CN119350502A - Antigen binding protein targeting FAP and its application - Google Patents

Abstract

The present application relates to an isolated antigen-binding protein, which can specifically bind to fibroblast activation protein (FAP). The present application also provides a preparation method of the isolated antigen-binding protein and its use.

Description

FAP-targeted antigen binding proteins and uses thereof

Technical Field

The application relates to the field of biological medicine, in particular to an antigen binding protein targeting FAP.

Background

Fibroblast activation protein (Fibroblast activation protein, FAP) is a membrane serine peptidase, one of the members of the serine protease family II, has dipeptidyl peptidase and collagenase activities, and has dual functions in tumor growth. The proteolytic enzyme activity of FAP can enhance the invasiveness of tumor cells to extracellular matrixes, and can promote the growth and proliferation of tumors. FAP is considered promising as an antigenic target for molecular imaging, diagnosis and treatment of a variety of cancers due to its expression in a variety of common cancers and limited expression in normal tissues.

Thus, there is a need in the art to develop antibodies capable of binding with high affinity to FAP.

Disclosure of Invention

The application provides an isolated antigen binding protein targeting Fibroblast Activation Protein (FAP), which has good specificity, high FAP affinity and stability.

In one aspect, the application provides an isolated antigen binding protein comprising CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence shown in SEQ ID NO. 2, said CDR2 comprising the amino acid sequence shown in SEQ ID NO. 13 and said CDR3 comprising the amino acid sequence of SEQ ID NO. 14.

In certain embodiments, it comprises CDR1, CDR2, and CDR3 selected from any one of the following groups:

1) The CDR1 comprises the amino acid sequence of SEQ ID NO. 2, the CDR2 comprises the amino acid sequence shown in SEQ ID NO. 3, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO. 4;

2) The CDR1 comprises the amino acid sequence of SEQ ID NO. 2, the CDR2 comprises the amino acid sequence shown as SEQ ID NO. 6, and the CDR3 comprises the amino acid sequence shown as SEQ ID NO. 7.

In certain embodiments, it comprises an antibody or antigen binding fragment thereof.

In certain embodiments, wherein the antigen binding fragment comprises a Fab, fab ', fv fragment, F (ab') 2,F (ab) 2, scFv, di-scFv, and/or dAb.

In certain embodiments, wherein the antibody comprises a single domain antibody.

In certain embodiments, it comprises a VHH comprising the amino acid sequence set forth in SEQ ID NO. 15.

In certain embodiments, it comprises a VHH comprising an amino acid sequence set forth in any one of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 9-13.

In certain embodiments, wherein the antibody is selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In certain embodiments, it is capable of specifically binding to fibroblast activation protein (Fibroblast activation protein, FAP).

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

In another aspect, the application provides a nucleic acid molecule encoding an isolated antigen binding protein of the application.

In another aspect, the application provides a vector comprising a nucleic acid molecule as described herein.

In another aspect, the application provides a cell comprising a nucleic acid molecule as described herein or a vector as described herein.

In another aspect, the application provides a pharmaceutical composition comprising an isolated antigen binding protein as described herein, an immunoconjugate as described herein, a nucleic acid molecule as described herein, a vector as described herein and/or a cell as described herein, and optionally a pharmaceutically acceptable carrier.

In another aspect, the application provides a method of making an isolated antigen binding protein of any of the application, comprising culturing a cell of the application under conditions such that the antigen binding protein is expressed.

In a further aspect, the application provides the use of an isolated antigen binding protein of the application, a polypeptide molecule of the application, an immunoconjugate of the application, a nucleic acid molecule of the application, a vector of the application, a cell of the application and/or a pharmaceutical composition of the application in the manufacture of a medicament for the prevention and/or treatment of a disease and/or disorder.

In certain embodiments, wherein the disease and/or disorder is caused or mediated by fibroblast activation protein.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the drawings and descriptions of the present application are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the application related to the application are shown in the appended claims. A better understanding of the features and advantages of the application in accordance with the present application will be obtained by reference to the exemplary embodiments and the accompanying drawings that are described in detail below. The drawings are briefly described as follows:

Fig. 1 shows the affinity of the antibodies of the application for human FAP, and fig. 1A and 1B show the results of the antibody and antigen affinity assays.

FIG. 2 shows the FACS detection results of antibodies of the application with FAP of different origins.

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

Definition of terms

In the present application, the term "fibroblast activation protein (Fibroblast activation protein, FAP)" also known as "isolated enzyme (Seprase)" or "prolyl endopeptidase FAP" generally refers to any native FAP from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term includes "full-length" unprocessed FAP as well as any form of FAP derived from processing in a cell. The term also encompasses naturally occurring variants of FAP, such as splice variants or allelic variants. For example, the amino acid sequence of human FAP may be Q12884 at UniProt accession number. For example, human FAP has genBank accession No. AAC 51668. For example, the antigen binding proteins of the application may bind to the extracellular domain of FAP.

In the present application, the term "isolated" generally refers to those obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" does not exclude the incorporation of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.

In the present application, the term "isolated antigen binding protein" generally refers to a protein having antigen binding ability that is free from its naturally occurring state. The "isolated antigen binding protein" may comprise an antigen-binding portion and optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen by the antigen-binding portion. The antigen binding proteins may comprise, for example, an antibody-derived protein Framework Region (FR) or an alternative protein framework region or artificial framework region with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, framework regions comprising antibody sources that are introduced, for example, to stabilize mutations in the three-dimensional structure of the antigen binding protein, as well as fully synthetic framework regions comprising, for example, biocompatible polymers. See, e.g., korndorfer et al, 2003, proteins: structure, function, andbrio information, 53 (1): 121-129 (2003); roque et al, biotechnol prog.20:639-654 (2004). Examples of antigen binding proteins include, but are not limited to, human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, single chain antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, fab ', fv fragments, F (ab') ₂,F(ab)₂, scFv, di-scFv, dAb, igD antibodies, igE antibodies, igM antibodies, igG1 antibodies, igG2 antibodies, igG3 antibodies, or IgG4 antibodies, and fragments thereof.

In the present application, the term "CDR" is also referred to as "complementarity determining region", and generally refers to a region in the variable domain of an antibody whose sequence is highly variable and/or forms a structurally defined loop. Typically, the antibody includes six CDRs, three in the VH (HCDR 1, HCDR2, HCDR 3), and three in the VL (LCDR 1, LCDR2, LCDR 3). In certain embodiments, naturally occurring camelid antibodies consisting of only heavy chains are also able to function normally and stably in the absence of light chains. See, e.g., hamers-CASTERMAN ET al, nature 363:446-448 (1993); SHERIFF ET AL, nature struct. Biol.3:733-736 (1996). Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, abM, chothia, IMGT, a combination of Kabat/Chothia et al. Such coding systems are known in the art and can be found, for example, in http:// www.bioinf.org.uk/abs/index. Html # kabatnum. For example, the amino acid sequence numbering of the antigen binding proteins may be according to IMGT numbering scheme (IMGT,the international ImMunoGeneTics information system@imgt.cines.fr;http：//imgt.cines.fr;Lefranc et al, 1999,Nucleic Acids Res.27:209-212; ruiz et al, 2000Nucleic Acids Res.28:219-221; lefranc et al, 2001,Nucleic Acids Res.29:207-209; lefranc et al, 2003,Nucleic Acids Res.31:307-310; lefranc et al, 2005,DevComp Immunol 29:185-203). For example, the CDRs of the antigen binding protein may be determined according to the Kabat numbering system (see, e.g., kabat EA & Wu TT (1971) ANN NY ACADSCI:190:382-391 and Kabat EAet al.,(1991)Sequences of Proteins of Immunological Interest,FifthEdition,U.S.Department of Health and Human Services,NIH Publication No.91-3242). for possible differences in the boundaries of CDRs of the variable regions of the same antibody based on different coding systems). When referring to defining antibodies with specific CDR sequences as defined in the present application, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the specific CDR sequences, but whose purported CDR boundaries differ from the specific CDR boundaries as defined in the present application by the application of different schemes (e.g., different coding systems). 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.

In the present application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain. The variable domains of the heavy and light chains may be referred to as "V _H" and "V _L", respectively (or "VH" and "VL", respectively). These domains are typically the most variable portions of an antibody (relative to other antibodies of the same type) and comprise antigen binding sites.

In the present application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ greatly in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains. It is typically concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, which form a circular connection and in some cases form part of a β -sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together promote the formation of the antigen binding site of the antibody (see Kabat et al,Sequences of Immunological Interest,Fifth Edition,National Institute of Health,Bethesda,Md.(1991)).

In the present application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibodies", for the purposes of the present application, the term "antibody" also includes antibody fragments such as Fab, F (ab') ₂, fv, scFv, fd, dAb, and other antibody fragments that retain antigen binding function (e.g., specifically bind FAP). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetramer units with another polypeptide called the J chain and contain 10 antigen binding sites, whereas IgA antibodies comprise 2-5 basic 4-chain units that can polymerize in conjunction with the J chain to form multivalent combinations. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains, followed by four CH domains for the mu and epsilon isoforms. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain of the heavy chain (CH 1). Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The VH and VL pairs together form a single antigen binding site. For the structure and properties of different classes of antibodies, see for example Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton&Lange,Norwalk,Conn.,1994,, page 71 and chapter 6. L chains from any vertebrate species can be divided into one of two distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes or isotypes based on the amino acid sequence of the constant domain of the heavy Chain (CH). There are five classes of immunoglobulins IgA, igD, igE, igG and IgM currently available, with heavy chains designated α, δ, ε, γ and μ, respectively.

In the present application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind an antigen (e.g., FAP). In the present application, the antigen binding fragment may comprise a Fab, fab ', F (ab) ₂, fv fragment, F (ab') ₂, scFv, di-scFv and/or dAb.

In the present application, the term "Fab" generally refers to an antigen-binding fragment of an antibody. As described above, papain can be used to digest intact antibodies. The antibodies, after digestion with papain, produce two identical antigen-binding fragments, a "Fab" fragment, and a residual "Fc" fragment (i.e., fc region, supra). The Fab fragment may consist of a complete L chain with a heavy chain variable region and the first constant region (C _H 1) of the H chain (V _H).

In the present application, the term "Fab' fragment" generally refers to a monovalent antigen binding fragment of a human monoclonal antibody that is slightly larger than the Fab fragment. For example, a Fab' fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of a heavy chain. For example, a Fab' fragment can also include part or all of the 220-330 amino acid residues of the heavy chain.

In the present application, the term "F (ab') 2" generally refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F (ab') 2 fragment contains two Fab fragments held together by disulfide bonds and a partial hinge region. F (ab') 2 fragments have divalent antigen binding activity and are capable of cross-linking antigens.

In the present application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy and light chain variable regions, and lacking the heavy and light chain constant regions. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, fv fragments comprise all or part of the amino terminal variable region of about 110 amino acids of the heavy and light chains.

In the present application, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and are capable of expression as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specifically stated otherwise, as used herein, an scFv may have the VL and VH variable regions described in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

In the present application, the term "dAb" generally refers to an antigen binding fragment having a VH domain, a VL domain or having a VH domain or a VL domain, reference to, for example, ward et al (Nature, 1989Oct 12;341 (6242): 544-6), reference to Holt et al, trends Biotechnol.,2003,21 (11): 484-490, and reference to other published patent applications such as WO 06/030220, WO 06/003388 and DomantisLtd. The term "dAb" generally includes sdabs. The term "sdAb" generally refers to a single domain antibody. Single domain antibodies generally refer to antibody fragments consisting of only the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL).

In the present application, the term "VHH" generally relates to variable antigen binding domains from heavy chain antibodies of the family camelidae (camel, dromedary, llama, alpaca, etc.) (see, nguyen v.k. Et al, 2000,The EMBO Journal,19,921-930;Muyldermans S, 2001,J Biotechnol, 74,277-302 and reviewed Vanlandschoot p. Et al, 2011,Antiviral Research 92,389-407). VHH may also be referred to as Nanobody (Nb).

In the present application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules consisting of single molecules. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" refers to the characteristics of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in the present application may be prepared in hybridoma cells or may be prepared by recombinant DNA methods.

In the present application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable regions are derived from antibodies in laboratory animals such as rodents ("parent antibodies") and the constant regions are derived from human antibodies such that the resulting chimeric antibodies have a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parent (e.g., alpaca-derived) antibody.

In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (e.g., alpaca antibody) are replaced with the corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible, provided that they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "humanized antibodies" retain antigen specificity similar to the original antibody. A "humanized" form of a non-human (e.g., alpaca) antibody may minimally comprise chimeric antibodies derived from sequences of non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) having the desired properties, affinity and/or ability, such as alpaca, mouse, rat, rabbit or non-human primate. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise amino acid modifications that are not in the recipient antibody or in the donor antibody. These modifications may be made to further improve the properties of the antibody, such as binding affinity.

The term "fully human antibody" generally refers to an antibody comprising only human immunoglobulin protein sequences. If it is produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell, the fully human antibody may contain a murine sugar chain. Similarly, "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans by phage display or other molecular biological methods in transgenic animals with human immunoglobulin germline sequences. Exemplary techniques that may be used to make antibodies are described in U.S. Pat. Nos. 6,150,584, 6,458,592, 6,420,140. Other techniques, such as the use of libraries, are known in the art.

In the present application, the term "nucleic acid molecule" generally refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analogue isolated from its natural environment or synthesized synthetically.

In the present application, the term "vector" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. For example, vectors may include plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda or M13 phages, animal viruses and the like. Animal virus species used as vectors may include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these.

In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that may or may not be the recipient of a subject plasmid or vector, which comprises a nucleic acid molecule according to the present application or a vector according to the present application. Cells may include progeny of a single cell. The offspring may not necessarily be identical to the original parent cell (either in the form of the total DNA complement or in the genome) due to natural, accidental or deliberate mutation. Cells may include cells transfected in vitro with the vectors of the application. The cells may be bacterial cells (e.g., E.coli), yeast cells, or other eukaryotic cells, such as COS cells, chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, heLa cells, HEK293 cells, COS-1 cells, NS0 cells.

In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugation (e.g., covalent attachment via a linker molecule) of the other agent (e.g., a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent) to the antibody or antigen-binding fragment thereof, which conjugate can specifically bind to an antigen on a target cell through the antibody or antigen-binding fragment thereof, delivering the other agent to the target cell (e.g., a tumor cell).

In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing/treating a disease or disorder. The pharmaceutical composition may comprise an isolated antigen binding protein of the application, a nucleic acid molecule of the application, a vector of the application and/or a cell of the application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) suitable formulations of carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the application include, but are not limited to, liquid, frozen and lyophilized compositions.

In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations employed. Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counter ions, such as sodium, and/or nonionic surfactants.

In the present application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds a target (which may be an epitope) may be an antibody that binds the target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In certain embodiments, the antibodies specifically bind to epitopes on proteins that are conserved among proteins of different species. In certain embodiments, specific binding may include, but is not required to be, exclusively binding.

In the present application, the term "subject" generally refers to a human or non-human animal, including but not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

In the context of the present application, reference to a protein, polypeptide and/or amino acid sequence is also to be understood as encompassing at least the scope of variants or homologues having the same or similar function as the protein or polypeptide.

In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof that specifically binds FAP). For example, the functional variant may comprise a protein or polypeptide that has been altered in amino acids by at least 1, such as 1-30, 1-20, or 1-10, and yet another such as 1,2,3, 4, or 5 amino acid substitutions, deletions, and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%,70%,80%,90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to alteration. For example, the substitution may be a conservative substitution.

In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof that specifically binds FAP).

In the present application, the homology generally refers to similarity, similarity or association between two or more sequences. The "percent sequence homology" may be calculated by comparing two sequences to be aligned in a comparison window, determining the number of positions in the two sequences where the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cys and Met) is present to give the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce the percent sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length sequence being compared or over the region of the target sequence. The homology can also be determined by FASTA and BLAST. A description of the FASTA algorithm can be found in "improved tools for biological sequence comparison" by W.R. Pearson and D.J. Lipman, proc. Natl. Acad. Sci., 85:2444-2448,1988, and "quick sensitive protein similarity search" by D.J. Lipman and W.R. Pearson, science,227:1435-1441,1989. A description of the BLAST algorithm can be found in "a basic local contrast (alignment) search tool", journal of molecular biology, 215:403-410,1990, U.S. Altschul, W.Gish, W.Miller, E.W.Myers, and D.Lipman.

In the present application, the term "comprising" generally means including, summarizing, containing or comprising. In some cases, the meaning of "as", "consisting of.

In the present application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

Detailed Description

Isolated antigen binding proteins

The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region may be contacted with an antigen or epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, chothia, IMGT, abM, a combination of Kabat/Chothia et al. Such coding systems are known in the art and can be found, for example, in http:// www.bioinf.org.uk/abs/index. Html # kabatnum. The CDR regions can be determined by one skilled in the art using different coding systems depending on the sequence and structure of the antibody. Using different coding systems, CDR regions may differ. In the present application, the CDRs encompass CDR sequences partitioned according to any CDR partitioning scheme, as well as variants thereof, including amino acid sequences of the CDRs that have been substituted, deleted and/or added with one or more amino acids. Such as 1-30, 1-20, or 1-10, and also such as 1,2, 3, 4, 5, 6,7,8, or 9 amino acid substitutions, deletions, and/or insertions, and homologues thereof, which may be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the CDR.

In one aspect, the application provides an isolated antigen binding protein comprising HCDR3. In some cases, the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 14. In some cases, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 4. In some cases, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 7.

In the present application, the isolated antigen binding protein may further comprise HCDR2. In some cases, the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 13. In some cases, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 3. In some cases, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO. 6.

In the present application, the isolated antigen binding protein may further comprise HCDR1. In some cases, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO. 2.

In the present application, the isolated antigen binding protein may further comprise HCDR1, HCDR2 and HCDR3. In some cases, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO. 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 13, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 14.

In the present application, the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence of SEQ ID No. 2, the HCDR2 comprising the amino acid sequence of SEQ ID No. 3 and the HCDR3 comprising the amino acid sequence of SEQ ID No. 4.

In the present application, the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence of SEQ ID No. 2, the HCDR2 comprising the amino acid sequence of SEQ ID No. 6 and the HCDR3 comprising the amino acid sequence of SEQ ID No. 7.

In the present application, the isolated antigen binding protein may comprise at least one CDR of the amino acid sequences shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 13 and SEQ ID NO. 14, which may comprise a CDR divided in any way. The CDRs delimited by any one of the means fall within the scope of the claims of the present application if the sequence is identical to SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 13 or SEQ ID NO. 14.

In the present application, the isolated antigen binding protein may comprise a VHH.

In some cases, CDR3 of the VHH can comprise the amino acid sequence shown in SEQ ID NO. 14, CDR2 of the VHH can comprise the amino acid sequence shown in SEQ ID NO. 13, and CDR1 of the VHH can comprise the amino acid sequence shown in SEQ ID NO. 2.

In some cases, CDR3 of the VHH can comprise the amino acid sequence shown in SEQ ID NO. 4, CDR2 of the VHH can comprise the amino acid sequence shown in SEQ ID NO. 3, and CDR1 of the VHH can comprise the amino acid sequence shown in SEQ ID NO. 2.

In some cases, CDR3 of the VHH can comprise the amino acid sequence shown in SEQ ID NO. 7, CDR2 of the VHH can comprise the amino acid sequence shown in SEQ ID NO. 6, and CDR1 of the VHH can comprise the amino acid sequence shown in SEQ ID NO. 2.

In the present application, the isolated antigen binding protein may comprise VH. In the present application, the isolated antigen binding protein may comprise a VHH. In some cases, the VHH may comprise an amino acid sequence as shown in SEQ ID NO. 15. In some cases, the VHH may comprise an amino acid sequence as set forth in any one of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 9-13.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region, which may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen binding protein may be derived from a human IgG1 heavy chain constant region.

In the present application, the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof.

In certain embodiments, the antigen binding fragment can comprise a Fab, fab ', fv fragment, F (ab') ₂,F(ab)₂, scFv, di-scFv, and/or dAb.

In certain embodiments, the antibodies may include monoclonal antibodies, chimeric antibodies, humanized antibodies, and/or fully human antibodies.

Furthermore, it is noted that the isolated antigen binding proteins of the application may comprise heavy and/or light chain sequences to which one or more conservative sequence modifications exist. By "conservative sequence modifications" is meant amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into the isolated antigen binding proteins of the application by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR regions of the isolated antigen binding proteins of the application may be replaced with other amino acid residues of the same side chain set. Those skilled in the art will appreciate that some conservative sequence modifications do not result in the loss of antigen binding, see, e.g. ,Brummell et al.,(1993)Biochem 32:1180-8;de Wildt et al.,(1997)Prot.Eng.10:835-41;Komissarov et al.,(1997)J.Biol.Chem.272:26864-26870;Hall et al.,(1992)J.Immunol.149:1605-12;Kelley and O'Connell(1993)Biochem.32:6862-35;Adib-Conquy et al.,(1998)Int.Immunol.10:341-6and Beers et al.,(2000)Clin.Can.Res.6:2835-43.

The FAP antigen binding proteins of the application may be identified, screened or characterized by various assays known in the art.

For example, the antigen binding activity of an antigen binding protein or fusion protein of the application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, and the like.

In the present application, the isolated antigen binding protein is capable of specifically binding to FAP. In certain embodiments, binding of the isolated antigen binding protein to FAP can be detected by ELISA methods. For example, the antigen binding proteins of the application may bind FAP at EC ₅₀ values of less than or equal to about 0.010 μg/mL, less than or equal to about 0.009 μg/mL, less than or equal to about 0.008 μg/mL, less than or equal to about 0.007 μg/mL, less than or equal to about 0.006 μg/mL, less than or equal to about 0.005 μg/mL, less than or equal to about 0.004 μg/mL, less than or equal to about 0.003 μg/mL, less than or equal to about 0.002 μg/mL, and less than or equal to about 0.001 μg/mL.

In the present application, the isolated antigen binding proteins are capable of preventing and/or treating diseases and/or disorders. In certain embodiments, the disease and/or disorder may be caused or mediated by expression of FAP. In certain embodiments, the disease and/or condition may include a tumor.

Nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions

In another aspect, the application provides isolated nucleic acid molecules that can encode the isolated antigen binding proteins of the application. For example, it may be produced or synthesized by (i) in vitro amplification, e.g., by Polymerase Chain Reaction (PCR) amplification, (ii) by clonal recombination, (iii) purification, e.g., by cleavage and gel electrophoresis fractionation, or (iv) synthesized, e.g., by chemical synthesis.

In another aspect, the application provides a vector which may comprise a nucleic acid molecule according to the application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not exclusively.

In another aspect, the application provides a cell, which may comprise a nucleic acid molecule according to the application or a vector according to the application. In certain embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors of the application. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors of the application. For example, the vectors of the application may be introduced into such host cells, e.g., eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. In certain embodiments, the cell may be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a 293T cell, a COS-1 cell, a SP2/0 cell, a NS0 cell, or a myeloma cell. The vectors of the application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like.

In another aspect, the application also provides immunoconjugates which may comprise the isolated antigen binding proteins of the application.

In certain embodiments, the isolated antigen binding proteins or fragments thereof of the present application may be linked to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, or the like. The linkage may be through one or more covalent bonds, or non-covalent interactions, and may include chelation. A variety of linkers (which may be known in the art) may be used to form the immunoconjugate. Furthermore, the immunoconjugate may be provided in the form of a fusion protein, which may be expressed from a polynucleotide encoding the immunoconjugate. The immunoconjugate may further comprise, for example, an antibody-drug conjugate (ADC). Suitable drugs may include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotics. In ADC, the antibody and therapeutic agent may be cross-linked by a linker that is cleavable, such as a peptide linker, disulfide linker, or hydrazone linker.

In another aspect, the application also provides a pharmaceutical composition, which may comprise an isolated antigen binding protein of the application, an immunoconjugate of the application, a nucleic acid molecule of the application, a vector of the application and/or a cell of the application, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) suitable formulations of adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutical compositions may also contain more than one active compound, typically those active compounds having complementary activity that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.

In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents compatible with drug administration, generally safe, non-toxic.

In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, endoluminal, intra-arterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient, as described in WO 2015/036583.

Preparation method

In another aspect, the application provides methods of making the isolated antigen binding proteins. The method may comprise culturing the host cell of the application under conditions such that the antigen binding protein is expressed. For example, such methods are known to those of ordinary skill in the art by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like.

Any method suitable for producing monoclonal antibodies may be used to produce the antigen binding proteins of the application. For example, animals may be immunized with linked or naturally occurring FAP or fragments thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used. In certain embodiments, isolated antigen binding proteins against FAP may be screened and enriched by extracting immune alpaca peripheral blood lymphocytes, extracting cell nucleic acid fragments, cloning into vectors, and screening by phage surface display systems.

Any suitable form of FAP can be used as an immunogen (antigen) for generating antibodies specific for FAP, and screening the antibodies for biological activity. For example, the priming immunogen may be full-length FAP, including natural homodimers, or a peptide containing single/multiple epitopes. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art.

Method and use

In a further aspect, the application provides the use of said isolated antigen binding protein, said nucleic acid molecule, said vector, said cell, said immunoconjugate and/or said pharmaceutical composition for the manufacture of a medicament for the prevention and/or treatment of a disease and/or disorder.

In another aspect, the application also provides a method of preventing and/or treating a disease and/or disorder, which method may comprise administering to a subject in need thereof the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition of the application.

In the present application, the use may be performed in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In another aspect, the isolated antigen binding proteins, the nucleic acid molecules, the vectors, the cells, the immunoconjugates and/or the pharmaceutical compositions of the application may be used for preventing and/or treating diseases and/or disorders.

In the present application, the disease and/or condition may be caused or mediated by FAP expression.

In the present application, the disease and/or condition includes a tumor.

In another aspect, the application also provides a method of detecting FAP in a sample, the method comprising administering the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition.

In some cases, the method of detecting FAP in a sample may be an in vitro method. For example, contacting an isolated antigen binding protein of the application with an ex vivo sample detects the presence and/or amount of FAP in the sample. In certain instances, the method of detecting FAP in a sample is for non-therapeutic purposes. In some cases, the method of detecting FAP in a sample is not a diagnostic method.

In another aspect, the application also provides a kit or kit for detecting FAP in a sample comprising the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition.

In a further aspect, the application also provides the use of the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition in the preparation of a kit for detecting the presence and/or amount of FAP in a sample.

Without intending to be limited by any theory, the following examples are presented merely to illustrate the antigen binding proteins, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the application.

Examples

EXAMPLE 1 expression of recombinant human FAP and related EGFP cell preparation

S1.1, obtaining an amino acid sequence of an extracellular domain of the human FAP (namely, 26 th residue to 760 th residue in Q12884) according to an amino acid sequence (Q12884) of the human FAP on a protein database Uniprot;

S1.2, obtaining the structural domain amino acid sequence of human IgG1-Fc (hFc) according to the constant region amino acid sequence (P01857) of human immunoglobulin gamma1 (IgG 1) on the protein database Uniprot (i.e. 104 th residue to 330 th residue in P01857);

S1.3, obtaining DNA fragments in the steps S1.1-S1.2 by means of artificial synthesis, subcloning the synthesized gene sequences into a commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20) through double enzyme digestion of HindIII and EcoRI of Fermentas company, and sequencing to verify the accuracy of constructing plasmids to obtain recombinant plasmid DNA, namely pcDNA4-hFAP-hFc;

S1.4, obtaining an enhanced green fluorescent protein EGFP amino acid sequence (C5 MKY 7), a human FAP amino acid sequence (Q12884), a mouse FAP amino acid sequence (P97321) and a monkey FAP amino acid sequence (A0A 7N9D0S 5) according to the information on a protein database Uniprot;

S1.5, obtaining the DNA fragment in the step S1.4 by an artificial synthesis mode, subcloning the synthesized gene sequence into a commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20) through double enzyme digestion of HindIII and EcoRI of Fermentas company, sequencing, verifying the accuracy of constructed plasmids, and obtaining recombinant plasmid DNA, namely pcDNA4-hFAP-EGFP, pcDNA4-mFAP-EGFP and pcDNA4-cynoFAP-EGFP.

S1.6, the EGFP recombinant plasmid of step S1.5 was transfected into HEK293 (ATCC, CRL-1573 ^TM) cells, and hFAP, mFAP, cynoFAP expression was confirmed by fluorescence activated signal sorting (FACS) 48h after transfection.

S1.7, pcDNA4-hFAP-Fc was transiently transfected into HEK293 cells for protein production. Diluting recombinant expression plasmids with Freestyle293 culture medium, adding PEI (Polyethylenimine) solution required for transformation, adding each group of plasmid/PEI mixture into cell suspension, placing in 37 ℃ and 10% CO ₂ and 90rpm for culture, collecting transient expression culture supernatant after 5-6 days of culture, and primarily purifying by a ProteinA affinity chromatography to obtain hFAP-Fc protein samples, which are used in the following examples.

EXAMPLE 2 screening of anti-FAP antibodies

HFAP-Fc was immunized against alpaca once for two weeks, 0.25mg each time, four times total immunization, finally two times of alpaca peripheral blood taking, alpaca peripheral blood lymphocytes were isolated, total RNA was extracted, the extracted RNA was reverse transcribed into cDNA, the gene of the alpaca heavy chain antibody variable region (VHH) was amplified by RT-PCR, the gene was cloned into phage vector, and E.coli competent cells were electrically transformed to construct phage display nanobody library against hFAP-Fc. And finally obtaining the anti-hFAP antibody, 329-VHH through carrying out panning and screening on the library by hFAP-Fc.

EXAMPLE 3 humanization of anti-FAP antibodies

NG and NHS on CDR2 and CDR3 of 329-VHH are deamidation and glycosylation sites, firstly, mutating the sites into NA and QHS respectively to obtain 329-VHH-NA-QHS, and then humanizing the sequences to obtain VHH1-5;

Affinity was detected by biological membrane interferometry (BLI), antibodies were immobilized to AHC biosensor (Sartorius, lot: 2201027311) at a final concentration of 1ug/ml, and antigens (FAP-his Acro, cat: FAP-H5244) were diluted to different concentrations (100 nM, 5 gradients 2-fold dilution) and the results are shown in FIGS. 1A and 1B.

EXAMPLE 4 binding of anti-FAP antibodies to human, mouse, cyno FAP-EGFP cells after humanization

HEK293 cells expressing hFAP-EGFP, mFAP-EGFP and cynoFAP-EGFP constructed in example 1 were resuspended in 0.5% PBS-BSA Buffer, and anti-hFAP mAb protein was added and incubated on ice for 20min. After washing, the eBioscience secondary antibody anti-hIg-PE was added and the mixture was kept on ice for 20min. After washing, the cells were resuspended in 500. Mu.l of 0.5% PBS-BSA Buffer and examined by flow cytometry. As a result, as shown in FIG. 2, the three antibodies (VHH-1, VHH-1 and VHH-1) each bound to hFAP-EGFP, mFAP-EGFP and cynoFAP-EGFP cells.

Claims (18)

1. An isolated antigen-binding protein comprising CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 13, and the CDR3 Contains the amino acid sequence of SEQ ID NO:14. 2. The isolated antigen-binding protein according to any one of claim 1, comprising a CDR1, a CDR2 and a CDR3 selected from any one of the following groups: 1) the CDR1 comprises the amino acid sequence of SEQ ID NO: 2, the CDR2 comprises the amino acid sequence of SEQ ID NO: 3, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 4; 2) The CDR1 comprises the amino acid sequence of SEQ ID NO:2, the CDR2 comprises the amino acid sequence of SEQ ID NO:6, and the CDR3 comprises the amino acid sequence of SEQ ID NO:7. 3. The isolated antigen-binding protein according to any one of claims 1-2, which comprises an antibody or an antigen-binding fragment thereof. 4. The isolated antigen-binding protein of claim 3, wherein the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab') ₂ , F(ab) ₂ , scFv, di-scFv and/or dAb. 5. The isolated antigen binding protein of any one of claims 3-4, wherein the antibody comprises a single domain antibody. 6. The isolated antigen-binding protein according to any one of claims 3 to 5, comprising a VHH comprising the amino acid sequence shown in SEQ ID NO: 15. 7. The isolated antigen-binding protein according to any one of claims 3 to 6, comprising a VHH comprising the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9 to 13. 8. The isolated antigen binding protein according to any one of claims 3 to 7, wherein the antibody is selected from one or more of the following groups: a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. 9. The isolated antigen binding protein according to any one of claims 1 to 8, which is capable of specifically binding to fibroblast activation protein (FAP). 10. An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-9. 11. A nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-9. 12. A vector comprising the nucleic acid molecule of claim 11. 13. A cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12. 14. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1 to 9, the immunoconjugate of claim 10, the nucleic acid molecule of claim 11, the vector of claim 12 and/or the cell of claim 13, and optionally a pharmaceutically acceptable carrier. 15. A method for preparing the isolated antigen binding protein of any one of claims 1-9, the method comprising culturing the cell of claim 13 under conditions such that the antigen binding protein is expressed. 16. Use of the isolated antigen-binding protein of any one of claims 1 to 9, the immunoconjugate of claim 10, the nucleic acid molecule of claim 11, the vector of claim 12, the cell of claim 13 and/or the pharmaceutical composition of claim 14 in the preparation of a medicament for preventing and/or treating a disease and/or condition. 17. Use according to claim 16, wherein the disease and/or disorder is caused or mediated by fibroblast activation protein. 18. The use according to any one of claims 16-17, wherein the disease and/or disorder comprises a tumor.