WO2023045997A1 - Anti-claudin18.2 antibody and use thereof - Google Patents

Abstract

Provided are an anti-Claudin18.2 antibody or an antigen binding fragment thereof and a use thereof. Further provided are a bispecific antibody, a multispecific antibody, a multi-functional fusion protein and a composition thereof containing the antibody, and a use in the preparation of a drug for treating cancer.

Description

A kind of anti-Claudin18.2 antibody and its application technical field

The invention belongs to the field of biomedicine, and in particular relates to an anti-Claudin18.2 antibody and its application.

Background technique

Intercellular tight junction is a kind of transmembrane protein complex. The stability of tight junction needs the coordinated activities of several different proteins to maintain, and Claudin protein is the main protein that ensures the specificity of tight junction permeability. So far, 24 Claudin family members have been found in mammals, and each protein is specifically expressed in a certain tissue. The extracellular loop structure of the claudin protein connects to this structure of adjacent cells, bridging the cell layer and regulating the parallel traffic between the compartment and the basal layer.

The structure of Claudin18 protein includes four transmembrane regions and two extracellular loops. Its N-terminal and C-terminal are in the cytoplasm. The molecular weight of the protein is about 26KD. The Claudin protein can be transformed into Claudin with different characteristics by selective cleavage. Isotypes: Claudin18.1 and Claudin18.2. Although there is only eight amino acid differences between the first extracellular domain of Claudin18.1 and Claudin18.2, the expression distribution is different. Claudin18.1 is selectively expressed in normal lung and gastric epithelium, while Claudin18.2 is only expressed in Expressed on transiently differentiated gastric epithelial cells, completely undetectable in any other normal human organ, but Claudin18.2 is significantly upregulated in a variety of malignant tumors, including 80% of gastrointestinal adenomas, 60% of pancreatic tumors , 30% esophageal cancer and 25% non-small cell lung cancer. In tumors, the tight junctions between cells are disrupted, and Claudin18.2 cannot perform its normal function. Therefore, Claudin18.2 is a suitable target for tumor therapy.

Ganymed Pharmaceuticals AG has developed Zolbetuximab (also known as IMAB362), an IgG1 chimeric antibody against Claudin18.2. IMAB362 recognizes the first extracellular domain (ECD1) of Claudin18.2 with high affinity and specificity. Following target binding, IMAB362 mediates cell killing via ADCC, CDC, induction of apoptosis via target cross-linking on the surface of tumor cells, and proliferation. Thus, IMAB362 efficiently lyses Claudin18.2-positive cells, including human gastric cancer cell lines in vitro and in vivo. Mice with Claudin18.2-positive cancer cell lines had a survival benefit, and when treated with IMAB362, up to 40% of mice showed regression of their tumors.

Gastric cancer and gastroesophageal junction (GEJ) adenocarcinoma is one of the most unmet medical needs of malignant tumors, and gastric cancer is also the third leading cause of cancer death worldwide. Therefore, the development of anti-Claudin18.2 antibodies and other drugs such as multifunctional and bifunctional antibodies has great significance for the treatment of some solid tumors, especially stomach-related tumors. However, there is currently no corresponding antibody developed and marketed.

Contents of the invention

Aiming at the deficiencies of existing problems, in this application, the inventors developed an anti-Claudin18.2 antibody with good performance, which does not bind to Claudin18.1, but can bind to Claudin18.2 with high affinity and specificity, and mediate Killing of Claudin 18.2 expressing target cells (eg tumor cells) by effector cells.

The present invention provides an anti-Claudin18.2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, The light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein,

(a) HCDR1 of the heavy chain variable region is selected from any amino acid sequence of SEQ ID NO: 1, 9, 15, 19, or has at least one amino acid sequence with any amino acid sequence of SEQ ID NO: 1, 9, 15, 19 A sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical, or to SEQ ID NO: 1, Amino acid sequences with one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of 9, 15 and 19;

(b) HCDR2 of the heavy chain variable region, selected from any amino acid sequence of SEQ ID NO: 2, 10, 16, 20, 21, 22, 23, 24, 25, or with SEQ ID NO: 2, 10, Any amino acid sequence of 16, 20, 21, 22, 23, 24, 25 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A sequence of 98%, 99% or more identity, or one or more (preferably 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions);

(c) HCDR3 of the heavy chain variable region, selected from any amino acid sequence of SEQ ID NO: 3, 11, 17, 26, 27, 63, or with SEQ ID NO: 3, 11, 17, 26, 27, Any amino acid sequence of 63 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical, Or an amino acid having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of SEQ ID NO: 3, 11, 17, 26, 27, 63 sequence;

(d) LCDR1 of the light chain variable region is selected from any amino acid sequence of SEQ ID NO: 4, 28, or has at least 80%, 85%, 90% of any amino acid sequence of SEQ ID NO: 4, 28 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared with any amino acid sequence of SEQ ID NO: 4, 28 Amino acid sequence with one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions);

(e) LCDR2 of the light chain variable region is selected from any amino acid sequence of SEQ ID NO: 5, or has at least 80%, 85%, 90%, 91%, any amino acid sequence with any amino acid sequence of SEQ ID NO: 5 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more ( Amino acid sequence of preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions); and/or

(f) LCDR3 of the light chain variable region is selected from any amino acid sequence of SEQ ID NO: 6, 12, or has at least 80%, 85%, 90% of any amino acid sequence of SEQ ID NO: 6, 12 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared with any amino acid sequence of SEQ ID NO: 6, 12 An amino acid sequence with one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are selected from any amino acid sequence in the following (1)-(11):

(1) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

(2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, HCDR3 shown in SEQ ID NO: 11, LCDR1 shown in SEQ ID NO: 4, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 12;

(3) HCDR1 shown in SEQ ID NO: 15, HCDR2 shown in SEQ ID NO: 16, HCDR3 shown in SEQ ID NO: 17, LCDR1 shown in SEQ ID NO: 4, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 12;

(4) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

(5) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 21, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

(6) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 22, HCDR3 shown in SEQ ID NO: 26, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

(7) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 23, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

(8) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

(9) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 25, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

(10) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 23, HCDR3 shown in SEQ ID NO: 27, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

(11) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 27, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO:6.

The present invention also provides an anti-Claudin18.2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

(a) said heavy chain variable region has any amino acid sequence given by SEQ ID NO: 7, 13, 18, 29, 30, 31, 33, 34, 35, 36, 37,

or have at least 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences,

Or have one or more (preferred 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions);

(b) said light chain variable region has any amino acid sequence given by SEQ ID NO: 8, 14, 32, 38;

Or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of any amino acid sequence given by SEQ ID NO: 8, 14, 32, 38 %, 98%, 99% or more identical sequences,

Or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acids compared with any amino acid sequence of SEQ ID NO: 8, 14, 32, 38 A mutated (preferably a substitution, insertion or deletion) amino acid sequence.

In some embodiments, the heavy chain variable region and the light chain variable region are selected from any amino acid sequence in the following (1)-(11):

(1) SEQ ID NO: 7 and SEQ ID NO: 8;

(2) SEQ ID NO: 13 and SEQ ID NO: 14;

(3) SEQ ID NO: 18 and SEQ ID NO: 14;

(4) SEQ ID NO: 29 and SEQ ID NO: 32;

(5) SEQ ID NO: 30 and SEQ ID NO: 32;

(6) SEQ ID NO: 31 and SEQ ID NO: 32;

(7) SEQ ID NO: 33 and SEQ ID NO: 38;

(8) SEQ ID NO: 34 and SEQ ID NO: 38;

(9) SEQ ID NO: 35 and SEQ ID NO: 38;

(10) SEQ ID NO: 36 and SEQ ID NO: 38;

(11) SEQ ID NO: 37 and SEQ ID NO: 38.

In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the anti-Claudin18.2 antibody or antigen-binding fragment thereof further comprises an Fc region selected from mouse IgG1, IgG2a, IgG2b and/or IgG3, or selected from rat IgG1, IgG2a , IgG2b and/or IgG2c.

In some embodiments, the anti-Claudin18.2 antibody or antigen-binding fragment thereof further comprises an Fc region selected from human IgG1, IgG2, IgG3 and/or IgG4.

The present invention also provides a nucleic acid molecule encoding the anti-Claudin18.2 antibody or antigen-binding fragment thereof described in any one of the above.

The present invention also provides a multifunctional fusion protein comprising the anti-Claudin18.2 antibody or antigen-binding fragment thereof described in any one of the above.

In some embodiments, the anti-Claudin18.2 antibody or antigen-binding fragment thereof further comprises one or more second antibodies or antigen-binding portions thereof that specifically bind to other antigens.

In some embodiments, the antigen that binds the second antibody or antigen-binding portion thereof is selected from tumor-associated antigens (TAAs) or immune checkpoints.

In some embodiments, the immune checkpoint is PD-L1, CTLA4, PD-L2, PD-1, 4-1BB, CD47, TIGIT, GITR, TIM3, ILT4, TNFR2, TREM2, LAG3, CD27, B7H3, or B7H4.

In some embodiments, cytokines are also included.

In some embodiments, the cytokine is selected from IL-1, IL-2, IL-2Rα, IL-2Rβ, IL-3, IL-3Rα, IL-4, IL-4Rα, IL-5, IL- 5Rα, IL-6, IL-6Rα, IL-7, IL-7Rα, IL-8, IL-9, IL-9Rα, IL-10, IL-10R1, IL-10R2, IL-11, IL-11Rα, IL-12, IL-12Rα, IL-12Rβ2, IL-12Rβ1, IL-13, IL-13Rα, IL-13Rα2, IL-14, IL-15, IL-15Rα sushi, IL-16, IL-17, IL- 18. IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL-21Rα, IL-22, IL-23, IL-23R, IL-27R, IL-31R, TGF, VEGF, IFNγ, IFNα or GM-CSF.

The present invention also provides the use of any one of the anti-Claudin18.2 antibodies or antigen-binding fragments thereof or any one of the multifunctional fusion proteins described above in the preparation of drugs for treating cancer.

In some embodiments, the cancer is lung cancer, liver cancer, melanoma, glioblastoma, head and neck cancer, esophageal cancer, gastric cancer, prostate cancer, ovarian cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, cervical cancer, or associated tumors.

In some embodiments, the use is achieved by one or more of tumor immunotherapy, cell therapy, and gene therapy.

The present invention also provides a pharmaceutical composition, which comprises the anti-Claudin18.2 antibody or antigen-binding fragment thereof described in any one of the above and a pharmaceutically acceptable carrier, diluent or excipient.

The present invention also provides a pharmaceutical composition, which comprises any one of the above-mentioned multifunctional fusion proteins and a pharmaceutically acceptable carrier, diluent or excipient.

Abbreviations and Definitions of Terms

The following abbreviations are used in this article:

mAb: monoclonal antibody

VH: Antibody heavy chain variable region

VL: antibody light chain variable region

CDR: Complementarity Determining Region in Immunoglobulin Variable Region

FR: Antibody framework region, that is, the amino acid residues in the antibody variable region except CDR residues

IgG: Immunoglobulin G

The term "antibody" refers to natural immunoglobulins or immunoglobulins prepared by partial or complete synthesis. Antibodies can be reconstituted and isolated from natural resources such as plasma or serum where the antibodies naturally exist, or culture supernatants of hybridoma cells producing antibodies, animal immune serum, or phage library screening. Alternatively, it can be partially or completely synthesized by using a technique of gene recombination or the like. Preferred antibodies include, for example, antibodies to immunoglobulin isotypes or subclasses of these isotypes. Known human immunoglobulins include nine classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Among these isotypes, antibodies of the invention may comprise IgG1, IgG2, IgG3 and/or IgG4.

The term "monoclonal antibody" refers to a homogeneous antibody directed only against a specific antigenic epitope. In contrast to common polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous character of the antibody and is not to be construed as requiring that the antibody be produced by any particular method. The monoclonal antibodies of the invention are preferably produced by recombinant DNA methods, or obtained by screening methods as described elsewhere herein.

The term "murine antibody" in the present invention refers to a monoclonal antibody prepared according to the knowledge and skills in the art. In preparation, test subjects are injected with the antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.

The term "chimeric antibody" is an antibody that fuses the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the variable region gene of the mouse The gene is connected with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences into human antibody variable region frameworks, that is, different types of human germline antibody framework sequences. It can overcome the strong heterologous reaction induced by chimeric antibodies due to carrying a large number of mouse protein components.

Partial antibodies as used herein are immunoglobulin molecules composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), or only a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as mediating immunoglobulin interactions with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement Binding of the first component (C1q) of the system. The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions (VH and VL) of each heavy chain/light chain pair form the antigen binding site, respectively.

The term "antigen-binding fragment" of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for binding of the antigen. specifically binds, which is also referred to as an "antigen-binding moiety". Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabody), linear antibody (linear antibody), nanobody (such as technology from Ablynx), domain antibody (such as technology from Domantis), and such polypeptides, which comprise at least a portion of an antibody sufficient to confer polypeptide-specific antigen-binding capacity .

The term "polypeptide" refers to a chain of amino acids of any length, regardless of modifications such as phosphorylation or glycosylation. The term polypeptide includes proteins and fragments thereof. Polypeptides may be "exogenous" in the sense that they are "heterologous", ie, foreign to the host cell utilized, eg, human polypeptides produced by bacterial cells. A polypeptide is disclosed herein as a sequence of amino acid residues. Those sequences are written left to right in amino-terminal to carboxy-terminal orientation. According to standard nomenclature, amino acid residue sequences are designated by three-letter or one-letter codes, as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), asparagine Aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F) , proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

The term "host cell" refers to a cell that has been or can be transformed with a nucleic acid sequence and thereby express a selected gene of interest. The term includes progeny of a parental cell, whether or not the progeny is morphologically or genetically identical to the original parental cell, as long as the progeny has the selected gene of interest. Commonly used host cells include bacteria, yeast, mammalian cells, and the like.

The term "vector" refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it has been linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked, and are referred to herein as "expression vectors."

The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline, water, and emulsions, as well as various types of wetting agents.

The term "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity . Alignment for purposes of determining percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for example, using publicly available computer software such as the BLAST software or the FASTA program package.

There are several methods/systems in the art to define and describe CDRs that have been developed and refined over the years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used, defining CDRs based on sequence variability; Chothia defines CDRs based on sequence variability based on the position of structural loop regions; IMGT system defines CDRs based on sequence variability and position within the variable domain structure; AbM is based on the Oxford Molecular Company The definition of AbM antibody modeling software is a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures, which is similar to Chothia in many respects.

The CDR of the anti-Claudin18.2 antibody of the present invention refers to the hypervariable region of the heavy chain and light chain of the immunoglobulin, wherein, SEQ ID NO: 1, 2, 3, 4, 5, 6, 9, 10, 11, 12 The CDR amino acid positions of , 15, 16, and 17 are defined according to Kabat, and the CDR amino acid positions of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, and 63 are defined according to AbM.

The term "specific" means that one of the molecules involved in specific binding does not show any significant binding to a molecule or molecules other than the binding partner molecules. In addition, the term is used when a domain comprising an antibody variable region is specific for a particular epitope among epitopes in an antigen. When the epitope to which an antibody variable region-containing domain binds is contained in several different antigens, an antigen-binding molecule comprising an antibody variable region-containing domain can bind various antigens having the epitope.

The term "epitope" refers to an antigenic determinant in an antigen, and refers to an antigenic site to which a domain of an antigen-binding molecule comprising an antibody variable region disclosed in this specification binds. Therefore, an epitope can be defined according to its structure. In addition, the epitope can also be defined based on the antigen-binding activity of the antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues forming the epitope; when the epitope is a sugar chain, the epitope can be determined by its specific sugar chain structure.

The term "positive control antibody" refers to natural or engineered cells capable of binding to or expressing a target protein.

The term "isotype control" refers to the use of the same species source, the same subtype, the same dose, the same immunoglobulin and subtype of immunoglobulin, the same marker, etc. as the experimental sample in the same experiment, used in the elimination experiment The effect of the experimental background of the non-specific binding sample on the experimental value is used as a negative control to illustrate the experimental effect.

The murine, chimeric or humanized monoclonal antibody combined with Claudin18.2 provided by the present invention has higher Claudin18.2 binding activity, ADCC activity and CDC activity than the prior art antibody. At the same time, the anti-Claudin18.2 antibody of the present invention is humanized, and the modified antibody has Claudin18.2 binding activity equivalent to that of the original antibody, and its affinity is relatively stable, which can ensure the specificity of the antibody. Its CDC activity is comparable to that of the original antibody. Also basically the same.

Description of drawings

Figure 1 shows the binding activity of murine antibodies 1, 2, and 3 to human Claudin18.2-PANC1.

Figure 2 shows the binding activity of chimeric antibodies 4 and 5 to human Claudin18.2-CHOK1.

Figure 3 shows the binding activity of chimeric antibody 6 to human Claudin18.2-CHOK1.

Figure 4 shows the binding activity of chimeric antibodies 4 and 5 to human Claudin18.2-PANC1.

Figure 5 shows the binding activity of humanized antibodies 7, 8, and 9 to human Claudin18.2-CHOK1.

Fig. 6 shows the binding activity of humanized antibody 10 to human Claudin18.2-CHOK1.

Fig. 7 shows the binding activity of humanized antibodies 11 and 12 to human Claudin18.2-CHOK1.

Figure 8 shows the CDC activity of murine antibodies 1, 2, and 3 against human Claudin18.2-CHOK1.

Figure 9 shows the CDC activity of chimeric antibodies 4 and 5 against human Claudin18.2-CHOK1.

Figure 10 shows the CDC activity of chimeric antibody 6 against human Claudin18.2-CHOK1.

Figure 11 shows the CDC activity of humanized antibody 10 against human Claudin18.2-CHOK1.

Figure 12 shows the CDC activity of humanized antibodies 11 and 12 against human Claudin18.2-CHOK1.

Figure 13 shows the ADCC activity of chimeric antibodies 4 and 5 against human Claudin18.2-PANC-1.

Figure 14 shows the ADCC activity of chimeric antibody 6 against human Claudin18.2-PANC-1.

Figure 15 shows the binding activity of chimeric antibodies A, B, C, D, E, F, G to human Claudin18.2-CHOK1.

Figure 16 shows the binding activity of humanized antibodies H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1, with Z1 as the control antibody.

Figure 17 shows the binding activity of humanized antibodies H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1, using IMAB362 as a control antibody.

Figure 18 is the CDC activity of humanized antibodies H, J, L on human Claudin18.2-CHOK1.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the protection content of the present invention is not limited to the following embodiments. It should also be understood that the terminology used in the embodiments of the present invention is for describing specific implementations, not for limiting the protection scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the protection scope of the present invention is the appended claims and any equivalents thereof. In the description and claims of the present invention, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the content specifically mentioned below, are common knowledge and common knowledge of those skilled in the art, and the present invention has no special limitation.

Example 1 Preparation of anti-Claudin18.2 antibody

293 cells overexpressing human Claudin18.2 were used as an immunogen to immunize Balb/c mice for 3-4 times, one week after each immunization with CHOK1 and cells coated with CHOK1 overexpressing human Claudin18.2 -ELISA to detect serum titer. After the titer reached the requirement for fusion, the cells were used for booster immunization 2 weeks after the last immunization. Three days later, the spleen was taken out, and spleen cells were fused with mouse myeloma cell SP2/0 by electrofusion, and cultured in medium containing HAT for 7-10 days, the culture supernatant was taken out, and human Claudin18.2 CHOK1 cell screening and reverse screening with CHOK1 and CHOK1 cells expressing human Claudin18.1 to obtain candidate antibody 1, candidate antibody 2 and candidate antibody 3 that specifically bind to human Claudin18.2. Construct chimeric antibodies 4, 5, and 6 on the basis of candidate antibodies (murine antibodies) 1, 2, and 3, respectively, and construct humanized antibodies 7, 8, and 9 on the basis of chimeric antibody 4. Construct humanized antibodies 10, 11, 12 on the basis of 5.

Table 1 Antibody sequence list

the	heavy chain amino acid sequence	light chain amino acid sequence
Antibody 1	SEQ ID NO: 7	SEQ ID NO: 8
antibody 2	SEQ ID NO: 13	SEQ ID NO: 14
Antibody 3	SEQ ID NO: 18	SEQ ID NO: 14
Antibody 4	SEQ ID NO: 39	SEQ ID NO: 40
Antibody 5	SEQ ID NO: 41	SEQ ID NO: 42
Antibody 6	SEQ ID NO: 43	SEQ ID NO: 42
Antibody 7	SEQ ID NO: 44	SEQ ID NO: 45
Antibody 8	SEQ ID NO: 44	SEQ ID NO: 46
Antibody 9	SEQ ID NO: 47	SEQ ID NO: 46
Antibody 10	SEQ ID NO: 48	SEQ ID NO: 49
Antibody 11	SEQ ID NO: 50	SEQ ID NO: 51

Antibody 12

SEQ ID NO: 50

SEQ ID NO: 49

Example 2 Detection of Antibody and Cell Binding Ability

Take the antibody to be tested, and dilute 6-8 gradients with 40ug/ml as the initial concentration. Take out the cells overexpressing human Claudin18.2 (human Claudin18.2-PANC1, human Claudin18.2-CHOK1) in the incubator, transfer the cell suspension to a 15ml centrifuge tube, centrifuge, resuspend in PBS and count. Set aside the blank control group (Blank), negative control group (NC), experimental group, irrelevant antibody group, and the control antibody for antibody 1-6 is the FUT8 gene silenced IMAB362 obtained by using the sugar knockout process described in patent application 202110752316.6, human source IMAB362 was used as the control antibody of Antibody 7-12. Spread the cell suspension in a 96-well plate at approximately 2×10 ⁵ cells/well. Centrifuge (1000rpm, 5min), then wash with PBS, and centrifuge again, repeat twice to remove medium residue. Discard the supernatant, add 100 μl of primary antibody solution and irrelevant antibody solution to the experimental group and irrelevant antibody group respectively, resuspend the cells, and incubate at room temperature for 1 h. Blank and NC groups were incubated with the same amount of PBS. After 1 h, centrifuge and add PBS to wash twice. After discarding the supernatant, add 100 μl of fluorescent secondary antibody diluent (goat anti-mouse IgGH&L-FITC from Abcam ab6785, goat anti-human IgG Fcγ-FITC from Biolegend 398006) except for the Blank group where 100 μl of PBS was added. After incubating at room temperature in the dark for 0.5 h, centrifuge and add PBS to wash twice. After the supernatant was discarded, 120 μl of PBS was added to resuspend, and the average fluorescence intensity was detected by flow cytometry in sequence. The results are shown in Figure 1-7.

The results showed that the binding curves of each mouse antibody, chimeric antibody and humanized antibody were concentration gradient dependent. Binding activity of murine antibody 1, 2, 3 to human Claudin18.2-PANC1, chimeric antibody to human Claudin18.2-PANC1 and human Claudin18.2-CHOK1, and humanized antibody to human Claudin18.2-CHOK1 The binding activity was significantly better than that of the control antibody.

The CDC activity experiment of embodiment 3 antibody

Take out the 96-well plate, and put the antibody to be tested and the control antibody (the control antibody of antibody 1-6 is the FUT8 gene silenced IMAB362 obtained by the sugar-knocking process described in patent application 202110752316.6, and the control antibody of humanized antibody 7-12 is IMAB362 ) and the isotype control antibody start at 10ug/ml, dilute 3 times, perform 7 serial dilutions, set 2 parallel wells, 50ul/well. Take out the CHOK1A2HIS cell count, set the cell number to 2×10 ⁴ /100ul/well and incubate with the antibody at 37°C for 15min. Add complement-preserved human serum at a concentration of 20%/50ul/well, that is, a final concentration of 5%/well. Incubate at 37°C for 1 hour. Add CCK-8, 20ul/well, incubate at 37°C for 4h, and read at a wavelength of 450nm with a microplate reader. The results are shown in Figure 8-12.

The results showed that the CDC activity of each mouse antibody, chimeric antibody and humanized antibody was higher than that of the control antibody.

The ADCC activity experiment of embodiment 4 antibody

Take out the 96-well plate, and start with 10ug/ml of the control antibody (IMAB362 of FUT8 gene silencing obtained by using the sugar-knocking process described in patent application 202110752316.6), isotype control antibody and chimeric antibody 4, 5, 6 at 10ug/ml, 5-fold dilution, Carry out 7 gradient dilutions, set up 2 parallel wells, 100ul/well. Take out the PANC-1 18.2 cell count and set the cell number to 1×10 ⁴ /50ul/well.

Take out the NK cell count of the target cells, and set the number of cells to 5×10 ⁴ /50ul/well. Centrifuge at 1000rpm for 4min. Incubate at 37°C for 3.25h. Set ST well (only target cells), SE well (only effector cells), BV well (200ul dilution), BM well (200ul dilution) and M well (50ul effector cells + 150ul dilution). Add 20ul of lysate to the BM and BV wells, and incubate at 37°C for 45min. Centrifuge at 1000rpm for 4min, take 50ul of supernatant, add 50ul of substrate and place it in a microplate plate, place it at room temperature for 30min, add 50ul of stop solution/well, and detect at 490nm with a microplate reader. The results are shown in Figures 13 and 14.

The results of ADCC activity showed that chimeric antibodies 4, 5, and 6 had similar inhibitory effects on ADCC activity in vitro as the control antibody. Among them, the control antibody is FUT8 gene-silenced IMAB362 obtained according to the sugar knockout process described in patent application 202110752316.6. Compared with IMAB362 without FUT8 gene knockout, the ADCC effect of FUT8 gene-silenced IMAB362 is increased by 5 times. On the basis of the 5-fold increase in the in vitro ADCC activity inhibitory effect of the control antibody, the in vitro ADCC activity inhibitory effects of chimeric antibodies 4, 5, and 6 are equivalent to those of the control antibody. It can be seen that the in vitro ADCC activity inhibitory effects of chimeric antibodies 4, 5, and 6 significantly.

CDR Transformation of Example 5 Sequence

Carry out CDR transformation on the mouse sequence of murine antibody 1 to obtain chimeric antibodies A, B, C, D, E, F, G and Z1 (reference product); carry out CDR on the humanized sequence of humanized antibody 7 Transformation to obtain humanized antibodies H, I, J, K, L, M, N, O, P, Q, R. The specific sequence of the antibody is shown in Table 2.

Table 2 Antibody Sequence List

Antibody name	Heavy chain SEQ ID NO.	Light chain SEQ ID NO.
Antibody A	52	61
Antibody B	53	40
Antibody C	53	61
Antibody D	54	40
Antibody E	54	61
Antibody F	55	40
Antibody G	55	61
Antibody H	44	62
Antibody I	56	45
Antibody J	56	62
Antibody K	57	45
Antibody L	57	62
Antibody M	58	45
Antibody N	58	62
Antibody O	59	45
Antibody P	59	62
Antibody Q	60	45
Antibody R	60	62
Z1 (reference product)	52	40

Example 6 Detection of Chimeric Antibodies A, B, C, D, E, F, G and Cell Binding Ability

Take the antibody to be tested, according to the initial concentration of 40ug/mL, first dilute 2 times, then dilute 4 times, and dilute 8 gradients. Take out the cells overexpressing human Claudin18.2 (human Claudin18.2-CHOK1) in the incubator, transfer the cell suspension to a 15ml centrifuge tube, centrifuge, resuspend in PBS and count. Set aside a blank control group (Blank), a negative control group (NC), an experimental group, and an irrelevant antibody group, and use Z1 as the control antibody. Spread the cell suspension in a 96-well plate at approximately 2×10 ⁵ cells/well. Centrifuge (1000rpm, 5min), wash with PBS, and centrifuge again, repeat twice to remove medium residue. Discard the supernatant, add 100 μL primary antibody solution and irrelevant antibody solution to the experimental group and irrelevant antibody group respectively, resuspend the cells, and incubate at room temperature for 1 h. Blank and NC groups were incubated with the same amount of PBS. After 1 h, centrifuge and add PBS to wash twice. After discarding the supernatant, add 100 μL of PBS to the blank group, and add 100 μL of fluorescent secondary antibody dilution (humanized antibody secondary antibody from Biolegend 398006) to the other sample groups, incubate at room temperature for 0.5 h in the dark, then centrifuge and add PBS to wash two times. Second-rate. After discarding the supernatant, add 120 μL of PBS to resuspend and perform flow cytometry in sequence to measure the average fluorescence intensity. The result is shown in Figure 15.

The results showed that the binding curves of each chimeric antibody were concentration gradient dependent. The binding activity of chimeric antibodies A, B, C, D, E to human Claudin18.2-CHOK1 was basically the same as that of the control antibody to human Claudin18.2-CHOK1, and the binding activity of chimeric antibody F and chimeric antibody G to human The binding activity of Claudin18.2-CHOK1 was lower than that of the control antibody to human Claudin18.2-CHOK1.

Example 7 Humanized Antibody H, I, J, K, L, M, N, O, P, Q, R Detection of Cell Binding Ability

Take the antibody to be tested, use 20ug/mL as the initial concentration, dilute 6 times, and dilute 6 gradients. Take out the cells overexpressing human Claudin18.2 (human Claudin18.2-CHOK1) in the incubator, transfer the cell suspension to a 15mL centrifuge tube, centrifuge, resuspend in PBS and count. Set aside a blank control group (Blank), a negative control group (NC), an experimental group, and an irrelevant antibody group, and use Z1 and IMAB362 as control antibodies. Spread the cell suspension in a 96-well plate at approximately 2×10 ⁵ cells/well. Centrifuge (1000rpm, 5min), then wash with PBS, and centrifuge again, repeat twice to remove medium residue. Discard the supernatant, add 100 μL primary antibody solution and irrelevant antibody solution to the experimental group and irrelevant antibody group respectively, resuspend the cells, and incubate at room temperature for 1 h. The blank group and the control group were incubated with the same amount of PBS. After 1 h, centrifuge and add PBS to wash twice. After discarding the supernatant, add 100 μL of PBS to the blank group, and add 100 μL of fluorescent secondary antibody dilution solution to the other sample groups (mouse secondary antibody is from Abcam ab6785, chimeric and humanized antibody secondary antibody is from Biolegend 398006), room temperature After incubation in the dark for 0.5 h, centrifuge and add PBS to wash twice. After discarding the supernatant, add 120 μL of PBS to resuspend and perform flow cytometry in sequence to measure the average fluorescence intensity. The results are shown in Figures 16 and 17.

The results showed that the binding curves of each humanized antibody were concentration gradient dependent. The binding activity of humanized anti-H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1 and the binding activity of control antibody Z1 to human Claudin18.2-CHOK1 Basically the same; the binding activity of humanized antibodies H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1 is the same as that of the control antibody IMAB362 to human Claudin18.2-CHOK1 The combined activity effect is basically the same.

Example 8 CDC Activity Experiment of Humanized Antibody H, J, L

Take out the 96-well plate, start with the antibody H, antibody J, antibody L, positive control antibody (using IMAB362) and isotype control antibody at 10ug/ml, dilute 4 times, perform 7 gradient dilutions, and set up 2 parallel wells , 50ul/well. Take out the CHOK1A2HIS cell count, set the cell number to 2×10 ⁴ /100ul/well and incubate with the antibody at 37°C for 15min. Add complement-preserved human serum at a concentration of 20%/50uL/well, that is, a final concentration of 5%/well. Incubate at 37°C for 1 hour. Add CCK-8, 20ul/well, incubate at 37°C for 4h, and read at a wavelength of 450nm with a microplate reader. The results are shown in Figure 18.

The results showed that the CDC activities of the humanized antibodies H, J, and L were basically the same as those of the control antibody.

The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are all included in the present invention, and the appended claims are the protection scope.

Claims (20)

1. An anti-Claudin18.2 antibody or an antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, The light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein,

   (a) HCDR1 of the heavy chain variable region is selected from any amino acid sequence of SEQ ID NO: 1, 9, 15, 19, or has at least one amino acid sequence with any amino acid sequence of SEQ ID NO: 1, 9, 15, 19 A sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical, or to SEQ ID NO: 1, Amino acid sequences with one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of 9, 15 and 19;

   (b) HCDR2 of the heavy chain variable region, selected from any amino acid sequence of SEQ ID NO: 2, 10, 16, 20, 21, 22, 23, 24, 25, or with SEQ ID NO: 2, 10, Any amino acid sequence of 16, 20, 21, 22, 23, 24, 25 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A sequence of 98%, 99% or more identity, or one or more (preferably 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions);

   (c) HCDR3 of the heavy chain variable region, selected from any amino acid sequence of SEQ ID NO: 3, 11, 17, 26, 27, 63, or with SEQ ID NO: 3, 11, 17, 26, 27, Any amino acid sequence of 63 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical, Or an amino acid having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of SEQ ID NO: 3, 11, 17, 26, 27, 63 sequence;

   (d) LCDR1 of the light chain variable region is selected from any amino acid sequence of SEQ ID NO: 4, 28, or has at least 80%, 85%, 90% of any amino acid sequence of SEQ ID NO: 4, 28 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared with any amino acid sequence of SEQ ID NO: 4, 28 Amino acid sequence with one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions);

   (e) LCDR2 of the light chain variable region is selected from any amino acid sequence of SEQ ID NO: 5, or has at least 80%, 85%, 90%, 91%, any amino acid sequence with any amino acid sequence of SEQ ID NO: 5 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more ( Amino acid sequence of preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions); and/or

   (f) LCDR3 of the light chain variable region is selected from any amino acid sequence of SEQ ID NO: 6, 12, or has at least 80%, 85%, 90% of any amino acid sequence of SEQ ID NO: 6, 12 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared with any amino acid sequence of SEQ ID NO: 6, 12 An amino acid sequence with one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions).
2. The anti-Claudin18.2 antibody or antigen-binding fragment thereof according to claim 1, wherein HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, and LCDR1, LCDR2, and LCDR3 of the light chain variable region are selected from Any amino acid sequence in the following (1)-(11):

   (1) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

   (2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, HCDR3 shown in SEQ ID NO: 11, LCDR1 shown in SEQ ID NO: 4, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 12;

   (3) HCDR1 shown in SEQ ID NO: 15, HCDR2 shown in SEQ ID NO: 16, HCDR3 shown in SEQ ID NO: 17, LCDR1 shown in SEQ ID NO: 4, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 12;

   (4) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

   (5) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 21, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

   (6) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 22, HCDR3 shown in SEQ ID NO: 26, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

   (7) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 23, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

   (8) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

   (9) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 25, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

   (10) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 23, HCDR3 shown in SEQ ID NO: 27, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;

   (11) HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 27, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO:6.
3. An anti-Claudin18.2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

   (a) said heavy chain variable region has any amino acid sequence given by SEQ ID NO: 7, 13, 18, 29, 30, 31, 33, 34, 35, 36, 37,

   or have at least 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences,

   Or have one or more (preferred 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions);

   (b) the light chain variable region has any amino acid sequence given by SEQ ID NO: 8, 14, 32, 38; or any amino acid sequence given by SEQ ID NO: 8, 14, 32, 38 A sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity,

   Or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acids compared with any amino acid sequence of SEQ ID NO: 8, 14, 32, 38 A mutated (preferably a substitution, insertion or deletion) amino acid sequence.
4. An anti-Claudin18.2 antibody or an antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region and the light chain variable region are selected from any of the following (1)-(11) An amino acid sequence:

   (1) SEQ ID NO: 7 and SEQ ID NO: 8;

   (2) SEQ ID NO: 13 and SEQ ID NO: 14;

   (3) SEQ ID NO: 18 and SEQ ID NO: 14;

   (4) SEQ ID NO: 29 and SEQ ID NO: 32;

   (5) SEQ ID NO: 30 and SEQ ID NO: 32;

   (6) SEQ ID NO: 31 and SEQ ID NO: 32;

   (7) SEQ ID NO: 33 and SEQ ID NO: 38;

   (8) SEQ ID NO: 34 and SEQ ID NO: 38;

   (9) SEQ ID NO: 35 and SEQ ID NO: 38;

   (10) SEQ ID NO: 36 and SEQ ID NO: 38;

   (11) SEQ ID NO: 37 and SEQ ID NO: 38.
5. The anti-Claudin18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
6. The anti-Claudin18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody is a monoclonal antibody.
7. The anti-Claudin18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-6, further comprising an Fc region selected from mouse IgG1, IgG2a, IgG2b and/or IgG3, Or selected from rat IgG1, IgG2a, IgG2b and/or IgG2c.
8. The anti-Claudin18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-6, further comprising an Fc region selected from human IgG1, IgG2, IgG3 and/or IgG4.
9. A nucleic acid molecule encoding the anti-Claudin18.2 antibody or antigen-binding fragment thereof of any one of claims 1-8.
10. A multifunctional fusion protein comprising the anti-Claudin18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-8.
11. The multifunctional fusion protein according to claim 10, further comprising one or more second antibodies or antigen-binding parts thereof that specifically bind to other antigens.
12. The multifunctional fusion protein according to claim 11, wherein the antigen that binds to the second antibody or its antigen-binding portion is selected from tumor-associated antigens (TAA) or immune checkpoints.
13. The multifunctional fusion protein according to claim 12, wherein the immune checkpoint is PD-L1, CTLA4, PD-L2, PD-1, 4-1BB, CD47, TIGIT, GITR, TIM3, ILT4, TNFR2, TREM2, LAG3, CD27, B7H3, or B7H4.
14. The multifunctional fusion protein according to any one of claims 10-13, further comprising cytokines.
15. The multifunctional fusion protein according to claim 14, wherein the cytokines are selected from IL-1, IL-2, IL-2 Rα, IL-2 Rβ, IL-3, IL-3 Rα, IL -4, IL-4 Rα, IL-5, IL-5 Rα, IL-6, IL-6 Rα, IL-7, IL-7 Rα, IL-8, IL-9, IL-9 Rα, IL- 10. IL-10R1, IL-10R2, IL-11, IL-11Rα, IL-12, IL-12Rα, IL-12Rβ2, IL-12Rβ1, IL-13, IL-13Rα, IL-13 Rα2, IL-14, IL-15, IL-15Rα sushi, IL-16, IL-17, IL-18, IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL-21 Rα, IL-22, IL-23, IL-23R, IL-27R, IL-31R, TGF, VEGF, IFNγ, IFNα, or GM-CSF.
16. Use of the anti-Claudin18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-8 or the multifunctional fusion protein according to any one of claims 10-15 in the preparation of a drug for treating cancer.
17. The use according to claim 16, wherein the cancer is lung cancer, liver cancer, melanoma, malignant glioma, head and neck cancer, esophageal cancer, gastric cancer, prostate cancer, ovarian cancer, bladder cancer, pancreatic cancer, gastric cancer , colon cancer, cervical cancer or related tumors.
18. The use according to claim 16 or 17, characterized in that the use is realized by one or more of tumor immunotherapy, cell therapy and gene therapy.
19. A pharmaceutical composition comprising the anti-Claudin18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-8 and a pharmaceutically acceptable carrier, diluent or excipient.
20. A pharmaceutical composition comprising the multifunctional fusion protein according to any one of claims 10-15 and a pharmaceutically acceptable carrier, diluent or excipient.

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