WO2023122882A1 - 双特异性t细胞衔接器、其重组溶瘤病毒及其用途 - Google Patents

双特异性t细胞衔接器、其重组溶瘤病毒及其用途 Download PDF

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WO2023122882A1
WO2023122882A1 PCT/CN2021/141634 CN2021141634W WO2023122882A1 WO 2023122882 A1 WO2023122882 A1 WO 2023122882A1 CN 2021141634 W CN2021141634 W CN 2021141634W WO 2023122882 A1 WO2023122882 A1 WO 2023122882A1
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virus
seq
αcd47
αcd3
sequence
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French (fr)
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徐建青
张晓燕
丁相卿
廖启彬
王诗语
周俊花
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上海鑫湾生物科技有限公司
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    • 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
    • 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
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • 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
    • C12N15/863—Poxviral vectors, e.g. entomopoxvirus
    • 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

Definitions

  • the invention belongs to the technical field of biomedicine and relates to a novel bispecific T cell engager (BiTE) comprising human CD47 and human CD3 specific binding fragments.
  • the present invention also relates to a recombinant oncolytic virus comprising the bispecific T cell engager (BiTE), and the present invention also provides the preparation method of the bispecific T cell engager and the oncolytic virus and their use in Antitumor applications.
  • Cancer is the second leading cause of death worldwide with an estimated 9.6 million deaths in 2018. Globally, cancer is responsible for about one in six deaths. The situation in China is particularly worrisome, due in part to rapid population growth and socioeconomic development. Although the incidence of cancer in China is lower than that in developed countries in Europe and America, the cancer mortality rate in China is 30% to 40% higher than that in Europe and the United States, and the prognosis is significantly worse.
  • cancer has attracted more and more attention in China, and related cancer treatment methods, especially tumor immunotherapy with the help of the body's own immune system, have increasingly become the focus of attention.
  • CD47 integrin-related protein
  • IRP ⁇ signaling regulatory protein alpha
  • CD47 antibody drug or SIRP ⁇ -Fc fusion protein binds to red blood cells, it can cause red blood cell agglutination, and then trigger the rupture of red blood cells; CD47 antibody drug with IgG1 Fc can also activate the phagocytosis of red blood cells by macrophages or antibody-dependent cells Antibody-dependent cell-mediated cytotoxicity (ADCC) further triggers the lysis of red blood cells, eventually leading to the occurrence of anemia.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Bispecific T-cell engager BiTE (bispecific T-cell engager) represents a class of bispecific antibodies with significant anti-tumor effects, which can target and activate self-T cells to kill tumor cells.
  • a BiTE consists of two single-chain variable fragments (scFv) connected in series by a flexible linker. One scFv recognizes the T cell surface protein CD3 ⁇ H, while the other scFv recognizes a specific tumor cell surface antigen.
  • BiTE The structure of BiTE and the ability to specifically bind proteins allow it to physically bridge T cells to tumor cells to form T cell-BiTE-tumor cell complexes, induce immune synapse formation, stimulate T cell activation, and produce tumor-killing tumor cells. cytokines. In recent years, BiTE has made significant progress in anti-tumor research and achieved ideal therapeutic effects in clinic.
  • oncolytic viruses benefit from their own characteristics of selectively infecting and lysing tumor cells locally in tumors, and are increasingly becoming the first choice for anti-tumor.
  • the tumor selectivity of oncolytic viruses can be either their own tumor tropism or the result of genetic modification.
  • Oncolytic viruses can act on multiple cellular pathways to reduce tumor resistance and induce different forms of cell death.
  • oncolytic viruses can break the immune tolerance of the tumor microenvironment and induce long-term tumor-specific immune responses.
  • Oncolytic viruses can specifically transport therapeutic proteins into tumor tissues, and the further replication of the virus increases the expression level in malignant tumor cells.
  • oncolytic viruses can be used in combination with chemotherapy, radiotherapy, and immunotherapy.
  • the oncolytic adenovirus product (oncorine) has been used in clinical treatment of nasopharyngeal carcinoma in China.
  • the oncolytic virus deletes the E1B-55kD gene of human adenovirus type 5, which can replicate and proliferate in cancer cells with p53 gene mutation and kill the host cells, resulting in oncolytic therapy; at the same time, the E3 region is deleted, so that the tumor antigen information can pass through The transmission of dendritic cells activates T cell immunity.
  • clinical data show that compared with radiation therapy, oncorine combined with chemotherapy has a weaker therapeutic effect on patients with nasopharyngeal carcinoma.
  • T-VEC herpes simplex virus
  • vaccinia virus Vaccinia virus, VV
  • WR Western reserve
  • Wyeth strain Wyeth strain
  • Copenhagen strain Lister strain
  • Tian Tan strain Tian Tan strain
  • vaccinia virus is used as the carrier of many recombinant vaccines such as influenza virus and human immunodeficiency virus.
  • oncolytic viruses most of them are currently in the preclinical research stage, and only a few have entered the clinical research stage.
  • JX-594 is based on the Wyeth strain virus, with hGM-CSF and LacZ genes inserted in the TK region. Due to the deletion of the thymokinase gene, JX-594 can express and replicate in cancer cells that express thymokinase at a high level, but it will not affect normal cells . At the same time, JX-594 can express GM-CSF in tumor cells because of the insertion of GM-CSF gene, and activate the body's anti-tumor immune response.
  • the oncolytic virus (GL-ONC1, also known as GLV-1h68) developed by Genelux in the United States is based on vaccinia virus (Lister strain), and its genes F14.5L, TK (encoding thymidine kinase) and HA (encoding hemagglutination) have been deleted.
  • F14.5L vaccinia virus
  • TK encoding thymidine kinase
  • HA encoding hemagglutination
  • GL-ONC1 has completed the phase I clinical trial of intravenous administration, showing good safety and efficacy, no dose-limiting toxicity, and no maximum tolerated dose. All patients had a neutralizing response to GL-ONC1; GL -ONC1 is currently undergoing an expanded
  • the life cycle of vaccinia virus is strictly carried out in the cytoplasm of the host cell, and the thymidine kinase gene of vaccinia virus is required for the genome of the progeny virus to replicate smoothly.
  • the synthesis of Thymidine Kinase (TK) occurs in the S phase of the cell division cycle. After the completion of cell division, Thymidine Kinase is degraded inside the cell, so the concentration of Thymidine Kinase in the cytoplasm lower.
  • tumor cells divide actively, and their thymidine kinase continues to be synthesized. Utilizing this feature, the thymidine kinase gene of the vaccinia virus is deleted, causing the vaccinia virus to amplify specifically in tumor cells and play the role of oncolysis.
  • vaccinia virus has initially shown certain advantages in tumor treatment. Most of the existing designs that use vaccinia virus as an oncolytic virus are relatively safe, and the clinical effectiveness needs to be further observed. However, further optimization is needed in terms of immune regulation and precise tumor targeting.
  • bispecific T cell engager that is particularly suitable for recombinant oncolytic viruses in the prior art.
  • safer and more targeted bispecific T cell engagers and oncolytic viruses containing them There is a need.
  • the purpose of the present invention is to provide a bispecific T cell engager for the deficiencies of the prior art.
  • the present invention also provides a recombinant oncolytic virus expressing the bispecific T cell engager (BiTE).
  • the bispecific T cell engager (BiTE) provided by the present invention overcomes the limitation of tumor immunoediting (immunosuppression and immune evasion) of human T cells by tumor cells of solid tumors, and can directly recruit human T cells to the inside of solid tumors to protect against tumors. Cells can kill tumor cells and have broad application prospects.
  • the present invention provides an ⁇ CD47 and ⁇ CD3 bispecific T cell engager ⁇ CD47- ⁇ CD3 BiTE, which comprises a fusion protein represented by any one of the following formulas:
  • VH ⁇ CD47 is the heavy chain variable region of the CD47 antibody, which includes the following three complementarity determining regions:
  • VH CDR1 which consists of the following sequence: SEQ ID NO: 1, or a sequence having one or several amino acid substitutions, deletions or additions thereto,
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 2, or a sequence having one or several amino acid substitutions, deletions or additions thereto, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 3, or a sequence having one or several amino acid substitutions, deletions or additions thereto;
  • substitutions described in any one of (i)-(iii) are conservative substitutions
  • the VH ⁇ CD47 comprises: VH CDR1 shown in SEQ ID NO:1, VH CDR2 shown in SEQ ID NO:2, VH CDR3 shown in SEQ ID NO:3;
  • VL ⁇ CD47 is the light chain variable region of the CD47 antibody, which includes the following three complementarity determining regions:
  • VL CDR1 which consists of the following sequence: shown in any one of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13 or SEQ ID NO:16, or A sequence having one or several amino acid substitutions, deletions or additions compared thereto,
  • VL CDR2 consisting of any one of SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:17, or a sequence having a substitution, deletion or addition of one or several amino acids compared to it, and
  • VL CDR3 consisting of any one of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15 or SEQ ID NO:18, or A sequence having one or several amino acid substitutions, deletions or additions compared thereto;
  • substitutions described in any one of (iv)-(vi) are conservative substitutions
  • the VL ⁇ CD47 comprises: VL CDR1 shown in SEQ ID NO:4, VL CDR2 shown in SEQ ID NO:5, VL CDR3 shown in SEQ ID NO:6;
  • VL ⁇ CD47 comprises: VL CDR1 shown in SEQ ID NO:7, VL CDR2 shown in SEQ ID NO:8, VL CDR3 shown in SEQ ID NO:9;
  • VL ⁇ CD47 comprises: VL CDR1 shown in SEQ ID NO:10, VL CDR2 shown in SEQ ID NO:11, VL CDR3 shown in SEQ ID NO:12;
  • VL ⁇ CD47 comprises: VL CDR1 shown in SEQ ID NO:13, VL CDR2 shown in SEQ ID NO:14, VL CDR3 shown in SEQ ID NO:15;
  • VL ⁇ CD47 comprises: VL CDR1 shown in SEQ ID NO:16, VL CDR2 shown in SEQ ID NO:17, VL CDR3 shown in SEQ ID NO:18.
  • VH ⁇ CD47 comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO:19;
  • the VL ⁇ CD47 comprises 3 CDRs contained in the light chain variable region shown in any one of SEQ ID NO:20-24;
  • the 3 CDRs contained in said heavy chain variable region, and/or the 3 CDRs contained in said light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.
  • VH ⁇ CD47 comprises an amino acid sequence selected from the following:
  • the VL ⁇ CD47 comprises an amino acid sequence selected from the group consisting of:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • VH ⁇ CD3 comprises an amino acid sequence selected from the following:
  • (iii) have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the sequence shown in SEQ ID NO: 25 or 27 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity;
  • the VL ⁇ CD3 comprises an amino acid sequence selected from the group consisting of:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • VH ⁇ CD47 , VH ⁇ CD3 , VL ⁇ CD47 and VL ⁇ CD3 are linked by a connecting peptide
  • VH ⁇ CD47 , VH ⁇ CD3 , VL ⁇ CD47 and VL ⁇ CD3 are optionally connected by one, two or three connecting peptides;
  • the bispecific T cell engager comprises a fusion protein represented by any one of the following formulae:
  • the L is KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GGGGGG, GGGGGGGG or (GGGGS) n ; further preferably, n is an integer of 1-5, most preferably, n is 3;
  • the bispecific T cell engager comprises a fusion protein represented by the following formula:
  • the VH ⁇ CD47 is shown in SEQ ID NO:19
  • the VL ⁇ CD47 is shown in SEQ ID NO:21 or 24
  • the VH ⁇ CD3 is shown in SEQ ID NO:25
  • the VL ⁇ CD3 is shown in SEQ ID NO: 26, and the L is GGGGSGGGGSGGGGS.
  • the present invention also provides an isolated nucleic acid molecule encoding the bispecific T cell engager
  • the nucleic acid molecule comprises the nucleic acid sequence shown in any one of SEQ ID NO: 31-35, which is the coding sequence of VL ⁇ CD47 ;
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 36, which is the coding sequence of VH ⁇ CD47 .
  • E1 is a CMV enhancer or/and other cis-acting elements; preferably comprising the nucleic acid sequence shown in SEQ ID NO: 37;
  • E2 is a promoter for recombinant expression, preferably a CMV promoter, more preferably comprising the nucleic acid sequence shown in SEQ ID NO: 38;
  • E3 is the 5' untranslated region, optionally does not include or includes an intron sequence, optionally includes one or more restriction enzyme sites; preferably includes the nucleic acid sequence shown in SEQ ID NO: 39;
  • E4 is the coding nucleotide sequence of the signal peptide of the BiTE protein; preferably, the signal peptide is derived from the signal peptide of humans or mice; preferably comprises the nucleic acid sequence shown in SEQ ID NO: 40;
  • E5 is the coding nucleotide sequence of the bispecific T cell adapter
  • E6 is the 3' end untranslated region, optionally comprising one or more restriction enzyme sites; preferably comprising the nucleic acid sequence shown in SEQ ID NO: 41;
  • E7 is the SV40 transcription termination signal region; preferably comprising the nucleic acid sequence shown in SEQ ID NO: 42.
  • the present invention provides a recombinant oncolytic virus, which is operably inserted into or contains the expression framework of the bispecific T cell adapter BiTE.
  • the expression framework is located in the thymidine kinase (TK) region of the recombinant oncolytic virus.
  • TK thymidine kinase
  • the expression framework can be expressed alone or fused with other genes or fragments.
  • the recombinant oncolytic virus also contains gene coding sequences of other immune regulatory factors, more preferably, the other immune regulatory factors include but not limited to IL-1, IL-2, IL-3, IL-7, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-33, IL-35, IL-37, GM-CSF, IFN- ⁇ , IFN- ⁇ , IFN- Gamma, anti-PD-1/PD-L1 antibody, anti-CTLA-4 antibody, anti-Lag-3 antibody, anti-TIGIT antibody, or anti-Tim-3 antibody; or
  • the recombinant oncolytic virus also includes gene coding sequences of proteins related to apoptosis and pyroptosis, and the proteins related to apoptosis and pyroptosis include but are not limited to apoptosis-related factor 1 (Apaf-1 ), interleukin-1 ⁇ -converting enzyme (ICE), Bcl-2 protein, Fas/APO-1, p53, myc, ataxia telangiectasia mutated gene (ATM), gasdermin D (gasdermin D), dermatin E (gasdermin E), etc.; or
  • the recombinant oncolytic virus also contains small RNA targeting immune regulation genes, apoptosis and pyroptosis genes.
  • the viral backbone of the oncolytic virus is derived from modified or transformed vaccinia virus Tiantan strain, vaccinia virus New York strain, vaccinia virus Copenhagen strain, vaccinia virus canary strain , vaccinia virus Ankara strain, adenovirus, adeno-associated virus, herpes simplex virus, varicella-zoster virus (VZV), respiratory syncytial virus (Respiratory Syncytial Virus, RSV), raw Ricky forest virus ( Semliki forest virus, SFV), Epstein-Barr virus, cytomegalovirus, human herpesvirus type 6, smallpox virus, vaccinia virus, molluscum contagiosum virus, oral ulcer virus, reovirus, rotavirus, enterovirus, plug Nekavirus, Poliovirus, Coxsackievirus, Rhinovirus, Hepatitis A virus, Foot-and-mouth disease virus, Tog
  • the backbone of the oncolytic virus is an intracellular mature virus, an intracellular packaging virus, a cell-associated packaging virus or an extracellular packaging virus.
  • the recombinant oncolytic virus according to the present invention wherein the oncolytic virus is a recombinant vaccinia virus Tiantan strain containing the gene encoding the bispecific T cell adapter ⁇ CD47- ⁇ CD3-BITE, named rTV- ⁇ CD47- ⁇ CD3- BITE, its deposit number is: CCTCC NO:V202081, the date of deposit is January 2, 2021, and the deposit address is China Center for Type Culture Collection.
  • the present invention provides a method for preparing the recombinant oncolytic virus, comprising the following steps:
  • step 2) Subcloning the expression framework obtained in step 1) into a shuttle plasmid of an oncolytic virus to construct a recombinant plasmid vector;
  • step 3 Transfect the recombinant plasmid vector obtained in step 2) into an oncolytic virus, and obtain a recombinant oncolytic virus through screening.
  • the obtained recombinant oncolytic virus is cultured.
  • the present invention provides a method for preparing recombinant vaccinia virus Tiantan strain, comprising the following steps:
  • the expression framework of the bispecific T cell engager ⁇ CD47- ⁇ CD3-BITE is controlled by the early/late promoter p7.5 of vaccinia virus.
  • the specific steps of utilizing VERO cells to amplify recombinant vaccinia virus include: when cultivating VERO cells to a density close to 100%, replacing the low-concentration fetal calf serum maintenance medium, adding oncolytic vaccinia virus (inoculum size is about 0.02MOI per 10cm culture plate), placed in an incubator for culture, after the amplification of the recombinant poxvirus is completed, the virus liquid is collected and repeatedly frozen and thawed, and purified by density gradient centrifugation with sucrose solution.
  • the present invention also provides the use of the bispecific T cell engager or recombinant oncolytic virus for the preparation of antitumor drugs; wherein, the tumor is selected from B-cell lymphoma, T-cell lymphoma, melanoma tumor, prostate cancer, renal cell carcinoma, sarcoma, glioma such as high-grade glioma, blastoma such as neuroblastoma, osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer such as small Cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, colorectal cancer, hematopoietic system cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related Lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell
  • the present invention provides a method for treating tumors, the method comprising administering a therapeutically effective amount of a bispecific T cell engager or a recombinant oncolytic virus to a subject in need.
  • the method further includes administering additional chemotherapeutic drugs, radiotherapy techniques, surgical treatment, immune cell drugs (including but not limited to CAR-T, NK, NKT, iNKT, CAR-NK, CAR-NKT, CAR-iNKT, etc.), other oncolytic viruses; preferably, the method is intravenous injection or intratumoral injection.
  • the present invention provides a novel ⁇ CD47- ⁇ CD3-BITE bispecific T cell engager, which has good anti-tumor activity in vitro. Furthermore, the present inventors took the vaccinia virus Tiantan strain as a research model and provided a vaccinia virus Tiantan strain equipped with the bispecific T cell engager of the present invention.
  • the inventors of the present invention found that the Tiantan strain of vaccinia virus itself has a strong anti-tumor effect; the bispecific T cell adapter equipped with ⁇ CD47- ⁇ CD3-BITE can avoid the binding of CD47 antibody to peripheral blood red blood cells, and utilize the broad-spectrum expression of CD47 The characteristics of CD3 identify tumor cells, and CD3 antibody activates T cells, thereby killing tumor cells, improving the effect of tumor immunotherapy and increasing safety.
  • the bispecific T cell engager (BiTE) provided by the present invention has a small molecular weight, can penetrate the intercellular space and reach the tumor microenvironment (TME) inside the solid tumor to play a role, and it can efficiently mediate the killing of cancer cells by T cells .
  • the present invention combines the bispecific T cell engager with an oncolytic virus to provide a vaccinia virus Tiantan strain that can efficiently express the bispecific T cell engager ⁇ CD47- ⁇ CD3-BITE gene.
  • the oncolytic virus When the oncolytic virus is administered to tumor lesions, it not only lyses tumor cells to exert an oncolytic effect, but also carries a highly lethal antibody against human CD47 targeting the tumor gene, blocks the combination of CD47 and SIRP ⁇ , and enhances the in vivo cytotoxicity. Phagocytic function of phagocytic cells to enhance the anti-tumor effect.
  • the oncolytic virus significantly enhances the ability to suppress malignant tumors.
  • the vaccinia virus Tiantan strain of the present invention can treat liver cancer and malignant lung cancer, and achieve good targeting and anti-tumor effects on tumors, and have relatively complete virus amplification and quality control
  • the system lays the foundation for further industrialization, therefore, the present invention has a good application prospect.
  • Figure 1 shows the construction of the shuttle plasmid vector ⁇ CD47- ⁇ CD3 BiTE hIgG1 WT of the present invention and the expression and purification of five corresponding BiTE proteins; wherein Figure 1A is the expression map of the shuttle plasmid hIgG1 WT integrated with the expression framework of ⁇ CD47- ⁇ CD3-BITE; Figure 1B is the result of protein purification using a nickel column in the culture supernatant of 293T transiently expressing ⁇ CD47- ⁇ CD3-BITE.
  • Figure 2 shows the effect of ⁇ CD47- ⁇ CD3-BITE mediated T cells against human ovarian adenocarcinoma cells in vitro; Among them, compared with the T cell control group, the CD47 antibody of clone 68 and the BiTE composed of ⁇ CD3 can all be well Mediates T cell killing of SK-OV3. It shows that ⁇ CD47- ⁇ CD3-BITE can well recognize the tumor cells of CD47 and activate T cells, thereby mediating the specific killing of the tumor cells by T cells; it shows that the bispecific T cells that contain human CD47 and human CD3 specifically bind BITE can well mediate the anti-tumor activity of T cells in vitro and has a broad spectrum.
  • FIG. 3 shows that ⁇ CD47- ⁇ CD3-BITE mediates the effect of T cells against human lung cancer cells in vitro; among them, compared with the T cell control group, the BiTE composed of the CD47 antibody of clone 101 and ⁇ CD3 can mediate T cells against NCI -H292 tumor cell killing, compared with the T cell group alone, the BiTE group had a significant killing effect on NCI-H292 cells; indicating that the bispecific T cell engager (BITE) containing human CD47 and human CD3 specific binding can work well in vitro It can mediate the anti-tumor activity of T cells and has a broad spectrum.
  • BITE bispecific T cell engager
  • Figure 4 shows the construction of the vaccinia virus Tiantan strain shuttle plasmid vector ( ⁇ CD47- ⁇ CD3-BITE) and the expression of ⁇ CD47- ⁇ CD3-BITE protein.
  • Figure 4A is the expression map of the vaccinia virus shuttle plasmid pSC65 integrated with the ⁇ CD47- ⁇ CD3-BITE gene;
  • Figure 4B is the PCR identification result of the insert fragment ⁇ CD47- ⁇ CD3-BITE in the recombinant vaccinia virus rTV- ⁇ CD47- ⁇ CD3-BITE;
  • Figure 4C It shows that there is expression of CD47- ⁇ CD3-BITE protein in the supernatant of VERO cells infected with recombinant vaccinia virus. It can be seen from Figure 4 that the ⁇ CD47- ⁇ CD3-BITE carried by the recombinant vaccinia virus rTV- ⁇ CD47- ⁇ CD3-BITE was successfully expressed.
  • Figure 5 shows that the ⁇ CD47- ⁇ CD3-BITE carried by the recombinant vaccinia virus rTV- ⁇ CD47- ⁇ CD3-BITE has a high affinity to the CD47 protein;
  • Figure 5 shows the cells collected from the recombinant vaccinia virus rTV- ⁇ CD47- ⁇ CD3-BITE infected VERO cells The flow cytometric results of affinity detection between the supernatant and the lung cancer cell A549 expressing human CD47; the results showed that compared with the wild-type vaccinia virus, the recombinant vaccinia virus rTV- ⁇ CD47- ⁇ CD3-BITE expressed protein had a binding rate of 85% to CD47. %above.
  • Figure 6 shows the in vitro killing effect of recombinant vaccinia virus of vaccinia virus Tiantan strain rTV- ⁇ CD47- ⁇ CD3-BITE on A549 cells.
  • the experiment was divided into four groups, including wild vaccinia virus infected supernatant control group, wild vaccinia virus infected supernatant + T cell group, recombinant vaccinia virus infected supernatant control group and recombinant vaccinia virus infected supernatant + T cell group . It can be seen from the figure that the recombinant vaccinia virus-infected supernatant+T cell group has a significant killing effect on lung cancer cells A549 cells compared with the blank control group.
  • Figure 7 shows the anticancer effects of ⁇ CD47- ⁇ CD3-BITE proteins with different structures.
  • Vaccinia virus Tiantan strain named rTV- ⁇ CD47- ⁇ CD3-BITE, its preservation number is: CCTCC NO:V202081, the preservation date is January 2, 2021, and the preservation address is China Type Culture Collection Center, address: Wuhan, Wuhan, China University.
  • Example 1 Construction and expression test of 293T recombinant expression plasmid of ⁇ CD47- ⁇ CD3-BITE certificate
  • the VH ⁇ CD47 is shown in SEQ ID NO:19
  • the VL ⁇ CD47 is shown in any one of SEQ ID NOs:20-24
  • the VH ⁇ CD3 is shown in SEQ ID NO:25
  • the VL ⁇ CD3 is shown in Shown in SEQ ID NO:26
  • said L is GGGGSGGGGSGGGGS.
  • Hu004-67 represents the ⁇ CD47- ⁇ CD3 BiTE comprising VL ⁇ CD47 shown in SEQ ID NO:20;
  • Hu004-68 represents the ⁇ CD47 - ⁇ CD3 BiTE comprising VL ⁇ CD47 shown in SEQ ID NO:21;
  • Hu004 -73 represents the ⁇ CD47- ⁇ CD3 BiTE comprising VL ⁇ CD47 shown in SEQ ID NO:22;
  • Hu004-100 represents the ⁇ CD47 - ⁇ CD3 BiTE comprising VL ⁇ CD47 shown in SEQ ID NO:23;
  • Hu004-101 represents the ⁇ CD47- ⁇ CD3 BiTE comprising SEQ ID NO: ⁇ CD47- ⁇ CD3 BiTE of VL ⁇ CD47 shown in 24, wherein WT Cloning vector AbVec-hIgG1 (GenBank: FJ475055.1).
  • the DNA sequence of ⁇ CD47- ⁇ CD3-BITE was artificially synthesized according to the structural formula, and the plasmid construction map is shown in Figure 1a, wherein the coding sequence of VL ⁇ CD47 is shown in any one of SEQ ID NO: 31-35, and the coding sequence of VH ⁇ CD47 is shown in Shown in SEQ ID NO:36.
  • the artificially synthesized hIgG1 WT vector containing the ⁇ CD47- ⁇ CD3-BITE target gene was transformed into Escherichia coli E.coli TOP10 (Video, Cat. No. DL1010S), and grown overnight on a culture plate containing ampicillin. On the second day, a single colony was randomly picked for overnight culture at 37°C, and a plasmid extraction kit (Qiagen, Cat. No. 27106) was used to extract the plasmid for 293T cell transfection.
  • 293T cell preparation Remove the supernatant from the cells grown in T175 culture flasks with a confluence of more than 90%, add 2ml 2.5% Trypsin-EDTA (GIBCO company, catalog number 25200072) to digest the cells until the cells are completely detached, add 10ml to complete Culture medium (DMEM medium + 10% FBS + 1% PS) to stop digestion, centrifuge at 150g for 5 minutes to collect cells, discard the supernatant, re-select cells with an appropriate amount of complete medium to make the cell concentration about 8 ⁇ 10 6 to 10 ⁇ 10 6 pieces/ml. The cell suspension was counted, and 1.3 ⁇ 10 7 cells were plated on a 150 mm cell culture dish (Thermo Scientific Company, Cat. No. 150468). The medium volume in the culture dish was 25 mL, and cultured until the next day for subsequent plasmid transfection.
  • Collect ⁇ CD47- ⁇ CD3-BITE expression supernatant collect the culture base in the expression culture dish into a 50mL centrifuge tube, centrifuge at 4000g for 5 minutes, and transfer the expression supernatant to a new 50mL centrifuge tube.
  • Example 2 ⁇ CD47- ⁇ CD3-BITE mediates the in vitro anti-human ovarian adenocarcinoma effect of T cells fruit
  • SK-OV3 cell plating SK-OV3 (stored in Shanghai Xinwan Biotechnology Co., Ltd.) is a Luciferase-expressing cell line. The cells were plated in a 96-well plate at a density of 1 ⁇ 104 /well, and incubated overnight at 37°C until they adhered to the wall.
  • B6H12- ⁇ CD3-BITE the heavy chain variable region and light chain variable region of B6H12 are shown in SEQ ID NO: 29 and 30 respectively, and the rest
  • the structure is the same as the BITE of Example 1 of the present invention
  • T cells Hu004-67- ⁇ CD3-BITE and T cells, Hu004-68- ⁇ CD3-BITE and T cells, Hu004-73- ⁇ CD3-BITE and T cells, Hu004 -100- ⁇ CD3-BITE and T cells, Hu004-101- ⁇ CD3-BITE and T cells.
  • B6H12 is a known CD47 antibody clone (US 9017675B2), as a positive control.
  • Hu004-68 and Hu004-101 are screened antibody clones with high affinity to CD47 protein (acquired by entrusting Takibi Biotechnology Company to conduct antibody screening). It can be seen from Figure 2 that for this tumor cell line, the anti-tumor activity of the CD47 antibody (scFv)- ⁇ CD3-BITE clone No. 68 was 3.5 times higher than that of the T cell control group, and compared with the clone No. 100 with lower killing ability There is a 2-fold increase, and a 1.5-fold increase compared with the positive control antibody, which can well mediate the in vitro anti-ovarian adenocarcinoma cell effect of T cells.
  • Example 3 ⁇ CD47- ⁇ CD3-BITE mediates the in vitro anti-human lung cancer cell effect of T cells
  • NCI-H292 cell plating NCI-H292 (stored in Shanghai Xinwan Biotechnology Co., Ltd.) is a Luciferase-expressing cell line. The cells were plated in a 96-well plate at a density of 1 ⁇ 104 /well, and incubated overnight at 37°C until they adhered to the wall.
  • T cells B6H12- ⁇ CD3-BITE and T cells, Hu004-67- ⁇ CD3-BITE and T cells, Hu004-68- ⁇ CD3-BITE and T cells, Hu004 -73- ⁇ CD3-BITE and T cells, Hu004-100- ⁇ CD3-BITE and T cells, Hu004-101- ⁇ CD3-BITE and T cells.
  • the measured results are shown in Figure 3.
  • the CD47 antibody (scFv)- ⁇ CD3-BITE of clone 101 can well mediate the in vitro anti-lung cancer cell effect of T cells.
  • the tumor-killing activity was increased by 4 times, compared with clone 100 with a lower killing ability, it was increased by 1.2 times, and compared with the positive control antibody, it was increased by 1.2 times, which can well mediate T cells in vitro Anti-ovarian adenocarcinoma cells.
  • the results in Figure 2 and Figure 3 together indicate that the bispecific T cell engager (BITE) containing human CD47 and human CD3 specific binding can well mediate the anti-tumor activity of T cells in vitro and has a broad spectrum.
  • Example 4 Construction and expression verification of recombinant vaccinia virus of ⁇ CD47- ⁇ CD3-BITE
  • the DNA sequence of ⁇ CD47- ⁇ CD3-BITE was artificially synthesized, and the synthetic DNA sequence was used as a template for PCR amplification using the following primers.
  • the primers for amplification are:
  • PCR reaction program pre-denaturation at 94°C for 5 minutes; denaturation at 98°C for 10 seconds, 58°C: annealing for 30 seconds, extension at 72°C for 1 minute, and 30 cycles of reaction; further extension at 72°C for 10 minutes, and stop at 25°C.
  • the target gene was separated in 2% agarose gel, and the pSC65 vector was digested with Sal I (Thermo Scientific Company, Cat. No. ER0642) to linearize the vector, and cut For gel recovery, use the Sanprep Column DNA Gel Recovery Kit (Promega, Cat. No. A9282) to recover PCR fragments and vector enzyme-digested fragments.
  • the recovered product of the gene was connected with the enzyme-cut linearized vector by homologous recombination (Novazyme, Cat. No. c112-02). The ligation product was transformed into E. coli TOP10 and grown overnight on ampicillin-containing culture plates.
  • Cell preparation Spread 143TK - cells in a 6-well plate, about 1 ⁇ 10 6 per well. Cultivate for about 24 hours, when the cells adhere to the wall and cover the entire bottom surface, proceed to the next step.
  • Vaccinia virus incubation Infect cells with wild-type vaccinia virus Tiantan strain at 0.0125/3PFU (PFU: plaque forming unit, virus liquid titer)/cell, incubate in a 37°C incubator for 1 hour, take it out, and suck off the supernatant , and washed once with 1mL PBS and then added 1mL complete medium.
  • PFU plaque forming unit, virus liquid titer
  • Plasmid transfection The above-mentioned shuttle plasmid PSC65- ⁇ CD47- ⁇ CD3-BITE was transfected into 143TK- cells. Cultivate in a 37°C incubator for about 48 hours, and the specific time depends on the condition of the cells.
  • the working concentration of BrdU is 50 ⁇ g/mL, put it in a 37°C incubator and incubate for about 48 hours, and spread the spots according to the formation of virus spots. This purification process needs to be carried out at least 5 times.
  • Sample collection Carefully aspirate 1 mL of the medium supernatant in the well. Blow down the cells with the remaining 1mL medium and store them in EP tubes, which can be used for subsequent genome extraction and amplification as virus seeds.
  • VERO cell plating 10cm dish, about 5 ⁇ 106 cells per dish, it is advisable to ensure that the cell density reaches 100% when inoculating vaccinia virus the next day;
  • Collect vaccinia virus Discard 8mL medium in the dish, take 2mL maintenance medium to blow off the remaining cells, and collect in a 15mL centrifuge tube;
  • 143TK - cells spread 143TK - cells in a 24-well plate, about 2 ⁇ 10 5 cells per well, and the cell density should reach 100% of the bottom area of the 24-well plate;
  • Spotting method prepare 8 mL of spotting medium containing 2 ⁇ DMEM medium + 4% FBS + 2% PS and 8 mL of low-melting point agarose that is melted in a boiling water bath and placed in a 37°C water bath. Mix, then add X-gal to the mixture, the final concentration is 200 ⁇ g/mL, set aside;
  • Virus spot counting first observe whether the number of virus spots is decreasing in a ten-fold ratio, and then count the number of single-digit blue spots in the two replicate wells of seed virus, and obtain the blue spots in the two wells. The sum of the values, multiplied by the reciprocal value of the dilution corresponding to the well is the titer of the virus in 1 mL.
  • maintenance medium DMEM medium + 2% FBS + 1% PS
  • Purify the virus supernatant for His-tagged protein use a syringe to manually purify, and fill the nickel beads with Ni-NTA Agarose (QIAGEN, Cat. No. 30210); 2 PO 4 ⁇ 2H 2 O, pH 8.0, in 300mM NaCl) to elute the non-specifically adsorbed protein , eluted at a natural flow rate, and collected the filtrate; ⁇ 2H 2 O, pH 8.0, 300mM NaCl) to elute the target protein, and collect the corresponding filtrate.
  • Example 5 Affinity detection of recombinant vaccinia virus rTV- ⁇ CD47- ⁇ CD3-BITE
  • MOI virus PFU/number of cells
  • CD47-A549 cells divide 2 ⁇ 10 6 cells into two EP tubes, centrifuge at 800g for 3 minutes, and discard the supernatant.
  • Fig. 5 shows that the cell supernatant expressed by vaccinia virus recombinant with ⁇ CD47- ⁇ CD3-BITE can bind to CD47, and the positive rate is 85.9% compared with wild-type vaccinia virus.
  • the positive clones were selected for passage stability test, and the strain with strong passage stability and high expression of the target protein was selected for preservation, named rTV- ⁇ CD47- ⁇ CD3-BITE, and its preservation number was: CCTCC NO:V202081, and the preservation date was On January 2, 2021, the preservation address is the Chinese Type Culture Collection Center, address: Wuhan University, Wuhan, China.
  • Example 6 Anti-lung cancer effect of recombinant vaccinia virus of ⁇ CD47- ⁇ CD3-BITE in vitro
  • 143TK - cell plating spread 143TK - cells in a 6-well plate, about 1 ⁇ 10 6 cells per well, and the cell density should reach 100% of the bottom area of the 6-well plate;
  • the in vitro killing experiment of A549 cells was carried out according to the instructions of the kit (Dojindo Company, Cat. No. CK17); pipette 100 ⁇ L of resuspended A549 cells into a 96-well plate, with 1 ⁇ 104 cells per well, at 37°C, 5 % CO 2 for overnight incubation.
  • ⁇ CD47- ⁇ CD3-BITE protein combined with T cells has a very strong killing activity on tumor cells, and the prepared recombinant vaccinia virus rTV- ⁇ CD47- ⁇ CD3-BITE, as an oncolytic virus, can also significantly control human lung cancer and many other diseases.
  • a solid tumor which has very high application value for tumor treatment and is easy to prepare, and is convenient for mass production and popularization.
  • Example 7 Different structural formulas of ⁇ CD47- ⁇ CD3-BITE mediate anti-human activity of T cells in vitro ovarian adenocarcinoma cells
  • the VH ⁇ CD47 is shown in SEQ ID NO:19
  • the VL ⁇ CD47 is shown in SEQ ID NO:21
  • the VH ⁇ CD3 is shown in SEQ ID NO:25
  • the VL ⁇ CD3 is shown in SEQ ID NO:26
  • said L is GGGGSGGGGSGGGGS.
  • the VH ⁇ CD47 is shown in SEQ ID NO:19
  • the VL ⁇ CD47 is shown in SEQ ID NO:21
  • the VH ⁇ CD3 is shown in SEQ ID NO:25
  • the VL ⁇ CD3 is shown in SEQ ID NO:26
  • said L is GGGGSGGGGSGGGGS.
  • the VH ⁇ CD47 is shown in SEQ ID NO:19
  • the VL ⁇ CD47 is shown in SEQ ID NO:21
  • the VH ⁇ CD3 is shown in SEQ ID NO:25
  • the VL ⁇ CD3 is shown in SEQ ID NO:26;
  • One connecting peptide is GGGGSGGGGSGGGGS.
  • BITE4 Same as BITE1, only the connecting peptide is replaced with GGGGGG;
  • BITE5 Same as BITE1, the VH ⁇ CD3 is shown in SEQ ID NO:27, and the VL ⁇ CD3 is shown in SEQ ID NO:28.
  • Embodiment is the same as embodiment 2 and 3, and the result is as shown in Figure 7.
  • VL ⁇ CD47 -L-VH ⁇ CD3 -L-VL ⁇ CD3 -L-VH ⁇ CD47 where L is (G 4 S) 3 , which is the optimal design of ⁇ CD47- ⁇ CD3-BITE, which can mediate the strong function of killing tumor cells and transform CD47 Or CD3 (such as clone number SP34) antibody clone species still retains a strong killing function;
  • VH ⁇ CD47-L-VH ⁇ CD3-L-VL ⁇ CD3-L-VL ⁇ CD47 where L is (G 4 S) 3 and is ⁇ CD47- ⁇ CD3-BITE suboptimal design, but still had similar tumor-killing activity to the control antibody.

Abstract

本发明提供了双特异性T细胞衔接器、其重组溶瘤病毒及其用途。本发明提供了一种αCD47和αCD3双特异性T细胞衔接器。本发明还提供了一种分离的核酸分子，其编码所述双特异性T细胞衔接器。本发明还提供了所述双特异性T细胞衔接器BiTE的表达框架。本发明还提供了一种重组溶瘤病毒，所述溶瘤病毒可操作地***或包含所述双特异性T细胞衔接器BiTE的表达框架。本发明将双特异性T细胞衔接器与溶瘤病毒结合，相对于单纯的基因疗法或者病毒疗法，所述溶瘤病毒显著增强对恶性肿瘤的抑制能力。

Description

双特异性T细胞衔接器、其重组溶瘤病毒及其用途 技术领域
本发明属于生物医药技术领域，涉及一种新的包含人CD47和人CD3特异性结合片段的双特异性T细胞衔接器(BiTE)。本发明还涉及一种包含所述双特异性T细胞衔接器(BiTE)的重组溶瘤病毒，本发明还提供了所属双特异性T细胞衔接器和所述溶瘤病毒的制备方法及其在抗肿瘤方面的应用。
背景技术
癌症是全球第二大死亡原因，2018年估计有960万人死于癌症。在全球范围内，大约六分之一的死亡是由癌症造成的。中国的情况尤其令人担忧，部分原因是由于快速的人口增长和社会经济发展。虽然中国的癌症发病率低于欧美发达国家，但是中国的癌症死亡率比欧美国家高30％～40％，而且预后明显较差。如今癌症在中国越来越受到关注，相关的癌症治疗方法，特别是借助人体自身免疫***的肿瘤免疫治疗日益成为人们关注的焦点。
人CD47即整合素相关蛋白，是一种细胞膜表面糖蛋白，属于免疫球蛋白超家族。CD47广泛表达于多种癌细胞表面，通过与肿瘤吞噬细胞表面信号调控蛋白α(signaling regulatory protein alpha，SIRPα)连接释放“别吃我”信号，阻止巨噬细胞吞噬作用。近年来，有多家公司投入大量人力物力研发CD47抗体，但是进入临床后收效甚微，目前CD47抗体的最好水平是可以进入组合药中。治疗效果不佳的主要原因是副作用难以控制，这归咎于CD47的致命弱点，即在红细胞上表达。CD47抗体药物或者SIRPα-Fc融合蛋白与红细胞结合后，可引起红细胞凝集，继而引发红细胞的破裂；带有IgG1的Fc的CD47抗体药物还能激活巨噬细胞对红细胞的吞噬作用或者抗体依赖的细胞介导的细胞毒性作用(antibody-dependent cell-mediated cytotoxicity，ADCC)，进一步引发红细胞的裂解，最终导致贫血的发生。
如何最大化利用CD47靶点在杀伤肿瘤细胞上优势，同时减少对红细胞的损伤，一直是成功开发针对CD47药物的关键所在。双特异性T细胞衔接器BiTE(bispecific T-cell engager)代表一类具有显著抗肿瘤效应的双特异性抗体，能够靶向性激活自身T细胞杀伤肿瘤细胞。BiTE由两个单链可变片段(scFv)组成，通过柔性接头串联连接。一个scFv识别T细胞表面蛋白CD3εH，而另一个scFv识别特异性肿瘤细胞表面抗原。BiTE的这种结构和特异性结合蛋白的能力允许它将T细胞物理性地桥接肿瘤细胞而形成T细 胞-BiTE-肿瘤细胞复合物，诱导免疫突触形成，刺激T细胞活化，产生杀伤肿瘤的细胞因子。近年来，BiTE在抗肿瘤研究中取得了显著进展，在临床上取得了理想的治疗效果。
由于标准肿瘤治疗方案在晚期肿瘤治疗中有限的治疗效果以及严重副作用，靶向肿瘤的溶瘤病毒在近几十年里引起了极大的关注。溶瘤病毒得益于其自身可选择性的在肿瘤局部感染并裂解肿瘤细胞的特性，正在越来越多的成为抗肿瘤的首选。溶瘤病毒的肿瘤选择性可以是本身的肿瘤趋向性，也可以是基因修饰的结果。溶瘤病毒可作用于多个细胞通路，以此减少肿瘤的抗性，还可以诱导不同形式的细胞死亡。另外，溶瘤病毒可以打破肿瘤微环境的免疫耐受，诱导长期的肿瘤特异性免疫应答。溶瘤病毒可特异性的转运治疗性蛋白进入肿瘤组织，伴随病毒的进一步复制增加在恶性肿瘤细胞中表达水平。另外，溶瘤病毒可以同化疗、放疗以及免疫治疗联合使用。
2006年，溶瘤腺病毒产品(oncorine)已在中国用于临床治疗鼻咽癌等。该溶瘤病毒删除了人5型腺病毒的E1B-55kD基因，可在p53基因突变的癌细胞中复制增殖并杀伤宿主细胞，产生溶瘤治疗作用；同时删除E3区，使肿瘤抗原信息能通过树突细胞的传递而激活T细胞的免疫。但临床数据显示，相较于放射治疗，溶瘤病毒oncorine联合化疗对鼻咽癌患者的治疗效果较弱。
2015年，安进公司的溶瘤单纯疱疹病毒(talimogene laherparepvec，T-VEC)获得美国FDA批准用于治疗黑色素瘤，同年12月又获得欧盟批准用于首次手术后复发的黑色素瘤患者不可切除的皮肤、皮下和***病灶的局部治疗。T-VEC临床研究结果极大地推动了溶瘤病毒在肿瘤治疗领域的研发进程。但是通过瘤内给药方式限制了治疗类型，只能应用在离体表近的便于手术的肿瘤类型中，对很多非浅表的实体瘤及转移瘤的治疗存在给药困难和治疗不彻底的问题，若能证实通过静脉给药仍具有较好的肿瘤治疗效果，将极大地提高其临床应用价值；由于疱疹病毒自身溶瘤作用的限制性，对体积大和/或转移的肿瘤清除不完全，所以还需要联合其他治疗手段来增强抗肿瘤作用。
痘苗病毒(Vaccinia virus，VV)的生物学性状和致病机制相对明晰，在消除天花的过程中起到了关键作用，它在人体的安全性也得到了充分证明。根据致病性，宿主范围等特点，痘苗病毒可分为WR(Western reserve)株、Wyeth株、Copenhagen株、Lister株、Tian Tan株等。因其具有宿主范围广，保守性高，安全性好及外源基因容量大等特点，痘苗病毒被用于流感病毒、人类免疫缺陷病毒等多个重组疫苗的载体。作为溶瘤病毒的研发，目前大多数处于临床前研究阶段，只有少数进入临床研究阶段。
利用痘苗病毒作为溶瘤病毒***的研究，目前进展最快的是美国Jennerex公司研发的Pexa-Vec(JX-594)。JX-594基于Wyeth株病毒，在TK区***hGM-CSF和LacZ基因，由于缺失胸腺激酶基因，JX-594可在高水平表达胸腺激酶的癌细胞中表达复制，但不会对正常细胞产生影响。同时JX-594因***了GM-CSF基因，可在肿瘤细胞中表达GM-CSF，激活机体的抗肿瘤免疫反应。JX-594多种肿瘤类型的临床试验结果证明，经瘤内给药或静脉滴注给药均具有良好的耐受性，Pexa-Vec联合sorafenib效果好于单独用药组。中期分析结果表明其延长患者生存期的可能性不高。原本计划2020上市的Pexa-Vec，临床III期试验提前终止。
美国Genelux公司研发的溶瘤病毒(GL-ONC1，又称为GLV-1h68)，是基于痘苗病毒(Lister株)，删除其基因F14.5L、TK(编码胸苷激酶)和HA(编码血凝素)以加强靶向肿瘤，并分别***luciferase-GFP融合蛋白、β-半乳糖苷转移酶和β-葡萄糖醛酸酶进行痘病毒的筛选与生产制备。GL-ONC1已经完成的静脉注射给药I期临床试验显示出良好的安全性和疗效，未出现剂量限制毒性，未到达最大耐受剂量，所有患者均出现针对GL-ONC1的中和反应；GL-ONC1目前正在进行静脉注射给药的扩大性实验。
痘苗病毒的生活周期严格在宿主细胞的细胞质中进行，且需要痘苗病毒的胸苷激酶基因作用，子代病毒的基因组才能顺利复制。而在正常的组织细胞周期中，细胞胸苷激酶(Thymidine Kinase，TK)的合成发生在细胞***周期的S期，细胞***完成后，胸苷激酶在细胞内部降解，因此细胞质中胸苷激酶浓度较低。但肿瘤细胞***活跃，其胸苷激酶持续合成。利用该特征，删除痘苗病毒的胸苷激酶基因，致使痘苗病毒在肿瘤细胞中特异性扩增，起到溶瘤的作用。
临床试验证据显示，痘苗病毒在肿瘤治疗方面已经初步显示了一定的优势。现有的大多数利用痘苗病毒作为溶瘤病毒设计安全性较高，临床有效性待进一步观察，但是在免疫调节与精准肿瘤靶向性上还需要进一步优化。
基于以上，现有技术中不存在特别适用于重组溶瘤病毒的双特异性T细胞衔接器，当前对更安全、靶向性更强的双特异性T细胞衔接器和包含其的溶瘤病毒存在需求。
发明简述
因此，本发明的目的是针对现有技术的不足，提供一种双特异性T细胞衔接器。本发明还提供了表达所述双特异性T细胞衔接器(BiTE)的重组溶瘤病毒。本发明提供的双特异性T细胞衔接器(BiTE)克服了实体瘤肿瘤 细胞对人类T细胞的肿瘤免疫编辑(免疫抑制和免疫逃避)的限制，能够直接募集人体T细胞到实体瘤内部对肿瘤细胞进行肿瘤细胞杀伤，有着广阔的应用前景。
本发明的目的是通过以下技术方案实现的：
一方面，本发明提供了一种αCD47和αCD3双特异性T细胞衔接器αCD47-αCD3 BiTE，其包含如下式中任一项所示的融合蛋白：
VL αCD47-L-VH αCD3-L-VL αCD3-L-VH αCD47；或
VH αCD47-L-VH αCD3-L-VL αCD3-L-VL αCD47
其中，所述VH αCD47为CD47抗体的重链可变区，其包含下述3个互补决定区：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:1，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:2，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列；
优选地，(i)-(iii)任一项中所述的置换为保守置换；
优选地，所述VH αCD47包含：如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:2所示的VH CDR2、如SEQ ID NO:3所示的VH CDR3；
其中，所述VL αCD47为CD47抗体的轻链可变区，其包含下述3个互补决定区：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10、SEQ ID NO:13或SEQ ID NO:16中任一项所示，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、SEQ ID NO:14或SEQ ID NO:17中任一项所示，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、SEQ ID NO:15或SEQ ID NO:18中任一项所示，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列；
优选地，(iv)-(vi)任一项中所述的置换为保守置换；
优选地，所述VL αCD47包含：如SEQ ID NO:4所示的VL CDR1、如SEQ ID NO:5所示的VL CDR2、如SEQ ID NO:6所示的VL CDR3；
或所述VL αCD47包含：如SEQ ID NO:7所示的VL CDR1、如SEQ ID NO:8所示的VL CDR2、如SEQ ID NO:9所示的VL CDR3；
或所述VL αCD47包含：如SEQ ID NO:10所示的VL CDR1、如SEQ ID NO:11所示的VL CDR2、如SEQ ID NO:12所示的VL CDR3；
或所述VL αCD47包含：如SEQ ID NO:13所示的VL CDR1、如SEQ ID NO:14所示的VL CDR2、如SEQ ID NO:15所示的VL CDR3；
或所述VL αCD47包含：如SEQ ID NO:16所示的VL CDR1、如SEQ ID NO:17所示的VL CDR2、如SEQ ID NO:18所示的VL CDR3。
根据本发明所述的双特异性T细胞衔接器，其中，所述VH αCD47包含SEQ ID NO:19所示的重链可变区中含有的3个CDR；
优选地，所述VL αCD47包含SEQ ID NO:20-24中任一项所示的轻链可变区中含有的3个CDR；
优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号***定义。
根据本发明所述的双特异性T细胞衔接器，其中，所述VH αCD47包含选自下列的氨基酸序列：
(i)SEQ ID NO:19所示的序列；
(ii)与SEQ ID NO:19所示的序列相比具有一个或几个氨基酸的置换、缺失或添加的序列；或
(iii)与SEQ ID NO:19所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
所述VL αCD47包含选自下列的氨基酸序列：
(iv)SEQ ID NO:20-24中任一项所示的序列；
(v)与SEQ ID NO:20-24中任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加的序列；或
(vi)与SEQ ID NO:20-24中任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
优选地，(ii)或(v)中所述的置换是保守置换。
根据本发明所述的双特异性T细胞衔接器，其中，所述VH αCD3包含选自下列的氨基酸序列：
(i)SEQ ID NO:25或27所示的序列；
(ii)与SEQ ID NO:25或27所示的序列相比具有一个或几个氨基酸的置换、缺失或添加的序列；或
(iii)与SEQ ID NO:25或27所示的序列具有至少80％、至少85％、至 少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
所述VL αCD3包含选自下列的氨基酸序列：
(iv)SEQ ID NO:26或28所示的序列；
(v)与SEQ ID NO:26或28所示的序列相比具有一个或几个氨基酸的置换、缺失或添加的序列；或
(vi)与SEQ ID NO:26或28所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
优选地，(ii)或(v)中所述的置换是保守置换。
根据本发明所述的双特异性T细胞衔接器，其中，所述VH αCD47、VH αCD3、VL αCD47和VL αCD3通过连接肽连接；
优选地，所述VH αCD47、VH αCD3、VL αCD47和VL αCD3之间任选地通过一条、两条或三条连接肽连接；
优选地，双特异性T细胞衔接器包含如下式中任一项所示的融合蛋白：
VL αCD47-L-VH αCD3-L-VL αCD3-L-VH αCD47；或
VH αCD47-L-VH αCD3-L-VL αCD3-L-VL αCD47
更优选地，所述L为KESGSVSSEQLAQFRSLD、EGKSSGSGSESKST、GGGGGG、GGGGGGGG或(GGGGS) n；进一步优选地，n为1-5的整数，最优选地，n为3；
优选地，所述双特异性T细胞衔接器包含如下式所示的融合蛋白：
VH αCD47-L-VH αCD3-L-VL αCD3-L-VL αCD47
其中，所述VH αCD47如SEQ ID NO:19所示，所述VL αCD47如SEQ ID NO:21或24所示，所述VH αCD3如SEQ ID NO:25所示，VL αCD3如SEQ ID NO:26所示，并且所述L为GGGGSGGGGSGGGGS。
本发明还提供了一种分离的核酸分子，其编码所述的双特异性T细胞衔接器；
优选地，所述核酸分子包含SEQ ID NO：31-35中任一项所示的核酸序列，所述核酸序列为VL αCD47的编码序列；
优选地，所述核酸分子包含如SEQ ID NO：36所示的核酸序列，所述核酸序列为VH αCD47的编码序列。
根据本发明所述的双特异性T细胞衔接器的表达框架：
5’-E1-E2-E3-E4-E5-E6-E7-3’
其中：
E1是CMV增强子或/和其它顺式作用元件；优选包含SEQ ID NO：37所示的核酸序列；
E2是重组表达的启动子，优选CMV启动子，更优选包含SEQ ID NO：38所示的核酸序列；
E3是5’端非翻译区，任选地不包含或包含一个内含子序列，任选地包含一个或多个限制酶位点；优选包含SEQ ID NO：39所示的核酸序列；
E4是BiTE蛋白的信号肽的编码核苷酸序列；优选地，所述信号肽源于人或小鼠的信号肽；优选包含SEQ ID NO：40所示的核酸序列；
E5是所述的双特异性T细胞衔接器的编码核苷酸序列；
E6是3’端非翻译区，任选地包含一个或多个限制酶位点；优选包含SEQ ID NO：41所示的核酸序列；
E7是SV40转录中止信号区；优选包含SEQ ID NO：42所示的核酸序列。
再一方面，本发明提供了一种重组溶瘤病毒，所述溶瘤病毒可操作地***或包含所述双特异性T细胞衔接器BiTE的表达框架。
优选地，所述表达框架位于所述重组溶瘤病毒的胸腺嘧啶核苷激酶(TK)区。
优选地，所述表达框架可单独表达、也可与其他基因或片段融合表达。
优选地，所述重组溶瘤病毒还包含其他免疫调节因子的基因编码序列，更优选地，所述其他免疫调节因子包括但不限于IL-1、IL-2、IL-3、IL-7、IL-11、IL-12、IL-15、IL-17、IL-18、IL-21、IL-33、IL-35、IL-37、GM-CSF、IFN-α、IFN-β、IFN-γ、抗PD-1/PD-L1抗体、抗CTLA-4抗体、抗Lag-3抗体、抗TIGIT抗体或抗Tim-3抗体；或
所述重组溶瘤病毒还包含与细胞凋亡和细胞焦亡相关的蛋白的基因编码序列，所述与细胞凋亡和细胞焦亡相关的蛋白包括但不限于凋亡相关因子1(Apaf-1)、白介素-1β转换酶(ICE)、Bcl-2蛋白、Fas/APO-1、p53、myc、共济失调毛细血管扩张突变基因(ATM)、消皮素D(gasdermin D)、消皮素E(gasdermin E)等；或
所述重组溶瘤病毒还包含靶向免疫调节基因、凋亡与焦亡基因的小RNA。
根据本发明所述的重组溶瘤病毒，其中，所述溶瘤病毒的病毒骨架来源于经修饰或经改造的痘苗病毒天坛株、痘苗病毒纽约株、痘苗病毒哥本哈根株、痘苗病毒金丝雀株、痘苗病毒安卡拉株、腺病毒、腺相关病毒、单纯疱疹病毒、水痘-带状疱疹病毒(varicella-zoster virus，VZV)、呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)、生里基森林病毒(Semliki forest virus， SFV)、EB病毒、巨细胞病毒、人疱疹病毒6型、天花病毒、痘苗病毒、***病毒、羊口疮病毒、呼肠孤病毒、轮状病毒、肠道病毒、塞内卡病毒、脊髓灰质炎病毒、柯萨奇病毒、鼻病毒、甲型肝炎病毒、***病毒、披膜病毒、甲病毒、塞姆利基森林病毒、东部马脑炎病毒、辛德毕斯病毒、风疹病毒、冠状病毒、黄病毒、丙型肝炎病毒、日本脑炎病毒、圣路易斯脑炎病毒、墨累谷热病毒、黄热病毒、西尼罗河病毒、寨卡病毒、登革病毒、埃博拉病毒、马尔堡病毒、沙粒病毒、拉沙热病毒、淋巴细胞性脉络丛脑膜炎病毒、皮钦德病毒、胡宁病毒、马丘波病毒、汉坦病毒、裂谷热病毒、副粘病毒、人副流感病毒、腮腺炎病毒、猴病毒5、麻疹病毒、水疱性口炎病毒、狂犬病病毒、正粘病毒、甲型流感病毒、乙型流感病毒、丙型流感病毒、丁型肝炎病毒、猴免疫缺陷病毒、人免疫缺陷病毒1型和人免疫缺陷病毒2型、劳氏肉瘤病毒、嗜人T细胞白血病病毒1型、猴泡沫病毒、乙型肝炎病毒、戊型肝炎病毒、人***瘤病毒或多瘤病毒。
优选地，所述溶瘤病毒骨架为细胞内成熟病毒、细胞内包装病毒、细胞相关包装病毒或细胞外包装病毒。
根据本发明所述的重组溶瘤病毒，其中，所述溶瘤病毒为包含双特异性T细胞衔接器αCD47-αCD3-BITE的编码基因的重组痘苗病毒天坛株，命名为rTV-αCD47-αCD3-BITE，其保藏编号为：CCTCC NO:V202081，保藏日期为2021年1月2日，保藏地址为中国典型培养物保藏中心。
再一方面，本发明提供了所述重组溶瘤病毒的制备方法，包括以下步骤：
1)合成所述双特异性T细胞衔接器BiTE的表达框架；
2)将步骤1)得到的表达框架亚克隆至溶瘤病毒的穿梭质粒中，构建重组质粒载体；
3)将步骤2)得到的重组质粒载体转染至溶瘤病毒中，经筛选获得重组溶瘤病毒。
任选地，培养获得的重组溶瘤病毒。
在一个具体的实施方案中，本发明提供了一种重组痘苗病毒天坛株的制备方法，包括以下步骤：
1)合成双特异性T细胞衔接器αCD47-αCD3-BITE的表达框架，其包含如SEQ ID NO：31-42中任一项所示的核酸序列；
2)将合成的表达框架亚克隆至痘苗病毒穿梭质粒(pSC65)的TK区中，构建重组质粒pSC65-αCD47-αCD3-BITE；
3)采用基因同源重组的方式，将pSC65-αCD47-αCD3-BITE质粒转染到已经被感染了野生型痘苗病毒的TK143 -细胞中，使两者同源重组，产生重组痘苗病毒rTV-αCD47-αCD3-BITE；经筛选获得所述TK区包含 αCD47-αCD3-BITE的编码序列的重组溶瘤痘苗病毒。
其中，双特异性T细胞衔接器αCD47-αCD3-BITE的表达框架由痘苗病毒的早/晚期启动子p7.5控制。
优选地，利用VERO细胞扩增重组痘苗病毒的具体步骤包括：培养VERO细胞至密度接近100％时，更换低浓度胎牛血清维持培养基，加入溶瘤痘苗病毒(接种量约为0.02MOI每10cm培养板)，放入孵箱培养，重组痘病毒扩增完毕后，收集病毒液再反复冻融后，用蔗糖溶液进行密度梯度离心纯化。
再一方面，本发明还提供了所述双特异性T细胞衔接器或重组溶瘤病毒用于制备抗肿瘤药物的用途；其中，所述肿瘤选自B细胞淋巴瘤、T细胞淋巴瘤、黑色素瘤、***癌、肾细胞癌、肉瘤、胶质瘤如高级别胶质瘤、母细胞瘤如神经母细胞瘤、骨肉瘤、浆细胞瘤、组织细胞瘤、胰腺癌、乳腺癌、肺癌诸如小细胞肺癌和非小细胞肺癌、胃癌、肝癌、结肠癌、直肠癌、食管癌、大肠癌、造血***癌、睾丸癌、***、卵巢癌、膀胱癌、鳞状细胞癌、腺癌、AIDS相关淋巴瘤、膀胱癌、脑癌、神经***癌、头颈癌、头颈部鳞状细胞癌、霍奇金氏淋巴瘤、非霍奇金氏淋巴瘤或血液致瘤疾病。
另一方面，本发明提供了一种***的方法，所述方法包括给予有需要的受试者治疗有效量的双特异性T细胞衔接器或重组溶瘤病毒。根据本发明所述的方法，其中所述方法还包括给予有需要的受试者另外的化疗药物、放疗技术、手术治疗、免疫细胞药物(包括但不限于CAR-T、NK、NKT、iNKT、CAR-NK、CAR-NKT、CAR-iNKT等)、其他溶瘤病毒；优选地，所述方法为静脉注射或瘤内注射。
本发明的发明构思如下：
本发明提供一种新的αCD47-αCD3-BITE双特异性T细胞衔接器，其在体外有良好的抗肿瘤活性。进一步地，本发明人以痘苗病毒天坛株为研究模式，提供了一种搭载本发明的双特异性T细胞衔接器的痘苗病毒天坛株。然后本发明的发明人发现痘苗病毒天坛株自身具有强大的抗肿瘤作用；搭载αCD47-αCD3-BITE的双特异性T细胞衔接器，可避免CD47抗体与外周血的红细胞结合，利用CD47广谱表达的特性识别肿瘤细胞，以及CD3抗体活化T细胞，进而起到杀伤肿瘤细胞的作用，提升肿瘤免疫治疗效果的同时，增加安全性。
本发明的有益效果是：
1.本发明提供的双特异性T细胞衔接器(BiTE)分子量小，能够穿透细胞间隙到达实体瘤内部的肿瘤微环境(TME)发挥作用，其能够高效介导T细胞对癌细胞的杀伤。
2.本发明将双特异性T细胞衔接器与溶瘤病毒结合，提供了一种可高效表达双特异性T细胞衔接器αCD47-αCD3-BITE基因的痘苗病毒天坛株。在将该溶瘤病毒投药至肿瘤病灶时，它在裂解肿瘤细胞发挥溶瘤效应的同时，还搭载靶向肿瘤的基因抗人CD47的高杀伤性抗体，阻断CD47与SIRPα结合，提升体内巨噬细胞吞噬功能，来增强抗肿瘤效果。相对于单纯的基因疗法或者病毒疗法，所述溶瘤病毒显著增强对恶性肿瘤的抑制能力。
3.根据本发明的体外实验研究，证明本发明的痘苗病毒天坛株能够治疗肝癌与恶性肺癌，并且实现了对肿瘤的良好靶向性及抗肿瘤效果，具有较为完备的病毒扩增及质量控制体系，为进一步的产业化奠定基础，因此，本发明具有良好的应用前景。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出了本发明的穿梭质粒载体αCD47-αCD3 BiTE hIgG1 WT的构建和5种相应BiTE蛋白的表达纯化；其中图1A为整合有αCD47-αCD3-BITE表达框架的穿梭质粒hIgG1 WT表达图谱；图1B为293T瞬时表达αCD47-αCD3-BITE的培养基上清，利用镍柱进行蛋白纯化后的结果。
图2示出了αCD47-αCD3-BITE介导T细胞的体外抗人卵巢腺癌细胞的效果；其中，相对于T细胞对照组，68号克隆的CD47抗体与αCD3组成的BiTE都能够很好地介导T细胞对SK-OV3的杀伤。说明αCD47-αCD3-BITE能够很好地识别CD47的肿瘤细胞且激活T细胞，从而介导T细胞对该肿瘤细胞的特异杀伤；表明包含人CD47和人CD3特异性结合的双特异性T细胞衔接器(BITE)在体外能够很好地介导T细胞的抗肿瘤活性且具有广谱性。
图3示出了αCD47-αCD3-BITE介导T细胞的体外抗人肺癌细胞的效果；其中，相对于T细胞对照组，101号克隆的CD47抗体与αCD3组成的BiTE能够介导T细胞对NCI-H292肿瘤细胞的杀伤，与单独T细胞组比较，BiTE组杀伤NCI-H292细胞效果明显；表明包含人CD47和人CD3特异性结合的双特异性T细胞衔接器(BITE)在体外能够很好得介导T细胞的抗肿瘤活性且具有广谱性。
图4显示了痘苗病毒天坛株穿梭质粒载体(αCD47-αCD3-BITE)的构建和αCD47-αCD3-BITE蛋白的表达。图4A为整合有αCD47-αCD3-BITE基因的痘苗病毒穿梭质粒pSC65表达图谱；图4B为所述重组痘苗病毒rTV-αCD47-αCD3-BITE中***片段αCD47-αCD3-BITE的PCR鉴定结果； 图4C显示了重组痘苗病毒感染VERO细胞的上清中有CD47-αCD3-BITE蛋白表达。从图4可知，重组痘苗病毒rTV-αCD47-αCD3-BITE所搭载的αCD47-αCD3-BITE成功表达。
图5显示了重组痘苗病毒rTV-αCD47-αCD3-BITE搭载的αCD47-αCD3-BITE与CD47蛋白具有很高的亲和力；图5中为重组痘苗病毒rTV-αCD47-αCD3-BITE感染VERO细胞收集的细胞上清与表达人源CD47的肺癌细胞A549细胞进行亲和检测的流式结果；结果显示与野生型痘苗病毒相比，重组痘苗病毒rTV-αCD47-αCD3-BITE表达蛋白与CD47的结合率在85％以上。
图6显示了痘苗病毒天坛株rTV-αCD47-αCD3-BITE的重组痘苗病毒的体外杀伤A549细胞作用。实验分为四组，包括野生痘苗病毒感染的上清对照组、野生痘苗病毒感染的上清+T细胞组、重组痘苗病毒感染的上清对照组和重组痘苗病毒感染的上清+T细胞组。从图中可知重组痘苗病毒感染的上清+T细胞组与空白对照组相比对肺癌细胞A549细胞具有显著的杀伤作用。
图7显示了不同结构的αCD47-αCD3-BITE蛋白的抗癌效果。
保藏信息
痘苗病毒天坛株，命名为rTV-αCD47-αCD3-BITE，其保藏编号为：CCTCC NO:V202081，保藏日期为2021年1月2日，保藏地址为中国典型培养物保藏中心，地址：中国武汉武汉大学。
实施发明的最佳方式
实施例1：αCD47-αCD3-BITE的293T重组表达质粒的构建以及表达验 证
1.1带αCD47-αCD3-BITE目的基因的hIgG1 WT载体构建
首先，筛选高亲和力的5个抗人CD47抗体，选取其重链可变区和轻链可变区，并按照下式构建融合蛋白：VH αCD47-L-VH αCD3-L-VL αCD3-L-VL αCD47；
其中，所述VH αCD47如SEQ ID NO:19所示，所述VL αCD47如SEQ ID NO:20-24中任一项所示，所述VH αCD3如SEQ ID NO:25所示，VL αCD3如SEQ ID NO:26所示，并且所述L为GGGGSGGGGSGGGGS。
并且，根据VL αCD47不同，Hu004-67表示包含SEQ ID NO:20所示的VL αCD47的αCD47-αCD3 BiTE；Hu004-68表示包含SEQ ID NO:21所示的VL αCD47的αCD47-αCD3 BiTE；Hu004-73表示包含SEQ ID NO:22所示的 VL αCD47的αCD47-αCD3 BiTE；Hu004-100表示包含SEQ ID NO:23所示的VL αCD47的αCD47-αCD3 BiTE；Hu004-101表示包含SEQ ID NO:24所示的VL αCD47的αCD47-αCD3 BiTE，其中WT Cloning vector AbVec-hIgG1(GenBank:FJ475055.1)。
按照结构式人工合成αCD47-αCD3-BITE的DNA序列，质粒构建图谱如图1a所示，其中为VL αCD47的编码序列如SEQ ID NO：31-35中任一项所示，VH αCD47的编码序列如SEQ ID NO：36所示。
将人工合成的包含αCD47-αCD3-BITE目的基因的hIgG1 WT载体转化至大肠杆菌E.coli TOP10(唯地公司，货号DL1010S)，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行37℃过夜培养，应用质粒抽提试剂盒(Qiagen公司，货号27106)提取质粒用于293T细胞转染。
1.2带αCD47-αCD3-BITE目的基因的293T细胞表达纯化
1. 293T细胞准备:将生长于T175培养瓶中汇合度超过90％的细胞移去上清，加入2ml 2.5％Trypsin-EDTA(GIBCO公司，货号25200072)消化细胞至细胞完全脱壁，加入10ml完全培养基(DMEM培养基+10％FBS+1％PS)中止消化，150g离心5分钟收集细胞，弃掉上清，用适量完全培养基重选细胞使细胞浓度约为8×10 6～10×10 6个/ml。将细胞悬液计数，并取1.3×10 7个细胞铺于150mm细胞培养皿(Thermo Scientific公司，货号150468)，培养皿中培养基体积为25mL，培养至第二天用于后续质粒转染。
2.αCD47-αCD3-BITE的hIgG1 WT穿梭质粒转染293T细胞：取1mL DMEM培养基于EP管中，加入20μgαCD47-αCD3-BITE的hIgG1 WT穿梭质粒Cloning vector AbVec-hIgG1(GenBank:FJ475055.1)，混匀后加入30μl FectoPro转染试剂(Polyplus公司，货号116-001)，之后立即置于漩涡振荡器上剧烈震荡15s，置于室温静置20分钟。培养箱中取出待转染的293T细胞，吸取并弃掉10mL培养基，边摇晃边加入静置后的转染试剂，置于37℃5％CO 2的培养箱中培养4-6小时。完全移除培养基，加入Expi293表达培养基(GIBCO公司，货号A1435102)，置于37℃5％CO 2的培养箱中培养4天。
3.收集αCD47-αCD3-BITE表达上清：收集表达培养皿中的培养基于50mL离心管中，4000g离心5分钟，将表达上清转移到新的50mL离心管中。
4.将表达上清进行His标签蛋白纯化：使用重力层析柱进行纯化，填充的镍珠为Ni-NTA Agarose(QIAGEN公司，货号30210)；装柱完成后，用5mM、10mM的咪唑(溶解在50mM NaH 2PO 4·2H 2O，pH 8.0，300mM NaCl中)洗脱非特异吸附的蛋白，自然流速洗脱，收集滤液；然后用20mM、100mM(分先后收集两管)、200mM(分先后收集两管)、500mM的咪唑(溶 解在50mM NaH 2PO 4·2H 2O，pH 8.0，300mM NaCl中)进行目的蛋白的洗脱，收集对应的滤液。
5.取20μL滤液制样，进行10％SDS-PAGE检测。如图所示，由5mM咪唑洗脱液到500mM咪唑洗脱液依次进行上样。由图1B所示100mM的洗脱液和200mM中出现目的蛋白，条带大小为54KD左右。
实施例2：αCD47-αCD3-BITE介导T细胞的体外抗人卵巢腺癌细胞效 果
1.SK-OV3细胞铺板：SK-OV3(保存于上海鑫湾生物科技有限公司)为Luciferase表达细胞系。将细胞以1×10 4个/孔的密度铺于96孔板中，37℃培养过夜待其贴壁。
2. 24小时后除去培养基，设7组，分别加入T细胞，B6H12-αCD3-BITE(B6H12的重链可变区和轻链可变区分别如SEQ ID NO：29和30所示，其余结构与本发明的实施例1的BITE相同)和T细胞，Hu004-67-αCD3-BITE和T细胞，Hu004-68-αCD3-BITE和T细胞，Hu004-73-αCD3-BITE和T细胞，Hu004-100-αCD3-BITE和T细胞，Hu004-101-αCD3-BITE和T细胞。
3. 37℃培养过夜进行杀伤。24小时后除去上清，每孔加入50μL细胞裂解液荧光素酶细胞裂解液(Promega公司，货号E1531)，室温震荡孵育30分钟，每孔加入30μL荧光素酶底物(Promega公司，货号E151A)。上机检测(GloMax Navigator Microplate Luminometer，Promega，Steady-Glo protocol)。
测得结果如图2。其中B6H12是已知的CD47抗体克隆(US 9017675B2)，作为阳性对照。Hu004-68和Hu004-101是筛选到的与CD47蛋白有高亲和力的抗体克隆(委托泷搌生物科技公司进行抗体筛选获得)。由图2可知，针对该种肿瘤细胞系，68号克隆的CD47抗体(scFv)-αCD3-BITE相比T细胞对照组，杀伤肿瘤活性有3.5倍的提升，相比较低杀伤能力的100号克隆有2倍的提升，与阳性对照抗体相比有1.5倍的提升，能够很好地介导T细胞的体外抗卵巢腺癌细胞效果。
实施例3：αCD47-αCD3-BITE介导T细胞的体外抗人肺癌细胞效果
1.NCI-H292细胞铺板：NCI-H292(保存于上海鑫湾生物科技有限公司)为Luciferase表达细胞系。将细胞以1×10 4个/孔的密度铺于96孔板中，37℃培养过夜待其贴壁。
2. 24小时后除去培养基，设7组，分别加入T细胞，B6H12-αCD3-BITE和T细胞，Hu004-67-αCD3-BITE和T细胞，Hu004-68-αCD3-BITE和T细 胞，Hu004-73-αCD3-BITE和T细胞，Hu004-100-αCD3-BITE和T细胞，Hu004-101-αCD3-BITE和T细胞。
3. 37℃培养过夜进行杀伤。24小时后除去上清，每孔加入50μL细胞裂解液(Promega公司，货号E1531)，室温震荡孵育30分钟，每孔加入30μL荧光素酶底物(Promega公司，货号E151A)。上机检测(GloMax Navigator Microplate Luminometer，Promega，Steady-Glo protocol)。
测得结果如图3所示，101号克隆的CD47抗体-αCD3-BITE能够很好的介导T细胞的体外抗肺癌细胞效果，101号克隆的CD47抗体(scFv)-αCD3-BITE相比T细胞对照组，杀伤肿瘤活性有4倍的提升，相比较低杀伤能力的100号克隆有1.2倍的提升，与阳性对照抗体相比有1.2倍的提升，能够很好的介导T细胞的体外抗卵巢腺癌细胞效果。图2和图3结果共同表明包含人CD47和人CD3特异性结合的双特异性T细胞衔接器(BITE)在体外能够很好得介导T细胞的抗肿瘤活性且具有广谱性。
实施例4：αCD47-αCD3-BITE的重组痘苗病毒的构建以及表达验证
4.1带αCD47-αCD3-BITE目的基因的pSC65载体构建
人工合成αCD47-αCD3-BITE的DNA序列，以合成的DNA序列为模版采用如下引物进行PCR扩增。
扩增的引物为：
αCD47-αCD3-BITE-F：SEQ ID NO:43
Figure PCTCN2021141634-appb-000001
αCD47-αCD3-BITE-R：SEQ ID NO:44
Figure PCTCN2021141634-appb-000002
PCR反应程序：94℃预变性5分钟；98℃变性10秒，58℃：退火30秒，72℃延伸1分钟，反应30个循环；72℃再充分延伸10分钟，终止于25℃。
PCR产物的回收与克隆构建：扩增结束后，在2％的琼脂糖凝胶中分离目的基因，同时将pSC65载体用Sal I酶切(Thermo Scientific公司，货号ER0642)线性化载体，并进行切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收PCR片断段与载体酶切片段。基因回收产物与酶切线性化载体用同源重组的方法连接(诺唯赞公司，货号c112-02)。将连接产物转化至大肠杆菌E.coli TOP10，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行测序，突变位点校正，验证全部序列正确后，成功克隆出αCD47-αCD3-BITE的pSC65穿梭质粒，质粒构建图谱如图4A所示。
4.2构建重组痘苗病毒αCD47-αCD3-BITE
1.细胞准备：将143TK -细胞铺在6孔板中，每孔约1×10 6个。培养24小时左右，当细胞贴壁并且铺满整个底面时，进行下一步操作。
2.痘苗病毒孵育：用0.0125/3PFU(PFU：空斑形成单位，病毒液滴度)/细胞的野生型痘苗病毒天坛株感染细胞，37℃孵箱中孵育1小时后取出，吸掉上清，并用1mL PBS冲洗一遍再加入1mL完全培养基。
3.质粒转染：将上述穿梭质粒PSC65-αCD47-αCD3-BITE转染143TK -细胞。37℃孵箱中培养48小时左右，具体时间根据细胞病变情况而定。
4.准备病毒铺斑用的2×DMEM维持培养基(含2％PS和4％FBS)，加入2％预热的低熔点琼脂糖再加X-gal(终浓度为200μg/mL)。
5.吸掉6孔板中的上清，将铺斑用的混合物加入6孔板中，每孔1mL。然后小心放入4℃冰箱，促进凝固，待低熔点琼脂糖凝固后再转入37℃孵箱中倒置培养直至出现清晰的蓝斑。
6.挑取蓝斑(重组的痘苗病毒rTV-αCD47-αCD3-BITE)到500μL的完全培养基。在-80℃反复冻融三次以上，使病毒尽量多的释放。
7.将143TK -细胞铺在6孔板中，每孔约1×10 6个。培养24小时左右，直至细胞贴壁并且铺满整个底面。
8.反复吹打EP管中的蓝斑，使其完全散开。
9.将完全培养基换成维持培养基然后将含有蓝斑的病毒液加入，37℃孵箱中孵育3-4小时。
10.加入筛选压力：BrdU工作浓度为50μg/mL，放入37℃孵箱中孵育48小时左右，根据病毒斑形成情况进行铺斑。该纯化过程至少需要进行5次。
11.然后进行重组痘苗病毒的小样扩增，铺143TK -细胞于六孔板，每孔1×10 6个细胞，使用时细胞约为孔板底面积的100％。
12.种毒前将孔中的培养基换成2mL维持培养基。将纯化得到的含有蓝斑的病毒液反复吹打至蓝斑散开。每孔加入100μL左右病毒液。37℃孵箱中孵育48小时左右，根据病毒斑形成情况收样。
13.收样：将孔里的培养基上清小心吸出1mL。用剩下的1mL培养基将细胞充分吹下，收于EP管中，可以用于后续基因组的提取以及作为毒种进行扩增。
4.3αCD47-αCD3-BITE的重组痘苗病毒的扩增与纯化
1.VERO细胞铺板：10cm皿，每个皿约5×10 6个细胞，保证第二天接种痘苗病毒时使细胞密度达100％为宜；
2.病毒接种前，需将完全培养基换成8mL维持培养基(DMEM培养 基+2％FBS+1％PS)，将病毒接种到维持培养基的细胞中，接种量约为0.02MOI(MOI＝病毒PFU/细胞数)。继续在37℃5％CO 2的孵箱中培养48小时左右，根据病毒斑形成情况收样；
3.收痘苗病毒：弃掉皿内8mL培养基，取用2mL维持培养基将剩余的细胞吹下，收于15mL离心管中；
4.冻存24小时后，将收得的病毒液再反复冻融2次，用36％的蔗糖溶液进行密度梯度离心，16000g 4℃离心90min，小心倒掉上清，用PBS缓冲液溶解离心管内的病毒沉淀，分装保存于-80℃，待测定病毒滴度。
4.4αCD47-αCD3-BITE的重组痘苗病毒的滴度测定
1. 143TK -细胞的准备：将143TK -细胞铺在24孔板中，每孔约为2×10 5个细胞，使用时细胞密度要达到24孔板底面积的100％；
2.稀释病毒，用维持培养基稀释痘苗病毒病毒液，从1:100开始，做10倍比稀释，终体积为1100μL；
3.弃掉24孔板中的完全培养基，取稀释好的病毒液500μL加入孔中，做两个复孔。37℃5％CO 2的孵箱中孵育48小时左右，根据病毒嗜斑形成情况决定铺斑时间；
4.铺斑方法：准备8mL含有2×DMEM培养基+4％FBS+2％PS的铺斑培养基和8mL沸水浴融化后也置于37℃水浴锅中的低熔点琼脂糖，将二者混合，再将X-gal加入混合物中，终浓度为200μg/mL，待用；
5.吸掉24孔板中的上清。步骤4中的铺斑混合物立刻加入24孔板中，每孔500μL。然后小心放入4℃冰箱，促进凝固，待低熔点琼脂糖凝固后再转入37℃孵箱中倒置培养直至出现清晰的蓝斑；
6.病毒斑计数：首先观察病毒嗜斑的数目是否呈十倍比的趋势递减，随后统计种毒的两个复孔中仅有个位数蓝斑的数量，得到的两个孔中蓝斑数值之和，乘以该孔所对应稀释度的倒数值即为1mL中病毒的滴度。
4.5重组痘苗病毒rTV-αCD47-αCD3-BITE的表达验证
1.收取病毒上清：取10cm培养皿，接种5×10 6个VERO细胞/皿，保证第二天接种痘苗病毒时使细胞密度达100％为宜。病毒接种前，需将完全培养基换成8mL维持培养基(DMEM培养基+2％FBS+1％PS)；然后加入病毒，接种量约为0.02MOI(MOI＝病毒PFU/细胞数)。在37℃5％CO 2的孵箱中培养48小时；根据病毒斑形成情况收取细胞上清，10000g离心5分钟，取上清到新的离心管中。
3.将病毒上清进行His标签蛋白纯化：使用注射器人工进行纯化，填充的镍珠为Ni-NTA Agarose(QIAGEN公司，货号30210)；装柱完成后用5nM、10nM的咪唑(溶解在50mM NaH 2PO 4·2H 2O，pH 8.0，300mM NaCl中) 洗脱非特异吸附的蛋白，自然流速洗脱，收集滤液；然后用20nM、100nM、200nM、500nM的咪唑(溶解在50mM NaH 2PO 4·2H 2O，pH 8.0，300mM NaCl中)进行目的蛋白的洗脱，收集对应的滤液。
4.取30μL滤液制样，进行10％SDS-PAGE检测，结果如图4B所示。由5mM咪唑洗脱液到500mM咪唑洗脱液依次进行上样。由图4C所示200nM的洗脱液中出现目的蛋白，条带大小为54kD左右。
实施例5：重组痘苗病毒rTV-αCD47-αCD3-BITE的亲和检测
1.收取病毒上清：取10cm培养皿，接种5×10 6个VERO细胞/皿，保证第二天接种痘苗病毒时使细胞密度达100％为宜。病毒接种前，需将完全培养基换成8mL维持培养基(DMEM培养基+2％FBS+1％PS)；然后加入病毒，接种量约为0.02MOI(MOI＝病毒PFU/细胞数)，同时设置野生型病毒对照；在37℃5％CO 2的孵箱中培养48小时；根据病毒斑形成情况收取细胞上清，10000g离心5分钟，取上清到新的离心管中进行His标签蛋白纯化。
2.His标签蛋白纯化，方法同上。
3.CD47-A549细胞的准备：取2×10 6个细胞分到两个EP管中，800g离心3分钟，弃上清。
4.用1mL的预冷清洗液(1×PBS+2％FBS)洗涤2遍，800g离心3分钟，弃上清。
5.取适量纯化蛋白与CD47-A549细胞共孵育，室温15分钟。
6.用1mL的预冷清洗液洗涤2遍，800g离心3分钟，弃上清。
7.每个样品加入0.1μL的PE标记的anti-His抗体，室温15分钟。
8.用1mL的预冷清洗液洗涤2遍，800g离心3分钟，弃上清。
9.每个样品加入200μL清洗液重悬，采用流式细胞术检测亲和力。
图5结果显示，重组有αCD47-αCD3-BITE的痘苗病毒表达的细胞上清与CD47能够结合，与野生型痘苗病毒相比，阳性率为85.9％。挑取阳性克隆进行传代稳定性测试，挑选出传代稳定性强，高表达目的蛋白的毒株进行保藏，命名为rTV-αCD47-αCD3-BITE，其保藏编号为：CCTCC NO:V202081，保藏日期为2021年1月2日，保藏地址为中国典型培养物保藏中心，地址：中国武汉武汉大学。
实施例6：αCD47-αCD3-BITE的重组痘苗病毒体外抗肺癌效果
1. 143TK -细胞铺板：将143TK -细胞铺在6孔板中，每孔约为1×10 6个细胞，使用时细胞密度要达到6孔板底面积的100％；
2.病毒接种前将完全培养基换成每孔2mL维持培养基(DMEM培养基+2％FBS+1％PS)，并接种病毒到该细胞中，接种量为0.02MOI。37℃5％CO 2的孵箱中孵育48小时左右，根据病毒嗜斑形成情况决定收上清时间。同时用野生痘苗病毒作为对照；
3.分别取上清100μL用来进行后续的A549体外杀伤实验；
4.A549细胞体外杀伤实验，按照试剂盒说明操作进行(Dojindo公司，货号CK17)；吸取100μL重悬好的A549细胞至96孔板中，每孔细胞为1×10 4个，37℃，5％CO 2培养过夜。
5.第二天，加入上清，37℃，5％CO 2培养1小时后，按照效靶比5：1加入T细胞，继续37℃，5％CO 2培养4小时。
6.在高对照孔中加入20μL裂解缓冲液后，在低对照孔和背景孔中加入20μL培养基，37℃，5％CO 2培养30分钟。
7.从每个孔中吸取100μL上清液至新的96孔板中。
8.在每个孔中加入100μL显色液后，避光室温反应5分钟。
9.最后在每个孔中加入50μL终止液终止，立即有酶标仪测定490nm的吸光度。
结果如图6所示，相比空白对照组，加载αCD47-αCD3-BITE的重组痘苗病毒在体外能够显著提高对于A549细胞的杀伤，有4倍之多的提升效果。
综上所述，αCD47-αCD3-BITE蛋白联合T细胞对肿瘤细胞有非常强的杀伤活性，且制备的重组痘苗病毒rTV-αCD47-αCD3-BITE作为溶瘤病毒，同样能够显著控制人肺癌等多种实体肿瘤，对于肿瘤的治疗有非常高的应用价值且制备简单，便于大量制备以及推广使用。
实施例7：不同结构式的αCD47-αCD3-BITE的介导T细胞的体外抗人 卵巢腺癌细胞效果
为了证明不同结构的αCD47-αCD3-BITE蛋白的抗癌效果，申请人分别设计了如下的BITE
BITE1：VL αCD47-L-VH αCD3-L-VL αCD3-L-VH αCD47
所述VH αCD47如SEQ ID NO:19所示，所述VL αCD47如SEQ ID NO:21所示，所述VH αCD3如SEQ ID NO:25所示，VL αCD3如SEQ ID NO:26所示，并且所述L为GGGGSGGGGSGGGGS。
BITE2：VH αCD47-L-VH αCD3-L-VL αCD3-L-VL αCD47
所述VH αCD47如SEQ ID NO:19所示，所述VL αCD47如SEQ ID NO:21所示，所述VH αCD3如SEQ ID NO:25所示，VL αCD3如SEQ ID NO:26所示，并且所述L为GGGGSGGGGSGGGGS。
BITE3：VH αCD47-L-VL αCD47-L-VH αCD3-L-VL αCD3
所述VH αCD47如SEQ ID NO:19所示，所述VL αCD47如SEQ ID NO:21所示，所述VH αCD3如SEQ ID NO:25所示，VL αCD3如SEQ ID NO:26所示；一条连接肽为GGGGSGGGGSGGGGS。
BITE4：同BITE1，仅连接肽更换为GGGGGG；
BITE5：同BITE1，所述VH αCD3如SEQ ID NO:27所示，VL αCD3如SEQ ID NO:28所示。
实施例同实施例2和3，结果如图7所示。VL αCD47-L-VH αCD3-L-VL αCD3-L-VH αCD47其中L为(G 4S) 3,为αCD47-αCD3-BITE的最优设计，能够介导强劲的杀伤肿瘤细胞功能，变换CD47或者CD3(如克隆号SP34)的抗体克隆种类，依然保有很强的杀伤功能；VHαCD47-L-VHαCD3-L-VLαCD3-L-VLαCD47其中L为(G 4S) 3,为αCD47-αCD3-BITE的次优设计，但是仍有跟对照抗体有类似的杀伤肿瘤活性。
上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (13)

  1. 一种αCD47和αCD3双特异性T细胞衔接器αCD47-αCD3 BiTE，其包含如下式中任一项所示的融合蛋白：
    VL αCD47-L-VH αCD3-L-VL αCD3-L-VH αCD47；或
    VH αCD47-L-VH αCD3-L-VL αCD3-L-VL αCD47
    其中，L表示连接肽；
    其中，所述VH αCD47为CD47抗体的重链可变区，其包含下述3个互补决定区：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:1，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:2，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列；
    优选地，(i)-(iii)任一项中所述的置换为保守置换；
    优选地，所述VH αCD47包含：如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:2所示的VH CDR2、如SEQ ID NO:3所示的VH CDR3；
    其中，所述VL αCD47为CD47抗体的轻链可变区，其包含下述3个互补决定区：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10、SEQ ID NO:13或SEQ ID NO:16中任一项所示，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、SEQ ID NO:14或SEQ ID NO:17中任一项所示，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、SEQ ID NO:15或SEQ ID NO:18中任一项所示，或与其相比具有一个或几个氨基酸的置换、缺失或添加的序列；
    优选地，(iv)-(vi)任一项中所述的置换为保守置换；
    优选地，所述VL αCD47包含：如SEQ ID NO:4所示的VL CDR1、如SEQ ID NO:5所示的VL CDR2、如SEQ ID NO:6所示的VL CDR3；
    或所述VL αCD47包含：如SEQ ID NO:7所示的VL CDR1、如SEQ ID NO:8所示的VL CDR2、如SEQ ID NO:9所示的VL CDR3；
    或所述VL αCD47包含：如SEQ ID NO:10所示的VL CDR1、如SEQ ID NO: 11所示的VL CDR2、如SEQ ID NO:12所示的VL CDR3；
    或所述VL αCD47包含：如SEQ ID NO:13所示的VL CDR1、如SEQ ID NO:14所示的VL CDR2、如SEQ ID NO:15所示的VL CDR3；
    或所述VL αCD47包含：如SEQ ID NO:16所示的VL CDR1、如SEQ ID NO:17所示的VL CDR2、如SEQ ID NO:18所示的VL CDR3。
  2. 根据权利要求1所述的双特异性T细胞衔接器，其中，所述VH αCD47包含SEQ ID NO:19所示的重链可变区中含有的3个CDR；
    优选地，所述VL αCD47包含SEQ ID NO:20-24中任一项所示的轻链可变区中含有的3个CDR；
    优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号***定义。
  3. 根据权利要求1或2所述的双特异性T细胞衔接器，其中，所述VH αCD47包含选自下列的氨基酸序列：
    (i)SEQ ID NO:19所示的序列；
    (ii)与SEQ ID NO:19所示的序列相比具有一个或几个氨基酸的置换、缺失或添加的序列；或
    (iii)与SEQ ID NO:19所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    所述VL αCD47包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:20-24中任一项所示的序列；
    (v)与SEQ ID NO:20-24中任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加的序列；或
    (vi)与SEQ ID NO:20-24中任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换。
  4. 根据权利要求1至3中任一项所述的双特异性T细胞衔接器，其中，所述VH αCD3包含选自下列的氨基酸序列：
    (i)SEQ ID NO:25或27所示的序列；
    (ii)与SEQ ID NO:25或27所示的序列相比具有一个或几个氨基酸的置换、缺失或添加的序列；或
    (iii)与SEQ ID NO:25或27所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、 至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    所述VL αCD3包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:26或28所示的序列；
    (v)与SEQ ID NO:26或28所示的序列相比具有一个或几个氨基酸的置换、缺失或添加的序列；或
    (vi)与SEQ ID NO:26或28所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换。
  5. [根据细则91更正 05.07.2022]
    根据权利要求1至5中任一项所述的双特异性T细胞衔接器，其中，所述VH αCD47、VH αCD3、VL αCD47和VL αCD3通过连接肽连接；
    优选地，所述VH αCD47、VH αCD3、VL αCD47和VL αCD3之间通过一条、两条或三条连接肽连接；
    更优选地，所述L为KESGSVSSEQLAQFRSLD、EGKSSGSGSESKST、GGGGGG、GGGGGGGG或(GGGGS) n；进一步优选地，n为1-5的整数，最优选地，n为3；
    优选地，所述双特异性T细胞衔接器包含如下式所示的融合蛋白：
    VH αCD47-L-VH αCD3-L-VL αCD3-L-VL αCD47
    其中，所述VH αCD47如SEQ ID NO:19所示，所述VL αCD47如SEQ ID NO:21或24所示，所述VH αCD3如SEQ ID NO:25所示，VL αCD3如SEQ ID NO:26所示，并且所述L为GGGGSGGGGSGGGGS。
  6. [根据细则91更正 05.07.2022]
    一种分离的核酸分子，其编码如权利要求1-6中任一项所述的双特异性T细胞衔接器；
    优选地，所述核酸分子包含SEQ ID NO：31-35中任一项所示的核酸序列，所述核酸序列为VL αCD47的编码序列；
    优选地，所述核酸分子包含如SEQ ID NO：36所示的核酸序列，所述核酸序列为VH αCD47的编码序列。
  7. [根据细则91更正 05.07.2022]
    如权利要求1-6中任一项所述的双特异性T细胞衔接器的表达框架：
    5’-E1-E2-E3-E4-E5-E6-E7-3’
    其中：
    E1是CMV增强子或/和其它顺式作用元件；优选包含SEQ ID NO：37所示的核酸序列；
    E2是重组表达的启动子，优选CMV启动子，更优选包含SEQ ID NO：38所示的核酸序列；
    E3是5’端非翻译区，任选地不包含或包含一个内含子序列，任选地包含一个或多个限制酶位点；优选包含SEQ ID NO：39所示的核酸序列；
    E4是BiTE蛋白的信号肽的编码核苷酸序列；优选地，所述信号肽源于人或小鼠的信号肽；优选包含SEQ ID NO：40所示的核酸序列；
    E5是如权利要求1至6中任一项所述的双特异性T细胞衔接器的编码核苷酸序列；
    E6是3’端非翻译区，任选地包含一个或多个限制酶位点；优选包含SEQ ID NO：41所示的核酸序列；
    E7是SV40转录中止信号区；优选包含SEQ ID NO：42所示的核酸序列。
  8. [根据细则91更正 05.07.2022]
    一种重组溶瘤病毒，所述溶瘤病毒可操作地***或包含如权利要求8所述的表达框架；
    优选地，所述表达框架位于所述重组溶瘤病毒的胸腺嘧啶核苷激酶(TK)区；
    优选地，所述表达框架可单独表达、与其他基因或片段融合表达；
    优选地，所述重组溶瘤病毒还包含其他免疫调节因子的基因编码序列，更优选地，所述其他免疫调节因子包括但不限于IL-1、IL-2、IL-3、IL-7、IL-11、IL-12、IL-15、IL-17、IL-18、IL-21、IL-33、IL-35、IL-37、GM-CSF、IFN-α、IFN-β、IFN-γ、抗PD-1/PD-L1抗体、抗CTLA-4抗体、抗Lag-3抗体、抗TIGIT抗体或抗Tim-3抗体；或
    所述重组溶瘤病毒还包含与细胞凋亡和细胞焦亡相关的蛋白的基因编码序列，所述与细胞凋亡和细胞焦亡相关的蛋白优选选自凋亡相关因子1、白介素-1β转换酶、Bcl-2蛋白、Fas/APO-1、p53、myc、共济失调毛细血管扩张突变基因、消皮素D或消皮素E；或
    所述重组溶瘤病毒还包含靶向免疫调节基因、凋亡与焦亡基因的小RNA。
  9. [根据细则91更正 05.07.2022]
    根据权利要求9所述的重组溶瘤病毒，其中，所述溶瘤病毒的病毒骨架来源于经修饰或经改造的痘苗病毒天坛株、痘苗病毒纽约株、痘苗病毒哥本哈根株、痘苗病毒金丝雀株、痘苗病毒安卡拉株、腺病毒、腺相关病毒、单纯疱疹病毒、水痘-带状疱疹病毒、呼吸道合胞病毒、生里基森林病毒、EB病毒、巨细胞病毒、人疱疹病毒6型、天花病毒、痘苗病毒、***病毒、羊口疮病毒、呼肠孤病毒、轮状病毒、肠道病毒、塞内卡病毒、脊髓灰质炎病毒、柯萨奇病毒、鼻病毒、甲型肝炎病毒、***病毒、披膜病毒、甲病毒、塞姆利基森林病毒、东部马脑炎病毒、辛德毕斯病毒、风疹病毒、冠状病毒、黄病毒、丙型肝炎病毒、日本脑炎病毒、圣路易斯脑炎病毒、 墨累谷热病毒、黄热病毒、西尼罗河病毒、寨卡病毒、登革病毒、埃博拉病毒、马尔堡病毒、沙粒病毒、拉沙热病毒、淋巴细胞性脉络丛脑膜炎病毒、皮钦德病毒、胡宁病毒、马丘波病毒、汉坦病毒、裂谷热病毒、副粘病毒、人副流感病毒、腮腺炎病毒、猴病毒5、麻疹病毒、水疱性口炎病毒、狂犬病病毒、正粘病毒、甲型流感病毒、乙型流感病毒、丙型流感病毒、丁型肝炎病毒、猴免疫缺陷病毒、人免疫缺陷病毒1型和人免疫缺陷病毒2型、劳氏肉瘤病毒、嗜人T细胞白血病病毒1型、猴泡沫病毒、乙型肝炎病毒、戊型肝炎病毒、人***瘤病毒或多瘤病毒；
    优选地，所述溶瘤病毒骨架为细胞内成熟病毒、细胞内包装病毒、细胞相关包装病毒或细胞外包装病毒。
  10. [根据细则91更正 05.07.2022]
    一种重组痘苗病毒天坛株rTV-αCD47-αCD3-BITE，其保藏编号为：CCTCC NO:V202081。
  11. [根据细则91更正 05.07.2022]
    如权利要求9至11中任一项所述的重组溶瘤病毒的制备方法，包括以下步骤：
    1)合成如权利要求8所述的双特异性T细胞衔接器BiTE的表达框架；
    2)将步骤1)得到的表达框架亚克隆至溶瘤病毒的穿梭质粒中，构建重组质粒载体；
    3)将步骤2)得到的重组质粒载体转染至溶瘤病毒中，经筛选获得重组溶瘤病毒；
    任选地，培养获得的重组溶瘤病毒；
    优选地，所述方法包括以下步骤：
    1)合成双特异性T细胞衔接器αCD47-αCD3-BITE的表达框架，其包含如SEQ ID NO：31-42中任一项所示的核酸序列；
    2)将合成的表达框架亚克隆至痘苗病毒穿梭质粒(pSC65)的TK区中，构建重组质粒pSC65-αCD47-αCD3-BITE；
    3)采用基因同源重组的方式，将pSC65-αCD47-αCD3-BITE质粒转染到已经被感染了野生型痘苗病毒的TK143 -细胞中，使两者同源重组，产生重组痘苗病毒rTV-αCD47-αCD3-BITE；经筛选获得所述TK区包含αCD47-αCD3-BITE的编码序列的重组溶瘤痘苗病毒；
    优选地，双特异性T细胞衔接器αCD47-αCD3-BITE的表达框架由痘苗病毒的早/晚期启动子p7.5控制。
  12. [根据细则91更正 05.07.2022]
    如权利要求1-6中任一项所述的双特异性T细胞衔接器或如权利要求9-11中任一项所述的重组溶瘤病毒用于制备抗肿瘤药物的用途；
    优选地，所述肿瘤选自B细胞淋巴瘤、T细胞淋巴瘤、黑色素瘤、***癌、肾细胞癌、肉瘤、胶质瘤如高级别胶质瘤、母细胞瘤如神经母细胞瘤、 骨肉瘤、浆细胞瘤、组织细胞瘤、胰腺癌、乳腺癌、肺癌诸如小细胞肺癌和非小细胞肺癌、胃癌、肝癌、结肠癌、直肠癌、食管癌、大肠癌、造血***癌、睾丸癌、***、卵巢癌、膀胱癌、鳞状细胞癌、腺癌、AIDS相关淋巴瘤、膀胱癌、脑癌、神经***癌、头颈癌、头颈部鳞状细胞癌、霍奇金氏淋巴瘤、非霍奇金氏淋巴瘤或血液致瘤疾病。
  13. [根据细则91更正 05.07.2022]
    一种***的方法，所述方法包括给与有需要的受试者治疗有效量的如权利要求1-6中任一项所述的双特异性T细胞衔接器或如权利要求9-11中任一项所述的重组溶瘤病毒。
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