CN114751984B

CN114751984B - Monoclonal antibody of targeted human Claudin18.2 protein and application thereof

Info

Publication number: CN114751984B
Application number: CN202210517876.8A
Authority: CN
Inventors: 谢海涛; 都晓龙; 马丽雅
Original assignee: Shenzhen Xiankangda Life Science Co ltd
Current assignee: Shenzhen Xiankangda Life Science Co ltd
Priority date: 2021-09-03
Filing date: 2021-09-03
Publication date: 2023-02-14
Anticipated expiration: 2041-09-03
Also published as: CN113788894A; CN114907482B; CN113788894B; CN114907482A; CN115057930B; CN114751984A; CN115057930A

Abstract

The invention relates to the technical field of biological antibodies, in particular to a monoclonal antibody targeting human Claudin18.2 protein and application thereof. The monoclonal antibody includes a heavy chain variable region HV and a light chain variable region LV, and the amino acid sequence and the nucleic acid sequence of the monoclonal antibody may be used in a single-stranded form or in a double-stranded form. The monoclonal antibody can specifically recognize human Claudin18.2 antigen and not recognize human Claudin18.1 antigen, has good specificity in human normal tissues, only combines with Claudin18.2 positive tissues, does not recognize other tissues and organs, and has no off-target risk. The monoclonal antibody has a specific antibody-mediated Complement Dependent (CDC) killing effect on Claudin18.2 positive target cells, and CAR-T prepared by the monoclonal antibody has a specific killing function on the Claudin18.2 positive target cells.

Description

Monoclonal antibody of targeted human Claudin18.2 protein and application thereof

The application is a divisional application with the application number of 202111031017X, the application date of 2021, 09.03 and the application name of the monoclonal antibody targeting human Claudin18.2 protein and the application thereof.

Technical Field

The invention relates to the technical field of biological cells, in particular to a monoclonal antibody of a targeted human Claudin18.2 protein, which can be used as a biological material to be applied to construction of other DNA vectors, expression of recombinant proteins, construction of cell lines and immune cells of chimeric antigen receptors; or coupling with other substances to be used as a biological agent, or directly using as the biological agent.

Background

Claudins is a family of proteins that function to maintain tight junctions that control the exchange of molecules between cells, are widely distributed in the stomach, pancreas and lung tissues, and can be used for disease diagnosis and treatment. The CLDN18.2 (i.e., claudin 18.2) subtype, as a gastric-specific subtype, has become an ideal target since Sahin found that CLDN18.2 is a highly selective molecule and is only widely expressed in cancer cells. CLDN18.2 is usually buried in the gastric mucosa, monoclonal antibodies in normal tissues are substantially inaccessible, and the development of malignant tumors can result in the disruption of tight junctions, exposing the CLDN18.2 epitope on the surface of tumor cells to become a specific target. Thus, CLDN18.2 confers specificity to targeted therapies. Expression recently found in pancreatic (50%), esophageal and lung cancers also show potential for diagnosis and treatment of other tumors.

Claudin-18 has two splice variants, claudin18.1 and Claudin18.2, respectively, with only eight amino acid differences between the sequences. The expression profiles of Claudin18.1 and Claudin18.2 are different, with Claudin18.1 being selectively expressed in cells of normal lung and Claudin18.2 being highly restricted in normal cells, but frequently ectopically activated and overexpressed in a variety of tumors (e.g., gastric, lung, pancreatic, etc.). The structure of Claudin protein includes four transmembrane regions, two extracellular loops, the N-terminal and C-terminal of which are in cytoplasm, and two extracellular loops which make it an ideal antibody target.

Disclosure of Invention

Based on this, there is a need to address the above problems, and one of the problems to be solved by the present invention is to provide a monoclonal antibody targeting the human claudin18.2 protein, which antibody specifically recognizes claudin18.2, but not claudin18.1.

The second problem to be solved by the present invention is to provide a biomaterial comprising the nucleic acid sequence or amino acid sequence of a monoclonal antibody;

the third problem to be solved by the present invention is to provide a biological agent comprising a monoclonal antibody.

The first scheme of the invention is as follows:

a monoclonal antibody targeting a human Claudin18.2 protein, comprising a heavy chain variable region HV and a light chain variable region LV, the amino acid sequence and the nucleic acid sequence of the monoclonal antibody are as follows:

a) HV nucleic acid sequence:

ATGGCTGTCCTGGCGCTACTCCTCTGCCTGGTGACTTTCCCAAGCTGTGCCCTGTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCTGGGTTCTCATTA AACAGCTATATTATAAACTGGGTTCGCCAGTCACCAGGAAAGGGTCTGGACTGGCTTGGAGTAATATGGACTGGTG GAGGCACAAATTATAATTCAGCGCTCAAATCCAGACTGAGCATCACCAAAGACAACTCCAAGAGTCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAGGGGCCTATTATGGTAATGCTATG GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCCA; wherein A-CDR1 representsGGGTTCTCATTAAACAGCTATATT(ii) a A-CDR2 representsATATGGACTGGTGGAGGCACA(ii) a A-CDR3 representsGCCAGAGGG GCCTATTATGGTAATGCTATGGACTAC；

a) HV amino acid sequence:

MAVLALLLCLVTFPSCALSQVQLKESGPGLVAPSQSLSITCTVSGFSLNSYIINWVRQSPGKGLDWLGVIWTGGGTNYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARGAYYGNAMDYWGQGTSVTVSS; wherein a-CDR1 representsGFSLNSYI(ii) a a-CDR2 representsIWTGGGT(ii) a a-CDR3 representsARGAYYGNAMDY；

B) LV nucleic acid sequence:

ATGGAATCACAGACTCAGGTCCTCATGTCCCTGCTGTTCTGGGTATCTGGTACCTGTGGGGACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAACTGCAAGTCCAGTCAGAGT CTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGCTTATAGTTATCCATTCACGTTCGGCTCGGAGACAAAGTTGGAATAAAA; wherein B-CDR1 representsCAGAGTCTGTTAAACAGTGGAAATCAAAAGAAC TAC(ii) a B-CDR2 representsTGGGCATCC(ii) a B-CDR3 representationCAGAATGCTTATAGTTATCCATTCACG；

b) LV amino acid sequence:

MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMNCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNAYSYPFTFGSETKLEIK; wherein b-CDR1 representsQSLLNSGNQKNY(ii) a b-CDR2 representsWAS(ii) a b-CDR3 representsQNAYSYPFT；

C) HV nucleic acid sequence:

ATGGCTGTCCTGGCGCTACTCCTCTGCCTGGTGACTTTCCCAAGCTGTGCCCTGTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCTGGGTTCTCATTA ACTAGTTATGTTATAAACTGGGTTCGCCAGCCACCAGGAAAGGGTCTGGAGTGGCTTGGAGTAATATGGACTGGTG GAGGCACAAATTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAAGACACCTCCAAGAGTCAAGTTTTCTTAAAGATGAACAGTCTGCAAACTGATGACACAGCCAGGTATTACTGTGCCAGAGGGGCCTACTATGGTAATGCTATG GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA; wherein C-CDR1 representsGGGTTCTCATTAACTAGTTATGTT(ii) a C-CDR2 representsATATGGACTGGTGGAGGCACA(ii) a C-CDR3 representationGCCAGAGGG GCCTACTATGGTAATGCTATGGACTAC；

c) HV amino acid sequence:

MAVLALLLCLVTFPSCALSQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYVINWVRQPPGKGLEWLGVIWTGGGTNYNSALKSRLSISKDTSKSQVFLKMNSLQTDDTARYYCARGAYYGNAMDYWGQGTSVTVSS; wherein c-CDR1 representsGFSLTSYV(ii) a c-CDR2 representsIWTGGGT(ii) a c-CDR3 representsARGAYYGNAMDY；

D) LV nucleic acid sequence:

ATGGAATCACAGACTCAGGTCCTCATGTCCCTGCTGTTCTGGGTATCTGGTACCTGTGGGGACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGT CTGTTAAACAGTGGAAATCAAAAGAACTACTTAACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAATTGTTGATCTATTGGGCAGCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATAGTTATCCATTCACGTTCGGCTCGGGGACAAAAATTGGAAATAAAA; wherein D-CDR1 representsCAGAGTCTGTTAAACAGTGGAAATCAAAAGAAC TAC；D-CDR2 representsTGGGCAGCC(ii) a D-CDR3 representationCAGAATGATTATAGTTATCCATTCACG；

d) LV amino acid sequence:

MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWAATRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK; wherein d-CDR1 representsQSLLNSGNQKNY(ii) a d-CDR2 representsWAA(ii) a d-CDR3 representsQNDYSYPFT；

E) HV nucleic acid sequence:

ATGGGATGGAGCTATATCATCCTCTTTTTGGTAGCAACATCTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCCTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACTTTC ACCAGCTACTGGATGCAGTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAATGACTCATCCTAACA GTGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACTGGGTCGGGGAAATGCTATG GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA; wherein E-CDR1 representsGGCTACACTTTCACCAGCTACTGG(ii) a E-CDR2 representationACTCATCCTAACAGTGGTGGTACT(ii) a E-CDR3 representationGCAAGA CTGGGTCGGGGAAATGCTATGGACTAC；

e) HV amino acid sequence:

MGWSYIILFLVATSTGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGMTHPNSGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARLGRGNAMDYWGQGTSVTVSS; wherein e-CDR1 representsGYTFTSYW(ii) a e-CDR2 representationTHPNSGGT(ii) a e-CDR3 representationARLGRGNAMDY；

F) LV nucleic acid sequence:

ATGGAATCACAGACTCAGGTCCTCATGTCCCTGCTGTTCTGGGTATCTGGTACCTGTGGGGACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGT CTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGACAATTTATTACTGTCAGAATGATTATAGTTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAATAAAA; wherein F-CDR1 representsCAGAGTCTGTTAAACAGTGGAAATCAAAAGAAC TAC(ii) a F-CDR2 representsTGGGCATCC(ii) a F-CDR3 representationCAGAATGATTATAGTTATCCATTCACG；

f) LV amino acid sequence:

MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRKSGVPDRFTGSGSGTDFTLTISSVQAEDLTIYYCQNDYSYPFTFGSGTKLEIK; wherein f-CDR1 representsQSLLNSGNQKNY(ii) a f-CDR2 representsWAS(ii) a f-CDR3 representsQNDYSYPFT；

G) HV nucleic acid sequence:

ATGAACTTCGGGCTCAGATTGATTTTCCTTGTCCTTACTTTAAAAGGTGTCCAGTGTGACGTGAAGTTGGTGGAGTCTGGGGAAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCGCTGGATTCACTTTC AGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTAGTGGTG GTGATTACATCTACTATGCAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAGGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTTCAAGACTCAGGCTACGTGGAGGAAAT GCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA; wherein G-CDR1 representsGGATTCACTTTCAGTAGCTATGCC(ii) a G-CDR2 representsATTAGTAGTGGTGGTGATTACATC(ii) a G-CDR3 representsTCAAGA CTCAGGCTACGTGGAGGAAATGCTATGGACTAC；

g) HV amino acid sequence:

MNFGLRLIFLVLTLKGVQCDVKLVESGEGLVKPGGSLKLSCAAAGFTFSSYAMSWVRQTPEKRLEWVAYISSGGDYIYYADTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCSRLRLRGGNAMDYWGQGTSVTVSS; wherein g-CDR1 representsGFTFSSYA(ii) a g-CDR2 representsISSGGDYI(ii) a g-CDR3 representsSRLRLRGGNAMDY；

H) LV nucleic acid sequence:

ATGGAATCACAGACTCAGGTCCTCATGTCCCTGCTGTTCTGGGTATCTGGTACCTGTGGGGACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAAGTCACTATGAGCTGCAAGTCCAGTCAGAGT CTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATGCTTATCCGCTCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAA; wherein H-CDR1 representsCAGAGTCTGTTAAACAGTGGAAATCAAAAGAAC TAC(ii) a H-CDR2 representationTGGGCATCC(ii) a H-CDR3 representationCAGAATGATTATGCTTATCCGCTCACG；

h) LV amino acid sequence:

MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYAYPLTFGTGTKLELK; wherein h-CDR1 representsQSLLNSGNQKNY(ii) a h-CDR2 representsWAS(ii) a h-CDR3 representsQNDYAYPLT。

Preferably, the monoclonal antibody has an amino acid sequence selected from the group consisting of: any one of the amino acid sequences a), c), e) and g) of the heavy chain variable region HV is double-stranded with any one of the amino acid sequences b), d), f) and h) of the light chain variable region LV, respectively; or any single strand of the amino acid sequences a), b), c), d), e), f), g), h).

Preferably, the monoclonal antibody has a nucleic acid sequence selected from the group consisting of: any one of the nucleic acid sequences A), C), E), G) of the heavy chain variable region HV is double-stranded in combination with any one of the nucleic acid sequences B), D), F), H) of the light chain variable region LV, respectively; or any single strand of the nucleic acid sequences A), B), C), D), E), F), G), H).

Preferably, the monoclonal antibody is of murine origin, and has three complementarity determining regions in the heavy chain variable region HV and the light chain variable region LV corresponding to the amino acid sequence and the nucleic acid sequence, respectively, and the complementarity determining regions are the amino acid sequence and the nucleic acid sequence corresponding to CDR1, CDR2, and CDR3, respectively.

Preferably, the monoclonal antibody, when humanized or otherwise engineered to be of animal origin, has no more than 20% change in the amino acid and/or nucleic acid sequence of the three complementarity determining regions CDR1, CDR2, CDR 3; or, when the monoclonal antibody is humanized or modified from other animal sources, the amino acid sequence and/or the nucleic acid sequence of CDR1, CDR2 and CDR3 of the three complementarity determining regions are changed into 0.

The invention also provides a biological material, which comprises an expression cassette, a recombinant vector, a recombinant protein, a recombinant microorganism or a recombinant cell line constructed by the nucleic acid sequence or the amino acid sequence, wherein the nucleic acid sequence or the amino acid sequence is derived from the monoclonal antibody.

Preferably, in the biological material, the recombinant vector comprises a gene recombinant expression vector and a chimeric antigen receptor.

The invention also provides a biological agent containing the monoclonal antibody.

Preferably, the biological preparation also comprises a reagent for detecting the concentration of the human Claudin18.2 protein, a reagent for detecting the expression degree of the human Claudin18.2 protein on the surface of tumor cells, a reagent for carrying out antibody-mediated complement-dependent or cell-dependent cytotoxic reaction on human Claudin18.2 positive cells, a reagent for killing the human Claudin18.2 positive cells by antibody-coupled toxins, a functional recombinant protein for targeting the human Claudin18.2 and other antigen polyclonal antibodies prepared by antibody coupling other antibodies, and a chimeric antigen receptor immune cell for targeting the human Claudin18.2 positive cells prepared by antibody coupling other proteins.

The main advantages of the monoclonal antibody of the invention include:

1. has the ability to specifically recognize human Claudin18.2 protein and not to recognize Claudin18.1;

2. the monoclonal antibody has good tissue specificity, only identifies and expresses Claudin18.2 tissues, and does not identify other tissues and organs, thereby showing that the monoclonal antibody has good detection application prospect;

3. the monoclonal antibody has a specific antibody-mediated Complement Dependent (CDC) killing effect on Claudin18.2 positive target cells, which indicates that the monoclonal antibody has a good application prospect of monoclonal antibody therapy;

4. the CAR-T prepared by the monoclonal antibody has a specific killing function on Claudin18.2 positive target cells, and the monoclonal antibody has a good application prospect in cell immunotherapy;

5. the monoclonal antibody has good targeting property in both a natural configuration state and a recombinant configuration state, and the monoclonal antibody has good application prospects of antibody drug conjugates, single-resistance drugs, multiple-resistance drugs and recombinant antibody proteins.

Drawings

FIGS. 1a, 1b, 1c, 1d, 1e and 1f show respectively the expression negative of 293T cell Claudin18.2, expression negative of 293T cell Claudin18.1, positive of 293T-hClaudin18.1, expression hClaudin18.1 of 293T-hClaudin18.2, expression hClaudin18.2 of 293T-hClaudin18.2, positive of 293T-mClaudin18.1, expression mClaudin18.1 of 293T-mClaudin18.2, and expression laudin18.2 of 293T-mClaudin 18.2;

FIG. 2 shows the affinity assay results of the murine mAb of example 1;

FIGS. 3a, 3b, 3c, 3d, and 3e show the species-specific detection results of the murine monoclonal antibodies; wherein, FIG. 3a is a graph of binding activity of murine mAb to 293T cells; FIG. 3b is a graph showing the binding activity of murine mAb to 293T-mClaudin18.1 cells; FIG. 3c is a graph showing the binding activity of murine mAb to 293T-mClaudin18.2 cells; FIG. 3d is a graph showing the binding activity of murine mAb to 293T-hClaudin18.1 cells; FIG. 3e is a graph showing the binding activity of murine mAb to 293T-hClaudin18.2 cells;

FIG. 4 shows the result of tissue-specific detection of murine monoclonal antibody;

FIGS. 5a, 5b, 5c, 5d, and 5e are the results of detecting CDC effect of murine monoclonal antibody; wherein, FIG. 5a is a 293T-hClaudin18.2 cell natural activity rate graph; FIG. 5B is a graph showing the activity rate of 293T-hClaudin18.2 cells and 1B8 murine monoclonal antibody; FIG. 5c is a graph showing the activity rate of 293T-hClaudin18.2 cells and 1B10 murine monoclonal antibody; FIG. 5d is a graph showing the activity rate of 293T-hClaudin18.2 cells and 1A6 murine monoclonal antibody; FIG. 5e is a graph showing the activity rate of 293T-hClaudin18.2 cells +2B5 murine mAb;

FIG. 6 is the CAR-Claudin18.2 structure;

FIG. 7 is an expansion curve of the immune cells of example 1;

FIGS. 8a, 8b, 8c, 8d, 8e, 8f are representations of CAR; wherein, fig. 8a is a NT cell (control cell) CAR expression negative map; FIG. 8b is a map of the positive rate of CAR expression in CAR-007 cells; FIG. 8c is a graph of the positive rate of CAR expression in CAR-1B8 cells; FIG. 8d is a map of the positive rate of CAR expression in CAR-1B10 cells; FIG. 8e is a graph of the positive rate of CAR expression in CAR-1A6 cells; FIG. 8f is a map of the positive rate of CAR expression in CAR-2B5 cells;

FIG. 9a is a graph of CAR-T vs. 293T cell killing;

FIG. 9b is a graph of CAR-T vs. 293T-Claudin18.2 cell killing.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following more detailed description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In the present invention, the monoclonal antibody (or abbreviated as monoclonal antibody) is derived from mouse source, and the technical scheme is illustrated and described.

In the present invention, the immune cell may be any one of T cell, NK cell, NKT cell, macrophage, gamma-delta T cell, TIL cell and TCR-T cell.

The invention provides a monoclonal antibody targeting human Claudin18.2 protein, which comprises a heavy chain variable region HV and a light chain variable region LV, and the amino acid sequence and the nucleic acid sequence of the monoclonal antibody are as follows:

the antibody sequences are shown in the following table 1:

TABLE 1 amino acid sequence and nucleic acid sequence listing of monoclonal antibodies

In the invention, letters such as A, B, C, D, E, F, G, H, a, B, C, D, E, F, G, H and the like before CDR1, CDR2 and CDR3 are only serial number identification numbers, are only limited in distinguishing action and have no other meanings.

For the amino acid sequences of monoclonal antibodies, the following corresponding combinatorial selections can be made at the time of use:

any one of the amino acid sequences a), c), e) and g) of the heavy chain variable region HV is double-stranded with any one of the amino acid sequences b), d), f) and h) of the light chain variable region LV, respectively; or any single strand of the amino acid sequences a), b), c), d), e), f), g), h).

For the amino acid double-strand combination use, for example, when the amino acid sequence of the heavy chain variable region HV is a), the amino acid sequence of the light chain variable region LV is the sequence b); or, when the amino acid sequence of the heavy chain variable region HV is c), the amino acid sequence of the light chain variable region LV is the sequence d); or, when the amino acid sequence of the heavy chain variable region HV is e), the amino acid sequence of the light chain variable region LV is the sequence f); alternatively, when the amino acid sequence of the heavy chain variable region HV is g), the amino acid sequence of the light chain variable region LV is the sequence h), and the like.

In another embodiment, the nucleic acid sequences of the monoclonal antibodies are selected for use in the corresponding combinations as follows: the nucleic acid sequence A), C), E) of the heavy chain variable region HV is used in combination with any of the nucleic acid sequences B), D), F) and H) of the light chain variable region LV; or any single strand of the nucleic acid sequences A), B), C), D), E), F), G), H).

For the nucleic acid double-stranded combination use, for example, when the nucleic acid sequence of the heavy chain variable region HV is A), the nucleic acid sequence of the light chain variable region LV is the sequence B); or, when the nucleic acid sequence of the heavy chain variable region HV is C), the nucleic acid sequence of the light chain variable region LV is sequence D); or, when the nucleic acid sequence of the heavy chain variable region HV is E), the nucleic acid sequence of the light chain variable region LV is the sequence F); alternatively, when the nucleic acid sequence of the heavy chain variable region HV is G), the nucleic acid sequence of the light chain variable region LV is the sequence H), and the like.

Specifically, for example, the amino acid sequences in the light and heavy chain variable regions of the monoclonal antibody are used in pair and expressed as murine monoclonal antibody, preferably the expression pairs are:

1) Sequence A and sequence B are paired, and the expression and purification mouse monoclonal antibody is named as 1B8;

2) Sequence C and sequence D are paired, and the expression and purification mouse monoclonal antibody is named as 1B10;

3) Sequence E and sequence F are paired, and the expression and purification mouse monoclonal antibody is named as 1A6;

4) Sequence G and sequence H were paired, and the expressed purified murine mab was named 2B5.

Preferably, since the monoclonal antibody is of murine origin, the heavy chain variable region HV and the light chain variable region LV have three complementarity determining regions, respectively, corresponding to the amino acid sequence and nucleic acid sequence, respectively, CDR1, CDR2, and CDR 3. Accordingly, the monoclonal antibody, when humanized or otherwise engineered to be of animal origin, does not alter the amino acid and/or nucleic acid sequence of the three

CDRs

1, 2, 3 by more than 20%; or, when the monoclonal antibody is humanized or modified from other animal sources, the amino acid sequence and/or the nucleic acid sequence of CDR1, CDR2 and CDR3 of the three complementarity determining regions are changed into 0.

The invention also provides a biological material, which comprises an expression cassette, a recombinant vector, a recombinant protein, a recombinant microorganism or a recombinant cell line which are constructed by the nucleic acid sequence or the amino acid sequence, wherein the nucleic acid sequence or the amino acid sequence is derived from the monoclonal antibody; wherein, in the biological material, the recombinant vector comprises a gene recombinant expression vector and a chimeric antigen receptor.

The invention also provides a biological preparation containing the monoclonal antibody, and the biological preparation also comprises a reagent for detecting the concentration of the human Claudin18.2 protein, a reagent for detecting the expression degree of the human Claudin18.2 protein on the surface of a tumor cell, a complement-dependent or cell-dependent cytotoxic reaction reagent for carrying out antibody-mediated reaction on a human Claudin18.2 positive cell, an antibody-coupled toxin for killing the human Claudin18.2 positive cell, and a chimeric antigen receptor immune cell which is prepared by antibody-coupled other proteins and targets the human Claudin18.2 positive cell.

The antibody development application of the present invention will be described in detail below typically using CAR-T cells as an example.

The antibody expansion application of the invention is not limited to CAR-T cells as described above and below, and immune cells have the same or similar technical features and benefits as CAR-T cells as described above and below. Specifically, when the immune cell expresses the chimeric antigen receptor CAR, the NK cells, NKT cells, TIL, gamma-delta T cells are identical to T cells (or T cells can replace NK cells).

The design of chimeric antigen receptors CARs goes through the following process:

the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and due to the single intracellular activation domain, it caused only transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and thus did not achieve good clinical efficacy.

The second generation CARs introduce a costimulatory molecule such as CD28, 4-1BB, OX40 and ICOS on the basis of the original structure, and compared with the first generation CARs, the function is greatly improved, and the persistence of CAR-T cells and the killing capability to tumor cells are further enhanced. On the basis of second generation CARs, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series.

Third and fourth generation CARs, and also dual or multiple CARs that express targeting 2 or more targets on the same cell, and the like

The Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain. Wherein the extracellular domain comprises an antigen binding domain, the intracellular domain comprises a costimulatory signaling region, the intracellular domain of a cytokine receptor, and a portion of the CD3 zeta chain, the costimulatory signaling region comprising a portion of the intracellular domain of a costimulatory molecule, and the costimulatory molecule is a cell surface molecule required for an effective response by lymphocytes to an antigen.

In a preferred embodiment, the extracellular domain of the CAR provided herein comprises the light and heavy chain variable region antigen binding domain of murine mab targeting claudin18.2. When expressed in T cells, the CARs of the invention are capable of antigen recognition based on antigen binding specificity or protein receptor binding. The antigen binding domain is preferably fused to an intracellular domain from a costimulatory molecule and the CD3 zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of CD28, 4-1BB, ICOS signaling domain, and CD3 zeta signaling domain, respectively.

In the CAR of the present invention, the intracellular domain includes the signaling domains of CD28, 4-1BB, ICOS, and CD3 zeta.

The vector for the expression cassette is selected from: DNA, RNA, plasmid, slow virus vector, adenovirus vector, retrovirus vector, transposon, other gene transfer system. Preferably, the vector is a viral vector.

The nucleic acid sequence of the vector encoding the desired molecule may be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The present invention also provides a vector for inserting an expression cassette, a vector derived from a retrovirus such as lentivirus, characterized by long-term, stable integration of a gene of interest into a cell; transducible non-proliferating cells, such as hepatocytes; low immunogenicity; the safety is high. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The expression vector may be provided to the cell in the form of a viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence, another example is elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV 40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebutan-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. A preferred method for introducing the polynucleotide into the host cell is lipofection. The nucleic acid may be associated with a lipid, the nucleic acid associated with a lipid may be encapsulated into the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising a lipid, the lipid being a fatty substance, which may be a naturally occurring or synthetic lipid. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

The invention provides CAR-T cells comprising the targeted claudin18.2 antibody as described above, and a pharmaceutically acceptable carrier, diluent, or excipient.

In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10 ³ ～1×10 ⁸ The concentration of CAR-T cells is more preferably 1X 10 ⁴ ～1×10 ⁷ Each/ml. In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

The invention includes therapeutic applications of cells (e.g., T cells) transduced with lentiviral vectors encoding the antibody constructs of the invention. The transduced T cells can elicit CAR-mediated T-cell responses. The injected cells are capable of killing the recipient's tumor cells, and the CAR-T cells are capable of replicating in vivo, resulting in long-term persistence that can lead to sustained tumor control. And the CAR-T cell membrane can express the scFv structural antigen chimeric receptor of the murine monoclonal antibody targeting Claudin18.2, specifically kills Claudin18.2 positive target cells and has no off-target risk.

In one embodiment, the immune cells of the invention can undergo robust T cell expansion in vivo and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, claudin18.2 CAR-T cells elicit a specific immune response against cells expressing claudin18.2.

Treatable conditions include tumor cells positively expressed by claudin18.2. Symptoms that may be caused by tumor cells include, but are not limited to, carcinomas, blastomas, sarcomas, and the like.

CAR immune cells using the antibodies of the invention, ex vivo procedures to modify T cells, at least one of the following occurs in vitro prior to administration of the cells into a human: i) Expanding the cell, ii) introducing into the cell a nucleic acid encoding the CAR and a nucleic acid encoding an immune checkpoint antibody protein fused to a cytokine receptor expressed on the cell membrane, and/or iii) cryopreserving the cell. Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells isolated from human peripheral blood are used to express the CARs disclosed herein and an immune checkpoint antibody protein fused to a cytokine receptor expressed on the cell membrane. T-modified cells can be administered to a recipient to provide a therapeutic benefit. Second, the immune cells may be autologous with respect to the recipient. Alternatively, the cell may be allogeneic, syngeneic (syngeneic) or xenogeneic with respect to the recipient.

Immune cell CAR-modified T cells using the antibodies of the invention can be administered alone or in combination with other drugs, pharmaceutical compositions, diluents, and/or with other components such as IL-2, IL-17, or other cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical composition made of the immune cells using the antibody of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, and by the clinical protocol. When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis and individual variability of the condition of the patient (subject). It is possible to generally point out: pharmaceutical compositions comprising T cells described herein may be at 1 × 10 ⁴ ～1×10 ⁹ Dosage per kg body weight, preferably 1X 10 ⁵ ～1×10 ⁶ Administered at a dose of one/kg body weight. The T cell composition may also be administered multiple times at these doses. Optimal dosages and treatment regimens for a particular patient may be determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the formulations of the invention may 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, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

The following are descriptions of specific embodiments.

Example 1

The preparation method and the functional verification of the antibody of the embodiment comprise the following steps:

s100: mouse immunization and mouse monoclonal antibody sequence acquisition

Balbc mice were immunized with the self-produced Claudin18.2 protein and immunized by subcutaneous multiple injections of 20 ug/mouse per dose, once per week. After 4 weeks of immunization, mice with blood antibody titers of 1k or more were sacrificed and spleen cells were fused with SP2.0 cells. Performing titer detection on the fusion cells, screening positive fusion cells, selecting monoclone by using a limiting dilution method, performing antibody fermentation and function evaluation, extracting genes from the finally screened murine monoclonal antibody, constructing antibody light and heavy chain expression plasmid fermentation purified antibodies, and constructing CAR plasmid.

S200: fermentation and purification of mouse monoclonal antibody

And (3) pairing the constructed light and heavy chain expression plasmids of the antibody, using a PEI reagent as a transfection reagent, simultaneously carrying out antibody protein fermentation on the light and heavy chain expression plasmids of a pair of transient transfection 293T cells, naturally pairing the light and heavy chains of the antibody in the fermentation process, and collecting a culture solution. The culture broth was filtered through a 0.22 μm sterile filter to obtain a culture supernatant, which was then purified by affinity chromatography.

The purification method comprises the following steps: (1) 20mM PB, pH7.0 equilibrium 1ml-Protein A affinity column, 1.0ml/min; (2) Loading the culture solution 50ml,1ml/min, and balancing with balancing solution; (3) Eluting with 0.1M glycine-HCl (pH2.7) at 1ml/min, and collecting 280nm absorption peak; (4) 20-50mM PB,150mM NaCl, pH6.7 balance Superdex 200Increatase 10/300GL column, 1ml/min; and (5) loading, and collecting 2ml of 280nm absorption peak.

And (4) carrying out HPLC analysis on the purified sample, detecting a receiving peak, detecting the concentration of the purified output peak, and calculating the amount of the protein.

S300: cell line culture

Cell lines that do not express claudin18.1 and claudin18.2: 293T (human embryonic kidney cell line), purchased from ATCC.

Cell line expressing human claudin18.1: 293T-hClaudin18.1, constructed by self-produced lentivirus infection.

Cell line expressing human claudin18.2: 293T-hClaudin18.2, constructed by self-produced lentivirus infection.

Cells expressing murine claudin18.1: 293T-mClaudin18.1, transient transformation from a self-produced plasmid.

Cells expressing murine claudin18.2: 293T-mClaudin18.2, self-produced plasmid transient.

Packaging cells: 293T (human embryonic kidney cell line) purchased from ATCC.

Establishment of cell lines overexpressing human claudin18.1 and human claudin18.2: cloning base sequences expressing hClaudin18.1 and hClaudin18.2 into a PHBV lentiviral vector skeleton, placing the skeleton under a promoter of EF1 alpha (EF-1 alpha) to form PHBV-EF 1 alpha-hClaudin18.1 and PHBV-EF 1 alpha-hClaudin18.2, and transferring three plasmids of PHBV-EF 1 alpha-hClaudin18.1 and PHBV-EF 1 alpha-hClaudin18.2, a lentiviral envelope Plasmid pMD2.G (Addge, plasmid # 12259) and a lentiviral packaging Plasmid psPAX2 (Addge Plasmid # 12260) into 293T by using Lipofectamine3000 to prepare a lentiviral complete expression vector; viral supernatants were collected at 48h and 72h, concentrated by ultracentrifugation (Merck Millipore); the concentrated virus was used to infect 293T and a 293T cell line, designated 293T-hClaudin18.1 and 293T-hClaudin18.2, which was either singly over-expressing hClaudin18.1 or hClaudin18.2 was finally obtained.

Cell construction expressing murine claudin18.1 and murine claudin18.2: cloning base sequences expressing the mClaudin18.1 and the mClaudin18.2 into a PHBV lentiviral vector skeleton, placing the skeleton under a promoter of EF1 alpha (EF-1 alpha) to form PHBV-EF 1 alpha-mClaudin18.1 and PHBV-EF 1 alpha-mClaudin18.2, and transferring the plasmids of the PHBV-EF 1 alpha-mClaudin18.1 and the PHBV-EF 1 alpha-mClaudin18.2 into 293T for transient expression by using Lipofectamine 3000; and detecting the cell positive rate after 72h to finally obtain 293T cells transiently expressing the mClaudin18.1 or the mClaudin18.2, and the 293T cells are named as 293T-mClaudin18.1 and 293T-mClaudin18.2 and used for detecting the antibody species specificity. All the above cell culture media were cultured: DMEM medium was used for the culture. All media were supplemented with 10% (v/v) fetal bovine serum.

FIGS. 1a, 1b, 1c, 1d, 1e and 1f show the expression negative for 293T cell Claudin18.2, expression negative for 293T cell Claudin18.1, expression positive for 293T-hClaudin18.1, expression positive for 293T-hClaudin18.2, expression positive for 293T-mClaudin18.1, and expression positive for 293T-mClaudin18.2.

FIG. 1a shows the result of detection of 293T cells using Claudin18.2 antibody, wherein the position of the peak pattern is shown on the left side of the vertical line, indicating that the detection result is negative to Claudin18.2; FIG. 1b shows the result of detection of 293T cells using the Claudin18.1 antibody, wherein the position of the peak pattern is on the left side of the vertical line, indicating that the detection result is negative to Claudin18.1; the results of the two FIG. 1a and FIG. 1b tests show that 293T cells show no expression of Claudin18.1 and Claudin18.2; FIG. 1c shows the result of detection of 293T-hClaudin18.1 cells using hClaudin18.1 antibody, in which the peak positions are on the right side of the vertical line, indicating that the detection result is positive for hClaudin 18.1; FIG. 1d shows the results of detection of 293T-hClaudin18.2 cells using hClaudin18.2 antibody. The position of the peak pattern in the figure is on the right side of the vertical line, and the detection result is positive for hClaudin18.2; in figure 1e, the 293T-mClaudin18.1 cell uses hClaudin18.1 antibody to detect the result, because the sequence of the mClaudin18.1 and hClaudin18.1 at the antibody detection site is completely consistent, the hClaudin18.1 antibody can be used to detect the mClaudin18.1, and the 293T-mClaudin18.1 cell is transient cell, the expression of which is not completely positive, the peak position in the figure is mClaudin18.1 positive at the right side of the vertical line, and is mClaudin18.1 negative at the left side of the vertical line, and the detection result shows that the 293T-mClaudin18.1 cell positive rate is 26.55%; in FIG. 1f, the result of detection of 293T-mClaudin18.2 cells by using the hClaudin18.2 antibody shows that the sequence of the mClaudin18.2 and the sequence of the hClaudin18.2 at the detection site of the antibody are completely consistent, the mClaudin18.2 antibody can be used for detecting the mClaudin18.2, the 293T-mClaudin18.2 cells are transient cells, the expression of the cells is not completely positive, the peak position in the figure is positive to the right side of the vertical line by the mClaudin18.2 and negative to the left side of the vertical line by the mClaudin18.2, and the result of detection shows that the positive rate of the 293T-mClaudin18.2 cells is 33.88%.

S400: evaluation of affinity and species specificity of mouse monoclonal antibody

The EC50 affinities of Claudin18.2 murine mAbs 1B8, 1B10, 1A6, 2B5 were determined using 293T-hClaudin18.2 cells as positive cells, mIgG2B as isotype control, 007 antibody as positive control, and the results are shown in FIG. 2. The results show that 1B8, 1B10, 1A6, 2B5 all had higher EC50 affinities than control antibody 007.

The species specificity of Claudin18.2 murine mAbs 1B8, 1B10, 1A6, 2B5 was determined using 293T, 293T-hClaudin18.1, 293T-hClaudin18.2, 293T-mClaudin18.1, 293T-mClaudin18.2 cells as target cells and mIgG2B as isotype control, and the results are shown in FIGS. 3a, 3B, 3c, 3d, 3e.

In FIG. 3a, the binding peaks of murine mAbs 1B8, 1B10, 1A6, 2B5 and isotype control mIgG2B and 293T cells, respectively, are on the left of the vertical line, indicating that they are negative; in FIG. 3B, 1B8, 1B10, 1A6, 2B5 and isotype control mIgG2B and 293T-mClaudin18.1 cell binding peak pattern are all on the left of the vertical line, indicating that the binding is negative; in fig. 3c, the binding peak pattern of isotype control mIgG2B and 293T-mclaudin18.2 cell is on the left of the vertical line, indicating that it is negative, the binding peak pattern of 1B8, 1B10, 1A6, 2B5 and 293T-mclaudin18.2 cell is negative partially on the left of the vertical line, positive partially on the right of the vertical line, the positive rate is consistent with the positive rate of 293T-mclaudin18.2 cell, and the binding cell of 1B8, 1B10, 1A6, 2B5 is determined to be positive 293T-mclaudin18.2 cell by detecting, indicating that the 1B8, 1B10, 1A6, 2B5 antibody can bind to mclaudin18.2; FIG. 3d shows that the binding peak patterns of 1B8, 1B10, 1A6, 2B5 and isotype control mIgG2B and 293T-hClaudin18.1 cells are on the left of the vertical line, indicating that the binding is negative; in FIG. 3e, the peaks of the isotype control mIgG2B binding to 293T-hClaudin18.1 cells are shown on the left of the vertical line, indicating that the binding is negative, and the peaks of the murine mAbs 1B8, 1B10, 1A6, 2B5 binding to 293T-hClaudin18.2 binding are shown on the right of the vertical line, indicating that the 1B8, 1B10, 1A6, 2B5 antibodies can bind to hClaudin18.2.

The above results indicate that murine monoclonal antibodies 1B8, 1B10, 1A6, 2B5 can specifically bind to human and murine Claudin18.2 and not to Claudin18.1. The antibody has the specificity of two human and mouse species, and can be used for primarily evaluating the pathological toxicological characteristics in a mouse animal experiment in a subsequent experiment.

S500: tissue specificity detection evaluation of mouse monoclonal antibody

Immunohistochemistry evaluation was performed on the tissue specificity of the murine mab using a human normal tissue chip, which included 37 normal tissue types: esophageal tissue, gastric tissue, small intestinal tissue, colon tissue, hepatic tissue, pancreatic tissue, appendiceal tissue, tongue tissue, salivary gland tissue, pharyngeal mucosa, lung tissue, testicular tissue, prostate tissue, breast tissue, ovarian tissue, endometrial tissue, cervical canal tissue, cervical tissue, renal cortex tissue, renal medullary tissue, bladder tissue, tonsil tissue, lymph node tissue, thymus tissue, spleen tissue, skin tissue, skeletal muscle tissue, arterial tissue, pleural mesothelial tissue, peripheral nerve tissue, cerebellar tissue, white matter of the brain, gray matter of the brain, adrenal tissue, thyroid tissue, myocardial tissue. There are 102 tissue points. The detection result is shown in figure 4, and the result shows that the staining effect is only on the cell membrane surface of esophagus and stomach tissue (Claudin18.2 positive expression), and no cell membrane surface staining is generated on the other normal tissues, thereby proving that the tissue specificity is good.

S600: CDC effect detection and evaluation of mouse monoclonal antibody

Preparing mouse monoclonal antibody 1B8, 1B10, 1A6 and 2B5 working solution by using DPBS (platelet-derived polysubtility) of 20% human plasma, co-incubating with 293T-hClaudin18.2 cells for 30min, washing the cells after incubation, and detecting the proportion of living cells in an up-flow manner. As shown in fig. 5a, 5b, 5c, 5d, and 5e, the in-frame selected cells in each map were in the ratio of 81.34%, 6.41%, 34.01%, 3.94%, and 21.66%, respectively, to viable cells. The live cell ratio of 293T-hClaudin18.2 cells incubated by mouse monoclonal antibody 1B8, 1B10, 1A6 and 2B5 and human plasma is greatly reduced, which indicates that the 1B8, 1B10, 1A6 and 2B5 have antibody-mediated Complement Dependent Cytotoxicity (CDC).

S700: peripheral blood PBMC isolation and T cell culture.

Separating monocytes from donor peripheral blood, performing density gradient centrifugation using ficol, and enriching T cells with a T cell sorting kit (CD 3 MicroBeads, human-lysohimized, 130-097-043), activating the cultured and expanded T cells using magnetic beads coupled with anti-CD3/anti-CD 28; the Medium was supplemented with TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) containing 10% FBS,2mM L-glutamine,100IU/ml rhIL2, all cells were placed at 37 ℃ and 5% CO ₂ Culturing in a constant temperature incubator to obtain T cells.

S800: CAR structural design and lentiviral packaging.

Claudin18.2-CAR structure, i.e. CAR structure targeting claudin 18.2:

in the invention, four pairs of 1B8/1B10/1A6/2B5 targeting Claudin18.2 murine monoclonal antibody light-heavy chain variable region scFv sequences are respectively constructed on an expression frame of the second generation CAR. The core structure of the CAR includes a secretion signal peptide sequence; a CD8 transmembrane region; intracellular domain stimulatory signal 4-1BB-CD3 ζ, named CAR-1B8, CAR-1B10, CAR-1A6, CAR-2B5, respectively, with the control targeting the light and heavy chain variable region scFv of the Claudin18.2 antibody to construct the expression cassette of CAR as a control, named CAR-007, as shown in FIG. 6.

As shown in FIG. 6, claudin18.2 scFv/TM/4-1BB/CD3 ζ represents a chimeric antigen receptor expressed on the cell membrane of an immune cell.

Cloning the expression cassette into PHBVLV lentiviral vector backbone, placing under the promoter of EF1 alpha (EF-1 alpha) to form PHBVV-EF 1 alpha-CAR-1B 8, PHBVV-EF 1 alpha-CAR-1B 10, PHBVV-EF 1 alpha-CAR-1A 6, PHBVV-EF 1 alpha-CAR-2B 5 and PHBVV-EF 1 alpha-CAR-007, transferring three plasmids of PHBVV-EF 1 alpha-CAR-1B 8, PHBVV-EF 1 alpha-CAR-1B 10, PHBV-EF 1 alpha-CAR-1A 6, PHBVV-EF 1 alpha-CAR-2B 5 and PHBVV-EF 1 alpha-CAR-007, lentivirus envelope Plasmid pMD2.G (Addgene, plasmid # 12259) and lentivirus packaging Plasmid PAX2 (Addgene pssmid # 60) into Lipofectamine3000 expression vector 293T using Lipofectamine; after culturing the cells for 48h and 72h, transferring the cells into a centrifuge tube for centrifugation, collecting virus supernatant after the centrifugation is stopped, and performing ultracentrifugation concentration on the supernatant (Merck Millipore); the concentrated virus is ready for infecting T cells.

S900: CAR-T cell preparation.

9.1 Lentiviral infection

After the primary T cells isolated and purified in step S700 were reactivated for 1 day, lentivirus vector infection was performed by MOI (1-10) using 5 lentiviruses (Lv-CAR-1B 8, lv-CAR-1B10, lv-CAR-1A6, lv-CAR-2B5, lv-CAR-007) packaged in step S800, and virus-infected T cells were transferred to cell culture flasks at 37 ℃ and 5 CO ₂ Culturing in a constant temperature incubator.

9.2 cell proliferation and CAR Positive Rate detection

And (3) sampling and detecting the number of the cells on 6 th, 9 th, 11 th and 13 th days after the T cells are infected, detecting the CAR positive rate of the T cells on the 6 th day respectively, and subculturing and supplementing the culture medium every 1-2 days.

Using the 5 lentiviral vectors of step S800, 5 CAR-T cells were successfully constructed, designated CAR-1B8, CAR-1B10, CAR-1A6, CAR-2B5, CAR-007 with no lentivirus infected T cells as controls (NT).

As shown in FIG. 7, the growth rates of 5 types of cells were not significantly different from each other under the same culture conditions.

As shown in FIGS. 8a, 8B, 8c, 8d, 8e, and 8f, which represent the expression of the tested CARs on day 6 of NT cells, CAR-007, CAR-1B8, CAR-1B10, CAR-1A6, and CAR-2B5, respectively, the positive rate of the CAR is shown on the right side of the vertical line in the figure, and it can be seen from the figure that different scFv have no significant effect on the positive rate of the CAR.

S1000: CAR-T to 293T-hClaudin18.2 cell killing

Performing an in vitro killing experiment on the 5 CAR-T cells obtained in the step S900, detecting the killing effect of the CAR-T cells by adopting an LDH method (promega: G1780), incubating the target cells and the effector cells for 6h, wherein the killing efficiency is obvious, and the result is shown in figure 9, wherein in figure 9a, CAR-007, CAR-1B8, CAR-1B10, CAR-1A6 and CAR-2B5 have no killing effect on 293T cells, which indicates that the cells have no killing activity on non-target cells; it is shown in FIG. 9B that CAR-1B8, CAR-1B10, CAR-1A6, CAR-2B5 all had essentially comparable or superior killing effects on 293T-hClaudin18.2 cells compared to the control CAR-007.

The above-mentioned embodiments only express one embodiment of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Sequence listing

<110> Shenzhen Shenxian Shen Shenzhong Shenda Life sciences Limited

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100 105 110

Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Glu Thr

115 120 125

Lys Leu Glu Ile Lys

130

<210> 3

<211> 411

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 3

atggctgtcc tggcgctact cctctgcctg gtgactttcc caagctgtgc cctgtcccag 60

gtgcagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcaca 120

tgcactgtct ctgggttctc attaactagt tatgttataa actgggttcg ccagccacca 180

ggaaagggtc tggagtggct tggagtaata tggactggtg gaggcacaaa ttataattca 240

gctctcaaat ccagactgag catcagcaaa gacacctcca agagtcaagt tttcttaaag 300

atgaacagtc tgcaaactga tgacacagcc aggtattact gtgccagagg ggcctactat 360

ggtaatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a 411

<210> 11

<211> 137

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)

<400> 11

Met Ala Val Leu Ala Leu Leu Leu Cys Leu Val Thr Phe Pro Ser Cys

1 5 10 15

Ala Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala

20 25 30

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Thr Ser Tyr Val Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser

65 70 75 80

Ala Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Thr Ser Lys Ser Gln

85 90 95

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr

100 105 110

Tyr Cys Ala Arg Gly Ala Tyr Tyr Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Ser Val Thr Val Ser Ser

130 135

<210> 4

<211> 399

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 4

atggaatcac agactcaggt cctcatgtcc ctgctgttct gggtatctgg tacctgtggg 60

gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 120

atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttaacc 180

tggtaccagc agaaaccagg gcagcctcct aaattgttga tctattgggc agccactagg 240

gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 300

atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 360

ccattcacgt tcggctcggg gacaaaattg gaaataaaa 399

<210> 12

<211> 133

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)

<400> 12

Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr

20 25 30

Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ala Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr

100 105 110

Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr

115 120 125

Lys Leu Glu Ile Lys

130

<210> 5

<211> 411

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 5

atgggatgga gctatatcat cctctttttg gtagcaacat ctacaggtgt ccactcccag 60

gtccaactgc agcagcctgg ggctgagctg gtaaagcctg gggcctcagt gaagttgtcc 120

tgcaaggctt ctggctacac tttcaccagc tactggatgc agtgggtgaa gcagaggcct 180

ggacaaggcc ttgagtggat tggaatgact catcctaaca gtggtggtac taactacaat 240

gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg 300

caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag actgggtcgg 360

ggaaatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a 411

<210> 13

<211> 137

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)

<400> 13

Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ser Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Met Thr His Pro Asn Ser Gly Gly Thr Asn Tyr Asn

65 70 75 80

Glu Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Arg Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Ser Val Thr Val Ser Ser

130 135

<210> 6

<211> 399

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 6

atggaatcac agactcaggt cctcatgtcc ctgctgttct gggtatctgg tacctgtggg 60

gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 120

atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 180

tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 240

aaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 300

atcagcagtg tgcaggctga agacctgaca atttattact gtcagaatga ttatagttat 360

ccattcacgt tcggctcggg gacaaagttg gaaataaaa 399

<210> 14

<211> 133

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)

<400> 14

Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr

20 25 30

Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

Lys Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Thr Ile Tyr

100 105 110

Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr

115 120 125

Lys Leu Glu Ile Lys

130

<210> 7

<211> 417

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 7

atgaacttcg ggctcagatt gattttcctt gtccttactt taaaaggtgt ccagtgtgac 60

gtgaagttgg tggagtctgg ggaaggctta gtgaagcctg gagggtccct gaaactctcc 120

tgtgcagccg ctggattcac tttcagtagc tatgccatgt cttgggttcg ccagactcca 180

gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtgattacat ctactatgca 240

gacactgtga agggccgatt caccatctcc agagacaatg ccaggaacac cctgtacctg 300

caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgttcaag actcaggcta 360

cgtggaggaa atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 417

<210> 15

<211> 139

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)

<400> 15

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Val Lys Leu Val Glu Ser Gly Glu Gly Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ala Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu

50 55 60

Glu Trp Val Ala Tyr Ile Ser Ser Gly Gly Asp Tyr Ile Tyr Tyr Ala

65 70 75 80

Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn

85 90 95

Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met

100 105 110

Tyr Tyr Cys Ser Arg Leu Arg Leu Arg Gly Gly Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135

<210> 8

<211> 399

<212> DNA

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 8

atggaatcac agactcaggt cctcatgtcc ctgctgttct gggtatctgg tacctgtggg 60

gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaagtcact 120

atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 180

tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 240

gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 300

atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatgcttat 360

ccgctcacgt tcggtactgg gaccaagctg gagctgaaa 399

<210> 16

<211> 133

<212> PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)

<400> 16

Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr

20 25 30

Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr

100 105 110

Tyr Cys Gln Asn Asp Tyr Ala Tyr Pro Leu Thr Phe Gly Thr Gly Thr

115 120 125

Lys Leu Glu Leu Lys

130

Claims

1. A monoclonal antibody targeting the human claudin18.2 protein, wherein said monoclonal antibody comprises a heavy chain variable region and a light chain variable region; wherein the amino acid sequence of the heavy chain variable region HCDR1 is GFSLTSYV, the amino acid sequence of the heavy chain variable region HCDR2 is IWTGGGT, and the amino acid sequence of the heavy chain variable region HCDR3 is ARGAYYGNAMDY; the amino acid sequence of the light chain variable region LCDR1 is QSLLNSGNQKNY, the amino acid sequence of the light chain variable region LCDR2 is WAA, and the amino acid sequence of the light chain variable region LCDR3 is QNDYSYPFT.

2. <xnotran> 1 , , MAVLALLLCLVTFPSCALSQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYVINWVRQPPGKGLEWLGVIWTGGGTNYNSALKSRLSISKDTSKSQVFLKMNSLQTDDTARYYCARGAYYGNAMDYWGQGTSVTVSS; </xnotran> <xnotran> MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWAATRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK. </xnotran>

3. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or 2.

4. An expression cassette comprising the nucleic acid of claim 3.

5. A recombinant vector comprising the nucleic acid molecule of claim 3.

6. A recombinant microorganism comprising the nucleic acid molecule of claim 3.

7. A recombinant cell line comprising the nucleic acid molecule of claim 3.

8. The recombinant vector according to claim 5, wherein the recombinant vector comprises a recombinant gene expression vector.

9. The monoclonal antibody of claim 1, wherein the source of the monoclonal antibody is murine.

10. A biological agent comprising the monoclonal antibody of claim 1 or 2.

11. The biological agent according to claim 10, wherein the biological agent is a reagent for detecting the concentration of human Claudin18.2 protein, or a reagent for detecting the degree of expression of human Claudin18.2 protein on the surface of tumor cells, or a reagent for antibody-mediated complement-dependent or cell-dependent cytotoxic reaction of human Claudin18.2 positive cells.