A B7H3 nanobody, a chimeric antigen receptor, and its applications and products

Abstract

This invention belongs to the field of antibody technology, specifically relating to a B7H3 nanobody, a chimeric antigen receptor, and their applications and products. The B7-H3 nanobody provided by this invention possesses a specific CDR sequence, exhibiting high affinity and strong specificity. A single-target chimeric antigen receptor constructed based on this nanobody can efficiently recognize B7-H3-positive tumor cells, mediating specific killing by immune cells. Further, this nanobody is tandemly linked with an IL13Rα2 nanobody to obtain a dual-target chimeric antigen receptor, capable of simultaneously recognizing both B7-H3 and IL13Rα2 antigens. This invention also includes nucleic acids encoding the above molecules, a vector, and CAR-T / CAR-γδT cells. The single-target formulation significantly kills tumors with high B7-H3 expression, while the dual-target formulation can simultaneously kill both single-positive and double-positive tumor cells, effectively overcoming antigenic heterogeneity and tumor escape, and can be used to prepare diagnostic reagents and therapeutic drugs for various malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a B7H3 nanobody, a chimeric antigen receptor, and their applications and products. Background Technology

[0002] Immunotherapy, especially chimeric antigen receptor T-cell (CAR-T) therapy, has shown significant potential in cancer treatment. In the development of CAR-T therapy, the CAR molecule is the most important structural element mediating the function of CAR-T, and the antigen recognition region is usually the scFv (single-chain variable fragment). Most of the CAR-T cell therapies reported so far use scFv derived from murine or human antibodies. However, traditional CARs rely on scFv as the antigen recognition domain, and its inherent defects may limit the development of dual-target therapies: (1) The murine component in murine or humanized antibody fragments can cause immune rejection in patients, thereby reducing the persistence of CAR-T in vivo and affecting efficacy. (2) The scFv size is too large, making it difficult to develop dual-target CAR structures. In the process of preparing dual-target CARs, if two scFvs are introduced to recognize two antigens, the gene of the entire CAR molecule will be too long, reducing the transduction efficiency. In addition, the variable regions of the heavy and light chains of the two scFvs may interact with each other, thus affecting the function of T cells. Therefore, the design is more difficult and requires more screening and optimization work. Compared with scFvs, alpaca-derived nanobodies (VHHs) have significant advantages: (1) high homology with human genes and low immunogenicity; (2) small size, suitable as the antigen-binding domain of dual-target CARs, and only contains one heavy chain, reducing the difficulty of development; (3) high affinity, and nanobodies that do not require affinity optimization can be screened, simplifying the development process; (4) can recognize cavities or hidden epitopes that traditional antibodies cannot recognize; (5) good permeability, which is expected to improve the ability of CAR-T cells to infiltrate tumors.

[0003] Currently, dozens of tumor antigens have been reported as potential targets for CAR-T therapy in hematological malignancies and solid tumors. Among them, B7 homologue 3 protein (B7-H3 / CD276) is an immune checkpoint molecule with dual regulatory functions, playing a co-stimulatory and co-inhibitory role in the immune response. Studies have found that B7-H3 is rarely expressed in normal human tissues, but is abnormally highly expressed in various malignant brain tumors such as glioblastoma, medulloblastoma, and ependymoblastoma, as well as in various tumor tissues such as non-small cell lung cancer, pancreatic cancer, primary liver cancer, colorectal cancer, breast cancer, prostate cancer, laryngeal cancer, and melanoma, thus making it a potential target for the treatment of various solid tumors. Interleukin-13 receptor α2 (IL13Rα2) is a transmembrane glycoprotein belonging to the cytokine receptor family, mainly participating in cell signaling by binding to interleukin-13 (IL-13). While its expression level is extremely low in normal tissues, it is overexpressed in melanoma, renal cell carcinoma (RCC), adrenocortical carcinoma (ACC), and various brain tumors. Furthermore, overexpression of IL13Rα2 is associated with advanced disease and poor prognosis in colorectal cancer (CRC), gastric cancer, breast cancer, clear cell ovarian cancer, lung cancer, ACC, papillary thyroid carcinoma, pancreatic ductal adenocarcinoma, and glioblastoma (GBM). Therefore, it serves as a biomarker for prognosis after treatment interventions in many solid tumors. Studies have found that both B7H3 and IL13Rα2 antigens are expressed simultaneously in various tumors, and the degree of overlap in their expression varies across different tumor types. For example, in glioblastoma, analysis of single-cell sequencing data revealed that they are expressed in different tumor cell populations. Therefore, developing single-target B7H3 and dual-target B7H3 and IL13Rα2 cell immunotherapies is of significant importance for the clinical treatment of various tumors. Summary of the Invention

[0004] To address the above shortcomings, this invention provides a B7H3 nanobody, a chimeric antigen receptor, and their applications and products.

[0005] The technical solution of this invention is as follows: Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0006] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0007] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0008] The term "T cell" in this invention refers to one of the important white blood cell types in the human immune system, playing a crucial role in the acquired immune response. Based on the composition of the T cell receptor (TCR), they are mainly divided into two categories: αβ T cells and γδ T cells. αβT cells, composed of the TCRα and TCRβ chains forming a heterodimer TCR, are the dominant T cell subset in peripheral blood. Their antigen recognition is restricted by the major histocompatibility complex (MHC), primarily recognizing antigenic peptides presented by MHC molecules, mediating specific cellular immune responses, including CD4+. + Helper T cells and CD8 + Cytotoxic T cells are the most commonly used effector cells in traditional CAR-T cell therapy.

[0009] γδT cells, composed of TCRγ and TCRδ chains, are mainly distributed in mucosal epithelial tissues and peripheral blood in the human body and belong to the innate lymphocyte category. Their antigen recognition is independent of MHC molecule presentation; they can directly recognize various ligands such as tumor antigens, stress molecules, and lipid antigens. They possess natural anti-tumor, broad-spectrum killing, and immunomodulatory properties, and are less likely to induce graft-versus-host disease (GVHD), making them highly advantageous effector cells in next-generation cell therapy.

[0010] In this invention, the term "antibody" refers to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or from the variable regions of the immunoglobulin light chain, thereby enabling specific binding to an antigen. The term "antibody" herein encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

[0011] In this invention, the terms "VHH domain" and "single-domain antibody" have the same meaning and can be used interchangeably. They refer to the variable region of a cloned heavy chain antibody, constructing a single-domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function.

[0012] The term "chimeric antigen receptor" in this invention refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells such as lymphocytes and specifically binds to antigens. It comprises at least (1) an extracellular antigen-binding region, such as a single-chain variable region fragment scFv, a single-domain antibody VHH, a ligand-binding domain, or an antigen-recognizing peptide; (2) a transmembrane region for anchoring and expressing the CAR molecule on the membrane of immune effector cells; and (3) an intracellular signal transduction domain responsible for initiating activation signals. The extracellular structure of the CAR may further include a hinge region / spacer region to enhance the flexibility and spatial accessibility of the antigen-binding region; the intracellular structure may further include one or more co-stimulatory molecular domains to form a co-stimulatory signal transduction domain to enhance the proliferation, survival, and killing efficacy of immune cells. CAR molecules can redirect immune effector cells such as T cells and NK cells to selected target cells, such as tumor cells, pathogen-infected cells, or autoimmune-related abnormal cells, in a non-MHC-restricted manner via their extracellular antigen-binding domain, thereby mediating a specific immune killing effect. In some embodiments of the present invention, the CAR includes, but is not limited to, first-generation, second-generation, third-generation, or fourth-generation CARs, as well as novel receptor structures based on the above-mentioned structural improvements or optimizations, such as STAR receptors, HIT receptors, and their functionally equivalent chimeric immune receptors.

[0013] The term "STAR receptor" as used in this invention refers to the Synthetic T cell receptor and Antigen Receptor, which is a synthetic chimeric immune receptor in the form of a double-stranded heterodimer. This receptor is typically composed of an α chain and a β chain, where the α chain contains a fusion structure of the antibody light chain variable region (VL) and the TCR α chain constant region (Cα), and the β chain contains a fusion structure of the antibody heavy chain variable region (VH) and the TCR β chain constant region (Cβ). The STAR receptor can assemble with the T cell's endogenous CD3 complex (CD3ε, CD3δ, CD3γ, CD3ζ) through its transmembrane region, specifically recognizing target antigens in a non-MHC-restricted manner and triggering physiological immune activation signals close to those of the natural TCR. It features high antigen sensitivity, precise signal regulation, and low risk of T cell exhaustion.

[0014] The term "HIT receptor" used in this invention refers to HLA-Independent TCR, or non-HLA-restricted T cell receptor, a type of artificial chimeric immune receptor that mimics the structure of the natural TCR but recognizes antigens without relying on HLA molecules. Its typical structure is a heterodimer, using an antibody-antigen binding domain to replace the variable region of the TCR, while retaining the constant region of the TCR to bind to the endogenous CD3 complex and transduce activation signals. The HIT receptor combines the non-MHC-restricted recognition characteristics of traditional CARs with the advantages of the natural TCR signaling pathway, enabling effective activation of immune effector cells under low antigen density conditions, and is particularly suitable for scenarios with low target antigen expression levels, such as solid tumors.

[0015] The term "TruC receptor" as used in this invention refers to a TCR fusion receptor, which is a type of artificial chimeric immune receptor designed with the natural TCR signaling pathway as its core. This receptor is typically a fusion structure, directly fusing an antigen-binding domain (such as nanobodies, scFv, etc.) to the constant region (Cα or Cβ) of the TCR α or β chain, without the need for additional artificial intracellular co-stimulatory domains. The TruC receptor can assemble with the endogenous TCR subunits and CD3 complex of T cells to form a functional receptor complex, specifically recognizing target antigens in a non-MHC-restricted manner and transducing immune activation signals through the natural TCR signaling pathway. It features high structural stability, signal transduction close to physiological states, and low risk of T cell exhaustion.

[0016] The term "TAC receptor" used in this invention refers to T cell antigen-coupled receptor, a type of multi-component artificial chimeric immune receptor. This receptor consists of an extracellular antigen recognition domain, a connector molecule binding domain, and an intracellular signal recruitment domain, independent of an artificial intracellular co-stimulatory signaling domain. After specifically binding to the target antigen through the extracellular antigen recognition domain, the TAC receptor recruits connector molecules and activates the endogenous TCR / CD3 signaling pathway of T cells, initiating the killing function of immune effector cells in a non-MHC-restricted manner. It features precise signal regulation, sustained immune cell activation, and strong adaptability to the tumor microenvironment.

[0017] The term "AbTCR receptor" as used in this invention refers to the antibody-TCR fusion receptor, a type of artificial chimeric immune receptor that combines antibody antigen recognition function with natural TCR signaling function. This receptor is typically a fusion structure, fusing an antibody antigen-binding domain (such as a single-domain antibody or scFv) with the constant region (Cα or Cβ) of the TCR α or β chain, retaining the TCR's ability to bind to the endogenous CD3 complex. AbTCR receptors can specifically recognize target antigens in a non-MHC-restricted manner and transduce activation signals through the natural TCR signaling pathway, combining the advantages of highly specific antibody binding and TCR physiological signal transduction, thereby enhancing the infiltration and killing efficacy of immune effector cells in the solid tumor microenvironment.

[0018] The term "nucleic acid" in this invention refers to any compound and / or substance comprising polymers containing nucleotides, such as polynucleotides. In this document, "nucleic acid," "polynucleotide," and "gene" are used synonymously. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby these bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'.

[0019] The terms "retrovirus" and "retroviral vector" in this invention refer to Retrovirus and Retroviral Vector, respectively. A retrovirus is a virus that can integrate a copy of its RNA genome into the DNA of the host cell it infects, thereby altering the host cell's genome.

[0020] The term "lentivirus" in this invention refers to a complex retrovirus that contains other genes with regulatory or structural functions in addition to the common retrovirus genes gag, pol, and env.

[0021] The term "lentiviral vector" in this invention refers to a vector created through multiple attenuations of HIV virulence genes using techniques such as gene editing and genetic engineering. For example, deleting genes like env, vif, vpr, vpu, and nef ensures the lentiviral vector's biocompatibility. Lentiviral vectors can stably integrate target genes, such as shuttle genes, into the chromosomes of target cells, allowing target cells to express the delivered shuttle genes long-term, providing a significant advantage for gene therapy. Furthermore, they do not transfer viral genes, thus avoiding the problem of transduced cells that can be destroyed by cytotoxic T cells. They also possess relatively high cloning capacity, sufficient to meet most anticipated clinical applications.

[0022] The term "signal peptide" in this invention refers to a short peptide (typically 16-30 amino acids long), sometimes also called a signal sequence, targeting signal, localization signal, localization sequence, transport peptide, leader sequence, or leader peptide.

[0023] The term "MOI" in this invention stands for "Multiplicity of Infection (MOI)," referring to the number of viral particles added to each cell during the infection process. For example, when one million viral particles are added to one million cells, MOI = 1.

[0024] The technical solution of this invention is as follows: On the one hand, the present invention provides a B7H3 nanobody targeting B7-H3, wherein the B7H3 nanobody comprises B-HCDR1, B-HCDR2 and B-HCDR3; The B-HCDR1 has an amino acid sequence as shown in any one of SEQ ID NO. 28-32; The B-HCDR2 has an amino acid sequence as shown in any one of SEQ ID NO. 33-37; The B-HCDR3 has an amino acid sequence as shown in any one of SEQ ID NO. 38-42.

[0025] Specifically, the B7H3 nanobody comprises B-HCDR1, B-HCDR2, and B-HCDR3 selected from any one or more of the following groups: (1) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.28, SEQ ID NO.33 and SEQ ID NO.38; (2) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.29, SEQ ID NO.34 and SEQ ID NO.39; (3) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.30, SEQ ID NO.35 and SEQ ID NO.40; (4) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.31, SEQ ID NO.36 and SEQ ID NO.41; (5) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.32, SEQ ID NO.37 and SEQ ID NO.42.

[0026] SEQ ID NO.28: GRAFSSIV.

[0027] SEQ ID NO.29: GSAFSIND.

[0028] SEQ ID NO.30: GGTFTRYD.

[0029] SEQ ID NO.31: GRAFSAYA.

[0030] SEQ ID NO.32: ASERAAHV.

[0031] SEQ ID NO.33: IKWDGRRS.

[0032] SEQ ID NO.34: ITSGGMST.

[0033] SEQ ID NO.35: INRTGMST.

[0034] SEQ ID NO.36: ITRTGRTT.

[0035] SEQ ID NO.37: GTNGRT.

[0036] SEQ ID NO.38: HAHLNKRVVL.

[0037] SEQ ID NO. 39: TARVQDEDTGSYYKVEYDY.

[0038] SEQ ID NO.40: AKDIVNGMDY.

[0039] SEQ ID NO. 41: NAKKKVFGRVNSY.

[0040] SEQ ID NO.42: NAVAPWAS.

[0041] Preferably, the B7H3 nanobody has an amino acid sequence as shown in any one of SEQ ID NO. 1-5, or contains partial segments of any one or more of the amino acid sequences shown in SEQ ID NO. 1-5; or has a sequence with more than 80% homology to any one of the amino acid sequences in SEQ ID NO. 1-5.

[0042] In another aspect, the present invention provides a chimeric immune receptor comprising the B7H3 nanobody described in any of the preceding claims.

[0043] Specifically, the chimeric immune receptor includes any one of the following: chimeric antigen receptor, STAR receptor, HIT receptor, TruC receptor, TAC receptor, and AbTCR receptor.

[0044] Preferably, the chimeric immune receptor is a chimeric antigen receptor, which includes: an antigen-binding region, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0045] More preferably, the hinge region is selected from the hinge regions of any one or more of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, or CD154.

[0046] More preferably, the transmembrane domain is selected from any one or more of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4- 1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2 B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2, or CS1.

[0047] More preferably, the intracellular co-stimulatory domain is selected from the intracellular co-stimulatory signal transduction domains of any one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, or MyD88.

[0048] More preferably, the intracellular signaling domain is selected from the intracellular signaling domains of any one or more of the following proteins: CD3, CD79a, CD79b, MP2A, DAP10, or DAP12.

[0049] Specifically, the chimeric antigen receptor also includes a signal peptide.

[0050] Preferably, the signal peptide is selected from the signal peptides of any one or more of the following proteins: LAMP1, CRT, IgE, Igκ, IgG1, tPA, IL-12, HLA-I, CD3, or CD8.

[0051] More preferably, the antigen-binding region is selected from any one or more of the following: (1) B7H3 nanobody; (2) B7H3 nanobody-Linker-IL13Rα2 nanobody; (3) IL13Rα2 nanobody-Linker-B7H3 nanobody.

[0052] More preferably, the IL13Rα2 nanobody has an amino acid sequence as shown in any one of SEQ ID NO. 6-10, or contains partial segments of any one or more of the amino acid sequences shown in SEQ ID NO. 6-10; or has a sequence with more than 80% homology to any one of the amino acid sequences in SEQ ID NO. 6-10.

[0053] More preferably, the Linker includes any one or more of flexible connectors and rigid connectors.

[0054] More preferably, the Linker is selected from any one or more of SL7, M288, G4S, and (G4S)3.

[0055] More preferably, the amino acid sequence of the Linker is shown in any one of SEQ ID NO.16-19.

[0056] In another aspect, the present invention provides a soluble protein comprising the B7H3 nanobody described in any of the preceding claims.

[0057] Preferably, the soluble protein includes any one of monospecific antibodies, bispecific antibodies, multispecific antibodies, T-cell binders, antibody-TCR fusion proteins, and antibody-drug conjugates.

[0058] Specifically, the soluble protein also includes the IL13Rα2 nanobody.

[0059] Preferably, the IL13Rα2 nanobody has an amino acid sequence as shown in any one of SEQ ID NO. 6-10, or contains partial segments of any one or more of the amino acid sequences shown in SEQ ID NO. 6-10; or has a sequence with more than 80% homology to any one of the amino acid sequences in SEQ ID NO. 6-10.

[0060] In another aspect, the present invention provides a nucleic acid molecule that encodes any of the B7H3 nanobody, chimeric immune receptor or soluble protein described above.

[0061] In another aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecules, wherein the vector includes any one or more of lentiviral vectors, retroviral vectors, and adenoviral vectors.

[0062] In another aspect, the present invention provides a chimeric antigen receptor cell comprising a vector expressing the above-described antigen.

[0063] Specifically, the chimeric antigen receptor cells include any one or more of T cells, NK cells, macrophages, monocytes, dendritic cells, NKT cells, B cells, and stem cells.

[0064] Preferably, the chimeric antigen receptor cell is a T cell.

[0065] More preferably, the T cells are selected from αβT cells and / or γδT cells.

[0066] In another aspect, the present invention provides applications of the above-mentioned B7H3 nanobody, chimeric immune receptor, soluble protein, nucleic acid molecule, carrier, or chimeric antigen receptor cell, wherein the applications include any one or more of the following: (1) Application in the preparation of B7H or B7H3 / IL13Rα23 detection products; (2) Application in the preparation of B7H3 or B7H3 / IL13Rα2 related disease and diagnostic products; (3) Application in the preparation of drugs for the treatment of B7H3 or B7H3 / IL13Rα2 related diseases.

[0067] Specifically, the B7H3-related diseases include any one or more of the following: tumors, infectious diseases, autoimmune diseases, and cardiovascular and cerebrovascular diseases.

[0068] Preferably, the tumor includes any one or more of glioblastoma, medulloblastoma, ependymoma, non-small cell lung cancer, pancreatic cancer, primary liver cancer, colorectal cancer, breast cancer, prostate cancer, laryngeal cancer, melanoma, acute myeloid leukemia, and hepatocellular carcinoma.

[0069] More preferably, the tumor is any one or more of glioblastoma, primary liver cancer, acute myeloid leukemia, and hepatocellular carcinoma.

[0070] In another aspect, the present invention provides a drug comprising the above-mentioned B7H3 nanobody, chimeric immune receptor, soluble protein, nucleic acid molecule, carrier or chimeric antigen receptor cell.

[0071] Specifically, the drug also includes at least one pharmaceutically acceptable excipient.

[0072] Preferably, the pharmaceutically acceptable excipients include any one or more of the following: fillers, binders, disintegrants, lubricants, emulsifiers, antioxidants, antibacterial agents, isotonic modifiers, suspending agents, solubilizers, cosolvents, preservatives, and flavoring agents.

[0073] Preferably, the dosage form of the drug includes ointment, suppository, aerosol, paste, gel, decoction, powder, pill, solution, syrup, emulsion, suspension, injection, nebulizer, and lyophilized agent.

[0074] In another aspect, the present invention provides a kit comprising the above-mentioned B7H3 nanobody, chimeric immune receptor, soluble protein, nucleic acid molecule, carrier or chimeric antigen receptor cell.

[0075] Specifically, the kit also includes a container for loading the antibody preparation.

[0076] The beneficial effects of this invention are as follows: This invention provides nanobodies (BNb1-BNb5) that specifically target B7-H3, exhibiting high antigen-binding activity and excellent affinity. Specifically, BNb5 has a dissociation equilibrium constant (KD) of 14.63 ± 0.32 nM for B7-H3 protein and an EC50 as low as 1.43 nM for B7-H3-positive target cells, enabling efficient and specific recognition of the B7-H3 antigen. Based on these nanobodies, this invention constructs a B7-H3 single-target chimeric antigen receptor and a B7-H3 / IL13Rα2 dual-target chimeric antigen receptor.

[0077] The B7-H3 single-target chimeric antigen receptor can be efficiently expressed on the surface of immune cells, specifically recognize and bind to B7-H3 positive tumor cells, effectively activate downstream signaling and mediate T cell-specific killing, and show significant anti-tumor activity in various tumor models with high B7-H3 expression. The single-target B7-H3 CAR-T / CAR-γδT cells constructed based on this receptor are normally expressed in human primary T cells and can efficiently eliminate B7-H3 high-expressing tumor cells.

[0078] The B7-H3 / IL13Rα2 dual-target chimeric antigen receptor, after optimization of the linker and linker sequence, achieves stable membrane expression and complete antigen recognition function. It can simultaneously recognize two tumor antigens, B7-H3 and IL13Rα2, while retaining the high efficiency of single-target recognition and killing ability. Based on this receptor, dual-target CAR-T / CAR-γδT cells can simultaneously kill B7-H3 single-positive, IL13Rα2 single-positive, and double-positive tumor cells. In mixed target cell systems and heterogeneous tumor models, the killing efficiency and persistence are superior to single-target CAR-T cells, which can effectively overcome the tumor escape problem caused by antigen heterogeneity.

[0079] The B7-H3 nanobody, chimeric immune receptor, and corresponding CAR-T cells of the present invention can be further used to prepare tumor detection reagents, diagnostic kits, and therapeutic drugs, and have important application value and clinical translation prospects in the field of precision tumor diagnosis and treatment. Attached Figure Description

[0080] Figure 1 This diagram illustrates the preparation and screening process for a natural nanobody library. Figure A shows the preparation process for an alpaca-derived natural nanobody-phage library; Figure B shows the nanobody screening process based on phage surface display technology.

[0081] Figure 2 The results show the phage binding ability detected by enzyme-linked immunosorbent assay (ELISA); A in the figure shows the binding detection results of monoclonal phage against B7-H3 nanobody; B shows the binding detection results of monoclonal phage against IL13Rα2 nanobody.

[0082] Figure 3 This study presents the results of CAR expression targeting B7H3 in primary T cells and the tumor-killing efficacy of CAR-T cells. Specifically, ELISA-positive phage clones (P / N>10) were selected and named B1-B15. The nanobody sequences within these clones were obtained, and CAR plasmids were constructed and subsequently transfected into primary T cells to obtain CAR-T cells. Figure A shows the results of flow cytometry analysis of CAR expression containing these nanobodies on the surface of primary T cells; Figure B shows the tumor-killing effect of CAR-T cells.

[0083] Figure 4This study presents the results of CAR expression targeting IL13Rα2 in primary T cells and the tumor-killing efficacy of CAR-T cells. Specifically, ELISA-positive (P / N>10) phage clones were selected and named I1-I34. The nanobody sequences within these clones were obtained, and CAR plasmids were constructed and subsequently transfected into primary T cells to obtain CAR-T cells. A shows the results of flow cytometry detection of CAR expression containing these nanobodies on the surface of primary T cells; B shows the results of co-culturing reporter cells with target cells after CAR transfection; C shows the tumor-killing effect of CAR-T cells.

[0084] Figure 5 The results show the functional assay of single-target CAR-T cells targeting B7H3; Figure A is a pseudo-color graph of the transduction efficiency and membrane expression level of BCAR1-5 in human primary T cells detected by flow cytometry; Figure B is a histogram of the statistical results of CAR membrane expression efficiency (VHH+ / RFP+) in human primary T cells expressing BCAR1-5; Figure C shows the cytotoxic function of BCAR1-5 against target cells in human primary T cells.

[0085] Figure 6 The figure shows the construction and screening results of dual-target BI-CARs with different linkers and linking sequences. Figure A is a schematic diagram of the dual-target CAR plasmid structure against B7H3 and IL13Rα2. Various linkers were constructed to link B7H3-specific VHH and IL13Rα2-specific VHH. For each linker, dual-target BI-CAR plasmids with different linking sequences of B7H3-specific VHH and IL13Rα2-specific VHH were constructed and named BI-CAR1-BI-CAR8. Figure B is a flow cytometry pseudocolor image showing the transduction efficiency and membrane expression level of BI-CAR1-BI-CAR8 in human primary T cells as detected by flow cytometry.

[0086] Figure 7 The figures show the cytotoxic effects of human γδT cells transfected with single-target and dual-target CARs on target cells expressing different antigens; AC in the figure represent single-target and dual-target CAR-γδT cells and U87-IL13Rα2 cells, respectively. KO Cells, U87-B7H3 KO The death rate of target cells after co-culturing cells or U87 cells with different ET ratios for 24 hours; D is the death rate of target cells after co-culturing single-target and dual-target CAR-γδT cells with mixed target cells at an E:T=1:1 effector-target ratio for different times.

[0087] Figure 8 This is to reproduce the results of the detection of the target cell killing function of other dual-target CARs, namely B7H3-specific VHH and IL13Rα2-specific VHH, in human primary T cells.

[0088] Figure 9 The results show the killing function of dual-target CAR-T cells against target cells of tumor models expressing two other antigens; A in the figure is OCI-AML-3, a human acute myeloid leukemia tumor model; B is Huh7, a human hepatocellular carcinoma tumor model.

[0089] Figure 10 The results show the affinity of B7-H3-specific recombinant single-chain nanobodies determined by flow cytometry. Figure A shows the affinity curves of different concentrations of BNb5 for B7-H3 protein determined by biomembrane interference technique; Figure B shows the affinity curves of different concentrations of BNb5 for B7-H3-positive target cells determined by flow cytometry. Detailed Implementation

[0090] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0091] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0092] Example 1: Formulation of main reagents for nanobody screening 1. 2×YT solid culture medium: Add 5g NaCl, 16g tryptone, 10g yeast extract, and 10g agar to 950 mL of ultrapure water, adjust the pH to 7.4, and bring the volume to 1L. Autoclave (121℃, 30 min). After cooling to 50-60℃, aseptically add 0.1% antibiotic, mix well, and store at 2-8℃.

[0093] 2. 2×YT liquid culture medium: Add 5g NaCl, 16g tryptone, and 10g yeast extract to 950mL of ultrapure water, adjust the pH to 7.4, and bring the volume to 1L. Autoclave. After cooling, store at 2-8℃. Add 0.1% antibiotic before use.

[0094] 3. Tris-base (2M): Weigh 48.456g of Tris, add 150mL of ultrapure water, stir magnetically to dissolve, bring the volume to 200mL, sterilize through a 0.22μm filter membrane, dispense, and store at room temperature.

[0095] 4. Upper culture medium for plaque culture: Add 0.25g NaCl, 0.8g tryptone, 0.5g yeast extract, and 0.4g agar to 45mL of ultrapure water, adjust the pH to 7.4, and bring the volume to 50mL. Autoclave. Equilibrate in a 60℃ water bath before use.

[0096] 5. Hypertonic 5×PEG8000: Dissolve 75g NaCl + 100g PEG8000 in 400mL of ultrapure water, bring the volume up to 500mL, autoclave, stir until homogeneous, and store at room temperature.

[0097] 6. 1×PBST: Take 50 μL of Tween-20 and add it to 50 mL of 1×PBS. Mix thoroughly and store at room temperature.

[0098] 7. 1×PBS-TB: This is 1×PBS containing 0.1% Tween-20 + 0.1% BSA. Take 29 mL of 1×PBST and add 1 mL of freshly prepared 3% BSA solution, then mix thoroughly. Prepare in the required proportions, and prepare fresh before use. Store at 2-8℃ for short periods.

[0099] 8. Gly-HCl (0.2M, pH 2.2): Dissolve 7.5g glycine in 400mL ultrapure water, adjust the pH to 2.2 with HCl, bring the volume to 500mL, and filter to sterilize.

[0100] Example 2: Construction of a natural nanobody phage display library 1. Peripheral blood was collected from unimmunized alpacas from multiple countries; PBMCs were isolated using Ficoll; mRNA was extracted from PBMCs, and the VHH gene sequence library was amplified using universal primers before and after VHH using the mRNA as a template; the VHH gene sequence library and phagemid vector were ligated to obtain a Phagemid library through homologous recombination; the Phagemid library was electroporated into competent E. coli cells, and after the bacteria were cultured to the logarithmic growth phase, phages were extracted to obtain a phage display library.

[0101] 2. Inoculate the bacterial culture from the nanobody bacterial library into 2×YT medium and incubate at 37°C with shaking until OD. 600 ≈0.8. After serial dilution, samples were plated on 2×YT-Kana and 2×YT-Amp plates, respectively. The initial volume (N0) was calculated, and the absence of Kan-resistant bacterial contamination was verified. The remaining bacterial culture was collected by centrifugation (3200×g, 20 min), aliquoted, and frozen in 2×YT medium containing 50% glycerol.

[0102] 3. Transfer the cryopreserved bacterial library at a ratio of 1:1000 to 2×YT medium. After incubation for 90 min, transfer to 2×YT medium containing 0.2% glucose and 100 μg / mL Amp, and incubate at 37°C until OD600≈0.5. Add M13KO7 helper phage (MOI=20), and incubate at 37°C for 1 hour, gently shaking to mix every 15 minutes. Plate samples onto 2×YT-Kana and 2×YT-Amp plates to determine the infection efficiency.

[0103] 4. Phage Amplification and Purification: Centrifuge the above bacterial culture (4000×g, 20 min), discard the supernatant, and resuspend the bacterial culture in fresh 2×YT medium containing Amp and Kan, and incubate overnight at 30℃. Centrifuge again to collect the supernatant, add 20% PEG8000-sodium chloride solution (final concentration 4%), and precipitate at 4℃ for 3 hours. Centrifuge again (3200×g, 20 min), resuspend the precipitate in PBS, and add 20% PEG8000 again for secondary precipitation purification. Finally, adjust the phage titer to 2×10⁻⁶ with PBS. 13 PFU / mL.

[0104] 5. Add an equal volume of sterile glycerol (final concentration 50%), dispense (1.5 mL / tube), and store at -20°C.

[0105] Figure 1 This diagram illustrates the preparation and screening process for a natural nanobody library. Figure A shows the preparation process of an alpaca-derived natural nanobody-phage library; Figure B shows the screening process for nanobodies based on phage surface display technology.

[0106] Example 3: Phage panning using B7-H3 or IL13Rα2 antigen 1. Reagent Preparation and Pre-blocking: Preparation before the experiment: First, seal all sterile 2mL centrifuge tubes used in the magnetic bead selection stage. Add 1mL of 3% BSA to each tube, then place them on a rotary mixer and mix at room temperature for 2 hours. Before use, briefly centrifuge the tubes to remove the 3% BSA before use.

[0107] 2. Blocking with cation exchange magnetic beads: Take 100 μL of magnetic beads (the amount used in the second and third rounds is changed to 50 μL) and place them in a 2 mL centrifuge tube. Add 1 mL of 1×PBST and mix well. Place the centrifuge tube on a magnetic rack for adsorption for 2 min, then discard the supernatant. Resuspend the magnetic beads in 1 mL of 3% BSA. Place it on a rotary mixer and incubate at room temperature for 2 h to ensure complete blocking.

[0108] 3. Blocking Negative Phage Selection: Add 500 μL of 3% BSA and 500 μL of the phage library to be screened to a 2 mL centrifuge tube for later use. Prepare another 2 mL centrifuge tube, add 100 μL of magnetic beads, and then add 1 mL of 1×PBST. Place the tube on a magnetic rack for adsorption for 2 min, then discard the supernatant. Resuspend the magnetic beads in the liquid from the centrifuge tube containing the mixture of 3% BSA and the phage library to be screened, and place it on a rotary mixer for incorporation at 4°C for 1 h.

[0109] 4. Blocking positive selector phages: After blocking with negative selector phages, place the centrifuge tube on a magnetic rack for 2 minutes to allow adsorption, then transfer the supernatant to a new centrifuge tube. Add biotinylated B7-H3 or IL13Rα2 (final concentration 200-300 nM) to the supernatant, place on a rotary mixer, and incubate at 4°C for 1 hour (for the second and third rounds of panning, change the final concentration of biotinylated B7-H3 or IL13Rα2 to 100-150 nM).

[0110] 5. Cathode Selection: After completing the above steps, perform cation selection. After blocking with magnetic beads, place the centrifuge tube on a magnetic rack for 2 minutes to absorb the phage, then discard the supernatant. Resuspend the magnetic beads in the blocked phage and place it on a rotary mixer at 4°C for 15 minutes. Then, place the mixture on a magnetic rack for 2 minutes to absorb the phage, and discard the supernatant. Resuspend the magnetic beads in 1 mL of 1×PBS-TB, place it on a magnetic rack for 2 minutes to absorb the phage, and discard the supernatant. Repeat this bead-washing process five times.

[0111] 6. Washing and Elution: Resuspend the magnetic beads in 1 mL of 1×PBS, place on a magnetic rack for 2 min, and discard the supernatant. Add 900 μL of Gly-HCl (pH=2.2) to resuspend the magnetic beads, place on a rotary mixer, and elute for 10 min at room temperature. Then, place the centrifuge tube on a magnetic rack for 2 min, transfer the supernatant (elution buffer) to a new centrifuge tube, and add 2M Tris to neutralize to a pH of 7.2-7.4. The neutralized mixture is the antigen-positive phage after this round of selection and should be stored at 4℃.

[0112] 7. Phage titer detection: TG1 competent cells were transferred to 50 mL of 2×YT medium at an appropriate ratio and cultured at 220 rpm and 37°C until the early logarithmic growth phase (OD). 600 =Approximately 0.5). Take 5 μL of the antigen-positive phage obtained from the panning process and add it to 45 μL of 2×YT medium, then dilute it to 10% of the original concentration. -4 (Second and third rounds of dilution factor 10) -65 μL of diluted phages were used to infect 495 μL of TG1 cells in the pre-log phase. After gentle mixing, the phages were incubated at 37°C for 30 min. 100 μL of each phage was then plated onto 2×YT-Amp plates and incubated overnight. The titer and the amount of phage recovered were calculated the following day based on the number of clones.

[0113] 8. Expanding antigen-positive phages: All remaining phages from the first round were expanded; half were used for expansion in the second and third rounds. The frozen TG1 competent cells were transferred and cultured at 220 rpm and 37°C until the logarithmic growth phase (OD200). 600 =0.6-0.8), add bacteriophage, mix well, and incubate at 37℃ for 30 min for infection. After infection, add Amp to a final concentration of 50 μg / mL, add 20% 1M glucose to 2%, and incubate at 37℃ and 220 rpm for 30 min. Add M13KO7 (MOI=20), incubate at 37℃ for 30 min for infection; after infection, incubate at 37℃ and 220 rpm for 30 min. After infection, centrifuge the bacterial culture at 3200 rpm for 20 min, discard the supernatant, and resuspend the bacterial pellet in 10 mL of 2×YT medium. Transfer the resuspended bacterial pellet to a shaker flask, add 90 mL of 2×YT medium, add 100 μL each of Amp and Kana stock solution, and incubate at 30℃ and 220 rpm for 16-18 hours. Transfer the overnight cultured bacterial suspension to a sterile centrifuge tube and centrifuge at 3200 rpm for 20 min. Transfer the supernatant to an Erlenmeyer flask pre-filled with 25 mL of hypertonic PEG8000, mix well, and incubate at 4°C for at least 2 h to allow complete phage precipitation. Transfer the precipitated phage solution to a sterile centrifuge bottle and centrifuge at 3200 rpm for 20 min, discarding the supernatant; centrifuge again for 2 min, aspirating the supernatant completely. Dissolve the phage precipitate with 0.5 mL of sterile PBS, being careful to avoid vigorous pipetting to prevent phage breakage. Transfer the dissolved phage to a 2 mL centrifuge tube and centrifuge at 12000 rpm for 2 min to remove impurities and precipitate. Transfer the supernatant to a new 15 mL centrifuge tube. Analyze the OD... 260 Measure the phage library titer and adjust the PBS volume to 1×10⁻⁶. 13 pfu / mL, store at 4℃.

[0114] 9. Repeat steps 1-8 for the next round of screening until the amount of phage recovered reaches the expected level.

[0115] Example 4: Detection of antigen and target cell binding 1. Phage culture: Take the final panning phages, pre-revive TG1 competent cells to a 5mL system, and culture until OD. 600=0.6. Based on the phage titer (pfu / mL), add 5 μL of phage to 45 μL of 2×YT medium and serially dilute to the appropriate ratio. Mix 5 μL of diluted phage with 495 μL of pre-logarithmic TG1, incubate at 37°C for 30 min, add 0.5 μL of Amp stock solution and 55.5 μL of 20% glucose solution, incubate at 37°C for 30 min at 220 rpm, then add M13KO7 (MOI=20), continue infection and incubate for another 30 min.

[0116] 2. Selecting single colonies for expansion: Prepare 100 mL of 2×YT liquid medium containing 1‰ Amp and 1‰ Kana. Add 750 μL of medium to each well of a 96-well plate. Select single colonies and transfer them to the 96-well plate. Mix well by pipetting and take 100 μL of each colony for storage. Incubate the remaining bacterial culture at 30℃ and 220 rpm for 16-18 h.

[0117] 3. Streptavidin coating: Dilute the streptavidin stock solution with 1×PBS to a final concentration of 5 ng / μL and add 100 μL / well to the sample wells and control wells of the ELISA plate. Incubate overnight at 4°C.

[0118] 4. ELISA positive rate: Sample preparation: Transfer the retained bacterial culture to 1 mL of 2×YT-AK medium and incubate for 6 hours until the logarithmic growth phase (OD200). 600 =0.6). Wash the coated plate three times with 1×PBST, add 200μL of 3% BSA for 1h, and wash the coated plate once with 1×PBST. Prepare a biotinylated B7-H3 or IL13Rα2 antigen solution with a final concentration of 0.5ng / μL, add 100μL / well to the sample wells, add 3% BSA (100μL / well) to the control wells, and incubate at room temperature for 1h. Centrifuge the bacterial culture (3200g / 10min), take the supernatant and add 200μL / well to the plate wells, and incubate at room temperature for 1h. After washing, add Anti-M13-HRP working solution (final concentration 0.5ng / μL), wash again after 30min, add TMB chromogenic solution (100μL / well), stop the reaction after 15min with 50μL of 2M HCl, and detect OD using a microplate reader. 450 Positive clones are identified by the ratio of OD450 values ​​between streptomycin wells and BSA wells (P / N>2 indicates a positive result, and P / N>5 is usually used).

[0119] 5. Positive monoclonal expansion and phage concentration: Based on an MOI of 20, add M13KO7 to the monoclonal bacterial culture, infect at 37°C for 1 hour, centrifuge, resuspend in 2×YT-AK medium, and incubate overnight at 30°C and 220 rpm. The next day, centrifuge the bacterial culture, mix the supernatant with hypertonic PEG8000 (incubate at 4°C for 2 hours), centrifuge, dissolve the precipitate in 0.1 mL of PBS, centrifuge at 12000 rpm for 2 minutes, and use the supernatant to detect OD. 260Calculate the titer and adjust it to 2 × 10⁻⁶. 11 pfu / mL is used for flow cytometry detection.

[0120] The results of detecting the phage-antigen binding ability by enzyme-linked immunosorbent assay (ELISA) are as follows: Figure 2 As shown in the figure, A represents the binding detection results of monoclonal phage against the B7-H3 nanobody; B represents the binding detection results of monoclonal phage against the IL13Rα2 nanobody. The horizontal axis represents the phage clone number, and the vertical axis represents P / N, which is the OD450 of the experimental group / OD450 of the negative antigen control group. The higher the P / N, the stronger the antigen binding positivity of the phage.

[0121] Example 5: CAR plasmid construction and lentivirus preparation 1. Phage particle extraction: TG1 competent cells were transferred to 2×YT liquid medium at a ratio of 1:100 and cultured at 37℃ with shaking at 220 rpm until the prolog phase (OD600≈0.5). 2×10⁻⁶ cells were then collected. 8p The antigen-positive phage of fu was added to the logarithmic-phase bacterial culture and incubated at 37°C for 30 min to complete infection. After infection, the bacterial culture was centrifuged at 3,200×g for 20 min (25°C), the supernatant was discarded, and the bacterial pellet was resuspended in 20 mL of 2×YT medium, with ampicillin added (final concentration 100 μg / mL). The resuspended bacterial culture was incubated overnight (16-18 h) at 37°C with shaking at 220 rpm. The overnight culture was centrifuged at 4,000×g for 30 min to collect the bacterial cells. Subsequently, plasmid extraction was performed according to the instructions of the Tiangen plasmid mini-extraction kit.

[0122] 2. CAR plasmid construction: (1) The single-target CAR used in this invention comprises: a signal peptide region, an extracellular antigen-binding region, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal domain.

[0123] The structure of the single-target CAR is: signal peptide-antigen binding region-hinge region-transmembrane region-intracellular co-stimulatory domain-intracellular signaling domain, wherein the antigen binding region is the single-domain antibody obtained by the above screening.

[0124] The amino acid sequence of the signal peptide region is shown in SEQ ID NO.11: MLLLVTSLLLCELPHPAFLLIPLE.

[0125] The amino acid sequence of the hinge region is shown in SEQ ID NO.12: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0126] The amino acid sequence of the transmembrane domain is shown in SEQ ID NO.13: IYIWAPLAGTCGVLLLSLVIT.

[0127] The amino acid sequence of the intracellular co-stimulatory domain is shown in SEQ ID NO.14: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0128] The amino acid sequence of the intracellular signaling domain is shown in SEQ ID NO.15: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0129] (2) The dual-target CAR used in this invention includes an extracellular antigen-binding region, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0130] The antigen-binding region consists of two single-domain antibodies (VVH1) recognizing B7H3 and IL13Rα2, respectively, linked by a linker (VVH1-Linker-VVH2 or VVH2-Linker-VVH1). To compare the effects of different linkers on the membrane expression and function of dual-target CARs, this invention constructed dual-target CAR plasmids containing several different linkers, including SL7 (SEQ ID NO. 16), M288 (SEQ ID NO. 17), G4S (SEQ ID NO. 18), and (G4S)3 (SEQ ID NO. 19).

[0131] The structure of the dual-target CAR is: VHH1-Linker-VHH2-hinge region-transmembrane region-intracellular co-stimulatory domain-intracellular CD3 signaling domain; or VHH2-Linker-VHH1-hinge region-transmembrane region-intracellular co-stimulatory domain-intracellular CD3 signaling domain.

[0132] SEQ ID NO.16: GTSGSSGSGSGGSGGGG; SEQ ID NO.17:GSGSSGKPGSGEGSTKG; SEQ ID NO.18: GGGGS; SEQ ID NO. 19: GGGGSGGGGSGGGGS.

[0133] Primers were designed based on the principle of homologous recombination, and the PCR system was prepared and PCR reaction was performed according to the KOD-DNA polymerase instructions to amplify the nanobody sequence in the phage particle. The CAR plasmid vector was double-digested with NEB restriction endonucleases according to the instructions, and the reaction was carried out at 37°C for 3 hours. PCR and digestion products were separated by agarose gel electrophoresis. Agarose gels with concentrations of 0.5%-2% were prepared according to the molecular weight range of the target fragment. After electrophoresis, gel blocks containing the target band were excised under UV light, and DNA fragments were purified using a gel extraction kit from Tiangen Biotech. The concentration of the recovered product was detected using a micro-spectrophotometer. The homologous recombination fragment and vector were ligated according to the instructions of the Vazyme ClonExpressII one-step cloning kit. Competent bacteria were thawed on ice, and then 20 μL of ligation product was added. The mixture was incubated on ice for 30 minutes and then transferred to a 42°C water bath for 45 seconds for heat shock. Immediately after heat shock, remove the cells and cool them on ice for 2 minutes. Add 400 μL of LB liquid medium, mix thoroughly, and incubate at 37°C in a shaker for 30-60 minutes to allow cell recovery. Spread 100 μL of the transformation product evenly onto LB agar plates containing the appropriate antibiotics, invert the plates, and incubate at 37°C for 12-16 hours. Pick 5-10 independent single colonies from the overnight culture plates and inoculate them into LB liquid medium containing antibiotics. Incubate at 37°C with shaking at 220 rpm for 6-7 hours. Perform initial colony PCR screening using specific primers. Select 3 PCR-positive clones, and send 1 mL of bacterial culture from each for commercial sequencing. Temporarily store the remaining bacterial culture at 4°C. After obtaining correct sequencing results, expand the culture of positive clones. Purify the plasmids using a medium- or large-scale plasmid extraction kit from Tiangen Biotech, depending on experimental requirements. Finally, determine the plasmid concentration (OD260) using a spectrophotometer.

[0134] 3. Lentiviral Packaging and Concentration Lenti-X293T cells with a confluence of 90%-95% were selected and passaged into 10cm culture dishes for expansion culture. Transfection was performed approximately 36 hours later, when the cell confluence reached 80%-90%. A transfection system was prepared using serum-free DMEM with a target plasmid:pMD2.G:PRSV-Rev:PRSV-Rev ratio of 4:1:1:2 and a plasmid:PEI ratio of 1:3. After gentle pipetting and mixing, the mixture was incubated at room temperature for 15 minutes. The system was then slowly added dropwise to the seeded Lenti-X293T cells, gently shaken, and incubated at 37°C. This was recorded as 0 hours. 14-16 hours after transfection, the medium was replaced with fresh DMEM complete medium. Viral supernatant was collected at 48h and 72h, centrifuged at 3000rpm for 5 minutes, and filtered through a 0.45μm sterile filter. Add 1 / 4 volume of 5×PEG8000 to the filtered virus supernatant and mix thoroughly by inverting. Mix by rotation at 4°C or let stand for at least 12 hours. Centrifuge at 3500 rpm for 30 minutes at 4°C; a white precipitate will be visible after centrifugation. Discard the supernatant and resuspend the precipitate in serum-free 1640 medium, repeatedly pipetting to ensure complete resuscitation. Centrifuge at 12000 rpm for 30-60 seconds, transfer the supernatant to a new tube, aliquot as needed, and store at -80°C, or use directly for infection.

[0135] Example 6: Construction of CAR-T cells and CAR-γδT cells 1. Construction of CAR-T cells (all T cells or αβ T cells): Dilute the CD3 antibody with 1×PBS to a final concentration of 5 μg / mL and add to a 24-well plate (300 μL / well) or a 48-well plate (200 μL / well). Coat overnight at 4°C. Recover frozen PBMCs, collect cells, centrifuge, resuspend, count, and adjust density to 1×10⁻⁶ cells / well. 6 Cells / mL. Remove the coating medium from the wells and seed with cell suspension (1 mL / well for 24-well plates, 500 μL / well for 48-well plates), and incubate at 37°C for activation. After 24 h of activation, add virus solution to the wells at MOI=10, gently shake to mix, and continue incubation at 37°C. Subsequently, use RPMI 1640 medium containing 10% inactivated serum and 200 IU / mL IL-2 for cell expansion and passage, maintaining a cell density of 1E6 / mL at each passage.

[0136] 2. Construction of CAR-γδT cells: The CD3 antibody was diluted to a final concentration of 5 μg / mL using 1×PBS and added to 48-well plates for overnight coating at 4°C. Frozen PBMCs were revived, cells were collected, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended and washed with PBSF (1×PBS plus 1% FBS). γδT cells were sorted according to the instructions of the Human γδT Cell Sorting Kit (StemCell). After counting the sorted γδT cells, the density was adjusted to 1E6 / mL. The antibody in the coated plate was discarded, and 200 μL of cell suspension was added to each well for activation at 37°C. After 48 h of activation, viral infection was performed by adding concentrated virus solution at an MOI of 20, gently shaking, and continuing culture. After infection, the cells were further expanded in RPMI 1640 medium containing 10% inactivated serum and 200 IU / mL IL-2 to maintain a cell density of 1-2E6 / mL.

[0137] Example 7: Detection and Functional Verification of CAR Expression in CAR-T Cells 1. CAR expression detection: Take an appropriate amount of CAR-T cells, centrifuge, and discard the supernatant. Wash once with 1×PBS and discard the supernatant. Add 100 μL of diluted antibody mixture (Anti-VHH-APC or biotin-conjugated B7-H3 and IL13Rα2 protein) and incubate on ice for 30 min. Centrifuge at 4°C for 5 min, discard the supernatant, and wash twice with PBS. If using biotin-conjugated B7-H3 and IL13Rα2 protein for detection, add diluted Anti-biotin-APC flow cytometry antibody after PBS washing, incubate on ice for 15 min, centrifuge at 4°C for 5 min, discard the supernatant, and wash twice with 200 μL of PBS. After staining, resuspend in PBS and transfer to flow cytometry tubes. Perform flow cytometry detection according to the BD Fortessa 5laser instrument manual. Since the plasmid carries RFP as a reporter gene, CAR infection efficiency can be assessed by RFP positivity, while antibody staining positivity indicates CAR membrane deposition efficiency. In some embodiments, the upper membrane expression rate of CAR is characterized by the ratio of the positive rate of Anti-VHH to the positive rate of RFP.

[0138] 2. Target cell construction: The tumor cells (i.e., target cells) used in this invention include: OCI-AML-3 cells (human acute myeloid leukemia cells, purchased from Wuhan Pronosei Biotechnology Co., Ltd., which are naturally B7H3 single-positive cells). Based on this, OCI-AML-3-Luc / GFP cells were obtained by stably expressing luciferase (Uniprotpk ID: P08659) and green fluorescent protein GFP (Uniprotpk ID: P42212) using a lentiviral system. Further overexpression of IL13Rα2 using a lentiviral system yielded OCI-AML3-IL13Rα2-Luc / GFP cells. U87 MG-Luc / GFP cells (U87 MG is a human astrocytoma cell line; U87 MG-Luc / GFP cells were purchased from Wuhan Pronosei Biotechnology Co., Ltd., and stably express luciferase (Uniprotpk ID: P08659) and green fluorescent protein GFP (Uniprotpk ID: P42212), hereinafter referred to as U87, and are naturally B7H3 and IL13Rα2 double-positive cells). Based on U87 cells, U87-B7H3KO and U87-IL13Rα2KO cells were obtained by knocking out the B7H3 gene or the IL13Rα2 gene using the CRISPR / Cas9 system. Huh7 cells (human hepatocellular carcinoma cells, purchased from the Chinese Academy of Sciences Type Culture Collection, catalog number SCSP-526) were used to stably express luciferase (Uniprotpk ID: P08659) and green fluorescent protein GFP (Uniprotpk ID: P42212) using a lentiviral system to obtain Huh7-Luc / GFP cells. Further overexpression of B7H3 and IL13Rα2 using a lentiviral system yielded Huh7-BI-Luc / GFP cells.

[0139] 3. Lethality test: When the target cell density reaches 80%-90%, collect and centrifuge according to the passage procedure, resuspend in culture medium, count, and adjust the density to 2×10⁶. 5Cells were seeded at a density of 500 μL / well in 48-well plates. Adherent cells were cultured overnight, and suspension cells were immediately used for co-culture with CAR-T cells. The CAR-T cells obtained above were co-cultured with corresponding target cells at specific effector-to-target ratios (1:1, 1:3, and 1:9 for U87 series cells; a single effector-to-target ratio of 1:3 was used in some examples; the effector-to-target ratio for Huh7 cells was 1:9, and for OCI-AML3 cells it was 1:3). After 24 hours, the number of surviving target cells was detected using a firefly luciferase kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), and the percentage of target cell death was calculated. In some examples, to evaluate the killing effect of dual-target and single-target CAR-T cells on heterogeneous tumors, U87, U87-B7H3KO, and U87-IL13Rα2KO were first mixed at a certain ratio (e.g., 2:1:1) before being co-cultured with CAR-T cells. In some embodiments, multiple series of co-culture times (e.g., 6 hours, 12 hours, 24 hours, 48 ​​hours) were set to evaluate the durability and differences in killing effects of dual-target and single-target CAR-T cells.

[0140] Figure 3 This study presents the results of CAR expression targeting B7H3 in primary T cells and the tumor-killing efficacy of CAR-T cells. Specifically, ELISA-positive phage clones (P / N>10) were selected and named B1-B15. The nanobody sequence was extracted, and a CAR plasmid was constructed, which was then transfected into primary T cells to obtain CAR-T cells. A shows the results of CAR expression on the surface of primary T cells detected by flow cytometry; B shows the tumor-killing effect of CAR-T cells.

[0141] from Figure 3 The experimental results in group A show that, except for B5, B7, and B9, other CARs have high membrane uptake efficiency. The results in group B show that B2, B3, B4, B8, and B11 have good tumor-killing efficacy.

[0142] The B11 anti-B7H3 nanobody is named BNb1 and has the amino acid sequence shown in SEQ ID NO.1; the B8 anti-B7H3 nanobody is named BNb2 and has the amino acid sequence shown in SEQ ID NO.2; the B2 anti-B7H3 nanobody is named BNb3 and has the amino acid sequence shown in SEQ ID NO.3; the B3 anti-B7H3 nanobody is named BNb4 and has the amino acid sequence shown in SEQ ID NO.4; and the B4 anti-B7H3 nanobody is named BNb5 and has the amino acid sequence shown in SEQ ID NO.5.

[0143] SEQ ID NO.1: DVQLQESGGGLVQAGGSLRLSCAASGRAFSSIVMGWFRQAPGKEREFVAAIKWDGRRSYYADSVKGRFTVTRDNSKDTAYLQMNNLKPDDTAVYYCHAHLNKRVVLKGRGTQVTVS。

[0144] SEQ ID NO.2: DVQLQESGGGLVQAGDSLRLSCAVSGSAFSINDMAWYRQAPGKERALVASITSGGMSTNYTDSVKGRFTISRDNAKNTMYLQMNSLKPDDTGVYYCTARVQDEDTGSYYKVEYDYWGQGTQVTVSS。

[0145] SEQ ID NO.3: DVQLQESGGGLVQAGGSLRLSCAASGGTFTRYDMGWFRQAPGKEREFVALINRTGMSTYYTASVKGRFIISRDNAKNTVYLQMNSLKSEDTAVYYCAKDIVNGMDYWGRGTQVTVSS。

[0146] SEQ ID NO.4: DVQLQESGGGSVQAGDSLRLSCVASGRAFSAYAMGWFRQAPGKEREFVAAITRTGRTTYYADSVRGRFTISRDNAKNTVYLQMNALKPEDTAVYYCNAKKKVFGRVNSYWGQGTQVTVSS。

[0147] SEQ ID NO.5: DVQLQESGGGSVQAGASLRLTCRASERAAHVMGWFRQAPGKERQFVAAIGTNGRTVYADSVKGRFIISRDNTKNIMWLQMNSLEPEDTAVYYCNAVAPWASWGQGTQVTVSS。

[0148] Figure 4This study presents the results of CAR expression targeting IL13Rα2 in primary T cells and the tumor-killing efficacy of CAR-T cells. Specifically, ELISA-positive (P / N>10) phage clones were selected and named I1-I34. The nanobody sequence was obtained from these clones, and a CAR plasmid was constructed and subsequently transfected into primary T cells to obtain CAR-T cells. A shows the results of CAR expression on the surface of primary T cells detected by flow cytometry; B shows the results of co-culturing reporter cells with target cells after CAR transfection; C shows the tumor-killing effect of CAR-T cells.

[0149] from Figure 4 The experimental results from the A group show that, except for I11, I14, I23, I27, I30-I32, and I34, other CARs all exhibit high membrane uptake efficiency. From... Figure 4 The experimental results for B show that I4, I6, I7, I10, I15, I22, I26, and I29 have high activation levels. From... Figure 4 The experimental results of C show that I6, I26, I10, I7, and I29 have good tumor-killing effects. The I6 anti-IL13Rα2 nanobody is named INb1, with the amino acid sequence shown in SEQ ID NO. 6; the I26 anti-IL13Rα2 nanobody is named INb2, with the amino acid sequence shown in SEQ ID NO. 7; the I10 anti-IL13Rα2 nanobody is named INb3, with the amino acid sequence shown in SEQ ID NO. 8; the I7 anti-IL13Rα2 nanobody is named INb4, with the amino acid sequence shown in SEQ ID NO. 9; and the I29 anti-IL13Rα2 nanobody is named INb5, with the amino acid sequence shown in SEQ ID NO. 10.

[0150] SEQ ID NO.6: DVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSS.

[0151] SEQ ID NO.7: DVQLQESGGGLVQAGGSLTLSCAASGWSLKNYPMGWFRQAPGKEREFVAAIDWGIFTTRYANSVKGRFTISRDNA*NTLDLQMNSLKPEDTAVYYCYAVGNALVHWGQGTQVTVSS.

[0152] SEQ ID NO.8: DVQLQESGGGLVQPGGSLTLSCTFPTTTFSINNMAWYRRAHGTARTLVAQIYGSGRTIYTESVKGRFTISRDNSKNTVYLQMNDLKPEDTAVYYCNSISDATRLFDWGQGTQVTVSS.

[0153] SEQ ID NO.9: DVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAVISRSGGTTYYTDSVKGRFAISRDNAKNTVYLEMNSLKPDDTAVYYCNARRIREDYWGQGTQVTVSS.

[0154] SEQ ID NO.10: DVQLQESGGGLVQAGGSLRLSCAVSGGNFNTYAMGWFRQAPGKEREFVAAITWGGDSTYFSNFVKGRFTISRDNAKNTVYLEMNSLKSEDSAVYVCKAVIKEFNDSRAVDYWGQGTQVTVSS.

[0155] Example 8 Antibody Affinity Detection 1. Construction of VHH recombinant antibody expression plasmid The VHH sequence of the screened positive antibody and the Fc gene sequence of human IgG1 were amplified by PCR using KOD-DNA polymerase, followed by DNA fragment purification using a gel extraction kit from Tiangen Biotech. Simultaneously, the pCDNA3.4 expression vector was double-digested with XbaI and BamHI, and the vector fragment was recovered and purified using a gel extraction kit from Tiangen Biotech. The VHH gene fragment, human IgG1 Fc gene fragment, and vector fragment were then ligated using a one-step cloning kit from Vazyme ClonExpressII. The ligation product was then transformed into competent E. coli cells, plated on ampicillin-resistant LB agar plates, and single clones were picked and sequenced. Clones with correct sequencing results were then inoculated for further culture and plasmid extraction.

[0156] 2. Antibody expression and purification The plasmid was transfected into 293F cells via PEI. After collecting the culture supernatant, the precipitate was removed by centrifugation. The antibody was initially purified by protein A / G column and then further purified by HPLC. Subsequently, the antibody was identified by SDA-PAGE and Western blot, thus obtaining the antibody for affinity detection.

[0157] 3. Cell-level antibody affinity detection Target cells expressing B7H3 and IL13Rα2 were cultured to approximately 80% confluence. Cells were collected according to passage procedures, washed once with 1×PBS, counted, and aliquoted into 96-well plates for assay, with 5E5 cells added to each well. Simultaneously, the recombinant antibody was serially diluted with 1×PBS: the final concentration from 10⁻⁵ M to 10⁻¹² M. After centrifuging to remove the supernatant, 100 μL of the diluted antibody solution was added to each well. The cells were stained on ice for 30 minutes, centrifuged to remove the supernatant, and washed twice with 1×PBS. Then, 100 μL of the diluted secondary antibody solution (anti-human IgG-APC antibody) was added to each well, stained on ice for 30 minutes, centrifuged to remove the supernatant, and washed twice with 1×PBS. The cells were then resuspended in 1×PBS and transferred to flow cytometry tubes for flow cytometry analysis.

[0158] The mean fluorescence intensity (MFI) of positive cells measured at each antibody concentration gradient was analyzed. Then, using Graph Pad Prism software, a dose-response curve for antibody-antigen binding was obtained by fitting the MFI values ​​at different concentrations and antibody concentrations. The antibody concentration corresponding to the half-capacity binding (i.e., (the highest MFI value achievable at binding saturation + the lowest MFI value at which binding is impossible) / 2) is the antibody affinity.

[0159] 4. Antibody affinity detection based on biomembrane interference technology Antibody affinity was determined using biomembrane interferometry, employing an Octet® RH16 molecular interaction analyzer. Biotinylated target antigens were diluted to 10 ng / μL with buffer and immobilized on the surface of a moistened streptavidin (SA) biosensor. Antibody samples were serially diluted in buffer at different concentrations, and binding and dissociation kinetics were measured sequentially in buffer and glycine-hydrochloric acid solution. Experimental data were fitted using Octet Anylsis Studio to obtain the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD). The buffer solution used in the above procedures was 2% BSA-1×PBS solution. The immobilization-binding-dissociation time was determined through preliminary experiments, and the experimental environment temperature was 4°C.

[0160] Example 9 Experimental Results 1. Results of functional assay of single-target CAR-T cells targeting B7H3 Figure 5 This section presents the functional assay results of single-target CAR-T cells targeting B7H3. B7H3-specific VHHs (BNb1-BNb5) were loaded into CAR plasmids, and CAR-T cells were constructed. The in vitro tumor-killing efficiency was then assessed. Figure 5 In the figure, A is a pseudo-color graph showing the transduction efficiency and membrane expression level of BCAR1-BCAR5 (containing BNb1-BNb5 respectively) in human primary T cells as detected by flow cytometry; B is a histogram showing the statistical results of CAR membrane expression efficiency (VHH+ / RFP+) in human primary T cells expressing BCAR1-BCAR5 (containing BNb1-BNb5 respectively); and C is the result of the cytotoxic function of BCAR1-BCAR5 (containing BNb1-BNb5 respectively) against target cells in human primary T cells.

[0161] The results showed that BCAR1-BCAR5 could be efficiently transduced into primary T cells with good membrane expression levels, and demonstrated a significant killing ability against B7H3-positive target cells compared to the control group.

[0162] 2. Construction and screening results of linkers and dual-target BI-CARs with different linker sequences. Figure 6 The results of construction and screening of linkers and dual-target BI-CARs with different linker sequences. Figure 6 A in the diagram is a schematic diagram of the dual-target CAR plasmid structure against B7H3 and IL13Rα2. Various linkers were constructed to connect B7H3-specific VHH and IL13Rα2-specific VHH. For each linker, dual-target BI-CAR plasmids with different connection sequences of B7H3-specific VHH and IL13Rα2-specific VHH were constructed and named BI-CAR1 to BI-CAR8. The amino acid sequence of the extracellular antigen-binding region of BI-CAR1 is shown in SEQ ID NO.20: DVQLQESGGGSVQAGASLRLTCRASERAAHVMGWFRQAPGKERQFVAAIGTNGRTVYADSVKGRFIISRDNTKNIMWLQMNSLEPEDTAVYYCNAVAPWASWGQGTQVTVSSGGGGSGGGGS GGGGSDVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSS.

[0163] The amino acid sequence of the extracellular antigen-binding region of BI-CAR2 is shown in SEQ ID NO.21: DVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSSGGGGS GGGGSGGGGSDVQLQESGGGSVQAGASLRLTCRASERAAHVMGWFRQAPGKERQFVAAIGTNGRTVYADSVKGRFIISRDNTKNIMWLQMNSLEPEDTAVYYCNAVAPWASWGQGTQVTVSS.

[0164] The amino acid sequence of the extracellular antigen-binding region of BI-CAR3 is shown in SEQ ID NO.22: DVQLQESGGGSVQAGASLRLTCRASERAAHVMGWFRQAPGKERQFVAAIGTNGRTVYADSVKGRFIISRDNTKNIMWLQMNSLEPEDTAVYYCNAVAPWASWGQGTQVTVSSGSTSGSGKPGSG EGSTKGDVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSS.

[0165] The amino acid sequence of the extracellular antigen-binding region of BI-CAR4 is shown in SEQ ID NO.23: DVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSSGSTSGSG KPGSGEGSTKGDVQLQESGGGSVQAGASLRLTCRASERAAHVMGWFRQAPGKERQFVAAIGTNGRTVYADSVKGRFIISRDNTKNIMWLQMNSLEPEDTAVYYCNAVAPWASWGQGTQVTVSS.

[0166] The amino acid sequence of the extracellular antigen-binding region of BI-CAR5 is shown in SEQ ID NO.24: DVQLQESGGGSVQAGASLRLTCRASERAAHVMGWFRQAPGKERQFVAAIGTNGRTVYADSVKGRFIISRDNTKNIMWLQMNSLEPEDTAVYYCNAVAPWASWGQGTQVTVSSGGGGS DVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSS.

[0167] The amino acid sequence of the extracellular antigen-binding region of BI-CAR6 is shown in SEQ ID NO.25: DVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSS GGGGSDVQLQESGGGSVQAGASLRLTCRASERAAHVMGWFRQAPGKERQFVAAIGTNGRTVYADSVKGRFIISRDNTKNIMWLQMNSLEPEDTAVYYCNAVAPWASWGQGTQVTVSS.

[0168] The amino acid sequence of the extracellular antigen-binding region of BI-CAR7 is shown in SEQ ID NO.26: DVQLQESGGGSVQAGASLRLTCRASERAAHVMGWFRQAPGKERQFVAAIGTNGRTVYADSVKGRFIISRDNTKNIMWLQMNSLEPEDTAVYYCNAVAPWASWGQGTQVTVSSGTSGSSGSGSGG SGSGGGGDVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSS.

[0169] The amino acid sequence of the extracellular antigen-binding region of BI-CAR8 is shown in SEQ ID NO.27: DVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSSGTSGSSG SGSGGSGSGGGGDVQLQESGGGSVQAGASLRLTCRASERAAHVMGWFRQAPGKERQFVAAIGTNGRTVYADSVKGRFIISRDNTKNIMWLQMNSLEPEDTAVYYCNAVAPWASWGQGTQVTVSS.

[0170] Figure 6 B in the figure is a flow cytometry pseudocolor image showing the transduction efficiency and membrane expression level of BI-CAR1-BI-CAR8 in human primary T cells as detected by flow cytometry. The results showed that BI-CAR1 to BI-CAR8 could be transduced normally in human primary T cells, except for BI-CAR-6; however, there were differences in membrane expression levels. BI-CAR-1, BI-CAR-3, and BI-CAR-7 were almost not expressed in human primary T cells, while BI-CAR-2, BI-CAR-4, BI-CAR-5, and BI-CAR-8 showed good membrane surface expression levels.

[0171] 3. Results of cytotoxicity assays of human γδT cells transfected with single-target and dual-target CARs against target cells expressing different antigens. Figure 7 The results show the killing function of human γδT cells transfected with single-target and dual-target CARs against target cells expressing different antigens. Figure 7 middle: B-CAR is a single-target CAR-γδT that targets B7H3, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.5; I-CAR is a single-target CAR-γδT that targets IL13Rα2, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.6; BI-CAR is a dual-target CAR-γδT that targets B7H3 and IL13Rα2, and its extracellular antigen-binding region has the amino acid sequence shown in SEQ ID NO. 23.

[0172] Figure 7 The AC values ​​represent single-target and dual-target CAR-γδT and U87-IL13Rα2, respectively. KO Cells, U87-B7H3 KO Cells or U87 cells, co-cultured for 24 hours at different ET ratios, representing the target cell death rate; D represents single-target and dual-target CAR-γδT and mixed target cells (U87:IL13Rα2). KO B7H3 KO =2:1:1) The target cell death rate after co-culturing for different times with an E:T=1:1 target ratio.

[0173] The results showed that for dual-target positive target cells, BI-CAR could essentially achieve the target-specific killing level of B-CAR under different effector-to-target ratios, demonstrating that the dual-target CAR model with VHH tandem still retains the antigen binding of a single VHH and mediates T cell cytotoxicity. For single-target positive target cells, BI-CAR exhibited stronger tumor-killing ability, and maintained highly efficient synergistic killing ability in mixed target cells with different target expressions, with significantly better killing power than B-CAR and I-CAR. This confirms that dual-target CAR-T has a better killing effect on tumors with target heterogeneity than single-target CAR-T, thus providing stronger resistance to tumor escape.

[0174] 4. Results of the detection of the cytotoxic function of dual-target CARs loaded with other B7H3-specific VHHs and IL13Rα2-specific VHHs in human primary T cells. Figure 8 To assess the cytotoxic function of dual-target CARs loaded with other B7H3-specific VHHs and IL13Rα2-specific VHHs in human primary γδT cells, the co-cultured cells had an effector-to-target ratio of 1:1 and a co-culture time of 24 hours. Figure 8 middle: BI-1 is a dual-target CAR that targets B7H3 and IL13Rα2, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.21; BI-2 is a dual-target CAR that targets B7H3 and IL13Rα2, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.23; BI-3 is a dual-target CAR that targets B7H3 and IL13Rα2, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.24; BI-4 is a dual-target CAR that targets B7H3 and IL13Rα2, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.27; B-CAR is a single-target CAR that targets B7H3, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.5; I-CAR is a single-target CAR that targets IL13Rα2, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.6.

[0175] The results showed that BI-1 to BI-4 exhibited tumor-killing capabilities comparable to or higher than those of B CAR in target cells expressing different target sites.

[0176] 5. Results of CAR-T cell killing function against target cells of tumor models expressing two other antigens. Figure 9 The results show the killing function of dual-target CAR-T cells against target cells of two other tumor models expressing two antigens (OCI-AML-3 is a human acute myeloid leukemia tumor model, and Huh7 is a human hepatocellular carcinoma tumor model). The effector-target ratio was 1:1, and the co-culture time was 24 hours. Figure 9 middle: B-CAR is a single-target CAR-γδT that targets B7H3, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.5; I-CAR is a single-target CAR-γδT that targets IL13Rα2, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.6; BI-CAR is a dual-target CAR-γδT that targets B7H3 and IL13Rα2, and its extracellular antigen-binding region has the amino acid sequence shown in SEQ ID NO. 23.

[0177] The results showed that in target cells of other tumor models expressing B7-H3 and IL13Rα2 antigens, BI-CAR exhibited better tumor-killing function compared with B-CAR and I-CAR, indicating that it can overcome the heterogeneity of tumor antigens in different tumors.

[0178] 6. Affinity results of B7H3-specific recombinant single-chain nanobodies Figure 10 The results show the affinity of B7-H3-specific recombinant single-chain nanobodies determined by flow cytometry. Figure A shows the affinity curves of different concentrations of BNb5 for B7-H3 protein determined by biomembrane interference technique; Figure B shows the affinity curves of different concentrations of BNb5 for B7-H3-positive target cells determined by flow cytometry.

[0179] The results showed that the dissociation equilibrium constant (KD) of different concentrations of BNb5 for B7-H3 protein, determined by biomembrane interference technology, was 14.63 ± 0.32 nM; and the half-maximum effect concentration (EC50) of different concentrations of BNb5 for binding to B7-H3 positive target cells, determined by flow cytometry, was 1.43 nM.

[0180] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A B7H3 nanobody targeting B7-H3, characterized in that, The B7H3 nanobody comprises B-HCDR1, B-HCDR2 and B-HCDR3; The B-HCDR1 has an amino acid sequence as shown in any one of SEQ ID NO. 28-32; The B-HCDR2 has an amino acid sequence as shown in any one of SEQ ID NO. 33-37; The B-HCDR3 has an amino acid sequence as shown in any one of SEQ ID NO. 38-42.

2. The B7H3 nanobody according to claim 1, characterized in that, The B7H3 nanobody comprises B-HCDR1, B-HCDR2, and B-HCDR3 selected from any one or more of the following groups: (1) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.28, SEQ ID NO.33 and SEQ ID NO.38; (2) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.29, SEQ ID NO.34 and SEQ ID NO.39; (3) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.30, SEQ ID NO.35 and SEQ ID NO.40; (4) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.31, SEQ ID NO.36 and SEQ ID NO.41; (5) B-HCDR1, B-HCDR2 and B-HCDR3 as shown in SEQ ID NO.32, SEQ ID NO.37 and SEQ ID NO.

42.

3. The B7H3 nanobody according to claim 2, characterized in that, The B7H3 nanobody has an amino acid sequence as shown in any one of SEQ ID NO. 1-5, or contains partial segments of any one or more of the amino acid sequences shown in SEQ ID NO. 1-5; or has a sequence with more than 80% homology to any one of the amino acid sequences in SEQ ID NO. 1-5.

4. A chimeric immune receptor, characterized in that, The chimeric immune receptor comprises the B7H3 nanobody according to any one of claims 1-3.

5. The chimeric immune receptor according to claim 4, characterized in that, The chimeric immune receptors mentioned include any one of the following: chimeric antigen receptor (CAR), STAR receptor, HIT receptor, TruC receptor, TAC receptor, and AbTCR receptor.

6. The chimeric immune receptor according to claim 4, characterized in that, The chimeric immune receptor is a chimeric antigen receptor, which includes: an antigen-binding region, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain.

7. The chimeric immune receptor according to claim 6, characterized in that, The hinge region is selected from the hinge regions of any one or more of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, or CD154. Alternatively, the transmembrane domain is selected from any one or more of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1B B. OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B 4. BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2 or CS1; Alternatively, the intracellular co-stimulatory domain is selected from the intracellular co-stimulatory signal transduction domains of any one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, or MyD88; Alternatively, the intracellular signaling domain is selected from the intracellular signaling domains of any one or more of the following proteins: CD3, CD79a, CD79b, MP2A, DAP10, or DAP12.

8. The chimeric immune receptor according to claim 6, characterized in that, The antigen-binding region is selected from any one or more of the following: (1) B7H3 nanobody; (2) B7H3 nanobody-Linker-IL13Rα2 nanobody; (3) IL13Rα2 nanobody-Linker-B7H3 nanobody.

9. The chimeric immune receptor according to claim 8, characterized in that, The IL13Rα2 nanobody has an amino acid sequence as shown in any one of SEQ ID NO. 6-10, or contains partial segments of any one or more of the amino acid sequences shown in SEQ ID NO. 6-10; or has a sequence with more than 80% homology to any one of the amino acid sequences in SEQ ID NO. 6-10.

10. The chimeric immune receptor according to claim 8, characterized in that, The Linker includes any one or more of flexible connectors and rigid connectors.

11. A soluble protein, characterized in that, The soluble protein comprises the B7H3 nanobody according to any one of claims 1-3.

12. The soluble protein according to claim 11, characterized in that, The soluble protein includes any one of monospecific antibodies, bispecific antibodies, multispecific antibodies, T-cell binders, antibody-TCR fusion proteins, and antibody-drug conjugates.

13. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the B7H3 nanobody according to any one of claims 1-3, the chimeric immune receptor according to any one of claims 4-10, or the soluble protein according to any one of claims 11-12.

14. A carrier comprising the nucleic acid molecule of claim 13, characterized in that, The vectors include any one or more of lentiviral vectors, retroviral vectors, and adenoviral vectors.

15. A chimeric antigen receptor cell, characterized in that, The chimeric antigen receptor cell comprises expressing the vector of claim 14.

16. The chimeric antigen receptor cell according to claim 15, characterized in that, The chimeric antigen receptor cells include any one or more of T cells, NK cells, macrophages, monocytes, dendritic cells, granulocytes, NKT cells, B cells, and stem cells; preferably, the chimeric antigen receptor cells are T cells; more preferably, the T cells are selected from αβT cells and / or γδT cells.

17. The application of the B7H3 nanobody according to any one of claims 1-3, the chimeric immune receptor according to any one of claims 4-10, the soluble protein according to any one of claims 11-12, the nucleic acid molecule according to claim 13, the carrier according to claim 14, or the chimeric antigen receptor cell according to any one of claims 15-16, characterized in that, The applications include any one or more of the following: (1) Application in the preparation of B7H or B7H3 / IL13Rα23 detection products; (2) Application in the preparation of B7H3 or B7H3 / IL13Rα2 related disease and diagnostic products; (3) Application in the preparation of drugs for the treatment of B7H3 or B7H3 / IL13Rα2 related diseases.

18. The application according to claim 17, characterized in that, The B7H3-related diseases include any one or more of the following: tumors, infectious diseases, autoimmune diseases, and cardiovascular and cerebrovascular diseases; the tumors include any one or more of the following: glioblastoma, medulloblastoma, ependymoblastoma, non-small cell lung cancer, pancreatic cancer, primary liver cancer, colorectal cancer, breast cancer, prostate cancer, laryngeal cancer, melanoma, acute myeloid leukemia, and hepatocellular carcinoma.

19. A drug, characterized in that, The drug comprises the B7H3 nanobody according to any one of claims 1-3, the chimeric immune receptor according to any one of claims 4-10, the soluble protein according to any one of claims 11-12, the nucleic acid molecule according to claim 13, the carrier according to claim 14, or the chimeric antigen receptor cell according to any one of claims 15-16.

20. A reagent kit, characterized in that, The kit comprises the B7H3 nanobody according to any one of claims 1-3, the chimeric immune receptor according to any one of claims 4-10, the soluble protein according to any one of claims 11-12, the nucleic acid molecule according to claim 13, the carrier according to claim 14, or the chimeric antigen receptor cell according to any one of claims 15-16.

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