ANTI-HUMAN CLAUDIN 18.2 ANTIBODY AND APPLICATION THEREOF

Abstract

Embodiments of the present disclosure provide an antibody that binds to human Claudin 18.2 or a fragment thereof, as well as encoded nucleic acids and the like thereof. The anti-human Claudin 18.2 antibody of embodiments of the present disclosure has strong affinity to an antigen Claudin 18.2 and significant complement-dependent cytotoxicity (CDC) activity and antibody-dependent cytotoxicity (ADCC) activity to target expression cells, and exhibits high specificity to human CLDN 18.2.

Claims

1. A preparation method of an anti-human Claudin 18.2 antibody, the preparation method including steps as follows: (1) immunizing an animal with cells expressing human Claudin 18.2 protein on surface as an immunogen; (2) preparing cell clones capable of producing antibodies using the animals immunized in step (1); (3) using cells expressing human Claudin 18.2 protein on surface as a positive screening antigen, screening antibodies having a binding activity to the positive screening antigen and cells producing the antibodies; (4) using the cells expressing human Claudin 18.1 protein on surface as a negative screening antigen, excluding antibodies having a binding activity to the negative screening antigen and cells producing the antibody.

2. The preparation method according to claim 1, wherein the cells expressing the human Claudin 18.2 protein in step (1) are derived from the same species as the animal immunized; preferably, the cells expressing the human Claudin 18.2 protein are mouse cells, and the animal immunized is a mouse.

3. The preparation method according to claim 1, wherein the cell clones capable of producing antibodies in step (2) are prepared by a technique selected from the group consisting of hybridoma technique and single cell amplification technique.

4. The preparation method according to claim 1, wherein the positive screening antigen in step (3) is the same as the immunogen in step (1); and the negative screening antigen in step (4) differs from the immunogen in step (1) only in that the protein expressed is human Claudin 18.1 protein.

5. The preparation method according to claim 1, wherein step (3) and step (4) each is conducted by a method selected from the group consisting of Enzyme-Linked Immunosorbent Assay (ELISA) and Fluorescence Resonance Energy Transfer (FRET).

6. An anti-human Claudin 18.2 antibody obtained according to the preparation method of any one of claims 1 to 6.

7. The anti-human Claudin 18.2 antibody according to claim 6, which specifically binds the N-terminus of human Claudin 18.2, preferably extracellular region at the N-terminus of human Claudin 18.2 comprising the first Extracellular Loop (ECL1).

8. The anti-human Claudin 18.2 antibody according to claim 6, which has a specific binding to human CLDN18.2 at nM scale; and which has no significant difference in binding to human CLDN18.1 as compared to an isotype negative antibody (or an unrelated antibody).

9. An antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of CDRs (VH-CDR1, VH-CDR2, VH-CDR3; and VL-CDR1, VL-CDR2, VL-CDR3) selected from the group consisting of: (1) VH-CDR1 as shown in SEQ ID NO. 31, VH-CDR2 as shown in SEQ ID NO. 32, and VH-CDR3 as shown in SEQ ID NO. 33; and VL-CDR1 as shown in SEQ ID NO. 34, VL-CDR2 as shown in SEQ ID NO. 35, and VL-CDR3 as shown in SEQ ID NO. 36; (2) VH-CDR1 as shown in SEQ ID NO. 37, VH-CDR2 as shown in SEQ ID NO. 38, and VH-CDR3 as shown in SEQ ID NO. 39, and VL-CDR1 as shown in SEQ ID NO. 40, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 42, (3) VH-CDR1 as shown in SEQ ID NO. 43, VH-CDR2 as shown in SEQ ID NO. 44, and VH-CDR3 as shown in SEQ ID NO. 45; and VL-CDR1 as shown in SEQ ID NO. 46, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 47; (4) VH-CDR1 as shown in SEQ ID NO. 48, VH-CDR2 as shown in SEQ ID NO. 49, and VH-CDR3 as shown in SEQ ID NO. 50; and VL-CDR1 as shown in SEQ ID NO. 40, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 51; (5) VH-CDR1 as shown in SEQ ID NO. 52, VH-CDR2 as shown in SEQ ID NO. 53, and VH-CDR3 as shown in SEQ ID NO. 54; and VL-CDR1 as shown in SEQ ID NO. 55, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 56; (6) VH-CDR1 as shown in SEQ ID NO. 57, VH-CDR2 as shown in SEQ ID NO. 58, and VH-CDR3 as shown in SEQ ID NO. 33; and VL-CDR1 as shown in SEQ ID NO. 34, VL-CDR2 as shown in SEQ ID NO. 59, and VL-CDR3 as shown in SEQ ID NO. 60; (7) VH-CDR1 as shown in SEQ ID NO. 61, VH-CDR2 as shown in SEQ ID NO. 62, and VH-CDR3 as shown in SEQ ID NO. 63; and VL-CDR1 as shown in SEQ ID NO. 46, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 64; (8) VH-CDR1 as shown in SEQ ID NO. 65, VH-CDR2 as shown in SEQ ID NO. 66, and VH-CDR3 as shown in SEQ ID NO. 67; and VL-CDR1 as shown in SEQ ID NO. 68, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 69; (9) VH-CDR1 as shown in SEQ ID NO. 65, VH-CDR2 as shown in SEQ ID NO. 70, and VH-CDR3 as shown in SEQ ID NO. 71; and VL-CDR1 as shown in SEQ ID NO. 72, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 73; (10) VH-CDR1 as shown in SEQ ID NO. 74, VH-CDR2 as shown in SEQ ID NO. 75, and VH-CDR3 as shown in SEQ ID NO. 76; and VL-CDR1 as shown in SEQ ID NO. 77, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 78; (11) VH-CDR1 as shown in SEQ ID NO. 79, VH-CDR2 as shown in SEQ ID NO. 80, and VH-CDR3 as shown in SEQ ID NO. 81; and VL-CDR1 as shown in SEQ ID NO. 82, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 83; (12) VH-CDR1 as shown in SEQ ID NO. 37, VH-CDR2 as shown in SEQ ID NO. 38, and VH-CDR3 as shown in SEQ ID NO. 39; and VL-CDR1 as shown in SEQ ID NO. 85, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 42; (13) VH-CDR1 as shown in SEQ ID NO. 37, VH-CDR2 as shown in SEQ ID NO. 38, and VH-CDR3 as shown in SEQ ID NO. 39; and VL-CDR1 as shown in SEQ ID NO. 85, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 86; (14) VH-CDR1 as shown in SEQ ID NO. 37, VH-CDR2 as shown in SEQ ID NO. 38, and VH-CDR3 as shown in SEQ ID NO. 39; and VL-CDR1 as shown in SEQ ID NO. 85, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 87; (15) VH-CDR1 as shown in SEQ ID NO. 74, VH-CDR2 as shown in SEQ ID NO. 75, and VH-CDR3 as shown in SEQ ID NO. 76; and VL-CDR1 as shown in SEQ ID NO. 89, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 90, and (16) VH-CDR1 as shown in SEQ ID NO. 79, VH-CDR2 as shown in SEQ ID NO. 91, and VH-CDR3 as shown in SEQ ID NO. 81; and VL-CDR1 as shown in SEQ ID NO. 92, VL-CDR2 as shown in SEQ ID NO. 41, and VL-CDR3 as shown in SEQ ID NO. 93.

10. The antibody or fragment thereof according to any one of claims 6 to 9, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 27, and SEQ ID NO. 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown; and/or the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 28, and SEQ ID NO. 30 is or an amino acid sequence having at least 75% identity to the amino acid sequence as shown

11. The antibody or fragment thereof according to any one of claims 6 to 10, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (1) an amino acid sequence as shown in SEQ ID NO. 1 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 1, and an amino acid sequence as shown in SEQ ID NO. 2 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 2; (2) an amino acid sequence as shown in SEQ ID NO. 3 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 3; and an amino acid sequence as shown in SEQ ID NO. 4 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 4; (3) an amino acid sequence as shown in SEQ ID NO. 5 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 5; and an amino acid sequence as shown in SEQ ID NO. 6 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 6; (4) an amino acid sequence as shown in SEQ ID NO. 7 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 7; and an amino acid sequence as shown in SEQ ID NO. 8 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 8; (5) an amino acid sequence as shown in SEQ ID NO. 9 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 9; and an amino acid sequence as shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 10; (6) an amino acid sequence as shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 11; and an amino acid sequence as shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 12; (7) an amino acid sequence as shown in SEQ ID NO. 13 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 13; and an amino acid sequence as shown in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 14; (8) an amino acid sequence as shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 15; and an amino acid sequence as shown in SEQ ID NO. 16 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 16; (9) an amino acid sequence as shown in SEQ ID NO. 17 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 17; and an amino acid sequence as shown in SEQ ID NO. 18 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 18; (10) an amino acid sequence as shown in SEQ ID NO. 19 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 19; and an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 20; (11) an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 21; and an amino acid sequence as shown in SEQ ID NO. 22 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 22; (12) an amino acid sequence as shown in SEQ ID NO. 23 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 23; and an amino acid sequence as shown in SEQ ID NO. 24 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 24; (13) an amino acid sequence as shown in SEQ ID NO. 23 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 23; and an amino acid sequence as shown in SEQ ID NO. 25 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 25; (14) an amino acid sequence as shown in SEQ ID NO. 23 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 23; and an amino acid sequence as shown in SEQ ID NO. 26 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 26; (15) an amino acid sequence as shown in SEQ ID NO. 27 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 27; and an amino acid sequence as shown in SEQ ID NO. 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 28; (16) an amino acid sequence as shown in SEQ ID NO. 29 or an amino acid sequence having at least 75% of identity to the amino acid sequence as shown in SEQ ID NO. 29; and an amino acid sequence as shown in SEQ ID NO. 30 or an amino acid sequence having at least 75% of identity to the amino acid sequence as shown in SEQ ID NO. 30.

12. The antibody or fragment thereof according to any one of claims 6 to 11, wherein the antibody or fragment thereof is in any form, e.g., a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody and the like; alternatively, the antibody or fragment thereof is a half-antibody or an antigen-binding fragment of the half-antibody, e.g., scFv, BsFv, dsFv, (dsFv).sub.2, Fab, Fab′, F(ab′).sub.2, or Fv; preferably, the antibody or fragment thereof further comprises a human or murine constant region, preferably a human or murine light chain constant region (CL) and/or heavy chain constant region (CH); more preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE and/or a kappa or lambda type light chain constant region.

13. The antibody or fragment thereof according to any one of claims 6 to 12, wherein the antibody is a monoclonal antibody, preferably a murine, chimeric, or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of IgG1 or IgG4 subtype and the light chain constant region of the monoclonal antibody is of kappa type; preferably, the heavy chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 124 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown; preferably, the light chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 125 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown.

14. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or fragment thereof according to any one of claims 6 to 13, or encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain comprised in the antibody or fragment thereof.

15. A vector comprising the nucleic acid molecule according to claim 14.

16. A host cell comprising the nucleic acid molecule according to claim 14 and/or the vector according to claim 15, or transformed or transfected with the nucleic acid molecule according to claim 14 and/or the vector according to claim 15.

17. A conjugate or fusion protein comprising the antibody or fragment thereof according to any one of claims 6 to 13.

18. A pharmaceutical composition comprising the antibody or fragment thereof according to any one of claims 6 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to claim 16, and/or the conjugate or fusion protein according to claim 17, and optionally a pharmaceutically acceptable excipient.

19. A kit comprising the antibody or fragment thereof according to any one of claims 6 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to claim 16, the conjugate or fusion protein according to claim 17, and/or the pharmaceutical composition according to claim 18.

20. Use of the antibody or fragment thereof according to any one of claims 6 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to is claim 16, the pharmaceutical composition according to claim 17, and/or the conjugate or fusion protein according to claim 18 in the manufacture of a medicament for the prevention and/or treatment of cancer.

21. Use of the antibody or fragment thereof according to any one of claims 6 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to claim 16, the pharmaceutical composition according to claim 17, and/or the conjugate or fusion protein according to claim 18 in the manufacture of an agent for the diagnosis of cancer.

22. Use of the antibody or fragment thereof according to any one of claims 6 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to claim 16, the pharmaceutical composition according to claim 17, and/or the conjugate or fusion protein according to claim 18 in the manufacture of CAR-T cells.

23. A method for preventing and/or treating cancer, the method including administering to a subject in need thereof the antibody or fragment thereof according to any one of claims 6 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to claim 16, the pharmaceutical composition according to claim 17, and/or the conjugate or fusion protein according to claim 18, and optionally other drugs or means.

24. A method for diagnosing cancer, the method including contacting the antibody or fragment thereof according to any one of claims 6 to 13, the nucleic acid molecule according to claim 14, the vector according to claim 15, the host cell according to claim 16, the pharmaceutical composition according to claim 17, and/or the conjugate or fusion protein according to claim 18 with a sample from a subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Embodiments of the present disclosure are described in detail below with reference to the attached figures, in which:

[0108] FIG. 1 shows the results of an in vitro cell binding assay of anti-human CLDN18.2 chimeric antibodies.

[0109] FIG. 2 shows the results of a Complement Dependent Cytotoxicity (CDC) assay of anti-human CLDN18.2 humanized antibodies, in which panel 2A: CHOK1; panel 2B: BxPC3; panel 2C: NCI-N87.

[0110] FIG. 3 shows the results of an Antibody Dependent Cellular Cytotoxicity (ADCC) assay of anti-human CLDN18.2 humanized antibodies, in which panel 3A: CHOK1; panel 3B: BxPC3; panel 3C: NCI-N87.

[0111] FIG. 4 shows the result of analysis of the binding characteristics of the anti-human CLDN18.2 humanized antibodies to proteins of the same family.

[0112] FIG. 5 shows the binding of anti-human CLDN18.2 humanized antibodies at different concentrations to human CLDN18.1 expressing cells.

[0113] FIG. 6 shows the results of analysis of species cross binding characteristics of anti-human CLDN18.2 humanized antibodies.

[0114] FIG. 7 shows the results of detected positive percentages of CAR-T cells prepared in different groups.

[0115] FIG. 8 shows different antigen expression profiles of different CHO cell strains constructed.

[0116] FIG. 9 shows the results of detected IFN-γ in supernatants after the incubation of CAR-T cells in different groups with target cells.

[0117] FIG. 10 shows the results of detected CD25 expression on CAR-T cells in different groups after incubation with target cells.

DETAILED DESCRIPTION

[0118] Embodiments of the present disclosure are illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the present disclosure and do not limit the scope of the present disclosure in any way.

[0119] The experimental procedures in the following examples are all conventional, unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products, unless otherwise specified.

[0120] The heavy and light chain sequences of IMAB362 are as shown in SEQ ID NO. 126 and SEQ ID NO. 127.

[0121] Antigen human CLDN18.2 is as shown in NP 001002026.1, and antigen human CLDN18.1 is as shown in NP 057453.1.

[0122] The negative control (Isotype control) in the figures is anti-CD33 IgG antibody Lintuzumab in full length.

Example 1 Screening for Murine Monoclonal Antibodies

[0123] Balb/c mice were immunized with CHOK1 cells stably expressing human CLDN18.2 protein on cell surfaces. One month later, sera from the mice were analyzed by flow cytometry (FACS), and spleens were taken from mice having high antibody titers in sera. The spleen cells isolated by standard methods were fused with myeloma cells P3X63Ag8.653 using PEG or electrofusion methods. The fused hybridoma cells were seeded in 384 well plates, and after 10-14 days of culture, supernatants obtained were analyzed by FACS for antibody secretion by the hybridoma cells. Several clones were obtained that were able to bind to CHOK1 cells stably expressing human CLDN18.2 protein on cell surfaces and were not able to bind to CHOK1 cells stably expressing human CLDN18.1 protein on cell surfaces. Single cells of the obtained clones were obtained by Limiting Dilution, and each of the monoclonal hybridoma cell clones obtained after being diluted three times secreted only one antibody.

[0124] The monoclonal hybridoma cells secreting anti human CLDN18.2 were subjected to expansion culture, and the total RNA of the cells was extracted using RNAfast200 Kit (Shanghai Flytech Biotechnology Co., Ltd.) according to the steps described in the Kit instructions. The total RNA of the hybridoma cells obtained was reverse transcribed to cDNA using 5 xPrimeScript RT Master Mix (Takara). And sequences of antibody light chain variable region IgVL (κ) and heavy chain variable region VH were amplified using degenerate primers (Anke Krebber., 1997) and Extaq PCR reagents (Takara). PCR amplification products were purified using PCR Clean-up Gel Extraction Kit (Macherey-Nagel GmbH & Co.), linked to T-vector using pClone007 Simple Vector Kit (Tsingke Biotechnology Co., Ltd.) according to Kit instructions, and transformed into competent Escherichia coli cells. Variable region sequences of the monoclonal antibodies were obtained by DNA sequencing after strain amplification and plasmid extraction.

TABLE-US-00001 TABLE 1 Light and heavy chain variable regions of murine antibodies Murine antibody Heavy chain variable region (VH) Light chain variable region (VL) 11M23 SEQ ID NO: 1 SEQ ID NO: 2 16K15 SEQ ID NO: 3 SEQ ID NO: 4 18B21 SEQ ID NO: 5 SEQ ID NO: 6 20L17 SEQ ID NO: 7 SEQ ID NO: 8 43B5 SEQ ID NO: 9 SEQ ID NO: 10 43L6 SEQ ID NO: 11 SEQ ID NO: 12 46J05 SEQ ID NO: 13 SEQ ID NO: 14 48G12 SEQ ID NO: 15 SEQ ID NO: 16 50C14 SEQ ID NO: 17 SEQ ID NO: 18 52E22 SEQ ID NO: 19 SEQ ID NO: 20 8K13 SEQ ID NO: 21 SEQ ID NO: 22 >11M23_vh (SEQ ID NO: 1) QVLQKESGPDLVAPSQSLSITCTVSGFSLTNYGVHWVRQPPGKGLEWLVVIWSDGRINYNSALKSRLSITKDNSKRQVFLK MNSLQIDDTAIYYCVRHPAFGPHAMDYWGQGISVTVSS >11M23_vl (SEQ ID NO: 2) DIVMTQDAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPHLLLYRMSNPASGAPDRFSGSGSGTEFTLRIS RVEAEDVGVYYCMQYLEYPPTFGAGTRLELK >16K15_vl (SEQ ID NO: 3) EVMLVESGGGLVRPGGSLKLSCAGSGITLSTYAMSWVRQTPERRLEWVASIISGGITYYLDSVKGRFTISRDNARNILYLQ MSSLRSEDTAIYYCARKYHGNALDYWGQGTSVTVSL >16K15_vl (SEQ ID NO: 4) DIVMTQSPSSLPVTAGETVTMRCKSSQSLLNSGNQRNYLTWYQRKPGQPPKKLIYWASTRESGVPDRFTGSGSGTDFTLTI SGVQAEDLAVYYCQNNYFYPLTFGAGTKLELK >18B21_vh (SEQ ID NO: 5) QIQMVQSGPELKKPGETVRISCKASGYSFTTAGMQWVRKMPGEGLKWIGWIIAHSGEPKYTEDFKGRFAFSLETSASTTYL QISNLKNEDTATYFCARWGKGNTMDYWGQGTSVIVSS >18B21_vl (SEQ ID NO: 6) DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNGGNQRNYLTWYQQKPGQPPKLLIYWAST RESGVPDRFTGSGSGTHFTLTISSVQAEDLAVYYCQNAYFFPLTFGAGTKLELK >20L17_vh (SEQ ID NO: 7) DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTIYYPDTVKGRFTVSRDNPKNTLFL QMTSLRSEDTAMYYCVRLGPRGNVMDHWGQGTSVTVSS >20L17_vl (SEQ ID NO: 8) DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQRNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTI SSVQAEDLAVYYCQNVYFYPLTFGTGTKLELR >43B5_vh (SEQ ID NO: 9) DVQLQESGPDLVKPSQSLSLTCTVSGYSISGAYNWHWIRQFPGNKLEWLAYMQYSGSSNYNPSFKSRISISRDTSKNQFFL QLKSVTTEDTATYYCARMYNGNSFLYWGQGTLVTVSA >43B5_vl (SEQ ID NO: 10) DIVMTQSPSSLTVTAGEKVTMNCKSSQSLFNSGNQKNYLTWYQQKPGQPPRLLIYWASTRESGVPDRFTGSGSGTDFTLTI SSVQAEDLSLYYCQNSYSYPLTFGAGTKLELK >43L6_vh (SEQ ID NO: 11) QVQLKESGPDLVAPSQSLSLTCSVSGFSLTSYGIHWVRQPPGKGLEWLVVIWSDGRTTYNSGLKSRLSISKDNSKSQVLLK MNSLRTDDTAIYYCVRHPAFGPHAMDYWGQGTSVTVSS >43L6_vl (SEQ ID NO: 12) DIVMTQAAPSVPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLRIS RVEAGDVGVYYCMQYLEYPVTFGAGTKLELK >46J05_vh (SEQ ID NO: 13) DVQLVESGGGLVQPGGSRKLSCAASGFTFSRFGMHWVRQAPKKGLEWVAYISSGSNTIYYADTVKGRFTISRDNPKNTLFL QTTSLRSEDTAIYYCGRLGFYGNSFDHWGQGTLVTVSA >46J05_vl (SEQ ID NO: 14) NILMTQSPSSLTVTAGEKVTMNCKSSQSLLNGGNQRNYLTWYQQKAGQPPKLLIYWASTRESGVPDRFTGGGSGTDFTLTI SSVQAEDLALYYCQNSYYYPLTFGAGTKLELK >48G12_vh (SEQ ID NO: 15) EVQLRQSGPELVKPGASVKMSCKASGYTFTTYIINWVKQKPGQGLEWIGYINPYNDDTRYNERVKGKATLTSDKSSSTAYM ELSSLTSEDSAVYYCARFYFGNSFTYWGQGTLVTVSA >48G12_vl (SEQ ID NO: 16) DIVMTQSPSSLPVTVGERVTMTCKSSQGLFNSGNQRNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTI SSVQAEDLAIYYCQNNYIYPLTFGAGTKLELK >50C14_vh (SEQ ID NO: 17) EVQLRQSGPELVKPGASVKMSCKASGYTFTTYIINWVKQKPGQGLEWIGYINPYNDGTRYNERVKGKATLTSDKSSSTAYM ELSSLTSEDSAVYYCARFHFGNSFTYWGQGTLVTVSA >50C14_vl (SEQ ID NO: 18) DIVMTQSPSSLPVTTGEKVTMTCKSSQGLFNNGNQRNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFIGSGSGTDFTLTI SSVQAEDLAIYYCQNNYIFPLTFGAGTKLELK >52E22_vh (SEQ ID NO: 19) QIQLVQSGPELKKPGETVKISCKASGYTLTNYGMNWVRQAPGKGLKWMGWIRPNTGEPTYAEDFKGRFVFSLETSAATAYL QITNLKSEDTSTYFCARLYRGNTLDNWGQGTSVIVSS >52E22_vl (SEQ ID NO: 20) DIVMTQSPSSLTVTTGEKVTMSCKSSQNLLNSGNQRNYLTWYQQKPGQSPKLLIYWASTRESGVPYRFTGSGSGTDFTLTI SSVQTDDLAIYYCQNGYSFPFTFGSGTKLEIK >8K13_vh (SEQ ID NO: 21) QVHLQQSGAELVRPGSSVKISCKASGYAFSNYWMNWVRQRPGQGLEWIGQIYPGNGDTKYSGKFNSKDTLTADKSSNTAYM QLNSLTSEDSAVYFCARFYYGNVMDYWGQGTSVTVSS >8K13_vl (SEQ ID NO: 22) DIVLTQSPSSLTVTAGEKVTMSCKSSQTLLNGGNQKNYLTWYQQKSGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTI SSVQAEDLAVYYCQNGYSYPLTFGVGTKLELK

Example 2 Preparation of Anti-Human CLDN18.2 Chimeric Antibodies

[0125] The heavy chain variable region sequence of each murine anti-human CLDN18.2 monoclonal antibody and the heavy chain constant region sequence of the published human monoclonal antibody IgG1 subclass (see SEQ ID NO: 124) were spliced together and constructed into a mammalian cell expression vector. And the light chain variable region sequence of each murine anti-human CLDN18.2 monoclonal antibody and the light chain constant region sequence of the published human monoclonal antibody kappa subclass (see SEQ ID NO: 125) were spliced together and constructed into a mammalian cell expression vector. The constructed heavy chain and light chain vectors of the anti-human CLDN18.2 chimeric antibodies were mixed in pairs, HEK293 cells were transfected with the vectors using Polyethyleneimine (PEI), and cell supernatants were collected about 7 days later. Anti-human CLDN18.2 chimeric antibody proteins were obtained via Protein A purification.

[0126] The chimeric antibodies of embodiments of the present disclosure were named following a format “murine antibody abbreviation-xiIgG”.

Example 3 Cell Binding Assay In Vitro of Anti-Human CLDN18.2 Chimeric Antibodies

[0127] The chimeric anti-human CLDN18.2 antibodies were diluted 2-fold in gradient from an initial concentration of 100 nM and solutions of each antibody of 16 concentrations were obtained. 10 μl of the solutions of each antibody of each concentration was added to a 384-well plate. CHOK1 cells expressing CLDN18.2 on the cell surface were collected by centrifugation for 5 min at 100 g at room temperature, and the cells were washed with PBS containing 0.5% BSA once and then were centrifuged for 5 min at 100 g at room temperature. The cells were resuspended at a density of about 2×10.sup.6 cells/ml, and 10 μl was added to each well of the 384-well plate to which the antibody had been added. After incubation for 1 hour at 4° C., fluorescently labeled secondary goat anti-human IgG antibody was added. After continued incubation for 1 hour at 4° C., the mean fluorescence readings of the cell populations were analyzed by a flow cytometer.

[0128] The results showed that the chimeric antibodies had specific binding to the cells expressing human CLDN18.2 at a nM scale. See FIG. 1.

Example 4 Humanization of Anti-Human CLDN18.2 Murine Antibodies

[0129] Based on a comprehensive analysis of Kabat and Chothia antibody coding schemes, amino acid sequence regions of 6 complementarity-determining regions (CDRs) and framework regions supporting the conserved three-dimensional conformation of the heavy and light chains of each murine antibody were determined. Subsequently, the heavy chain variable region of the human antibody which mostly resembles the murine antibody, such as IGHV1|IGHJ4*01, was searched for and selected in known human antibody sequences. The framework region sequences were selected as a template, and the heavy chain CDRs of the murine antibody were combined with the framework regions of the human antibody, and a humanized heavy chain variable region sequence was ultimately produced. In the same manner, a humanized light chain variable region sequence was produced.

[0130] An Antibody with murine CDRs grafted directly to its human framework regions often exhibits a dramatic decrease in binding activity, thus requiring the conversion of individual amino acids in the framework regions from being human back to murine. In order to determine which positions need to be reverted to original murine residues, the designed humanized antibody sequence and the original murine antibody sequence should be compared to check for differences in the amino acids, and to check whether those different amino acids are important for supporting the antibody structure or for binding to the antigen. The sequences obtained by humanization design need to be checked for potential post-Translational Modification Sites (PTMs), such as an N (asparagine) glycosylation site, an N-deamidation site, a D (aspartic acid) isomerization site, etc.

[0131] The gene of each humanized heavy chain variable region sequence was constructed into a mammalian cell expression vector comprising the gene of the heavy chain constant region sequence of the human monoclonal antibody IgG1 subclass. And the gene of each humanized light chain variable region sequence was constructed into a mammalian cell expression vector comprising the gene of the light chain constant region sequence of the human monoclonal antibody kappa subclass. The constructed heavy chain and light chain vectors of the humanized anti-human CLDN18.2 antibodies were mixed in pairs, HEK293 cells were transfected with the vectors using Polyethyleneimine (PEI), and cell supernatants were collected about 7 days later. Anti-human CLDN18.2 humanized antibody proteins were obtained via Protein A purification.

[0132] Humanized antibodies of embodiments of the present disclosure were named following the format “murine antibody abbreviation-hz”. Antibodies having CDRs from murine antibodies grafted directly to their human framework regions were named following the format “murine antibody abbreviation-hz00”; and antibodies further engineered were numbered with the numbers of the humanized sequences.

[0133] The binding kinetic parameters of the chimeric antibodies and the humanized antibodies thereof to the antigen human CLDN18.2 were analyzed by a Fortebio (BLITZ pro1.1.0.28) instrument. Before performing the assay, an NTA bioprobe was soaked in PBS for 10 min, and the probe was then placed in PBS containing 100 nM antigen for 300 s to capture the His-tagged antigen. The probe was further subjected to a binding reaction with 100 nM antibody for a binding time of 400 s, and the probe was then transferred to PBS and subjected to a dissociation reaction for 600 s. When the assay was finished, data from which the response value of a blank control had been deducted were fitted to a 1:1 Langmuir binding model using software, and then the kinetic constants for antigen-antibody binding were calculated. The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Comparison of binding kinetics parameters following the humanization of murine antibodies K.sub.D K.sub.on K.sub.off K.sub.off/ Ab ID PTM Response (M) (1/Ms) (s.sup.−1) K.sub.off(Xi-IgG) 8K13-xiIgG Yes 0.134 3.82E−10 2.98E+05 1.14E−04 1.00 8K13-hz00 Yes 0.133 7.44E−10 2.66E+05 1.98E−04 1.74 8K13-hz11 Yes 0.139 2.97E−10 2.27E+05 6.73E−05 0.59 8K13-hz24 No 0.133 1.51E−09 2.64E+05 3.99E−04 3.50 11M23-xiIgG Yes 0.184 2.93E−10 3.66E+05 1.07E−04 1.00 11M23-hz00 Yes 0.164 1.49E−09 2.18E+05 3.24E−04 3.03 11M23-hz11 Yes 0.174 1.29E−09 3.56E+05 4.60E−04 4.30 11M23-hz22 No 0.136 2.92E−09 6.70E+05 1.95E−03 18.22 16K15-xiIgG Yes 0.261 5.57E−10 2.76E+05 1.54E−04 1.00 16K15-hz00 Yes 0.176 8.95E−11 2.40E+05 2.15E−05 0.14 16K15-hz11 Yes 0.17 5.35E−10 1.88E+05 1.00E−04 0.65 16K15-hz22 No 0.266 1.10E−09 2.59E+05 2.86E−04 1.86 52E22-xiIgG Yes 0.223 9.96E−11 3.57E+05 3.55E−05 1.00 52E22-hz00 Yes 0.188 3.09E−10 1.98E+05 6.11E−05 1.72 52E22-hz11 Yes 0.238 5.42E−10 3.63E+05 1.97E−04 5.55 52E22-hz12 No 0.226 3.60E−10 2.18E+05 7.83E−05 2.21

[0134] The results showed that the dissociation constant of 11M23-hz22 relative to the murine chimeric antibody was raised more than 10 fold after humanization and removal of the critical post translational modification site (PTM) and was therefore eliminated from the list of candidates. The dissociation constants of the remaining 8K13-hz24, 16K15-hz22 and 52E22-hz12 were raised 1 to 4 fold, compared to original murine chimeric antibodies and could be used as lead molecules for subsequent studies. The light chain amino acid sequence of 16K15-hz22 contained two consecutive “NN” residues, and further optimized mutant antibodies 16K15-hz22_2 and 16K15-hz22_3 were obtained by changing the “NN” residues to “SN” and “QN” residues, respectively.

TABLE-US-00003 TABLE 3 Light and heavy chain variable regions of humanized antibodies Humanized light chain Humanized heavy chain Humanized Murine antibody variable region variable region antibody 16K15 SEQ ID NO. 128 SEQ ID NO. 129 16K15-hz00 SEQ ID NO. 128, having SEQ ID NO. 129, having 16K15-hz11 reverse mutations at reverse mutation at position 24 positions 52 and 89 SEQ ID NO. 24 SEQ ID NO. 23 16K15-hz22 SEQ ID NO. 25 16K15-hz22_2 SEQ ID NO. 26 16K15-hz22_3 52E22 SEQ ID NO. 130 SEQ ID NO. 131 52E22-hz00 SEQ ID NO. 130, having SEQ ID NO. 27 52E22-hz11 reverse mutation at position 89 SEQ ID NO. 28 SEQ ID NO. 27 52E22-hz12 8K13 SEQ ID NO. 132 SEQ ID NO. 133 8K13-hz00 SEQ ID NO. 132, having SEQ ID NO. 133, having 8K13-hz11 reverse mutations at reverse mutations at positions positions 4 and 89 74 and 77 SEQ ID NO. 30 SEQ ID NO. 29 8K13-hz24 11M23 SEQ ID NO. 88 SEQ ID NO. 84 11M23-hz00 SEQ ID NO. 88, having SEQ ID NO. 84, having 11M23-hz11 reverse mutation at reverse mutation at position position 41 71 SEQ ID NO. 88, having SEQ ID NO. 84, having 11M23-hz22 reverse mutation at reverse mutation at position position 41 as well as 71 as well as D54E and N60Q N31S and N33S >16K15_vl_hz2 (SEQ ID NO: 24) DIVMTQSPDSLAVSLGERATINCKSSQSLLSSGNQRNYLTWYQQKPGQPPKKLIYWASTRESGVPDRFSGSGSGTDFTLTISS LQAEDLAVYYCQNNYRYPLTFGQGTKLEIK >16K15_vl_hz2_N-S (SEQ ID NO: 25) DIVMTQSPDSLAVSLGERATINCKSSQSLLSSGNQRNYLTWYQQKPGQPPKKLIYWASTRESGVPDRFSGSGSGTDFTLTISS LQAEDLAVYYCQSNYFYPLTFGQGTKLEIK >16K15_vl_hz2_N-Q (SEQ ID NO: 26) DIVMTQSPDSLAVSLGERATINCKSSQSLLSSGNQRNYLTWYQQKPGQPPKKLIYWASTRESGVPDRFSGSGSGTDFTLTISS LQAEDLAVYYCQQNYFYPLTFGQGTKLEIK >16k15_vh_hz2 (SEQ ID NO: 23) EVQLVESGGGLVQPGGSLRLSCAGSGITLSTYAMSWVRQAPGKGLEWVSSIISGGITYYLDSVKGRFTISRDNAKNTLYLQMN SLRAEDTAVYYCARKYHGNALDYWGQGLTVTVSS >52E22_vl_hz2 (SEQ ID NO: 28) DIVMTQSPDSLAVSLGERATINCKSSQNLLSSGNQRNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS LQAEDLAVYYCQQGYSFPFTFGQGTKLEIK >52E22_vh_hz1 (SEQ ID NO: 27) QIQLVQSGSELKKPGASVKVSCKASGYTLTNYGMNWVRQAPGQGLEWMGWIRPNTGEPTYAEDFKGRFVFSLDTSVATAYLQI TSLKAEDTAVYYCARLYRGNTLDNWGQGTLVTVSS >8K13_vl_hz4_N-S_N-Q (SEQ ID NO: 30) DIVLTQSPDSLAVSLGERATINCKSSQTLLSGGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS LQAEDLAVYYCQQGYSYPLTFGQGTKLEIK >8K13_vh_hz2 (SEQ ID NO: 29) QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYWMNWVRQAPGQGLEWMGQIYPGSGDTKYSGKFQSRVTITADKSTNTAYMEL SSLRSEDTAVYYCARFYYGNVMDYWGQGTLVTVSS

Example 5 Kinetic Experiment of Binding Affinity In Vitro of Anti-Human CLDN18.2 Humanized Antibodies

[0135] Antibody-antigen interactions were measured using BIAcore 5200 from GE Healthcare.

[0136] Referring to the instructions provided in the Human Antibody Capture Kit (cat No. BR-1008-39, Lot 10261753) from GE Healthcare, the analytical channel and the control sample channel on a CM5 sensor chip were first saturated and coupled with the maximum amount of anti-human Fc antibody, then buffers containing anti-human CLDN18.2 chimeric antibodies, humanized antibodies or a control antibody IMAB362 at 7.5 μg/ml were allowed to flow through the analytical channel and uniformly distribute. Finally, antigen samples diluted in gradient (the initial concentration was 20 nM and diluted by 1:3 to get 8 concentrations, and the concentration 0.741 nM was set to be repeated) were allowed to flow through both the analytical channel and the sample channel, and the photoreactions upon the binding of antibody to antigen were measured. Subsequently, the association constant Kon and dissociation constant Koff and affinity constant KD of each antibody were finally obtained by instrument software fitting analysis.

[0137] The results showed that the in vitro binding affinity constants of the anti-human CLDN18.2 humanized antibodies were not significantly different from those of the original murine antibodies, but were lower than that of IMAB362 by one order of magnitude. See Table 4.

TABLE-US-00004 TABLE 4 Binding kinetics of antibodies of the present disclousre Antibody ka (M−1s−1) kd (s−1) KD (M) 8K13-xiIgG 8.19E+04 5.73E−04 6.99E−09 8K13-hz24 7.09E+04 2.02E−03 2.85E−08 16K15-xiIgG 1.07E+05 1.44E−03 1.35E−08 16K15-hz22 5.71E+04 5.63E−04 9.85E−09 52E22-xiIgG 4.90E+05 5.45E−04 1.11E−09 52E22-hz12 1.46E+05 1.72E−04 1.18E−09 IMAB362 1.48E+06 0.201 1.36E−07

Example 6 Cytology Assay In Vitro of Anti-Human CLDN18.2 Humanized Antibodies

[0138] 6.1 Complement Dependent Cytotoxicity (CDC)

[0139] The humanized antibodies of embodiments of the present disclosure were analyzed for their ability to induce complement-dependent cytotoxicity (CDC) on CHOK1, BxPC3 and NCI-N87 cells stably expressing human CLDN18.2 using a commercial human serum whole complement available by Quidel, Inc.

[0140] Cells were mixed with the antibody to be tested at a final concentration ranging from 250m/ml to 3.8 ng/ml. Whole human serum at a concentration of 6.25% dissolved in cell culture medium RPMI-1640 was added into the mixture which was then incubated for 3 hours at 37° C. Cytotoxicity was measured by a CCK-8 kit. Finally, absorbance at 450 nm was measured by a MD plate reader. EC50 values of the samples were calculated by plotting 4-parameter fitting curves with the absorbance values using the softmax pro? software.

[0141] The results showed that the anti-human CLDN18.2 humanized antibodies had specific Complement Dependent Cytotoxicity (CDC) against target expressing cells and their cell killing activities were significantly superior to that of IMAB362. See FIG. 2 and Table 5.

TABLE-US-00005 TABLE 5 CDC of the antibodies of embodiments of the present disclosure Target expressing cells a. CHOK1 b. BxPC3 c. NCI-N87 Antibody EC50 (nM) EC50 (nM) EC50 (nM) 8K13-hz24 IgG 21.27 28.53 57.7  16K15-hz22 IgG 5.2 12.22 17.36 52E22-hz12 IgG 2.08 NA NA IMAB362 30.45 52.23 474.2 

[0142] 6.2 Antibody Dependent Cellular Cytotoxicity (ADCC)

[0143] Engineered Jurkat cells stably expressing a FcγRIIIa-FcεIαγ hybrid receptor and expressing firefly Luciferase under driving by the NFAT response element were used as effector cells. The biological activity of an antibody in an ADCC mechanism is quantified by Luciferase produced by activation of the NFAT pathway. 1.5E5 effector cells were mixed with the antibody to be tested at a final concentration ranging from 33 μg/ml to 85 pg/ml, and then 2.5E4 target cells were added into the mixture (effector to target E: T ratio was 6:1), which was then incubated for 16 hours at 37° C. Cytotoxicity was measured by a kit, Bio-Glo™ Luciferase Assay System from Promega; and LUM values were finally determined by a MD plate reader.

[0144] The data were processed as follows: Fold induction=(reading value of the well detected−background value)/(reading value of the negative control well−background value). EC50 values of the samples were calculated by plotting 4-parameter fitting curves with the data.

[0145] The results showed that the anti-human CLDN18.2 humanized antibodies had specific Antibody Dependent Cellular Cytotoxicity (ADCC) against target expressing cells and their cell killing activities were comparable to that of IMAB362. See FIG. 3 and Table 6.

TABLE-US-00006 TABLE 6 ADCC of the antibodies of embodiments of the present disclosure Target expressing cells a. CHOK1 b. BxPC3 c. NCI-N87 Antibody EC50 (nM) EC50 (nM) EC50 (nM) 8K13-hz24 IgG 0.5894 0.3045 0.1499 16K15-hz22 IgG 0.7122 0.1759 0.0677 52E22-hz12 IgG 0.5672 NA NA IMAB362 0.5929 0.1056 0.0909

Example 7 Analysis of Binding Characteristics of the Anti-Human CLDN18.2 Humanized Antibodies to Proteins of the Same Family

[0146] The genes of human CLDN18.2 and CLDN18.1 were constructed, respectively, into eukaryotic expression vectors, and HEK293 cells were transfected with the vectors using Polyethyleneimine (PEI). Three days after the transfection, the cells were collected by centrifugation, washed with PBS once, and resuspended at a cell density of 2×10.sup.6/ml. 10 μl was added to each well of a 384-well plate and then humanized antibodies at different concentrations were added into the wells. After incubation for 1 hour at 4° C., fluorescently labeled secondary goat anti-human IgG antibody was added. After continued incubation for 1 hour at 4° C., the mean fluorescence readings of the 384-well plate were read by a flow cytometer. The data were analyzed to obtain the binding characteristics of the cells to the anti-human CLDN18.2 humanized antibodies. The positive control used for the experiment was the commercial anti-human CLDN18 rabbit mAb 34H14L15 (available from Abcam corporation) and the negative Isotype control was the anti-CD33 IgG antibody Lintuzumab in full-length.

[0147] The results showed that the anti-human CLDN18.2 humanized antibodies exhibited characteristics of binding to human CLDN18.2 but not to human CLDN18.1. See FIG. 4. And the non-specific binding to human CLDN18.1 cells at low, medium and high concentrations was all lower than that of IMAB362. See FIG. 5.

Example 8 Analysis of Human, Monkey, and Murine Species Cross Binding Characteristics of the Anti-Human CLDN18.2 Humanized Antibodies

[0148] The genes of human, murine and Rhesus CLDN18.2 were constructed, respectively, into eukaryotic expression vectors, and HEK293 cells were transfected with the vectors using Polyethyleneimine (PEI). The cells were collected 2 days later. The binding specificity of the cells to anti-human CLDN18.2 humanized antibodies was analyzed using flow cytometry. See Example 7 for the procedure of flow cytometry.

[0149] The results showed that the anti-human CLDN18.2 humanized antibody 16K15-22 IgG bound to CLDN18.2 of all three species, and 8K13-24 IgG and 52E22-12 IgG bound to Rhesus CLDN18.2 but not to mouse CLDN18.2. The results are shown in FIG. 6.

Example 9 Preparation of CAR-T Cells Using Anti-Human CLDN18.2 Humanized Antibodies and Activation Thereof

[0150] 9.1 Lentivirus Packaging

[0151] Lentivirus packaging was performed according to the grouping shown in Table 7. Lentiviral vector plasmids pLTR containing different antibody genes were first constructed, and their plasmid DNA was extracted using a plasmid extraction kit from Qiagen after the DNA was confirmed correct by sequencing. The plasmid DNA was dissolved in sterile TE, and its concentration and purity were determined by UV light absorption, ensuring A260/A280 of the extracted plasmid DNA was between 1.8 and 2.0. The DNA of two helper packaging element plasmids, pCMV-VSV-G and pCMV-dR8.2, was extracted as well. HEK293T cells for transfection were prepared to obtain freshly passaged cells which should grow to a confluency of about 60%. Three plasmids were co-transferred into HEK293T cells using calcium phosphate as a transfection reagent. 48 hours after transfection, cell supernatant containing the packaged virus was collected by centrifugation at low temperature, and cell debris was removed using a 0.45 μm filter. The virus was concentrated using an ultrafiltration centrifuge tube, and sub-packaged and stored in a refrigerator at −80° C. A small amount of the virus concentrate was taken and the virus titer was determined by FACS.

TABLE-US-00007 TABLE 7 Lentivirus groupings No. Virus Virus titer Group 135 LV-135.N 8.75E+06 Negative control 417 LV-417.N 1.65E+07 16k15 (16K15-hz22) scfv* 418 LV-418.N 1.38E+07 52E22 (52E22-hz12) scfv* 419 LV-419.N 5.13E+07 8K13 (8K13-hz24) scfv* 420 LV-420.N 1.50E+07 Positive control

[0152] The negative control shown in Table 7 was another antibody not binding CLDN18.2 and the positive control was antibody hu8E5 from Carsgen Therapeutics (see WO2018006882A1). *: The single chain antibody was formed by linking humanized VH and VL via a short peptide (GST SGGGSGGGSGGGGS S).

[0153] 9.2 Preparation of CAR-T Cells

[0154] A healthy donor detected negative for HBV, HCV and HIV was selected. 100 ml of blood was collected from the antecubital vein, and Ficoll density gradient centrifugation was performed to isolate the white layer containing PBMCs. CD3+ T cells were isolated using DynaBeads CD3/CD28 (LifeTechnologies, Cat. No. 40203D) at a ratio of 3:1 of DynaBeads: CD3+ T cells. 24 hours after activation, the percentage of CD25+CD69+ T cells was detected by flow cytometry. When activated, the CD3+ T cells were transduced with the lentiviruses at an MOI of 5. A 24-well plate was coated with Novonectin for 2 hours at 37° C.; the cell suspension obtained by the procedure above was formulated into a suspension for transduction with various lentiviruses (MOI=5), SYNPERONIC® F108 (Sigma, Cat. No. 07579-250G-F), and Tscm (2 U/ml). The suspension for transduction was added into the 24-well plate with the density of cells adjusted to 1.0E+06/ml. The plate was centrifuged at 500 g for 30 min, and placed in an incubator at 37° C., 5% CO.sub.2 for static culture for 48 h. After the transduction, the cells were cultured in 5% FBS X-vivol5 medium (LONZA, Cat. No. 04-418Q), supplemented with Tscm (at a final concentration of 2 U/ml) on alternate days. The cells were counted, adjusted to 0.5E+06/ml, and harvested on Day 8-10 of culture.

[0155] The results of positive percentages of CAR-T cells in each group obtained are shown in FIG. 7.

[0156] 9.3 Activation of CAR-T Cells

[0157] CHO cells expressing antigen human CLDN18.1 and antigen human CLDN18.2 were constructed and used as target cells.

[0158] Detection results of antigen expression of the cells are shown in FIG. 8. CHO-BLANK (blank control) and CHO-CLDN18.1 had no expression of antigen CLDN18.2, and CHO-CLDN18.2 had a high expression of antigen CLDN18.2.

[0159] The effector cells with density adjusted as described above, including each group of CAR-T cells and T cells without lentivirus transduction were mixed with the target cells in 1.5 mL centrifuge tubes, respectively, at an effective to target ratio of 16:1. Total volumes were replenished to 200 μL using T cell broth X-vivol5 (free of autologous serum and Tscm); and then 200 μL of the systems was respectively transferred into a 96-well plate V-bottom for a 24-hour co-incubation.

[0160] After the incubation, the supernatant of each culture system was taken for the detection of human IFNγ. A significant increase in IFNγ release was found in the CAR-T cells in group 417 after incubation with CHO-CLDN18.1 and CHO-CLDN18.2; and an increase in IFNγ release was found in the CAR-T cells in groups 418, 419, and 420 only after incubation with CHO-CLDN18.2. The results are shown in Table 8 and FIG. 9.

TABLE-US-00008 TABLE 8 Human IFNγ detected in the supernatants of the culture systems Group T: CHO-BLANK T: CHO-claduin18.1 T: CHO-claduin18.2 nc 1020.1 912 1095.2 135 1441.2 1458.6 1877 417 9016 60328.2 79178.6 418 3940.8 4540 78903.4 419 4486.2 4670.6 75516.8 420 7381.1 6973.2 78247

[0161] The CAR-T cells from each group were detected for T-cell activation marker proteins CD3/CD25. CD25 expression was found to be up-regulated in CAR-T cells in group 417 after incubation with CHO-CLDN18.1 and CHO-CLDN18.2, and up-regulated in CAR-T cells in groups 418, 419, and 420 CART only after incubation with CHO-CLDN18.2. See FIG. 10.

[0162] The above description of embodiments of the present disclosure is not intended to limit the present disclosure, and those skilled in the art may make various changes and modifications to the present disclosure without departing from the spirit of the present disclosure, which should fall within the scope of the appended claims.