CN112940124B

CN112940124B - Humanized monoclonal antibody targeting Claudin18.2 and preparation method and application thereof

Info

Publication number: CN112940124B
Application number: CN202110397828.5A
Authority: CN
Inventors: 代红久; 徐慧; 王梦瑶
Original assignee: Nanjing Kaidi Medical Technology Co ltd
Current assignee: Nanjing Kaidi Medical Technology Co ltd
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2022-04-05
Anticipated expiration: 2041-04-14
Also published as: CN112940124A

Abstract

The invention relates to a humanized monoclonal antibody targeting Claudin18.2, a preparation method and application thereof; according to the invention, through cell fusion and hybridoma technology, the obtained Claudin18.2 humanized monoclonal antibody can be combined with CHO-Claudin18.2 cells with high specificity, but hardly combined with CHO-Claudin18.1 cells, the preparation method of the humanized monoclonal antibody of the Claudin18.2 is simple in steps, and the obtained Claudin18.2 humanized monoclonal antibody has good ADCC and CDC activities on expressing Claudin18.2 positive tumor cells. The humanized monoclonal antibody of Claudin18.2 has high specificity and small side effect, and has good prospect in the application of treating and/or preventing or diagnosing diseases such as tumors related to Claudin18.2.

Description

Humanized monoclonal antibody targeting Claudin18.2 and preparation method and application thereof

Technical Field

The invention belongs to the technical field of immunotherapy biomedicine, and relates to a humanized monoclonal antibody targeting Claudin18.2, a preparation method and application thereof, in particular to application in treating and/or preventing or diagnosing diseases such as tumors related to Claudin18.2.

Background

Gastric cancer is one of the most common cancers worldwide, and accounts for the fourth (male) and fifth (female) among cancer-related death factors in developed countries. Most gastric cancer patients are already in an advanced stage after diagnosis. Early gastric cancer may be cured by gastrectomy, with a recurrence rate still reaching 50%. So far, advanced gastric cancer is incurable, and median survival time is 8-10 months after chemotherapy. Multiple chemotherapy regimens were studied to improve response and tolerance, however 5-year survival remains elusive.

Claudin-18(CLDN18) is a protein encoded by the CLDN18 gene in humans. It belongs to the claudins class. Claudin-18 belongs to a large family of Claudin proteins, which form tight connecting chains in epithelial cells. Claudin-18 plays a major role in the specific clearance of tight junctions from the intercellular spaces by calcium-independent cell adhesion activity.

The data presented show that Claudin18.2, as a highly specific cell surface molecule, is hardly expressed in normal tissues, only on differentiated gastric mucosal epithelial cells. The Claudin18.2 molecule is mostly expressed in primary gastric cancer and cancer types after metastasis, and activated expression of Claudin18.2 is also frequently observed in pancreatic cancer, esophageal cancer, ovarian cancer and lung cancer. Aberrant expression of claudin18.2 alters the normal structure and function of the claudin, thereby causing loss of cell polarity and allowing the diffusion of nutrients and other factors essential for tumor cell survival and growth, playing an important role in tumor cell metastasis and tumor cell nutrient supply. Meanwhile, Claudin18.2 acts as a cell membrane surface protein, and the exposed extracellular structure allows binding of antibodies, and these characteristics indicate that Claudin18.2 is an ideal target for development of a therapeutic monoclonal antibody (see Thorsten Klamp et al, Cancer Research (2011)).

Furthermore, for Claudin-18, there are two splice variants, Claudin18.1 and Claudin18.2, which differ only in eight amino acids between their first extracellular domains, and in the tissue site of expression. Therefore, in the development process of the Claudin18.2 medicament, the problem that the Claudin18.1 is easy to cause cross reaction to generate side effect is noticed. The unique expression profile of Claudin18.2 in normal tissues, as well as abnormal expression profiles in a variety of tumors, make it a very attractive target for anti-cancer therapy. Therefore, development of therapeutic antibodies against claudin18.2 has greater anticancer potential, lower toxicity, and greater space for optimal dosing.

Disclosure of Invention

In view of the above-mentioned problems and/or other problems of the related art, it is an object of the present invention to provide a humanized monoclonal antibody targeting claudin18.2 with good specificity, higher affinity and stability.

Another object of the present invention is to provide a gene encoding the above humanized monoclonal antibody targeting Claudin18.2.

The invention also aims to provide a preparation method of the humanized monoclonal antibody targeting Claudin18.2.

It is still another object of the present invention to provide an anti-tumor agent.

It is yet another object of the present invention to provide a diagnostic test agent.

In particular, it relates to

A humanized monoclonal antibody or a functional fragment thereof targeting claudin18.2 comprising a heavy chain variable region comprising amino acid sequences at least 80% identical in the following HCDR1, HCDR2 and HCDR3 sequences respectively and a light chain variable region comprising amino acid sequences at least 80% identical in the following LCDR1, LCDR2 and LCDR3 sequences respectively:

the amino acid sequence of HCDR1 is GFTFSSFG;

the amino acid sequence of HCDR2 is ISSGSHTI;

the amino acid sequence of HCDR3 is FQYGNSFDY;

the amino acid sequence of LCDR1 is QSLLNSGNQKNY;

the amino acid sequence of LCDR2 is WAS;

the amino acid sequence of LCDR3 is QNTYSFPLT;

or

The amino acid sequence of HCDR1 is GFTFSKFG;

the amino acid sequence of HCDR2 is FSSGGDY;

the amino acid sequence of HCDR3 is AKLYYGNSMDS;

the amino acid sequence of LCDR1 is QSLLNSGNQRNY;

the amino acid sequence of LCDR2 is WAS;

the amino acid sequence of LCDR3 was QNAYYYPFT.

The two sets of CDR sequences have partially identical characteristic sequences and can be combined with Claudin18.2, so that the requirement of uniqueness is met.

In one aspect, the present invention provides a humanized monoclonal antibody or a functional fragment thereof targeting claudin18.2, comprising a heavy chain variable region comprising an amino acid sequence with 1, 2 or 3 amino acid residues substituted, inserted or deleted in the following HCDR1, HCDR2 and HCDR3 sequences, respectively, and a light chain variable region comprising an amino acid sequence with 1, 2 or 3 amino acid residues substituted, inserted or deleted in the following LCDR1, LCDR2 and LCDR3 sequences, respectively:

the amino acid sequence of HCDR1 is GFTFSSFG;

the amino acid sequence of HCDR2 is ISSGSHTI;

the amino acid sequence of HCDR3 is FQYGNSFDY;

the amino acid sequence of LCDR1 is QSLLNSGNQKNY;

the amino acid sequence of LCDR2 is WAS;

the amino acid sequence of LCDR3 is QNTYSFPLT;

in a further aspect the present invention provides a humanized monoclonal antibody or a functional fragment thereof targeting claudin18.2, comprising a heavy chain variable region comprising an amino acid sequence with 1, 2 or 3 amino acid residues substituted, inserted or deleted in the following HCDR1, HCDR2 and HCDR3 sequences, respectively, and a light chain variable region comprising an amino acid sequence with 1, 2 or 3 amino acid residues substituted, inserted or deleted in the following LCDR1, LCDR2 and LCDR3 sequences, respectively:

the amino acid sequence of HCDR1 is GFTFSKFG;

the amino acid sequence of HCDR2 is FSSGGDY;

the amino acid sequence of HCDR3 is AKLYYGNSMDS;

the amino acid sequence of LCDR1 is QSLLNSGNQRNY;

the amino acid sequence of LCDR2 is WAS;

the amino acid sequence of LCDR3 was QNAYYYPFT.

In one aspect, the invention provides a humanized monoclonal antibody or functional fragment thereof targeting claudin18.2 comprising a heavy chain variable region comprising an amino acid sequence at least 80% identical in the sequence of HCDR1, HCDR2 and HCDR3, respectively, as described above, and a light chain variable region comprising an amino acid sequence at least 80% identical in the sequence of LCDR1, LCDR2 and LCDR3, respectively, as described above.

In one embodiment, the heavy chain variable region comprises an amino acid sequence having at least 81% to 99% identity to the HCDR1, HCDR2 and HCDR3 sequences described above, respectively.

In one embodiment, the heavy chain variable region comprises an amino acid sequence having at least 81% -99% identity to the LCDR1, LCDR2, and LCDR3 sequences described above, respectively.

In one embodiment, the present invention provides a humanized monoclonal antibody or functional fragment thereof targeting claudin18.2 comprising a heavy chain variable region comprising amino acid sequences as set forth in the following HCDR1, HCDR2 and HCDR3 sequences, respectively, and a light chain variable region comprising amino acid sequences as set forth in the following LCDR1, LCDR2 and LCDR3 sequences, respectively:

the amino acid sequence of HCDR1 is GFTFSSFG;

the amino acid sequence of HCDR2 is ISSGSHTI;

the amino acid sequence of HCDR3 is FQYGNSFDY;

the amino acid sequence of LCDR1 is QSLLNSGNQKNY;

the amino acid sequence of LCDR2 is WAS;

the amino acid sequence of LCDR3 is QNTYSFPLT;

in yet another embodiment, the present invention provides a humanized monoclonal antibody or functional fragment thereof targeting claudin18.2, comprising a heavy chain variable region comprising amino acid sequences as shown in the following HCDR1, HCDR2 and HCDR3 sequences, respectively, and a light chain variable region comprising amino acid sequences as shown in the following LCDR1, LCDR2 and LCDR3 sequences, respectively:

the amino acid sequence of HCDR1 is GFTFSKFG;

the amino acid sequence of HCDR2 is FSSGGDY;

the amino acid sequence of HCDR3 is AKLYYGNSMDS;

the amino acid sequence of LCDR1 is QSLLNSGNQRNY;

the amino acid sequence of LCDR2 is WAS;

the amino acid sequence of LCDR3 was QNAYYYPFT.

In one embodiment, the heavy chain variable region amino acid sequence is selected from the group consisting of: SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.19, SEQ ID NO.20 or SEQ ID NO. 21.

In one embodiment, the light chain variable region amino acid sequence is selected from the group consisting of: SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.23 or SEQ ID NO. 24.

In one embodiment, the humanized monoclonal antibody or functional fragment thereof targeting claudin18.2, its heavy chain variable region and light chain variable region are selected from the group consisting of:

the heavy chain variable region is SEQ ID NO.15 and the light chain variable region is SEQ ID NO. 17;

the heavy chain variable region is SEQ ID NO.15 and the light chain variable region is SEQ ID NO. 18;

the heavy chain variable region is SEQ ID NO.16 and the light chain variable region is SEQ ID NO. 17;

the heavy chain variable region is SEQ ID NO.16 and the light chain variable region is SEQ ID NO. 18;

the heavy chain variable region is SEQ ID NO.19 and the light chain variable region is SEQ ID NO. 22;

the heavy chain variable region is SEQ ID NO.19 and the light chain variable region is SEQ ID NO. 23;

the heavy chain variable region is SEQ ID NO.19 and the light chain variable region is SEQ ID NO. 24;

the heavy chain variable region is SEQ ID NO.20 and the light chain variable region is SEQ ID NO. 22;

the heavy chain variable region is SEQ ID NO.20 and the light chain variable region is SEQ ID NO. 23;

the heavy chain variable region is SEQ ID NO.20 and the light chain variable region is SEQ ID NO. 24;

the heavy chain variable region is SEQ ID NO.21 and the light chain variable region is SEQ ID NO. 22;

the heavy chain variable region is SEQ ID NO.21 and the light chain variable region is SEQ ID NO. 23; or

The heavy chain variable region is SEQ ID NO.21 and the light chain variable region is SEQ ID NO. 24.

In a preferred embodiment, the humanized monoclonal antibody or functional fragment thereof targeting claudin18.2, whose heavy chain variable region and light chain variable region are selected from the group consisting of:

the heavy chain variable region is SEQ ID NO.20 and the light chain variable region is SEQ ID NO. 22; or

The heavy chain variable region is SEQ ID NO.21 and the light chain variable region is SEQ ID NO. 23.

In a more preferred embodiment, the humanized monoclonal antibody or functional fragment thereof targeting claudin18.2, whose heavy chain variable region and light chain variable region are selected from the group consisting of:

In one embodiment, the invention provides an isolated polynucleotide encoding a humanized monoclonal antibody targeted to claudin18.2 of the present invention or a functional fragment thereof.

In one embodiment, the polynucleotide comprises a heavy chain coding sequence encoding the heavy chain variable region of the humanized monoclonal antibody targeted to claudin18.2 as described herein, and a light chain coding sequence encoding the light chain variable region of the humanized monoclonal antibody targeted to claudin18.2 as described herein.

In another aspect, the present invention provides an expression vector comprising said polynucleotide.

In another aspect, the invention provides a host cell comprising the expression vector.

In one embodiment, the host cell is a 293F cell.

In another aspect, the invention provides the use of said humanized monoclonal antibody or functional fragment thereof targeting claudin18.2, said polynucleotide, said expression vector or said host cell for the preparation of a medicament for the treatment of a tumor positive to claudin 18.2.

In another aspect, the invention provides the use of said humanized monoclonal antibody or functional fragment thereof targeting claudin18.2, said polynucleotide, said expression vector or said host cell for the preparation of a medicament for the treatment of tumors.

In another aspect, the invention provides the humanized monoclonal antibody or functional fragment thereof targeting Claudin18.2, the polynucleotide, the expression vector or the host cell for preparing a medicament for treating tumors.

In another aspect, the present invention provides an anti-tumor pharmaceutical composition comprising an effective amount of said humanized monoclonal antibody or functional fragment thereof targeting claudin18.2, and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a diagnostic kit or kit comprising an effective amount of said humanized monoclonal antibody or functional fragment thereof targeting claudin18.2 for use in the in vitro (e.g. cell or tissue) or in vivo (e.g. human or animal model) diagnosis of a disease associated with claudin18.2 (e.g. a tumor or a viral infection, such as a viral infection with high expression of claudin18.2 or a tumor with high expression of claudin 18.2).

In another aspect, the present invention provides a method for preparing said humanized monoclonal antibody or functional fragment thereof targeting claudin18.2, comprising:

(1) humanizing a murine antibody to obtain variable region coding sequences of heavy chains and light chains of the humanized monoclonal antibody or a functional fragment thereof targeting Claudin18.2;

(2) and (3) carrying out recombinant antibody production by using the variable region coding sequence to obtain the functional humanized monoclonal antibody or functional fragment thereof targeting Claudin18.2.

A method for preparing a humanized monoclonal antibody targeting Claudin18.2 is characterized in that a recombinant expression vector containing the coding gene is transfected into a competent cell and cultured to obtain the humanized monoclonal antibody targeting Claudin18.2.

The humanized monoclonal antibody targeting Claudin18.2 is obtained by the technical personnel of the invention through the following method:

1) using an immune mouse which recombinantly expresses human Claudin18.2 to obtain an immune response against the human Claudin18.2;

2) taking spleen cells of the mouse in the step 1) for fusion, and screening obtained hybridoma cells to obtain positive mother clone for specifically identifying human Claudin18.2;

3) subcloning the positive parent clone obtained in the step 2) to obtain a stable hybridoma cell strain;

4) sequencing the hybridoma cell strain obtained in the step 3) to obtain variable region coding sequences of the light chain and the heavy chain of the antibody.

5) And (4) selecting the optimal mouse monoclonal antibody to carry out humanization according to the results to obtain a humanized antibody sequence.

The humanized monoclonal antibody can be specifically combined with human Claudin18.2, and can greatly reduce the immunogenicity of a mouse monoclonal antibody.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Principle of action

The invention firstly obtains the monoclonal antibody of the mouse source through screening, the antibody has high affinity and high specificity, but because the monoclonal antibody of the mouse source can cause human immune reaction in actual clinical use, the humanization is very necessary. The invention carries out humanized modification on the murine monoclonal antibody to obtain a series of Claudin18.2 humanized monoclonal antibodies, wherein most of the antibodies have high affinity and high specificity to Claudin18.2 and have good ADCC and CDC effects on expressing Claudin18.2 positive tumor cells. Therefore, the target Claudin18.2 humanized monoclonal antibody provided by the invention can realize the aim of tumor immunotherapy by recognizing the Claudin18.2 antigen on the surface of a tumor cell.

Advantageous effects

According to the invention, through cell fusion and hybridoma technology, the obtained Claudin18.2 humanized monoclonal antibody can be combined with CHO-Claudin18.2 cells with high specificity, but hardly combined with CHO-Claudin18.1 cells, the preparation method of the humanized monoclonal antibody of the Claudin18.2 is simple in steps, and the obtained Claudin18.2 humanized monoclonal antibody has good ADCC and CDC activities on expressing Claudin18.2 positive tumor cells. The humanized monoclonal antibody of Claudin18.2 has high specificity and small side effect, and has good prospect in the application of treating and/or preventing or diagnosing diseases such as tumors related to Claudin18.2.

Drawings

FIG. 1 shows the results of the measurement of serum titer of mice after immunization in example 3. FIG. 1A shows the binding of immunized mice to CHO-Claudin18.2; FIG. 1B shows the binding of immunized mice to CHO cells.

FIG. 2 shows the results of affinity flow assay of hybridoma clones in example 5. FIG. 2A shows the binding of hybridoma clones to CHO-Claudin18.2 cells, FIG. 2B shows the binding of hybridoma clones to CHO-Claudin18.1 cells, and FIG. 2C shows the binding of hybridoma clones to CHO cells.

FIG. 3 shows the binding of hybridoma subclones to CHO-Claudin18.2 cells in example 6.

FIG. 4 shows the binding of hybridoma subclones to CHO-Claudin18.1 cells in example 6.

FIG. 5 shows a schematic diagram of the chimeric antibody expression vector in example 8.

FIG. 6 shows the flow verification results of the chimeric antibody of example 8, in which FIG. 6A shows the binding of the hybridoma clone to CHO-Claudin18.2 cell, FIG. 6B shows the binding of the hybridoma clone to CHO-Claudin18.1 cell, and FIG. 6C shows the binding of the hybridoma clone to CHO cell.

FIG. 7 shows the results of flow assay of 1-C4-2-F8-G6 hybridoma clone and humanized antibody expression thereof in example 11.

FIG. 8 shows the results of flow assay of 7-C5-1-C10-1-G7 hybridoma clone and humanized antibody expression thereof in example 11.

Fig. 9 shows the results of measuring the affinity and the specificity of the humanized monoclonal antibody in example 12, fig. 9A is the result of measuring the affinity of the humanized monoclonal antibody, and fig. 9B is the result of measuring the specificity of the humanized monoclonal antibody.

FIG. 10 shows the case of detecting the expression of human Claudin18.2 antigen on different tumor cells by the humanized monoclonal antibody in example 13.

FIG. 11 shows the luciferase labeling assay for ADCC activity in vitro of the humanized monoclonal antibody in example 14.

Fig. 12 shows luciferase labeling assay for CDC activity of humanized monoclonal antibody in vitro in example 15.

Detailed Description

The inventor finds that the antibody specifically combined with Claudin18.2 can be applied to the preparation of various targeting antitumor drugs and drugs for diagnosing tumors through extensive and intensive research. The present invention has been completed based on this finding.

The scientific terms used herein have the same or similar meanings as commonly understood by one of ordinary skill in the art. To facilitate understanding of the invention, some terms are defined below.

The term "Claudin18.2" as used herein, refers to a type III transmembrane protein composed of 184 amino acid residues (NCBI Reference Sequence: NP-001183.2). In the examples, Claudin18.2 refers to human Claudin18.2.

The term "antibody" herein refers to an antigen binding protein of the immune system. The term "antibody" as referred to herein includes the complete full-length antibody having an antigen-binding region and any fragment thereof, or single chains thereof, such as single chain variable fragments (scFv), wherein the "antigen-binding portion" or "antigen-binding region" remains. A natural antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding fragments thereof, interconnected by disulfide bonds. The term "antibody" also includes all recombinant forms of antibodies (particularly antibodies described herein), such as those expressed in prokaryotic cells, unglycosylated antibodies as well as antibody fragments that bind antigen and derivatives referred to below. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. VH and VL can be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs) interspersed with more conserved regions known as Framework Regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

Antibody fragments include, but are not limited to: (i) fab fragments consisting of the VL, VH, CL and CH1 domains, including Fab 'and Fab' -SH, (ii) Fd fragments consisting of the VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region (Ward et al, 1989, Nature 341: 544-546); (v) a F (ab') 2 fragment, a bivalent fragment comprising 2 linked Fab fragments; (vi) a single chain Fv molecule antigen binding site; (vii) bispecific single chain Fv dimers; (viii) "diabody" or "trisomy", multivalent or multispecific fragments constructed by gene fusion; and (ix) scFv genetically fused to the same or a different antibody.

The term "Fc" or "Fc region" herein includes polypeptides comprising an antibody constant region other than a first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinges at the N-terminus of these domains. For IgA and IgM, Fc may comprise J chains. For IgG, Fc includes immunoglobulin domains C γ 2 and C γ 3 and a hinge between C γ 1 and C γ 2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as comprising residues C226 or P230 at its carboxy-terminus, where the numbering is according to the EU index of Kabat. For human IgG1, Fc is defined herein as comprising residue P232 to its carboxy terminus, wherein the numbering is according to the EU index in Kabat. Fc may refer to this region in isolation, or in the environment of an Fc polypeptide, such as an antibody. The "hinge" described above includes a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain terminates at EU220 and the IgG CH2 domain begins at residue EU 237. Thus, for IgG, an antibody hinge is defined herein to include positions 221 (D221 of IgG 1) to 231 (a 231 of IgG 1), where the numbering is according to the EU index of Kabat.

The term "parent antibody" or "parent immunoglobulin" as used herein includes unmodified antibodies which are subsequently modified to produce variants. The parent antibody may be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody. A parent antibody may refer to the antibody itself, a composition comprising the parent antibody, or an amino acid sequence encoding the same. The term "parent antibody" or "parent immunoglobulin" as used herein includes murine or chimeric antibodies that are subsequently modified to produce humanized antibodies.

The term "variant antibody" or "antibody variant" as used herein includes antibody sequences that differ from a parent antibody sequence by at least one amino acid modification compared to the parent. The variant antibody sequences herein preferably have at least about 80%, most preferably at least about 90%, more preferably at least about 95% amino acid sequence identity to the parent antibody sequence. An antibody variant may refer to the antibody itself, a composition comprising the parent antibody, or an amino acid sequence encoding the same.

The term "variant" as used herein includes antibody sequences that differ from a parent antibody sequence by at least one amino acid modification compared to the parent. In particular embodiments, the variant antibody sequences herein have at least about 80%, preferably at least about 90%, more preferably at least about 95%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% amino acid sequence identity to the parent antibody sequence. An antibody variant may refer to the antibody itself, a composition comprising the parent antibody, or an amino acid sequence encoding the same. The term "amino acid modification" includes amino acid substitutions, additions and/or deletions, and "amino acid substitution" means the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, the substitution R94K refers to the substitution of arginine at position 94 with lysine, and "amino acid insertion" as used herein means the addition of an amino acid at a particular position in the parent polypeptide sequence. "amino acid deletion" or "deletion" as used herein means the removal of an amino acid at a particular position in a parent polypeptide sequence.

The term "conservative modification" or "conservative sequence modification" as used herein means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged acute side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions or in the framework regions of the antibody of the invention may be replaced with amino acid residues of other identical side chain families, and the altered antibody (variant antibody) may be tested for retained function.

All immunoglobulin heavy chain constant region positions discussed herein are numbered according to the EU index of Kabat (Kabat et al, 1991, sequences of proteins of immunological interest, 5 th edition, United States Public Health Service, National Institutes of Health, Bethesda, incorporated by reference in its entirety). The "EU index of Kabat" refers to the residue numbering of the human IgG1EU antibody, as described by Edelman et al, 1969, Biochemistry 63: 78-85.

The term "epitope", also called epitope, as used herein, can consist of a continuous sequence of the Claudin18.2 protein sequence, or can consist of a discontinuous three-dimensional structure of the Claudin18.2 protein sequence.

The antibody or the variant thereof can be applied to the preparation of various targeting antitumor drugs and drugs for diagnosing tumors, and particularly applied to the preparation of immune effector cells targeting Claudin18.2.

The present invention relates to a humanized monoclonal antibody targeting Claudin18.2 with functionality, and embodiments of the present invention will be described in detail below with reference to examples. Unless defined otherwise, 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. Unless otherwise indicated, the methods and materials of the examples described below are all conventional products available on the market. Those skilled in the art to which the invention pertains will appreciate that the methods and materials described below are illustrative only and should not be taken as limiting the scope of the invention.

Example 1: antigen preparation

According to Claudin18.2 sequence information, after codon optimization, gene synthesis is carried out, subcloning is carried out to an adenovirus shuttle vector pShuttle-CMV-GFP, after a shuttle plasmid pShuttle-CMV-Claudin18.2 is obtained, in vitro recombination is carried out with an adenovirus framework plasmid pAD-Backbone, and a recombinant adenovirus plasmid is obtained.

The recombinant adenovirus vector was purified and transfected into 293A cells using lvfransm transfection reagent after purification to prepare adenovirus seeds. Based on seed virus, large-scale amplification is carried out, adenovirus is obtained through purification, and titer determination is carried out by using Anti-Hexon antibody.

Example 2: immunization of mice

All mice were housed in a barrier system using sterilized pelleted feed and autoclaved drinking water. 8 Balb/c mice (SPF grade) were labeled with ear tags and used purified recombinant adenovirus according to a classical immunization protocol at 1X 10⁷pfu/mouse dose, mice were immunized by intramuscular injection.

TABLE 1 Claudin18.2 recombinant adenovirus animal immunization protocol

Example 3: serum titer detection in immunized mice

1. Taking out the mouse from the cage, disinfecting the tail of the mouse by using a 75% medical alcohol cotton ball, and pricking a small wound on the tail of the mouse by using a blood taking needle;

2. collecting blood drops by using a capillary glass blood collection tube;

3. after blood collection, the blood collection points are slightly pressed by using a dry sterile cotton ball to stop bleeding, and the mice are returned to the cage for slight observation;

4. placing the centrifuge tube with the collected blood sample in an incubator at 37 ℃ for 1 hour; the blood samples were then transferred to 4 ℃ overnight.

5. Separating serum from blood clot, transferring into a new sterile centrifuge tube, centrifuging at 4 deg.C and 10000g for 10 min;

6. the serum was diluted at 1:1000 and FACS-detected with CHO-Claudin18.2 cells and control cells, CHO, respectively.

The result of serum titer detection of immunized mice is shown in FIG. 1, wherein FIG. 1A shows the binding of 8 immunized mice to CHO-Claudin18.2 cells; FIG. 1B shows the binding of 8 immunized mice to CHO cells. The results show that both immunized mice can bind to the native claudin18.2 protein, and mice nos. 80 and 82 were selected for subsequent cell fusion experiments.

NC in the figure: PBS.

Example 4: hybridoma fusion and screening

1. Taking the mouse with the best immune titer detection, dislocating and killing the neck of the mouse, obtaining the spleen of the mouse under the aseptic condition, preparing B cell single cell suspension, mixing the B cell single cell suspension with non-secretory SP2/0 myeloma cells according to the proportion of 1:1, and performing cell fusion by using a BTX cell electrofusion apparatus.

2. Immediately after electrofusion, all cells were suspended in complete media (DMEM, 20% FBS and HAT) and seeded into 96-well plates.

3. The culture medium was changed to HT medium about 10 days after the fusion, and after two days, the supernatant was taken to detect the specific antibody.

4. 120uL of the culture supernatant was removed from each well of the 96-well plate while each well was supplemented with fresh HT medium. The culture supernatant was incubated with ELISA plates precoated with the antigen of interest and identified according to standard ELISA procedures.

And selecting the wells with higher O.D values for subcloning, and performing ELISA identification by referring to the same steps after each round of subcloning until a single clone is formed.

Example 5: hybridoma clone affinity flow assay

1. CHO-Claudin18.1 and CHO-Claudin18.2 cell lines were recovered from liquid nitrogen and cell status was adjusted to logarithmic growth phase using F12K, 10% FBS complete medium.

2. The CHO-Claudin18.1 and CHO-Claudin18.2 cell lines were divided into several portions, each of which had a number of cells of 3X 10⁵Each cell was incubated with 100ul of hybridoma supernatant for 1 hour at room temperature after mixing well.

3. After centrifugation at 800g for 5 minutes at room temperature, the supernatant containing the antibody was removed, and the cells were washed 3 times with PBS.

4. Adding PE-labeled Anti-mouse IgG (1:1000), fully and uniformly mixing, and incubating for 30 minutes at room temperature in a dark place;

5. centrifuging at 800g for 5 minutes at room temperature, removing the supernatant containing the secondary antibody, and washing the cells 3 times with PBS;

6. cells were resuspended using 200uL PBS and the fluorescence intensity was read by flow cytometry for recheck of fusion clone titers.

The affinity flow assay for hybridoma clones is shown in FIG. 2, where FIG. 2A shows the binding of hybridoma clones to CHO-Claudin18.2 cells, FIG. 2B shows the binding of hybridoma clones to CHO-Claudin18.1 cells, and FIG. 2C shows the binding of hybridoma clones to CHO cells, and clones that bind only to CHO-Claudin18.2 cells are selected for subcloning.

Note: R2-NC in the figure: PBS control group, R2-PC was a 1:1000 dilution immune serum control group.

Example 6: hybridoma clone subclone affinity flow assay

1. Collecting hybridoma cell strain with good growth state, and making into single cell suspension.

2. Counting was performed using a hemocytometer and 100 cells were taken and diluted to 10 ml.

3. A96-well plate was taken and 100ul of cell suspension was added to each well, and the volume was finally replenished to 200 ul.

4. The cells were placed in a 37 ℃ incubator and labeled.

5. After 3 days of continuous culture, the monoclonals were selected under a microscope.

6. Culture to day 7, change fresh medium.

7. Wells that flow-bound to positive immune sera (1:1000) were selected for re-subcloning by FACS detection up to day 8.

The results of affinity flow assay for hybridoma subclones are shown in FIGS. 3 and 4, where FIG. 3 shows the binding of hybridoma subclones to CHO-Claudin18.2 cells and FIG. 4 shows the binding of hybridoma subclones to CHO-Claudin18.1 cells. According to the detection result, 7 positive antibodies specifically recognizing 18.2 are obtained in total, and hybridoma sequencing is carried out.

Example 7: sequencing of hybridomas

1. Take 2X 10⁶The resulting hybridoma cells were finally selected, lysed using Trizol and total RNA extracted from the hybridoma cells according to standard methods.

2. After the total RNA is reversely transcribed into a polynucleotide sample by using a reverse transcription kit, a heavy chain variable region and a light chain variable region of an antibody are amplified by using a hybridoma sequencing primer through a PCR method, TA cloning is carried out, and a fragment obtained by the PCR is subcloned into a pMD-19T vector. After screening with blue and white spots, 10 clones were picked for sequencing for each chain, and the finally obtained antibody sequences were analyzed.

After sequencing, a murine anti-human Claudin18.2 antibody sequence was obtained, as shown in the following table:

table 2 murine anti-human claudin18.2 antibody heavy chain complementary region sequence information:

table 3 sequence information of the light chain complementary region of the murine anti-human claudin18.2 antibody:

example 8: antibody expression vector construction and validation

The heavy chain variable region of the antibody obtained by sequencing the hybridoma was subcloned into pcDNA3.1-IgG1Fc expression vector (the schematic vector diagram is shown in FIG. 5B), and the light chain variable region was subcloned into pcDNA3.1-IgKc expression vector (the schematic vector diagram is shown in FIG. 5A). The constructed light chain and heavy chain expression vectors were transiently co-transformed into 293F cells (serum-free culture), and 72 hours after transfection, the expressed supernatant was collected for FACS.

The flow verification results of the chimeric antibody are shown in FIG. 6, and FIG. 6A shows the binding condition of 7 hybridoma clones and the formed hybridoma subclones with CHO-Claudin18.2 cells, wherein R2-NC is not bound, the hybridoma clone with the number of 12-H9-2-D9-C2 is not bound, and the binding rate of other hybridoma clones is more than 89%. FIG. 6B shows the binding of 7 hybridoma clones and the resulting hybridoma subclones to CHO-Claudin18.1 cells, wherein all hybridoma clones were not bound. FIG. 6C shows binding of 3 hybridoma clones to CHO cells, wherein 3 hybridoma clones did not bind to CHO cells. Among them, 1-C4-2-F8-G6 and 7-C5-1-C10-1-G7 hybridoma clones bound strongly to Claudin18.2.

Example 9: humanized antibody sequence design

The amino acid sequence information of the heavy chain and the light chain of the antibody of 1-C4-2-F8-G6 and 7-C5-1-C10-1-G7 is humanized and designed, the CDR region sequences of the original antibody are kept unchanged, different humanized antibody templates are respectively selected for the heavy chain and the light chain according to the germline alignment result and the antibody structure simulation result, and the back mutation is carried out on the framework region after the humanization, so that the following candidate humanized monoclonal antibody sequences targeting Claudin18.2 are designed.

TABLE 3 humanized monoclonal antibody sequences targeting Claudin18.2

Note: in the table, the variable region (CDR) sequences in the heavy or light chains were not changed, but the Framework Regions (FR) were humanized

Example 10: humanized antibody gene synthesis and expression vector construction

The humanized single-chain antibody fragment is subjected to gene synthesis, the heavy chain is subcloned into a pcDNA3.1-IgG1Fc expression vector, and the light chain variable region is subcloned into a pcDNA3.1-IgKc expression vector. After the vector was verified to be free of errors by sequencing, endotoxin-free plasmids were prepared using a Qiagen plasmid macrodrawer.

Example 11: humanized monoclonal antibody expression detection

1. After thawing the transfection reagent LVTransm and the single-chain antibody expression vector at room temperature, the mixture is blown up and down by a pipette gun and is mixed evenly. The PBS buffer was removed and warmed to room temperature. And (3) adding 0.5mL of PBS into one hole of a 24-hole plate, respectively adding 2ug of heavy chain and 4ug of light chain, blowing and beating up and down a pipette gun to be fully and uniformly mixed, adding 18uL of LVTransm, immediately blowing and beating up and down by using a pipette to be uniformly mixed, and standing at room temperature for 10 minutes.

2. The DNA/LVTransm complex was added to 1.5mL 293F cells, and mixed well by gentle shaking. The cells were incubated at 37 ℃ with 5% CO₂After culturing at 130rpm for 6-8 hours in the incubator, 1mL of fresh 293F medium was added, and the cells were returned to the incubator for further culture.

3. After continuous culture for 3 days, the culture medium supernatant was collected by centrifugation and subjected to flow assay.

The results of expression flow assay of humanized monoclonal antibody are shown in FIGS. 7 and 8, in which FIG. 7 shows the results of expression flow assay of 1-C4-2-F8-G6 hybridoma clone and humanized antibody thereof, and FIG. 8 shows the results of expression flow assay of 7-C5-1-C10-1-G7 hybridoma clone and humanized antibody.

Note: R2-NC in the figure: PBS control group.

Example 12: flow detection of affinity and specificity of humanized monoclonal antibody

1. The CHO-Claudin18.1 and CHO-Claudin18.2 recombinant cell lines were recovered from liquid nitrogen and the cell status was adjusted to logarithmic growth phase using F12K, 10% FBS complete medium.

2. Dividing two kinds of cells into several parts, each part having a number of 5 × 10⁵The cells were resuspended in 100uL PBS, humanized antibodies at different concentrations were added, mixed well and incubated at room temperature for half an hour.

3. Centrifuging at 800g for 5 minutes at room temperature, removing the supernatant containing the antibody, and washing the cells 3 times with PBS;

4. adding 0.5uL PE-labeled Anti-human IgG (eBioscience, A11013), mixing, and incubating at room temperature in dark for 30 min;

6. the cells were resuspended using 500uL PBS and flow analyzed.

The detection result of the humanized monoclonal antibody EC50 is shown in the following table:

TABLE 4 results of EC50 detection of humanized monoclonal antibody

Wherein C4-2-F8-G6 Chimeric is formed by the CDR region of 1-C4-2-F8-G6 and Fc chimera of human IgG, and 7-C5-1-C10-1-G7 Chimeric is formed in the same manner, aiming at verifying the effect of Fc of human IgG and humanized monoclonal antibody on affinity.

The results of the affinity and specificity detection of the humanized monoclonal antibody are shown in fig. 9, and fig. 9A shows the results of the affinity detection of the humanized monoclonal antibody. FIG. 9B shows the specificity of the humanized monoclonal antibody, both the humanized monoclonal antibody and the positive control have high specificity; and, after humanization, has an effect on the affinity of the antibody, some humanizations may reduce the affinity, but some increase the affinity, except that the affinity of C4-2-F8-G6VH2-VL1 is too low, and others do not differ much from the positive control, particularly 7-C5-1-C10-1-G7 VH2-VL1 has an affinity that exceeds that of positive controls IMAB362 and AB011 (see CN112138171A patent and Hua Jiang et al, sequence of the National Cancer Institute (2019), see example 8 for construction methods)

Example 13: humanized monoclonal antibody for detecting human Claudin18.2 antigen expression on different tumor cells

Human gastric cancer cells MKN-28-18.2 and N87-18.2 expressing Claudin18.2, human renal epithelial cells 293T-18.1 expressing Claudin18.1, human gastric cancer cells NUGC-4 and negative control cells for detection were collected and washed 3 times with PBS. Counting to adjust cell densityIs 2 x 10⁵cells/100. mu.L, 100ul were taken as a control group and an experimental group, respectively. The experimental group was incubated at 4 ℃ for 1 hour in the dark with primary antibody (humanized monoclonal antibody) added to the cell suspension at a dilution ratio of 1: 50. After the incubation, the experimental group washed the cells 3 times with PBS, and both the control group and the experimental group were incubated at 4 ℃ for 1 hour with goat anti-mouse IgG secondary antibody added at the same time according to the dilution ratio specified in the specification. Finally, cells were washed 3 times with PBS and signals were read with FACS BD Calibur. The detection is shown in fig. 10: the humanized monoclonal antibody has strong binding force with human gastric cancer cells MKN-28-18.2 and N87-18.2 expressing Claudin18.2, almost does not bind with human renal epithelial cells 293T expressing Claudin18.1, and has certain binding with human gastric cancer cells NUGC-4, which indicates that the humanized monoclonal antibody has good specificity.

Example 14: luciferase labelling assay for in vitro ADCC Activity of humanized antibodies

Jurkat-NFAT-Luciferase-CD16 effector cells were co-cultured with CHO-Claudin18.2 target cell line at 1X 10⁵The effective target ratio is 5:1, 2 humanized antibodies (7-C5-1-C10-1-G7-VH2-VL1, 7-C5-1-C10-1-G7-VH3-VL2), 1 Chimeric antibody (7-C5-1-C10-1-G7-Chimeric) and positive control antibodies (0, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1 μ G/ml) of Kejisheng AB011 and AnsteelaimaB 362 are added respectively at different concentrations, and each group has 2 multiple wells. After co-culturing the target cells for 18h, 20ul of One-Glo reagent was added, luciferase activity was detected, and EC50 values were calculated. The results of in vitro ADCC activity assay of the humanized antibodies are shown in fig. 11: the humanized antibody is consistent with the chimeric antibody EC50, wherein 7-C5-1-C10-1-G7 VH3-VL2 has stronger ADCC effect. Although this example employs two humanized antibody experiments, it is known that other humanized antibodies have similar ADCC activity against the above-mentioned target cells based on the principle of antigen-antibody affinity.

Example 15: luciferase labelling assay of humanized antibodies for in vitro CDC Activity

Taking CHO-Claudin18.2Cells, seeded at a cell mass of 4X 10⁴Each of the cells was inoculated into a 96-well plate for 90. mu.l, and 2 humanized antibodies (7-C5-1-C10-1-G7-VH2-VL1, 7-C5-1-C10-1-G7-VH3-VL2), 1 Chimeric antibody (7-C5-1-C10-1-G7-Chimeric), and 100. mu.l of a positive control antibody (0, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100. mu.g/ml) of Kejisheng AB011 and Ansteela IMAB362 were added at different concentrations, while 10. mu.l of human complement serum was added to the corresponding well. After 18 hours of co-culture, 50ul CellCounting-Lite 3D reagent was added, lysis of the target cells was reflected by detecting the change in luminescence, and EC50 value was calculated. The results of the humanized antibody CDC activity assay in vitro are shown in fig. 12: the CDC effect of 7-C5-1-C10-1-G7 VH3-VL2 is consistent with that of chimeric antibodies. Although this example employed two humanized antibody experiments, it was found that other humanized antibodies had similar CDC activity to the above-mentioned target cells based on the principle of antigen-antibody affinity.

In conclusion, the humanized monoclonal antibody or the functional fragment thereof targeting Claudin18.2 constructed by the invention can be applied to the treatment and/or prevention or diagnosis of diseases related to Claudin18.2, such as tumor and the like.

It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.

The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Sequence listing

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Ala Arg Ser Gly Leu Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

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Asp Thr Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

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Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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<213> R3-5-E6-B5-B7 murine antibody heavy chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

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<213> R3-5-E6-B5-B7 murine antibody light chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

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Asp Thr Val Met Thr Gln Ser Pro Ser Ser Leu Pro Val Thr Ala Gly

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Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

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<213> R9-4-A4-D12-F8 murine antibody heavy chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

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Ser Arg Leu Thr Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

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Lys Met Asn Arg Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

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Arg Ser Tyr Tyr Gly Asn Ser Phe Glu Tyr Trp Gly Gln Gly Thr Leu

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<213> R9-4-A4-D12-F8 murine antibody light chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

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

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Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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Val Tyr Tyr Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

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<213> F4-2-H3-H8-C1 murine antibody heavy chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

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Val Ile His Trp Val Ser Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

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Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Asn Thr Val Tyr

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Leu Glu Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Arg Asp Asn Tyr Gly Tyr Tyr Trp Phe Ala Tyr Trp Gly Gln Gly

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115

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

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Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

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

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Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

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Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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<213> 1-C4-2-F8-G6-VH1 humanized antibody heavy chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

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Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Phe Ile Ser Ser Gly Ser His Thr Ile Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Phe Gln Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

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Leu Val Thr Val Ser Ser

115

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<213> 1-C4-2-F8-G6-VH2 humanized antibody heavy chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 16

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Phe Ile Ser Ser Gly Ser His Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Phe Gln Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

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115

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<213> 1-C4-2-F8-G6-VL1 humanized antibody light chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 17

Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

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

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

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Thr Tyr Ser Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

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Lys

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<211> 113

<212> PRT

<213> 1-C4-2-F8-G6-VL2 humanized antibody light chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 18

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

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

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

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

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn

85 90 95

Thr Tyr Ser Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 19

<211> 118

<212> PRT

<213> 7-C5-1-C10-1-G7-VH1 humanized antibody heavy chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 19

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Phe Ser Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Leu Tyr Tyr Gly Asn Ser Met Asp Ser Trp Ser Gln Gly Leu

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 20

<211> 118

<212> PRT

<213> 7-C5-1-C10-1-G7-VH2 humanized antibody heavy chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Phe Ser Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Leu Tyr Tyr Gly Asn Ser Met Asp Ser Trp Ser Gln Gly Leu

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 21

<211> 118

<212> PRT

<213> 7-C5-1-C10-1-G7-VH3 humanized antibody heavy chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 21

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Phe Ser Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Lys Leu Tyr Tyr Gly Asn Ser Met Asp Ser Trp Ser Gln Gly Leu

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 22

<211> 113

<212> PRT

<213> 7-C5-1-C10-1-G7-VL1 humanized antibody light chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 22

Asp Ile Val Ile Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Tyr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 23

<211> 113

<212> PRT

<213> 7-C5-1-C10-1-G7-VL2 humanized antibody light chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 23

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

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

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Tyr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 24

<211> 113

<212> PRT

<213> 7-C5-1-C10-1-G7-VL3 humanized antibody light chain variable region sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 24

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

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

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Tyr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

Claims

1. A humanized monoclonal antibody targeting claudin18.2 comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 sequences, said heavy and light chain variable regions comprising 6 CDR sequences:

the amino acid sequence of HCDR1 is GFTFSSFG;

the amino acid sequence of HCDR2 is ISSGSHTI;

the amino acid sequence of HCDR3 is FQYGNSFDY;

the amino acid sequence of LCDR1 is QSLLNSGNQKNY;

the amino acid sequence of LCDR2 is WAS;

the amino acid sequence of LCDR3 is QNTYSFPLT;

or the amino acid sequence of HCDR1 is GFTFSKFG;

the amino acid sequence of HCDR2 is FSSGGDY;

the amino acid sequence of HCDR3 is AKLYYGNSMDS;

the amino acid sequence of LCDR1 is QSLLNSGNQRNY;

the amino acid sequence of LCDR2 is WAS;

the amino acid sequence of LCDR3 was QNAYYYPFT.

2. The humanized monoclonal antibody targeted to claudin18.2 of claim 1, wherein the heavy chain variable region and the light chain variable region of the humanized monoclonal antibody are any one of the following combinations:

3. A nucleic acid molecule encoding a humanized monoclonal antibody targeting claudin18.2 according to claim 1 or 2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. Use of a humanized monoclonal antibody targeting claudin18.2 according to claim 1 or claim 2, a nucleic acid molecule according to claim 3, an expression vector according to claim 4 or a host cell according to claim 5 for the preparation of a medicament for the treatment of a positive tumor in claudin 18.2.

7. A pharmaceutical composition comprising the humanized monoclonal antibody targeted against claudin18.2 according to claim 1 or 2 and pharmaceutically acceptable excipients.

8. A diagnostic kit characterized in that it contains a humanized monoclonal antibody targeting claudin18.2 according to claim 1 or 2.