Anti-claudin 18.2 nanobody and preparation method and application thereof

Abstract

The application provides an anti-CLDN18.2 nanobody and a preparation method and application thereof. Specifically, the application provides an anti-CLDN18.2 nanobody, an anti-CLDN18.2 antibody and a corresponding CLDN18.2 stable cell strain and the like, and a preparation method and application. The anti-CLDN18.2 nanobody of the application can reach a protein concealed epitope which cannot be accessed by a conventional antibody, and can reach a higher affinity compared with the prior art, is superior to a positive control Zolbetuximab, and has higher drug-making value and potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical or biopharmaceutical technology, and more specifically to an anti-CLDN18.2 nanobody, its preparation method, and its application. Background Technology

[0002] The Claudin (CLDN) family is a class of integrin membrane proteins found in tight junctions between epithelial and endothelial cells, with a molecular weight of approximately 23 kDa. CLDNs are among the most critical proteins in the formation of tight junctions, effectively maintaining the polarity of epithelial and endothelial cells and regulating the permeability of barrier structures. Claudin18 (CLDN18) is an important member of the CLDN protein family, capable of forming two different splicing mutants that encode two distinct tight junction proteins: CLDN18.1 and CLDN18.2. These two proteins exhibit different cellular lineages: in normal healthy tissues, CLDN18.1 is strictly expressed in lung epithelial cells, while CLDN18.2 is limited to epithelial cells differentiated in the stomach, such as mucous cells, parietal cells, and chief cells.

[0003] CLDN18.2 is a highly selective marker protein expressed only in differentiated gastric mucosal epithelial cells in normal healthy tissues. It is typically found at tight junctions of gastric mucosal cells, maintaining the barrier function of the gastric mucosa and preventing H+ leakage of gastric acid via the paracellular pathway. However, CLDN18.2 has been found to be aberrantly expressed in various cancers, including metastatic gastric cancer, pancreatic cancer, esophageal adenocarcinoma, and a small number of non-small cell lung cancers, and is involved in tumor cell proliferation, differentiation, and migration. Due to its specific expression pattern, CLDN18.2 has become a unique molecule for targeted therapy in various cancers and is a pan-cancer target suitable for the development of therapeutic antibodies.

[0004] Zolbetuximab (IMAB362, claudixmab) is a first-in-class human-mouse chimeric IgG1 monoclonal antibody (mAb) targeting Claudin18.2 (CLDN18.2), developed by Astellas Pharma. It is the world's first approved antibody targeting CLDN18.2 and the world's first IgG1 monoclonal antibody targeting CLDN18.2, used as first-line treatment for patients with CLDN18.2-positive, HER2-negative locally advanced unresectable or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma. It specifically binds to CLDN18.2 on the surface of tumor cells, subsequently inducing cancer cell death by activating two distinct immune system pathways—antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Preclinical studies have successfully demonstrated its powerful ability to eliminate cancer cells and control disease.

[0005] There are no reports of marketed drugs based on CLDN18.2 nanobodies, both domestically and internationally. Nanobodies can reach protein epitopes that are inaccessible to conventional antibodies and possess high affinity, resulting in better tumor penetration and faster tumor uptake. Furthermore, compared to traditional antibodies, nanobodies offer the following advantages: high stability and refolding ability under adverse conditions of pH, temperature, and even protein exposure; increased solubility and reduced aggregation due to the absence of light chain contacts and the presence of hydrophilic residues in FR2; low immunogenicity due to the high sequence similarity between the camel-derived VHH and human VH domains; ease of expression and preparation in microbial systems; and ease of antibody modification, allowing for the construction of various forms of high-affinity bispecific and trispecific antibodies.

[0006] Therefore, there is a need in this field to develop nanobodies against CLDN18.2. Summary of the Invention

[0007] The purpose of this invention is to provide an anti-CLDN18.2 nanobody, its preparation method, and its application.

[0008] Another objective of this invention is to provide antibodies based on anti-CLDN18.2 nanobodies, CLDN18.2 stable cell lines, etc.

[0009] Another object of the present invention is to provide methods for preventing and / or treating diseases with high CLDN18.2 expression, as well as diagnostic methods for diseases with high CLDN18.2 expression.

[0010] In a first aspect of the invention, an anti-CLDN18.2 nanobody is provided, the anti-CLDN18.2 nanobody having one or more complementarity-determining regions (CDRs) selected from the group consisting of:

[0011] (1) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:5 and CDR3 shown in SEQ ID NO:7;

[0012] (2) CDR1 shown in SEQ ID NO:11, CDR2 shown in SEQ ID NO:13 and CDR3 shown in SEQ ID NO:15;

[0013] (3) CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:21 and CDR3 shown in SEQ ID NO:23;

[0014] (4) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:28 and CDR3 shown in SEQ ID NO:30;

[0015] (5) CDR1 shown in SEQ ID NO:33, CDR2 shown in SEQ ID NO:35 and CDR3 shown in SEQ ID NO:37;

[0016] (6) CDR1 shown in SEQ ID NO:40, CDR2 shown in SEQ ID NO:42 and CDR3 shown in SEQ ID NO:43;

[0017] (7) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:5 and CDR3 shown in SEQ ID NO:47;

[0018] (8) CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:52 and CDR3 shown in SEQ ID NO:54;

[0019] (9) CDR1 shown in SEQ ID NO:57, CDR2 shown in SEQ ID NO:59 and CDR3 shown in SEQ ID NO:61;

[0020] (10) CDR1 shown in SEQ ID NO:64, CDR2 shown in SEQ ID NO:65 and CDR3 shown in SEQ ID NO:67;

[0021] (11) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:71 and CDR3 shown in SEQ ID NO:73;

[0022] (12) CDR1 shown in SEQ ID NO:76, CDR2 shown in SEQ ID NO:77 and CDR3 shown in SEQ ID NO:78;

[0023] (13) CDR1 shown in SEQ ID NO:81, CDR2 shown in SEQ ID NO:82 and CDR3 shown in SEQ ID NO:84;

[0024] (14) CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:87 and CDR3 shown in SEQ ID NO:89;

[0025] (15) CDR1 shown in SEQ ID NO:92, CDR2 shown in SEQ ID NO:94 and CDR3 shown in SEQ ID NO:96;

[0026] (16) CDR1 shown in SEQ ID NO:99, CDR2 shown in SEQ ID NO:101 and CDR3 shown in SEQ ID NO:102;

[0027] (17) CDR1 shown in SEQ ID NO:99, CDR2 shown in SEQ ID NO:101 and CDR3 shown in SEQ ID NO:104;

[0028] (18) CDR1 shown in SEQ ID NO:99, CDR2 shown in SEQ ID NO:107 and CDR3 shown in SEQ ID NO:109;

[0029] (19) CDR1 shown in SEQ ID NO:112, CDR2 shown in SEQ ID NO:114 and CDR3 shown in SEQ ID NO:116;

[0030] (20) CDR1 shown in SEQ ID NO:119, CDR2 shown in SEQ ID NO:121 and CDR3 shown in SEQ ID NO:122;

[0031] (21) CDR1 shown in SEQ ID NO:125, CDR2 shown in SEQ ID NO:127 and CDR3 shown in SEQ ID NO:129;

[0032] (22) CDR1 shown in SEQ ID NO:132, CDR2 shown in SEQ ID NO:133 and CDR3 shown in SEQ ID NO:135;

[0033] (23) CDR1 shown in SEQ ID NO:138, CDR2 shown in SEQ ID NO:101 and CDR3 shown in SEQ ID NO:141;

[0034] (24) CDR1 shown in SEQ ID NO:144, CDR2 shown in SEQ ID NO:146 and CDR3 shown in SEQ ID NO:148.

[0035] In another preferred embodiment, any of the above-mentioned amino acid sequences further includes a derived sequence which has optionally been added, deleted, modified and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and retains the ability to specifically bind to CLDN18.2.

[0036] In another preferred embodiment, the derived sequence, which has been added, deleted, modified, and / or substituted with at least one amino acid and is capable of retaining the ability to specifically bind to CLDN18.2, is an amino acid sequence with homology or sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0037] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the backbone regions FR1, FR2, FR3 and FR4 of the VHH chain, respectively.

[0038] In another preferred embodiment, the anti-CLDN18.2 nanobody has the structure shown in formula (I):

[0039] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 formula (I)

[0040] Each of the "-" symbols independently represents a linking peptide or a peptide bond.

[0041] In another preferred embodiment, intercyclic disulfide bonds exist between the CDR1 and CDR3 regions of the anti-CLDN18.2 nanobody.

[0042] In another preferred embodiment, the anti-CLDN18.2 nanobody further includes a backbone region FR.

[0043] In another preferred embodiment, the skeleton region FR is selected from one or more of the following groups:

[0044] (1) FR1 shown in SEQ ID NO:2, FR2 shown in SEQ ID NO:4, FR3 shown in SEQ ID NO:6 and FR4 shown in SEQ ID NO:8;

[0045] (2) FR1 shown in SEQ ID NO:10, FR2 shown in SEQ ID NO:12, FR3 shown in SEQ ID NO:14 and FR4 shown in SEQ ID NO:16;

[0046] (3) FR1 shown in SEQ ID NO:18, FR2 shown in SEQ ID NO:20, FR3 shown in SEQ ID NO:22 and FR4 shown in SEQ ID NO:8;

[0047] (4) FR1 shown in SEQ ID NO:25, FR2 shown in SEQ ID NO:27, FR3 shown in SEQ ID NO:29 and FR4 shown in SEQ ID NO:16;

[0048] (5) FR1 shown in SEQ ID NO:32, FR2 shown in SEQ ID NO:34, FR3 shown in SEQ ID NO:36 and FR4 shown in SEQ ID NO:16;

[0049] (6) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:41, FR3 shown in SEQ ID NO:6 and FR4 shown in SEQ ID NO:16;

[0050] (7) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:4, FR3 shown in SEQ ID NO:6 and FR4 shown in SEQ ID NO:16;

[0051] (8) FR1 shown in SEQ ID NO:46, FR2 shown in SEQ ID NO:4, FR3 shown in SEQ ID NO:6 and FR4 shown in SEQ ID NO:48;

[0052] (9) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:51, FR3 shown in SEQ ID NO:53 and FR4 shown in SEQ ID NO:16;

[0053] (10) FR1 shown in SEQ ID NO:56, FR2 shown in SEQ ID NO:58, FR3 shown in SEQ ID NO:60 and FR4 shown in SEQ ID NO:16;

[0054] (11) FR1 shown in SEQ ID NO:63, FR2 shown in SEQ ID NO:58, FR3 shown in SEQ ID NO:66 and FR4 shown in SEQ ID NO:16;

[0055] (12) FR1 shown in SEQ ID NO:69, FR2 shown in SEQ ID NO:58, FR3 shown in SEQ ID NO:72 and FR4 shown in SEQ ID NO:16;

[0056] (13) FR1 shown in SEQ ID NO:75, FR2 shown in SEQ ID NO:58, FR3 shown in SEQ ID NO:6 and FR4 shown in SEQ ID NO:16;

[0057] (14) FR1 shown in SEQ ID NO:80, FR2 shown in SEQ ID NO:58, FR3 shown in SEQ ID NO:83 and FR4 shown in SEQ ID NO:16;

[0058] (15) FR1 shown in SEQ ID NO:32, FR2 shown in SEQ ID NO:58, FR3 shown in SEQ ID NO:88 and FR4 shown in SEQ ID NO:16;

[0059] (16) FR1 shown in SEQ ID NO:91, FR2 shown in SEQ ID NO:93, FR3 shown in SEQ ID NO:95 and FR4 shown in SEQ ID NO:16;

[0060] (17) FR1 shown in SEQ ID NO:98, FR2 shown in SEQ ID NO:100, FR3 shown in SEQ ID NO:6 and FR4 shown in SEQ ID NO:8;

[0061] (18) FR1 shown in SEQ ID NO:106, FR2 shown in SEQ ID NO:58, FR3 shown in SEQ ID NO:108 and FR4 shown in SEQ ID NO:16;

[0062] (19) FR1 shown in SEQ ID NO:111, FR2 shown in SEQ ID NO:113, FR3 shown in SEQ ID NO:115 and FR4 shown in SEQ ID NO:117;

[0063] (20) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:120, FR3 shown in SEQ ID NO:6 and FR4 shown in SEQ ID NO:16;

[0064] (21) FR1 shown in SEQ ID NO:124, FR2 shown in SEQ ID NO:126, FR3 shown in SEQ ID NO:128 and FR4 shown in SEQ ID NO:16;

[0065] (22) FR1 shown in SEQ ID NO:131, FR2 shown in SEQ ID NO:58, FR3 shown in SEQ ID NO:134 and FR4 shown in SEQ ID NO:16;

[0066] (23) FR1 shown in SEQ ID NO:137, FR2 shown in SEQ ID NO:139, FR3 shown in SEQ ID NO:140 and FR4 shown in SEQ ID NO:8;

[0067] (24) FR1 shown in SEQ ID NO:143, FR2 shown in SEQ ID NO:145, FR3 shown in SEQ ID NO:147 and FR4 shown in SEQ ID NO:16.

[0068] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-CLDN18.2 nanobody is selected from one or more of SEQ ID NO: 1, 9, 17, 24, 31, 38, 44, 45, 49, 55, 62, 68, 74, 79, 85, 90, 97, 103, 105, 110, 118, 123, 130, 136, 142.

[0069] In another preferred embodiment, the anti-CLDN18.2 nanobody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0070] In a second aspect of the invention, an anti-CLDN18.2 antibody is provided, the antibody comprising one or more anti-CLDN18.2 nanobodies as described in the first aspect of the invention.

[0071] In another preferred embodiment, the antibody comprises one or more VHH chains of the anti-CLDN18.2 nanobody as described in the first aspect of the invention.

[0072] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-CLDN18.2 nanobody is selected from one or more of SEQ ID NO: 1, 9, 17, 24, 31, 38, 44, 45, 49, 55, 62, 68, 74, 79, 85, 90, 97, 103, 105, 110, 118, 123, 130, 136, 142.

[0073] In another preferred embodiment, the anti-CLDN18.2 antibody may be a monomer, a bivalent antibody, and / or a multivalent antibody.

[0074] In another preferred embodiment, the anti-CLDN18.2 antibody is a bivalent antibody.

[0075] In another preferred embodiment, the bivalent antibody further includes an antigen-binding domain that targets a second target.

[0076] In another preferred embodiment, the second target is selected from the group consisting of BCMA, CD16a, CD19, CD20, CD138, CD3, CD47, CD5, or a combination thereof.

[0077] In a third aspect of the invention, a humanized antibody is provided, which is obtained by humanization of the anti-CLDN18.2 nanobody described in the first aspect or the anti-CLDN18.2 antibody described in the second aspect.

[0078] In a fourth aspect of the invention, a chimeric antigen receptor (CAR) is provided, the CAR containing an extracellular domain comprising an anti-CLDN18.2 nanobody as described in the first aspect of the invention, an anti-CLDN18.2 antibody as described in the second aspect of the invention, or a humanized antibody as described in the third aspect of the invention.

[0079] In another preferred embodiment, the extracellular domain further includes a signal peptide.

[0080] In another preferred embodiment, the extracellular domain also includes other exogenous proteins.

[0081] In another preferred embodiment, the CAR has the structure shown in Formula Ia:

[0082] L-Nb-H-TM-C-CD3ζ (Ia)

[0083] In the formula,

[0084] L represents the absence of a signal peptide sequence;

[0085] Nb is a specific binding domain;

[0086] H represents the area with no hinge or no connection.

[0087] TM represents a transmembrane domain;

[0088] C represents the co-stimulation signal structure domain;

[0089] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers);

[0090] The "-" indicates a linking peptide or peptide bond.

[0091] In another preferred embodiment, the L is selected from the signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or combinations thereof.

[0092] In another preferred embodiment, the Nb targets CLDN18.2.

[0093] In another preferred embodiment, the Nb is an anti-CLDN18.2 nanobody.

[0094] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0095] In another preferred embodiment, H is the human IgG1 Fc hinge region.

[0096] In another preferred embodiment, the TM is selected from the transmembrane regions of the following histones: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants / modified forms thereof, or combinations thereof.

[0097] In another preferred embodiment, C is selected from the co-stimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0098] In another preferred embodiment, the CAR includes the intracellular domain of a cytokine.

[0099] In another preferred embodiment, the cytokines include: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), growth factors, chemokines, or combinations thereof; preferably, the cytokines are interleukins.

[0100] In another preferred embodiment, the CAR includes the intracellular domain of interleukin.

[0101] In another preferred embodiment, the interleukin is selected from the group consisting of IL-12, IL-2, IL-15, IL-21, or combinations thereof; preferably IL-12.

[0102] In a fifth aspect of the invention, a fusion protein is provided, the fusion protein having:

[0103] (Z1) First protein, the first protein comprising: anti-CLDN18.2 nanobody as described in the first aspect of the present invention, anti-CLDN18.2 antibody as described in the second aspect of the present invention, humanized antibody or its active fragment as described in the third aspect of the present invention;

[0104] (Z2) Second protein, which includes cytokines; and

[0105] (Z3) An optional linker located between the first protein and the second protein.

[0106] In another preferred embodiment, the cytokines include: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), growth factors, chemokines, or combinations thereof.

[0107] In another preferred embodiment, the second protein is interleukin.

[0108] In another preferred embodiment, the first protein is an anti-CLDN18.2 nanobody as described in the first aspect of the present invention, and the second protein is an interleukin.

[0109] In another preferred embodiment, the interleukin is selected from the group consisting of IL-12, IL-2, IL-15, IL-21, or combinations thereof.

[0110] In another preferred embodiment, the interleukin is IL-12.

[0111] In a sixth aspect of the invention, a recombinant protein is provided, the recombinant protein having:

[0112] (i) the anti-CLDN18.2 nanobody as described in the first aspect of the present invention, the anti-CLDN18.2 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, or an active fragment thereof; and

[0113] (ii) Optional tag sequences to assist in expression and / or purification.

[0114] In another preferred embodiment, the tag includes an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

[0115] In another preferred embodiment, the recombinant protein specifically binds to CLDN18.2.

[0116] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0117] In another preferred embodiment, the label is an Fc label.

[0118] In a seventh aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a protein selected from the group consisting of: an anti-CLDN18.2 nanobody as described in the first aspect of the invention, an anti-CLDN18.2 antibody as described in the second aspect of the invention, a humanized antibody as described in the third aspect of the invention, a chimeric antigen receptor as described in the fourth aspect of the invention, a fusion protein as described in the fifth aspect of the invention, a recombinant protein as described in the sixth aspect of the invention, or a combination thereof.

[0119] In another preferred embodiment, the polynucleotide is RNA, DNA, or cDNA.

[0120] In an eighth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the seventh aspect of the invention.

[0121] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector includes viral vectors, such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0122] In another preferred embodiment, the expression vector is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0123] In another preferred embodiment, the expression vector is pcDNA3.1 vector, pMES4 vector, or pABG1 vector (including pABG1-Fc vector).

[0124] In another preferred embodiment, the expression vector further includes a selection from the group consisting of: promoters, transcriptional enhancement elements (WPREs), long terminal repeat sequences (LTRs), etc.

[0125] In a ninth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the eighth aspect of the invention, or having a genome containing polynucleotides as described in the seventh aspect of the invention.

[0126] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0127] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0128] In a tenth aspect of the present invention, an engineered immune cell is provided, the engineered immune cell containing an expression vector as described in the eighth aspect of the present invention or having an exogenous polynucleotide as described in the seventh aspect of the present invention integrated into its genome, or expressing a chimeric antigen receptor as described in the fourth aspect of the present invention.

[0129] In another preferred embodiment, the engineered immune cells are selected from the group consisting of:

[0130] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0131] (ii) Chimeric antigen receptor γδ T cells (CAR-T cells);

[0132] (iii) Chimeric antigen receptor NKT cells (CAR-NKT cells);

[0133] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0134] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or combinations thereof.

[0135] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0136] In an eleventh aspect of the present invention, a method for generating anti-CLDN18.2 nanobodies is provided, the method comprising the steps of:

[0137] (a) Under conditions suitable for the production of nanobodies, host cells as described in the ninth aspect of the present invention are cultured to obtain a culture containing anti-CLDN18.2 nanobodies;

[0138] (b) Isolate and / or recover the anti-CLDN18.2 nanobody from the culture; and

[0139] (c) Optionally, the anti-CLDN18.2 nanobody obtained in step (b) is purified and / or modified.

[0140] In a twelfth aspect of the present invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0141] (a) the anti-CLDN18.2 nanobody as described in the first aspect of the present invention, the anti-CLDN18.2 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, or the recombinant protein as described in the sixth aspect of the present invention; and

[0142] (b) The conjugate selected from the group consisting of: detectable markers, drugs, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0143] In another preferred embodiment, part (a) is an anti-CLDN18.2 nanobody as described in the first aspect of the present invention, an anti-CLDN18.2 antibody as described in the second aspect of the present invention, or a humanized antibody as described in the third aspect of the present invention.

[0144] In another preferred embodiment, the (a) portion is coupled to the coupling portion by a chemical bond or a connector.

[0145] In another preferred embodiment, the radionuclide includes:

[0146] (i) a diagnostic isotope selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and / or

[0147] (ii) Therapeutic isotopes selected from the group consisting of: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or combinations thereof.

[0148] In another preferred embodiment, the coupling portion is a drug or toxin.

[0149] In another preferred embodiment, the drug is a drug for targeted treatment of diseases with high CLDN18.2 expression.

[0150] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to, solid tumors, hematologic malignancies, immune diseases, or combinations thereof.

[0151] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and bile duct cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, or combinations thereof.

[0152] In another preferred embodiment, the drug is a cytotoxic drug.

[0153] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0154] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, docarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0155] In another preferred embodiment, the toxin is selected from the group consisting of: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, methemosiderin, pyrethroids, pyrethroid A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxychloroquine. Anthraxone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, jatropha toxin, croton toxin, chachomycin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0156] In another preferred embodiment, the coupling portion is a detectable marker.

[0157] In another preferred embodiment, the coupling portion is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), or any form of nanoparticles.

[0158] In another preferred embodiment, the immunoconjugate contains a multivalent (e.g., bivalent) VHH chain of the anti-CLDN18.2 nanobody as described in the first aspect of the invention.

[0159] In another preferred embodiment, the polyvalent means that the amino acid sequence of the immunoconjugate contains a plurality of repeating identical or different VHH chains of the anti-CLDN18.2 nanobody as described in the first aspect of the invention.

[0160] In a thirteenth aspect of the present invention, there is provided a use of an active ingredient selected from the group consisting of: anti-CLDN18.2 nanobodies as described in the first aspect of the present invention, anti-CLDN18.2 antibodies as described in the second aspect of the present invention, humanized antibodies as described in the third aspect of the present invention, chimeric antigen receptors as described in the fourth aspect of the present invention, fusion proteins as described in the fifth aspect of the present invention, recombinant proteins as described in the sixth aspect of the present invention, host cells as described in the ninth aspect of the present invention, engineered immune cells as described in the tenth aspect of the present invention, immunoconjugates as described in the twelfth aspect of the present invention, or combinations thereof, wherein the active ingredient is used to prepare:

[0161] (a) Medications for the prevention and / or treatment of diseases with high CLDN18.2 expression;

[0162] (b) Reagents for detecting diseases with high CLDN18.2 expression.

[0163] In another preferred embodiment, the reagent is a diagnostic reagent, preferably a test strip or test plate.

[0164] In another preferred embodiment, the diagnostic reagent is used to detect CLDN18.2 protein or fragments thereof in a sample.

[0165] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to, solid tumors, hematologic malignancies, immune diseases, or combinations thereof.

[0166] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and bile duct cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, or combinations thereof.

[0167] In a fourteenth aspect of the invention, a method for in vitro detection of CLDN18.2 protein or fragments thereof in a sample is provided, the method comprising the steps of:

[0168] (1) In vitro, the sample is contacted with anti-CLDN18.2 nanobody as described in the first aspect of the present invention, anti-CLDN18.2 antibody as described in the second aspect of the present invention, humanized antibody as described in the third aspect of the present invention, chimeric antigen receptor as described in the fourth aspect of the present invention, fusion protein as described in the fifth aspect of the present invention, recombinant protein as described in the sixth aspect of the present invention, host cell as described in the ninth aspect of the present invention, engineered immune cell as described in the tenth aspect of the present invention, immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof;

[0169] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CLDN18.2 protein or fragments thereof in the sample.

[0170] In another preferred embodiment, the detection includes diagnostic or non-diagnostic methods.

[0171] In a fifteenth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0172] (i) the anti-CLDN18.2 nanobody as described in the first aspect of the present invention, the anti-CLDN18.2 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the host cell as described in the ninth aspect of the present invention, the engineered immune cell as described in the tenth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof as an active ingredient; and

[0173] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0174] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections and lyophilized preparations.

[0175] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the anti-CLDN18.2 nanobody as described in the first aspect of the present invention, the anti-CLDN18.2 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the host cell as described in the ninth aspect of the present invention, the engineered immune cell as described in the tenth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0176] In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1 × 10⁻⁶. 3 -1×10 8 cells / mL, preferably 1×10⁻⁶. 4 -1×10 7 Cells / mL.

[0177] A sixteenth aspect of the present invention provides a kit comprising:

[0178] (1) A first container containing, as described in the first aspect of the present invention, an anti-CLDN18.2 nanobody, as described in the second aspect of the present invention, a humanized antibody, as described in the third aspect of the present invention, a chimeric antigen receptor, as described in the fourth aspect of the present invention, a fusion protein, as described in the fifth aspect of the present invention, a recombinant protein, as described in the sixth aspect of the present invention, a host cell, an engineered immune cell, as described in the tenth aspect of the present invention, an immunoconjugate, or a combination thereof, as described in the twelfth aspect of the present invention; and / or

[0179] (2) A second container, wherein the second container contains a secondary antibody against the contents of the first container;

[0180] or,

[0181] The kit contains a detection plate, which includes a substrate (support plate) and a test strip. The test strip contains an anti-CLDN18.2 nanobody as described in the first aspect of the present invention, an anti-CLDN18.2 antibody as described in the second aspect of the present invention, a humanized antibody as described in the third aspect of the present invention, a fusion protein as described in the fifth aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention, an immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof.

[0182] In another preferred embodiment, the kit also includes an instruction manual, according to which the kit is used for non-invasive detection of CLDN18.2 expression in a test subject.

[0183] In another preferred embodiment, the kit is used for the detection of diseases with high CLDN18.2 expression.

[0184] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to, solid tumors, hematologic malignancies, immune diseases, or combinations thereof.

[0185] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and bile duct cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, or combinations thereof.

[0186] In a seventeenth aspect of the invention, a method for preventing and / or treating diseases with high CLDN18.2 expression is provided, the method comprising: administering to a desired subject an anti-CLDN18.2 nanobody as described in a first aspect of the invention, an anti-CLDN18.2 antibody as described in a second aspect of the invention, a humanized antibody as described in a third aspect of the invention, a chimeric antigen receptor as described in a fourth aspect of the invention, a fusion protein as described in a fifth aspect of the invention, a recombinant protein as described in a sixth aspect of the invention, a host cell as described in a ninth aspect of the invention, engineered immune cells as described in a tenth aspect of the invention, an immunoconjugate as described in a twelfth aspect of the invention, a pharmaceutical composition as described in a fifteenth aspect of the invention, or a combination thereof.

[0187] In another preferred embodiment, the object includes mammals, such as humans.

[0188] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to, solid tumors, hematologic malignancies, immune diseases, or combinations thereof.

[0189] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and bile duct cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer.

[0190] In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from the subject (autologous cells).

[0191] In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from a healthy individual (allogeneic cells).

[0192] In another preferred embodiment, the method may be used in combination with other treatment methods.

[0193] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy, etc.

[0194] In an eighteenth aspect of the present invention, a diagnostic method for diseases with high expression of anti-CLDN18.2 is provided, comprising the steps of:

[0195] (i) Obtaining a sample from a diagnostic subject and contacting the sample with an anti-CLDN18.2 nanobody as described in the first aspect of the present invention, an anti-CLDN18.2 antibody as described in the second aspect of the present invention, a humanized antibody as described in the third aspect of the present invention, a chimeric antigen receptor as described in the fourth aspect of the present invention, a fusion protein as described in the fifth aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention, a host cell as described in the ninth aspect of the present invention, an engineered immune cell as described in the tenth aspect of the present invention, an immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof; and

[0196] (ii) Detect whether an antigen-antibody complex is formed, wherein the formation of a complex indicates that the subject is a confirmed patient with a disease characterized by high expression of CLDN18.2.

[0197] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues or organs.

[0198] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to, solid tumors, hematologic malignancies, immune diseases, or combinations thereof.

[0199] In another preferred embodiment, the diseases in which CLDN18.2 is highly expressed include, but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and bile duct cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer.

[0200] In a nineteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, wherein the recombinant polypeptide is an anti-CLDN18.2 nanobody as described in the first aspect of the present invention, an anti-CLDN18.2 antibody as described in the second aspect of the present invention, a humanized antibody as described in the third aspect of the present invention, a chimeric antigen receptor as described in the fourth aspect of the present invention, a fusion protein as described in the fifth aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention, or a combination thereof, and the method comprises:

[0201] (a) Culture host cells as described in the ninth aspect of the present invention under suitable expression conditions; and

[0202] (b) Isolate the recombinant polypeptide from the culture.

[0203] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0204] Figure 1 This shows the binding level of positive antibodies to CHO-K1 stable cell lines expressing CLDN18.2.

[0205] Figure 2 The binding level of the HEK 293T stable cell line expressing CLDN18.2 to the positive antibody was shown.

[0206] Figure 3 The agarose gel assay results of PCR-amplified CALL fragments are shown, where lane M is the marker, lane 1 is the blank control, and lanes 2-12 are the CALL fragments.

[0207] Figure 4 The agarose gel verification results of PCR amplification of the VHH fragment are shown, where lane M is the marker, lane 1 is the blank control, and lanes 2-12 are the VHH fragments.

[0208] Figure 5 The results of sequencing verification of positive monoclonal samples are shown, where lane M is the marker, lane 1 is the blank control, and lanes 2-24 are the VHH bands after bacterial P.

[0209] Figure 6 The binding levels of each serially diluted VHH to CHO-CLDN18.2 cells were shown.

[0210] Figure 7 The binding levels of serially diluted VHH-Fc and the positive control Zolbetuximab to CHO-CLDN18.2 cells were shown.

[0211] Figure 8 The binding specificity of serially diluted VHH-Fc and the positive control Zolbetuximab to CLDN18.1 protein is shown.

[0212] Figure 9 The ADCC effect induced by the nanobody of the present invention is demonstrated. Compared with the positive control Zolbetuximab, the nanobody of the present invention induces a stronger ADCC effect. Detailed Implementation

[0213] Through extensive and in-depth research and numerous screenings, the inventors unexpectedly obtained, for the first time, an anti-CLDN18.2 nanobody with high affinity and high specificity. Specifically, this invention develops nanobody drugs targeting CLDN18.2. By preparing stable CLDN18.2 cell lines, immunizing alpacas, and utilizing phage display technology, nanobodies specifically binding to human CLDN18.2 were screened. Protein-level and cellular-level binding assays showed that the anti-CLDN18.2 nanobody of this invention has a good binding capacity to CLDN18.2, superior to the positive control Zolbetuximab, and possesses higher drug value and potential, providing a better treatment and diagnostic tool for diseases with high CLDN18.2 expression. This invention was completed based on these findings.

[0214] the term

[0215] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0216] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0217] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0218] The terms "nanobody", "single-domain antibody", "antibody of the present invention", and "nanobody of the present invention" are used interchangeably and all refer to the nanobody as described in the first aspect of the present invention that specifically recognizes and binds to the CLDN18.2 protein (including human CLDN18.2 protein).

[0219] The antibody numbers and corresponding sequence numbers of the nanobodies of this invention are shown in Table A below.

[0220] Table A

[0221]

[0222] Note: Each value in the table represents a sequence number, i.e., "1" represents "SEQ ID NO:1". The sequence numbers of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4 shown in the table are the sequence numbers of their amino acid sequences.

[0223] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0224] As used herein, the terms "single-domain antibody," "VHH," "nanobody," and "single-domain antibody (sdAb, or nanobody)" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain.

[0225] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called backbone regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally β-sheeted and linked by three CDRs forming a linking loop, and in some cases, partially β-sheeted structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0226] As those skilled in the art will recognize, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the nanobody or fragments thereof targeting CLDN18.2.

[0227] As used in this article, the terms “hypervariant region”, “highly variable region”, “complementarity determining region” and “complementarity determining region (CDR)” are used interchangeably.

[0228] In a preferred embodiment of the present invention, the nanobody or the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0229] In a preferred embodiment of the present invention, the nanobody or the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0230] In this invention, the terms "nanobody of the present invention," "antibody of the present invention," "protein of the present invention," or "peptide of the present invention" are used interchangeably and all refer to peptides that specifically bind to the CLDN18.2 protein, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0231] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0232] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four backbone regions (FR). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of similar antibodies can be compared to determine which amino acids constitute the FR or CDR regions.

[0233] The heavy chain variable regions of nanobodies or antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology to the CDRs identified herein.

[0234] As used herein, the term "humanized antibody" has the meaning conventionally understood by those skilled in the art, referring to antibodies obtained by humanizing alpaca-derived antibodies, thereby reducing the immunogenicity of murine antibodies. Based on the anti-CLDN18.2 nanobody of this invention, those skilled in the art will know how to humanize it to obtain a humanized antibody.

[0235] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0236] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0237] The antibody of this invention refers to a polypeptide having CLDN18.2 protein-binding activity and including the aforementioned CDR region. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibody of this invention.

[0238] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0239] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0240] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table B.

[0241] Table B

[0242]

[0243] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0244] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0245] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0246] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0247] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0248] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0249] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0250] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0251] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0252] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0253] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0254] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0255] The nanobodies or antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified portions, or any combination of the above substances.

[0256] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0257] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0258] CLDN18.2 related diseases

[0259] Based on the teachings of this invention, those skilled in the art will recognize that the nanobodies, their active fragments, or derivatives of this invention can be used to treat CLDN18.2-related diseases.

[0260] The CLDN18.2-related diseases are those with high CLDN18.2 expression. In a specific implementation, these CLDN18.2-related diseases are tumors that express CLDN18.2; including but not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and bile duct cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer.

[0261] Labeled antibodies

[0262] In a preferred embodiment of the invention, the nanobody or antibody may carry a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0263] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-CLDN18.2 nanobody or antibody is labeled with colloidal gold to obtain a colloidal gold-labeled antibody. The anti-CLDN18.2 nanobody or antibody of the present invention can effectively bind to the CLDN18.2 protein.

[0264] Detection methods

[0265] The present invention also relates to a method for detecting CLDN18.2 protein or fragments thereof. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of CLDN18.2 protein in the dissolved samples.

[0266] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0267] Reagent test kit

[0268] The present invention also provides a kit containing the anti-CLDN18.2 nanobody or antibody (or fragment thereof) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0269] This invention also provides a detection kit for detecting CLDN18.2 protein levels. The kit includes an antibody that recognizes CLDN18.2 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0270] Pharmaceutical Composition

[0271] The present invention also provides a pharmaceutical composition comprising, as an active ingredient, an anti-CLDN18.2 nanobody as described in the first aspect of the present invention, an anti-CLDN18.2 antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the third aspect of the present invention, a fusion protein as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, a host cell as described in the eighth aspect of the present invention, an engineered immune cell as described in the ninth aspect of the present invention, an immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof, and a pharmaceutically acceptable carrier.

[0272] These substances are typically formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH usually around 5-8, preferably around 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0273] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0274] When using the pharmaceutical composition, a safe and effective amount of the antibody (or its conjugate) described above is administered to mammals, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0275] application

[0276] As described above, the nanobodies and antibodies of the present invention have broad biological and clinical application value, and their applications involve multiple fields such as the diagnosis and treatment of diseases related to the CLDN18.2 protein, basic medical research, and biological research. A preferred application is for the clinical diagnosis, prevention, and treatment of the CLDN18.2 protein.

[0277] The present invention also provides a method for stimulating T-cell-mediated immune responses targeting mammalian tumor cell populations or tissues, comprising the following steps: administering the CAR-T cells of the present invention to a mammal.

[0278] In one embodiment, the present invention includes a type of cell therapy in which patient-associated (or allogeneic) T cells are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This approach results in an extremely low probability of graft-versus-host disease, and the antigen is recognized by the T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-lasting, durable antibodies that lead to sustained tumor control.

[0279] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and persist for months to years. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-T cells can induce a specific immune response against tumor cells that highly express antigens recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention elicit a specific immune response against tumor cells that highly express CLDN18.2.

[0280] Treatable cancers include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. Types of cancer treated with the CAR of this invention include, but are not limited to: breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small bowel cancer, large bowel cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, etc.

[0281] Typically, activated and expanded cells, as described herein, can be used to treat and prevent diseases such as tumors. Therefore, this invention provides a method for treating cancer comprising administering a therapeutically effective amount of the CAR-T cells of this invention to a subject in need of treatment.

[0282] The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may include target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0283] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the treatment (or prevention) of a disease. The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.

[0284] When referring to "immunologically effective dose," "antitumor effective dose," "tumor-inhibitory effective dose," or "therapeutic dose," the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. Pharmaceutical compositions including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 7 Administered at a dose of cells per kg body weight (inclusive of all integer values ​​within the range). The T-cell composition may also be administered multiple times at these doses. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a medical professional by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0285] The composition can be administered in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient via intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered via intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0286] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded immune cells of the present invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0287] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 5 One to 1×10 10 The modified T cells of this invention are administered to a patient, for example, via intravenous infusion.

[0288] Main advantages of the invention

[0289] 1. Existing CLDN18.2 recombinant antibodies have large molecular weights and are not easy to bind to cryptic epitopes, while the CLDN18.2 nanobody of the present invention can reach cryptic protein epitopes that conventional antibodies cannot access.

[0290] 2. Currently, there are no drug-grade nanobodies targeting CLDN18.2, but the CLDN18.2 nanobody of this invention can achieve higher affinity than existing technologies, which is superior to the positive control Zolbetuximab, and has higher drug value and potential.

[0291] 3. The CLDN18.2 nanobody of the present invention can also be linked to Fc to have an ADCC effect and be used to kill tumor cells.

[0292] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0293] sequence list

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300] Example 1: Construction of a stable CLDN18.2 cell line

[0301] The CLDN18.2 gene was derived from NCBI (ACCESSION: NP_001002026; VERSION: NP_001002026.1), synthesized as a full-length (1-261aa) gene, and a His tag was attached to the C-terminus. The gene fragment was ligated into the eukaryotic expression vector pCDNA3.1+. The target gene plasmid PGMLV-EF1A-18.2-his-PGK-Puro was constructed for lentiviral packaging, and the plasmid was extracted after construction.

[0302] A stable transfectant expressing CLDN18.2 was constructed using a lentiviral infection-drug screening method. The density of 293T cells was adjusted to 5 × 10^5 / mL, and 10 mL of HEK 293T cells were added to a 10 cm cell culture dish and incubated overnight at 37°C with 5% CO2. Cell status was observed the following day; transfection was indicated when cell confluence reached 90%. In the lentiviral three-plasmid transfection system, the mass ratio of the three plasmids was 4:3:1 (target gene plasmid: packaging plasmid psPAX2: envelope plasmid VSVG), according to Lipofectamine. TMFollowing the instructions for the 3000 transfection reagent, collect the supernatant 48 hours after transfection and replace it with complete culture medium. Store the supernatant at 4°C. After 72 hours, collect the supernatant again and mix it with the previous supernatant. Centrifuge to remove cell debris and impurities. Add reagent to the collected viral supernatant according to the Lenti-X Concentrator instructions, adjusting the volume accordingly, and concentrate overnight at 4°C. Determine the approximate viral titer using a lentivirus titer rapid detection card (specifically for virus packaging). Infect target cells (CHO-K1 cells or HEK 293T cells) with lentivirus at a predetermined MOI using polybrene and incubate at 37°C in a 5% CO2 incubator. Replace with fresh complete culture medium after 48 hours, and perform flow cytometry analysis after 96 hours.

[0303] Flow cytometry employs an indirect staining method, using the positive control antibody Zolbetuximab as the primary antibody and Alexa Fluor. The labeled Fc secondary antibody was analyzed by FACS. The results are as follows: Figure 1 , Figure 2 As shown, CHO-K1 cells and HEK 293T cells expressing CLDN18.2 bind well to the positive antibody and are significantly different from the negative control, indicating that the stable CHO-K1 and HEK 293T cell lines expressing CLDN18.2 have been successfully constructed.

[0304] Example 2: CLDN18.2 Cellular Immunization

[0305] Adult healthy alpacas were immunized with 1.0 × 10^7 CHO-CLDN18.2 cells established in Example 1 using Freund's adjuvant as an immunostimulant. Immunization was repeated every two weeks for a total of five immunizations. Peripheral blood from the jugular vein was collected seven days post-immunization to measure the titer.

[0306] The results showed that the three-free vaccine had the highest potency, with a potency of 1:24300.

[0307] Example 3: Lymphocyte Extraction

[0308] Using 50 mL peripheral blood lymphocyte separation tubes, add 10 mL of triple-immunized alpaca blood to each tube, centrifuge at 800 × g for 15 min. Carefully aspirate the milky white ring-shaped material with a pipette tip, add 10 mL of PBS, centrifuge at 250 × g for 10 min to wash, repeat the washing three times, resuspend in a small amount of PBS for counting, dilute the cell density to 5 × 10^6 cells / mL using RNAkeeper, and freeze at -80℃.

[0309] Example 4: Database Construction

[0310] 1. RNA extraction. RNA was extracted from lymphocytes using the Novizan RNA Extraction Kit (catalog number RC101-01). After RNA extraction, a portion was immediately subjected to electrophoresis, and the remainder was immediately subjected to reverse transcription.

[0311] 2. Reverse transcription into cDNA. Using the Hiscriptr III ISt strand cDNA synthesis Kit (Novozymes Reverse Transcription Kit, catalog number R312-02), the extracted RNA was reverse transcribed into cDNA.

[0312] 3. Using cDNA as a template, nested PCR was used to amplify the CALL fragment, and the band of approximately 700 bp was recovered by gel electrophoresis. The preparation system is as follows:

[0313] name Dosage cDNA 50μg 2×master Mix 425μL upstream primer 35μL Downstream primer 35μL water to 850μL

[0314] Mix well and dispense 50 μL / tube. The negative control group is prepared by omitting the cDNA template from the above system.

[0315] The PCR reaction procedure is as follows:

[0316]

[0317] After PCR, the gel was run on a 1.5% agarose gel for 40 minutes. The results are as follows. Figure 3 As shown, the CALL fragment exhibits a band of approximately 700 bp, indicating complete amplification, and is ready for subsequent gel extraction and recovery. Gel extraction was performed using the Tiangen Biotech Gel Extraction Kit.

[0318] 4. Amplify the VHH fragment using CALL as a template and recover the band of approximately 450 bp using gel electrophoresis:

[0319] Homologous recombination primers were used in this amplification to facilitate seamless cloning with the vector in the subsequent process.

[0320] The preparation system is as follows:

[0321] name Dosage 2×Mix 2mL upstream primer 100μL Downstream primer 100μL template 450ng water to 4mL

[0322] Mix well and dispense 50 μL / tube. The negative control group is prepared by omitting the cDNA template from the above system.

[0323] PCR procedure:

[0324]

[0325] After running the PCR, run the 2% agarose gel for 40 minutes. The results are as follows: Figure 4 As shown, the VHH fragment is approximately 450 bp, with a clear and uniform band, suitable for subsequent gel recovery. Gel recovery was performed using the Tiangen Biotech Gel Recovery Kit.

[0326] 5. The PMES4 vector was digested using two enzymes, PstI and BstEII, and then validated.

[0327] The enzyme digestion system is as follows:

[0328] name Dosage PMES4 10μg BstEII 4μL PstI 4μL 10×cutsmart 4μL water to 40μL

[0329] The above system was digested overnight at 37°C. After digestion, the plasmid was recovered by gel extraction. Solution I enzyme was used to ligate and transform the recovered plasmid. The plasmid was incubated overnight at 37°C. The next day, no bacteria were observed to grow on the ampicillin plate, indicating that the digested and recovered plasmid was complete and could be used for subsequent ligation experiments.

[0330] 6. Connect the power switch:

[0331] Using homologous recombinase, small amounts of the recovered VHH and PMES4 were ligated.

[0332] Connection system:

[0333] name Dosage carrier 900ng VHH 300ng Yisheng homologous recombinase 25μL water to 50μL

[0334] The above-mentioned linkage system was linked at 50°C for 30 min. The reaction products were directly converted or frozen at -20°C.

[0335] After successful validation of transformant strain P, a large number of strains were ligated and column-recovered.

[0336] After determining the concentration of the recovered product, preheat the SOC medium to 37°C. Wash the electroporation vessel with ultrapure water, air dry, and irradiate with UV for 30 min. Cool on ice for 5 min. Place competent cells on ice for 5 min, add the ligation product to the competent cells, and let stand on ice for 10 min. Add the mixture to the electroporation vessel, ensuring there are no air bubbles. Select the preset program for the electroporator, in the bacteria section, with settings of 2.5 kV, 25 μF, 200 Ω, competent cell volume of 50 μL, and an electroporation vessel size of 2 mm. Electroporate. Immediately after electroporation, add 1 mL of SOC medium and centrifuge in a 15 mL tube at 37°C for 2 h.

[0337] Take 500 μL of bacteria and add it to 500 μL of glycerol for cryopreservation. Take 100 μL of bacteria, dilute it to the power of 10^6, and then take 100 μL to plate. The number of single colonies on the next day is 116. Therefore, the library capacity for this construction is 1.16 × 10^9 cfu / mL.

[0338] Calculate storage capacity = dilution factor × number of colonies × 10.

[0339] Positive rate of bacterial P verification: 23 single clones were selected for bacterial P verification, and the results are as follows. Figure 5 As shown, the positivity rate of 23 monoclonal antibodies was 100%.

[0340] Twelve clones preceding the P criterion were randomly selected from the above positive monoclonal samples and sent for sequencing to test diversity, which was 100%. The library volume was 1.16 × 10^9 cfu / mL.

[0341] Take another 500 μL of the electroporated bacteria and add it to 10 mL of 2YT medium containing 2% glucose and 1 / 1000 ampicillin. Shake until OD 0.6-0.8. Add M13K07 at a ratio of 20:1 and incubate for 1 h. Centrifuge at 4000 rpm for 10 min to remove the supernatant. Resuspend in 10 mL of glucose-free 2YT medium and incubate at 37°C and 220 rpm for 1 h. Transfer to 100 mL of glucose-free 2YT medium and add ampicillin, kanamycin, and IPTG at ratios of 1:1000, 1:1000, and 1:2000, respectively. Shake at 37°C for 1 h and then transfer to 30°C for overnight incubation.

[0342] Example 5: Screening Container

[0343] 1. Precipitate bacteriophages:

[0344] Centrifuge the bacterial culture at 3000 rpm for 10 min, collect the supernatant, and add PEG / NaCl at a 4:1 ratio. Incubate on ice for 2 h. Then centrifuge the phage at 10000 rpm, 4℃ for 15 min, discard the supernatant, drain the water, resuspend in 2 mL of PBS, filter through a 0.45 μm filter, and measure the titer. The amplified phage titer was determined to be 2.5 × 10^12 pfu / mL.

[0345] 2. Select phage antibodies using cell panning:

[0346] Take 1×10^13 pfu phage and add 4% BSA-PBS solution to make a total volume of 2 mL. Prepare two aliquots and incubate at 37℃ for 30 min. Digest HEK 293T and HEK 293T-CLDN18.2 cells, centrifuge to remove the supernatant, and wash twice with PBS. Set aside. Resuspend 5×10^7 HEK 293T cells in the incubated phage solution and incubate at 4℃ with shaking for 1 h. Centrifuge to collect unbound phage from the supernatant. Discard the subtracted phage and resuspend 1×10^7 HEK 293T-CLDN18.2 cells and HEK 293T cells respectively, incubate at 4℃ with shaking for 1 h, and incubate statically for 30 min. Centrifuge to discard the supernatant, retaining the cells. Resuspend the cells in PBS and centrifuge to collect the cells. Wash 3 times in the first round, 5 times in the second round, and 8 times in the third round. Resuspend the cells in 1 mL of 0.1 M Gly-HCl solution (pH = 2.2), elute by shaking at room temperature for 5 min, centrifuge to remove the supernatant, and immediately add about 100 μL of 1 M Tris-HCl solution (pH = 9.1) to neutralize to about 7.0. Filter through a 0.45 μm filter membrane for later use.

[0347] Take 10 μL of eluted phage to infect 200 μL of fresh TG1 (using glucose 2YT medium), incubate at 37℃ for 1 h, and take 100 μL to make 10-fold, 100-fold, and 1000-fold dilutions to plate and measure the titer (the plates are ampicillin 2YT solid medium with glucose).

[0348] Add 500 μL of eluted phage to 4 mL of glucose 2YT medium and incubate for 1 h. Then add helper phage at a ratio of 20:1 and incubate for 1 h of superstaining. Centrifuge to remove supernatant and glucose, resuspend in 10 mL of glucose-free 2YT medium, add ampicillin at the specified ratio, shake at 37°C for 1 h, then add to 50 mL of glucose-free 2YT medium. Supplement with ampicillin and IPTG, and incubate overnight at 30°C.

[0349] The next day, the phages were precipitated again, and the above library screening operation was repeated. The specific conditions and results are shown in Table 1 below.

[0350] Table 1

[0351]

[0352]

[0353] 3. Monoclonal ELISA:

[0354] 526 monoclonal antibodies were picked from the titer plate and subjected to monoclonal ELISA.

[0355] Pick a single clone and incubate it in 200 μL of 2YT-amphetamine medium at 37°C and 200 rpm for 2-3 hours until the OD value is around 0.6.

[0356] Take 50 μL and add 50 μL of glycerol to preserve bacteria at 4°C.

[0357] Add 10^9 M13K07 cells per well (100 μL per well), incubate for 30 min, then add kanamycin IPTG (prepared beforehand, then add 200 μL per well: 90 μL kanamycin, 50 μL IPTG, 40 mL culture medium). Incubate at 37°C for 1 h, then at 30°C overnight. Add 10^5 293T-CLDN18.2 cells per well (100 μL per well) to three 96-well plates; also, plate three separate plates with 293T cells as a negative control.

[0358] The following day, discard the cell supernatant, add 100 μL of cold methanol to each well to fix the cells, incubate at 4°C for 15 min, discard the methanol, add 200 μL of PBS to each well and wash twice with shaking, 5 min each time. Block with 5% skim milk powder, 200 μL / well, at 37°C for 2 h. Simultaneously, centrifuge the picked single clones and collect the supernatant. After complete blocking, discard the blocking solution, and add 100 μL of the single clone supernatant to three 96-well plates, labeling them. Perform the same operation on the three BSA-coated plates. Simultaneously, provide negative controls of M13K07 and TG1 supernatants. After incubating at 37°C for 1 h, wash twice with PBST with shaking, 5 min each time. Add anti-M13 secondary antibody, incubate for 1 h, and wash three times with PBST with shaking, 5 min each time. Add 100 μL / well of TMB chromogenic solution, incubate at room temperature in the dark for 10 min, then add sulfuric acid to stop the reaction, and read the OD at 450 nm.

[0359] Single clones with an OD value greater than 1 and a P / N ratio greater than 2.1 were sent for sequencing.

[0360] Example 6: Expression and purification of nanobodies

[0361] After comparing the sequencing results and expressing and purifying the nanobodies, 25 nanobodies with different amino acid sequences were obtained: #20, #21, #22, #31, #49, #19, #170, #171, #173, #189, #191, #194, #195, #8, #57, #27, #60, #100, #128, #201, #215, #251, #257, #258, and #265. Their sequence information is shown in the sequence listing and Table A.

[0362] The obtained plasmid was transformed into BL21. After a single colony was correctly sequenced, expression was induced using 800 mL of IPTG. The next day, periplasmic proteins were extracted using TES, purified using a Ni column, and then run on a protein gel.

[0363] Example 7: Detection of Nanobody Cell-Level Binding Ability

[0364] CHO-CLDN18.2 cells in logarithmic growth phase were collected and centrifuged at 1300 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 1×PBS and washed twice. The cell density was adjusted to 3×10^6 / mL, and 100 μL was seeded per well. The primary antibody was diluted with FACS buffer, and the nanobody was diluted five-fold from a concentration of 30 μg / mL. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μL / well. The cells were incubated at 4°C for 60 min. The cells were washed twice with 200 μL / well of 1×PBS, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The anti-His-tagged fluorescent secondary antibody was diluted with FACS buffer, and the cells were resuspended in 100 μL / well. The cells were incubated at 4°C for 60 min. The cells were washed twice with 200 μL / well of 1×PBS, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 100 μL / well of PBS and analyzed.

[0365] The results are as follows Figure 6 As shown, most nanobodies can bind to cells expressing the CLDN18.2 protein. Experiments were then conducted using nanobodies #19, #20, #31, #49, #189, #194, #27, #100, #251, and #258.

[0366] Example 8: Functional Experiment

[0367] 1. Construct VHH-Fc:

[0368] Nanobodies were fused with IgG1-Fc, and the fusion protein could exert tumor-killing effects through antibody targeting. VHH-Fc was constructed separately. The constructed molecular plasmids were transfected into 293F cells, expressed and purified, and then subjected to cell binding activity experiments.

[0369] 2. Detection of VHH-Fc cell-level binding affinity

[0370] The primary antibody for flow cytometry was VHH-Fc, and the positive control antibody Zolbetuximab was diluted five-fold from a concentration of 30 μg / mL. The secondary antibody was diluted with FACS buffer to obtain the anti-Fc-tagged fluorescent secondary antibody. All other procedures were the same as in Example 7.

[0371] The results are as follows Figure 7 As shown in Table 2, the EC50 values ​​are as follows. It is shown that, except for 251-Fc, whose EC50 value is slightly higher than the positive control antibody Zolbetuximab, the EC50 values ​​of the other VHH-Fc cells are lower than the positive control antibody.

[0372] Table 2

[0373] #19 #20 #31 #49 #189 #194 EC50 (μg / mL) 0.4810 0.4064 0.4498 0.5057 0.3951 0.4194 #27 #100 #251 #258 Zmab EC50 (μg / mL) 0.4425 0.4395 0.9326 0.5869 0.8591

[0374] 3. VHH-Fc specific binding assay

[0375] The ELISA method was used to primarily detect whether each VHH-Fc cell bound to the CLDN18.1 protein.

[0376] like Figure 8 As shown, none of the VHH-Fc cells bind to the CLDN18.1 protein, indicating that the screened nanobodies have high specificity.

[0377] 4. ADCC Experiment:

[0378] Mix CHO-K1 and NK media at a 1:1 ratio. Use the mixed medium to serially dilute VHH-Fc five-fold, starting at 200 μg / mL, for a total of eight concentration gradients. Add 50 μL to region D of each well. Adjust the CHO-CLDN18.2 cell density to 4E5 cells / mL using the mixed medium, and add 25 μL to regions B, C, and D of each well. Adjust the CHO-CLDN18.2 cell density to 4E6 cells / mL using the mixed medium (effect-to-target ratio of 10:1), and add 25 μL to regions C and D of each well. Make up to 100 μL of mixed medium in all wells. Mix the cell culture plates by shaking and incubate at 37°C in a 5% CO2 incubator for 20 h.

[0379] The next day, 10 μL of CCK8 solution was added to each well, and the absorbance at 450 nm was measured using a microplate reader at 2 h. The readings were taken every 1 h.

[0380] Data is processed according to the lethality formula. Lethality (%) = (CD) / (BA)

[0381] A: 100 μL of mixed culture medium;

[0382] B: 25 μL of CHO-CLDN18.2 cells + 75 μL of mixed culture medium;

[0383] C: 25 μL of CHO-CLDN18.2 cells + 25 μL of NK cells + 50 μL of mixed culture medium;

[0384] D: 25 μL of CHO-CLDN18.2 cells + 25 μL of NK cells + 50 μL of VHH-Fc or positive control antibody diluted in mixed culture medium.

[0385] The results are as follows Figure 9 As shown, the killing effect of 19-Fc and 31-Fc of the present invention is better than that of the positive control Zolbetuximab, and can induce a stronger ADCC effect.

[0386] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-CLDN18.2 Nanobody, characterized in that, The anti-CLDN18.2 Nanobody has the following complementarity determining regions (CDRs): CDR1 as shown in SEQ ID NO: 40, CDR2 as shown in SEQ ID NO: 42, and CDR3 as shown in SEQ ID NO:

43.

2. A humanized Nanobody, which is obtained by humanizing the anti-CLDN18.2 Nanobody of claim 1.

3. A recombinant protein, characterized in that, The recombinant protein has: (i) the anti-CLDN18.2 Nanobody of claim 1, or the humanized Nanobody of claim 2; and (ii) an optional tag sequence to assist expression and / or purification.

4. A polynucleotide, comprising: The polynucleotide encodes a protein selected from the group consisting of the anti-CLDN18.2 Nanobody of claim 1, or the humanized Nanobody of claim 2, the recombinant protein of claim 3, or a combination thereof.

5. An expression vector, characterized by, The expression vector contains the polynucleotide of claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector of claim 5, or the polynucleotide of claim 4 is integrated into the genome of the host cell.

7. A method of producing an anti-CLDN18.2 Nanobody, characterized in that, The method comprises the steps of: (a) culturing the host cell of claim 6 under suitable conditions for producing a Nanobody, thereby obtaining a culture containing the anti-CLDN18.2 Nanobody; (b) isolating and / or recovering the anti-CLDN18.2 Nanobody from the culture.

8. The method of claim 7, wherein, The method further comprises the step of: (c) purifying the anti-CLDN18.2 Nanobody obtained in step (b). The recombinant protein has: (i) the anti-CLDN18.2 Nanobody of claim 1, or the humanized Nanobody of claim 2; and (ii) an optional tag sequence to assist expression and / or purification. The polynucleotide encodes a protein selected from the group consisting of the anti-CLDN18.2 Nanobody of claim 1, or the humanized Nanobody of claim 2, the recombinant protein of claim 3, or a combination thereof. The expression vector contains the polynucleotide of claim 4. The host cell contains the expression vector of claim 5, or the polynucleotide of claim 4 is integrated into the genome of the host cell. The method comprises the steps of: (a) culturing the host cell of claim 6 under suitable conditions for producing a Nanobody, thereby obtaining a culture containing the anti-CLDN18.2 Nanobody; (b) isolating and / or recovering the anti-CLDN18.2 Nanobody from the culture. The method further comprises the step of: (c) purifying the anti-CLDN18.2 Nanobody obtained in step (b).

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