Vgamma9Vdelta2 T cell for expressing B7-H3-targeted chimeric antigen receptor and application of Vgamma9Vdelta2 T cell

Abstract

The invention belongs to the field of immunotherapy, relates to a Vgamma9Vdelta2T cell expressing a B7-H3-targeted chimeric antigen receptor and application of the Vgamma9Vdelta2T cell, and particularly provides an antibody specifically bound with B7-H3 or an antigen binding fragment of the antibody, the chimeric antigen receptor containing the antibody or the antigen binding fragment of the antibody, and application of the antibody or the antigen binding fragment in preparation of drugs for treating diseases. The CAR-Vgamma9Vdelta2T cell prepared by the screened CAR sequence disclosed by the invention is normal in proliferation, the suicide injury phenomenon is not observed, the cell survival rate level is relatively high, and the CAR-Vgamma9Vdelta2T cell can effectively kill tumor cells in both in-vivo and in-vitro environments.

Description

Technical Field

[0001] The present disclosure belongs to the field of immunotherapy, and specifically relates to a Vγ9Vδ2 T cell expressing a chimeric antigen receptor targeting B7-H3 and an application thereof. Background Art

[0002] Cancer is one of the major diseases that threaten human health. Common cancer therapies include radiotherapy, chemotherapy, targeted therapy, etc., and immunotherapy is gradually becoming a mainstream therapy. Chimeric antigen receptor-mediated cell therapy is a type of immunotherapy. Currently, there are 9 chimeric antigen receptor-mediated autologous T cell therapies on the market worldwide. They have achieved outstanding results in the treatment of myeloma and B cell-related tumors, and have achieved cure in some patients (no tumor recurrence for 5 years). At the same time, it has also promoted the rapid development of this therapy in the field of blood cancer. However, due to factors such as the long preparation cycle of autologous cells, worsening of the patient's condition during the waiting period, and failure of preparation due to substandard patient cell quality, some patients participating in this therapy cannot receive timely treatment. In addition, chimeric antigen receptor-mediated cell therapy still performs poorly in the treatment of solid tumors. Therefore, the development of universal T cells with chimeric antigen receptors targeting new targets is particularly critical to solving this problem.

[0003] B7-H3 is a membrane protein of the B7 immunomodulator family. There are two subtypes in the human body, 2Ig-B7-H3 and 4Ig-B7-H3, while there is only 2Ig-B7-H3 in mice. Some literature reports that B7-H3 can reduce the release of type I interferon by T cells and inhibit the killing function of natural killer cells. Therefore, B7-H3 is considered to be an immunosuppressive factor. Other literature has also confirmed that B7-H3 can inhibit diseases such as graft-versus-host reaction, heart transplant rejection, airway inflammation, and autoimmune encephalomyelitis. However, there are also literatures that describe B7-H3 as a T cell co-stimulatory factor in in vitro and in vivo autoimmune models.

[0004] B7-H3 is only expressed at very low levels in normal human tissues, but is often overexpressed in tumors (ranging from 74% to 94%). In addition, studies have found that B7-H3 is also highly expressed in tumor-associated blood vessels and fibroblasts in tumor stroma. High expression of B7-H3 is often closely related to fewer tumor-infiltrating lymphocytes in tumors, rapid development of cancer, and poor clinical treatment outcomes. It is common in cancers such as gliomas, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer, and kidney cancer. Since B7-H3 is highly expressed in a variety of solid tumors, it can be used as a good target for the treatment of a variety of solid tumors. At the same time, since B7-H3 is expressed on the cell surface, it can be targeted using chimeric antigen receptor-mediated cell therapy to achieve the purpose of killing tumor cells without damaging normal cells.

[0005] T cells are divided into two categories, αβT cells and γδT cells, according to different TCRs. Human peripheral blood lymphocytes are mainly αβT cells, and γδT cells generally account for only 1%-5%. The most important γδT cell subset is Vγ9Vδ2 T cells, accounting for about 90%. γδT cells can directly kill tumor cells through NK cell receptors on their cell surface, ADCC effects, and cytokines (IFN-γ, TNF-α) secreted by them. γδT cells can also activate B / DC / αβT / NK cells in other ways (such as acting as antigen presenting cells to activate αβT cells, or inducing NK-mediated anti-tumor cytotoxicity through the 4-1BB co-stimulation pathway, etc.), thereby achieving indirect killing of tumors.

[0006] Patients with γδT cell-infiltrated tumors have the best prognosis compared to patients with other immune cell-infiltrated tumors. And unlike conventional cancer therapies (such as chemotherapy and radiotherapy), there have been no serious adverse reactions associated with γδT cell therapy to date. γδTCR is naturally not restricted by MHC (histocompatibility complex), which means that if the donor's γδT cells are transferred to the patient (allogeneic cell therapy), the γδT cells will not recognize the patient's body as a foreign object, and therefore will not cause GvHD (graft-versus-host disease), which is a source of high-quality allogeneic cell products. However, there are not many publicly available research projects on Vγ9Vδ2T cells, and most of them are not modified. Moreover, the current general cell production process has many limitations for the amplified production of Vγ9Vδ2 T cells expressing chimeric antigen receptors. The single cell amount from the peripheral blood of healthy people is low, and the initial activation and amplification are difficult. However, the success rate of CAR virus transduction of Vγ9Vδ2 T cells is low, and the large amount of virus used leads to increased costs. In addition, after CAR virus transfection, most T cells are exhausted due to their own recognition and killing, and cannot meet the requirements of subsequent expansion and preparation.

[0007] Therefore, there is an urgent need to develop a new type of T cell to further enhance the efficacy and biocompatibility of cellular immunotherapy by combining a chimeric antigen receptor targeting B7-H3 with γδT cells. Summary of the invention

[0008] In order to solve the problems existing in the prior art, the purpose of the present disclosure is to provide a Vγ9Vδ2 T cell expressing a chimeric antigen receptor targeting B7-H3 and its application.

[0009] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to B7-H3, wherein the antibody or antigen-binding fragment comprises a light chain variable region VL and a heavy chain variable region VH,

[0010] The light chain variable region comprises: LCDR1 having an amino acid sequence as shown in SEQ ID NO.5 or any variant thereof, LCDR2 having an amino acid sequence as shown in SEQ ID NO.6 or any variant thereof, and LCDR3 having an amino acid sequence as shown in SEQ ID NO.7 or any variant thereof;

[0011] The heavy chain variable region comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO.10 or any variant thereof, HCDR2 having the amino acid sequence shown in SEQ ID NO.11 or any variant thereof, and HCDR3 having the amino acid sequence shown in SEQ ID NO.12 or any variant thereof.

[0012] In another aspect, the present disclosure provides a chimeric antigen receptor comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises the aforementioned antibody or antigen-binding fragment thereof.

[0013] In another aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding the aforementioned antibody or antigen-binding fragment thereof or the aforementioned chimeric antigen receptor

[0014] In another aspect, the present disclosure provides an expression vector comprising the aforementioned polynucleotide.

[0015] In another aspect, the present disclosure provides a cell introduced with or containing the aforementioned polynucleotide or the aforementioned expression vector.

[0016] In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector, and the aforementioned cell.

[0017] In another aspect, the present disclosure provides a use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector, and the aforementioned cell in preparing a drug for treating a disease.

[0018] The present disclosure has at least the following beneficial effects:

[0019] (1) The CAR-Vγ9Vδ2 T cells prepared by the CAR sequence screened in the present disclosure proliferated normally, no suicide injury was observed, and the cell survival rate could be maintained at a high level.

[0020] (2) The present invention discloses the preparation of Vγ9Vδ2 T cells with chimeric antigen receptors that can target B7-H3, which can effectively kill tumor cells both in vivo and in vitro. Experiments in an in vivo mouse brain glioma model show that Vγ9Vδ2 T cells with B7-H3 chimeric antigen receptors can eliminate tumors in mice and control tumor recurrence for at least 70 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The expression of B7-H3 in different tumor cell lines.

[0022] Figure 2 shows the expression of B7-H3 in brain glioma samples and ovarian cancer samples. Figure 2A The expression of B7-H3 in brain glioma samples. Figure 2B The expression of B7-H3 in ovarian cancer samples.

[0023] Figure 3 This is the basic structure of the B7-H3 chimeric antigen receptor.

[0024] Figure 4 shows the culture of Vγ9Vδ2 T cells with different B7-H3 chimeric antigen receptors. Figure 4A The microscopic morphology of Vγ9Vδ2 T cells with different B7-H3 chimeric antigen receptors. Figure 4B The mean cell viability of different control groups.

[0025] Figure 5 shows the expression of B7-H3 chimeric antigen receptor on Vγ9Vδ2 T cells. Figure 5A The positive transduction results of different chimeric antigen receptors. Figure 5B The graph shows the statistical results of positive transduction rates of different chimeric antigen receptors.

[0026] Figure 6 shows the co-culture of B7-H3 chimeric antigen receptor-T cells and tumor cells. Fig. 6A To detect the ratio of T cells to tumor cells after co-culture by flow cytometry, Figure 6B The killing statistics are for different effect-target ratios.

[0027] Figure 7 It is the cytokine level when B7-H3 chimeric antigen receptor-T cells kill tumors.

[0028] Figure 8 The killing effect of B7-H3 chimeric antigen receptor-T cells on brain glioma in vivo (N=8, 0-35 days).

[0029] Fig. 9 The killing effect of B7-H3 chimeric antigen receptor-T cells on brain glioma in vivo (N=3, 0-70 days). DETAILED DESCRIPTION

[0030] I. Terminology

[0031] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0032] As used herein, the articles "a" and "an" refer to one or more than one (ie, at least one) of the grammatical object to which the article refers. For example, "an element" means one element or more than one element.

[0033] As used herein, the term "about" represents and encompasses a specified value and a range greater than and less than that value. In certain embodiments, the term "about" represents a specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term "about" represents a specified value ± one standard deviation of that value.

[0034] As used herein, the term "activation" refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function.

[0035] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct that comprises at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") (including a functional signaling domain).

[0036] As used herein, the term "nucleic acid", "nucleotide" or "oligonucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single-stranded or double-stranded form. Unless explicitly limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0037] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be a complete immunoglobulin derived from a natural source or from a recombinant source, and may be an immunoreactive portion of a complete immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The antibodies in the present disclosure may exist in a variety of forms, including but not limited to: polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, etc., as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci., USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426).

[0038] As used herein, the term "antibody fragment" or "antigen binding fragment" refers to any portion of a full-length antibody that is less than the full length, but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites), and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody. Therefore, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p3-25, Kipriyanov). The fragment may include multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), obtains an antibody that immunospecifically binds (i.e., exhibits a Ka of at least or at least about 107-108M-1) to an antigen. "Functional fragments" or "analogs of anti-B7-H3 antibodies" are fragments or analogs that prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction. As used herein, functional fragments generally have the same meaning as "antibody fragments", and in the case of antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. "Fv" fragments consist of a dimer (VH-VL dimer) formed by non-covalent binding of the variable domain of a heavy chain and the variable domain of a light chain. In this configuration, the three CDRs of each variable domain interact to determine the target binding site on the surface of the VH-VL dimer, as in the case of an intact antibody. The six CDRs together confer target binding specificity to the intact antibody. However, even a single variable domain (or half of an Fv comprising only three target-specific CDRs) can still have the ability to recognize and bind to a target.

[0039] As used herein, the term "monoclonal antibody" refers to a population of identical antibodies, indicating that each individual antibody molecule in a monoclonal antibody population is identical to other antibody molecules. This characteristic is in contrast to the characteristic of a polyclonal population of antibodies, which comprises antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming host cells with a plasmid carrying an artificial sequence of nucleotides encoding antibodies.

[0040] As used herein, the term "heavy chain" refers to the larger chain of the two types of polypeptide chains present in all antibody molecules in a naturally occurring conformation.

[0041] As used herein, the term "light chain" refers to the smaller of the two types of polypeptide chains present in their naturally occurring conformations in all antibody molecules, with kappa and lambda light chains referring to the two major antibody light chain isotypes.

[0042] As used herein, the term "scFv" refers to a fusion protein comprising at least one variable region antibody fragment including a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacent via a short flexible polypeptide linker and can be expressed in a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used herein, scFv can have the VL and VH variable regions in either order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0043] As used herein, the term "costimulatory factor" refers to an associated binding partner on an immune cell (such as a T cell), which specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by an immune cell, such as but not limited to proliferation and survival. The costimulatory signaling region may be derived from at least one of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88 and 4-1BB. In some embodiments, the costimulatory signaling region is derived from 4-1BB. In some embodiments, the 4-1BB costimulatory signaling region includes the amino acid sequence shown in SEQ ID NO.26.

[0044] As used herein, the term "fratricide" refers to the recognition and killing of other T cells during T cell proliferation. Specifically, the expression of target antigens on CAR-T cells can induce fratricide and efficacy loss of CAR-T cells, reducing their clinical benefits.

[0045] As used herein, the term "anti-tumor effect" or "anti-tumor activity" refers to a biological effect that can be manifested in a variety of ways, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor cell proliferation, a reduction in tumor cell survival, or an improvement in a variety of physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure to prevent the occurrence of tumors in the first place, such as in a preventive therapy or treatment. Cancers include, but are not limited to, B cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease and mu chain disease, benign monoclonal gammopathy, and immune cell amyloidosis, melanoma, breast cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, glioma, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, blood tissue cancer, etc. Other non-limiting examples of cancer types suitable for the methods encompassed by the present disclosure include human sarcomas and cancers such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, breast cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, hepatoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, In some embodiments, the cancer is melanoma, glioma, cervical cancer, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer, renal cell carcinoma, ovarian cancer, ovarian cancer, cervical cancer, cervical cancer, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer, and renal cancer.

[0046] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be interpreted to include non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0047] As used herein, the term "autologous" refers to any material derived from the same subject that is subsequently reintroduced into the subject.

[0048] As used herein, the term "allogeneic" refers to any material derived from different animals of the same species as the subject, which is introduced into the subject. When the genes at one or more loci are not the same, two or more subjects are referred to as allogeneic to each other. In some embodiments, the allogeneic material from individuals of the same species may be sufficient to interact with antigens to different degrees genetically (e.g., at specific genes, such as MHC alleles). In some embodiments, the allogeneic material from individuals of the same species may be sufficient to be unable to interact with antigens to a genetically similar degree (e.g., at specific genes, such as MHC alleles).

[0049] The isolated nucleic acid molecules of the present disclosure include any nucleic acid molecules encoding polypeptides of the present disclosure or fragments thereof. Such nucleic acid molecules do not need to be 100% homologous or identical to endogenous nucleic acid sequences, but will generally show basic identity. Nucleic acids having "basic identity" or "basic homology" with endogenous sequences are generally capable of hybridizing with at least one chain of a double-stranded nucleic acid molecule. As used herein, "hybridization" refers to pairing under various stringency conditions to form double-stranded molecules between complementary polynucleotide sequences (e.g., genes described herein) or their parts. For example, stringent salt concentrations can be less than about 750mM NaCl and 75mM trisodium citrate, less than about 500mM NaCl and 50mM trisodium citrate, or less than about 250mM NaCl and 25mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can be obtained in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions will generally include a temperature of at least about 30° C., at least about 37° C., or at least about 42° C. Various other parameters, such as hybridization time, concentration of detergents (e.g., sodium dodecyl sulfate (SDS)), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Different levels of stringency can be achieved by combining these different conditions as desired.

[0050] By "substantially identical" or "substantially homologous" is meant a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence is at least about 60%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% homologous or identical to the sequence used for comparison at the amino acid level or nucleic acid. Sequence identity is typically measured using sequence analysis software (e.g., sequence analysis software package of Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and / or other modifications. Conservative substitutions generally include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, where probability scores between e-3 and e-100 represent closely related sequences.

[0051] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny thereof. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, this is clear from the context.

[0052] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not occur.

[0053] "Pharmaceutical composition" means containing one or more modified T cells described herein, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of active ingredients, and thus exert biological activity.

[0054] "Administering," "giving," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0055] "Treatment" means administering an internal or external therapeutic agent, such as any modified T cell disclosed herein, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated patient or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although the embodiments of the present disclosure (e.g., treatment methods or articles of manufacture) may not be effective in alleviating the target disease symptoms in every patient, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0056] The term "consisting essentially of" or variations thereof as used throughout the specification and claims means including all recited elements or groups of elements, and optionally including other elements of similar or different properties to the recited elements, which other elements do not significantly alter the basic or novel properties of a specified dosing regimen, method, or composition.

[0057] II. Detailed description of specific implementation scheme

[0058] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to B7-H3, wherein the antibody or antigen-binding fragment comprises a light chain variable region VL and a heavy chain variable region VH,

[0059] The light chain variable region comprises: LCDR1 having an amino acid sequence as shown in SEQ ID NO.5 or any variant thereof, LCDR2 having an amino acid sequence as shown in SEQ ID NO.6 or any variant thereof, and LCDR3 having an amino acid sequence as shown in SEQ ID NO.7 or any variant thereof;

[0060] The heavy chain variable region comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO.10 or any variant thereof, HCDR2 having the amino acid sequence shown in SEQ ID NO.11 or any variant thereof, and HCDR3 having the amino acid sequence shown in SEQ ID NO.12 or any variant thereof.

[0061] In some embodiments, the light chain variable region comprises LCDR1 having the amino acid sequence shown in SEQ ID NO.5, LCDR2 having the amino acid sequence shown in SEQ ID NO.6, and LCDR3 having the amino acid sequence shown in SEQ ID NO.7; the heavy chain variable region comprises HCDR1 having the amino acid sequence shown in SEQ ID NO.10, HCDR2 having the amino acid sequence shown in SEQ ID NO.11, and HCDR3 having the amino acid sequence shown in SEQ ID NO.12.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to B7-H3 comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO.4, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the aforementioned sequence; and / or

[0063] A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO.9, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the aforementioned sequence.

[0064] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to B7-H3 comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO.4, and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO.9.

[0065] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to B7-H3 is edited or modified by genetic engineering.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof is a single chain antibody.

[0067] In some embodiments, the amino acid sequence of the single-chain antibody is as shown in SEQ ID NO.2.

[0068] In another aspect, the present disclosure provides a chimeric antigen receptor comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises the aforementioned antibody or antigen-binding fragment thereof.

[0069] In some embodiments, the chimeric antigen receptor comprises an extracellular antigen binding domain, and the extracellular antigen binding domain comprises a single-chain antibody with an amino acid sequence as shown in SEQ ID NO.2.

[0070] In some embodiments, the chimeric antigen receptor further comprises a hinge region, a transmembrane region, a co-stimulatory domain, and a signaling domain.

[0071] In some embodiments, the chimeric antigen receptor comprises a single-chain antibody, a hinge region, a transmembrane region, a co-stimulatory domain, and a signaling domain from N-terminus to C-terminus, respectively.

[0072] In some embodiments, the hinge region is selected from CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

[0073] In some embodiments, the hinge region is CD8α. In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO.20.

[0074] In some embodiments, the transmembrane region is selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD4 9D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0075] In some embodiments, the transmembrane region is CD8α.In some embodiments, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO.22.

[0076] In some embodiments, the co-stimulatory domain is selected from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, 4-1BB, or any combination thereof.

[0077] In some embodiments, the costimulatory domain is CD28 or 4-1BB. In some embodiments, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO.24 or SEQ ID NO.26.

[0078] In some embodiments, the signal transduction region is selected from CD2, CD3ζ, CD7, CD27, CD28, CD137, CD134, LCK, TNFR-1, TNFR-1, FasNKG2D, DAP10, DAP12, B7-H3, TLR2, TLR4, IL7R or any combination thereof.

[0079] In some embodiments, the signal transduction region is CD3ζ. In some embodiments, the signal transduction region comprises the amino acid sequence shown in SEQ ID NO.28.

[0080] In some embodiments, the chimeric antigen receptor comprises, from N-terminus to C-terminus, a B7-H3 single-chain antibody, a CD8α hinge region, a CD8α transmembrane region, a CD28 or 4-1BB co-stimulatory domain, and a CD3ζ signaling region, respectively.

[0081] In some embodiments, the chimeric antigen receptor further comprises any one or any two or more of a membrane protein, a secretory protein, an intracellular protein, a small molecule drug, and a cytotoxic drug.

[0082] In another aspect, the present disclosure provides a polynucleotide comprising the nucleotide sequence of the aforementioned antibody or antigen-binding fragment thereof or the aforementioned chimeric antigen receptor. In another aspect, the present disclosure provides an expression vector comprising the aforementioned polynucleotide.

[0083] In another aspect, the present disclosure provides a cell introduced with or containing the aforementioned polynucleotide or the aforementioned expression vector.

[0084] In some embodiments, the cell is one or more of autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and red blood cells.

[0085] In some embodiments, the cell is a T cell.

[0086] In some embodiments, the T cells include αβ T cells, γδ T cells, and regulatory T cells.

[0087] In some embodiments, the T cells are Vγ9Vδ2 T cells.

[0088] On the other hand, the present disclosure provides a pharmaceutical composition comprising one or more of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector, and the aforementioned cell.

[0089] On the other hand, the present disclosure provides a use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector, and the aforementioned cell in preparing a drug for treating a disease.

[0090] In some embodiments, the disease is cancer; in some embodiments, the cancer is a cancer with high expression of B7-H3.

[0091] In some embodiments, the cancer includes, but is not limited to, B cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease and mu chain disease, benign monoclonal gammopathy, and immune cell amyloidosis, melanoma, breast cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, glioma, bladder cancer, cancer of the brain or central nervous system, cancer of the peripheral nervous system, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, blood tissue cancer, etc. Other non-limiting examples of cancer types suitable for the methods encompassed by the present disclosure include human sarcomas and cancers such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, breast cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, hepatoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, testicular cancer, small cell lung cancer, bladder cancer, epithelial cancers, gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, retinoblastomas; leukemias such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemias); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); as well as polycythemia vera, lymphomas (Hodgkin's and non-Hodgkin's), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.

[0092] In some embodiments, the cancer is glioma, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer, and kidney cancer.

[0093] In some embodiments, the brain glioma includes glioblastoma, medulloblastoma, astrocytoma, oligodendroglioma.

[0094] On the other hand, the present disclosure provides a method of administering a therapeutically effective amount of the aforementioned Vγ9Vδ2 T cells or pharmaceutical compositions expressing a chimeric antigen receptor targeting B7-H3 to a subject to treat a disease or condition, wherein the method further comprises administering a second drug or therapy. In some embodiments of the present disclosure, the disease or condition is cancer or a related disease.

[0095] In some embodiments of the present disclosure, the Vγ9Vδ2 T cells or pharmaceutical compositions expressing a chimeric antigen receptor targeting B7-H3 are administered simultaneously with a second drug or therapy. In some embodiments of the present disclosure, the Vγ9Vδ2 T cells or pharmaceutical compositions expressing a chimeric antigen receptor targeting B7-H3 are administered sequentially with a second drug or therapy.

[0096] In another aspect, the present disclosure provides a method for treating a subject, comprising administering to the subject a therapeutically effective amount of the aforementioned Vγ9Vδ2 T cell expressing a chimeric antigen receptor targeting B7-H3 or a pharmaceutical composition.

[0097] On the other hand, the present disclosure provides a method for detecting B7-H3, which comprises the step of using the aforementioned antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned cell to detect B7-H3 in a sample.

[0098] On the other hand, the present disclosure provides a kit for detecting B7-H3, which comprises the aforementioned antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned cell.

[0099] On the other hand, the present disclosure provides a method for enhancing cell function in vitro, comprising: contacting cells with the aforementioned antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned cell.

[0100] Example

[0101] A further understanding of the present disclosure can be obtained by referring to some specific embodiments given herein, which are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Obviously, various modifications and changes can be made to the present disclosure without departing from the essence of the present disclosure, and therefore, these modifications and changes are also within the scope of the protection required by the present application.

[0102] Example 1: Cell culture

[0103] The cell lines used in the present disclosure are all adherent cells, including 293T cells, WM-266-4, SK-OV-3, LN-229, U-87MG, and Hela cells, which are cultured in DMDM ​​medium containing 10% FBS, 1% GlutaMAX, and 1% double antibody. Among them, 293T (kindly donated by Zhao Bin Laboratory, Zhejiang University) is a human embryonic kidney cell line, which is commonly used for virus preparation. WM-266-4 (kindly donated by Zhao Bin Laboratory, Zhejiang University) is a human melanoma cell line, SK-OV-3 (kindly donated by Zhao Bin Laboratory, Zhejiang University) is a human ovarian cancer cell line, LN-229 (kindly donated by Zhao Bin Laboratory, Zhejiang University) and U-87MG (kindly donated by Zhao Bin Laboratory, Zhejiang University) are both human glioblastoma cell lines, and Hela is a human cervical cancer cell line. GFP-positive cells are monoclonal stable cell lines prepared by infecting target cells with retrovirus encoding GFP, and the tumor cell line used for mice is a cell line infected with a lentivirus encoding Luciferase. The retrovirus encoding GFP was prepared by transiently transfecting 293T cells using a three-plasmid system (SFG-GFP, MMLV-gag-pol and pVSV-G). The lentivirus encoding Luciferase was prepared by transiently transfecting 293T cells with SFG-GFP-FFLuc, MMLV-gag-pol and pVSV-G.

[0104] Example 2: Detection of B7-H3 expression in tumors

[0105] In order to determine the expression of B7-H3 antigen in tumors, the applicant first selected different tumor cell lines for detection. At the same time, in order to further verify the expression of B7-H3 in primary tumors, the applicant detected the expression of B7-H3 in sections containing brain glioma tissues and ovarian cancer tissues from multiple patients.

[0106] The specific experimental process is as follows:

[0107] 1. Detection of B7-H3 expression in tumor cell lines

[0108] Tumor cells (human melanoma cells WM-266-4, human ovarian cancer cells SK-OV-3, human glioblastoma cells LN-229, human brain glioblastoma cells U-87MG, human cervical cancer cells Hela) were collected and washed three times with 1xPBS, then incubated with B7-H3 antibody ID35 (1:100, diluted in 1xPBS) at 2-8°C for 30 min, washed twice with 1xPBS, and then APC-conjugated goat anti-human secondary antibody (Jackson ImmunoResearch, 109-605-044) was added at a dilution of 1:100 in 1xPBS, incubated at 2-8°C for 30 min, and washed with 1xPBS, and the expression of B7-H3 on tumor cells was detected by flow cytometry.

[0109] 2. Detection of B7-H3 expression in tumor tissue sections (immunohistochemical staining)

[0110] After paraffin sections were dewaxed with xylene and rehydrated with alcohol, antigen retrieval was performed using sodium citrate. Then, they were blocked in serum for 1 hour. B7-H3 antibody ID35 staining was performed at 2-8°C overnight, and then incubated with recombinant Anti-HLA Class 1ABC antibody (abcam, ab225636) for 30 minutes. After washing, DAB was used for color development, and then hematoxylin was used for staining. After staining, the sections were observed and photographed under a microscope.

[0111] The results of flow cytometry were shown in Figure 1 .from Figure 1 It can be seen that B7-H3 is highly expressed in various tumor cells. The results of sections of brain glioma tissue and ovarian cancer tissue are shown in Figure 2A , 2B Compared with the negative control in 2B, different levels of B7-H3 expression were detected in all glioma samples and most ovarian cancer samples. This indicates that B7-H3 is a good target for solid tumor therapy.

[0112] Example 3: Construction of chimeric antigen receptor

[0113] The present invention is based on the sequence of the monoclonal antibody ID35 generated against the B7-H3 protein fragment, and is transformed into a second-generation chimeric antigen receptor, the structure of which is as shown in the attached Figure 3 As shown, it contains a single-chain antibody region scFv (SEQ ID NO: 1, 2), a hinge region and a transmembrane region of CD8α, a costimulatory region of CD28 or 4-1BB, and a CD3ζ signal transduction region. Other selected sequence structures in the patent are also shown in the attached Figure 3 As shown. The single-chain antibody region of the chimeric antigen receptor is derived from antibodies against B7-H3 produced by each monoclonal hybridoma cell. The chimeric antigen receptor is produced by gene synthesis and then cloned into a lentiviral vector. After cloning, the correctness of its sequence is confirmed by sequencing. In addition to ID35, the scFV also includes the amino acid sequences shown in SEQ ID NO: 14, 16, and 18, which are reference sequences.

[0114] The specific sequence information can be found in Table 1 below.

[0115] Table 1. Chimeric antigen receptor related sequence information

[0116]

[0117]

[0118]

[0119]

[0120] Example 4: Preparation of chimeric antigen receptor lentivirus

[0121] Based on the chimeric antigen receptor obtained in Example 3, this example further prepared lentiviral particles containing the chimeric antigen receptor. The specific experimental details are as follows:

[0122] On the first day, 6x10 6 293T cells of cells were cultured in 100mm cell culture dishes with a culture volume of 10mL. After 24h, a three-plasmid system (6μg, 4μg, 2μg) was used, in which the expression plasmid backbone came from pCDH-EF1a-eFFly-eGFPplasmid (Addgene, Plasmid#104834), the auxiliary plasmid pspAx2 (Addgene, #12260) and the auxiliary plasmid pCMV-VSV-G (Addgene, Plasmid#8454), the three plasmids were mixed in 500μL transfection reagent buffer, 20uljetPRIME auxiliary transfection reagent was added and mixed, and 293T cells were transiently transfected. After 6 hours, replace with new DMEM medium. After 48 and 72 hours of transfection, collect the culture supernatant (the 48-hour supernatant can be stored at 4°C and co-processed with the 72-hour supernatant), filter with a 0.45 μm needle filter, and centrifuge at 70,000 rcf for 2 hours (centrifuge speed 9: speed increase and 3: speed decrease). After centrifugation, add RPMI-1640 medium with 1% volume of the initial virus solution to resuspend the virus particles and store at -80°C for use.

[0123] Example 5: Preparation of T cells expressing chimeric antigen receptor Vγ9Vδ2 targeting B7-H3

[0124] This example further prepared chimeric antigen receptor Vγ9Vδ2 T cells expressing B7-H3 targeting. The specific experimental method is as follows:

[0125] 1. Expansion and viral transduction of Vγ9Vδ2 T cells

[0126] Peripheral blood mononuclear cells were isolated from healthy donors (subjects were informed and consented). The lymphocyte density gradient separation method was used to separate the mononuclear cells. The isolated mononuclear cells were cultured in RPMI-1640 medium containing 10% FBS, 1% glutaMAX, 1% double antibody, and 100U / mL rhIL-2, and the initial cell density was controlled to 2x10 6cells / mL, inoculated in a 24-well plate, added 5 μM zoledronic acid (ZOL) to activate Vγ9Vδ2 T cells, and replaced half of the medium every 48 hours. After 4 days, the cell density was adjusted to 1x10 6 cells / mL and continue culturing, changing the medium every 48 h.

[0127] The Vγ9Vδ2 T cells activated and cultured in vitro were collected and the cell density was adjusted to 1x10 6 cells / mL, inoculated in a 24-well plate (1 mL). 1x10 7 TU lentivirus was cultured in a cell culture incubator. Half of the medium was replaced after 24 hours, and then 1x10 6 The medium was changed and cultured for 10 min at 4 °C for 2-3 s. The subsequent expansion culture was used to detect the expression of chimeric antigen receptor and tumor killing ability. The cell counter used AOPI dye to count the number of T cells and cell viability.

[0128] 2. B7-H3 chimeric antigen receptor expression detection

[0129] Charge 1x10 6 The expression of B7-H3 chimeric antigen receptor was detected by using the fusion protein of B7-H3 and antibody Fc fragment (1:100, diluted in 1xPBS) for amplified CAR-Vγ9Vδ2T cells. The cells were incubated at room temperature for 30 minutes. The secondary antibody was APC fluorescent dye-labeled goat anti-human antibody Fc binding protein (1:100, diluted in 1xPBS) and incubated at 2-8°C for 30 minutes. The expression of chimeric antigen receptor on T cells was detected by flow cytometry.

[0130] Lentivirus was prepared in 293T cells using a lentiviral vector containing the chimeric antigen receptor and infected with human Vγ9Vδ2 T cells (including different healthy donors) stimulated with zoledronic acid (ZOL). The culture of Vγ9Vδ2 T cells with different B7-H3 chimeric antigen receptors is shown in Figure 4. Figure 4A It can be seen that Vγ9Vδ2 T cells expressing different chimeric antigen receptors have different proliferation states. The cell viability was counted by repeated experiments. The results show that Figure 4B, there are significant differences in the viability of Vγ9Vδ2 T cells expressing different chimeric antigen receptors. Compared with the NT group without viral transduction and the control group transduced with GFP virus, the T cells prepared by other reference chimeric antigen receptors with the same target all showed a large amount of exhaustion due to mutual recognition and killing, and could not meet the requirements of subsequent amplification and preparation viability and quantity. The CAR-Vγ9Vδ2 T cells prepared by the CAR sequence screened in the present disclosure proliferated normally, no suicide phenomenon was observed, and the cell viability could be maintained at a high level. After CAR viral transduction, flow cytometry was used to verify the expression of B7-H3 chimeric antigen receptor on the surface of Vγ9Vδ2 T cells. The results of B7-H3 chimeric antigen receptor expression detection are shown in Figure 5. As can be seen from Figure 5, >50% of T cells express the chimeric antigen receptor of the present disclosure, that is, the CAR positive transduction rate is >50%, which can not only better meet the needs of functional detection experiments, but also provide guarantees for further application in production and preparation. This result was also verified in T cells from multiple other healthy donors.

[0131] Example 6: Killing of tumor cells by CAR-Vγ9Vδ2 T cells in an in vitro co-culture system

[0132] In order to further determine whether the chimeric antigen receptor of the present disclosure can mediate Vγ9Vδ2 T cells to kill tumor cells with high expression of B7-H3, the applicant designed a co-culture experiment to test the killing of various tumor cells by the T cells. After the tumor cells adhered to the wall, T cells expressing control and B7-H3 chimeric antigen receptors were added respectively, and then the killing of various tumor cells by each test group was detected by flow cytometry.

[0133] The specific test process is as follows:

[0134] 1. Long-term cytotoxicity test (cell co-culture)

[0135] Inoculate 1-2 x 10 cells per well in a 24-well plate 5 GFP-labeled tumor cells (WM-266-4, SK-OV-3, U-87MG, Hela) were added 24 hours later, and the corresponding number of T cells were added according to the effector-target ratio of 1:1, 1:2, and 1:5. The cells in the wells were collected 2 days later, and the proportion of residual tumor cells was detected by flow cytometry. Vγ9Vδ2 T cells were detected by CD3, and tumor cells were detected by GFP.

[0136] 2. Enzyme-linked immunosorbent assay (ELISA)

[0137] In the experiment of co-culture of tumor cells and chimeric antigen receptor-T cells, CAR-Vγ9Vδ2 T cells were added 24 hours later, and the culture supernatant was collected in a 96-well plate and stored at -80°C. The cytokines released in the supernatant were detected using ELISA detection kits (INF-γ detection kit, R&D, DY285B, IL-2 detection kit, R&D, DY202).

[0138] The experimental results of flow cytometry are shown in Figure 6. Fig. 6A It can be seen that in a 1:2 effector-target ratio, flow cytometry detection revealed that the control group T cells had no killing effect on any tumor cells, while the B7-H3 chimeric antigen receptor T cells disclosed in the present invention could effectively kill tumor cells in co-culture with various target-positive tumor cells. Figure 6B The flow cytometry analysis summarizes the residual cell ratios in multiple effector-target ratio experimental groups. It can be seen that the B7-H3 chimeric antigen receptor T cells disclosed in the present invention have obvious and effective killing of tumor cells at different effector-target ratios. The results of cytokine release during T cell killing are shown in Figure 7 ,Depend on Figure 7 It can be seen that the levels of cytokines released by T cells in the control group were all below the detection range, while the T cells of the B7-H3 chimeric antigen receptor disclosed in the present invention released a large amount of Th1 cytokines (including IFNγ and IL-2) during the killing process, indicating that the CAR-Vγ9Vδ2 T cells prepared in the present invention have the ability to kill tumor cells and can also activate other tumor killing mechanisms.

[0139] Example 7: Experiment on orthotopic xenograft tumor in mice

[0140] In order to further determine the killing effect of T cells expressing B7-H3 chimeric antigen receptors in the present disclosure on tumors in vivo, the applicant selected a male nude mouse (Shanghai Slake, BALB / c) glioma model. Luciferase-labeled human tumor LN-229 cells were injected into the brain of 6-8 week-old nude mice, and solvent PBS, control NT, and B7-H3-CAR-T cells were injected in situ after tumor formation, and then imaging was performed to detect the clearance of tumors by T cells and the survival of treated mice.

[0141] The specific experimental process is as follows:

[0142] The in vivo experiment of CAR-Vγ9Vδ2 T cells targeting brain glioma was completed in 6-8 weeks old nude mice. Luciferase-labeled tumor cell line LN-229 was first implanted into the nude mouse's skull in a stereotaxic manner. After 2 weeks, the successful tumor transplantation was confirmed in the in vivo imaging system, and then control or B7-H3 chimeric antigen receptor-Vγ9Vδ2 T cells were injected in situ. After that, imaging was performed once a week to observe the killing of T cells on the tumor. A total of two experiments were conducted, and the initial experimental results are shown in Figure 8 The results of repeated experiments are shown in Fig. 9 .

[0143] The results of B7-H3 chimeric antigen receptor-Vγ9Vδ2 T cells killing brain glioma in mice are shown in the attached figure. Figure 8 , 9 As shown in the figure, the detection signal is getting stronger and stronger, which can represent the continuous proliferation of intracranial tumor cells in mice. Figure 8 The light blue diffuse signal patches in the figure are background noise. Fig. 9 The red signal in the middle indicates that the upper limit of the detection threshold of the imager has been reached, and the imaging missing position indicates that the mouse died naturally or was euthanized in accordance with animal ethics requirements. The experimental results show that compared with the control group in which the tumor continued to grow and died after 6 weeks, the Vγ9Vδ2 T cells of the B7-H3 chimeric antigen receptor disclosed in the present invention were able to clear tumors in most mice and control tumor recurrence within 2 months. This result shows that the T cells of the B7-H3 chimeric antigen receptor can kill tumors in vivo and ensure that the tumor does not recur for at least 70 days, proving that the T cells of the B7-H3 chimeric antigen receptor disclosed in the present invention can be used as a potential tumor treatment method.

[0144] The above is only an embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and equivalent changes made by using the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to B7-H3, wherein: The antibody or antigen-binding fragment comprises a light chain variable region VL and a heavy chain variable region VH, The light chain variable region comprises: LCDR1 having an amino acid sequence as shown in SEQ ID NO.5 or any variant thereof, LCDR2 having an amino acid sequence as shown in SEQ ID NO.6 or any variant thereof, and LCDR3 having an amino acid sequence as shown in SEQ ID NO.7 or any variant thereof; The heavy chain variable region comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO.10 or any variant thereof, HCDR2 having the amino acid sequence shown in SEQ ID NO.11 or any variant thereof, and HCDR3 having the amino acid sequence shown in SEQ ID NO.12 or any variant thereof.

2. The antibody or antigen-binding fragment thereof that specifically binds to B7-H3 according to claim 1, wherein: The light chain variable region comprises LCDR1 having the amino acid sequence shown in SEQ ID NO.5, LCDR2 having the amino acid sequence shown in SEQ ID NO.6 and LCDR3 having the amino acid sequence shown in SEQ ID NO.7; the heavy chain variable region comprises HCDR1 having the amino acid sequence shown in SEQ ID NO.10, HCDR2 having the amino acid sequence shown in SEQ ID NO.11 and HCDR3 having the amino acid sequence shown in SEQ ID NO.

12.

3. The antibody or antigen-binding fragment thereof that specifically binds to B7-H3 according to claim 1 or 2, comprising a light chain variable region of the amino acid sequence shown in SEQ ID NO.4, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the aforementioned sequence; and / or A heavy chain variable region comprising the amino acid sequence of SEQ ID NO.9, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the aforementioned sequence; Preferably, the antibody or antigen-binding fragment thereof that specifically binds to B7-H3 comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO.4, and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO.

9.

4. The antibody or antigen-binding fragment thereof that specifically binds to B7-H3 according to any one of claims 1 to 3, wherein: The antibody or antigen-binding fragment thereof is a single-chain antibody, and the amino acid sequence of the single-chain antibody is shown in SEQ ID NO.

2.

5. A chimeric antigen receptor comprising an extracellular antigen binding domain, wherein the extracellular antigen binding domain comprises the antibody or antigen binding fragment thereof according to any one of claims 1 to 4.

6. The chimeric antigen receptor of claim 5, further comprising a hinge region, a transmembrane region, a co-stimulatory domain, and a signaling domain; Preferably, the hinge region is selected from CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154; Preferably, the transmembrane region is selected from the group consisting of α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D , ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, S LAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C; Preferably, the costimulatory domain is selected from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, 4-1BB or any combination thereof; Preferably, the signal transduction region is selected from CD2, CD3ζ, CD7, CD27, CD28, CD137, CD134, LCK, TNFR-1, TNFR-1, FasNKG2D, DAP10, DAP12, B7-H3, TLR2, TLR4, IL7R or any combination thereof.

7. A polynucleotide comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the chimeric antigen receptor according to any one of claims 5 to 6. An expression vector comprising the polynucleotide according to claim 7 .

9. A cell introduced with or containing the polynucleotide according to claim 7 or the expression vector according to claim 8.

10. The cell according to claim 9, wherein the cell is one or more of autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and erythrocytes; Preferably, the cell is a T cell; Preferably, the T cells include αβT cells, γδT cells, and regulatory T cells; Preferably, the T cells are γδ T cells.

11. A pharmaceutical composition comprising one or more of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the chimeric antigen receptor according to any one of claims 5 to 6, the polynucleotide according to claim 7, the expression vector according to claim 8, and the cell according to any one of claims 9 to 10.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the chimeric antigen receptor according to any one of claims 5 to 6, the polynucleotide according to claim 7, the expression vector according to claim 8, or the cell according to any one of claims 9 to 10 in the preparation of a drug for treating a disease; Preferably, the disease is cancer; Preferably, the cancer is selected from one or more of melanoma, glioma, cervical cancer, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer and kidney cancer.

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