WO2022268200A1

WO2022268200A1 - Antibody against claudin 18.2 and use thereof

Info

Publication number: WO2022268200A1
Application number: PCT/CN2022/101080
Authority: WO
Inventors: 周帅祥; 李莉; 王杰
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2021-06-25
Filing date: 2022-06-24
Publication date: 2022-12-29

Abstract

Provided are a novel antibody and antibody fragment that specifically bind claudin (CLDN) 18.2 and a conjugate containing the antibody or the antibody fragment. In addition, provided are a nucleic acid encoding the antibody or the antibody fragment thereof and a host cell comprising same, and related use. The antibody or an antigen binding fragment or the conjugate can be used for diagnosing cancer and/or determining whether cancer cells express CLDN18.2.

Description

Antibodies against claudin 18.2 and uses thereof

This application is based on the application with CN application number 202110710398.8 and the application date is June 25, 2021, and claims its priority. The disclosure content of this CN application is incorporated into this application as a whole this time.

technical field

The present invention relates generally to the fields of immunology and antibody engineering. In particular, the present invention relates to novel antibodies or antigen-binding fragments thereof that specifically bind to CLDN18.2 and conjugates containing said antibodies or antigen-binding fragments thereof. Furthermore, the present invention relates to the use of these antibodies or antigen-binding fragments or conjugates thereof for diagnosing cancer and/or determining whether cancer cells express CLDN18.2.

Background technique

Claudin 18 (CLDN18) belongs to the tight junction protein family and is an essential component of cell tight binding, and plays an important role in maintaining epithelial cell polarity, controlling paracellular diffusion, and regulating cell growth and differentiation. There are two splice forms of CLDN18. CLDN18.1 is selectively expressed in normal lung epithelium. CLDN18.2 expression is also highly restricted in normal tissues, expressed only in differentiated short-lived cells of the gastric epithelium, but not in the gastric stem cell zone. However, CLDN18.2 is abundantly present in many primary gastric cancers and their metastases, including gastric, esophageal, pancreatic, and lung cancers. Taking gastric cancer as an example, studies have shown (Christoph Pohde et al., Comparison of claudin 18.2 expression in primary tumors and lymph node metastases in Japanese patients with gastric adenocarcinoma, 2019, 1-7) 87% (n=228/ 262) The patients were positive for CLDN18.2 expression, and more than 51.5% (n=135/262) of the patients had high expression of CLDN18.2 (40% tumor cells were ≥2+IHC membrane staining), while gastric cancer patients with lymphatic metastasis 45% (n=61/135) of them were highly expressed in CLDN18.2. In conclusion, CLDN18.2 is an ideal target for targeted therapy of gastric cancer. Antibodies against CLDN18.2 have been used in cancer research. Clinical studies have shown that the clinical efficacy is related to the expression abundance of CLDN18.2 (assessed by immunohistochemistry), and the higher the expression level of CLDN18.2 protein, the better the patient response. Therefore, immunohistochemical detection of claudin 18.2 expression abundance in patients participating in clinical trials to improve treatment accuracy is an indispensable part of claudin 18.2 drug clinical trials. Therefore, it is necessary to develop anti-CLDN18.2 diagnostic antibodies with excellent staining intensity and good specificity.

Contents of the invention

The present invention provides a novel antibody or antigen-binding fragment thereof that binds to CLDN18.2.

In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to CLDN18.2, comprising a heavy chain variable region VH and/or a light chain variable region VL, wherein,

1) The VH comprises three complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:1, and the VL comprises the VL contained in SEQ ID NO:2 LCDR1, LCDR2 and LCDR3;

2) The VH comprises three complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:3, and the VL comprises the VL shown in SEQ ID NO:4 LCDR1, LCDR2 and LCDR3;

3) The VH comprises three complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:5, and the VL comprises the VL shown in SEQ ID NO:6 LCDR1, LCDR2, and LCDR3.

In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CLDN18.2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein,

(i) the VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 22 or consists of said amino acid sequence; HCDR2 comprises SEQ ID The amino acid sequence shown in NO:14 or SEQ ID NO:23 or consists of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:24 or consists of said amino acid sequence;

(ii) wherein said VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises SEQ ID NO: 16, the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 25 or consists of said amino acids Sequence composition; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:20 or is made up of said amino acid sequence; LCDR3 comprises the aminoacid sequence shown in SEQ ID NO:18 or SEQ ID NO:21 or is made up of said aminoacid sequence The above amino acid sequence composition.

1) The VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:13; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:14 sequence or consist of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15 or consists of said amino acid sequence;

The VL comprises complementary determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16 or consists of the amino acid sequence; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17 or Consists of the amino acid sequence; LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18 or consists of the amino acid sequence;

2) The VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:13; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:14 sequence or consist of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15 or consists of said amino acid sequence;

The VL comprises complementarity determining regions (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:19 or consists of said amino acid sequence; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:20 or Consists of the amino acid sequence; LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 21 or consists of the amino acid sequence;

3) The VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:22; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23 sequence or consist of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24 or consists of said amino acid sequence;

The VL comprises complementarity determining regions (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:25 or consists of said amino acid sequence; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:20 or Composed of said amino acid sequence; LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 21 or consists of said amino acid sequence.

(a) heavy chain variable region VH

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical or consist of said amino acid sequences; or

(ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; or

(iii) comprising or consisting of an amino acid sequence having 1-10 amino acid substitutions, insertions or deletions compared to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 ;

(b) light chain variable region VL

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical or consist of said amino acid sequences;

(ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; or

(iii) comprising or consisting of an amino acid sequence having 1-10 amino acid substitutions, insertions or deletions compared to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 .

In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds CLDN18.2, comprising

(1) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:1 sequence or a heavy chain variable region VH consisting of said amino acid sequence, and comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO:2 %, 97%, 98% or 99% identical amino acid sequence or light chain variable region VL consisting of said amino acid sequence;

(2) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:3 sequence or a heavy chain variable region VH consisting of said amino acid sequence, and comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or light chain variable region VL consisting of said amino acid sequence;

(3) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:5 sequence or a heavy chain variable region VH consisting of said amino acid sequence, and comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO:6 %, 97%, 98% or 99% identical amino acid sequence or light chain variable region VL consisting of said amino acid sequence.

(1) A heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 1 or consisting of said amino acid sequence and comprising an amino acid sequence shown in SEQ ID NO: 2 or a light chain consisting of said amino acid sequence Variable region VL;

(2) A heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 3 or consisting of said amino acid sequence and comprising an amino acid sequence shown in SEQ ID NO: 4 or a light chain consisting of said amino acid sequence Variable region VL;

(3) The heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 5 or consisting of said amino acid sequence and the light chain comprising the amino acid sequence shown in SEQ ID NO: 6 or consisting of said amino acid sequence Variable region VL.

In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds CLDN18.2 comprising a heavy chain and a light chain, wherein

(a) heavy chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, Amino acid sequences that are 96%, 97%, 98% or 99% identical or consist of;

(ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5) compared with the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 , 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region of the heavy chain, more preferably, the said amino acid changes do not occur in the heavy chain variable region;

(b) light chain

(ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5) compared to the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12 , 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region of the light chain, more preferably, all The amino acid changes described above do not occur in the light chain variable region.

(1) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:7 A specific amino acid sequence or a heavy chain consisting of said amino acid sequence, and comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO:8 , 96%, 97%, 98% or 99% identical amino acid sequence or a light chain consisting of said amino acid sequence;

(2) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:9 A specific amino acid sequence or a heavy chain consisting of said amino acid sequence, and comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO:10 , 96%, 97%, 98% or 99% identical amino acid sequence or a light chain consisting of said amino acid sequence;

(3) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 11 A specific amino acid sequence or a heavy chain consisting of said amino acid sequence, and comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 12 , 96%, 97%, 98% or 99% identical amino acid sequence or a light chain consisting of said amino acid sequence.

(1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:7 or consisting of said amino acid sequence and comprising an amino acid sequence shown in SEQ ID NO:8 or a light chain consisting of said amino acid sequence;

(2) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9 or consisting of the amino acid sequence and a light chain comprising the amino acid sequence shown in SEQ ID NO: 10 or consisting of the amino acid sequence;

(3) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 11 or consisting of the amino acid sequence and a light chain comprising the amino acid sequence shown in SEQ ID NO: 12 or consisting of the amino acid sequence.

In some embodiments, the invention provides an isolated nucleic acid encoding a CLDN18.2-binding antibody or antigen-binding fragment thereof of the invention, a vector comprising the nucleic acid, a host cell comprising the nucleic acid or the vector.

In some embodiments, the present invention provides a method for preparing the CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention, the method comprising culturing the present invention under conditions suitable for expressing the nucleic acid encoding the present invention. host cells.

In some embodiments, the invention provides a conjugate comprising said antibody or antigen-binding fragment thereof coupled to at least one detectable label.

In some embodiments, the present invention provides the use of the above-mentioned antibody binding to CLDN18.2 or an antigen-binding fragment thereof, or the above-mentioned conjugate in the manufacture of a diagnostic test kit for diagnosing, detecting or monitoring cancer, the diagnostic , Detecting or monitoring cancer involves the following steps:

(i) contacting a biological sample with said antibody or antigen-binding fragment thereof, or with said conjugate, and

(ii) detecting the formation of a complex between said antibody or antigen-binding fragment thereof, or said conjugate and CLDN18.2 and/or determining the amount of the complex.

Further, the cancer is a patient with positive expression of CLDN18.2, such as gastric cancer, pancreatic cancer, and gastroesophageal junction adenocarcinoma.

In some embodiments, the present invention also provides a diagnostic test kit, which comprises the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned conjugate.

The CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention has the following advantages: excellent staining intensity, clear and sharp edges, strong resolution and no non-specific background staining, greatly improving the accuracy of CLDN18.2 expression abundance determination in clinical diagnosis Accuracy.

The invention is further illustrated in the following figures and specific embodiments. However, these drawings and specific embodiments should not be considered as limiting the scope of the present invention, and changes easily conceived by those skilled in the art will be included in the spirit of the present invention and the protection scope of the appended claims.

Description of drawings

Figure 1 shows the staining of the antibody on DAN-G claudin18.2 cell tumor (pancreatic cancer).

Figure 2 shows the staining of antibodies on SNU-620 cell tumor (gastric cancer).

detailed description

I. Definition

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In order to explain this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit of 5% less and an upper limit of 5% greater than the stated numerical value.

As used herein, the term "and/or" means either one of the alternatives or both of the alternatives.

As used herein, the term "comprising" or "comprising" means including stated elements, integers or steps, but not excluding any other elements, integers or steps. Herein, when the term "comprising" or "comprises" is used, unless otherwise specified, it also covers the situation of combining the mentioned elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region that consists of that particular sequence.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody constructs including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (e.g. , bispecific antibody), single-chain antibody, whole antibody, or antibody fragment thereof that exhibits the desired antigen-binding activity. A whole antibody will usually comprise at least two full-length heavy chains and two full-length light chains, but in some cases may comprise fewer chains, for example antibodies naturally occurring in camelids may comprise only heavy chains.

The term "antibody fragment" includes any portion of the above-mentioned antibodies, preferably their antigen-binding or variable regions.

The term "antigen-binding fragment" refers to a molecule, distinct from an intact antibody, that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies (dAbs); linear antibodies; single chain antibodies (e.g. scFv); Antibodies (single domain antibodies); antigen-binding fragments of bivalent or bispecific antibodies; camelid antibodies; and other fragments that exhibit the desired ability to bind CLDN18.2.

The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially any protein or peptide, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA.

As used herein, the term "epitope" refers to the portion of an antigen (eg, CLDN18.2) that specifically interacts with an antibody molecule.

The terms "full length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain comprising an Fc region as defined herein .

As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition (i.e., they are produced by the same type of immune cells, which are all clones of a single parental cell, and thus the molecules are identical ). Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology such as CDR grafting, or combinations of these or other techniques known in the art.

As used herein, the terms "bind" and "specifically bind" mean that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or optical interferometry of biofilm layers (ForteBio) or other conventional binding assays known in the art. By way of example of the present invention is meant that an antibody or antigen-binding fragment binds to an antigenic epitope in an in vitro assay, preferably in optical interferometry of biofilm layers using purified wild-type antigen. In certain embodiments, an antibody or antigen-binding fragment is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are divided into "classes": IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses, eg, IgG1, IgG2, IgG3 and IgG4, IgA1, and IgA2. The heavy-chain constant regions that correspond to the different antibody classes are called α, δ, ε, γ, and μ, respectively. The light chain constant regions (CL) found in all five antibody classes are called kappa and lambda. Within full-length light and heavy chains, typically the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See, eg, Fundamental Immunology, Ch. 7 (Paul, W. ed., 2nd ed., Raven Press, N.Y. (1989)) (which is hereby incorporated by reference in its entirety for all purposes). The variable region of each light chain/heavy chain pair generally forms the antigen binding site.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5 ^th Ed. Public Health Service, As described in National Institutes of Health, Bethesda, MD, 1991.

The term "variable region" or "variable domain" refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies typically have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (see, e.g., Kindt et al. Kuby Immunology, 6 ^th ed., WH Freeman and Co. p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind a particular antigen can be used to isolate antibodies that bind that antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

Variable regions generally exhibit the same general structure of relatively conserved framework regions (FRs) joined by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from the two chains of each pair, which allow binding of specific epitopes, are usually aligned by the framework regions. Both light and heavy chain variable regions generally comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus.

A "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" (used interchangeably herein with a hypervariable region "HVR"), is an antibody variable domain that is hypervariable in sequence and Structurally defined loops ("hypervariable loops") and/or regions containing antigen contact residues ("antigen contact points") are formed. The CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus. The CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (International Immunogenetics Information System, World Wide Web imgt.cines.fr/), and based on the use of a large number of crystal structures based on neighbor propagation clustering (affinity Propagation clustering) North CDR definition (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

For example, differently defined ranges of CDR regions using Kabat and Chothia numbering.

CDRs can also be determined based on having the same Kabat numbering position as the reference CDR sequence.

Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the above ways.

Unless otherwise stated, in the present invention, when referring to residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues), it is meant according to the Kabat numbering system ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th ^Ed . Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

In one embodiment, HCDR1 of an antibody of the invention is bounded by AbM rules, and HCDR2, HCDR3, and LCDR1-3 are bounded by Kabat rules, for example as shown in Table A below.

However, it should be noted that the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment systems may differ. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different. Thus, where reference is made to defining antibodies with a particular CDR sequence as defined in the present invention, the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the methods of Kabat, Chothia, AbM, Contact and North, the region of minimal overlap can be determined, thereby providing a "minimum binding unit" for antigen binding. A minimal binding unit may be a subsection of a CDR. As will be apparent to those skilled in the art, the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conserved amino acid residues.

The term "antibody in IgG form" refers to the IgG form to which the heavy chain constant region of the antibody belongs. The heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different. For example, an antibody in IgG1 form refers to an Ig domain whose heavy chain constant region Ig domain is IgG1.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity in originally transformed cells are included herein.

As used herein, the term "multispecific" antibody refers to an antibody that has at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope. Antigen binding to different epitopes. Multispecific antibodies are antibodies that have binding specificities for at least two different antigenic epitopes. In one embodiment, provided herein are bispecific antibodies that have binding specificities for a first antigen and a second antigen.

The term "effector functions" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) , cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (eg, B-cell receptors), and B-cell activation.

The term "cytokine" is a general term for proteins released by one cell population to act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7. IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL) and gamma-interferon. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines, including small molecular entities produced artificially, and their pharmaceutically acceptable derivatives and salts.

The term "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other Human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

The term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of a non-human antibody, and all Or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

The term "chimeric antibody" is an antibody molecule in which (a) the constant region or part thereof is altered, replaced or exchanged such that the antigen binding site is of a different or altered class, effector function and/or species constant region or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) etc. that confers new properties on the chimeric antibody; Changes, substitutions, or exchanges of the variable regions of the For example, mouse antibodies can be modified by exchanging their constant regions with those from human immunoglobulins. Due to the exchange of human constant regions, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in humans as compared to the original mouse antibody.

The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.

The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive when referred to herein.

The term "conjugate" is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.

As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent to which it is conjugated or fused. Labels can themselves be detectable (eg, radioisotopic or fluorescent labels) or, in the case of enzymatic labels, can catalyze the chemical alteration of a detectable substrate compound or composition. The term is intended to encompass both direct labeling of a probe or antibody by conjugating (ie, physically linking) a detectable substance to the probe or antibody as well as indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled. Examples of indirect labeling include detection of primary antibodies using fluorescently labeled secondary antibodies and end-labeling of DNA probes with biotin so that they can be detected with fluorescently labeled streptavidin.

The term "isolated" antibody is an antibody that has been separated from a component of its natural environment. In some embodiments, antibodies are purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase determined by HPLC). For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B848:79-87 (2007).

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating 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 integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

"Isolated nucleic acid encoding an antibody that binds CLDN18.2 or an antigen-binding fragment thereof" refers to one or more nucleic acid molecules encoding an antibody heavy or light chain (or an antigen-binding fragment thereof), included in a single vector or in separate Such nucleic acid molecules in vectors, and such nucleic acid molecules present at one or more locations in the host cell.

A "sample" may be any sample usable according to the invention, in particular a biological sample, such as a tissue sample (including body fluids) and/or a cell sample, and may be obtained in a conventional manner, such as by tissue biopsy (including punch biopsy) and by Collection of blood, bronchial aspirates, sputum, urine, faeces, or other bodily fluids. According to the invention, the term "sample" also includes processed samples, eg fractions or isolates of biological samples, eg nucleic acid and peptide/protein isolates. Preferably, the sample comprises cells or tissue of the organ to be tested (eg, to be diagnosed with cancer).

Calculation of sequence identity between sequences is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a first and second amino acid sequence or nucleic acid sequence may be placed between a first and a second amino acid sequence or nucleic acid sequence for optimal alignment). Gaps may be introduced in one or both or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

The comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm (available at http://www.gcg.com available), use the Blossum 62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 to determine the distance between two amino acid sequences. percent identity. In yet another preferred embodiment, using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and Length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

It is also possible to use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), utilize the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11- 17) Determining the percent identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.

II. Antibodies

As used herein, the term "antibody that binds CLDN18.2", "anti-CLDN18.2", "CLDN18.2 antibody" or "anti-CLDN18.2 antibody" refers to an antibody that is capable of Binding to CLDN18.2 protein or an antigen-binding fragment thereof such that the antibody can be used as a diagnostic and/or therapeutic agent targeting CLDN18.2. In one embodiment, the antibody that binds CLDN18.2 is associated with The non-CLDN18.2 protein binds to a degree less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% of the binding of the antibody to CLDN18.2 Or about 90% or more. In some embodiments, the CLDN18.2 is human CLDN18.2.

In some embodiments, a CLDN18.2-binding antibody or antigen-binding fragment thereof of the invention binds CLDN18.2 (e.g., human CLDN18.2) with sufficient affinity, e.g., with the following equilibrium dissociation constant (K _D ) with CLDN18 .2 binding, said _KD is less than about 10 nM, preferably, less than or equal to about 7 nM, more preferably less than or equal to about 6 nM, more preferably less than or equal to about 5 nM, 4.9 nM, 4.8 nM, 4.7 nM, 4.6 nM , 4.5nM, 4nM, or 3nM, most preferably, said K _D is less than or equal to about 2.5nM, 2.4nM, 2.3nM, 2.2nM, 2.1nM. In some embodiments, the CLDN18.2-binding antibody of the invention binds _CLDN18.2 with a KD of 1 nM-6 nM, preferably 1.5 nM-5 nM, 1.7 nM-5 nM, 1.8 nM-5 nM, 1.9 nM-5 nM. In some embodiments, CLDN18.2 is human CLDN18.2. In some embodiments, antibody binding affinity is determined using biofilm layer optical interferometry.

In some embodiments, an antibody of the invention, or antigen-binding fragment thereof, binds to cells expressing CLDN18.2, e.g., at less than or equal to about 2 nM, 1.9 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 _EC50 in nM, 0.5 nM, 0.4 nM. In some embodiments, the binding is determined using flow cytometry (eg, FACS). In some embodiments, the CLDN18.2-expressing cell is a CLDN18.2-expressing Chinese Hamster Ovary (CHO) cell (eg, a CHO-S cell). In some embodiments, CLDN18.2 is human CLDN18.2.

In some embodiments, the CLDN18.2-binding antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein

(a) said VH comprises:

(i) three complementarity determining regions (CDRs) contained in the VH of any antibody listed in Table B;

with

(b) said VL comprises:

(i) Three complementarity determining regions (CDRs) contained in the VL of any of the antibodies listed in Table B.

In a preferred embodiment, VH comprises the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or consists of said amino acid sequence.

In a preferred embodiment, VL comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or consists of said amino acid sequence.

In some embodiments, the CLDN18.2-binding antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein,

In some embodiments, the CLDN18.2-binding antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

(i) the VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 22, or consists of the amino acid sequence; HCDR2 comprises SEQ ID NO: The amino acid sequence shown in ID NO:14 or SEQ ID NO:23, or consists of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:24 or consists of said amino acid sequence;

In a preferred embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to CLDN18.2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

1) The VH comprises complementary determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13, or consists of the amino acid sequence; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14 Amino acid sequence, or consists of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15 or consists of said amino acid sequence; said VL comprises complementarity determining regions (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises The amino acid sequence shown in SEQ ID NO:16 or consists of said amino acid sequence; LCDR2 comprises or consists of said amino acid sequence shown in SEQ ID NO:17; LCDR3 comprises the amino acid sequence shown in SEQ ID NO:18 or consist of said amino acid sequence;

2) The VH comprises complementary determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13, or consists of the amino acid sequence; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14 Amino acid sequence, or consists of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15 or consists of said amino acid sequence; said VL comprises complementarity determining regions (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises The amino acid sequence shown in SEQ ID NO:19 or consists of said amino acid sequence; LCDR2 comprises or consists of said amino acid sequence shown in SEQ ID NO:20; LCDR3 comprises the amino acid sequence shown in SEQ ID NO:21 or consist of said amino acid sequence;

3) The VH comprises complementary determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22, or consists of the amino acid sequence; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23 Amino acid sequence, or is made up of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24 or is made up of said amino acid sequence; Described VL comprises complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises The amino acid sequence shown in SEQ ID NO:25 or consists of said amino acid sequence; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:20 or consists of said amino acid sequence; LCDR3 comprises the amino acid sequence shown in SEQ ID NO:21 Or consist of said amino acid sequence.

In a preferred embodiment, the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in the CLDN18.2-binding antibody or antigen-binding fragment thereof provided by the present invention is shown in the following table (Table A):

Table A: Exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 in antibodies of the invention or antigen-binding fragments thereof

In some embodiments, the CLDN18.2-binding antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein,

(a) heavy chain variable region VH

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical or consist of; or

(ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; or

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) the amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region;

with

(b) light chain variable region VL

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical or consist of;

(ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; or

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region.

In some embodiments, a CLDN18.2-binding antibody or antigen-binding fragment thereof of the invention comprises

(1) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:1 The heavy chain variable region VH of the sequence, and comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO:2 or the light chain variable region VL of an amino acid sequence of 99% identity;

(2) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:3 The heavy chain variable region VH of the sequence, and comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO:4 or the light chain variable region VL of an amino acid sequence of 99% identity;

(3) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:5 The heavy chain variable region VH of the sequence, and comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO:6 or 99% identical amino acid sequence of the light chain variable region VL.

In a preferred embodiment, the antibody or antigen-binding fragment thereof that binds to CLDN18.2 provided by the invention comprises

(1) heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:1 and light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO:2;

(2) heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:3 and light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO:4;

(3) The heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:5 and the light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO:6.

In a preferred embodiment, the combination of the heavy chain variable region VH and the light chain variable region VL contained in the CLDN18.2-binding antibody or antigen-binding fragment thereof provided by the present invention is shown in the following table (Table B):

Table B: Exemplary combinations of heavy chain variable region VH and light chain variable region VL in antibodies of the invention or antigen-binding fragments thereof

In some embodiments, a CLDN18.2-binding antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and a light chain, wherein

(a) heavy chain

with

(b) light chain

(ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO:8, SEQ IN NO:10 or SEQ ID NO:12; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5) compared to the amino acid sequence shown in SEQ ID NO:8, SEQ IN NO:10 or SEQ ID NO:12 , 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region of the light chain, more preferably, all The amino acid changes described above do not occur in the light chain variable region.

(1) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:7 A heavy chain having a specific amino acid sequence, and comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the light chain of the amino acid sequence;

(2) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:9 A heavy chain having a specific amino acid sequence, and comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the light chain of the amino acid sequence;

(3) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 11 A heavy chain having a specific amino acid sequence, and comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the light chain.

(1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:7 and a light chain comprising the amino acid sequence shown in SEQ ID NO:8;

(2) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:9 and a light chain comprising the amino acid sequence shown in SEQ ID NO:10;

(3) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 11 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 12.

In a preferred embodiment, the antibody or antigen-binding fragment thereof that binds to CLDN18.2 provided by the present invention includes a combination of heavy chain and light chain as shown in the following table (Table C):

Table C: Exemplary combinations of heavy and light chains in antibodies of the invention or antigen-binding fragments thereof

In one embodiment of the invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.

In some embodiments, the substitutions are conservative substitutions. A conservative substitution is one in which an amino acid is replaced by another within the same class, such as one acidic amino acid by another acidic amino acid, one basic amino acid by another basic amino acid, or one neutral amino acid by another neutral amino acid replacement. Exemplary permutations are shown in Table D below:

Form D

In certain embodiments, substitutions occur in the CDR regions of the antibody. Typically, the resulting variant is modified (eg, improved) relative to the parent antibody in certain biological properties (eg, increased affinity) and/or will have certain biological properties of the parent antibody that are substantially retained. Exemplary substitutional variants are affinity matured antibodies.

In some embodiments, a CLDN18.2-binding antibody of the invention is an IgG1 antibody, or an IgG2a antibody.

In some embodiments, the antibody that binds CLDN18.2 is a monoclonal antibody.

In some embodiments, the antibody that binds CLDN18.2 is a murine antibody.

In some embodiments, the antibody that binds CLDN18.2 is a chimeric antibody.

In some embodiments, the antibody that binds CLDN18.2 is humanized. Different methods for humanizing antibodies are known to the skilled person as described in the review by Almagro & Fransson, the content of which is hereby incorporated by reference in its entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13:1619-1633 ).

In some embodiments, the antibody that binds CLDN18.2 is a human antibody. Human antibodies can be prepared using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol 20:450-459 (2008).

In one embodiment, the CLDN18.2-binding antibody of the present invention also encompasses antibody fragments thereof, preferably antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, Single chain antibodies (eg scFv), single domain antibodies, diabodies (dAbs) or linear antibodies.

III. Nucleic acids of the invention and vectors and host cells comprising them

In one aspect, the invention provides nucleic acid encoding any of the above antibodies or antigen-binding fragments thereof that bind to CLDN18.2 or any chain thereof. The nucleic acid may encode an amino acid sequence comprising a light chain variable region and a heavy chain variable region of an antibody, or an amino acid sequence comprising both a light chain and a heavy chain of an antibody. The invention also encompasses nucleic acids that hybridize under stringent conditions to the aforementioned nucleic acids or that have one or more substitutions (eg, conservative substitutions), deletions or insertions compared to the aforementioned nucleic acids.

In one embodiment, the invention provides one or more vectors comprising said nucleic acid. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). In one embodiment, the vector is a pcDNA3.1 vector.

Once the expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, eg, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, biolistic, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the produced transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the particular expression vector and Mammalian host cell alteration or optimization.

Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. A marker can, for example, confer prototrophy, biocidal (eg, antibiotic) or heavy metal (eg, copper) resistance, etc. to an auxotrophic host. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splicing signals, as well as transcriptional promoters, enhancers and termination signals.

In one embodiment, the invention provides a host cell comprising said nucleic acid or said vector. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523, see also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003) , pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells), or other cells suitable for the production of antibodies or antigen-binding fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains in which glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006). Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (293HEK or 293F or 293 cells, as for example Graham et al., J. Gen Virol. 1977) and others. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:216 (1980), CHO-S cells, etc.; and bone marrow Tumor cell lines such as Y0, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa , NJ), pp. 255-268 (2003). In another embodiment, the host cell is prokaryotic, such as E. coli.

IV. Production and Purification of Antibody Molecules of the Invention

In one embodiment, the present invention provides a method for preparing an antibody or fragment thereof (preferably an antigen-binding fragment) that binds to CLDN18.2, wherein the method comprises expression in an antibody or fragment thereof (preferably an antigen-binding fragment) suitable for expressing the antibody or fragment thereof (preferably an antigen-binding fragment) The host cell is cultured under conditions for the nucleic acid, and the antibody or fragment thereof (preferably an antigen-binding fragment) is optionally isolated. In a certain embodiment, the method further comprises recovering the antibody or fragment thereof (preferably an antigen-binding fragment) that binds CLDN18.2 from the host cell.

In one embodiment, a method for preparing an antibody that binds to CLDN18.2 is provided, wherein the method includes, under conditions suitable for antibody expression, cultivating chain) or a host cell comprising an expression vector for said nucleic acid, as provided above, and optionally recovering said antibody from said host cell (or host cell culture medium).

For the recombinant production of an antibody that binds CLDN18.2, nucleic acid encoding an antibody (e.g., an antibody described above, e.g., any polypeptide chain and/or multiple polypeptide chains) is isolated and inserted into one or more vectors for Further cloning and/or expression in host cells. Such nucleic acids are readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the antibody heavy and light chains).

Antibody molecules prepared as described herein can be purified by known art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on such factors as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. The purity of antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

V. Assay

Antibodies provided herein that bind to CLDN18.2 can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, the antibodies of the present invention are tested for their antigen-binding activity, for example, by known methods such as ELISA, Western blotting, and the like. Binding to CLDN18.2 can be assayed using methods known in the art, exemplary methods are disclosed herein. In some embodiments, biofilm layer optical interferometry or MSD assays are used.

It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to antibodies that bind CLDN18.2.

It will be appreciated that any of the above assays can be performed using antibodies that bind CLDN18.2 and additional active agents.

VI. Immunoconjugates

In some embodiments, the invention provides immunoconjugates comprising any of the CLDN18.2-binding antibodies provided herein and other substances, which may be immunofluorescent substances or secondary antibodies.

In some embodiments, the immunoconjugates are used to detect tumors or cancer.

IX. METHODS FOR DIAGNOSIS AND DETECTION

In certain embodiments, any of the CLDN18.2-binding antibodies, or antigen-binding fragments thereof, or immunoconjugates provided herein can be used to detect the presence of CLDN18.2 in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg, RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, a biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

In one embodiment, an antibody or antigen-binding fragment thereof, or an immunoconjugate that binds CLDN18.2 for use in a method of diagnosis or detection is provided. In another aspect, methods of detecting the presence of CLDN18.2 in a biological sample are provided. In certain embodiments, the methods comprise detecting the presence of CLDN18.2 protein in a biological sample. In certain embodiments, CLDN18.2 is human CLDN18.2. In certain embodiments, the method comprises combining a biological sample with a CLDN18.2-binding antibody or antigen-binding fragment thereof or immunoconjugate as described herein in a manner that allows the CLDN18.2-binding antibody or antigen-binding fragment thereof or The immunoconjugate is contacted under conditions that bind to CLDN18.2, and it is detected whether a complex is formed between the antibody or antigen-binding fragment thereof or the immunoconjugate that binds CLDN18.2 and CLDN18.2. Complex formation indicates the presence of CLDN18.2. The method can be an in vitro or in vivo method. In one embodiment, an antibody or antigen-binding fragment thereof, or immunoconjugate that binds CLDN18.2 is used to select a suitable subject for detection or diagnosis, e.g., wherein CLDN18.2 is used to select said subject of biomarkers.

In one embodiment, an antibody of the invention or an antigen-binding fragment thereof, or an immunoconjugate may be used to diagnose a CLDN18.2-associated disease or disorder such as cancer or a tumor, e.g., to evaluate (e.g., monitor) a CLDN18.2-associated disease herein in a subject or the treatment or progression of a disorder, its diagnosis and/or staging. In certain embodiments, a labeled CLDN18.2-binding antibody or antigen-binding fragment thereof, or immunoconjugate is provided.

In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with an antibody or antigen-binding fragment thereof, and an immunoconjugate that binds CLDN18.2. In some embodiments, the sample is obtained prior to treatment with a drug for a CLDN18.2-associated disease or disorder. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin-fixed, paraffin-coated (FFPE). In some embodiments, the sample is a biopsy (eg, a core biopsy), a surgical specimen (eg, a specimen from a surgical resection), or a fine needle aspirate.

In some embodiments, CLDN18.2 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

The subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, a patient suffering from or at risk of having a disease described herein). In one embodiment, the subject has or is at risk of having a disease described herein. In certain embodiments, the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy.

In other aspects, the present invention provides the use of an antibody that binds to CLDN18.2, or an antigen-binding fragment thereof, or an immunoconjugate, in the production or preparation of a kit for detecting or diagnosing the CLDN18.2-related compounds mentioned herein. disease or condition. The diagnostic composition or test kit may be used in a method of the invention, eg, a method of diagnosis, detection or monitoring of the invention. These kits may optionally contain detectable labels such as indicator enzymes, radiolabels, fluorophores or paramagnetic particles. The kit can include an informational leaflet, eg, a manual illustrating how to use the reagents to practice the methods disclosed herein.

In some embodiments, a tumor or cancer described herein. In some embodiments, the tumor or cancer is gastric cancer, pancreatic cancer, gastric junction adenocarcinoma.

In one embodiment, the tumor or cancer is a cancer that expresses elevated levels of CLDN18.2 and/or CLDN18.2.

It will be appreciated that any detection or diagnosis can be performed using an immunoconjugate of the invention in place of or in addition to an antibody that binds CLDN18.2.

X. Exemplary anti-CLDN18.2 antibodies of the invention

Table 1 The amino acid sequence of the CDR of the exemplary antibody of the present invention

Note: HCDR1 is determined according to Abm rules, HCDR2, 3 and LCDR1-3 are determined according to Kabat rules.

Table 2 Light chain variable region (VL) and heavy chain variable region (VH) of the exemplary antibody of the present invention

Table 3 Amino acid sequences of heavy chain (HC) and light chain (LC) of exemplary antibodies of the present invention

抗体名称Antibody name	HCHC	LCLC
50D650D6	SEQ ID NO:7SEQ ID NO:7	SEQ ID NO:8SEQ ID NO:8
50D1250D12	SEQ ID NO:9SEQ ID NO:9	SEQ ID NO:10SEQ ID NO:10
33B233B2	SEQ ID NO:11SEQ ID NO:11	SEQ ID NO:12SEQ ID NO:12

The above specific amino acid sequence is as follows:

Example

Embodiment 1, the preparation of hybridoma cell

Expression and purification of antigen GST-CLDN18(M191-V261)

The C-terminal M191-V261 peptide (SEQ ID NO: 26) of the CLDN18.2 gene was fused to the C-terminal of the GST protein, named GST-CLDN18.2 (SEQ ID NO: 27), through 5'NcoI and 3' HindIII was cloned into the vector pET-28a(+), and the expression plasmid GST-CLDN18.2(M191-V261) was constructed.

Sequence 1: CLDN18.2 (M191-V261)

Sequence 2.GST-CLDN18.2 (M191-V261)

Transform the recombinant plasmid into Escherichia coli BL21(DE3), pick a single clone into a 5ml shake tube, culture overnight, transfer to 400ml LB medium the next day, culture at 37°C until the OD600 is 0.4-0.8, add the final concentration Induced by 1 mM IPTG, culture was continued at 20°C for 20 h. Collect the bacteria, centrifuge at 8000g for 10min, then resuspend the bacteria in PBS, break up the bacteria by ultrasonic (ultrasonic conditions: 150w, sonicate for 2s, stop for 5s, operate on ice), sonicate until the bacteria are clear, centrifuge at 12000g for 20min, and use the supernatant Filter through a 0.22um membrane for later use. The supernatant was purified with a column packed with Glutathione Sepharose 4B (GE, 17-0756-01). The operation process is as follows: equilibrate the packing column with 5 times column volume of equilibration solution PBS (Gibco, 70011-044) before purification; pass the supernatant through the column, and then wash the packing column with 10 times column volume of equilibration solution to remove non-specific binding proteins ; Rinse the filler with 5 times column volume of elution buffer, namely PBS solution containing 10 mM reduced glutathione (Sigma-Aldrich, G4251-100G), and collect the eluate. The collected protein was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), and the protein concentration was determined and stored in aliquots.

immunity

The GST-CLDN18.2 (M191-V261) prepared above was emulsified with complete Freund's adjuvant (sigma, Cat#F5881) and then immunized with Balb/c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) After two weeks, it was emulsified with Freund's incomplete adjuvant (sigma, F5506) for three times and injected intramuscularly once every two weeks (50ug polypeptide per mouse).

cell fusion

When the serum titer meets the requirements, the spleen of the mouse is removed to prepare a suspension of B lymphocytes, which is mixed with SP2/0 myeloma cells (ATCC) at a ratio of 1:2 to 1:1 and then electrofused. Transfer the fused cells from the electrode dish into a 50ml centrifuge tube, dilute the cells with HAT ^- containing medium to 1-2×104 cells/ml, and add 100 μl of cell suspension to each well of a 96-well plate. The selection medium was replaced on the 7th day after the fusion, and after the 10th day of culture (or longer, depending on the cell growth state), the enzyme-linked immunosorbent reaction (Elisa) test was performed to screen positive clones.

Screening of hybridoma positive clones

Dilute NeutrAvidin Biotin Binding protein (Thermo, Cat#3100) to 2ug/mL with coating solution, spread 100uL/well on a 96-well plate, and incubate at 37°C for 1 hour; wash three times with 200μl PBST, shake off the liquid in the plate; add 200μl blocking solution, incubate at 37°C for 1 hour; wash with 200μl PBST three times, shake off the liquid in the plate; dilute Biotin-CLDN18 (M191-V261) (Nanjing GenScript Synthetic) to 25ng/ml, 100uL/well, 37°C Incubate for 1 hour; wash three times with 200 μl PBST, shake off the liquid in the plate; add 100 uL/well of hybridoma supernatant according to the experimental plan, and incubate at 37°C for 1 hour; wash three times with 200 μl PBST, shake off the liquid in the plate; put goat anti-mouse IgG antibody (Biolegend, Cat#405306) was diluted with 1% BSA (1:5000), 100μl/well, incubated at 37°C for 1 hour; washed three times with 200μl PBST, shaken off the liquid in the plate; added 100μl TMB to each well, and protected from light for color development 3min; add 50μl of ELISA stop solution to each well, and read the photometric value at OD450nm.

The positive clones were then re-screened for Control-peptide-GST using the same method as above (target irrelevant polypeptides, with GST tags, prepared in-house). Clones that specifically bind to CLDN18.2(M191-V261) and not to Control-peptide were subcloned.

Positive hybridoma subclones

Subcloning step: prepare a 96-well plate, add 200 μl of medium to each well, and replace HAT with HT (Gibco, Cat#11067-030) on the basis of the screening medium, and the rest of the formula is the same. Make a cell suspension from the cells in the positive wells screened out above, take 100ul and add them to each well in the first row and mix well, then take 100μl of the cell suspension in the first row and add it to the second row, mix well Then take 100 μl and add it to the next row; repeat the above steps, let the 96-well plate stand for 30 minutes, observe and count under the microscope. Take the volume corresponding to 100 cells and add 20ml of medium, mix well and plate, 200μl per well. One week later, observe under the microscope, judge and mark the monoclonal wells. When the confluence of cells in each hole reaches more than 50%, detect with the above-mentioned screening method of Eilsa, pick out the positive hole of the target, and adopt the biofilm thin layer interferometry technique (ForteBio) to measure the hybridoma cell supernatant obtained and the antigen ratio. affinity. There are 3 clones that specifically bind to CLDN18.2 (M191-V261) and do not bind to control-peptide, and all the detection results are shown in Table 4:

Table 4: Supernatant detection results of hybridoma subclones

Example 2, Preparation of Recombinant Mouse Antibody

The antibody light and heavy chain gene sequences were retrieved from the hybridoma candidate clones 50D12, 33B2 and 50D6 obtained in Example 1. Take about 5×10 ⁶ freshly cultured cells per strain, and extract RNA (Macherey-Nagel, Cat#740984.250). cDNA was obtained by reverse transcription using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). The upstream primer is designed with the base sequence located in the 5' end FR1 region, and the downstream primer is designed with the base sequence located in the antibody constant region or FR4 region to amplify the antibody light chain and heavy chain variable region gene fragments. It was connected to a T vector (Mighty TA-cloning Kit, Takara), single clones were picked for sequencing, and the sequencing results were analyzed and compared using MEGA7 software. Finally, according to the specificity of the variable region sequence of the light and heavy chains of the antibody and the affinity of the antibody, three clones, 50D12, 33B2 and 50D6, were constructed for recombinant murine antibody.

Homologous recombinase

II (Nanjing Novizan, C112-01) was connected to the pcDNA3.1 vector, in which the mIgG2a subtype was selected for the constant region, and the expression plasmids of the light chain and heavy chain antibodies were obtained. The sequence of the control antibody 43-14A is derived from the patent US9512232.

Then the light chain plasmid and heavy chain plasmid of the same antibody were mixed at a molar ratio of 1:1, and transfected into 293F cells with polyethyleneimine (PEI) (Polysciences, Cat#23966). After 5-7 days of culture, the cell viability was low At 60%, the cell culture supernatant was collected and the monoclonal antibody was purified with Protein A affinity column.

Example 3 Biomembrane Thin Layer Interferometry Determination of the Binding Kinetics of Antibodies of the Present Invention and Antigens

The equilibrium dissociation constant (KD) of the antibody of the present invention binding to GST-CLDN18 (M191-V261) was determined by biofilm thin layer interferometry technique (ForteBio). ForteBio affinity determination was carried out according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5 (2): p. 270-8).

Briefly, the AMQ (Pall, 1506091) sensor was equilibrated offline for 30 minutes in assay buffer, followed by online detection for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor (ForteBio) for ForteBio Affinity measurement. The sensor with loaded antibody was then exposed to the antigen GST-CLDN18 (M191-V261 ), after which the sensor was transferred to assay buffer for off-rate measurement. KD values were analyzed using ForteBio analysis software. The detection results of antibody affinity are shown in Table 5:

Table 5 ForteBio detects the binding affinity of antigen and antibody (equilibrium dissociation constant KD)

From the above affinity data, it can be seen that the antibody obtained by our hybridoma has a good affinity with GST-CLDN18 (M191-V261). affinity.

Example 4 Immunohistochemical staining analysis of mouse tumor tissue sections

We used DAN-G claudin18.2 (DSMZ, product number ACC 249) and SNU-620 (JCRB CELL BANK, product number JCRB0834) cells to inoculate NOG mice. SPF grade female NOG mice (18-20 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used in the experiment, and the certificate numbers are NO.110011211104489938 and NO.110011211104633423.

Cells were routinely subcultured for subsequent experiments. Collect the cells by centrifugation, disperse the cells with PBS and Matrigel at a ratio of 1:1, and prepare cell suspensions with cell concentrations of 5x10 ⁶ cells/ml, 3x10 ⁷ cells/ml, and 3x10 ⁷ cells/ml, respectively. On day 0, take 0.2ml of cell suspension and subcutaneously inoculate the right dorsal and abdominal area of NOG mice to establish the tumor-bearing mouse model of each cell, that is, the inoculation amount is DAN-G claudin18.2 1×10 ⁶ cells/mouse and SNU-620 6×10 ⁶ cells/mouse.

On the 7th day after tumor cell inoculation, the tumors of all mice were removed and fixed in neutral formalin (Sigma, product number HT501128-4L) for 48 hours, and then fully automatic dehydrator (SAKURA Tissue-Tek

6AI) After dehydration and immersion in wax, wax blocks (Leica Histore Arcadia H) and slices (Leica RM2255) were made.

The staining of the four clones 50D12, 33B2, 50D6 and 43-14A in tumor tissues of each mouse was then evaluated by immunohistochemical staining. The specific experimental steps are as follows:

1. The paraffin sections of mouse tumor tissue were soaked in xylene (Sinopharm, Cat. No. 10023418) for 10min-5min-5min in the order of dewaxing.

2. Soak the dewaxed slices for 5 min-95% for 5 min-80% for 5 min-75% for 5 min-ddH2O for 5 min in 100% absolute ethanol (National Medicine, product number 10009218) for rehydration.

3. Put the Tris-EDTA buffer solution pH 9.0 (abcam, product number ab93684) into the pressure cooker and heat it to 60 degrees, and place the slices in high-pressure repair for 10 minutes. After repairing, take out the slices from the pressure cooker, wait for the repairing solution to cool down to 60 degrees, take out, and soak in ddH2O for 5 minutes.

4. Place the slices in a wet box, add 3% hydrogen peroxide (Gene Technology, Cat. No. GT100535) dropwise to cover the entire tissue, and inactivate at room temperature for 15 minutes in the dark.

5. Discard the hydrogen peroxide on the slices, immerse them in 1ⅹTTBS (Scytek, product number TBT999) and wash them three times, 5 minutes each time. Goat serum (Jackson Immuno research, Cat. No. 005-000-121) was diluted to 10% with 1ⅹ TTBS, an appropriate amount of 10% goat serum was added dropwise to cover the whole tissue, and incubated at room temperature for 1 h.

6. Dilute the primary antibodies (50D12, 33B2, 50D6, and 43-14A) to 10ug/ml with antibody diluent (abcam, Cat. No. ab64211), discard the serum after blocking, and directly add an appropriate amount of primary antibody to cover The whole tissue was incubated overnight at 4°C.

7. Discard the primary antibody on the slice and wash it three times by immersing in 1ⅹTTBS, 5min each time. Dilute the corresponding goat anti-mouse secondary antibody (Jackson ImmunoResearch, Cat. No. 115-035-003) with 1ⅹPBS (Sangon Biotech, Cat. No. E607008-0500) to a concentration of 5ug/ml, add an appropriate amount of secondary antibody to cover the entire tissue, and incubate at room temperature 30min.

8. Discard the secondary antibody on the slice and wash it three times by immersing in 1ⅹTTBS, 5min each time. Dilute the DAB chromogenic solution (abcam, product number ab64238) at a ratio of 1:200, drop an appropriate amount on the tissue and observe under a microscope. After the color development, immerse in ddH2O to terminate the reaction.

9. Immerse the slices after color development in hematoxylin (sigma, product number 51275). After counterstaining for 5-10 seconds, quickly immerse the slices in tap water and rinse with running water for 15 minutes to return to blue.

10. After the bluing, the slices were soaked in 75% ethanol for 5 minutes-80% ethanol for 5 minutes-95% ethanol for 5 minutes-100% ethanol for 5 minutes for gradient dehydration of the slices.

11. Immerse the dehydrated slices in xylene for 5 minutes to make them transparent.

12. Seal the transparent slices with neutral gum (Sinopharm, Cat. No. 10004160). After sealing, place them in a fume hood to dry and scan the slices for analysis (Leica Aperio VERSA 8).

The experimental results are shown in Figures 1 and 2. The staining intensity and clarity of the three clones 50D12, 33B2 and 50D6 in the two mouse tumor tissues were significantly better than that of the control 43-14A, and the specificity was also significantly better than that of 43-14A , which can clearly distinguish the negative and positive areas. Under the same experimental conditions, the latter staining is not clear enough and has strong non-specific background staining (marked in red boxes in Figure 1 and Figure 2).

Claims

An antibody or antigen-binding fragment thereof that binds to CLDN18.2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein,

1) The VH comprises three complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:1, and the VL comprises the VL contained in SEQ ID NO:2 LCDR1, LCDR2 and LCDR3;

2) The VH comprises three complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:3, and the VL comprises the VL shown in SEQ ID NO:4 LCDR1, LCDR2 and LCDR3;

3) The VH comprises three complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO:5, and the VL comprises the VL shown in SEQ ID NO:6 LCDR1, LCDR2, and LCDR3.
An antibody or antigen-binding fragment thereof that binds to CLDN18.2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein,

(i) the VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 22 or consists of said amino acid sequence; HCDR2 comprises SEQ ID The amino acid sequence shown in NO:14 or SEQ ID NO:23 or consists of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:24 or consists of said amino acid sequence;

with

(ii) wherein said VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises SEQ ID NO: 16, the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 25 or consists of said amino acids Sequence composition; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:20 or is made up of said amino acid sequence; LCDR3 comprises the aminoacid sequence shown in SEQ ID NO:18 or SEQ ID NO:21 or is made up of said aminoacid sequence The above amino acid sequence composition.
The antibody or antigen-binding fragment thereof according to claim 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein,

1) The VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:13; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:14 Sequence or be made up of described aminoacid sequence; HCDR3 comprises the aminoacid sequence shown in SEQ ID NO:15 or is made up of described aminoacid sequence; Described VL comprises complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises SEQ ID The amino acid sequence shown in NO:16 or consists of said amino acid sequence; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:17 or consists of said amino acid sequence; LCDR3 comprises the amino acid sequence shown in SEQ ID NO:18 or consists of said amino acid sequence composition;

2) The VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:13; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:14 Sequence or be made up of described aminoacid sequence; HCDR3 comprises the aminoacid sequence shown in SEQ ID NO:15 or is made up of described aminoacid sequence; Described VL comprises complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, and wherein LCDR1 comprises SEQ ID The amino acid sequence shown in NO:19 or consists of said amino acid sequence; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:20 or consists of said amino acid sequence; LCDR3 comprises the amino acid sequence shown in SEQ ID NO:21 or consists of said amino acid sequence composition;

3) The VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:22; HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23 sequence or consist of said amino acid sequence; HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24 or consists of said amino acid sequence; said VL comprises complementarity determining regions (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises SEQ ID The amino acid sequence shown in NO:25 or consists of said amino acid sequence; LCDR2 comprises the amino acid sequence shown in SEQ ID NO:20 or consists of said amino acid sequence; LCDR3 comprises the amino acid sequence shown in SEQ ID NO:21 or consists of The amino acid sequence composition.
The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a heavy chain variable region VH and a light chain variable region VL, wherein,

(a) heavy chain variable region VH

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical or consist of said amino acid sequences; or

(ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; or

(iii) comprising or consisting of an amino acid sequence having 1-10 amino acid substitutions, insertions or deletions compared to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 ;

with

(b) light chain variable region VL

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical or consist of said amino acid sequences;

(ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; or

(iii) comprising or consisting of an amino acid sequence having 1-10 amino acid substitutions, insertions or deletions compared to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 .
The antibody or antigen-binding fragment thereof according to claim 4, comprising

(1) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:1 sequence or a heavy chain variable region VH consisting of said amino acid sequence, and comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO:2 %, 97%, 98% or 99% identical amino acid sequence or light chain variable region VL consisting of said amino acid sequence;

(2) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:3 sequence or a heavy chain variable region VH consisting of said amino acid sequence, and comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or light chain variable region VL consisting of said amino acid sequence;

(3) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:5 sequence or a heavy chain variable region VH consisting of said amino acid sequence, and comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO:6 %, 97%, 98% or 99% identical amino acid sequence or light chain variable region VL consisting of said amino acid sequence.
The antibody or antigen-binding fragment thereof according to claim 5, comprising

(1) A heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 1 or consisting of said amino acid sequence and comprising an amino acid sequence shown in SEQ ID NO: 2 or a light chain consisting of said amino acid sequence Variable region VL;

(2) A heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 3 or consisting of said amino acid sequence and comprising an amino acid sequence shown in SEQ ID NO: 4 or a light chain consisting of said amino acid sequence Variable region VL;

(3) The heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 5 or consisting of said amino acid sequence and the light chain comprising the amino acid sequence shown in SEQ ID NO: 6 or consisting of said amino acid sequence Variable region VL.
The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, comprising a heavy chain and a light chain, wherein

(a) heavy chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, Amino acid sequences that are 96%, 97%, 98% or 99% identical or consist of said amino acid sequences;

(ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11; or

(iii) comprising or consisting of an amino acid sequence having 1-10 amino acid substitutions, insertions or deletions compared to the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 ;

with

(b) light chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, Amino acid sequences that are 96%, 97%, 98% or 99% identical or consist of said amino acid sequences;

(ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12; or

(iii) comprising or consisting of an amino acid sequence having 1-10 amino acid substitutions, insertions or deletions compared to the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12 .
The antibody or antigen-binding fragment thereof according to claim 7, comprising

(1) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:7 A specific amino acid sequence or a heavy chain consisting of said amino acid sequence, and comprising at least 85%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or a light chain consisting of said amino acid sequence;

(2) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:9 A specific amino acid sequence or a heavy chain consisting of said amino acid sequence, and comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO:10 , 96%, 97%, 98% or 99% identical amino acid sequence or a light chain consisting of said amino acid sequence;

(3) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 11 A specific amino acid sequence or a heavy chain consisting of said amino acid sequence, and comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 12 , 96%, 97%, 98% or 99% identical amino acid sequence or a light chain consisting of said amino acid sequence.
The antibody or antigen-binding fragment thereof according to claim 8, comprising

(1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:7 or consisting of said amino acid sequence and comprising an amino acid sequence shown in SEQ ID NO:8 or a light chain consisting of said amino acid sequence;

(2) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9 or consisting of the amino acid sequence and a light chain comprising the amino acid sequence shown in SEQ ID NO: 10 or consisting of the amino acid sequence;

(3) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 11 or consisting of the amino acid sequence and a light chain comprising the amino acid sequence shown in SEQ ID NO: 12 or consisting of the amino acid sequence.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is an antibody in IgGl, IgG2, IgG3, IgG4 format or an antibody in IgG2a format.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody is a monoclonal antibody, a murine antibody, a chimeric antibody, a humanized antibody or a human antibody, a bispecific or A multispecific antibody molecule, said antigen-binding fragment being an antibody fragment of Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv) or (Fab') 2 .
An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-11.
A vector comprising the nucleic acid according to claim 12, preferably, the vector is an expression vector.
A host cell comprising a nucleic acid according to claim 12 or a vector according to claim 13.
The host cell according to claim 14, wherein said host cell is prokaryotic or eukaryotic.
The host cell according to claim 15, wherein the host cell is a 293 cell or a CHO cell.
A method for preparing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell according to any one of claims 14-16 under conditions suitable for expressing the nucleic acid encoding the nucleic acid according to claim 12, optionally by The method further comprises isolating said antibody or antigen-binding fragment thereof, optionally said method further comprises recovering said antibody or antigen-binding fragment thereof from said host cell.
A conjugate comprising the antibody or antigen-binding fragment thereof of claims 1-11 coupled to at least one detectable label.
Use of the antibody or antigen-binding fragment thereof according to claims 1-11, or the conjugate according to claim 18, in the manufacture of a diagnostic test kit for diagnosing, detecting or monitoring cancer, the diagnosis, detection Or monitoring for cancer involves the following steps:

(i) contacting a biological sample with said antibody or antigen-binding fragment thereof, or with said conjugate, and

(ii) detecting the formation of a complex between said antibody or antigen-binding fragment thereof, or said conjugate and CLDN18.2 and/or determining the amount of the complex.
The use according to claim 19, wherein the cancer is a patient with positive expression of CLDN18.2, such as gastric cancer, pancreatic cancer, gastroesophageal junction adenocarcinoma.
A diagnostic test kit comprising the antibody or antigen-binding fragment thereof of claims 1-11, or the conjugate of claim 18.