Anti-Claudin18.2 nanoantibodies, chimeric antigen receptors and their applications

Abstract

The present application relates to anti-Claudin18.2 nanobodies, chimeric antigen receptors and their applications. The present application provides nanobodies or antigen-binding fragments thereof, chimeric antigen receptors, host cells, immunoconjugates, pharmaceutical compositions, kits, and related methods and uses that specifically bind to Claudin18.2.

Description

Technical Field

[0001] The present application relates to the field of immunology, and in particular, to nanobodies or antigen-binding fragments thereof, chimeric antigen receptors, host cells, immunoconjugates, pharmaceutical compositions, kits, and related methods and uses that specifically bind to Claudin18.2. Background Art

[0002] Claudin18.2 (CLDN18.2) is a tumor-specific antigen expressed in a variety of cancers, including pancreatic cancer, gastric cancer, colon cancer, etc. (Zhang, D., Huang, G., Liu, J., & Wei, W. Claudin18.2-targeted cancer theranostics. Am J Nucl Med Mol Imaging. 64–69. (2023).). In cancer treatment, targeted drugs targeting CLDN18.2 are a hot research direction. Some targeted drugs have entered the clinical trial stage, including but not limited to antibodies, CAR-T, ADC, such as ADC drug CMG901 targeting Claudin 18.2.

[0003] Among them, pancreatic cancer is a common pancreatic tumor. It is a highly malignant digestive tract malignant tumor that is difficult to diagnose and treat. About 90% of pancreatic cancers are ductal adenocarcinomas that originate from the glandular duct epithelium. Its morbidity and mortality have increased significantly in recent years, and its 5-year survival rate is very low. It is one of the malignant tumors with the worst prognosis (Zhao, Z., & Liu, W. Pancreatic Cancer: A Review of Risk Factors, Diagnosis, and Treatment. Technol Cancer Res Treat. 1533033820962117. (2020).). The clinical symptoms of pancreatic cancer are hidden and atypical, the early diagnosis rate is not high, the surgical mortality rate is high, and the cure rate is almost zero. There is an urgent need to develop new treatment options.

[0004] Cell therapy refers to the use of bioengineering methods to obtain cells with specific functions, and after in vitro expansion, special culture and other treatments, these cells have the functions of enhancing immunity, killing pathogens and tumor cells, so as to achieve the purpose of treating a certain disease. Cell therapy mainly includes immune cell therapy and stem cell therapy. Immune cell therapy generally refers to the targeted treatment of certain types of immune cells such as T cells and NK cells in vitro and then returning them to the human body to make them show functions such as killing tumor cells and clearing viruses (Albinger, N., Hartmann, J., & Ullrich, E. Current status and perspective of CAR-T and CAR-NK cell therapy trials in Germany. (2021). 513–527. (2021).). Among them, the modification of chimeric antigen receptors (CAR) can give immune cells more powerful anti-tumor functions. The structure of CAR includes: extracellular domain, transmembrane domain and intracellular domain. The extracellular domain is derived from antibodies, which determines the specificity and affinity of antigen recognition and is the basis for the safety and effectiveness of CAR-T therapy. Currently, the most widely used method is to specifically recognize the target protein based on the single chain variable region (ScFv) of monoclonal antibodies of human or other species. However, there are the following defects: on the basis of the comparable affinity between ScFv and nano antibodies, ScFv has a larger molecular weight than nano antibodies, and has certain limitations in molecular expression and function; ScFv is derived from its parent monoclonal antibody, and may have certain deficiencies in terms of activity and stability.

[0005] Heavy-chain antibody (HCAb; heavy-chain antibody) is a naturally light-chain-deficient antibody found in the peripheral blood of alpacas, containing only one heavy-chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning its variable region, a single-domain antibody (sdAb) consisting only of the heavy-chain variable region can be obtained, called a nanobody (VHH). The molecular weight of VHH is only 15KD, which is the smallest unit known to bind to the target antigen (Yang, EY, & Shah, K. Nanobodies: NextGeneration of Cancer Diagnostics and Therapeutics. Front Oncol. 1182. (2020).). The advantages of nano antibodies mainly include: 1. Small size and stronger penetration: Nano antibodies can easily penetrate into human tissues, including tumors, due to their small size, and reach places that traditional antibodies cannot reach; 2. Stronger thermal and chemical stability: Nano antibodies can still maintain stability under high temperature and extreme pH conditions, and can even work at 90°C; 3. Stronger affinity: Nano antibodies can recognize hidden epitopes and have stronger affinity; 4. Avoid complement reaction: Nano antibodies have only one domain and do not have the Fc segment of traditional antibodies, which can avoid complement reactions caused by the Fc segment. 5. Lower immunogenicity: Nano antibodies have lower immunogenicity, which makes them have a lower risk of immune response in clinical applications; 6. Easy to produce and transform: Nano antibodies can be expressed in large quantities in microbial systems such as yeast, and are easy to transform and optimize. Therefore, nano antibodies have broad application prospects in tumor treatment, neurological disease treatment, infectious disease treatment, etc., and are gradually becoming an emerging force in the new generation of biomedicine and clinical diagnostic reagents.

[0006] In order to provide safer and more effective disease therapies, there is a need in the art for new anti-Claudin18.2 nanobodies. Summary of the invention

[0007] In a first aspect, the present application provides a nanobody or an antigen-binding fragment thereof that specifically binds to Claudin18.2, wherein the nanobody or the antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3,

[0008] The amino acid sequence of the CDR1 is shown in SEQ ID NO. 7, the amino acid sequence of the CDR2 is shown in SEQ ID NO. 8, and the amino acid sequence of the CDR3 is shown in SEQ ID NO. 9.

[0009] In a second aspect, the present application provides a chimeric antigen receptor, which comprises the nanobody or antigen-binding fragment thereof described in the first aspect of the present application.

[0010] In a third aspect, the present application provides a nucleic acid encoding the nanobody or antigen-binding fragment thereof described in the first aspect of the present application or the chimeric antigen receptor described in the second aspect of the present application.

[0011] In a fourth aspect, the present application provides a vector comprising the nucleic acid described in the third aspect of the present application.

[0012] In a fifth aspect, the present application provides a host cell comprising the nucleic acid described in the third aspect of the present application or the vector described in the fourth aspect of the present application.

[0013] In a sixth aspect, the present application provides an immunoconjugate, which comprises the nanobody or antigen-binding fragment thereof described in the first aspect of the present application or the chimeric antigen receptor described in the second aspect of the present application, and a payload connected thereto.

[0014] In the seventh aspect, the present application provides a pharmaceutical composition, which comprises the nanoantibody or antigen-binding fragment thereof described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application or the immunoconjugate described in the sixth aspect of the present application, and a pharmaceutically acceptable carrier.

[0015] In the eighth aspect, the present application provides the use of the nanoantibody or its antigen-binding fragment described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application, the immunoconjugate described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application in the preparation of a medicament for treating diseases related to abnormal expression of Claudin18.2.

[0016] In the ninth aspect, the present application provides a method for treating a disease associated with abnormal expression of Claudin18.2, the method comprising administering to a subject in need thereof a therapeutically effective amount of the nanobody or antigen-binding fragment thereof described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application, the immunoconjugate described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application.

[0017] In the tenth aspect, the present application provides a kit, which comprises the nanoantibody or antigen-binding fragment thereof described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application, the immunoconjugate described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application.

[0018] In the eleventh aspect, the present application provides the use of the nanoantibody or its antigen-binding fragment described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application, the immunoconjugate described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application in the preparation of a reagent for detecting or diagnosing a disease related to abnormal expression of Claudin18.2.

[0019] In the twelfth aspect, the present application provides a method for detecting the expression level of Claudin18.2 protein in a sample, the method comprising: contacting the sample with the nanobody or its antigen-binding fragment described in the first aspect of the present application, and detecting the binding of the nanobody or its antigen-binding fragment to the Claudin18.2 protein.

[0020] The nano-antibodies or antigen-binding fragments thereof that specifically bind to Claudin18.2 provided in the present application have good binding ability to Claudin18.2 or tumor cells expressing Claudin18.2, with high specificity and affinity. In addition, CAR-T and CAR-NK formed based on the nano-antibodies or antigen-binding fragments that specifically bind to Claudin18.2 provided in the present application can significantly inhibit tumor cell proliferation, significantly reduce tumor volume, and improve survival rate, thereby being able to more effectively treat diseases related to abnormal expression of Claudin18.2. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The technical roadmap for obtaining CLDN18.2 nanoantibodies (A) and the SDS-PAGE (polyacrylamide) protein electrophoresis identification results of 6 antibody clones (B) are shown.

[0022] Figure 2The binding specificity of 2-F09, 2-B08, 11-F09, 4-H09, 2-A12, 6-H09, positive control (PC), and negative control (NC) to CHO-K1 cells, CHO-K1 cells stably transfected with the human CLDN18.1 coding sequence (CHO-K1 / 18.1), CHO-K1 cells stably transfected with the human CLDN18.2 coding sequence (CHO-K1 / 18.2), and CHO-K1 cells stably transfected with the mouse CLDN18.2 coding sequence (CHO-K1 / mouse 18.2), respectively, is shown.

[0023] Figure 3 The relative affinity of the VHH of 2-B08, 11-F09, 4-H09 and the ScFv of KLST-18.2 after chimerization with the human IgG1 Fc part to CHO-K1 cells stably transfected with the human CLDN18.2 coding sequence (A) and the mouse CLDN18.2 coding sequence (B) is shown.

[0024] Figure 4 The relative affinity of humanized 2-B08-HM1, 2-B08-HM2, 2-B08-HM3, 2-B08-HM4 and non-humanized 2-B08 after chimerization with human IgG1 Fc part to CHO cells stably transfected with human CLDN18.2 coding sequence (A) and mouse CLDN8.2 coding sequence (B) is shown.

[0025] Figure 5 The relative affinities of humanized 2-B08-HM2, 2-B08-HM4 and non-humanized 2-B08 after chimerization with human IgG1 Fc part to CLDN18.2 protein were compared.

[0026] Figure 6 Plasmid maps for constructing CAR-NK (A) and CAR-T (B) cells.

[0027] Figure 7 The positive rate of HM1, HM2, HM3, HM4, 2B08, 11F09, 4H09 and 8E5-2I CAR-NK was determined.

[0028] Figure 8 The killing activities of NK cells 102NK and 108NK from two healthy donors, as well as HM1, HM2, HM3, HM4, 2B08, 11F09, 4H09 and 8E5-2I CAR-NK cells against the NUGC-4-luciferase cell line at different time points in vitro were compared.

[0029] Fig. 9To determine the positive rate of 2B08 CAR-NK92, 2B08-HM2 CAR-NK92, and 2B08-HM4 CAR-NK92.

[0030] Fig.10 The results of in vitro killing activity (A) and cytokine secretion (B) of 2B08 CAR-NK92, 2B08-HM2 CAR-NK92, and 2B08-HM4 CAR-NK92 cells against NUGC-4-luciferase cell lines were compared.

[0031] Fig.11 To determine the positive rate of HM2 CAR-T, 8E5-2I CAR-T and flag CAR-T.

[0032] Fig.12 The in vitro cytotoxic activity of HM2, 8E5-2I and flag CAR-T cells against tumor cell lines was compared.

[0033] Fig.13 The results of secretion assay of HM2, 8E5-2I and flag CAR-T cell factors.

[0034] Fig.14 The comparison graph of the effect of CLDN18.2-CAR T on tumor volume over time (A) and the comparison graph of survival (B) in the in vivo treatment of pancreatic cancer CLDN18.2-AsPC-1 immunodeficient mouse subcutaneous transplant tumor model are described.

[0035] Fig.15 A comparison of the effects of CLDN18.2-CAR T in vivo treatment on tumor volume (A) and survival (B) in a CLDN18.2 CXCL9-AsPC-1 immunodeficient mouse subcutaneous xenograft tumor model expressing the chemokine CXCL9 is shown. DETAILED DESCRIPTION

[0036] Unless otherwise stated, the terms used herein have the usual understanding meanings for 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 operations of organic chemistry, medicinal chemistry, and biology described herein are well known in the art and are generally used 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. In the case where there are multiple definitions of the terms used herein, unless otherwise stated, the definitions of this section shall prevail.

[0037] 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.

[0038] As used herein, the terms “comprises,” “includes,” and “comprising” mean that in addition to the listed elements, other elements are not excluded.

[0039] In a first aspect, the present application provides a nanobody or an antigen-binding fragment thereof that specifically binds to Claudin18.2, wherein the nanobody or the antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3,

[0040] The amino acid sequence of the CDR1 is shown in SEQ ID NO. 7, the amino acid sequence of the CDR2 is shown in SEQ ID NO. 8, and the amino acid sequence of the CDR3 is shown in SEQ ID NO. 9.

[0041] The nanoantibodies or antigen-binding fragments thereof that specifically bind to Claudin18.2 provided in the present application have good binding ability to Claudin18.2 or tumor cells expressing Claudin18.2, and have high specificity and affinity, thereby being able to more effectively treat diseases related to abnormal expression of Claudin18.2.

[0042] In this application, the terms "nanobody", "VHH", "single domain antibody", "sdAb" are used indiscriminately and represent the variable domain of a single heavy chain of antibodies of those types found in camelids. In the absence of a light chain, each nanobody has three CDRs, denoted CDR1, CDR2 and CDR3. It is the smallest antigen-binding fragment with complete function. Usually, an antibody naturally lacking 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 (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 cloned from the heavy chain variable region (VHH) of natural heavy chain antibodies of camelids. It has excellent biological properties, a molecular weight of 12-15kDa, which is one-tenth of a complete antibody, and has good tissue penetration, high specificity and good water solubility.

[0043] As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that retain the binding specificity to a target antigen. Antigen-binding fragments include, but are not limited to, VHH, fragments containing CDRs, and the like.

[0044] In some embodiments, the nano antibody or its antigen binding fragment can be a humanized antibody or a chimeric antibody. In some embodiments, the nano antibody or its antigen binding fragment can be a monoclonal antibody. In some embodiments, the nano antibody or its antigen binding fragment can be a monovalent antibody, a bivalent antibody or a multivalent antibody. In some embodiments, the nano antibody or its antigen binding fragment can be a part of a bispecific antibody or a multispecific antibody. In some embodiments, the nano antibody or its antigen binding fragment can be fused with other fragments or molecules. In some embodiments, the antigen binding fragment can be a Fab.

[0045] The term "specific binding" as used herein 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) of 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 binding and dissociation rates. (See Nature 361: 186-87 (1993)).

[0046] In the present application, 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, such as the binding ability between the nanobody of the present application and the Claudin18.2 protein. The affinity of the nanobody described in the present application can be measured in vitro by several methods, including surface plasmon resonance or by ELISA.

[0047] In this application, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. The variability is concentrated in three segments in the heavy chain variable region called complementary determining regions (CDRs) or hypervariable regions. The variable region of a natural heavy chain each contains four FR regions (the more conservative parts of the variable region), which are generally in a β-folded configuration, connected by three CDRs that form a connecting loop, and can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region 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 antibodies to antigens, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of antibodies.

[0048] It is well known to those skilled in the art that the amino acid positions / boundaries of variable regions and CDRs in antibody sequences can be determined using various rules known in the art (e.g., Kabat, IMGT, Chothia, AbM, Contact, etc. numbering systems), and when different numbering systems are used, the amino acid positions / boundaries of variable regions and CDRs may change accordingly. When used herein, unless otherwise indicated, the positions / boundaries of variable regions and CDRs are determined according to the IMGT numbering scheme.

[0049] In some embodiments, the heavy chain variable region of the Nanobody or its antigen-binding fragment comprises the amino acid sequence shown in any one of SEQ ID NOs. 1-6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs. 1-6.

[0050] In some embodiments, the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs. 1-6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with an amino acid sequence as shown in any one of SEQ ID NOs. 1-6.

[0051] When used in this article, the term "identity" refers to the degree of similarity between a pair of sequences (nucleotides or amino acids). Identity is determined by dividing the number of identical residues by the total number of residues, and multiplying the quotient by 100 to obtain a percentage. Gaps are not counted when evaluating identity. Therefore, two copies of identical sequences have 100% identity, but sequences with deletions, additions or substitutions may have a lower degree of identity. It is known to those skilled in the art that there are some computer programs that can be used to determine the identity of a sequence, such as those using algorithms such as BLAST. BLAST nucleotide searches are performed using the NBLAST program, and BLAST protein searches are performed using the BLASTP program, wherein the default parameters of each program have been used.

[0052] 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.

[0053] Nanobodies or antigen-binding fragments thereof disclosed herein can be conjugated to other factors chemically or by genetic engineering. These factors provide the effect of targeting the antibody to a desired functional site or improve or provide other properties for the antibody.

[0054] Nanobodies or antigen-binding fragments thereof disclosed herein can be chemically or genetically labeled to provide detectable nanobodies, detectable antibodies comprising detectable moieties. 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.

[0055] 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, fluorescent 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.

[0056] In a second aspect, the present application provides a chimeric antigen receptor, which comprises the nanobody or antigen-binding fragment thereof described in the first aspect of the present application.

[0057] CAR-T and CAR-NK formed based on the nanoantibodies or antigen-binding fragments thereof that specifically bind to Claudin18.2 provided in the present application can significantly inhibit tumor cell proliferation, significantly reduce tumor volume, and improve survival rate, thereby more effectively treating diseases related to abnormal Claudin18.2 expression.

[0058] In some embodiments, the chimeric antigen receptor further comprises one or more selected from the following: a signal peptide, a hinge region, a transmembrane domain, an intracellular signaling region (co-stimulatory signaling domain, intracellular signaling domain), a self-cleavage peptide, a detection tag, and a detection tag signal peptide.

[0059] In some embodiments, 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.

[0060] In some embodiments, 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.

[0061] In some embodiments, the transmembrane domain includes the transmembrane domain of the following molecules: CD8, CD28, CD27, CD137, IgG1, IgG4, 4-1BB, PD-1, CD34, CD3ε, CD8α, IL-2 receptor, IL-7 receptor, IL-11 receptor.

[0062] In some embodiments, the costimulatory signaling domain includes the costimulatory signaling domain of the following molecules: 4-1BB, CD28, ICOS, CD27, CD19, LIGHT, CD40, OX40, CD2, CD226, CD278.

[0063] In some embodiments, the intracellular signaling domain includes the intracellular signaling domain of the following molecules: CD3ζ, FcεRIγ, CD27, CD28, CD134, CD137, MyD88, CD40, OX40, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, FYN.

[0064] In some embodiments, the self-cleaving peptide is selected from P2A, T2A, E2A, F2A.

[0065] In some embodiments, the detection tag is tEGFR.

[0066] In some embodiments, the detection tag signal peptide is a tEGFR signal peptide.

[0067] In some embodiments, the chimeric antigen receptor comprises the nanobody or antigen-binding fragment thereof, the transmembrane region and the intracellular signaling region described in the first aspect of the present application connected in series.

[0068] In some embodiments, the chimeric antigen receptor comprises the nanobody or antigen-binding fragment thereof described in the first aspect of the present application, a CD8 transmembrane region, and a CD3ζ intracellular signaling region, which are connected in series.

[0069] In some embodiments, the chimeric antigen receptor comprises the nanobody or antigen-binding fragment thereof described in the first aspect of the present application, a CD8 transmembrane region, an OX40 and a CD3ζ intracellular signaling region connected in series.

[0070] In some embodiments, the chimeric antigen receptor comprises the nanobody or antigen-binding fragment thereof described in the first aspect of the present application, a CD8 transmembrane region, and CD137 and CD3ζ intracellular signaling regions connected in series.

[0071] In some embodiments, the chimeric antigen receptor comprises the Nanobody or antigen-binding fragment thereof described in the first aspect of the present application, a CD28 transmembrane region, and CD28 and CD3ζ intracellular signaling regions connected in series.

[0072] In some embodiments, the chimeric antigen receptor comprises the nanobody or antigen-binding fragment thereof described in the first aspect of the present application, a CD28 transmembrane region, and CD28, CD137 and CD3ζ intracellular signaling regions connected in series.

[0073] In a third aspect, the present application provides a nucleic acid encoding the nanobody or antigen-binding fragment thereof described in the first aspect of the present application or the chimeric antigen receptor described in the second aspect of the present application.

[0074] As used herein, the term "nucleic acid" refers to polynucleotides, which are deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded forms. Nucleic acids can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas, cDNA encoding the light and heavy chains of the antibodies produced by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding antibodies can be recovered from a variety of phage clones as library members.

[0075] In some embodiments, the nucleic acid comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 10-15 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs. 10-15.

[0076] In a fourth aspect, the present application provides a vector comprising the nucleic acid described in the third aspect of the present application.

[0077] Various vectors can be used to express nucleic acids encoding antibodies or their antigen-binding fragments, chimeric antigen receptors of the present application. Vectors include cloning vectors, expression vectors. Both viral and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically with expression cassettes for expressing proteins or RNA) and human artificial chromosomes (see, for example, Harrington et al., Nat Genet. [Natural Genetics] 15: 345, 1997, the contents of which are hereby incorporated by reference herein). Virus-derived vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxvirus vectors, herpesvirus vectors.

[0078] In a fifth aspect, the present application provides a host cell comprising the nucleic acid described in the third aspect of the present application or the vector described in the fourth aspect of the present application.

[0079] In some embodiments, mammalian host cells are used to express and produce the nano antibodies or their antigen-binding fragments, chimeric antigen receptors of the present application. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines containing exogenous expression vectors. These include any normal non-immortal or normal or abnormal immortal animals or human cells. For example, many suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. Exemplary host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells (e.g., HEK293, HEK293T, HEK293F), monkey kidney (COS) cells (e.g., COS-1, COS-7), baby hamster kidney (BHK) cells (e.g., BHK-21), African green monkey kidney cells (e.g., BSC-1), HeLa cells, human hepatocellular carcinoma cells (e.g., Hep G2), myeloma cells (e.g., NS0, 653, SP2 / 0), lymphoma cells, oocytes, and cells from transgenic animals (e.g., mammary epithelial cells), or any derivative, immortalized, or transformed cells thereof.

[0080] In some embodiments, the host cell is an immune cell, including but not limited to T cells, B cells, NK cells, NKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof. In some embodiments, the immune cell is a T cell or a NK cell.

[0081] In some embodiments, the host cell also carries an exogenous cytokine coding sequence. In some embodiments, the host cell also expresses another chimeric antigen receptor that recognizes a different tumor antigen; or it also expresses a chemokine receptor; or it also expresses a protein that can block TGF beta signaling; or it also expresses a safety switch. In some embodiments, the chemokine receptors include: CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CCR4, CCR5, CCR6.

[0082] In a sixth aspect, the present application provides an immunoconjugate, which comprises the nanobody or antigen-binding fragment thereof described in the first aspect of the present application or the chimeric antigen receptor described in the second aspect of the present application, and a payload connected thereto.

[0083] As used herein, the term "immunoconjugate" is intended to cover the bioactive substances formed by linking the nanobody or antigen-binding fragment thereof described in the first aspect of the present application or the chimeric antigen receptor described in the second aspect of the present application with a payload in any manner, including but not limited to antibody-drug conjugates (ADCs), proteolysis-targeted chimeras (PROTACs)-antibody conjugates (DACs), targeted nuclear drugs (RDCs) and fusion proteins.

[0084] The nanobody or its antigen binding fragment or chimeric antigen receptor that specifically binds to Claudin18.2 can be directly connected or indirectly connected to the payload. In some embodiments, the nanobody or its antigen binding fragment or chimeric antigen receptor that specifically binds to Claudin18.2 is indirectly connected to the payload through a linker. Therefore, in some embodiments, the immunoconjugate further comprises a linker for connecting the nanobody or its antigen binding fragment or chimeric antigen receptor that specifically binds to Claudin18.2 to the payload.

[0085] The linker can be a cleavable linker or a non-cleavable linker. For example, an acid-labile linker (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020) can be used.

[0086] The cleavable linker may include a peptide unit comprising 2-20 amino acids, preferably a peptide unit selected from -valine-citrulline-(-Val-Cit-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -valine-alanine-(-Val-Ala-), -valine-lysine-(-Val-Lys-), -valine-arginine-(-Val-Arg-), -phenylalanine-citrulline-(-Phe-Cit-), -phenylalanine-lysine-(-Phe-Lys-), -phenylalanine-arginine-(-Phe-Arg-) and combinations thereof.

[0087] In some embodiments, the payload comprises a toxin, a PROTAC, a molecular glue, a polymer, a protein (including but not limited to an antibody or an antigen-binding fragment thereof), a drug, a radionuclide, a nucleic acid compound, a glucocorticoid, or a detectable marker.

[0088] As used herein, toxins (or cytotoxins) are intended to encompass any agent that inhibits or prevents cell function and / or causes cell destruction. Representative toxins include, but are not limited to, antibiotics, tubulin inhibitors, DNA damaging agents, and agents that disrupt protein synthesis or essential protein functions (such as protein kinases, phosphatases, topoisomers, enzymes, and cell cycle proteins). Tubulin inhibitors include, but are not limited to, dolastatin and auristatin cytotoxic molecules, maytansine cytotoxic molecules; DNA damaging agents include, but are not limited to, calicheamicin, duocarmycin, anthramycin derivatives PBD, camptothecins, and camptothecin derivatives (SN-38, Dxd); auristatin cytokine molecules include, but are not limited to, MMAE or MMAF or their derivatives, and maytansine cytotoxic molecules include, but are not limited to, DM1, DM4, or their derivatives.

[0089] As used herein, PROTAC refers to a heterobifunctional degrader, one end of which is connected to a ligand that binds to a target protein, and the other end is connected to a ligand of an E3 ligase, connected in the middle by a suitable spacer group. PROTAC degrades the target protein through the ubiquitin proteasome system (UPS), and the general process is as follows: PROTAC molecules bind to the target protein and the E3 ligase to form a ternary complex, which ubiquitinates the target protein, and then the ubiquitinated target protein is recognized and degraded by the proteasome 26S in the cell.

[0090] As used herein, molecular glue refers to a small molecule degrader that interacts with an E3 ligase or a target protein to form a ternary complex by inducing or stabilizing the protein-protein interaction between the E3 ligase and the target protein, thereby causing ubiquitination and degradation of the target protein.

[0091] In some embodiments, the nucleic acid compound includes any one or more of sgRNA, mRNA, siRNA, shRNA and antisense RNA.

[0092] When the payload is an antibody or an antigen-binding fragment thereof, the immunoconjugate is a fusion protein, such as a bispecific antibody, a multispecific antibody, a bivalent antibody, a multivalent antibody, etc. In some embodiments, the payload is a molecule targeting a tumor surface marker or a molecule targeting a surface marker of an immune cell.

[0093] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), a targeted nuclear drug (RDC), or a fusion protein.

[0094] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the payload includes a small molecule drug with biological activity, which may include but is not limited to toxins, cell differentiation factors, stem cell trophic factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs, drugs for treating infectious diseases, cytokines, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, pro-apoptotic agents, glucocorticoids, nucleic acid compounds, polymers. Small molecule drugs with biological activity may include existing active drug units used in ADCs, such as those disclosed in the Chinese invention patent with patent publication number CN110997010A.

[0095] In some embodiments, the immunoconjugate is a PROTAC-antibody conjugate (DAC), wherein the payload comprises a proteolysis targeting chimera (PROTAC).

[0096] In some embodiments, the immunoconjugate is a targeted nuclear medicine (RDC, or radionuclide conjugated drug), wherein the payload includes a radionuclide, which achieves the therapeutic purpose through the irradiation of its α, β or Auger rays. Commonly used radionuclides include, but are not limited to, iodine-131, radium-223, thallium-201, arsenic-211, rhodium-188, and lutetium-177.

[0097] In some embodiments, the immunoconjugate is a bispecific antibody, a multispecific antibody, a bivalent antibody or a multivalent antibody. In some embodiments, the molecule targeting the surface marker of an immune cell is an antibody that binds to a T cell surface marker, which forms a bifunctional antibody that T cells participate in with the nanobody or its antigen-binding fragment described in the first aspect of the present application.

[0098] In the seventh aspect, the present application provides a pharmaceutical composition, which comprises the nanoantibody or antigen-binding fragment thereof described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application or the immunoconjugate described in the sixth aspect of the present application, and a pharmaceutically acceptable carrier.

[0099] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, which are involved in carrying or delivering the antibody or antigen-binding fragment thereof of the present application in the subject or carrying or delivering the antibody or antigen-binding fragment thereof of the present application to the subject so that it can perform its intended function. Each 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 solution; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; perfuming agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; fixatives; suspending agents; surfactants; humectants; carriers; stabilizers; and other nontoxic, compatible substances used in pharmaceutical preparations, or any combination thereof.

[0100] The pharmaceutical composition may also include an excipient. Specific excipients include buffers, surfactants, preservatives, fillers, polymers and stabilizers, which can be used with the antibody or its antigen-binding fragment. Buffers are used to control the pH of the composition. Surfactants are used to stabilize proteins, inhibit protein aggregation, inhibit protein adsorption to the surface, and assist protein refolding. Exemplary surfactants include Tween 80, Tween 20, Brij 35, Triton X-10, Pluronic F127 and sodium dodecyl sulfate. Preservatives are used to inhibit microbial growth. Examples of preservatives include benzyl alcohol, metacresol and phenol. Fillers are used during the freeze-drying process to increase the volume. Hydrophilic polymers (such as dextran, hydroxyethyl starch, polyethylene glycol, gelatin) can be used to stabilize proteins. Polymers with non-polar parts (such as polyethylene glycol polymers) can also be used as surfactants. Protein stabilizers may include polyols, sugars, amino acids, amines and salts. Suitable sugars include sucrose and trehalose. Amino acids include histidine, arginine, glycine, methionine, proline, lysine, glutamic acid, and mixtures thereof. Proteins such as human serum albumin can also competitively adsorb to the surface and reduce aggregation of antibodies. It should be noted that specific molecules can be used for multiple purposes. For example, histidine can be used as a buffer and antioxidant. Glycine can be used as a buffer and a filler.

[0101] The antibodies or antigen-binding fragments thereof, chimeric antigen receptors, nucleic acids, vectors, host cells, immunoconjugates, and pharmaceutical compositions of the present application can be appropriately developed to be suitable for the following routes of administration, including inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, ​​vaginal (e.g., transvaginal and intravaginal), (trans)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, sachets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, slurries, lozenges, creams, pastes, ointments, lotions, disks, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol formulations for inhalation, compositions and formulations for intravesical administration, and the like.

[0102] In the eighth aspect, the present application provides the use of the nanoantibody or its antigen-binding fragment described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application, the immunoconjugate described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application in the preparation of a medicament for treating diseases related to abnormal expression of Claudin18.2.

[0103] In some embodiments, the disease associated with abnormal expression of Claudin18.2 includes cancer. In some embodiments, the disease associated with abnormal expression of Claudin18.2 includes solid tumors.

[0104] In some embodiments, the diseases associated with abnormal expression of Claudin18.2 include gastric cancer (e.g., gastrointestinal cancer), pancreatic cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma or lung squamous cell carcinoma), esophageal cancer, liver cancer (e.g., hepatocellular carcinoma / hepatoma), squamous cell carcinoma, peritoneal cancer, brain tumor (e.g., glioblastoma / glioblastoma multiforme (GBM), non-glioblastoma brain tumor or meningioma), glioma (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma or mixed glioma, such as oligoastrocytoma), cervical cancer, Ovarian cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma / hepatoma, hepatoma), bladder cancer (e.g., urothelial carcinoma), breast cancer, colon cancer, colorectal cancer, rectal cancer, endometrial cancer, or uterine cancer, salivary gland cancer, kidney cancer (e.g., renal rhabdoid tumor), prostate cancer, vulvar cancer, penis cancer, anal cancer (e.g., anal squamous cell carcinoma), thyroid cancer, head and neck cancer (e.g., nasopharyngeal cancer), skin cancer (e.g., melanoma or squamous cell carcinoma), osteosarcoma, Ewing's sarcoma, chondrosarcoma, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma), carcinoid tumor, eye cancer (e.g., retinoblastoma) , mesothelioma, lymphocytic / lymphoblastic leukemias (e.g., acute lymphocytic / lymphoblastic leukemia (ALL) of the T-cell lineage and B-cell precursor lineage, chronic lymphoblastic / lymphocytic leukemia (CLL), acute myeloid / myeloblastic leukemia (AML), including mast cell leukemia, chronic myeloid / myelocytic / myeloblastic leukemia (CML), hairy cell leukemia (HCL), Hodgkin's disease, non-Hodgkin's lymphoma, chronic myelomonocytic leukemia (CMML), follicular lymphoma (FL), diffuse large B-cell lymphoma ( The following are examples of cancers that may be treated: leukemia, leukemia, leukemia (DLCL), mantle cell lymphoma (MCL), Burkitt's lymphoma (BL), mycosis fungoides, Sezary syndrome, cutaneous T-cell lymphoma, mastocytoma, medulloblastoma, nephroblastoma, solitary plasmacytoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, central nervous system tumors, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumors, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, gallbladder cancer, idiopathic myelofibrosis, and pediatric cancers, such as pediatric sarcomas (e.g., neuroblastoma, rhabdomyosarcoma, and osteosarcoma).

[0105] In some embodiments, the diseases associated with abnormal expression of Claudin18.2 include gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, Wilms' tumor, lung cancer, ovarian cancer, colon cancer, rectal cancer, liver cancer, head and neck cancer, chronic myeloid leukemia or gallbladder cancer.

[0106] As used herein, the term "treatment" refers to the administration of the Nanobodies or antigen-binding fragments thereof, chimeric antigen receptors, nucleic acids, vectors, host cells, pharmaceutical compositions or immunoconjugates that specifically bind to Claudin18.2 described herein to prevent, improve or eliminate a disease or one or more symptoms associated with the disease, and includes but is not limited to: (i) preventing the occurrence of a disease or disease state in a mammal, especially when such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state; (ii) inhibiting a disease or disease state, i.e., curbing its development; (iii) alleviating a disease or disease state, even if the disease or disease state regresses; (iv) reducing any direct or indirect pathological consequences of a disease or disease state.

[0107] In the context of cancer, the term "treat" includes any or all of the following: inhibiting or slowing the growth of cancer cells, inhibiting the proliferation of cancer cells, reducing the number of cancer cells, reducing the rate of cancer cell infiltration into surrounding organs, reducing the rate or extent of tumor metastasis, reducing the overall tumor burden (reducing tumor volume and / or tumor weight), and improving one or more symptoms associated with cancer.

[0108] As used herein, the term "cancer" has the same meaning as the term "tumor" unless clearly contradicted by the context or indicated otherwise.

[0109] In the ninth aspect, the present application provides a method for treating a disease associated with abnormal expression of Claudin18.2, the method comprising administering to a subject in need thereof a therapeutically effective amount of the nanobody or antigen-binding fragment thereof described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application, the immunoconjugate described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application.

[0110] 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).

[0111] As used herein, the term "therapeutically effective amount" refers to any amount of a drug or agent that provides a therapeutic effect when used alone or in combination with another therapeutic agent, such as protecting a subject from the onset of a disease or promoting regression of a disease. The "therapeutically effective amount" may vary depending on many factors, such as the disease state and its severity, the mode of administration, and the age of the mammal to be treated, and can be determined by a person skilled in the art based on his or her own knowledge in combination with information such as the disclosure of this application.

[0112] In the tenth aspect, the present application provides a kit, which comprises the nanoantibody or antigen-binding fragment thereof described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application, the immunoconjugate described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application.

[0113] In some embodiments, the kit is used to detect the presence and / or expression abundance of Claudin18.2 in a sample.

[0114] As used herein, "sample" means a portion of a larger element in this article. Preferably, a sample is a substance of biological origin. It contains cells and / or other molecular entities to be characterized and / or identified according to, for example, physical, biochemical, chemical and / or physiological characteristics. For example, it refers to any sample derived from a subject of interest, which 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, lymphatic 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 tissues, tumor tissues, cell extracts, and combinations thereof.

[0115] In the eleventh aspect, the present application provides the use of the nanoantibody or its antigen-binding fragment described in the first aspect of the present application, the chimeric antigen receptor described in the second aspect of the present application, the nucleic acid described in the third aspect of the present application, the vector described in the fourth aspect of the present application, the host cell described in the fifth aspect of the present application, the immunoconjugate described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application in the preparation of a reagent for detecting or diagnosing a disease related to abnormal expression of Claudin18.2.

[0116] In the present application, the reagent can be used alone or in combination with an instrument, an apparatus, a device or a system. In the process of disease prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation and prediction of genetic diseases, the reagent can be used to perform in vitro detection on human samples (e.g., various body fluids, cells, tissue samples, etc.).

[0117] In the twelfth aspect, the present application provides a method for detecting the expression level of Claudin18.2 protein in a sample, the method comprising: contacting the sample with the nanobody or its antigen-binding fragment described in the first aspect of the present application, and detecting the binding of the nanobody or its antigen-binding fragment to the Claudin18.2 protein.

[0118] The various embodiments and preferences provided above for the present application can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments formed by such combination are all considered as part of the disclosure of the present application.

[0119] Example

[0120] The following is a description of 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 to be merely exemplary and are by no means intended to limit the scope of protection of the present application. The scope of protection of the present application is limited only by the claims. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0121] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0122] Example 1. Preparation and screening of anti-Claudin18.2 nanobodies

[0123] Antibody fragments were obtained using standard biological protocols. Figure 1 The technical roadmap for obtaining CLDN18.2 nanoantibodies shown in A in the middle, construct mammalian cells that highly express human CLDN18.2 to immunize alpacas, subcutaneously multiple immune sites, immunize 4-5 times, and monitor the immune titer. Collect peripheral blood from alpacas, separate lymphocytes (PBMC), lyse, extract RNA, reverse transcribe into cDNA, amplify VHH sequences, and subclone into phage display vectors, electrotransform competent E. coli cells, and construct nanoantibody phage display libraries. Mammalian cells that highly express human CLDN18.1 are used for negative screening to remove nonspecific clones that can bind to blank cells and human CLDN18.1 in the display library, and then mammalian cells that highly express human CLDN18.2 are used to enrich candidate antibodies, and the above process is repeated 3-4 times to enrich positive clones. Phage ELISA determines the monoclonal positive rate after four rounds of panning, selects positive nanoantibody clones that can bind to CLDN18.2 for sequencing analysis, and obtains the nucleic acid and amino acid sequences of candidate antibodies that bind to the target protein CLDN18.2.

[0124] The candidate antibody clones were expressed in vitro and the identification results were as follows Figure 1Shown in B: SDS-PAGE (polyacrylamide) protein electrophoresis identification results of 2-F09 (hereinafter also referred to as 2F09), 2-B08 (hereinafter also referred to as 2B08), 6-H09 (hereinafter also referred to as 6H09), 11-F09 (hereinafter also referred to as 11F09), 4-H09 (hereinafter also referred to as 4H09), and 2-A12 (hereinafter also referred to as 2A12) antibody clones.

[0125] The antibody binding cell specificity was evaluated by flow cytometry using CHO-K1 cells (Cell Bank of the Chinese Academy of Sciences, SCSP-507), CHO-K1 cells stably transfected with the human CLDN18.1 coding sequence (SEQ ID NO: 20) (CHO-K1 / 18.1), CHO-K1 cells stably transfected with the human CLDN18.2 coding sequence (SEQ ID NO: 21) (CHO-K1 / 18.2), and CHO-K1 cells stably transfected with the mouse CLDN18.2 coding sequence (SEQ ID NO: 22) (CHO-K1 / mouse 18.2). The experimental results are shown in Figure 2. Figure 2 As shown; flow cytometry was then used to compare the affinity of the antibody to CHO-K1, CHO-K1 / 18.1, CHO-K1 / 18.2, and CHO-K1 / mouse 18.2. The experimental results are shown in Figure 3 shown.

[0126] Figure 2 The results show that: the positive control (PC, containing the KLST-18.2 coding sequence of SEQ ID NO:23), 2-B08, 11-F09, and 4-H09 subclones specifically bind to CHO-K1 / 18.2 and CHO-K1 / mouse 18.2, but not to CHO-K1 and CHO-K1 / 18.1; the 2-F09 and 2-A12 subclones also bind to CHO-K1 / 18.2 and CHO-K1 / mouse 18.2, but not to CHO-K1, but have weak binding to CHO-K1 / 18.1; the negative control (NC, containing the sequence of SEQ ID NO:24), 6-H09 do not bind to CHO-K1, CHO-K1 / 18.1, CHO-K1 / 18.2, and CHO-K1 / mouse 18.2.

[0127] Figure 3 It shows that after the VHH of 2-B08, 11-F09, 4-H09 and the ScFv of KLST-18.2 were chimerized with the human IgG1 Fc part (SEQID NO: 25), the relative affinity of binding to CHO-K1 cells stably transfected with the human CLDN18.2 coding sequence (A) and the mouse CLDN18.2 coding sequence (B) was relatively ideal.

[0128] The relevant amino acid sequence information of 2-B08 is shown in Table 1 below, and the relevant nucleotide sequence information is shown in Table 2 below.

[0129] Table 1. Amino acid sequence information of 2-B08

[0130]

[0131] Table 2. Related nucleotide sequence information of 2-B08

[0132]

[0133] Example 2. Preparation, screening and identification of humanized antibodies against 2B08

[0134] According to the screening experiment results of Example 1, nano antibody 2B08 was selected, and humanization was performed using CDR&SDR Grafting and Backmutation. After synthesizing the humanized antibody sequence, a humanized antibody expression vector was constructed. The expression vector prepared above was subjected to a large plasmid extraction to prepare a transfection-grade plasmid. In the design stage, the glycosylation sites, antibody aggregation, immunogenic sites, post-translational modifications, etc. that may exist in humanized antibodies were model predicted. The possible glycosylation sites, immunogenic sites, deamidation, isomerization oxidation and glycosylation were transformed through structural models to avoid physicochemical properties that are not conducive to antibody development, such as antibody aggregation, isoelectric point, etc., to improve the binding activity and developability of the modified antibody. The humanized antibody expression vector prepared above was transiently transfected into mammalian cells 293F, and the recombinant antibody was purified using Protein A, and quantified using the BCA method after concentration.

[0135] The purified antibodies were used for FACS to detect the binding of humanized antibodies to cells overexpressing CLDN18.2, confirming the specific binding of candidate antibodies to the target protein before and after humanization. After gradient dilution of the candidate humanized nanoantibodies, FACS was performed with the overexpressing cell lines, and a curve was drawn with the antibody molar concentration as the horizontal axis and the MFI value as the vertical axis, and the molar concentration of each antibody was calculated; the four humanized antibodies were compared with the original antibody 2B08 by flow cytometry, and the experimental results are as follows Figure 4 The results showed that the binding strength of humanized 2-B08-HM1 (hereinafter referred to as HM1), 2-B08-HM2 (hereinafter referred to as HM2), 2-B08-HM3 (hereinafter referred to as HM3), 2-B08-HM4 (hereinafter referred to as HM4) and CHO-CLDN18.2 and CHO-mouse CLDN18.2 was consistent with that of the original antibody 2B08.

[0136] The binding strengths of humanized antibodies 2-B08-HM2, 2-B08-HM4 and non-humanized antibody 2-B08 to CLDN18.2 protein (Bioscience Biotech Co., Ltd., CL2-H85D4) were further compared by ELISA. The experimental results are shown in Figure 5 The results showed that 2-B08-HM2, 2-B08-HM4 and the non-humanized antibody 2B08 had similar target binding abilities.

[0137] The relevant amino acid sequence information of 2-B08-HM1, 2-B08-HM2, 2-B08-HM3, 2-B08-HM4, and 2-B08-HM5 is shown in Table 3 below, and the relevant nucleotide sequence information is shown in Table 4 below.

[0138] Table 3. Amino acid sequence information of 2-B08-HM1, 2-B08-HM2, 2-B08-HM3, 2-B08-HM4, and 2-B08-HM5

[0139]

[0140] Table 4. Related nucleotide sequence information of 2-B08-HM1, 2-B08-HM2, 2-B08-HM3, 2-B08-HM4, and 2-B08-HM5

[0141]

[0142] Example 3. Construction of chimeric antigen receptor plasmid (CAR plasmid) and viral packaging

[0143] Construction of NK cell chimeric antigen receptor plasmid (CAR plasmid), such as Figure 6The plasmid map shown in A in the figure shows that pRV-P2A-puro (synthesized by Nanjing GenScript Biotechnology Co., Ltd., SEQ ID NO:26) was used as a vector to construct a retroviral plasmid expressing chimeric antigen receptors of humanized antibodies HM1, HM2, HM3, HM4 and non-human antibodies 2B08, 11F09, 4H09, 8E5-2I (amino acid sequence as shown in SEQ ID NO:16, nucleotide sequence as shown in SEQ ID NO:17), flag (amino acid sequence as shown in SEQ ID NO:18, nucleotide sequence as shown in SEQ ID NO:19), including CD8a SP (SEQ ID NO:27), CD8 Hinge (SEQ ID NO:28), CD8 TM (SEQ ID NO:29), OX40 (SEQ ID NO:30), CD3zeta (SEQ ID NO:31), T2A (SEQ ID NO:32), IgE SP (SEQ ID NO:33), IL15 (SEQ ID NO:34), Linker (SEQ ID NO:35), IL-15 (SEQ ID NO:36), IL-25 (SEQ ID NO:37), IL-36 (SEQ ID NO:38), IL-40 (SEQ ID NO:39), IL-50 (SEQ ID NO:40), IL-60 (SEQ ID NO:41), IL-70 (SEQ ID NO:42), IL-80 (SEQ ID NO:43), IL-90 (SEQ ID NO:44), IL-10 (SEQ ID NO:45), IL-110 (SEQ ID NO:46), IL-111 (SEQ ID NO:47), IL-112 (SEQ ID NO:48), IL-113 (SEQ ID NO:49), IL-114 (SEQ ID NO:49), IL-115 (SEQ ID NO:41), IL-116 (SEQ ID NO:42), IL NO:35), IL15RA (SEQ ID NO:36), P2A (SEQ ID NO:37), puro (SEQ ID NO:38).

[0144] Construction of T cell chimeric antigen receptor plasmid (CAR plasmid), such as Figure 6 The plasmid map shown in B is a retroviral plasmid expressing the chimeric antigen receptor of humanized antibody HM2 and control antibody 8E5-2I and flag, using pRV-P2A-puro as a vector, including CD8a SP, CD8 Hinge, CD8 TM, OX40, CD3 zeta, P2A, and puro.

[0145] 1×10 6 293T cells (Cell Bank of the Chinese Academy of Sciences, SCSP-502) cultured to the 6th to 10th passage were inoculated at a density of 1.5 μg / mL in a 6-well culture plate and cultured overnight at 37°C, 5% CO2 in preparation for transfection. The culture medium was DMEM containing 10% fetal bovine serum (Gibico).

[0146] Dissolve 1.5 μg of CAR retroviral plasmids expressing HM1, HM2, HM3, HM4, 11F09, 4H09, 8E5-2I and flag and 1.5 μg of packaging plasmid pCL-10A1 (Novus Biologicals, NBP2-29542) in 150 μL blank DMEM culture medium; dissolve 6 μg PEI (1 μg / μl) in 150 μl serum-free DMEM culture medium and mix well.

[0147] Add the plasmid mixture to the PEI mixture, mix well and incubate at room temperature for 20 minutes to form a transfection complex; add 300 μL of the transfection complex dropwise into a 6-well plate containing 2 ml of 10% FBS DMEM medium; after 6-8 hours, replace the transfected 293T cells with 10% FBS DMEM medium, incubate at 37°C for 72 hours, and collect the virus supernatant.

[0148] Example 4. Construction of CAR NK cells and cytotoxicity assay

[0149] 1. Preparation of CAR-positive NK cell lines

[0150] 1.1 Retronectin coated 12-well plate: Add 1 ml of 25 μg / ml retronectin solution (PBS) to each well, incubate overnight at 4 degrees, and inoculate NK cells cultured for 1 week (peripheral blood mononuclear cells were purchased from Zhejiang Free Trade Zone Maishun Biotechnology Co., Ltd., catalog number PB003F-W, refer to the method published in https: / / doi.org / 10.1084 / jem.20201529 to obtain NK cells from peripheral blood mononuclear cells.) into a 6-well plate coated with retronectin, with 5×10^5 cells per well and a culture medium volume of 1 ml;

[0151] 1.2 Add 2 ml of retroviral supernatant to each well, centrifuge at 32 degrees for 90 minutes and transfer to a cell culture incubator;

[0152] 1.3 Overnight, discard 1.7 ml of supernatant, add 1.7 ml of retroviral supernatant to each well, centrifuge at 32 degrees for 90 minutes, and transfer to a cell culture incubator;

[0153] 1.4 Expansion culture: The infected cells were passaged every other day at a density of 5×10^5 / mL, and recombinant human IL-2 (Novoprotein, GMP-CD66-1mg) was added to the lymphocyte culture medium at a final concentration of 1000U / mL.

[0154] 2. Expression of chimeric antigen receptor on NK cells

[0155] 2.1 On the 15th day of culture, 1×10^6 cells of retrovirus-infected NK cells were collected by centrifugation in a centrifuge tube;

[0156] 2.2 The cells in the control group were added with 100ul Rabbit Anti-Camelid VHH Cocktail [PE] (Nanjing GenScript Biotechnology Co., Ltd., A02018-200) (1.5:100 dilution) antibody and incubated on ice. After resuspending, they were used as the control. The cells in the detection group were added with 100μl Rabbit Anti-Camelid VHH Cocktail [PE] (1.5:100 dilution) antibody and incubated on ice for 30min.

[0157] 2.3 Add 1 ml PBS (2% FBS) to resuspend the cells, centrifuge at 4°C and discard the supernatant; add 200 μl PBS (2% FBS) and transfer to a flow cytometer. Detect the PE channel by flow cytometry to determine the proportion of CAR-positive NK cells. The results are as follows: Figure 7 As shown, HM1, HM2, HM3, HM4 and 8E5-2I are humanized antibodies and therefore cannot be detected using Rabbit Anti-Camelid VHHCocktail [PE].

[0158] 2.4 Cytotoxicity assay: 5×10^4 cells of NUGC-4 target cells expressing the luciferase coding sequence (SEQ ID NO:39) (Nanjing Kebai Biotechnology Co., Ltd., CBP60493) were inoculated in 96-well plates; HM1, HM2, HM3, HM4, 2B08, 11F09, 4H09, 8E5-2I NK cells and NK cells 102NK and 108NK (effector cells) from two healthy donors were added at effector-target ratios of 1:1, 1:4, 1:16, and 1:64; 3 replicate wells were set up in each group, and the average of the 3 replicate wells was taken. The detection time was 48h, 96h, 144h, 192h, 240h, and 288h. The experimental groups and control groups are as follows: experimental groups: target cells + the above-mentioned effector cells; target cell group: target cells spontaneously release luciferase signals; blank group (no target cells and no effector cells, the rest are the same as the experimental groups): culture medium luciferase signal.

[0159] 3. Luciferase detection of target cells in the experimental group

[0160] Detection method: GMOne-Step luciferase reporter gene detection kit (Jiman Biotechnology Co., Ltd.) was used. Please refer to the instructions of GMOne-Step luciferase reporter gene detection kit for details. The cytotoxicity calculation formula is:

[0161] .

[0162] The results are as follows Figure 8As shown. The results showed that HM1, HM2, HM3, HM4, 2B08, 11F09, 4H09 and 8E5-2I NK cells had significant in vitro killing activity against NUGC-4 cells that weakly expressed CLDN18.2. Since NK cells are natural killer cells, there is a killing mechanism for NUGC-4 cells that is independent of CAR signals. In the early stage, NK cells 102NK and 108NK from two healthy donors also had strong killing toxicity. However, after prolonging the killing time, it can be observed that the killing ability of NK cells is decreasing, and at this time, CAR-NK cells still maintain a high ability to kill target cells.

[0163] Example 5. Cytotoxicity and cytokine secretion assay of CAR NK92 cells

[0164] 1. Preparation of CAR-positive NK 92 cell line

[0165] 1.1 Retronectin coated 12-well plate: add 1 ml of 25 μg / ml retronectin solution (PBS) to each well and incubate at 4 degrees overnight. Inoculate NK 92 cells (Nanjing Kebai Biotechnology Co., Ltd., CBP60980) into 6-well plates coated with retronectin, with 6×10^6 cells per well and 3 ml of culture medium.

[0166] 1.2 Add 2B08-CAR, 2B08-HM2-CAR, and 2B08-HM4-CAR viruses at 200ul per well, centrifuge at 32 degrees for 90 minutes, and transfer to a cell culture incubator;

[0167] 1.3 Expansion culture: The infected cells were passaged every other day at a density of 5×10^5 / mL, and recombinant human IL-2 (Novoprotein, GMP-CD66-1mg) was added to the lymphocyte culture medium at a final concentration of 1000U / mL.

[0168] 2. Expression of chimeric antigen receptor on NK92 cells

[0169] 2.1 96 h after virus infection of NK92 cells, 2B08 CAR-NK92, 2B08-HM2 CAR-NK92, and 2B08-HM4CAR-NK92 were obtained and stained with cetuximab (MedChemExpress, HY-P9905) and anti-Human-Fc-PE (ThermoFisher, 12-4998-82);

[0170] 2.2 The NK92 cells of the non-transfected group were used as a control to detect the positive rate of CLDN18.2 CAR-NK92. The results are as follows Fig. 9The results showed that the positive rates of 2B08 CAR-NK92, 2B08-HM2 CAR-NK92, and 2B08-HM4 CAR-NK92 were 39.2%, 22.98%, and 20.1%, respectively.

[0171] 2.3 Cytotoxicity assay: The CLDN18.2 expression level of NUGC-4 target cells expressing luciferase coding sequence (Nanjing Kebai Biotechnology Co., Ltd., CBP60493) was 88.6%. NUGC-4 target cells were inoculated with 1×10^4 cells in 96-well plates; 2B08 CAR-NK92, 2B08-HM2 CAR-NK92, and 2B08-HM4CAR-NK92 cells (effector cells) were added at effector-target ratios of 1:1, 2.5:1, 5:1, and 10:1; 3 replicate wells were set up in each group, and the average value of the 3 replicate wells was taken, and non-transfected NK92 was selected as the control. The detection time was 6 hours of co-culture. The experimental groups and control groups were as follows: experimental groups: target cells + the above-mentioned effector cells; target cell group: target cells spontaneously released luciferase signals; blank group: culture medium luciferase signals. Read the fluorescence value, and the luciferase activity was detected to reflect the lysis ability of recombinant CAR-T cells on target cells.

[0172] 3. Luciferase detection of target cells in the experimental group

[0173] Detection method: GMOne-Step luciferase reporter gene detection kit (Jiman Biotechnology Co., Ltd.) was used. Please refer to the instructions of GMOne-Step luciferase reporter gene detection kit for details. The cytotoxicity calculation formula is:

[0174] .

[0175] The results are as follows Fig.10 As shown in A. The results showed that 2B08 CAR-NK92, 2B08-HM2 CAR-NK92 and 2B08-HM4CAR-NK92 cells had significant in vitro killing activity against NUGC-4-luciferase cells that were positive for CLDN18.2 expression, which was significantly better than NK92 cells.

[0176] 4. In vitro cytokine release assay induced by CLDN18.2 CAR-NK92 cells

[0177] 4.1 In order to verify the secretion of cytokines by the constructed CAR-NK92 cells after co-incubation with target cells, the cytokines released by 2B08 CAR-NK92, 2B08-HM2 CAR-NK92 and 2B08-HM4 CAR-NK92 cells were detected respectively. The supernatant was collected after co-culture for 18 h at effector-target ratios of 1:1, 2.5:1, 5:1 and 10:1, and the IFN-γ released by the supernatant during the co-culture of CAR NK92 lymphocytes and target cells was detected by sandwich ELISA.

[0178] The experimental results are as follows Fig.10 As shown in B. The results showed that when co-incubated with NUGC-4-luciferase cells expressing CLDN18.2, 2B08 CAR-NK92, 2B08-HM2 CAR-NK92 and 2B08-HM4 CAR-NK92 cells were able to release IFN-γ, while the NK92 control group could not release IFN-γ, and there were significant differences.

[0179] Example 6. Cytotoxicity and cytokine secretion assay of CAR-T cells

[0180] 1. Retrovirus infection of T lymphocytes

[0181] 1.1 About 1.46×10 6 / mL density was added to lymphocyte culture medium (Gibco, 0870112DK) for culture, and Dynabeads magnetic beads (gibco) and recombinant human IL-2 (Novoprotein, GMP-CD66-1mg) at a final concentration of 250U / mL were added at a ratio of 1:1 for stimulation and culture for 48h;

[0182] 1.2 Retronectin coated 24-well plate: add 0.5 ml of 25 μg / ml retronectin solution (PBS) to each well, incubate at 4 degrees overnight, discard the retronectin solution (PBS) in the 24-well plate, wash twice with 1 ml PBS, add 1 ml of retroviral supernatant to each well of the retronectin-coated 24-well plate, and centrifuge at 32 degrees for 90 minutes;

[0183] 1.3 Aspirate the viral supernatant and inoculate the cells into 24-well plates coated with retronectin at a cell number of 5 × 10 per well. 5 Each well was cultured in 0.5 ml of culture medium. 0.5 ml of retroviral supernatant was added to each well, centrifuged at 32 degrees for 90 minutes and transferred to a cell culture incubator;

[0184] 1.4 Expansion culture: After infection, 5×10 cells were cultured every other day.5 / mL for passage, and at the same time, recombinant human IL-2 at a final concentration of 250U / mL was added to the lymphocyte culture medium.

[0185] 2. Expression of chimeric antigen receptor on T lymphocytes

[0186] 2.1 On the 11th day of culture, take 1×10^6 cells of retrovirus-infected T cells, centrifuge at 400g, 4℃ for 5 minutes, and discard the supernatant;

[0187] 2.2 In the control group 1, cells were added with 100ul Rabbit Anti-Camelid VHH Cocktail [PE] (3:200 dilution) antibody and incubated on ice. After resuspending, they were used as the control. In the HM2 group, cells were added with 100μl Rabbit Anti-Camelid VHH Cocktail [PE] (3:200 dilution) antibody and incubated on ice for 30 min. In the control group 2, cells were added with anti-G4S (CST, 69782S) (1:50 dilution) antibody and incubated on ice. After resuspending, they were used as the control. In the 8E5-2I group, cells were added with 100μl anti-G4S (1:50 dilution) antibody and incubated on ice for 30 min. In the control group 3, cells were added with anti-DYKDDDDK (Biolegend, 637322) (1:50 dilution) antibody and incubated on ice. After resuspending, they were used as the control. In the flag group, cells were added with 100μl anti-DYKDDDDK (1:50 dilution) antibody and incubated on ice for 30 min.

[0188] 2.3 Add 1 ml PBS (2% FBS) to resuspend the cells, centrifuge at 4°C and discard the supernatant; add 200 μl PBS (2% FBS) and transfer to a flow cytometer. Detect the proportion of CAR-positive T cells by flow cytometry. The results are as follows: Fig.11 The results showed that the infection rates of HM2 CAR-T, 8E5-2I CAR-T, and flag CAR-T (control) were 96.4%, 97.52%, and 36.18%, respectively.

[0189] 3. Cytotoxicity assay of CAR T cells targeting CLDN18.2

[0190] 3.1 Target cells: AsPC-1 cells expressing human Claudin 18.2 and luciferase coding sequences (Chinese Academy of Sciences Cell Bank, SCSP-5080) had a CLDN18.2 expression level of 87.07%. CLDN18.2 AsPC-1-luciferase cells were seeded with 5×10 4 cells in 96-well plates.

[0191] 3.2 Effector cells: HM2 CAR-T, 8E5-2I CAR-T, and flag CAR-T were added at an effector-target ratio of 4:1, 1:1, or 1:4, with 3 replicate wells set up and the average of the 3 replicate wells taken. The detection time was 24 hours.

[0192] 3.3 The experimental groups and control groups are as follows: HM2 cell experimental group: target cells + HM2 CAR T cells; 8E5-2I cell experimental group: target cells + 8E5-2I CAR T cells; target cell group: fluorescence value released by control plated target cells; flag group: target cells + flag CAR-T cells; blank group: fluorescence value of culture medium.

[0193] 3.4 Detection method: GMOne-Step luciferase reporter gene detection kit (Jiman Biotechnology Co., Ltd.) was used. Please refer to the instructions of GMOne-Step luciferase reporter gene detection kit for details. The cytotoxicity calculation formula is:

[0194] .

[0195] 3.5 Cell killing results of different effector-target ratios Fig.12 As shown. The results showed that HM2 CAR-T and 8E5-2I CAR-T showed good killing effects on AsPC-1 cells with positive expression of CLDN18.2. Among them, HM2 CAR-T cells killed more than 65% of AsPC-1 cells when the effector-target ratio was 4:1. At the same time, the results also showed that the killing effect of flag CAR-T cells on cells with positive expression of CLDN18A2 was not obvious.

[0196] 4. In vitro cytokine release assay induced by CLDN18.2 CAR-T cells

[0197] 4.1 In order to verify the secretion of cytokines by the constructed CAR-T cells after co-incubation with target cells, the cytokines released by T cells transfected with empty vectors (flag CAR-T cells), HM2 CAR-T cells, and 8E5-2I CAR-T cells were detected respectively. The supernatant was collected after co-culture for 18 h at an effector-target ratio of 1:1, and the IFN-γ, TNF-α, and GM-CSF released during the co-culture of CAR T lymphocytes and target cells in the supernatant were detected by sandwich ELISA.

[0198] 4.2 Experimental results Fig.13As shown. The results showed that when co-incubated with target cells, HM2 CAR-T cells and 8E5-2ICAR-T cells released IFN-γ, TNF-α and GM-CSF cytokines, and the release amount of HM2 CAR-T cells was significantly higher than that of 8E5-2I CAR-T cells, while the flag CAR-T cell control group could not release the above cytokines, and there were significant statistical differences. Regarding the release of IFN-γ, TNF-α and GM-CSF cytokines, compared with flag CAR-T cells, the P values ​​of HM2CAR-T cells were 0.0080, 0.0018 and 0.0006, respectively.

[0199] Example 7. In vivo activity of CLDN18.2 CAR-T cells in mice carrying AsPC-1

[0200] The anti-tumor therapeutic effect of empty vector-transfected T cells (flag CAR-T cells) and HM2 CAR-T cells on subcutaneous transplanted tumors of CLDN18.2 AsPC-1 cells was tested.

[0201] 1) Inoculation of CLDN18.2 AsPC-1 subcutaneous transplanted tumors: AsPC-1 cells expressing the coding sequence of human Claudin 18.2 (Chinese Academy of Sciences Cell Bank, SCSP-5080) have a CLDN18.2 expression level of 99.3%. Collect CLDN18.2 AsPC-1 cells in the logarithmic growth phase and in good growth condition and adjust the density to 6×10 using DPBS. 7 / mL, the injection volume of cell suspension was 50μL (3×10 6 / mouse) was injected subcutaneously in the axilla of the right front leg of 8-week-old female NPSG mice (purchased from Phenotype Biotechnology (Shanghai) Co., Ltd.) on the day of tumor cell inoculation as day D0.

[0202] 2) Experimental grouping: On day 10 after tumor inoculation, the tumor size was measured and the NPSG mice were divided into the experimental group HM2 group / negative control group flag group according to the tumor size, with 5 mice in the negative control flag cell group and 6 mice in the experimental group HM2 cell group.

[0203] 3) Adoptive transfer of T cells: 1.0×10 7 CAR-T cells were revived overnight. T cells transfected with empty vector (flag group) were used as control to observe and measure the growth of subcutaneous transplanted tumors.

[0204] The experimental results are as follows Fig.14As shown in A and B. The results showed that on the 17th day of HM2 CAR T cell treatment, the tumor inhibition rate of CLDN18.2 AsPC-1 transplanted tumors was 100%; according to ethics, the tumor volume of mice was higher than 2000mm 3 Determined as dead, on the 21st day of tumor inoculation and growth, mice in the flag group began to die, with a survival rate of 80%, and one mouse in the HM2 CAR T treatment group had a tumor volume of 0. On the 41st day of tumor inoculation and growth, all mice in the flag control group died, and the tumor volume of all mice in the HM2 CAR T treatment group was 0, which lasted until the 50th day of tumor inoculation and growth.

[0205] Example 8. In vivo activity of CLDN18.2 CAR-T cells in mice carrying CLDN18.2 CXCL9-AsPC-1

[0206] The anti-tumor therapeutic effect of empty vector-transfected T cells (flag CAR-T cells), HM2 CAR-T cells, and 8E5-2I CAR-T cells on subcutaneous transplanted tumors of CLDN18.2 CXCL9-AsPC-1 cells was tested.

[0207] 1) Inoculation of CLDN18.2 CXCL9-AsPC-1 subcutaneous transplanted tumors: AsPC-1 cells (SCSP-5080, Chinese Academy of Sciences Cell Bank) expressing human Claudin 18.2 and human CXCL9 coding sequence (SEQ ID NO: 40) have a CLDN18.2 expression level of 99.9%. Collect CLDN18.2 CXCL9-AsPC-1 cells in logarithmic growth phase and in good growth condition and adjust the density to 6×10 using DPBS. 7 / mL, the injection volume of cell suspension was 50μL (3×10 6 / mouse) were injected subcutaneously in the axilla of the right foreleg of 8-week-old female NPSG mice on the day of tumor cell inoculation as D0.

[0208] 2) Experimental grouping: On day 6 of tumor inoculation, the tumor size was measured and the NPSG mice were divided into experimental group HM2 group / positive control group 8E5-2I group / negative control group flag group according to the tumor size, with 5 mice in each group, divided into negative T cell flag group, positive control group 8E5-2I cell group and experimental group HM2 cell group.

[0209] 3) Adoptive transfer of T cells: 1.0×10 T cells were infused via tail vein on D7. 7 Cryopreserved and revived CAR-T cells (HM2CAR-T, 8E5-2I CAR-T, flag CAR-T) were used to observe and measure the growth of subcutaneous transplanted tumors.

[0210] The experimental results are as follows Fig.15 The results showed that the inhibition rate of HM2 CAR T cells on CLDN18.2 CXCL9-AsPC-1 transplanted tumors was 100%; according to ethics, the tumor volume of mice was higher than 2000mm 3 Death was determined when mice in the flag group began to die on the 24th day of tumor growth (15B). On the 28th day, the therapeutic effect of the HM2 group on CLDN18.2 AsPC-1 transplanted tumors was significantly different from that of the flagCAR-T group, with a P value of 0.0030.

Claims

1. A nanobody or antigen-binding fragment thereof that specifically binds to Claudin18.2, wherein the nanobody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, wherein The amino acid sequence of the CDR1 is shown in SEQ ID NO. 7, the amino acid sequence of the CDR2 is shown in SEQ ID NO. 8, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.

9.

2. The Nanobody or antigen-binding fragment thereof according to claim 1, wherein the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs. 1-6 or an amino acid sequence having at least 80% sequence identity with an amino acid sequence as shown in any one of SEQ ID NOs. 1-6.

3. A chimeric antigen receptor comprising the Nanobody or antigen-binding fragment thereof according to claim 1 or 2.

4. A nucleic acid capable of encoding the Nanobody or antigen-binding fragment thereof according to any one of claims 1 or 2 or the chimeric antigen receptor according to claim 3.

5. The nucleic acid according to claim 4, comprising a nucleotide sequence as shown in any one of SEQ ID NOs. 10-15 or a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence as shown in any one of SEQ ID NOs. 10-15. A vector comprising the nucleic acid according to claim 4 or 5.

7. A host cell comprising the nucleic acid according to claim 4 or 5 or the vector according to claim 6.

8. An immunoconjugate comprising the Nanobody or antigen-binding fragment thereof according to claim 1 or 2 or the chimeric antigen receptor according to claim 3, and a payload connected thereto.

9. A pharmaceutical composition comprising the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, the chimeric antigen receptor according to claim 3, the nucleic acid according to claim 4 or 5, the vector according to claim 6, the host cell according to claim 7 or the immunoconjugate according to claim 8, and a pharmaceutically acceptable carrier.

10. Use of the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, the chimeric antigen receptor according to claim 3, the nucleic acid according to claim 4 or 5, the vector according to claim 6, the host cell according to claim 7, the immunoconjugate according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating gastric cancer or pancreatic cancer associated with abnormal expression of Claudin18.

2.

11. A kit comprising the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, the chimeric antigen receptor according to claim 3, the nucleic acid according to claim 4 or 5, the vector according to claim 6, the host cell according to claim 7, the immunoconjugate according to claim 8, or the pharmaceutical composition according to claim 9.

12. A method for detecting the expression level of Claudin18.2 protein in a sample for non-diagnostic purposes, the method comprising: The sample is contacted with the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, and the binding of the Nanobody or antigen-binding fragment thereof to the Claudin18.2 protein is detected.

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