Anti-CLDN18.2 nanobody, chimeric antigen receptor, and uses thereof

Abstract

The present invention provides an anti-CLDN18.2 nanobody, a chimeric antigen receptor, and uses thereof, and specifically provides a single-domain nanobody and a bispecific nanobody specifically binding to CLDN18.2 or an antigen binding fragment thereof, a chimeric antigen receptor based on the nanobody specifically binding to CLDN18.2, an engineered host cell, a nanobody-drug conjugate or conjugate or composition, a pharmaceutical composition, a kit, and related methods and uses.

Description

Anti-CLDN18.2 nanoantibodies, chimeric antigen receptors and their applications

[0001] Cross-references

[0002] This application claims priority to Chinese patent application No. CN202410288737.1 filed on March 13, 2024. The entire contents of the prior application are deemed to be the disclosure contents of this application and are incorporated herein in their entirety.

[0003] Sequence Listing Reference

[0004] The Sequence Listing, created on February 20, 2024, which is 49,377 bytes in size and named D-CF231747-sequence-listing-20240220.xml, is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to the field of biomedicine technology. Specifically, the present invention relates to nanobodies or antigen-binding fragments thereof that specifically bind to CLDN18.2, chimeric antigen receptors based on nanobodies or antigen-binding fragments thereof that specifically bind to CLDN18.2, and related applications thereof. Background Art

[0006] Claudin18.2 (CLDN18.2) is a splice variant 2 of claudin18, a member of the tight junction protein family. CLDN18.2 is a highly selective marker protein that is only expressed in differentiated gastric mucosal epithelial cells, with very limited expression in normal healthy tissues and no expression in undifferentiated gastric stem cells. Studies have shown that CLDN18.2 is abnormally expressed in the occurrence and development of various tumors, such as gastric cancer, gastroesophageal cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, and ovarian cancer. This makes CLDN18.2 a marker of interest in the field of tumor-targeted immunotherapy. Existing clinical studies have covered monoclonal antibodies, bispecific antibodies, chimeric antigen receptor-T (CAR-T) derived from CLDN18.2, and antibody-drug conjugates (ADCs). Compared with traditional antibodies, nanoantibodies have the advantages of small size, high stability, easy production through microbial systems, easy modification, simple humanization, and the ability to reach more sites of action. There is currently a demand for the development of anti-CLDN18.2 nanoantibodies.

[0007] Claudin 18.1 (CLDN18.1), a splice variant of Claudin18, is structurally similar to CLDN18.2, differing only by seven amino acid residues in the extracellular domain ECL1 sequence. Developing antibodies that specifically target CLDN18.2 but not CLDN18.1 has been challenging.

[0008] In recent years, there has been a growing interest in translational medicine within the field of immunology, particularly in tumor immunotherapy. CAR-T cell immunotherapy is a widely used strategy in tumor immunotherapy. This immunotherapy strategy achieved promising results in translational medicine in the early 1990s. Effective stimulation of T cells in the normal body and their immunological function require the regulation of multiple signals, including recognition of the T cell receptor by the MHC-antigen peptide complex as the first signal, recognition and activation of co-stimulatory molecules on the T cell surface as the second signal, and even the involvement of cytokines as the third signal. Chimeric antigen receptors (CARs) primarily incorporate proteins required for T cell stimulation and activation into a tandem array, thereby promoting T cell activation and specific killing. The specific killing of CAR-T cells primarily relies on recognition and binding of the antibody molecule at the front end. Currently, the most widely used approach is to specifically recognize the target protein using the single-chain variable region (scFv) of a monoclonal antibody derived from human or other species. However, there are the following defects: while ScFv and nanoantibodies have comparable affinities, ScFv has a larger molecular weight than nanoantibodies, and has certain limitations in molecular expression and functional performance; ScFv is derived from its parent monoclonal antibody and may have certain deficiencies in activity and stability.

[0009] In view of this, it is necessary to provide new and effective anti-CLDN18.2 nanoantibodies, as well as new chimeric antigen receptors and / or fusion proteins to address the shortcomings of the existing technology. Summary of the Invention

[0010] To address the above technical problems, the present disclosure provides novel single and double nanobodies that specifically bind to CLDN18.2. The present disclosure also provides chimeric antigen receptors (CARs) based on nanobodies and chimeric antigen receptor immune cells. The single and double nanobody CAR-T cells provided herein specifically kill cell lines expressing CLDN18.2.

[0011] In one aspect, the present disclosure provides a Nanobody or an antigen-binding fragment thereof that specifically binds to CLDN18.2, said Nanobody or antigen-binding fragment thereof comprising at least one of the following (a)-(c):

[0012] (a) VHH10, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, or amino acid sequences that are at least 75% identical to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively;

[0013] (b) VHH12, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or are amino acid sequences that are at least 75% identical to SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively;

[0014] (c) said VHH10 and VHH12.

[0015] In one aspect, the present disclosure provides a Nanobody or an antigen-binding fragment thereof that specifically binds to CLDN18.2, said Nanobody or antigen-binding fragment thereof comprising at least one of the following (a)-(c):

[0016] (a) VHH10, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively, or SEQ ID NO: 1 with 1, 2, 3, or 4 conservative substitutions, SEQ ID NO: 2 with 1, 2, 3, or 4 conservative substitutions, or SEQ ID NO: 3 with 1, 2, 3, or 4 conservative substitutions, respectively;

[0017] (b) VHH12, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, respectively, or SEQ ID NO: 4 with 1, 2, 3, or 4 conservative substitutions, SEQ ID NO: 5 with 1, 2, 3, or 4 conservative substitutions, or SEQ ID NO: 6 with 1, 2, 3, or 4 conservative substitutions, respectively;

[0018] (c) said VHH10 and VHH12.

[0019] In one aspect, the present disclosure provides a Nanobody or antigen-binding fragment thereof that specifically binds to CLDN18.2, said Nanobody or antigen-binding fragment thereof comprising at least one of the following (a)-(c):

[0020] (a) VHH10, the amino acid sequence of the heavy chain variable region of VHH10 being as shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity with SEQ ID NO: 13;

[0021] (b) VHH12, the amino acid sequence of the heavy chain variable region of VHH12 being as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with SEQ ID NO: 14;

[0022] (c) said VHH10 and VHH12.

[0023] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) based on a Nanobody or an antigen-binding fragment thereof that specifically binds to CLDN18.2, the chimeric antigen receptor comprising at least a Nanobody or an antigen-binding fragment thereof disclosed herein, preferably, the chimeric antigen receptor further comprises one or more of the following: a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, an intracellular signaling domain, a self-cleavage peptide, a detection tag, a detection tag signal peptide;

[0024] Preferably, the signal peptide includes the signal peptides of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6;

[0025] Preferably, the hinge region includes the hinge region of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, IL-11 receptor;

[0026] Preferably, the transmembrane domain includes the transmembrane domain of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, CD3ε, CD8α, IL-2 receptor, IL-7 receptor, IL-11 receptor;

[0027] Preferably, the costimulatory signaling domain includes the costimulatory signaling domains of the following molecules: 4-1BB, CD28, ICOS, CD27, CD19, CD4, CD8α, CD8β, HVEM, LIGHT, CD40, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, CD278;

[0028] Preferably, the intracellular signaling domain includes the intracellular signaling domain of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, FYN;

[0029] Preferably, the self-cleaving peptide is selected from P2A, T2A, E2A, and F2A;

[0030] Preferably, the detection tag is tEGFR;

[0031] Preferably, the detection tag signal peptide is a tEGFR signal peptide;

[0032] Most preferably, the chimeric antigen receptor is a signal peptide, a Nanobody disclosed herein or an antigen-binding fragment thereof, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signaling domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and a tEGFR connected in series;

[0033] Most preferably, the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO: 22 or has at least 75% sequence identity with SEQ ID NO: 22;

[0034] Most preferably, the amino acid sequence of the CD8 transmembrane domain is as shown in SEQ ID NO: 24 or has at least 75% sequence identity with SEQ ID NO: 24;

[0035] Most preferably, the amino acid sequence of the 4-1BB costimulatory signaling domain is as shown in SEQ ID NO: 26 or has at least 75% sequence identity with SEQ ID NO: 26;

[0036] Most preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 28 or has at least 75% sequence identity with SEQ ID NO: 28;

[0037] Most preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 18 or has at least 75% sequence identity with SEQ ID NO: 18;

[0038] Most preferably, the amino acid sequence of T2A is as shown in SEQ ID NO: 30 or has at least 75% sequence identity with SEQ ID NO: 30;

[0039] Most preferably, the amino acid sequence of the tEGFR signal peptide is as shown in SEQ ID NO: 18 or has at least 75% sequence identity with SEQ ID NO: 18;

[0040] Most preferably, the amino acid sequence of tEGFR is as shown in SEQ ID NO: 32 or has at least 75% sequence identity with SEQ ID NO: 32.

[0041] In another aspect, the disclosure provides an isolated nucleic acid encoding a Nanobody or antigen-binding fragment thereof disclosed herein, or a Chimeric Antigen Receptor disclosed herein.

[0042] In one aspect, the disclosure provides an isolated nucleic acid encoding a Nanobody or antigen-binding fragment thereof disclosed herein, said isolated nucleic acid comprising a nucleotide sequence encoding the CDR1, CDR2, CDR3 of VHH10 disclosed herein and / or a nucleotide sequence encoding the CDR1, CDR2, CDR3 of VHH12 disclosed herein,

[0043] Preferably, the nucleotide sequences encoding CDR1, CDR2, and CDR3 of VHH10 are respectively shown as SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, or are nucleotide sequences that are at least 75% identical to SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; the nucleotide sequences encoding CDR1, CDR2, and CDR3 of VHH12 are respectively shown as SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or are nucleotide sequences that are at least 75% identical to SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0044] In one aspect, the disclosure provides an isolated nucleic acid encoding a Nanobody or antigen-binding fragment thereof disclosed herein, said isolated nucleic acid comprising a nucleotide sequence encoding the heavy chain variable region of VHH10 disclosed herein and / or a nucleotide sequence encoding the heavy chain variable region of VHH12 disclosed herein,

[0045] Preferably, the nucleotide sequence encoding the heavy chain variable region of VHH10 is shown as SEQ ID NO: 15 or a nucleotide sequence having at least 75% identity with SEQ ID NO: 15; the nucleotide sequence encoding the heavy chain variable region of VHH12 is shown as SEQ ID NO: 16 or a nucleotide sequence having at least 75% identity with SEQ ID NO: 16.

[0046] In one aspect, the present disclosure provides an isolated nucleic acid encoding a chimeric antigen receptor disclosed herein, said isolated nucleic acid encoding a chimeric antigen receptor comprising at least an isolated nucleic acid encoding a Nanobody or an antigen-binding fragment thereof disclosed herein, preferably, said isolated nucleic acid encoding a chimeric antigen receptor further comprises one or more of the following: a promoter nucleotide sequence, a signal peptide coding sequence, a hinge region coding sequence, a transmembrane domain coding sequence, a co-stimulatory domain coding sequence, an intracellular signaling domain coding sequence, a self-cleavage peptide coding sequence, a detection tag signal peptide coding sequence, a detection tag coding sequence;

[0047] Preferably, the promoter nucleotide sequence is the EF1α promoter sequence shown in SEQ ID NO: 17;

[0048] Preferably, the signal peptide coding sequence is the signal peptide coding sequence shown in SEQ ID NO: 19;

[0049] Preferably, the hinge region coding sequence is the CD8α hinge region coding sequence shown in SEQ ID NO: 23;

[0050] Preferably, the transmembrane domain coding sequence is the CD8α transmembrane domain coding sequence shown in SEQ ID NO: 25;

[0051] Preferably, the costimulatory domain encoding sequence is the 4-1BB costimulatory domain encoding sequence shown in SEQ ID NO: 27;

[0052] Preferably, the intracellular signaling domain encoding sequence is the CD3ζ signaling domain encoding sequence shown in SEQ ID NO: 29;

[0053] Preferably, the self-cleaving peptide coding sequence is the T2A coding sequence shown in SEQ ID NO: 31;

[0054] Preferably, the coding sequence of the detection tag signal peptide is the tEGFR signal peptide coding sequence as shown in SEQ ID NO: 34;

[0055] Preferably, the detection tag coding sequence is the tEGFR coding sequence shown in SEQ ID NO: 33;

[0056] More preferably, the isolated nucleic acid encoding the chimeric antigen receptor is a promoter nucleotide sequence, a signal peptide coding sequence, an isolated nucleic acid encoding a nanobody or an antigen-binding fragment thereof, a hinge region coding sequence, a transmembrane domain coding sequence, a co-stimulatory domain coding sequence, an intracellular signaling domain coding sequence, a self-cleavage peptide coding sequence, a detection tag signal peptide coding sequence, and a detection tag coding sequence, which are sequentially connected in series.

[0057] In one aspect, the present disclosure provides an isolated polynucleotide having a sequence that is an isolated nucleic acid sequence encoding a chimeric antigen receptor disclosed herein, or a complementary sequence thereof.

[0058] In another aspect, the present disclosure provides a vector comprising an isolated nucleic acid or isolated polynucleotide disclosed herein. In one embodiment, the vector comprises a cloning vector or an expression vector. Preferably, the vector comprises a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector. More preferably, the viral vector comprises a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, or a herpesvirus vector.

[0059] In yet another aspect, the disclosure provides engineered host cells, wherein the engineered host cells express an isolated nucleic acid, an isolated polynucleotide, or a vector as provided herein. In one embodiment, the host cells include eukaryotic cells or prokaryotic cells. In one embodiment, the host cells are immune cells comprising T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, or any combination thereof. In a preferred embodiment, the host cells are T cells.

[0060] In yet another aspect, the present disclosure provides derivatives comprising a detectably labeled chimeric antigen receptor, isolated nucleic acid, or isolated polynucleotide disclosed herein, a chimeric antigen receptor, isolated nucleic acid, or isolated polynucleotide disclosed herein that confers antibiotic resistance, or a chimeric antigen receptor, isolated nucleic acid, or isolated polynucleotide disclosed herein coupled or conjugated to a therapeutic agent. In one embodiment, the detectable label comprises a fluorescent dye, a chemiluminescent marker, colloidal gold, or a chemiluminescent catalyst. In one embodiment, the chemiluminescent marker comprises luminol and its derivatives, acridinium esters or their derivatives, adamantane, isoluminol and its derivatives, rare earth elements, or ruthenium bipyridine complexes; the chemiluminescent catalyst comprises horseradish peroxidase or alkaline phosphatase. In one embodiment, the antibiotic resistance gene comprises a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, or a kanamycin resistance gene. In one embodiment, the therapeutic agent comprises a radionuclide, a cytokine, gold nanoparticles, viral particles, liposomes, nanomagnetic particles, a prodrug-activating enzyme, or a chemotherapeutic agent. In one embodiment, the cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF, M-CSF; the chemotherapeutic agents include cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, oxaliplatin, and oxaliplatin.

[0061] In one aspect, the disclosure provides a Nanobody-drug conjugate or conjugate or composition comprising a Nanobody or antigen-binding fragment thereof disclosed herein and a therapeutic agent.

[0062] In yet another aspect, the disclosure provides pharmaceutical compositions comprising a Nanobody or antigen-binding fragment thereof, a chimeric antigen receptor, an isolated nucleic acid, an isolated polynucleotide, a vector, a host cell or, a derivative or a Nanobody-drug conjugate or conjugate or composition disclosed herein, and a pharmaceutically acceptable carrier.

[0063] In one aspect, the present disclosure provides a detection product for detecting CLDN18.2 protein, wherein the detection product comprises the Nanobody or antigen-binding fragment thereof provided by the present disclosure.

[0064] In another aspect, the present disclosure provides a kit comprising an isolated nucleic acid, isolated polynucleotide, or vector disclosed herein. In one embodiment, the kit further comprises reagents for introducing the isolated nucleic acid, isolated polynucleotide, or vector into a host cell. In one embodiment, the kit further comprises instructions for introducing the isolated nucleic acid, isolated polynucleotide, or vector into a host cell.

[0065] In yet another aspect, the present disclosure provides any of the following methods, comprising:

[0066] (a) a method for preparing an engineered host cell disclosed herein, comprising the steps of introducing an isolated nucleic acid, an isolated polynucleotide or a vector provided herein into a host cell; preferably, the method of introduction comprises lipofection, microinjection, electroporation, a DNA vector, an RNA vector, a retroviral vector, a lentiviral vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector, or an adeno-associated virus vector;

[0067] (b) a method for stimulating a target cell population or tissue in a subject to produce an immune response, the method comprising the steps of: administering to the subject an engineered host cell disclosed herein;

[0068] (c) a method for producing a Nanobody or an antigen-binding fragment thereof disclosed herein, said method comprising the steps of culturing an engineered host cell disclosed herein, and isolating the Nanobody or an antigen-binding fragment thereof disclosed herein from the culture;

[0069] (d) a method for detecting CLDN18.2 in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising the steps of: contacting the test sample with a Nanobody or antigen-binding fragment thereof as disclosed herein, and detecting the formation of a complex between the Nanobody or antigen-binding fragment thereof and CLDN18.2;

[0070] Preferably, the Nanobody or antigen-binding fragment thereof is a Nanobody or antigen-binding fragment thereof labeled with a detectable marker;

[0071] More preferably, the markers that can be used for detection include fluorescent dyes, avidin, paramagnetic atoms, and radioactive isotopes;

[0072] Most preferably, the fluorescent pigment is fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, or peridinin-chlorophyll protein;

[0073] Most preferably, the avidin is biotin, egg white avidin, streptavidin, egg yolk avidin, or avidin-like;

[0074] Most preferably, the radioisotope is radioactive iodine, radioactive cesium, radioactive iridium, or radioactive cobalt;

[0075] (e) a method for preventing or treating a disease associated with abnormal expression of CLDN18.2, the method comprising administering to a subject in need thereof a therapeutically effective amount of a chimeric antigen receptor, isolated nucleic acid, isolated polynucleotide, vector, host cell, derivative, nanobody-drug conjugate or conjugate or composition or pharmaceutical composition disclosed herein;

[0076] Preferably, the disease associated with abnormal expression of CLDN18.2 includes solid tumors, preferably gastric cancer, gastroesophageal cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, and ovarian cancer.

[0077] In another aspect, the present invention discloses an application of any of the following aspects, comprising:

[0078] (a) Use of the Nanobodies or antigen-binding fragments thereof disclosed herein in the preparation of a detection product for detecting CLDN18.2 protein;

[0079] (b) Use of the Nanobodies or antigen-binding fragments thereof, chimeric antigen receptors, isolated nucleic acids, isolated polynucleotides, vectors, engineered host cells, derivatives, Nanobody-drug conjugates or conjugates or compositions, or pharmaceutical compositions disclosed herein for the preparation of a medicament for preventing or treating a disease associated with abnormal CLDN18.2 expression;

[0080] (c) Use of the chimeric antigen receptors, isolated nucleic acids, isolated polynucleotides, vectors, engineered host cells, and derivatives disclosed herein in the preparation of a kit for preparing immune cells for preventing or treating diseases associated with abnormal CLDN18.2 expression;

[0081] (d) Use of the kit disclosed herein in preparing immune cells for preventing or treating diseases associated with abnormal CLDN18.2 expression;

[0082] Preferably, the disease associated with abnormal expression of CLDN18.2 includes solid tumors, preferably gastric cancer, gastroesophageal cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, and ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 shows the alpaca immunization process, nanoantibody library construction process, antibody library enrichment and screening process, and monoclonal identification process in Example 1.

[0084] FIG2 shows the enrichment and screening results of the antibody library in Example 2.

[0085] Figure 3 shows the OD450 results of the screening and identification of monoclonal clones Nb015-10 and Nb015-12 in Example 1. Con: blank control.

[0086] FIG4 shows the flow cytometry identification results of the CLDN18.1 and CLDN18.2 overexpressing cell lines in Example 2.

[0087] Figure 5A shows a schematic diagram of the structural components of a single VHH CAR. Figure 5B shows a schematic diagram of the structural components of a single VHH CAR on the cell membrane.

[0088] Figure 6 shows the transduction efficiency test results of single VHH CAR-T cell culture in Example 3. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T.

[0089] Figure 7 shows the CD4 / CD8 ratio after culture of single VHH CAR-T cells in Example 3. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T.

[0090] Figure 8 shows the amplification curves of the single VHH CAR-T cells in Example 3. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T.

[0091] Figure 9 shows the in vitro killing results of NALM6, NALM6-CLDN18.1, and NALM6-CLDN18.2 by the single VHH CAR-T in Example 3. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T.

[0092] Figure 10A shows the amount of IFN-γ released during co-incubation of single VHH CAR-T cells with different target cells in Example 3. Figure 10B shows the amount of IFN-α released during co-incubation of single VHH CAR-T cells with different target cells in Example 3. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T.

[0093] Figure 11A shows a schematic diagram of the structural components of the dual VHH CAR in Example 4, and Figure 11B shows a schematic diagram of the structural components of the dual VHH CAR in Example 4 on the cell membrane.

[0094] Figure 12 shows the transduction efficiency test results of single and dual VHH CAR-T cell culture in Example 4. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T; dNMCO15-A: dNMCO15-A CAR-T.

[0095] Figure 13 shows the CD4 / CD8 ratios of single and dual VHH CAR-T cells after culture in Example 4. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T; dNMCO15-A: dNMCO15-A CAR-T.

[0096] Figure 14 shows the amplification curves of single and dual VHH CAR-T cells in Example 4. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T; dNMCO15-A: dNMCO15-A CAR-T.

[0097] Figure 15 shows the in vitro killing results of single and dual VHH CAR-T cells against NALM6, NALM6-CLDN18.1, and NALM6-CLDN18.2 in Example 4. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T; dNMCO15-A: dNMCO15-A CAR-T.

[0098] Figure 16A shows the amount of IFN-γ released during co-incubation of single and dual VHH CAR-T cells with different target cells in Example 4. Figure 16B shows the amount of IFN-α released during co-incubation of single and dual VHH CAR-T cells with different target cells in Example 4. Con: blank T cell control; NMCO15-10: NMCO15-10 CAR-T; NMCO15-12: NMCO15-12 CAR-T; dNMCO15-A: dNMCO15-A CAR-T.

[0099] Figure 17A shows the affinity results of Nb015-10 and CLDN18.2 antigen in Example 5. Figure 17B shows the affinity results of Nb015-12 and CLDN18.2 antigen in Example 5. Figure 17C shows the affinity results of dNb015-A Nanobody and CLDN18.2 antigen in Example 5.

[0100] FIG18 shows the specificity verification results of Nb015-10, Nb015-12, and dNb015-A for CLDN18.2 in Example 5.

[0101] Figure 19A shows the changes in tumor volume over time in mice after administration of dNMC015-A CAR-T or cryopreserved solution CS in Example 6; Figure 19B shows the changes in survival rate over time in tumor-bearing mice after administration of dNMC015-A CAR-T or cryopreserved solution CS in Example 6. Group 1 is the vehicle group using cryopreserved solution CS, and 0.2 ml of cryopreserved solution was injected into the tail vein. Group 2 is the dNMC015-A CAR-T cell group, and 0.75×10 7 CAR T cells / cell (0.2ml). DETAILED DESCRIPTION

[0102] Unless otherwise stated, the terms used herein have the usual understanding meaning to those skilled in the art. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained by the application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or the manner understood by those skilled in the art. In general, the nomenclature used herein and the experimental procedures of organic chemistry, medicinal chemistry, biology described herein are well known in the art and are generally adopted in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings that are commonly understood by those of ordinary skill in the art of the application. Where there are multiple definitions for the terms used herein, unless otherwise stated, the definitions in this section shall prevail.

[0103] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meanings of similar expressions can be deduced analogously.

[0104] As used herein, the terms "comprises," "includes," and "comprising" mean that in addition to the listed elements, other elements are not excluded.

[0105] As used herein, a "conservative amino acid substitution" can generally be described as an amino acid substitution in which one amino acid residue is replaced by another amino acid having a similar chemical structure and / or similar chemical properties, and which has little effect on the function, activity or other biological properties of a peptide, such as a Nanobody or an antigen-binding fragment thereof. Such conservative amino acid substitutions are well known in the art. Such conservative substitutions can, for example, be a substitution of one amino acid from the following groups (a)-(e) by another amino acid from the same group: (a) small aliphatic, non-polar or weakly polar amino acid residues: Ala, Ser, Thr, Pro and Gly; (b) negatively charged polar amino acid residues and their amides: Asp, Asn, Glu and Gln; (c) positively charged polar amino acid residues: His, Arg and Lys; (d) large aliphatic, non-polar amino acid residues: Met, Leu, Ile, Val and Cys; (e) aromatic amino acid residues: Phe, Tyr and Trp. Conservative amino acid substitutions can be substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0106] As used herein, the term "therapeutically effective amount" refers to an amount that results in a benefit or treatment of a disease compared to a corresponding subject not receiving that amount, but which is sufficiently low within the scope of sound medical judgment to avoid serious side effects. The therapeutically effective amount of the chimeric antigen receptor, isolated nucleic acid, isolated polynucleotide, vector, host cell, derivative, nanobody-drug conjugate or conjugate or composition or pharmaceutical composition described herein will vary with the chimeric antigen receptor, isolated nucleic acid, isolated polynucleotide, vector, host cell, derivative, nanobody-drug conjugate or conjugate or composition or pharmaceutical composition chosen; the route of administration; the severity of the disease being treated; the age, size, weight and physical condition of the patient being treated; the medical history of the patient being treated; the duration of treatment; the nature of concurrent treatment; the desired therapeutic effect, etc., but can still be determined in a routine manner by those skilled in the art.

[0107] As used herein, a "nucleic acid" may comprise deoxyribonucleotides, ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides; a "nucleotide sequence" may comprise deoxyribonucleotides, ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides.

[0108] As used herein, the term "identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is generally expressed as a percentage. Preferably, identity is determined over the entire length of the compared sequences. Therefore, two copies of the identical sequence have 100% identity. Those skilled in the art will recognize that some algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and Clustal W.

[0109] In the present invention, the terms "nanobody" and "VHH" are used indiscriminately and represent the variable domains of a single heavy chain of antibodies of the type found in camelids. In the absence of a light chain, each nanobody has three CDRs, represented as CDR1, CDR2 and CDR3. It is the smallest antigen-binding fragment with complete function. Usually, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (Nb) consisting of only one heavy chain variable region, also known as VHH. Nanobody / single domain antibody, as a new type of small molecule antibody fragment, is obtained by cloning the heavy chain variable region (VHH) of the natural heavy chain antibody of camels. It has excellent biological properties, a molecular weight of 12-15kDa, which is one-tenth of a complete antibody, and has good tissue penetrability, high specificity and good water solubility.

[0110] As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that retain binding specificity to a target antigen. Antigen-binding fragments include, but are not limited to, VHHs, CDR-containing fragments, and the like.

[0111] As used herein, the term "specific binding" refers to the non-covalent interaction between a Nanobody or its antigen-binding fragment and an antigen. The strength or affinity of the interaction can be expressed by the equilibrium dissociation constant (KD or Kd) between the antigen and the corresponding antibody: the smaller the KD value, the stronger the binding strength between the epitope and the antibody. The equilibrium dissociation constant (KD) is calculated as the ratio of Koff / kon, where the "association rate constant (Kon)" and "dissociation rate constant (Koff)" can be determined by calculating the concentration and the actual association and dissociation rates. (See Nature 361:186-87 (1993)).

[0112] In certain embodiments of the present disclosure, when the dissociation constant (KD) is less than or equal to 1×10 -6 M, in certain embodiments less than or equal to 1X10 -7 M, in certain embodiments less than or equal to 1X10 -8 M, in certain embodiments less than or equal to 1X10 -9 M, and in certain embodiments between 1×10 -8 Up to 1X10 -12 When the antibody or antigen-binding fragment thereof is between 1 and 2, the antibody or antigen-binding fragment thereof is considered to specifically bind to the antigen.

[0113] In the present invention, the term "affinity" refers to the binding ability between a macromolecule and its bound antigen, in particular the binding ability between a Nanobody and its bound antigen, for example the binding ability between the Nanobody of the present invention and the CLDN18.2 protein. The affinity of the Nanobodies of the present invention can be measured in vitro by several methods, including surface plasmon resonance or ELISA.

[0114] In the present invention, the term "variable" refers to the fact that certain parts of the variable region of an antibody differ in sequence, which contribute to the binding and specificity of each specific antibody to its specific antigen. The variability is concentrated in three segments of the heavy chain variable region called the complementarity determining regions (CDRs) or hypervariable regions. The variable region of a natural heavy chain each contains four FR regions (the more conserved parts of the variable region), which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, forming a partial β-pleated structure. The CDRs in each chain are closely together through the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody. The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in the antibody's antibody-dependent cytotoxicity.

[0115] As used herein, "sample" means a portion of a larger element. Preferably, a sample is a substance of biological origin. It contains cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical and / or physiological characteristics. For example, it refers to any sample derived from a subject of interest that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, tissue culture fluids, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0116] In the present invention, the term "administer" refers to the physical introduction of the product of the present invention into the subject using any of the various methods and delivery systems known to those skilled in the art, including but not limited to systemic administration and local administration, such as subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral, transdermal, pulmonary, ocular and topical administration, etc.

[0117] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can work together or independently to initiate transcription; an example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably connected to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include but are not limited to metallothionein promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters.

[0118] The CAR modified T cells provided by the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Briefly, the pharmaceutical composition of the present invention may include a target cell group as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0119] The mode of administering the product described in the present invention includes any convenient mode, including by spraying, injection, swallowing, infusion, implantation or transplantation. The composition described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention, such as a CAR-T composition, is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of T cells can be directly injected into a tumor, lymph node or infection site. In addition, the pharmaceutical composition, CAR-modified T cells, derivatives, etc. provided by the present invention can also be administered together with other therapeutic agents. Preferred examples of the other therapeutic agents include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, E), vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNA enzymes and RNA enzymes; radionuclides, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4 and melanoma growth stimulating protein. In addition, the pharmaceutical composition of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0120] As used herein, the term "subject" includes animals, such as vertebrates, preferably mammals, such as dogs, cats, pigs, cows, sheep, horses, rodents (e.g., mice, rats, or guinea pigs), or primates (e.g., gorillas, chimpanzees, and humans).

[0121] As used herein, the term "treat," ...

[0122] As used herein, an "isolated" nucleic acid consisting of a nucleotide sequence encoding a protein, or a portion or fragment thereof, is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in its production environment. Preferably, the isolated nucleic acid is free from all components of its production environment. The isolated nucleic acids encoding the proteins, or portions or fragments thereof, described herein are in a form or environment different from that in which they are found in nature. Thus, an isolated nucleic acid is distinguished from a nucleic acid encoding a protein, or portion or fragment thereof, described herein, that is naturally present in a cell.

[0123] As used herein, an "isolated" polynucleotide consisting of a nucleotide sequence encoding a protein, or a portion or fragment thereof, has been identified and separated from at least one contaminating polynucleotide with which it is normally associated in its production environment. Preferably, the isolated polynucleotide is free from association with any components of the production environment. The isolated polynucleotides encoding a protein, or a portion or fragment thereof, described herein, are in a form or environment different from that in which they are found in nature. Thus, an isolated polynucleotide is distinguished from a polynucleotide encoding a protein, or a portion or fragment thereof, described herein, that is naturally present in a cell.

[0124] As used herein, the CDRs may be numbered according to Kabat, IMGT, Chothia, or other commonly used systems. In a specific embodiment, the CDR numbering is based on the IMGT system.

[0125] As commonly used in the art, the left-hand end of a polypeptide sequence or amino acid sequence as used herein corresponds to the amino terminus or N-terminus, and the right-hand end corresponds to the carboxyl terminus or C-terminus. Similarly, the left-hand end of a single-stranded polynucleotide or nucleic acid sequence corresponds to the 5' end, the left-hand end of a double-stranded polynucleotide or nucleic acid sequence corresponds to the 5' direction, the right-hand end of a single-stranded polynucleotide or nucleic acid sequence corresponds to the 3' end, and the right-hand end of a double-stranded polynucleotide or nucleic acid sequence corresponds to the 3' direction.

[0126] Nanobodies or antigen-binding fragments thereof

[0127] The present disclosure provides Nanobodies or antigen-binding fragments thereof that specifically bind to CLDN18.2, said Nanobodies or antigen-binding fragments thereof comprising at least one of the following (a)-(c):

[0128] (a) VHH10, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, or amino acid sequences that are at least 75% identical to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively;

[0129] (b) VHH12, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or are amino acid sequences that are at least 75% identical to SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively;

[0130] (c) said VHH10 and VHH12.

[0131] In one embodiment, the Nanobodies or antigen-binding fragments thereof that specifically bind to CLDN18.2 provided herein comprise VHH10 and VHH12, wherein VHH10 comprises CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or are amino acid sequences having at least 75% identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively; and VHH12 comprises CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or are amino acid sequences having at least 75% identity to SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, respectively, and wherein VHH10 and VHH12 are directly connected or indirectly connected via a linker. In a preferred embodiment, the linker is G4S. In a more preferred embodiment, the linker is 3, 4, or 5 G4S. In a more preferred embodiment, the linker is represented by GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 20).

[0132] The present disclosure provides Nanobodies or antigen-binding fragments thereof that specifically bind to CLDN18.2, said Nanobodies or antigen-binding fragments thereof comprising at least one of the following (a)-(c):

[0133] (a) VHH10, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively, or SEQ ID NO: 1 with 1, 2, 3, or 4 conservative substitutions, SEQ ID NO: 2 with 1, 2, 3, or 4 conservative substitutions, or SEQ ID NO: 3 with 1, 2, 3, or 4 conservative substitutions, respectively;

[0134] (b) VHH12, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, respectively, or SEQ ID NO: 4 with 1, 2, 3, or 4 conservative substitutions, SEQ ID NO: 5 with 1, 2, 3, or 4 conservative substitutions, or SEQ ID NO: 6 with 1, 2, 3, or 4 conservative substitutions, respectively;

[0135] (c) said VHH10 and VHH12.

[0136] In one embodiment, the Nanobodies or antigen-binding fragments thereof that specifically bind to CLDN18.2 provided herein comprise VHH10 and VHH12, wherein the VHH10 comprises CDR1, CDR2, and CDR3, and the amino acid sequences of the CDR1-3 are as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively, or SEQ ID NO: 1 with 1, 2, 3, or 4 conservative substitutions, SEQ ID NO: 2 with 1, 2, 3, or 4 conservative substitutions, or SEQ ID NO: 3 with 1, 2, 3, or 4 conservative substitutions, respectively; and the VHH12 comprises CDR1, CDR2, and CDR3, and the amino acid sequences of the CDR1-3 are as shown in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, respectively, or SEQ ID NO: 4 with 1, 2, 3, or 4 conservative substitutions, SEQ ID NO: 5 with 1, 2, 3, or 4 conservative substitutions, or SEQ ID NO: 6 with 1, 2, 3, or 4 conservative substitutions, respectively. NO:6, and the VHH10 and VHH12 are directly connected or indirectly connected via a linker. In a preferred embodiment, the linker is G4S. In a more preferred embodiment, the linker is 3, 4 or 5 G4S. In a more preferred embodiment, the linker is represented by GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:20).

[0137] The present disclosure provides Nanobodies or antigen-binding fragments thereof that specifically bind to CLDN18.2. Said Nanobodies or antigen-binding fragments thereof comprise at least one of the following (a)-(c):

[0138] (a) VHH10, the amino acid sequence of the heavy chain variable region of VHH10 being as shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity with SEQ ID NO: 13;

[0139] (b) VHH12, the amino acid sequence of the heavy chain variable region of VHH12 being as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with SEQ ID NO: 14;

[0140] (c) said VHH10 and VHH12.

[0141] In one embodiment, the Nanobodies or antigen-binding fragments thereof that specifically bind to CLDN18.2 provided herein comprise VHH10 and VHH12, the amino acid sequence of the heavy chain variable region of VHH10 being as shown in SEQ ID NO: 13 or being an amino acid sequence having at least 75% identity to SEQ ID NO: 13, the amino acid sequence of the heavy chain variable region of VHH12 being as shown in SEQ ID NO: 14 or being an amino acid sequence having at least 75% identity to SEQ ID NO: 14, and the VHH10 and VHH12 being directly connected or indirectly connected via a linker. In a preferred embodiment, the linker is (G4S)n. In one embodiment, n is an integer from 1 to 8. In a preferred embodiment, n is an integer from 1 to 5. In a more preferred embodiment, n is 5, and the linker is as shown in GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 20).

[0142] In a specific embodiment, the full-length sequence of CLDN18.2 is MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV, SEQ ID NO: 49.

[0143] Furthermore, antibodies comprising conservative sequence variants of the amino acid sequences of preferred antibodies are also included within the scope of the present invention. Conservative amino acid sequence variants include modifications of the amino acid sequence that do not significantly alter the binding and neutralizing properties of the monoclonal neutralizing antibodies of the present invention, such as variants resulting from similar amino acid substitutions well known in the art, and variants resulting from amino acid deletions and additions are all within the scope of protection of the present invention. In addition, the Nanobodies of the present invention also include human and non-human antibodies, as well as all antibodies that have the same function as the monoclonal antibodies of the present invention or have been modified and optimized.

[0144] The Nanobodies or antigen-binding fragments thereof disclosed herein may comprise one or more glycosylation sites in the heavy chain variable region. As is well known to those skilled in the art, the presence of one or more glycosylation sites in the heavy chain variable region may enhance antibody immunogenicity or alter the pharmacokinetics of the antibody due to altered antigen binding.

[0145] Nanobodies or antigen-binding fragments thereof disclosed herein can be conjugated to other factors chemically or by genetic engineering. These factors provide the ability to target the antibody to a desired functional site or to enhance or provide other properties to the antibody.

[0146] The Nanobodies or antigen-binding fragments thereof disclosed herein can be labeled chemically or by genetic engineering to provide detectable Nanobodies, and detectable antibodies include a detectable moiety. Detectable moieties include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions.

[0147] The label used for detection, analysis and / or diagnostic purposes depends on the specific detection, analysis and / or diagnostic method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, laser scanning cytometry detection, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), bioassay (e.g., phagocytosis assay), Western blot application, etc. Suitable labels for detection, analysis, diagnostic methods known in the art are well known to those skilled in the art.

[0148] Chimeric Antigen Receptor (CAR)

[0149] The present disclosure provides a chimeric antigen receptor based on a Nanobody or an antigen-binding fragment thereof that specifically binds to CLDN18.2, the chimeric antigen receptor comprising at least a Nanobody or an antigen-binding fragment thereof disclosed herein. In a preferred embodiment, the chimeric antigen receptor based on a Nanobody or an antigen-binding fragment thereof that specifically binds to CLDN18.2 comprises a Nanobody or an antigen-binding fragment thereof disclosed herein, and further comprises one or more of the following: a signal peptide, a hinge region, a transmembrane domain, a costimulatory signaling domain, an intracellular signaling domain, a self-cleavage peptide, a detection tag, and a detection tag signal peptide.

[0150] In one embodiment, the signal peptide comprises the signal peptide of the following molecules: the α chain and β chain of the T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6. In a preferred embodiment, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 18 or has at least 75% sequence identity with SEQ ID NO: 18.

[0151] In one embodiment, the hinge region comprises the hinge region of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, IL-11 receptor. In a preferred embodiment, the hinge region comprises the CD8 hinge region. In a preferred embodiment, the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO: 22 or has at least 75% sequence identity with SEQ ID NO: 22.

[0152] In one embodiment, the transmembrane domain comprises the transmembrane domain of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, CD3ε, CD8α, IL-2 receptor, IL-7 receptor, IL-11 receptor. In a preferred embodiment, the transmembrane domain comprises the CD8 transmembrane domain. In a preferred embodiment, the amino acid sequence of the CD8 transmembrane domain is as shown in SEQ ID NO: 24 or has at least 75% sequence identity with SEQ ID NO: 24.

[0153] In one embodiment, the costimulatory signaling domain includes the costimulatory signaling domain of the following molecules: 4-1BB, CD28, ICOS, CD27, CD19, CD4, CD8α, CD8β, HVEM, LIGHT, CD40, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, CD278. In a preferred embodiment, the costimulatory signaling domain includes the 4-1BB costimulatory signaling domain. In a preferred embodiment, the amino acid sequence of the 4-1BB costimulatory signaling domain is as shown in SEQ ID NO: 26 or has at least 75% sequence identity with SEQ ID NO: 26.

[0154] In one embodiment, the intracellular signaling domain comprises the intracellular signaling domain of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, FYN. In a preferred embodiment, the intracellular signaling domain comprises the CD3ζ intracellular signaling domain. In a preferred embodiment, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 28 or has at least 75% sequence identity to SEQ ID NO: 28.

[0155] In one embodiment, the self-cleaving peptide is selected from P2A, T2A, E2A, and F2A. In a preferred embodiment, the self-cleaving peptide comprises T2A. In a preferred embodiment, the amino acid sequence of T2A is as shown in SEQ ID NO:30 or has at least 75% sequence identity with SEQ ID NO:30.

[0156] In one embodiment, the detection tag is tEGFR. In a preferred embodiment, the amino acid sequence of tEGFR is as shown in SEQ ID NO: 32 or has at least 75% sequence identity with SEQ ID NO: 32.

[0157] In one embodiment, the detection tag signal peptide is a tEGFR signal peptide. In a preferred embodiment, the amino acid sequence of the tEGFR signal peptide is as shown in SEQ ID NO: 18 or has at least 75% sequence identity with SEQ ID NO: 18.

[0158] In a preferred embodiment, the chimeric antigen receptor based on the Nanobody or its antigen-binding fragment that specifically binds to CLDN18.2 provided herein is a signal peptide, the Nanobody or its antigen-binding fragment disclosed herein, the CD8 hinge region, the CD8 transmembrane domain, the 4-1BB co-stimulatory signaling domain, the CD3ζ intracellular signaling domain, T2A, the tEGFR signal peptide, and the tEGFR are sequentially connected in series.

[0159] In a more preferred embodiment, the chimeric antigen receptor based on the Nanobody or antigen-binding fragment thereof that specifically binds to CLDN18.2 provided herein is a signal peptide, VHH10 disclosed herein, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signaling domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and a tEGFR sequenced in series.

[0160] In a more preferred embodiment, the chimeric antigen receptor based on the Nanobody or antigen-binding fragment thereof that specifically binds to CLDN18.2 provided herein is a signal peptide, VHH12 disclosed herein, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signaling domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and a tEGFR sequence connected in series.

[0161] In a more preferred embodiment, the chimeric antigen receptor based on the nanobody or antigen-binding fragment thereof that specifically binds to CLDN18.2 provided herein is a signal peptide, VHH10 and VHH12 disclosed herein, CD8 hinge region, CD8 transmembrane domain, 4-1BB costimulatory signaling domain, CD3ζ intracellular signaling domain, T2A, tEGFR signal peptide, tEGFR are sequentially connected in series. In one embodiment, the VHH10 and VHH12 are directly connected or indirectly connected through a linker, preferably through (G4S)n connection, wherein n is an integer of 1-8. In a more preferred embodiment, the VHH10 and VHH12 are linked by a linker shown in SEQ ID NO: 20.

[0162] Isolated nucleic acid, isolated polynucleotide

[0163] The present disclosure provides isolated nucleic acids encoding the Nanobodies or antigen-binding fragments thereof or chimeric antigen receptors disclosed herein.

[0164] The present disclosure provides an isolated nucleic acid encoding a Nanobody or antigen-binding fragment thereof disclosed herein, the isolated nucleic acid comprising a nucleotide sequence encoding the CDR1, CDR2, and CDR3 of VHH10 disclosed herein and / or a nucleotide sequence encoding the CDR1, CDR2, and CDR3 of VHH12 disclosed herein. In one embodiment, the nucleotide sequence encoding the CDR1, CDR2, and CDR3 of VHH10 is as shown in SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, respectively, or a nucleotide sequence having at least 75% identity to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, respectively; the nucleotide sequence encoding the CDR1, CDR2, and CDR3 of VHH12 is as shown in SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, respectively, or a nucleotide sequence having at least 75% identity to SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, respectively.

[0165] The present disclosure provides an isolated nucleic acid encoding a Nanobody or antigen-binding fragment thereof disclosed herein, the isolated nucleic acid comprising a nucleotide sequence encoding the heavy chain variable region of VHH10 disclosed herein and / or a nucleotide sequence encoding the heavy chain variable region of VHH12 disclosed herein. In one embodiment, the nucleotide sequence encoding the heavy chain variable region of VHH10 is as shown in SEQ ID NO: 15 or is a nucleotide sequence having at least 75% identity to SEQ ID NO: 15; the nucleotide sequence encoding the heavy chain variable region of VHH12 is as shown in SEQ ID NO: 16 or is a nucleotide sequence having at least 75% identity to SEQ ID NO: 16.

[0166] In one embodiment, when the Nanobody or antigen-binding fragment thereof disclosed herein comprises VHH10 and VHH12 disclosed herein, the isolated nucleic acid encoding the Nanobody or antigen-binding fragment thereof may optionally comprise a nucleotide sequence encoding a linker connecting said VHH10 and VHH12. In a preferred embodiment, when the Nanobody or antigen-binding fragment thereof disclosed herein comprises VHH10 and VHH12 disclosed herein, the isolated nucleic acid encoding the Nanobody or antigen-binding fragment thereof comprises a nucleotide sequence encoding a linker connecting said VHH10 and VHH12, and the nucleotide sequence of the linker is shown in SEQ ID NO: 21.

[0167] The present disclosure provides an isolated nucleic acid encoding a chimeric antigen receptor disclosed herein, the isolated nucleic acid encoding the chimeric antigen receptor comprising at least an isolated nucleic acid encoding a nanobody or an antigen-binding fragment thereof disclosed herein. In a preferred embodiment, the isolated nucleic acid encoding the chimeric antigen receptor comprises an isolated nucleic acid encoding a nanobody or an antigen-binding fragment thereof disclosed herein, and further comprises one or more of the following: a promoter nucleotide sequence, a signal peptide coding sequence, a hinge region coding sequence, a transmembrane domain coding sequence, a costimulatory domain coding sequence, an intracellular signaling domain coding sequence, a self-cleavage peptide coding sequence, a coding sequence for a detection tag signal peptide, and a detection tag coding sequence.

[0168] In one embodiment, the promoter nucleotide sequence is the EF1α promoter sequence shown in SEQ ID NO: 17.

[0169] In one embodiment, the signal peptide coding sequence is the signal peptide coding sequence shown in SEQ ID NO: 19.

[0170] In one embodiment, the hinge region coding sequence is a CD8α hinge region coding sequence, preferably, a CD8α hinge region coding sequence as shown in SEQ ID NO: 23;

[0171] In one embodiment, the transmembrane domain encoding sequence is a CD8α transmembrane domain encoding sequence, preferably, a CD8α transmembrane domain encoding sequence as shown in SEQ ID NO:25.

[0172] In one embodiment, the costimulatory domain coding sequence is a 4-1BB costimulatory domain coding sequence, preferably, a 4-1BB costimulatory domain coding sequence as shown in SEQ ID NO: 27.

[0173] In one embodiment, the intracellular signaling domain encoding sequence is a CD3ζ signaling domain encoding sequence, more preferably, a CD3ζ signaling domain encoding sequence as shown in SEQ ID NO: 29.

[0174] In one embodiment, the self-cleaving peptide coding sequence is a T2A coding sequence, preferably, the T2A coding sequence shown in SEQ ID NO: 31.

[0175] In one embodiment, the coding sequence of the detection tag signal peptide is a tEGFR signal peptide coding sequence, preferably, a tEGFR signal peptide coding sequence as shown in SEQ ID NO: 34.

[0176] In one embodiment, the detection tag coding sequence is a tEGFR coding sequence, preferably, a tEGFR coding sequence as shown in SEQ ID NO: 33.

[0177] In a preferred embodiment, the isolated nucleic acid encoding the chimeric antigen receptor provided herein is a promoter nucleotide sequence, a signal peptide coding sequence, an isolated nucleic acid encoding a nanobody or an antigen-binding fragment thereof, a hinge region coding sequence, a transmembrane domain coding sequence, a co-stimulatory domain coding sequence, an intracellular signaling domain coding sequence, a self-cleavage peptide coding sequence, a coding sequence of a detection tag signal peptide, and a detection tag coding sequence, which are sequentially connected in series.

[0178] In a more preferred embodiment, the isolated nucleic acid encoding the chimeric antigen receptor provided herein is a promoter nucleotide sequence, a signal peptide coding sequence, an isolated nucleic acid encoding VHH10 disclosed herein, a hinge region coding sequence, a transmembrane domain coding sequence, a co-stimulatory domain coding sequence, an intracellular signaling domain coding sequence, a self-cleavage peptide coding sequence, a detection tag signal peptide coding sequence, and a detection tag coding sequence, which are sequentially connected in series.

[0179] In a more preferred embodiment, the isolated nucleic acid encoding the chimeric antigen receptor provided herein is a promoter nucleotide sequence, a signal peptide coding sequence, an isolated nucleic acid encoding VHH12 disclosed herein, a hinge region coding sequence, a transmembrane domain coding sequence, a co-stimulatory domain coding sequence, an intracellular signaling domain coding sequence, a self-cleavage peptide coding sequence, a detection tag signal peptide coding sequence, and a detection tag coding sequence, which are sequentially connected in series.

[0180] In a more preferred embodiment, the isolated nucleic acid encoding the chimeric antigen receptor provided herein is a promoter nucleotide sequence, a signal peptide coding sequence, an isolated nucleic acid encoding VHH10 and VHH12 disclosed herein, a hinge region coding sequence, a transmembrane domain coding sequence, a costimulatory domain coding sequence, an intracellular signaling domain coding sequence, a self-cleavage peptide coding sequence, a detection tag signal peptide coding sequence, and a detection tag coding sequence, which are sequentially connected in series. In one embodiment, the isolated nucleic acids encoding VHH10 and VHH12 are directly linked or indirectly linked via a nucleotide encoding a linker, preferably via a nucleotide encoding (G4S)n, wherein n is an integer of 1-8. In a more preferred embodiment, the isolated nucleic acids encoding VHH10 and VHH12 are linked via a nucleotide as shown in SEQ ID NO: 21.

[0181] The present disclosure provides an isolated polynucleotide, the sequence of which is an isolated nucleic acid sequence encoding a chimeric antigen receptor disclosed herein, or a complementary sequence thereof. In one embodiment, when the chimeric antigen receptor comprises a Nanobody or antigen-binding fragment thereof comprising VHH10 and VHH12 disclosed herein, the VHH10 and VHH12 are connected by a linker, the nucleotide sequence of the linker being as shown in SEQ ID NO: 21.

[0182] Furthermore, the isolated nucleic acids encoding the Nanobodies or Chimeric Antigen Receptors provided herein include nucleic acids having conservative nucleotide sequence variants of the above-mentioned nucleotide sequences. The so-called conservative nucleotide sequence variants arise from variants of the degeneracy and silence of the genetic code, and substitutions, deletions and additions of nucleotides are also included within the scope of protection of the present invention, as long as the nucleic acids comprising conservative nucleotide sequence variants of the above-mentioned nucleotide sequences can encode the Nanobodies or Chimeric Antigen Receptors provided herein.

[0183] In specific embodiments of the present invention, the isolated nucleic acids and isolated polynucleotides of the present invention can be synthesized, for example, by standard chemical synthesis methods and / or recombinant methods, or produced semi-synthetically, for example, by combining chemical synthesis and recombinant methods. The coding sequence can be linked to transcriptional regulatory elements and / or to other amino acid coding sequences using established methods, such as restriction enzyme digestion, ligation, and molecular cloning.

[0184] carrier

[0185] The present disclosure provides a vector comprising an isolated nucleic acid or isolated polynucleotide disclosed herein. In one embodiment, the vector comprises a cloning vector, an expression vector. In one embodiment, the vector includes but is not limited to a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector. In one embodiment, the viral vector includes but is not limited to a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, or a herpesvirus vector.

[0186] Furthermore, in addition to the isolated nucleic acid or isolated polynucleotide disclosed herein, the vector also comprises an expression control sequence operably linked to the isolated nucleic acid or isolated polynucleotide sequence.

[0187] An expression vector is a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted, leading to protein expression. A vector can transform, transduce, or transfect host cells, allowing the genetic material it carries to be expressed within the host cell. Vectors include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, and other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and be stable within the host. In addition to an origin of replication, an expression vector may also contain a marker gene and other translation control elements.

[0188] host cells

[0189] The present disclosure provides engineered host cells, and the engineered host cells express the isolated nucleic acids provided herein, the isolated polynucleotides provided herein, or the vectors provided herein. In one embodiment, the host cells include eukaryotic cells and prokaryotic cells. In one embodiment, the eukaryotic cells include animal cells (e.g., mammals, insects), plant cells, and yeast cells. In one embodiment, prokaryotic cells include mycoplasmas, chlamydia, rickettsiae, bacteria, actinomycetes, and cyanobacteria. In one embodiment, the host cells are immune cells, including but not limited to T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, or any combination thereof. In a preferred embodiment, the immune cells are T cells.

[0190] derivative

[0191] The present disclosure provides derivatives, comprising a detectably labeled chimeric antigen receptor disclosed herein, an isolated nucleic acid, or an isolated polynucleotide, a chimeric antigen receptor disclosed herein that confers antibiotic resistance, an isolated nucleic acid, or an isolated polynucleotide, and a chimeric antigen receptor disclosed herein, an isolated nucleic acid, or an isolated polynucleotide coupled or conjugated to a therapeutic agent. In one embodiment, coupling means connecting by supply. In one embodiment, conjugation means linking by a linker (e.g., a cleavable or non-cleavable linker). In one embodiment, the detectable label comprises a fluorescent dye, a chemiluminescent marker, colloidal gold, or a chemiluminescent catalyst. In one embodiment, the chemiluminescent marker comprises luminol and its derivatives, acridinium esters or their derivatives, adamantane, isoluminol and its derivatives, rare earth elements, and bipyridine ruthenium complexes; the chemiluminescent catalyst comprises horseradish peroxidase and alkaline phosphatase. In one embodiment, the antibiotic resistance gene comprises a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, or a kanamycin resistance gene. In one embodiment, the therapeutic agent comprises a radionuclide, a cytokine, a gold nanoparticle, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme, or a chemotherapeutic agent. In one embodiment, the cytokine comprises IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF, or M-CSF; and the chemotherapeutic agent comprises cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, oxaliplatin, or eloxatin.

[0192] Nanobody-drug conjugate or conjugate or composition

[0193] The present disclosure provides Nanobody-drug conjugates or conjugates or compositions comprising a Nanobody or antigen-binding fragment thereof disclosed herein and a therapeutic agent.

[0194] In one embodiment, the Nanobody-drug conjugate is formed by covalently attaching a Nanobody disclosed herein to a therapeutic agent.

[0195] In one embodiment, the Nanobody-drug conjugate is formed by linking a Nanobody disclosed herein to a therapeutic agent via a linker. The linker may be a cleavable linker or a non-cleavable linker.

[0196] In one embodiment, the Nanobody-drug composition is formed by mixing a Nanobody disclosed herein with a therapeutic agent.

[0197] The therapeutic agent can be any therapeutic agent, including small molecules and macromolecules. Macromolecules include, but are not limited to, antibodies. Small molecule drugs include, but are not limited to, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenic agents, antiestrogens, antiandrogens, and immunomodulators. The alkylating agents include, but are not limited to, chlorambucil, melphalan, propranolol, chlorambucil, estramustine, cyclophosphamide, hexamethylmefidine, cyclophosphamide, ifosfamide, triamcinol, carmustine, streptozotocin, fortepdin, cyclohexyl lomustine, busulfan, thiosulfate, improsulfan, dacarbazine, cisplatin, oxaliplatin, and carboplatin. The antimetabolites include but are not limited to methotrexate, 5-fluorouracil, fluorouracil, 5-fluorodeoxyuracil, capecitabine, cytarabine, fludarabine, cytarabine, 6-mercaptopurine (6-MP), 6-mercaptoguanine (6-TG), 2-chlorodeoxyadenosine, 5-azacytidine, 2,2-difluorodeoxycytidine, cladribine, deoxycoformycin, and pentostatin. The antitumor antibiotics include but are not limited to doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone hydrochloride, dactinomycin, mithramycin, mithramycin, mitomycin C, bleomycin, and procarbazine. The mitotic inhibitors include but are not limited to paclitaxel, docetaxel, vinblastine, vincristine, vinamide, and vinorelbine. The chromatin function inhibitors include but are not limited to topotecan, irinotecan, etopoxetine, etopoxetine phosphate, and teniposide. The anti-angiogenic agent includes but is not limited to aprotinoxate, marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-27023A, bromochloropiperaqua, COL-3, neivastat, BMS-275291, and thalidomide. The anti-estrogen includes but is not limited to anastrozole, letrozole, tamoxifen, toremifene, raloxifene, droloxifene, odoxifene, and exemestane. The anti-androgen includes but is not limited to flutamide, nilutamide, bicalutamide, spironolactone, cyproterone acetate, finasteride, and cimetidine. The immunomodulator includes but is not limited to interferon, interleukin, tumor necrosis factor, mushroom polysaccharide, cizosin, roquine, pidomod, methoxypolyethylene glycol succinamide adenosine deaminase, and thymosin preparation.

[0198] Pharmaceutical composition

[0199] In yet another aspect, the disclosure provides pharmaceutical compositions comprising a Nanobody or antigen-binding fragment thereof, a chimeric antigen receptor, an isolated nucleic acid, an isolated polynucleotide, a vector, a host cell or, a derivative or a Nanobody-drug conjugate or conjugate or composition disclosed herein, and a pharmaceutically acceptable carrier.

[0200] As used herein, the term "pharmaceutical composition" refers to a mixture of a Nanobody or antigen-binding fragment thereof, a chimeric antigen receptor, an isolated nucleic acid, an isolated polynucleotide, a vector, a host cell, or a derivative or a Nanobody-drug conjugate or a composition disclosed herein and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. There are many ways of administering pharmaceutical compositions in the art, including but not limited to subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, pulmonary administration, ocular administration, and topical administration.

[0201] In the present application, the pharmaceutical composition can be configured into a dosage form suitable for administration to a subject via a desired route of administration, including but not limited to tablets, capsules, caplets, pills, lozenges, powders, syrups, brews, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalants, creams, ointments, lotions, pastes, sprays, freeze-dried solutions, injections, and gels.

[0202] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, which are involved in carrying or transporting the peptides of the present disclosure within a subject or carrying or transporting the peptides of the present disclosure to a subject so that it can perform its intended function. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate; Esters and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavorings; perfuming agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardening agents; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other nontoxic, compatible substances used in pharmaceutical formulations, or any combination thereof.

[0203] Testing products

[0204] The present disclosure provides detection products for detecting CLDN18.2 protein, comprising the Nanobodies or antigen-binding fragments thereof provided herein. Such detection products include, but are not limited to, detection reagents, kits, chips, or test strips. Any detection product that includes the Nanobodies or antigen-binding fragments disclosed herein and is capable of detecting CLDN18.2 is included within the scope of protection of the present disclosure.

[0205] Reagent test kit

[0206] The present disclosure provides a kit comprising an isolated nucleic acid, isolated polynucleotide, or vector disclosed herein. In one embodiment, the kit further comprises reagents for introducing the isolated nucleic acid, isolated polynucleotide, or vector into a host cell. In one embodiment, the kit further comprises instructions for introducing the isolated nucleic acid, isolated polynucleotide, or vector into a host cell.

[0207] Methods and uses

[0208] The present disclosure provides any of the following methods, comprising:

[0209] (a) A method for preparing an engineered host cell disclosed herein, the method comprising the following steps: introducing the isolated nucleic acid, isolated polynucleotide or vector provided herein into a host cell. The method of introduction comprises a physical method, a chemical method, and a biological method. The physical method comprises calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. The chemical method comprises a colloidal dispersion system and a lipid-based system; the colloidal dispersion system comprises a macromolecular complex, a nanocapsule, a microsphere, and a bead; the lipid-based system comprises an oil-in-water emulsion, a micelle, a mixed micelle, and a liposome. The biological method comprises a DNA vector, an RNA vector, a lentiviral vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector, and an adeno-associated virus vector. In a preferred embodiment, the method of introduction comprises lipofection, microinjection, electroporation, a DNA vector, an RNA vector, a retroviral vector, a lentiviral vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector, and an adeno-associated virus vector;

[0210] (b) a method for stimulating a target cell population or tissue in a subject to produce an immune response, the method comprising the steps of: administering to the subject an engineered host cell disclosed herein;

[0211] (c) a method for producing a Nanobody or an antigen-binding fragment thereof disclosed herein, said method comprising the steps of culturing an engineered host cell disclosed herein, and isolating the Nanobody or an antigen-binding fragment thereof disclosed herein from the culture;

[0212] (d) a method for detecting CLDN18.2 in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising the steps of: contacting the test sample with a Nanobody or antigen-binding fragment thereof as disclosed herein, and detecting the formation of a complex between the Nanobody or antigen-binding fragment thereof and CLDN18.2;

[0213] Preferably, the Nanobody or antigen-binding fragment thereof is a Nanobody or antigen-binding fragment thereof labeled with a detectable marker;

[0214] More preferably, the markers that can be used for detection include fluorescent dyes, avidin, paramagnetic atoms, and radioactive isotopes;

[0215] Most preferably, the fluorescent pigment is fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, or peridinin-chlorophyll protein;

[0216] Most preferably, the avidin is biotin, egg white avidin, streptavidin, egg yolk avidin, or avidin-like;

[0217] Most preferably, the radioisotope is radioactive iodine, radioactive cesium, radioactive iridium, or radioactive cobalt;

[0218] (e) a method for preventing or treating a disease associated with abnormal expression of CLDN18.2, the method comprising administering to a subject in need thereof a therapeutically effective amount of a chimeric antigen receptor, isolated nucleic acid, isolated polynucleotide, vector, host cell, derivative, nanobody-drug conjugate or conjugate or composition or pharmaceutical composition disclosed herein;

[0219] Preferably, the disease associated with abnormal expression of CLDN18.2 includes solid tumors, preferably gastric cancer, gastroesophageal cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, and ovarian cancer.

[0220] This article provides any of the following applications, including:

[0221] (a) Use of the Nanobodies or antigen-binding fragments thereof disclosed herein in the preparation of a detection product for detecting CLDN18.2 protein;

[0222] (b) Use of the Nanobodies or antigen-binding fragments thereof, chimeric antigen receptors, isolated nucleic acids, isolated polynucleotides, vectors, engineered host cells, derivatives, Nanobody-drug conjugates or conjugates or compositions, or pharmaceutical compositions disclosed herein for the preparation of a medicament for preventing or treating a disease associated with abnormal CLDN18.2 expression;

[0223] (c) Use of the chimeric antigen receptors, isolated nucleic acids, isolated polynucleotides, vectors, engineered host cells, and derivatives disclosed herein in the preparation of a kit for preparing immune cells for preventing or treating diseases associated with abnormal CLDN18.2 expression;

[0224] (d) Use of the kit disclosed herein in preparing immune cells for preventing or treating diseases associated with abnormal CLDN18.2 expression;

[0225] Preferably, the disease associated with abnormal expression of CLDN18.2 includes solid tumors, preferably gastric cancer, gastroesophageal cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, and ovarian cancer.

[0226] Example

[0227] The following describes exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, it should be appreciated by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0228] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0229] Example 1. Screening of anti-CLDN18.2 nanobodies

[0230] 1. Animal immunization and titer determination

[0231] Alpacas were immunized using VLP-CLDN18.2 full-length protein (Cat. No. CLD-HE1822) purchased from Kaika Biopharm. The specific alpaca immunization process is shown in Figure 1. Immunizations were performed once a week for a total of six consecutive immunizations. Before the sixth immunization, 5 ml of blood was drawn for titer pre-testing. Seven days after the final immunization, 100 ml of peripheral blood was collected for antibody library construction and screening.

[0232] 2. Construction and enrichment screening of nanoantibody library

[0233] 2.1 Construction of Nanobody Library

[0234] Seven days after the last immunization, 100 mL of peripheral blood was collected from the alpacas, and peripheral blood mononuclear cells were separated by Ficoll density gradient centrifugation, and RNA was extracted and cDNA was prepared using a reverse transcription kit.

[0235] Using cDNA as a template, immunoglobulin heavy chain-specific primers CALL001 and CALL002 were used to amplify the variable regions of all immunoglobulin heavy chains (VHs and VHHs). The approximately 700 bp fragment represents the heavy chain-only antibody repertoire, while the approximately 1000 bp fragment corresponds to the heavy chain of a conventional antibody. The amplified products were analyzed on a 1% (wt / vol) agarose gel. The approximately 700 bp product was recovered by gel extraction.

[0236] Using the gel-recovered product as a template, the VHHs sequence was specifically amplified using the degenerate primers VHH-BACK and VHH-FOR. The amplified product was analyzed on a 1% (wt / vol) agarose gel. It was then ligated into the pMES4 phage display vector, and the ligation product was electroporated into electrocompetent Escherichia coli cells TG1. The resulting bacterial library was the constructed BCMA single-domain heavy chain antibody phage display library. After library construction was completed, 25 clones were randomly selected for colony PCR using primers MP57 and GⅢ to test the insertion efficiency of the library. The PCR products were then subjected to Sanger sequencing. The construction process of the nanobody library is shown in Figure 1.

[0237] The results showed that a nanoantibody library for CLDN18.2 was successfully constructed with a library capacity of 1.05E8 and an insertion rate of nearly 95%.

[0238] Table 1 provides the primer information for the construction of single VHH CAR constructs.

[0239] Table 1. Primers for single VHH CAR construction

[0240] 2.2 Enrichment and screening of antibody library

[0241] The TG1 E. coli nanobody library was transferred to 2-YT liquid medium and cultured at 37°C and 200 rpm to an OD value of 0.5. Helper phage VCSM13 was then added to the cells for infection. After gentle mixing, the cells were incubated at 37°C for 30 minutes. The culture was centrifuged to remove traces of glucose, and the pellet was resuspended in 2-YT medium supplemented with ampicillin and kanamycin resistance medium and incubated overnight at 37°C and 200 rpm to amplify the nanobody-displaying phage. The overnight culture was transferred to a 50 mL centrifuge tube, the supernatant was centrifuged, and a 20% (wt / vol) PEG6000 / 2.5 M NaCl solution was added to precipitate the phage. The supernatant was discarded after centrifugation, and the pellet was resuspended in 1 mL of PBS. After centrifugation, the supernatant was transferred to a new centrifuge tube, glycerol was added to a final concentration of 20% and stored at -80°C; the titer of the phage nanoantibody library was determined by diluting the phage in a 10-fold gradient, and using phages of different dilution multiples to infect TG1 bacteria in the logarithmic growth phase, culturing at 37°C overnight, and estimating the titer of the phage nanoantibody library by the number of plaques on the second day.

[0242] Nanobodies were selected by ELISA. The full-length VLP-CLDN18.2 protein was coated on an ELISA plate and incubated overnight at 4°C. The plate was washed three times with 250 μL PBST, 200 μL blocking solution was added, and the plate was incubated at room temperature for 2 hours. The corresponding phage was added to each well and incubated at room temperature for 2 hours. The plate was washed 15 times with 250 μL PBST. 100 μL trypsin was added to each well and incubated at room temperature at 700 rpm for 0.5 hours. The phage was eluted with AEBSF. The eluted phage was titered and amplified for phage infection. The number of eluted phage was positive; when the number of negative phage was ≥100, the panning was stopped. The enrichment and screening process of the antibody library is shown in Figure 1.

[0243] The panning results are shown in Figure 2. After two rounds of enrichment and screening, the final positive:negative phage ratio reached 8120-fold, meeting the standard for screening single clones. Therefore, after two rounds of panning, the panning was stopped and the next step of single clone screening and identification was carried out.

[0244] 3. Monoclonal Identification

[0245] Single clones were selected from the TG1 E. coli library obtained after two rounds of screening and expanded. These clones were then infected with helper phage VCSM13 to prepare monoclonal phage. The monoclonal phage was added to a microtiter plate coated with BCMA protein and blocked with 2% skim milk powder and incubated at room temperature for 2 hours. After washing the plate with PBST, HA-HRP antibody (GenScript, Catalog No. A01296) was added and incubated at room temperature for 1 hour. After washing the plate with PBST, 100 μL of TMB single-component colorimetric solution was added, incubated at room temperature for 30 minutes, and then 100 μL of stop solution was added. Absorbance at 450 nm was measured using a microplate reader. A positive clone was identified when the OD450 value of the sample well compared to the blank control was greater than 2. The monoclonal identification process is shown in Figure 1. Positive clones were subjected to bacterial PCR and Sanger sequencing. Sequences of the Sanger-sequenced clones were then aligned using DNAMAN software. Sequence-specific clones were then screened.

[0246] After sequence identification, two clones with specific sequences and high OD450 values ​​as determined by ELISA were identified and named Nb015-10 (VHH10) and Nb015-12 (VHH12). The OD450 results are shown in Figure 3.

[0247] The amino acid sequence information of Nb015-10 (VHH10) and Nb015-12 (VHH12) is shown in Table 2. The nucleic acid sequence information of Nb015-10 (VHH10) and Nb015-12 (VHH12) is shown in Table 3. The numbering system used is IMGT.

[0248] Table 2. Related amino acid sequences of Nb015-10 (VHH10) and Nb015-12 (VHH12)

[0249] Table 3. Related nucleic acid sequences of Nb015-10 (VHH10) and Nb015-12 (VHH12)

[0250] Example 2. Construction of 293T-CLDN18.1, 293T-CLDN18.2, NALM6-CLDN18.1, and NALM6-CLDN18.1 Stable Cell Lines

[0251] 1. Experimental methods

[0252] Using the plasmid purchased from Sino Biological (Cat. No. HG20047-U) as a template, the full-length sequence of CLDN18.1 was obtained by PCR, and the sequence was inserted into the lentiviral vector pLVX-Puro by double enzyme digestion and ligation with Xho I and EcoR I restriction sites at both ends. The full-length sequence of CLDN18.2 (MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV, SEQ ID NO: 49) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and flanked by Xho I and EcoR I restriction site, and the sequence was inserted into the lentiviral vector pLVX-Puro through double enzyme digestion and ligation. The recombinant plasmids were named pLVX-CLDN18.1-Puro and pLVX-CLDN18.2-Puro, respectively. The recombinant plasmids use the CMV promoter and carry a puromycin resistance gene.

[0253] The target plasmids pLVX-CLDN18.1-Puro and pLVX-CLDN18.2-Puro were packaged with helper plasmids for lentiviral packaging. Prior to CAR-T cell preparation, lentiviral packaging was performed: the target plasmid and three helper plasmids (pMD2.G, pRSV-REV, and pMDLg / RRE) were co-transfected into NALM6 and 293T cells using PEI-Pro. The medium was changed 6 hours after packaging, and the lentiviral stock was harvested 48 hours after packaging. The harvested lentiviral stock was concentrated by ultracentrifugation, and the lentiviral particles were resuspended in DMEM high-glucose medium and aliquoted for use.

[0254] 2. Experimental results

[0255] The results are shown in Figure 4, which show that the constructed NALM6-CLDN18.1, 293T-CLDN18.1 and NALM6-CLDN18.2, 293T-CLDN18.2 stable cell lines highly expressed CLDN18.1 and CLDN18.2, that is, the present invention successfully constructed NALM6-CLDN18.1, 293T-CLDN18.1 and NALM6-CLDN18.2, 293T-CLDN18.2 stable cell lines.

[0256] Example 3. Preparation and in vitro functional verification of single VHH CAR-T cells

[0257] 1. Construction of single VHH CAR structure

[0258] Sequence-specific clones were used to construct a single VHH CAR construct. First, the VHH sequence of the positive clone was amplified using primers NCAR-F1 and NCAR-R1, using the sequenced plasmid as a template. After the first round of PCR, a second round of PCR was performed using the first-round PCR product as a template and primers NCAR-F2 and NCAR-R2. The second-round PCR product was ligated into the vector Sen1-S88BZ via homologous recombination, and the vector was digested with Not I. Thus, a CAR construct containing a single VHH targeting CLDN18.2 was successfully constructed. The structural diagram is shown in Figure 5, and the primer sequences are shown in Table 4.

[0259] Table 4. Primer list for single VHH CAR structure construction

[0260] Two single VHH CAR structures were constructed, named NMC015-10 and NMC015-12, respectively. The structures are shown in Figure 5. EF1α is the promoter of elongation factor 1α, Leader is the coding sequence of the signal peptide, VHH is the coding sequence of the anti-CLDN18.2 nanobody, CD8αH+TM is the hinge region and transmembrane region of CD8α, 4-1BB and CD3ζ intracellular signaling regions are intracellular costimulatory domains, and the tEGFR extracellular region is expressed through a T2A peptide connection to facilitate detection of CAR expression after lentiviral transduction.

[0261] The amino acid sequences of the relevant elements used in the single VHH CAR are shown in Table 5 below. The nucleic acid sequences of the relevant elements used in the single VHH CAR are shown in Table 6 below.

[0262] Table 5. Amino acid sequences of relevant elements used in single VHH CARs

[0263] Table 6. Nucleic acid sequences of relevant elements used in single VHH CARs

[0264] 2. Lentiviral packaging

[0265] Before preparing CAR-T cells, lentivirus packaging was first performed: the target plasmid and three helper plasmids (pMD2.G, pRSV-REV, and pMDLg / RRE) were co-transfected into 293T cells under the action of PEI-Pro; the medium was changed 6 hours after packaging; the lentivirus was harvested 48 hours after packaging; the harvested lentivirus stock solution was concentrated by ultracentrifugation, and the lentiviral particles were resuspended in DMEM high-glucose medium and aliquoted for use.

[0266] 3. Single VHH CAR-T cell preparation

[0267] After lentiviral packaging, CAR-T cell preparation was performed: peripheral blood mononuclear cells (PBMCs) were collected from patients or healthy donors; αβ T cells were sorted using CD3 magnetic beads; the sorted αβ T cells were cultured in TexMACS GMP medium (MACS); lentiviral transduction was performed 2 days later; culture was continued for 12-14 days before CAR-T cell harvesting to obtain single VHH CAR-T cells targeting CLDN18.2 (designated NMC015-10 CAR-T and NMC015-12 CAR-T, respectively). Flow cytometry was performed on days 6 and 13 of culture to determine the proportion of CAR+ cells, and tEGFR expression was detected using an anti-EGFR antibody. Cell counts were performed on days 6, 9, and 13 of culture to measure cell proliferation, and samples were taken on days 6 and 13 of culture for flow cytometry to determine the CD4 / CD8 ratio of CAR-T cells.

[0268] 4. In vitro functional validation of single VHH CAR-T cells

[0269] In order to verify the in vitro biological activity of the anti-CLDN18.2 VHH CAR-T cells prepared in this example, an in vitro killing experiment was performed during the culture process: first, the target cells were collected, and the overexpression cell lines NALM6-CLDN18.1 and NALM6-CLDN18.2 described in Example 2, as well as the blank cell line NALM6, were collected, centrifuged at 2000 rpm for 5 min, resuspended in DPBS, and counted. The cells were counted at 1×10 5 The number of cells / well was added to a 96-well plate. Then, according to different effector-target ratios (E:T = 0.3:1, 1:1, 3:1), appropriate amount of effector cells were added to the target cells, mixed and incubated for 4 hours, and the cell killing ratio was detected by flow cytometry.

[0270] Take target cells and adjust the cell density to 2×106 100 μL was added to each well of a 96-well plate at a density of 2 × 10 5 The cells were cultured in a 4% flask at 4 °C for 24 h. An appropriate amount of effector cells was added to the target cells in each well according to the effector cell: target cell (E:T) ratio of 3:1. The cells were mixed and incubated for 18 h. The supernatant was collected to detect the secretion of IFN-γ and TNF-α.

[0271] 5. Experimental results

[0272] CAR-T cells were prepared by transducing the single VHH construct into T cells. Flow cytometry analysis of the CAR-T cells after 6 and 13 days of culture is shown in Figure 6. The results show that both NMC015-10 and NMC015-12 CAR-T cells achieved high transduction rates, and the transduction rates were stable during culture. This demonstrates that the CAR construct in the single VHH CAR construct was successfully expressed.

[0273] During the culture of single VHH CAR-T cells, statistical analysis of cell expansion folds and CD4 / CD8 ratios was performed. The CD4 / CD8 ratio during cell culture is shown in Figure 7. While the CD4 / CD8 ratio varied during the culture process, at the final harvest, the CD4 / CD8 ratios for NMC015-10 and NMC015-12 cells were 1.72 and 1.79, respectively, while the CD4 / CD8 ratio for naive T cells was 1.46. Figure 8 shows the expansion curves of single VHH CAR-T cells. From the start of culture to harvest on day 13, the expansion folds for NMC015-10, NMC015-12, and naive T cells were 40, 36, and 89, respectively. These results demonstrate that single VHH CAR-T cells were successfully expanded, and that the CD4 / CD8 ratio remained stable during expansion.

[0274] The in vitro biological activity of single VHH CAR-T cells was verified by killing cell lines overexpressing NALM6-CLDN18.1 and NALM6-CLDN18.2, as well as the blank cell line NALM6. Figure 9 shows the in vitro killing results of single VHH CAR-T against NALM6, NALM6-CLDN18.1, and NALM6-CLDN18.2. At killing ratios of 0.3:1, 1:1, and 3:1, NMC015-10 and NMC015-12 both produced specific killing against NALM6-CLDN18.2, and the killing rate gradually increased with increasing effector-target ratios. No significant killing was produced against NALM6, NALM6-CLDN18.1, and blank T. These results indicate that single VHH CAR-T cells have produced specific killing against cell lines positive for CLDN18.2 expression.

[0275] The in vitro biological activity of single-VHH CAR-T cells was further verified by cytokine analysis. As shown in Figures 10A-B, co-incubation of NMC015-10 and NMC015-12 with target cells NALM6-CLDN18.2 and 293T-CLDN18.2 significantly increased the release of IFN-γ and TNF-α, demonstrating that single-VHH CAR-T cells specifically kill CLDN18.2-positive cell lines.

[0276] Example 4. Preparation and functional verification of dual VHH CAR-T (dNMC015-A) cells

[0277] 1. dNMC015-A structure construction

[0278] The nanoantibodies Nb015-10 and Nb015-12 screened by the present invention were used to construct a dual VHH CAR structure target plasmid, the structural schematic of which is shown in Figures 11A-B. First, the VHH12 sequence was amplified by PCR; after the first round of PCR, the second round of PCR was performed using the first round PCR product as a template. The primer sequences are shown in Table 7. Then, the second round of PCR product was connected to the vector NMC015-10 by homologous recombination. The vector was digested with Not I, and the recombinant plasmid was named dNMC015-A. The structural schematic is shown in Figures 11A-B.

[0279] Table 7. Primer list for dual VHH CAR structure construction

[0280] EF1α is the promoter of elongation factor 1α, Leader is the coding sequence of the signal peptide, VHH10 and VHH12 are the coding sequences of the nanoantibody against CLDN18.2, CD8αH+TM is the hinge region and transmembrane region of CD8α, 4-1BB and CD3ζ intracellular signal regions are intracellular co-stimulatory domains, and the tEGFR extracellular region is expressed by connecting with the T2A peptide to detect the expression of CAR after lentiviral transduction.

[0281] The amino acid sequences of the relevant elements used in dNMC015-A are shown in Table 8. The nucleic acid sequences of the relevant elements used in the single VHH CAR are shown in Table 9.

[0282] Table 8. Amino acid sequences of relevant elements used in dNMC015-A

[0283] Table 9. Nucleic acid sequences of related elements used in dNMC015-A

[0284] 2. Preparation of dNMC015-A CAR-T cells

[0285] CAR-T cells were cultured according to the preparation process in Example 3. dNMC015-A, NMC015-10, and NMC015-12 CAR-T cells were also cultured. Flow cytometry was performed on days 6 and 15 of culture to determine the proportion of CAR+ cells. TEGFR expression was detected using an anti-EGFR antibody, and the CD4 / CD8 ratio of CAR-T cells was calculated. Cell counts were performed on days 6, 9, and 15 of culture to measure cell proliferation and the proportion of viable cells.

[0286] 3. In vitro functional validation of dNMC015-A CAR-T cells

[0287] In order to verify the in vitro biological activity of dNMC015-A, NMC015-10, and NMC015-12 CAR-T cells prepared in this example, an in vitro killing experiment was performed during the culture process: first, target cells were collected, and NALM6-CLDN18.2, NALM6-CLDN18.1, and NALM6 were collected respectively, centrifuged at 2000 rpm for 5 min, resuspended in DPBS, and counted. The cells were counted at 1×10 5 The number of cells / well was added to a 96-well plate. Then, according to different effector-target ratios (E:T = 0.3:1, 1:1, 3:1), appropriate amount of effector cells were added to the target cells, mixed and incubated for 4 hours, and the cell killing ratio was detected by flow cytometry.

[0288] Take target cells and adjust the cell density to 2×10 6 100 μL was added to each well of a 96-well plate at a density of 2 × 10 5 The cells were cultured in a 4% flask at 4 °C for 24 h. An appropriate amount of effector cells was added to the target cells in each well according to the effector cell: target cell (E:T) ratio of 3:1. The cells were mixed and incubated for 18 h. The supernatant was collected to detect the secretion of IFN-γ and TNF-α.

[0289] 4. Experimental results

[0290] CAR-T cells were prepared by transducing the dual VHH construct into T cells. Flow cytometry results after 6 and 15 days of CAR-T cell culture are shown in Figure 12. These results show that both NMC015-10, NMC015-12, and dNMC015-A CAR-T cells achieved high transduction rates, and the transduction rates were stable during culture. This demonstrates that the CAR structure in the dual VHH CAR construct was successfully expressed.

[0291] During the CAR-T cell culture process of this embodiment, the cell expansion multiple and CD4 / CD8 ratio were statistically analyzed. The CD4 / CD8 ratio during cell culture is shown in Figure 13. The CD4 / CD8 ratio decreased during the culture process. At the final harvest, the CD4 / CD8 ratios of NMC015-10, NMC015-12, and dNMC015-A were 0.47, 0.45, and 0.34, respectively, which were at a comparable level (0.23) with blank T cells. Figure 14 shows the NMC015-10, NMC015-12, and dNMC015-A CAR-T amplification curves. As can be seen from the figure, by day 15, the amplification multiple of NMC015-10 was 65.0 times, the amplification multiple of NMC015-12 was 79.2 times, the amplification multiple of dNMC015-A was 96.6 times, and the amplification multiple of blank T cells was 99.4 times. The above results proved that dual VHH CAR-T cells were successfully expanded, and the CD4 / CD8 ratio tended to normal levels compared with blank T cells.

[0292] The in vitro biological activity of single and dual VHH CAR-T cells was verified by killing cell lines overexpressing NALM6-CLDN18.1 and NALM6-CLDN18.2, as well as the blank cell line NALM6. Figure 15 shows the in vitro killing results of single and dual VHH CAR-T cells against NALM6, NALM6-CLDN18.1, and NALM6-CLDN18.2. At killing ratios of 0.3:1, 1:1, and 3:1, NMC015-10, NMC015-12, and dNMC015-A all showed specific killing against NALM6-CLDN18.2, and the killing rate gradually increased with increasing effector-target ratios. NMC015-10, NMC015-12, and dNMC015-A did not show significant killing effects against NALM6-CLDN18.1 or blank T cells. These results demonstrate that both single- and dual-VHH CAR-T cells specifically kill CLDN18.2-positive cell lines. Furthermore, the dNMC015-A construct exhibited higher cytotoxicity than NMC015-10 and NMC015-12, demonstrating that dual-VHH constructs are functionally superior to single-VHH constructs.

[0293] The in vitro biological activity of single and dual VHH CAR-T cells was further verified by cytokine analysis. As shown in Figures 16A-B, co-incubation of NMC015-10, NMC015-12, and dNMC015-A with target cells NALM6-CLDN18.2 and 293T-CLDN18.2 significantly increased the release of IFN-γ and TNF-α, demonstrating that both single and dual VHH CAR-T cells specifically kill CLDN18.2-positive cell lines.

[0294] Example 5. Antibody Characterization

[0295] 1. Recombinant Antibody Expression

[0296] In order to further confirm the characteristics of the nanobodies Nb015-10, Nb015-12, and the diabody dNb015-A screened in Example 1, these nanobodies were expressed and purified in vitro.

[0297] First, the nanobodies Nb015-10, Nb015-12 and dNb015-A were inserted between the BamH I and NHE I restriction sites of the pET-28a-Sumo-Nb-Fc plasmid (retained in this laboratory) by molecular cloning. The N-terminus of the nanobody sequence includes a SUMO tag (SUMO tag protein is a small molecule ubiquitin-like modified protein. Studies have found that SUMO can be used as a fusion tag and molecular chaperone for recombinant protein expression, which can not only further increase the expression level of the fusion protein, but also has the functions of resisting protease hydrolysis, promoting the correct folding of the target protein, and improving the solubility of the recombinant protein). The C-terminus of the nanobody sequence carries a human IgG1 Fc (GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, SEQ ID NO: 50) tag for protein purification. After sequencing was confirmed, the plasmid was extracted and transformed into the E. coli strain BL21. Protein expression was induced by IPTG. After expression, the cells were harvested and lysed by ultrasonication to obtain crude protein. Purification by Protein A affinity chromatography yielded highly pure nanobodies. The purified nanobodies were named Nb015-10-Fc, Nb015-12-Fc, and dNb015-A-Fc.

[0298] 2. Affinity detection

[0299] The expressed and purified Nanobodies Nb015-10-Fc, Nb015-12-Fc, and dNb015-A-Fc were used for SPR analysis. The affinity of Nanobodies Nb015-10-Fc, Nb015-12-Fc, and dNb015-A-Fc for human CLDN18.2 was determined by SPR using a Biacore T200 analysis system.

[0300] The affinity detection method for each nanobody to CLDN18.2 is as follows: First, CLDN18.2 is immobilized on a CM5 chip using the capture method; then, serially diluted Nb015-10-Fc, Nb015-12-Fc, and dNb015-A-Fc are injected as the mobile phase at a flow rate of 30 μl / min, followed by dissociation; finally, the results are analyzed using the Biacore T200 Evaluation Software Kinetic 1:1 Binding mode.

[0301] 3. Specific detection method

[0302] Flow cytometry was used to determine the specificity of Nb015-10, Nb015-12, and dNb015-A for CLDN18.2. Flow cytometry was also used to determine the specificity of Nb015-10-Fc, Nb015-12-Fc, and dNb015-A-Fc for CLDN18.2-positive cell lines. Flow cytometry was used to determine the specificity of each nanobody for the human CLDN18.2 antigen by binding to the human CLDN18.2-positive cell lines NALM6-CLDN18.2 and 293T-CLDN18.2, and the CLDN18.2-negative cell lines NALM6 and 293T, using biotinylated, purified nanobodies Nb015-10-Fc, Nb015-12-Fc, and dNb015-A-Fc as primary antibodies and SA-PE antibody as secondary antibody.

[0303] 4. Experimental results

[0304] The results of antibody affinity activity detected by SPR are shown in Table 10 and Figure 17. The affinity constant between Nb015-10 and human CLDN18.2 is 3.43×10 -9 The affinity constant between Nb015-12 and human CLDN18.2 is 2.71×10 -9 The affinity constant between dNb015-A and human CLDN18.2 is 3.43×10 -9 .

[0305] The above results show that the two anti-CLDN18.2 nanobodies Nb015-10 and Nb015-12 obtained by screening and identification in the present invention have good affinity to human CLDN18.2; dNb015-A double nanobody has good affinity to human CLDN18.2 and tends to be higher than that of a single nanobody.

[0306] Table 10. Statistics of antibody affinity determination using the SPR method

[0307] As shown in the flow cytometry results in Figure 18 , Nb015-10, Nb015-12, and dNb015-A did not specifically bind to the CLDN18.2-negative cell lines NALM6 and 293T, but all did specifically bind to the CLDN18.2-positive cell lines NALM6-CLDN18.2 and 293T-CLDN18.2, demonstrating that Nb015-10, Nb015-12, and dNb015-A are CLDN18.2-specific antibodies.

[0308] Example 6. Verification of the killing activity of dNMC015-A CAR-T cells in vivo

[0309] 1. Experimental methods

[0310] 6-8 week old M-NSG male mice (Shanghai Model Organisms Science Co., Ltd.) were used for modeling. NUGC-4-CLDN18.2 tumor cells (NUGC-4 (Purutin Biotechnology (Beijing) Co., Ltd.) were transformed by Yikang (Beijing) Pharmaceutical Technology Co., Ltd.) were used at 1×10 7 Cells were inoculated into the subcutaneous tissue of the ribs of mice. When the average tumor volume reached 40-60 mm 3 Around 6 days later, the mice were randomly divided into 2 groups, with 3 mice in each group for group administration. Group 1 was the vehicle group (cryopreservative solution CS), which was injected with 0.2 ml of the cryopreservative solution per mouse through the tail vein. Group 2 was the dNMC015-ACAR-T cell group, which was injected with 0.75×10 7 CAR T cells were added to each mouse (0.2 ml). The growth of subcutaneous transplanted tumors and the survival time of mice were observed and measured.

[0311] 2. Experimental results

[0312] The experimental results are shown in Figure 19. Starting from day 18 after administration, tumors in the dNMC015-A CAR-T cell group were under control, and by day 25, the tumors were completely eliminated, with a tumor inhibition rate of 100%. Furthermore, there was no tumor recurrence through day 60. Compared with the vehicle group, the dNMC015-A CAR-T cell group significantly prolonged the survival of mice.

Claims

1. A nanobody or antigen-binding fragment thereof that specifically binds to CLDN18.2, characterized in that The Nanobody or antigen-binding fragment thereof comprises at least one of the following (a)-(c): (a) VHH10, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, or amino acid sequences that are at least 75% identical to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; (b) VHH12, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or are amino acid sequences that are at least 75% identical to SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; (c) said VHH10 and VHH12.

2. A nanobody or antigen-binding fragment thereof that specifically binds to CLDN18.2, characterized in that: The Nanobody or antigen-binding fragment thereof comprises at least one of the following (a)-(c): (a) VHH10, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively, or SEQ ID NO: 1 with 1, 2, 3, or 4 conservative substitutions, SEQ ID NO: 2 with 1, 2, 3, or 4 conservative substitutions, or SEQ ID NO: 3 with 1, 2, 3, or 4 conservative substitutions, respectively; (b) VHH12, comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1-3 are as shown in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, respectively, or SEQ ID NO: 4 with 1, 2, 3, or 4 conservative substitutions, SEQ ID NO: 5 with 1, 2, 3, or 4 conservative substitutions, or SEQ ID NO: 6 with 1, 2, 3, or 4 conservative substitutions, respectively; (c) said VHH10 and VHH12.

3. The Nanobody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The amino acid sequence of the heavy chain variable region of VHH10 is shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity with SEQ ID NO: 13; the amino acid sequence of the heavy chain variable region of VHH12 is shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with SEQ ID NO:

14.

4. The Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3, when the Nanobody or antigen-binding fragment thereof comprises VHH10 and VHH12, the VHH10 and VHH12 are directly linked or indirectly linked via a linker, preferably via (G4S)n, where n is an integer from 1 to 8.

5. A chimeric antigen receptor (CAR) based on a nanobody or antigen-binding fragment thereof that specifically binds to CLDN18.2, characterized in that: The chimeric antigen receptor comprises at least the Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 4, preferably, the chimeric antigen receptor further comprises one or more of the following: a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, an intracellular signaling domain, a self-cleavage peptide, a detection tag, a detection tag signal peptide; Preferably, the signal peptide includes the signal peptides of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6; Preferably, the hinge region includes the hinge region of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, IL-11 receptor; Preferably, the transmembrane domain includes the transmembrane domain of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, CD3ε, CD8α, IL-2 receptor, IL-7 receptor, IL-11 receptor; Preferably, the costimulatory signaling domain includes the costimulatory signaling domains of the following molecules: 4-1BB, CD28, ICOS, CD27, CD19, CD4, CD8α, CD8β, HVEM, LIGHT, CD40, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, CD278; Preferably, the intracellular signaling domain includes the intracellular signaling domain of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, FYN; Preferably, the self-cleaving peptide is selected from P2A, T2A, E2A, and F2A; Preferably, the detection tag is tEGFR; Preferably, the detection tag signal peptide is a tEGFR signal peptide; Most preferably, the chimeric antigen receptor is a signal peptide, a Nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 4, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory signaling domain, a CD3ζ intracellular signaling domain, T2A, a tEGFR signal peptide, and tEGFR in series; Most preferably, the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO: 22 or has at least 75% sequence identity with SEQ ID NO: 22; Most preferably, the amino acid sequence of the CD8 transmembrane domain is as shown in SEQ ID NO: 24 or has at least 75% sequence identity with SEQ ID NO: 24; Most preferably, the amino acid sequence of the 4-1BB costimulatory signaling domain is as shown in SEQ ID NO: 26 or has at least 75% sequence identity with SEQ ID NO: 26; Most preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 28 or has at least 75% sequence identity with SEQ ID NO: 28; Most preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 18 or has at least 75% sequence identity with SEQ ID NO: 18; Most preferably, the amino acid sequence of T2A is as shown in SEQ ID NO: 30 or has at least 75% sequence identity with SEQ ID NO: 30; Most preferably, the amino acid sequence of the tEGFR signal peptide is as shown in SEQ ID NO: 18 or has at least 75% sequence identity with SEQ ID NO: 18; Most preferably, the amino acid sequence of tEGFR is as shown in SEQ ID NO: 32 or has at least 75% sequence identity with SEQ ID NO:

32.

6. An isolated nucleic acid encoding the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the Chimeric Antigen Receptor according to claim 5, said nucleic acid comprising deoxyribonucleotides, ribonucleotides or a mixture of deoxyribonucleotides and ribonucleotides.

7. An isolated nucleic acid encoding a Nanobody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The isolated nucleic acid comprises a nucleotide sequence encoding CDR1, CDR2, and CDR3 of VHH10 and / or a nucleotide sequence encoding CDR1, CDR2, and CDR3 of VHH12, wherein the nucleotide sequence comprises deoxyribonucleotides, ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides. Preferably, the nucleotide sequences encoding CDR1, CDR2, and CDR3 of VHH10 are respectively shown as SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, or are nucleotide sequences that are at least 75% identical to SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; the nucleotide sequences encoding CDR1, CDR2, and CDR3 of VHH12 are respectively shown as SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or are nucleotide sequences that are at least 75% identical to SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

8. An isolated nucleic acid encoding a Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that The isolated nucleic acid comprises a nucleotide sequence encoding the heavy chain variable region of VHH10 and / or a nucleotide sequence encoding the heavy chain variable region of VHH12, wherein the nucleotide sequence comprises deoxyribonucleotides, ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides. Preferably, the nucleotide sequence encoding the heavy chain variable region of VHH10 is shown as SEQ ID NO: 15 or a nucleotide sequence having at least 75% identity with SEQ ID NO: 15; the nucleotide sequence encoding the heavy chain variable region of VHH12 is shown as SEQ ID NO: 16 or a nucleotide sequence having at least 75% identity with SEQ ID NO:

16.

9. The isolated nucleic acid encoding a Nanobody or an antigen-binding fragment thereof according to claim 7 or 8, when the Nanobody or the antigen-binding fragment thereof comprises VHH10 and VHH12, the isolated nucleic acid encoding the Nanobody or the antigen-binding fragment thereof may optionally comprise a nucleotide sequence encoding a linker connecting VHH10 and VHH12, preferably, the nucleotide sequence of the linker is as shown in SEQ ID NO:

21.

10. The isolated nucleic acid encoding the chimeric antigen receptor of claim 5, wherein The isolated nucleic acid encoding the chimeric antigen receptor comprises at least the isolated nucleic acid encoding the Nanobody or antigen-binding fragment thereof as described in any one of claims 6 to 9, preferably, the isolated nucleic acid encoding the chimeric antigen receptor further comprises one or more of the following: a promoter nucleotide sequence, a signal peptide coding sequence, a hinge region coding sequence, a transmembrane domain coding sequence, a co-stimulatory domain coding sequence, an intracellular signaling domain coding sequence, a self-cleavage peptide coding sequence, a detection tag signal peptide coding sequence, a detection tag coding sequence; Preferably, the promoter nucleotide sequence is the EF1α promoter sequence shown in SEQ ID NO: 17; Preferably, the signal peptide coding sequence is the signal peptide coding sequence shown in SEQ ID NO: 19; Preferably, the hinge region coding sequence is the CD8α hinge region coding sequence shown in SEQ ID NO: 23; Preferably, the transmembrane domain coding sequence is the CD8α transmembrane domain coding sequence shown in SEQ ID NO: 25; Preferably, the costimulatory domain encoding sequence is the 4-1BB costimulatory domain encoding sequence shown in SEQ ID NO: 27; Preferably, the intracellular signaling domain encoding sequence is the CD3ζ signaling domain encoding sequence shown in SEQ ID NO: 29; Preferably, the self-cleaving peptide coding sequence is the T2A coding sequence shown in SEQ ID NO: 31; Preferably, the coding sequence of the detection tag signal peptide is the tEGFR signal peptide coding sequence as shown in SEQ ID NO: 34; Preferably, the detection tag coding sequence is the tEGFR coding sequence shown in SEQ ID NO: 33; More preferably, the isolated nucleic acid encoding the chimeric antigen receptor is a promoter nucleotide sequence, a signal peptide coding sequence, an isolated nucleic acid encoding a nanobody or an antigen-binding fragment thereof, a hinge region coding sequence, a transmembrane domain coding sequence, a co-stimulatory domain coding sequence, an intracellular signaling domain coding sequence, a self-cleavage peptide coding sequence, a detection tag signal peptide coding sequence, and a detection tag coding sequence, which are sequentially connected in series.

11. An isolated polynucleotide, characterized in that The sequence of the isolated polynucleotide is the isolated nucleic acid sequence encoding the chimeric antigen receptor according to claim 10, or its complementary sequence, and the isolated polynucleotide comprises deoxyribonucleotides, ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides.

12. The isolated polynucleotide according to claim 11, when the chimeric antigen receptor comprises a Nanobody or an antigen-binding fragment thereof comprising VHH10 and VHH12, said VHH10 and VHH12 are connected by a linker, the nucleotide sequence of the linker being shown in SEQ ID NO:

21.

13. A carrier, characterized in that The vector comprises the isolated nucleic acid of any one of claims 6 to 10 or the isolated polynucleotide of claim 11 or 12.

14. An engineered host cell, characterized in that The engineered host cell expresses the isolated nucleic acid according to any one of claims 6 to 10, the isolated polynucleotide according to claim 11 or 12, or the vector according to claim 13. Preferably, the host cell is a T cell.

15. A derivative, characterized in that The derivatives include a detectably labeled chimeric antigen receptor according to claim 5, an isolated nucleic acid according to claim 6 or 10, or an isolated polynucleotide according to claim 11 or 12, a chimeric antigen receptor according to claim 5, an isolated nucleic acid according to claim 6 or 10, or an isolated polynucleotide according to claim 11 or 12 that confers antibiotic resistance, and a chimeric antigen receptor according to claim 5, an isolated nucleic acid according to claim 6 or 10, or an isolated polynucleotide according to claim 11 or 12 coupled or conjugated to a therapeutic agent.

16. A nanobody-drug conjugate or conjugate or composition, characterized in that The Nanobody-drug conjugate or conjugate or composition comprises the Nanobody or antigen-binding fragment thereof as claimed in any one of claims 1 to 4 and a therapeutic agent.

17. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a Nanobody or antigen-binding fragment thereof as claimed in any one of claims 1 to 4, a Chimeric Antigen Receptor as claimed in claim 5, an isolated nucleic acid as claimed in any one of claims 6 to 10, an isolated polynucleotide as claimed in claim 11 or 12, a vector as claimed in claim 13, a host cell as claimed in claim 14 or, a derivative as claimed in claim 15 or a Nanobody-drug conjugate or conjugate or composition as claimed in claim 16, and a pharmaceutically acceptable carrier.

18. A detection product for detecting CLDN18.2 protein, characterized in that: The detection product comprises the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4.

19. A kit, characterized in that The kit comprises the isolated nucleic acid of claim 6 or 10, the isolated polynucleotide of claim 11 or 12, or the vector of claim 13; Preferably, the kit further comprises reagents for introducing the isolated nucleic acid, isolated polynucleotide or vector into a host cell; Preferably, the kit further comprises instructions for introducing the isolated nucleic acid, isolated polynucleotide or vector into a host cell.

20. Any of the following methods, characterized in that: The method comprises: (a) A method for preparing the engineered host cell according to claim 14, characterized in that the method comprises the following steps: introducing the isolated nucleic acid according to claim 6 or 10, the isolated polynucleotide according to claim 11 or 12, or the vector according to claim 13 into the host cell; (b) A method for stimulating a target cell population or tissue in a subject to produce an immune response, characterized in that the method comprises the following steps: administering to the subject an effective amount of the engineered host cell according to claim 14; (c) a method for producing a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that the method comprises the steps of: culturing the engineered host cell according to claim 14, and isolating the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 from the culture; (d) a method for detecting CLDN18.2 in a test sample for non-diagnostic and non-therapeutic purposes, characterized in that the method comprises the following steps: contacting the test sample with the Nanobody or its antigen-binding fragment according to any one of claims 1 to 4, and detecting the formation of a complex between the Nanobody or its antigen-binding fragment and CLDN18.2; Preferably, the Nanobody or antigen-binding fragment thereof is a Nanobody or antigen-binding fragment thereof labeled with a detectable marker; (e) a method for preventing or treating a disease associated with abnormal expression of CLDN18.2, characterized in that the method comprises administering to a subject in need thereof a therapeutically effective amount of a chimeric antigen receptor according to claim 5, an isolated nucleic acid according to claim 6 or 10, an isolated polynucleotide according to claim 11 or 12, a vector according to claim 13, a host cell according to claim 14, a derivative according to claim 15, a Nanobody-drug conjugate or conjugate or composition according to claim 16, or a pharmaceutical composition according to claim 17; Preferably, the disease associated with abnormal expression of CLDN18.2 includes solid tumors, preferably gastric cancer, gastroesophageal cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, and ovarian cancer.

21. The application of any one of the following aspects, characterized in that: The applications include: (a) Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a detection product for detecting CLDN18.2 protein; (b) Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, the Chimeric Antigen Receptor according to claim 5, the isolated nucleic acid according to any one of claims 6 to 10, the isolated polynucleotide according to claim 11 or 12, the vector according to claim 13, the engineered host cell according to claim 14, the derivative according to claim 15, the Nanobody-drug conjugate or conjugate or composition according to claim 16, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for preventing or treating a disease associated with abnormal expression of CLDN18.2; (c) Use of the chimeric antigen receptor of claim 5, the isolated nucleic acid of claim 6 or 10, the isolated polynucleotide of claim 11 or 12, the vector of claim 13, the engineered host cell of claim 14, or the derivative of claim 15 in the preparation of a kit for preparing immune cells for preventing or treating diseases associated with abnormal CLDN18.2 expression; (d) Use of the kit according to claim 19 in the preparation of immune cells for preventing or treating diseases associated with abnormal CLDN18.2 expression; Preferably, the disease associated with abnormal expression of CLDN18.2 includes solid tumors, preferably gastric cancer, gastroesophageal cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, and ovarian cancer.

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