Anti-CLDN18.2 nano antibody as well as preparation method and application thereof

Abstract

The invention provides an anti-CLDN18.2 nano antibody as well as a preparation method and application thereof. Specifically, the invention provides an anti-CLDN18.2 nano antibody, an anti-CLDN18.2 antibody, a corresponding CLDN18.2 stably transfected cell strain and the like, and a preparation method and application of the anti-CLDN18.2 nano antibody and the anti-CLDN18.2 antibody. The anti-CLDN18.2 nano antibody disclosed by the invention can reach a protein concealment epitope which cannot be approached by a conventional antibody, can reach higher affinity compared with the prior art, is superior to a positive control Zolbetuximab, and has higher patent medicine value and potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to an anti-CLDN18.2 nanobody and a preparation method and application thereof. Background Art

[0002] The Claudin (CLDN) family is a class of integrin membrane proteins present in the tight junctions of epithelial and endothelial cells, with a molecular weight of approximately 23kDa. CLDN is one of the most critical proteins constituting tight junctions, which can effectively maintain the polarity of epithelial and endothelial cells and regulate the permeability of barrier structures. Claudin18 (CLDN18) is an important member of the CLDN protein family. It can form two different splice mutants, encoding two different tight junction proteins, CLDN18.1 and CLDN18.2 respectively; and the two have different cell lineages. In normal healthy tissues, CLDN18.1 is strictly expressed in epithelial cells of lung tissue, and CLDN18.2 is limited to differentiated epithelial cells in the stomach, such as mucus cells, gastric parietal cells, and chief cells.

[0003] CLDN18.2 is a highly selective marker protein that is only expressed in differentiated gastric mucosal epithelial cells in normal healthy tissues. It is usually present in the tight junctions of gastric mucosal cells, maintaining the barrier function of the gastric mucosa and preventing H+ from leaking gastric acid through the paracellular pathway. However, CLDN18.2 has been found to be abnormally expressed in a variety of cancers, including diffuse gastric cancer, pancreatic cancer, esophageal adenocarcinoma, and a small number of non-small cell lung cancers, and is involved in the proliferation, differentiation, and migration of tumor cells. With its specific expression pattern, CLDN18.2 has become a unique molecule for targeted therapy of different cancers and 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. It is also the world's first approved antibody targeting CLDN18.2 and the world's first IgG1 monoclonal antibody against CLDN18.2. It is used as a first-line treatment for patients with CLDN18.2-positive, HER2-negative locally advanced unresectable or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma. It can specifically bind to CLDN18.2 on the surface of tumor cells and then induce cancer cell death by activating two different immune system pathways - antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Preclinical studies have successfully demonstrated its powerful ability to eliminate cancer cells and control the disease.

[0005] There is no report on the marketed drugs of CLDN18.2 nanobodies at home and abroad. Nanobodies can reach the protein hidden epitopes that conventional antibodies cannot access and have high affinity, and can have better tumor penetration and faster tumor uptake. In addition, compared with traditional antibodies, nanobodies also have the following advantages: showing high stability and refolding ability under adverse conditions such as pH, temperature, and even protein exposure; the absence of the light chain contact surface in FR2 and the presence of hydrophilic residues will increase solubility and make it less likely to aggregate; the high sequence similarity between camel-derived VHH and human VH domains results in low immunogenicity; it is easy to express and prepare in microbial systems, and antibody modification is easy, and it can be used to construct various forms of bispecific and trispecific antibodies with high affinity.

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

[0007] The object of the present invention is to provide an anti-CLDN18.2 nanobody and its preparation method and application.

[0008] Another object of the present 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 a method for preventing and / or treating CLDN18.2 overexpression diseases, as well as a diagnostic method for CLDN18.2 overexpression diseases.

[0010] In the first aspect of the present invention, an anti-CLDN18.2 nanobody is provided, and the anti-CLDN18.2 nanobody has one or more complementarity-determining regions CDR selected from the following groups:

[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 as shown in SEQ ID NO:99, CDR2 as shown in SEQ ID NO:101, and CDR3 as shown in SEQ ID NO:102;

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

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

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

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

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

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

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

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

[0035] In another preferred example, any one of the above amino acid sequences further includes a derivative sequence that optionally has at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid added, deleted, modified, and / or substituted and can retain the ability to specifically bind to CLDN18.2.

[0036] In another preferred example, the derivative sequence that has at least one amino acid added, deleted, modified and / or substituted and is capable of retaining the ability to specifically bind to CLDN18.2 is an amino acid sequence with a 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 example, the CDR1, CDR2 and CDR3 are separated in sequence by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain, respectively.

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

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

[0040] Wherein, each of the "-" is independently a linker peptide or a peptide bond.

[0041] In another preferred example, there is an inter-loop disulfide bond between the CDR1 region and the CDR3 region of the anti-CLDN18.2 nanobody.

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

[0043] In another preferred example, the framework region FR is one or more selected from 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 example, 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 example, the anti-CLDN18.2 nanobody includes a humanized antibody, a camel-derived antibody, and a chimeric antibody.

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

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

[0072] In another preferred example, 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 example, the anti-CLDN18.2 antibody can be a monomer, a bivalent antibody, and / or a multivalent antibody.

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

[0075] In another preferred example, the bivalent antibody further includes an antigen-binding domain targeting 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 present invention, there is provided a humanized antibody, which is obtained by humanizing the anti-CLDN18.2 nanobody described in the first aspect or the anti-CLDN18.2 antibody described in the second aspect of the present invention.

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

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

[0080] In another preferred embodiment, the extracellular domain further comprises other foreign 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 is absent or a signal peptide sequence;

[0085] Nb is a specific binding domain;

[0086] H is absent or a hinge region;

[0087] TM is a transmembrane domain;

[0088] C is a co-stimulatory signal domain;

[0089] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or its mutant / modified form);

[0090] The "-" is a linker peptide or a peptide bond.

[0091] In another preferred embodiment, the L is respectively selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, or their mutants / modified forms, or a combination 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 regions of the following proteins: CD8, CD28, CD137, IgG, or a combination thereof.

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

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

[0097] In another preferred embodiment, the C is selected from the co-stimulatory domains of the following proteins: 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 a mutant / modified form thereof, or a combination thereof.

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

[0099] In another preferred embodiment, the cytokine includes: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), growth factor, chemokine, or a combination thereof; preferably, the cytokine is interleukin.

[0100] In another preferred embodiment, the CAR comprises an 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 a combination thereof; preferably IL-12.

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

[0103] (Z1) A first protein, the first protein comprising: a nanobody against CLDN18.2 as described in the first aspect of the present invention, an antibody against CLDN18.2 as described in the second aspect of the present invention, a humanized antibody as described in the third aspect of the present invention, or an active fragment thereof;

[0104] (Z2) A second protein, the second protein comprising a cytokine; and

[0105] (Z3) Optionally, a linker located between the first protein and the second protein.

[0106] In another preferred embodiment, the cytokine comprises: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), growth factor, chemokine, or a combination thereof.

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

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

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

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

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

[0112] (i) A nanobody against CLDN18.2 as described in the first aspect of the present invention, an antibody against CLDN18.2 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, or an active fragment thereof; and

[0113] (ii) Optionally, a tag sequence for assisting expression and / or purification.

[0114] In another preferred embodiment, the tag comprises 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 multimer.

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

[0118] In the seventh aspect of the present invention, a polynucleotide is provided, which encodes a protein selected from the group consisting 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, or a combination thereof.

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

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

[0121] In another preferred example, the expression vector is selected from the group consisting of: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof. Preferably, the expression vector includes a viral vector, such as a lentivirus, an adenovirus, an AAV virus, a retrovirus, or a combination thereof.

[0122] In another preferred example, 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 example, the expression vector is a pcDNA3.1 vector, a pMES4 vector, a pABG1 vector (including the pABG1-Fc vector).

[0124] In another preferred example, the expression vector further includes a promoter, a transcriptional enhancer element WPRE, a long terminal repeat sequence LTR, etc., selected from the group consisting of.

[0125] In the ninth aspect of the present invention, a host cell is provided, which contains the expression vector as described in the eighth aspect of the present invention, or the polynucleotide as described in the seventh aspect of the present invention is integrated into its genome.

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

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

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

[0129] In another preferred example, 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 example, the engineered immune cells include autologous or allogeneic αβ T cells, γδ T cells, NKT cells, NK cells, or a combination thereof.

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

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

[0137] (a) Culturing the host cell as described in the ninth aspect of the present invention under conditions suitable for generating nanobodies, thereby obtaining a culture containing anti-CLDN18.2 nanobody;

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

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

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

[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) A conjugate moiety selected from the group consisting of: detectable markers, drugs, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral coat proteins or VLPs, or a combination thereof.

[0143] In another preferred example, the part (a) is a nanobody against CLDN18.2 as described in the first aspect of the present invention, an antibody against CLDN18.2 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 example, the part (a) is coupled to the coupling part through a chemical bond or a linker.

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

[0146] (i) Diagnostic isotopes 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 a combination 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 a combination thereof.

[0148] In another preferred example, the coupling part is a drug or a toxin.

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

[0150] In another preferred example, the diseases with high expression of CLDN18.2 include but are not limited to: solid tumors, hematological tumors, immune diseases, or a combination thereof.

[0151] In another preferred example, the diseases with high expression of CLDN18.2 include but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and biliary tract cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, or a combination thereof.

[0152] In another preferred example, 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 classes of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Representative cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, 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: auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), calicheamicin, maytansinol, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, anthramycin, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A-chain, momordin A-chain, alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, croton toxin, calicheamicin, saponaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0156] In another preferred embodiment, the conjugate moiety is a detectable label.

[0157] In another preferred embodiment, the coupling moiety 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, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), or any form of nanoparticles.

[0158] In another preferred embodiment, the immunoconjugate contains: multivalent (such as divalent) VHH chains of the anti-CLDN18.2 nanobody as described in the first aspect of the present invention.

[0159] In another preferred embodiment, the multivalent refers to containing multiple repeats of the same or different VHH chains of the anti-CLDN18.2 nanobody as described in the first aspect of the present invention in the amino acid sequence of the immunoconjugate.

[0160] In the thirteenth aspect of the present invention, there is provided the use of an active ingredient selected from the group consisting 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 the active ingredient is used for preparing:

[0161] (a) A drug for preventing and / or treating CLDN18.2 overexpression diseases;

[0162] (b) A reagent for detecting CLDN18.2 overexpression diseases.

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

[0164] In another preferred embodiment, the diagnostic reagent is used for: detecting CLDN18.2 protein or its fragment in a sample.

[0165] In another preferred embodiment, the CLDN18.2 overexpression diseases include but are not limited to: solid tumors, hematological tumors, immune diseases, or a combination thereof.

[0166] In another preferred example, the CLDN18.2 highly expressed diseases include, but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and biliary tract 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 present invention, there is provided a method for in vitro detecting CLDN18.2 protein or its fragment in a sample, the method comprising the steps of:

[0168] (1) In vitro, contacting the sample with 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;

[0169] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CLDN18.2 protein or its fragment in the sample.

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

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

[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) A pharmaceutically acceptable carrier, diluent or excipient.

[0174] In another preferred example, the dosage form of the pharmaceutical composition is selected from the group consisting of: injection, lyophilized product.

[0175] In another preferred example, the pharmaceutical composition comprises 0.01-99.99% 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 0.01-99.99% of a pharmaceutically acceptable carrier, and the percentages are by mass of the pharmaceutical composition.

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

[0177] In the sixteenth aspect of the present invention, a kit is provided, which comprises:

[0178] (1) A first container containing 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 / or

[0179] (2) A second container containing a secondary antibody against the content of the first container;

[0180] Alternatively,

[0181] The kit contains a test plate, which comprises: a substrate (support plate) and a test strip, and the test strip contains 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, the recombinant protein as described in the sixth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof.

[0182] In another preferred embodiment, the kit further contains an instruction manual, according to which the kit is used for non-invasively detecting the expression of CLDN18.2 in a subject to be tested.

[0183] In another preferred embodiment, the kit is used for detecting CLDN18.2 overexpressing diseases.

[0184] In another preferred embodiment, the CLDN18.2 overexpressing diseases include but are not limited to: solid tumors, hematological malignancies, immune diseases, or combinations thereof.

[0185] In another preferred embodiment, the CLDN18.2 overexpressing diseases include but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and biliary tract 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 the seventeenth aspect of the present invention, a method for preventing and / or treating CLDN18.2 overexpressing diseases is provided, the method comprising: administering to a subject in need 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, a pharmaceutical composition as described in the fifteenth aspect of the present invention, or combinations thereof.

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

[0188] In another preferred embodiment, the CLDN18.2 overexpressing diseases include but are not limited to: solid tumors, hematological malignancies, immune diseases, or combinations thereof.

[0189] In another preferred embodiment, the CLDN18.2 overexpressing diseases include but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and biliary tract cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, etc.

[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 example, the method can be used in combination with other treatment methods.

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

[0194] In the eighteenth aspect of the present invention, a method for diagnosing a disease with high CLDN18.2 expression is provided, comprising the steps of:

[0195] (i) Obtaining a sample from a diagnostic subject, and contacting the sample with 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

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

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

[0198] In another preferred example, the diseases with high CLDN18.2 expression include but are not limited to: solid tumors, hematological tumors, immune diseases, or combinations thereof.

[0199] In another preferred example, the diseases with high CLDN18.2 expression include but are not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and biliary tract cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, etc.

[0200] In the nineteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the recombinant polypeptide being 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, or a combination thereof, the method comprising:

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

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

[0203] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (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 elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0204] Figure 1 Showing the binding level of a CHO-K1 stable cell line expressing CLDN18.2 with a positive antibody.

[0205] Figure 2 Showing the binding level of a HEK 293T stable cell line expressing CLDN18.2 with a positive antibody.

[0206] Figure 3 Showing the agarose gel verification results of PCR amplification of the CALL fragment, where lane M is the Marker, lane 1 is the blank control, and lanes 2 - 12 are the CALL fragments.

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

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

[0209] Figure 6 Showing the binding levels of each gradient-diluted VHH with CHO-CLDN18.2 cells.

[0210] Figure 7 Showing the binding levels of gradient-diluted VHH-Fc and the positive control Zolbetuximab with CHO-CLDN18.2 cells.

[0211] Figure 8 Showing the binding specificity detection of gradient-diluted VHH-Fc and the positive control Zolbetuximab with CLDN18.1 protein..

[0212] Figure 9 Showing the ADCC effect induced by the nanobody of the present invention. Compared with the positive control Zolbetuximab, the nanobody of the present invention induces a stronger ADCC effect. DETAILED DESCRIPTION OF THE INVENTION

[0213] After extensive and in-depth research and a large number of screenings, the inventor of the present invention unexpectedly obtained, for the first time, a nanobody against CLDN18.2 with high affinity and high specificity. Specifically, the present invention develops a nanobody drug targeting the CLDN18.2 target. By preparing a CLDN18.2 stable cell line, immunizing alpacas, and using phage display technology, a nanobody specifically binding to human CLDN18.2 was screened. Binding detection at the protein level and cell level shows that the anti-CLDN18.2 nanobody of the present invention has a good binding ability to CLDN18.2, and is superior to the positive control Zolbetuximab, with higher drug development value and potential, providing a better treatment and diagnosis means for CLDN18.2 high-expression diseases. On this basis, the present invention was completed.

[0214] Term

[0215] To make the present disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0216] The term "about" may refer to a value or a component within an acceptable error range of a specific value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component is measured or determined.

[0217] The term "administer" refers to physically introducing the product of the present invention into a subject using any of a variety of 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", "nanobody of the present invention", etc. 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 the present invention are shown in Table A below.

[0220] Table A

[0221]

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

[0223] As used herein, the terms "antibody" or "immunoglobulin" refer to heterotetrameric glycoproteins of approximately 150,000 daltons with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a number of constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0224] As used herein, the terms "single-domain antibody", "VHH", "Nanobody", "single-domain antibody (sdAb, or Nanobody)" have the same meaning and are used interchangeably, referring to the variable region of the cloned antibody heavy chain, constructing a single-domain antibody (VHH) composed only of one variable region of the heavy chain, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant region of the heavy chain (CH1), the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) composed only of one variable region of the heavy chain.

[0225] As used herein, the term "variable" means that some parts of the variable regions in antibodies are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the light chain and the heavy chain. The more conserved parts in the variable regions are called framework regions (FRs). The variable regions of the native heavy chain and light chain each contain four FR regions, which are generally in a β-sheet configuration, connected by three CDRs that form connecting loops and can form a partial β-sheet structure in some cases. The CDRs in each chain are closely juxtaposed 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)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell cytotoxicity.

[0226] As is known to those skilled in the art, 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 that bind to the nanobodies or fragments thereof against CLDN18.2.

[0227] As used herein, the terms "hypervariable region", "highly variable region", "complementary 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 comprises three complementary determining regions CDR1, CDR2, and CDR3.

[0229] In a preferred embodiment of the present invention, the heavy chain of the nanobody or the antibody comprises the above-mentioned heavy chain variable region and a heavy chain constant region.

[0230] In the present invention, the terms "the nanobody of the present invention", "the antibody of the present invention", "the protein of the present invention", or "the polypeptide of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to the CLDN18.2 protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain a starting 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, as long as the variable region is the same as or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibody of the present invention.

[0232] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the heavy chain variable region, called variable regions (CDR), which divide this segment into four framework 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 loop structures, and the β-sheets formed by the intervening FRs are close to each other in the spatial structure. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

[0233] The nanobodies or heavy chain variable regions of the antibodies of the present invention are of particular interest because at least some of them are involved in antigen binding. Accordingly, the present invention encompasses molecules having a heavy chain variable region of an antibody with CDRs, provided that the CDRs thereof have a homology of more than 90% (preferably more than 95%, most preferably more than 98%) with the CDRs identified herein.

[0234] As used herein, the term "humanized antibody" has the meaning conventionally understood by those skilled in the art, which refers to an antibody obtained by humanizing a camelid-derived antibody, thereby being able to reduce the immunogenicity of a murine antibody. Based on the anti-CLDN18.2 nanobody of the present invention, those skilled in the art know how to perform humanization to obtain a humanized antibody.

[0235] The present invention includes not only intact antibodies, but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives, and analogs of the said antibodies.

[0236] As used herein, the terms "fragment", "derivative", and "analog" refer to polypeptides that substantially retain the same biological function or activity of 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 conservative or non-conservative amino acid residues (preferably conservative 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 (such as a compound that extends the polypeptide half-life, for example, polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0237] The antibody of the present invention refers to a polypeptide having CLDN18.2 protein binding activity and including the above CDR regions. This term also includes variant forms of the polypeptide containing the above CDR regions that have the same function as the antibody of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1 - 50, preferably 1 - 30, more preferably 1 - 20, most preferably 1 - 10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar or close properties, the function of the protein is usually not changed. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. This term also includes active fragments and active derivatives of the antibody of the present invention.

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

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

[0240] In the present invention, the "conservative variant of the antibody of the present invention" refers to a polypeptide in which, compared with the amino acid sequence of the antibody of the present invention, at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table B.

[0241] Table B

[0242]

[0243] The present invention also provides polynucleotide molecules encoding the above antibody or its fragments or its fusion proteins. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0244] The polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; the coding sequences of the mature polypeptides and various additional coding sequences; the coding sequences of the mature polypeptides (and optional additional coding sequences) and non-coding sequences.

[0245] The term "polynucleotide encoding a polypeptide" may be a polynucleotide including the polynucleotide encoding this polypeptide, or may also be a polynucleotide further including additional coding and / or non-coding sequences.

[0246] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides described in the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least above 90%, preferably above 95%. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0247] The full-length nucleotide sequences or fragments of the antibodies of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis methods. A feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0248] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually to clone them into a vector, then transfer them into cells, and then isolate the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0249] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then, this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0250] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0251] The host cell can be a prokaryotic cell, such as a bacterial cell; or 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; animal cells such as CHO, COS7, 293 cells, etc.

[0252] Transformation of the host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

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

[0254] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods using its 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 renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0255] The nanobody or antibody of the present invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety or a combination of any of the above substances.

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

[0257] Therapeutic agents that can bind or conjugate with the antibodies of the present invention include, but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles / nanorods; 5. Virus particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0258] CLDN18.2-related diseases

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

[0260] The CLDN18.2-related diseases are CLDN18.2 overexpression diseases. In a specific embodiment, the CLDN18.2 overexpression diseases are tumors expressing CLDN18.2; including but not limited to: gastric cancer, pancreatic cancer, esophageal cancer, gallbladder and biliary tract cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, etc.

[0261] Labeled antibody

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

[0263] Colloidal gold labeling can be carried out by 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 method

[0265] The present invention also relates to a method for detecting CLDN18.2 protein or its fragment. The steps of this method are generally as follows: obtaining a cell and / or tissue sample; dissolving the sample in a medium; detecting the level of CLDN18.2 protein in the dissolved sample.

[0266] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.

[0267] Kit

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

[0269] The present invention also provides a detection kit for detecting the CLDN18.2 protein level. The kit includes an antibody that recognizes the CLDN18.2 protein, a lysis medium for lysing the sample, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0270] Drug composition

[0271] The present invention also provides a drug composition. It contains, as active ingredients, 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 chimeric antigen receptor as described in the third aspect of the present invention, the fusion protein as described in the fourth aspect of the present invention, the recombinant protein as described in the fifth aspect of the present invention, the host cell as described in the eighth aspect of the present invention, the engineered immune cell as described in the ninth aspect of the present invention, the immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof, and a pharmaceutically acceptable carrier.

[0272] Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5 - 8, preferably about 6 - 8, although the pH value can vary depending on the nature of the substance being formulated and the condition to be treated. The formulated drug composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0273] The drug composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the above-mentioned antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The drug composition of the present invention can be made into an injectable form, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectables and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0274] When using the pharmaceutical composition, a safe and effective amount of the above-mentioned antibody (or its conjugate) of the present invention is administered to a mammal, 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, the dose is about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0275] Application

[0276] As described above, the nanobody and antibody of the present invention have extensive biological and clinical application values, 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 an immune response mediated by T cells targeting a mammalian tumor cell population or tissue, which comprises 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 autologous T cells (or allogeneic donors) of a patient are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient. This method has a very low probability of graft-versus-host reaction, and the antigen is recognized by T cells in an MHC-unrestricted manner. In addition, one type of CAR-T can treat all cancers expressing the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo and produce long-term persistence that can lead to continuous control of tumors.

[0279] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and can last for several months to several years. In addition, 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 with high expression of the antigen recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention induce a specific immune response against tumor cells with high expression of CLDN18.2.

[0280] The cancers that can be treated include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. The types of cancers treated with the CAR of the present invention include, but are not limited to: breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small intestine cancer, large intestine cancer, cholangiocarcinoma, cervical cancer, lymphoma, esophageal cancer, etc.

[0281] Typically, the cells activated and expanded as described herein can be used for treating and preventing diseases such as tumors. Accordingly, the present invention provides a method for treating cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-T cells of the present invention.

[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 with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention can comprise a population of target cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

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

[0284] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition of the patient (subject). A pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 4 to 10 9 cells / kg body weight, preferably at a dose of 10 5 to 10 7 cells / kg body weight (including all integer values within the range). The T cell composition can also be administered multiple times at these doses. The cells can be administered by 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 particular patient can be readily determined by those skilled in the medical art by monitoring the signs of the patient's disease and adjusting the treatment accordingly.

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

[0286] In certain embodiments of the present 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 (e.g., before, simultaneously, or after) with any number of relevant treatment modalities, which treatment modalities include, but are not limited to, treatment with the following reagents: such reagents as antiviral therapy, cidofovir, and interleukin-2, cytarabine (also known as ARA-C), or treatment with natalizumab for MS patients or efalizumab for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with: chemotherapy, radiation, immunosuppressive agents, such as, cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination (e.g., before, simultaneously, or after) with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide. For example, in one embodiment, a subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0287] The doses of the above treatments administered to a patient will vary with the precise nature of the disorder being treated and the recipient of the treatment. Dosage ratios for human administration can be practiced according to accepted practice in the art. Generally, for each treatment or each course of treatment, 1×10 5 to 1×10 10 modified T cells of the present invention can be administered to a patient, for example, by intravenous infusion.

[0288] The main advantages of the present invention

[0289] 1. Existing CLDN18.2 recombinant antibodies have a large molecular weight and are not easily bound to cryptic epitopes, while the CLDN18.2 nanobody of the present invention can reach protein cryptic epitopes that are inaccessible to conventional antibodies.

[0290] 2. Currently, there are no nanobodies targeting CLDN18.2 that have been developed into drugs, while the CLDN18.2 nanobody of the present invention can achieve a higher affinity compared to the prior art, is superior to the positive control Zolbetuximab, and has higher drug development value and potential.

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

[0292] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out 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 according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight percentage and weight parts.

[0293] Sequence Listing

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

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

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

[0302] A stable cell line with high expression of CLDN18.2 is constructed by the lentivirus infection-drug screening method. The density of 293T cells is adjusted to 5×10^5 / mL, and 10 mL of HEK 293T cells are added to a 10 cm cell culture dish and cultured overnight in a 37°C, 5% CO2 incubator. Observe the cell state the next day, and transfection can be carried out when the cell confluence reaches 90%. In the transfection system of the three plasmids for lentivirus expression, the mass ratio of the three plasmids is according to the ratio of the target gene plasmid: packaging plasmid psPAX2: envelope plasmid VSVG of 4:3:1. According to Lipofectamine TMOperate according to the instruction manual of the 3000 transfection reagent. Collect the supernatant 48 hours after transfection and replace it with complete medium. Store the supernatant temporarily at 4°C. Collect the supernatant again 72 hours later and mix it with the previous supernatant. Centrifuge to remove cell debris and impurities. For the collected viral supernatant, add the reagent according to the actual volume with reference to the instruction manual of Lenti-X Concentrator and concentrate it overnight at 4°C. Detect the approximate viral titer using a rapid lentivirus titer detection card (specially for virus packaging). Use polybrene to infect the target cells (CHO-K1 cells or HEK 293T cells) with lentivirus according to a certain MOI value and culture them in an incubator at 37°C and 5% CO2. Replace the fresh complete medium 48 hours later and collect the cells for flow cytometry detection 96 hours later.

[0303] Flow cytometry uses an indirect staining method, that is, using the positive control antibody Zolbetuximab as the primary antibody and using Alexa Fluor labeled Fc secondary antibody for FACS analysis. The results are as Figure 1 , Figure 2 shown: The CHO-K1 cells and HEK 293T cells expressing CLDN18.2 bind well to the positive antibody and are significantly distinguishable from the negative control, indicating that the stable transfected cell lines of CHO-K1 and HEK 293T expressing CLDN18.2 are successfully constructed.

[0304] Example 2: CLDN18.2 cell immunization

[0305] Using Freund's adjuvant as an immune enhancer, immunize adult healthy alpacas with 1.0×10^7 CHO-CLDN18.2 cells established in Example 1. Immunize once every two weeks for a total of five times. Collect peripheral blood from the jugular vein 7 days after immunization to measure the titer.

[0306] The titer is the highest after the third immunization, and the titer is 1:24300.

[0307] Example 3: Lymphocyte extraction

[0308] Use a 50 mL peripheral blood lymphocyte separation tube. Add 10 mL of the blood from the alpaca after the third immunization to each tube and centrifuge at 800×g for 15 minutes. Carefully aspirate the milky white ring with a pipette tip, add 10 mL of PBS, centrifuge at 250×g for 10 minutes for washing, repeat the washing three times, resuspend and count with a small amount of PBS, and dilute the cell density to 5×10^6 cells / mL using RNAkeeper and freeze at -80°C.

[0309] Example 4: Library construction

[0310] 1. RNA extraction. Use the Novizan RNA extraction kit (product number RC101-01) to extract the RNA of lymphocytes. After the RNA extraction, immediately take a part for electrophoresis and perform reverse transcription on the remaining part immediately.

[0311] 2. Reverse transcription into cDNA. Use the Hiscriptr III ISt strand cDNA synthesis Kit (Novizan reverse transcription kit, product number R312-02) to reverse transcribe the extracted RNA into cDNA.

[0312] 3. Use cDNA as a template to amplify the CALL fragment by nested PCR and gel-recover the band around 700 bp. The reaction system is prepared as follows:

[0313] Name Dosage cDNA 50 μg 2×master Mix 425 μL Forward primer 35 μL Reverse primer 35 μL Water to 850 μL

[0314] Mix well and dispense 50 μL per tube. In addition, the negative control group is prepared with the above system without adding cDNA template.

[0315] The PCR reaction program is as follows:

[0316]

[0317] After running PCR, run on a 1.5% agarose gel for 40 min. The result is as Figure 3 shown, showing that the CALL fragment has a band around 700 bp, and the amplification is complete, so subsequent gel recovery can be carried out. Use the Tiangen Biotech gel recovery kit for gel recovery.

[0318] 4. Use CALL as a template to amplify the VHH fragment and gel-recover the band around 450 bp:

[0319] In this amplification, homologous recombination primers are used to facilitate seamless cloning with the vector in the subsequent steps.

[0320] The reaction system is prepared as follows:

[0321] Name Dosage 2×Mix 2 mL Forward primer 100 μL Reverse primer 100 μL Template 450 ng Water to 4 mL

[0322] Mix well and dispense 50 μL per tube. In addition, the negative control group is prepared with the above system without adding cDNA template.

[0323] PCR program:

[0324]

[0325] After running PCR, run on a 2% agarose gel for 40 min. The result is as Figure 4 shown, showing that the VHH fragment is around 450 bp, and the band is clear and single, so subsequent gel recovery can be carried out. Use the Tiangen Biotech gel recovery kit for gel recovery.

[0326] 5. Digest the PMES4 vector with two enzymes, PstI and BstEII, and perform verification:

[0327] The digestion system is as follows:

[0328] Name Dosage PMES4 10 μg BstEII 4 μL PstI 4 μL 10×cutsmart 4 μL Water to 40 μL

[0329] Digest the above system overnight at 37°C. After digestion, run gel electrophoresis and recover the gel. Use solutionI enzyme to ligate and transform the plasmid recovered from the gel. Incubate overnight in a 37°C incubator. The next day, no colonies grew on the ampicillin plate, indicating that the plasmid recovered by digestion was complete and could be used for subsequent ligation experiments.

[0330] 6. Ligation and electroporation:

[0331] Use homologous recombination enzyme to ligate a small amount of the recovered VHH and PMES4.

[0332] Ligation system:

[0333] Name Dosage Vector 900 ng VHH 300 ng Yeasen homologous recombinase 25 μL Water to 50 μL

[0334] Ligate the above ligation system at 50°C for 30 min. The reaction product can be directly transformed or frozen at -20°C.

[0335] After successful verification of the transformed bacteria P, perform large-scale ligation and column recovery.

[0336] Preheat the SOC medium at 37°C when measuring the concentration of the recovered product. Wash the electroporation cuvette with ultrapure water, dry it, and irradiate with ultraviolet light for 30 min. Cool it on ice for 5 min. Take the competent cells and place them on ice for 5 min. Add the ligation product to the competent cells and let it stand on ice for 10 min. Add the mixture to the electroporation cuvette without bubbles. Select the preset program for bacteria on the electroporator, 2.5 KV, 25 μF, 200 Ω, competent cell volume 50 μL, electroporation cuvette 2 mm. Perform electroporation. Immediately add 1 mL of SOC medium after electroporation and shake in a 15 mL centrifuge tube at 37°C for 2 h.

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

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

[0339] Positive rate of bacteria P verification: Pick 23 monoclonal colonies for bacteria P verification. The results are as Figure 5 shown, indicating that the positive rate of the 23 monoclonal colonies was 100%.

[0340] Randomly select 12 clones before bacteria P from the above-mentioned positive monoclonal antibodies for sequencing to test the diversity, and the diversity is 100%. The library capacity is 1.16×10^9 cfu / mL.

[0341] Take another 500 μL of the bacteria after electroporation and add it to 10 mL of 2YT medium containing 2% glucose and one-thousandth ampicillin, and shake until the OD is 0.6 - 0.8. Add M13K07 at a ratio of 20:1 and let it stand for infection for 1 h. Centrifuge at 4000 rpm for 10 min to remove the supernatant, resuspend with 10 mL of 2YT without glucose, culture at 37°C and 220 rpm for 1 h, then transfer it to 100 mL of 2YT medium without glucose, and add ampicillin, kanamycin, and IPTG at ratios of 1:1000; 1:1000; 1:2000 respectively. First shake at 37°C for 1 h and then transfer to 30°C for overnight culture.

[0342] Example 5: Library screening

[0343] 1. Precipitate phage:

[0344] Centrifuge the bacterial liquid in the above step at 3000 rpm for 10 min, collect the supernatant, add PEG / NaCl at a ratio of 4:1 and incubate on ice for 2 h. Then centrifuge the phage at 10000 rpm, 4°C for 15 min, discard the supernatant, drain the water, add 2 mL of PBS to resuspend, and take it out to measure the titer after passing through a 0.45 μm filter membrane. After measurement, the titer of the amplified phage is 2.5×10^12 pfu / mL.

[0345] 2. Select phage antibodies by cell panning method:

[0346] Take 1×10^13 pfu of phage, add 4% BSA-PBS solution to make the total volume 2 mL. Prepare two portions in total and incubate at 37°C 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 with the incubated phage solution and incubate with shaking at 4°C for 1 h. Centrifuge to collect the phage in the supernatant that has not bound to the cells. Take the phage obtained after subtraction, resuspend 1×10^7 HEK 293T-CLDN18.2 cells and HEK 293T cells respectively, incubate with shaking at 4°C for 1 h, and then incubate statically for 30 min. Centrifuge to discard the supernatant and retain the cells. Resuspend the cells with PBS and centrifuge to collect the cells. Wash three times in the first round, five times in the second round, and eight times in the third round. Add 1 mL of 0.1 M Gly-HCl solution (pH = 2.2) to resuspend the cells, oscillate and elute at room temperature for 5 min, centrifuge to aspirate the supernatant, immediately add about 100 μL of 1 M Tris-HCl solution (pH = 9.1) to neutralize to about pH 7.0, and pass through a 0.45 μm filter membrane for standby.

[0347] Take 10 μL of the eluted phage to infect 200 μL of fresh TG1 (using glucose 2YT medium), let it stand and infect at 37 °C for 1 h. Take 100 μL and make 10-fold, 100-fold, and 1000-fold dilutions to plate and measure the titer (the plate is ampicillin 2YT solid medium with glucose).

[0348] Take 500 μL of the eluted phage and add it to 4 mL of glucose 2YT medium, let it stand and infect for 1 h. Then add helper phage at a ratio of 20:1, and let it stand for superinfection for 1 h. Centrifuge to remove the supernatant and glucose, resuspend with 10 mL of 2YT medium without glucose, add ampicillin and kanamycin in proportion, shake at 37 °C for 1 h and then add it to 50 mL of 2YT medium without glucose, supplement with ampicillin, kanamycin and IPTG, and shake the bacteria overnight at 30 °C.

[0349] Precipitate the phage again the next day, and repeat the above library screening operation. The specific conditions and results are shown in Table 1 below.

[0350] Table 1

[0351]

[0352]

[0353] 3. Monoclonal ELISA:

[0354] Pick 526 monoclonal colonies from the titer measurement plate for monoclonal ELISA.

[0355] Pick monoclonal colonies into 200 μL of 2YT-ampicillin medium, culture at 37 °C and 200 rpm for 2 - 3 h until the OD is about 0.6.

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

[0357] Add M13K07 at 10^9 / well, 100 μL per well. Let it stand and infect for 30 min, then add kanamycin and IPTG. First prepare it and then add 200 μL / well (90 μL of kanamycin, 50 μL of IPTG, 40 mL of medium). After 1 h at 37 °C, change to overnight incubation at 30 °C. Add 293T-CLDN18.2 cells at 10^5 / 100 μL / well to 96-well plates, a total of three plates; take 293T cells and plate three plates in the same way as negative controls.

[0358] The next 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, then discard the methanol. Add 200 μL of PBS to each well and wash twice by oscillation for 5 min each time. Block with 5% skim milk, 200 μL per well, at 37 °C for 2 h. Meanwhile, centrifuge the picked monoclonal colonies and collect the supernatant. After complete blocking, discard the blocking solution. Take 100 μL of each monoclonal supernatant and add them to a total of three 96-well plates, and make good marks. Perform the same operation on the three plates coated with BSA. At the same time, use M13K07 and TG1 supernatants as negative controls. After incubating at 37 °C for 1 h, wash twice by oscillation with PBST for 5 min each time. Add the anti-M13 secondary antibody and incubate for 1 h, then wash three times by oscillation with PBST for 5 min each time. Add 100 μL of TMB chromogenic solution per well, incubate at room temperature in the dark for 10 min, then add sulfuric acid to terminate the reaction, and read the OD value at 450 nm.

[0359] Send the monoclonal colonies with OD value greater than 1 and P / N > 2.1 for sequencing.

[0360] Example 6: Nanobody expression and purification

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

[0362] Transform the obtained plasmid into BL21. After picking a single colony and sending it for correct sequencing, induce expression with IPTG for 800 mL. The next day, extract the periplasmic protein using TES, purify it using an Ni column, and then run a protein gel.

[0363] Example 7: Detection of nanobody binding ability at the cellular level

[0364] Collect CHO-CLDN18.2 cells in the logarithmic growth phase, centrifuge at 1300 rpm for 5 min, discard the supernatant, and resuspend the cells with 1×PBS and wash twice. Adjust the cell density to 3×10^6 / mL and plate 100 μL per well. Dilute the primary antibody with FACS buffer, starting from a concentration of 30 μg / mL of the nanobody and diluting it five-fold. After centrifuging the cells at 1500 rpm for 5 min, discard the supernatant, resuspend the cells with 100 μL per well, and incubate at 4°C for 60 min. Wash twice with 200 μL per well of 1×PBS, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Dilute the anti-his-tag fluorescent secondary antibody with FACS buffer, resuspend the cells with 100 μL per well, and incubate at 4°C for 60 min. Wash twice with 200 μL per well of 1×PBS, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Resuspend the cells with 100 μL per well of PBS and perform on-machine detection.

[0365] The results are as Figure 6 shown, indicating that most nanobodies can bind to cells expressing the CLDN18.2 protein. Comprehensively select #19, #20, #31, #49, #189, #194, #27, #100, #251, #258 to continue the experiment.

[0366] Example 8: Functional experiment

[0367] 1. Construction of VHH-Fc:

[0368] Fuse the nanobody with IgG1-Fc. The fusion protein can exert tumor killing effects through antibody targeting, and construct VHH-Fc respectively. Transfect the constructed molecular plasmid into 293F, and perform cell binding activity experiments after expression and purification.

[0369] 2. Detection of the binding ability of VHH-Fc at the cell level

[0370] For the flow cytometry experiment, the primary antibodies are VHH-Fc and the positive control antibody Zolbetuximab, starting from a concentration of 30 μg / mL and diluting it five-fold; the secondary antibody is diluted with FACS buffer to the anti-Fc-tag fluorescent secondary antibody. All other operations are the same as in Example 7.

[0371] The results are as Figure 7 shown, and the EC50 values are shown in Table 2. It shows that except for the EC50 value of 251-Fc being slightly higher than that of the positive control antibody Zolbetuximab, the EC50 values of the other VHH-Fc are lower than that of 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. Specific binding experiment of VHH-Fc

[0375] By ELISA method, mainly detect whether each VHH-Fc binds to CLDN18.1 protein:

[0376] As Figure 8 shown, it can be seen that all VHH-Fc do not bind to CLDN18.1 protein, indicating that the selected nanobodies have high specificity.

[0377] 4. ADCC experiment:

[0378] Mix CHO-K1 medium and NK medium at a ratio of 1:1, and serially dilute VHH-Fc with the mixed medium starting from 200 μg / mL in 5-fold gradients, with a total of 8 concentration gradients. Add 50 μL per well to area D; adjust the density of CHO-CLDN18.2 cells to 4E5 cells / mL with the mixed medium and add 25 μL per well to areas B, C, and D; adjust the density of CHO-CLDN18.2 cells to 4E6 cells / mL (effector-to-target ratio of 10:1) with the mixed medium and add 25 μL per well to areas C and D; make up all wells to 100 μL with the mixed medium. Shake the cell culture plate well and culture it in an incubator at 37 °C and 5% CO2 for 20 h.

[0379] The next day, add 10 μL of CCK8 solution to each well, measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader at 2 h, and read the value every 1 h.

[0380] Process the data according to the killing rate formula. Killing rate (%) = (C - D) / (B - A)

[0381] A: 100 μL of mixed medium;

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

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

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

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

[0386] All documents mentioned in this invention are cited herein for reference as if each individual document was cited for reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An anti-CLDN18.2 nanobody, characterized in that, The anti-CLDN18.2 nanobody has one or more complementarity-determining regions (CDRs) selected from the following groups: (1) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:7; (2) CDR1 shown in SEQ ID NO:11, CDR2 shown in SEQ ID NO:13, and CDR3 shown in SEQ ID NO:15; (3) CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:21, and CDR3 shown in SEQ ID NO:23; (4) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:28, and CDR3 shown in SEQ ID NO:30; (5) CDR1 shown in SEQ ID NO:33, CDR2 shown in SEQ ID NO:35, and CDR3 shown in SEQ ID NO:37; (6) CDR1 shown in SEQ ID NO:40, CDR2 shown in SEQ ID NO:42, and CDR3 shown in SEQ ID NO:43; (7) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:47; (8) CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:54; (9) CDR1 shown in SEQ ID NO:57, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:61; (10) CDR1 shown in SEQ ID NO:64, CDR2 shown in SEQ ID NO:65, and CDR3 shown in SEQ ID NO:67; (11) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:71, and CDR3 shown in SEQ ID NO:73; (12) CDR1 shown in SEQ ID NO:76, CDR2 shown in SEQ ID NO:77, and CDR3 shown in SEQ ID NO:78; (13) CDR1 shown in SEQ ID NO:81, CDR2 shown in SEQ ID NO:82, and CDR3 shown in SEQ ID NO:84; (14) CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:87, and CDR3 shown in SEQ ID NO:89; (15) CDR1 shown in SEQ ID NO:92, CDR2 shown in SEQ ID NO:94, and CDR3 shown in SEQ ID NO:96; (16)CDR1 shown in SEQ ID NO:99, CDR2 shown in SEQ ID NO:101, and CDR3 shown in SEQ ID NO:102; (17)CDR1 shown in SEQ ID NO:99, CDR2 shown in SEQ ID NO:101, and CDR3 shown in SEQ ID NO:104; (18)CDR1 shown in SEQ ID NO:99, CDR2 shown in SEQ ID NO:107, and CDR3 shown in SEQ ID NO:109; (19)CDR1 shown in SEQ ID NO:112, CDR2 shown in SEQ ID NO:114, and CDR3 shown in SEQ ID NO:116; (20)CDR1 shown in SEQ ID NO:119, CDR2 shown in SEQ ID NO:121, and CDR3 shown in SEQ ID NO:122; (21)CDR1 shown in SEQ ID NO:125, CDR2 shown in SEQ ID NO:127, and CDR3 shown in SEQ ID NO:129; (22)CDR1 shown in SEQ ID NO:132, CDR2 shown in SEQ ID NO:133, and CDR3 shown in SEQ ID NO:135; (23)CDR1 shown in SEQ ID NO:138, CDR2 shown in SEQ ID NO:101, and CDR3 shown in SEQ ID NO:141; (24)CDR1 shown in SEQ ID NO:144, CDR2 shown in SEQ ID NO:146, and CDR3 shown in SEQ ID NO:

148.

2. An anti-CLDN18.2 antibody, characterized in that, The antibody comprises one or more anti-CLDN18.2 nanobodies as claimed in claim 1.

3. A humanized antibody, which is obtained by humanizing the anti-CLDN18.2 nanobody as claimed in claim 1 or the anti-CLDN18.2 antibody as claimed in claim 2.

4. A chimeric antigen receptor (CAR), characterized in that, The CAR contains an extracellular domain, and the extracellular domain comprises the anti-CLDN18.2 nanobody as claimed in claim 1, the anti-CLDN18.2 antibody as claimed in claim 2, or the humanized antibody as claimed in claim 3.

5. A fusion protein, characterized in that, The fusion protein has: (Z1) A first protein, which comprises: the anti-CLDN18.2 nanobody as claimed in claim 1, the anti-CLDN18.2 antibody as claimed in claim 2, or the humanized antibody as claimed in claim 3 or an active fragment thereof; (Z2) A second protein, which comprises a cytokine; and (Z3) Optionally, a linker located between the first protein and the second protein.

6. A recombinant protein, characterized in that, The recombinant protein has: (i) The anti-CLDN18.2 nanobody according to claim 1, the anti-CLDN18.2 antibody according to claim 2, or the humanized antibody according to claim 3, the fusion protein according to claim 5, or an active fragment thereof; and (ii) Optionally, a tag sequence for assisting expression and / or purification.

7. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of: the anti-CLDN18.2 nanobody according to claim 1, the anti-CLDN18.2 antibody according to claim 2, or the humanized antibody according to claim 3, the chimeric antigen receptor according to claim 4, the fusion protein according to claim 5, the recombinant protein according to claim 6, or a combination thereof.

8. An expression vector, characterized in that, The expression vector contains the polynucleotide according to claim 7.

9. A host cell, characterized in that, The host cell contains the expression vector according to claim 8, or the polynucleotide according to claim 7 is integrated into its genome.

10. An engineered immune cell, characterized in that, The engineered immune cell contains the expression vector according to claim 8 or the exogenous polynucleotide according to claim 7 is integrated into its genome, or expresses the chimeric antigen receptor according to claim 4.

11. A method for generating anti-CLDN18.2 nanobodies, characterized in that, The method comprises the steps of: (a) Culturing the host cell according to claim 9 under conditions suitable for generating nanobodies, thereby obtaining a culture containing the anti-CLDN18.2 nanobody; (b) Isolating and / or recovering the anti-CLDN18.2 nanobody from the culture; and (c) Optionally, purifying and / or modifying the anti-CLDN18.2 nanobody obtained in step (b).

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