A nanobody targeting il13rα2, chimeric immunoreceptors, and applications and products thereof

Abstract

This invention belongs to the field of antibody technology, specifically relating to an IL13Rα2 nanobody targeting IL13Rα2, a chimeric immune receptor, and their applications and products. This invention is the first to discover an IL13Rα2 nanobody with a specific complementarity-determining region amino acid sequence. The nanobody possesses advantages such as high affinity, high specificity, low immunogenicity, small molecular weight, and strong tissue penetration. This invention also provides a chimeric immune receptor comprising the nanobody, recombinant protein, nucleic acid molecule, expression vector, engineered immune cells, antibody preparation, kit, and pharmaceutical composition. CAR-T cells constructed based on the nanobody exhibit highly efficient and specific killing ability against IL13Rα2-positive tumor cells and can effectively overcome tumor antigen heterogeneity. They can be used to prepare drugs or kits for the diagnosis or treatment of IL13Rα2-related diseases, showing promising clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a nanobody targeting IL13Rα2, a chimeric immune receptor, and their applications and products. Background Technology

[0002] Immunotherapy, particularly 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 CAR-T function, with the antigen recognition region typically being the scFv (single-chain variable fragment). Most reported CAR-T cell therapies currently utilize scFv derived from murine or human antibodies. However, traditional CARs rely on scFv as the antigen recognition domain, and its inherent limitations severely restrict the efficacy of CAR-T therapy: on the one hand, the murine component in murine or humanized antibody fragments can induce immune rejection in patients, thereby reducing the persistence of CAR-T in vivo and affecting efficacy; on the other hand, scFv is large in size, has a high molecular weight, lacks flexibility, and is prone to aggregation, resulting not only in a short in vivo half-life and weak ability to penetrate solid tumors, but also potentially causing abnormal leakage of the CAR molecule's "background" signal, increasing the risk of T cell exhaustion and non-specific activation, further limiting its application in CAR-T therapy.

[0003] Compared with scFv, alpaca-derived nanobodies (VHH, also known as single-domain antibodies) have significant advantages as the smallest antigen-binding fragment composed only of the variable region of the heavy chain. They have become an ideal choice for the extracellular antigen-binding region of CAR and are expected to solve many problems of traditional scFv: (1) They have high homology with human genes and low immunogenicity, which can reduce the immune rejection reaction in patients and prolong the in vivo survival time of CAR-T cells; (2) They are small in size and structurally stable, making them suitable as the antigen-binding domain of CAR. They contain only one heavy chain and do not need to consider the interaction between the light chain and the heavy chain, which reduces the difficulty of designing and preparing CAR molecules; (3) They have high affinity and can screen nanobodies that do not require affinity optimization, which simplifies the development process of CAR-T therapy; (4) They can recognize cavities or hidden epitopes that traditional antibodies cannot recognize, which can better approach the hidden antigens on the surface of tumor cells and improve the ability to recognize tumor cells; (5) They have good permeability and are expected to improve the infiltration ability of CAR-T cells into tumor tissues and improve the problem of low migration and infiltration efficiency of CAR-T cells in the treatment of solid tumors.

[0004] Currently, dozens of tumor antigens have been reported as potential targets for CAR-T therapy in hematological malignancies and solid tumors. Among them, interleukin-13 receptor α2 (IL13Rα2) is a transmembrane glycoprotein belonging to the cytokine receptor family. It mainly participates in cell signaling by binding to interleukin-13 (IL-13). Its expression level is extremely low in normal human tissues, but 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, papillary thyroid carcinoma, pancreatic ductal adenocarcinoma, and glioblastoma (GBM). Therefore, it has become a biomarker for prognosis after treatment intervention in many solid tumors and is also a highly promising tumor-specific therapeutic target, providing an important site of action for precision targeted therapy of tumors.

[0005] Currently, although research on targeted therapy for IL13Rα2 has been conducted, there are still significant shortcomings: existing anti-IL13Rα2 antibodies are mostly traditional monoclonal antibodies or scFv, which are limited by the inherent defects of scFv, resulting in limited targeting, affinity and tumor penetration ability, making it difficult to meet clinical treatment needs; at the same time, existing cell therapies targeting IL13Rα2, such as CAR-T therapy, mostly use scFv as the antigen recognition domain, which also faces problems such as immune rejection, poor stability of CAR molecules, T cell exhaustion and tumor escape caused by tumor target heterogeneity, which seriously affect the efficacy of treatment.

[0006] Therefore, screening for IL13Rα2 nanobodies with high specificity and high affinity, constructing chimeric immune receptor molecules based on these nanobodies, and developing IL13Rα2-specific cell therapies have become technical challenges that urgently need to be addressed by those skilled in the art. Summary of the Invention

[0007] To address the above shortcomings, this invention provides a nanobody targeting IL13Rα2, a chimeric immune receptor, and its applications and products.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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 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. They are the most commonly used effector cells in traditional CAR-T cell therapy.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] The term "nucleic acid" in this invention refers to any compound and / or substance comprising polymers containing nucleotides, such as nucleic acid molecules. In this document, "nucleic acid," "nucleic acid molecule," 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'.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] In this invention, the term "IL13Rα2" refers to interleukin-13 receptor subunit alpha-2, a transmembrane glycoprotein belonging to the cytokine receptor family. IL13Rα2 primarily participates in cell signaling regulation by specifically binding to interleukin-13 (IL-13). Its expression level is extremely low in normal human tissues, but it is highly expressed in various malignant tumors such as glioblastoma, melanoma, renal cell carcinoma, colorectal cancer, gastric cancer, breast cancer, lung cancer, pancreatic cancer, liver cancer, head and neck cancer, and adrenocortical carcinoma. High expression is usually closely related to tumor progression, invasion and metastasis, and poor prognosis, making it an important tumor-specific target and prognostic biomarker.

[0028] The technical solution of the present invention: On one hand, the present invention provides an IL13Rα2 nanobody targeting IL13Rα2, wherein the complementarity-determining region of the IL13Rα2 nanobody includes: I-HCDR1, I-HCDR2 and I-HCDR3; The I-HCDR1 has an amino acid sequence as shown in any one of SEQ ID NO. 11-15; The I-HCDR2 has an amino acid sequence as shown in any one of SEQ ID NO.16-20; The I-HCDR3 has an amino acid sequence as shown in any one of SEQ ID NO.21-25.

[0029] SEQ ID NO.11: TTTFSINT.

[0030] SEQ ID NO.12: GWSLKNYP.

[0031] SEQ ID NO.13: TTTFSINN.

[0032] SEQ ID NO.14: GRTFSSYA.

[0033] SEQ ID NO.15: GGNFNTYA.

[0034] SEQ ID NO.16: IYGDGR.

[0035] SEQ ID NO.17: IDWGIFTT.

[0036] SEQ ID NO.18: IYGSGRT.

[0037] SEQ ID NO.19: ISSRGGTT.

[0038] SEQ ID NO.20: ITWGGDST.

[0039] SEQ ID NO.21: NSISAATRLFD.

[0040] SEQ ID NO.22: YAVGNALVH.

[0041] SEQ ID NO.23: NSISDATRLFD.

[0042] SEQ ID NO.24: NARRIREDY.

[0043] SEQ ID NO. 25: KAVIKEFNDSRAVDY.

[0044] Specifically, the complementarity-determining region of the IL13Rα2 nanobody is selected from any one or more of the following: (1) I-HCDR1 as shown in SEQ ID NO.11, I-HCDR2 as shown in SEQ ID NO.16, and I-HCDR3 as shown in SEQ ID NO.21; (2) I-HCDR1 as shown in SEQ ID NO.12, I-HCDR2 as shown in SEQ ID NO.17, and I-HCDR3 as shown in SEQ ID NO.22; (3) I-HCDR1 as shown in SEQ ID NO.13, I-HCDR2 as shown in SEQ ID NO.18, and I-HCDR3 as shown in SEQ ID NO.23; (4) I-HCDR1 as shown in SEQ ID NO.14, I-HCDR2 as shown in SEQ ID NO.19, and I-HCDR3 as shown in SEQ ID NO.24; (5) I-HCDR1 as shown in SEQ ID NO.15, I-HCDR2 as shown in SEQ ID NO.20, and I-HCDR3 as shown in SEQ ID NO.25.

[0045] Preferably, the IL13Rα2 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.

[0046] In another aspect, the present invention provides a chimeric immune receptor comprising the IL13Rα2 nanobody described in any of the preceding claims.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] Specifically, the chimeric antigen receptor also includes a signal peptide.

[0054] 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.

[0055] In another aspect, the present invention provides a recombinant protein, characterized in that the recombinant protein has: (1) The IL13Rα2 nanobody described in any of the above items; (2) Bioactive proteins or their functional fragments that assist in the expression and / or secretion of IL13Rα2 nanobodies or prolong their half-life in vivo.

[0056] In another aspect, the present invention provides a nucleic acid molecule that encodes any of the IL13Rα2 nanobody, chimeric immune receptor or recombinant protein described above.

[0057] In another aspect, the present invention provides an expression vector containing any of the nucleic acid molecules described above.

[0058] Preferably, the expression vector includes any one or more of lentiviral vectors, retroviral vectors, and adenoviral vectors.

[0059] In another aspect, the present invention provides an engineered immune cell comprising the expression vector described in any of the preceding claims.

[0060] Specifically, the engineered immune cells include chimeric immune receptor cells.

[0061] Preferably, the chimeric immune receptor cell source includes any one or more of T cells, NK cells, macrophages, monocytes, dendritic cells, granulocytes, NKT cells, B cells, and stem cells.

[0062] More preferably, the chimeric immune receptor cells are derived from T cells.

[0063] More preferably, the T cells are selected from αβT cells and / or γδT cells.

[0064] In another aspect, the present invention provides an antibody preparation comprising any of the IL13Rα2 nanobodies, chimeric immune receptors, recombinant proteins, nucleic acid molecules, expression vectors, or engineered immune cells described above.

[0065] Specifically, the antibody formulation also includes a pharmaceutically acceptable carrier.

[0066] Preferably, the pharmaceutically acceptable carrier buffer, protectant, surfactant, isotonic regulator, antioxidant, pH adjuster, chelating agent, solubilizer, filler, and preservative are selected from any one or more of these.

[0067] In another aspect, the present invention provides a kit comprising any of the IL13Rα2 nanobody, chimeric immune receptor, recombinant protein, nucleic acid molecule, expression vector, engineered immune cell or antibody preparation described above.

[0068] Specifically, the kit is used for IL13Rα23 detection and / or the diagnosis of IL13Rα2-related diseases.

[0069] Preferably, the IL13Rα2-related diseases include any one or more of tumors, infectious diseases, autoimmune diseases, and cardiovascular and cerebrovascular diseases.

[0070] More preferably, the tumor includes any one or more of 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.

[0071] In another aspect, the present invention provides a pharmaceutical composition comprising any of the above-described IL13Rα2 nanobody, chimeric immune receptor, recombinant protein, nucleic acid molecule, expression vector, engineered immune cell or antibody preparation.

[0072] Specifically, the pharmaceutical composition further includes at least one pharmaceutically acceptable excipient.

[0073] Preferably, the excipients include any one or more of the following: solvent, diluent, disintegrant, precipitation inhibitor, surfactant, flow aid, binder, lubricant, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, stabilizer, hydrating agent, emulsification accelerator, buffer, absorbent, colorant, flavoring agent, sweetener, ion exchanger, release agent, coating agent, flavoring agent, and antioxidant.

[0074] In another aspect, the present invention provides a method for in vitro detection of IL13Rα2 in samples for non-diagnostic purposes, the method comprising using any of the above-described IL13Rα2 nanobodies, chimeric immune receptors, recombinant proteins, nucleic acid molecules, expression vectors, engineered immune cells, or kits.

[0075] The beneficial effects of this invention are: This invention successfully screened and identified five specific nanobodies targeting IL13Rα2 by constructing a natural nanobody phage display library. These antibodies exhibited extremely high binding affinity to both IL13Rα2 protein and positive cells, with a dissociation equilibrium constant (KD) of up to 4.11 nM and a half-maximal effective concentration (EC50) as low as 0.47 nM, providing high-quality targeting molecules for subsequent applications.

[0076] This invention uses alpaca-derived nanobodies to replace traditional scFv as the antigen recognition domain for CARs. Nanobodies have advantages such as small molecular weight, low immunogenicity, good stability, and strong tissue penetration, which can effectively reduce the immune rejection response of patients, prolong the in vivo persistence of CAR-T cells, and enhance the infiltration ability of solid tumors, thereby solving the problems of immune rejection, T cell depletion, and difficulty in penetrating solid tumors in traditional CAR-T therapy.

[0077] CAR-T cells (ICAR1-ICAR5) constructed based on the aforementioned nanobodies exhibited significant anti-tumor activity in in vitro experiments. Experiments demonstrated that these CAR-T cells could efficiently and specifically kill various IL13Rα2-positive tumor cells (such as glioblastoma U87, acute myeloid leukemia OCI-AML-3, and hepatocellular carcinoma Huh7), while showing no significant killing effect on IL13Rα2-negative cells, demonstrating high targeting specificity and powerful killing function.

[0078] This invention not only provides nanobodies and CAR-T cells targeting IL13Rα2, but also encompasses recombinant proteins, nucleic acid molecules, expression vectors, engineered immune cells (such as T cells and γδT cells), antibody formulations, kits, and pharmaceutical compositions containing these components. These products and applications provide comprehensive technical solutions for the diagnosis, treatment, and research of IL13Rα2-related diseases, particularly various tumors that highly express IL13Rα2, such as glioblastoma, pancreatic cancer, colorectal cancer, and breast cancer. Attached Figure Description

[0079] 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.

[0080] Figure 2 To demonstrate the binding detection results of monoclonal phages against the anti-IL13Rα2 nanobody, the results of phage binding detection were presented using enzyme-linked immunosorbent assay (ELISA) to assess the ability of phages to bind to antigens.

[0081] Figure 3 This 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.

[0082] Figure 4 The results show the functional assay of CAR-T cells targeting IL13Rα2; Figure A is a pseudo-color graph of the transduction efficiency and membrane expression level of ICAR1-5 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 ICAR1-5; Figure C shows the results of the killing function of ICAR1-5 on target cells in human primary T cells.

[0083] Figure 5 The figure shows the cytotoxic function of human γδT cells transfected with CAR targeting IL13Rα2 against target cells expressing different antigens; AC in the figure represent CAR-γδT and U87-IL13Rα2, respectively. KO Cells, U87-B7H3 KOThe death rate of target cells after co-culturing cells or U87 cells at different ET ratios for 24 hours.

[0084] Figure 6 The results show the killing function of CAR-T cells against target cells of tumor models expressing 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.

[0085] Figure 7 The results of flow cytometry determination of the affinity of IL13Rα2-specific recombinant single-chain nanobody are shown. In the figure, A is the affinity curve of different concentrations of INb1 to IL13Rα2 protein determined by biomembrane interference technique; B is the affinity curve of different concentrations of INb1 to IL13Rα2-positive target cells determined by flow cytometry. Detailed Implementation

[0086] 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.

[0087] 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.

[0088] 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℃.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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 2×10¹³ PFU / mL with PBS.

[0100] 5. Add an equal volume of sterile glycerol (final concentration 50%), dispense (1.5 mL / tube), and store at -20°C.

[0101] 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.

[0102] Example 3: Phage panning using 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.

[0103] 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.

[0104] 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.

[0105] 4. Blocking positive phages: After blocking with negative 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 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 IL13Rα2 to 100-150 nM).

[0106] 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.

[0107] 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℃.

[0108] 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.

[0109] 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℃.

[0110] 9. Repeat steps 1-8 for the next round of screening until the amount of phage recovered reaches the expected level.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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 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).

[0115] 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.

[0116] 6. Phage flow cytometry detection. Cell preparation: Collect cells according to the passage procedure, centrifuge, resuspend in PBS containing 5% FBS, and adjust the density to 2×10⁶ cells / mL. 7 / mL, add 50μL to each well of a U-shaped 96-well plate. Phage binding and staining: add 100μL of phage (2×10⁶ / mL) to each well. 10 Incubate with either M13KO7 (NC wells) or M13KO7 (NC wells) at 4°C for 2 hours. After washing four times, add 100 μL of diluted anti-M13-PE antibody and incubate in the dark for 30 minutes. After washing three times, resuspend in fixative and transfer to flow cytometry tubes for detection. PE-positive results indicate a positive binding clone.

[0117] The results of detecting the phage-antigen binding ability by enzyme-linked immunosorbent assay (ELISA) are as follows: Figure 2 As shown, Figure 2 The results of phage binding assays for the anti-IL13Rα2 nanobody are presented. The horizontal axis represents the phage clone number, and the vertical axis represents the P / N ratio, which is the OD450 of the experimental group versus the OD450 of the negative antigen control group. A higher P / N ratio indicates a stronger positive antigen binding of the phage.

[0118] Example 5: Construction of IL13Rα2-targeted CAR plasmid and preparation of lentivirus 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. 8 PFU antigen-positive phages were added to 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 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. Plasmid extraction was then performed according to the instructions of the Tiangen plasmid mini-extraction kit.

[0119] 2. Construction of IL13Rα2-targeted CAR plasmid: The IL13Rα2-targeted CAR used in this invention comprises: an extracellular antigen-binding region, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0120] 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.

[0121] The amino acid sequence of the signal peptide region is shown in SEQ ID NO.6: MLLLVTSLLLCELPHPAFLLIPLE.

[0122] The amino acid sequence of the hinge region is shown in SEQ ID NO.7: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0123] The amino acid sequence of the transmembrane domain is shown in SEQ ID NO.8: IYIWAPLAGTCGVLLLSLVIT.

[0124] The amino acid sequence of the intracellular co-stimulatory domain is shown in SEQ ID NO.9: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0125] The amino acid sequence of the intracellular signal transduction region is shown in SEQ ID NO.10: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0126] 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.

[0127] 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.

[0128] 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 24-well plates (300 μL / well) or 48-well plates (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.

[0129] 2. Construction of CAR-γδT cells: CD3 antibody was diluted to a final concentration of 5 μg / mL using 1×PBS and added to 48-well plates. The plates were incubated overnight 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. The cells were activated at 37°C. After 48 h of activation, viral infection was performed by adding concentrated virus solution at an MOI of 20, gently mixing, and continuing culture. Following infection, the cells were further cultured in RPMI 1640 medium containing 10% inactivated serum and 200 IU / mL IL-2 to maintain a cell density of 1-2E6 / mL.

[0130] 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 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 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 5 laser 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.

[0131] 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.). 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 human astrocytoma cell; 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, which are naturally IL13Rα2 positive cells). Based on U87 cells, U87-IL13Rα2 was obtained by knocking out the IL13Rα2 gene using the CRISPR / Cas9 system. KO Cells. Huh7 cells (human hepatocellular carcinoma cells, purchased from the Cell Bank of Type Culture Collection, Chinese Academy of Sciences, 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.

[0132] 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. 5 Cells were seeded at a density of 500 μL / well in 48-well plates and cultured overnight. Suspension cells were immediately used for co-culture with CAR-T cells. The obtained CAR-T cells 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; 1:9 for Huh7 cells and 1:3 for OCI-AML3 cells). 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 dead target cells was calculated.

[0133] 4. Detect CAR activation levels using reporter cells. The activation level of CAR molecules was detected using a reporter gene assay. The CAR gene to be tested was transduced into the Jurkat-NFAT cell line, which stably expresses the NFAT response element driving the firefly luciferase reporter gene, to obtain CAR-Jurkat reporter cells. These reporter cells were co-cultured with antigen-positive target cells at a certain ratio (e.g., an effector-to-target ratio of 1:1), while a negative control was set up (target cells were cultured alone or co-cultured with Jurkat-NFAT cells without CAR transduction). After 24 hours of co-culture, cells were collected and chemiluminescence values ​​were measured using a firefly luciferase assay kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.). The level of chemiluminescence reflects the degree of NFAT pathway activation, thereby evaluating the activation level of CAR.

[0134] Figure 3 This 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.

[0135] from Figure 3 The experimental results in group A show that, except for I11, I14, I23, I27, I30-I32, and I34, other CARs all have high membrane uptake efficiency. The experimental results in group B show that I4, I6, I7, I10, I15, I22, I26, and I29 have high activation levels. The experimental results in group C show that I6, I26, I10, I7, and I29 have good tumor-killing efficacy. The I6 anti-IL13Rα2 nanobody is named INb1 and has the amino acid sequence shown in SEQ ID NO.1; the I26 anti-IL13Rα2 nanobody is named INb2 and has the amino acid sequence shown in SEQ ID NO.2; the I10 anti-IL13Rα2 nanobody is named INb3 and has the amino acid sequence shown in SEQ ID NO.3; the I7 anti-IL13Rα2 nanobody is named INb4 and has the amino acid sequence shown in SEQ ID NO.4; and the I29 anti-IL13Rα2 nanobody is named INb5 and has the amino acid sequence shown in SEQ ID NO.5.

[0136] SEQ ID NO.1: DVQLQESGGGLVQPGGSLTLSCTFPTTTFSINTMAWYRRAHGTARTLVAQIYGDGRTTYTESVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNSISAATRLFDWGQGTQVTVSS。

[0137] SEQ ID NO.2: DVQLQESGGGLVQAGGSLTLSCAASGWSLKNYPMGWFRQAPGKEREFVAAIDWGIFTTRYANSVKGRFTISRDNA*NTLDLQMNSLKPEDTAVYYCYAVGNALVHWGQGTQVTVSS。

[0138] SEQ ID NO.3: DVQLQESGGGLVQPGGSLTLSCTFPTTTFSINNMAWYRRAHGTARTLVAQIYGSGRTIYTESVKGRFTISRDNSKNTVYLQMNDLKPEDTAVYYCNSISDATRLFDWGQGTQVTVSS。

[0139] SEQ ID NO.4: DVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAVISRSGGTTYYTDSVKGRFAISRDNAKNTVYLEMNSLKPDDTAVYYCNARRIREDYWGQGTQVTVSS。

[0140] SEQ ID NO.5: DVQLQESGGGLVQAGGSLRLSCAVSGGNFNTYAMGWFRQAPGKEREFVAAITWGGDSTYFSNFVKGRFTISRDNAKNTVYLEMNSLKSEDSAVYVCKAVIKEFNDSRAVDYWGQGTQVTVSS。

[0141] 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.

[0142] 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.

[0143] 3. Affinity detection of nanobodies at the cell level Target cells expressing IL13Rα2 were cultured until 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, with the final concentration decreasing from high to low by 10. -5 M to 10 -12 M. After centrifuging and removing the supernatant, add 100 μL of diluted antibody solution to each well, stain on ice for 30 minutes, centrifuge to remove the supernatant, wash twice with 1×PBS, add 100 μL of diluted secondary antibody solution (anti-human IgG-APC antibody) to each well, stain on ice for 30 minutes, centrifuge to remove the supernatant, wash twice with 1×PBS, resuspend in 1×PBS, and transfer to flow cytometry tubes for flow cytometry detection. Analyze the mean fluorescence intensity (MFI) of positive cells measured at each antibody concentration gradient, and then use GraphPad Prism software to perform fitting plots for different MFI concentrations and antibody concentrations to obtain the dose-response curve for antibody-antigen binding in flow cytometry. The antibody concentration corresponding to the half-maximal binding (i.e., (the highest MFI value achievable at binding saturation + the lowest MFI value at non-binding) / 2) is the antibody affinity.

[0144] 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.

[0145] Example 9 Experimental Results 1. Results of IL13Rα2-targeted CAR-T cell function assay Figure 4 The results show the functional assay of IL13Rα2-targeted CAR-T cells. IL13Rα2 (INb1-INb5) specific VHHs were loaded into CAR plasmids and transfected into human primary αβT cells to construct CAR-T cells, and the tumor-killing efficiency in vitro was then assessed. Figure 4 In the figure, A is a pseudo-color graph showing the transduction efficiency and membrane expression level of ICAR1-ICAR5 (containing INb1-INb5) 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 ICAR1-ICAR5 (containing INb1-INb5); and C is the result of the cytotoxic function of ICAR1-ICAR5 (containing INb1-INb5) against target cells in human primary T cells.

[0146] The results showed that ICAR1-ICAR5 could be normally transduced into primary T cells with good membrane expression levels, and demonstrated a significant killing ability against IL13Rα2-positive target cells compared to the control group.

[0147] 2. Results of cytotoxic function of human γδT cells transfected with IL13Rα2-targeted CAR against target cells expressing different antigens Figure 5 The results show the killing function of human γδT cells transfected with IL13Rα2-targeted CARs against target cells expressing different antigens. Figure 5 The ICAR is an IL13Rα2-targeting CAR-γδT, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.1.

[0148] Figure 5The AC values ​​in the text represent IL13Rα2 targeting CAR-γδT and U87-IL13Rα2, respectively. KO Cells, U87-B7H3 KO The death rate of target cells after co-culturing U87 cells with different ET ratios for 24 hours.

[0149] The results showed that ICAR-γδT cells were more effective against wild-type U87 cells and B7H3 knockout U87-B7H3 cells. KO Cells exhibited significant and effector-to-target-dependent killing effects; while U87-IL13Rα2 knockout cells showed significant and effector-to-target-dependent killing effects. KO The absence of non-specific cell killing indicates that its cytotoxic activity is strictly dependent on the expression of the IL13Rα2 antigen on the surface of the target cells, demonstrating high antigen specificity.

[0150] 3. Results of CAR-T cell killing function against target cells of tumor models expressing other antigens Figure 6 The results show the killing function of CAR-T cells against target cells of other tumor models expressing 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 6 In this study, ICAR is a CAR-γδT targeting IL13Rα2, and its extracellular antigen-binding region has an amino acid sequence as shown in SEQ ID NO.1. The results showed that ICAR exhibited good tumor-killing activity in target cells of other tumor models expressing IL13Rα2 antigen, indicating that it can show excellent killing ability in different tumors.

[0151] 5. Affinity results of IL13Rα2-specific recombinant single-chain nanobodies Figure 7 The affinity of IL13Rα2-specific recombinant single-chain nanobodies was determined by flow cytometry. Figure A shows the affinity curves of different concentrations of INb1 for IL13Rα2 protein determined by biomembrane interferometry; Figure B shows the affinity curves of different concentrations of INb1 for IL13Rα2-positive target cells determined by flow cytometry. The results showed that the dissociation equilibrium constant (KD) of different concentrations of INb1 for B7-H3 protein, determined by biomembrane interferometry, was 4.11 ± 0.27 nM; Figure B shows the half-maximum effective concentration (EC50) of different concentrations of INb1 for IL13Rα2-positive target cells, determined by flow cytometry. 50 The value is 0.47 nM.

[0152] 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. An IL13Rα2 nanobody targeting IL13Rα2, characterized in that, The complementarity-determining regions of the IL13Rα2 nanobody include: I-HCDR1, I-HCDR2 and I-HCDR3; The I-HCDR1 has an amino acid sequence as shown in any one of SEQ ID NO. 11-15; The I-HCDR2 has an amino acid sequence as shown in any one of SEQ ID NO.16-20; The I-HCDR3 has an amino acid sequence as shown in any one of SEQ ID NO.21-25.

2. The IL13Rα2 nanobody according to claim 1, characterized in that, The complementarity-determining region of the IL13Rα2 nanobody is selected from any one or more of the following: (1) I-HCDR1 as shown in SEQ ID NO.11, I-HCDR2 as shown in SEQ ID NO.16, and I-HCDR3 as shown in SEQ ID NO.21; (2) I-HCDR1 as shown in SEQ ID NO.12, I-HCDR2 as shown in SEQ ID NO.17, and I-HCDR3 as shown in SEQ ID NO.22; (3) I-HCDR1 as shown in SEQ ID NO.13, I-HCDR2 as shown in SEQ ID NO.18, and I-HCDR3 as shown in SEQ ID NO.23; (4) I-HCDR1 as shown in SEQ ID NO.14, I-HCDR2 as shown in SEQ ID NO.19, and I-HCDR3 as shown in SEQ ID NO.24; (5) I-HCDR1 as shown in SEQ ID NO.15, I-HCDR2 as shown in SEQ ID NO.20, and I-HCDR3 as shown in SEQ ID NO.

25.

3. The IL13Rα2 nanobody according to claim 1, characterized in that, The IL13Rα2 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 amino acid sequences as shown in SEQ ID NO. 1-5; or has a sequence with more than 80% homology to any one amino acid sequence in SEQ ID NO. 1-5.

4. A chimeric immune receptor, characterized in that, The chimeric immune receptor comprises the IL13Rα2 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 5, 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. 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, FcεRIγ, 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.

7. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (1) The IL13Rα2 nanobody as described in any one of claims 1-3; (2) Bioactive proteins or their functional fragments that assist in the expression and / or secretion of IL13Rα2 nanobodies or prolong their half-life in vivo.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the IL13Rα2 nanobody according to any one of claims 1-3, the chimeric immune receptor according to any one of claims 4-6, or the recombinant protein according to claim 7.

9. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 8.

10. The expression vector according to claim 9, characterized in that, The expression vectors include any one or more of lentiviral vectors, retroviral vectors, and adenoviral vectors.

11. An engineered immune cell, characterized in that, The engineered immune cells comprise the expression vector as described in any one of claims 9-10.

12. The engineered immune cells according to claim 11, characterized in that, The engineered immune cells mentioned above are any one or more of the following: T cells, NK cells, macrophages, monocytes, dendritic cells, granulocytes, NKT cells, B cells, and stem cells.

13. An antibody preparation, characterized in that, The antibody formulation comprises the IL13Rα2 nanobody according to any one of claims 1-3, the chimeric immune receptor according to any one of claims 4-6, the recombinant protein according to claim 7, the nucleic acid molecule according to claim 8, the expression vector according to any one of claims 9-10, or the engineered immune cell according to any one of claims 11-12.

14. A reagent kit, characterized in that, The kit comprises the IL13Rα2 nanobody according to any one of claims 1-3, the chimeric immune receptor according to any one of claims 4-6, the recombinant protein according to claim 7, the nucleic acid molecule according to claim 8, the expression vector according to any one of claims 9-10, the engineered immune cells according to any one of claims 11-12, or the antibody preparation according to claim 13.

15. The kit according to claim 14, characterized in that, The kit is used for IL13Rα2 detection and / or the diagnosis of IL13Rα2-related diseases, including any one or more of tumors, infectious diseases, autoimmune diseases, and cardiovascular and cerebrovascular diseases. The tumors mentioned include any one or more of the following: glioblastoma, medulloblastoma, ependymoma, non-small cell lung cancer, pancreatic cancer, primary liver cancer, colorectal cancer, breast cancer, prostate cancer, pharyngeal cancer, melanoma, acute myeloid leukemia, and hepatocellular carcinoma.

16. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the IL13Rα2 nanobody according to any one of claims 1-3, the chimeric immune receptor according to any one of claims 4-6, the recombinant protein according to claim 7, the nucleic acid molecule according to claim 8, the expression vector according to any one of claims 9-10, the engineered immune cell according to any one of claims 11-12, or the antibody preparation according to claim 13.

17. A method for in vitro detection of IL13Rα2 in samples for non-diagnostic purposes, characterized in that, The method includes using the IL13Rα2 nanobody of any one of claims 1-3, the chimeric immune receptor of any one of claims 4-6, the recombinant protein of claim 7, the nucleic acid molecule of claim 8, the expression vector of any one of claims 9-10, the engineered immune cells of any one of claims 11-12, the antibody preparation of claim 13, or the kit of any one of claims 14-15.

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