WO2021239026A1

WO2021239026A1 - Antibody against claudin18.2 and use thereof

Info

Publication number: WO2021239026A1
Application number: PCT/CN2021/096205
Authority: WO
Inventors: 李朝辉; 吕裕斌; 吴敏; 张骞; 方和娣; 方杰
Original assignee: 杭州邦顺制药有限公司
Priority date: 2020-05-29
Filing date: 2021-05-27
Publication date: 2021-12-02
Also published as: CN113735974A; CN113735974B

Abstract

The present application provides an isolated antigen-binding protein, having the ability to specifically bind to Claudin18.2 on a cell surface, showing a CDC effect on Claudin18.2 positive tumor cells, showing an ADCC effect on Claudin18.2 positive tumor cells, and having the effect of inhibiting the growth of Claudin18.2 positive tumors.

Description

Antibodies against Claudin 18.2 and their uses

Technical field

This application relates to the field of biomedicine, in particular to an antibody against Claudin 18.2 and its use.

Background technique

Claudin is a family of cell surface proteins that establishes paracellular barriers and controls the flow of molecules between cells. Claudin is a necessary component of tight binding, which plays an important role in maintaining the polarity of epithelial cells, controlling paracellular proliferation, and regulating cell growth and differentiation. Different Claudins are expressed in different tissues, and their altered functions are related to the formation of cancer in each tissue. Claudin-1 expression has been shown to have prognostic value in colon cancer, Claudin-18 in gastric cancer, and Claudin-10 in hepatocellular carcinoma. Therefore claudins has also become a promising therapeutic target. There are 2 variants of Claudin18, among which Claudin 18.1 is selectively expressed in the epithelium of normal lung and stomach, and Claudin 18.2 is only expressed in trace amounts in differentiated short-lived cells of normal gastric epithelium, but Claudin18 can be found in a variety of tumors. .2 is strongly expressed, such as 75% of gastric cancer patients, 50% of pancreatic cancer patients, 30% of esophageal cancer patients, and lung cancer patients.

CN103509114A discloses a monoclonal antibody against claudin-18 for the treatment of cancer. The 175D10 antibody disclosed therein is the IMAB362 currently used in clinical trials by Astellas, which exhibits specific binding to CLD18.2 and mediates killing Activity of cells expressing CLD18.2. The development of antibodies specific to Claudin 18.2 complements the unmet medical needs. There is also a need for antibodies against Claudin 18.2 with better activity, including binding activity, effector cell activity, tumor-killing activity, pharmacodynamics, etc., to meet the treatment of patients in this field and related treatment needs.

Summary of the invention

This application provides an antibody against Claudin 18.2 and uses thereof, including isolated antigen binding proteins, nucleic acid molecules, carriers, cells with high specific activity against Claudin 18.2, and preparation methods, pharmaceutical compositions and uses thereof.

This application provides an isolated antigen binding protein, which has one or more of the following properties:

1) In the FACS assay, it can specifically bind to Claudin 18.2 on the cell surface;

2) In the FACS assay, it does not bind to Claudin 18.1 on the cell surface;

3) Show CDC effect on Claudin 18.2 positive tumor cells;

4) Does not show CDC effect on Claudin 18.1 positive tumor cells;

5) Induce ADCC effect on Claudin 18.2 positive tumor cells;

6) Inhibit the growth of Claudin 18.2 positive tumors.

In some embodiments, the isolated antigen binding protein is selected from antibodies or antigen binding fragments thereof.

In some embodiments, the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody, preferably a fully human antibody.

In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv, or dAb.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH, and the VH comprises at least one of the following HCDRs:

HCDR1, whose amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 12, or includes the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12;

HCDR2, whose amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 13, or includes the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 13;

HCDR3, whose amino acid sequence is shown in SEQ ID NO: 4 or SEQ ID NO: 14, or includes the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 14;

In some embodiments, the isolated antigen binding protein comprises a light chain variable region VL, and the VL comprises at least one LCDR as follows:

LCDR1, whose amino acid sequence is shown in SEQ ID NO: 7 or SEQ ID NO: 17, or includes the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 17;

LCDR2, whose amino acid sequence is shown in SEQ ID NO: 8 or SEQ ID NO: 18, or includes the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 18;

LCDR3, whose amino acid sequence is shown in SEQ ID NO: 9 or SEQ ID NO: 19, or includes the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 19.

In some embodiments, the VH includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

In some embodiments, the VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

In some embodiments, the VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

In some embodiments, the VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

In some embodiments, the VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, and the VL includes SEQ ID NO: 7. LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 8 and SEQ ID NO: 9.

In some embodiments, the VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; and the VL includes SEQ ID NO: 17. LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 18 and SEQ ID NO: 19.

In some embodiments, the VH of the isolated antigen binding protein includes framework regions H-FR1, H-FR2, H-FR3 and H-FR4.

In some embodiments, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes the amino acid sequence shown in SEQ ID NO: 21 or 29.

In some embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 22 or 30.

In some embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 23 or 31.

In some embodiments, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 24 or 32.

In some embodiments, the VL of the isolated antigen binding protein includes the framework regions L-FR1, L-FR2, L-FR3 and L-FR4.

In some embodiments, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 25 or 33.

In some embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 26 or 34.

In some embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 27 or 35.

In some embodiments, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 28 or 36.

In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:1 or 11.

In some embodiments, the VL comprises the amino acid sequence shown in SEQ ID NO: 6 or 16.

In some embodiments, the amino acid sequence of the VH is shown in SEQ ID NO: 1, and the amino acid sequence of the VL is shown in SEQ ID NO: 6.

In some embodiments, the amino acid sequence of the VH is shown in SEQ ID NO: 11, and the amino acid sequence of the VL is shown in SEQ ID NO: 16.

In certain embodiments, the isolated antigen binding protein further comprises an antibody heavy chain constant region selected from the group consisting of a human IgG constant region, an IgA constant region, an IgM constant region, an IgD constant region, or an IgE constant region. Region, the human IgG constant region is further selected from a human IgG1 constant region, an IgG2 constant region, an IgG3 constant region or an IgG4 constant region;

In some embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 37;

In some embodiments, the isolated antigen binding protein comprises an antibody light chain constant region selected from a human Igκ constant region or a human Igλ constant region.

In some embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 38 or 39.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain HC and an antibody light chain LC.

In some embodiments, the HC comprises the amino acid sequence shown in SEQ ID NO: 5 or 15.

In some embodiments, the LC comprises the amino acid sequence shown in SEQ ID NO: 10 or 20.

In some embodiments, the HC includes the amino acid sequence shown in SEQ ID NO: 5, and the LC includes the amino acid sequence shown in SEQ ID NO: 10.

In some embodiments, the HC includes the amino acid sequence shown in SEQ ID NO: 15, and the LC includes the amino acid sequence shown in SEQ ID NO: 20.

In another aspect, the present application provides an immunoconjugate comprising the isolated antigen binding protein described in any one of the present application.

In another aspect, the present application provides an isolated nucleic acid molecule that encodes the isolated antigen binding protein or the immunoconjugate described in any one of the present application.

In another aspect, the present application provides a vector, which contains the nucleic acid molecule described in the present application.

In another aspect, the present application provides a cell, which contains the nucleic acid molecule or the vector described in the present application.

In another aspect, the present application provides a method for preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions that allow the expression of the isolated antigen binding protein described in this application.

In another aspect, the present application provides a pharmaceutical composition, which comprises the isolated antigen binding protein, the immunoconjugate, the nucleic acid molecule, the carrier or the cell described in the present application, and Optionally a pharmaceutically acceptable adjuvant.

On the other hand, the present application provides the isolated antigen binding protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell or the pharmaceutical composition for preparing medicines. The medicine is used to prevent, diagnose, alleviate or treat tumors.

In another aspect, the present application provides the isolated antigen binding protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in For use in the preparation of medicines, the tumors include solid tumors and/or hematological tumors.

In another aspect, the present application provides the isolated antigen binding protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in For use in the preparation of medicines, the solid tumors include lung cancer, colon cancer, liver cancer, esophageal cancer, ovarian cancer, bladder cancer, stomach cancer, kidney cancer and/or pancreatic cancer.

On the other hand, the present application provides a method of preventing, diagnosing, alleviating and/or treating tumors, which includes administering the isolated antigen binding protein, the immunoconjugate, and the immunoconjugate in an amount effective to treat the cancer to a subject in need thereof Substance, said nucleic acid molecule, said vector, said cell or said pharmaceutical composition.

In another aspect, the application provides the administration of the isolated antigen binding protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition A method for preventing, diagnosing, alleviating and/or treating tumors, the tumors including solid tumors and/or hematological tumors.

On the other hand, the application provides for the administration of the isolated antigen binding protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell or the pharmaceutical composition for A method of preventing, diagnosing, alleviating and/or treating tumors, the solid tumors including lung cancer, colon cancer, liver cancer, esophageal cancer, ovarian cancer, bladder cancer, stomach cancer, kidney cancer and/or pancreatic cancer.

On the other hand, the present application provides the isolated antigen binding protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell or the pharmaceutical composition, which is used For the prevention, diagnosis, alleviation and/or treatment of tumors.

On the other hand, the present application provides the isolated antigen binding protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell or the pharmaceutical composition, which is used For the prevention, diagnosis, alleviation and/or treatment of tumors, the tumors include solid tumors and/or hematological tumors.

On the other hand, the present application provides the isolated antigen binding protein, the immunoconjugate, the nucleic acid molecule, the carrier, the cell or the pharmaceutical composition, which is used For the prevention, diagnosis, alleviation or treatment of tumors, the solid tumors include lung cancer, colon cancer, liver cancer, esophageal cancer, ovarian cancer, bladder cancer, gastric cancer, kidney cancer and/or pancreatic cancer.

In another aspect, the present application provides a method for inhibiting the growth of Claudin 18.2 positive tumor cells and/or killing the Claudin 18.2 positive tumor cells, comprising contacting the Claudin 18.2 positive tumor cells with the isolated antigen binding Protein or said immunoconjugate.

The beneficial effects of the isolated antigen binding protein provided in this application include one or more of the following: it can specifically bind to Claudin 18.2 on the cell surface, Claudin 18.2 that does not bind to the cell surface, and it shows CDC on Claudin 18.2 positive tumor cells. It does not show CDC effect on Claudin 18.1 positive tumor cells, induces ADCC effect on Claudin 18.2 positive tumor cells and/or inhibits the growth of Claudin 18.2 positive tumors.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Correspondingly, the drawings and descriptions in the specification of the present application are merely exemplary, rather than restrictive.

Description of the drawings

Figure 1 shows the affinity of the anti-Claudin 18.2 antibody described in this application with Claudin 18.2 positive HEK293 cells.

Figure 2 shows the affinity of the antibody HDR002C04 described in this application to Claudin 18.1 positive HEK293 cells or Claudin 18.2 positive HEK293 cells.

Figure 3 shows the affinity of the antibody HDR002C06 described in this application with Claudin 18.1 positive HEK293 cells or Claudin 18.2 positive HEK293 cells.

Figure 4 shows that the anti-Claudin 18.2 antibody described in this application exerts CDC activity on Claudin 18.2 positive HEK293 cells.

Figure 5 shows that the anti-Claudin 18.2 antibody described in this application exerts CDC activity on Claudin 18.2 positive BxPC3 cells.

Figure 6 shows that the anti-Claudin 18.2 antibody described in this application exerts CDC activity on Claudin 18.2 positive N87 cells.

Figure 7 shows the specific CDC activity of the antibody HDR002C04 described in this application.

Figure 8 shows the specific CDC activity of the antibody HDR002C06 described in this application.

Figure 9 shows that the anti-Claudin 18.2 antibody described in this application exerts ADCC activity on Claudin 18.2 positive BxPC3 cells.

Figure 10 shows that the anti-Claudin 18.2 antibody described in this application exerts ADCC activity on Claudin 18.2 positive N87 cells.

Figure 11 shows the effect of the antibody HDR002C04 in this application on inhibiting tumor volume growth.

Figure 12 shows the effect of the antibody HDR002C04 in this application on inhibiting tumor weight increase.

Figure 13 shows the effect of the antibody HDR002C06 in this application on inhibiting tumor volume growth.

Figure 14 shows the effect of the antibody HDR002C06 in this application on inhibiting the increase in tumor weight.

Detailed ways

The following specific examples illustrate the implementation of the invention of this application. Those familiar with this technology can easily understand the other advantages and effects of the invention of this application from the content disclosed in this specification.

Definition of Terms

To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this document, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

The term "Claudin 18.2 protein" in this application is a transmembrane egg located on the cell membrane, which is only expressed on differentiated gastric mucosal epithelial cells in normal tissues, and is mostly expressed in primary gastric cancer and metastatic cancers. In addition, the activated expression of Claudin 18.2 protein can also be observed in lung cancer, pancreatic cancer, and ovarian cancer.

In this application, the term "does not bind" or "substantially does not bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to it with high affinity, that is, the K _D of the ^{binding protein or cell is 1.0x10 -6} M or more, can be 1.0x10 ^-5 M or more, can be 1.0x10 ^-4 M or more, 1.0x10 ^-3 M or more, or can be 1.0x10 ^-2 M or more.

In this application, the term "reference antibody" generally refers to a variant or homologue that has the same or similar functions as the protein, polypeptide, and/or amino acid sequence involved in this application. Competitively bind to the antigen Claudin 18.2 protein than the antibody. The term "isolated antigen binding protein" in the present application refers to an antigen binding protein that is substantially free of other antigen binding proteins with different antigen specificities. For example, an isolated antigen binding protein that specifically binds to Claudin 18.2 protein does not substantially contain an antigen binding protein that specifically binds to antigens other than Claudin 18.2 protein. However, the isolated antigen binding protein that specifically binds to human Claudin 18.2 protein may have cross-binding to other antigens, such as Claudin 18.2 protein of other species.

In this application, the term "constant region" generally refers to a part of an immunoglobulin molecule that has a more conservative amino acid sequence relative to other parts of the immunoglobulin molecule or the variable region containing an antigen binding site. The constant region contains the CH1, CH2, and CH3 domains of the heavy chain and the CL domain of the light chain.

In this application, the term "diagnosis" includes, for example, diagnosis or detection of the presence of disorders related to or mediated by Claudin 18.2 expression of pathological hyperproliferative tumor formation, monitoring of disease progression, and identification or detection of indications Cells or samples of disorders related to Claudin 18.2 expression. The terms "diagnosis", "detection", "identification", etc. are used interchangeably herein.

In this application, the term "alleviation" refers to the reduction, reduction or elimination of a certain condition, disease, disorder or phenotype, including malformations or symptoms.

In this application, the terms "antibody", "Ig" or "immunoglobulin" are meant to include full-length antibodies and any antigen-binding fragments (ie, antigen-binding portions) or single chains thereof. Such antibodies include, but are not limited to, fully human antibodies, primatized antibodies, chimeric antibodies, monoclonal antibodies, monospecific antibodies, polyclonal antibodies, multispecific antibodies, non-specific antibodies, bispecific antibodies, and polyclonal antibodies. Specific antibodies, humanized antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, graft-coupled antibodies (i.e. antibodies conjugated or fused to other proteins, radioactive markers, cytotoxins) and generated in vitro Antibody. Antibodies can be from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody may also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of the present invention can be derived from any species, including but not limited to mice, humans, camels, llamas, fish, sharks, goats, rabbits, chickens, and cattle. The constant region of the antibody can be changed, such as mutation, to modify the characteristics of the antibody (for example, to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine residues, effect Organ cell function, or complement function). Generally, antibodies specifically bind to predetermined antigens, such as antigens associated with disorders, such as inflammatory, immune, autoimmune, neurodegenerative, metabolic, and/or malignant disorders. A full-length antibody is a glycoprotein containing at least two heavy chains (HC) and two light chains (LC), the heavy and light chains are connected by disulfide bonds. Each heavy chain and a heavy chain constant region of a heavy chain variable region (abbreviated VH or V _H). The heavy chain constant region is composed of three domains, namely CH1, CH2 and CH3. Each light chain and light chain constant region is comprised of a light chain variable region (abbreviated VL or V _L). The constant region of the light chain consists of a domain CL. The VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDR), which are separated by more conservative framework regions (FR) regions. Each VH and VL are composed of three CDRs and four FRs, and are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, FR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including a variety of immune system cells (for example, effector cells) and the first component (C1q) of the traditional complement system.

In this application, the term "chimeric antibody" generally refers to an antibody whose light chain and heavy chain genes have been constructed from immunoglobulin gene segments belonging to different species through genetic engineering. For example, the variable region (V) segment of a mouse monoclonal antibody gene may bind to human constant (C) segments such as IgG1 and IgG4. For example, it may be human isotype IgG1. Therefore, a typical chimeric antibody is a hybrid protein consisting of mouse antibody V or antigen binding domain and human antibody C or effector domain.

In this application, the term "humanized antibody" refers to an antibody that is compared with the CDR of the parental immunoglobulin in which the framework or "complementarity determining region" (CDR) has been modified to include immunoglobulins of different specificities. Protein CDR. In another embodiment, mouse CDRs are grafted into the framework regions of human antibodies to prepare the "humanized antibodies". See, for example, Riechmann, L, et al., Nature332 (1988) 323-327; and Neuberger, M.S., et al., Nature314 (1985) 268-270. In addition, the CDRs correspond to those that recognize the above-mentioned antigen sequences for chimeric and bifunctional antibodies. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or changed from the constant region of the original antibody to produce the characteristics according to the present invention, particularly with regard to Clq binding and/or Fc receptor ( FcR) those antibodies that have the characteristics of binding.

In this application, the term "antigen-binding protein" refers to a molecule composed of one or more polypeptides that recognize and specifically bind to a target, such as Claudin 18.2, such as an anti-Claudin 18.2 antibody or an antigen-binding fragment thereof.

In this application, the term "antigen-binding fragment" (or simply "antibody portion") refers to one or more fragments that retain the ability of an antibody to specifically bind to an antigen (for example, Claudin 18.2 protein). It has been confirmed that the antigen-binding function of antibodies can be implemented by fragments of full-length antibodies. Examples of the binding fragment contained in the "antigen binding portion" of the antibody include Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv and/or dAb. A number of antigen binding fragments are listed below: (i) Fab fragment, a monovalent fragment composed of VL, VH, CL and CH1; (ii) F(ab′) ₂ fragment, containing two Fabs connected by a disulfide bridge in the hinge region bivalent fragment fragments; (iii) consisting of the VH and CH1 Fd fragment; (iv) Fv fragments consisting of a single arm of an antibody V _L and V _H; (v) dAb fragment consisting of V _H; (vi) separation The complementarity determining region (CDR) of the CDR; and (vii) Nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains V _L and V _H Fv fragment, a synthetic linker which can be a single chain via a protein so that the two encoded by separate genes by recombination are connected, wherein V _L and V _H regions pair to form monovalent Molecule (called single-chain Fv (scFv)). These single chain antibodies have also been included in the meaning of the term. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

In this application, the terms "variable region" or "variable domain" are used interchangeably, and generally refer to a part of the light or heavy chain of an antibody, and generally refer to the amino terminus of the antibody. It can contain about 100-130 amino acids in the heavy chain or about 90-115 amino acids in the light chain, the sequence of which varies greatly between antibodies, and is used for the binding specificity of a specific antibody to a specific antigen. Sequence variability is concentrated in those regions called complementarity determining regions (CDR), while the more highly conserved regions in variable domains are called framework regions (FR). The CDRs of the light chain and the heavy chain are mainly responsible for the interaction and specificity between the antibody and the antigen.

In this application, the term "framework region" refers to the part of the antibody variable region recognized in the art that exists between the more divergent (ie hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and provide a framework for presenting six CDRs (three from the heavy chain and three from the light chain) in a three-dimensional space to Form the antigen binding surface.

In this application, the term "immunoconjugate" or "antibody conjugate" generally refers to the connection of an antibody or antibody fragment thereof with other active agents, such as chemotherapeutics, toxins, immunotherapeutics, imaging probes, and spectroscopic probes. Needle, wait. The connection may be a covalent bond, or a non-covalent interaction such as through electrostatic forces. A variety of linkers known in the art can be used to form immunoconjugates. In addition, the immunoconjugate can be provided in the form of a fusion protein, which can be expressed from a polynucleotide encoding the immunoconjugate. "Fusion protein" as used herein refers to a protein produced by linking two or more genes or gene fragments that originally coded for independent proteins (including peptides and polypeptides). The translation of the fusion gene produces a single protein with functional properties derived from each original protein.

In this application, the term "light chain" generally refers to any polypeptide that has sufficient variable region sequence to impart specificity for a particular antigen. The full-length light chain includes the variable region domain VL, and the constant region domain CL. Like the heavy chain, the variable domain of the light chain is located at the amino terminus of the polypeptide. The light chain includes kappa chain and lambda chain.

In this application, the term "fully human antibody" refers to an antibody containing only human immunoglobulin protein sequences. Phage display or other molecular biology methods can be used to generate fully human antibodies in humans and in transgenic animals with human immunoglobulin germline sequences. Phage antibody expression technology allows the production of specific antibodies without animal immunity, as described in U.S. Patent No. 6,946,546, which is hereby incorporated by reference in its entirety. These techniques are further described in Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994) and Schier et al. (1996), which are incorporated herein by reference in their entirety. Phage display methods (see U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO98/46645, WO98/50433, WO98/24893, WO98/16654, WO96/34096, WO96/33735 and WO91/10741). Other techniques, such as the use of libraries, are known in the art.

In this application, the term "binding" may refer to specific binding, and "specific binding" means that the binding of an agent (such as an antibody) to its specific target (such as an epitope) is stronger than its binding to other targets. _{If the dissociation constant (K D} ) of the reagent binding to the first target is lower than the dissociation constant of the second target, its binding to the first target is stronger than the binding to the second target. In addition, the dissociation constant (K _D ) of the target specifically bound by the reagent is less than _{1/10 of the dissociation constant (K D} ) of the target non-specifically bound by the reagent, can be less than 1/20, can be 1/ Below 50, it can even be 1/100, 1/200, 1/500 or 1/1000 or less.

In this application, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such names include progeny. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.

In this application, the term "efficiency to target ratio" refers to the ratio of the number of effector cells to target cells.

In this application, the term "inhibition of growth" (for example, involving cells) is meant to include any measurable measure of cell growth upon contact with the anti-Claudin 18.2 antibody compared to the growth of the same cell that has not been exposed to the anti-Claudin 18.2 antibody. Decrease, for example, the growth of cells is inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

In this application, the term "drug" generally refers to a chemical compound or composition that can induce a desired therapeutic effect when it is properly administered to a patient.

In this application, the term "pharmaceutical composition" means a mixture containing one or more of the compounds described in this application or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as Physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredients and then exert the biological activity. The therapeutic composition should generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high antibody concentration. Sterile injectable solutions can be prepared by incorporating the active compound (ie antibody or antibody portion) in the required amount together with one of the ingredients or combinations of ingredients listed above in a suitable solvent, as required, followed by filtration and sterilization. .

In this application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be ligated. Another type of vector is a viral vector, in which other DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome, such as naked RNA polynucleotides, naked DNA polynucleotides that cannot replicate autonomously, Polynucleotides composed of DNA and RNA in the chain, poly-lysine-coupled DNA or RNA, peptide-coupled DNA or RNA, liposome-coupled DNA, etc. In addition, certain vectors can direct the expression of genes effectively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally speaking, expression vectors used in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmid is the most commonly used form of vector.

In this application, the term "directly or indirectly connected" refers to the covalent linkage (directly or indirectly) of amino acids. For example, at least one domain of a ligand (such as HGF) that is operatively linked to at least one amino acid encoded by an intron of a gene encoding a ligand means that the amino acid from the domain of the ligand is covalently linked to the The amino acid coded by the intron. This type of connection is typically achieved directly through a peptide bond, but can also be achieved indirectly, for example through a linker or through a non-peptide linkage. Therefore, a polypeptide containing at least one domain of a ligand that is operatively linked to at least one amino acid encoded by an intron of a gene encoding a cell surface receptor may be an intron fusion protein. When intron sequences are spliced, or covalently linked to exon sequences (domains encoding cell surface receptors) in-frame, nucleic acids encoding such polypeptides can be produced. The translation of the nucleic acid molecule produces the following polypeptide, in which the intron coding portion of the amino acid (at least containing the stop codon encoded by the intron sequence) is covalently linked to the ligand domain. They can also be produced synthetically by linking the part containing exons to the part containing introns, including chimeric intron fusion proteins, where the exons are derived from different parts of the introns. The isotype is encoded by the gene, and the isotype includes different ligand isotypes or cell surface receptor isotypes, and vice versa.

In this application, the term "treatment" means administering an internal or external therapeutic agent, such as a composition containing any one of the binding compounds of this application, to a patient who has one or more disease symptoms, and the patient is known to The therapeutic agent has a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered to the patient or population to be treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. The therapeutic effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, reduction or alleviation of the disease state, and alleviation or improvement The prognosis.

In this application, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition in mammals that is usually characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors) , Gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma and melanoma. "Tumor cell" further includes "solid tumor", which refers to a tumor selected from the group consisting of gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, Hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer (hepatic cancer), anal cancer, Penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancer may be lung cancer, colon cancer, liver cancer, esophageal cancer, ovarian cancer, bladder cancer, stomach cancer, kidney cancer, and/or pancreatic cancer.

In this application, the term "heavy chain" generally refers to a full-length heavy chain and a fragment thereof with sufficient variable region sequence to confer binding specificity. Mammalian full-length heavy chain antibodies generally include the variable region domain VH and three constant region domains CH1, CH2, and CH3. The VH domain faces the amino terminus of the polypeptide, and the CH domain faces the carboxy terminus, where CH3 is closest to the carboxy terminus of the polypeptide. Human heavy chains can generally be isotypes including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

In this application, the term "adjuvant" generally refers to any substance that assists or modulates the action of a drug, including but not limited to immunological adjuvants, which enhance or diversify immune responses to antigens.

In this application, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, and may be mammals, Examples include non-human primates, sheep, dogs, cats, cows, and horses.

In the present application, the term "therapeutically effective amount" refers to the amount of the antibody of the present application that is sufficient to prevent or alleviate the symptoms associated with a disease or disorder (e.g., cancer). The therapeutically effective amount is related to the disease to be treated, and those skilled in the art can easily distinguish the actual effective amount.

In this application, the terms "ADCC effect", "antibody-dependent cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refer to cell-mediated immune defense in which the immune system effector cells actively The cell membrane surface antigen is lysed with an antibody, such as Claudin 18.2 antibody, and bound target cells such as cancer cells.

In this application, the terms "CDC effect", "complement-dependent cytotoxicity" or "CDC" refer to the effector function of IgG and IgM antibodies, which when combined with surface antigens trigger a typical complement pathway, including the formation of a membrane attack complex And target cell lysis. When the antigen binding protein of the present application binds to Claudin 18.2, it triggers CDC on cancer cells.

In this application, the term "CDR" and its plural form "CDRs" usually refer to the complementarity determining region (CDR), where the three constitute the binding properties of the light chain variable region (LCDR1, LCDR2 and LCDR3); the three constitute the heavy The binding properties of chain variable regions (HCDR1, HCDR2 and HCDR3). CDR contributes to the functional activity of the antibody molecule and is separated by an amino acid sequence containing a backbone or framework region.

In this application, the terms “Claudin 18.1”, “claudin 18.1”, “CLD 18.1” or “claudin 18.1” include Claudin 18. The term includes variants, homologs, orthologs and paralogs.

In this application, the term "Claudin 18.1 positive tumor cell" refers to a cell that expresses Claudin 18.1 on its surface.

In this application, the terms "Claudin18.2", "claudin18.2", "CLD18.2" or "claudin 18.2" include Claudin18 type 2. The term includes variants, homologs, orthologs and paralogs.

In this application, the term "Claudin 18.2 positive tumor" refers to a tumor that expresses Claudin 18.2 protein.

In this application, the term "Claudin 18.2 positive tumor cell" refers to a tumor cell that expresses Claudin 18.2 on its surface.

In this application, the term "FACS" or "flow cytometry" refers to a tool for querying cell phenotypes and characteristics. It senses the cells or particles as they move in the liquid stream through the laser (amplified by the stimulated emission of radiation)/beam passing through the sensing area. Measure the relative light scattering of microscopic particles and the color to distinguish fluorescence. Cell flow analysis and differentiation are based on size, granularity, and whether the cells carry fluorescent molecules in the form of antibodies or dyes. When the cell passes through the laser beam, the light is scattered in all directions, and the light scattered in the forward direction at a low angle (0.5-10°) from the axis is proportional to the square of the radius of the sphere, and is therefore proportional to the cell or particle Is proportional to the size. Light can enter cells; therefore, 90° light (right angle, side) scattering can be labeled with fluorescent dye-linked antibodies, or stained with fluorescent membrane, cytoplasmic or nuclear dyes. Therefore, the differentiation of cell types, the presence of membrane receptors and antigens, membrane potential, pH, enzyme activity and DNA content can be promoted. Flow cytometry is multi-parameter, recording several measurements for each cell; therefore, it is possible to identify homogeneous subpopulations within a heterogeneous population. Fluorescence-activated cell sorting (FACS) that allows the separation of different cell populations that are too similar in physical characteristics to be separated by size or density, uses fluorescent tags to detect differentially expressed surface proteins, and allows for physically homogeneous cell populations The subtle difference.

In this application, the term "between" usually means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and the N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment. Indirect connection. In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly connected to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly connected to the N-terminus of the LCDR2. For another example, the N-terminus of the L-FR3 is directly or indirectly connected to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly connected to the N-terminus of the LCDR3. In the heavy chain, for example, the N-terminus of the H-FR2 is directly or indirectly connected to the C-terminus of the HCDR1, and the C-terminus of the H-FR2 is directly or indirectly connected to the N-terminus of the HCDR2. For another example, the N-terminus of the H-FR3 is directly or indirectly connected to the C-terminus of the HCDR2, and the C-terminus of the H-FR3 is directly or indirectly connected to the N-terminus of the HCDR3. In this application, the "first amino acid fragment" and the "second amino acid fragment" can be any amino acid fragment that is the same or different.

In this application, the term "light chain constant region" refers to a region containing the light chain constant domain CL. The light chains of human immunoglobulins are generally classified into κ and λ light chains, and these chains each contain a variable domain and a constant domain. The term "Igκ constant region" or "Igλ constant region" corresponds to the immunoglobulin κ light chain, respectively. The region of the constant domain CL or the region of the immunoglobulin lambda light chain constant domain CL.

In this application, the term "pfu" means plaque forming unit, which is a measure of the number of infectious phage particles (virions) or phage titer.

Antigen binding protein

In one aspect, the application provides an antigen binding protein comprising at least one CDR in the variable region VL of an antibody light chain.

In the present application, the antigen binding protein may include LCDR1, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 7 or 17.

In the present application, the antigen binding protein may include LCDR2, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 8 or 18.

In the present application, the antigen binding protein may include LCDR3, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 9 or 19.

In the present application, the antigen binding protein may include a framework region L-FR1, the C-terminus of L-FR1 is directly or indirectly connected to the N-terminus of LCDR1, and the L-FR1 may include SEQ ID NO : 25 or 33 amino acid sequence.

In this application, the antigen binding protein may include the framework region L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may include SEQ ID NO: 26 or 34 shown in the amino acid sequence.

In the present application, the antigen binding protein may include the framework region L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may include SEQ ID NO: 27 or 35 shown in the amino acid sequence.

In the present application, the antigen binding protein may include a framework region L-FR4, the N-terminus of L-FR4 is directly or indirectly connected to the C-terminus of LCDR3, and the L-FR4 may include SEQ ID NO: 28 or 36 amino acid sequence.

In the present application, the antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 6 or 16.

In the present application, the antigen binding protein may include a light chain constant region CL, and the antibody light chain constant region includes a human Igκ constant region or a human Igλ constant region, for example, the CL may include SEQ ID NO: 38 or 39 The amino acid sequence shown.

In the present application, the antigen binding protein may include a light chain LC, and the LC may include the amino acid sequence shown in SEQ ID NO: 10 or 20.

The antigen-binding protein described in the antigen-binding protein of the present application may comprise at least one CDR in the VH of the variable region of the antibody heavy chain.

In the present application, the antigen binding protein may include HCDR1, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 2 or 12.

In the present application, the antigen binding protein may include HCDR2, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 3 or 13.

In the present application, the antigen binding protein may include HCDR3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 4 or 14.

In the present application, the antigen binding protein may include the framework region H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may include SEQ ID NO : The amino acid sequence shown in 21 or 29.

In the present application, the antigen binding protein may include the framework region H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may include SEQ ID NO: 22 or The amino acid sequence shown at 30.

In the present application, the antigen binding protein may include the framework region H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may include SEQ ID NO: 23 or The amino acid sequence shown in 31.

In the present application, the antigen binding protein may include a framework region H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 may include SEQ ID NO: 24 or 32 amino acid sequence.

In the present application, the antigen binding protein may include the heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 1 or 11.

In the present application, the antigen binding protein may include a heavy chain constant region CH, and the antibody heavy chain constant region includes a human IgG constant region. Preferably, the antibody heavy chain constant region described in the present application includes a human IgG1 constant region, For example, the CH may include the amino acid sequence shown in SEQ ID NO: 37.

In the present application, the antigen binding protein may include a heavy chain HC, and the HC may include the amino acid sequence shown in SEQ ID NO: 5 or 15.

In the present application, the isolated antigen binding protein may include LCDR1-3, wherein the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 7 or 17, and the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 8 or 18. And the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 9 or 19.

For example, the antigen binding protein described in the present application may include LCDR1-3 which is the same as HDR002C04, wherein the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 7; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 8 And the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 9.

For example, the antigen binding protein described in the present application may include LCDR1-3 which is the same as HDR002C06, wherein the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 17; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 18 And the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 19.

In the present application, the isolated antigen binding protein may comprise L-FR1-4, wherein the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 25 or 33; the L-FR2 comprises SEQ ID NO The amino acid sequence shown in: 26 or 34; the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 27 or 35; and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 28 or 36.

For example, the antigen-binding protein described in the present application may include the same L-FR1-4 as HDR002C04, wherein the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 25, and the L-FR2 may include SEQ ID NO The amino acid sequence shown in: 26, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 27, and L-FR4 may include the amino acid sequence shown in SEQ ID NO: 28.

For example, the antigen binding protein described in the present application may include the same L-FR1-4 as HDR002C06, wherein the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 33, and the L-FR2 may include SEQ ID NO The amino acid sequence shown in: 34, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 35, and L-FR4 may include the amino acid sequence shown in SEQ ID NO: 36.

In this application, the isolated antigen binding protein may comprise HCDR1-3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2 or 12; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3 or 13 And the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 4 or 14.

For example, the antigen binding protein described in this application may include the same HCDR1-3 as HDR002C04, wherein the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 2; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 3 The amino acid sequence of; and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 4.

For example, the antigen binding protein described in the present application may include the same HCDR1-3 as HDR002C06, wherein the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 12; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 13 And the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 14.

In this application, the isolated antigen binding protein may comprise H-FR1-4, wherein the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 21 or 29; and the H-FR2 comprises SEQ ID NO : The amino acid sequence shown in 22 or 30; the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 23 or 31; and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 24 or 32.

For example, the antigen binding protein described in the present application may include the same H-FR1-4 as HDR002C04, wherein the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 21, and the H-FR2 may include SEQ ID NO The amino acid sequence shown in: 22, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 23, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 24.

For example, the antigen binding protein described in the present application may include the same H-FR1-4 as HDR002C06, wherein the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 29, and the H-FR2 may include SEQ ID NO The amino acid sequence shown in: 30, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 31, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 32.

In the present application, the isolated antigen binding protein may include LCDR1-3 and HCDR1-3, wherein the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 7 or 17; the LCDR2 includes SEQ ID NO: 8 Or the amino acid sequence shown in 18; the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 9 or 19; the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2 or 12; the HCDR2 includes SEQ ID NO: The amino acid sequence shown in 3 or 13; and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 4 or 14.

For example, the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as HDR002C04, wherein the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 7; the LCDR2 may include SEQ ID NO The amino acid sequence shown in: 8; the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 9; the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 2; the HCDR2 may include SEQ ID NO: 3 The amino acid sequence shown; and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 4.

For example, the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 that are the same as HDR002C06, wherein the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 17; the LCDR2 may include SEQ ID NO The amino acid sequence shown in: 18; the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 19; the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 12; the HCDR2 may include SEQ ID NO: 13 The amino acid sequence shown; and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 14.

In the present application, the antigen binding protein may include a light chain variable region VL and a heavy chain variable region VH, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 6 or 16, and the VH It may include the amino acid sequence shown in SEQ ID NO: 1 or 11.

For example, the antigen binding protein described in the present application may include the same light chain variable region VL and heavy chain variable region VH as HDR002C04, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 6, and The VH may include the amino acid sequence shown in SEQ ID NO:1.

For example, the antigen binding protein described in the present application may include the same light chain variable region VL and heavy chain variable region VH as HDR002C06, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 16, and The VH may include the amino acid sequence shown in SEQ ID NO: 11.

In the present application, the antigen binding protein may include a light chain constant region CL and a heavy chain constant region CH, wherein the CL may include the amino acid sequence shown in SEQ ID NO: 38 or 39, and the CH may include The amino acid sequence shown in SEQ ID NO: 37.

For example, the antigen binding protein described in the present application may include the same light chain constant region CL and heavy chain constant region CH as HDR002C04, wherein the CL may include the amino acid sequence shown in SEQ ID NO: 38, and the CH It may include the amino acid sequence shown in SEQ ID NO: 37.

For example, the antigen binding protein described in the present application may include the same light chain constant region CL and heavy chain constant region CH as HDR002C06, wherein the CL may include the amino acid sequence shown in SEQ ID NO: 39, and the CH It may include the amino acid sequence shown in SEQ ID NO: 37.

In the present application, the antigen binding protein may include an antibody light chain LC and an antibody heavy chain HC, where the LC may include the amino acid sequence shown in SEQ ID NO: 10 or 20, and the HC may include SEQ ID NO : The amino acid sequence shown in 5 or 15.

For example, the antigen binding protein described in the present application may include the same antibody light chain LC and antibody heavy chain HC as HDR002C04, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 10, and the HC may include The amino acid sequence shown in SEQ ID NO: 5.

For example, the antigen binding protein described in the present application may include the same antibody light chain LC and antibody heavy chain HC as HDR002C06, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 20, and the HC may include The amino acid sequence shown in SEQ ID NO: 15.

The antigen binding protein described in this application may include LCDR1-3 and L-FR1-4, wherein the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 7; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 8 And the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 9; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 25, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 26 In the amino acid sequence shown, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 27, and L-FR4 may include the amino acid sequence shown in SEQ ID NO: 28. The antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 6, and the CL may include the amino acid sequence shown in SEQ ID NO: 38. The antigen binding protein may also include HCDR1-3 and H-FR1-4, wherein the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 2; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 3 Sequence; and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 4; the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 21, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 22 The amino acid sequence, H-FR3 may include the amino acid sequence shown in SEQ ID NO:23, and H-FR4 may include the amino acid sequence shown in SEQ ID NO:24. The antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 1, and the CH may include the amino acid sequence shown in SEQ ID NO: 37. And the antigen binding protein may include LC and HC, where the LC may include the amino acid sequence shown in SEQ ID NO: 10, and the HC may include the amino acid sequence shown in SEQ ID NO: 5. For example, the antigen binding protein may comprise the same antibody light chain and antibody heavy chain as HDR002C04.

The antigen binding protein described in this application may include LCDR1-3 and L-FR1-4, wherein the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 17; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 18 And the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 19; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 33, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 34 In the amino acid sequence shown, L-FR3 may include the amino acid sequence shown in SEQ ID NO: 35, and L-FR4 may include the amino acid sequence shown in SEQ ID NO: 36. The antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 16, and the CL may include the amino acid sequence shown in SEQ ID NO: 39. The antigen binding protein may also include HCDR1-3 and H-FR1-4, wherein the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 12; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 13 Sequence; and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 14; the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 29, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 30 The amino acid sequence, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 31, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 32. The antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 11, and the CH may include the amino acid sequence shown in SEQ ID NO: 37. And the antigen binding protein may include LC and HC, where the LC may include the amino acid sequence shown in SEQ ID NO: 20, and the HC may include the amino acid sequence shown in SEQ ID NO: 15. For example, the antigen binding protein may comprise the same antibody light chain and antibody heavy chain as HDR002C06.

Reference antibody

The protein, polypeptide and/or amino acid sequence involved in this application should also be understood to include at least the following range: variants or homologues that have the same or similar functions as the protein or polypeptide and are included in this application Within the scope of protection, it is called a reference antibody.

The isolated antigen binding protein described in this application can compete with the reference antibody for binding to the Claudin 18.2. In this application, the reference antibody may comprise LCDR1-3. Wherein, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 7 or 17; the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 8 or 18; and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 9 or 19 The amino acid sequence.

In this application, the reference antibody may comprise HCDR1-3. Wherein, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2 or 12; the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3 or 13; and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 4 or 14 The amino acid sequence.

In this application, the reference antibody may comprise LCDR1-3 and HCDR1-3. Wherein, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 7 or 17; the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 8 or 18; the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 9 or 19 Amino acid sequence; the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2 or 12; the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3 or 13; and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 4 or 14 The amino acid sequence shown.

In this application, the reference antibody may comprise a light chain variable region VL and a heavy chain variable region VH. Wherein, the VL may include the amino acid sequence shown in SEQ ID NO: 6 or 16, and the VH may include the amino acid sequence shown in SEQ ID NO: 1 or 11.

Detection method

The physical/chemical properties and/or biological activity of the Claudin 18.2 antigen binding protein described in this application can be identified, screened or characterized by various assays known in the art.

Nucleic acid, vector, host cell and preparation method

In another aspect, the present application also provides one or more isolated nucleic acid molecules. The one or more nucleic acid molecules may encode the antigen binding protein described in this application. For example, each nucleic acid molecule of the one or more nucleic acid molecules may encode the complete antigen binding protein, or may encode a part of it (for example, HCDR1-3, LCDR1-3, VL, VH, light chain Or one or more of the heavy chain).

The nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesis, for example, by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

In this application, the nucleic acid encoding the antibody and its antigen-binding fragment can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the use of restriction fragment operations or the use of synthetic oligonucleotides. Overlapping extension PCR. For specific operations, please refer to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.

In another aspect, this application provides one or more vectors, which comprise one or more nucleic acid molecules described in this application. Each vector may contain one or more of the nucleic acid molecules. In addition, the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions. In addition, the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence can vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc. For example, the 5' non-transcriptional expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In this application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoters, CMV promoters and artificial hybrid promoters (such as CMV), wherein A certain part may be fused with a certain part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), and it may or may not contain additional introns. One or more nucleic acid molecules described in this application can be operably linked to the expression control element.

The vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

In another aspect, the application provides a host cell, which may comprise one or more nucleic acid molecules described in this application and/or one or more vectors described in this application. In certain embodiments, each or each host cell may contain one or one of the nucleic acid molecules or vectors described in this application. In certain embodiments, each or each host cell may contain multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described in the present application. For example, the vector described in the present application can be introduced into the host cell, such as a eukaryotic cell, such as a plant-derived cell, fungus, or yeast cell. The vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.

In another aspect, the application provides a method for preparing the antibody or antigen-binding fragment thereof. The method may include culturing the host cell described in the present application under conditions that allow the expression of the antibody or antigen-binding fragment thereof. For example, it is possible to use an appropriate culture medium, an appropriate temperature, a culture time, etc., and these methods are understood by those of ordinary skill in the art.

In some cases, the method may further include the step of isolating and/or purifying the antibody or antigen-binding fragment thereof. For example, protein G-Sepharose or Protein A-Sepharose can be used for affinity chromatography, and gel electrophoresis and/or high performance liquid chromatography can also be used to purify and separate the antibodies or antigen-binding fragments described in this application. .

Pharmaceutical composition, method, use

In another aspect, the present application provides a pharmaceutical composition, which may comprise the antigen binding protein and/or the immunoconjugate described in the present application, the nucleic acid molecule, the carrier, the Host cell, and optionally a pharmaceutically acceptable adjuvant. The pharmaceutical composition described in the present application may include a preventive and/or therapeutically effective amount of the antibody or antigen-binding fragment thereof. The prophylactic and/or therapeutically effective amount is a dose required to prevent and/or treat (at least partially treat) a disease or disorder and/or any complications thereof in a subject suffering from or at risk of development. The pharmaceutically acceptable adjuvant is non-toxic to the recipient at the dose and concentration used, and may include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine Acid; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride) chloride), phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol ); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gel or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamyl acid, Aspartic acid, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol , Trehalose or sorbitol; salt-forming counterions, such as sodium ions; metal complexes (for example, Zn-protein complexes); and/or nonionic surfactants, such as TWEEN ^TM , PLURONICS ^TM or polyethylene Alcohol (PEG). The pharmaceutical composition in this application may also contain more than one active compound, usually those active compounds with complementary activities that do not adversely affect each other. The type and effective amount of such drugs depend on, for example, the amount and type of antagonist present in the formulation, and the clinical parameters of the subject.

This application also provides the method for detecting Claudin 18.2 expression in biological samples as described below. In some cases, the method includes contacting a biological sample with the antigen-binding protein described in the present application under conditions that allow the antigen-binding protein to bind Claudin 18.2, and detecting the relationship between the antigen-binding protein and Claudin 18.2 Whether to form a complex between. For example, the tumor or cancer is a tumor or cancer in which the expression of Claudin 18.2 is increased compared to a non-tumor or cancer sample. Such methods can be in vitro or in vivo methods. The antigen-binding protein described in this application can be used in, for example, immunoassays including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (for example, western blotting), flow cytometry (for example, FACS ) And enzyme-linked immunosorbent assay (ELISA). In some cases, such as when Claudin 18.2 is a biomarker for selecting patients, the antigen binding protein is used to select subjects suitable for therapy with the antigen binding protein described in this application.

The pharmaceutical composition can be used to inhibit tumor growth. For example, the pharmaceutical composition of the present application can inhibit or delay the development or progression of the disease, can reduce the tumor size (or even substantially eliminate the tumor), and/or can reduce and/or stabilize the disease state. Examples of tumors include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors) , Gastrinoma and islet cell carcinoma), mesothelioma, schwannomas (including acoustic neuroma), meningioma, adenocarcinoma and melanoma. "Tumor cell" further includes "solid tumor", which refers to a tumor selected from the group consisting of gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, Hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer (hepatic cancer), anal cancer, Penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancer may be lung cancer, colon cancer, liver cancer, esophageal cancer, ovarian cancer, bladder cancer, stomach cancer, kidney cancer, and/or pancreatic cancer.

On the other hand, the present application provides a method for treating cancer in a subject, inhibiting tumor growth in a subject, and/or inhibiting tumor cell proliferation, including administering the method of the present application to a subject in need or the tumor cell The antigen-binding fragment and/or the immunoconjugate, the molecular nucleic acid, the carrier, the host cell and/or the pharmaceutical composition. It can be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, transcutaneously, intraarterially, and intraperitoneally. , Intra-injury, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, and rectal , Locally, intratumorally, peritoneally, subconjunctivally, intracapsular, mucosal, intrapericardial, intraumbilical, intraocular, intraorbital, orally Way, by topical way, by transdermal way, by intravitreal way (for example, by intravitreal injection), by eye drops, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by direct Bathe the local perfusion of target cells, through a catheter, through lavage, in the form of a cream or in the form of a lipid composition. The composition used in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (for example, the compound or composition being administered and the severity of the condition, disease, or disorder being treated). In certain embodiments, in intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, implantation, inhalation , Intrathecal, intraventricular, or intranasal administration of anti-cancer therapy (e.g., anti-Claudin 18.2 antibody). Depending in part on whether the administration is short-lived or long-term, the administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection. Various dosing schedules are covered herein, including but not limited to a single administration or multiple administrations at various time points, bolus administrations, and pulse infusions.

On the other hand, the application provides the use of the antigen binding protein in the preparation of medicines. The medicine is used for diagnosing cancer, treating cancer, inhibiting tumor growth and/or inhibiting tumor cell proliferation. In certain embodiments, the tumor or cancer comprises colorectal tumor or cancer. In some embodiments, the tumor or cancer is a tumor or cancer with abnormal expression of Claudin 18.2.

The application also provides the use of the antigen binding protein in a method for diagnosing a subject suffering from a disorder (for example, cancer or immune dysfunction), the method comprising: binding a sample to the antigen of the present invention The protein contacts and detects the presence of the bound antigen binding protein to determine the presence or expression level of Claudin 18.2 in the sample obtained from the subject. In some cases, the sample can be selected from the group consisting of a tissue sample, a whole blood sample, a serum sample, and a plasma sample. In some cases, the tissue sample may be a tumor sample. In some cases, the tumor sample may include tumor infiltrating immune cells, tumor cells, stromal cells, and any combination thereof. In some cases, biological samples include tissue or cell samples. For example, a biological sample may include cells or tissues from normal or cancer patients. In some cases, the source of the tissue or cell sample can be, for example, solid tissue from fresh, frozen, and/or preserved organ or tissue samples or biopsy or aspirate; blood or any blood component; body fluids, such as cerebrospinal fluid, Amniotic fluid, peritoneal fluid, or interstitial fluid; cells from the subject at any time during pregnancy or development. In other cases, the biological sample is obtained from an in vitro tissue or cell culture. Examples of biological samples in this application include, but are not limited to, tumor biopsy, circulating tumor cells, serum or plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and Preserved tumor samples, such as formalin-fixed paraffin-embedded tumor samples or frozen tumor samples.

The antigen binding protein or pharmaceutical composition described herein can be formulated, administered, and administered in a manner consistent with good medical practice. The considerations in this situation include the specific condition being treated, the specific mammal being treated, the clinical condition of a single patient, the cause of the condition, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner . The therapeutic agent (e.g., anti-Claudin 18.2 antibody) need not be, but optionally, formulated and/or administered simultaneously with one or more agents currently used to prevent or treat the condition in question. The effective amount of such other agents depends on the amount of therapeutic agent (e.g., anti-Claudin 18.2 antibody) present in the formulation, the type of disorder or treatment, and other factors discussed above. These agents can generally be used in any dose empirically/clinically determined to be appropriate and through any route empirically/clinically determined to be appropriate. Compared with a single treatment, the dose of the antibody administered in the combination treatment can be reduced. It is easy to monitor the progress of this therapy by conventional techniques.

And the Claudin 18.2 protein in this application is preferably human Claudin 18.2 protein, GenBank accession number NP_001002026; Claudin 18.2 positive cells refer to transfecting a vector containing a cDNA sequence encoding human Claudin 18.2 into a blank cell line to produce A stable cell line that overexpresses human Claudin 18.2, for example, Claudin 18.2 positive HEK293 cells means that a vector encoding human Claudin 18.2 cDNA sequence is transfected into a blank HEK293 cell line to generate HEK293 cells that overexpress human Claudin 18.2 Strains; Claudin 18.1 positive cells refer to the transfection of a vector containing the cDNA sequence encoding human Claudin 18.1 (GenBank accession number NP_057453) into a blank cell line to generate a stable cell line overexpressing human Claudin 18.1, such as Claudin 18.1 A positive HEK293 cell means that a vector encoding the cDNA sequence of human Claudin 18.1 is transfected into a blank HEK293 cell line to generate a HEK293 cell line overexpressing human Claudin 18.1. In a similar way, CHO positive cells, BxPC3 positive cells, and N87 positive cells expressing human Claudin 18.1 or human Claudin 18.2 were prepared.

Familiarity is not intended to be limited by any theory. The following examples are only used to illustrate the antigen binding protein, preparation method, and use of the present application, and are not used to limit the scope of the present application. Those who know this technology can easily understand the other advantages and effects of the invention of this application from the content disclosed in this specification. The experimental methods without specific conditions in the embodiments of the present invention usually follow conventional conditions or the conditions recommended by raw material or commodity manufacturers. The reagents without specific sources are the conventional reagents purchased on the market.

Example

Example 1 Screening of specific anti-Claudin 18.2 antibodies

The standard natural fully human antibody library phage display technology was used to screen and obtain antibodies against Claudin 18.2. To put it simply, human Claudin 18.2 protein or Claudin 18.2 positive cells are used to screen out Claudin 18.2 positive antibodies in the fully human phage library, and Claudin 18.1 positive cells are used to exclude non-specific binding to Claudin 18.1 anti-Claudin 18.2 antibodies. Obtain fully human monoclonal antibodies. The process is as follows: the whole human phage library (GenScript: Kappa and Lambda) is precipitated with PEG/NaCl and resuspended in PBS, and then screened by the following two methods: Method 1: 2 rounds of protein screening plus 1 round of cell screening; Method 2: 3- 4 rounds of cell screening.

The protein screening process is as follows: human Claudin 18.2 protein is coated on the ELISA plate, 2×10 ¹² pfu phage is diluted with 5% (w/v) skimmed milk powder dissolved in PBS, and then added to the blank wells coated with 3% skimmed milk powder Incubate to remove non-specific binding phages, and then transfer the phages to human Claudin 18.2 protein-coated wells. After incubating at room temperature, the antigen-binding phages are collected after washing with 0.05% PBST and PBS.

The cell selection process is as follows: After the phage is blocked with 3% (w/v) skimmed milk powder, it is incubated with Claudin 18.1 positive HEK293 cells at room temperature for 45 minutes. ^{The supernatant phage was collected by centrifugation, and incubated with 2×10 7} Claudin 18.2 positive CHO cells or Claudin 18.2 positive HEK293 cells blocked with 3% (w/v) skimmed milk powder at room temperature. After washing with 1% BSA-PBS buffer, the cell-bound phage was eluted with 0.1M TEA, and neutralized with 1M Tris-HCl (pH 7.4). After the eluted phage infects competent cells TG1, it is released by M13K07 (NEB, Cat. No.: N0315S) to help phage release. The screened phage infected competent cells TG1 and then selected a single clone for enzyme-linked immunoassay (ELISA) detection: the recombinant Claudin 18.2 protein was coated overnight at 4°C, washed and blocked, and then added to the phage supernatant, followed by conjugated spicy Root peroxidase mouse anti-M13IgG (Sino Biological) was used as the detection antibody, 3,3'5,5"-tetramethylbenzidine (TMB) was developed, and the plate was read at 450nm with a microplate reader after termination.

The positive single clones tested by ELISA were tested by flow cytometry (FACS), and the supernatant of HEK293 cells with Claudin 18.2 positive or Claudin 18.1 positive was used to detect the supernatant. Anti-fd Bacteriophage-Biotin (B2661) , Sigma-Aldrich) after incubation and washing, FACS was used to read the signal, and FACS positive clones were selected for DNA sequencing. The full-length DNA sequence of light and heavy chain obtained by sequencing and expressing Claudin 18.2 antibody were inserted into pCDNA3.4, respectively, to obtain the expression plasmid of full-length antibody. The heavy chain and light chain expression plasmids were co-transfected into Expi293F cells, and the supernatant was harvested for Protein A purification after 6-7 days of culture.

Table 1a Screened anti-Claudin 18.2 antibodies and their sequence numbers

Table 1b Screened anti-Claudin 18.2 antibodies and their sequence numbers

IMAB362 is the 175D10 in the patent application CN103509114A1. The antibody is currently used in the Phase 3 clinical trial of Astellas. According to the sequence and method disclosed in the patent application, the anti-Claudin 18.2 control antibody IMAB362 was prepared.

Example 2 Affinity detection of anti-Claudin 18.2 antibody and Claudin 18.2 positive cell line

The binding ability of the antibody of this application to Claudin 18.2 positive HEK293 cells was detected by flow cytometry. Plate Claudin 18.2 positive HEK293 cells or Claudin 18.1 positive HEK293 cells in a 96-well plate, and add gradient dilutions (0.1μg/mL, 0.3μg/mL, 1.0μg/mL, 3.0μg/mL 10μg/mL) to the plate. mL) of the antibody HDR002C04, HDR002C06 or control antibody IMAB362 of the application, no antibody was added to the blank group. Incubate for 30 minutes at 4°C, wash with PBS, add FITC-labeled goat anti-human IgG secondary antibody (1:100, Jackson ImmunoResearch), incubate at 4°C for 30 minutes, wash with PBS, and then use FACS instrument (Beckman) to detect cell fluorescence.

As shown in Figure 1, the affinity of HDR002C04 to Claudin 18.2 positive HEK293 cells is similar to that of the control antibody IMAB362, and the affinity of HDR002C06 is significantly stronger than IMAB362. Figure 2 shows the binding of HDR002C04 to Claudin 18.1 positive HEK293 cells or Claudin 18.2 positive HEK293 cells. Figure 3 shows the binding of HDR002C06 to Claudin 18.1 positive HEK293 cells or Claudin 18.2 positive HEK293 cells. The results show that HDR002C04 and HDR002C06 Like the blank group, it did not bind to Claudin 18.1 positive HEK293 cells; HDR002C04 and HDR002C06 had a strong affinity for Claudin 18.2 positive HEK293 cells.

Example 3 CDC effect of anti-Claudin 18.2 antibody on Claudin 18.2 positive cell line

Blood was drawn from healthy volunteers, and the whole blood was left standing and centrifuged to prepare normal human serum. Using the CCK-8 detection kit (CK04, Tongren Chemical), the antibodies HDR002C04 and HDR002C06 of the application and the control antibody IMAB362 were tested against human Claudin 18.2 positive HEK293 cells, Claudin 18.2 positive BxPC3 cells and Claudin 18.2 positive N87 cells. The ability of cells to trigger CDC effects.

Claudin 18.2 positive HEK293 cells, Claudin 18.2 positive BxPC3 or Claudin 18.2 positive N87 cells were centrifuged at 1000 rpm for 5 minutes, and then resuspended in complete medium (Gibco). The target cells are added to a 96-well plate in a quantity of 20,000 cells/well, and the antibody or control antibody of the application is diluted to different concentrations and added to the detection well. After incubating the antibody or control antibody of this application with the target cells for 1 hour, add the above-mentioned normal human serum with a final concentration of 20%, incubate at 37°C for 1-3 hours, and add CCK-8 reagent (Dongren Chemical) at a concentration of 10μl/well. Then continue to incubate in a 37°C incubator. Measure the absorbance at 450nm with a microplate reader.

The test results are shown in Figure 4, Figure 5 and Figure 6. The antibodies HDR002C04, HDR002C06 and the control antibody of the application can induce Claudin 18.2 positive HEK293 cells, Claudin 18.2 positive BxPC3 or Claudin 18.2 positive N87 cells in a dose-dependent manner. Strong CDC effect, and the CDC activity of the antibodies HDR002C04 and HDR002C06 of the application on Claudin 18.2 positive cells is stronger than that of the control antibody IMAB362.

Example 4 CDC effect of anti-Claudin 18.2 antibody on Claudin 18.1 positive cell line

In order to determine that the antibodies HDR002C04 and HDR002C06 of the present application specifically exhibit CDC effects on target cells expressing Claudin 18.2, but do not have CDC effects on cells expressing Claudin 18.1, Claudin 18.1 positive HEK293 cells or Claudin 18.2 positive HEK293 were used. Cells are used as target cells for CDC activity detection.

First, the target cells were centrifuged at 1000 rpm for 5 minutes, and then resuspended in FreeStyle 293 Expression Medium (Gibco). Add target cells to a 96-well plate at a rate of 20,000 cells/well, add the dilutions of the antibody HDR002C04 or HDR002C06 of the application at a final concentration of 10 μg/mL, incubate at 37°C for 1 hour, and add the normal human serum described in Example 3 After incubating at 37°C, the color was developed with CCK-8, and the plate was read by a microplate reader.

The test results are shown in Figure 7 and Figure 8. The experimental results show that the antibodies HDR002C04 and HDR002C06 of the present application did not show CDC activity on Claudin 18.1 positive HEK293 cells, but showed strong CDC activity on Claudin 18.2 positive HEK293 cells, indicating The CDC activity effect caused by the antibody of this application is highly specific to Claudin 18.2 positive cell lines.

Example 5 ADCC effect of anti-Claudin 18.2 antibody on Claudin 18.2 positive cell line

Blood was drawn from healthy volunteers, and normal human peripheral blood mononuclear cells (PBMC) were extracted by density gradient centrifugation. Lactate dehydrogenase (LDH) detection kit (Roche) was used to detect the ability of the antibodies HDR002C04, HDR002C06 and the control antibody IMAB362 of the present application against the ADCC effect induced by Claudin 18.2 positive BxPC3 cells or Claudin 18.2 positive N87 cells.

The antibodies HDR002C04, HDR002C06 and the control antibody IMAB362 of the application were diluted to different concentrations and added to a 96-well plate. The target cells Claudin 18.2 positive BxPC3 cells, Claudin 18.2 positive N87 cells and effector cell PBMC were collected into clean centrifuge tubes at 2500 rpm. Centrifuge for 5 minutes, then resuspend in phenol red-free RPMI 1640 medium (Gibco). Human PBMC cells and target cells (one of Claudin18.2 positive BxPC3 cells or Claudin18.2 positive N87 cells) were mixed evenly according to the effective target ratio of 24:1, plated on the detection well, incubated at 37°C for 20-24 hours, and added LDH color developing solution, keep it at room temperature and avoid light for 10-20 minutes, and read the plate with MD SpectrsMax 190 microplate reader after termination.

As shown in Figure 9 and Figure 10, all antibodies of the application and control antibodies can induce a strong ADCC effect on Claudin 18.2 positive cell lines in a dose-dependent manner, and the ADCC activity of antibodies HDR002C04 and HDR002C06 of the application on Claudin 18.2 positive cells It is stronger than the control antibody IMAB362.

Example 6 Anti-tumor growth effect of antibody HDR002C04 in vivo

BALB/c nude mice were randomly divided into groups. On day 0, 5×10 ⁶ Claudin 18.2 positive N87 cells were subcutaneously inoculated into the axilla of nude mice. A few hours after tumor inoculation, intravenous administration was given on the same day. The experimental group was given the antibody HDR002C04 or the control antibody IMAB362 (diluted in PBS solution) at a dose of 10 mg/kg; then intravenous injection and intraperitoneal injection were performed alternately, and the drug was administered in one week. Times for 3 weeks. The blank group was injected with PBS in the same way. Monitor the tumor growth over time, measure the length and width of the tumor with a vernier caliper, and calculate the tumor volume through the formula: tumor volume = (length × width ² )/2; at the end of the experiment, take out and measure the tumor weight; calculate the size of each group The average volume and weight of mouse tumors.

As shown in Figure 11 and Figure 12, at a dose of 10 mg/kg, the control antibody IMAB362 was not active, and there was no significant difference between the tumor weight and the blank group at the end of the experiment, while the antibody HDR002C04 of the present application showed good anti-tumor activity.

Example 7 Anti-tumor growth effect of antibody HDR002C06 in vivo

BALB/c nude mice were randomly divided into groups. On day 0, 5×10 ⁶ Claudin 18.2 positive BxPC3 cells were subcutaneously inoculated into the armpits of nude mice. On the 3rd day after tumor inoculation, the experimental group was given the antibody HDR002C06 or the control antibody IMAB362 (diluted in PBS solution) at a dose of 10 mg/kg to each animal in the tail vein; then intravenous injection and intraperitoneal injection were alternately administered for one week 2 times, continuous administration for 3 weeks. The blank group was injected with PBS in the same way. Monitor the tumor growth over time, measure the length and width of the tumor with a vernier caliper, and calculate the tumor volume through the formula: tumor volume = (length × width ² )/2; at the end of the experiment, take out and measure the tumor weight; calculate the size of each group The average volume and weight of mouse tumors.

As shown in Figure 13 and Figure 14, at a dose of 10 mg/kg, HDR002C06 is better than the positive control IMAB362 in inhibiting tumor growth in vivo.

Claims

An isolated antigen binding protein, which comprises:

The heavy chain variable region VH, the VH includes at least one of the following HCDRs:

HCDR1, whose amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 12, or includes the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12;

HCDR2, whose amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 13, or includes the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 13;

HCDR3, whose amino acid sequence is shown in SEQ ID NO: 4 or SEQ ID NO: 14, or includes the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 14;

and / or

The light chain variable region VL, the VL includes at least one of the following LCDRs:

LCDR1, whose amino acid sequence is shown in SEQ ID NO: 7 or SEQ ID NO: 17, or includes the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 17;

LCDR2, whose amino acid sequence is shown in SEQ ID NO: 8 or SEQ ID NO: 18, or includes the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 18;

LCDR3, whose amino acid sequence is shown in SEQ ID NO: 9 or SEQ ID NO: 19, or includes the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 19.
The isolated antigen binding protein of claim 1, wherein the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively; or, the VH includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively;

and / or

The VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9; or, the VL includes SEQ ID NO: 17, SEQ ID NO: 18 and the LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 19.
The isolated antigen binding protein of claim 2, wherein the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the VL Including LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 respectively;

or,

The VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and the VL includes SEQ ID NO: 17, SEQ ID NO: 18, respectively. And LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 19.
The isolated antigen binding protein of any one of claims 1-3, wherein:

The VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes SEQ ID NO: 21 or 29; the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 22 or 30; the H- FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 23 or 31; the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3 , And the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 24 or 32;

and / or

The VL includes framework regions L-FR1, L-FR2, L-FR3, and L-FR4, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes SEQ ID NO: 25 or 33; the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 26 or 34; the L- FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 27 or 35; the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3 , And the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 28 or 36.
The isolated antigen binding protein of any one of claims 1 to 4, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 1 or 11; and/or the VL comprises the amino acid sequence shown in SEQ ID NO: 6 or 16. Show the amino acid sequence;

Preferably, the amino acid sequence of the VH is shown in SEQ ID NO: 1, and the amino acid sequence of the VL is shown in SEQ ID NO: 6; or

The amino acid sequence of the VH is shown in SEQ ID NO: 11, and the amino acid sequence of the VL is shown in SEQ ID NO: 16.
The isolated antigen binding protein of any one of claims 1-5, which further comprises:

An antibody heavy chain constant region, the antibody heavy chain constant region is selected from a human IgG constant region, an IgA constant region, an IgM constant region, an IgD constant region, or an IgE constant region, and the human IgG constant region is further selected from a human IgG1 constant region , IgG2 constant region, IgG3 constant region or IgG4 constant region; preferably, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 37;

and / or

An antibody light chain constant region, the antibody light chain constant region is selected from a human Igκ constant region or a human Igλ constant region; preferably, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 38 or 39.
The isolated antigen binding protein of any one of claims 1 to 6, which comprises an antibody heavy chain HC and an antibody light chain LC, wherein the HC comprises the amino acid sequence shown in SEQ ID NO: 5 or 15; and/or The LC includes the amino acid sequence shown in SEQ ID NO: 10 or 20;

Preferably, the HC includes the amino acid sequence shown in SEQ ID NO: 5, and the LC includes the amino acid sequence shown in SEQ ID NO: 10; or

The HC includes the amino acid sequence shown in SEQ ID NO: 15, and the LC includes the amino acid sequence shown in SEQ ID NO: 20.
The isolated antigen binding protein of any one of claims 1-7, which has one or more of the following properties:

1) In the FACS assay, it can specifically bind to Claudin 18.2 on the cell surface;

2) In the FACS assay, it does not bind to Claudin 18.1 on the cell surface;

3) Show CDC effect on Claudin 18.2 positive tumor cells;

4) Does not show CDC effect on Claudin 18.1 positive tumor cells;

5) Induce ADCC effect on Claudin 18.2 positive tumor cells;

6) Inhibit the growth of Claudin 18.2 positive tumors.
The isolated antigen-binding protein of any one of claims 1-8, which is selected from an antibody or an antigen-binding fragment thereof, preferably, the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody, More preferably, it is a fully human antibody; the antigen-binding fragment is selected from Fab, Fab', F(ab )2 , Fv fragment, F(ab' )2 , scFv, di-scFv or dAb.
An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-9.
An isolated nucleic acid molecule that encodes the isolated antigen binding protein of any one of claims 1-9 or encodes the immunoconjugate of claim 10.
A vector comprising the nucleic acid molecule according to claim 11.
A cell comprising the nucleic acid molecule according to claim 11 or the vector according to claim 12.
A method for preparing the isolated antigen binding protein of any one of claims 1-9, the method comprising culturing the isolated antigen binding protein of any one of claims 1-9 under conditions The cell of claim 13.
A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-9, the immunoconjugate of claim 10, the nucleic acid molecule of claim 11, and the protein of claim 12 The carrier or the cell of claim 13, and optionally a pharmaceutically acceptable adjuvant.
The isolated antigen binding protein according to any one of claims 1-9, the immunoconjugate according to claim 10, the nucleic acid molecule according to claim 11, the carrier according to claim 12, the immunoconjugate according to claim 13 Use of the cell or the pharmaceutical composition of claim 15 in the preparation of a medicament for inhibiting the growth of Claudin 18.2 positive tumor cells or killing Claudin 18.2 positive tumor cells, the medicament being used for prevention, diagnosis, alleviation or treatment Tumors; preferably, the tumors are selected from solid tumors or hematological tumors; more preferably, the solid tumors are selected from lung cancer, colon cancer, liver cancer, esophageal cancer, ovarian cancer, bladder cancer, stomach cancer, kidney cancer or pancreatic cancer.
A method for inhibiting the growth of Claudin 18.2 positive tumor cells, killing the Claudin 18.2 positive tumor cells, or preventing, diagnosing, alleviating or treating tumors, comprising contacting the Claudin 18.2 positive tumor cells with any of claims 1-9 One of the isolated antigen binding protein, the immunoconjugate of claim 10, the nucleic acid molecule of claim 11, the carrier of claim 12, the cell of claim 13, or the cell of claim 15 The pharmaceutical composition; preferably, the tumor is selected from solid tumors or hematological tumors; more preferably, the solid tumor is selected from lung cancer, colon cancer, liver cancer, esophageal cancer, ovarian cancer, bladder cancer, gastric cancer, kidney Cancer or pancreatic cancer.