A chimeric antigen receptor (Vγ9Vδ2) T cell expressing B7-H3 and its application

Abstract

This disclosure pertains to the field of immunotherapy and relates to a Vγ9Vδ2T cell expressing a chimeric antigen receptor targeting B7-H3 and its application. Specifically, it provides an antibody that specifically binds to B7-H3 or its antigen-binding fragment, a chimeric antigen receptor comprising the aforementioned antibody or its antigen-binding fragment, and its use in the preparation of drugs for treating diseases. CAR-Vγ9Vδ2T cells prepared from the CAR sequence screened in this disclosure proliferate normally, no suicide attack was observed, cell viability is high, and they can effectively kill tumor cells both in vivo and in vitro.

Description

Technical Field

[0001] This disclosure pertains to the field of immunotherapy, specifically relating to a Vγ9Vδ2 T cell expressing a chimeric antigen receptor targeting B7-H3 and its applications. Background Technology

[0002] Cancer is one of the major diseases threatening human health. Common cancer treatments include radiotherapy, chemotherapy, and targeted therapy, while immunotherapy is gradually becoming a mainstream treatment. Chimeric antigen receptor-mediated cell therapy is a type of immunotherapy. Currently, nine chimeric antigen receptor-mediated autologous T-cell therapies are available globally, achieving remarkable success in treating myeloma and B-cell-related tumors, with some patients achieving cure (no recurrence for 5 years). This has also driven the rapid development of this therapy in the field of hematological malignancies. However, due to factors such as the long autologous cell preparation cycle, patient disease deterioration during the waiting period, and failure to prepare the cells 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 shows poor performance in treating solid tumors. Therefore, the development of universal T-cells targeting new chimeric antigen receptors is crucial to solving this problem.

[0003] B7-H3 is a membrane protein belonging to the B7 immunomodulatory factor family. In humans, two subtypes exist: 2Ig-B7-H3 and 4Ig-B7-H3, while mice only have the 2Ig-B7-H3 subtype. Literature reports that B7-H3 can reduce the release of interferon-1 from T cells and inhibit the killing function of natural killer cells; therefore, B7-H3 is considered an immunosuppressive factor. Other literature has also confirmed that B7-H3 can inhibit graft-versus-host disease, heart transplant rejection, airway inflammation, and autoimmune encephalomyelitis. However, some literature describes B7-H3 as a T cell co-stimulatory factor in in vitro and in vivo autoimmune models.

[0004] B7-H3 is expressed at extremely low levels in normal human tissues, but is often overexpressed in tumors (ranging from 74% to 94%). Furthermore, studies have found high expression of B7-H3 in tumor-associated blood vessels and fibroblasts of the tumor stroma. High expression of B7-H3 is often closely associated with fewer tumor-infiltrating lymphocytes within the tumor, rapid cancer progression, and poor clinical treatment outcomes, and is commonly found in cancers such as glioma, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer, and kidney cancer. Because B7-H3 is highly expressed in various solid tumors, it can serve as a good target for the treatment of many solid tumors. Simultaneously, since B7-H3 is expressed on the cell surface, chimeric antigen receptor-mediated cell therapy can be used to target it, aiming to kill tumor cells without harming normal cells.

[0005] T cells are divided into two main categories based on their TCR: αβ T cells and γδ T cells. Human peripheral blood lymphocytes are predominantly αβ T cells, with γδ T cells typically accounting for only 1%-5%. The most prevalent γδ T cell subset is Vγ9Vδ2 T cells, comprising approximately 90%. γδ T cells can directly kill tumor cells through their cell surface NK cell receptors, ADCC effects, and secreted cytokines (IFN-γ, TNF-α). γδ T cells can also indirectly kill tumor cells by activating B / DC / αβ T / NK cells through other mechanisms (e.g., acting as antigen-presenting cells to activate αβ T cells, or inducing NK-mediated anti-tumor cytotoxicity via the 4-1BB co-stimulatory pathway).

[0006] Patients with tumors infiltrated by γδ T cells have the best prognosis compared to those with tumors infiltrated by other immune cells. Furthermore, unlike conventional cancer therapies (such as chemotherapy and radiotherapy), there have been no serious adverse reactions associated with γδ T cell therapy to date. γδ TCRs are inherently unrestricted by the MHC (histocompatibility complex), meaning that if donor γδ T cells are transferred to a patient (allogeneic cell therapy), the γδ T cells will not recognize the patient's body as foreign, thus avoiding GvHD (graft-versus-host disease), making it a high-quality source of allogeneic cell products. However, there are currently few publicly disclosed research projects on Vγ9Vδ2 T cells, and most are not modified. Moreover, current general cell production processes have significant limitations in scaling up the production of Vγ9Vδ2 T cells expressing chimeric antigen receptors. The single-dose cell quantity from peripheral blood of healthy individuals is low, making initial activation and expansion difficult. Furthermore, CAR virus transduction of Vγ9Vδ2 T cells has a low success rate, and the large amount of virus used increases costs. In addition, after CAR virus transfection, most T cells are exhausted due to their own recognition and killing, and cannot meet the requirements for subsequent expansion and preparation.

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

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

[0009] In one aspect, this disclosure provides 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.

[0010] The light chain variable region comprises: LCDR1 having the amino acid sequence shown in SEQ ID NO.5 or any variant thereof, LCDR2 having the amino acid sequence shown in SEQ ID NO.6 or any variant thereof, and LCDR3 having the amino acid sequence 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, this disclosure provides a chimeric antigen receptor comprising an extracellular antigen-binding domain, said extracellular antigen-binding domain comprising the aforementioned antibody or an antigen-binding fragment thereof.

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

[0014] In another respect, this disclosure provides an expression vector comprising the aforementioned polynucleotides.

[0015] In another aspect, this disclosure provides a cell that contains or contains the aforementioned polynucleotides or the aforementioned expression vectors.

[0016] In another aspect, this 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 cells.

[0017] In another aspect, this disclosure provides the use of the aforementioned antibody or its antigen-binding fragment, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector, and the aforementioned cells in the preparation of a drug for treating diseases.

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

[0019] (1) CAR-Vγ9Vδ2 T cells prepared from the CAR sequence screened in this publication have normal proliferation, no suicide injury phenomenon was observed, and the cell survival rate can be maintained at a high level.

[0020] (2) The present invention prepares Vγ9Vδ2 T cells that can target the chimeric antigen receptor of B7-H3. These cells can effectively kill tumor cells in vivo and in vitro. Experiments in a mouse glioma model in vivo show that Vγ9Vδ2 T cells with the chimeric antigen receptor of B7-H3 can clear tumors in mice and control tumor recurrence for at least 70 days. Attached Figure Description

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

[0022] Figure 2 shows the expression of B7-H3 in glioma and ovarian cancer samples. Figure 2A The expression of B7-H3 in 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 status of Vγ9Vδ2 T cells with different B7-H3 chimeric antigen receptors. Figure 4A Microscopic morphology of Vγ9Vδ2 T cells with different B7-H3 chimeric antigen receptors. Figure 4B The mean cell viability is denoted as .

[0025] Figure 5 shows the expression of the B7-H3 chimeric antigen receptor on Vγ9Vδ2 T cells. Among them, Figure 5A Positive transduction results for different chimeric antigen receptors. Figure 5B Statistical results of positive transduction rates for different chimeric antigen receptors.

[0026] Figure 6 shows the co-culture of B7-H3 chimeric antigen receptor-T cells and tumor cells. Figure 6A To detect the ratio of T cells to tumor cells after co-culture by flow cytometry. Figure 6B This is a statistical representation of the kill effect at different effective-to-target ratios.

[0027] Figure 7 This represents the cytokine levels during tumor killing by B7-H3 chimeric antigen receptor-T cells.

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

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

[0030] I. Terminology

[0031] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless it is obvious elsewhere in this document that they are explicitly defined, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0032] The articles “a” and “a kind” used in this article refer to one or more (i.e., at least one) grammatical objects. For example, “a kind of element” means one element or more elements.

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

[0034] As used in this article, the term "activation" refers to the state of T cells that have been adequately stimulated to induce detectable cell proliferation. Activation can 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 contains at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signal transduction domain (also referred to herein as an “intracellular signal transduction domain”) (including the functional signal transduction domain).

[0036] As used herein, the terms “nucleic acid,” “nucleotide,” or “oligonucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, as well as polymers thereof. Unless explicitly limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conserved variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixture of bases 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. Antibodies can be complete immunoglobulins derived from natural or recombinant sources and can be the immunoreactive portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in this disclosure can exist in a variety of forms, including but not limited to: polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, 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 full-length but contains at least a portion of the antibody's variable region (e.g., one or more CDRs and / or one or more antibody-binding sites) that binds to an antigen, and thus retains binding specificity and at least a portion of the full-length antibody's specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as the derived antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant 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, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p3-25, Kipriyanov). The fragment may comprise 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, upon insertion into an antibody framework (e.g., by replacing the corresponding region), acquires an antibody that specifically binds to (i.e., exhibits a Ka of at least or at least about 107-108 M⁻¹) an antigen. A “functional fragment” or “analog of an anti-B7-H3 antibody” is a fragment or analogue that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as “antibody fragment”, and, in the context of an antibody, may refer to a fragment that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. An “Fv” fragment consists of a dimer (VH-VL dimer) formed by the non-covalent binding of a variable domain of a heavy chain and a 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 is the case with the intact antibody. The six CDRs collectively confer target-binding specificity to the intact antibody. However, even a single variable domain (or half the Fv of a domain containing 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, meaning that each individual antibody molecule in a population of monoclonal antibodies is identical to the others. This characteristic contrasts with that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of 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 from immortalized B cells, for instance, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibody.

[0040] As used in this article, the term "heavy chain" refers to the larger of the two types of polypeptide chains present in the spontaneous conformation of all antibody molecules.

[0041] As used in this article, the term "light chain" refers to the smaller chain in the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation, with κ and λ light chains referring to the two main isotypes of antibody light chains.

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

[0043] As used herein, the term "co-stimulatory factor" refers to an associated binding chaperone on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand, thereby enabling the immune cell to mediate a co-stimulatory response, such as, but not limited to, proliferation and survival. The co-stimulatory signaling region may be derived from an intracellular signaling region of 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 co-stimulatory signaling region is derived from 4-1BB. In some embodiments, the 4-1BB co-stimulatory signaling region comprises the amino acid sequence shown in SEQ ID NO. 26.

[0044] As used in this article, the term "fratricide" refers to the phenomenon in which T cells recognize and kill other T cells during their proliferation. Specifically, the expression of target antigens on CAR-T cells can induce CAR-T cells to kill each other and lose their efficacy, thus reducing their clinical benefits.

[0045] As used herein, the terms "antitumor effect" or "antitumor activity" refer to biological effects that can be manifested in a variety of ways, including but not limited to, for example, tumor volume reduction, tumor cell number reduction, metastasis reduction, increased life expectancy, reduced tumor cell proliferation, decreased tumor cell survival, or improvement of various physiological symptoms associated with cancer. "Antitumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of this disclosure to prevent the occurrence of tumors in the primary sense (e.g., in prophylactic therapy or treatment). Cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenström macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease and μ chain disease, benign monoclonal gammopathy, immune cell amyloidosis, melanoma, breast cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, glioma, bladder cancer, cancers of the brain or central nervous system, cancers of the peripheral nervous system, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancers 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, and hematologic malignancies. Other non-limiting examples of cancer types applicable to the methods included in this disclosure include human sarcomas and cancers such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovoma, 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, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumors, and testicular tumors. Testicular cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); as well as polycythemia vera, lymphoma (Hodgkin's and non-Hodgkin's), multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease. In some embodiments, the cancers are melanoma, glioma, cervical cancer, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer, and kidney cancer.

[0046] A "vector" is a composition of substances containing isolated nucleic acids and capable of delivering those isolated nucleic acids into cells. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0047] As used in this article, the term “self” refers to any material derived from the same subject and subsequently reintroduced into that subject.

[0048] As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species as the subject, which is introduced into the subject. Two or more subjects are referred to as allogeneic to each other when the genes at one or more loci are different. In some embodiments, allogeneic material from individuals of the same species may be genetically different to a degree (e.g., at a particular gene, such as an MHC allele) sufficient to allow antigen-antigen interactions. In some embodiments, allogeneic material from individuals of the same species may be genetically similar to a degree (e.g., at a particular gene, such as an MHC allele) sufficient to prevent antigen-antigen interactions.

[0049] The isolated nucleic acid molecules of this disclosure include any nucleic acid molecule encoding a polypeptide or fragment thereof. Such nucleic acid molecules do not need to be 100% homologous or identical to endogenous nucleic acid sequences, but will generally exhibit substantially similarity. Nucleic acids having “substantially similarity” or “substantially homology” to endogenous sequences are generally capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. As used herein, “hybridization” refers to pairing under various stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof. For example, stringent salt concentrations can be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low-stringency hybridization can be obtained in the absence of organic solvents (e.g., formamide), while high-stringency hybridization can be obtained in the presence of at least about 35% or at least about 50% formamide. Strict temperature conditions typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Various other parameters, such as hybridization time, detergent concentration (e.g., sodium dodecyl sulfate (SDS)), and inclusion or exclusion of vector DNA, are well known to those skilled in the art. Different levels of stringency can be achieved by combining these different conditions as needed.

[0050] The terms "substantially identical" or "substantially homologous" mean that the polypeptide or nucleic acid molecule exhibits at least about 50% homology or similarity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or 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 similar at the amino acid level or nucleic acid level to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (e.g., the 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 typically 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 for determining the degree of identity, the BLAST procedure can be used, where probability scores between e-3 and e-100 represent closely related sequences.

[0051] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include their progeny. Therefore, the words “transformation” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are used, the context will be clear.

[0052] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0053] "Pharmaceutical composition" means containing one or more of the modified T cells described herein, as well as other components such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0054] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0055] "Treatment" means administering an oral or topical therapeutic agent, such as a modified T cell comprising any of the present disclosure, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of a disease, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measured degree. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. While the disclosed embodiments (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease present in every patient, they should reduce the symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student 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 “consistently of” or variations thereof, used throughout the specification and claims, means that all said elements or groups of elements are included, and optionally include other elements that are similar to or different in nature from said elements, which do not significantly alter or introduce new properties to the specified dosing regimen, method or composition.

[0057] II. Detailed Implementation Plan

[0058] In one aspect, this disclosure provides 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.

[0059] The light chain variable region comprises: LCDR1 having the amino acid sequence shown in SEQ ID NO.5 or any variant thereof, LCDR2 having the amino acid sequence shown in SEQ ID NO.6 or any variant thereof, and LCDR3 having the amino acid sequence 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 that specifically binds to B7-H3 or its antigen-binding fragment 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] The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO.9, or the 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 that specifically binds to B7-H3 or its antigen-binding fragment comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO.4 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO.9.

[0065] In some implementations, the antibody that specifically binds to B7-H3 or its antigen-binding fragment has been genetically engineered or modified.

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

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

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

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

[0070] In some implementations, the chimeric antigen receptor further includes a hinge region, a transmembrane region, a co-stimulatory domain, and a signal transduction domain.

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

[0072] In some implementations, 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 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 co-stimulatory domain is CD28 or 4-1BB. In some embodiments, the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO.24 or SEQ ID NO.26.

[0078] In some implementations, 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 the N-terminus to the 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 signal transduction region.

[0081] In some embodiments, the chimeric antigen receptor further comprises one or more of membrane proteins, secretory proteins, intracellular proteins, small molecule drugs, and cytotoxic drugs.

[0082] On the other hand, this disclosure provides a polynucleotide comprising the aforementioned antibody or its antigen-binding fragment, or the aforementioned chimeric antigen receptor nucleotide sequence. On the other hand, this disclosure provides an expression vector comprising the aforementioned polynucleotide.

[0083] On the other hand, this disclosure provides a cell that contains or contains the aforementioned polynucleotides or the aforementioned expression vectors.

[0084] In some embodiments, the cells are one or more of the following: 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.

[0085] In some implementations, the cells are T cells.

[0086] In some implementations, the T cells include αβT cells, γδT cells, and regulatory T cells.

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

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

[0089] On the other hand, this disclosure provides the use of the aforementioned antibody or its antigen-binding fragment, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector, and the aforementioned cells in the preparation of a drug for treating diseases.

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

[0091] In some implementations, the cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenström macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease, and μ 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, cancers of the brain or central nervous system, cancers of the peripheral nervous system, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancers of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small bowel or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, and hematologic malignancies. Other non-limiting examples of cancer types applicable to the methods included in this disclosure include human sarcomas and cancers such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovoma, 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, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumors, and testicular tumors. Testicular cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemia such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); as well as polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia and heavy chain disease.

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

[0093] In some implementations, the glioma includes glioblastoma, medulloblastoma, astrocytoma, and oligodendroglioma.

[0094] On the other hand, this disclosure provides a method for treating a disease or condition by administering to a subject a therapeutically effective amount of the aforementioned Vγ9Vδ2 T cell or pharmaceutical composition expressing a chimeric antigen receptor targeting B7-H3, wherein the method further includes administering a second drug or therapy. In some embodiments of this disclosure, the disease or condition is cancer or a related disease.

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

[0096] On the other hand, this disclosure provides a method for treating a subject, the method comprising administering to the subject a therapeutically effective amount of the aforementioned Vγ9Vδ2 T cells or pharmaceutical composition expressing a chimeric antigen receptor targeting B7-H3.

[0097] On the other hand, this disclosure provides a method for detecting B7-H3, which includes the steps of using the aforementioned antibody or its antigen-binding fragment, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned cells to detect B7-H3 in a sample.

[0098] On the other hand, this disclosure provides a kit for detecting B7-H3, which includes the aforementioned antibody or its antigen-binding fragment, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned expression vector or the aforementioned cells.

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

[0100] Example

[0101] A further understanding of this disclosure can be obtained by referring to some specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. It will be apparent that various modifications and variations can be made to this disclosure without departing from its essence, and therefore such modifications and variations are also within the scope of protection claimed in this application.

[0102] Example 1: Cell Culture

[0103] The cell lines used in this disclosure are all adherent cells, including 293T cells, WM-266-4, SK-OV-3, LN-229, U-87 MG, and HeLa cells, cultured in DMDM ​​medium containing 10% FBS, 1% GlutaMAX, and 1% penicillin-dextrose antibodies. Among them, 293T (a gift from the laboratory of Zhao Bin, Zhejiang University) is a human embryonic kidney cell line, commonly used for virus preparation. WM-266-4 (a gift from the laboratory of Zhao Bin, Zhejiang University) is a human melanoma cell line, SK-OV-3 (a gift from the laboratory of Zhao Bin, Zhejiang University) is a human ovarian cancer cell line, LN-229 (a gift from the laboratory of Zhao Bin, Zhejiang University) and U-87 MG (a gift from the laboratory of Zhao Bin, 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 a GFP-encoding retrovirus. The tumor cell lines used in mice are cell lines obtained by infecting target cells with a lentivirus encoding Luciferase. Retroviruses encoding GFP were prepared by transiently transfecting 293T cells using a three-plasmid system (SFG-GFP, MMLV-gag-pol, and pVSV-G). Lentivirals encoding Luciferase were prepared by transiently transfecting 293T cells using SFG-GFP-FFLuc, MMLV-gag-pol, and pVSV-G.

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

[0105] To determine the expression of the B7-H3 antigen in tumors, the applicant first selected different tumor cell lines for testing. Simultaneously, to further verify the expression of B7-H3 in primary tumors, the applicant examined the expression of B7-H3 in sections containing glioma tissue and ovarian cancer tissue from multiple patients.

[0106] The specific experimental procedure 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 glioblastoma cells U-87MG, and human cervical cancer cells HeLa) were collected and washed three times with 1xPBS. Then, they were incubated with B7-H3 antibody ID35 (1:100, diluted with 1xPBS) at 2-8℃ for 30 min. After washing twice with 1xPBS, APC-conjugated goat anti-human secondary antibody (Jackson ImmunoResearch, 109-605-044) was added at 1:100 with 1xPBS and incubated at 2-8℃ for 30 min. After washing with 1xPBS, the expression of B7-H3 on the tumor cells was detected by flow cytometry.

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

[0110] Paraffin sections were dewaxed with xylene and rehydrated with alcohol, followed by antigen retrieval using sodium citrate. They were then blocked in serum for 1 hour. B7-H3 antibody ID35 staining was performed overnight at 2-8°C, followed by incubation with recombinant Anti-HLA Class 1 ABC antibody (abcam, ab225636) for 30 minutes. After washing, DAB staining was performed, followed by hematoxylin staining. The sections were then observed and photographed under a microscope after staining.

[0111] The results of flow cytometry analysis are shown below. Figure 1 .from Figure 1 It was found that B7-H3 was highly expressed in various tumor cell types. The section results of glioma and ovarian cancer tissues are shown below. Figure 2A , 2B Compared to the negative control in 2B, varying degrees of B7-H3 expression were detected in all glioma samples and most ovarian cancer samples. This suggests that B7-H3 is a promising target for solid tumor therapy.

[0112] Example 3: Construction of chimeric antigen receptors

[0113] This disclosure describes a second-generation chimeric antigen receptor, based on the sequence of the monoclonal antibody ID35 generated against the B7-H3 protein fragment, and its structure is shown in the attached figure. Figure 3 As shown, it includes the single-chain antibody region scFv (SEQ ID NO: 1, 2), the hinge region and transmembrane region of CD8α, the co-stimulatory region of CD28 or 4-1BB, and the CD3ζ signal transduction region. Other patented intra-sequence structures selected are also shown in the appendix. Figure 3As shown. The single-chain antibody region of the chimeric antigen receptor is derived from antibodies against B7-H3 produced by various monoclonal hybridoma cells. The chimeric antigen receptor is produced by gene synthesis and then cloned into a lentiviral vector. After cloning, its sequence accuracy 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] Example 4: Preparation of chimeric antigen receptor lentivirus

[0118] Based on the chimeric antigen receptor obtained in Example 3, this example further prepared lentiviral particles containing the chimeric antigen receptor. Specific experimental details are as follows:

[0119] Day 1, 6 x 10 6 293T cells were cultured in 100 mm cell culture dishes with a volume of 10 mL. After 24 h, a three-plasmid system (6 μg, 4 μg, 2 μg) was used to transiently transfect 293T cells. The expression plasmid backbone was derived from pCDH-EF1a-eFFly-eGFP plasmid (Addgene, Plasmid #104834)), the helper plasmid pspAx2 (Addgene, #12260) and the helper plasmid pCMV-VSV-G (Addgene, Plasmid #8454). The three plasmids were mixed in 500 μL of transfection reagent buffer, and 20 μL of jet PRIME transfection aid was added and mixed well. Replace with fresh DMEM medium after 6 hours. Collect the supernatant 48 and 72 hours after transfection (the 48-hour supernatant can be stored at 4°C and co-processed with the 72-hour supernatant). Filter through a 0.45 μm syringe filter and centrifuge at 70,000 rcf for 2 hours (centrifuge speed 9, deceleration 3). After centrifugation, resuspend the virus particles in 1% volume of RPMI-1640 medium from the initial virus solution and store at -80°C for later use.

[0120] Example 5: Preparation of chimeric antigen receptor Vγ9Vδ2 T cells expressing B7-H3

[0121] This embodiment further prepared chimeric antigen receptor Vγ9Vδ2 T cells expressing B7-H3. The specific experimental methods are as follows:

[0122] 1. Amplification of Vγ9Vδ2 T cells and viral transduction

[0123] Peripheral blood mononuclear cells were isolated from donated blood from healthy individuals (with informed consent from the subjects). Monocytes were then isolated using a lymphocyte density gradient separation method. The isolated mononuclear cells were cultured in RPMI-1640 medium containing 10% FBS, 1% glutaMAX, 1% penicillin-dextrin antibiotics, and 100 U / mL rhIL-2, with an initial cell density controlled at 2 x 10⁻⁶ cells / mL. 6 Cells / mL were seeded in 24-well plates, and Vγ9Vδ2 T cells were activated with 5 μM zoledronic acid (ZOL). Half the medium was changed every 48 hours. After 4 days, the cell density was adjusted to 1 x 10⁻⁶ cells / mL. 6 Continue culturing at cells / mL, changing the medium every 48 hours.

[0124] Collect Vγ9Vδ2 T cells activated and cultured in vitro, and adjust the cell density to 1x10 after cell counting. 6 Cells / mL, seeded into 24-well plates (1 mL). Add 1 x 10⁻⁶ cells / mL to each well. 7 TU lentivirus was cultured in a cell culture incubator, and half of the medium was replaced after 24 hours. Thereafter, the medium was increased by 1 x 10⁻⁶ cells every 48 hours. 6 The cells / mL were cultured in a different medium, and subsequent expansion cultures were used to detect chimeric antigen receptor expression and tumor-killing ability. T cell counts and cell viability were determined using an AOPI dye analyzer.

[0125] 2. Detection of B7-H3 chimeric antigen receptor expression

[0126] Receive 1x10 6 The expression of the B7-H3 chimeric antigen receptor was detected in expanded CAR-Vγ9Vδ2T cells using a fusion protein of B7-H3 and antibody Fc fragment (1:100, 1xPBS dilution). The cells were incubated at room temperature for 30 min. The secondary antibody was APC fluorescent dye-labeled goat anti-human antibody Fc binding protein (1:100, 1xPBS dilution), and the cells were incubated at 2-8℃ for 30 min. Flow cytometry was used to detect the expression of the chimeric antigen receptor on T cells.

[0127] Lentiviral cells were prepared in 293T using a lentiviral vector containing the chimeric antigen receptor and then infected with human Vγ9Vδ2 T cells (from various 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 exhibit different proliferation states. Repeated experiments were conducted, and cell viability was statistically analyzed. The results show... Figure 4B Significant differences in the viability of Vγ9Vδ2 T cells expressing different chimeric antigen receptors were observed. Compared with the untransduced NT group and the GFP-transduced control group, T cells prepared from other reference chimeric antigen receptors targeting the same target showed significant depletion due to mutual recognition and killing, failing to meet the requirements for viability and quantity in subsequent expansion. However, CAR-Vγ9Vδ2 T cells prepared from the CAR sequence screened in this disclosure proliferated normally, with no observed suicide attack, and maintained a high viability. Flow cytometry was used to verify the expression of the B7-H3 chimeric antigen receptor on the surface of Vγ9Vδ2 T cells after CAR virus transduction. The results of B7-H3 chimeric antigen receptor expression detection are shown in Figure 5. As shown in Figure 5, >50% of T cells expressed the chimeric antigen receptor disclosed in this disclosure, i.e., the CAR positive transduction rate was >50%, which not only satisfies the needs of functional testing experiments but also provides a guarantee for further application in production. This result was also verified in T cells from several other healthy donors.

[0128] Example 6: Killing of tumor cells by CAR-Vγ9Vδ2 T cells in an in vitro co-culture system

[0129] To further determine whether the chimeric antigen receptor disclosed herein can mediate the killing of tumor cells with high B7-H3 expression by Vγ9Vδ2 T cells, the applicant designed a co-culture experiment to test the killing effect of the T cells on various tumor cells. After the tumor cells adhered, T cells expressing control and B7-H3 chimeric antigen receptors were added, respectively. Then, the killing effect of each experimental group on various tumor cells was detected by flow cytometry.

[0130] The specific experimental procedure is as follows:

[0131] 1. Long-term cytotoxicity testing (cell co-culture)

[0132] In a 24-well plate, seed 1-2 x 10⁻⁶ cells per well. 5 GFP-labeled tumor cells (WM-266-4, SK-OV-3, U-87MG, HeLa) were added 24 hours later with appropriate numbers of T cells at effector-to-target ratios of 1:1, 1:2, and 1:5. Cells were collected from the wells two days later, and the proportion of residual tumor cells was detected by flow cytometry. Vγ9Vδ2 T cells were detected using CD3+, and tumor cells were detected using GFP.

[0133] 2. Enzyme-linked immunosorbent assay (ELISA)

[0134] In the co-culture experiment of tumor cells and chimeric antigen receptor-T cells, CAR-Vγ9Vδ2 T cells were added. After 24 hours, the supernatant of the culture medium was collected in 96-well plates and stored at -80°C. The cytokines released in the supernatant were detected using ELISA kits (INF-γ assay kit, R&D, DY285B; IL-2 assay kit, R&D, DY202).

[0135] The results of the flow cytometry experiment are shown in Figure 6. Figure 6A It can be seen that, in a 1:2 effector-to-target ratio, flow cytometry analysis revealed that the control group T cells had no killing effect on any type of tumor cell, while the B7-H3 chimeric antigen receptor T cells disclosed herein could effectively kill tumor cells in co-culture with various target-positive tumor cells. Figure 6B A summary of residual cell proportions from multiple effector-to-target ratio experimental groups was obtained by flow cytometry. It was observed that the B7-H3 chimeric antigen receptor T cells disclosed herein effectively killed tumor cells at different effector-to-target ratios. The results of cytokine release during T cell killing are shown in [Figure number missing]. Figure 7 ,Depend on Figure 7 It is known that the levels of cytokines released by the control group T cells were all below the detection range, while the B7-H3 chimeric antigen receptor T cells disclosed in this paper released a large number of Th1 cytokines (including IFNγ and IL-2) during the killing process, indicating that the CAR-Vγ9Vδ2 T cells prepared in this paper have the ability to kill tumor cells and can also activate other tumor killing mechanisms.

[0136] Example 7: Xenograft orthotopic tumor mouse experiment

[0137] To further determine the tumor-killing effect of T cells expressing the B7-H3 chimeric antigen receptor as disclosed in this disclosure, the applicant selected a glioma model in male nude mice (Shanghai Slack, BALB / c). Luciferase-labeled human tumor LN-229 cells were injected into the brains of nude mice at 6-8 weeks of age. After tumor formation, PBS, control NT, and B7-H3-CAR-T cells were injected in situ. Imaging was then used to detect the tumor clearance by T cells and the survival of the treated mice.

[0138] The specific experimental procedure is as follows:

[0139] In vivo experiments of CAR-Vγ9Vδ2 T cell targeting glioma were conducted in nude mice aged 6-8 weeks. Luciferase-labeled tumor cell line LN-229 was stereotactically implanted into the brains of nude mice. Two weeks later, after confirming successful tumor transplantation using an in vivo imaging system, control or B7-H3 chimeric antigen receptor-Vγ9Vδ2 T cells were injected in situ. Imaging was then performed weekly to observe the tumor-killing effect of T cells. Two experiments were conducted in total; the initial results are shown below. Figure 8 The results of repeated experiments are shown in Figure 9 .

[0140] The results of B7-H3 chimeric antigen receptor-Vγ9Vδ2 T cell killing of gliomas in mice are shown in the attached figure. Figure 8 , 9 As shown, the increasingly stronger detection signal indicates the continuous proliferation of intracranial tumor cells in mice, among which... Figure 8 The pale blue diffuse signal patch in the middle represents background noise. Figure 9 The red signal indicates that the upper limit of the imaging threshold has been reached, and the imaging defect represents natural death of the mouse or euthanasia in accordance with animal ethics requirements. Experimental results showed that, compared with the control group whose tumors continued to grow and died after 6 weeks, the B7-H3 chimeric antigen receptor Vγ9Vδ2 T cells disclosed herein were able to clear tumors in most mice and control tumor recurrence within 2 months. This result indicates that B7-H3 chimeric antigen receptor T cells can kill tumors in vivo and ensure tumor recurrence-free status for at least 70 days, demonstrating that the B7-H3 chimeric antigen receptor T cells disclosed herein can serve as a potential cancer treatment method.

[0141] The above description is merely an embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A chimeric antigen receptor targeting B7-H3, wherein, The chimeric antigen receptor comprises an anti-B7-H3 antibody domain, a hinge region, a transmembrane region, a co-stimulatory domain, and a signal transduction domain. The amino acid sequence of the anti-B7-H3 antibody is shown in SEQ ID NO.2; The hinge region is a CD8α hinge region, and the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.20; The transmembrane region is the CD8α transmembrane region, and the amino acid sequence of the CD8α transmembrane region is shown in SEQ ID NO.22; The co-stimulatory domain is CD28 or 4-1BB, the amino acid sequence of CD28 is shown in SEQ ID NO.24, and the amino acid sequence of 4-1BB is shown in SEQ ID NO.26; The signal transduction domain is CD3ζ, and the amino acid sequence of CD3ζ is shown in SEQ ID NO.

28.

2. A polynucleotide, wherein, The polynucleotide comprises a nucleotide sequence encoding the chimeric antigen receptor of claim 1.

3. An expression carrier, wherein, The expression vector comprises the polynucleotide of claim 2.

4. A type of cell, in which, The cells are introduced into or contain the polynucleotide of claim 2 or the expression vector of claim 3, and the cells are T cells.

5. The cell according to claim 4, wherein, The T cells include αβT cells, γδT cells, and regulatory T cells.

6. The cell according to claim 5, wherein, The T cells mentioned are γδT cells.

7. A pharmaceutical composition comprising one or more of the chimeric antigen receptor of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, and the cells of any one of claims 4-6.

8. Use of the chimeric antigen receptor of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, and the cell of any one of claims 4-6 in the preparation of a medicament for treating diseases; The disease in question is melanoma, glioma, cervical cancer, or ovarian cancer.

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