Fusion protein for activating immune cells to kill tumor cells and recombinant virus and application thereof

Abstract

The invention provides a fusion protein for activating immune cells to kill tumor cells and a recombinant virus and application thereof. The fusion protein comprises an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain, wherein the extracellular domain is a polypeptide or an active fragment thereof which is specifically combined with an immune cell surface antigen, and the killing function of the immune cell is activated by specifically combining the extracellular domain with the immune cell surface antigen; the transmembrane domain is a polypeptide that anchors the fusion protein on a cell membrane of a tumor cell. The fusion protein and the recombinant virus can effectively kill tumor cells, and have broad-spectrum killing activity for various tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to a fusion protein that is anchored on tumor cell membranes and activates immune cells to kill tumor cells. The present invention also relates to oncolytic viruses and lentiviruses containing this fusion protein that is anchored on tumor cell membranes and activates immune cells to kill tumor cells. The present invention also relates to the use of the fusion protein and its oncolytic and lentiviruses in tumor treatment. Background Art

[0002] Cancer has become one of the most serious diseases threatening human health. In 2021, the global total number of malignant tumor cases reached 19.74 million, with approximately 9.31 million deaths. With the aging population and the impact of social and environmental factors, the number of cancer cases is expected to continue to rise. It is estimated that by 2030, the number of cases will reach 24.04 million, and the number of deaths will reach 12.94 million. To improve survival rates, it is necessary not only to carry out cancer prevention and screening, but also to continuously develop new, effective and affordable cancer treatments.

[0003] The body's immune system inherently possesses powerful anti-tumor capabilities, including T cells, NK cells, NKT cells, iNKT cells, mucosal-associated invariant T (MAIT) cells, γδT cells, K cells, monocytes, macrophages, and neutrophils. T cells, among others, play a crucial role in the immune system, capable of identifying and eliminating abnormal cells, such as tumor cells. Tumors are composed of cells that grow and divide abnormally, and T cells can combat them in a variety of ways.

[0004] One way is through cytotoxic T cells (CTLs), which can recognize and kill tumor cells. CTLs recognize antigens on the surface of tumor cells, which are related to proteins abnormally expressed by tumor cells, and then release cytotoxins to kill the tumor cells. In addition, CTLs can also secrete cytokines, such as interferon and tumor necrosis factor, to further inhibit tumor growth.

[0005] Another way is through helper T cells (Th cells), which regulate the immune response. Th cells can differentiate into different subtypes, such as Th1 and Th2. Th1 cells produce cytokines such as interferon, which activate CTLs and macrophages, thereby strengthening the attack on tumor cells. Th2 cells produce certain cytokines that help B cells produce antibodies, providing specific recognition of tumor antigens.

[0006] NK cells play a key role in both innate and adaptive immune responses, possessing multiple cytotoxic mechanisms and capable of regulating immune responses through the production of cytokines. NK cells, short for "Natural Killer cells," are a specialized type of cell in the immune system. Unlike other immune cells, NK cells can selectively lyse cells without prior activation, inherently priming them to kill tumor cells.

[0007] How to fully utilize or mobilize the anti-tumor cells in the body's immune system to exert anti-tumor functions is a very important direction for the development of new anti-tumor drugs.

[0008] CD3 is a classic signaling target. Currently, more than half of the antibodies studied in bispecific antibodies activate T cells through CD3. CD3 is an important biomarker on the T cell membrane and consists of four protein chains, namely CD3δ, CD3ε, CD3γ, and CD3ζ. Among them, two pairs of heterodimers, namely CD3δ / CD3ε and CD3γ / CD3ε, can form TCR / CD3 complexes with T cell receptors, participating in T cell antigen recognition, signal transduction, and the regulation of T cell development. The intracellular domain of TCR is very short and cannot complete signal transduction independently, such as in the form of chimeric antigen receptor CAR-T cell therapy. Compared with CAR-T, bispecific antibodies can be dose-controlled to reduce toxicity. Therefore, CD3 is a very promising tumor treatment target in the field of bispecific antibodies.

[0009] However, while bispecific antibodies focus on tumor targeting and specificity, they also lose their broad spectrum. A CD3 bispecific antibody often targets only one target and has a relatively narrow disease indication, which increases product development costs and prolongs the time to market.

[0010] Therefore, in response to the above-mentioned defects, it is necessary to fully develop a broader-spectrum, safer and more effective T cell activation strategy targeting the CD3 molecule to enhance anti-tumor activity.

[0011] Oncolytic viruses are a type of virus that can infect tumor cells and lyse and destroy tumors. Commonly used oncolytic viruses rely on their tumor cell infection specificity to selectively infect tumor tissues, or replicate in large quantities in tumor cells, or release cytotoxic factors without non-specifically killing and destroying normal cells, ultimately causing tumor cell lysis. The progeny viruses released after oncolysis continue to infect adjacent tumor cells. Alternatively, the target gene carried by the oncolytic virus expresses the corresponding protein to enhance the body's anti-tumor response. After the death of tumor cells, the tumor cells release tumor-associated antigens, inducing a systemic anti-tumor immune response, causing distant tumors that have not been exposed to the virus to regress. Currently, oncolytic virus drugs have been clinically tested in multiple solid tumor fields, including melanoma, head and neck cancer, bladder cancer, prostate cancer, glioblastoma and other malignant tumors.

[0012] Viruses are an effective choice for killing tumor cells due to their natural cell lysis function. From the original natural viruses to gene-edited viruses, the types of oncolytic viruses have also evolved from the original herpes viruses to more than ten commonly used viruses. Among them, adenovirus has become the most commonly used oncolytic virus due to its ease of gene editing and flexibility of use. Other commonly used viruses include herpes simplex virus, vaccinia virus, Newcastle disease virus, measles virus, reovirus, coxsackie virus, polio virus, etc. The types of oncolytic viruses range from single-stranded viruses to double-stranded viruses, from RNA viruses to DNA viruses, and from natural viruses to gene-edited viruses, which greatly enhances the flexibility of this therapy in the clinical treatment of solid tumors.

[0013] Genetically modified oncolytic viruses can overcome problems such as weak receptor affinity, low infection efficiency, and non-specific infection of natural oncolytic viruses, and can target and infect tumor cells more effectively and safely.

[0014] However, oncolytic viruses as therapeutic drugs still face many challenges. One of them is that the overall efficacy of single drugs is limited. According to existing data, the overall efficacy of oncolytic viruses is less than 30%. Another is that based on its anti-tumor mechanism and its own characteristics, the route of administration is limited to intratumoral injection. However, systemic blood delivery is still required for the treatment of metastatic cancer. The main reason why the route of administration of oncolytic viruses is limited to intratumoral injection is that: viral membrane receptors are widely present in the human body, which makes the tumor specificity of virus infection low, and it is very likely to infect and destroy normal cell tissues; after the oncolytic virus enters the blood, it will be neutralized by the virus-specific antibodies in the serum and then removed; the blood will have a dilution effect on the virus, and the tumor microenvironment will inhibit the effective infiltration of the virus into the tumor tissue.

[0015] Therefore, there is a need to develop more effective and easier-to-administer oncolytic viruses. Summary of the Invention

[0016] In response to the shortcomings of the prior art, the present invention aims to provide a fusion protein that activates immune cells to kill tumor cells, a recombinant virus thereof, and its uses. On the one hand, the present invention develops a broader, safer, and more effective immune cell activation strategy targeting immune cell surface antigens, thereby enhancing anti-tumor activity. For example, the present invention develops a fusion protein targeting the CD3 molecule on the surface of T cells that anchors CD3-activating antibodies to the tumor cell membrane, thereby activating T cells by activating the CD3 molecule. On the other hand, the present invention also develops recombinant viral vectors such as adenovirus vectors, vaccinia virus vectors, and lentivirus vectors for the fusion protein, using recombinant viral vectors to target the fusion protein to tumor tissue cells, thereby broadening the route of administration of the fusion protein.

[0017] The purpose of the present invention is achieved through the following technical solutions:

[0018] The first aspect of the present invention provides a fusion protein that activates immune cells to kill tumor cells, which comprises an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain; wherein the extracellular domain is a polypeptide or an active fragment thereof that specifically binds to an immune cell surface antigen, and activates the killing function of the immune cells by specifically binding to the immune cell surface antigen; the transmembrane domain is a polypeptide that anchors the fusion protein to the cell membrane of the tumor cell; the hinge region connects the extracellular domain and the transmembrane domain.

[0019] According to the fusion protein of the present invention, the immune cells are selected from one or more of T cells, NK cells, NKT cells, iNKT cells, mucosa-associated invariant T cells, γδT cells, K cells, monocytes, macrophages and neutrophils; the immune cell surface antigens are selected from one or more of CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD45, CD49, CD56, CD62L, CD94, CD122, CD16, CD32, CD64, CD68, CD80, CD86, CD127, CD137, CD152, CD197, CD369, α4-1BB, NKp46 and SIRPα.

[0020] Preferably, the immune cells are selected from one or more of T cells, NK cells and macrophages; and the immune cell surface antigens are selected from one or more of CD3, CD28, α4-1BB, NKp46, CD16 and SIRPα.

[0021] According to the fusion protein of the present invention, the extracellular domain comprises one or more selected from the following:

[0022] The amino acid sequence shown in SEQ ID NO: 8 or an active fragment thereof;

[0023] The amino acid sequence shown in SEQ ID NO: 32 or an active fragment thereof;

[0024] The amino acid sequence shown in SEQ ID NO: 34 or an active fragment thereof

[0025] The amino acid sequence shown in SEQ ID NO: 36 or an active fragment thereof;

[0026] The amino acid sequence shown in SEQ ID NO: 38 or an active fragment thereof;

[0027] The amino acid sequence shown in SEQ ID NO: 40 or an active fragment thereof; and

[0028] The amino acid sequence shown in SEQ ID NO: 42 or an active fragment thereof.

[0029] According to the fusion protein of the present invention, the hinge region is selected from one or more of the following:

[0030] (1) the hinge region of antibody IgG4 or a mutant thereof;

[0031] (2) the hinge region of antibody IgG4 or its mutant, and CH2 region;

[0032] (3) the hinge region, CH2 region, and CH3 region of an IgG4 antibody or a mutant thereof;

[0033] (4) the hinge region of antibody IgG1 or its mutant;

[0034] (5) the hinge region of antibody IgG1 or its mutant, and CH2 region;

[0035] (6) the hinge region, CH2 region, and CH3 region of an IgG1 antibody or a mutant thereof;

[0036] (7) Hinge region of immunoglobulin Fc receptor: FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcαR (CD89), FcεRI or FcεRII (CD23);

[0037] (8) co-stimulatory molecule CD28 hinge region, CD137 hinge region, CD8 hinge region, CD4 hinge region, PD-1 hinge region or CTLA-4 hinge region; and

[0038] (9) Any combination of the above different hinge regions.

[0039] Preferably, the hinge region is the CD8 hinge region.

[0040] More preferably, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 10 or an active fragment thereof.

[0041] According to the fusion protein of the present invention, the transmembrane domain is selected from one or more of the following: the transmembrane domain of the CD3ξ chain of the T cell receptor complex, the CD28 transmembrane domain, the immunoglobulin Fc receptor transmembrane domain, the CD4 transmembrane domain, the CD8 transmembrane domain, the CD16 transmembrane domain, the CD137 transmembrane domain, the CTLA-4 transmembrane domain, the PD-1 transmembrane domain, the LAG-3 transmembrane domain, the VISTA transmembrane domain and combinations thereof.

[0042] Preferably, the transmembrane domain is the CD8 transmembrane domain.

[0043] More preferably, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 12 or an active fragment thereof.

[0044] According to the fusion protein of the present invention, wherein the intracellular domain is a specific region of the fusion protein present in tumor cells. Specifically, the intracellular domain is a kinase domain that participates in cell signal transduction and is responsible for phosphorylating specific substrate proteins, or a DNA binding domain that binds to DNA and regulates gene expression, or a GTPase domain that participates in cell signal transduction and is responsible for GTP hydrolysis reactions, or a transcription factor domain that regulates the transcription process of genes, or a hydrophilic sequence that does not play any biological function and only serves to anchor membrane proteins. In practical applications, those skilled in the art can select a suitable intracellular domain according to their needs.

[0045] According to the fusion protein of the present invention, its extracellular domain may include different polypeptides that activate the same immune cell (i.e., a combination of domains that activate mutually synergistic signaling pathways on the same immune cell), or it may include polypeptides that activate different immune cells.

[0046] In a preferred embodiment, the extracellular domain of the fusion protein is a polypeptide that specifically binds to the T cell surface antigen CD3, 4-1BB or CD28, and the amino acid sequence is shown in SEQ ID NO: 8, SEQ ID NO: 32 or SEQ ID NO: 34, respectively. Alternatively, the extracellular domain of the fusion protein includes a polypeptide that specifically binds to the T cell surface antigen CD3 and a polypeptide that specifically binds to the T cell surface antigen 4-1BB or CD28.

[0047] In a more preferred embodiment, the extracellular domain of the fusion protein is a polypeptide that specifically binds to the T cell surface antigen CD3, the hinge region is the CD8 hinge region, and the transmembrane domain is the CD8 transmembrane domain. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 29.

[0048] In another preferred embodiment, the extracellular domain of the fusion protein is a polypeptide that specifically binds to the NK cell surface antigen NKp46 or CD16, and the amino acid sequence is shown in SEQ ID NO: 38 or SEQ ID NO: 36, respectively.

[0049] In another preferred embodiment, the extracellular domain of the fusion protein is a polypeptide that specifically binds to the macrophage surface antigen SIRPα, and the amino acid sequence is shown in SEQ ID NO: 40.

[0050] The fusion protein of the present invention can be used alone or in combination with other immune function molecules. Therefore, the second aspect of the present invention provides a pharmaceutical composition comprising the fusion protein of the present invention and an immune function molecule, wherein the immune function molecule is selected from one or more of a cytokine, a chemokine, and an immune checkpoint blocking antibody.

[0051] Preferably, the cytokine is selected from one or more of GM-CSF, IL-2, IL-3, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-24, IL-33, IL-35, IL-37, IFN-α, IFN-β, IFN-γ and MALP-2; more preferably, the cytokine is IL-21, whose amino acid sequence is shown in SEQ ID NO: 19.

[0052] Preferably, the chemokine is selected from one or more of CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL4, CCL19, CCL20, CCL21 and CX3CL1; more preferably, the chemokine is CXCL13, the amino acid sequence of which is shown in SEQ ID NO: 23.

[0053] Preferably, the immune checkpoint blocking antibody is selected from one or more of CTLA-4 blocking antibody, CD47 blocking antibody, SIRPα blocking antibody, PD-1 blocking antibody, PD-L1 blocking antibody, LAG-3 blocking antibody and Tim-3 blocking antibody.

[0054] In a more preferred embodiment, the fusion protein of the present invention responds to a hypoxic environment. Accordingly, the pharmaceutical composition according to the present invention further comprises an oxygen-sensitive sequence that is degraded under normoxic conditions but is stable under hypoxic conditions; preferably, the oxygen-sensitive sequence comprises the amino acid sequence shown in SEQ ID NO: 15 or an active fragment thereof.

[0055] It should be noted that in the pharmaceutical composition according to the present invention, the fusion protein, the immune function molecule, and the oxygen-sensitive sequence are each contained in the pharmaceutical composition as a separate component; alternatively, any two or more of the fusion protein, the immune function molecule, and the oxygen-sensitive sequence are connected together via a linker. The linker can be any linker commonly used in the art, such as a T2A cleavage peptide, a P2A cleavage peptide, or an IRES sequence, wherein the amino acid sequence of the T2A cleavage peptide is shown in SEQ ID NO: 21, and the amino acid sequence of the P2A cleavage peptide is shown in SEQ ID NO: 17.

[0056] The third aspect of the present invention provides a polynucleotide encoding the fusion protein of the present invention. Specifically, the polynucleotide comprises a nucleotide sequence encoding an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain.

[0057] In a specific embodiment, the coding sequence of the extracellular domain that specifically binds to CD3, α4-1BB, CD28, CD16, NKp46 or SIRPα is shown in SEQ ID NO: 7, 31, 33, 35, 37 or 39, respectively.

[0058] In another specific embodiment, the coding sequence of the hinge region is shown in SEQ ID NO:9.

[0059] In another specific embodiment, the coding sequence of the transmembrane domain is shown in SEQ ID NO:11.

[0060] In a preferred embodiment, the polynucleotide is the nucleotide sequence shown in SEQ ID NO: 28.

[0061] The fusion protein of the present invention can be delivered to tumor cells using different delivery vectors, including but not limited to: recombinant viral vectors, mRNA / DNA, protein molecule carriers, lipid nanoparticles, extracellular vesicles (EVs), etc. Therefore, the fourth aspect of the present invention provides a recombinant viral vector comprising the polynucleotide of the present invention. The recombinant viral vector of the present invention can be prepared by inserting the polynucleotide into a viral vector.

[0062] Preferably, the viral backbone of the recombinant viral vector is derived from a recombinant adenovirus, a recombinant vaccinia virus, a recombinant herpes simplex virus, an adeno-associated virus, varicella-zoster virus, respiratory syncytial virus, Serioke Forest virus, Epstein-Barr virus, cytomegalovirus, human herpesvirus type 6, smallpox virus, vaccinia virus, molluscum contagiosum virus, canker sore virus, reovirus, rotavirus, enterovirus, Seneca virus, poliovirus, coxsackie virus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus, togavirus, alphavirus, Semlicke 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, virus, Marburg virus, arenavirus, Lassa fever virus, lymphocytic choriomeningitis virus, Pichinde virus, Junin virus, Machupo virus, Hantavirus, Rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, simian virus 5, measles virus, vesicular stomatitis virus, rabies virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, lentivirus, simian immunodeficiency virus, human immunodeficiency virus type 1, 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 papillomavirus or polyomavirus; more preferably, the viral backbone of the recombinant viral vector is an intracellular mature virus, an intracellular packaged virus, a cell-associated packaged virus or an extracellular packaged virus.

[0063] In order to be able to simultaneously deliver the fusion protein and immune function molecules of the present invention to tumor cells, the recombinant viral vector of the present invention also contains a polynucleotide expressing the immune function molecule; wherein the immune function molecule is selected from one or more of cytokines, chemokines and immune checkpoint blocking antibodies.

[0064] Preferably, the cytokine is selected from one or more of GM-CSF, IL-2, IL-3, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-24, IL-33, IL-35, IL-37, IFN-α, IFN-β, IFN-γ, and MALP-2. More preferably, the cytokine is IL-21, and the polynucleotide encoding the cytokine is shown in SEQ ID NO: 18.

[0065] Preferably, the chemokine is selected from one or more of CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL4, CCL19, CCL20, CCL21 and CX3CL1. More preferably, the chemokine is CXCL13, and the polynucleotide encoding the chemokine is shown in SEQ ID NO: 22.

[0066] Preferably, the immune checkpoint blocking antibody is selected from one or more of CTLA-4 blocking antibody, CD47 blocking antibody, SIRPα blocking antibody, PD-1 blocking antibody, PD-L1 blocking antibody, LAG-3 blocking antibody and Tim-3 blocking antibody.

[0067] In a preferred embodiment, the present invention provides a recombinant adenoviral vector derived from the chimpanzee adenoviral vector AdC68XY-R1, wherein the promoter of the chimpanzee adenoviral vector AdC68XY-R1 is constructed as a tumor-specific promoter and a hypoxia-responsive promoter, and / or an expression framework containing a hypoxia regulatory element is inserted downstream of the E1A gene of the chimpanzee adenovirus AdC68XY-R1.

[0068] Preferably, the tumor-specific promoter is the human telomerase reverse transcriptase (hTERT) promoter, the sequence of which is shown in SEQ ID NO: 1.

[0069] Preferably, the tumor-specific promoter can also be selected from carcinoembryonic antigen (CEA) promoter, alpha-fetoprotein (AFP) promoter, human prostate-specific antigen (PSA) promoter, cyclooxygenase-2 (COX-2) promoter, apoptosis inhibitor protein (survivin) promoter or human intestinal tissue-specific antigen (A33) promoter, etc.

[0070] Preferably, the hypoxia-responsive promoter is a promoter of a transcriptional control system triggered in response to hypoxia, which comprises a polynucleotide encoding an oxygen-sensitive sequence, for example, it comprises a hypoxia regulatory element HRE. For example, the sequence of the hypoxia-responsive promoter is as shown in SEQ ID NO: 2, which is a tandem element of five HREs (5HRE); or, the sequence of the hypoxia-responsive promoter is as shown in SEQ ID NO: 3, which is a tandem element of four HREs (4HRE).

[0071] As a preferred example, the recombinant adenoviral vector contains nucleotide sequences expressing a fusion protein specifically binding to CD3, IL-21, and CXCL13. These inserted nucleotide sequences (also referred to as insertion elements) are regulated by a combination of the 4HRE, 5HRE, and the truncated CMV promoters cmd3 or miniP. The sequence of the cmd3 promoter is shown in SEQ ID NO: 4, and the sequence of the miniP promoter is shown in SEQ ID NO: 5.

[0072] Preferably, the recombinant adenoviral vector further contains nucleic acids encoding elements that regulate transcription initiation or termination, such as 5'UTR, 3'UTR, and BGH poly A. The sequence of the 5'UTR is shown in SEQ ID NO: 6, the sequence of the 3'UTR is shown in SEQ ID NO: 24, and the sequence of the BGH poly A is shown in SEQ ID NO: 25.

[0073] Preferably, a nucleotide sequence for expressing an oxygen-sensitive sequence is further inserted into the recombinant adenoviral vector, which may be the nucleotide sequence shown in SEQ ID NO: 14.

[0074] Preferably, the sequences inserted into the recombinant adenoviral vector can be connected by a linker sequence, such as a T2A cleavage peptide, a P2A cleavage peptide, or an IRES sequence, wherein the nucleic acid encoding the T2A cleavage peptide is shown in SEQ ID NO: 20, and the nucleic acid encoding the P2A cleavage peptide is shown in SEQ ID NO: 16. In addition, the linker sequence between the sequences can also be a nucleotide sequence shown in SEQ ID NO: 13.

[0075] It will be understood by those skilled in the art that the target genes such as the above-mentioned chemokines, cytokines and fusion proteins of the present invention are regulated by the hypoxia-responsive promoter with HRE as the core: under normoxia, the activity of the promoter is extremely low and cannot effectively initiate the transcription and translation of the target gene; while under hypoxic conditions, the hypoxia inducible factor-1 (HIF-1) complex enters the nucleus and binds to the HRE element, effectively activating the promoter activity and promoting the efficient expression of the target gene.

[0076] At the same time, the oxygen-sensitive sequence coupled to the fusion protein is degraded under normoxia but stable under hypoxic conditions, which can further enhance the specificity of hypoxic expression of the target gene.

[0077] As a particularly preferred example, the recombinant adenoviral vector of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 27.

[0078] The recombinant adenoviral vector of the present invention can be prepared using the genome of the chimpanzee adenoviral vector AdC68XY-R1 as a basic framework. Specifically, the recombinant adenoviral vector of the present invention can be prepared by a method comprising the following steps:

[0079] Using the genome of chimpanzee adenovirus vector AdC68XY-R1 as a basic framework, the polynucleotide of the present invention, and optionally the polynucleotide expressing the immune function molecule, is cloned into the replicative chimpanzee adenovirus vector AdC68XY-R1; and / or

[0080] The promoter of the chimpanzee adenovirus vector AdC68XY-R1 is replaced with the tumor-specific promoter and the hypoxia-responsive promoter; and / or an expression framework of a hypoxia regulatory element is inserted downstream of the E1A gene of the chimpanzee adenovirus vector AdC68XY-R1.

[0081] In the present invention, the chimpanzee adenovirus vector AdC68XY-R1 can be purchased from the market, for example, from Suzhou Xiangyi Biotechnology Co., Ltd.

[0082] As an example, the recombinant adenoviral vector of the present invention can be prepared by a method comprising the following steps:

[0083] (1) Artificial synthesis of hypoxia-responsive exogenous gene sequences

[0084] 5HRE-cmd3-CD3TM-odd22-P2A-IL21-T2A-CXCL13 or

[0085] 4HRE-miniP-CD3TM-odd22-P2A-IL21-T2A-CXCL13;

[0086] (2) cloning the exogenous gene sequence into the AdC68XY-R1 vector to obtain the recombinant plasmid pAdC68XY-R1-5HRE-cmd3-CD3TM-odd22-P2A-IL21-T2A-CXCL13 or pAdC68XY-R1-4HRE-miniP-CD3TM-odd22-P2A-IL21-T2A-CXCL13;

[0087] (3) transfecting the recombinant plasmid into HEK293 cells and packaging the recombinant adenovirus;

[0088] (4) purifying and identifying the recombinant adenovirus plaques; and

[0089] (5) Verify the function of the recombinant adenovirus.

[0090] It should be noted that, in the present invention, "recombinant adenovirus" and "recombinant oncolytic adenovirus" have the same meaning and can be used interchangeably.

[0091] In another preferred embodiment, the present invention provides a recombinant vaccinia virus vector. Preferably, the recombinant vaccinia virus vector is derived from a vaccinia virus strain Tiantan vector, such as vaccinia virus strain Tiantan vector 752-1 (purchased from Shanghai Xinwan Biotechnology Co., Ltd.). More preferably, the recombinant vaccinia virus vector comprises a nucleotide sequence as set forth in SEQ ID NO: 30 inserted into the C9 region of a vaccinia virus strain Tiantan vector.

[0092] The recombinant vaccinia virus vector of the present invention can be prepared by a method comprising the following steps:

[0093] Subcloning the nucleotide sequence shown in SEQ ID NO: 30 into the C9 region of the vaccinia virus shuttle plasmid pSC65 to construct a recombinant plasmid; and

[0094] The recombinant plasmid is transfected into human thymidine kinase-deficient cells that have been infected with wild-type vaccinia virus by means of gene homologous recombination, so that the two are homologously recombined to obtain the recombinant vaccinia virus vector.

[0095] As an example, the recombinant vaccinia virus vector of the present invention can be prepared by a method comprising the following steps:

[0096] (1) Synthesizing the exogenous gene sequence 6*loxp-P11-αCD3-TM as shown in SEQ ID NO: 30;

[0097] (2) The exogenous gene sequence 6*loxp-P11-αCD3-TM was subcloned into the C9 region of the vaccinia virus shuttle plasmid pSC65 to construct the recombinant plasmid pSC65-C9-6*loxp-P11-αCD3-TM;

[0098] (3) using homologous recombination to transfect the pSC65-C9-6*loxp-P11-αCD3-TM recombinant plasmid into human thymidine kinase-deficient cells (TK143 cells) that have been infected with wild-type vaccinia virus, allowing the two to homologously recombine to produce recombinant vaccinia virus rTV-pSC65-C9-6*loxp-P11-αCD3-TM; and

[0099] (4) Screening was performed to obtain a recombinant oncolytic vaccinia virus whose C9 region contained the coding sequence shown in SEQ ID NO.30.

[0100] It should be noted that, in the present invention, "recombinant vaccinia virus" and "recombinant oncolytic vaccinia virus" have the same meaning and can be used interchangeably.

[0101] In another preferred embodiment, the present invention provides a recombinant lentiviral vector, wherein the lentiviral vector is a viral vector system modified based on the human immunodeficiency virus (HIV-1 virus).

[0102] In the present invention, the lentiviral vector can be purchased from the market. For example, the lentiviral vector can be synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0103] Preferably, the recombinant lentiviral vector comprises the nucleotide sequence shown in SEQ ID NO: 28.

[0104] The recombinant lentiviral vector of the present invention can be prepared by cloning the nucleotide sequence shown in SEQ ID NO: 28 into a blank lentiviral expression plasmid pXW-EF1α-MCS.

[0105] As an example, the recombinant lentiviral vector of the present invention can be prepared by a method comprising the following steps:

[0106] (1) Synthesizing the exogenous gene sequence αCD3-CD8 TM as shown in SEQ ID NO. 28;

[0107] (2) cloning the exogenous gene sequence αCD3-CD8 TM into a lentiviral expression vector to obtain an αCD3-CD8 TM lentiviral expression plasmid; and

[0108] (3) The lentiviral expression plasmid, backbone plasmid and envelope plasmid are co-transfected into HEK293T cells, and lentiviral particles are packaged and obtained. The lentiviral concentrate is obtained by centrifugation and concentration, which is the recombinant lentiviral vector.

[0109] The fifth aspect of the present invention provides a recombinant virus that can deliver the above-mentioned fusion protein or the above-mentioned pharmaceutical composition to tumor cells. The recombinant virus can be obtained by packaging the recombinant viral vector of the present invention.

[0110] It should be noted here that, in certain circumstances, a strict distinction may not be made between the recombinant viral vector and the recombinant virus of the present invention.

[0111] The sixth aspect of the present invention provides a pharmaceutical composition comprising the recombinant viral vector or recombinant virus of the present invention.

[0112] The recombinant viral vector or recombinant virus of the present invention can be used alone or in combination with immune cells. Therefore, the pharmaceutical composition of the present invention also contains immune cells.

[0113] Preferably, the immune cells are selected from one or more of T cells, CAR-T cells, iNKT cells, NK cells, K cells, macrophages, CAR-iNKT cells, CAR-NK cells and CAR-macrophages.

[0114] More preferably, the T cells are selected from one or more of unsorted and purified T cells, sorted and purified T cells, sorted and purified PD-1+T cells, sorted and purified CD137+T cells, sorted and purified CD160+T cells, sorted and purified naive T cells (Tnaive), sorted and purified central memory T cells (TCM), sorted and purified effector memory T cells (TEM), sorted and purified effector T cells (TEM RA), sorted and purified transitional memory T cells (Transitional Memory T cells, TTM) and sorted and purified tissue memory T cells (Tissue residential memory T cells, TRM).

[0115] The seventh aspect of the present invention provides a method for treating tumors, which comprises administering a therapeutically effective amount of the fusion protein, polynucleotide, recombinant viral vector, recombinant virus or pharmaceutical composition of the present invention to a subject in need thereof.

[0116] The eighth aspect of the present invention provides use of the fusion protein of the present invention, the polynucleotide of the present invention, the recombinant viral vector of the present invention, the recombinant virus of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for treating tumors.

[0117] Preferably, the tumor is selected from one or more of B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high-grade glioma, blastoma 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 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, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or blood oncogenic diseases.

[0118] The beneficial effects of the present invention are:

[0119] The oncolytic virus of the fusion protein that is anchored on the tumor cell membrane and can activate immune cells to kill tumor cells provided by the present invention overcomes the limitation of tumor immunosuppression and immune escape of solid tumor tumor cells to human immune cells (such as T cells). It can directly recruit human immune cells (such as T cells) into the inside of solid tumors, thereby killing tumor cells. The oncolytic virus of the fusion protein that can activate immune cells to kill tumor cells provided by the present invention also overcomes the limitation of targets in anti-tumor drugs, has a broad spectrum killing activity of multiple tumors, and has broad application prospects.

[0120] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 Schematic diagrams of the construction of two recombinant adenoviral vectors according to the embodiments of the present invention, wherein A is a schematic diagram of the construction of pAdC68XY-R1-5HRE-cmd3-CD3TM-odd22-P2A-IL21-T2A-CXCL13, and B is a schematic diagram of the construction of pAdC68XY-R1-4HRE-miniP-CD3TM-odd22-P2A-IL21-T2A-CXCL13.

[0122] Figure 2 The results of the enzyme digestion identification of the recombinant adenovirus vector according to the embodiment of the present invention by BglII, Mfe1 and Xho1 are shown, which indicate that the recombinant plasmid bands are all correct.

[0123] Figure 3 The plaques of the recombinant adenovirus according to the embodiment of the present invention are shown. Both recombinant adenoviruses can produce obvious plaques in HEK293 cells.

[0124] Figure 4 The figure shows the expression of recombinant adenovirus according to an embodiment of the present invention in human ovarian cancer cells (SKOV3) under normoxic and hypoxic conditions. As shown in the figure, in human ovarian cancer cells, both IL-21 and CXCL13 show high expression under hypoxic conditions and low expression under normoxic conditions.

[0125] Figure 5 The figure shows the differential expression of CD3 antibodies in human ovarian cancer cells (SKOV3) under normoxic and hypoxic conditions by recombinant adenoviruses according to an embodiment of the present invention. As shown in the figure, CD3 antibodies are highly expressed in human ovarian cancer cells under hypoxic conditions and lowly expressed under normoxic conditions.

[0126] Figure 6 The figure shows the killing effect of recombinant adenoviruses according to embodiments of the present invention on human ovarian cancer cells (SKOV3) under normoxic and hypoxic conditions. As shown in the figure, the recombinant adenoviruses of the present invention can achieve high-efficiency tumor cell killing under hypoxic conditions, while achieving low-level killing under normoxic conditions.

[0127] Figure 7 The figure shows the differential expression of CD3 antibodies in human cervical cancer cells (HeLa) under normoxic and hypoxic conditions by the recombinant adenovirus according to an embodiment of the present invention. As shown in the figure, in human cervical cancer cells, CD3 antibodies are highly expressed under hypoxic conditions and lowly expressed under normoxic conditions.

[0128] Figure 8 The figure shows the killing effect of the recombinant adenovirus of the present invention on human cervical cancer cells (HeLa) under normoxic and hypoxic conditions. As shown in the figure, the recombinant adenovirus of the present invention can achieve high-efficiency killing of tumor cells under hypoxic conditions, while achieving low-level killing of tumor cells under normoxic conditions.

[0129] Figure 9 Schematic diagram of the construction of the pSC65-C9-6*loxp-P11-αCD3-TM recombinant vaccinia virus vector according to an embodiment of the present invention.

[0130] Figure 10 The figure shows the killing effect of a recombinant vaccinia virus carrying 6*loxp-P11-αCD3-TM according to an embodiment of the present invention on human ovarian cancer cells (SKOV3) under normoxic and hypoxic conditions. As shown, the recombinant vaccinia virus of the present invention can achieve high tumor cell killing under hypoxic conditions, while achieving low-level killing under normoxic conditions.

[0131] Figure 11 Schematic diagram of the construction of the pXW-EF1α-αCD3-TM recombinant lentiviral vector according to an embodiment of the present invention.

[0132] Figure 12 It was shown that the lentivirus carrying αCD3-TM according to the embodiment of the present invention can highly express CD3 antibodies on the cell membrane after infecting 293T cells.

[0133] Figure 13 It was shown that the lentivirus carrying αCD3-TM according to the embodiment of the present invention can mediate the efficient killing effect of T cells on SKOV3 after infecting human ovarian cancer cells (SKOV3).

[0134] Figure 14Schematic diagrams of the construction of two eukaryotic expression plasmid vectors according to the embodiments of the present invention, wherein A is a schematic diagram of the construction of pXW-CMV-αCD28-TM, and B is a schematic diagram of the construction of pXW-CMV-α4-1BB-TM.

[0135] Figure 15 It was shown that the plasmids carrying αCD28-TM and α4-1BB-TM according to the embodiment of the present invention or respectively combined with αCD3-TM were transfected into human ovarian cancer cells (SKOV3) and could mediate the efficient killing effect of T cells on SKOV3.

[0136] Figure 16 Schematic diagrams of the construction of two eukaryotic expression plasmid vectors according to the embodiments of the present invention, wherein A is a schematic diagram of the construction of pXW-CMV-αNKp46-TM, and B is a schematic diagram of the construction of pXW-CMV-αCD16-TM.

[0137] Figure 17 It was shown that the plasmid carrying αNKp46-TM and αCD16-TM according to the embodiment of the present invention can mediate the efficient killing effect of NK cells on SKOV3 after transfection into human ovarian cancer cells (SKOV3).

[0138] Figure 18 Schematic diagrams of the construction of two eukaryotic expression plasmid vectors according to the embodiments of the present invention, wherein A is a schematic diagram of the construction of pXW-CMV-αSIRPα-TM, and B is a schematic diagram of the construction of pXW-CMV-αSIRPα-Fc.

[0139] Figure 19 It was shown that the plasmid carrying αSIRPα-TM or αSIRPα-Fc according to the embodiment of the present invention can mediate the efficient killing effect of macrophages on SKOV3 after being transfected into human ovarian cancer cells (SKOV3). DETAILED DESCRIPTION

[0140] The following examples are only used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0141] All references cited herein are incorporated herein by reference in their entirety as if fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0142] Example

[0143] Example 1: Construction and functional verification of αCD3-TM recombinant adenovirus

[0144] The DNA sequences of artificially synthesized hypoxia-responsive 5HRE-cmd3-CD3TM-odd22-P2A-IL21-T2A-CXCL13 and 4HRE-miniP-CD3TM-odd22-P2A-IL21-T2A-CXCL13 are shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively.

[0145] The chimpanzee adenovirus AdC68XY-R1 plasmid was digested with PI-SceI (NEB, Catalog No. R0696S) at 37°C for 4 hours and then inactivated at 65°C for 20 minutes. The exogenous sequence was ligated to the linearized vector using seamless cloning (Novagen, Catalog No. c112-02) to construct the exogenous sequence behind the E1A gene of the pAdC68 vector. The resulting ligation product was transformed into Escherichia coli Stbl2 (VideoBio, Catalog No. DL1045) and grown overnight on ampicillin-containing plates.

[0146] Single colonies were randomly picked and sequenced by PCR using specific primers. After verifying that all sequences were correct, the endotoxin-free recombinant adenoviral plasmid A: pAdC68XY-R1-5HRE-cmd3-CD3TM-odd22-P2A-IL21-T2A-CXCL13 and recombinant adenoviral plasmid B: pAdC68XY-R1-4HRE-miniP-CD3TM-odd22-P2A-IL21-T2A-CXCL13 were extracted from the culture. The construction maps of the two plasmids are shown in Figure 2. Figure 1 The target genes carried by plasmid A and plasmid B are both under the control of tumor-specific promoter (as shown in SEQ ID NO: 1).

[0147] Example 2: Enzyme digestion and identification of recombinant adenovirus plasmid

[0148] The recombinant adenovirus plasmid obtained in Example 1 and sequenced correctly was digested and identified using three restriction endonucleases: Mfei (NEB, Catalog No. R3589S), BglII (NEB, Catalog No. R0144S), and XhoI (NEB, Catalog No. R0146S). 1% agarose gel electrophoresis was performed to verify that the molecular clone was complete and correct, as evidenced by DNA bands. The digestion patterns of the two plasmids are shown in Figure 2. Figure 2 shown.

[0149] Example 3: Packaging and amplification of recombinant oncolytic adenovirus

[0150] 3.1 Viral Packaging: 2.5 μg of each of the two recombinant adenoviral plasmids obtained in Example 1 were linearized using PacI (NEB, Catalog No. R0696S). Enzymatic digestion was performed at 37°C for 4 h, followed by inactivation at 65°C for 20 min. Following the instructions for the Lipofectamine 3000 transfection kit (Invitrogen, Catalog No. L3000015), the two linearized recombinant plasmids were transfected into HEK293 cells at a confluence of 60%-70% in 6-well plates. DNA / lipofectamine complexes were added for transfection. Six hours after transfection, the culture medium was replaced with DMEM supplemented with 5% FBS, 1% penicillin, and streptomycin. Cytopathic effects were observed daily under a microscope until numerous plaques appeared on the HEK293 cells after 5-10 days. P0 passage viruses were harvested and stored in a -80°C freezer. Figure 3 The development of cytopathic effects on day 7 is shown.

[0151] 3.2 Virus amplification: The collected P0 virus was repeatedly frozen and thawed three times. Centrifuged at 5000g for 5 minutes, and then an appropriate amount of the frozen and thawed virus supernatant was taken to infect HEK293 cells (Shanghai Xinwan Biotechnology Co., Ltd.) and serially passaged. The virus TCID was determined in HEK293 cells. 50 (50% infectious dose of tissue cells), calculated using the Reed-Muench method.

[0152] Example 4: Detection of protein expression in SK-OV3 cells infected with oncolytic adenovirus under normoxia and hypoxia

[0153] 4.1 SK-OV3-Luc cells (human ovarian cancer cells modified with firefly luciferase gene, Shanghai Xinwan Biotechnology Co., Ltd.) were plated in two 24-well plates with a cell number of approximately 1×10 5 After culturing for about 24 hours, when the cell confluence was approximately 80%-90%, two 24-well plates were inoculated with the two viruses obtained in Example 1 at infection doses of MOI 5 and 50, respectively, and infected at 37°C for 2 hours. The two plates were then placed in normoxic (21% O2) and hypoxic (1% O2) incubators, respectively. The cell supernatant was collected after 48 hours and centrifuged at 12,000 g for 5 minutes. The supernatant was transferred to new EP tubes and placed in a -80°C refrigerator for testing.

[0154] 4.2 ELISA kits for IL21 (BioLegend, Catalog No. 433804) and CXCL13 (R&D, Catalog No. DCX130) were used to detect the supernatants collected under normoxia and hypoxia, respectively. The specific detection methods were referred to the kit instructions.

[0155] Test results such as Figure 4 As shown. Figure 4It can be seen that under hypoxic conditions, the expressions of IL-21 and CXCL13 of the two recombinant adenovirus vectors A and B were higher than those under normoxic conditions.

[0156] The IL-21 detection results showed that for IL21 expression of virus A, when MOI = 5, the normoxic OD value was 0.28, and the hypoxic OD value was 1.48, which was 5.2 times the normoxic OD value; when MOI = 50, the normoxic OD value was 0.27, and the hypoxic OD value was 2.85, which was 10.5 times the normoxic OD value. For IL21 expression of virus B, when MOI = 5, the normoxic OD value was 0.23, and the hypoxic OD value was 2.96, which was 12.9 times the normoxic OD value; when MOI = 50, the normoxic OD value was 0.59, and the hypoxic OD value was 2.95, which was 5 times the normoxic OD value.

[0157] CXCL13 detection showed that for CXCL13 expression in virus A, at an MOI of 5, the normoxic OD value was 0.02, and the hypoxic OD value was 0.37, which was 18.5 times the normoxic OD value. At an MOI of 50, the normoxic OD value was 0.13, and the hypoxic OD value was 3.37, which was 25.9 times the normoxic OD value. For CXCL13 expression in virus B, at an MOI of 5, the normoxic OD value was 0.03, and the hypoxic OD value was 3.06, which was 102 times the normoxic OD value. At an MOI of 50, the normoxic OD value was 1.03, and the hypoxic OD value was 3.4, which was 3.3 times the normoxic OD value.

[0158] Example 5: Detection of CD3 Antibodies on the Membrane of SKOV3 Cells Infected with Oncolytic Adenovirus under Normoxia and Hypoxia

[0159] SKOV3 cells were plated in two 96-well black plates, with a cell number of approximately 1×10 4 After approximately 24 hours of culture, when the cell confluence reached approximately 80%-90%, two black plates were inoculated with the two viruses obtained in Example 1 at an MOI of 50 and infected at 37°C for 2 hours. The two plates were then placed in a normoxic (21% O2) and anoxic (1% O2) incubator, respectively. 16-24 hours after viral infection, CD3 antibody flow cytometry was performed.

[0160] The results are as follows Figure 5 As shown. Figure 5As can be seen, both recombinant adenovirus vectors A and B expressed higher CD3 antibodies under hypoxic conditions than under normoxic conditions. Under hypoxic conditions, when virus A infected SKOV3 cells, 93.7% of target cells expressed CD3 antibodies on their surfaces; when virus B infected SKOV3 cells, 72.4% of target cells expressed CD3 antibodies on their surfaces. Under normoxic conditions, no CD3 antibody expression was detected in SKOV3 cells infected with either virus A or virus B.

[0161] Example 6: Detection of oncolytic adenovirus infection and killing of SKOV3 under normoxia and hypoxia

[0162] 6.1 Killing Assay: Activated untransduced T cells (UTD) were added to the cell plates containing SKOV3 cells infected with adenoviruses (AdC68XY-R1, adenovirus A, and adenovirus B) for 16-24 hours as described in Example 5. The effector cell to target cell ratios (E:T) were 4:1, 2:1, and 1:1, respectively. Control wells containing AdC68XY-R1 virus, UTD cells, and control wells without UTD cells were also set up. The cell plates were then incubated overnight at 37°C in a 21% O2 / 1% O2 incubator for approximately 18 hours for killing assays to be performed.

[0163] 6.2 Killing Assay: Remove the supernatant from the cell plate and add 50 μL of cell lysis buffer (Promega, Cat. No. E1531) to each well. Incubate at room temperature with shaking for 30 min. Add 30 μL of luciferase substrate (Promega, Cat. No. E151A) to each well. Detection was performed using a GloMax Navigator Microplate Luminometer (Promega, Steady-Glo protocol).

[0164] The formula for calculating the cell killing rate is as follows:

[0165] Cell killing rate (%) = [(luciferase activity value of virus + target cells without T cells added) - (luciferase activity value of virus + target cells + T cells group)] / (luciferase activity value of virus + target cells without T cells added) × 100%.

[0166] The results are as follows Figure 6 As shown, from Figure 6It can be seen that when the effector-target ratio (E:T) = 4:1, the killing rate (i.e., cytotoxicity) of virus A against SKOV3 target cells under hypoxia was approximately 93.3%, while the killing rate of virus A against SKOV3 target cells under normoxia was approximately 20%. When the effector-target ratio (E:T) = 2:1, the killing rate of virus A against SKOV3 target cells under hypoxia was approximately 82%, while the killing rate of virus A against SKOV3 target cells under normoxia was approximately 3.5%. When the effector-target ratio (E:T) = 1:1, the killing rate of virus A against SKOV3 target cells under hypoxia was approximately 58%, while the killing rate of virus A against SKOV3 target cells under normoxia was approximately 7%. The results show that at the three effector-target ratios, virus A has significantly differentiated cytotoxic activity against SKOV3 cells under both hypoxia and normoxia.

[0167] from Figure 6 It can be seen that when the effector-target ratio (E:T) = 4:1, the killing rate of virus B against SKOV3 target cells under hypoxia was approximately 90%, while the killing rate under normoxia was approximately 16%. When the effector-target ratio (E:T) = 2:1, the killing rate of virus B against SKOV3 target cells under hypoxia was approximately 73%, while the killing rate under normoxia was approximately 5.5%. When the effector-target ratio (E:T) = 1:1, the killing rate of virus B against SKOV3 target cells under hypoxia was approximately 50%, while the killing rate under normoxia was approximately 1.5%. The results show that at all three effector-target ratios, virus B exhibits significantly differentiated cytotoxic activity against SKOV3 cells under hypoxia and normoxia.

[0168] Example 7: Detection of CD3 Antibodies on HeLa Cell Membranes Infected by Oncolytic Adenovirus under Normoxia and Hypoxia

[0169] HeLa cells (Shanghai Xinwan Biotechnology Co., Ltd.) were plated in two 96-well black plates, with a cell number of approximately 1×10 4 After approximately 24 hours of culture, when the cell confluence reached approximately 80%-90%, two black plates were inoculated with the two viruses obtained in Example 1 at an MOI of 50 and infected at 37°C for 2 hours. The two plates were then placed in a normoxic (21% O2) and anoxic (1% O2) incubator, respectively. 16-24 hours after viral infection, CD3 antibody flow cytometry was performed.

[0170] The results are as follows Figure 7 As shown. Figure 7As can be seen, both recombinant adenovirus vectors A and B expressed higher CD3 antibodies under hypoxic conditions than under normoxic conditions. Under hypoxic conditions, when virus A infected HeLa cells, 76.5% of target cells expressed CD3 antibodies on their surfaces; when virus B infected HeLa cells, 46.7% of target cells expressed CD3 antibodies on their surfaces. Under normoxic conditions, no CD3 antibody expression was detected in HeLa cells infected with either virus A or virus B.

[0171] Example 8: Detection of HeLa cell killing by oncolytic adenovirus infection under normoxia and hypoxia

[0172] Killing assay: Activated T cells were added to the cell plates containing HeLa cells infected with adenoviruses (AdC68XY-R1, adenovirus A, and adenovirus B) for 16-24 hours as described in Example 7. The effector cell to target cell ratio (E:T) was 4:1, 2:1, and 1:1, respectively. Control wells containing AdC68XY-R1 virus, UTD cells, and control wells without UTD cells were also set up. The cell plates were then incubated overnight at 37°C in a 21% O2 and 1% O2 incubator for approximately 18 hours. Killing assays were then performed using the same method as in Example 6.

[0173] The results are as follows Figure 8 shown.

[0174] from Figure 8 It can be seen that when the effector-target ratio E:T = 4:1, virus A had a killing rate of approximately 94% against HeLa target cells under hypoxia and approximately 14.2% under normoxia. When the effector-target ratio E:T = 2:1, virus A had a killing rate of approximately 80% against HeLa target cells under hypoxia and approximately 5.7% under normoxia. When the effector-target ratio E:T = 1:1, virus A had a killing rate of approximately 60% against HeLa target cells under hypoxia and approximately 1.8% under normoxia. The results show that virus A has significantly differentiated killing activity against HeLa cells under both hypoxia and normoxia conditions at the three effector-target ratios.

[0175] from Figure 8It can be seen that when the effector-target ratio E:T = 4:1, the killing rate of virus B against HeLa target cells under hypoxia is approximately 77%, and the killing rate of virus B against HeLa target cells under normoxia is approximately 16%. When the effector-target ratio E:T = 2:1, the killing rate of virus B against HeLa target cells under hypoxia is approximately 52%, and the killing rate of virus B against HeLa target cells under normoxia is approximately 2%. When the effector-target ratio E:T = 1:1, the killing rate of virus B against HeLa target cells under hypoxia is approximately 31%, and the killing rate of virus B against HeLa target cells under normoxia is approximately 1%. The results show that at all three effector-target ratios, virus B has significantly differentiated killing activity against HeLa cells under hypoxia and normoxia.

[0176] Example 9: Construction of recombinant vaccinia virus containing αCD3-TM

[0177] 9.1 Construction of pSC65 vector carrying αCD3-TM target gene

[0178] The hypoxia-responsive 6*loxp-P11-αCD3-TM DNA sequence was artificially synthesized, as shown in SEQ ID NO: 30, and cloned into the shuttle plasmid pSC65 at the vaccinia virus C9 gene site. The construction map of the pSC65-C9-6*loxp-P11-αCD3-TM plasmid is shown in FIG. Figure 9 shown.

[0179] 9.2 Construction of recombinant vaccinia virus 6*loxp-P11-αCD3-TM

[0180] 9.2.1 Cell preparation: 143TK cells were plated in 6-well plates, with approximately 1×10 cells per well. 6 Incubate the 6-well plate for about 24 hours. When the cells adhere to the wall and cover the entire bottom surface, proceed to the next step.

[0181] 9.2.2 Vaccinia Virus Incubation: Infect cells with 0.0125 / 3 PFU (Plaque Forming Units, viral titer) / cell of the Tiantan strain of vaccinia virus, which is TK-deficient and overexpresses the hypoxia-responsive protein dNLS-CRE-VP64 at the C16L-VGF1 locus. Incubate in a 37°C incubator for 1 h, remove cells, aspirate the supernatant, rinse once with 1 mL of PBS, and then add 1 mL of complete DMEM medium (DMEM medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin antibiotics (PS)). For specific procedures, refer to Chinese invention patent application No. 202211562658.2, the contents of which are incorporated herein by reference.

[0182] 9.2.3 Plasmid Transfection: Transfect 143TK- cells with the shuttle plasmid pSC65-C9-αCD3-TM. Incubate at 37°C for approximately 48 hours, depending on the severity of the cytopathic effect.

[0183] 9.2.4 Prepare 2× DMEM maintenance medium (containing 2% PS and 4% FBS) for virus plating, add 2% preheated low-melting-point agarose, and then add X-gal (final concentration of 200 μg / mL).

[0184] 9.2.5 Aspirate the supernatant from the 6-well plate and add 1 mL of the plating mixture to each well. Carefully place the plate in a 4°C refrigerator to promote solidification. Once the low-melting-point agarose has solidified, transfer the plate to a 37°C incubator and incubate upside down until clear blue fluorescent plaques appear.

[0185] 9.2.6 Pick the blue fluorescent virus plaques (recombinant vaccinia virus rTV-6*loxp-P11-αCD3-TM) and add them to 500 μL of complete DMEM medium. Freeze and thaw repeatedly at -80°C for at least three times to release as much virus as possible.

[0186] 9.2.7 143TK cells were plated in 6-well plates, with approximately 1×10 cells per well. 6 Culture for about 24 hours until the cells adhere to the wall and cover the entire bottom surface.

[0187] 9.2.8 Repeatedly blow the virus solution in the EP tube to disperse it completely.

[0188] 9.2.9 Replace the complete medium with maintenance medium, then add the virus solution containing blue fluorescence and incubate in a 37°C incubator for 3-4 hours.

[0189] 9.2.10 Add screening pressure: BrdU working concentration is 50 μg / mL. Incubate in a 37°C incubator for approximately 48 hours. Spread the plaques based on the appearance of viral plaques. This purification process should be repeated at least five times.

[0190] 9.2.11 Perform small-scale amplification of recombinant vaccinia virus. Plate 143TK- cells in a six-well plate, with 1×10 cells per well. 6 When used, the cells should occupy approximately 100% of the bottom area of ​​the well plate.

[0191] 9.2.12 Before inoculation, replace the culture medium in the wells with 2 mL of maintenance medium. Repeatedly pipette the purified blue fluorescent virus solution until dispersed. Add approximately 100 μL of the virus solution to each well. Incubate in a 37°C incubator for approximately 48 hours. Collect samples based on the formation of viral plaques.

[0192] 9.2.13 Sample Collection: Carefully aspirate 1 mL of the culture supernatant from the well. Use the remaining 1 mL of culture medium to thoroughly blow off the cells and collect them in an EP tube. This can be used for subsequent genome extraction and as a virus seed amplification. 9.36 Amplification and Purification of Recombinant Vaccinia Virus Containing Loxp-P11-αCD3-TM

[0193] 9.3.1 VERO cell plating: Take 10 cm culture dishes and plate approximately 5 × 10 6 cells to ensure that the cell density reaches 100% when the vaccinia virus is inoculated the next day.

[0194] 9.3.2 Prior to virus inoculation, replace the complete culture medium with 8 mL of maintenance medium (DMEM + 2% FBS + 1% PS). Inoculate the cells in the maintenance medium with virus at an MOI of approximately 0.02 (MOI = viral PFU / cell number). Continue incubating at 37°C in a 5% CO2 incubator for approximately 48 hours. Collect samples based on the formation of viral plaques.

[0195] 9.3.3 Collecting Vaccinia Virus: Discard 8 mL of culture medium from the culture dish, use 2 mL of maintenance medium to blow off the remaining cells, and collect the cells in a 15 mL centrifuge tube.

[0196] 9.3.4 After 24 hours of frozen storage, freeze-thaw the harvested virus solution twice more and perform density gradient centrifugation using a 36% sucrose solution at 16,000g for 90 minutes at 4°C. Carefully discard the supernatant and dissolve the virus pellet in the centrifuge tube with PBS buffer. Aliquot and store at -80°C until the virus titer is determined.

[0197] 9.4 Titer determination of 6*loxp-P11-αCD3-TM recombinant vaccinia virus

[0198] 9.4.1 Preparation of 143TK cells: 143TK cells were plated in a 24-well plate, with approximately 2 × 10 cells per well. 5 When using, the cell density should reach 100% of the bottom area of ​​24-well plates.

[0199] 9.4.2 Virus dilution: Dilute the vaccinia virus solution with maintenance medium, starting from 1:100, and make 10-fold dilutions to a final volume of 1100 μL.

[0200] 9.4.3 Discard the complete medium in the 24-well plate, take 500 μL of the diluted virus solution and add it to the wells, making two replicates. Incubate in a 37°C 5% CO2 incubator for about 48 hours. The plaque formation time is determined based on the virus plaque formation.

[0201] 9.4.4 Plating Method: Prepare 8 mL of plating medium containing 2× DMEM medium + 4% FBS + 2% PS and 8 mL of low-melting-point agarose melted in a boiling water bath and placed in a 37°C water bath. Mix the two and then add X-gal to the mixture to a final concentration of 200 μg / mL. Set aside.

[0202] 9.4.5 Aspirate the supernatant from the 24-well plate. Immediately add the plating mixture from 9.4.4 to the 24-well plate, 500 μL per well. Then carefully place the 24-well plate in a 4°C refrigerator to promote solidification. After the low-melting-point agarose solidifies, transfer the 24-well plate to a 37°C incubator and incubate inverted until clear blue fluorescent plaques appear.

[0203] 9.4.6 Virus plaque counting: First, observe whether the number of virus plaques decreases tenfold. Then, count the number of single-digit blue fluorescent spots in the two replicate wells inoculated with the virus. The sum of the blue fluorescent plaque counts in the two wells multiplied by the reciprocal value of the dilution corresponding to the well is the virus titer in 1 mL.

[0204] Example 10: Effect of 6*loxp-P11-αCD3-TM recombinant vaccinia virus on ovarian cancer under normoxic and hypoxic conditions Cellular effects

[0205] 10.1 Target cell plating: SKOV3 (Shanghai Xinwan Biotechnology Co., Ltd.) is a luciferase-expressing cell line. 4 The cells were plated at a density of 1000 cells / well in a 96-well plate and cultured at 37°C overnight until they adhered to the wall.

[0206] 10.2 HIF-1α Inhibitor Treatment: After 24 hours, the 96-well plates were evenly divided into a normoxic culture group and a HIF-1α inhibitor molidustat (MCE, Cat. No. 1154026-82-6) treatment group. Molidustat is a hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor. By stabilizing HIF and inhibiting its degradation, it can simulate a hypoxic microenvironment. The 96-well plates were incubated at 37°C overnight.

[0207] 10.3 Virus infection: 24 hours later, cells were infected with the 6*loxp-P11-αCD3-TM recombinant vaccinia virus at MOIs of 0.3, 1, and 3, and incubated at 37°C overnight for infection.

[0208] 10.4 Killing experiment: After 24 hours, activated T cells were added again with an effector-target ratio of 1:1. T cell and no T cell control groups were also set up.

[0209] 10.5 Killing Assay: Perform killing assays approximately 24 hours after incubation. Remove the supernatant from the wells of the 96-well plate and add 50 μL of cell lysis buffer (Promega, Cat. No. E1531) to each well. Incubate at room temperature with shaking for 30 minutes. Add 30 μL of luciferase substrate (Promega, Cat. No. E151A) to each well. Detection is performed using a GloMax Navigator Microplate Luminometer (Promega, Steady-Glo protocol).

[0210] The formula for calculating the cell killing rate is as follows:

[0211] Cell killing rate (%) = [(luciferase activity value of target cell + T cell group) - (luciferase activity value of infection well of experimental group + target cell + T cell group)] / (luciferase activity value of target cell + T cell group) × 100%

[0212] The results are as follows Figure 10 As shown. Figure 10 As can be seen, the hypoxia-responsive 6*loxp-P11-αCD3-TM recombinant vaccinia virus not only exhibits strong cytotoxicity but also exhibits hypoxia responsiveness, with low expression under normoxic conditions and high expression under hypoxia. Under normoxic conditions, at an MOI of 0.3, the recombinant vaccinia virus had a target cell killing rate of only 5%; at an MOI of 1, the killing rate was less than 20%; and at an MOI of 3, the killing rate was less than 30%. Under hypoxia, however, at an MOI of 0.3, the killing rate of the recombinant vaccinia virus reached 38%; at an MOI of 1, the killing rate reached 40%; and at an MOI of 3, the killing rate exceeded 40%. These results demonstrate that the recombinant vaccinia virus exhibits distinctly differentiated cytotoxic activity against tumor cells under both hypoxic and normoxic conditions.

[0213] Example 11: Chronic Diseases poison Construction of expression plasmid

[0214] The exogenous gene αCD3-CD8 TM was synthesized by Shanghai Qingke Biotechnology Co., Ltd., and its nucleic acid sequence is shown in SEQ ID NO: 28. The exogenous gene αCD3-CD8 TM was cloned into the blank lentiviral expression plasmid pXW-EF1α-MCS to obtain the pXW-EF1α-αCD3-TM recombinant lentiviral expression plasmid. The plasmid map is shown in Figure 11 shown.

[0215] Example 12: Packaging, concentration and titer determination of lentivirus

[0216] 12.1 Lentivirus Packaging

[0217] HEK293T cell treatment: 24 h before transfection, HEK293T cells in the logarithmic growth phase were collected and seeded into 10 cm cell culture dishes (6-8×10 6 Cells were grown in 10 mL of complete DMEM medium in a 37°C, 5% CO2 incubator for 18-24 hours. Plasmid transfection was performed when the cell density reached 70%-90%.

[0218] HEK293T cell transfection: Add 1 mL of basal DMEM medium to a 15 mL centrifuge tube and prepare a transfection mix at a mass ratio of lentiviral expression plasmid: packaging plasmid: envelope plasmid = 1:3:1, for a total plasmid volume of 15 μg / dish. Add 30 μL of TurboFect transfection reagent at a ratio of plasmid (μg) to transfection reagent (μL) = 1:2. Incubate at room temperature for 15-20 minutes, then add to a dish containing HEK293T cells. Incubate in a 37°C, 5% CO2 incubator for 48 hours, then collect the viral supernatant. Centrifuge at 1000 × g at 4°C for 10 minutes, discard the pellet, and collect the viral supernatant.

[0219] 12.2 Concentration of Lentivirus

[0220] Filter the collected viral supernatant through a 0.45 μm filter and add 1 / 3 the volume of Lenti-X Lentivirus Concentration Reagent (Clontech, Cat. No. 631232). Mix by inversion several times and incubate overnight at 4°C. Centrifuge at 2000 × g for 45 minutes at 4°C. A white precipitate will form at the bottom of the tube, representing the concentrated viral particles. Carefully discard the supernatant and resuspend the white precipitate in RPMI-1640 medium (1 / 50–1 / 100 the volume of the original viral supernatant). Aliquot and freeze at -80°C until needed.

[0221] 12.3 Lentiviral Titer Determination

[0222] Jurkat T cells were cultured at a rate of 1×10 5 Inoculate cells / well in a 96-well U-bottom plate and dilute the collected lentiviral concentrate in 10-fold increments. Add 100 μL of the viral dilution to the corresponding well. Add the infection-enhancing reagent protamine sulfate and adjust the concentration to 10 μg / mL. Centrifuge at 1000 × g at 32°C for 90 minutes. After overnight incubation, replace the 96-well U-bottom plate with fresh RPMI1640 complete medium and continue incubation for 48 hours. Detect the proportion of fluorescent-positive cells by flow cytometry.

[0223] The virus titer was calculated using the following formula:

[0224] Virus titer (TU / mL) = [1 × 105 × the ratio of fluorescence-positive cells / 100] × 1000 × the corresponding dilution factor.

[0225] Example 13: Detection of αCD3-TM expression on cell membrane

[0226] The lentiviral expression plasmid of Example 11 and the lentiviral vector preparation method of Example 12 were used to carry EF1α-αCD3-TM lentivirus. The lentivirus was added to a 1×10 5 To a 24-well flat-bottom plate containing 293T cells, add the infection-promoting reagent protamine sulfate and adjust its working concentration to 10 μg / mL. Infection is then centrifuged at 1000 × g at 32°C for 90 minutes. Continue incubation for 24 hours. Afterward, the cells are harvested and eluted once with FACS buffer. Add 2 μg / mL of the flow cytometry antibody PE-anti-DYKDDDDK (Biolegend, Cat. No. 637310) and incubate at room temperature in the dark for 20 minutes. After incubation, the cells are eluted twice with FACS buffer, resuspended and mixed thoroughly in 300 μL of FACS buffer, and then analyzed for αCD3-TM expression using a flow cytometer.

[0227] The results are as follows Figure 12 Flow cytometry results showed that compared with the blank control group, αCD3-TM was highly expressed on the 293T cell membrane, with a positive rate of 90.5%.

[0228] Example 14: Killing of tumor cells by αCD3-TM carried by lentiviral vector

[0229] Tumor cell killing efficiency was assessed by a luciferase-based cytotoxicity assay. The details are as follows:

[0230] First, 1×10 4 SKOV3-Luc (human ovarian cancer cells modified with the firefly luciferase gene) were inoculated on a 96-well flat-bottom black plate with 100 μL of culture medium per well. The 96-well flat-bottom black plate was placed in a cell culture incubator at 37°C and 5% CO2 for 18 hours. The next day, the target cells were divided into two groups, the experimental group and the control group. The experimental group added lentivirus carrying αCD3-TM and the infection-promoting reagent protamine sulfate and adjusted its working concentration to 10 μg / mL, and centrifuged at 1000×g at 32°C for 90 minutes; the control group was not treated. After culturing for 24 hours, activated T cells were added to the wells containing target cells according to the ratio of effector cells to target cells (E:T) of 4:1, 2:1 and 1:1, respectively, and cultured for another 20 hours. After the co-culture is completed, use The luciferase activity of target cells was detected by a microplate luminescence detector.

[0231] The formula for calculating the cell killing rate is as follows:

[0232] Killing rate of cells in the lentivirus-infected group (%) = (luciferase activity value of the lentivirus-infected group without T cells added - luciferase activity value of the experimental group) / luciferase activity value of the lentivirus-infected group without T cells added × 100%

[0233] Cell killing rate of the non-lentivirus-infected group (%) = (luciferase activity value of the non-lentivirus-infected group without T cells added - luciferase activity value of the experimental group) / luciferase activity value of the non-lentivirus-infected group without T cells added × 100%

[0234] The results are as follows Figure 13 As shown. Figure 13 As can be seen, SKOV3 tumor cells infected with the αCD3-TM lentivirus were efficiently killed by T cells. At an effector-to-target ratio (E:T) of 1:1, the killing rate was 92.5%. Further decreasing the E:T ratio maintained high killing efficiency, achieving a high killing rate of 75% at an E:T ratio of 1:4.

[0235] Example 15: αCD28-TM and α4-1BB-TM carried by eukaryotic expression plasmid vector or combined with αCD3- TM activates T cells to mediate tumor cell killing

[0236] Tumor cell killing efficiency was assessed by a luciferase-based cytotoxicity assay. The details are as follows:

[0237] First, 1×10 4 SKOV3-Luc (human ovarian cancer cells modified with the firefly luciferase gene) were seeded onto a 96-well flat-bottomed black plate with 100 μL of culture medium per well. The 96-well flat-bottomed black plate was placed in a 37°C, 5% CO2 cell culture incubator for 18 hours. The next day, the target cells were divided into two groups, the experimental group and the control group. The experimental group was transfected with the αCD28-TM plasmid (see Figure 2). Figure 14 A), α4-1BB-TM plasmid (map as shown Figure 14B), or a plasmid of αCD28-TM combined with αCD3-TM, or a plasmid of α4-1BB-TM combined with αCD3-TM. The antibody sequence of CD28 refers to patent US20200299388A1, which is included herein by reference. For example, the encoding nucleic acid of αCD28 is shown in SEQ ID NO: 33, and the amino acid sequence of αCD28 is shown in SEQ ID NO: 34. The antibody sequence of 4-1BB refers to the monoclonal antibody drug Utomilumab, for example, the encoding nucleic acid of α4-1BB is shown in SEQ ID NO: 31, and the amino acid sequence of α4-1BB is shown in SEQ ID NO: 32.

[0238] After 24 hours of culture, activated T cells were added to the wells containing target cells at a ratio of 1:1 between effector cells and target cells (E:T). The control group was not treated with T cells and cultured for another 20 hours. The luciferase activity of target cells was detected by a microplate luminescence detector.

[0239] The formula for calculating the cell killing rate is as follows:

[0240] Plasmid-transfected cell killing rate (%) = (luciferase activity value of the plasmid-transfected group without T cells - luciferase activity value of the experimental group) / luciferase activity value of the plasmid-transfected group without T cells × 100%

[0241] The results are as follows Figure 15 As shown. Figure 15 As can be seen, SKOV3 tumor cells transfected with the αCD28-TM plasmid, the α4-1BB-TM plasmid, or a combination of the αCD28-TM and αCD3-TM plasmids, or the α4-1BB-TM and αCD3-TM plasmids, were efficiently killed by T cells. At an effector-target ratio (E:T) of 1:1, transfection with the αCD28-TM plasmid resulted in a killing rate of nearly 70%, while the combination of the αCD28-TM and αCD3-TM plasmids increased this to 84%. Transfection with the α4-1BB-TM plasmid resulted in a killing rate of 54%, while the combination of the α4-1BB-TM and αCD3-TM plasmids increased this to 86%.

[0242] Example 16: Eukaryotic expression plasmid vector carrying α NKp46-TM or α CD16-TM activated NK cells mediate Tumor cell killing

[0243] Tumor cell killing efficiency was assessed by a luciferase-based cytotoxicity assay. The details are as follows:

[0244] First, 1×10 4 SKOV3-Luc (human ovarian cancer cells modified with the firefly luciferase gene) were seeded onto a 96-well flat-bottomed black plate with 100 μL of culture medium per well. The 96-well flat-bottomed black plate was placed in a 37°C, 5% CO2 cell culture incubator for 18 hours. The next day, the target cells were divided into two groups, the experimental group and the control group. The experimental group was transfected with αNKp46-TM (see Figure 2). Figure 16 A) or αCD16-TM plasmid (map as shown in Figure 16 For antibody sequences related to NKp46, reference is made to patent CA3216053A1, which is incorporated herein by reference. For example, the nucleic acid encoding NKp46 is shown in SEQ ID NO: 37, and the amino acid sequence of NKp46 is shown in SEQ ID NO: 38. For antibody sequences related to CD16, reference is made to patent CN111465618A, which is incorporated herein by reference. For example, the nucleic acid encoding CD16 is shown in SEQ ID NO: 35, and the amino acid sequence of CD16 is shown in SEQ ID NO: 36.

[0245] After 24 hours of culture, activated NK cells were added to the wells containing target cells at a ratio of 1:1 between effector cells and target cells (E:T). The control group was not treated with NK cells and cultured for another 20 hours. The luciferase activity of target cells was detected by a microplate luminescence detector.

[0246] The formula for calculating the cell killing rate is as follows:

[0247] Plasmid transfection cell killing rate (%) = (luciferase activity value of the plasmid transfection group without NK cells - luciferase activity value of the experimental group) / luciferase activity value of the plasmid transfection group without NK cells × 100%

[0248] The results are as follows Figure 17 As shown. Figure 17 As can be seen, SKOV3 tumor cells transfected with plasmids carrying either αNKp46-TM or αCD16-TM were efficiently killed by NK cells. At an effector-target ratio (E:T) of 1:1, the killing rate mediated by the αNKp46-TM plasmid reached 77%, and the killing rate mediated by the αCD16-TM plasmid reached 82%.

[0249] Example 17: Eukaryotic expression plasmid vector carrying α SIRP α -TM or α SIRP α -Fc activated macrophages Killing tumor cells

[0250] Tumor cell killing efficiency was assessed by a luciferase-based cytotoxicity assay. The details are as follows:

[0251] First, 1×10 4 SKOV3-Luc (human ovarian cancer cells modified with firefly luciferase gene) were seeded on a 96-well flat-bottom black plate with 100 μL culture medium per well. The 96-well flat-bottom black plate was placed in a 37°C, 5% CO2 cell culture incubator for 18 hours. The next day, the target cells were divided into two groups, the experimental group and the control group. The experimental group was transfected with αSIRPα-TM (Figure 1). Figure 18 A) or αSIRPα-Fc plasmid (map as shown in Figure 18 (B) The antibody sequences of SIRPα are shown in JP6606505B2, which is incorporated herein by reference. For example, the nucleic acid encoding SIRPα-TM is shown in SEQ ID NO: 39, the amino acid sequence of SIRPα-TM is shown in SEQ ID NO: 40, the nucleic acid encoding SIRPα-Fc is shown in SEQ ID NO: 41, and the amino acid sequence of SIRPα-Fc is shown in SEQ ID NO: 42.

[0252] After 24 hours of culture, activated macrophages were added to the wells containing target cells at a ratio of 1:1 between effector cells and target cells (E:T). The control group was not treated with macrophages and cultured for another 20 hours. The luciferase activity of target cells was detected by a microplate luminescence detector.

[0253] The formula for calculating the cell killing rate is as follows:

[0254] Plasmid-transfected cell killing rate (%) = (luciferase activity value of the group transfected with plasmid without macrophages - luciferase activity value of the experimental group) / luciferase activity value of the group transfected with plasmid without macrophages × 100%

[0255] The results are as follows Figure 19 As shown. Figure 19 As can be seen, SKOV3 tumor cells transfected with plasmids carrying αSIRPα-TM or αSIRPα-Fc were also efficiently killed by macrophages. At an effector-target ratio (E:T) of 1:1, the killing rate mediated by the αSIRPα-TM plasmid reached 60%, and the killing rate mediated by the αSIRPα-Fc plasmid was 65%.

[0256] The above embodiments are exemplary and should not be construed as limiting the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A fusion protein for activating immune cells to kill tumor cells, comprising an extracellular domain, a hinge region, a transmembrane domain, and an intracellular domain; wherein: The extracellular domain is a polypeptide or an active fragment thereof that specifically binds to immune cell surface antigens, and activates the killing function of immune cells by specifically binding to the immune cell surface antigens; the transmembrane domain is a polypeptide that anchors the fusion protein to the cell membrane of tumor cells.

2. The fusion protein according to claim 1, wherein The immune cells are selected from one or more of T cells, NK cells, NKT cells, iNKT cells, mucosal-associated invariant T cells, γδT cells, K cells, monocytes, macrophages and neutrophils; preferably, the immune cell surface antigens are selected from one or more of CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD45, CD49, CD56, CD62L, CD94, CD122, CD16, CD32, CD64, CD68, CD80, CD86, CD127, CD137, CD152, CD197, CD369, α4-1BB, NKp46 and SIRPα; More preferably, the immune cells are selected from one or more of T cells, NK cells and macrophages; further preferably, the immune cell surface antigens are selected from one or more of CD3, CD28, α4-1BB, NKp46, CD16 and SIRPα.

3. The fusion protein according to claim 2, wherein The extracellular domain comprises one or more selected from the following: The amino acid sequence shown in SEQ ID NO: 8 or an active fragment thereof; The amino acid sequence shown in SEQ ID NO: 32 or an active fragment thereof The amino acid sequence shown in SEQ ID NO: 34 or an active fragment thereof; The amino acid sequence shown in SEQ ID NO: 36 or an active fragment thereof; The amino acid sequence shown in SEQ ID NO: 38 or an active fragment thereof; The amino acid sequence shown in SEQ ID NO: 40 or an active fragment thereof; and The amino acid sequence shown in SEQ ID NO: 42 or an active fragment thereof.

4. The fusion protein according to claim 1, wherein The hinge region is selected from one or more of the following: (1) the hinge region of antibody IgG4 or a mutant thereof; (2) the hinge region of antibody IgG4 or its mutant, and CH2 region; (3) the hinge region, CH2 region, and CH3 region of an IgG4 antibody or a mutant thereof; (4) the hinge region of antibody IgG1 or its mutant; (5) the hinge region of antibody IgG1 or its mutant, and CH2 region; (6) the hinge region, CH2 region, and CH3 region of an IgG1 antibody or a mutant thereof; (7) Hinge region of immunoglobulin Fc receptor: FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcαR (CD89), FcεRI or FcεRII (CD23); (8) co-stimulatory molecule CD28 hinge region, CD137 hinge region, CD8 hinge region, CD4 hinge region, PD-1 hinge region or CTLA-4 hinge region; and (9) any combination of the above different hinge regions; Preferably, the hinge region is the CD8 hinge region; More preferably, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 10 or an active fragment thereof.

5. The fusion protein according to claim 1, wherein The transmembrane domain is selected from one or more of the following: the transmembrane domain of the CD3ζ chain of the T cell receptor complex, the CD28 transmembrane domain, the immunoglobulin Fc receptor transmembrane domain, the CD4 transmembrane domain, the CD8 transmembrane domain, the CD16 transmembrane domain, the CD137 transmembrane domain, the CTLA-4 transmembrane domain, the PD-1 transmembrane domain, the LAG-3 transmembrane domain, the VISTA transmembrane domain, and combinations thereof; Preferably, the transmembrane domain is the CD8 transmembrane domain; More preferably, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 12 or an active fragment thereof.

6. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 5 and an immune function molecule, wherein the immune function molecule is selected from one or more of a cytokine, a chemokine, and an immune checkpoint blocking antibody; Preferably, the cytokine is selected from one or more of GM-CSF, IL-2, IL-3, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-24, IL-33, IL-35, IL-37, IFN-α, IFN-β, IFN-γ and MALP-2; Preferably, the chemokine is selected from one or more of CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL4, CCL19, CCL20, CCL21 and CX3CL1; Preferably, the immune checkpoint blocking antibody is selected from one or more of CTLA-4 blocking antibody, CD47 blocking antibody, SIRPα blocking antibody, PD-1 blocking antibody, PD-L1 blocking antibody, LAG-3 blocking antibody and Tim-3 blocking antibody; Optionally, the composition further comprises an oxygen-sensitive sequence that degrades under normoxic conditions but is stable under hypoxic conditions; preferably, the oxygen-sensitive sequence comprises the amino acid sequence shown in SEQ ID NO: 15 or an active fragment thereof; Optionally, the fusion protein, the immune function molecule and the oxygen-sensitive sequence are respectively contained in the pharmaceutical composition as separate components; or, any two or more of the fusion protein, the immune function molecule and the oxygen-sensitive sequence are connected together via a linker. A polynucleotide encoding the fusion protein according to any one of claims 1 to 5.

8. A recombinant viral vector comprising the polynucleotide according to claim 7; Preferably, the viral backbone of the recombinant viral vector is derived from recombinant adenovirus, recombinant vaccinia virus, recombinant herpes simplex virus, adeno-associated virus, varicella-zoster virus, respiratory syncytial virus, Semlicke Forest virus, Epstein-Barr virus, cytomegalovirus, human herpes virus type 6, smallpox virus, vaccinia virus, molluscum contagiosum virus, canker sore virus, reovirus, rotavirus, enterovirus, Seneca virus, poliovirus, coxsackievirus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus, togavirus, alphavirus, Semlicke 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, Junin virus, Machupo virus, Hantavirus, Rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, simian virus 5, measles virus, vesicular stomatitis virus, rabies virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, lentivirus, simian immunodeficiency virus, human immunodeficiency virus type 1, 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 papillomavirus, or polyomavirus; More preferably, the viral backbone of the recombinant viral vector is an intracellular mature virus, an intracellular packaged virus, a cell-associated packaged virus, or an extracellular packaged virus; Optionally, the recombinant viral vector further comprises a polynucleotide expressing an immune function molecule; wherein, The immune function molecules are selected from one or more of cytokines, chemokines and immune checkpoint blocking antibodies; Preferably, the cytokine is selected from one or more of GM-CSF, IL-2, IL-3, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-24, IL-33, IL-35, IL-37, IFN-α, IFN-β, IFN-γ and MALP-2; Preferably, the chemokine is selected from one or more of CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL4, CCL19, CCL20, CCL21 and CX3CL1; Preferably, the immune checkpoint blocking antibody is selected from one or more of CTLA-4 blocking antibody, CD47 blocking antibody, SIRPα blocking antibody, PD-1 blocking antibody, PD-L1 blocking antibody, LAG-3 blocking antibody and Tim-3 blocking antibody.

9. The recombinant viral vector according to claim 8, wherein The recombinant viral vector is a recombinant adenoviral vector, which is derived from the chimpanzee adenoviral vector AdC68XY-R1, and the promoter of the chimpanzee adenoviral vector AdC68XY-R1 is constructed as a tumor-specific promoter and a hypoxia-responsive promoter, and / or an expression framework of a hypoxia regulatory element is inserted downstream of the E1A gene of the chimpanzee adenovirus AdC68XY-R1.

10. The recombinant viral vector according to claim 9, wherein The tumor-specific promoter is selected from the group consisting of human telomerase reverse transcriptase promoter, carcinoembryonic antigen promoter, alpha-fetoprotein promoter, human prostate-specific antigen promoter, cyclooxygenase-2 promoter, apoptosis inhibitory protein promoter, and human intestinal tissue-specific antigen promoter; preferably, the tumor-specific promoter comprises the nucleotide sequence shown in SEQ ID NO: 1; Optionally, the hypoxia-responsive promoter is a promoter of a transcriptional control system triggered in response to hypoxia; preferably, the hypoxia-responsive promoter comprises the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO:

3.

11. The recombinant viral vector according to claim 10, wherein The recombinant viral vector comprises the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO:

27.

12. The recombinant viral vector according to claim 8, wherein The recombinant viral vector is a recombinant vaccinia virus vector; preferably, the recombinant vaccinia virus vector is derived from the vaccinia virus Tiantan strain vector; more preferably, the recombinant vaccinia virus vector comprises the nucleotide sequence shown in SEQ ID NO: 30 in the C9 region.

13. The recombinant viral vector according to claim 8, wherein The recombinant viral vector is a lentiviral vector; preferably, the recombinant lentiviral vector comprises the nucleotide sequence shown in SEQ ID NO:

28.

14. A method for preparing the recombinant viral vector according to any one of claims 8 to 13, comprising inserting the polynucleotide according to claim 7 into a viral vector; Preferably, the viral vector is a chimpanzee adenovirus vector AdC68XY-R1. More preferably, the method comprises: Using the genome of chimpanzee adenovirus vector AdC68XY-R1 as a basic framework, the polynucleotide of claim 7, and optionally the polynucleotide expressing the immune function molecule, is cloned into the replicative chimpanzee adenovirus vector AdC68XY-R1; and / or Replacing the promoter of the chimpanzee adenovirus vector AdC68XY-R1 with the tumor-specific promoter and the hypoxia-responsive promoter; and / or inserting an expression framework of a hypoxia regulatory element downstream of the E1A gene of the chimpanzee adenovirus vector AdC68XY-R1; or, Preferably, the viral vector is the vaccinia virus shuttle plasmid pSC65, and the polynucleotide is the nucleotide sequence shown in SEQ ID NO:

30. More preferably, the method comprises: Subcloning the nucleotide sequence shown in SEQ ID NO: 30 into the C9 region of the vaccinia virus shuttle plasmid pSC65 to construct a recombinant plasmid; and The recombinant plasmid is transfected into human thymidine kinase-deficient cells that have been infected with wild-type vaccinia virus by means of gene homologous recombination, so that the two cells undergo homologous recombination to obtain the recombinant vaccinia virus vector; or, Preferably, the viral vector is a blank lentiviral expression plasmid pXW-EF1α-MCS, and the polynucleotide is the nucleotide sequence shown in SEQ ID NO:

28. More preferably, the method comprises: cloning the nucleotide sequence shown in SEQ ID NO: 28 into a blank lentiviral expression plasmid pXW-EF1α-MCS.

15. A recombinant virus for delivering the fusion protein according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 to tumor cells, which is obtained by packaging the recombinant viral vector according to any one of claims 8 to 13.

16. A pharmaceutical composition comprising the recombinant viral vector according to any one of claims 8 to 13 or the recombinant virus according to claim 15; Preferably, the pharmaceutical composition further comprises immune cells; more preferably, the immune cells are selected from one or more of T cells, CAR-T cells, iNKT cells, NK cells, K cells, macrophages, CAR-iNKT cells, CAR-NK cells and CAR-macrophages; further preferably, the T cells are selected from one or more of unsorted and purified T cells, sorted and purified T cells, sorted and purified PD-1+T cells, sorted and purified CD137+T cells, sorted and purified CD160+T cells, sorted and purified naive T cells, sorted and purified central memory T cells, sorted and purified effector memory T cells, sorted and purified effector T cells, sorted and purified transitional memory T cells and sorted and purified tissue memory T cells.

17. Use of the fusion protein of any one of claims 1 to 5, the pharmaceutical composition of claim 6, the polynucleotide of claim 7, the recombinant viral vector of any one of claims 8 to 13, the recombinant virus of claim 15, or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating tumors; Preferably, the tumor is selected from one or more of B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high-grade glioma, blastoma 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 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, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or blood oncogenic diseases.

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