  • SEQ ID NO.7 ie AC_000008.1nt 923-nt 946
  • AC_000008.1 is NCBI (ie, National Center for Biotechnology Information, website: https://www. ncbi.nlm.nih.gov) GenBank number
  • the Rb protein binding region deletion type encodes a mutated E1A protein, and the mutated E1A protein is shown in SEQ ID NO.6.
  • the present invention utilizes its own E1B gene regulatory element in the oncolytic virus genome to regulate the expression of exogenous genes, avoiding possible interference of the inserted exogenous gene regulatory elements on the expression of the viral genome, and thus affecting the effective replication of the virus.
  • the length of the inserted foreign gene fragment can also be increased, so that the oncolytic virus vector can carry more foreign gene load.
  • the oncolytic adenovirus constructed in the present invention lacks the coding region of the E1B gene.
  • E1B-19K protein can inhibit apoptosis induced by tumor necrosis factor and FAS pathways, making infected cells resistant to T cell killing. Deletion of the E1B-19K gene increases the sensitivity of infected tumor cells to killer T cells. Deletion of the E1B-55K gene increases the tumor cell oncolytic selectivity of oncolytic adenoviruses.
  • E1A-12S 243R
  • E1A-13S 289R
  • 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, they show significant differences in biological activity .
  • E1A-13S is the major viral protein involved in activating viral gene expression through its CR3 interaction with multiple transcription factors.
  • the E1A289R-expressing virus drives viral gene and protein expression more efficiently and replicates its genome more quickly and more efficiently than the E1A-12S-expressing adenovirus.
  • co-expression of E1A-12S (lack of CR3) inhibited the transcriptional activation function of E1A-13S, while the transcriptional repression of E1A-12S was at its N-terminus and correlated with its ability to bind p300/CBP.
  • the present invention finds that, in the genome of the oncolytic adenovirus, the cDNA sequence of E1A that transcribes E1A-13S mRNA is designed upstream of the exogenous gene instead of the E1A genome gene, so that the present invention can only transcribe E1A-13S and avoid transcribing E1A- 12S, thereby increasing the expression of foreign genes and enhancing the replication of the viral genome.
  • the core site of adenovirus E1A protein binding to Rb protein is Leu-122-X-Cys-X-Glu (X represents any amino acid residue).
  • the E1A protein lacking this amino acid sequence cannot bind to the Rb protein, resulting in oncolytic adenoviruses selectively replicating and lysing tumor cells in Rb/E2F1 pathway-deficient tumor cells. Therefore, in the genome of the recombinant oncolytic adenovirus of the present invention, when the cDNA sequence of E1A transcribing E1A 13s mRNA is the Rb protein binding region deletion type, in one embodiment, the Rb protein binding region deletion type is the The wild-type cDNA has the nucleotide sequence shown in SEQ ID NO.
  • L-T-C-H-E-A-G-F (Leu-Thr-Cys-His-Glu-Ala-Gly-Phe) is missing in the amino acid sequence of the E1A protein encoded by the Rb protein-binding region-deleted cDNA.
  • the Rb protein binding region deletion type encodes a mutated E1A protein, and the mutated E1A protein is shown in SEQ ID NO. 6, wherein the mutation sites are L122V, C124S and E126D.
  • the adenovirus is human type C adenovirus, including human type 2 adenovirus and human type 5 adenovirus.
  • nucleotide sequence of the E1B promoter is shown in SEQ ID NO.
  • the nucleotide addition signal sequence ie E1B/pIX polyadenylation signal sequence
  • SEQ ID NO. 2 ie AC_000008.1nt 4038-nt4043
  • 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.
  • 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).
  • the start site of the exogenous gene comprises a Kozak sequence, preferably, the Kozak sequence is shown in SEQ ID NO. 4 (ie GCCRCC ATG G, R is a purine (A or G)).
  • nucleotide sequence of the wild-type cDNA is shown in SEQ ID NO. 5 (i.e. AC_000008.1nt 560-nt 1545 removed nt 1113-nt 1228).
  • the cDNA of E1A transcribing E1A 13s mRNA is located upstream of the E1B promoter, and since the E1B promoter contains the added pA signal sequence of the endogenous E1A gene (AC_000008. 1nt 1611-nt1616) and pA addition site (AC_000008.1nt 1632), and part of the E1A gene sequence (nt 1336-nt 1552), therefore, the cDNA of E1A that transcribes E1A 13s mRNA and the nucleosides of the E1B promoter The acid sequences partially overlap.
  • the genome of the recombinant oncolytic adenovirus comprises the nucleotide sequence shown in SEQ ID NO. 2:
  • the nucleotide sequence shown in SEQ ID NO.2 comprises: the cDNA (marked with underline) of E1A transcribing the E1A 13s mRNA of the Rb protein binding region deletion type, and the E1B promoter (marked with italics). And, the nucleotide sequence shown in SEQ ID NO. 2 is located between the site of the endogenous E1A promoter/enhancer or the exogenous promoter and the start site of the exogenous gene.
  • 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 endogenous E1A promoter/enhancer
  • the nucleotide sequence of the sexual E1A promoter/enhancer is shown in SEQ ID NO. 8 (i.e. AC_000008.1nt 1-nt 499).
  • the E1A cDNA sequence transcribing the E1A 13s mRNA is under the control of an exogenous promoter.
  • an exogenous promoter is inserted after these positions.
  • the nucleotide sequence shown in SEQ ID NO. 9 ie AC_000008.1nt 376-nt 559 is removed from the genome of the recombinant oncolytic adenovirus, and the A foreign promoter is inserted into the genome from this removed site.
  • the exogenous promoter includes EF-1 ⁇ promoter, CMV promoter, PKG promoter, E2F promoter, AFP promoter and TERT promoter.
  • the exogenous genes include: HLA protein coding sequence, marker polypeptide coding sequence, HLA protein coding sequence and marker polypeptide coding sequence, HLA protein coding sequence and ⁇ 2-microglobulin coding sequence , or HLA protein coding sequence, ⁇ 2-microglobulin coding sequence and marker polypeptide coding sequence.
  • the present invention refers to "HLA protein coding sequence and marker polypeptide coding sequence", "HLA protein coding sequence and ⁇ 2-microglobulin coding sequence” and "HLA protein coding sequence, ⁇ 2-microglobulin coding sequence and marker polypeptide” "coding sequence” refers to the simultaneous insertion of the HLA protein coding sequence and the marker polypeptide coding sequence, or the simultaneous insertion of the HLA protein coding sequence and the ⁇ 2-microglobulin coding sequence, or the simultaneous insertion of HLA into the genome of the oncolytic adenovirus. Protein coding sequences, ⁇ 2-microglobulin coding sequences and marker polypeptide coding sequences.
  • the HLA protein comprises HLA class I molecules including HLA-A, HLA-B and HLA-C.
  • the HLA protein is HLA-A*02:01 protein, and the amino acid sequence (including the signal peptide sequence) is shown in SEQ ID NO.12.
  • HLA-C The expression of HLA-C on the cell surface is low.
  • at least one of the following mutations is performed on the inserted exogenous HLA-C: 1)
  • the 2nd arginine is mutated to alanine. In this way, after the initiator methionine is removed, Ala at the N-terminus will be acetylated, making the newly synthesized HLA-C more stable and not degraded.
  • the 4th nucleotide of the nucleotide sequence encoding the HLA-C protein is mutated from C to G, and the 5th nucleotide is mutated from G to C, which can form a strong Kozak sequence (GCCRCC ATG G, R is purine (A or G) to enhance protein translation and expression. 3) Isoleucine at position 362 is mutated to threonine; 4) Glutamic acid at position 359 is mutated to valine.
  • the C-terminus of HLA-C protein contains a dihydrophobic (LI) internalization and lysosomal targeting signal (DxSLI), and isoleucine at position 362 is a key amino acid affecting the activity of this motif.
  • LI dihydrophobic
  • DxSLI lysosomal targeting signal
  • isoleucine at position 362 of the HLA-C tail to threonine (I362T) in the tails of HLA-A and B, and/or glutamic acid at position 359 to valine increases the Surface expression of HLA-C. Since these mutations are not located in the antigen polypeptide presentation region, they will not affect the antigen polypeptide presentation and TCR recognition.
  • the HLA protein is HLA-C with the I362T point mutation. More preferably, the HLA protein is HLA-C*08:02 protein, and its amino acid sequence comprises R2A point mutation, I362T point mutation and E359V point mutation, and its amino acid sequence is shown in SEQ ID NO.13.
  • HLA proteins include but are not limited to HLA-A*01:01 protein, HLA-A*02:03 protein, HLA-A*02:06 protein, HLA-A*03:01 protein, HLA-A*11:01 protein, HLA-A*24:02 protein, HLA-A*30:01 protein, HLA-A*68:01 protein, HLA-B*08:01 protein, HLA-B*14:02 protein, HLA-B *1501, HLA-B*58:01, HLA-C*07:01 protein, HLA-C*01:02 protein.
  • the HLA protein coding sequence is expressed under the control of optional exogenous gene expression regulatory elements including promoters, enhancers, enhancers, silencers and polyadenylation signals, or the pharmaceutically acceptable vector itself Gene expression regulatory elements.
  • the antigenic protein expressed in the cytoplasm can enter the MHC class I antigen presentation pathway.
  • the formed short peptide (containing the antigenic epitope polypeptide) is transduced into the endoplasmic reticulum by TAP molecules on the membrane of the endoplasmic reticulum.
  • TAP molecules on the membrane of the endoplasmic reticulum.
  • trimers with HLA protein and ⁇ 2 -microglobulin and is presented on the cell surface (where HLA protein and ⁇ 2 -microglobulin pair to form MHC class I molecules), thereby being immune Cell identification.
  • the tumor antigens expressed in the cytoplasm cannot effectively form epitope polypeptides or enter the endoplasmic reticulum and combine with HLA and ⁇ 2-microglobulin to form a complex.
  • the exogenous marker polypeptide expressed in the tumor cells and/or cancer cells can enter the MHC class I antigen presentation Therefore, the expression of HLA/antigen epitope polypeptide complexes on the surface of tumor cells is increased, thereby enhancing the recognition sensitivity of the T cell receptor-modified immune cells to tumor cells and/or cancer cells.
  • the marker polypeptide comprises operably linked, in series, the following amino acid sequences: the amino acid sequence of an N-terminal signal peptide, the amino acid sequence of one or more epitope polypeptides, an optional C-terminal endoplasmic The amino acid sequence of the net retention signal, wherein when the marker polypeptide includes a plurality of amino acid sequences of the antigenic epitope polypeptides, the amino acid sequences of every two adjacent antigenic epitope polypeptides are connected by cleavage The amino acid sequence of the polypeptide is linked; the amino acid sequence of the epitope polypeptide and the amino acid sequence of the optional C-terminal endoplasmic reticulum retention signal can be linked by the amino acid sequence of the cleavable linking polypeptide.
  • the marker polypeptide comprises the amino acid sequence of the C-terminal endoplasmic reticulum retention signal.
  • the cleavable linking polypeptide is a furin cleavage recognition polypeptide.
  • the amino acid sequence of the antigenic epitope polypeptide can be derived from the amino acid sequence of a protein that exists in nature, or is an artificially synthesized amino acid sequence that does not exist in nature.
  • the naturally occurring proteins include human-derived proteins and proteins of species other than humans.
  • the amino acid sequences of the epitope polypeptides are derived from the amino acid sequences of tumor-associated antigens, tumor-specific antigens, and tumor neo-antigens containing mutation points.
  • Tumor-associated antigens generally refer to normal proteins derived from self, but overexpressed or abnormally expressed in tumor cells, including carcinoembryonic antigens, tumor-testis antigens (CT antigens), and the like.
  • Tumor-specific antigens generally refer to self-derived mutant proteins, or heterologous viral proteins associated with tumorigenesis and progression.
  • 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 the Cancer Antigenic Peptide Database (https://caped.icp.ucl.ac.be).
  • the tumor antigen may be a tumor antigen as described in Table 1 below.
  • the epitope polypeptide can be a peptide segment of 8-11 amino acids that can be presented by MHC class I molecules.
  • the epitope polypeptide may be an epitope polypeptide as described in the Cancer Antigenic Peptide Database (https://caped.icp.ucl.ac.be).
  • the epitope polypeptide may be an epitope polypeptide as described in Table 1 below.
  • the epitope polypeptide is 4-9 consecutive identical amino acids (eg, 4, 5, 6, 7, 8, or 9 consecutive) as the epitope polypeptides described in Table 1 below. epitope polypeptides of the same amino acids), and these polypeptides are 8-11 amino acids in length.
  • Table 1 Preferred tumor antigen and antigenic epitope polypeptide list
  • both ends of each of the epitope polypeptides have flexible linking fragments, which serve as cleavage sites for proteolytic enzymes in the cytoplasm to release the epitope polypeptide.
  • the flexible linker segments include GSGSR, AGSGSR and AGSGS.
  • the marker polypeptide has a signal peptide at the amino terminus of the one or more epitope polypeptide amino acid sequences that can introduce the marker polypeptide into the endoplasmic reticulum.
  • the core of the signal peptide contains long stretches of hydrophobic amino acids forming a single ⁇ -helix.
  • the amino-terminus of the signal peptide usually begins with a short positively charged amino acid sequence, and there is usually an amino acid cleavage site at the end of the signal peptide that is recognized and cleaved by signal peptidase.
  • the signal peptide can be a signal peptide (SEQ ID NO. 14) consisting of amino acids 1-22 of the amino terminal of insulin-like protein (INSL5).
  • the marker polypeptide has a plurality of the epitope polypeptides
  • every two of the epitope polypeptides may be linked by a cleavable linker polypeptide.
  • Cleavable linker polypeptides include furin cleavage recognition polypeptides, which have a standard four-amino acid motif that can be cleaved by furin enzymes, namely the RX-[KR]-R amino acid sequence (see document "Molecular Therapy 2007; vol.15no.6, 1153–1159”).
  • the amino acid sequence of the cleavable linking polypeptide is RRKR.
  • the antigenic epitope polypeptide connected by the RX-[KR]-R amino acid sequence is cleaved and hydrolyzed by furin enzyme in the endoplasmic reticulum, and the antigenic epitope polypeptide is released, which is combined with the endoplasmic reticulum.
  • HLA and ⁇ 2 - microglobulin in the endoplasmic reticulum form antigenic complexes.
  • Aminopeptidases and carboxypeptidases in the endoplasmic reticulum may also be involved in the enzymatic hydrolysis and release of epitope polypeptides (see the document "J Immunol. 2009 November 1; 183(9): 5526-5536"), therefore, cleavable Sex-linking polypeptides may also include aminopeptidase and carboxypeptidase cleavage recognition polypeptides.
  • the marker polypeptide has an endoplasmic reticulum retention signal peptide at the carboxy terminus of the one or more epitope polypeptide amino acid sequences.
  • the amino acid sequence of the endoplasmic reticulum retention signal (ER retention signal) of soluble polypeptides (ie, non-transmembrane proteins) is KDEL, and the endoplasmic reticulum retention signal of ER membrane proteins is KKXX (see the document "Molecular Biology of the Cell. 2003; 14" (3):889–902”).
  • the marker polypeptide is a soluble polypeptide. It is therefore preferred that the endoplasmic reticulum retention signal peptide is a K-D-E-L fragment consisting of lysine-aspartic acid-glutamic acid-leucine residues.
  • the tumor-associated antigen is selected from NY-ESO-1 157-165, NY-ESO-1 1-11, NY-ESO-1 53-62, NY-ESO-1 18-27, Her2/ neu 369-377, SSX-2 41-49, MAGE-A4 230-239, MAGE-A10 254-262, MAGE-C2 336-344, MAGE-C2 191-200, MAGE-C2 307-315, MAGE-C2 42-50, MAGE-A1 120-129, MAGE-A1 230-238, MAGE-A1 161-169, KK-LC-1 76-84, p53 99-107, PRAME 301-309, alpha-fetoprotein 158-166 , HPV16-E6 29-38, HPV16-E7 11-19, EBV-LMP1 51-59, EBV-LMP1 125-133, KRAS: G12D 10-18, KRAS: G12D 8-16, KRAS: G12D 7-16
  • the epitope polypeptide is NY-ESO-1 157-165 as shown in SEQ ID NO.10 or KRAS:G12D 10-18 as shown in SEQ ID NO.11.
  • the marker polypeptides include 3-8 NY-ESO-1 157-165 linked by a cleavable linking polypeptide or 3-8 KRAS:G12D10-18 linked by a cleavable linking polypeptide.
  • the exogenous gene includes the HLA protein coding sequence and the marker polypeptide coding sequence, the HLA protein coding sequence and the The marker polypeptide coding sequences are under the control of their 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 cleavably linked to the polypeptide coding sequence is operably linked to the marker polypeptide coding sequence.
  • the phenotype of the HLA protein is consistent with the phenotype of the HLA protein to which the marker polypeptide can bind.
  • the promoter may be a eukaryotic promoter, including a continuous expression promoter and an inducible expression promoter, including, for example: PGK1 promoter, EF-1 ⁇ promoter, CMV promoter, SV40 promoter, Ubc promoter , CAG promoter, TRE promoter, CaMKIIa promoter, human beta actin (human beta actin) promoter.
  • cleavable linking polypeptide linking the HLA protein and the marker polypeptide are known in the art, such as 2A polypeptides, 2A polypeptides including but not limited to F2A polypeptides from picornavirus, and Similar class 2A polypeptides from other viruses; also Furin-F2A linker fragments.
  • the combination of the HLA protein and the marker polypeptide is listed as follows: HLA-A-02:01 protein with The marker polypeptide of the antigenic epitope peptide NY-ESO-1 157-165, as shown in SEQ ID NO.15; HLA-C*08:02 protein and the marker polypeptide comprising the antigenic epitope peptide KRAS: G12D 10-18 , as shown in SEQ ID NO.16; HLA-A-01:01 protein and a marker polypeptide comprising antigenic epitope peptides KRAS: Q61H 55-64, KRAS: Q61L 55-64 or KRAS: Q61R 55-64; HLA -A-02:01, HLA-A-02:03 or HLA-A-02:06 proteins with epitope-containing peptides NY-ESO-1 157-165, Her2/neu 369-377
  • the exogenous gene includes the HLA protein coding sequence and the ⁇ 2-microglobulin coding sequence
  • the HLA protein coding sequence and the ⁇ 2-microglobulin coding sequence are under the control of respective promoters, or
  • the HLA protein coding sequence is under the control of the same promoter as the ⁇ 2-microglobulin coding sequence, and the HLA protein coding sequence is operably linked to the ⁇ 2-microglobulin coding sequence by cleavably linking the polypeptide coding sequence connect.
  • This embodiment applies to tumors lacking ⁇ 2-microglobulin expression.
  • the promoter may be a eukaryotic promoter, including a continuous expression promoter and an inducible expression promoter, including, for example: PGK1 promoter, EF-1 ⁇ promoter, CMV promoter, SV40 promoter, Ubc promoter , CAG promoter, TRE promoter, CaMKIIa promoter, human beta actin (human beta actin) promoter.
  • cleavable linking polypeptide linking the HLA protein and the marker polypeptide are known in the art, such as 2A polypeptides, 2A polypeptides including but not limited to F2A polypeptides from picornavirus, and Similar class 2A polypeptides from other viruses; also Furin-F2A linker fragments.
  • the ⁇ 2-microglobulin is a human protein or a murine protein.
  • amino acid sequence of the ⁇ 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 E1B gene regulatory element, the E1B gene coding region is deleted, and is upstream of the foreign gene Contains the cDNA sequence of E1A that transcribes the E1A 13s mRNA.
  • the vector may be backboned by pShuttle.
  • the pShuttle plasmid is derived from the pBR322 plasmid and contains the kanamycin resistance gene from pZero 2.1 and a multiple cloning site for insertion of the foreign gene.
  • the two ends of the multiple cloning site are homologous recombination sequences of the type 5 adenovirus genome.
  • the homologous recombination sequence at the left end is adenovirus type 5 nucleic acid sequence 34,931–35,935.
  • the right-hand homologous recombination sequence is adenovirus type 5 nucleic acid sequence 3,534–5,790.
  • upstream of the E1A 13s sequence is the exogenous promoter.
  • the oncolytic adenovirus constructed in the present invention can be used alone as a tumor vaccine, or can be used in combination with immune cells modified by T cell receptors.
  • the exogenous gene of the oncolytic adenovirus is selected from a marker polypeptide coding sequence, and an HLA protein coding sequence and a marker polypeptide coding sequence.
  • the present invention also provides a therapeutic agent for treating tumor and/or cancer, comprising:
  • a first composition wherein the first composition comprises a first active ingredient in a first pharmaceutically acceptable carrier, the first active ingredient comprising or containing an agent according to the present invention for introduction into tumor cells and/or cancer cells
  • the first composition comprises a first active ingredient in a first pharmaceutically acceptable carrier, the first active ingredient comprising or containing an agent according to the present invention for introduction into tumor cells and/or cancer cells
  • an agent according to the present invention for introduction into tumor cells and/or cancer cells
  • composition comprising a second active ingredient in a second pharmaceutically acceptable carrier, the second active ingredient comprising T cell receptor-modified immune cells.
  • tumors and/or cancers suitable for use in the therapeutic agent of the present invention should express an epitope-providing polypeptide within the An endogenous tumor antigen, expressing or not expressing an endogenous HLA protein that can present the epitope polypeptide.
  • tumors and/or cancers suitable for use in the therapeutic agent of the invention should express an endogenous polypeptide capable of presenting the epitope in the marker polypeptide HLA protein, expressing or not expressing the endogenous tumor antigen that can provide the epitope polypeptide.
  • the oncolytic adenovirus expresses HLA protein and marker polypeptide
  • the oncolytic adenovirus expresses HLA protein, ⁇ 2-microglobulin and marker polypeptide
  • the tumor and/or cancer of the therapeutic agent may or may not express the endogenous tumor antigen that provides the epitope polypeptide, and may or may not express the endogenous HLA protein that can present the epitope polypeptide.
  • the exogenous gene of the oncolytic adenoviral vector is selected from the group consisting of marker polypeptide coding sequences and HLA protein coding sequences and marker polypeptide coding sequences, and the marker polypeptide has one or more antigenic expression
  • the amino acid sequence of the epitope polypeptide which can be presented on the surface of the tumor cell and/or cancer cell by MHC class I molecules; and the immune cell modified by the T cell receptor can specifically recognize and bind to the The epitope polypeptide presented by the MHC class I molecule.
  • the first composition and the second composition are each independently present in the therapeutic agent without mixing with each other.
  • the immune cells modified with T cell receptors include naive T cells or their precursor cells, NKT cells, or T cell strains.
  • the T cell receptor includes at least one of an alpha chain and a beta chain, both of which contain variable and constant regions, and the T cell receptor is capable of specifically recognizing tumor cells and/or cancer cells the epitope polypeptide on the cell surface.
  • variable regions of the TCR alpha and beta chains are used to bind antigenic polypeptides/major histocompatibility complex (MHC I), and include three hypervariable regions or called complementarity determining regions (CDRs), respectively, namely, CDR1, CDR2, CDR3.
  • CDRs complementarity determining regions
  • the CDR3 region is crucial for the specific recognition of antigenic polypeptides presented by MHC molecules.
  • the TCR ⁇ chain is formed by recombination of different V and J gene segments, and the ⁇ chain is formed by the recombination of different V, D and J gene segments.
  • the corresponding CDR3 region formed by the recombination of specific gene fragments, as well as the palindromic and random nucleotide additions of the binding region form the specificity of TCR for antigen polypeptide recognition (see the document "Immunobiology: The immune system”). in health and disease. 5th edition, Chapter 4, The generation of Lymphocyte antigen receptors”).
  • the MHC class I molecules include human HLA.
  • the HLAs include: HLA-A, B, and C.
  • the exogenous TCR ⁇ and ⁇ chains expressed by T cells may be mismatched with the ⁇ and ⁇ chains of the T cell’s own TCR, which not only dilutes the expression of correctly paired foreign TCRs, but also reduces the antigen specificity of mismatched TCRs.
  • TCR alpha and beta chains it is preferable to modify the constant regions of the TCR alpha and beta chains to reduce or avoid mismatches.
  • the constant region of the ⁇ chain and/or the constant region of the ⁇ chain is derived from human; preferably, the present invention finds that the constant region of the ⁇ chain may be wholly or partially may be replaced in whole or in part by homologous sequences derived from other species, and/or the constant regions of the beta strands may be replaced in whole or in part by homologous sequences derived from other species. More preferably, the other species is mouse.
  • the replacement can increase the expression of TCR in cells, and can further improve the specificity of cells modified by the TCR for Her2/neu antigen.
  • 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, eg 1 or 2.
  • TCRs modified in two different ways are prepared, one way is to add a disulfide bond in the constant region of the TCR by point mutation, the method is described in the document "Cancer Res. 2007 Apr 15; 67(8): 3898-903.", which is incorporated by reference in its entirety.
  • Methods for replacing the corresponding human TCR constant region sequences with mouse TCR constant region sequences are described in "Eur. J. Immunol. 2006 36:3052-3059", which is incorporated herein by reference in its entirety.
  • the first composition comprises a therapeutically effective amount of the oncolytic adenovirus.
  • the administration dose of the oncolytic adenovirus is 5 ⁇ 10 7 -5 ⁇ 10 12 vp/day, 1-2 times a day, for 1-7 consecutive days.
  • the second composition comprises a therapeutically effective amount of the T cell receptor-modified immune cells.
  • the T cell receptor-modified immune cells are contained in a total dose ranging from 1 ⁇ 10 3 to 1 ⁇ 10 9 cells/Kg body weight per course of treatment.
  • the oncolytic adenovirus can be formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid administration, or intravenous administration.
  • the immune cells can be formulated for administration by arterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular, or intraperitoneal.
  • the therapeutic agent consists of the first composition and the second composition.
  • the therapeutic agent of the present invention may further comprise suitable pharmaceutically acceptable excipients, including pharmaceutical or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives , including carbohydrates, lipids, polypeptides, amino acids, antioxidants, adjuvants, preservatives, etc.
  • suitable pharmaceutically acceptable excipients including pharmaceutical or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives , including carbohydrates, lipids, polypeptides, amino acids, antioxidants, adjuvants, preservatives, etc.
  • the present invention also provides a combination medicine kit with synergistic effect for the treatment of tumor and/or cancer, comprising:
  • 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 separate;
  • the present invention also provides the use of the oncolytic adenovirus of the present invention in preparing a medicament for treating tumors and/or cancers.
  • the foreign gene of the oncolytic adenovirus is selected from the group consisting of HLA protein coding sequences, marker polypeptide coding sequences, HLA protein coding sequences and marker polypeptide coding sequences, HLA protein coding sequences and ⁇ 2-microbe globulin coding sequences, or HLA protein coding sequences, ⁇ 2-microglobulin coding sequences and marker polypeptide coding sequences.
  • the tumors and/or cancers include: breast cancer, head and neck cancer, 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder cancer, ureteral cancer, glioma, neuroblastoma Tumor, Meningioma, Spinal Cord Tumor, Osteochondroma, Chondrosarcoma, Ewing's Sarcoma, Carcinoma of Unknown Primary Site, Carcinoid, Fibrosarcoma, Paget's Disease, Cervical Cancer, Gallbladder Cancer, Eye Cancer, Kaposi's Sarcoma , prostate cancer, testicular cancer, squamous cell carcinoma of the
  • the present invention also provides the use of the therapeutic agent of the present invention in the preparation of a medicament for treating tumors and/or cancers.
  • the tumors and/or cancers include: breast cancer, head and neck cancer, 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder cancer, ureteral cancer, glioma, neuroblastoma Tumor, Meningioma, Spinal Cord Tumor, Osteochondroma, Chondrosarcoma, Ewing's Sarcoma, Carcinoma of Unknown Primary Site, Carcinoid, Fibrosarcoma, Paget's Disease, Cervical Cancer, Gallbladder Cancer, Eye Cancer, Kaposi's Sarcoma , prostate cancer, testicular cancer, squamous cell carcinoma of the
  • the present invention also provides the use of the kit of the present invention in preparing a medicament for treating tumors and/or cancers.
  • the tumors and/or cancers include: breast cancer, head and neck cancer, 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder cancer, ureteral cancer, glioma, neuroblastoma Tumor, Meningioma, Spinal Cord Tumor, Osteochondroma, Chondrosarcoma, Ewing's Sarcoma, Carcinoma of Unknown Primary Site, Carcinoid, Fibrosarcoma, Paget's Disease, Cervical Cancer, Gallbladder Cancer, Eye Cancer, Kaposi's Sarcoma , prostate cancer, testicular cancer, squamous cell carcinoma of the
  • the present invention also provides a method of treating tumor and/or cancer, comprising administering the oncolytic adenovirus according to the present invention to a tumor and/or cancer patient.
  • the foreign gene of the oncolytic adenovirus is selected from HLA protein coding sequence, marker polypeptide coding sequence, HLA protein coding sequence and marker polypeptide coding sequence, HLA protein coding sequence and ⁇ 2-microglobulin coding sequence sequence, or HLA protein coding sequence, ⁇ 2-microglobulin coding sequence and marker polypeptide coding sequence.
  • the tumors and/or cancers include: breast cancer, head and neck cancer, 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder cancer, ureteral cancer, glioma, neuroblastoma Tumor, Meningioma, Spinal Cord Tumor, Osteochondroma, Chondrosarcoma, Ewing's Sarcoma, Carcinoma of Unknown Primary Site, Carcinoid, Fibrosarcoma, Paget's Disease, Cervical Cancer, Gallbladder Cancer, Eye Cancer, Kaposi's Sarcoma , prostate cancer, testicular cancer, squamous cell carcinoma of the
  • the tumor and/or cancer expresses an endogenous tumor antigen that provides an epitope polypeptide, and at the same time expresses an endogenous HLA-class I molecule that presents the epitope polypeptide.
  • the oncolytic adenovirus is administered in a therapeutically effective amount.
  • the administration dose of the oncolytic adenovirus is 5 ⁇ 10 7 -5 ⁇ 10 12 vp/day, 1-2 times a day, for 1-7 consecutive days.
  • the oncolytic adenovirus can be formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid administration, or intravenous administration.
  • the present invention also provides a method for treating tumor and/or cancer, comprising:
  • the second composition of the therapeutic agent according to the present invention is administered to the tumor and/or cancer patient.
  • the first composition and the second composition in the therapeutic agent can be administered simultaneously (e.g., injected intratumorally at the same time as a mixture), separately but simultaneously (e.g., administered by intratumoral and intravenous injection, respectively), or sequentially (e.g., The first composition is applied first, followed by the second composition; or the second composition is applied first, then the first composition).
  • the method comprises the following steps in sequence:
  • the second composition of the therapeutic agent is administered to the tumor and/or cancer patient on days 1-30 after the first administration of the first composition.
  • administering the second composition of the therapeutic agent to the tumor and/or cancer patient on days 1-30 after the first administration of the first composition means that the first administration of the second composition and the The time interval between the first administration of the first composition is 1-30 days (eg, 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 first application of the second composition that is most adjacent to the first
  • the compositions are administered at intervals of 1-30 days (eg, 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).
  • the time interval between the first administration of the second composition and the most adjacent administration of the first composition immediately preceding it is 3-14 days (eg, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14 days).
  • the first composition comprises the oncolytic adenovirus, and the oncolytic adenovirus is administered at a dose of 5 ⁇ 10 7 -5 ⁇ 10 12 vp/day, 1-2 times a day , administered continuously for 1-7 days, or any value in the above range.
  • the administered dose of the T cell receptor-modified immune cells is in the range of 1 ⁇ 10 3 -1 ⁇ 10 9 cells/Kg body weight for each course of treatment. Preferably, it is administered 1-3 times a day for 1-7 consecutive days.
  • the methods of treating tumors and/or cancer further comprise administering to the patient other drugs for treating tumors and/or cancers, and/or drugs for modulating the patient's immune system, to enhance the Number and function of T cell receptor-modified immune cells in vivo.
  • Said other drugs for the treatment of tumors and/or cancers include, but are not limited to: chemotherapeutic drugs, such as cyclophosphamide, fludarabine; radiotherapy drugs; immunosuppressants, such as cyclosporine, azathioprine, Methotrexate, mycophenolate, FK50; antibodies, eg, anti-CD3, IL-2, IL-6, IL-17, TNF[alpha].
  • the method of treating tumors and/or cancer further comprises administering to the patient other drugs for treating tumors and/or cancer, and/or drugs for modulating the patient's immune system, for use when all When the T cell receptor-modified immune cells produce serious toxic side effects, the number and function of the T cell receptor-modified immune cells carrying the suicide gene in the body are eliminated.
  • Said other drugs for the treatment of tumors and/or cancers include, but are not limited to: chemically induced dimerization (CID) drugs, AP1903, phosphorylated ganciclovir, anti-Cd20 antibody, anti-CMYC antibody, anti-EGFR antibody.
  • the oncolytic adenovirus can be formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid administration, or intravenous administration.
  • the T cell receptor-modified immune cells can be formulated for administration by arterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular, or intraperitoneal.
  • the tumors and/or cancers include: breast cancer, head and neck cancer, 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder cancer, ureteral cancer, glioma, neuroblastoma Tumor, Meningioma, Spinal Cord Tumor, Osteochondroma, Chondrosarcoma, Ewing's Sarcoma, Carcinoma of Unknown Primary Site, Carcinoid, Fibrosarcoma, Paget's Disease, Cervical Cancer, Gallbladder Cancer, Eye Cancer, Kaposi's Sarcoma , prostate cancer, testicular cancer, squamous cell carcinoma of the
  • tumors and/or cancers suitable for use in the therapeutic agent of the present invention should express an epitope-providing polypeptide within the An endogenous tumor antigen, expressing or not expressing an endogenous HLA protein that can present the epitope polypeptide.
  • tumors and/or cancers suitable for use in the therapeutic agent of the invention should express an endogenous polypeptide capable of presenting the epitope in the marker polypeptide HLA protein, expressing or not expressing the endogenous tumor antigen that can provide the epitope polypeptide.
  • the oncolytic adenovirus expresses HLA protein and marker polypeptide
  • the oncolytic adenovirus expresses HLA protein, ⁇ 2-microglobulin and marker polypeptide
  • the tumor and/or cancer of the therapeutic agent may or may not express the endogenous tumor antigen that provides the epitope polypeptide, and may or may not express the endogenous HLA protein that can present the epitope polypeptide.
  • Methods within the scope of the present invention may be provided to patients according to their own actual conditions of tumor and/or cancer.
  • the patient can be subjected to HLA-I typing and tumor antigen expression detection before using the above-mentioned treatment.
  • HLA-I molecules can be used to detect the matching of HLA-I molecules, for example, serological matching, PCR-SSP (sequence-specific priming PCR), PCR-SSOP (sequence-specific oligonucleotide probes), PCR- RFLP (restriction fragment length polymorphism), PCR-SBT (sequencing-based typing), PCR-RSCA (reference strand-mediated conformational analysis), and next-generation sequencing (NGS)-based HLA typing.
  • serological matching for example, serological matching, PCR-SSP (sequence-specific priming PCR), PCR-SSOP (sequence-specific oligonucleotide probes), PCR- RFLP (restriction fragment length polymorphism), PCR-SBT (sequencing-based typing), PCR-RSCA (reference strand-mediated conformational analysis), and next-generation sequencing (NGS)-based HLA typing.
  • PCR-SSP sequence
  • NGS next-generation sequencing
  • recombinant plasmids and viral vectors, or polypeptides and proteins can be made using standard recombinant methods (Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 2012) using the methods described herein. Recombinant nucleic acid production.
  • the percentage concentration (%) of each reagent refers to the volume percentage concentration (% (v/v)) of the reagent.
  • the cell line used to make lentiviral particles or used as target cells was 293T cells (ATCC CRL-3216).
  • the presenting cell line used to present antigenic peptides was 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 cancer cell line SKOV3 (ATCC HTB-77), Human lung cancer cell line H1299 (ATCC CRL-5803), human osteosarcoma cell line HOS-C1 (ATCC CRL-1547), human lung cancer cell line A549 (ATCC CCL-185).
  • human melanoma cell line A375 ATCC CRL-1619
  • human fibrosarcoma cell line HT1080 ATCC CCL-121
  • human ovarian cancer cell line SKOV3 ATCC HTB-77
  • Human lung cancer cell line H1299 ATCC CRL-5803
  • human osteosarcoma cell line HOS-C1 ATCC CRL-1547
  • human lung cancer cell line A549 ATCC CCL-185.
  • the plasmid pCDNA3.3-NY encoding the NY-ESO-1 protein or the plasmid pCDNA3.3-kras/g12d encoding the KRAS G12D protein was constructed from the pCDNA3.3 plasmid (Thermo Fisher K830001) by conventional methods, and transfected into SKOV3 cells, respectively. And selection with 500 ⁇ 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 multiple cancer types. The method for making pCDNA3.3-NY is described below.
  • 293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine in high glucose (VWR Cat# VWRL0101-0500).
  • DMEM Dulbecco's Modified Eagle's Medium
  • Other cell lines were supplemented with fetal bovine serum (ATCC 30-2020), 2mmol/L L-glutamic acid, 1 ⁇ essential amino acids 50 ⁇ (Invitrogen 11130-051), 1 ⁇ streptomycin/penicillin 100 ⁇ (Invitrogen 15140- 122), 1 ⁇ sodium pyruvate 100 ⁇ (Invitrogen 11360-070) and 1 ⁇ 2-mercaptoethanol 1000 ⁇ (Thermo fisher 21985023) in RPMI-1640 complete medium (Lonza, cat. no. 12-115F).
  • DMEM Dulbecco's Modified Eagle's Medium
  • fetal bovine serum ATCC 30-2020
  • PBMC peripheral blood mononuclear cells
  • Expression plasmids include plasmids encoding the marker polypeptides, exogenous HLA proteins or amino acid sequences described in this application. Methods for preparing plasmids are described below. If a plasmid containing the adenoviral genome was used to transfect 293T cells, the plasmid was pre-digested with Pad enzyme (New England Biolabs, R0547S) to release the adenoviral genome. 48-72 hours after transfection, cells transiently expressing the exogenous protein can be used as target cells.
  • Pad enzyme New England Biolabs, R0547S
  • SKOV3 cells were incubated with medium supplemented with 500 ⁇ g/mL geneticin cultivate together.
  • PBMC cells in 24-well plates were purified with RPMI-1640 supplemented with 2 ⁇ g/ml anti-human CD3 antibody (Biolegend 317303) and 2 ⁇ g/ml anti-human CD28 antibody (Biolegend 302914) Media were incubated together for 24 hours, or PBMCs were treated with human T-CD3/CD8 magnetic beads (Thermo Fisher 11131D) according to the manufacturer's instructions. After 24 hours, cells were cultured with complete RPMI-1640 medium supplemented with IL-2 100IU/ml, IL-7 5ng/ml, IL-15 5ng/ml.
  • beta chain deficient mutant JRT J.RT3-T3.5 cells from the Jurkat cell line were cultured with RPMI-1640 complete medium.
  • activated PBMC or JRT cells were resuspended in 1 ml freshly prepared lentiviral supernatant in a 24-well plate and Polybrene (Santa Cruz Biotechnology sc) was added at 25°C -134220). The final concentration is 5–8 ⁇ g/ml. Cells were centrifuged at 1000 g and 32°C for 2 hours.
  • RPMI-1640 medium supplemented with IL-2 100IU/ml, IL-7 5ng/ml, IL-15 5ng/ml.
  • Cells can also be transfected using RetroNectin dishes (RetroNectin pre-dish, 35 mm ⁇ ) (Takara T110A) according to the manufacturer's instructions.
  • the flow cytometer was a MACSQuant Analyzer 10 (Miltenyi Biotec Corporation) and the results were analyzed by Flowjo software (Flowjo Corporation).
  • MACSQuant Analyzer 10 Miltenyi Biotec Corporation
  • Flowjo software Flowjo Corporation
  • TCR gene-modified JRT cells were co-cultured for 16 hours in duplicate wells of 96-well plates with target cells, e.g., with T2 cells loaded with different concentrations of antigenic peptide concentrations, using the methods described in the present application.
  • IFN- ⁇ secretion by specific T cells following antigen stimulation was measured by IFN- ⁇ ELISA (enzyme-linked immunosorbent assay). Briefly, TCR gene-modified PBMCs were co-cultured with the target cells described above in duplicate or triplicate wells of a 96-well plate. Cell supernatants were collected within 18-24 hrs for use with the IFN-gamma ELISA Read-Set-Go Kit (eBioscience 88-7316) or the Human IFN-gamma DuoSet ELISA Kit (R&D Systems DY285B) according to the manufacturer's instructions IFN- ⁇ ELISA analysis was performed.
  • IFN- ⁇ ELISA enzyme-linked immunosorbent assay
  • the TCR gene was cloned into a replication-deficient lentiviral vector pCDH-EF1 ⁇ -MCS-PGK-GFP (System Biosciences CD811A-1). By removing the PGK promoter and GFP gene on the vector pCDH-EF1 ⁇ -MCS-PGK-GFP, the GFP-free vector pCDH-EF1 ⁇ -MCS was generated.
  • the NY-ESO-1 157-165 table in the context of HLA-A*02:01 was determined according to the sequences described in patent application publication US8143376B2, WO2018099402A1 or reference "J Immunol 2010; 184(9), 4936-46" Sequences of the peptide-specific TCR- ⁇ -VDJ and TCR- ⁇ -VDJ regions (denoted as TCR-NY-LY for US8143376B2 and TCR-NY-LI for WO2018099402A1, for reference J Immunol 2010; 184(9 ), 4936-46 is represented as TCR-NY-AE).
  • TCR- ⁇ -VDJ and TCR- ⁇ -VDJ regions specific for the KRAS:G12D 10-18 peptide in the context of HLA-C*08:02 are according to patent application publication WO2018026691 (denoted as TCR- RAS G12D) determined.
  • the sequences of mouse TCR-alpha constant chain and mouse TCR-beta constant chain were determined from reference sequences (GeneBank KU254562 and EF154514.1, respectively).
  • Nucleic acid sequences of TCR-NY-LY, TCR-NY-AE and TCR-NY-LI against NY-ESO-1 157-165, nucleic acid sequences of TCR-RAS-G12D against KRAS:G12D 10-18 are shown in SEQ respectively ID No. 18, 19, 20 or 21 and synthesized by Integrated DNA Technologies or LifeSct LLC.
  • the synthesized nucleic acid was cloned into the multiple cloning site downstream of the EF-1 ⁇ promoter of the lentiviral vector pCDH-EF1 ⁇ -MCS-PGK-GFP or pCDH-EF1 ⁇ -MCS (without GFP) according to the manufacturer's instructions.
  • the lentiviral vector expressing the TCR against the NY-ESO-1 157-165 epitope is called pCDH-EF1 ⁇ -TCR-NY.
  • the inserted nucleic acid was sequenced and found no errors or mutations.
  • the lentiviral vector plasmids were transformed into competent bacteria stellar (Takara Bio, 636763) to prepare plasmid stocks for the preparation of lentiviral particles.
  • TCR lentiviral particles were produced from 293T or 293FT cells (Thermo Fisher R70007) transfected with a lentiviral vector plasmid containing the TCR gene. Briefly, 6-well plates were co-transfected with TCR lentiviral vector plasmids and pPACKH1-lentivector packaging kit (System Biosciences LV500A-1) using Lipofectaine 3000 transfection reagent (invitrogen, 11668019) according to the manufacturer’s instructions Growing 293T or 293FT cells. After 48 hours of incubation, the supernatant was collected and filtered through a 0.4 ⁇ m filter.
  • Viral supernatants were concentrated using a Lenti-X TM concentrator (Takara, 631231) according to the manufacturer's instructions. Freshly prepared TCR-lentiviruses were used to infect JRT cells or activated PBMCs.
  • RNA purified from HT1080 cells (NY-ESO-1+, KRAS wild-type) using the PureLink TM RNA Mini Kit (Thermo Fisher 12183020) according to the manufacturer's instructions was used as a PCR assay using the PrimeScript TM RT-PCR Kit ( Takara RR014A) template for generating RT-PCR products.
  • the NY-ESO-1 full-length gene was generated by the following PCR primer pairs: 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 pairs: 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. Use according to manufacturer's instructions High-Fidelity 2X PCR Master Mix (New England Biolabs M0541L) performed the PCR described in this application.
  • the replication deficient recombinant adenovirus system described in this application is based on the AdEasy system (Nature Protocols 2007; 2: 1236-1247).
  • the nucleic acid of the EF1 ⁇ promoter was cloned from the pCDH-EF1 ⁇ -MCS-PGK-GFP plasmid (System Biosciences, CD811A-1) by the following PCR primer pair: 5′-CTCATAGCGCGTAATGGCTCCGGTGCCCGTCAGTGGGCAG-3′ (SEQ ID NO. 26) and 5'-GAATTCGCTAGCTCTAGATCACGACACCTGAAATGGAAG-3' (SEQ ID NO.
  • the cDNA encoding full-length HLA-A*02:01 (as shown in SEQ ID NO. 28) and the cDNA encoding human ⁇ 2-microglobulin were generated from T2 cells and cloned into pcDNA3.3- according to the manufacturer's instructions TOPO carrier.
  • nucleic acid encoding a marker polypeptide connecting HLA-A*201 protein and a linker peptide as shown in SEQ ID NO. 31 were synthesized by Integrated DNA Technologies.
  • the following steps were performed: using the synthetic nucleic acid encoding the marker polypeptide as a template, primer pair 5 '-AGAGCTAGCGAATTCAACATGAAAGGTTCCATCTTCAC-3' (SEQ ID NO. 32) and 5'-ACACTGTGTAATCCACATCAATAGCGATCTCTTTC-3' (SEQ ID NO.
  • PCR was performed using the synthetic nucleic acid encoding the linker peptide as template with the following primer pairs: 5'-TGGATTACACAGTGTCGTCGTAAGCGATCCGGAAGCGCG-3' (SEQ ID NO.
  • 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 the encoding Nucleic acid of labeled polypeptide linking HLA-A*201 protein to linker peptide.
  • the resulting nucleic acid was cloned into the multiple cloning site of the pShuttle-EF1 ⁇ vector using conventional gene cloning techniques in the art to generate pShuttle-EF1 ⁇ -NY-A2.
  • the pShuttle vector was linearized with PmeI (NEB Biolabs, R0560s). After purification, the vector was transferred into the electroactive BJ5183-AD-1 bacterial strain (Agilent) in a Bio-Rad Gene Concentration Loading Generator electroporator at a frequency of 2500 V, 200 ⁇ and 25 ⁇ F, following the manufacturer's instructions. Technologies, 200157). Restriction digestion with Pad (NEB Biolabs R0547S) was used to screen for potential adenoviral recombinants. Correct recombinants usually yield a larger fragment (about 30 kb), and a smaller fragment of 3.0 or 4.5 kb.
  • the pShuttle vector which comprises the adenovirus E1A gene driven by an exogenous promoter and a nucleic acid encoding an HLA molecule bound to the marker polypeptide or an HLA molecule bound to ⁇ 2-microglobulin driven by the native E1B promoter
  • the pShuttle vector the following procedure was performed: the sequence shown in SEQ ID NO.2 was synthesized by Integrated DNA Technologies, which contained the natural E1B promoter sequence and encoded E1A- containing a 24 base pair deletion (E1A 122-129 deletion) 13s protein nucleic acid sequence (native E1A polyA signal sequence with E1A polyA addition site).
  • PCR was carried out using the following primer pairs: 5'-ATGAGACATATTATCTGCCACGGAG-3' (SEQ ID NO. 46) and 5'-CATGGTGGCGAGGTCAGATGTAAC-3' (SEQ ID NO. 47), obtaining a sequence containing 24
  • the base pair deleted E1A-13s nucleic acid sequence and the gene fragment of the native E1B promoter sequence (denoted as E1Ad24).
  • PCR was performed using the cDNA encoding full-length HLA-A*02:01 or the cDNA encoding human ⁇ -2 microglobulin as a template (the Kozak sequence was introduced at this time), and the HLA-A*02:01 gene fragment (denoted as A2) and the ⁇ -2 microglobulin gene fragment (denoted BM).
  • the synthetic mutant HLA-C*08:02 gene is shown in SEQ ID NO.49 (represented as C08), and the synthetic nucleic acid encodes a marker polypeptide comprising 3 NY-ESO-1 157-165 epitope peptides, as shown in SEQ ID NO.
  • SEQ ID NO.29 (denoted as NY), or encoding a marker polypeptide comprising 3 KRAS:G12D 10-18 epitope peptides, as shown in SEQ ID NO.50 (denoted as RAS), or a synthetic nucleic acid encoding A linker peptide, such as SEQ ID NO. 30 (denoted as F2A), was used as a template for PCR to obtain the corresponding gene fragment (Kozak sequence was introduced when PCR was performed using the mutant HLA-C*08:02 gene as a template) .
  • E1Ad24-A2-F2A-NY, E1Ad24-C08-F2A-RAS, E1Ad24-A2-F2A-BM and E1Ad24-C08-F2A were generated using the In-Fusion HD Cloning Plus Kit (Takara 638909) according to the manufacturer's instructions - Combinations of nucleic acid fragments of BM.
  • the resulting gene combination was cloned downstream of the EF1 ⁇ promoter in the pShuttle-EF1 ⁇ vector to generate pShuttle-EF1 ⁇ -E1Ad24-A2-F2A-NY, pShuttle-EF1 ⁇ -E1Ad24-C08-F2A-RAS, pShuttle-EF1 ⁇ -E1Ad24-C08 - F2A-BM and pShuttle-EF1 ⁇ -E1Ad24-A2-F2A-BM.
  • the pShuttle vector was linearized with PmeI and transformed into electroactive BJ5183 as described above by delivering concentration loading in a Bio-Rad Gene concentration loading electroporator at frequencies of 2500 V, 200 ⁇ and 25 ⁇ F. -AD-1.
  • the resulting recombinant oncolytic adenovirus plasmids were denoted pAd-EF1 ⁇ -E1Ad24-A2-F2A-NY, pAd-EF1 ⁇ -E1Ad24-C08-F2A-RAS, pAd-EF1 ⁇ -E1Ad24-C08-F2A-BM, and pAd-EF1 ⁇ - E1Ad24-A2-F2A-BM.
  • the replication-defective recombinant adenovirus plasmid obtained by the above method and the recombinant oncolytic adenovirus plasmid with conditional replication ability are used to prepare the corresponding recombinant adenovirus by this method.
  • adenoviral plasmids were digested with Pac I (NEB Biolabs R0547S) to release adenoviral genomic DNA.
  • the linearized plasmid was purified by phenol/chloroform extraction and transfected into ADENO-X 293 cells (Takara 632271) with Lipofectaine 3000 transfection reagent (Thermo Fisher L3000001) according to the manufacturer's instructions. Transfected cells were maintained in a 37°C, 5% CO2 incubator for 14–20 days until cytopathic effect (CPE) was observed. Four cycles of freeze-thaw vortexing were performed to release adenovirus from cells and obtain viral particles.
  • CPE cytopathic effect
  • Adenovirus titers were determined by Adeno-X GoStix kit (Takara 632270) according to the manufacturer's instructions.
  • MOI multiplicity of infection, which refers to the number of viral particles per cell infected.
  • the expression of foreign genes can be detected 3-4 days after infection.
  • Human ovarian cancer cell line SKOV3, human lung cancer cell line H1299, and human osteosarcoma cell line HOS C1 were purchased from ATCC;
  • the human melanoma cell line A375 was purchased from the Cell Bank of the Chinese Academy of Sciences;
  • Human IFN-gamma ELISA kit was purchased from R&D Company;
  • xCELLigence RTCA S16 real-time label-free cell function analyzer was purchased from ACEA Bio;
  • APC-bound anti-human HLA-A2 antibody was purchased from BD company, and PE-bound anti-human CD3 antibody was purchased from BD company;
  • hrIL-2 was purchased from Kingsley Company, and hrIL-7 and hrIL-15 were purchased from Nearshore Protein Company;
  • Flow cytometer Novocyte was purchased from ACEA Bio Company.
  • FIG. 1A shows a schematic representation of the constructs described in this application.
  • pAdEasy-EF1 ⁇ -NY-A2 is a replication-deficient adenovirus vector expressing the marked polypeptide shown in SEQ ID NO.51, which contains NY-ESO-1 157- 165 epitope peptide, and HLA-A2 protein as shown in SEQ ID NO.12.
  • the marker polypeptide and the HLA-A2 gene are linked by the F2A sequence.
  • the expression unit of the marker polypeptide is flanked by the exogenous EF-1 ⁇ promoter and the SV40 poly(A) signal sequence.
  • pAd-EF1 ⁇ -E1A-A2-F2A-NY is a replication competent adenoviral vector containing nucleic acid encoding the marker polypeptide of the HLA-A2 gene and three NY-ESO-1 157-165 epitope peptides.
  • pAd-EF1 ⁇ -E1A-A2-F2A-BM is a replication competent adenoviral vector containing nucleic acids encoding the HLA-A2 gene and human ⁇ 2-microglobulin.
  • Both the pAd-EF1 ⁇ -E1Ad24-A2-F2A-NY and pAd-EF1 ⁇ -E1Ad24-A2-F2A-BM constructs contain nucleic acid encoding the E1-13S mutein (ie, the cDNA sequence of E1A transcribing the E1A 13s mRNA), the E1A The protein contains a deletion of 24 residues as shown in SEQ ID NO.43.
  • the E1A-13s gene is flanked by an exogenous EF-1 ⁇ promoter and a native E1A poly(A) signal sequence, respectively.
  • FIG. 1B is a schematic diagram of the lentiviral vectors pCDH-EF1 ⁇ -TCR-NY-LY, pCDH-EF1 ⁇ -TCR-NY-AE and pCDH-EF1 ⁇ -TCR-NY-LI, which contain encoding each NY-ESO-1 specific TCR ⁇ Nucleic acids of chain and alpha chain polypeptides (the polypeptides are shown in SEQ ID NO.
  • TCR beta and alpha chains were replaced by murine TCR constant region sequences.
  • the TCR ⁇ chain and ⁇ chain are connected by polypeptide Furin protease cleavage point and F2A linking sequence, and the two ends are EF-1 ⁇ promoter and lentiviral post-transcriptional regulatory element (WPRE).
  • pShuttle-EF1 ⁇ -NY-A2 pShuttle-EF1 ⁇ -E1Ad24-A2-F2A-NY or pShuttle-EF1 ⁇ -E1Ad24-A2-F2A-BM containing expression units (which On both sides are the genome sequences homologous to adenovirus type 5) transfected 293T cells with low expression of endogenous HLA-A2 on the cell surface. Cells were stained with anti-HLA-A2 antibody and HLA-A2 expression was assessed by flow cytometry.
  • Figure 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 function to drive the expression of exogenous proteins and are linked by Furin protease and F2A sequence, HLA-A2 protein can be isolated and expressed on the cell surface.
  • the fluorescence intensity of HLA-A2 expression in pShuttle-EF1 ⁇ -NY-A2 transduced 293T cells was lower than that in pShuttle-EF1 ⁇ -E1Ad24-A2-F2A-NY and pShuttle-EF1 ⁇ -E1Ad24-A2-F2A-BM transduced 293T cells , indicating that the native E1B promoter and E1B poly(A) signal may more efficiently drive the expression of foreign genes in the context of adenovirus genomes.
  • lentiviral particles expressing NY-ESO-1-specific TCR were prepared from 293T cells transfected with pCDH-EF1 ⁇ -TCR-NY vector and used to infect J.RT3-T3 .5 cells (JRT cells, ATCC TIB153). Infected JRT cells were stained with anti-CD8 antibody and NY-ESO-1 157-165/HLA-A2 tetramer, which can specifically bind and recognize NY-ESO-1 presented by HLA-A2 TCR of 157-165 epitope polypeptides.
  • FIG. 1D shows that JRT cells transfected with lentivirus express different TCRs capable of binding the NY-ESO-1 157-165/HLA-A2 tetramer, indicating that the recombinant lentivirus expresses a specific TCR consisting of TCR alpha and beta chains.
  • the alpha and beta chains are paired by the included murine constant regions to form the TCR/CD3 complex and expressed on the surface of JRT cells.
  • JRT cells were first transfected with recombinant lentiviruses co-transfected with pCDH-EF1 ⁇ - 293T cell production of TCR-NY-LY, pCDH-EF1 ⁇ -TCR-NY-AE or pCDH-EF1 ⁇ -TCR-NY-LI and the above packaging vector. JRT cells expressing specific TCRs were co-cultured with HLA-A2 positive T2 cells, and a 10-fold dilution series of NY-ESO-1 157-165 polypeptide starting at 1 ⁇ g/ml was added to the culture wells.
  • FIG. 2A shows that JRT cells expressing specific TCR express CD69 after stimulation with antigenic peptide, indicating that JRT cells are activated by HLA-A2-presented NY-ESO-1 157-165 peptide, and its activity exhibits an antigen dose-dependent relationship .
  • different TCRs also showed differences in the recognition sensitivity of different TCRs to the NY-ESO-1 157-165 epitope.
  • the -1 157-165 epitope polypeptide is about 10 times less sensitive.
  • JRT cells transfected with NY-ESO-1 157-165 antigen polypeptide-specific TCR were used as effector cells, and HLA-A2 negative and NY- ESO-1-negative 293T cells were used as target cells, and 293T cells transduced with empty pShuttle vector were used as control target cells to assess whether target cells transfected with vectors expressing marker polypeptides and exogenous HLA-A2 were in target cells
  • the surface expresses epitope polypeptides that are recognized by specific T cells.
  • Figure 2B shows that both pShuttle-EF1a-NY-A2 or pShuttle-EF1a-E1Ad24-A2-NY transfected 293T cells can activate JRT cells expressing different NY-ESO-1 157-165 polypeptide-specific TCRs.
  • the percentage of CD69+ JRT cells was significantly increased against the 293T target cells expressing the above marker polypeptides and HLA-A2 compared to the control target 293T transduced with the empty pShuttle vector (Student's t-test, p ⁇ 0.01).
  • 293T cells were co-transfected with pCDNA3.3-NY and pShuttle-EF1a-E1A d 24- A2-F2A-BM, and used as target cells.
  • pCDNA3.3-NY encodes the full-length NY-ESO-1 protein, allowing 293T cells to express the NY-ESO-1 protein as an endogenous tumor antigen, thereby producing the NY-ESO-1 157-165 epitope peptide.
  • the target cells can be expressed by specific TCRs. recognized by JRT cells.
  • 293T cells transfected only with pCDNA3.3-NY or pShuttle-EF1a-E1Ad24-A2-F2A-BM were used as negative controls.
  • Figure 2C shows that 293T cells transduced with pShuttle-EF1a-E1A 24-A2-F2A-BM alone were unable to activate JRT cells expressing NY-ESO-1 specific TCR.
  • the NY-ESO-1 157-165 epitope polypeptide can be produced by the endogenous HLA class I processing mechanism and be recognized by the specific TCR after being presented by exogenous HLA-A2. Therefore, it is feasible to introduce exogenous HLA class I molecules as allogeneic HLA (allo-HLA) into tumor cells to present epitope peptides from endogenous proteins.
  • the endogenous protein that produces the antigenic epitope polypeptide may be an overexpressed tumor-associated antigen or a tumor neoantigen (neo-antigen) produced by a mutant protein.
  • the adenoviral vector pAd-EF1a-E1Ad24-A2-NY was digested with Pad to Recombinant adenovirus genomic DNA was obtained, comprising exogenous nucleic acid encoding HLA-A2 protein and marker polypeptide having NY-ESO-1 157-165 epitope polypeptide.
  • pAd-EF1a-E1Ad 24-A2-BM was also digested with Pad to obtain adenovirus genomic DNA expressing exogenous HLA-A2 and ⁇ 2-microglobulin.
  • 293T cells were transfected with adenoviral DNA and used as target cells 48 h later to stimulate JRT cells expressing NY-ESO-1-specific TCR.
  • Figure 2D shows that pAd-EF1a-E1Ad24-A2-BM expresses exogenous HLA-A2 in 293T cells and is not only able to present the NY-ESO-1 157-165 epitope polypeptide loaded at a concentration of 1 ⁇ g/ml, but also Capable of presenting the NY-ESO-1 157-165 epitope polypeptide from the NY-ESO-1 protein.
  • CD69+ JRT cells The percentage of CD69+ JRT cells was significantly increased compared to control target cells transfected only with pAd-EF1a-E1Ad24-A2-BM (Student's t-test, p ⁇ 0.01).
  • 293T cells transfected with adenovirus DNA of pAd-EF1a-E1Ad24-A2-NY express both the labeled polypeptide with NY-ESO-1 157-165 epitope and exogenous HLA-A2, and can also activate the expression of NY- JRT cells of ESO-1-specific TCR.
  • epitope peptides can be presented by exogenous HLA class I molecules in tumor cells when tumor cells are transduced with a genetic construct containing the nucleic acid encoding the marker polypeptide and HLA class I protein. Regardless of whether tumor cells express specific tumor antigens or have HLA class I alleles that present specific epitope polypeptides, the expression of said marker polypeptides and exogenous HLA class I molecules enables tumor cells to be recognized by specific T cells target cells. In addition, the expression of exogenous allogeneic HLA class I proteins can not only increase the immunogenicity of tumor cells, but also present epitope peptides from endogenous tumor antigens and activate HLA-restricted epitope-specific peptides. TCR.
  • A375 is a human melanoma cell line representing HLA-A2 + and NY-ESO-1 + tumor cells.
  • SKOV3 is a human ovarian cancer cell line representing HLA - A2- and NY-ESO- 1- double negative tumor cells.
  • SKOV3 cells transduced with pCDNA3.3-NY and stably expressing NY-ESO-1 protein represented 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, used as target cells, and co-cultured with JRT cells expressing the NY-ESO-1 specific TCR.
  • Figure 3 shows that both JRT-TCR-NY-LY and JRT-TCR-NY-AE can be activated by A375 cells, suggesting that the NY-ESO-1 157-165 epitope peptide can be generated from endogenous NY-ESO-1 protein, and presented by its own HLA-A2 molecule.
  • the amount of HLA-A2 complex presented on A375 cells is not limited by the amount of HLA-A2, but by the amount of NY-ESO-1 157-165 polypeptide produced by the HLA class I antigen processing machinery.
  • TEPs transport proteins
  • pShuttle-EF1a-E1Ad24-A2-NY allowed SKOV3 cells to be recognized by JRT cells expressing NY-ESO-1-specific TCR (Student's t-test, p ⁇ 0.01), indicating that regardless of endogenous antigen or matched HLA class I What is the expression state of the allele, the antigenic epitope peptide expressed after the oncolytic virus vector transfects the tumor cells can be combined with the HLA-A2 molecule and be recognized by the specific TCR.
  • SKOV3-NY cells transduced by pShuttle-EF1a-E1Ad24-A2-BM can activate JRT cells with NY-ESO-1-specific TCR, indicating that exogenous HLA class I molecules introduced into tumor cells can also make tumor cells susceptible to specific TCR recognition, but tumor cells must express tumor antigens as a source of antigenic peptides.
  • a complete antigen processing and presentation mechanism is required in tumor cells in order to process tumor antigen proteins to generate epitope peptides and be presented by exogenous HLA class I molecules.
  • Target cells transfected with pShuttle-A2-F2A-NY activated more JRT-TCR-NY-LY cells than target cells SKOV3-NY transfected with pShuttle-A2-F2A-BM (Student's t-test, p ⁇ 0.05), indicating that compared with the endogenous antigen protein, the labeled polypeptide produces more NY-ESO-1 157-165 epitope polypeptides.
  • This example shows that tumor cells transfected with the nucleic acids encoding marker polypeptides and/or exogenous HLA class I molecules can be recognized by primary T cells expressing specific TCRs.
  • HLA-A2-negative PBMCs were 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-CD8-APC and NY-ESO-1 157-165 tetramer-PE.
  • Figure 4A shows that T cells transduced with all three TCR genes can express the NY-ESO-1 157-165-specific TCR tagged with the NY-ESO-1157-165 tetramer.
  • NY-ESO-1 157-165 tetramer positive cells were observed in both CD8 + and CD8- cell populations.
  • CD8 + cells are NY-ESO-1 - specific killer T cells (CTLs)
  • CD8- cells may be CD4 + T helper cells expressing NY-ESO-1-specific TCRs. This result indicates that TCR-NY-LY, TCR-NY-AE and TCR-NY-LI have high binding affinity to the NY-ESO-1 157-165/HLA-A2 complex and are independent of CD8 helper function.
  • PBMCs expressing NY-ESO-1 specific TCRs were used as effector cells to assess the recognition sensitivity of tumor cells expressing the marker polypeptides and exogenous HLA class I molecules to specific T cells.
  • A375 cell line, SKOV3 cell line and SKV3-NY cells expressing full-length NY-ESO-1 protein represent NY-ESO-1 and HLA-A2 double positive, NY-ESO-1 and HLA-A2 double negative and NY- ESO-1 single positive target cells.
  • These target cells were transfected with pShuttle-EF1a-E1Ad24-A2-NY or pShuttle-EF1a-E1A d24-A2-BM as target cells to evaluate expression of the marker polypeptide and exogenous HLA-A2 for tumor cells Recognition function of specific TCRs expressed on generation T cells.
  • Recombinant lentivirus-transfected PBMCs were co-incubated with target cells at an E:T ratio of 10:1 for 16-24 hours.
  • the secretion of IFN- ⁇ in the supernatant was examined to evaluate the ability of target cells to stimulate T.
  • Figure 4B shows that all target cells transfected with pShuttle-EF1a-E1A d24-A2-BM can efficiently present the NY-ESO-1 156-165 polypeptide and activate T cells expressing specific TCRs, indicating that pShuttle -
  • the exogenous HLA-A2 protein expressed by the EF1a-E1A d24-A2-BM vector binds to the exogenous NY-ESO-1 156-165 polypeptide to form a NY-ESO-1 156-165/HLA-A2 complex and is expressed NY-ESO-1-specific TCR recognized by primary T cells.
  • 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.
  • SKOV3 cells were transfected with pShuttle-EF1a-E1Ad24-A2-NY, which expressed both HLA-A2 molecules and NY-ESO-1 157-165 epitope peptides, could they be recognized by T cells expressing specific TCRs.
  • the oncolytic virus vector expressing the marker polypeptide containing antigenic epitope peptide and exogenous HLA class I molecule can not only make tumor cells recognized by JRT cells expressing specific TCR, but also can be recognized by JRT cells expressing specific TCR. identified by primary T cells.
  • the human lung cancer cell line H1299 (NY-ESO-1 + /HLA-A2 ⁇ ), could be transduced by nucleic acids encoding the marker polypeptides and/or exogenous HLA class I molecules
  • the human osteosarcoma cell line HOS-C1 (NY-ESO-1 low/HLA-A2 + ) and the human lung cancer cell line A549 (NY-ESO-1 ⁇ /HLA-A2 ⁇ ) were transduced with the pShuttle-NY-A2 vector and used as target cells.
  • PBMCs transfected with recombinant lentiviruses expressing TCR-NY-LY or TCR-NY-LI were used as effector cells.
  • TCR-NY-LY or TCR-NY-LI produced substantial amounts of IFN- ⁇ (Student's t-test, p ⁇ 0.01) compared to controls with blank control T cells ), indicating that the tested tumor cells can be labeled by expressing marker polypeptides containing NY-ESO-1 157-165 epitope peptide and exogenous HLA-A2 protein and express NY-ESO-1 specific TCR recognized by T cells.
  • Example 5 Expression of HLA-A2 after infection of SKOV3 cells with recombinant oncolytic adenovirus OAd-NY/A2
  • the recombinant oncolytic adenovirus "OAd-NY/A2" described below is a recombinant oncolytic adenovirus obtained by packaging the plasmid pAd-EF1 ⁇ -E1Ad24-A2-F2A-NY according to the aforementioned method.
  • HLA-A2 can be expressed on the cell surface.
  • 24-well plates were seeded at 5 x 104 SKOV3 cells/well.
  • recombinant oncolytic adenovirus OAd-NY/A2 was added to SKOV3 cells at MOI of 5, 10, 20, 50, and 100, respectively. After an additional 48 hours of culture, the cells were harvested.
  • the cells were stained with APC-conjugated anti-human HLA-A2 antibody (BD company) (1:50 dilution), and the expression of HLA-A2 on the cell surface was detected by flow cytometer Novocyte (purchased from ACEA Bio company).
  • APC-conjugated anti-human HLA-A2 antibody (BD company) (1:50 dilution)
  • flow cytometer Novocyte purchased from ACEA Bio company
  • T cell culture medium is AIM V+5%FBS+100IU/ml hrIL-2+10ng/ml hrIL-7+10ng/ml hrIL-15.
  • revive human PBMCs obtained from Hangzhou Kangwanda Pharmaceutical Technology Co., Ltd.
  • inoculate PBMCs into one well of a 24-well plate according to 5 ⁇ 10 5 cells/ml, 1 ml/well, according to T:beads 1 :1 Join dynabeads.
  • Example 6 Cytokine secretion after co-culture of TCR-T targeting NY-ESO-1 and recombinant oncolytic adenovirus OAd-NY/A2 infection-labeled tumor cells
  • the secretion of IFN ⁇ was detected by ELISA after overnight co-culture of TCR-T cells targeting NY-ESO-1 and tumor cells infected with recombinant oncolytic adenovirus OAd-NY/A2.
  • the detected tumor cell lines include human melanoma cell line A375, human lung cancer cell line H1299, human ovarian cancer cell line SKOV3, and human osteosarcoma cell line HOSC1.
  • 12-well plates were seeded at 1 x 105 tumor cells/well.
  • 80 ⁇ l of recombinant oncolytic adenovirus OAd-NY/A2 was added to the corresponding tumor cells.
  • tumor cells and tumor cells infected with recombinant oncolytic adenovirus OAd-NY/A2 were digested and counted with trypsin, and 1 ⁇ 10 5 tumor cells/well were inoculated into a 96-well plate.
  • TCR-T cells or Mock-T cells targeting NY-ESO-1 (expressing GFP, as a negative control group for TCR-T) were seeded in corresponding wells of a 96-well plate at 3 ⁇ 10 5 cells/well.
  • the ratio of effector cells to target cells (E:T) was 3:1, and each group consisted of 3 replicate wells.
  • human IFN- ⁇ DuoSet ELISA purchased from R&D Company was used to detect the content of IFN ⁇ in the culture supernatant.
  • TCR-T cells since A375 cells were NY-ESO-1 positive and HLA-A2 positive, targeting NY-ESO- 1 TCR-T cells (shown as A375+TCR-T in the figure) can secrete IFN ⁇ after being stimulated by A375. Infection of A375 with recombinant oncolytic adenovirus OAd-NY/A2 did not induce the secretion of IFN ⁇ . If T cells were co-cultured with recombinant oncolytic adenovirus OAd-NY/A2-infected A375 cells, after being stimulated by OAd-NY/A2-labeled A375, they were compared with Mock-T of the negative control group (shown as A375+ in the figure).
  • TCR-T cells targeting NY-ESO-1 could secrete more IFN ⁇ .
  • H1299 cells were NY-ESO-1 positive and HLA-A2 negative
  • targeting NY-ESO- 1 TCR-T cells did not secrete IFN ⁇ after being stimulated by H1299.
  • Infection of H1299 with recombinant oncolytic adenovirus OAd-NY/A2 did not induce the secretion of IFN ⁇ .
  • T cells were co-cultured with recombinant oncolytic adenovirus OAd-NY/A2-infected H1299 cells, after being stimulated by OAd-NY/A2-labeled H1299, they were compared with Mock-T of the negative control group (shown as H1299+ in the figure). Compared with Mock-T+OAd-NY/A2), TCR-T cells targeting NY-ESO-1 (shown as H1299+TCR-T+OAd-NY/A2 in the figure) could secrete more IFN ⁇ .
  • T cells were co-cultured with recombinant oncolytic adenovirus OAd-NY/A2-infected SKOV3 cells, after being stimulated by OAd-NY/A2-labeled SKOV3, compared with Mock-T of the negative control group (shown as SKOV3+ in the figure) Compared with Mock-T+OAd-NY/A2), TCR-T cells targeting NY-ESO-1 (shown as SKOV3+TCR-T+OAd-NY/A2 in the figure) could secrete more IFN ⁇ .
  • HOSC1 cells are weakly positive for NY-ESO-1 and positive for HLA-A2
  • Mock-T in the negative control group shown as HOSC1+Mock-T in the figure
  • targeting NY-ESO -1 TCR-T cells shown as HOSC1+TCR-T in the figure
  • TCR-T secreted a small amount of IFN ⁇ .
  • Recombinant oncolytic adenovirus OAd-NY/A2 infected HOSC1 also did not induce the secretion of IFN ⁇ .
  • T cells were co-cultured with recombinant oncolytic adenovirus OAd-NY/A2-infected HOSC1 cells, after being stimulated by OAd-NY/A2-labeled HOSC1, compared with Mock-T of the negative control group (shown as HOSC1+ in the figure) Compared with Mock-T+OAd-NY/A2), TCR-T cells targeting NY-ESO-1 (shown as HOSC1+TCR-T+OAd-NY/A2 in the figure) could secrete more IFN ⁇ .
  • the TCR targeting NY-ESO-1 recognizes the target cell surface HLA-A2 and the NY-ESO-1 peptide molecule it presents. If the tumor cells themselves are HLA-A2-positive and NY-ESO-1-positive, the recombinant oncolytic adenovirus OAd-NY/A2 can improve the targeting of TCR-T targeting NY-ESO-1 to a certain extent. identification of cells. If the tumor cells themselves are HLA-A2 negative or NY-ESO-1 negative or double negative, after the recombinant oncolytic adenovirus OAd-NY/A2 infects the labeled tumor cells, it can make the original TCR-T unable to recognize the target cells to become able Identify and attack tumor cells.
  • Example 7 In vitro combined killing of SKOV3 by recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1
  • the combined killing effect of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T cells targeting NY-ESO-1 on human ovarian cancer cell line SKOV3 in vitro was detected by RTCA real-time killing monitoring method.
  • 5 x 103 tumor cells/well were seeded into 16-well E-plates.
  • the recombinant oncolytic adenovirus OAd-NY/A2 was added to the corresponding wells at an MOI of 10.
  • NY-ESO-1-targeting TCR-T cells or Mock-T cells (expressing GFP, as a negative control for TCR-T) were added to the corresponding wells at 5 ⁇ 10 3 T cells/well. , 2 duplicate wells in each group.
  • the growth of SKOV3 cells was monitored by the xCELLigence RTCA S16 real-time label-free cell function analyzer.
  • SKOV3 cells continued to grow after inoculation (shown as SKOV3 in the figure), and the addition of recombinant oncolytic adenovirus OAd-NY/A2 alone did not affect the growth of SKOV3 cells (shown as SKOV3+OAd-NY/A2 in the figure) ), only adding TCR-T cells or Mock-T cells targeting NY-ESO-1 had a weak effect on the growth of SKOV3 cells (shown as SKOV3+Mock-T or SKOV3+TCR-T in the figure).
  • the combined effect of recombinant oncolytic adenovirus OAd-NY/A2 and Mock-T can slow down the growth of SKOV3 cells, while the recombinant oncolytic adenovirus OAd-NY/
  • the combined effect of A2 and TCR-T cells targeting NY-ESO-1 shown as SKOV3+OAd-NY/A2+TCR-T in the figure) showed a very significant killing effect on SKOV3 cells and significantly reduced the number of SKOV3 cells .
  • 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 with the formula: 100% ⁇ (experimental group cell index-SKOV3 cell index)/SKOV3 cell index (cell index (Cell index) obtained by RTCA instrument).
  • Example 8 In vitro combined killing of H1299 by recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T targeting NY-ESO-1
  • the combined killing effect of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T cells targeting NY-ESO-1 on human lung cancer cell line H1299 in vitro was detected by RTCA real-time killing monitoring method.
  • 5 x 103 tumor cells/well were seeded into 16-well E-plates.
  • the recombinant oncolytic adenovirus OAd-NY/A2 was added to the corresponding wells at an MOI of 10.
  • NY-ESO-1-targeting TCR-T cells or Mock-T cells (expressing GFP, as a negative control for TCR-T) were added to the corresponding wells at 1 ⁇ 10 4 T cells/well. , 2 duplicate wells in each group. Growth of H1299 cells was monitored by the xCELLigence RTCA S16 real-time label-free cell function analyzer.
  • H1299 cells continued to grow after inoculation (shown as H1299 in the figure), and the addition of recombinant oncolytic adenovirus OAd-NY/A2 hardly affected the growth of H1299 cells (shown as H1299+OAd-NY/ A2), only adding TCR-T cells or Mock-T cells targeting NY-ESO-1 had a significant effect on the growth of H1299 cells (shown as H1299+Mock-T or H1299+TCR-T in the figure).
  • the combination of recombinant oncolytic adenovirus OAd-NY/A2 and Mock-T can effectively kill H1299 cells, while the recombinant oncolytic adenovirus OAd-NY/
  • the combined effect of A2 and TCR-T cells targeting NY-ESO-1 shown as H1299+OAd-NY/A2+TCR-T in the figure) showed a very significant killing effect on H1299 cells, making the number of H1299 cells more significant reduce.
  • the cell growth index of the experimental node was analyzed for 61.14 hours, and the tumor growth inhibition rate (IR%) was calculated with the formula: 100% ⁇ (cell index of experimental group-H1299 cell index)/H1299 cell index.
  • the results are shown in Figure 8B, the recombinant oncolytic adenovirus, Mock-T or TCR-T alone can only inhibit the growth of H1299 cells to a certain extent, and the combined effect of recombinant oncolytic adenovirus and Mock-T can reduce H1299
  • the number of cells, and the combined effect of recombinant oncolytic adenovirus and TCR-T can significantly reduce the number of H1299 cells, and its killing effect is greater than the superposition of the effect of recombinant oncolytic adenovirus and TCR-T alone, thus resulting in 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
  • the combined killing effect of recombinant oncolytic adenovirus OAd-NY/A2 and TCR-T cells targeting NY-ESO-1 on human osteosarcoma cell line HOS C1 in vitro was detected by RTCA real-time killing monitoring method.
  • 5 x 103 tumor cells/well were seeded into 16-well E-plates.
  • the recombinant oncolytic adenovirus OAd-NY/A2 was added to the corresponding wells at an MOI of 30.
  • NY-ESO-1-targeting TCR-T cells or Mock-T cells (expressing GFP, as a negative control for TCR-T) were added to the corresponding wells at 1.5 ⁇ 10 4 T cells/well. , 2 duplicate wells in each group. Growth of HOS C1 cells was monitored by the xCELLigence RTCA S16 real-time label-free cell function analyzer.
  • HOS C1 cells continued to grow after inoculation (shown as HOS C1 in the figure), and only the addition of recombinant oncolytic adenovirus OAd-NY/A2 weakly affected the growth of HOS C1 cells (shown as HOS C1+ in the figure) OAd-NY/A2), only adding TCR-T cells or Mock-T cells targeting NY-ESO-1 also inhibited the growth of HOS C1 cells (shown as HOS C1+Mock-T or HOS C1 in the figure). +TCR-T).
  • the combined effect of recombinant oncolytic adenovirus OAd-NY/A2 and Mock-T can reduce the number of cells in HOS C1, while the recombinant oncolytic adenovirus OAd
  • the combined effect of -NY/A2 and TCR-T cells targeting NY-ESO-1 shown as HOS C1+OAd-NY/A2+TCR-T in the figure
  • the number of HOS C1 cells was significantly reduced.
  • the cell growth index at the end of the experiment at 61.14 hours was analyzed, and the tumor growth inhibition rate was calculated with the formula: 100% ⁇ (experimental group cell index-HOS C1 cell index)/HOS C1 cell index (IR%).
  • the results are shown in Figure 9B, the recombinant oncolytic adenovirus, Mock-T or TCR-T alone had only a weak inhibitory effect on the growth of HOS C1 cells, and the combined effect of the recombinant oncolytic adenovirus and Mock-T could be weak.
  • the combination of recombinant oncolytic adenovirus and TCR-T can significantly reduce the number of HOS C1 cells, and its killing effect is greater than the superposition of the effects of recombinant oncolytic adenovirus and TCR-T alone. , resulting in a synergistic killing effect.
  • the vector may be a plasmid vector, recombinant virus, nanoparticle or naked DNA or RNA
  • the epitope-containing Peptides and/or marker polypeptides of exogenous HLA class I molecules can enable tumor cells to be recognized by T cells expressing specific TCRs.
  • combination therapy for the treatment of solid tumors could expand the reach of adoptive T-cell therapy, often in patients who may not be included in trials, such as those whose HLA-type mismatches (despite their tumor cells expressing specific of tumor antigens) can also benefit from treatment.
  • a nucleic acid encoding a marker polypeptide containing an epitope and an HLA class I molecule that can present the epitope peptide can be delivered into the It can be recognized by adoptive specific T cells in tumor cells, which can become a general method for the treatment of various solid tumors.
  • off-target toxicities may occur if normal cells are also labeled with labeled nucleic acids and targeted for adoptive transfer of T cells.
  • the delivery vehicle can selectively express marker epitope polypeptides or exogenous HLA molecules in tumor cells, for example using an oncolytic virus capable of selective replication of tumor cells as suggested in this application, or Controllable.
  • the oncolytic virus vector of the present invention enables tumor cells to directly express the labeling of the HLAI class molecules that contain antigenic epitopes and can present antigenic epitope peptides peptides and make them targets for adoptive transfusion of T cells.
  • allogeneic HLA class I molecules are delivered to tumor cells harboring the most common tumor driver mutations (such as KRAS or p53 mutations), rendering them incapable of being presented by endogenous HLA I
  • Tumor antigen neo-epitopes presented by T-like molecules are recognized and cleared by adoptively transfused specific T cells. This combination therapy is a promising treatment that could benefit more cancer patients.

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Abstract

提供了分离的能够用于表达外源基因的溶瘤腺病毒、载体、治疗剂及其用途。所述溶瘤腺病毒为对腺病毒进行基因改造而得到的选择复制型重组溶瘤腺病毒，其基因组具有以下特征：1)包含E1B基因调控元件；2)缺失了E1B基因编码区，并且在该位点插入外源基因，该外源基因位于E1B启动子之后，受E1B基因调控元件的控制；3)外源基因的上游包含转录E1A 13s mRNA的E1A的cDNA，该cDNA为野生型或Rb蛋白结合区域缺失型。所述溶瘤腺病毒能更有效地将具有标记性多肽编码序列的核酸导入肿瘤和/或癌细胞，更加高效地在肿瘤和/或癌细胞内表达外源标记性多肽，使其进入MHC I类抗原提呈途径，增强T细胞受体修饰的免疫细胞对肿瘤和/或癌细胞的识别敏感性。

Description

分离的能够用于表达外源基因的溶瘤腺病毒、载体、治疗剂及其用途 技术领域
本发明属于生物技术领域，具体而言，涉及分离的用于表达外源基因的溶瘤腺病毒、载体、治疗剂及其用途。
背景技术
随着针对免疫检查点抑制剂在临床试验中表现出显著的抗肿瘤疗效(参见文献“Cancer Cell 27,450–461(2015)”)，以及以CAR-T(chimeric antigen receptor(CAR)T cells)为代表的过继T细胞疗法对血液肿瘤所表现出的长期有效的治疗效果，肿瘤免疫治疗已成为最具发展前景的领域之一。过继细胞治疗(ACT)，包括过继转输肿瘤浸润淋巴细胞(TIL)，表达外源的肿瘤特异性T细胞受体的T细胞(TCR-T)，或CAR-T细胞，已被证明是最有前途的一种癌症的免疫疗法(参见文献“N Engl J Med 2017；377:2545-2554”)。
虽然靶向CD19或BCMA的CAR-T疗法对B细胞型血液肿瘤已显示出显著的临床疗效，CAR-T尚未对实体瘤患者显示出明显的临床益处(参见文献“J Immunother.2019；42：126-135”)。而过继转输TIL或转输经过肿瘤抗原特异性TCR基因修饰的TCR-T细胞均对实体瘤患者显示出临床疗效(参见文献“Adv Immunol.2016；130:279-94”)。目前，TCR-T治疗各种实体瘤的临床试验处于不同阶段(参见文献“Technol Cancer Res Treat。2019；1-13”)。尽管过继T细胞疗法是治疗实体瘤的一种有前途的方法，进一步提高其疗效仍然面临一些障碍。其中之一就是肿瘤组织异质性，表现为被T细胞特异性识别的肿瘤抗原在肿瘤细胞中的表达不均一(参见文献“Int J Cancer。2001 Jun 15；92(6)：856-60”)。部分肿瘤细胞中靶抗原的表达缺失或不足会使它们逃逸过继转输的T细胞的识别和杀伤作用。肿瘤发生和发展过程中，肿瘤细胞中的抗原加工和提 呈机制常发生异常，表现为HLA I类分子突变或表达降低，β2-微球蛋白，TAP，tapasin酶，LMP复合物等表达降低，使得肿瘤抗原不能有效提呈到细胞表面而逃避被肿瘤抗原特异性T细胞所识别和清除(参见文献“J Natl Cancer Inst.2013；105(16)：1172-87”)。另外，肿瘤特异性T细胞识别的是由主要组织相容性复合物(MHC，人的MHC为HLA分子)所提呈的抗原表位肽(epitope peptide)，T细胞识别抗原的功能受到MHC的限制，只能识别被特定MHC分子所提呈的肿瘤抗原表位肽。肿瘤患者的肿瘤细胞表达特异性肿瘤抗原并同时具有特定的HLA等位基因，才能成为过继T细胞治疗的适用人群，因而MHC的限制性明显限制了过继T细胞疗法的应用范围。影响过继转输T细胞的抗肿瘤疗效的另一个因素是肿瘤微环境(TME)的免疫抑制性，会影响过继T细胞的增殖，分化，细胞毒性和归巢(参见文献“Curr Opin Immunol.2016 Apr；39：1-6”)。
针对上述过继T细胞疗法所面临的问题，一个解决策略是利用溶瘤病毒作为载体来标记肿瘤细胞，使其表达可被过继T细胞特异性识别的靶抗原，并利用病毒自身的免疫原性来改善肿瘤微环境的免疫抑制性。溶瘤病毒感染肿瘤细胞后选择性在肿瘤细胞中进行复制，通过子病毒的大量增殖从而裂解肿瘤细胞达到特异性杀伤肿瘤细胞的作用。释放的子病毒又可以选择性感染并裂解其他肿瘤细胞从而最大程度地清除肿瘤组织(参见文献“Nat Biotechnol.2012 Jul 10；30(7):658-70”)。肿瘤细胞中异常的RAS、TP53、RB1、PTEN、WNT等信号传导途径影响了细胞自身的抗病毒机制，使得病毒在肿瘤细胞中更容易复制，是形成肿瘤选择性的主要原因(参见文献“Nat Rev Cancer.2017 11；17(11):633”)。除了本身的溶瘤作用外，溶瘤病毒还可以改变肿瘤组织的微环境，主要是通过诱导分泌细胞因子，吸引天然免疫细胞，释放肿瘤抗原，提供免疫危险信号等，从而增强肿瘤局部的抗肿瘤免疫反应(参见文献“J.Clin.Invest.2018；128,1258-1260”)。腺病毒作为载体是较早被研发的溶瘤病毒，基于E1B55K gene缺陷的Ad5型腺病毒H101是第一个批准上市的溶瘤病毒产品(参见文献“Hum Gene Ther.2018 Feb；29(2):151-159”)。腺 病毒的分子结构和生物特性的研究比较深入，使得腺病毒更容易通过基因工程改造成为溶瘤病毒。溶瘤病毒的基因组常被修饰以提高溶瘤病毒的肿瘤细胞的选择性。另外，利用肿瘤特异性基因启动子来驱动腺病毒复制所必需的基因也可以提供肿瘤的选择性。
然而，对溶瘤病毒基因组的修饰势必会影响病毒在细胞内的转录复制以及裂解细胞的能力，使得被病毒感染的肿瘤细胞不能被裂解。另外，溶瘤病毒完成复制周期需要依赖宿主肿瘤细胞的大量细胞组分的参与并受特有的分子机制调控(参见文献“J Virol.2008 Aug；82(15):7252-63”)，而每个肿瘤细胞的基因调控和蛋白表达各异，决定了不同生长状态和性质的肿瘤细胞被溶瘤病毒感染后，有些肿瘤细胞不能完成复制周期并裂解细胞(参见文献“Nat Rev Cancer.2002；2(12):938-950”)。另外，宿主产生的针对溶瘤病毒的中和抗体以及肿瘤组织对病毒颗粒扩散的限制都可降低溶瘤病毒的溶瘤作用。
因此，目前仍迫切需要提高过继转输T细胞的抗肿瘤疗效和溶瘤病毒的抗肿瘤疗效。
发明内容
为解决上述现有技术中所存在的问题，本发明提出，采用溶瘤病毒联合应用过继T细胞，以增强过继T细胞的杀瘤功能和适用范围，以及通过T细胞来清除不能完成病毒复制周期的肿瘤细胞。为此，本发明进一步提供能够在肿瘤细胞中有效表达被特异性T细胞所识别的靶抗原的溶瘤病毒，并能够联合应用过继TCR-T细胞疗法，以进一步增强实体肿瘤免疫治疗的疗效。
具体而言，为解决上述现有技术中所存在的问题，本发明提供了分离的用于表达外源基因的溶瘤腺病毒、载体、治疗剂及其用途。
具体而言，本发明提供了：
(1)一种分离的用于表达外源基因的溶瘤腺病毒，其中所述溶瘤腺病毒为对腺病毒进行基因改造而得到的选择复制型重组溶瘤腺病毒，该重组溶瘤腺病毒的基因组具有以下特征：
1)包含E1B基因调控元件，该元件包括E1B启动子和E1B与 pIX共用的多聚腺苷酸添加信号序列；
2)缺失了E1B基因编码区，并且，当需要插入所述外源基因时，在该E1B基因编码区位点插入所述外源基因，并且该外源基因位于所述E1B启动子之后，并受所述E1B基因调控元件的控制；
3)在所述外源基因的上游，包含转录E1A 13s mRNA的E1A的cDNA序列，并且该cDNA为野生型或Rb蛋白结合区域缺失型，该Rb蛋白结合区域缺失型为所述野生型cDNA去除了如SEQ ID NO.7所示的核苷酸序列，或该Rb蛋白结合区域缺失型编码突变的E1A蛋白，所述突变的E1A蛋白如SEQ ID NO.6所示。
(2)根据(1)所述的溶瘤腺病毒，其中所述E1B启动子的核苷酸序列如SEQ ID NO.1所示，所述E1B与pIX共用的多聚腺苷酸添加信号序列如aataaa所示。
(3)根据(1)所述的溶瘤腺病毒，其中所述E1B基因包括E1B-55K基因和E1B-19K基因。
(4)根据(1)所述的溶瘤腺病毒，其中所述E1B基因编码区的核苷酸序列如SEQ ID NO.3所示。
(5)根据(1)所述的溶瘤腺病毒，其中所述外源基因的起始位点包含Kozak序列，优选地，所述Kozak序列如SEQ ID NO.4所示。
(6)根据(1)所述的溶瘤腺病毒，其中所述野生型E1A cDNA的核苷酸序列如SEQ ID NO.5所示。
(7)根据(1)所述的溶瘤腺病毒，其中所述转录E1A 13s mRNA的E1A的cDNA位于所述E1B启动子的上游，并且与所述E1B启动子的核苷酸序列部分重合。
(8)根据(1)所述的溶瘤腺病毒，其中所述转录E1A 13s mRNA的E1A的cDNA序列在内源性E1A启动子/增强子的控制下，或者在外源启动子的控制下；优选地，所述内源性E1A启动子/增强子的核苷酸序列如SEQ ID NO.8所示。
(9)根据(1)所述的溶瘤腺病毒，其中所述转录E1A 13s mRNA的E1A的cDNA序列在外源启动子的控制下，所述重组溶瘤腺病毒的基因组中去除了如SEQ ID NO.9所示的核苷酸序列，并且在该去除 的位点处插入所述外源启动子核苷酸序列。
(10)根据(8)或(9)所述的溶瘤腺病毒，其中所述外源启动子包括EF-1α启动子、CMV启动子、PKG启动子、E2F启动子、AFP启动子和TERT启动子。
(11)根据(1)所述的溶瘤腺病毒，其中所述外源基因包括：HLA蛋白编码序列，标记性多肽编码序列，HLA蛋白编码序列及标记性多肽编码序列，HLA蛋白编码序列及β2-微球蛋白编码序列，或HLA蛋白编码序列、β2-微球蛋白编码序列及标记性多肽编码序列。
(12)根据(11)所述的溶瘤腺病毒，其中所述HLA蛋白包括HLA I类分子，该HLA I类分子包括HLA-A、HLA-B和HLA-C。
(13)根据(12)所述的溶瘤腺病毒，其中所述HLA-C包括野生型分子，或以下突变中的至少一种：1)第2位精氨酸突变为丙氨酸；2)编码该HLA-C蛋白的核苷酸序列的第4位核苷酸由C突变为G，第5位核苷酸由G突变为C；3)第362位异亮氨酸突变为苏氨酸；4)第359位谷氨酸突变为缬氨酸。
(14)根据(11)所述的溶瘤腺病毒，其中所述标记性多肽包括可操作地连接的、依次串联的以下氨基酸序列：N端信号肽的氨基酸序列、一个或多个抗原表位多肽的氨基酸序列、可任选的C端内质网滞留信号的氨基酸序列，其中当所述标记性多肽包括多个所述抗原表位多肽的氨基酸序列时，每两个相邻的所述抗原表位多肽的氨基酸序列之间由可切割性连接多肽的氨基酸序列连接；优选地，所述可切割性连接多肽是furin酶切识别多肽。
(15)根据(14)所述的溶瘤腺病毒，其中所述抗原表位多肽的氨基酸序列来源于自然界存在的蛋白的氨基酸序列，或者为人工合成的自然界不存在的氨基酸序列；优选地，所述自然界存在的蛋白包括人源蛋白和除人以外的其它物种的蛋白。
(16)根据(14)所述的溶瘤腺病毒，其中所述抗原表位多肽的氨基酸序列来源于肿瘤相关抗原或肿瘤特异性抗原的氨基酸序列。
(17)根据(16)所述的溶瘤腺病毒，其中所述肿瘤相关抗原选自NY-ESO-1 157-165、NY-ESO-1 1-11、NY-ESO-1 53-62、NY-ESO-1  18-27、Her2/neu 369-377、SSX-2 41-49、MAGE-A4 230-239、MAGE-A10 254-262、MAGE-C2 336-344、MAGE-C2 191-200、MAGE-C2 307-315、MAGE-C2 42-50、MAGE-A1 120-129、MAGE-A1 230-238、MAGE-A1 161-169、KK-LC-1 76-84、p53 99-107、PRAME 301-309、甲胎蛋白158-166、HPV16-E6 29-38、HPV16-E7 11-19、EBV-LMP1 51-59、EBV-LMP1 125-133、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：Q61R 55-64、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、TP53：V157G 156-164、TP53：R248Q 240-249、TP53：R248W 240-249、TP53：G245S 240-249、TP53：V157F 156-164、TP53：V157F 149-158、TP53：Y163C 156-164、TP53：R248Q 247-255、TP53：R248Q 245-254、TP53：R248W 245-254、TP53：G245S 245-254、TP53：G249S 245-254、TP53：Y220C 217-225、TP53：R175H 168-176、TP53：R248W 240-249、TP53：K132N 125-134、CDC73：Q254E 248-256、CYP2A6：N438Y 436-444、CTNNB1：T41A 41-49、CTNNB1：S45P 41-49、CTNNB1：T41A 34-43、CTNNB1：S37Y 30-39、CTNNB1：S33C 30-39、CTNNB1：S45P 40-49、EGFR：L858R 852-860、EGFR：T790M 790-799、PIK3CA：E542K 533-542、PIK3CA：H1047R 1046-1055、GNAS：R201H 197-205、CDK4：R24C 23-32、H3.3：K27M 26-35、BRAF：V600E 591-601、CHD4：K73Rfs 141-148、NRAS：Q61R 55-64、IDH1：R132H 126-135、TVP23C：C51Y 51-59、TVP23C：C51Y 42-51和TVP23C：C51Y 45-53。
(18)根据(14)所述的溶瘤腺病毒，其中所述抗原表位多肽为如SEQ ID NO.10所示的NY-ESO-1 157-165或如SEQ ID NO.11所示的KRAS：G12D 10-18。
(19)一种用于制备(1)-(18)中任一项所述的溶瘤腺病毒的载体，其中所述载体包含所述E1B基因调控元件、缺失了所述E1B 基因编码区、并且在所述外源基因的上游包含所述的转录E1A 13s mRNA的E1A的cDNA序列。
(20)一种用于治疗肿瘤和/或癌症的治疗剂，包含：
(a)第一组合物，其中该第一组合物包含位于第一可药用载体中的第一活性成分，该第一活性成分包括或含有用于导入肿瘤细胞和/或癌细胞的根据(1)-(18)中任一项所述的溶瘤腺病毒；和
(b)第二组合物，其中该第二组合物包含位于第二可药用载体中的第二活性成分，该第二活性成分包含T细胞受体修饰的免疫细胞。
(21)根据(20)所述的治疗剂，其中所述第一组合物和所述第二组合物各自独立地存在于所述治疗剂中而互不混合。
(22)根据(20)所述的治疗剂，其中所述免疫细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。
(23)根据(20)所述的治疗剂，其中所述第一组合物包含治疗有效量的所述溶瘤腺病毒。
(24)根据(20)所述的治疗剂，其中所述第二组合物包含治疗有效量的所述的T细胞受体修饰的免疫细胞。
(25)根据(20)所述的治疗剂，其中所述溶瘤腺病毒配制成通过瘤内注射给药、腹膜内给药、蛛网膜下腔内给药、或静脉给药。
(26)根据(20)所述的治疗剂，其中所述免疫细胞配制成通过动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
(27)根据(1)-(18)中任一项所述的溶瘤腺病毒在制备用于治疗肿瘤和/或癌症的药物中的用途。
(28)根据(27)所述的用途，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发 部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
(29)根据(20)-(26)中任一项所述的治疗剂在制备用于治疗肿瘤和/或癌症的药物中的用途。
(30)根据(29)所述的用途，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
(31)一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据(1)-(18)中任一项所述的溶瘤腺病毒。
(32)根据(31)所述的方法，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖 端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
(33)一种治疗肿瘤和/或癌症的方法，包括：
对肿瘤和/或癌症患者施用根据(20)-(26)中任一项所述的治疗剂中的第一组合物；和
对所述肿瘤和/或癌症患者施用根据(20)-(26)中任一项所述的治疗剂中的第二组合物。
(34)根据(33)所述的方法，包括以下依次进行的步骤：
1)对所述肿瘤和/或癌症患者施用所述第一组合物；和
2)在施用所述第一组合物之后，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物。
(35)根据(34)所述的方法，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
本发明与现有技术相比具有以下优点和积极效果：
本发明构建的用于表达外源基因的溶瘤腺病毒能够选择性地在肿瘤细胞和/或癌细胞中复制，并且更加高效地表达外源基因。具体地，本发明利用溶瘤病毒基因组中自身的E1B基因调控元件来调控 外源基因的表达，避免插入的外源基因调控元件对病毒基因组表达所造成的可能干扰，而影响病毒的有效复制，以及下游基因的表达。另外，也可以增加插入的外源基因片段的长度，使得溶瘤病毒载体可以携带更多外源基因负荷。另一方面，本发明构建的溶瘤腺病毒缺失了E1B基因编码区。E1B-19K蛋白可以抑制通过肿瘤坏死因子和FAS通路所诱导的细胞凋亡，使得感染的细胞耐受T细胞的杀伤作用。去除E1B-19K基因可以增加被感染的肿瘤细胞对杀伤性T细胞的敏感性。去除E1B-55K基因可以增加溶瘤腺病毒的肿瘤细胞溶瘤选择性。另外，本发明发现，在溶瘤腺病毒的基因组中，通过在外源基因的上游设计转录E1A-13S mRNA的E1A的cDNA序列而不是E1A基因组基因，使得本发明可以只转录E1A-13S而避免转录E1A-12S，从而增加外源基因的表达，以及加强病毒基因组的复制。
本发明构建的用于表达外源基因的溶瘤腺病毒通过巧妙的设计，比现有技术中类似的用于表达外源基因的溶瘤腺病毒能够更加有效地实现将具有标记性多肽编码序列的核酸导入肿瘤细胞和/或癌细胞，并更加高效地在肿瘤细胞和/或癌细胞内表达外源标记性多肽，尤其来源于基因突变的新生抗原(neo-antigen)的表位多肽，使其进入MHC I类抗原提呈途径，肿瘤细胞表面HLA/抗原表位多肽复合物的表达量得到了显著增加，由此进一步增强了所述T细胞受体修饰的免疫细胞对肿瘤细胞和/或癌细胞的识别敏感性。另外，通过融瘤病毒载体携带的异种HLA I类分子(allo-HLA class I)来标记肿瘤细胞，也大大增加了过继T细胞疗法的适用范围。
本发明所构建的用于表达外源基因的溶瘤腺病毒可更加高效地在肿瘤细胞内表达外源抗原表位肽、β2-微球蛋白和/或外源MHC I类分子，以显著加强肿瘤细胞表面的抗原表位肽/MHC I类分子复合物的数量，并且可以采用特异性针对该抗原表位肽的TCR修饰的免疫细胞进行联合治疗。当通过所述溶瘤腺病毒作为载体介导外源抗原表位肽、β2-微球蛋白和/或外源MHC I类分子在肿瘤细胞内的表达时，溶瘤病毒对肿瘤的杀伤与TCR修饰的免疫细胞对肿瘤的杀伤还能够产生协同治疗效果。
本发明通过所构建的溶瘤腺病毒将标记性多肽、β2-微球蛋白和/或HLA蛋白编码核酸导入肿瘤细胞和/或癌细胞，在发挥溶瘤病毒杀伤肿瘤细胞和/或癌细胞的作用的同时，进一步增强了上述显著加强外源抗原表位肽在肿瘤细胞表面的提呈与T细胞受体修饰的免疫细胞联合所达到的协同治疗效果。另外，溶瘤病毒在杀伤肿瘤的同时还可以缓解肿瘤微环境的免疫抑制状态，提高T细胞受体修饰的免疫细胞的归巢；此外，T细胞受体修饰的免疫细胞还可以有效清除被溶瘤病毒感染后，不能完成复制周期并产生足够数量的子病毒而发生裂解的肿瘤细胞；由此实现了进一步的协同作用。此外，被溶瘤病毒裂解的肿瘤细胞所释放的抗原可进一步激活机体自身的抗肿瘤免疫，从而可实现比单独使用溶瘤病毒或T细胞受体修饰的免疫细胞更好的肿瘤杀伤效果，实现了协同治疗效果。
定义
在本发明中，词语“肿瘤”、“癌症”、“肿瘤细胞”、“癌细胞”涵盖本领域通常认为的含义。
本文所用的词语“溶瘤病毒”是指能够选择性地在肿瘤细胞中复制并裂解肿瘤细胞的病毒。
本文所用的词语“治疗有效量”是指功能药剂或药物组合物能够表现出可检测的治疗效果或抑制效果的量，或者起到抗肿瘤效果的量。所述效果可以通过本领域任何已知的检验方法检测。
本文所用的词语“给药”或“施用”是指向受试者提供化合物、复合物或组合物(包括病毒和细胞)。
本文所用的词语“患者”是指人或非人类生物。因此，本文所述的方法和组合物适用于人类疾病和兽类疾病。在一些实施方案中，患者患有肿瘤。在一些例子中，患者同时患有一种或多种类型的癌症。
本文所用的词语“协同效果”是指两种或多种药剂共同起到的效果，该效果大于其中各药剂的单独效果的总和。
根据本发明，“异常表达”或“异常表达”是指与非致瘤性正常细胞或健康个体(即，没有疾病的个体)相比，表达被改变，优选 增加与某些蛋白质，例如肿瘤相关抗原的异常或异常表达有关。表达的增加是指增加至少10％，特别是至少20％，至少50％或至少100％或更多。在一个实施方案中，仅在患病组织中发现表达，而在健康组织中表达被抑制。
如本文所用，术语“大约”是指在20％之内，或在某些情况下在10％之内，或在某些情况下在5％之内，或在某些情况下在1％之内，或在某些情况下在0.1之内给定值或范围的％，因为这样的变化适合于执行所公开的方法或适合于所公开的组合物的预期目的。
术语“活性成分”是指药物产品组合物中具有生物活性或具有预期药物作用的成分。
术语“过继细胞转移”或“过继细胞疗法”或“ACT”是指这样的免疫疗法，其中收集受试者或患者自身的免疫细胞(例如自体T细胞)或来自健康供体的免疫细胞(例如同种异体T细胞)，以治疗其癌症。TCR-T细胞疗法是ACT的一种。
本文中所使用的术语“和/或”包括“和”，“或”和“由所述术语连接的元件的所有或任何其他组合”的含义。
术语“抗肿瘤”是指可以通过各种方式表现的生物学效应，包括但不限于例如肿瘤体积的减少，肿瘤细胞数量的减少，肿瘤细胞转移的数量的减少，预期寿命的增加，肿瘤细胞增殖的减少，肿瘤细胞存活率的降低或与癌症状况相关的各种生理症状的改善。“抗肿瘤作用”也可以通过本发明的肽，多核苷酸，细胞和抗体首先预防肿瘤发生的能力来表现。
如本文所用，术语“抗体”是指衍生自与抗原特异性结合的免疫球蛋白分子的蛋白质或多肽序列。抗体可以是多克隆或单克隆，多链或单链或完整的免疫球蛋白，并且可以衍生自天然来源或重组来源。抗体可以是免疫球蛋白分子的四聚体。
如本文所用，术语“抗原”或“Ag”被定义为引起免疫应答的分子。这种免疫应答可能涉及抗体的产生，或特定免疫学上具有免疫力的细胞的激活，或两者都涉及。技术人员将理解，任何大分子，包括实际上所有的蛋白质或肽，都可以用作抗原。此外，抗原可以衍生 自重组DNA或基因组DNA。本领域技术人员将理解，包含编码引发免疫应答的蛋白质的核苷酸序列或部分核苷酸序列的任何DNA均编码本文所用术语“抗原”。此外，本领域技术人员将理解抗原不必仅由基因的全长核苷酸序列编码。显而易见，本发明包括但不限于使用一个以上基因的部分核苷酸序列，并且这些核苷酸序列以各种组合排列以编码引发所需免疫应答的多肽。此外，技术人员将理解，抗原根本不需要由“基因”编码。显而易见的是，抗原可以合成产生或可以源自生物样品，或者可以是多肽以外的大分子。这样的生物样品可以包括但不限于组织样品，肿瘤样品，细胞或具有其他生物成分的流体。
术语“抗原提呈机制”(或“抗原呈递机制”)是指加工和制备抗原以呈递给T淋巴细胞的免疫分子或细胞。抗原提呈机制涉及两种不同的途径，用于处理来自生物体自身(自身)蛋白质或细胞内病原体(例如病毒)或吞噬细胞的病原体(例如细菌)的抗原；这些抗原在I类或I类主要组织相容性复合物(MHC)分子上的后续呈递取决于所使用的途径。MHC I类和II类都必须先结合抗原，然后才能在细胞表面稳定表达。MHC I抗原提呈通常涉及抗原加工的内源性途径，而MHC II抗原提呈涉及抗原加工的外源性途径。
术语“自体的”是指源自同一个体的任何材料，随后将其重新引入该个体。
术语“β2-微球蛋白”或“β 2-微球蛋白”是MHC I类分子的组分，MHC I类分子具有存在于所有有核细胞(不包括红细胞)上的α1，α2和α3蛋白。在人类中，β2微球蛋白由B2M基因编码。
术语“C末端”(也称为C末端，羧基末端，羧基末端，C末端尾部，C末端或COOH末端)是指氨基酸链的末端(蛋白质或多肽)，以游离羧基(-COOH)终止。当蛋白质从信使RNA翻译时，是从N端到C端产生的。编写肽序列的惯例是将C-在右侧的末端插入从N端到C端的序列。
如本文所用，术语“组合”是指各种组分(例如溶瘤病毒和一种或多种在抗癌疗法中有效的物质)可能的任何布置。这样的布置包括所述组分的混合物以及用于同时或顺序施用的单独的组合。本发明 包括具有不同有效剂量的组合。应当理解，组合的每种成分的最佳剂量可以由本领域技术人员确定。
如本文所用，术语“组合物”或“药物组合物”是指适合于以预期的药物作用(例如，预防和治疗作用)施用于受试者或患者的化学和/或生物学组合物。适合于此类治疗应用的组合物的实例包括用于肠胃外，皮下，经皮，皮内，肌内，冠状内，心肌内，脑内，肿瘤内，腹膜内，静脉内(例如可注射)或气管内给药的制剂，例如无菌混悬剂，乳剂和气溶胶。气管内给药可涉及使肺组织(例如肺泡)接触或暴露于治疗剂，所述治疗剂包含处于药物载体中的治疗有效量的核酸和/或免疫细胞(例如T细胞(例如TCR修饰的T细胞))。在一些情况下，适合于治疗应用的药物组合物可以与一种或多种药学上可接受的赋形剂，稀释剂或载体(如无菌水，生理盐水，葡萄糖等)混合。
如本文所用，当用于定义产品，组合物和方法时，术语“包括”，“具有”，“包含”或“含有”是开放式的，不排除其他未引用的元素或方法步骤。因此，当氨基酸序列可能是多肽的最终氨基酸序列的一部分时，该多肽“包含”该氨基酸序列。这样的多肽可以具有多达数百个额外的氨基酸残基。“基本上由...组成”是指排除任何具有重要意义的其他组分或步骤。因此，基本上由所述组分组成的组合物将不排除痕量污染物和药学上可接受的载体。当存在这样的氨基酸序列时，该多肽“基本上由”氨基酸序列组成，最终仅具有几个额外的氨基酸残基。“由...组成”是指排除其他组分的痕量元素或步骤。例如，当多肽除列举的氨基酸序列之外不包含其它任何氨基酸时，该多肽“由”氨基酸序列组成。
术语“有条件复制能力的病毒”或“有条件复制病毒”或“CRV”或“选择复制型病毒”是指病毒被设计为能够在肿瘤细胞中选择性复制，导致其破坏，同时保留正常细胞。
术语“组成型启动子”是指核苷酸序列，当其与编码或指定基因产物的多核苷酸可操作地连接时，导致在细胞的大多数或全部生理条件下在细胞中产生该基因产物。
术语“死亡受体”是指肿瘤坏死因子受体超家族的成员，其特征在于称为“死亡域”的细胞质区域，当与同源配体结合时，该细胞质区域使受体能够启动细胞毒性信号。
术语“获自”，“起源自”或“衍生自”用于鉴定组分(例如多肽，核酸分子，氨基酸序列)的原始来源，但无意限制制备组分的方法，该方法可以是例如通过化学合成或重组手段的方法。
术语“可药用的”或“药学上可接受的”是指适合用于给予患者或受试者以达到预期的药物或药物作用而没有不适当的不利副作用(例如毒性，刺激和过敏)，例如，具有合理的收益/风险比。
如本文所用，“可药用载体”或“药学上可接受的媒介物”或“药学上可接受的载体”是指用于给予治疗剂的媒介物或载体，所述治疗剂适合于人和/或哺乳动物而没有过度的不利副作用((例如毒性，刺激性和过敏性)，并具有合理的收益/风险比。如本文所用，“可药用载体”包括可用于递送和/或表达核酸的任何媒介物或载体，包括纳米颗粒，脂质，质粒，病毒或细胞。
当应用于多核苷酸时，术语“编码”是指如果在其天然状态或通过本领域技术人员公知的方法操作时可以“编码”多肽的多核苷酸，其可被转录和/或翻译以产生用于多肽和/或其片段的mRNA。反义链是这种核酸的互补物，并且可以从其推导编码序列。“编码”肽的核酸或核酸序列是指包含该肽的编码序列的核酸。“编码”肽的氨基酸序列是指含有肽序列的氨基酸序列。
术语“内质网保留信号序列”(“内质网滞留信号序列”)是指在折叠成ER驻留蛋白后使蛋白质保留在内质网或ER中的信号序列。经典的ER保留信号是C端KDEL(Lys-Asp-Glu-Leu)序列。
术语“抗原表位”，“表位”或“抗原决定簇”是指被免疫系统(特别是抗体，B细胞或T细胞)识别并结合的抗原部分。术语“表位肽”或“抗原表位肽”是指呈肽形式的表位或抗原表位。
当在本文中使用时，术语“赋形剂”或“添加剂”旨在表示药物制剂中不是活性成分的所有物质，例如载体(例如载体DNA，质粒，载体病毒)，结合剂，润滑剂，增稠剂，表面活性剂，防腐剂， 乳化剂，缓冲剂，调味剂或着色剂。
术语“外源的”是指从生物，细胞，组织或系统引入或在生物，细胞，组织或系统外部产生的任何材料。例如，本文使用的术语“外源HLA蛋白”是指来自受试者或患者外部的HLA蛋白，并且“外源HLA蛋白”可以由受试者或患者的细胞或组织产生或可以不产生。
术语“内源的”是指来自生物体，细胞，组织或系统内部或在其内部产生的任何材料。
如本文所用，“表达”是指多核苷酸被转录成mRNA的过程和/或转录的mRNA随后被翻译成肽，多肽或蛋白质的过程。如果多核苷酸衍生自基因组DNA，则表达可包括在真核细胞中剪接mRNA。
如本文所用，“基因表达”是指将基因中的遗传信息，DNA碱基对的序列制成功能性基因产物(例如蛋白质或RNA)的过程。基本过程是将DNA转录为RNA，然后将RNA翻译为蛋白质。
术语“人类白细胞抗原(HLA)”是指编码人类主要组织相容性复合物(MHC)蛋白的基因复合物或系统，也称为“HLA蛋白”。MHC蛋白是负责调节人体免疫系统的细胞表面蛋白。HLA基因复合体位于6p21号染色体上3Mbp的片段上，具有高度多态性，这意味着它们具有许多不同的等位基因，从而可以微调适应性免疫系统。HLA对应MHC I类(A，B和C)，从细胞内部到T淋巴细胞都提呈外源抗原(例如病毒抗原)，HLA I类/抗原肽复合物可以刺激细胞毒性T细胞(也称为CTL)，从而反过来杀死靶细胞。
术语“HLA I类蛋白”是指人MHC I类蛋白或分子。MHC I类分子是跨膜蛋白，由单个α链组成，并与β2-微球蛋白缔合，可正确折叠并运输到细胞表面。
术语“免疫细胞”是指免疫系统的细胞，其可以被分类为淋巴细胞(T细胞，B细胞和NK细胞)，嗜中性粒细胞和单核细胞/巨噬细胞。这些是所有类型的白细胞。
术语“免疫危险信号”是指当组织细胞由于损伤，感染等而陷入困境时，它们开始在其表面上分泌或表达表示“危险”的分子，或者入侵生物的成分(例如，病毒DNA或RNA)也被免疫系统敏化为 危险信号。
术语“免疫原”是指能够引发免疫应答的特定类型的抗原。
术语“肿瘤微环境(TME)”或“免疫抑制性肿瘤微环境”是指肿瘤周围的环境，包括周围的血管，免疫细胞，成纤维细胞，信号分子和细胞外基质(ECM)。肿瘤与周围的微环境密切相关并不断相互作用。肿瘤可以通过释放细胞外信号来抑制免疫反应，促进肿瘤血管生成并诱导外周免疫耐受，从而影响微环境，而微环境中的免疫细胞可以影响癌细胞的生长和进化。
诸如“增加”或“增强”的术语优选地涉及增加或增强约至少5％，优选至少10％，优选至少20％，优选至少30％，更优选至少40％，更优选至少50％，甚至更优选至少80％，还优选至少100％。这些术语也可能与以下情况有关，其中在零时没有某种化合物或条件的可检测信号，而在特定时间点晚于零时有某种化合物或条件的可检测信号。
术语“诱导型启动子”是指核苷酸序列，当其与编码或指定基因产物的多核苷酸可操作地连接时，基本上仅当对应于启动子的诱导剂存在于细胞时才导致在细胞中产生基因产物。
如本文所用，术语“分离的”是指从其天然环境中移去的细胞，蛋白质，多肽，肽，多核苷酸，载体等(即与至少一种其他组分分离，该其他组分与它是自然关联或在自然界中发现的)。
慢病毒载体是一种逆转录病毒，其可以感染分裂细胞和非分裂细胞，因为它们的整合前复合物(病毒“壳”)可以穿过靶细胞核的完整膜。慢病毒载体衍生自人免疫缺陷病毒。
如本文所用，“接头肽”或“接头序列”是指连接两个其他氨基酸序列的氨基酸序列。例如，HLA I类蛋白的一部分可以通过接头序列与一部分肿瘤相关抗原序列，例如表位序列连接。
术语“杂合性丧失”或“LOH”是指一个亲本对细胞的贡献的丧失，可能是由于直接缺失，由于不平衡的重排，基因转化，有丝分裂重组α或染色体丢失(单染色体)引起的。
术语“主要组织相容性复合物”或“MHC”是指一组基因，其 编码对于获得的免疫系统识别脊椎动物中的外来分子而言必不可少的细胞表面蛋白，这反过来决定了组织相容性。MHC分子与病原体产生的抗原结合并在细胞表面展示，以被适当的T细胞识别，MHC分子介导白细胞(WBC)与其他白细胞的相互作用。MHC决定供体器官移植的相容性，以及通过交叉反应免疫对自身免疫疾病的敏感性，人类MHC也称为HLA(人类白细胞抗原)复合物(通常简称HLA)。MHC是使免疫系统(更具体而言是T细胞)结合，识别和耐受自身(自动识别)的组织抗原。MHC还是作为潜在的外源抗原与MHC结合并提呈给T细胞受体(TCR)的细胞内肽的伴侣分子。MHC与TCR及其共受体相互作用，以从抗原结合亲和力和特异性以及信号转导效率的角度优化TCR-抗原相互作用的结合条件。
术语“MHC I类蛋白”或“MHC I类分子”是指主要的组织相容性复合体(MHC)分子或糖蛋白或蛋白质的两个主要类别之一(另一类为MHC II类)，存在于脊椎动物体内所有有核细胞的细胞表面。MHC I蛋白在机体大多数有核细胞上形成功能性受体。主要的组织相容性复合体(MHC)I类分子负责将肽表位呈递给细胞毒性T细胞。在人类中，人类白细胞抗原(HLA)系统是编码I类和MHC类MHC分子的基因位点。HLA-A，-B和-C基因编码MHC I类(MHCI)蛋白。通常长度为8-11个氨基酸的肽将通过与位于反平行β折叠上方的两个α螺旋形成的凹槽相互作用而结合MHC I分子。肽-MHC I类(pMHCI)分子的加工和呈递涉及一系列连续阶段，包括：a)蛋白酶介导的蛋白质消化；b)由与抗原加工(TAP)相关的转运蛋白介导的肽转运进入内质网(ER)；c)使用新合成的MHC I分子形成pMHCI；d)将pMHCI运输到细胞表面。在细胞表面，pMHCI将通过T细胞受体(TCR)与细胞毒性T细胞相互作用。在复杂的pMHCI-TCR相互作用之后，非自体抗原的鉴定可能通过一系列由相关酶，共受体，衔接子分子和转录因子介导的生化事件导致细胞毒性T细胞活化。活化的细胞毒性T细胞将增殖，以产生大量的效应T细胞，这些T细胞表达对所鉴定的免疫原性肽表位具有特异性的TCR。对鉴定出的非自身表位具有TCR特异性的T细胞的扩增会导 致免疫介导的细胞凋亡，从而显示出活化的非自身表位。
术语“MHC蛋白”是指由MHC基因编码的蛋白。
免疫抑制肿瘤微环境(TME)：肿瘤促进耐受性微环境的能力以及多种免疫抑制机制的激活，这些机制可能共同作用以抵消有效的免疫反应，例如肿瘤引起的抗原提呈受损，负性共刺激信号的激活以及复杂化免疫抑制因子。
术语“突变体”或“突变体类型”是指由突变实例产生或产生的菌株，基因或特征，其通常是生物体的基因组或染色体的DNA序列的改变。
术语“N-末端信号肽”是指存在于大多数新合成的蛋白质的N末端的信号肽(通常长16-30个氨基酸)，这些蛋白质预定朝向分泌途径。这些蛋白质包括驻留在某些细胞器(内质网，高尔基体或内体)中，从细胞分泌或插入大多数细胞膜中的蛋白质。信号肽的功能是促使细胞将蛋白质易位，通常转移至细胞膜。
术语“新抗原”是指以前未被免疫系统识别的新形成的抗原。例如，新抗原可以源自由于肿瘤基因改变而形成的改变的肿瘤蛋白，包括点突变，插入/缺失，扩增/融合，翻译后修饰，或源自病毒蛋白。
如本文所用，“核酸”与术语“多核苷酸”可互换，并且通常是指任何多核糖核苷酸或多脱氧核糖核苷酸，其可以是未修饰的RNA或DNA或修饰的RNA或DNA或任何它们的组合。“核酸”包括但不限于单链和双链核酸。如本文所用，术语“核酸”还包括含有一个或多个修饰碱基的如上所述的DNA或RNA。因此，具有因稳定性或其他原因而修饰的主链的DNA或RNA是“核酸”。本文所用的术语“核酸”包括核酸的这种化学修饰、酶促修饰或代谢修饰形式，以及病毒和细胞(包括例如简单和复杂细胞)特有的DNA和RNA的化学形式。“核酸”或“核酸序列”还可包括单链或双链RNA或DNA或任何组合的区域。
如本文所用，术语“编码核酸”，“编码核酸分子”，“DNA序列编码”和“DNA编码”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或序列。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋 白质)链的氨基酸顺序。因此，核酸序列编码氨基酸序列。
如本文所用，术语“溶瘤病毒”是指能够在体外或体内，在肿瘤细胞(例如，诸如癌细胞的增殖性细胞)中选择性复制以减慢所述分裂细胞的生长和/或裂解的病毒，而在正常细胞中则没有或只有很少的复制。通常，溶瘤病毒包含包装到病毒颗粒(或病毒粒子)中的病毒基因组并且具有感染性(即能够感染并进入宿主细胞或受试者)。如本文所用，该术语涵盖DNA或RNA载体(取决于所讨论的病毒)以及由其产生的病毒颗粒。
术语“一个或多个”是指一个或大于一个的数字(例如2、3、4、5等)。
短语“可操作地连接”或“可操作地连接”是指两个或更多个多核苷酸(例如，DNA)区段之间的功能关系。通常，它是指转录调节序列与转录序列的功能关系。例如，如果启动子或增强子序列在合适的宿主细胞或其他表达系统中刺激或调节编码序列的转录，则将其与编码序列可操作地连接。通常，与转录序列可操作连接的启动子转录调控序列在物理上与该转录序列邻接，即它们是顺式作用的。但是，某些转录调控序列，例如增强子，在物理上不必是连续的或位于其增强转录的编码序列的附近。
术语“多肽”，“肽”和“蛋白质”是指氨基酸残基的聚合物，其包含通过肽键结合的多个氨基酸。该聚合物可以是直链，支链或环状的，并且可以包含天然存在的和/或氨基酸类似物，并且可以被非氨基酸中断。
术语“质粒”是指在细胞中发现的“额外的”自我复制遗传元件。质粒用于基因工程中以产生重组DNA，并作为在生物体之间转移基因的机制。
点突变或取代是遗传突变，其中单个核苷酸碱基从DNA或RNA序列中被改变，插入或缺失。
术语“多聚腺苷酸添加信号”或“多聚腺苷酸添加信号序列”或“多聚腺苷酸化信号”或“多聚腺苷酸化信号序列”或“多聚腺苷酸信号”或“多聚腺苷酸信号序列”是指由RNA切割复合物识别的 序列基序，其在真核生物组之间变化。大多数人聚腺苷酸化信号包含AAUAAA序列。
如本文所用，术语“预防”是指旨在抑制不希望的生理变化或病症或病状发展的预防或预防措施。预防疾病或病状可以包括在疾病或病状(或其症状)出现或存在之前的时间开始施用根据本文提供的方法获得的T细胞，使得不会出现疾病或病状，或其病理特征，后果或不良影响。
术语“启动子”是指被细胞的合成机制或引入的合成机制识别的，启动多核苷酸序列的特异性转录所需的DNA序列。
如本文所用，术语“受体”是指在细胞表面表达的分子，其中所述分子能够结合细胞配体。当合适的配体与受体结合时，本文所用的受体-配体结合优选能够初始化或抑制生化途径和/或信号级联。
术语“重组”是指涉及或表示使用重组技术形成的生物，细胞，蛋白质，遗传材料，DNA或RNA。
术语“重组DNA”是指通过组合来自两种或更多种来源的DNA制成的DNA或rDNA。使用限制性内切酶(也称为限制性内切核酸酶)将DNA片段从染色体的正常位置切下，然后使用称为连接酶的酶将其插入其他染色体或DNA分子中。
术语“重组TCR”是指通过重组技术制备的TCR。
如本文所用，术语“具有复制能力”或“具有复制能力的病毒”是指具有复制能力的病毒或其复制取决于癌细胞中的因子(例如上调因子)的病毒。
在本说明书的上下文中，术语“具有复制能力的病毒”是指具有在体外和体内在细胞中复制的所有必需机制的病毒，即无需包装细胞系的辅助。在本文中，能够在互补包装细胞系中复制的病毒载体(例如至少在E1A区域中缺失的病毒载体)，不是具有复制能力的病毒。
在本说明书的上下文中，术语“复制缺陷型病毒”是指需要包装细胞系(包含转基因)复制的病毒。
术语“逆转录病毒载体”是指可以容纳目的基因以允许将两者掺入靶细胞的前病毒序列。载体还可以包含病毒和细胞基因启动子， 例如CMV启动子，以增强目的基因在靶细胞中的表达。
术语“自切割接头肽”是指存在于蛋白质(例如重组蛋白)中的短肽(例如18-22个氨基酸长的肽)，在细胞中其可以触发蛋白质的自切割。裂解在蛋白质翻译后开始。自切割接头肽的确切分子机制仍不确定。然而，据信其涉及甘氨酰脯氨酰肽键形成的核糖体“跳跃”而不是真正的蛋白水解切割。
自灭活慢病毒载体已用于将基因导入成熟的T细胞中，以通过递送嵌合抗原受体(CARs)或克隆的T细胞受体产生对癌症的免疫力。
术语“自身蛋白质”是指通常由特定生物体产生的蛋白质。特定生物体的免疫系统应能耐受自身蛋白质。如果没有，则有自身免疫。
如本文所用，术语“沉默子”是指能够结合转录调节因子的DNA序列，称为阻遏物。DNA包含提供产生信使RNA(mRNA)模板的基因。然后将该mRNA翻译成蛋白质。当阻遏蛋白结合至DNA的沉默子区域时，可防止RNA聚合酶将RNA序列转录为RNA。由于转录受阻，因此无法将RNA翻译成蛋白质。因此，沉默子阻止基因被表达为蛋白质。
术语“实体瘤”是指通常不包含囊肿或液体区域的组织或肿瘤的异常块。实体瘤可能是良性的(非癌性)或恶性的(癌性)。
如本文所用，术语“受试者”或“患者”可互换使用，并且可涵盖任何脊椎动物，包括但不限于人，哺乳动物，爬行动物，两栖动物和鱼类。然而，有利地，受试者或患者是诸如人的哺乳动物，或诸如驯养的哺乳动物(例如狗，猫，马等)或家畜(例如牛，绵羊，猪等)的哺乳动物。在示例性实施方式中，受试者是人。如本文所用，短语“需要”表示受试者的状态，其中需要治疗或预防措施。这样的状态可以包括但不限于患有诸如癌症的疾病或状况的受试者。
术语“自杀基因”是指编码能够将药物前体转化为细胞毒性化合物的蛋白质的基因。自杀基因包括但不限于编码具有胞嘧啶脱氨酶活性，胸苷激酶活性，尿嘧啶磷酸核糖基转移酶活性，嘌呤核苷磷酸化酶活性和胸苷酸激酶活性的蛋白质的基因。下表中公开了自杀基因 和包含一个核碱基部分的药物的相应前体的实例。
术语“表面表达”是指目的蛋白与宿主细胞的天然表面蛋白的融合体。这导致重组蛋白被运输到宿主表面并随后展示在宿主表面上。
术语“T细胞”是指在胸腺中发育的一种淋巴细胞(因此得名)，并在免疫应答中起关键作用。通过T细胞受体在细胞表面上的存在，可以将T细胞与其他淋巴细胞区分开。“细胞毒性T细胞或CD8+T细胞或杀伤细胞能够直接杀死被病毒感染的细胞以及癌细胞。CD8+T细胞还能够利用称为细胞因子的小信号蛋白在免疫接种时募集其他细胞。辅助性T细胞或CD4+T细胞通过间接杀死被识别为外来的细胞而发挥功能：它们确定免疫系统的其他部分是否以及如何响应特定的感知威胁。
短语“串联”是指两个或多个实体(例如多核苷酸(例如DNA)和多肽)之间的空间关系，其排列方式使得它们一个接一个地放置。
如本文所用，术语“靶标”是指免疫应答所针对的分子(例如蛋白质或肽)，细胞或组织或生物。
术语“靶抗原”是指期望针对其产生免疫应答的任何物质，但是通常，靶抗原是蛋白质或肽。靶抗原可以包含诱导免疫应答的全长蛋白质或其片段(即，免疫原性片段)。可以修饰靶抗原或其片段，例如以降低靶抗原的一种或多种生物学活性或增强其免疫原性。
术语“靶细胞”是指期望对其产生免疫应答或可以被免疫细胞例如(T细胞)特异性识别的任何细胞。
如本文所用，术语“T细胞受体”或“TCR”是指在T细胞或T淋巴细胞表面上发现的分子，其负责将抗原片段识别为与主要组织相容性复合物(MHC)分子结合的肽。
除非另有说明，否则根据常规用法使用本文的技术术语。分子生物学中常用术语的定义可以在以下文献中找到：本杰明·莱温的《基因VII》，牛津大学出版社出版，1999年；Kendrew等编辑，《分子生物学百科全书》，Blackwell Science Ltd.出版，1994年；和罗伯特·迈耶斯(Robert A.Meyers)编辑，《分子生物学和生物技术：综合参 考书》，由VCH Publishers，Inc.出版，1995年；和其他类似参考。
本文所用的术语“治疗的”是指治疗。通过减少，抑制，缓解，减轻，预防或根除疾病状态可获得治疗效果。
如本文所用，术语“治疗有效剂量”或“治疗有效量”或“有效量”或“有效剂量”是指单独或与其他剂量组合导致所需反应或在治疗特定疾病或特定病症的情况下，期望的反应与疾病进展的抑制有关，这可能包括疾病进展的减速，特别是对疾病进展的破坏。治疗疾病或病症的所需反应还可以是延迟疾病或病症的发生或抑制疾病或病症的发生。有效量的本发明组合物取决于状况或疾病，疾病的严重程度，患者的各个参数，包括年龄，生理状况，身高和体重，持续时间治疗，可选的伴随疗法的类型，具体的给药途径和类似因素。如果患者对初始剂量的反应不充分，则可以采用多剂量或更高剂量(或更高的有效剂量，可以通过更局限的给药途径实现)。
术语“疗法”(以及疗法的任何形式，例如“治疗”)是指治疗和预防或预防措施，其目的是预防或减缓(减轻)不希望的生理变化或病理性疾病。治疗癌症可包括但不限于减轻一种或多种临床适应症，降低肿瘤生长或肿瘤细胞增殖，降低一种或多种癌症病症的临床适应症的严重性，减轻病症的程度，稳定受试者的疾病状态(即不恶化)，延迟或减慢，停止或逆转癌症进展，并实现部分或完全缓解。如果与根据标准医学实践(而不结合根据本文提供的方法获得的T细胞)进行治疗的预后相比，治疗癌症还包括将生存期延长几天，几周，几个月或几年。需要治疗的受试者可以包括已经患有或被诊断出患有癌症的受试者，以及易于，可能发展或怀疑患有癌症(例如淋巴瘤或多发性骨髓瘤)或感染的受试者。
术语“肿瘤抗原”是指在肿瘤细胞中产生的抗原性物质，即它在宿主中触发免疫反应。肿瘤抗原是用于通过诊断测试鉴定肿瘤细胞的有用肿瘤标志物，并且是用于癌症治疗的潜在候选物。
术语“肿瘤相关抗原”是指存在于一些肿瘤细胞以及某些正常细胞上的抗原。
“肿瘤浸润淋巴细胞(TIL)”是指已经浸润了受试者肿瘤的受 试者自身的天然T细胞。可以将其收获，激活，扩展和重新引入(例如重新输注)到受试者中，在那里其可以寻找并消灭肿瘤，作为癌症或肿瘤治疗的一部分。
术语“肿瘤特异性抗原”是指存在于肿瘤细胞上而不存在于任何其他细胞上的抗原。
术语“载体”是指能够转运已与其连接的另一核酸的核酸分子。
术语“表达载体”包括任何载体(例如质粒，粘粒或噬菌体染色体)，其包含基因构建体，其形式适合于细胞表达(例如，与启动子连接)。
在本说明书中，“质粒”和“载体”可互换使用，因为质粒是载体的常用形式。此外，本发明旨在包括具有等效功能的其他载体。
术语“病毒载体”定义为病毒或病毒颗粒，其包含待体内，离体或体外递送至宿主细胞的多核苷酸。病毒载体的实例包括逆转录病毒载体，慢病毒载体，腺病毒载体，腺伴随病毒载体，α病毒载体等。还开发了α病毒载体，例如基于塞姆利基森林病毒的载体和基于Sindbis病毒的载体，用于基因治疗和免疫治疗。参见文献：“Schlesinger and Dubensky(1999)Cur.Opin.Biotechnol.5:434-439”；以及“Ying,et al.(1999)Nat.Med.5(7):823-827”。
术语“vp/天”是指每天的病毒颗粒。
术语“野生的”或“野生型”是指与非典型突变体类型不同的，在自然条件下普遍存在的品系，基因或特征。
附图说明
图1A显示了本文所述的重组溶瘤腺病毒构建体的示意图。pAdEasy-NY-A2(也即pAdEasy-EF1α-NY-A2)代表复制缺陷型腺病毒载体，其表达所述标记性多肽和HLA-A2蛋白，该标记性多肽包含NY-ESO-1 157-165表位肽。该构建体的主链衍生自具有E1区缺失(E1del)和E3区缺失(E3 del)的5型腺病毒基因组DNA。在E1A区域中，结合了表达单元。该表达单元包含标记性多肽编码序列和HLA-A2蛋白，该标记性多肽编码序列包括INSL5信号肽(INSL5 SP)，通过Furin蛋白酶切割位点连接的三个NY-ESO-1 157-165表 位肽(NY表位x3)，接头肽(Furin-F2A)，表达单元的两侧是EF1α启动子(EF1αpro)和SV40poly(A)信号序列(SV40pA)。LITR和RITR分别代表左端反向末端重复序列(ITR)和右端反向末端重复序列。pAd-EF1α-E1A-A2-F2A-NY(也即pAd-EF1α-E1Ad24-A2-F2A-NY)和pAd-EF1α-E1A-A2-F2A-BM(也即pAd-EF1α-E1Ad24-A2-F2A-BM)代表有条件复制能力的腺病毒载体，分别包含HLA-A2基因和编码所述含有NY-ESO-1 157-165表位肽的标记性多肽，或带有人β2-微球蛋白基因。这些构建体的骨架是带有E1和E3区缺失的5型腺病毒的基因组DNA。在E1A区域并入具有122-129缺失的突变E1A基因(E1A del24)，并在两侧分别具有EF-1α启动子和内源E1A多聚A信号(E1A pA)。编码HLA-A2和带有ER保留信号(KDEL)的标记性多肽的表达单元的两侧是5'端内源性E1B启动子(E1B pro)和3'端pIX基因区域，包括内源性E1B/IX poly A信号序列(E1B/IX pA)。另一方面，E1B基因区域被删除。
图1B显示了用于表达TCR的慢病毒载体的示意图。pCDH-EF1α-TCR-NY代表一种基于HIV的慢病毒载体，表达特异性针对NY-ESO-1 157-165表位肽的TCR。U3区增强子的缺失(U3del)确保了慢病毒构建体的自我灭活。TCR基因编码具有可变序列(TCR-βV)和鼠恒定序列(鼠TCR-βC)的β链和具有可变序列(TCR-αV)和鼠恒定序列(鼠TCR-αC)的α链)，两侧是EF1α启动子和慢病毒WPRE区。
图1C显示了用pShuttle载体转染后293T细胞的HLA-A2表达，该载体表达了所述包含NY-ESO-1 157-165表位肽的标记性多肽和HLA-A2蛋白。293T细胞用FITC标记的抗HLA-A2抗体染色，并用流式细胞仪分析。深色粗线显示了在用编码HLA-A2蛋白的载体转导的293T细胞上HLA-A2的表达。浅灰线是用空载体转导的对照293T细胞。门控群体(gated population)的平均荧光强度(Geom均值)显示在流式细胞仪图上。左图显示pShuttle-EF1α-NY-A2转导的293T细胞的HLA-A2表达，中间图显示 pShuttle-EF1α-E1Ad24-A2-F2A-NY转导的293T细胞的HLA-A2表达，右图显示了pShuttle-EF1α-E1Ad24-A2-F2A-BM转导的293T细胞中HLA-A2的表达。
图1D显示了在慢病毒转染后，JRT细胞表达了NY-ESO-1特异性TCR，所述慢病毒在HLA-A2的背景下表达了对NY-ESO-1157-165表位肽具有特异性的不同TCR。将JRT细胞转染重组慢病毒并在7-10天内进行分析。JRT细胞用APC标记的抗CD8抗体和PE标记的NY-ESO-1 157-165/HLA-A2四聚体染色，并通过流式细胞仪进行分析。在流式细胞仪图上显示了门控JRT细胞群中四聚体阳性细胞的百分比。从左自右第1图示出的对照组是未用慢病毒转导的JRT细胞。从左自右第2图示出的“TCR-NY-LY”组是用由pCDH-EF1α-TCR-NY-LY产生的慢病毒转染的JRT细胞。从左自右第3图示出的“TCR-NY-AE”组是用由pCDH-EF1α-TCR-NY-AE产生的慢病毒转染的JRT细胞。从左自右第4图示出的“TCR-NY-LI”组是用由pCDH-EF1α-TCR-NY-LI产生的慢病毒转染的JRT细胞。
图2A显示了NY-ESO-1特异性TCR可以识别HLA-A2在T2细胞上呈递的NY-ESO-1 157-165表位肽。由pCDH-EF1α-TCR-NY-LY(JRT-TCR-NY-LY)，pCDH-EF1α-TCR-NY-AE(JRT-TCR-NY-AE)或pCDH-EF1α-TCR-NY-LI(JRT-TCR-NY-LI)产生的重组慢病毒转导的JRT细胞与T2细胞一起培养，该T2细胞用从1μg/ml开始10倍稀释的NY-ESO-1 157-165表位肽系列稀释液浓度加载处理16小时。收获细胞并用抗CD69抗体染色以通过流式细胞术分析CD69的表达。X轴是用一系列浓度的NY-ESO-1 157-165肽浓度加载处理的T2靶细胞，Y轴是门控JRT细胞中CD69 +细胞的百分比。
图2B显示表达含有NY-ESO-1 157-165表位肽的标记性多肽和HLA-A2的293T细胞可以激活JRT细胞上的NY-ESO-1特异性TCR。在重复的孔中，将用重组慢病毒转导以表达对NY-ESO-1特异性的不同TCR的JRT细胞与用pShuttle-EF1a-NY-A2或pShuttle-EF1a-E1Ad24-A2-NY转导的293T细胞孵育持续16小时， 收获细胞并用抗CD69抗体染色以通过流式细胞术分析CD69的表达。X轴显示JRT细胞表达特异性针对NY-ESO-1的不同TCR，包括JRT-TCR-NY-LY，JRT-TCR-NY-AE和JRT-TCR-NY-LI。Y轴是门控JRT细胞中CD69 +细胞的百分比(平均值±SD；n＝2)。“对照”代表用空pShuttle载体转导的293T靶细胞；“pShuttle-EF1a-NY-A2”代表用pShuttle-EF1a-NY-A2载体转导的293T靶细胞；“pShuttle-EF1a-E1Ad24-A2-NY”代表用pShuttle-EF1a-E1Ad24-A2-NY载体转导的293T靶细胞。用Student's t检验分析数据，**代表p<0.01，*代表p<0.05。
图2C显示外源性HLA-A2可以呈递源自NY-ESO-1蛋白的NY-ESO-1 157-165表位肽，以激活JRT细胞上的NY-ESO-1特异性TCR。在重复的孔中，将用表达全长NY-ESO-1蛋白的pCDNA3.3载体(pCDNA3.3-NY)和表达外源HLA-A2蛋白的pshuttle载体(pShuttle-EF1a-E1Ad24-A2-F2A-BM)共转染的293T细胞用作靶细胞以刺激用NY-ESO-1特异性TCR转导的JRT细胞。单独用pCDNA3.3-NY或pShuttle-EF1a-E1Ad24-A2-F2A-BM转导的293T细胞为阴性对照。单独用pShuttle-EF1a-E1Ad24-A2-F2A-BM转导并以1μg/ml的NY-ESO-1 157-165表位肽浓度加载处理的293T细胞作为阳性对照。在16小时内收获细胞，用抗CD69抗体染色并通过流式细胞术分析。X轴显示JRT细胞表达对NY-ESO-1特异性的不同TCR，包括JRT-TCR-NY-LY，JRT-TCR-NY-AE和JRT-TCR-NY-LI。Y轴是门控JRT细胞中CD69 +细胞的百分比(平均值±SD；n＝2)。“pCDNA3.3-NY”代表仅用pCDNA3.3-NY转导的293T靶细胞；“pShuttle-EF1a-E1Ad24-A2-F2A-BM”代表仅通过pShuttle-EF1a-E1Ad24-A2-F2A-BM转导的293T靶细胞；“pShuttle-EF1a-E1Ad24-A2-F2A-BM+pCDNA3.3-NY”代表用pShuttle-EF1a-E1Ad24-A2-F2A-BM和pCDNA3.3-NY共转导的293T靶细胞；“pShuttle-EF1a-E1Ad24-A2-F2A-BM+NY-ESO-1多肽”代表用pShuttle-EF1a-E1Ad24-A2-F2A-BM转导并用NY-ESO-1 157-165肽浓度加载处理的靶293T细胞。用Student's t检验分析数据，**代 表p<0.01，*代表p<0.05。
图2D显示了重组溶瘤腺病毒DNA中编码外源肽和蛋白质的核酸可以转导293T细胞以表达包含NY-ESO-1 157-165表位肽的标记性多肽和外源HLA-A2。用重组溶瘤腺病毒载体pAd-EF1a-E1Ad24-A2-NY和pAd-EF1a-E1Ad24-A2-BM转导293T细胞，并将其用作靶细胞刺激表达NY-ESO-1特异性TCR的JRT细胞。在重复的孔中，将混合培养的细胞温育16小时，并收集以通过流式细胞术分析CD69的表达。X轴显示JRT细胞表达特异性针NY-ESO-1的不同TCR，包括JRT-TCR-NY-LY和JRT-TCR-NY-AE。Y轴是门控JRT细胞中CD69 +细胞的百分比(平均值±SD；n＝2)。“对照”是没有转染的293T细胞；“pAd-EF1a-E1Ad24-A2-F2A-NY”和“pAd-EF1a-E1Ad24-A2-F2A-BM”代表分别用pAd-EF1a-E1Ad24-A2-F2A-NY或pAd-EF1a-E1Ad24-A2-F2A-BM转导的293T靶细胞。“pAd-EF1a-E1Ad24-A2-F2A-BM+pCDNA3.3-NY”代表用pAd-EF1a-E1Ad24-A2-F2A-BM和pCDNA3.3-NY共转导的293T靶细胞；“pAd-EF1a-E1Ad24-A2-F2A-BM+NY-ESO-1多肽”代表用pAd-EF1a-E1A d 24-A2-F2A-BM转导并用NY-ESO-1 157-165肽浓度加载处理的293T靶细胞。用Student's t检验分析数据，**代表p<0.01，*代表p<0.05。
图3显示可以通过JRT细胞上的NY-ESO-1特异性TCR识别包含NY-ESO-1表位肽的标记性多肽和外源性HLA-A2来敏化肿瘤细胞。用pShuttle-A2-F2A-NY或pShuttle-A2-F2A-BM转导肿瘤细胞系A375，SKOV3和SKOV3-NY(用pCDNA3.3-NY转导并稳定表达NY-ESO-1蛋白的SKOV3细胞)，并用作刺激JRT细胞的靶细胞，所述JRT细胞表达NY-ESO-1特异性TCR。在重复的孔中，将肿瘤细胞和JRT细胞混合培养16小时，收集细胞以通过流式细胞术分析CD69的表达。X轴显示JRT细胞表达对NY-ESO-1有特异性的不同TCR，包括JRT-TCR-NY-LY和JRT-TCR-NY-AE。Y轴是门控JRT细胞中CD69 +细胞的百分比(平均值±SD；n＝2)。“A375”，“SKOV3” 和“SKOV3-NY”代表未转导的靶细胞；“A375-pShuttle-A2-F2A-BM”，“SKOV3-pShuttle-A2-F2A-BM”和“SKOV3-NY-pShuttle-A2-F2A-BM”代表用pShuttle-A2-F2A-BM转导的肿瘤靶细胞；“A375-pShuttle-A2-F2A-NY”，“SKOV3-pShuttle-A2-F2A-NY”和“SKOV3-NY-pShuttle-A2-F2A-NY”代表用pShuttle-A2-F2A-NY转导的肿瘤靶细胞。用Student's t检验分析数据，**代表p<0.01，*代表p<0.05。
图4A显示，在HLA-A2的背景下，用编码NY-ESO-1特异性TCR的重组慢病毒转染的PBMC可以表达特异性针对NY-ESO-1157-165肽的TCR。用编码不同NY-ESO-1特异性TCR(包括TCR-NY-LY(左图)，TCR-NY-AE(中图)和TCR-NY-LI(右图))的新鲜制备的慢病毒感染PBMC细胞。在7-10天内收获细胞，用APC标记的抗CD8抗体和PE标记的NY-ESO-1 157-165/HLA-A2四聚体染色，并通过流式细胞术进行分析。流式细胞术图显示了基于前向散射和侧向散射的门控淋巴细胞群中CD8 +，四聚体 +细胞和CD8 -且四聚体 +细胞的百分比。
图4B显示可以通过包含NY-ESO-1表位肽的标记性多肽和/或外源性HLA-A2敏化肿瘤细胞，以被初级T细胞上的NY-ESO-1特异性TCR识别。用pShuttle-A2-F2A-NY或pShuttle-A2-F2A-BM转导了肿瘤细胞系A375，SKOV3和SKOV3-NY，并将其用作靶细胞来刺激表达NY-ESO-1特异性TCR的HLA-A2-PBMC细胞。在重复的孔中，将肿瘤细胞和PBMC细胞混合培养24小时，E:T比为10:1。温育后，收集上清液以评估T细胞分泌IFN-γ。X轴显示表达针对NY-ESO-1的不同TCR的PBMC细胞，包括PBMC-TCR-NY-LY，PBMC-TCR-NY-AE和PBMC-TCR-NY-LI。Y轴显示由特异性T细胞产生的IFN-γ的浓度(平均值±SD；n＝2)。“A375”，“SKOV3”和“SKOV3-NY”代表未转导的靶细胞；“A375+A2BM”，“SKOV3+A2BM”和“SKOV3-NY+A2BM”代表用pShuttle-A2-F2A-BM转导的肿瘤靶细胞；“A375+A2NY”，“SKOV3+A2NY”和“SKOV3-NY+A2NY”代表用pShuttle-A2-F2A-NY转导的肿瘤靶细 胞；“A375+A2BM+NY多肽”，“SKOV3+A2BM+NY肽”和“SKOV3-NY+A2BM+NY多肽”代表用pShuttle-A2-F2A-BM转导并用1μg/ml的NY-ESO-1 157-165肽浓度加载处理的肿瘤靶细胞。用Student's t检验分析数据，**代表p<0.01，*代表p<0.05。
图4C显示，更多的肿瘤细胞系可被含有NY-ESO-1表位肽的标记性多肽和外源性HLA-A2敏化，并被原代T细胞上的NY-ESO-1特异性TCR识别。用pShuttle-NY-A2转导肿瘤细胞系A549，H1299和HOS-C1(分别得到“A549-NY”，“H1299-NY”和“HOS-NY”)，并将其用作靶细胞以刺激用NY-ESO-1特异性TCR基因转导的PBMC细胞。在一式三份的孔中，将肿瘤细胞和PBMC细胞混合培养24小时，E:T比为5:1。温育后，收集上清液以评估T细胞分泌IFN-γ。X轴显示效应细胞和靶细胞的组合，Y轴显示特定T细胞产生的IFN-γ的浓度(平均值±SD；n＝3)。模拟效应细胞是用空的慢病毒转导的PBMC细胞。其他效应细胞是用TCR-NY-LY基因转导的PBMC和用TCR-NY-LI转导的PBMC。用Student's t检验分析数据，**代表p<0.01，*代表p<0.05。
图5示出本发明实施例5中，用不同MOI的重组溶瘤腺病毒OAd-NY/A2感染SKOV3细胞48小时后，通过流式细胞术检测细胞表面HLA-A2表达的结果。横坐标为重组溶瘤腺病毒OAd-NY/A2的MOI，纵坐标为HLA-A2的表达百分比。
图6示出本发明的实施例6中，Mock-T或靶向NY-ESO-1的TCR-T与SKOV3或重组溶瘤腺病毒OAd-NY/A2感染的SKOV3共培养后，释放的IFN-γ的结果。图6A为人黑素瘤细胞株A375(NY-ESO-1阳性和HLA-A2阳性)的结果，图6B为人肺癌细胞株H1299(NY-ESO-1阳性和HLA-A2阴性)的结果，图6C为人卵巢癌细胞株SKOV3(NY-ESO-1阴性和HLA-A2阴性)的结果，图6D为人骨肉瘤细胞株HOSC1(NY-ESO-1弱阳性和HLA-A2阳性)的结果。图中“Mock-T”表示表达GFP的T细胞组(对照组)，“TCR-T”表示表达靶向NY-ESO-1TCR的T细胞组。图中横坐标代表不同实验组，纵坐标代表IFN-γ的浓度(pg/ml)。
图7示出本发明实施例7中重组溶瘤腺病毒OAd-NY/A2和靶向NY-ESO-1的TCR-T对人卵巢癌细胞株SKOV3的体外联合杀伤结果。图7A为实时杀伤的结果，横坐标为肿瘤细胞铺板后(即实验开始后)的时间，以小时(h)计，纵坐标为归一的细胞指数，图中垂直向下的箭头所指分别为加入OAd-NY/A2以及Mock-T或TCR-T的时间点。图7B是对图7A实验终点(90.8小时)各组的细胞指数进行分析，计算出的肿瘤生长抑制率，横坐标为不同实验组，纵坐标为肿瘤生长抑制率IR(％)。
图8示出本发明实施例8中重组溶瘤腺病毒OAd-NY/A2和靶向NY-ESO-1的TCR-T对人肺癌细胞株H1299的体外联合杀伤结果。图8A为实时杀伤的结果，横坐标为肿瘤细胞铺板后(即实验开始后)的时间，以小时(h)计，纵坐标为归一的细胞指数，图中垂直向下的箭头所指分别为加入OAd-NY/A2以及Mock-T或TCR-T的时间点。图8B是对图8A的第61.14小时各组的细胞指数进行分析，计算出的肿瘤生长抑制率，横坐标为不同实验组，纵坐标为肿瘤生长抑制率IR(％)。
图9示出本发明实施例9中重组溶瘤腺病毒OAd-NY/A2和靶向NY-ESO-1的TCR-T对人骨肉瘤细胞株HOS C1的体外联合杀伤结果。图9A为实时杀伤的结果，横坐标为肿瘤细胞铺板后(即实验开始后)的时间，以小时(h)计，纵坐标为归一的细胞指数，图中垂直向下的箭头所指分别为加入OAd-NY/A2以及Mock-T或TCR-T的时间点。图9B是对图9A的第64.44小时各组的细胞指数进行分析，计算出的肿瘤生长抑制率，横坐标为不同实验组，纵坐标为肿瘤生长抑制率IR(％)。
具体实施方式
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
本发明提供了一种分离的用于表达外源基因的溶瘤腺病毒，其中所述溶瘤腺病毒为对腺病毒进行基因改造而得到的选择复制型重组 溶瘤腺病毒，该重组溶瘤腺病毒的基因组具有以下特征：
1)包含E1B基因调控元件，该元件包括E1B启动子和E1B与pIX共用的多聚腺苷酸添加信号序列；
2)缺失了E1B基因编码区，并且，当需要插入所述外源基因时，在该E1B基因编码区位点插入所述外源基因，并且该外源基因位于所述E1B启动子之后，受所述E1B基因调控元件的控制；
3)在所述外源基因的上游，包含转录E1A 13s mRNA的E1A的cDNA序列，并且该cDNA为野生型或Rb蛋白结合区域缺失型，该Rb蛋白结合区域缺失型为所述野生型cDNA去除了如SEQ ID NO.7(即AC_000008.1nt 923-nt 946)所示的核苷酸序列(所述AC_000008.1为NCBI(即，美国国立生物技术信息中心，网址：https://www.ncbi.nlm.nih.gov)的GenBank编号)，或该Rb蛋白结合区域缺失型编码突变的E1A蛋白，所述突变的E1A蛋白如SEQ ID NO.6所示。
研究发现，5型腺病毒的E1A基因内含子2的部分序列和下游E1B启动子重合，而E1A mRNA的通读转录(read-through transcription)是病毒感染早期E1B启动子活化所必需的。如果终止E1A基因的通读转录，会大大降低下游E1b基因的顺式(in cis)表达。因此，本发明利用溶瘤病毒基因组中自身的E1B基因调控元件来调控外源基因的表达，避免插入的外源基因调控元件对病毒基因组表达所造成的可能干扰，而影响病毒的有效复制。另外，也可以增加插入的外源基因片段的长度，使得溶瘤病毒载体可以携带更多外源基因负荷。另一方面，本发明构建的溶瘤腺病毒缺失了E1B基因编码区。E1B-19K蛋白可以抑制通过肿瘤坏死因子和FAS通路所诱导的细胞凋亡，使得感染的细胞耐受T细胞的杀伤作用。去除E1B-19K基因可以增加被感染的肿瘤细胞对杀伤性T细胞的敏感性。去除E1B-55K基因可以增加溶瘤腺病毒的肿瘤细胞溶瘤选择性。另一方面，在腺病毒感染早期，E1A基因mRNA转录物的不同剪接会合成两种主要的E1A蛋白E1A-12S(243R)和E1A-13S(289R)。E1A-13S(289R)蛋白在保守区(CR3)具有特有的46个氨基酸，尽管E1A-12S(243R) 和E1A-13S(289R)非常相似，但两者在生物学活性方面显示出显着差异。E1A-13S是主要的病毒蛋白，通过其CR3与多种转录因子相互作用参与激活病毒基因表达。和表达E1A-12S的腺病毒相比，表达E1A289R的病毒能够更有效地驱动病毒基因和蛋白质表达，并且能够更快且更多地复制其基因组。另外，共表达E1A-12S(缺少CR3)可抑制E1A-13S的转录激活功能，而E1A-12S对转录的抑制作用作用在其N末端，并与结合p300/CBP的能力相关。本发明发现，在溶瘤腺病毒的基因组中，通过在外源基因的上游设计转录E1A-13S mRNA的E1A的cDNA序列而不是E1A基因组基因，使得本发明可以只转录E1A-13S而避免转录E1A-12S，从而增加外源基因的表达，以及加强病毒基因组的复制。
腺病毒E1A蛋白结合Rb蛋白的核心位点是Leu-122-X-Cys-X-Glu(X表示任何氨基酸残基)。缺失这段氨基酸序列的E1A蛋白不能结合Rb蛋白，导致溶瘤腺病毒选择性地在Rb/E2F1通路缺陷的肿瘤细胞中复制并裂解肿瘤细胞。因此，在本发明的重组溶瘤腺病毒的基因组中，当转录E1A 13s mRNA的E1A的cDNA序列为Rb蛋白结合区域缺失型时，在一个实施方案中，该Rb蛋白结合区域缺失型为所述野生型cDNA去除了如SEQ ID NO.7(即AC_000008.1nt 923-nt 946)所示的核苷酸序列。相应地，所述Rb蛋白结合区域缺失型cDNA所编码的E1A蛋白的氨基酸序列缺失的是L-T-C-H-E-A-G-F(Leu-Thr-Cys-His-Glu-Ala-Gly-Phe)。在另一个实施方案中，该Rb蛋白结合区域缺失型编码突变的E1A蛋白，所述突变的E1A蛋白如SEQ ID NO.6所示，其中突变位点为L122V、C124S和E126D。
优选地，所述腺病毒为人C型腺病毒，包括人2型腺病毒和人5型腺病毒。
在本发明一个优选的具体实施方案中，所述E1B启动子的核苷酸序列如SEQ ID NO.1(即AC_000008.1nt 1336-nt 1702)所示，所述E1B与pIX共用的多聚腺苷酸添加信号序列(即E1B/pIX多聚腺苷酸添加信号序列)如SEQ ID NO.2(即AC_000008.1nt 4038-nt4043) 所示。
在本发明中，优选地，缺失E1B基因编码区包括缺失E1B-55K基因和E1B-19K基因的编码区。
更优选地，所述E1B基因编码区的核苷酸序列如SEQ ID NO.3(即AC_000008.1nt 1714-nt 3509)所示。
优选地，所述外源基因的起始位点包含Kozak序列，优选地，所述Kozak序列如SEQ ID NO.4(即GCCRCC ATGG，R为嘌呤(A or G))所示。
优选地，所述野生型cDNA的核苷酸序列如SEQ ID NO.5(即AC_000008.1nt 560-nt 1545去除nt 1113-nt 1228)所示。
在本发明一个优选的具体实施方案中，所述转录E1A 13s mRNA的E1A的cDNA位于所述E1B启动子的上游，并且由于E1B启动子内包含内源的E1A基因的添加pA信号序列(AC_000008.1nt 1611-nt1616)和pA添加位点(AC_000008.1nt 1632)，以及部分E1A基因序列(nt 1336-nt 1552)，因此，该转录E1A 13s mRNA的E1A的cDNA与所述E1B启动子的核苷酸序列部分重合。在一个实施方案中，重组溶瘤腺病毒的基因组包含如SEQ ID NO.2所示的核苷酸序列：
Figure PCTCN2021117571-appb-000001
Figure PCTCN2021117571-appb-000002
其中，如SEQ ID NO.2所示的核苷酸序列包含：Rb蛋白结合区域缺失型的转录E1A 13s mRNA的E1A的cDNA(用下划线标记)，以及E1B启动子(用斜体标记)。并且，如SEQ ID NO.2所示的核苷酸序列位于在内源性E1A启动子/增强子或外源启动子的位点与所述外源基因的起始位点之间。
在本发明的一些实施方案中，所述转录E1A 13s mRNA的E1A的cDNA序列在内源性E1A启动子/增强子的控制下，或者在外源启动子的控制下；优选地，所述内源性E1A启动子/增强子的核苷酸序列如SEQ ID NO.8(即AC_000008.1nt 1-nt 499)所示。
在本发明的另一些实施方案中，所述转录E1A 13s mRNA的E1A的cDNA序列在外源启动子的控制下。在具体的实施方案中，由于溶瘤腺病毒基因组的起始位置(例如AC_000008.1nt 1-nt 375)包含病毒的ITR、AdΨ和包装元件，因此，在这些位置之后插入外源启动子。在人5型腺病毒的实施方案中，所述重组溶瘤腺病毒的基因组中去除了如SEQ ID NO.9(即AC_000008.1nt 376-nt 559)所示的核苷酸序列，并且所述外源启动子由该去除的位点插入所述基因组。
优选地，所述外源启动子包括EF-1α启动子、CMV启动子、PKG启动子、E2F启动子、AFP启动子和TERT启动子。
在本发明优选的实施方案中，所述外源基因包括：HLA蛋白编码序列、标记性多肽编码序列、HLA蛋白编码序列及标记性多肽编码序列、HLA蛋白编码序列及β2-微球蛋白编码序列、或HLA蛋白编码序列、β2-微球蛋白编码序列及标记性多肽编码序列。
本发明在提及“HLA蛋白编码序列及标记性多肽编码序列”、“HLA蛋白编码序列及β2-微球蛋白编码序列”和“HLA蛋白编码序列、β2-微球蛋白编码序列及标记性多肽编码序列”时，是指在所述溶瘤腺病毒的基因组中同时插入HLA蛋白编码序列和标记性多肽编码 序列，或者同时插入HLA蛋白编码序列和β2-微球蛋白编码序列、或者同时插入HLA蛋白编码序列、β2-微球蛋白编码序列和标记性多肽编码序列。
优选地，所述HLA蛋白包括HLA I类分子，该HLA I类分子包括HLA-A、HLA-B和HLA-C。
优选地，HLA蛋白是HLA-A*02：01蛋白，并且氨基酸序列(含信号肽序列)如SEQ ID NO.12所示。
HLA-C在细胞表面的表达较低，为了增加HLA-C的表达，对插入的外源HLA-C进行以下突变中的至少一种：1)第2位精氨酸突变为丙氨酸。这样去除起始子甲硫氨酸后，N端的Ala就会乙酰化，使得新合成的HLA-C更稳定而不被降解。2)编码该HLA-C蛋白的核苷酸序列的第4位核苷酸由C突变为G，第5位核苷酸由G突变为C，这样可形成强的Kozak序列(GCCRCC ATGG，R为嘌呤(A or G)，以增强蛋白的翻译和表达。3)第362位异亮氨酸突变为苏氨酸；4)第359位谷氨酸突变为缬氨酸。HLA-C蛋白的C端包含二疏水(dihydrophobic，LI)内在化和溶酶体靶向信号(DxSLI)，并且第362位的异亮氨酸是影响该基序活性的关键氨基酸。因此，将HLA-C尾部的362位的异亮氨酸更改为HLA-A和B尾部中的苏氨酸(I362T)，和/或将第359位谷氨酸突变为缬氨酸，可提高HLA-C的表面表达。由于这些突变不是位于抗原多肽提呈区域，因而不会影响抗原多肽的提呈和TCR的识别。
优选地，HLA蛋白是具有I362T点突变的HLA-C。更优选地，HLA蛋白是HLA-C*08：02蛋白，其氨基酸序列包含R2A点突变、I362T点突变和E359V点突变，其氨基酸序列如SEQ ID NO.13所示。
其他HLA蛋白包括但不限于HLA-A*01：01蛋白，HLA-A*02：03蛋白，HLA-A*02：06蛋白，HLA-A*03：01蛋白，HLA-A*11：01蛋白，HLA-A*24：02蛋白，HLA-A*30：01蛋白，HLA-A*68：01蛋白，HLA-B*08：01蛋白，HLA-B*14：02蛋白，HLA-B*1501，HLA-B*58：01，HLA-C*07：01蛋白，HLA-C*01：02蛋白。
HLA蛋白的编码序列在任选的外源基因表达调控元件的控制下 表达，所述调控元件包括启动子，增强子，增强子，沉默子和聚腺苷酸化信号，或可药用载体自身的基因表达调控元件。
通常，表达在细胞浆内的抗原蛋白可进入MHC I类抗原提呈途径，经系列蛋白酶水解后，形成的短肽(含有抗原表位多肽)通过内质网膜上的TAP分子转导进入内质网内，并与其中的HLA蛋白和β 2-微球蛋白形成三聚体后被提呈在细胞表面(其中HLA蛋白和β 2-微球蛋白配对形成MHC I类分子)，从而被免疫细胞识别。由于肿瘤细胞中常发生MHC I类抗原提呈途径的功能缺失，导致细胞浆内表达的肿瘤抗原不能有效形成表位多肽或进入内质网并与HLA和β2-微球蛋白结合形成复合物。
在本发明中，通过将所述具有标记性多肽编码序列的核酸导入肿瘤细胞和/或癌细胞，表达在肿瘤细胞和/或癌细胞内的外源标记性多肽可进入MHC I类抗原提呈途径，从而增加了肿瘤细胞表面HLA/抗原表位多肽复合物的表达量，由此增强了所述T细胞受体修饰的免疫细胞对肿瘤细胞和/或癌细胞的识别敏感性。
优选地，所述标记性多肽包括可操作地连接的、依次串联的以下氨基酸序列：N端信号肽的氨基酸序列、一个或多个抗原表位多肽的氨基酸序列、可任选的C端内质网滞留信号的氨基酸序列，其中当所述标记性多肽包括多个所述抗原表位多肽的氨基酸序列时，每两个相邻的所述抗原表位多肽的氨基酸序列之间由可切割性连接多肽的氨基酸序列连接；所述抗原表位多肽的氨基酸序列和可任选的C端内质网滞留信号的氨基酸序列可以由可切割性连接多肽的氨基酸序列连接。优选的是，所述标记性多肽包括所述C端内质网滞留信号的氨基酸序列。优选地，所述可切割性连接多肽是furin酶切识别多肽。
所述抗原表位多肽的氨基酸序列可以来源于自然界存在的蛋白的氨基酸序列，或者为人工合成的自然界不存在的氨基酸序列。优选地，所述自然界存在的蛋白包括人源蛋白和除人以外的其它物种的蛋白。更优选地，所述抗原表位多肽的氨基酸序列来源于肿瘤相关抗原、肿瘤特异性抗原、和含有突变点的肿瘤新抗原(neo-antigen)的氨基 酸序列。
“肿瘤相关抗原”通常指来源于自身的正常蛋白，但在肿瘤细胞中过度表达或异常表达，其包括癌胚抗原、肿瘤-睾丸抗原(CT抗原)等。
“肿瘤特异性抗原”通常指来源于自身的突变蛋白，或异体的与肿瘤发生和发展相关的病毒蛋白。
在本发明中，有时将“肿瘤相关抗原”和“肿瘤特异性抗原”笼统地称为“肿瘤抗原”。
所述肿瘤抗原可以是如癌症抗原性多肽数据库(Cancer Antigenic Peptide Database)(网址https://caped.icp.ucl.ac.be)中所述的肿瘤抗原。优选的是，所述肿瘤抗原可以是如下表1中所述的肿瘤抗原。
所述抗原表位多肽可以是能够被MHC I类分子提呈的具有8-11个氨基酸的肽段。所述抗原表位多肽可以是如癌症抗原性多肽数据库(Cancer Antigenic Peptide Database)(网址https://caped.icp.ucl.ac.be)中所述的抗原表位多肽。优选的是，所述抗原表位多肽可以是如下表1中所述的抗原表位多肽。在其它实施方案中，所述抗原表位多肽为与如下表1中所述的抗原表位多肽具有4-9个连续的相同氨基酸(例如，4、5、6、7、8或9个连续的相同氨基酸)的抗原表位多肽，并且这些多肽的长度为8-11个氨基酸。
表1优选的肿瘤抗原及抗原表位多肽表
肿瘤抗原名称 HLA分型 氨基酸序列位置
N-ras A1 55-64
MART2 A1 446-455
MATN A11 226-234
CDKN2A A11 125-133
CDK12 A11 924-932
k-ras A2 224-232
hsp70-2 A2 286-295
HAUS3 A2 154-162
GAS7 A2 141-150
CSNK1A1 A2 26-34
CLPP A2 240-248
CDK4 A2 23-32
α-辅肌动蛋白-4 A2 118-127
β-连环蛋白 A24 29-37
SIRT2 A3 192-200
GPNMB A3 179-188
EFTUD2 A3 668-677
MUM-3 A68 322-330
延伸因子2 A68 581-589
CASP-8 B35 476-484
SNRPD1 B38 19-Oct
OS-9 B44 438-446
MUM-2 B44 123-133
MUM-1 B44 30-38
KIAAO205 B44 262-270
NFYC B52 275-282
RBAF600 B7 329-337
HSDL1 Cw14 20-27
MUM-2 Cw6 126-134
K-ras Cw8 (10-18)
K-ras Cw8 (10-19)
MAGE-A3 A1 168-176
MAGE-A1 A1 161-169
SSX-2 A2 41-49
NY-ESO-1/LAGE-2 A2 157-165
NY-ESO-1/LAGE-2 A2 (1-11)
MAGE-C2 A2 336-344
MAGE-C2 A2 191-200
MAGE-A10 A2 254-262
LAGE-1 A2 (1-11)
HERV-K-MEL A2 (1-9)
GAGE-3,4,5,6,7 A29 (10-18)
NY-ESO-1/LAGE-2 A31 53-62
NY-ESO-1/LAGE-2 A31 (18-27)
LAGE-1 A31 (18-27)
MAGE-A6 A34 290-298
KK-LC-1 B15 76-84
MAGE-A6 B35 168-176
MAGE-A6 B37 127-136
MAGE-A3 B37 127-136
MAGE-A2 B37 127-136
MAGE-A1 B37 120-129
MAGE-C2 B44 307-315
MAGE-C2 B57 42-50
MAGE-A6 Cw16 293-301
MAGE-A1 Cw16 230-238
BAGE-1 Cw16 (2-10)
GAGE-1,2,8 Cw6 (9-16)
MAGE-A12m Cw7 170-178
酪氨酸酶 A1 243-251
酪氨酸酶 A1 146-156
酪氨酸酶 A2 (1-9)
酪氨酸酶 A2 369-377
Melan-A/MART-1 A2 32-40
Melan-A/MART-1 A2 26(27)-35
酪氨酸酶 A24 368-373和336-340e
酪氨酸酶 A24 206-214
酪氨酸酶 A26 90-98
TRP-2 A31 197-205
TRP-2 A33 197-205
酪氨酸酶 B35 312-320
酪氨酸酶 B35 309-320
Melan-A/MART-1 B35 26-35
酪氨酸酶 B38 388-397
酪氨酸酶 B44 192-200
Melan-A/MART-1 B45 24-33(34)
TRP-2 Cw8 387-395
MMP-2 A2 560-568
HER-2/neu A2 369-377
CPSF A2 1360-1369
CPSF A2 250-258
CALCA A2 16-25
PRAME A24 301-309
FGF5 A3 172-176和217-220
p53 B46 99-107
PBF B55 499-510
H3.3K27M A2 26-35
HPV16-E6   29-38
HPV16-E7   11–19
HPV16-E7   11-19
EBV-LMP1   51-59
EBV-LMP1   125-133
在某些实施方案中，每个所述抗原表位多肽的两端具有柔性连接片段，作为细胞浆内蛋白水解酶的酶切位点，以释放出该抗原表位 多肽。所述柔性连接片段包括GSGSR、AGSGSR和AGSGS。
在某些实施方案中，所述标记性多肽在所述一个或多个抗原表位多肽氨基酸序列的氨基端具有可把该标记性多肽导入内质网的信号肽。信号肽的核心含有长段疏水性氨基酸，形成单个α-螺旋。信号肽氨基端常以短的带正电荷的氨基酸序列开始，信号肽末端通常存在一段被信号肽酶识别和切割的氨基酸切割位点。所连接的外源多肽进入内质网后，信号肽被信号肽酶识别和切割，在内质网内释放出外源多肽。因此，携带信号肽的标记性多肽可以不通过MHC I类抗原提呈途径中的蛋白酶水解以及TAP分子的转运，即可直接进入内质网。所述信号肽可以为由胰岛素样蛋白(INSL5)氨基端第1-22氨基酸所组成的信号肽(SEQ ID NO.14)。
在所述标记性多肽具有多个所述抗原表位多肽的情况中，每两个所述抗原表位多肽可由可切割性连接多肽所连接。可切割性连接多肽包括furin酶切识别多肽，其具有可被Furin酶切割的标准的四氨基酸基序，即R-X-[KR]-R氨基酸序列(参见文献“Molecular Therapy2007；vol.15no.6,1153–1159”)。优选的是，所述可切割性连接多肽的氨基酸序列为R-R-K-R。所述标记性多肽被上述信号肽导入内质网后，由R-X-[KR]-R氨基酸序列连接的抗原表位多肽被内质网中的furin酶切割水解，释放出抗原表位多肽，与内质网中的HLA和β 2-微球蛋白形成抗原复合物。内质网内的氨肽酶和羧肽酶也可能参与抗原表位多肽的酶解和释放(参见文献“J Immunol.2009 November 1；183(9):5526–5536”)，因此，可切割性连接多肽还可以包括氨肽酶和羧肽酶酶切识别多肽。
为了使被信号肽导入内质网的标记性多肽能够滞留在内质网腔内，以利于释放出抗原表位多肽并与HLA和β2-微球蛋白结合形成抗原复合物，在某些实施方案中，所述标记性多肽在所述一个或多个抗原表位多肽氨基酸序列的羧基端具有内质网滞留信号肽。可溶性多肽(即非跨膜蛋白)的内质网滞留信号(ER retention signal)的氨基酸序列是KDEL，ER膜蛋白的内质网滞留信号是KKXX(参见文献“Molecular Biology of the Cell.2003；14(3):889–902”)。在本发明 中，所述标记性多肽是可溶性多肽。因此优选的是，所述内质网滞留信号肽是由赖氨酸-天冬氨酸-谷氨酸-亮氨酸残基组成的K-D-E-L片段。
更优选的是，所述肿瘤相关抗原选自NY-ESO-1 157-165、NY-ESO-1 1-11、NY-ESO-1 53-62、NY-ESO-1 18-27、Her2/neu 369-377、SSX-2 41-49、MAGE-A4 230-239、MAGE-A10 254-262、MAGE-C2 336-344、MAGE-C2 191-200、MAGE-C2 307-315、MAGE-C2 42-50、MAGE-A1 120-129、MAGE-A1 230-238、MAGE-A1 161-169、KK-LC-1 76-84、p53 99-107、PRAME 301-309、甲胎蛋白158-166、HPV16-E6 29-38、HPV16-E7 11-19、EBV-LMP1 51-59、EBV-LMP1 125-133、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：Q61R 55-64、KRAS：G12D 5-14、KRAS：G1 3 D 5-14、KRAS：G12 A 5-14、KRAS：G12C 5-14、KRAS：G12S 5-14、KRAS：G12 R 5-14、KRAS：G12D 10-19、TP53：V157G 156-164、TP53：R248Q 240-249、TP53：R248W 240-249、TP53：G245S 240-249、TP53：V157F 156-164、TP53：V157F 149-158、TP53：Y163C 156-164、TP53：R248Q 247-255、TP53：R248Q 245-254、TP53：R248W 245-254、TP53：G245S 245-254、TP53：G249S 245-254、TP53：Y220C 217-225、TP53：R175H 168-176、TP53：R248W 240-249、TP53：K132N 125-134、CDC73：Q254E 248-256、CYP2A6：N438Y 436-444、CTNNB1：T41A 41-49、CTNNB1：S45P 41-49、CTNNB1：T41A 34-43、CTNNB1：S37Y 30-39、CTNNB1：S3 3C 30-39、CTNNB1：S45P 40-49、EGFR：L858R 852-860、EGFR：T790M 790-799、PIK3CA：E542K 533-542、PIK3CA：H1047R 1046-1055、GNAS：R201H 197-205、CDK4：R24C 23-32、H3.3：K27M 26-35、BRAF：V600E 591-601、CHD4：K73Rfs 141-148、NRAS：Q61R 55-64、IDH1：R132H 126-135、TVP23C：C51Y 51-59、TVP23C：C51Y 42-51和TVP23C：C51Y：45-53。
更优选的是，所述抗原表位多肽为如SEQ ID NO.10所示的NY-ESO-1 157-165或如SEQ ID NO.11所示的KRAS：G12D 10-18。
优选地，所述标记性多肽包括由可切割性连接多肽R-R-K-R所连接的n个所述抗原表位多肽，其中n为大于等于2的整数，例如n＝2、3、4、5、6、7、8、9、10、11、12…。优选的是，n为2-20之间的整数；还优选的是，n为2-10之间的整数；还优选的是，n＝3-8。例如，所述标记性多肽包括由可切割性连接多肽连接的3-8个NY-ESO-1 157-165或由可切割性连接多肽连接的3-8个KRAS：G12D10-18。
考虑到肿瘤细胞和/或癌细胞中的HLA蛋白也常出现表达缺陷，包括完全缺失，单倍型缺失或等位基因缺失，不同肿瘤类型缺失的范围从65％到90％不等(参见文献“Immunol Today.1997；18:89–95”)，因此，在一些实施方案中，所述外源基因包括所述HLA蛋白编码序列及标记性多肽编码序列，所述HLA蛋白编码序列与所述标记性多肽编码序列分别在各自的启动子控制之下，或者该HLA蛋白编码序列与所述标记性多肽编码序列在同一启动子控制之下并且该HLA蛋白编码序列通过可切割性连接多肽编码序列与所述标记性多肽编码序列可操作地连接。
优选的是，该HLA蛋白的表型与所述标记性多肽能够结合的HLA蛋白的表型一致。所述启动子可以是真核细胞启动子，包括持续表达启动子和可诱导表达启动子，包括(例如)：PGK1启动子、EF-1α启动子、CMV启动子、SV40启动子、Ubc启动子、CAG启动子、TRE启动子、CaMKIIa启动子、人β肌动蛋白(human beta actin)启动子。
连接所述HLA蛋白与所述标记性多肽之间的所述可切割性连接多肽的例子是本领域已知的，例如2A多肽，2A多肽包括但不限于来自微小核糖核酸病毒的F2A多肽、以及来自其它病毒的相似的2A类多肽；还可以是Furin-F2A连接片段。
在所述外源基因包括所述HLA蛋白编码序列及标记性多肽编码序列的一些实施方案中，优选地，HLA蛋白和标记性多肽的组合如下 所列：HLA-A-02：01蛋白与包含抗原表位肽NY-ESO-1 157-165的标记性多肽，如SEQ ID NO.15所示；HLA-C*08：02蛋白与包含抗原表位肽KRAS：G12D 10-18的标记性多肽，如SEQ ID NO.16所示；HLA-A-01：01蛋白与包含抗原表位肽KRAS：Q61H 55-64、KRAS：Q61L 55-64或KRAS：Q61R 55-64的标记性多肽；HLA-A-02：01、HLA-A-02：03或HLA-A-02：06蛋白与包含抗原表位肽NY-ESO-1 157-165、Her2/neu 369-377、NY-ESO-1 1-11、NY-ESO-1 53-62、NY-ESO-1 18-27、SSX-2 41-49、MAGE-A4 230-239、MAGE-A10 254-262、MAGE-C2 336-344、MAGE-C2 191-200、MAGE-C2 307-315、MAGE-C2 42-50、MAGE-A1 120-129、MAGE-A1 230-238、MAGE-A1 161-169、KK-LC-1 76-84、p53 99-107、PRAME 301-309、甲胎蛋白158-166、HPV16-E6 29-38、HPV16-E7 11-19、EBV-LMP1 51-59、EBV-LMP1 125-133、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、TP53：R248Q 247-255、TP53：R248Q 245-254、TP53：R248W 245-254、TP53：G245S 245-254、TP53：G249S 245-254、TP53：Y240C 217-225、TP53：R175H 168-176、CTNNB1：T41A 34-43、CTNNB1：S37Y 30-39、CTNNB1：S33C 30-39、EGFR：T790M 790-799、GNAS：R201H 197-205、CDK4：R24C 23-32、H3.3：K28M 27-36、TVP23C：C51Y 51-59或CDC73：Q254E的标记性多肽；HLA-A-03：01蛋白与包含抗原决定簇肽KRAS：G12V 8-16、KRAS：G12V 7-16、CTNNB1：S45P 41-49、CTNNB1：S45P 40-49、BRAF：V600E 591-601或TP53-V157G 156-164的标记性多肽；HLA-A11：01蛋白与包含抗原决定簇肽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、TP53：R248Q 240-249、TP53：R248W 240-249、TP53：G245S 240-249、TP53：V157F 156-164、TP53：V157F 149-158、TP53：Y163C 156-164、CTNNB1：T41A 41-49、CTNNB1：S45P 41-49、EGFR：L858R 852-860或PIK3CA：E542K 533-542的标记性多肽；HLA-A24：02蛋白与包含抗原表位肽TP53：K132N 125-134 的标记性多肽；HLA-A68：01蛋白与包含抗原表位肽TP53：R248W 240-249的标记性多肽；HLA-B-08：01蛋白与包含抗原表位肽CHD4 K73Rfs 141-148的标记性多肽；HLA-B-15：01蛋白与包含抗原表位肽TVP23C：C51Y 42-51或IDH1：R132H 126-135的标记性多肽；HLA-B58：01蛋白与包含抗原表位肽TVP23C：C51Y 45-53的标记性多肽；包含I362T和E359V突变的HLA-C-01：02蛋白与包含抗原表位肽KRAS：G12V 11-19的标记性多肽；包含I362T和E359V突变的HLA-C-07：01蛋白与包含抗原表位肽PIK3CA：H1047R 1046-1055的标记性多肽；包含I362T和E359V突变的HLA-C-08：02蛋白与包含抗原表位肽KRAS：G12D 10-19或KRAS：G12D 10-18的标记性多肽。
当所述外源基因包括所述HLA蛋白编码序列及β2-微球蛋白编码序列时，所述HLA蛋白编码序列与所述β2-微球蛋白编码序列分别在各自的启动子控制之下，或者该HLA蛋白编码序列与所述β2-微球蛋白编码序列在同一启动子控制之下，并且该HLA蛋白编码序列通过可切割性连接多肽编码序列与所述β2-微球蛋白编码序列可操作地连接。
该实施方案适用于β2-微球蛋白表达缺失的肿瘤。
所述启动子可以是真核细胞启动子，包括持续表达启动子和可诱导表达启动子，包括(例如)：PGK1启动子、EF-1α启动子、CMV启动子、SV40启动子、Ubc启动子、CAG启动子、TRE启动子、CaMKIIa启动子、人β肌动蛋白(human beta actin)启动子。
连接所述HLA蛋白与所述标记性多肽之间的所述可切割性连接多肽的例子是本领域已知的，例如2A多肽，2A多肽包括但不限于来自微小核糖核酸病毒的F2A多肽、以及来自其它病毒的相似的2A类多肽；还可以是Furin-F2A连接片段。
优选地，所述β2-微球蛋白是人源蛋白或鼠源蛋白。
还优选地，所述β2-微球蛋白的氨基酸序列如SEQ ID NO.17所示。
本发明还提供了一种用于制备所述的溶瘤腺病毒的载体，其中所述载体包含所述E1B基因调控元件、缺失了所述E1B基因编码区、 并且在所述外源基因的上游包含所述的转录E1A 13s mRNA的E1A的cDNA序列。在一个实施方案中，所述载体可以以pShuttle为骨架。pShuttle质粒来源于pBR322质粒，包含源自pZero 2.1的卡那霉素抗性基因以及插入外源基因的多克隆位点。多克隆位点两端为5型腺病毒基因组的同源重组序列。左端同源重组序列为5型腺病毒核酸序列34,931–35,935。右端同源重组序列为5型腺病毒核酸序列3,534–5,790。在一个实施方案中，所述E1A 13s序列上游为所述外源启动子。
本发明构建的溶瘤腺病毒可作为肿瘤疫苗单独使用，也可联合T细胞受体修饰的免疫细胞使用。
在溶瘤腺病毒作为肿瘤疫苗单独使用时，优选地，所述溶瘤腺病毒的所述外源基因选自标记性多肽编码序列、和HLA蛋白编码序列及标记性多肽编码序列。
本发明还提供了一种用于治疗肿瘤和/或癌症的治疗剂，包含：
(a)第一组合物，其中该第一组合物包含位于第一可药用载体中的第一活性成分，该第一活性成分包括或含有用于导入肿瘤细胞和/或癌细胞的根据本发明所述的溶瘤腺病毒；和
(b)第二组合物，其中该第二组合物包含位于第二可药用载体中的第二活性成分，该第二活性成分包含T细胞受体修饰的免疫细胞。
优选地，在所述溶瘤腺病毒表达HLA蛋白、和表达HLA蛋白及β2-微球蛋白的情况中，适用于本发明的治疗剂的肿瘤和/或癌症应表达提供抗原表位多肽的内源性肿瘤抗原，表达或不表达能提呈该抗原表位多肽的内源性HLA蛋白。
优选地，在所述溶瘤腺病毒表达标记性多肽的情况中，适用于本发明的治疗剂的肿瘤和/或癌症应表达能提呈所述标记性多肽中的抗原表位多肽的内源性HLA蛋白，表达或不表达能提供该抗原表位多肽的内源性肿瘤抗原。
优选地，在所述溶瘤腺病毒表达HLA蛋白及标记性多肽的情况中，以及在所述溶瘤腺病毒表达HLA蛋白、β2-微球蛋白及标记性多 肽的情况中，适用于本发明的治疗剂的肿瘤和/或癌症表达或不表达提供抗原表位多肽的内源性肿瘤抗原均可，并且表达或不表达能提呈该抗原表位多肽的内源性HLA蛋白均可。
在一些优选的实施方案中，所述溶瘤腺病毒载体的外源基因选自标记性多肽编码序列和HLA蛋白编码序列及标记性多肽编码序列，所述标记性多肽具有一个或多个抗原表位多肽的氨基酸序列，所述抗原表位多肽能够被MHC I类分子提呈在所述肿瘤细胞和/或癌细胞表面；并且所述T细胞受体修饰的免疫细胞能够特异性识别并结合被所述MHC I类分子提呈的所述抗原表位多肽。
优选地，所述第一组合物和所述第二组合物各自独立地存在于所述治疗剂中而互不混合。
在本发明所述的治疗剂中，被T细胞受体修饰的所述免疫细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。
所述T细胞受体包括α链和β链中的至少一者，所述α链和β链均包含可变区和恒定区，所述T细胞受体能够特异性识别肿瘤细胞和/或癌细胞表面上的所述抗原表位多肽。
TCRα链和β链的可变区用于结合抗原多肽/主要组织相容性复合体(MHC I)，分别包括三个超变区或称为互补决定区(complementarity determining regions，CDRs)，即，CDR1、CDR2、CDR3。其中CDR3区域对特异性识别被MHC分子提呈的抗原多肽至关重要。TCRα链是不同的V和J基因片段重组而成，β链则是不同的V、D和J基因片段重组而成。特定基因片段重组结合所形成的相应CDR3区域，以及结合区域回文以及随机插入的核苷酸(palindromic and random nucleotide additions)形成了TCR对抗原多肽识别的特异性(参见文献“Immunobiology:The immune system in health and disease.5 th edition,Chapter 4,The generation of Lymphocyte antigen receptors”)。所述MHC I类分子包括人HLA。所述HLA包括：HLA-A、B、C。
T细胞表达的外源TCRα链和β链有可能和T细胞本身TCR的α链和β链发生错配，不仅会稀释正确配对的外源TCR的表达量，错配TCR 的抗原特异性也不明确，因而有识别自身抗原的潜在危险，因此优选将TCRα链和β链的恒定区修饰以减少或避免错配。
在一个实施方案中，所述α链的所述恒定区和/或所述β链的所述恒定区来源于人；优选地，本发明发现所述α链的所述恒定区可以全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链的所述恒定区可以全部或部分地被来源于其它物种的同源序列所替换。更优选地，所述其它物种为小鼠。
所述替换可以增加细胞中TCR的表达量，并且可以进一步提高被该TCR修饰的细胞对Her2/neu抗原的特异性。
所述α链的所述恒定区可以修饰有一个或多个二硫键，并且/或者所述β链的所述恒定区可以修饰有一个或多个二硫键，例如1个或2个。
在具体的实施方式中，制备了两种不同方式修饰的TCR，一种方式是通过点突变在TCR恒定区增加一个二硫键，方法在文献“Cancer Res.2007 Apr 15；67(8):3898-903.”中描述，其全文通过引用方式并入本文。用小鼠TCR恒定区序列置换相应的人TCR恒定区序列的方法在文献“Eur.J.Immunol.2006 36:3052–3059”中描述，其全文通过引用方式并入本文。
优选地，所述第一组合物包含治疗有效量的所述溶瘤腺病毒。优选地，所述溶瘤腺病毒的施用剂量为5×10 7-5×10 12vp/天，每天1-2次，连续施用1-7天。
还优选地，所述第二组合物包含治疗有效量的所述的T细胞受体修饰的免疫细胞。优选地，包含每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重的所述T细胞受体修饰的免疫细胞。
所述溶瘤腺病毒可以配制成通过瘤内注射给药、腹膜内给药、蛛网膜下腔内给药、或静脉给药。
所述免疫细胞可以配制成通过动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
优选地，所述治疗剂由所述第一组合物和所述第二组合物组成。
本领域的技术人员可以理解，本发明的治疗剂还可包含合适的 可药用的辅料，包括药用或生理载体、赋形剂、稀释剂(包括生理盐水、PBS溶液)、以及各种添加剂，包括糖类、脂类、多肽、氨基酸、抗氧化剂、佐剂、保鲜剂等。
本发明还提供了一种用于治疗肿瘤和/或癌症的具有协同作用的联合药物的药盒，包括：
第一容器，该第一容器装有根据本发明所述的治疗剂中的第一组合物；
第二容器，该第二容器装有根据本发明所述的治疗剂中的第二组合物，其中所述第一容器和所述第二容器是独立的；以及
载明给药时机和给药方式的说明书。
本发明还提供了本发明所述的溶瘤腺病毒在制备用于治疗肿瘤和/或癌症的药物中的用途。
在一些实施方案中，所述溶瘤腺病毒的所述外源基因选自HLA蛋白编码序列，标记性多肽编码序列，HLA蛋白编码序列及标记性多肽编码序列，HLA蛋白编码序列及β2-微球蛋白编码序列，或HLA蛋白编码序列、β2-微球蛋白编码序列及标记性多肽编码序列。
所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
本发明还提供了本发明所述的治疗剂在制备用于治疗肿瘤和/或癌症的药物中的用途。
所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
本发明还提供了本发明所述的药盒在制备用于治疗肿瘤和/或癌症的药物中的用途。
所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
本发明还提供了一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据本发明所述的溶瘤腺病毒。
优选地，所述溶瘤腺病毒的所述外源基因选自HLA蛋白编码序列，标记性多肽编码序列，HLA蛋白编码序列及标记性多肽编码序列， HLA蛋白编码序列及β2-微球蛋白编码序列，或HLA蛋白编码序列、β2-微球蛋白编码序列及标记性多肽编码序列。
所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
优选地，所述肿瘤和/或癌症可表达提供抗原表位多肽的内源性肿瘤抗原，同时可表达提呈该抗原表位多肽的内源性HLA-I类分子。
所述溶瘤腺病毒的施用剂量为治疗有效量。优选地，所述溶瘤腺病毒的施用剂量为5×10 7-5×10 12vp/天，每天1-2次，连续施用1-7天。
所述溶瘤腺病毒可以配制成通过瘤内注射给药、腹膜内给药、蛛网膜下腔内给药、或静脉给药。
本发明还提供了一种治疗肿瘤和/或癌症的方法，包括：
对肿瘤和/或癌症患者施用根据本发明所述的治疗剂中的第一组合物；和
对所述肿瘤和/或癌症患者施用根据本发明所述的治疗剂中的第二组合物。
所述治疗剂中的第一组合物和第二组合物可以同时(例如，作为混合物同时瘤内注射)、分开但同时(例如，分别通过瘤内和静脉注射给药)或依次施用(例如，首先施用第一组合物，然后施用第二组合物；或者首先施用第二组合物，然后施用第一组合物)。
优选地，所述方法包括以下依次进行的步骤：
1)对所述肿瘤和/或癌症患者首先施用所述第一组合物；和
2)在施用所述第一组合物之后，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物。
优选地，在首先施用所述第一组合物之后的第1-30天，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物。
“在首先施用所述第一组合物之后的第1-30天，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物”是指首次第二组合物的施用与首次第一组合物施用的时间间隔为1-30天(例如，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天)，或首次第二组合物的施用与在其之前最相邻一次的所述第一组合物施用的时间间隔为1-30天(例如，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天)。优选地，首次第二组合物的施用与在其之前最相邻一次的所述第一组合物施用的时间间隔为3-14天(例如，3、4、5、6、7、8、9、10、11、12、13、14天)。
在本发明的一个优选实施方案中，第一组合物包含所述溶瘤腺病毒，所述溶瘤腺病毒的施用剂量为5×10 7-5×10 12vp/天，每天1-2次，连续施用1-7天，或上述范围间的任何值。
在本发明的一个优选实施方案中，所述T细胞受体修饰的免疫细胞的施用剂量为，每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重。优选地，1天施用1-3次，连续施用1-7天。
在某些实施方案中，所述治疗肿瘤和/或癌症的方法还包括对患者施用其它用于治疗肿瘤和/或癌症的药物，和/或用于调节患者免疫系统的药物，以增强所述T细胞受体修饰的免疫细胞在体内的数量和功能。所述其它用于治疗肿瘤和/或癌症的药物包括但不限于：化疗药物，例如环磷酰胺、氟达拉滨(fludarabine)；放疗药物；免疫抑制剂，例如环孢素、硫唑嘌呤、甲氨蝶呤、麦考酚酯(mycophenolate)、FK50；抗体，例如抗CD3、IL-2、IL-6、IL-17、 TNFα的抗体。
在某些实施方案中，所述治疗肿瘤和/或癌症的方法还包括对患者施用其它用于治疗肿瘤和/或癌症的药物，和/或用于调节患者免疫系统的药物，用于当所述T细胞受体修饰的免疫细胞产生严重毒副作用时，清除携带自杀基因的所述T细胞受体修饰的免疫细胞在体内的数量和功能。所述其它用于治疗肿瘤和/或癌症的药物包括但不限于：化学诱导二聚化(CID)药物、AP1903、磷酸化更昔洛韦(ganciclovir)、抗Cd20抗体、抗CMYC抗体、抗EGFR抗体。
所述溶瘤腺病毒可以配制成通过瘤内注射给药、腹膜内给药、蛛网膜下腔内给药、或静脉给药。
所述T细胞受体修饰的免疫细胞可以配制成通过动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
优选地，在所述溶瘤腺病毒表达HLA蛋白、和表达HLA蛋白及β2-微球蛋白的情况中，适用于本发明的治疗剂的肿瘤和/或癌症应表达提供抗原表位多肽的内源性肿瘤抗原，表达或不表达能提呈该抗原表位多肽的内源性HLA蛋白。
优选地，在所述溶瘤腺病毒表达标记性多肽的情况中，适用于本 发明的治疗剂的肿瘤和/或癌症应表达能提呈所述标记性多肽中的抗原表位多肽的内源性HLA蛋白，表达或不表达能提供该抗原表位多肽的内源性肿瘤抗原。
优选地，在所述溶瘤腺病毒表达HLA蛋白及标记性多肽的情况中，以及在所述溶瘤腺病毒表达HLA蛋白、β2-微球蛋白及标记性多肽的情况中，适用于本发明的治疗剂的肿瘤和/或癌症表达或不表达提供抗原表位多肽的内源性肿瘤抗原均可，并且表达或不表达能提呈该抗原表位多肽的内源性HLA蛋白均可。
可以根据肿瘤和/或癌症患者的自身实际情况，为患者提供本发明范围内的方法。
为了选择适用于上述溶瘤腺病毒、治疗剂和药盒治疗的患者，可以在使用上述治疗前对患者进行HLA-I分型和肿瘤抗原表达检测。
检测HLA-I类分子的配型可采用现有技术，例如，血清学配型、PCR-SSP(序列特异性priming PCR)、PCR-SSOP(序列特异性寡核苷酸探针)、PCR-RFLP(限制性片段长度多态性)、PCR-SBT(基于测序的分型)、PCR-RSCA(参考链介导的构像分析)、以及基于下一代测序(NGS)的HLA分型。
检测肿瘤抗原表达可采用现有技术，例如，检测特定肿瘤抗原的基因过度表达的qPCR和FISH(荧光原位杂交)等、检测特定肿瘤抗原的蛋白过度表达的免疫组化、流式细胞等检测手段。检测是否携带含特定点突变的肿瘤新抗原主要通过基于下一代测序(NGS)技术，包括全基因组测序和/或RNA测序，通过对比肿瘤组织和对应正常组织的序列获得somatic变体，以确定肿瘤是否携带和表达含有特定突变点的新抗原(Frnt.Immunol 2019,(24)；10:1392)。
以下通过例子的方式进一步解释或说明本发明的内容，但这些例子不应被理解为对本发明的保护范围的限制。
例子
除非特别说明，否则以下例子中所用实验方法均使用生物工程 领域的常规实验流程、操作、材料和条件进行。例如，重组质粒和病毒载体，或多肽和蛋白质可以使用标准重组方法(Green and Sambrook，分子克隆：实验室手册，第4版，冷泉港出版社，冷泉港，NY，2012)使用本文所述的核酸重组产生。
以下除非特别说明，否则各试剂的百分浓度(％)均指该试剂的体积百分浓度(％(v/v))。
实施例1-4所用材料和方法如下：
1.细胞系：
用于制备慢病毒颗粒或用作靶细胞的细胞系是293T细胞(ATCC CRL-3216)。用于呈递抗原肽的呈递细胞系是T2细胞(174xCEM.T2，ATCC CRL-1992)。用于TCR基因表达和功能分析的细胞系是JRT细胞(J.RT3-T3.5，
Figure PCTCN2021117571-appb-000003
TIB-153)。用作靶细胞的肿瘤细胞系包括：人黑素瘤细胞系A375(ATCC CRL-1619)，人纤维肉瘤细胞系HT1080(ATCC CCL-121)，人卵巢癌细胞系SKOV3(ATCC HTB-77)，人肺癌细胞系H1299(ATCC CRL-5803)，人骨肉瘤细胞系HOS-C1(ATCC CRL-1547)，人肺癌细胞系A549(ATCC CCL-185)。采用常规方法由pCDNA3.3质粒(Thermo Fisher K830001)构建得到编码NY-ESO-1蛋白的质粒pCDNA3.3-NY或编码KRAS G12D蛋白的质粒pCDNA3.3-kras/g12d，分别转染SKOV3细胞，并用500μg/mL遗传霉素(Thermo Fisher 10131027)进行选择，可以生成表达NY-ESO-1蛋白或具有G12D点突变的KRAS突变蛋白的SKOV3细胞系。NY-ESO-1(或CTAG-1B)是一种众所周知的癌-睾丸抗原(CTA)，在多种癌症类型中均过表达。下文描述了制备pCDNA3.3-NY的方法。
2.细胞培养基：
293T细胞在补充有10％胎牛血清，2mM L-谷氨酰胺的高葡萄糖(VWR目录号VWRL0101-0500)的Dulbecco改良Eagle培养基(DMEM)中培养。其他细胞系在补充胎牛血清(ATCC 30-2020)，2mmol/L L-谷氨酸，1×必需氨基酸50×(Invitrogen 11130-051)，1×链霉素/青霉素100×(Invitrogen 15140-122)，1×丙酮酸钠100×(Invitrogen 11360-070)和1×2-巯基乙醇1000×(Thermo fisher  21985023)的RPMI-1640完全培养基(Lonza，目录号12-115F)中培养。
3.外周血：
健康供体的人外周血产物来自斯坦福血液中心。根据制造商的说明，使用Ficoll-Paque PLUS密度梯度介质(GE Healthcare17144002)通过提取法(LRS小室，产品代码A1012)从残留的白细胞中产生外周血单核细胞(PBMC)。
4.表达外源蛋白的靶细胞的制备：
根据制造商的说明，用Lipofectamine 3000(Thermo Fisher L3000015)转染293T细胞或肿瘤细胞。表达质粒包括编码所述标记性多肽，外源HLA蛋白或本申请中描述的氨基酸序列的质粒。制备质粒的方法描述如下。如果将含有腺病毒基因组的质粒用于转染293T细胞，则用PacI酶(New England Biolabs，R0547S)预先消化该质粒，以释放腺病毒基因组。转染后48-72小时，瞬时表达外源蛋白的细胞可用作靶细胞。为了产生表达NY-ESO-1蛋白或具有G12D点突变的突变KRAS蛋白的稳定SKOV3细胞系，在用编码相应蛋白质的质粒转染后72小时，将SKOV3细胞与补充500μg/mL遗传素的培养基一起培养。
5.表达重组TCR的T细胞的制备：
为了产生表达TCR的活化的人T细胞，将24孔板中的PBMC细胞与补充有2μg/ml抗人CD3抗体(Biolegend 317303)和2μg/ml抗人CD28抗体(Biolegend 302914)的RPMI-1640完全培养基一起培养，持续24小时，或根据制造商的说明书用人T-CD3/CD8磁珠(Thermo Fisher 11131D)处理PBMC。24小时后，将细胞用补充有IL-2 100IU/ml，IL-7 5ng/ml，IL-15 5ng/ml的RPMI-1640完全培养基培养。为了产生用于表达TCR的T细胞系，将来自Jurkat细胞系的β链缺陷突变体JRT(J.RT3-T3.5)细胞与RPMI-1640完全培养基一起培养。为了用编码所述TCR的慢病毒感染T细胞，将活化的PBMC或JRT细胞用1ml新鲜制备的慢病毒上清液重悬于24孔板中，并在25℃下加入Polybrene(Santa Cruz Biotechnology sc-134220)。最终浓度为5–8μg/ml。将细胞在1000g和32℃下离心2小时。6小时后，将培养基更换为补充 有IL-2 100IU/ml，IL-7 5ng/ml，IL-15 5ng/ml的RPMI-1640完全培养基。也可以根据制造商的说明，使用RetroNectin皿(RetroNectin预涂皿，35mmφ)(Takara T110A)转染细胞。
6.通过流式细胞术的细胞表型分析：
为了分析用编码外源TCR的慢病毒转染的PBMC或JRT细胞表达TCR，将细胞重悬于含1％FBS的DPBS缓冲液(2.7mM KCl，1.5mM KH 2PO 4、136.9mM NaCl，8.9mM Na 2HPO 4·7H 2O，pH 7.4)中，并用APC标记的抗人CD8抗体(Biolegend 300912)和iTAg四聚体/PE-HLA-A*02：01NY-ESO-1(SLLMWITQC)(MBL International TB-M011-1)染色。流式细胞仪为MACSQuant Analyzer 10(Miltenyi Biotec Corporation)，并且通过Flowjo软件(Flowjo Corporation)分析结果。为了分析转染了编码HLA-A2蛋白的质粒的293T细胞的HLA-A2表达，将细胞用FITC抗人HLA-A2抗体(Biolegend 343303)染色并通过流式细胞术进行分析。
7.T细胞功能分析：
为了评估由JRT细胞表达的TCR的特异性和功能，根据本领域的方法，通过流式细胞术评估抗原刺激后的CD69表达(参见文献“Cytometry.1996；26(4)：305-10”)。简而言之，在96孔板的重复孔中，将TCR基因修饰的JRT细胞与靶细胞共培养16小时，例如，与以不同浓度的抗原肽浓度加载处理的T2细胞、用本申请中描述的核酸转导的293T细胞或肿瘤混合培养。将细胞用抗CD69抗体(Biolegend 310905)染色，并根据制造商的说明通过流式细胞仪分析CD69 +JRT细胞的频率。为了评估转导到PBMC中的TCR的特异性和功能，通过IFN-γELISA(酶联免疫吸附测定)测量了抗原刺激后特定T细胞的IFN-γ分泌。简而言之，在96孔板的一式两份或一式三份的孔中，将TCR基因修饰的PBMC与上述靶细胞共培养。在18-24小时内收集细胞上清液，以使用IFN-gamma ELISA Read-Set-Go试剂盒(eBioscience 88-7316)或人IFN-gamma DuoSet ELISA试剂盒(R&D Systems DY285B)根据制造商的说明进行IFN-γELISA分析。
8.重组TCR慢病毒载体的制备：
将所述TCR基因克隆到复制缺陷型慢病毒载体pCDH-EF1α-MCS-PGK-GFP(System Biosciences CD811A-1)中。通过去除载体pCDH-EF1α-MCS-PGK-GFP上的PGK启动子和GFP基因，产生了没有GFP的载体pCDH-EF1α-MCS。根据在专利申请公开US8143376B2、WO2018099402A1或参考文献“J Immunol 2010；184(9),4936-46”中描述的序列确定在HLA-A*02：01背景下对NY-ESO-1 157-165表位肽特异的TCR-α-V-D-J区域和TCR-β-V-D-J区域的序列(对于US8143376B2表示为TCR-NY-LY，对于WO2018099402A1表示为TCR-NY-LI，对于参考文献J Immunol 2010；184(9),4936-46表示为TCR-NY-AE)。在HLA-C*08：02的背景下，对KRAS：G12D 10-18肽具有特异性的TCR-α-V-D-J区和TCR-β-V-D-J区的序列是根据专利申请公开WO2018026691(表示为TCR-RAS G12D)确定的。根据参考序列(分别为GeneBank KU254562和EF154514.1)确定小鼠TCR-α恒定链和小鼠TCR-β恒定链的序列。针对NY-ESO-1 157-165的TCR-NY-LY、TCR-NY-AE和TCR-NY-LI的核酸序列，针对KRAS:G12D 10-18的TCR-RAS-G12D的核酸序列(分别包含具有小鼠TCR-β恒定链的TCRβ链、具有小鼠TCR-α恒定链的TCRα链、编码furin蛋白酶切割肽的接头核酸、以及TCRα和β链之间的F2A肽的核酸)分别显示在SEQ ID NO.18、19、20或21中，并且由Integrated DNA Technologies或LifeSct LLC合成。根据制造商的说明，将合成的核酸克隆到慢病毒载体pCDH-EF1α-MCS-PGK-GFP或pCDH-EF1α-MCS(不含GFP)的EF-1α启动子下游的多克隆位点。表达针对NY-ESO-1 157-165表位的TCR的慢病毒载体称为pCDH-EF1α-TCR-NY。对插入的核酸进行测序，未发现错误和突变。将慢病毒载体质粒转化到感受态细菌stellar(Takara Bio，636763)中，以制备用于制备慢病毒颗粒的质粒储备液。
9.重组TCR慢病毒颗粒的制备：
TCR慢病毒颗粒是由293T或293FT细胞(Thermo Fisher R70007)产生的，所述细胞被含有所述TCR基因的慢病毒载体质粒转染。简而言之，按照制造商的说明，使用Lipofectaine 3000转染试剂(invitrogen， 11668019)，用TCR慢病毒载体质粒和pPACKH1-lentivector包装试剂盒(System Biosciences LV500A-1)共转染6孔板中生长的293T或293FT细胞。培养48小时后，收集上清液并通过0.4μm滤膜过滤。根据制造商的说明，用Lenti-X TM浓缩器(Takara，631231)浓缩病毒上清液。新鲜制得的TCR-慢病毒用于感染JRT细胞或活化的PBMC。
10.编码NY-ESO-1蛋白或突变体KRAS G12D蛋白的表达载体的制备：
将根据制造商的说明，使用PureLink TMRNA Mini试剂盒(Thermo Fisher 12183020)从HT1080细胞(NY-ESO-1+，KRAS野生型)中纯化的总RNA用作使用PrimeScript TMRT-PCR试剂盒(Takara RR014A)产生RT-PCR产物的模板。NY-ESO-1全长基因是通过以下PCR引物对产生的：5'-TATATAAGAGCAGAGCTGCCACCATGCAGGCCGAAGGCCGGGGCA-3'(SEQ ID NO.22)和5'-TGATTGTCGACGCCCTTAGCGCCTCTGCCCTGAGGGAGGCTG-3'(SEQ ID NO.23)。KRAS G12D全长基因是通过以下PCR引物对产生的：5'-ATGACTGAATATAAACTTGTGGTAGTTGGAGCTGACGGCGTAGGCAAGAGTGCCTTG-3'(SEQ ID NO.24)和5'-TGATTGTCGACGCCCTTACATAATTACACACTTTGTCTTTGACTTC-3'(SEQ ID NO.25)。根据制造商的说明，将所得基因克隆到pCDNA3.3载体(Thermo Fisher K830001)的TOPO克隆位点。根据制造商的说明，使用
Figure PCTCN2021117571-appb-000004
High-Fidelity 2X PCR Master Mix(New England Biolabs M0541L)进行本申请中描述的PCR。
11.编码所述标记性多肽和/或外源HLA I类分子的复制缺陷型重组腺病毒质粒的制备：
本申请中描述的复制缺陷型重组腺病毒系统基于AdEasy系统(Nature Protocols 2007；2：1236-1247)。EF1α启动子的核酸通过以下PCR引物对从pCDH-EF1α-MCS-PGK-GFP质粒(System Biosciences，CD811A-1)中克隆，引物对为：5'- CTCATAGCGCGTAATGGCTCCGGTGCCCGTCAGTGGGCAG-3'(SEQ ID NO.26)和5'-GAATTCGCTAGCTCTAGATCACGACACCTGAAATGGAAG-3’(SEQ ID NO.27)，并且将其整合到pShuttle-CMV载体(Agilent Technologies，目录号24007)中以取代CMV启动子以产生pShuttle-EF1α载体。编码全长HLA-A*02：01的cDNA(如SEQ ID NO.28中所示)和编码人β2-微球蛋白的cDNA由T2细胞产生，并根据制造商的说明克隆到pcDNA3.3-TOPO载体中。SEQ ID NO.29所示的编码包含3个抗原表位肽NY-ESO-1 157-165的标记性多肽的核酸和SEQ ID NO.30所示的编码接头肽的核酸由Integrated DNA Technologies合成。为了制备如SEQ ID NO.31所示的编码将HLA-A*201蛋白与接头肽连接的标记性多肽的核酸，进行如下步骤：使用合成的编码标记性多肽的核酸作为模板，用引物对5'-AGAGCTAGCGAATTCAACATGAAAGGTTCCATCTTCAC-3'(SEQ ID NO.32)和5'-ACACTGTGTAATCCACATCAATAGCGATCTCTTTC-3'(SEQ ID NO.33)进行PCR，以产生编码带有NY-ESO-1 157-165的标记性多肽的核酸片段(表示为NY)；使用合成的编码接头肽的核酸作为模板，以以下引物对进行PCR：5'-TGGATTACACAGTGTCGTCGTAAGCGATCCGGAAGCGCG-3'(SEQ ID NO.34)和5'-CGCCATGACGGCCATGGGCCCAGGGTTGGACTCGACGTC-3'(SEQ ID NO.35)，以生成编码接头肽的核酸；然后使用编码全长HLA-A*02：01的cDNA对5'-ATGGCCGTCATGGCGCCCCGA-3'(SEQ ID NO.36)和5'-TCACACTTTACAAGCTGTGAGAGACAC-3'(SEQ ID NO.37)引物进行PCR生成HLA-A*201基因(称为A2)。混合以上纯化的PCR产物作为模板，以使用以下引物对进行PCR：5'-ATGAAAGGTTCCATCTTCACATTGTTTTTGTTC-3'(SEQ ID NO.38)和5'-CGCCATGACGGCCATGGGCCCAGGGTTGGACTCGACGTC-3'(SEQ ID NO.39)，以生成编码将HLA-A*201蛋白与接头肽连接的标记性多肽的核酸。用本领域常规基因克隆技术将所得核酸克隆到 pShuttle-EF1α载体的多克隆位点，以产生pShuttle-EF1α-NY-A2。
12.重组腺病毒质粒的制备
pShuttle载体用PmeI(NEB Biolabs，R0560s)线性化。纯化后，按照生产商的说明在Bio-Rad Gene浓度加载发生器电穿孔仪中以2500V，200Ω和25μF的频率输送浓度加载，将载体转入具有电活性的BJ5183-AD-1细菌菌株(Agilent Technologies，200157)。用PacI(NEB Biolabs R0547S)限制性消化以筛选潜在的腺病毒重组体。正确的重组体通常会产生一个较大的片段(约30kb)，和一个3.0或4.5kb的较小片段。
13.编码所述标记性多肽和/或外源性HLA I类分子的有条件复制能力的腺病毒质粒的制备：
为了产生pShuttle载体，其包含由外源启动子驱动的腺病毒E1A基因和由天然E1B启动子驱动的编码与所述标记性多肽结合的HLA分子或与β2-微球蛋白结合的HLA分子的核酸的pShuttle载体，进行如下程序：由Integrated DNA Technologies合成如SEQ ID NO.2所示的序列，其中包含天然E1B启动子序列和编码含有24个碱基对缺失(E1A 122-129缺失)的E1A-13s蛋白核酸序列(具有E1A polyA添加位点的天然E1A polyA信号序列)。以合成的基因序列作为模板，使用以下引物对进行PCR：5'-ATGAGACATATTATCTGCCACGGAG-3'(SEQ ID NO.46)和5'-CATGGTGGCGAGGTCAGATGTAAC-3'(SEQ ID NO.47)，获得包含具有24个碱基对缺失的E1A-13s核酸序列和天然E1B启动子序列的基因片段(表示为E1Ad24)。使用编码全长HLA-A*02：01的cDNA或编码人β-2微球蛋白的cDNA作为模板进行PCR(此时引入Kozak序列)，获得了HLA-A*02：01基因片段(表示为A2)和β-2微球蛋白基因片段(表示为BM)。合成的突变HLA-C*08：02基因如SEQ ID NO.49所示(表示为C08)，合成的核酸编码包含3个NY-ESO-1 157-165表位肽的标记性多肽，如SEQ ID NO.29所示(表示为NY)，或编码包含3个KRAS：G12D 10-18表位肽的标记性多肽，如SEQ ID NO.50所示(表示为RAS)，或合成的核酸编码接头肽，如SEQ ID NO.30(表示为F2A)，将其用作模板以 进行PCR以获取相应的基因片段(将突变HLA-C*08：02基因用作模板进行PCR时引入Kozak序列)。根据制造商的说明，使用In-Fusion HD Cloning Plus试剂盒(Takara 638909)生成包含E1Ad24-A2-F2A-NY，E1Ad24-C08-F2A-RAS，E1Ad24-A2-F2A-BM和E1Ad24-C08-F2A-BM的核酸片段的组合。将所得基因组合克隆到pShuttle-EF1α载体中EF1α启动子的下游，以生成pShuttle-EF1α-E1Ad24-A2-F2A-NY，pShuttle-EF1α-E1Ad24-C08-F2A-RAS，pShuttle-EF1α-E1Ad24-C08-F2A-BM和pShuttle-EF1α-E1Ad24-A2-F2A-BM。为了产生重组腺病毒质粒，如上所述，通过在Bio-Rad Gene浓度加载电穿孔仪中以2500V，200Ω和25μF的频率输送浓度加载，将pShuttle载体用PmeI线性化并转入具有电活性的BJ5183-AD-1。所得重组溶瘤腺病毒质粒表示为pAd-EF1α-E1Ad24-A2-F2A-NY，pAd-EF1α-E1Ad24-C08-F2A-RAS，pAd-EF1α-E1Ad24-C08-F2A-BM，和pAd-EF1α-E1Ad24-A2-F2A-BM。
14.编码所述标记性多肽和/或外源HLA I类分子的重组腺病毒的制备：
将采用上述方法得到的复制缺陷型重组腺病毒质粒和有条件复制能力的重组溶瘤腺病毒质粒，通过本方法制备得到相应的重组腺病毒。
用Pac I(NEB Biolabs R0547S)消化重组腺病毒质粒以释放腺病毒基因组DNA。线性化的质粒通过苯酚/氯仿提取进行纯化，并根据制造商的说明用Lipofectaine 3000转染试剂(Thermo Fisher L3000001)转染ADENO-X 293细胞(Takara 632271)。转染的细胞在37℃，5％CO 2的培养箱中保持14–20天，直到观察到细胞病变效应(CPE)。进行四个冻融涡旋循环以从细胞中释放腺病毒并获得病毒颗粒。通常需要两到四轮扩增才能生成大规模制备的高滴度病毒。制备大规模腺病毒的程序遵循参考文献中所述的方法(Nat Protoc 2007；2(5),1236-47)。腺病毒滴度由Adeno-X GoStix试剂盒(Takara 632270)根据制造商的说明确定。为了用重组腺病毒感染靶细胞，基于确定的MOI(感染复数，指每个细胞感染的病毒颗粒的数量)确定病毒滴度的数量和靶细 胞的数量。通常，感染后3-4天可以检测到外源基因的表达。
实施例5-9和制备例1所用材料和方法如下：
人卵巢癌细胞株SKOV3、人肺癌细胞株H1299、人骨肉瘤细胞株HOS C1购自ATCC；
人黑素瘤细胞株A375购自中国科学院细胞库；
人IFN-gamma ELISA试剂盒购自R&D公司；
xCELLigence RTCA S16实时无标记细胞功能分析仪购自ACEA Bio公司；
APC结合的抗人HLA-A2的抗体购自BD公司，PE结合的抗人CD3的抗体购自BD公司；
Dynabeads TM Human T-Activator CD3/CD28购自Thermofisher公司；
hrIL-2购自金丝利公司，hrIL-7和hrIL-15购自近岸蛋白公司；
流式细胞仪Novocyte购自ACEA Bio公司。
实施例1
该实施例证明，外源基因可以通过本申请中描述的基因构建体有效表达，所述构建体包括包含编码所述标记性多肽和/或外源HLA I类分子的核酸的腺病毒载体；以及包括编码T细胞受体的核酸的慢病毒载体。图1A示出了本申请中描述的构建体的示意图。pAdEasy-EF1α-NY-A2是复制缺陷型腺病毒载体，表达如SEQ ID NO.51所示的所述标记性多肽，其中包含3个Furin蛋白酶切位点所联接的NY-ESO-1 157-165表位肽，以及如SEQ ID NO.12所示的HLA-A2蛋白。标记性多肽和HLA-A2基因之间有F2A序列所连接。所述标记性多肽的表达单元的两侧是外源EF-1α启动子和SV40 poly(A)信号序列。pAd-EF1α-E1A-A2-F2A-NY是有复制能力的腺病毒载体，其包含编码HLA-A2基因和3个NY-ESO-1 157-165表位肽的所述标记性多肽的核酸。pAd-EF1α-E1A-A2-F2A-BM是具有复制能力的腺病毒载体，其包含编码HLA-A2基因和人β2-微球蛋白的核酸。pAd-EF1α-E1Ad24-A2-F2A-NY和pAd-EF1α-E1Ad24-A2-F2A-BM构建 体均包含编码E1-13S突变蛋白的核酸(即转录E1A 13s mRNA的E1A的cDNA序列)，该E1A蛋白含有24个残基的缺失，如SEQ ID NO.43所示。所述E1A-13s基因两侧为分别为外源EF-1α启动子和天然E1A poly(A)信号序列。在这两个有复制能力的构建体中，删除了E1B基因区域，并插入所述编码标记性多肽和其他外源基因的核酸到该区域中，并利用天然E1B调控元件，包括天然E1B启动子和E1B/IX poly(A)信号，驱动外源基因的表达。图1B为慢病毒载体pCDH-EF1α-TCR-NY-LY，pCDH-EF1α-TCR-NY-AE和pCDH-EF1α-TCR-NY-LI的示意图，其中包含编码各NY-ESO-1特异性TCRβ链和α链多肽的核酸(所述多肽分别如SEQ ID NO.52、53、54所示)。TCRβ链和α链的恒定区均被鼠TCR恒定区序列取代。TCRβ链和α链用多肽Furin蛋白酶切点和F2A联接序列所联接，其两端是EF-1α启动子和慢病毒转录后调控元件(WPRE)。
为了评估重组构建体是否可以表达外源基因，用包含表达单元的pShuttle-EF1α-NY-A2，pShuttle-EF1α-E1Ad24-A2-F2A-NY或pShuttle-EF1α-E1Ad24-A2-F2A-BM(它们的两侧分别是与5型腺病毒同源的基因组序列)转染内源HLA-A2在细胞表面低表达的293T细胞。用抗HLA-A2抗体对细胞染色，并通过流式细胞术评估HLA-A2的表达。图1C显示，用包含HLA-A2基因的所有三种构建体转染的293T细胞可以表达HLA-A2，表明构建体中的调控元件具有驱动外源蛋白表达的功能，并且通过Furin蛋白酶和F2A联接序，HLA-A2蛋白可以被分离出来并表达在细胞表面。pShuttle-EF1α-NY-A2转导的293T细胞中HLA-A2表达的荧光强度低于pShuttle-EF1α-E1Ad24-A2-F2A-NY和pShuttle-EF1α-E1Ad24-A2-F2A-BM转导的293T细胞的荧光强度，表明天然E1B启动子和E1B poly(A)信号可能在腺病毒基因组的背景下更有效地驱动外源基因的表达。
为了评估所构建的重组慢病毒载体是否表达TCR，通过pCDH-EF1α-TCR-NY载体转染的293T细胞制备表达NY-ESO-1特异性TCR的慢病毒颗粒，并用于感染J.RT3-T3.5细胞(JRT细胞，ATCC TIB153)。感染的JRT细胞用抗CD8抗体和NY-ESO-1 157-165/ HLA-A2四聚体染色，该四聚体可以特异性结合可特异性识别被HLA-A2提呈的NY-ESO-1 157-165表位多肽的TCR。图1D显示用慢病毒转染的JRT细胞表达能够结合NY-ESO-1 157-165/HLA-A2四聚体的不同TCR，这表明重组慢病毒表达由TCRα和β链组成的特异性TCR。α和β链通过包含的鼠恒定区配对以形成TCR/CD3复合物并在JRT细胞的表面表达。
实施例2
该实施例证明，所述构建体载体一旦表达标记性多肽和外源HLA I类蛋白，抗原表位肽便可以从标记性多肽中释放并由外源HLA I类分子呈递，形成抗原肽/HLA I类复合物，该复合物可被特异性TCR识别。
为了评估JRT细胞所表达的特异性TCR识别由外源HLA I类分子提呈的抗原表位肽的能力，首先用重组慢病毒转染JRT细胞，所述重组慢病毒由共转染pCDH-EF1α-TCR-NY-LY，pCDH-EF1α-TCR-NY-AE或pCDH-EF1α-TCR-NY-LI和上述包装载体的293T细胞产生。将表达特异性TCR的JRT细胞与HLA-A2阳性的T2细胞共培养，在培养孔中加入从1μg/ml开始的10倍稀释系列的NY-ESO-1 157-165多肽。抗原刺激16-24小时后，通过流式细胞术分析CD69阳性的JRT细胞的百分比。图2A显示，表达特异性TCR的JRT细胞在用抗原肽刺激后表达CD69，表明JRT细胞被HLA-A2呈递的NY-ESO-1 157-165肽所激活，其活性表现出抗原剂量依赖的关系。另外，也显示出不同TCR对NY-ESO-1 157-165抗原表位识别敏感性的差异，TCR-NY-LY和TCR-NY-AE识别活性相似，但TCR-NY-LI对NY-ESO-1 157-165抗原表位多肽的敏感性低约10倍。
用NY-ESO-1 157-165抗原多肽特异性TCR转染的JRT细胞作为效应细胞，用pShuttle-EF1a-NY-A2或pShuttle-EF1a-E1Ad24-A2-NY转染HLA-A2阴性和NY-ESO-1阴性的293T细胞作为靶细胞，用空的pShuttle载体转导的293T细胞用作对照靶细胞，以评估转染表达标记性多肽和外源HLA-A2的载体的靶细胞是否在靶细胞表面表达被特异性T细胞所识别的抗原表位多肽。图2B显示pShuttle-EF1a-NY-A2或 pShuttle-EF1a-E1Ad24-A2-NY转染的293T细胞均可激活表达不同的NY-ESO-1 157-165多肽特异性TCR的JRT细胞。与用空的pShuttle载体转导的对照靶293T相比，针对表达上述标记性多肽和HLA-A2的293T靶细胞，CD69+JRT细胞的百分比显着增加(学生t检验，p<0.01)。结果显示标记性多肽和外源HLA-A2在293T细胞中分别表达，并形成可被特异性TCR所识别的HLA—A2/NY-ESO-1 157-165多肽复合物。在表达TCR-NY-LY和TCR-NY-AE的JRT细胞为效应细胞的组中，与pShuttle-EF1α-NY-A2转染的靶细胞相比，用pShuttle-EF1α-E1Ad24-A2-F2A-NY转染的靶细胞能够诱导出更多的CD69+JRT细胞(学生t检验，p<0.05)。此结果与pShuttle-EF1α-E1Ad24-A2-F2A-NY转染293T细胞后HLA-A2的高表达相一致，这表明腺病毒基因组DNA中，天然的E1B启动子和E1B poly(A)信号可以有效地驱动外源基因的表达。
为了评估所述构建体表达的外源HLA I类分子提呈来自内源性抗原蛋白的抗原表位肽的功能，将293T细胞共转染pCDNA3.3-NY和pShuttle-EF1a-E1A d 24-A2-F2A-BM，并用作靶细胞。pCDNA3.3-NY编码全长NY-ESO-1蛋白，使293T细胞表达NY-ESO-1蛋白作为内源性肿瘤抗原，从而产生NY-ESO-1 157-165表位肽。如果NY-ESO-1蛋白可以通过HLA I类抗原加工途径提供NY-ESO-1 157-165表位多肽，并被外源性HLA-A2所提呈，则靶细胞可以被表达特异性TCR的JRT细胞所识别。用只转染pCDNA3.3-NY或pShuttle-EF1a-E1Ad24-A2-F2A-BM的293T细胞为阴性对照。图2C显示，单独用pShuttle-EF1a-E1A 24-A2-F2A-BM转导的293T细胞不能激活表达NY-ESO-1特异性TCR的JRT细胞。然而，当靶细胞被1μg/ml浓度的NY-ESO-1 157-165表位多肽加载处理时，可以诱导大量表达各种TCR的JRT细胞表达CD69，这表明外源性HLA-A2在靶细胞中表达，并可呈递抗原多肽以激活T细胞。此外，与阴性对照靶细胞相比，当靶细胞同时表达NY-ESO-1蛋白和HLA-A2分子时，表达NY-ESO-1特异性TCR的JRT细胞混合培养物，可以诱导出更多的CD69+JRT细胞(学生t检验，p<0.05)。结果表明，NY-ESO-1 157-165表位多 肽可以通过内源性HLA I类加工机制产生，并由外源性HLA-A2提呈后被特异性TCR所识别。因此，将外源性HLA I类分子作为同种异体HLA(allo-HLA)引入肿瘤细胞，以提呈来自内源性蛋白的抗原表位肽是可行的。所述产生抗原表位多肽的内源性蛋白可以是过度表达的肿瘤相关抗原或由突变蛋白产生的肿瘤新生抗原(neo-antigen)。
为了进一步评估在重组腺病毒基因组DNA框架内编码标记性多肽和/或HLA I类分子的核酸是否可以表达外源多肽和蛋白质，腺病毒载体pAd-EF1a-E1Ad24-A2-NY用PacI消化，以获得重组腺病毒基因组DNA，包含外源的编码HLA-A2蛋白和具有NY-ESO-1 157-165表位多肽的标记性多肽的核酸。pAd-EF1a-E1A d 24-A2-BM也用PacI消化，以获得表达外源HLA-A2和β2-微球蛋白的腺病毒基因组DNA。用腺病毒DNA转染293T细胞，48小时后用作靶细胞，以刺激表达NY-ESO-1特异性TCR的JRT细胞。图2D显示pAd-EF1a-E1Ad24-A2-BM在293T细胞中表达外源HLA-A2，并且不仅能够提呈以1μg/ml浓度所加载的NY-ESO-1 157-165表位多肽，而且还能够提呈来自NY-ESO-1蛋白的NY-ESO-1 157-165表位多肽。与仅转染pAd-EF1a-E1Ad24-A2-BM的对照靶细胞相比，CD69+JRT细胞的百分比显著增加(学生t检验，p<0.01)。转染pAd-EF1a-E1Ad24-A2-NY的腺病毒DNA的293T细胞同时表达带有NY-ESO-1 157-165表位的标记性多肽和外源性HLA-A2，也可以激活表达NY-ESO-1特异性TCR的JRT细胞。与同时转染pCDNA3.3-NY/pAd-EF1a-E1Ad24-A2-BM的靶细胞相比，转染pAd-EF1a-E1Ad24-A2-NY的靶细胞活化JRT-TCR-NY-LY细胞的能力相似。尽管更多的JRT-TCR-NY-AE细胞被pAd-EF1a-E1Ad24-A2-NY的靶细胞所激活，但两组之间没有显著性差异。这些结果表明，如果在靶细胞中的HLA I类抗原加工和呈递机制和功能完整，则抗原表位肽可以由内源性蛋白质所产生，被所述载体引入靶细胞的外源HLA I类分子有效呈递，并被特异性T细胞所识别。外源HLA I类分子可以是自体HLA(auto-HLA)也可以是异体HLA(allo-HLA)
实施例3
该实施例证实了当用含有编码所述标记性多肽和HLA I类蛋白的核酸的基因构建体转导肿瘤细胞时，抗原表位肽可由肿瘤细胞中的外源HLA I类分子呈递。无论肿瘤细胞是否表达特定的肿瘤抗原或具有提呈特定抗原表位多肽的HLA I类等位基因，表达所述标记性多肽和外源HLA I类分子可以使肿瘤细胞成为被特异性T细胞识别的靶细胞。此外，表达外源的异体HLA I类蛋白，不仅可以增加肿瘤细胞的免疫原性，还可以提呈来自内源性肿瘤抗原的抗原表位肽，并激活HLA限制的抗原表位多肽特异性的TCR。
A375是人黑素瘤细胞系，代表HLA-A2 +和NY-ESO-1 +肿瘤细胞。SKOV3是人类卵巢癌细胞系，代表HLA-A2 -和NY-ESO-1 -双阴性肿瘤细胞。转导pCDNA3.3-NY并稳定表达NY-ESO-1蛋白的SKOV3细胞代表HLA-A2阴性和NY-ESO-1阳性肿瘤细胞。用pShuttle-EF1a-E1Ad24-A2-NY或pShuttle-EF1a-E1Ad24-A2-BM转染这些肿瘤细胞，用作靶细胞，与表达NY-ESO-1特异性TCR的JRT细胞共培养。
图3显示JRT-TCR-NY-LY和JRT-TCR-NY-AE均可被A375细胞激活，这表明可由内源性NY-ESO-1蛋白产生NY-ESO-1 157-165表位肽，并由其自身的HLA-A2分子所提呈。尽管用pShuttle-EF1a-E1Ad24-A2-BM转染的A375细胞表达的HLA-A2较多，但不能显著提高该细胞被特异性TCR识别的敏感性，这表明NY-ESO-1157-165肽/HLA-A2复合物在A375细胞上呈递的数量不受HLA-A2数量的限制，但受HLA I类抗原加工机制所产生的NY-ESO-1 157-165多肽的数量限制。与对照A375细胞相比，用pShuttle-EF1a-E1A d24-A2-NY转导的A375细胞显著增加了CD69 +JRT-TCR-NY-LY细胞的百分比(学生t检验，p<0.05)，表明所述标记性多肽可以避开与抗原加工有关的转运蛋白(TAP)，将抗原肽递送到内质网(ER)的腔中，并被结合到HLA I类分子上。NY-ESO-1 -/HLA-A2 -双阴性SKOV3细胞和用pShuttle-EF1a-E1Ad24-A2-BM转导得到的NY-ESO-1 -/HLA-A2 +SKOV3细胞不能激活JRT-TCR-NY-LY和 JRT-TCR-NY-AE细胞。pShuttle-EF1a-E1Ad24-A2-NY可以使SKOV3细胞被表达NY-ESO-1特异性TCR的JRT细胞所识别(学生t检验，p<0.01)，表明无论内源性抗原或匹配的HLA I类等位基因的表达状态如何，所述溶瘤病毒载体转染肿瘤细胞后所表达的抗原表位肽能够和HLA-A2分子相结合并被特异性TCR所识别。pShuttle-EF1a-E1Ad24-A2-BM转导的SKOV3-NY细胞可以激活NY-ESO-1特异性TCR的JRT细胞，表明引入肿瘤细胞的外源HLA I类分子也可以使肿瘤细胞被特异性TCR识别，但肿瘤细胞必需表达肿瘤抗原以作为产生抗原肽的来源。另外，肿瘤细胞中还需要有完整的抗原加工和呈递机制，以便处理肿瘤抗原蛋白以产生抗原表位肽并被外源HLA I类分子所提呈。与转染pShuttle-A2-F2A-BM的靶细胞SKOV3-NY相比，转染pShuttle-A2-F2A-NY的靶细胞激活更多的JRT-TCR-NY-LY细胞(学生t检验，p<0.05)，说明和内源抗原蛋白相比，标记性多肽产生更多的NY-ESO-1 157-165表位多肽。
实施例4
该实施例表明，用所述编码标记性多肽和/或外源HLA I类分子的核酸转染的肿瘤细胞可以被表达特异性TCR的原代T细胞(primary T cell)识别。
用表达NY-ESO-1特异性TCR的重组慢病毒(包括TCR-NY-LY，TCR-NY-AE或TCR-NY-LI)转染HLA-A2阴性PBMC。转染后7-10天，PBMC用抗CD8-APC和NY-ESO-1 157-165四聚体-PE染色。图4A显示了用所有三个TCR基因转导的T细胞可以表达被NY-ESO-1157-165四聚体标记的NY-ESO-1 157-165特异性TCR。在CD8 +和CD8 -细胞群体中均观察到NY-ESO-1 157-165四聚体阳性细胞。虽然CD8 +细胞是NY-ESO-1特异的杀伤性T细胞(CTL)，CD8 -细胞可能是表达NY-ESO-1特异性TCR的CD4 +T辅助细胞。该结果表明TCR-NY-LY，TCR-NY-AE和TCR-NY-LI与NY-ESO-1 157-165/HLA-A2复合物具有高结合亲和力，并且不依赖CD8的辅助功能。将表达NY-ESO-1特异性TCR的PBMC用作效应细胞，以评估表达所述 标记性多肽和外源HLA I类分子的肿瘤细胞的对特异性T细胞的识别敏感性。
A375细胞系，SKOV3细胞系和表达全长NY-ESO-1蛋白的SKV3-NY细胞分别代表NY-ESO-1和HLA-A2双阳性，NY-ESO-1和HLA-A2双阴性和NY-ESO-1单阳性的靶细胞。用pShuttle-EF1a-E1Ad24-A2-NY或pShuttle-EF1a-E1A d24-A2-BM转染这些靶细胞，作为靶细胞以评估表达所述标记性多肽和外源性HLA-A2使肿瘤细胞被原代T细胞上表达的特异性TCR的识别功能。将重组慢病毒转染的PBMC与靶细胞以10:1的E:T比率共孵育16-24小时。检测上清液中IFN-γ的分泌，以评价靶细胞刺激T的能力。图4B显示，用pShuttle-EF1a-E1A d24-A2-BM转染的所有靶细胞都可以有效地提呈NY-ESO-1 156-165多肽，并激活表达特异性TCR的T细胞，表明由pShuttle-EF1a-E1A d24-A2-BM载体表达的外源HLA-A2蛋白结合外源的NY-ESO-1 156-165多肽形成NY-ESO-1 156-165/HLA-A2复合物，并被表达NY-ESO-1特异性TCR的原代T细胞所识别。转染pShuttle-EF1a-E1A d24-A2-NY和pShuttle-EF1a-E1A d24-A2-BM的A375和SKOV3-NY细胞均可以被表达特异性TCR的T细胞所识别。而SKOV3细胞只有转染同时表达HLA-A2分子和NY-ESO-1 157-165表位肽的pShuttle-EF1a-E1Ad24-A2-NY时才能被表达特异性TCR的T细胞所识别。结果证实，所述表达含抗原表位肽的标记性多肽以及外源HLA I类分子的溶瘤病毒载体不仅可以使肿瘤细胞被表达特异性TCR的JRT细胞所识别，还可以被表达特异性TCR的原代T细胞所识别。
为了评估是否可以通过用编码所述标记性多肽和/或外源HLA I类分子的核酸转导其他类型的肿瘤细胞，人肺癌细胞系H1299(NY-ESO-1 +/HLA-A2 -)，人骨肉瘤细胞系HOS-C1(NY-ESO-1低/HLA-A2 +)和人肺癌细胞系A549(NY-ESO-1 -/HLA-A2 -)用pShuttle-NY-A2载体转导并用作靶细胞。用表达TCR-NY-LY或TCR-NY-LI的重组慢病毒转染的PBMC作为效应细胞。空白对照效应细胞是用空的慢病毒转染的PBMC。将效应细胞或空白对照细胞与靶细胞以E:T＝5:1 的比例孵育24小时。用ELISA检测上清液中IFN-γ的分泌。如图4C所示，与用空白对照T细胞的对照组相比，表达TCR-NY-LY或TCR-NY-LI的特异性T细胞产生了大量的IFN-γ(学生t检验，p<0.01)，表明所测试的肿瘤细胞都能够被表达含NY-ESO-1 157-165抗原表位肽的标记性多肽和外源性HLA-A2蛋白所标记并被表达NY-ESO-1特异性TCR的T细胞所识别。
实施例5：重组溶瘤腺病毒OAd-NY/A2感染SKOV3细胞后HLA-A2的表达
下文所述重组溶瘤腺病毒“OAd-NY/A2”均是根据前述方法，将质粒pAd-EF1α-E1Ad24-A2-F2A-NY包装得到的重组溶瘤腺病毒。
本实施例验证了重组溶瘤腺病毒OAd-NY/A2感染肿瘤细胞后，细胞表面可以表达HLA-A2。第0天，按照5×10 4个SKOV3细胞/孔接种24孔板。第1天，分别按照5、10、20、50、100MOI向SKOV3细胞中加入重组溶瘤腺病毒OAd-NY/A2。继续培养48小时后，收获细胞。用APC偶联的抗人HLA-A2抗体(BD公司)(1:50稀释)进行染色，使用流式细胞仪Novocyte(购自ACEA Bio公司)检测细胞表面HLA-A2的表达。
结果如图5所示，可成功地检测到HLA-A2的表达，并且随着MOI的增加，HLA-A2的表达比例也增加。
制备例1：Mock-T和靶向NY-ESO-1的TCR-T细胞的制备
T细胞培养液为AIM V+5％FBS+100IU/ml hrIL-2+10ng/ml hrIL-7+10ng/ml hrIL-15。第0天，复苏人PBMC(得自杭州康万达医药科技有限公司)，按照5×10 5个细胞/ml将PBMC接种至24孔板的一个孔中，1ml/孔，按照T:珠子＝1:1加入dynabeads。活化6小时后，按照MOI＝5加入GFP慢病毒或TCR-NY-AE慢病毒，分别用于制备下述实施例中的Mock-T和靶向NY-ESO-1的TCR-T细胞。第3天，更换全部培养基，按照5×10 5-1×10 6个细胞/ml调整细胞密度。之后每隔1-2天，补液，按照5×10 5-1×10 6个细胞/ml调整细胞密度。培养到 第10天，收获全部细胞，计数，通过流式细胞术检测Mock-T以及TCR-T细胞中GFP的表达比例，利用PE结合的抗人CD3抗体检测Mock-T以及TCR-T细胞中CD3的表达比例。
实施例6：靶向NY-ESO-1的TCR-T与重组溶瘤腺病毒OAd-NY/A2感染标记的肿瘤细胞共培养后细胞因子的分泌
本实施例通过ELISA检测了靶向NY-ESO-1的TCR-T细胞与重组溶瘤腺病毒OAd-NY/A2感染标记的肿瘤细胞共培养过夜后，IFNγ的分泌。检测的肿瘤细胞株包括人黑素瘤细胞株A375，人肺癌细胞株H1299，人卵巢癌细胞株SKOV3，人骨肉瘤细胞株HOSC1。第0天，按照1×10 5个肿瘤细胞/孔接种12孔板。第1天，向相应的肿瘤细胞中加入80μl的重组溶瘤腺病毒OAd-NY/A2。第2天，用胰蛋白酶将肿瘤细胞以及重组溶瘤腺病毒OAd-NY/A2感染标记的肿瘤细胞消化计数，按照1×10 5个肿瘤细胞/孔接种于96孔板。将靶向NY-ESO-1的TCR-T细胞或Mock-T细胞(表达GFP，作为TCR-T的阴性对照组)按照3×10 5个/孔接种于96孔板相应的孔中，上述效应细胞与靶标细胞的个数比例(E:T)为3:1，每组3个复孔。共培养过夜后，用人IFN-γDuoSet ELISA(购自R&D公司)检测培养上清中IFNγ的含量。
如图6A所示，由于A375细胞是NY-ESO-1阳性和HLA-A2阳性，与阴性对照组的Mock-T(图中显示为A375+Mock-T)相比，靶向NY-ESO-1的TCR-T细胞(图中显示为A375+TCR-T)受到A375刺激后，可以分泌IFNγ。重组溶瘤腺病毒OAd-NY/A2感染A375不会引起IFNγ的分泌。如果将T细胞与重组溶瘤腺病毒OAd-NY/A2感染的A375细胞共培养，受到OAd-NY/A2感染标记的A375刺激后，与阴性对照组的Mock-T(图中显示为A375+Mock-T+OAd-NY/A2)相比，靶向NY-ESO-1的TCR-T细胞(图中显示为A375+TCR-T+OAd-NY/A2)可以分泌更多的IFNγ。如图6B所示，由于H1299细胞是NY-ESO-1阳性和HLA-A2阴性，与阴性对照组的Mock-T(图中显示为H1299+Mock-T)相比，靶向NY-ESO-1的TCR-T细胞(图中显示为H1299+TCR-T)受到H1299刺激后，没有分泌IFNγ。重组溶瘤腺病毒 OAd-NY/A2感染H1299也不会引起IFNγ的分泌。如果将T细胞与重组溶瘤腺病毒OAd-NY/A2感染的H1299细胞共培养，受到OAd-NY/A2感染标记的H1299刺激后，与阴性对照组的Mock-T(图中显示为H1299+Mock-T+OAd-NY/A2)相比，靶向NY-ESO-1的TCR-T细胞(图中显示为H1299+TCR-T+OAd-NY/A2)可以分泌更多的IFNγ。如图6C所示，由于SKOV3细胞是NY-ESO-1阴性和HLA-A2阴性，与阴性对照组的Mock-T(图中显示为SKOV3+Mock-T)相比，靶向NY-ESO-1的TCR-T细胞(图中显示为SKOV3+TCR-T)受到SKOV3刺激后，没有分泌IFNγ。重组溶瘤腺病毒OAd-NY/A2感染SKOV3也不会引起IFNγ的分泌。如果将T细胞与重组溶瘤腺病毒OAd-NY/A2感染的SKOV3细胞共培养，受到OAd-NY/A2感染标记的SKOV3刺激后，与阴性对照组的Mock-T(图中显示为SKOV3+Mock-T+OAd-NY/A2)相比，靶向NY-ESO-1的TCR-T细胞(图中显示为SKOV3+TCR-T+OAd-NY/A2)可以分泌更多的IFNγ。如图6D所示，由于HOSC1细胞是NY-ESO-1弱阳性和HLA-A2阳性，与阴性对照组的Mock-T(图中显示为HOSC1+Mock-T)相比，靶向NY-ESO-1的TCR-T细胞(图中显示为HOSC1+TCR-T)受到HOSC1刺激后，TCR-T分泌少量的IFNγ。重组溶瘤腺病毒OAd-NY/A2感染HOSC1也不会引起IFNγ的分泌。如果将T细胞与重组溶瘤腺病毒OAd-NY/A2感染的HOSC1细胞共培养，受到OAd-NY/A2感染标记的HOSC1刺激后，与阴性对照组的Mock-T(图中显示为HOSC1+Mock-T+OAd-NY/A2)相比，靶向NY-ESO-1的TCR-T细胞(图中显示为HOSC1+TCR-T+OAd-NY/A2)可以分泌更多的IFNγ。
靶向NY-ESO-1的TCR识别的是靶细胞表面HLA-A2及其呈递的NY-ESO-1短肽分子。如果肿瘤细胞本身是HLA-A2阳性以及NY-ESO-1阳性，重组溶瘤腺病毒OAd-NY/A2感染标记后，可以在一定程度上提高靶向NY-ESO-1的TCR-T对靶细胞的识别。如果肿瘤细胞本身是HLA-A2阴性或NY-ESO-1阴性或双阴性，重组溶瘤腺病毒OAd-NY/A2感染标记肿瘤细胞后，可以使原本的TCR-T无法识别靶细胞变成可以识别并对肿瘤细胞发起攻击。
实施例7：重组溶瘤腺病毒OAd-NY/A2与靶向NY-ESO-1的TCR-T对SKOV3的体外联合杀伤
本实施例通过RTCA实时杀伤监测的方法检测了重组溶瘤腺病毒OAd-NY/A2与靶向NY-ESO-1的TCR-T细胞对人卵巢癌细胞株SKOV3的体外联合杀伤效果。第0天，按照5×10 3个肿瘤细胞/孔接种至16孔E-plate中。第1天，向相应的孔中加入重组溶瘤腺病毒OAd-NY/A2，MOI为10。第2天，向相应的孔中按5×10 3个T细胞/孔加入靶向NY-ESO-1的TCR-T细胞或Mock-T细胞(表达GFP，作为TCR-T的阴性对照组)，每组2个复孔。通过xCELLigence RTCA S16实时无标记细胞功能分析仪监测SKOV3细胞的生长。
如图7A所示，SKOV3细胞接种后持续生长(图中显示为SKOV3)，只加入重组溶瘤腺病毒OAd-NY/A2没有影响SKOV3细胞的生长(图中显示为SKOV3+OAd-NY/A2)，只加入靶向NY-ESO-1的TCR-T细胞或Mock-T细胞对SKOV3细胞的生长有微弱的影响(图中显示为SKOV3+Mock-T或SKOV3+TCR-T)。重组溶瘤腺病毒OAd-NY/A2和Mock-T联合作用(图中显示为SKOV3+OAd-NY/A2+Mock-T)可以减缓SKOV3细胞的生长，而重组溶瘤腺病毒OAd-NY/A2和靶向NY-ESO-1的TCR-T细胞联合作用(图中显示为SKOV3+OAd-NY/A2+TCR-T)对SKOV3细胞体现出非常显著的杀伤作用，使SKOV3细胞数量显著减少。对实验终点90.8个小时的细胞生长指数进行分析，计算肿瘤生长抑制率(IR％)，公式为：100％×(实验组细胞指数-SKOV3细胞指数)/SKOV3细胞指数(细胞指数(Cell index)由RTCA仪得到)。结果如图7B所示，重组溶瘤腺病毒、Mock-T或TCR-T单独作用，仅对SKOV3细胞的生长产生了微弱的抑制作用，重组溶瘤腺病毒和Mock-T联合作用对SKOV3细胞的生长产生了更大的抑制作用，而重组溶瘤腺病毒和TCR-T联合作用组可以显著减少SKOV3细胞的数量，其杀伤的效果大于重组溶瘤腺病毒和TCR-T单独作用的效果的叠加，因此产生了协同杀伤的效果。
实施例8：重组溶瘤腺病毒OAd-NY/A2与靶向NY-ESO-1的TCR-T对H1299的体外联合杀伤
本实施例通过RTCA实时杀伤监测的方法检测了重组溶瘤腺病毒OAd-NY/A2与靶向NY-ESO-1的TCR-T细胞对人肺癌细胞株H1299的体外联合杀伤效果。第0天，按照5×10 3个肿瘤细胞/孔接种至16孔E-plate中。第1天，向相应的孔中加入重组溶瘤腺病毒OAd-NY/A2，MOI为10。第2天，向相应的孔中按1×10 4个T细胞/孔加入靶向NY-ESO-1的TCR-T细胞或Mock-T细胞(表达GFP，作为TCR-T的阴性对照组)，每组2个复孔。通过xCELLigence RTCA S16实时无标记细胞功能分析仪监测H1299细胞的生长。
如图8A所示，H1299细胞接种后持续生长(图中显示为H1299)，只加入重组溶瘤腺病毒OAd-NY/A2几乎没有影响H1299细胞的生长(图中显示为H1299+OAd-NY/A2)，只加入靶向NY-ESO-1的TCR-T细胞或Mock-T细胞对H1299细胞的生长有显著的影响(图中显示为H1299+Mock-T或H1299+TCR-T)。重组溶瘤腺病毒OAd-NY/A2和Mock-T联合作用(图中显示为H1299+OAd-NY/A2+Mock-T)可以有效的杀伤H1299细胞，而重组溶瘤腺病毒OAd-NY/A2和靶向NY-ESO-1的TCR-T细胞联合作用(图中显示为H1299+OAd-NY/A2+TCR-T)对H1299细胞体现出非常显著的杀伤作用，使H1299细胞数量更加显著减少。对实验节点61.14个小时的细胞生长指数进行分析，计算肿瘤生长抑制率(IR％)，公式为:100％×(实验组细胞指数-H1299细胞指数)/H1299细胞指数。结果如图8B所示，重组溶瘤腺病毒、Mock-T或TCR-T单独作用，仅对H1299细胞的生长产生了一定的抑制作用，重组溶瘤腺病毒和Mock-T联合作用可以减少H1299细胞的数量，而重组溶瘤腺病毒和TCR-T联合作用组可以显著减少H1299细胞数量，其杀伤的效果大于重组溶瘤腺病毒和TCR-T单独作用的效果的叠加，因此产生了协同杀伤的效果。
实施例9：重组溶瘤腺病毒OAd-NY/A2与靶向NY-ESO-1的TCR-T对HOSC1的体外联合杀伤
本实施例通过RTCA实时杀伤监测的方法检测了重组溶瘤腺病毒OAd-NY/A2与靶向NY-ESO-1的TCR-T细胞对人骨肉瘤细胞株HOS C1的体外联合杀伤效果。第0天，按照5×10 3个肿瘤细胞/孔接种至16孔E-plate中。第1天，向相应的孔中加入重组溶瘤腺病毒OAd-NY/A2，MOI为30。第2天，向相应的孔中按1.5×10 4个T细胞/孔加入靶向NY-ESO-1的TCR-T细胞或Mock-T细胞(表达GFP，作为TCR-T的阴性对照组)，每组2个复孔。通过xCELLigence RTCA S16实时无标记细胞功能分析仪监测HOS C1细胞的生长。
如图9A所示，HOS C1细胞接种后持续生长(图中显示为HOS C1)，只加入重组溶瘤腺病毒OAd-NY/A2微弱地影响HOS C1细胞的生长(图中显示为HOS C1+OAd-NY/A2)，只加入靶向NY-ESO-1的TCR-T细胞或Mock-T细胞对HOS C1细胞的生长也有一定的抑制(图中显示为HOS C1+Mock-T或HOS C1+TCR-T)。重组溶瘤腺病毒OAd-NY/A2和Mock-T联合作用(图中显示为HOS C1+OAd-NY/A2+Mock-T)可以使HOS C1的细胞数量减少，而重组溶瘤腺病毒OAd-NY/A2和靶向NY-ESO-1的TCR-T细胞联合作用(图中显示为HOS C1+OAd-NY/A2+TCR-T)对HOS C1细胞体现出非常显著的杀伤作用，使HOS C1细胞数量显著减少。对实验终点61.14个小时的细胞生长指数进行分析，计算肿瘤生长抑制率，公式为：100％×(实验组细胞指数-HOS C1细胞指数)/HOS C1细胞指数(IR％)。结果如图9B所示，重组溶瘤腺病毒、Mock-T或TCR-T单独作用，仅对HOS C1细胞的生长产生了微弱的抑制作用，重组溶瘤腺病毒和Mock-T联合作用可以微弱的减少HOS C1的细胞数量，而重组溶瘤腺病毒和TCR-T联合作用组可以显著减少HOS C1细胞的数量，其杀伤的效果大于重组溶瘤腺病毒和TCR-T单独作用的效果的叠加，因此产生了协同杀伤的效果。
结论
一旦将编码所述外源抗原表位肽或HLA I类蛋白的核酸通过载体(载体可以是质粒载体，重组病毒，纳米颗粒或裸露的DNA或RNA) 递送入肿瘤细胞，所述包含抗原表位肽和/或外源HLA I类分子的标记性多肽即可以使肿瘤细胞被表达特异性TCR的T细胞所识别。如本申请中所建议的，用于治疗实体瘤的联合疗法可以使过继T细胞疗法的应用范围得到扩展，通常可能不被纳入试验的患者，例如其HLA类型不匹配(尽管其肿瘤细胞表达特定的肿瘤抗原)的患者也可接受治疗而获益。理论上，无论肿瘤细胞的HLA I类的配型和肿瘤细胞中的抗原表达水平如何，将编码包含抗原表位的标记性多肽和可以呈递该抗原表位肽的HLA I类分子的核酸递送入肿瘤细胞中，并使其被过继转输的特异性T细胞所识别，可以成为一种治疗各种实体肿瘤的通用方法。然而，如果正常细胞也被标记性核酸所标记并成为过继转输T细胞的靶标，就可能会产生脱靶毒副作用。如果递送载体可以在肿瘤细胞中选择性地表达标记性表位多肽或外源HLA分子，例如使用如本申请中所建议的具有肿瘤细胞选择性复制能力的溶瘤病毒，则该风险可以避免或可控。另外，针对肿瘤细胞中常发生的抗原加工和呈递机制的缺陷，本发明所述的溶瘤病毒载体使肿瘤细胞直接表达所述包含抗原表位以及可以呈递抗原表位肽的HLAI类分子的标记性多肽，并使之成为过继转输T细胞的靶标。另外，如本发明所建议的，将同种异体HLA I类分子递送到携带最常见的肿瘤驱动基因突变(例如KRAS或p53突变)的肿瘤细胞中，使之提呈不能被内源性HLA I类分子所提呈的肿瘤抗原新表位，并被过继转输的特异性T细胞所识别并清除。这种联合疗法是使更多癌症患者受益的颇具前途的治疗方法。

Claims (35)

  1. 一种分离的用于表达外源基因的溶瘤腺病毒，其中所述溶瘤腺病毒为对腺病毒进行基因改造而得到的选择复制型重组溶瘤腺病毒，该重组溶瘤腺病毒的基因组具有以下特征：
    1)包含E1B基因调控元件，该元件包括E1B启动子和E1B与pIX共用的多聚腺苷酸添加信号序列；
    2)缺失了E1B基因编码区，并且，当需要插入所述外源基因时，在该E1B基因编码区位点插入所述外源基因，并且该外源基因位于所述E1B启动子之后，并受所述E1B基因调控元件的控制；
    3)在所述外源基因的上游，包含转录E1A 13s mRNA的E1A的cDNA序列，并且该cDNA为野生型或Rb蛋白结合区域缺失型，该Rb蛋白结合区域缺失型为所述野生型cDNA去除了如SEQ ID NO.7所示的核苷酸序列，或该Rb蛋白结合区域缺失型编码突变的E1A蛋白，所述突变的E1A蛋白如SEQ ID NO.6所示。
  2. 根据权利要求1所述的溶瘤腺病毒，其中所述E1B启动子的核苷酸序列如SEQ ID NO.1所示，所述E1B与pIX共用的多聚腺苷酸添加信号序列如aataaa所示。
  3. 根据权利要求1所述的溶瘤腺病毒，其中所述E1B基因包括E1B-55K基因和E1B-19K基因。
  4. 根据权利要求1所述的溶瘤腺病毒，其中所述E1B基因编码区的核苷酸序列如SEQ ID NO.3所示。
  5. 根据权利要求1所述的溶瘤腺病毒，其中所述外源基因的起始位点包含Kozak序列，优选地，所述Kozak序列如SEQ ID NO.4所示。
  6. 根据权利要求1所述的溶瘤腺病毒，其中所述野生型E1A cDNA的核苷酸序列如SEQ ID NO.5所示。
  7. 根据权利要求1所述的溶瘤腺病毒，其中所述转录E1A 13s mRNA的E1A的cDNA位于所述E1B启动子的上游，并且与所述E1B启动子的核苷酸序列部分重合。
  8. 根据权利要求1所述的溶瘤腺病毒，其中所述转录E1A 13s mRNA的E1A的cDNA序列在内源性E1A启动子/增强子的控制下，或者在外源启动子的控制下；优选地，所述内源性E1A启动子/增强子的核苷酸序列如SEQ ID NO.8所示。
  9. 根据权利要求1所述的溶瘤腺病毒，其中所述转录E1A 13s mRNA的E1A的cDNA序列在外源启动子的控制下，所述重组溶瘤腺病毒的基因组中去除了如SEQ ID NO.9所示的核苷酸序列，并且在该去除的位点处插入所述外源启动子核苷酸序列。
  10. 根据权利要求8或9所述的溶瘤腺病毒，其中所述外源启动子包括EF-1α启动子、CMV启动子、PKG启动子、E2F启动子、AFP启动子和TERT启动子。
  11. 根据权利要求1所述的溶瘤腺病毒，其中所述外源基因包括：HLA蛋白编码序列，标记性多肽编码序列，HLA蛋白编码序列及标记性多肽编码序列，HLA蛋白编码序列及β2-微球蛋白编码序列，或HLA蛋白编码序列、β2-微球蛋白编码序列及标记性多肽编码序列。
  12. 根据权利要求11所述的溶瘤腺病毒，其中所述HLA蛋白包括HLA I类分子，该HLA I类分子包括HLA-A、HLA-B和HLA-C。
  13. 根据权利要求12所述的溶瘤腺病毒，其中所述HLA-C包括 野生型分子，或以下突变中的至少一种：1)第2位精氨酸突变为丙氨酸；2)编码该HLA-C蛋白的核苷酸序列的第4位核苷酸由C突变为G，第5位核苷酸由G突变为C；3)第362位异亮氨酸突变为苏氨酸；4)第359位谷氨酸突变为缬氨酸。
  14. 根据权利要求11所述的溶瘤腺病毒，其中所述标记性多肽包括可操作地连接的、依次串联的以下氨基酸序列：N端信号肽的氨基酸序列、一个或多个抗原表位多肽的氨基酸序列、可任选的C端内质网滞留信号的氨基酸序列，其中当所述标记性多肽包括多个所述抗原表位多肽的氨基酸序列时，每两个相邻的所述抗原表位多肽的氨基酸序列之间由可切割性连接多肽的氨基酸序列连接；优选地，所述可切割性连接多肽是furin酶切识别多肽。
  15. 根据权利要求14所述的溶瘤腺病毒，其中所述抗原表位多肽的氨基酸序列来源于自然界存在的蛋白的氨基酸序列，或者为人工合成的自然界不存在的氨基酸序列；优选地，所述自然界存在的蛋白包括人源蛋白和除人以外的其它物种的蛋白。
  16. 根据权利要求14所述的溶瘤腺病毒，其中所述抗原表位多肽的氨基酸序列来源于肿瘤相关抗原或肿瘤特异性抗原的氨基酸序列。
  17. 根据权利要求16所述的溶瘤腺病毒，其中所述肿瘤相关抗原选自NY-ESO-1 157-165、NY-ESO-1 1-11、NY-ESO-1 53-62、NY-ESO-1 18-27、Her2/neu 369-377、SSX-2 41-49、MAGE-A4 230-239、MAGE-A10 254-262、MAGE-C2 336-344、MAGE-C2 191-200、MAGE-C2 307-315、MAGE-C2 42-50、MAGE-A1 120-129、MAGE-A1 230-238、MAGE-A1 161-169、KK-LC-1 76-84、p53 99-107、PRAME 301-309、甲胎蛋白158-166、HPV16-E6 29-38、HPV16-E7 11-19、EBV-LMP1 51-59、EBV-LMP1 125-133、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：Q61R 55-64、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、TP53：V157G 156-164、TP53：R248Q 240-249、TP53：R248W 240-249、TP53：G245S 240-249、TP53：V157F 156-164、TP53：V157F 149-158、TP53：Y163C 156-164、TP53：R248Q 247-255、TP53：R248Q 245-254、TP53：R248W 245-254、TP53：G245S 245-254、TP53：G249S 245-254、TP53：Y220C 217-225、TP53：R175H 168-176、TP53：R248W 240-249、TP53：K132N 125-134、CDC73：Q254E 248-256、CYP2A6：N438Y 436-444、CTNNB1：T41A 41-49、CTNNB1：S45P 41-49、CTNNB1：T41A 34-43、CTNNB1：S37Y 30-39、CTNNB1：S33C 30-39、CTNNB1：S45P 40-49、EGFR：L858R 852-860、EGFR：T790M 790-799、PIK3CA：E542K 533-542、PIK3CA：H1047R 1046-1055、GNAS：R201H 197-205、CDK4：R24C 23-32、H3.3：K27M 26-35、BRAF：V600E 591-601、CHD4：K73Rfs 141-148、NRAS：Q61R 55-64、IDH1：R132H 126-135、TVP23C：C51Y 51-59、TVP23C：C51Y 42-51和TVP23C：C51Y 45-53。
  18. 根据权利要求14所述的溶瘤腺病毒，其中所述抗原表位多肽为如SEQ ID NO.10所示的NY-ESO-1 157-165或如SEQ ID NO.11所示的KRAS：G12D 10-18。
  19. 一种用于制备权利要求1-18中任一项所述的溶瘤腺病毒的载体，其中所述载体包含所述E1B基因调控元件、缺失了所述E1B基因编码区、并且在所述外源基因的上游包含所述的转录E1A 13s mRNA的E1A的cDNA序列。
  20. 一种用于治疗肿瘤和/或癌症的治疗剂，包含：
    (a)第一组合物，其中该第一组合物包含位于第一可药用载体 中的第一活性成分，该第一活性成分包括或含有用于导入肿瘤细胞和/或癌细胞的根据权利要求1-18中任一项所述的溶瘤腺病毒；和
    (b)第二组合物，其中该第二组合物包含位于第二可药用载体中的第二活性成分，该第二活性成分包含T细胞受体修饰的免疫细胞。
  21. 根据权利要求20所述的治疗剂，其中所述第一组合物和所述第二组合物各自独立地存在于所述治疗剂中而互不混合。
  22. 根据权利要求20所述的治疗剂，其中所述免疫细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。
  23. 根据权利要求20所述的治疗剂，其中所述第一组合物包含治疗有效量的所述溶瘤腺病毒。
  24. 根据权利要求20所述的治疗剂，其中所述第二组合物包含治疗有效量的所述的T细胞受体修饰的免疫细胞。
  25. 根据权利要求20所述的治疗剂，其中所述溶瘤腺病毒配制成通过瘤内注射给药、腹膜内给药、蛛网膜下腔内给药、或静脉给药。
  26. 根据权利要求20所述的治疗剂，其中所述免疫细胞配制成通过动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内或腹膜内给药。
  27. 根据权利要求1-18中任一项所述的溶瘤腺病毒在制备用于治疗肿瘤和/或癌症的药物中的用途。
  28. 根据权利要求27所述的用途，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌， 食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
  29. 根据权利要求20-26中任一项所述的治疗剂在制备用于治疗肿瘤和/或癌症的药物中的用途。
  30. 根据权利要求29所述的用途，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
  31. 一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据权利要求1-18中任一项所述的溶瘤腺病毒。
  32. 根据权利要求31所述的方法，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
  33. 一种治疗肿瘤和/或癌症的方法，包括：
    对肿瘤和/或癌症患者施用根据权利要求20-26中任一项所述的治疗剂中的第一组合物；和
    对所述肿瘤和/或癌症患者施用根据权利要求20-26中任一项所述的治疗剂中的第二组合物。
  34. 根据权利要求33所述的方法，包括以下依次进行的步骤：
    1)对所述肿瘤和/或癌症患者施用所述第一组合物；和
    2)在施用所述第一组合物之后，对所述肿瘤和/或癌症患者施用所述的治疗剂中的第二组合物。
  35. 根据权利要求34所述的方法，其中所述肿瘤和/或癌症包括：乳腺癌，头颈部肿瘤，滑膜癌，肾癌，结缔组织癌，黑色素瘤，肺癌，食管癌，结肠癌，直肠癌，脑癌，肝癌，骨癌，绒毛膜癌，胃泌素瘤，嗜铬细胞瘤，催乳素瘤，von Hippel-Lindau病，Zollinger-Ellison综合征，肛门癌，胆管癌，膀胱癌，输尿管癌，神经胶质瘤，神经母细胞瘤，脑膜瘤，脊髓肿瘤，骨软骨瘤，软骨肉瘤，尤文氏肉瘤，原发 部位不明癌，类癌，纤维肉瘤，佩吉特病，宫颈癌，胆囊癌，眼癌，卡波西氏肉瘤，前列腺癌，睾丸癌，皮肤鳞状细胞癌，间皮瘤，多尖端骨髓瘤，卵巢癌，胰腺内分泌瘤，胰高血糖素瘤，胰腺癌，阴茎癌，垂体癌，软组织肉瘤，视网膜母细胞瘤，小肠癌，胃癌，胸腺癌，滋养细胞癌，葡萄胎，子宫内膜癌，阴道癌，外阴癌，蕈样真菌病，胰岛素瘤，心脏癌，脑膜癌，血液癌，腹膜癌和胸膜癌。
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