CN114249836A

CN114249836A - Bispecific T cell engagers, recombinant oncolytic viruses thereof and uses thereof

Info

Publication number: CN114249836A
Application number: CN202111615701.2A
Authority: CN
Inventors: 徐建青; 张晓燕; 丁相卿; 廖启彬; 王诗语; 周俊花
Original assignee: Shanghai Sinobay Bio Tech Co ltd
Current assignee: Shanghai Sinobay Bio Tech Co ltd
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2022-03-29

Abstract

The present invention provides bispecific T cell adaptors, recombinant oncolytic viruses thereof, and uses thereof. The invention provides an alpha CD47 and alpha CD3 bispecific T cell adaptor. The invention also provides an isolated nucleic acid molecule encoding the bispecific T cell engager. The invention also provides an expression framework of the bispecific T cell adaptor BiTE. The invention also provides a recombinant oncolytic virus operably inserted into or comprising the expression framework of the bispecific T cell engager BiTE. The present invention combines a bispecific T cell engager with an oncolytic virus that significantly enhances the ability to suppress malignant tumors relative to simple gene therapy or viral therapy.

Description

Bispecific T cell engagers, recombinant oncolytic viruses thereof and uses thereof

Technical Field

The invention belongs to the technical field of biomedicine, and relates to a novel bispecific T cell adaptor (BiTE) containing specific binding fragments of human CD47 and human CD 3. The invention also relates to a recombinant oncolytic virus comprising the bispecific T cell adaptor (BiTE), and also provides the preparation method of the bispecific T cell adaptor and the oncolytic virus and application thereof in the aspect of tumor resistance.

Background

Cancer is the second leading cause of death worldwide, with 960 million estimated to die of cancer in 2018. Around one sixth of deaths worldwide are caused by cancer. Related cancer therapies, particularly tumor immunotherapy via the human autoimmune system, are becoming a focus of increasing attention.

Human CD47, an integrin-associated protein, is a cell membrane surface glycoprotein and belongs to the immunoglobulin superfamily. CD47 is widely expressed on the surface of various cancer cells, releases an 'other eating me' signal by connecting with a signaling regulatory protein alpha (SIRP alpha) on the surface of a tumor phagocyte, and prevents phagocytosis of the macrophage. In recent years, many companies have invested a lot of manpower and material resources to develop CD47 antibody, but the effect after clinical treatment is very small, and the best level of CD47 antibody can be put into combination drug at present. The main reason for the poor therapeutic effect is the difficulty in controlling the side effects due to the fatal weakness of CD47, i.e., its expression on erythrocytes. After the CD47 antibody drug or the SIRP alpha-Fc fusion protein is combined with red blood cells, the agglutination of the red blood cells can be caused, and then the rupture of the red blood cells is triggered; the CD47 antibody drug with Fc of IgG1 can also activate phagocytosis of erythrocytes by macrophages or antibody-dependent cell-mediated cytotoxicity (ADCC), further induce lysis of erythrocytes and finally cause anemia.

How to maximize the advantages of the CD47 target on killing tumor cells and simultaneously reducing damage to red blood cells is always the key point for successfully developing CD47 medicaments. The bispecific T-cell engager BiTE (bispecific T-cell engage) represents a bispecific antibody with a remarkable anti-tumor effect, and can be used for targeting activation of self T cells to kill tumor cells. 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 epsilon H, while the other scFv recognizes a specific tumor cell surface antigen. This structure of BiTE and the ability to specifically bind proteins allows it to physically bridge T cells to tumor cells to form a T cell-BiTE-tumor cell complex, induce immune synapse formation, stimulate T cell activation, and produce tumor-killing cytokines. In recent years, BiTE has made remarkable progress in antitumor studies, and has achieved clinically desirable therapeutic effects.

Oncolytic viruses targeting tumors have attracted considerable attention in recent decades due to the limited therapeutic efficacy of standard tumor treatment regimens in the treatment of advanced tumors and severe side effects. Oncolytic viruses, which benefit from their own ability to selectively infect and lyse tumor cells locally in tumors, are becoming increasingly the first choice for anti-tumor. The tumor selectivity of oncolytic viruses can be the tumor tropism itself or the result of genetic modification. Oncolytic viruses act on multiple cellular pathways, thereby reducing tumor resistance and also inducing different forms of cell death. In addition, oncolytic viruses can break immune tolerance of tumor microenvironment and induce long-term tumor specific immune response. Oncolytic viruses can specifically transport therapeutic proteins into tumor tissue, with further replication of the virus increasing the level of expression in malignant cells. Alternatively, the oncolytic virus may be used in combination with chemotherapy, radiotherapy and immunotherapy.

In 2006, oncolytic adenovirus product (oncorine) has been used in china for clinical treatment of nasopharyngeal carcinoma and the like. The oncolytic virus deletes the E1B-55kD gene of the human adenovirus type 5, can replicate and proliferate in cancer cells with p53 gene mutation and kill host cells to generate an oncolytic therapeutic effect; the deletion of the E3 region allows tumor antigen information to be transmitted by dendritic cells to activate T cell immunity. However, clinical data show that oncolytic virus oncorine in combination with chemotherapy is less effective in treating patients with nasopharyngeal carcinoma than radiotherapy.

In 2015, the american FDA approval for melanoma was obtained by the oncolytic herpes simplex virus (T-VEC) of the ann company, and again in 12 months of the year, the european union was approved for local treatment of unresectable skin, subcutaneous and lymph node lesions in melanoma patients who had relapsed after the first surgery. The results of clinical studies on T-VEC have greatly promoted the development of oncolytic viruses in the field of tumor therapy. However, the treatment type is limited by the intratumoral administration mode, the method can only be applied to tumor types which are close to the body surface and are convenient for operation, the problems of difficult administration and incomplete treatment exist in the treatment of a plurality of non-superficial solid tumors and metastatic tumors, and if the method can be proved to have good tumor treatment effect by intravenous administration, the clinical application value of the method can be greatly improved; because of the limited oncolytic effect of herpes viruses, incomplete clearance of bulky and/or metastatic tumors, there is a need for additional therapeutic approaches to enhance the anti-neoplastic effect.

The biological properties and pathogenic mechanism of Vaccinia Virus (VV) are relatively clear, plays a key role in eliminating smallpox, and the safety of the Vaccinia virus in human bodies is fully proved. Vaccinia viruses are classified into WR (Western Reserve) strain, Wyeth strain, Copenhagen strain, Lister strain, Tian Tan strain, etc., according to pathogenicity, host range, etc. Because of its wide host range, high conservation, good safety and large foreign gene capacity, vaccinia virus is used as a vector for a plurality of recombinant vaccines such as influenza virus and human immunodeficiency virus. Most of the oncolytic viruses are currently in preclinical research phase as research and development of oncolytic viruses, and only a few enter clinical research phase.

The research on the use of vaccinia virus as an oncolytic virus for tumor therapy is currently the most rapidly progressing Pexa-Vec (JX-594) developed by Jennerex, USA. JX-594 is based on Wyeth strain virus, hGM-CSF and LacZ genes are inserted into the TK region, and JX-594 can express and replicate in cancer cells expressing high level of thymokinase due to deletion of the thymokinase gene, but does not affect normal cells. Meanwhile, the JX-594 can express GM-CSF in tumor cells due to the insertion of GM-CSF gene, and can activate the anti-tumor immune response of the body. The results of clinical trials of JX-594 tumor types prove that the drug combination has good tolerance after intratumoral administration or intravenous drip administration, and the effect of the combination of the Pexa-Vec and the sorafenib is better than that of a single drug combination. The interim analysis results indicate that it is not highly likely to prolong patient survival. Pexa-Vec originally planned to be marketed 2020, the clinical phase III trial was terminated early.

Oncolytic virus (GL-ONC1, also called GLV-1h68) developed by Genelux corporation, USA, is based on vaccinia virus (Lister strain), whose genes F14.5L, TK (encoding thymidine kinase) and HA (encoding hemagglutinin) are deleted to enhance targeting of tumor, and luciferase-GFP fusion protein, beta-galactosyltransferase and beta-glucuronidase are inserted respectively for screening and production preparation of poxvirus. The already completed clinical trial of i.v. administration of GL-ONC1 showed good safety and efficacy, no dose limiting toxicity, no maximum tolerated dose reached, and all patients had a neutralization response against GL-ONC 1; GL-ONC1 is currently undergoing extensive experimentation with intravenous administration.

The life cycle of vaccinia virus is strictly carried out in the cytoplasm of host cells, and the thymidine kinase gene of vaccinia virus is required for the genome of progeny virus to be successfully replicated. In the normal tissue cell cycle, however, synthesis of Thymidine Kinase (TK) occurs in the S phase of the cell division cycle, and after cell division is complete, Thymidine Kinase degrades inside the cell, so the concentration of Thymidine Kinase in the cytoplasm is low. However, tumor cells divide actively and thymidine kinase continues to be synthesized. By utilizing the characteristics, the thymidine kinase gene of the vaccinia virus is deleted, so that the vaccinia virus is specifically amplified in tumor cells to play a role in dissolving tumor.

Clinical trial evidence shows that vaccinia virus has shown some initial advantages in tumor treatment. Most of the existing vaccinia viruses used as oncolytic viruses are high in safety and clinical effectiveness to be further observed, but further optimization is needed in immune regulation and accurate tumor targeting.

Based on the above, there is no bispecific T cell engager in the prior art that is particularly suitable for recombinant oncolytic viruses, and there is currently a need for safer, more targeted bispecific T cell engagers and oncolytic viruses comprising the same.

Disclosure of Invention

Accordingly, it is an object of the present invention to address the deficiencies of the prior art by providing a bispecific T cell engager. The invention also provides recombinant oncolytic viruses expressing the bispecific T cell engagers (bites). The bispecific T cell adaptor (BiTE) provided by the invention overcomes the limitation of tumor immune editing (immunosuppression and immune evasion) of solid tumor cells on human T cells, can directly recruit the human T cells into the solid tumor to kill the tumor cells, and has wide application prospect.

The purpose of the invention is realized by the following technical scheme:

in one aspect, the invention provides an α CD47 and α CD3 bispecific T cell engager α CD47- α CD3 BiTE comprising a fusion protein represented by any one of the following formulae:

VL_αCD47-L-VH_αCD3-L-VL_αCD3-L-VH_αCD47(ii) a Or

VH_αCD47-L-VH_αCD3-L-VL_αCD3-L-VL_αCD47

Wherein said VH_αCD47Is the heavy chain variable region of the CD47 antibody, which comprises the following 3 complementarity determining regions:

(i) a VH CDR1, consisting of the sequence: 1, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto,

(ii) a VH CDR2, consisting of the sequence: 2, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto, and

(iii) a VH CDR3, consisting of the sequence: 3, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto;

preferably, the substitution recited in any one of (i) - (iii) is a conservative substitution;

preferably, said VH_αCD47Comprises the following steps: VH CDR1 shown in SEQ ID NO. 1, VH CDR2 shown in SEQ ID NO. 2, VH CDR3 shown in SEQ ID NO. 3;

wherein, the VL is_αCD47Is the light chain variable region of the CD47 antibody, which comprises the following 3 complementarity determining regions:

(iv) a VL CDR1, consisting of the sequence: 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 substitution, deletion or addition of one or more amino acids compared thereto,

(v) a VL CDR2, consisting of the sequence: a sequence shown in 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 having substitution, deletion or addition of one or more amino acids compared thereto, and

(vi) a VL CDR3, consisting of the sequence: 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 substitution, deletion or addition of one or more amino acids compared thereto;

preferably, the substitution recited in any one of (iv) - (vi) is a conservative substitution;

preferably, said VL_αCD47Comprises the following steps:VL CDR1 shown in SEQ ID NO. 4, VL CDR2 shown in SEQ ID NO. 5, VL CDR3 shown in SEQ ID NO. 6;

or the VL_αCD47Comprises the following steps: VL CDR1 shown in SEQ ID NO. 7, VL CDR2 shown in SEQ ID NO. 8, VL CDR3 shown in SEQ ID NO. 9;

or the VL_αCD47Comprises the following steps: VL CDR1 shown in SEQ ID NO. 10, VL CDR2 shown in SEQ ID NO. 11, VL CDR3 shown in SEQ ID NO. 12;

or the VL_αCD47Comprises the following steps: VL CDR1 shown in SEQ ID NO. 13, VL CDR2 shown in SEQ ID NO. 14, VL CDR3 shown in SEQ ID NO. 15;

or the VL_αCD47Comprises the following steps: VL CDR1 shown in SEQ ID NO. 16, VL CDR2 shown in SEQ ID NO. 17, and VL CDR3 shown in SEQ ID NO. 18.

The bispecific T cell engager according to the present invention, wherein the VH_αCD47Comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 19;

preferably, said VL_αCD47Comprises 3 CDRs contained in a light chain variable region represented by any one of SEQ ID NOS 20-24;

preferably, the 3 CDRs contained in the heavy chain variable region, and/or the 3 CDRs contained in the light chain variable region, are defined by the Kabat, Chothia or IMGT numbering system.

The bispecific T cell engager according to the present invention, wherein the VH_αCD47Comprising an amino acid sequence selected from the group consisting of:

(i) 19 in SEQ ID NO;

(ii) a sequence having substitution, deletion or addition of one or several amino acids as compared with the sequence shown in SEQ ID NO. 19; or

(iii) A sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO. 19;

and/or the presence of a gas in the gas,

the VL_αCD47Comprising an amino acid sequence selected from the group consisting of:

(iv) a sequence set forth in any one of SEQ ID NOs 20-24;

(v) a sequence having substitution, deletion or addition of one or several amino acids compared to the sequence shown in any one of SEQ ID NOs 20 to 24; or

(vi) A sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in any one of SEQ ID NOS 20-24;

preferably, the substitutions described in (ii) or (v) are conservative substitutions.

The bispecific T cell engager according to the present invention, wherein the VH_αCD3Comprising an amino acid sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO 25 or 27;

(ii) a sequence having substitution, deletion or addition of one or several amino acids compared to the sequence shown in SEQ ID NO 25 or 27; or

(iii) A sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in SEQ ID NO 25 or 27;

and/or the presence of a gas in the gas,

the VL_αCD3Comprising an amino acid sequence selected from the group consisting of:

(iv) 26 or 28;

(v) a sequence having substitution, deletion or addition of one or several amino acids compared with the sequence shown in SEQ ID NO. 26 or 28; or

(vi) A sequence having 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in SEQ ID NO 26 or 28;

The bispecific T cell engager according to the present invention, wherein the VH_αCD47、VH_αCD3、 VL_αCD47And VL_αCD3Linked by a linker peptide;

preferably, said VH_αCD47、VH_αCD3、VL_αCD47And VL_αCD3Optionally connected by one, two or three connecting peptides;

preferably, the bispecific T cell engager comprises a fusion protein according to any one of the following formulae:

VL_αCD47-L-VH_αCD3-L-VL_αCD3-L-VH_αCD47(ii) a Or

VH_αCD47-L-VH_αCD3-L-VL_αCD3-L-VL_αCD47

More preferably, L is KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GGGGGG, GGGGGGGG or (GGGGS)_n(ii) a Further preferably, n is an integer from 1 to 5, most preferably, n is 3;

preferably, the bispecific T cell engager comprises a fusion protein represented by the formula:

VH_αCD47-L-VH_αCD3-L-VL_αCD3-L-VL_αCD47

wherein said VH_αCD47As shown in SEQ ID NO 19, the VL_αCD4721 or 24, the VH_αCD3As shown in SEQ ID NO. 25, VL_αCD326, and the L is GGGGSGGGGSGGGGS.

The invention also provides an isolated nucleic acid molecule encoding the bispecific T cell adaptor;

preferably, the nucleic acid molecule comprises SEQ ID NO: 31-35, said nucleic acid sequence being a VL_αCD47The coding sequence of (a);

preferably, the nucleic acid molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 36 positionThe nucleic acid sequence shown is VH_αCD47The coding sequence of (a).

Expression framework for the bispecific T cell engager according to the present invention:

5’-E1-E2-E3-E4-E5-E6-E7-3’

wherein:

e1 is a CMV enhancer or/and other cis-acting elements; preferably comprises SEQ ID NO: 37;

e2 is a promoter for recombinant expression, preferably a CMV promoter, more preferably a promoter comprising SEQ ID NO: 38;

e3 is a 5' untranslated region, optionally containing no or one intron sequence, optionally containing one or more restriction enzyme sites; preferably comprises 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 a human or mouse signal peptide; preferably comprises SEQ ID NO: 40;

e5 is the coding nucleotide sequence of the bispecific T cell engager;

e6 is a 3' untranslated region, optionally comprising one or more restriction enzyme sites; preferably comprises SEQ ID NO: 41;

e7 is the SV40 transcriptional stop signal region; preferably comprises SEQ ID NO: 42, or a nucleotide sequence shown in the specification.

In a further aspect, the present invention provides a recombinant oncolytic virus operably inserted into or comprising the expression framework of the bispecific T cell engager BiTE.

Preferably, the expression cassette is located in the Thymidine Kinase (TK) region of the recombinant oncolytic virus.

Preferably, the expression frame can be expressed alone or in fusion with other genes or fragments.

Preferably, the recombinant oncolytic virus further comprises gene coding sequences for other immunomodulatory factors, more preferably, the other immunomodulatory factors include, but are 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- α, IFN- β, IFN- γ, anti-PD-1/PD-L1 antibodies, anti-CTLA-4 antibodies, anti-Lag-3 antibodies, anti-TIGIT antibodies, or anti-Tim-3 antibodies; or

The recombinant oncolytic virus further comprises gene coding sequences for apoptosis and apoptosis-related proteins including, but not limited to, apoptosis-related factor 1(Apaf-1), interleukin-1 beta converting enzyme (ICE), Bcl-2 protein, Fas/APO-1, p53, myc, ataxia telangiectasia mutated gene (ATM), corticotropin d (gasdermin d), corticotropin e (gasdermin e), and the like; or

The recombinant oncolytic virus also comprises small RNA of a targeted immune regulation gene and an apoptosis gene.

The recombinant oncolytic Virus of the invention, wherein the viral backbone of the oncolytic Virus is derived from a modified or modified 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 (RSV), Shenglikichen Virus (SFV), EB Virus, cytomegalovirus, human herpesvirus type 6, smallpox Virus, infectious molluscum Virus, orf Virus, reovirus, rotavirus, enterovirus, seneca Virus, poliovirus, coxsackie Virus, rhinovirus, hepatitis A Virus, foot and mouth disease Virus, Togavirus, alphavirus, semliki forest virus, eastern equine encephalitis virus, sindbis virus, rubella virus, coronavirus, flavivirus, hepatitis c virus, japanese encephalitis virus, st louis encephalitis virus, murray valley fever virus, yellow fever virus, west nile virus, zika virus, dengue virus, ebola virus, marburg virus, arenavirus, lassa fever virus, lymphocytic choriomeningitis virus, pichinde virus, hunin virus, marjo virus, hantaan virus, rift valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, monkey virus 5, measles virus, vesicular stomatitis virus, rabies virus, orthomyxovirus, influenza a virus, influenza b virus, influenza c virus, hepatitis d virus, monkey immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, Rous sarcoma virus, human T cell leukemia virus type 1, simian foamy virus, hepatitis B virus, hepatitis E virus, human papilloma virus or polyoma virus.

Preferably, the oncolytic viral scaffold is an intracellular maturation virus, an intracellular packaging virus, a cell-associated packaging virus or an extracellular packaging virus.

The recombinant oncolytic virus is a recombinant vaccinia virus Tiantan strain containing a coding gene of a bispecific T cell adaptor alpha CD 47-alpha CD3-BITE, named as rTV-alpha CD 47-alpha CD3-BITE, and has a preservation number of: CCTCC NO of V202081, the preservation date is 2021 year, 1 month and 2 days, and the preservation address is China center for type culture Collection.

In still another aspect, the present invention provides a method for preparing the recombinant oncolytic virus, comprising the steps of:

1) synthesizing an expression framework for the bispecific T cell engager BiTE;

2) subcloning the expression framework obtained in the step 1) into a shuttle plasmid of an oncolytic virus to construct a recombinant plasmid vector;

3) transfecting the recombinant plasmid vector obtained in the step 2) into the oncolytic virus, and screening to obtain the recombinant oncolytic virus.

Optionally, the obtained recombinant oncolytic virus is cultured.

In a specific embodiment, the invention provides a method for preparing a recombinant vaccinia virus Tiantan strain, comprising the following steps:

1) an expression framework for the synthetic bispecific T cell engager α CD47- α CD3-BITE comprising the amino acid sequence as set forth in SEQ ID NO: 31-42;

2) subcloning the synthesized expression framework into TK region of vaccinia virus shuttle plasmid (pSC65) to construct recombinant plasmid pSC 65-alpha CD 47-alpha CD 3-BITE;

3) by means of gene homologous recombination, pSC 65-alpha CD 47-alpha CD3-BITE plasmid is transfected into TK143 infected by wild type vaccinia virus^-In cells, the two are subjected to homologous recombination to generate recombinant vaccinia virus rTV-alpha CD 47-alpha CD 3-BITE; screening to obtain the recombinant oncolytic vaccinia virus of which the TK region comprises the coding sequence of alpha CD 47-alpha CD 3-BITE.

Wherein the expression frame of the bispecific T cell adaptor alpha CD 47-alpha CD3-BITE is controlled by the early/late promoter p7.5 of vaccinia virus.

Preferably, the specific steps of expanding the recombinant vaccinia virus using VERO cells comprise: culturing VERO cells until the density is close to 100%, replacing low-concentration fetal calf serum to maintain the culture medium, adding oncolytic vaccinia virus (the inoculum size is about 0.02MOI per 10cm of culture plate), culturing in an incubator, collecting virus liquid after the recombinant poxvirus is amplified, repeatedly freezing and thawing, and performing density gradient centrifugation purification by using sucrose solution.

In still another aspect, the present invention also provides the use of the bispecific T cell engager or the recombinant oncolytic virus for the preparation of an anti-tumor medicament; wherein the tumor is selected from the group consisting of a B cell lymphoma, T cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma such as high grade glioma, parent cell tumor 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, large intestine cancer, hematopoietic cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell cancer, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck squamous cell cancer, hodgkin's lymphoma, non-hodgkin's lymphoma, or hematologic malignancy.

In another aspect, the invention provides a method of treating a tumor, the method comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific T cell engager or a recombinant oncolytic virus. The method according to the present invention, wherein the method further comprises administering to a subject in need thereof additional chemotherapeutic drugs, radiotherapeutic techniques, surgical treatments, immunocytotic 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 invention concept of the invention is as follows:

the invention provides a novel alpha CD 47-alpha CD3-BITE bispecific T cell adaptor which has good antitumor activity in vitro. Furthermore, the inventor provides a vaccinia virus Tiantan strain carrying the bispecific T cell adaptor of the invention by taking the vaccinia virus Tiantan strain as a research model. Then, the inventor finds that the vaccinia virus Tiantan strain has a strong anti-tumor effect; the bispecific T cell adaptor carrying the alpha CD 47-alpha CD3-BITE can avoid the combination of the CD47 antibody and red blood cells of peripheral blood, recognize tumor cells by utilizing the broad-spectrum expression characteristic of CD47 and activate T cells by utilizing the CD3 antibody, thereby playing a role in killing the tumor cells, improving the tumor immunotherapy effect and simultaneously increasing the safety.

The invention has the beneficial effects that:

1. the bispecific T cell adaptor (BiTE) provided by the invention has small molecular weight, can penetrate through the cell gap to reach the Tumor Microenvironment (TME) inside the solid tumor to play a role, and can efficiently mediate the killing of T cells on cancer cells.

2. The invention combines the bispecific T cell adapter with the oncolytic virus and provides a vaccinia virus Tiantan strain capable of efficiently expressing the bispecific T cell adapter alpha CD 47-alpha CD3-BITE gene. When the oncolytic virus is administrated to tumor focus, the oncolytic virus can crack tumor cells to exert an oncolytic effect, and simultaneously, a high-killing antibody of a tumor-targeting gene anti-human CD47 is carried to block the combination of CD47 and SIRP alpha, so that the phagocytic function of macrophages in vivo is improved, and the anti-tumor effect is enhanced. The oncolytic virus significantly enhances the suppression of malignant tumors relative to gene therapy or viral therapy alone.

3. According to the in vitro experimental study, the vaccinia virus Tiantan strain disclosed by the invention is proved to be capable of treating liver cancer and malignant lung cancer, realizing good targeting and anti-tumor effects on tumors, having a relatively complete virus amplification and quality control system and laying a foundation for further industrialization, so that the vaccinia virus Tiantan strain has a good application prospect.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the construction of the shuttle plasmid vector α CD47- α CD3 BiTE hIgG1 WT and the expression purification of 5 corresponding BiTE proteins of the present invention; wherein FIG. 1A is an expression map of shuttle plasmid hIgG1 WT integrated with α CD47- α CD3-BITE expression framework; FIG. 1B shows the result of 293T culture supernatant transiently expressing α CD47- α CD3-BITE after protein purification using a nickel column.

FIG. 2 shows the effect of α CD47- α CD3-BITE on anti-human ovarian adenocarcinoma cells in vitro; among them, the CD47 antibody cloned No. 68 and BiTE composed of alpha CD3 were both able to mediate the killing of T cells to SK-OV3 well relative to the T cell control group. The alpha CD 47-alpha CD3-BITE can well recognize the tumor cells of CD47 and activate T cells, so that the specific killing of the T cells to the tumor cells is mediated; bispecific T cell engagers (BITE) comprising specific binding of human CD47 and human CD3 were shown to mediate well the anti-tumor activity of T cells in vitro and to have a broad spectrum.

FIG. 3 shows the effect of α CD47- α CD3-BITE on in vitro anti-human lung cancer cells; wherein, compared with a T cell control group, the BiTE consisting of the CD47 antibody cloned in No. 101 and the alpha CD3 can mediate the T cells to kill the NCI-H292 tumor cells, and compared with a single T cell group, the BiTE group has obvious effect of killing the NCI-H292 cells; it was shown that bispecific T cell engagers (BITE) comprising specific binding of human CD47 and human CD3 mediate the antitumor activity of T cells well in vitro and with a broad spectrum.

FIG. 4 shows the construction of a shuttle plasmid vector (α CD47- α CD3-BITE) of vaccinia virus Tiantan strain and the expression of α CD47- α CD3-BITE protein. FIG. 4A is a diagram of the expression map of shuttle plasmid pSC65 of vaccinia virus incorporating the gene α CD47- α CD 3-BITE; FIG. 4B shows the result of PCR identification of the recombinant vaccinia virus rTV- α CD47- α CD3-BITE inserted with the fragment α CD47- α CD 3-BITE; FIG. 4C shows expression of CD47- α CD3-BITE protein in the supernatant of VERO cells infected with recombinant vaccinia virus. As shown in FIG. 4, the recombinant vaccinia virus rTV- α CD47- α CD3-BITE carried α CD47- α CD3-BITE was successfully expressed.

FIG. 5 shows that recombinant vaccinia virus rTV- α CD47- α CD 3-BITE-loaded α CD47- α CD3-BITE has high affinity for CD47 protein; FIG. 5 shows the flow-through results of affinity assay of cell supernatants collected from recombinant vaccinia virus rTV- α CD47- α CD3-BITE infected VERO cells and human CD47 expressing lung cancer cell A549 cells; the result shows that compared with the wild type vaccinia virus, the binding rate of the recombinant vaccinia virus rTV-alpha CD 47-alpha CD3-BITE expression protein and CD47 is more than 85%.

FIG. 6 shows the in vitro killing of A549 cells by recombinant vaccinia virus, i.e., vaccinia virus Tiantan strain rTV- α CD47- α CD 3-BITE. The experiment was divided into four groups, including the control group of supernatants infected with wild vaccinia virus, the group of supernatants + T cells infected with wild vaccinia virus, the control group of supernatants infected with recombinant vaccinia virus, and the group of supernatants + T cells infected with recombinant vaccinia virus. From the figure, it can be seen that the supernatant + T cell group infected by the recombinant vaccinia virus has a significant killing effect on the lung cancer a549 cells compared with the blank control group.

FIG. 7 shows the anti-cancer effect of α CD47- α CD3-BITE protein of different structures.

Preservation information

The vaccinia virus Tiantan strain is named as rTV-alpha CD 47-alpha CD3-BITE, and the preservation number is as follows: CCTCC NO of V202081, the preservation date is 2021 year 1 month 2 days, the preservation address is China center for type culture Collection, the address is: wuhan university in Wuhan, China.

Detailed Description

Example 1: construction and expression verification of 293T recombinant expression plasmid of alpha CD 47-alpha CD3-BITE

1.1 hIgG1 WT vector construction with alpha CD 47-alpha CD3-BITE target gene

First, high affinity 5 anti-human CD were screened47 antibody, the heavy chain variable region and the light chain variable region are selected, and the fusion protein is constructed according to the following formula: VH_αCD47-L-VH_αCD3-L-VL_αCD3-L-VL_αCD47；

Wherein said VH_αCD47As shown in SEQ ID NO 19, the VL_αCD4720-24, said VH_αCD3As shown in SEQ ID NO. 25, VL_αCD326, and the L is GGGGSGGGGSGGGGS.

And according to VL_αCD47In contrast, Hu004-67 shows a VL comprising SEQ ID NO:20_αCD47α CD47- α CD3 BiTE; hu004-68 shows a VL comprising SEQ ID NO:21_αCD47α CD47- α CD3 BiTE; hu004-73 represents a polypeptide comprising the VL shown in SEQ ID NO:22_αCD47α CD47- α CD3 BiTE; hu004-100 shows a VL comprising SEQ ID NO:23_αCD47α CD47- α CD3 BiTE; hu004-101 represents a polypeptide comprising the VL shown in SEQ ID NO:24_αCD47In alpha CD 47-alpha CD3 BiTE, wherein WT Cloning vector AbVec-hIgG1(GenBank: FJ 475055.1).

The DNA sequence of alpha CD 47-alpha CD3-BITE was artificially synthesized according to the structural formula, and the plasmid construction map is shown in FIG. 1a, wherein VL is_αCD47The coding sequence of (A) is shown in SEQ ID NO: 31-35 any of claims VH_αCD47The coding sequence of (A) is shown in SEQ ID NO: shown at 36.

The artificially synthesized hIgG1 WT vector containing the target gene of alpha CD 47-alpha CD3-BITE was transformed into E.coli TOP10 (Dimocha, cat # DL1010S) and grown overnight on a culture plate containing ampicillin. On day 2, single colonies were randomly picked for overnight culture at 37 ℃ and plasmids were extracted using a plasmid extraction kit (Qiagen, cat. No. 27106) for transfection of 293T cells.

1.2 expression and purification of 293T cells with alpha CD 47-alpha CD3-BITE target genes

1.293T cell preparation cells grown in T175 flasks with confluency of more than 90% were removed from the supernatant, added with 2ml of 2.5% Trypsin-EDTA (GIBCO Co., Ltd., cat # 25200072) to digest the cells until the cells were completely detached, and added to 10ml of complete medium (DMEM medium + 10% FBS + 1% PS)Stopping digestion, centrifuging at 150g for 5 min to collect cells, discarding supernatant, and reselecting cells to a cell concentration of about 8X 10 using an appropriate amount of complete medium⁶～10×10⁶One per ml. The cell suspension was counted and 1.3X 10 cells were taken⁷The cells were plated on 150mm cell culture dishes (Thermo Scientific, cat # 150468) with a medium volume of 25mL and cultured the next day for subsequent plasmid transfection.

2. hIgG1 WT shuttle plasmid of α CD47- α CD3-BITE transfected 293T cells: a1 mL DMEM-based culture medium was taken out of an EP tube, and 20. mu.g of hIgG1 WT shuttle plasmid Cloning vector AbVec-hIgG1(GenBank: FJ475055.1) of. alpha. CD 47-. alpha. CD3-BITE was added thereto, and after mixing, 30. mu.l of FectoPro transfection reagent (Polyplus, cat. 116-001) was added thereto, and immediately thereafter, the mixture was shaken vigorously on a vortex shaker for 15 seconds and allowed to stand at room temperature for 20 minutes. Taking out 293T cells to be transfected in an incubator, sucking and discarding 10mL of culture medium, adding the immobilized transfection reagent while shaking, and placing at 37 ℃ in 5% CO₂Cultured in an incubator for 4-6 hours. The medium was completely removed, Expi293 expression medium (GIBCO Co., Ltd., Cat. A1435102) was added, and the mixture was left at 37 ℃ with 5% CO₂Cultured in an incubator for 4 days.

3. Collecting the alpha CD 47-alpha CD3-BITE expression supernatant: the culture medium in the expression dish was collected in a 50mL centrifuge tube, centrifuged at 4000g for 5 minutes, and the expression supernatant was transferred to a new 50mL centrifuge tube.

4. The expression supernatant was subjected to His-tag protein purification: the purification was performed using a gravity chromatography column packed with nickel beads as Ni-NTAAgarose (QIAGEN, cat # 30210); after completion of column packing, 5mM, 10mM imidazole (dissolved in 50mM NaH) was used₂PO₄·2H₂O, pH 8.0, 300mM NaCl) to elute the non-specifically adsorbed protein, eluting at a natural flow rate, and collecting the filtrate; then using 20mM, 100mM (collecting two tubes in sequence), 200mM (collecting two tubes in sequence), 500mM imidazole (dissolved in 50mM NaH)₂PO₄·2H₂O, pH 8.0, 300mM NaCl) to elute the target protein, and collecting the corresponding filtrate.

5. mu.L of the filtrate was sampled and examined by 10% SDS-PAGE. As shown, the sample was sequentially loaded from 5mM imidazole eluent to 500mM imidazole eluent. The target protein appears in the 100mM eluate and 200mM eluate shown in FIG. 1B, and the band size is about 54 kD.

Example 2: alpha CD 47-alpha CD3-BITE mediated T cell in vitro anti-human ovarian adenocarcinoma cell effect

SK-OV3 cell plating: SK-OV3 (deposited at Shanghai Xinwan Biotech, Inc.) is a Luciferase expressing cell line. The cells were cultured at 1X 10⁴The cells/well are plated in 96-well plates and incubated overnight at 37 ℃ until they adhere.

After 2.24 hours, the medium was removed, and 7 groups were set, to which T cells, B6H12- α CD3-BITE (the heavy chain variable region and the light chain variable region of B6H12 are shown in SEQ ID NOS: 29 and 30, respectively, and the remaining structures are the same as those of BITE of example 1 of the present invention), T cells, Hu004-67- α CD3-BITE and T cells, Hu004-68- α CD3-BITE and T cells, Hu004-73- α CD3-BITE and T cells, Hu004-100- α CD3-BITE and T cells, Hu004-101- α CD3-BITE and T cells, respectively, were added.

Killing was performed by overnight incubation at 3.37 ℃. After 24 hours, the supernatant was removed, 50. mu.L of cell lysate luciferase cell lysate (Promega, cat. No. E1531) was added to each well, and incubated at room temperature with shaking for 30 minutes, and 30. mu.L of luciferase substrate (Promega, cat. No. E151A) was added to each well. On-line assay (GloMax Navigator Microplate Luminometer, Promega, Steady-Glo protocol).

The results are shown in FIG. 2. Wherein B6H12 is a known CD47 antibody clone (US 9017675B2) as a positive control. Hu004-68 and Hu004-101 are selected antibody clones with high affinity to CD47 protein (obtained by selecting antibodies from Hexagrammos otakii wipe Biotech). As can be seen from FIG. 2, for this tumor cell line, the CD47 antibody (scFv) - α CD3-BITE of clone No. 68 has a 3.5-fold increase in tumor killing activity compared to the T cell control group, a 2-fold increase compared to clone No. 100 with a lower killing ability, and a 1.5-fold increase compared to the positive control antibody, and can well mediate the effect of the T cell on anti-ovarian adenocarcinoma cells in vitro.

Example 3: in-vitro anti-human lung cancer cell effect of alpha CD 47-alpha CD3-BITE mediated T cells

NCI-H292 cell plating: NCI-H292 (deposited at Shanghai Xinwan Biotech, Inc.) is a Luciferase expressing cell line. The cells were cultured at 1X 10⁴The density of each well was plated in 96-well plates and incubated overnight at 37 ℃ until they adhered.

After 2.24 hours the medium was removed and 7 groups were added T cells, B6H12- α CD3-BITE and T cells, Hu004-67- α CD3-BITE and T cells, Hu004-68- α CD3-BITE and T cells, Hu004-73- α CD3-BITE and T cells, Hu004-100- α CD3-BITE and T cells, Hu004-101- α CD3-BITE and T cells, respectively.

Killing was performed by overnight incubation at 3.37 ℃. After 24 hours, the supernatant was removed, 50. mu.L of cell lysate (Promega, cat. No. E1531) was added to each well, and incubated at room temperature for 30 minutes with shaking, and 30. mu.L of luciferase substrate (Promega, cat. No. E151A) was added to each well. Detection on a machine (GloMax Navigator Microplate Luminometer, Promega, Steady-Glo protocol).

The measured results are shown in fig. 3, CD47 antibody- α CD3-BITE of clone No. 101 can well mediate the in vitro anti-lung cancer cell effect of T cells, CD47 antibody (scFv) - α CD3-BITE of clone No. 101 has a 4-fold increase in the killing tumor activity compared with a T cell control group, has a 1.2-fold increase compared with clone No. 100 with low killing ability, and has a 1.2-fold increase compared with a positive control antibody, and can well mediate the in vitro anti-ovarian adenocarcinoma cell effect of T cells. Together, the results of fig. 2 and 3 indicate that bispecific T cell engagers (BITE) comprising specific binding of human CD47 and human CD3 mediate the anti-tumor activity of T cells well in vitro and with a broad spectrum.

Example 4: construction and expression verification of recombinant vaccinia virus of alpha CD 47-alpha CD3-BITE

4.1 construction of pSC65 vector with alpha CD 47-alpha CD3-BITE target Gene

The DNA sequence of alpha CD 47-alpha CD3-BITE was artificially synthesized, and PCR amplification was performed using the synthesized DNA sequence as a template and the following primers.

The primers for amplification were:

αCD47-αCD3-BITE-F：SEQ ID NO:43

GTACCAGGCCTAGTACTATGGAGAGGACCCTTGTCTG

αCD47-αCD3-BITE-R：SEQ ID NO:44

AATAAGCTCGAAGTCGAC CTAGGAGAGATGCTGATG

PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10 seconds, 58 ℃: annealing for 30 seconds, extending for 1 minute at 72 ℃, and reacting for 30 cycles; a further 10 minutes of extension at 72 ℃ was carried out, terminating at 25 ℃.

Recovery and cloning construction of PCR products: after completion of amplification, the desired gene was isolated on 2% agarose gel, and the vector pSC65 was linearized by digestion with Sal I (Thermo Scientific, cat. No. ER0642), and recovered by gel cutting, and the PCR fragment and the vector digested fragment were recovered using a Sanprep column DNA gel recovery kit (Promega, cat. No. A9282). The gene recovery product was ligated with the enzyme-digested linearized vector by homologous recombination (Novozak, cat. c 112-02). The ligation products were transformed into E.coli TOP10 and grown overnight on ampicillin-containing plates. On day 2, single colonies were randomly picked for sequencing, mutation site correction was performed, and after the complete sequence was verified to be correct, the shuttle plasmid pSC65 of α CD47- α CD3-BITE was successfully cloned, and the plasmid construction map is shown in FIG. 4A.

4.2 construction of recombinant vaccinia Virus α CD47- α CD3-BITE

1. Cell preparation: will be 143TK^-Cells were plated in 6-well plates at approximately 1X 10 per well⁶And (4) respectively. The culture was carried out for about 24 hours, and when the cells adhered to the wall and spread over the entire bottom surface, the next operation was carried out. 2. Incubation of the vaccinia virus: the cells were infected with 0.0125/3PFU (PFU: plaque Forming Unit, virus titer)/wild type vaccinia virus Tiantan strain of the cells, incubated at 37 ℃ for 1 hour in an incubator, removed, the supernatant aspirated, washed once with 1mL of PBS and then added with 1mL of complete medium.

3. Plasmid transfection: the shuttle plasmid PSC 65-alpha CD 47-alpha CD3-BITE was transfected into 143TK^-A cell. Culturing in 37 ℃ incubator for about 48 hours, wherein the specific time is determined according to the cytopathic condition.

4.2 XDMEM maintenance medium (containing 2% PS and 4% FBS) for virus spotting was prepared, 2% pre-warmed low melting agarose was added, and X-gal (final concentration of 200. mu.g/mL) was added.

5. The supernatant in the 6-well plate was aspirated off, and the spotting mixture was added to the 6-well plate at 1mL per well. Then carefully placing the mixture into a refrigerator at 4 ℃ to promote solidification, and transferring the mixture into a incubator at 37 ℃ to perform inverted culture after the low-melting-point agarose is solidified until clear blue spots appear.

6. Bluespots (recombinant vaccinia virus rTV- α CD47- α CD3-BITE) were picked to 500 μ L of complete medium. Repeatedly freezing and thawing at-80 deg.C for more than three times to release virus as much as possible.

7. Will be 143TK^-Cells were plated in 6-well plates at approximately 1X 10 per well⁶And (4) respectively. Culture for about 24 hours until the cells adhere to the wall and spread over the entire floor.

8. Repeatedly blow the blue spots in the EP tube to completely disperse the blue spots.

9. The complete medium was changed to the maintenance medium and then the viral fluid containing the blue spots was added and incubated in an incubator at 37 ℃ for 3-4 hours.

10. Adding screening pressure: the BrdU working concentration is 50 mug/mL, and the BrdU working concentration is placed into an incubator at 37 ℃ for incubation for about 48 hours, and spot paving is carried out according to the virus spot forming condition. The purification process needs to be performed at least 5 times.

11. Then performing small sample amplification of the recombinant vaccinia virus, and paving 143TK^-Cells were plated in six well plates, 1X 10 per well⁶And (3) cells, wherein the cells are about 100% of the bottom area of the pore plate when in use.

12. The medium in the wells was changed to 2mL of maintenance medium before inoculation. Repeatedly blowing and beating the virus liquid containing the blue spots obtained by purification until the blue spots are scattered. Approximately 100. mu.L of virus solution was added to each well. Incubating in an incubator at 37 ℃ for about 48 hours, and collecting samples according to the virus spot forming condition.

13. Collecting a sample: the culture supernatant in the wells was carefully aspirated off 1 mL. The cells were blown down well with the remaining 1mL of medium, harvested in EP tubes and used for subsequent genome extraction and amplification as a seed virus.

4.3 amplification and purification of recombinant vaccinia Virus of α CD47- α CD3-BITE

VERO cell plating: 10cm dishAbout 5X 10 per dish⁶Ensuring that the cell density reaches 100 percent when the vaccinia virus is inoculated on the next day;

2. before virus inoculation, the complete medium was replaced with 8mL of maintenance medium (DMEM medium + 2% FBS + 1% PS), and the virus was inoculated into cells in the maintenance medium in an amount of about 0.02MOI (MOI ═ virus PFU/cell count). Continuing at 37 ℃ with 5% CO₂Culturing in an incubator for about 48 hours, and collecting samples according to the virus spot forming condition;

3. collecting vaccinia virus: discarding 8mL of culture medium in the dish, blowing down the rest cells by using 2mL of maintenance culture medium, and collecting in a 15mL centrifuge tube;

4. after being frozen and stored for 24 hours, the collected virus liquid is repeatedly frozen and thawed for 2 times, the density gradient centrifugation is carried out by 36 percent of sucrose solution, the centrifugation is carried out for 90 minutes at 16000g and 4 ℃, the supernatant is carefully poured off, the virus precipitate in a centrifugal tube is dissolved by PBS buffer solution, and the virus precipitate is subpackaged and stored at-80 ℃ to be tested for the virus titer.

4.4 titer determination of recombinant vaccinia Virus of α CD47- α CD3-BITE

1.143TK^-Preparation of cells: will be 143TK^-Cells were plated in 24-well plates at approximately 2X 10 per well⁵The cell density is 100 percent of the bottom area of the 24-pore plate when the cell is used;

2. diluting the virus, namely diluting vaccinia virus solution by using a maintenance medium, and diluting the solution by 10 times from 1:100 to a final volume of 1100 mu L;

3. the complete medium in the 24-well plate was discarded, and 500. mu.L of diluted virus solution was added to the wells to make two duplicate wells. 5% CO at 37 ℃₂Incubating in the incubator for about 48 hours, and determining the plaque paving time according to the virus plaque forming condition;

4. the spot paving method comprises the following steps: preparing 8mL of spot-paving culture medium containing 2 XDMEM culture medium, 4% FBS and 2% PS and 8mL of low-melting-point agarose which is placed in a water bath kettle at 37 ℃ after being melted in a boiling water bath, mixing the two, and adding X-gal into the mixture to obtain the final concentration of 200 mu g/mL for later use;

5. the supernatant in the 24-well plate was aspirated off. The spotting mixture of step 4 was immediately added to a 24-well plate at 500. mu.L per well. Carefully placing the agarose gel into a refrigerator at 4 ℃ to promote solidification, transferring the agarose gel into an incubator at 37 ℃ after the agarose gel with low melting point is solidified, and performing inverted culture until clear blue spots appear;

6. and (3) counting virus plaques: firstly, observing whether the number of virus plaques is decreased in a ten-fold ratio trend, then counting the number of single-digit blue plaques in two multiple virus wells, and multiplying the sum of the obtained blue plaque values in the two wells by the reciprocal value of the corresponding dilution of the well to obtain the titer of the virus in 1 mL.

4.5 expression verification of recombinant vaccinia virus rTV- α CD47- α CD3-BITE

1. Collecting virus supernatant: inoculating 5X 10cm of culture dish⁶VERO cells/dish, ensuring that the cell density reaches 100% when vaccinating vaccinia virus the next day. Before virus inoculation, the complete medium needs to be changed into 8mL of maintenance medium (DMEM medium + 2% FBS + 1% PS); the virus was then added at approximately 0.02MOI (MOI ═ virus PFU/cell number). 5% CO at 37 ℃₂Culturing in an incubator for 48 hours; collecting cell supernatant according to virus spot formation condition, centrifuging for 5 min at 10000g, and taking supernatant to a new centrifuge tube.

3. Viral supernatants were purified for His-tag protein: purification was performed manually using a syringe, and the packed nickel beads were Ni-NTAAgarose (QIAGEN, cat # 30210); after completion of column packing, 5nM, 10nM imidazole (dissolved in 50mM NaH) was used₂PO₄·2H₂O, pH 8.0, 300mM NaCl) to elute the non-specifically adsorbed protein, eluting at a natural flow rate, and collecting the filtrate; then 20nM, 100nM, 200nM, 500nM imidazole (dissolved in 50mM NaH)₂PO₄·2H₂O, pH 8.0, 300mM NaCl) to elute the protein of interest, and collecting the corresponding filtrate.

4. 30 μ L of the filtrate was sampled and examined by 10% SDS-PAGE, and the results are shown in FIG. 4B. The loading was carried out from 5mM imidazole eluent to 500mM imidazole eluent in sequence. The target protein appears in 200nM eluate as shown in FIG. 4C, with a band size of about 54 kD.

Example 5: affinity detection of recombinant vaccinia virus rTV-alpha CD 47-alpha CD3-BITE

1. Collecting virus supernatant: inoculating 5X 10cm of culture dish⁶VERO cells/dish, ensuring that the cell density reaches 100% when vaccinating vaccinia virus the next day. Before virus inoculation, the complete medium needs to be changed into 8mL of maintenance medium (DMEM medium + 2% FBS + 1% PS); then virus was added at approximately 0.02MOI (MOI ═ virus PFU/cell number) while wild-type virus controls were set; 5% CO at 37 ℃₂Culturing in an incubator for 48 hours; collecting cell supernatant according to virus spot formation condition, centrifuging at 10000g for 5 min, and collecting supernatant to a new centrifuge tube for His tag protein purification.

His-tagged protein purification was performed as described above.

Preparation of CD47-A549 cells: take 2X 10⁶The cells were split into two EP tubes, centrifuged at 800g for 3 min and the supernatant discarded.

4. The cells were washed 2 times with 1mL of a pre-cooled wash solution (1 XPBS + 2% FBS), centrifuged at 800g for 3 minutes, and the supernatant discarded.

5. A suitable amount of purified protein was incubated with CD47-A549 cells at room temperature for 15 minutes.

6. Washed 2 times with 1mL of precooled wash, centrifuged at 800g for 3 minutes, and the supernatant discarded.

7. 0.1. mu.L of PE-tagged anti-His antibody was added to each sample for 15 minutes at room temperature.

8. Washed 2 times with 1mL of precooled wash, centrifuged at 800g for 3 minutes, and the supernatant discarded.

9. Each sample was resuspended by adding 200. mu.L of wash solution and the affinity was measured by flow cytometry.

The results in FIG. 5 show that the cell supernatants expressed by vaccinia virus recombinant with α CD47- α CD3-BITE bound to CD47 with a positive rate of 85.9% compared to wild-type vaccinia virus. Selecting positive clone to carry out passage stability test, selecting a strain with strong passage stability and high expression target protein to preserve, and naming the strain as rTV-alpha CD 47-alpha CD3-BITE, wherein the preservation number is as follows: CCTCC NO:

v202081, preservation date 1/2/2021, preservation address is chinese type culture collection center, address: wuhan university in Wuhan, China.

Example 6: recombinant vaccinia virus of alpha CD 47-alpha CD3-BITE with in vitro anti-lung cancer effect

1.143TK^-Cell plating: will be 143TK^-Cells were plated in 6-well plates at approximately 1X 10 per well⁶The cell density is 100% of the bottom area of the 6-pore plate when the cell is used;

2. complete medium was replaced with 2mL of maintenance medium per well (DMEM medium + 2% FBS + 1% PS) before virus inoculation, and virus was inoculated into the cells at an inoculum size of 0.02 MOI. 5% CO at 37 ℃₂The incubation time in the incubator is about 48 hours, and the supernatant collection time is determined according to the virus plaque formation condition. Meanwhile, wild vaccinia virus was used as a control;

3. respectively taking 100 mu L of supernatant to carry out subsequent A549 in vitro killing experiments;

an A549 cell in vitro killing experiment performed according to the kit instructions (Dojindo, K17); pipette 100. mu.L of resuspended A549 cells into a 96-well plate at 1X 10 cells per well⁴37 ℃ and 5% CO₂The culture was carried out overnight.

5. The next day, the supernatant was added at 37 ℃ with 5% CO₂After 1 hour of incubation, the ratio of effective target was 5: 1 addition of T cells, continued at 37 ℃ with 5% CO₂The culture was carried out for 4 hours.

6. After 20. mu.L lysis buffer was added to the high control wells, 20. mu.L medium was added to the low control and background wells at 37 ℃ with 5% CO₂Incubate for 30 minutes.

7. Pipette 100 μ L of supernatant from each well into a new 96 well plate.

8. After adding 100. mu.L of a developing solution to each well, the reaction was carried out for 5 minutes in the dark at room temperature.

9. Finally, 50. mu.L of stop buffer was added to each well, and the absorbance at 490nm was immediately measured by a microplate reader.

The result is shown in figure 6, compared with a blank control group, the recombinant vaccinia virus loaded with the alpha CD 47-alpha CD3-BITE can remarkably improve the killing effect on A549 cells in vitro, and has the improvement effect of 4 times.

In conclusion, the alpha CD 47-alpha CD3-BITE protein combined with T cells has very strong killing activity on tumor cells, and the prepared recombinant vaccinia virus rTV-alpha CD 47-alpha CD3-BITE as an oncolytic virus can also obviously control various solid tumors such as human lung cancer and the like, has very high application value for treating tumors, is simple to prepare, and is convenient for large-scale preparation and popularization.

Example 7: t cell mediated in vitro anti-human ovarian adenocarcinoma cell of different structural formulas alpha CD 47-alpha CD3-BITE Effect

To demonstrate the anticancer effects of the different structures of α CD47- α CD3-BITE protein, the applicants designed the following BITE proteins, respectively

BITE1：VL_αCD47-L-VH_αCD3-L-VL_αCD3-L-VH_αCD47

Said VH_αCD47As shown in SEQ ID NO 19, the VL_αCD4721, the VH shown as SEQ ID NO_αCD3As shown in SEQ ID NO. 25, VL_αCD326, and the L is GGGGSGGGGSGGGGS.

BITE2：VH_αCD47-L-VH_αCD3-L-VL_αCD3-L-VL_αCD47

BITE3：VH_αCD47-L-VL_αCD47-L-VH_αCD3-L-VL_αCD3

Said VH_αCD47As shown in SEQ ID NO 19, the VL_αCD4721, the VH shown as SEQ ID NO_αCD3As shown in SEQ ID NO. 25, VL_αCD3As shown in SEQ ID NO. 26; one linker peptide is GGGGSGGGGSGGS.

BITE 4: like BITE1, only the linker peptide was changed to GGGG;

BITE 5: as with BITE1, the VH_αCD3As shown in SEQ ID NO 27, VL_αCD3As shown in SEQ ID NO 28.

Practice ofThe results are shown in FIG. 7, which are the same as in examples 2 and 3. VL_αCD47-L-VH_αCD3-L-VL_αCD3-L -VH_αCD47Wherein L is (G)₄S)₃The method is the optimal design of alpha CD 47-alpha CD3-BITE, can mediate strong tumor cell killing function, changes the antibody clone species of CD47 or CD3 (such as clone number SP34), and still has strong killing function;

VH α CD47-L-VH α CD3-L-VL α CD3-L-VL α CD47 wherein L is (G)₄S)₃It is a suboptimal design for α CD47- α CD3-BITE, but still has similar tumor killing activity as the control antibody.

The above-described embodiments are illustrative, and should not be construed as limiting the invention, and variations, modifications, substitutions, and alterations may be made therein by those of ordinary skill in the art without departing from the scope of the invention.

Sequence listing

<110> Shanghai Xinwan Biotech Co., Ltd

<120> bispecific T cell engagers, recombinant oncolytic viruses thereof and uses thereof

<130> DIC21110049

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<213> Artificial Sequence (Artificial Sequence)

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Gln His Arg Gly Thr

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<213> Artificial Sequence (Artificial Sequence)

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Arg Ala Ser Gln Gly Ile Ser Ser Arg Leu Ala

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Gln Gln Ala Asn Ser

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Gln His Arg Val Thr

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 20

<211> 103

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Arg Arg Ala Phe Gly Gln

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 21

<211> 103

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Glu Ile Val Leu Thr Gln Ser Pro Val Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Thr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Arg Gly Thr Phe Gly Gln

85 90 95

Gly Thr Lys Val Asp Ile Lys

100

<210> 22

<211> 103

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Gly Gly

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 23

<211> 103

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Thr Thr Leu Ser Ala Ser Val Gly

1 5 10 15

His Arg Val Thr Ile Asn Cys Arg Ala Ser Gln Ser Ile Ser Asn Trp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Ala Gly Ser Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu Ala Asn Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Ile Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln His Arg Val Thr Phe Gly Pro

85 90 95

Gly Thr Lys Val Asp Leu Lys

100

<210> 24

<211> 103

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Gly Thr Phe Gly Gln

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 25

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 26

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

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

35 40 45

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

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 27

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Val Arg His Gly Asn Phe Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 28

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Gly Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro

1 5 10 15

Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr

20 25 30

Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe

35 40 45

Thr Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Leu Ile Gly Lys Ala Ala Leu Thr Ile Thr Gly

65 70 75 80

Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 29

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

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

35 40 45

Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 30

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Asn Gly His Gly Phe Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 31

<211> 309

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gacattcaga tgacacagtc tccatctaca ctgtccgcct ccgtgggcga ccgggtgacc 60

attacatgcc gcgcctctca gtctattagc tcttggctcg cctggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatttacaag gcctccagtc tggaatctgg cgtgccatct 180

aggttcagcg gctctggcag cggcaccgag ttcacactca caatttcttc cttacagccc 240

gacgacttcg ccacatacta ctgccagcac cggagagcct tcggccaggg cacaaaggtg 300

gagatcaag 309

<210> 32

<211> 309

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gagatcgtgc tgacccagtc tccagtgaca ctgagcgtgt cccctggcga gagagccacc 60

ctgtcttgca gagcctctca gggcgtgaca tctaacctcg cctggtatca gcagaagcct 120

ggccaggccc caagactcct gatttacggc gcctctacac gcgccacagg cgtgcccgcc 180

cggttcagcg gctctggctc cggcaccgag ttcacactca caattagctc tcttcagtcc 240

gaggacttcg ccgtgtacta ctgccagcac cgcggcacat tcggccaggg cacaaaggtg 300

gacattaag 309

<210> 33

<211> 309

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gacattcaga tgacacagtc cccatctagc gtgtctgcct ccgtgggcga ccgggtgaca 60

attacatgcc gcgcctctca gggcattagc tcacggctcg cctggtatca gcagaagcct 120

ggcaaggccc ctaagctgct gatttacgcc gcctcttccc tccagagcgg cgtgccatct 180

aggttctctg gctccggctc tggcaccgac ttcacactga ccatttcttc ccttcaaccc 240

gaggacttcg ccacatacta ctgccagcag gccaactcct tcggcggcgg cacaaaggtg 300

gagatcaag 309

<210> 34

<211> 309

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gacatccaga tgacacagtc tcctacaaca ctgagcgcct ccgtgggcca cagagtgaca 60

attaactgcc gcgcctctca gtctatctct aactgggtgg cctggtatca gcagaaggcc 120

ggctccgccc caaagctgct gatttacaag gcctctacac tcgccaacgg cgtgcctagc 180

cggttcagcg gctccggctc tggcaccgag ttcattctca ccatttcttc cttgcaaccc 240

gaggacgtgg ccacatacta ctgccagcac agggtgacct tcggcccagg caccaaggtg 300

gacctcaag 309

<210> 35

<211> 309

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gacattcaga tgacacagtc cccatccacc ctgagcgcct ccgtgggcga cagagtgacc 60

attacatgcc gcgcctctca gtctatttcc aactggctcg cctggtatca gcagaagcca 120

ggcgaggccc ctaagctgct gatttacaag gcctctaact tggagtctgg cgtgccatct 180

aggttctccg gcagcggctc cggcaccgag ttcacactca caattagctc tttacaaccc 240

gacgacttcg ccacatacta ctgccagcag ttcggcacct tcggccaggg cacaaaggtg 300

gagatcaag 309

<210> 36

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gaggtgcagc tggtggagtc tggcggcggc ctcgtgcagc ctggcggctc cctgagactg 60

tcctgcgccg cctctggctt cacattctcc ggctacggca tgtcttgggt gagacaggcc 120

ccaggcaagg gcctagagtg ggtggccaca attacatccg gcggcacata cacctactac 180

cccgacagcg tgaagggcag attcaccatt tcccgcgaca acgccaagaa ctctctgtac 240

ctccagatga acagcctgag agccgaggac accgccgtgt actactgcgc ccggtcactc 300

gccggcaacg ctatggacta ctggggccag ggcacactcg tgaccgtgtc tagc 354

<210> 37

<211> 380

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg 380

<210> 38

<211> 204

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 60

ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120

tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 180

tgggaggtct atataagcag agct 204

<210> 39

<211> 331

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga 60

agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc 120

cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttcg 180

ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga 240

cactatagaa taacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac 300

tgcacctcgg ttctatcgat tgaattccac c 331

<210> 40

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

atgggatggt catgtatcat cctttttcta gtagcaactg caacc 45

<210> 41

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

aagcttggcc gccatggccc 20

<210> 42

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

tatcatgtct ggatc 135

<210> 43

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

gtaccaggcc tagtactatg gagaggaccc ttgtctg 37

<210> 44

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

aataagctcg aagtcgacct aggagagatg ctgatg 36

Claims

1. An α CD47 and α CD3 bispecific T cell engager α CD47- α CD3 BiTE comprising a fusion protein of any one of the following formulae:

VL_αCD47-L-VH_αCD3-L-VL_αCD3-L-VH_αCD47(ii) a Or

VH_αCD47-L-VH_αCD3-L-VL_αCD3-L-VL_αCD47

Wherein L represents a linker peptide;

preferably, said VL_αCD47Comprises the following steps: VL CDR1 shown in SEQ ID NO. 4, VL CDR2 shown in SEQ ID NO. 5, VL CDR3 shown in SEQ ID NO. 6;

or the VL_αCD47Comprises the following steps: VL CDR1 as shown in SEQ ID NO. 16, VL CD as shown in SEQ ID NO. 17R2, VL CDR3 as shown in SEQ ID NO: 18.

2. The bispecific T cell engager of claim 1, wherein the VH_αCD47Comprises 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO. 19;

preferably, said VL_αCD47Comprises 3 CDRs contained in the light chain variable region set forth in any one of SEQ ID NOs 20-24;

3. The bispecific T-cell engager of claim 1 or 2, wherein the VH_αCD47Comprising an amino acid sequence selected from the group consisting of:

(i) 19 in SEQ ID NO;

and/or the presence of a gas in the gas,

(iv) a sequence set forth in any one of SEQ ID NOs 20-24;

4. The bispecific T-cell engager of any one of claims 1 to 3, wherein said VH_αCD3Comprising an amino acid sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO 25 or 27;

(ii) a sequence having substitution, deletion or addition of one or several amino acids compared with the sequence shown in SEQ ID NO. 25 or 27; or

and/or the presence of a gas in the gas,

(iv) 26 or 28;

5. The bispecific T-cell engager of any one of claims 1 to 4, wherein said VH_αCD47、VH_αCD3、VL_αCD47And VL_αCD3Linked by a linker peptide;

preferably, said VH_αCD47、VH_αCD3、VL_αCD47And VL_αCD3Connected with each other through one, two or three connecting peptides;

VH_αCD47-L-VH_αCD3-L-VL_αCD3-L-VL_αCD47

6. An isolated nucleic acid molecule encoding the bispecific T-cell engager of any one of claims 1-5;

preferably, the nucleic acid molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 36, said nucleic acid sequence being VH_αCD47The coding sequence of (a).

7. The expression framework of the bispecific T-cell engager of any one of claims 1-5:

5’-E1-E2-E3-E4-E5-E6-E7-3’

wherein:

e5 is a nucleotide sequence encoding the bispecific T cell engager of any one of claims 1 to 5;

e7 is the SV40 transcriptional stop signal region; preferably comprises SEQ ID NO: 42.

8. A recombinant oncolytic virus operably inserted or comprising the expression framework of claim 7;

preferably, the expression cassette is located in the Thymidine Kinase (TK) region of the recombinant oncolytic virus;

preferably, the expression frame can be expressed alone, or in fusion with other genes or fragments;

The recombinant oncolytic virus further comprises gene coding sequences for proteins associated with apoptosis and apoptosis of cells, preferably selected from apoptosis-related factor 1, interleukin-1 beta converting enzyme, Bcl-2 protein, Fas/APO-1, p53, myc, ataxia telangiectasia mutated gene, corticotropin D or corticotropin E; or

9. The recombinant oncolytic virus of claim 8, wherein the viral backbone of the oncolytic virus is derived from a modified or engineered 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, respiratory syncytial virus, Sendzein virus, EB virus, cytomegalovirus, human herpesvirus 6, variola virus, vaccinia virus, molluscum contagiosum virus, orf virus, reovirus, rotavirus, enterovirus, Seneca virus, polio virus, Coxsackie virus, rhinovirus, hepatitis A virus, foot and mouth disease virus, togavirus, alphavirus, Selim virus, eastern equine encephalitis virus, Sindbis virus, Simmondsi virus, Va virus, Coxsackie virus, rhinovirus, hepatitis A virus, foot and mouth disease virus, Tokyo virus, Cinders virus, and Addison virus, Rubella virus, coronavirus, flavivirus, hepatitis C virus, Japanese encephalitis virus, St.Louis encephalitis virus, Murray Valley fever virus, yellow fever virus, West Nile virus, Zika virus, dengue virus, Ebola virus, Marburg virus, arenavirus, Lassa virus, lymphocytic choriomeningitis virus, Pichimede virus, Hunning virus, Martha virus, Hantaan virus, rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, monkey virus 5, measles virus, vesicular stomatitis virus, rabies virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, monkey immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, Rous sarcoma virus, human T cell leukemia virus type 1, simian foamy virus, herpes virus, Rous sarcoma virus, Rous virus, Ro, Hepatitis b virus, hepatitis e virus, human papilloma virus or polyoma virus;

10. A recombinant vaccinia virus Tiantan strain rTV-alpha CD 47-alpha CD3-BITE, which has a preservation number of: CCTCC NO: V202081.

11. A method of producing a recombinant oncolytic virus according to any one of claims 8 to 10, comprising the steps of:

1) synthesizing an expression framework for the bispecific T cell engager BiTE of claim 7;

3) transfecting the recombinant plasmid vector obtained in the step 2) into an oncolytic virus, and screening to obtain a recombinant oncolytic virus;

optionally, culturing the obtained recombinant oncolytic virus;

preferably, the method comprises the steps of:

3) by means of gene homologous recombination, pSC 65-alpha CD 47-alpha CD3-BITE plasmid is transfected into TK143 infected by wild type vaccinia virus^-In cells, the two are subjected to homologous recombination to generate recombinant vaccinia virus rTV-alpha CD 47-alpha CD 3-BITE; screening to obtain the recombinant oncolytic vaccinia virus of which the TK region comprises the coding sequence of alpha CD 47-alpha CD 3-BITE;

preferably, the expression frame of the bispecific T-cell engager α CD47- α CD3-BITE is controlled by the early/late promoter p7.5 of vaccinia virus.

12. Use of the bispecific T-cell engager of any one of claims 1-5 or the recombinant oncolytic virus of any one of claims 8-10 for the preparation of an anti-tumor medicament;

preferably, the tumour is selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, gliomas 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, oesophageal cancer, large bowel cancer, hematopoietic cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell cancer, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell cancer of the head and neck, hodgkin's lymphoma, non-hodgkin's lymphoma or hematological tumour disease.