CN116813782A

CN116813782A - Claudin-18.2 specific antibody and application thereof

Info

Publication number: CN116813782A
Application number: CN202311006962.3A
Authority: CN
Inventors: 毛天强; 任文林; 朱学刚
Original assignee: Beijing Baipusai Biotechnology Co ltd
Current assignee: Beijing Baipusai Biotechnology Co ltd
Priority date: 2023-08-10
Filing date: 2023-08-10
Publication date: 2023-09-29
Anticipated expiration: 2043-08-10
Also published as: CN116813782B; WO2024146419A1

Abstract

The application relates to the technical field of antibodies, in particular to a Claudin-18.2 specific antibody and application thereof, wherein the antibody serving as the specific antibody for Claudin-18.2 antigen detection can be used for quantitatively detecting expressed Claudin-18.2 or for carrying out diagnosis detection in preclinical and clinical samples.

Description

Claudin-18.2 specific antibody and application thereof

Technical Field

The application relates to the technical field of antibodies, in particular to a Claudin-18.2 specific antibody and application thereof.

Background

Claudin-18.2 protein is a subtype of Claudin 18, a highly selective biomarker, and is limited in expression in normal tissues, and is frequently expressed abnormally during the development of various primary malignancies such as gastric cancer/gastroesophageal junction (GC/GEJ) cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchus cancer and non-small cell lung cancer. Claudin-18.2 plays an important role in the development and metastasis of tumors and participates in the biological processes such as proliferation, differentiation and migration of tumor cells. Claudin-18.2 is considered as a potential target for tumor treatment because of its high expression specificity in tumors.

More recently, claudin-18.2 expression has been found to be a potential specific marker for diagnosis of neoplastic disease, and the present application has been developed in view of this.

Summary of The Invention

Aiming at the technical problems, the application uses Claudin-18.2 protein constructed in vitro as immunogen to carry out mouse immunity, and the hybridoma cell strain expressing the antibody is obtained through cell fusion and screening; experiments prove that the antibody can specifically identify Claudin-18.2 antigen recognition sites, has an effect obviously better than that of the currently commercialized antibody, and can be applied to various detection methods such as ELISA, flow assay, IHC and the like, so that the application at least comprises the following purposes:

the first object of the present application is to provide an antibody or antigen-binding fragment thereof specific for Claudin-18.2, and the corresponding products;

the second object of the present application is to provide an application of the above antibody or antigen binding fragment in detection, including qualitative or quantitative detection before and after clinical use.

In order to achieve the above purpose, the present application specifically adopts the following technical scheme:

the application firstly provides an antibody or antigen binding fragment thereof for resisting Claudin-18.2, which comprises 3 CDRs in the amino acid sequence of a heavy chain variable region shown in SEQ ID NO.7 and 3 CDRs in the amino acid sequence of a light chain variable region shown in SEQ ID NO. 8;

Alternatively, variants having single or multiple CDRs with the light and heavy chain CDR regions described above that do not vary more than 2 amino acids per CDR region.

Further, when encoding an antibody HCDRs according to the Kabat encoding rule, the antibody or antigen binding fragment specifically comprises the following CDR sequences:

i. the amino acid sequences of complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3, respectively;

the amino acid sequences of complementarity determining regions LCDR1, LCDR2, LCDR3 of the light chain variable region are shown in SEQ ID NO.4, 5, 6, respectively;

alternatively, variants having single or multiple CDRs of no more than 2 amino acid conservative changes per CDR region with the 6 CDR regions of i-ii above.

Further, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, the sequences being selected from the group consisting of:

a) The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.7, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO. 7;

b) The amino acid sequence of the light chain variable region is shown as SEQ ID NO.8 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO. 8.

Further, the antibody or antigen binding fragment has a coupling moiety attached thereto, the coupling moiety being selected from one or more of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein, a lipid, and biotin, wherein the antibody or antigen binding fragment is optionally attached to the coupling moiety via a linker; preferably, the linker is a peptide or polypeptide.

Further, the antibody or antigen binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antisera, chimeric antibodies, humanized antibodies, and human antibodies; preferably, the antibody is selected from the group consisting of multispecific antibodies, single chain Fv (scFv), single chain antibodies, anti-idiotype (anti-Id) antibodies, diabodies, minibodies, nanobodies, single domain antibodies, fab fragments, F (ab') fragments, disulfide-linked bispecific Fv (sdFv) and intracellular antibodies.

The application also provides an isolated polynucleotide encoding an antibody or antigen binding fragment of any one of the above.

The application also provides a recombinant vector comprising the polynucleotide described above, and optionally regulatory sequences;

further preferably, the recombinant vector is a cloning vector or an expression vector;

still further preferred, the regulatory sequence is selected from a leader sequence, polyadenylation sequence, propeptide sequence, promoter, signal sequence, transcription terminator, or any combination thereof.

The application also provides a product comprising one or more of an antibody or antigen-binding fragment, polynucleotide or recombinant vector as described in any of the above, and contained in a suitable container.

Further, the product is in the form of a kit;

further preferred, the kit includes, but is not limited to: ELISA, flow sorting, IHC kits, and the like.

The application provides any of the following uses of an antibody or antigen binding fragment of any of the above:

(1) The application in detection, identification or screening of Claudin-18.2 protein or in preparation of Claudin-18.2 detection, identification or screening product;

preferably, the detection comprises a quantitative or qualitative detection;

more preferably, the detection comprises an immunogenicity detection;

further preferred, the assay includes, but is not limited to, a flow assay, an ELISA assay, or an IHC assay;

(2) The application in the diagnosis or the accompanying diagnosis of the tumor or the application in the preparation of a diagnosis or an accompanying diagnosis kit of the tumor;

(3) The application of the Claudin-18.2 in preparing a preclinical and clinical detection reagent.

Drawings

FIG. 1 IHC assay results for sensitivity, specificity and cross-reactivity of antibody Claudin18.2 (3B 10);

FIG. 2 IHC detection results for different antibody sensitivities and specificities.

Detailed Description

The present application discloses an isolated antibody or antigen binding fragment thereof, and uses thereof will be made by those skilled in the art with reference to the disclosure herein, and it is specifically noted that all such substitutions and modifications as would be apparent to those skilled in the art are deemed to be included in the present application. While the present application has been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations and modifications can be made in the methods and applications of the present application, and that the techniques of the application can be implemented and practiced without departing from the spirit and scope of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The following terms or definitions are provided solely to aid in the understanding of the application. These definitions should not be construed to have a scope less than understood by those skilled in the art.

Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the application are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.

The terms "about" and "substantially" in this application mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

The terms "or more", "at least", "exceeding", etc., such as "at least one" should be understood to include, but not be limited to, values of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 600, 700, 900, or 5000. But also any larger numbers or scores therebetween.

Conversely, the term "no more than" includes every value that is less than the recited value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 nucleotides. But also any smaller numbers or scores therebetween.

The terms "plurality," "at least two," "two or more," "at least a second," and the like should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 600, 700, 900, or more, 5000, or more. But also any larger numbers or scores therebetween.

Definition of terms:

as used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that is capable of non-covalently, reversibly and in a specific manner binding to a corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain CL. VH and VL regions can be further subdivided into regions of hypervariability known as Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved, known as Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including different cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). "antibodies" include, but are not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the present disclosure). Antibodies can be of any isotype/class (e.g., igG, igE, igM, igD, igA and IgY) or subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2).

Antibodies comprise globular regions of heavy or light chain polypeptides known as "domains". The domain may comprise peptide loops, typically 3 to 4 loops, which are stabilised, for example, by β -sheet and/or intrachain disulphide bonds. Based on the relative lack of sequence variation within the domains of the members of different classes in the case of "constant" domains, or significant variation within the domains of the members of different classes in the case of "variable" domains, the domains are often referred to as "constant" or "variable". An antibody or polypeptide "domain" is often interchangeably referred to in the art as an antibody or polypeptide "region".

The term "monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition, and does not relate to a method of preparation thereof. Monoclonal antibodies or immunologically active fragments thereof may be produced by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques, and the like, or other production techniques known in the art, and methods of the application involving monoclonal antibody production include in vitro culture of hybridoma cells or by DNA recombination techniques. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Each monoclonal antibody is directed against a single determinant on the antigen.

The term "antigen" is an entity (e.g., a protein entity or peptide) to which an immunoglobulin or antibody (or antigen binding fragment thereof) specifically binds.

The term "fragment" refers to a portion or portion of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with the portion when present in the intact antibody. Fragments may be obtained by chemical or enzymatic treatment of whole or complete antibodies or antibody chains. Fragments may also be obtained recombinantly.

The term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

"complementarity determining domain" or "complementarity determining region" ("CDR") interchangeably refer to the hypervariable regions of VL and VH. CDRs are target protein binding sites of antibody chains carrying the specificity of such target proteins. Three CDRs (CDRs 1-3, numbered sequentially from the N-terminus) are present in each human VL or VH, totaling about 15-20% of the variable domains. CRD may be referred to by its region and sequence. For example, "VHCDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. CDRs are structurally complementary to epitopes of the target protein and are therefore directly responsible for binding specificity. The remaining stretches of VL or VH (so-called framework regions) exhibit little variation in amino acid sequence (Kuby, immunology, 4 th edition, chapter 4 w.h. frieman (w.h.freeman & co.), new york, 2000).

In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al (1989) Nature 342:877-883, al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", journal of Molecular Biology,273,927-948 (1997)) based on the three-dimensional structure of antibodies and topology of CDR loops, kabat (Kabat et al Sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT), and North CDR definitions based on neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures.

However, it should be noted that the boundaries of CDRs of the variable regions of the same antibody obtained based on different assignment systems may differ. I.e. the CDR sequences of the same antibody variable region defined under different assignment systems are different. For example, the ranges of residues under the definition of the different assignment systems for CDR regions numbered using Kabat, abM, chothia, IMGT, contact are shown in the table below.

CDR residue ranges under different assignment system definitions

Thus, when referring to defining antibodies with a particular CDR sequence as defined herein, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the particular CDR sequence, but whose purported CDR boundaries differ from the particular CDR boundaries defined herein by the application of different protocols (e.g., different assignment system rules or combinations).

The CDRs of the antibodies of the application can be evaluated manually to determine boundaries according to any protocol or combination of protocols in the art. In the present application, unless otherwise indicated, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways. That is, the CDR sequences in the present application include 3 CDRs defined on the basis of any one of the heavy chain variable region amino acid sequences shown in SEQ ID NO.7 and 3 CDs defined on the basis of any one of the light chain variable region amino acid sequences shown in SEQ ID NO. 8. For example, in some particular embodiments of the application, when the CDRs of an antibody are encoded according to the Kabat encoding rules, the CDR sequences are: i. the amino acid sequences of complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3 respectively; the amino acid sequences of the complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO.4, 5 and 6, respectively.

Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelbody antibodies, affibodies, fab fragments, F (ab') 2 fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies), miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics"), and antigen binding fragments of any of the above.

The term "antigen binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) with an epitope of an antigen. Examples of binding fragments include, but are not limited to: single chain Fv (scFv), disulfide linked Fv (sdFv), fab fragments, F (ab') fragments (i.e., monovalent fragments consisting of VL, VH, CL and CH1 domains); f (ab) 2 fragments (i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region); fd fragment consisting of VH and CH1 domains; fv fragments consisting of the VL and VH domains of the antibody single arm; dAb fragments consisting of VH domains (Ward et al Nature 341:544-546,1989); and isolated Complementarity Determining Regions (CDRs) or other epitope-binding fragments of antibodies. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these two domains can be joined, using recombinant methods, by a synthetic linker that enables the two domains to become a single protein chain in which pairs of VL and VH regions are used to form monovalent molecules, known as single chain Fv ("scFv"); see, e.g., bird et al, science 242:423-426,1988; huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883,1988). Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment". These antigen binding fragments are obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as whole antibodies.

Antigen binding fragments may also be incorporated into single domain antibodies, large antibodies, minibodies, nanobodies, intracellular antibodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFv (see, e.g., hollinger and Hudson, nature Biotechnology (Nature Biotechnology) 23:1126-1136,2005). Antigen binding fragments may be grafted into a scaffold based on a polypeptide such as fibronectin type III (Fn 3) (see us patent No. 6,703,199, which describes a fibronectin polypeptide mab). Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH 1-VH-CH 1) that together with a complementary light chain polypeptide form a pair of antigen binding regions (Zapata et al, protein engineering (Protein Eng.)) 8:1057-1062,1995, and U.S. Pat. No. 5,641,870.

The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to sequences of a non-human antibody and all or substantially all of the FRs correspond to sequences of a human antibody.

The term "affinity" refers to the strength of interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at a number of sites by weak non-covalent forces; the more interactions, the stronger the affinity.

The term "competition" herein refers to competition between antigen binding proteins, as used in the context of antigen binding proteins (e.g. neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, as determined by the following assay: in such assays, the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen.

As used herein, the term "variant" refers to a heavy chain variable region or a light chain variable region that has been modified by at least one, e.g., 1, 2, or 3 amino acid substitution, deletion, or addition, wherein a modified antigen binding protein comprising a heavy chain or light chain variant substantially retains the biological characteristics of the antigen binding protein prior to modification. In one embodiment, the antigen binding protein comprising a variable heavy chain variable region or light chain variable region sequence retains 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein prior to modification. It will be appreciated that each heavy chain variable region or light chain variable region may be modified either alone or in combination with another heavy chain variable region or light chain variable region. The antigen binding proteins of the present disclosure comprise a heavy chain variable region amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to a heavy chain variable region amino acid sequence described herein. The antigen binding proteins of the present disclosure include light chain variable region amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequences described herein. The percent homology may be over the entire heavy chain variable region and/or the entire light chain variable region, or the percent homology may be limited to framework regions, while the sequences corresponding to CDRs have 100% identity to CDRs disclosed herein within the heavy chain variable region and/or the light chain variable region. As used herein, the term "CDR variant" refers to a CDR that has been modified by at least one, e.g., 1, 2, or 3 amino acid substitution, deletion, or addition, wherein the modified antigen binding protein comprising the CDR variant substantially retains the biological characteristics of the pre-modified antigen binding protein. In one embodiment, the antigen binding protein comprising variant CDRs retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein prior to modification.

Amino acid changes, preferably conservative changes, in the above amino acid homology may include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitutions refer to the substitution of one amino acid with another within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the following table (amino acid substitutions).

In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FR). More preferably, the amino acid changes described in the present application occur in the Fc region.

The term "vector" as used herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked by transformation. The term includes vectors that are self-replicating nucleic acid structures and that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "host cell" refers to a cell that is foreign to nucleic acid, including the progeny of such a cell. And is capable of expressing the exogenous nucleic acid in a cell or cell membrane or released outside the cell.

The term "subject" encompasses both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein, except when indicated.

1. The specific antibodies or antigen binding fragments of the application directed against Claudin-18.2

The terms "specific antibody against Claudin-18.2", "specific antibody specific for Claudin-18.2" or "specific antibody binding to Claudin-18.2" are used interchangeably herein to refer to an antibody or antigen-binding fragment of the application which is capable of binding to Claudin-18.2 protein or antigen in a specific manner, whereby the antibody antigen-binding fragment can be used for detection, identification or screening of Claudin-18.2 or the like.

The antibodies and antigen binding fragments of the application specifically bind to the Claudin-18.2 protein with high affinity, and in some embodiments, the specific antibodies or antigen binding fragments of the application are of the class of sequences as follows from the different analytical methods for CDRs in the definition above:

comprises 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO.7 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO. 8; or variants having single or multiple CDRs with the light and heavy chain CDR regions described above that do not vary more than 2 amino acids per CDR region.

In some embodiments, when encoding the antibody CDRs according to the Kabat encoding rule, the antibody or antigen binding fragment comprises the following CDR sequences:

i. the amino acid sequences of complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3 respectively;

the amino acid sequences of complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO.4, 5 and 6 respectively;

In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, the sequences being selected from the group consisting of:

b) The amino acid sequence of the light chain variable region is shown as SEQ ID NO.8, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO. 8;

in some specific embodiments, the antibody or antigen binding fragment further comprises a heavy chain constant region as set forth in SEQ ID No.9, and a light chain constant region as set forth in SEQ ID No. 10;

in some more specific embodiments, the heavy chain full length sequence of the antibody or antigen binding fragment is set forth in SEQ ID No.10 and the light chain full length sequence is set forth in SEQ ID No. 11.

In some embodiments, the antibody or antigen binding fragment may be further linked to a coupling moiety selected from one or more of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein, a lipid, and biotin, wherein the polypeptide or antibody and the coupling moiety may be optionally linked by a linker, preferably the linker is a peptide or polypeptide.

In some embodiments, the antibody or antigen binding fragment may be selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antisera, chimeric antibodies, humanized antibodies, and human antibodies; more preferably, the antibody is selected from the group consisting of multispecific antibodies, single chain Fv (scFv), single chain antibodies, anti-idiotype (anti-Id) antibodies, diabodies, minibodies, nanobodies, single domain antibodies, fab fragments, F (ab') fragments, disulfide-linked bispecific Fv (sdFv) and intracellular antibodies.

In some embodiments, the antibodies or antigen binding fragments thereof of the application may be produced by recombinant expression. The above-described nucleic acids encoding the light and heavy chain variable regions optionally linked to constant regions may be inserted into expression vectors. Vectors comprising nucleic acids encoding antibodies described herein are themselves an aspect of the application. The light and heavy chains may be cloned into the same or different expression vectors. The nucleic acids encoding the antibody chains described herein may be operably linked to one or more control sequences in an expression vector to ensure expression of the antibody chains. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Such vectors may be incorporated into a suitable host whereby the host is maintained under conditions suitable for high level expression of the nucleotide sequences and for collection and purification of the antibodies.

2. Nucleic acids, corresponding vectors, host cells, and the like of the application

The present application provides a nucleic acid encoding any of the above Claudin-18.2 antibodies or antigen binding fragments thereof or any one of the strands thereof, a vector comprising said nucleic acid, a host cell comprising said nucleic acid or said vector.

The nucleic acid of the present application is a nucleic acid encoding an anti-Claudin-18.2 protein antibody or antigen-binding fragment thereof, or a VH or VL domain thereof; it is understood that any nucleic acid capable of encoding an antibody or antigen-binding fragment thereof, or a VH or VL domain thereof, as described above is within the scope of the application.

Of course, the nucleic acid of the application may also be a nucleic acid sequence having codon-degeneracy with any of the above, such as a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above sequence.

The vectors of the present application comprising one or more of the above-described nucleic acids encoding the antibodies described herein may be cloning vectors or expression vectors, and are not limited herein.

In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs), and the like.

In one embodiment, the vector comprises optional regulatory sequences; in some embodiments, the regulatory sequence may be selected from a leader sequence, polyadenylation sequence, propeptide sequence, promoter, signal sequence, transcription terminator, or any combination thereof, without limitation.

The host cells of the application, such as host cells, comprising the expression vectors are yeast cells, mammalian cells or other cells suitable for the preparation of antibodies or antigen-binding fragments thereof. In some embodiments, suitable host cells include prokaryotic microorganisms, such as E.coli. The host cell may also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, e.g., insect cells, etc. Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth may be used. Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney (HEK 293 or 293F cells), 293 cells, baby Hamster Kidney (BHK), monkey kidney (CV 1), african green monkey kidney (VERO-76), human cervical cancer (HELA), canine kidney (MDCK), buffalo rat liver (BRL 3A), human lung (W138), human liver (Hep G2), chinese Hamster Ovary (CHO) cells, CHOs cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63, and Sp2/0, and the like. For reviews of mammalian host cell lines suitable for the production of proteins see, for example, yazaki and Wu, methods in Molecular Biology, volume 248 (b.K.C.Lo et al, humana Press, totowa, NJ), pages 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or 293 cell.

Vectors described herein comprising polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics, or virus-based transfection may be used for other cellular hosts. (see generally Green and Sambrook, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual) (Cold spring harbor Press, 4 th edition, 2012.) other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection (see generally Sambrook et al, supra.) to produce transgenic animals, the transgene may be microinjected into fertilized oocytes, or it may be integrated into the genome of embryonic stem cells, and the nuclei of these cells transferred into enucleated oocytes.

3. Methods of making, producing and purifying antibodies or antigen binding fragments of the application

The present application provides a method of making a Claudin-18.2 antibody or antigen binding fragment, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the antibody or antigen binding fragment, and optionally isolating the antibody or antigen binding fragment. In a certain embodiment, the method further comprises recovering and purifying the corresponding antibody or antigen binding fragment from the host cell (or host cell culture medium).

In some embodiments, the method may comprise transferring a vector comprising one or more nucleic acids encoding an anti-Claudin-18.2 protein antibody or antigen-binding fragment thereof or antibody chain thereof as described above into a host cell as described herein, culturing the host cell culture under conditions allowing expression of the nucleic acids, and recovering the expressed anti-Claudin-18.2 protein antibody or antigen-binding fragment thereof. Any suitable method known in the art may be employed.

In some embodiments, the antibodies or antigen binding fragments prepared as described herein may be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art. The purity of the antibodies of the application may be determined by any of a variety of well-known analytical methods including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

In some embodiments, the Claudin-18.2 antibodies provided herein can be identified, screened, or characterized for physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, antibodies of the application are tested for their antigen binding activity, for example by known methods such as ELISA, western blot, IHC, and the like. Binding to the Claudin-18.2 protein may be determined using methods known in the art, and in some embodiments, SPR or biofilm layer interferometry may be used to determine binding of the Claudin-18.2 antibody of the application to the Claudin-18.2 protein.

4. Detection and diagnostic uses of antibodies or antigen-binding fragments thereof of the application

The antibody or antigen binding fragment provided by the application can be used for detecting the existence or the content of Claudin-18.2 in a biological sample, namely, the antibody or antigen binding fragment can be used for qualitative detection and quantitative detection. And further can be used for preclinical, middle and back analyses, such as immunoassays, immunogenicity assays, etc., and even for diagnosis of diseases or concomitant diagnosis.

The Claudin-18.2 antibody or antigen binding fragment thereof provided by the application can be conveniently used in a kit, and Claudin-18.2 in vivo or in vitro biological fluids or on tissues can be detected by the antibody or antigen binding fragment thereof provided by the application. Can be used for any sample containing a detectable amount of Claudin-18.2 protein; the sample may be any stage before or after the clinic, and is not limited herein.

The test sample according to the present application may include, but is not limited to, liquids such as urine, saliva, cerebral spinal fluid, blood, serum and the like, or the test sample according to the present application may be solid or semi-solid such as tissues, feces and the like, or may be solid tissues such as those commonly used in histological diagnosis.

The term "kit" is used to refer to a combination of reagents and other materials that facilitate analysis of a sample. In some embodiments, an immunoassay kit described herein includes a suitable antigen, a binding agent comprising a detectable moiety, and a detection reagent. The system for amplifying the signal generated by the detectable moiety may or may not also be included in the kit. Furthermore, in other embodiments, the kits include, but are not limited to, components such as devices for sample collection, sample tubes, racks, trays, shelves, plates, instructions for the kit user, solutions or other chemical reagents, and samples for normalization, and/or control.

The term "detection" as used herein includes quantitative or qualitative detection, exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays. In some embodiments, the Claudin-18.2 antibodies or antigen binding fragments thereof of the application can be conjugated to a detectable label such as luciferase, biotin, or the like, in a liquid or solid phase for use in direct or indirect immunoassays of FACS, IHC, ELISA, including competitive or non-competitive, and the like.

Illustratively, the present application provides a method of detecting the presence of Claudin-18.2 in a biological sample, the method comprising detecting the presence of Claudin-18.2 protein in the biological sample. In certain embodiments, the method comprises contacting the biological sample with a Claudin-18.2 antibody or antigen binding fragment as described herein under conditions that allow binding of the Claudin-18.2 antibody or antigen binding fragment to the Claudin-18.2 protein, and detecting whether a complex is formed between the Claudin-18.2 antibody or antigen binding fragment and the Claudin-18.2 protein. The formation of the complex indicates the presence of Claudin-18.2. The method may be an in vitro or an in vivo method.

The assays of the application are useful for diagnosis of related diseases such as various types of neoplastic diseases or cancers including gastric cancer/gastroesophageal junction (GC/GEJ) cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, and the like, which are closely related to Claudin-18.2, including, for example, contacting a sample obtained from a patient with a Claudin-18.2 antibody or antigen binding fragment of the application, wherein the antibody or antigen binding fragment is labeled with a detectable label or reporter or used as a capture ligand to selectively isolate Claudin-18.2 from a patient sample. The detectable label or reporter may be a radioisotope, such as 3H, 14C, 32P, 35S or 125I; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine, or enzymes such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure Claudin-18.2 in a sample include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunohistochemistry (IHC), fluorescence Activated Cell Sorting (FACS), and the like.

Samples that can be used in the diagnostic disease assay according to the application include any biological sample obtainable from a patient that comprises a detectable amount of the Claudin-18.2 protein or fragment thereof under normal or physiological conditions. In some embodiments, including but not limited to liquids such as urine, saliva, cerebral spinal fluid, blood, serum and the like, or the sample may be a solid or semi-solid such as tissue, stool and the like, or may be a solid tissue such as those commonly used in histological diagnosis. Generally, the level of Claudin-18.2 protein in a particular sample obtained from a healthy patient will be measured to initially establish a baseline or standard level. This baseline or standard level may then be compared to a measured level of Claudin-18.2 protein in a sample obtained from an individual suspected of having the associated disease.

5. Other aspects of the antibodies or antigen binding fragments thereof of the application find use

It will be appreciated that the antibodies or antigen binding fragments thereof of the present application may also find application in screening, purification or preparation of Claudin-18.2 protein, and the like, based on the basic antigen-antibody binding activity of the antibody.

The technical solutions of the present application will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

EXAMPLE 1 preparation of specific Claudin-18.2 antibodies

In this example, claudin-18.2 specific antibodies were prepared as follows:

1) Claudin-18.2 protein immunogen preparation: the expression plasmid capable of expressing human Claudin-18.2 protein, his and Twain-Strep Tag is constructed by a gene synthesis method, the expression plasmid is transfected into HEK293 cells by using Invitrogen Lipofectamine transfection reagent, culture supernatant is harvested after 48 hours, and then purified Claudin-18.2 (DDM) protein is obtained by an affinity chromatography method.

2) Immunization of mice: the Claudin-18.2 (DDM) protein prepared in the step 1 is used as an immunogen, 3 Balb/c mice are immunized by the Claudin-18.2 (DDM) protein, and a manganese adjuvant immunization means is adopted. After the immunization, the immune response level was determined by detecting serum of the immunized animal by ELISA. After conventional immunization, cell fusion can be performed if the immunized animal is able to reach an immune response level against the immunogen (OD value >1.0, titer 1:8,000).

3) Cell fusion and plating: cell fusion was performed 1 time using electrofusion method. Half of the fused cells were plated in semi-solid medium and the other half of the fused cells were frozen.

4) Picking up monoclonal cell strains: single cell clusters cultured in semi-solid medium were picked up and cultured in 96-well plates.

5) Screening: the supernatant of the fused cells was screened by ELISA to select cells positive for both Claudin-18.2 (DDM) and Claudin-18.2 (VLP) protein binding.

6) Clone expansion culture and retesting: positive clone cells were transferred to 48 well plates for expansion, and 0.2 ml of supernatant was collected for each expanded clone for detection by the indirect ELISA method.

7) Clone expansion culture and retesting: positive clonal cells were transferred to a 12-well plate for expansion, and 1 ml of supernatant was collected for each expanded clone for detection by an indirect ELISA method. Based on antigen recognition confirmation, 2 stable cell lines (clone numbers 3B10 and 12C 11) were selected for low temperature storage. 5 ml of supernatant was collected for each clone before cryopreservation and all clones were subtype identified and stored.

8. Hybridoma cell antibody gene sequencing: total RNA of the hybridoma cells is extracted, and the RNA is reversely transcribed into cDNA through an RT-PCR reaction. Cloning antibody light chain and heavy chain sequences, constructing the antibody light chain and heavy chain sequences on a T vector, and then carrying out DNA sequencing analysis to obtain antibody gene sequences.

The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of clone No. 3B10 antibody are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4. 5 and 6; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: shown in figure 7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 8.

The specific sequences are shown in the following table:

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9) Antibody production and purification: and (3) transfecting the antibody gene sequence obtained in the step (8) into HEK293 cells, performing amplification culture, purifying the antibody by adopting a protein A/G affinity chromatography method, and storing the purified antibody in Phosphate Buffer (PBS) by adopting a dialysis method.

EXAMPLE 2Claudin-18.2 antibody 3B10 specificity assay

In this example, monoclonal antibody 3B10 obtained according to the method of example 1 above was specifically analyzed by an enzyme-linked immunosorbent assay as follows:

1) Claudin-18.2 (DDM), claudin-18.2 (VLP), claudin-18.1, claudin-6 proteins were diluted to 5. Mu.g/mL with PBS and added to the wells of the microplate at 100. Mu.L per well. The plates were sealed and left to stand overnight (about 16 h) at 4 ℃.

2) The wells were discarded, the ELISA plates were dried by pipetting, washed with PBST wash, 300. Mu.L/Kong Jinpao S, and the plates were dried by pipetting and washed 3 times.

3) mu.L of blocking reagent (PBST wash containing 5% BSA) was added to each well, the plates were sealed, and incubated at 37℃for 1.5h.

4) The plate is washed 3 times by repeating the step 2.

5) The Claudin-18.2 antibody was diluted to 1. Mu.g/mL with a sample diluent (PBST wash containing 0.5% BSA). Add to the elisa plate 100 μl per well. Sealing plates are used for sealing plates, and the plates are placed at 37 ℃ for incubation for 1.0h.

6) The plate is washed 3 times by repeating the step 2.

7) HRP-coat-Anti-Mouse IgG was diluted 1:20000 times with sample dilution, 100. Mu.L per well was added, membrane sealed with sealing plate, and incubated at 37℃for 1.0h.

8) The plate is washed 3 times by repeating the step 2.

9) Adding 100 μl of color development liquid into each well, sealing with sealing plate membrane, and incubating at 37deg.C in dark place for 20min.

10 Add 50 μl of stop solution per well, gently shake the elisa plate until color development is uniform.

11 Reading with enzyme-labeled instrumentThe absorbance values at 450nm and 630nm were taken and the OD was used ₄₅₀ Knot subtracts OD ₆₃₀ The values give absorbance values (OD values).

The absorbance (OD) of the monoclonal antibody is shown in table 1.

ELISA detection of the antibodies of Table 1 OD values

The results of the experiment show that the antibodies have the best specificity and highest activity against Claudin18.2 protein and do not cross react with Claudin18.1 and Claudin 6 proteins.

Example 3Claudin-18.2 antibody 3B10 Performance analysis (sensitivity, specificity, cross-reactivity)

The antibody Claudin-18.2 (3B 10) is diluted by a diluent according to a certain proportion to prepare a primary antibody working solution.

1) The FFPE normal stomach tissue is fixed by formalin, paraffin is embedded, and the slice sample is restored to room temperature for standby.

2) Dewaxing, hydration and antigen retrieval are carried out on the tissue sections.

3) 150. Mu.L of endogenous peroxidase blocker was added until the tissue was completely covered, and after incubation for 5 minutes at room temperature, PBS water was rinsed 3 times,

4) 150. Mu.L of primary antibody working solution was added, and after incubation for 30 minutes at room temperature, PBS was washed 3 times.

5) Adding 150 mu L of secondary antibody, and incubating for 8 minutes at room temperature; PBS was washed 3 times for 2 minutes each and purified water was washed 1 time.

7) 150. Mu. LDAB chromogenic solution was added to completely cover the tissue, incubated at room temperature for 10 minutes, and rinsed 3 times with purified water.

8) mu.L of hematoxylin was added, incubated at room temperature for 5 minutes, rinsed 1 time with purified water, rinsed 1 time with PBS, and rinsed 1 time with purified water.

Sequentially soaking gradient alcohol 85%,95% and 100% for 3 min each time; the xylenes were transparent twice for 15 minutes each.

9) The samples were flaked with neutral gum.

10 Immunohistochemical detection results were observed and judged by a qualified pathologist under a microscope on the stained sections. Under the microscope, dark brown staining was observed at the specific cell location corresponding to the antigen, with no background staining.

The immunohistochemical staining results for each monoclonal antibody are shown in FIG. 1.

Experimental results show that in the aspects of sensitivity and specificity, claudin18.2 (3B 10) primary antibody stains gastric gland cytoplasma and/or cell membrane with strong positive staining, and other tissue cells with negative staining, and the monoclonal antibody has very high sensitivity and specificity for Claudin18.2 protein. Moreover, in terms of cross-reactivity, the above-described cloned antibodies stained lung tissue without a positive signal, considering that the lung tissue was CLDN18.1 positive, demonstrated that they did not cross-react with the claudin18.1 protein.

Example 4 comparison of Properties with commercial antibodies

The antibody Claudin-18.2 (3B 10) of the present application and the commercial antibody Claudin-18.2 (ABCAM EPR 19202) of the company ABCAM were diluted with a diluent in proportion to prepare a primary antibody working solution (used at the same concentration in use), and the primary antibody working solution was tested in the following steps.

9) The samples were flaked with neutral gum.

The results of the two antibody staining were compared and analyzed, and the results of immunohistochemical staining with each monoclonal antibody are shown in FIG. 2.

Experimental results show that the specificity of the 2 cloned antibodies for Claudin18.2 protein detection meets the use requirement of the antibodies. However, the positive signal of the antibody Caudin18.2 (3B 10) is far stronger than that of the commercial antibody Claudin18.2 (EPR 19202) in terms of sensitivity, and the antibody Caudin18.2 (3B 10) has obvious and unexpected advantages compared with the antibody Claudin18.2 (EPR 19202).

The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the application and its practical application to thereby enable one skilled in the art to make and utilize the application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. An antibody against Claudin-18.2 or an antigen-binding fragment thereof, comprising 3 CDRs in the heavy chain variable region amino acid sequence as set forth in SEQ ID No.7 and 3 CDRs in the light chain variable region amino acid sequence as set forth in SEQ ID No. 8;

or variants having single or multiple CDRs with the light and heavy chain CDR regions described above that do not vary more than 2 amino acids per CDR region.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the following CDR sequences when encoded for antibody CDRs according to the Kabat encoding rule:

3. The antibody or antigen-binding fragment of any one of claims 1-2, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the sequence is selected from the group consisting of seq id nos:

4. The antibody or antigen-binding fragment of any one of claims 1-3, wherein the antibody or antigen-binding fragment has a conjugated moiety attached thereto, the conjugated moiety selected from one or more of a radionuclide, a drug, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein, a lipid, and biotin;

Preferably, the antibody or antigen binding fragment is optionally linked to the coupling moiety by a linker;

more preferably, the linker is a peptide or polypeptide.

5. The antibody or antigen-binding fragment of any one of claims 1-4, wherein the antibody or antigen-binding fragment is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antisera, a chimeric antibody, a humanized antibody, and a human antibody; preferably, the antibody is selected from the group consisting of multispecific antibodies, single chain Fv (scFv), single chain antibodies, anti-idiotype (anti-Id) antibodies, diabodies, minibodies, nanobodies, single domain antibodies, fab fragments, F (ab') fragments, disulfide-linked bispecific Fv (sdFv) and intracellular antibodies.

6. An isolated polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1-5.

7. A recombinant vector comprising the polynucleotide of claim 6, and optionally regulatory sequences;

preferably, the recombinant vector is a cloning vector or an expression vector;

more preferably, the regulatory sequence is selected from a leader sequence, polyadenylation sequence, propeptide sequence, promoter, signal sequence, transcription terminator, or any combination thereof.

8. A product comprising one or more of the antibodies or antigen-binding fragments, polynucleotides or recombinant vectors of any one of claims 1-7, and contained in a suitable container.

9. The product of claim 8, wherein the product is in the form of a kit;

preferably, the kit includes, but is not limited to: ELISA kit, flow sorting kit or IHC kit.

10. The antibody or antigen binding fragment of any one of claims 1-5 for use as follows:

preferably, the detection comprises a quantitative or qualitative detection;

more preferably, the detection comprises an immunogenicity detection;

further preferred, the means of detection include, but are not limited to, flow detection, ELISA detection or IHC detection;

(2) The application in the diagnosis or the accompanying diagnosis of the tumor or the application in the preparation of a diagnosis or an accompanying diagnosis kit of the tumor.