Anti-claudin18.2 antibody and use thereof

Abstract

An isolated antibody or antigen-binding domain targeting Claudin18.2 protein, and a chimeric antigen receptor comprising the antibody or antigen-binding domain. The present invention also relates to an isolated nucleic acid molecule, a vector, a cell, and a pharmaceutical composition, and use thereof in the preparation of a drug for treating a disease.

Description

Anti-Claudin18.2 antibodies and their applications

[0001] priority

[0002] This application claims the rights and priority of Chinese application No. 2025101248036, filed on January 26, 2025. The entire contents of Chinese application No. 2025101248036 are incorporated herein by reference for all purposes. Technical Field

[0003] This application relates to antibodies targeting Claudin18.2 and their use, particularly in medicaments for the treatment and / or prevention or diagnosis of diseases related to Claudin18.2. Background Technology

[0004] According to the latest global cancer statistics released by the International Agency for Research on Cancer (IARC) in 2024, an estimated 19,976,499 new cancer cases and 9,743,832 cancer deaths occurred globally in 2022, with gastrointestinal tumors accounting for the leading cause. Colorectal cancer (9.6%) and stomach cancer (4.9%) ranked third and fifth in incidence, respectively. Colorectal cancer (9.3%), liver cancer (7.8%), and stomach cancer (6.8%) were among the top five causes of cancer death (Bray F, et al. CA Cancer J Clin. 2024 May-Jun, 74(3), 229-263.). In my country, the incidence of gastrointestinal tumors is very high, accounting for more than 50% of new cases worldwide each year. Colorectal cancer has the highest diagnosis rate and mortality rate among gastrointestinal cancers, followed by pancreatic cancer, then liver cancer, stomach cancer, and esophageal cancer (Shen L.Int J Cancer.2023 Dec 1, 153(11), 1875-1876.). The high mortality rate of gastrointestinal tumors is mainly due to the fact that most patients are diagnosed at an advanced stage, rendering existing drugs ineffective. Therefore, there is an urgent need for new methods to improve the survival rate of patients with advanced gastrointestinal tumors.

[0005] Claudin18.2 is a subtype of Claudin 18 tight junction protein, composed of 261 amino acids, with four transmembrane domains. The inner membrane contains a short N-terminus and a C-terminus, while the outer membrane contains two domains, ECL1 and ECL2. Claudin18.2 is expressed in normal gastric mucosal epithelial cells and plays a role in maintaining the gastric mucosal barrier (Hayashi D, et al. Gastroenterol. 2012, 142(2), 292-304.). In addition, Claudin 18.2 is highly expressed in gastric cancer, pancreatic cancer, esophageal cancer, and lung cancer tissues (Sahin U, et al. Clin Cancer Res. 2008 Dec 1, 14(23), 7624-34.). Because Claudin18.2 is embedded in the gastric mucosa in normal tissues, it cannot be accessed or recognized by antibodies in small quantities. However, in tumor tissues, cancer cells disrupt tight junctions, leading to the exposure of Claudin18.2, making it a highly promising target for cancer therapy. Astellas' zolbetuximab was the first monoclonal antibody developed targeting Claudin18.2, demonstrating good efficacy and safety in combination therapy clinical trials for gastric cancer (Shitara K, et al. Lancet. 2023, 401(10389): 1655-1668.). Subsequently, CARsgen's monoclonal antibody AB011 and Amgen and BeiGene's bispecific antibody AMG-910 entered clinical trials. In cell therapy, CARsgen's CT041 is the world's first and most advanced CAR-T drug targeting Claudin18.2, and has been approved for Phase I / II clinical trials in multiple countries. In addition, Legend Biotech's Claudin18.2-CAR-T drug LB1908, prepared using a high-affinity VHH antibody, is currently undergoing clinical trials in China and the United States. Although CAR-T drugs targeting Claudin18.2 have shown some efficacy in treating gastric cancer (CARsgen Therapeutics' CT041 achieved an ORR of 57.1% in treating gastric cancer patients with high antigen density), their efficacy and applicability still need improvement.The key points in the development of Claudin18.2 antibodies mainly include two aspects: Firstly, due to the heterogeneity of tumor antigen expression, the expression of Claudin18.2 varies in gastric cancer (Rohde C, et al. Jpn J Clin Oncol. 2019, 49(9), 870-876; Türeci O, et al. Ann Oncol. 2019, 30(9), 1487-1495; Sahin U, et al. Ann Oncol. 2021, 32(5), 609-619.). Therefore, obtaining antibodies with high affinity is particularly important for eliminating tumor cells with low target expression. On the other hand, the other splice variant of Claudin 18, Claudin18.1, is structurally highly similar to Claudin18.2, differing only by 7 amino acid residues in the extracellular domain ECL1 sequence (Hashimoto I, et al. Cancers (Basel). 2022 Jan 7, 14(2), 290.), making the development of antibodies that specifically bind to Claudin18.2 somewhat difficult. Therefore, finding anti-Claudin18.2 antibodies with high specificity and affinity remains a significant challenge.

[0006] Chimeric antigen receptors (CARs) are synthetic receptors that guide lymphocytes to specifically recognize and eliminate tumor cells expressing homologous target ligands (Jackson HJ, et al. Nat Rev Clin Oncol. 2016 Jun, 13(6), 370-83.). The structure of a CAR mainly includes the following components: antigen-binding domain, hinge region, transmembrane region, co-stimulatory signaling region, and CD3ζ signaling activation region (Rafiq S, et al. Nat Rev Clin Oncol. 2020 Mar, 17(3), 147-167.). Chimeric antigen receptor T cells (CAR-T) are T cells in which CAR molecules are expressed on the patient's own T cells. Through their antigen-binding domains, they specifically recognize tumor antigens, triggering activation of intracellular co-stimulatory signaling regions and CD3ζ signaling regions. This, in turn, activates downstream signaling pathways and induces the expression of cytokines, granzymes, and perforin, ultimately lysing and killing tumor cells (Jackson HJ, et al. Nat Rev Clin Oncol. 2016 Jun, 13(6), 370-83.). The antigen-binding domain, usually derived from a single-chain antibody fragment (scFv), is a key component affecting the specificity and effectiveness of CAR-T in killing tumor cells. Therefore, developing antibodies against Claudin18.2 is crucial for researching corresponding CAR-T cell therapies. Summary of the Invention

[0007] This application provides isolated antibody or antigen-binding domains targeting the Claudin18.2 protein, comprising a heavy chain variable region VH and a light chain variable region VL, wherein...

[0008] The VH includes: CDR1 containing or composed of the amino acid sequence shown in SEQ ID NO: 13, CDR2 containing or composed of the amino acid sequence shown in SEQ ID NO: 14, and CDR3 containing or composed of the amino acid sequence shown in SEQ ID NO: 15; the VL includes: CDR1 containing or composed of the amino acid sequence shown in SEQ ID NO: 16, CDR2 containing or composed of the amino acid sequence shown in SEQ ID NO: 17, and CDR3 containing or composed of the amino acid sequence shown in SEQ ID NO: 18.

[0009] In some specific embodiments, the VH in the antibody or antigen binding domain includes: CDR1 as shown in SEQ ID NO: 13, CDR2 as shown in SEQ ID NO: 14, and CDR3 as shown in SEQ ID NO: 15, and the VL includes: CDR1 as shown in SEQ ID NO: 16, CDR2 as shown in SEQ ID NO: 17, and CDR3 as shown in SEQ ID NO: 18.

[0010] In some embodiments, in the antibody or antigen-binding domain, the VH comprises the amino acid sequence shown in SEQ ID NO: 19 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 19, and the VL comprises the amino acid sequence shown in SEQ ID NO: 20 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 20.

[0011] In some specific embodiments, in the antibody or antigen-binding domain, the VH contains the amino acid sequence shown in SEQ ID NO: 19, and the VL contains the amino acid sequence shown in SEQ ID NO: 20.

[0012] In some specific embodiments, in the antibody or antigen-binding domain, the VH is an amino acid sequence as shown in SEQ ID NO: 19, and the VL is an amino acid sequence as shown in SEQ ID NO: 20.

[0013] In some specific embodiments, in the antibody or antigen-binding domain, the VH is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 19, and the VL is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 20.

[0014] In the antibody or antigen-binding domain of this application, VH and VL can be linked by a linker peptide. Specifically, the C-terminus of VH can be linked to the N-terminus of VL via a linker peptide, or the C-terminus of VL can be linked to the N-terminus of VH via a linker peptide.

[0015] In some specific embodiments, the VH in the antibody or antigen-binding domain is a sequence as shown in SEQ ID NO: 19, and the VL is a sequence as shown in SEQ ID NO: 20, wherein the C-terminus of the VH is connected to the N-terminus of the VL via a linker peptide, or the C-terminus of the VL is connected to the N-terminus of the VH via a linker peptide.

[0016] In some specific embodiments, the linker peptide comprises a sequence as shown in SEQ ID NO: 21 or a sequence as shown in (G4S)n, where n is an integer from 1 to 4.

[0017] In some specific embodiments, the antibody or antigen-binding domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 22-23 or 39-63, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0018] In some specific embodiments, the antibody or antigen-binding domain is selected from the amino acid sequence shown in any one of SEQ ID NOs: 22-23 or 39-63, or an amino acid sequence having at least 75% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0019] In some specific implementations, the antibody or antigen-binding domain is scFv, di-scFv, Fab, or F(ab').

[0020] In some specific implementations, the antibody or antigen-binding domain is a rabbit-derived, human-derived, chimeric, or humanized antibody.

[0021] In some specific embodiments, the humanized antibody, wherein the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 29-33 or comprises an amino acid sequence having at least 75% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it, and the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 34-38 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0022] In some specific embodiments, the humanized antibody, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 29 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it, and the VL comprises the amino acid sequence shown in SEQ ID NO: 34 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0023] In some specific embodiments, the humanized antibody contains the amino acid sequence shown in SEQ ID NO: 29 for the VH and the amino acid sequence shown in SEQ ID NO: 34 for the VL.

[0024] In some specific embodiments, in the humanized antibody, VH is the amino acid sequence shown in SEQ ID NO: 29, and VL is the amino acid sequence shown in SEQ ID NO: 34.

[0025] In some specific embodiments, in the humanized antibody, the VH is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 29, and the VL is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 34.

[0026] In some specific embodiments, the humanized antibody, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 30 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it, and the VL comprises the amino acid sequence shown in SEQ ID NO: 35 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0027] In some specific embodiments, the humanized antibody contains the VH amino acid sequence shown in SEQ ID NO: 30 and the VL amino acid sequence shown in SEQ ID NO: 35.

[0028] In some specific embodiments, in the humanized antibody, VH is the amino acid sequence shown in SEQ ID NO: 30, and VL is the amino acid sequence shown in SEQ ID NO: 35.

[0029] In some specific embodiments, in the humanized antibody, the VH is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 35, and the VL is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 35.

[0030] In some specific embodiments, the humanized antibody, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 31 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it, and the VL comprises the amino acid sequence shown in SEQ ID NO: 36 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0031] In some specific embodiments, the humanized antibody contains the amino acid sequence shown in SEQ ID NO: 31 for the VH and the amino acid sequence shown in SEQ ID NO: 36 for the VL.

[0032] In some specific embodiments, in the humanized antibody, VH is the amino acid sequence shown in SEQ ID NO: 31, and VL is the amino acid sequence shown in SEQ ID NO: 36.

[0033] In some specific embodiments, in the humanized antibody, the VH is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 31, and the VL is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 36.

[0034] In some specific embodiments, the humanized antibody, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 32 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it, and the VL comprises the amino acid sequence shown in SEQ ID NO: 37 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0035] In some specific embodiments, the humanized antibody contains the VH amino acid sequence shown in SEQ ID NO: 32 and the VL amino acid sequence shown in SEQ ID NO: 37.

[0036] In some specific embodiments, in the humanized antibody, VH is the amino acid sequence shown in SEQ ID NO: 32, and VL is the amino acid sequence shown in SEQ ID NO: 37.

[0037] In some specific embodiments, in the humanized antibody, the VH is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 37, and the VL is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 37.

[0038] In some specific embodiments, the humanized antibody, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 33 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it, and the VL comprises the amino acid sequence shown in SEQ ID NO: 38 or comprises an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0039] In some specific embodiments, the humanized antibody contains the amino acid sequence shown in SEQ ID NO: 33 for the VH and the amino acid sequence shown in SEQ ID NO: 38 for the VL.

[0040] In some specific embodiments, in the humanized antibody, VH is the amino acid sequence shown in SEQ ID NO: 33, and VL is the amino acid sequence shown in SEQ ID NO: 38.

[0041] In some specific embodiments, in the humanized antibody, the VH is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 33, and the VL is an amino acid sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 38.

[0042] In some specific embodiments, the humanized antibody comprises a humanized IgG Fc segment, preferably the humanized IgG Fc segment shown in SEQ ID NO: 69.

[0043] In some specific embodiments, the humanized antibody comprises an amino acid sequence selected from any one of SEQ ID NOs: 39-63 or an amino acid sequence having at least 75% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0044] In some specific embodiments, the humanized antibody is selected from the amino acid sequence shown in any one of SEQ ID NOs: 39-63 or contains an amino acid sequence having at least 75% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0045] On the other hand, this application provides a chimeric antigen receptor targeting the Claudin18.2 protein, which comprises an antibody or antigen-binding domain as described above or a humanized antibody as described above.

[0046] In some specific embodiments, the chimeric antigen receptor further includes a co-stimulatory domain and / or an intracellular signaling domain; preferably, the co-stimulatory domain is selected from CD28, 4-1BB, or a combination thereof, and / or the intracellular signaling domain is selected from the CD3ζ intracellular signaling domain.

[0047] In some specific embodiments, the co-stimulatory domain is selected from CD28, and preferably, the CD28 co-stimulatory domain comprises or consists of the sequence shown in SEQ ID NO: 25.

[0048] In some specific embodiments, the CD3ζ intracellular signaling domain comprises or consists of a sequence as shown in SEQ ID NO: 26.

[0049] In some specific embodiments, the chimeric antigen receptor further includes a hinge region and a transmembrane region, preferably the hinge region and transmembrane region are selected from the hinge region and transmembrane region of IgG1, IgG4, CD8α, and CD28.

[0050] In some specific embodiments, the hinge region and transmembrane region are selected from CD28, and preferably the CD28 hinge region and transmembrane region contain or consist of the amino acid sequence shown in SEQ ID NO: 24.

[0051] In some specific embodiments, the chimeric antigen receptor comprises an amino acid sequence selected from any one of SEQ ID NO: 27-28 or 64-68, or an amino acid sequence having at least 75% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

[0052] On the other hand, this application provides isolated nucleic acid molecules that encode antibody or antigen-binding domains as described above, or chimeric antigen receptors as described above.

[0053] On the other hand, this application provides a vector comprising isolated nucleic acid molecules as described above; preferably, the vector is selected from one or more of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, and retroviral vectors.

[0054] On the other hand, this application provides cells that contain isolated nucleic acid molecules or vectors as described above.

[0055] In some specific implementations, the cells are T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, monocytes, or macrophages.

[0056] On the other hand, this application provides a pharmaceutical composition comprising one or more of the following:

[0057] i) The isolated antibody or antigen-binding domain as described above, or the humanized antibody as described above;

[0058] ii) Chimeric antigen receptors as described above;

[0059] iii) Nucleic acid molecules isolated as described above;

[0060] iv) The carrier as described above; and

[0061] v) Cells as described above;

[0062] In addition, pharmaceutically acceptable carriers, diluents, or excipients.

[0063] On the other hand, this application provides a method for preparing cells as described above, comprising: introducing a nucleic acid of a chimeric antigen receptor as described above into the cells.

[0064] On the other hand, this application provides the use of the isolated antibody or antigen-binding domain, humanized antibody, chimeric antigen receptor, nucleic acid molecule, carrier, cell or pharmaceutical composition as described above in the preparation of a medicament for treating a disease, preferably the disease being selected from tumors, autoimmune toxins or infectious diseases caused by bacteria.

[0065] On the other hand, this application provides a method for performing cell immunotherapy on a subject suffering from a disease, comprising administering to the subject a pharmaceutical composition or cells as described above, preferably the disease being selected from tumors, autoimmune toxins, or infectious diseases caused by bacteria.

[0066] In some specific implementations, the tumor is selected from at least one of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer, rectal cancer, liver cancer, hepatobiliary cancer, head and neck cancer, ovarian cancer, and gallbladder cancer. Attached Figure Description

[0067] Figure 1 shows the binding ability of the antibody to 293T cells, 293T cells overexpressing Claudin18.2 (293T-CLDN18.2) and Claudin18.1 (293T-CLDN18.1).

[0068] Figure 2 shows the SDS-PAGE gel electrophoresis analysis of the antibodies. The left image shows the reduced antibody, the middle image shows the non-reduced antibody, and the right image shows the antibody names for each lane.

[0069] Figure 3 shows the binding ability of different Claudin18.2 scFv-Fc fusion antibodies to cells stably overexpressing Claudin18.2 or Claudin18.1 after co-incubation.

[0070] Figure 4 shows the proportion of CAR-positive expression in each group of Claudin18.2 CAR-T cells as detected by flow cytometry.

[0071] Figure 5 illustrates the cytotoxic effects of CAR-T cells containing different Claudin18.2 antigen recognition domains on tumor cells.

[0072] Figure 6 shows the affinity of the humanized antibody 11G9VLVH in 293T-CLDN18.2 and HGC-27-CLDN18.2 cells, using zotocimab (Astellas Pharma, IMAB362) as the standard.

[0073] Figure 7 shows the proportion of CAR-positive expression in each group of Claudin18.2 CAR-T cells as detected by flow cytometry.

[0074] Figure 8 illustrates the cytotoxic effects of CAR-T cells containing different Claudin18.2 antigen recognition domains on target cells.

[0075] Figure 9 shows the remaining number of CAR-T cells and tumor cells after multiple killings of target cells by CAR-T cells containing different Claudin18.2 antigen recognition domains.

[0076] Figure 10 illustrates the tumor suppression and CAR-T amplification capabilities of CAR-T cells containing different Claudin18.2 antigen recognition domains in a mouse model of gastric cancer.

[0077] Figure 11 illustrates the tumor suppression and CAR-T amplification capabilities of CAR-T cells containing different Claudin18.2 antigen recognition domains in a mouse model of pancreatic cancer. Detailed Implementation

[0078] The following description of this application is merely intended to illustrate various embodiments of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0079] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0080] As used in this disclosure, the term "Claudin18.2" is intended to include any form of Claudin18.2, such as 1) a naturally occurring, untreated Claudin18.2 molecule, a "full-length" Claudin18.2 chain, or a naturally occurring Claudin18.2 variant, including, for example, a splice variant or allelic variant; 2) any form of Claudin18.2 produced after intracellular treatment; or 3) a full-length, fragmented (e.g., truncated, extracellular / transmembrane domain), or modified (e.g., mutant, glycosylated / pegylated, His-tag / immunofluorescence fusion) Claudin18.2 subunit produced by recombinant methods. An exemplary "Claudin18.2" in this disclosure refers to a type III transmembrane protein (NCBI Reference Sequence: NP_001183.2) consisting of 184 amino acid residues. As used in this disclosure, the term "targeting Claudin18.2" refers to a substance that specifically binds to Claudin18.2 (such as human Claudin18.2). For example, a chimeric antigen receptor targeting Claudin18.2 refers to a chimeric antigen receptor that specifically binds to Claudin18.2; and an scFv targeting Claudin18.2 refers to a single-chain antibody that specifically binds to Claudin18.2 (such as human Claudin18.2).

[0081] The CDRs of the antibody and antigen-binding domains disclosed in this disclosure are defined or identified by IMGT numbers.

[0082] As used in this disclosure, the term "humanized" refers to antibodies or antibody peptides, including CDRs derived from non-human animals, FWR regions derived from humans, and constant regions derived from humans where applicable.

[0083] The numerical limits or ranges described in this article include endpoints, specifically all values ​​and subranges within the numerical limits or ranges.

[0084] Example

[0085] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0086] Example 1: Generation of rabbit antibody against Claudin18.2

[0087] To construct stable cell lines expressing Claudin 18.2 and Claudin 18.1 for rabbit immunization and antibody selection, the following methods were employed: Claudin 18.2 and Claudin 18.1 genes were synthesized (Beijing Bomaide Gene Technology Co., Ltd.). The Claudin 18.1 and Claudin 18.2 genes were inserted into the lentiviral vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) using PCR and enzyme digestion ligation methods, thus constructing plasmids containing the Claudin 18.2 and Claudin 18.1 genes. These plasmids, along with the lentiviral packaging plasmids pLP / VSVG (Thermo Fisher, Waltham, MA, USA) and pLP1 / MDK (Thermo Fisher, Waltham, MA, USA), were transfected into HEK293T cells to prepare lentiviruses carrying the Claudin 18.2 or Claudin 18.1 genes. Cells were digested with trypsin (Gibco, CAT: 25300062), centrifuged at 400g for 5 minutes, and the supernatant was discarded. An appropriate amount of complete culture medium (DMEM medium supplemented with 10% fetal bovine serum; DMEM medium, Gibco, CAT: 10566016; fetal bovine serum, Gibco, CAT: A5256701) was taken, resuspended, and cell counts were performed at a rate of 3 × 10⁶ cells / year. 5Add cells to a T25 cell culture flask at a density appropriate to the specified concentration, mix gently, and incubate at 37°C with 5% CO2 for 24 hours. When cell confluence reaches approximately 70-80%, add an appropriate amount of lentivirus carrying Claudin18.2 or Claudin18.1 evenly to the cell supernatant and mix gently. Once the cells have grown to a sufficient quantity, use flow cytometry to sort out Claudin18.2 or Claudin18.1 positive cells. Based on the above methods, mouse fibroblast cell line L929 (Pronosai, CAT: CL-0137) cells (L929-Claudin18.2) that stably expresses Claudin18.2 were constructed, as were human gastric cancer cell line HGC-27 (National Experimental Cell Resource Sharing Service Platform) cells (HGC-27-CLDN18.2) cells overexpressing Claudin18.2, human pancreatic cancer cell line Panc-1 (National Experimental Cell Resource Sharing Service Platform) cells (Panc-1-CLDN18.2) cells overexpressing Claudin18.2, 293T (National Experimental Cell Resource Sharing Service Platform) cells overexpressing Claudin18.2 (293T-CLDN18.2) cells, and 293T cells overexpressing Claudin18.1 (293T-CLDN18.1).

[0088] Rabbits were immunized with L929-Claudin18.2 cells, with each rabbit receiving 1 × 10⁻⁶ cells per immunization. 8 Each cell was immunized every two weeks for a total of six immunizations. Rabbit serum was collected for ELISA testing, and the titer was greater than 200,000. Following standard biological protocols, the rabbits were euthanized and the spleens were harvested to prepare a cell suspension. The spleen cells were cultured in B cell culture medium (Shangen Biotechnology, CAT: SNPM-H284).

[0089] Six-well plates were coated with tagged human Claudin18.2 protein (Claudin18.2-His, Twin-Strep Tag, Acro BIOSYSTEMS, CL2-H5587) or human full-length Claudin18.2 protein-VLP (Claudin18.2-VLP, Acro BIOSYSTEMS, CL2-H52P7) and incubated overnight at 4°C. The cultured splenocytes were then incubated with blocking peptides WG-01734M-1 (SEQ ID NO: 70) and WG-01734M-2 (SEQ ID NO: 71) at room temperature for 15 minutes to block the Claudin18.1 binding site. After incubation, splenocytes were incubated in 6-well plates pre-coated with human Claudin18.2-His, Twin-Strep Tag, or human Claudin18.2-VLP at 37°C and 5% CO2 for 60 minutes. After washing, the splenocytes on the surface of the plates were digested with trypsin and collected. Single B cells were then sorted by flow cytometry and collected into 96-well plates (1 cell / well) for culture. After 12 days of culture, the B cell supernatant was collected. HGC-27-Claudin18.2 cells and HGC-27 cells not expressing Claudin18.2 were added to 96-well plates at a ratio of 5E5 / well, 100 μl / well, and centrifuged at 400g for 3 minutes, discarding the supernatant. A 10-fold diluted B cell supernatant was added to 96-well plates at 100 μl / well and mixed with the cells, then incubated at room temperature for 20 minutes. The 96-well plates were centrifuged at 400g for 5 minutes, discarding the supernatant. The fluorescent secondary antibody Goat anti-Rabbit IgG-APC (Jackson ImmunoResearch, CAT: 111-606-046) was diluted 1:500 with 1xPBS + 0.5% BSA. 100 μl of the diluted antibody was added to each well of the corresponding sample and mixed well. The cells were incubated at room temperature for 20 minutes. The 96-well plates were centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1xPBS + 0.5% BSA for analysis. Nine positive clones binding to Claudin18.2 were identified using the human Claudin18.2-His Twin-Strep Tag, and 39 positive clones binding to Claudin18.2 were identified using the human Claudin18.2-VLP.

[0090] RNA binding to Claudin18.2 in single B cells was extracted using an RNA extraction kit (ABclonal, CAT: RK30120), and reverse transcription was performed using a reverse transcription kit (ABclonal, CAT: RK21400). After cDNA synthesis, VH and VL genes were amplified using 2×Gloria Nova (ABclonal, CAT: RK20715). After amplification, electrophoresis was performed on a 1% agarose gel, and the target band size was approximately 500 bp. The VH / VL fragments were recovered using a gel recovery kit (Tiangen Biotech, CAT: DP204-3). The VH and VL fragments were then constructed into a linear expression module (LEM) plasmid using 2×Vazyme LAmp Master Mix (Novazyme, CAT: P311-02). LEM vector was transfected into 293T cells. After 48 hours of culture, cell supernatants were collected, and flow cytometry was used to detect the binding ability of the antibodies in the supernatant to 293T-CLDN18.2 cells, 293T-CLDN18.1 cells, and 293T cells. The specific steps were the same as those for B cell supernatant detection described above. As shown in Figure 1, based on the criterion of specific binding to human 293T-CLDN18.2 cells but not to 293T-CLDN18.1 cells, a total of 5 clones were screened: 8D5, 11G9, 1H3, 27B3, and 17E3 (sequence information is shown in Table 1). Among them, 11G9 and 17E3 have the same sequence. The binding ability of 8D5 and 11G9 to 293T-CLDN18.2 cells was not lower than that of the control antibody zotuximab (Zolbetuximab, Astellas Pharma, IMAB362).

[0091] Example 2: Construction of Claudin18.2 scFv-Fc fusion antibody

[0092] Rabbit monoclonal antibody genes containing the heavy chain variable region (VH) and light chain variable region (VL) of 8D5, 11G9, 1H3, and 27B3 (ABclonal) were synthesized. A linker peptide (SEQ ID NO: 21) was introduced to form VLVHscFv and VHVL scFv, respectively. The synthesized scFv and the pcDNA3.4 vector containing the antibody Fc portion were double-digested using EcoRI (ABclonal, CAT: RK21102) and BamHI (ABclonal, CAT: RK21101). The scFv and vector were ligated using T4 ligase (ABclonal, CAT: RK21501) to obtain the scFv-Fc fusion antibody. 5 μL of the ligation mixture was added to 50 μL of thawed competent cells and incubated on ice for 30 minutes. The mixture was then heat-shocked at 42°C for 1 minute, followed by an ice incubation for 2 minutes. Add 400 μL of preheated LB medium (37°C) to the centrifuge tubes and incubate at 37°C and 220 rpm for 1 hour. After incubation, centrifuge at 1000g for 3 minutes, discard 300 μL of supernatant, and resuspend the bacterial pellet in the remaining LB medium. Spread the bacterial culture onto preheated ampicillin-resistant LB agar plates (Biofroxx, CAT: 1146GR005) at 37°C and incubate overnight at 37°C. Pick 2-3 single colonies from each cell and inoculate them into LB medium, incubating overnight at 37°C and 220 rpm. Dilute the total plasmid in sterile tubes with Opti-MEM medium (ThermoFisher, CAT: 31985070). Add the transfection reagent (pH 7.1) to the diluted total plasmid at a concentration of 1 mg / ml, mix immediately by vortexing, inverting, or pipetting, and incubate at room temperature for 15 minutes. Add the mixture to the treated cells, gently swirling to mix thoroughly. Collect the cell supernatant 72-96 hours after transfection.

[0093] Take 300 μL of Protein A Agarose suspension (Tiandi Renhe, CAT: SA023100) into a 1.5 mL EP tube, centrifuge and discard the supernatant, then wash twice with PBS. Add the washed Protein A Agarose suspension to the collected supernatant, vortex and incubate for 3-4 hours, centrifuge at 400g, carefully remove the supernatant, transfer the Protein A Agarose suspension to an empty column, and wash twice with PBS. Add elution buffer to the resin, centrifuge at 400g to collect the supernatant, and measure the antibody concentration using Nanodrop (Ausun, Nano-300). Dialyze using a dialysis apparatus for at least 5 hours or overnight, collect the dialyzed sample, and remeasure the concentration using Nanodrop. Qualitatively analyze the obtained antibody by SDS-PAGE gel electrophoresis. Prepare a 10% separating gel + 4% stacking gel for running reducing samples, and a 6% separating gel + 4% stacking gel for running non-reducing samples. Preparation of reduced samples: 5 μg protein sample + 5 μl 4× reduced protein loading buffer + PBS to a total volume of 20 μl, mix well, and heat at 95℃ for 10 minutes; Preparation of non-reduced samples: 5 μg protein sample + 5 μl 4× non-reduced protein loading buffer + PBS to a total volume of 20 μl, mix well, and heat at 37℃ for 10 minutes. After electrophoresis, staining, and destaining, the samples were placed in an imaging system for photography. A schematic diagram of the purification process is shown in Figure 2.

[0094] The specificity and affinity of the Claudin18.2 scFv-Fc fusion antibody were detected by flow cytometry. HGC-27, 293T, 293T-CLDN18.2, 293T-CLDN18.1, and HGC-27-CLDN18.2 cells were resuspended in PBS containing 0.5% BSA. Cells were added to 96-well plates at a concentration of 5E5 cells / well and centrifuged at 400g for 3 minutes, then the supernatant was discarded. The Claudin18.2 scFv-Fc fusion antibody was diluted to 1 μg / mL, 5 μg / mL, and 10 μg / mL. 100 μL of the diluted antibody was added to each well and mixed with the cells. The mixture was incubated at room temperature for 20 minutes, centrifuged at 400g for 5 minutes, and the supernatant was discarded. Alexa Fluor 647 AffiniPure F(ab')2Fragment Goat Anti-Human IgG(H+L)-APC (Jackson ImmunoResearch, CAT: 109-606-088) was diluted 1:500 with PBS containing 0.5% BSA. 100 μL of the diluted fluorescent secondary antibody was mixed with the corresponding sample and incubated at room temperature for 20 minutes. The cells were centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS containing 0.5% BSA before being analyzed and read.

[0095] The results are shown in Figure 3. 8D5 VLVH, 8D5 VHVL, 11G9 VLVH, and 11G9 VHVL showed significant signals against cell lines overexpressing Claudin18.2 (HGC-27-CLDN18.2, 293T-CLDN18.2), but no significant signals against cells not expressing Claudin18.2 (HGC-27, 293T) or cells overexpressing Claudin18.1 (293T-CLDN18.1). Among these, 11G9 VLVH and 11G9 VHVL showed higher signal intensity against the HGC-27-CLDN18.2 cell line than Zolbetuximab (Astellas Pharma, IMAB362).

[0096] Example 3: Construction of chimeric antigen receptor T cells based on rabbit-derived antibodies

[0097] 3.1 Construction of chimeric antigen receptor (CAR) plasmids

[0098] The sequences encoding scFvs targeting Claudin18.2 (including 1H3 VLVH, 1H3 VHVL, 27B3 VLVH, 27B3 VHVL, 8D5 VLVH, 8D5 VHVL, 11G9 VLVH, and 11G9 VHVL) were synthesized into the pcDNA3.1 vector (Beijing Bomaide Gene Technology Co., Ltd.). The sequences encoding the CD28 hinge region and transmembrane region were synthesized into the pcDNA3.1 vector using gene synthesis methods. The sequences encoding the CD28 intracellular region and CD3ζ signaling domain were also synthesized into the pcDNA3.1 vector using gene synthesis methods.

[0099] The lentiviral vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) was double-digested with XbaI and SalI (Thermo Fisher, Waltham, MA, USA), and the 6330bp vector fragment was recovered using agarose gel electrophoresis and a DNA gel recovery kit (Beijing TransGen Biotech).

[0100] The sequences encoding 1H3 VLVH scFv, the gene encoding the CD28 hinge region and transmembrane region, and the gene encoding the CD28 intracellular region and CD3ζ signaling domain were cloned from the pcDNA3.1 vector and then combined with the 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion to obtain the complete 1H3 VLVH CAR vector through homologous recombination using the 2×Seamless Cloning Mix kit (Bomaide, CL117-01).

[0101] The genes encoding 1H3 VHVL scFv, CD28 hinge region and transmembrane region, and CD28 intracellular region and CD3ζ signaling domain were cloned from the pcDNA3.1 vector and then combined with a 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion to obtain the complete 1H3 VHVL CAR vector through homologous recombination using a 2×Seamless Cloning Mix kit (Bomaide, CL117-01).

[0102] The genes encoding 27B3 VLVH scFv, CD28 hinge region and transmembrane region, and CD28 intracellular region and CD3ζ signaling domain were cloned from the pcDNA3.1 vector and then combined with the 6330bpp Lenti6.3 / V5 vector fragment recovered after enzyme digestion to obtain the complete 27B3 VLVH CAR vector through homologous recombination using the 2×Seamless Cloning Mix kit (Bomaide, CL117-01).

[0103] The genes encoding 27B3 VHVL scFv, CD28 hinge region and transmembrane region, and CD28 intracellular region and CD3ζ signaling domain were cloned from the pcDNA3.1 vector and then combined with a 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion to obtain a complete 27B3 VHVL CAR vector through homologous recombination using a 2×Seamless Cloning Mix kit (Bomaide, CL117-01).

[0104] The genes encoding 8D5 VLVH scFv, CD28 hinge region and transmembrane region, and CD28 intracellular region and CD3ζ signaling domain were cloned from the pcDNA3.1 vector and then combined with a 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion to obtain a complete 8D5 VLVH CAR vector through homologous recombination using a 2×Seamless Cloning Mix kit (Bomaide, CL117-01).

[0105] The genes encoding 8D5 VHVL scFv, CD28 hinge region and transmembrane region, and CD28 intracellular region and CD3ζ signaling domain were cloned from the pcDNA3.1 vector and then combined with a 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion to obtain a complete 8D5 VHVL CAR vector through homologous recombination using a 2×Seamless Cloning Mix kit (Bomaide, CL117-01).

[0106] The genes encoding 11G9 VLVH scFv, CD28 hinge region and transmembrane region, and CD28 intracellular region and CD3ζ signaling domain were cloned from the pcDNA3.1 vector and then combined with a 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion to obtain a complete 8D5 VLVH CAR vector through homologous recombination using a 2×Seamless Cloning Mix kit (Bomaide, CL117-01).

[0107] The genes encoding 11G9 VHVL scFv, CD28 hinge region and transmembrane region, and CD28 intracellular region and CD3ζ signaling domain were cloned from the pcDNA3.1 vector and then combined with the pLenti6.3 / V5 vector fragment recovered after enzyme digestion to obtain the complete 8D5 VHVL CAR vector through homologous recombination using the 2×Seamless Cloning Mix kit (Bomaide, CL117-01).

[0108] 3.2 Lentiviral Packaging

[0109] Lentiviral packaging plasmids pLP / VSVG, pLP1 / MDK, and pLP2 / RSK (Thermo Fisher, Waltham, MA, USA) were combined with the CAR plasmid obtained in the previous step and transfected into HEK293T cells using liposome Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA). After 48 hours, the culture medium was collected, and cell debris was removed by centrifugation at 300×g. The cells were then centrifuged at 25,000 rpm for 3 hours. The precipitate was dissolved in 1 mL of physiological saline to obtain the desired lentiviral vector.

[0110] 3.3 Lentiviral transfection of T lymphocytes

[0111] T cells were isolated from apheresis blood of healthy volunteers. The specific steps were as follows: CD3 / CD28 Dynabeads (Thermo Fisher, CAT: 40203D) were added at a ratio of 1.5:1, and the cells were incubated with gentle shaking for 30 minutes. After adsorption using a magnetic rack (Thermo Fisher, CAT: 12301D), T lymphocytes were obtained. Complete culture medium X-VIVO 15 + 500 IU / mL IL-2 (Shuanglu, National Drug Approval Number S19991010) was added to culture and expand the T cells. After 24 hours of culture, the aforementioned lentivirus was added at an MOI of 1 to infect the T cells. 24 hours after viral infection, the culture medium was replaced with fresh complete culture medium, and the cells were cultured again. On day 4 of culture, all cells were collected, and the Dynabeads were removed from the culture system using a magnetic rack. At different culture days, CAR-T cells were incubated with PE-labeled Whitlow / 218 Linker (E3U7Q) Rabbit mAb (Cell Signaling Technology) at room temperature for 20 minutes, and the proportion of CAR-positive cells in each group was detected by flow cytometry (ACEA Biosciences, NovoCyte 2060R).

[0112] As shown in Figure 4, the CAR positivity rates of 1H3VLVH, 8D5VLVH, 11G9VLVH, and 11G9VHVL increased with the number of days, while the CAR positivity rates of the other groups decreased with the number of days.

[0113] Example 4: In vitro evaluation of the cytotoxicity of rabbit-derived scFv-constructed CAR-T cells

[0114] 4.1 Real-time kill experiment

[0115] Add 50 μL of tumor cell culture medium to each well of the E-Plate 96-well plate that comes with the Agilent xCELLigence RTCA SP instrument. Place the plate on the RTCA Station and perform an instrument self-test. After the self-test is complete, remove the E-plate 96-well plate and add 100 μL of a solution containing 10% ... 4 A suspension of tumor cells was incubated at room temperature for 30 minutes, and then the E-plate 96 was placed on the RTCA Station in the incubator overnight to detect the cell proliferation curve.

[0116] The following day, CAR-T cells were collected, centrifuged at 400×g for 5 minutes, and resuspended in X-VIVO 15 medium without IL-2. CAR expression rate was detected by flow cytometry. CAR-T cell suspensions were prepared in triplicate for each group at an effector-to-target ratio of 1:20. The real-time cell killing instrument was paused, and the E-plate 96 was removed and placed in a biosafety cabinet. 50 μL of supernatant was pipetted from each well, and 100 μL of the mixed CAR-T cell suspension from each group was added to the corresponding well. The E-Plate 96 detection plate was placed on the real-time cell killing instrument to begin monitoring and observe the CAR-T cell-killing effect on tumor cells. A lower HGC-27-Claudin18.2-LE cell index indicates a stronger CAR-T cell-killing ability against tumor cells. The results are shown in Figure 5, where the 11G9VLVH CAR-T group showed superior tumor-killing ability compared to other groups.

[0117] Example 5: Preparation of humanized antibodies

[0118] The variable region of the 11G9VLVH antibody was submitted to an antibody database and compared with human antibody sequences to select several human antibody sequences with the highest homology. Furthermore, the frame region and CDR region (SEQ ID NOs: 13-18) within the variable region of the 11G9 antibody were predicted using a series of bioinformatics methods. The CDR region of the 11G9 antibody was grafted into the frame region of highly homologous human antibodies, resulting in 5 light chain mutants and 5 heavy chain mutants (SEQ ID NOs: 29-38). The humanized light and heavy chain antibodies were paired to obtain 25 humanized scFvs (SEQ ID NOs: 39-63). Following the description in Example 2, the humanized scFv-Fc fusion antibody was constructed, expressed, purified, and its affinity was verified.

[0119] The results are shown in Figure 6. Using zotuximab (Astellas Pharma, IMAB362) as the standard, the humanized antibodies with higher affinity than zotuximab in both cell lines were 11G9-L4H4, 11G9-L5H4, and 11G9-L5H5.

[0120] Example 6: Construction of CAR-T cells based on humanized antibody scFv

[0121] 6.1 Construction of chimeric antigen receptor (CAR) plasmids

[0122] Five humanized scFv strains—11G9-L1H5, 11G9-L4H4, 11G9-L5H5, 11G9-L5H4, and 11G9-L4H3—were selected for CAR gene construction. The specific steps are as follows:

[0123] The sequences encoding humanized scFvs (11G9-L1H5, 11G9-L4H4, 11G9-L5H5, 11G9-L5H4, 11G9-L4H3) targeting Claudin18.2 were synthesized into the pcDNA3.1 vector. The sequences encoding the CD28 hinge region and transmembrane region were synthesized into the pcDNA3.1 vector using gene synthesis methods. The sequences encoding the CD28 intracellular region and CD3ζ signaling domain were synthesized into the pcDNA3.1 vector using gene synthesis methods (Beijing Bomaide Gene Technology Co., Ltd.).

[0124] The lentiviral vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) was double-digested with XbaI and SalI (Thermo Fisher, Waltham, MA, USA), and the 6330bp vector fragment was recovered using agarose gel electrophoresis and a DNA gel recovery kit (Beijing TransGen Biotech).

[0125] The gene encoding 11G9-L1H5 scFv, the gene encoding the CD28 hinge region and transmembrane region, the gene encoding the CD28 intracellular region and CD3 signaling domain, and the 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion were combined with homologous recombination using a 2×Seamless Cloning Mix kit (Bomaide, CL117-01) to obtain the Hu-9212-CAR vector.

[0126] The Hu-9213-CAR vector was obtained by homologous recombination using a 2×Seamless Cloning Mix kit with the gene encoding 11G9-L4H4 scFv, the gene encoding the CD28 hinge region and transmembrane region, the gene encoding the CD28 intracellular region and CD3 signaling domain, and the 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion.

[0127] The Hu-9214-CAR vector was obtained by homologous recombination using a 2×Seamless Cloning Mix kit with the gene encoding 11G9-L5H5 scFv, the gene encoding the CD28 hinge region and transmembrane region, the gene encoding the CD28 intracellular region and CD3 signaling domain, and the 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion.

[0128] The Hu-9216-CAR vector was obtained by homologous recombination using a 2×Seamless Cloning Mix kit with the gene encoding 11G9-L5H4 scFv, the gene encoding the CD28 hinge region and transmembrane region, the gene encoding the CD28 intracellular region and CD3 signaling domain, and the 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion.

[0129] The Hu-9218-CAR vector was obtained by homologous recombination using a 2×Seamless Cloning Mix kit with the gene encoding 11G9-L4H3 scFv, the gene encoding the CD28 hinge region and transmembrane region, the gene encoding the CD28 intracellular region and CD3 signaling domain, and the 6330bp pLenti6.3 / V5 vector fragment recovered after enzyme digestion.

[0130] 6.2 Lentiviral Packaging

[0131] The lentivirus vector was obtained in accordance with the method described in Example 3.2.

[0132] 6.3 Lentiviral transfection of T lymphocytes

[0133] Lentiviral transfection of T lymphocytes was performed as described in Example 3.3. The CAR-positive rate in each group was detected by flow cytometry (ACEA Biosciences, NovoCyte 2060R) on days 6, 8, 11, and 14 of culture. The results are shown in Figure 7; the CAR-positive rate in all groups was higher than 20%.

[0134] Example 7: Evaluation of the antitumor effect of Claudin18.2-targeted CAR-T in a gastric cancer model

[0135] 7.1 Real-time kill experiment

[0136] Real-time killing experiments were conducted as described in Example 4.1. The results are shown in Figure 8; all groups of CAR-T cells exhibited killing activity against target cells. Among them, Hu-9218 showed the fastest killing rate.

[0137] 7.2 Target Cell Repetitive Stimulation Experiment

[0138] 300 μL containing 5 × 10 4Cell suspensions of HGC-27-CLDN18.2 tumor cells were added to 48-well plates and cultured overnight. The next day, CAR-T cells were collected, centrifuged at 400×g for 5 minutes, and resuspended in X-VIVO 15 medium without IL-2. CAR expression rate was detected by flow cytometry. CAR-T cell suspensions were prepared in quadruplicates per group at an effector-to-target ratio of 2:1. 500 μL of each group's CAR-T cell suspension was added to the corresponding well. The remaining tumor cells were observed. After complete tumor cell killing, the CAR-T cells in the wells were collected and added to a new round of tumor cells for repeated stimulation experiments. When at least one group had remaining tumor cells, the samples were collected for flow cytometry analysis. The fewer the remaining tumor cells and the more remaining CAR-T cells, the stronger the CAR-T's ability to kill target cells. As shown in Figure 9, the Hu-9218 group had the fewest remaining tumor cells and the most remaining CAR-T cells.

[0139] 7.3 In vivo efficacy experiments

[0140] CAR-T cells of Hu-9212, Hu-9213, Hu-9214, Hu-9216, and Hu-9218 were obtained in accordance with the method described in Example 6. On day 8, the cells were harvested, counted, and their CAR expression rate was analyzed by flow cytometry.

[0141] Beijing Bomeide Gene Technology Co., Ltd. was commissioned to synthesize the luciferase gene (GenBank: AFE85520.1) and the fluorescent protein GFP gene (GenBank: AAG49427.1). The luciferase and GFP genes were inserted into the lentiviral vector pLenti6.3 / V5 using PCR and enzyme digestion ligation methods to construct a plasmid containing the luciferase and GFP genes. HEK293T cells were transfected with this plasmid and the lentiviral packaging plasmids pLP / VSVG and pLP1 / MDK to prepare lentivirus carrying the luciferase and GFP genes. The lentivirus was used to infect HGC-27-claudin18.2 cells, and GFP-positive cells were sorted by flow cytometry and named HGC-27-claudin18.2-LAE. These cells simultaneously expressed GFP and luciferase.

[0142] 150 μL containing 3 × 10 6A suspension of HGC-27-claudin18.2-LAE tumor cells was subcutaneously injected into 5-6 week old NPG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.). Seven days later, the mice underwent luciferase in vivo imaging (Lumina II small animal in vivo imaging system, PerkinElmer, USA) and were grouped as follows: PBS group, T group, Hu-9212 group, Hu-9213 group, Hu-9214 group, Hu-9216 group, and Hu-9218 group. Each group of mice received the corresponding treatment via intravenous injection, with the CAR-T group receiving 5 × 10⁻⁶ cells / mL. 6 Each mouse in the CAR-T group received one CAR-T cell. T-group mice were injected with T cells containing the same total number of untransfected CAR molecules as the CAR-T group. In vivo imaging analysis was performed on mice at days 0, 8, 12, 16, and 21 after CAR-T cell and T cell injection. Peripheral blood CAR-T levels were measured on days 2, 5, 7, 9, 12, and 16 after CAR-T cell injection. The results are shown in Figure 10. The Hu-9218 group exhibited the best tumor suppression effect and a longer duration of tumor suppression, while the remaining number of CAR-T cells showed no significant advantage compared to other groups.

[0143] Example 8: Evaluation of the antitumor effect of Claudin18.2-targeted CAR-T in a pancreatic cancer model

[0144] Hu-9218 CAR-T cells were obtained as described in Example 7.3. On day 8, the cells were harvested, counted, and their CAR expression rate was analyzed by flow cytometry.

[0145] The pancreatic cancer cell line Panc-1-CLDN18.2-LAE, overexpressing GFP and luciferase, was obtained in accordance with the method described in Example 7.3.

[0146] The mouse tumor model and CAR-T cell reinfusion were performed as described in Example 7.3. In vivo imaging analysis was performed on mice on days 0, 4, and 9 after CAR-T cell and T cell injection, and peripheral blood CAR-T levels were detected on days 2, 7, 10, and 14 after CAR-T cell injection. The results, as shown in Figure 11, indicate that Hu-9218 CAR-T cells can be effectively expanded in mice and can effectively inhibit the growth of pancreatic cancer tumors.

[0147] Table 1. Sequence Information

Claims

1. An isolated antibody or antigen-binding domain targeting the Claudin18.2 protein, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein... The VH includes: The VL comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 13 or composed of the sequence, CDR2 containing the amino acid sequence shown in SEQ ID NO: 14 or composed of the sequence, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 15 or composed of the sequence, and the VL comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 16 or composed of the sequence, CDR2 containing the amino acid sequence shown in SEQ ID NO: 17 or composed of the sequence, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 18 or composed of the sequence.

2. The antibody or antigen-binding domain as described in claim 1, wherein... The VH is a sequence containing the amino acid sequence shown in SEQ ID NO: 19 or a sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it, and the VL is a sequence containing the amino acid sequence shown in SEQ ID NO: 20 or a sequence having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

3. The antibody or antigen-binding domain as described in claim 1 or 2, wherein the C-terminus of VH is connected to the N-terminus of VL via a linker peptide, or the C-terminus of VL is connected to the N-terminus of VH via a linker peptide. Preferably, the linker peptide is a sequence as shown in SEQ ID NO: 21 or a sequence as shown in (G4S)n, where n is any integer from 1 to 4.

4. The antibody or antigen-binding domain according to any one of claims 1-3, wherein the antibody or antigen-binding domain comprises an amino acid sequence selected from SEQ ID NOs: 22-23 or 39-63, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

5. The antibody or antigen-binding domain according to any one of claims 1-4, wherein the antibody or antigen-binding domain is scFv, di-scFv, Fab, or F(ab').

6. The antibody or antigen-binding domain according to any one of claims 1-5, wherein the antibody is a rabbit-derived, human-derived, chimeric, or humanized antibody.

7. The antibody or antigen-binding domain according to any one of claims 1-6, wherein in the humanized antibody, the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 29-33 or having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it, and the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 34-38 or having at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with it.

8. The antibody or antigen-binding domain of any one of claims 1-7, wherein the humanized antibody further comprises a humanized IgG Fc fragment.

9. The antibody or antigen-binding domain of any one of claims 1-8, wherein the humanized IgG Fc segment is shown in SEQ ID NO:

69.

10. The antibody or antigen-binding domain of any one of claims 1-9, wherein the humanized antibody comprises an amino acid sequence selected from any one of SEQ ID NOs: 39-63.

11. A chimeric antigen receptor targeting the isolated Claudin18.2 protein, comprising an antibody or antigen-binding domain as described in any one of claims 1-10.

12. The chimeric antigen receptor of claim 11, further comprising a co-stimulatory domain and an intracellular signaling domain, preferably wherein the co-stimulatory domain is selected from CD28, 4-1BB, or a combination thereof, and the intracellular signaling domain is selected from the CD3ζ intracellular signaling domain.

13. The chimeric antigen receptor as described in any one of claims 11-12, further comprising a hinge region and a transmembrane domain. Preferably, the hinge region and transmembrane domain are selected from the hinge regions and transmembrane domains of IgG1, IgG4, CD8α, and CD28.

14. The chimeric antigen receptor according to any one of claims 11-13, wherein the chimeric antigen receptor comprises a sequence selected from any one of SEQ ID NOs: 27-28 or 64-68 or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

15. An isolated nucleic acid molecule encoding an antibody or antigen-binding domain as described in any one of claims 1-10, or a chimeric antigen receptor as described in any one of claims 11-14.

16. A vector comprising the isolated nucleic acid molecule of claim 15, preferably, said vector being selected from one or more of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, and retroviral vectors.

17. A cell comprising the isolated nucleic acid molecule as claimed in claim 15 or the carrier as claimed in claim 16, preferably, the cell being a T lymphocyte, B lymphocyte, natural killer cell, dendritic cell, monocyte or macrophage.

18. A pharmaceutical composition comprising one or more of the following: i) the isolated antigen-binding domain as described in any one of claims 1-10; ii) The chimeric antigen receptor according to any one of claims 11-14; iii) The isolated nucleic acid molecule as described in claim 15; iv) The carrier as described in claim 16; and v) The cell as described in claim 17; In addition, pharmaceutically acceptable carriers, diluents, or excipients.

19. Use of the isolated antibody or antigen-binding domain of any one of claims 1-10, the chimeric antigen receptor of any one of claims 11-14, the nucleic acid molecule of claim 15, the carrier of claim 16, the cell of claim 17, or the pharmaceutical composition of claim 18 in the preparation of a medicament for treating a disease selected from tumors, autoimmune diseases, or infectious diseases caused by bacteria.

20. The use as described in claim 19, wherein the tumor is a gastrointestinal tumor, more preferably selected from at least one of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer, rectal cancer, liver cancer, hepatobiliary cancer, head and neck cancer, ovarian cancer, and gallbladder cancer.

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