Antigen binding protein targeting FAP and application thereof

Abstract

The present application relates to an isolated antigen binding protein capable of specifically binding to a fibrocyte-activated protein (FAP). The invention also provides a preparation method and application of the separated antigen binding protein.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting FAP. Background Art

[0002] Fibroblast activation protein (FAP) is a membrane serine peptidase and a member of the type II serine protease family. It has dipeptidyl peptidase and collagenase activities and has dual functions in tumor growth. The proteolytic activity of FAP can enhance the invasiveness of tumor cells to the extracellular matrix and also promote tumor growth and proliferation. Due to its expression in many common cancers and limited expression in normal tissues, FAP is considered to be a promising antigen target for molecular imaging, diagnosis and treatment of various cancers.

[0003] Therefore, there is a need in the art to develop antibodies that can bind to FAP with high affinity. Summary of the invention

[0004] The present application provides an isolated antigen-binding protein targeting fibroblast activation protein (FAP), which has good specificity, high FAP affinity and stability.

[0005] On the one hand, the present application provides 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 comprises the amino acid sequence shown in SEQ ID NO: 14.

[0006] In certain embodiments, it comprises a CDR1, a CDR2 and a CDR3 selected from any one of the following groups:

[0007] 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;

[0008] 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.

[0009] In certain embodiments, it comprises an antibody or an antigen-binding fragment thereof.

[0010] In certain embodiments, the antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb.

[0011] In certain embodiments, the antibody comprises a single domain antibody.

[0012] In certain embodiments, it comprises a VHH comprising the amino acid sequence shown in SEQ ID NO:15.

[0013] In certain embodiments, it comprises a VHH comprising the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9-13.

[0014] In certain embodiments, 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.

[0015] In certain embodiments, it can specifically bind to fibroblast activation protein (FAP).

[0016] In another aspect, the present application provides an immunoconjugate comprising the isolated antigen-binding protein described in the present application.

[0017] In another aspect, the present application provides a nucleic acid molecule encoding the isolated antigen-binding protein described in the present application.

[0018] In another aspect, the present application provides a vector comprising the nucleic acid molecule described in the present application.

[0019] In another aspect, the present application provides a cell comprising the nucleic acid molecule described in the present application or the vector described in the present application.

[0020] On the other hand, the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein described in the present application, the immunoconjugate described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and / or the cell described in the present application, and optionally a pharmaceutically acceptable carrier.

[0021] On the other hand, the present application provides a method for preparing the isolated antigen-binding protein described in any one of the present applications, the method comprising culturing the cells described in the present application under conditions allowing the antigen-binding protein to be expressed.

[0022] On the other hand, the present application provides a use of the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the immunoconjugate described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application and / or the pharmaceutical composition described in the present application in the preparation of a drug, wherein the drug is used to prevent and / or treat a disease and / or a condition.

[0023] In certain embodiments, the disease and / or condition is caused or mediated by fibroblast activation protein.

[0024] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

[0026] FIG1 shows the affinity of the antibodies described in the present application to human FAP. Figure 1A and Figure 1B Displays antibody and antigen affinity test results.

[0027] Figure 2 Shown are the FACS detection results of the antibodies described in this application and FAP from different sources. DETAILED DESCRIPTION

[0028] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0029] Definition of terms

[0030] In the present application, the term "Fibroblast activation protein (FAP)", also known as "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 cells. 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 can be Q12884 in UniProt accession number. For example, the genBank accession number of human FAP is AAC 51668. For example, the antigen-binding protein of the present invention can bind to the extracellular domain of FAP.

[0031] In this application, the term "isolated" generally refers to an artificially obtained substance from a natural state. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0032] In the present application, the term "isolated antigen-binding protein" generally refers to a protein with antigen binding ability that has been separated from its naturally occurring state. The "isolated antigen-binding protein" may include a portion that binds to an antigen and, optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the antigen-binding portion to bind to an antigen. The antigen-binding protein may include, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or an artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions that include mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic framework regions that include, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 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, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgG1 antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.

[0033] In this application, the term "CDR", also known as "complementarity determining region", generally refers to a region in an antibody variable domain whose sequence is highly variable and / or forms a structurally defined loop. Typically, an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camel antibodies consisting only of heavy chains can function normally and stably in the absence of light chains. See, for example, 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, Kabat / Chothia, etc. These coding systems are known in the art, and specific references include, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnumFor example, the amino acid sequence of the antigen binding protein can be numbered according to the 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., 2000 Nucleic 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, Dev Comp Immunol 29:185-203). For example, the CDR of the antigen-binding protein can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The boundaries of the CDR of the variable region of the same antibody obtained based on different coding systems may be different. That is, the CDR sequences of the variable region of the same antibody defined under different coding systems are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers such antibodies, whose variable region sequences contain the specific CDR sequences, but due to the application of different schemes (such as different coding systems), the CDR boundaries claimed are different from the specific CDR boundaries defined in the present invention. The CDR of the antibody of the present invention can be manually evaluated and determined according to any scheme or combination thereof in the art. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways.

[0034] In this application, the term "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy chain and / or light chain. The variable domains of the heavy chain and light chain may be referred to as "V H ” and “V L” (or “VH” and “VL”, respectively). These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.

[0035] In this application, the term "variable" generally refers to certain segments of the variable domain between antibodies that may have large differences in sequence. The variable domain mediates antigen binding and determines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domain. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light chain and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, most of which adopt a β-folded configuration, connected by three CDRs, which form a ring connection and form a part of the β-folded structure in some cases. The CDRs in each chain are kept together in close proximity by the FR region, 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)).

[0036] In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, covering any polypeptide including an antigen binding site, whether it is 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, mutated and transplanted antibodies. Unless otherwise modified by the term "complete", as in "complete antibody", for the purposes of the present invention, 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., specific binding to FAP). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units with an additional polypeptide called J chain and contain 10 antigen binding sites, while IgA antibodies include 2-5 basic 4-chain units that can be combined with J chains to form multivalent combinations. For IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds depending on the isotype of the H chain. 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 α and γ chains, and four CH domains for the μ and ε isotypes. 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 (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. VH and VL pair together to 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. The L chain from any vertebrate species can be divided into one of two distinct types based on the amino acid sequence of its constant domain, called κ and λ. According to the amino acid sequence of the heavy chain (CH) constant domain, immunoglobulins can be divided into different classes or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains named α, δ, ε, γ and μ, respectively.

[0037] In the present application, the term "antigen binding fragment" generally refers to one or more fragments that have the ability to specifically bind to an antigen (e.g., FAP). In the present application, the antigen binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0038] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, a whole antibody can be digested with papain. After papain digestion of an antibody, two identical antigen-binding fragments are produced, namely, a "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, as above). The Fab fragment can be composed of a complete L chain with the variable region of a heavy chain and the H chain (V H )'s first constant region (C H 1) Composition.

[0039] In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is slightly larger than a Fab fragment. For example, a Fab' fragment may include all of the light chain, all of the heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0040] 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 a disulfide bond and a portion of the hinge region. The F(ab')2 fragment has a divalent antigen binding activity and is capable of cross-linking antigens.

[0041] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the heavy chain variable region and the light chain variable region, and lacking the heavy chain constant region and the light chain constant region. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, an Fv fragment includes all or part of the amino terminal variable regions of about 110 amino acids of the heavy chain and the light chain.

[0042] In the present application, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment including a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker), and can be expressed in a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used in the present application, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0043] 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, with reference to, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), with reference to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and with reference to, for example, WO 06 / 030220, WO 06 / 003388, and other published patent applications of Domantis Ltd. The term "dAb" generally includes sdAb. The term "sdAb" generally refers to a single-domain antibody. A single-domain antibody generally refers to an antibody fragment consisting only of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL).

[0044] In the present application, the term "VHH" generally refers to the variable antigen binding domains of heavy chain antibodies from Camelidae (camel, dromedary, llama, alpaca, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH may also be referred to as Nanobody (Nb).

[0045] In the present application, the term "monoclonal antibody" generally refers to an antibody molecule preparation of a single molecule composition. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (generally having different antibodies for different determinants), each monoclonal antibody is for a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins. The modifier "monoclonal" represents the characteristics of an antibody obtained from a substantially homogeneous antibody population, and is not to be construed as requiring the production of antibodies by any ad hoc method. For example, the monoclonal antibodies used in the present application can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.

[0046] In this 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 region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is less likely to induce an adverse immune response in a human individual than the parent (e.g., alpaca-derived) antibody.

[0047] In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., an alpaca antibody) are replaced by corresponding amino acids derived from human immunoglobulins. In the CDR region, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permitted, as long as they still retain the ability of the antibody to bind to a specific antigen. Humanized antibodies may optionally contain at least a portion of a human immunoglobulin constant region. "Humanized antibodies" retain antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., alpaca) antibody may minimally contain a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as alpaca, mouse, rat, rabbit or non-human primate) having desired properties, affinity and / or ability. In some cases, FR region residues of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity.

[0048] The term "fully human antibody" generally refers to an antibody that contains 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, a fully human antibody may contain mouse sugar chains. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be generated in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are described in U.S. Patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as the use of libraries, are known in the art.

[0049] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.

[0050] In the present application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries are expressed in the host cell. For example, the vector may include: plasmid; phagemid; cosmid; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. The types of animal viruses used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). 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 initiation site. A vector may also include components that assist it in entering cells, such as viral particles, liposomes, or protein coats, but is not limited to these substances.

[0051] In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. Cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original parent cell (in the form of total DNA complement or in the genome). Cells may include cells transfected in vitro with the vectors described in the present application. Cells may be bacterial cells (e.g., Escherichia 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.

[0052] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating (e.g., covalently linked via a linker molecule) other agents (e.g., chemotherapeutic agents, radioactive elements, cytostatic agents and cytotoxic agents) to the antibody or its antigen-binding fragment, which can deliver the other agents to target cells (e.g., tumor cells) through the antibody or its antigen-binding fragment specifically binding to antigens on target cells.

[0053] In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may include the separated antigen-binding proteins described herein, the nucleic acid molecules described herein, the carriers described herein and / or the cells described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also include a suitable formulation of one or more (pharmaceutically effective) 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 dosage and concentration used. The pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0054] In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers may 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 counterions, such as sodium; and / or nonionic surfactants.

[0055] In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which 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 to a target (which may be an epitope) may be an antibody that binds to the target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other targets. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In certain embodiments, specific binding may include but does not require exclusive binding.

[0056] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat or monkey.

[0057] In the present application, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs having the same or similar functions as the proteins or polypeptides.

[0058] 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 to FAP). For example, the functional variant may comprise a protein or polypeptide that has been subjected to amino acid changes by at least 1, for example, 1-30, 1-20 or 1-10, for example, 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 polypeptide before the change (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 ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0059] In the present application, the homolog can be a protein or polypeptide having at least about 85% (for example, 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 with the amino acid sequence of the protein and / or the polypeptide (for example, an antibody or a fragment thereof that specifically binds to FAP).

[0060] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences. " sequence homology percentage ratio " can be calculated in the following manner: two sequences to be compared are compared in a comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number of positions in two sequences to obtain the number of matching positions, the number of matching positions is divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce sequence homology percentage ratio.Comparison carried out in order to determine the sequence homology percentage ratio can be realized 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. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and DJ. Lipman, "Improved tools for biological sequence comparisons," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and DJ. Lipman and W. R. Earson, "Rapid and sensitive protein similarity searches," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers and D. Lipman, "A basic local alignment search tool," Journal of Molecular Biology, 215: 403-410, 1990.

[0061] In this application, the term "comprising" generally means including, encompassing, containing or encompassing. In some cases, it also means "for", "consisting of...".

[0062] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 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 a specified value. DETAILED DESCRIPTION OF THE INVENTION

[0064] Isolated antigen binding protein

[0065] The CDR of an antibody is also called the complementary determining region, which is a part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. The antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, Kabat / Chothia, etc. These coding systems are known in the art, and specific references can be made to, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In the present application, the CDR covers the CDR sequence obtained by any CDR division method; it also covers its variants, which include the amino acid sequence of the CDR being substituted, deleted and / or added with one or more amino acids. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs may be amino acid sequences having at least about 85% (e.g., 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 with the amino acid sequence of the CDR.

[0066] In one aspect, the present application provides an isolated antigen-binding protein comprising a HCDR3. In some cases, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 14. In some cases, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 4. In some cases, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 7.

[0067] In the present application, the isolated antigen-binding protein may further comprise HCDR2. In some cases, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 13. In some cases, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3. In some cases, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6.

[0068] In the present application, the isolated antigen-binding protein may further comprise HCDR1. In certain cases, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:2.

[0069] 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 of SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 13, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 14.

[0070] In the present application, the isolated antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:2, the HCDR2 comprises the amino acid sequence of SEQ ID NO:3, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:4.

[0071] In the present application, the isolated antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:2, the HCDR2 comprises the amino acid sequence of SEQ ID NO:6, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:7.

[0072] In the present application, the isolated antigen-binding protein may comprise at least one CDR in the amino acid sequence 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, and the CDR may include CDRs obtained by division in any manner. CDRs delineated in any manner, if the sequence is the same as 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, all fall within the protection scope of the claims of the present application.

[0073] In the present application, the isolated antigen binding protein may comprise VHH.

[0074] In some cases, the CDR3 of the VHH may comprise the amino acid sequence shown in SEQ ID NO:14, the CDR2 of the VHH may comprise the amino acid sequence shown in SEQ ID NO:13, and the CDR1 of the VHH may comprise the amino acid sequence shown in SEQ ID NO:2.

[0075] In some cases, the CDR3 of the VHH may comprise the amino acid sequence shown in SEQ ID NO:4, the CDR2 of the VHH may comprise the amino acid sequence shown in SEQ ID NO:3, and the CDR1 of the VHH may comprise the amino acid sequence shown in SEQ ID NO:2.

[0076] In some cases, the CDR3 of the VHH may comprise the amino acid sequence shown in SEQ ID NO:7, the CDR2 of the VHH may comprise the amino acid sequence shown in SEQ ID NO:6, and the CDR1 of the VHH may comprise the amino acid sequence shown in SEQ ID NO:2.

[0077] In the present application, the isolated antigen-binding protein may comprise a 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 shown in any one of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9-13.

[0078] In the present application, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region 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.

[0079] In the present application, the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.

[0080] In certain embodiments, the antigen binding fragment may include Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb.

[0081] In certain embodiments, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0082] In addition, it should be noted that the antigen-binding proteins separated as described herein may include heavy and / or light chain sequences with which there are one or more conservative sequence modifications. The so-called "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antigen-binding proteins separated as described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are amino acid residues that are replaced with amino acid residues with similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with 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), β-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 region of the isolated antigen-binding protein described herein can be replaced with other amino acid residues of the same side chain group. Those skilled in the art know that some conservative sequence modifications will not eliminate antigen binding. For details, see, for example, 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. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0083] The FAP antigen binding proteins described herein can be identified, screened, or characterized by various assays known in the art.

[0084] For example, the antigen binding activity of the antigen binding protein or fusion protein of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0085] In the present application, the isolated antigen-binding protein can specifically bind to FAP. In certain embodiments, the binding of the isolated antigen-binding protein to FAP can be detected by an ELISA method. For example, the antigen-binding protein described in the present application can be 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 EC 50 Value combined with FAP.

[0086] In the present application, the isolated antigen binding protein can prevent and / or treat a disease and / or condition. In certain embodiments, the disease and / or condition can be caused or mediated by the expression of FAP. In certain embodiments, the disease and / or condition can include a tumor.

[0087] Nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions

[0088] On the other hand, the present application provides an isolated nucleic acid molecule that can encode an isolated antigen-binding protein described herein. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification; (ii) produced by cloning and recombination; (iii) purified, such as by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesized, such as by chemical synthesis.

[0089] On the other hand, the present application provides a vector, which may include the nucleic acid molecules described in the present application. In addition, the vector may also include other genes, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions. In addition, the vector may also include expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The vector may be expressed in the host cell by transforming, transducing, or transfecting the host cell. The vector may include, for example, plasmids, cosmids, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may also include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not only these substances.

[0090] On the other hand, the application provides a kind of cell, it can comprise the nucleic acid molecule described in the application or the vector described in the application.In some embodiments, each or each host cell can comprise one or a nucleic acid molecule or vector described in the application.In some embodiments, each or each host cell can comprise multiple (for example, 2 or more) or multiple (for example, 2 or more) nucleic acid molecules or vectors described in the application.For example, the vector described in the application can be introduced into the host cell, such as eukaryotic cells, such as cells from plants, fungi or yeast cells, etc.In some embodiments, the cell can be a bacterial cell (for example, Escherichia coli), a yeast cell or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NS0 cells or myeloma cells.The vector described in the application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0091] On the other hand, the present application also provides an immunoconjugate, which may comprise the isolated antigen-binding protein described in the present application.

[0092] In certain embodiments, the antigen binding protein or fragment thereof separated as described herein can be connected with another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, a spectroscopic probe, etc. The connection can be through one or more covalent bonds, or non-covalent interactions, and can include chelation. A variety of joints (the joints can be known in the art) can be used to form an immunoconjugate. In addition, an immunoconjugate can be provided in the form of a fusion protein, which can be expressed by a polynucleotide encoding an immunoconjugate. The immunoconjugate can also include, for example, an antibody-drug conjugate (ADC). Suitable drugs can include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA crosslinking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADC, antibodies and therapeutic agents can be cross-linked by joints, which can be cut, such as peptide joints, disulfide joints, or hydrazone joints.

[0093] On the other hand, the present application also provides a pharmaceutical composition, which may contain the isolated antigen-binding protein described in the present application, the immunoconjugate described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and / or the cell described in the present application, and optionally a pharmaceutically acceptable carrier.

[0094] In certain embodiments, the pharmaceutical composition may also include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0095] In certain embodiments, the pharmaceutical composition may also contain more than one active compound, generally those with complementary activities 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.

[0096] In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe and non-toxic.

[0097] In certain embodiments, the pharmaceutical composition may include parenteral, percutaneous, intracavitary, 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 in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered uninterruptedly. 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 WO2015 / 036583.

[0098] Preparation method

[0099] In another aspect, the present application provides a method for preparing the isolated antigen-binding protein. The method may include culturing the host cell described in the present application under conditions that allow the antigen-binding protein to be expressed. For example, the method may be performed by using an appropriate culture medium, an appropriate temperature and a culture time, etc., which methods are known to those of ordinary skill in the art.

[0100] Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application. For example, animals can be immunized with linked or naturally occurring FAP or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used. In certain embodiments, the isolated antigen binding proteins for FAP can be screened and enriched by extracting peripheral blood lymphocytes from immune alpacas, extracting cell nucleic acid fragments and cloning them into vectors, and then screened and enriched by phage surface display systems.

[0101] Any suitable form of FAP can be used as an immunogen (antigen) to generate antibodies specific to FAP and screen the biological activity of the antibodies. For example, the stimulating immunogen can be a full-length FAP, including a natural homodimer, or a peptide containing a single / multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0102] Methods and uses

[0103] On the other hand, the present application 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 medicament for preventing and / or treating a disease and / or condition.

[0104] On the other hand, the present application also provides a method for preventing and / or treating a disease and / or condition, which may include administering the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described in the present application to a subject in need.

[0105] In the present application, the use can be carried out by different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0106] On the other hand, the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described in the present application can be used to prevent and / or treat diseases and / or disorders.

[0107] In the present application, the diseases and / or disorders may be caused or mediated by FAP expression.

[0108] In the present application, the diseases and / or disorders include tumors.

[0109] On the other hand, the present application also provides a method for detecting FAP in a sample, which comprises administering the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition.

[0110] In some cases, the method for detecting FAP in a sample can be an in vitro method. For example, the isolated antigen binding protein described in the present application is contacted with an in vitro sample to detect the presence and / or content of FAP in the sample. In some cases, the method for detecting FAP in a sample is for non-therapeutic purposes. In some cases, the method for detecting FAP in a sample is not a diagnostic method.

[0111] On the other hand, the present application also provides a reagent or kit for detecting FAP in a sample, which comprises the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition.

[0112] On the other hand, the present 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 preparing a kit for detecting the presence and / or content of FAP in a sample.

[0113] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods and uses of the present application, and are not intended to limit the scope of the present invention.

[0114] Example

[0115] Example 1 Expression of recombinant human FAP and preparation of related EGFP cells

[0116] S1.1. According to the amino acid sequence of human FAP (Q12884) on the protein database Uniprot, the amino acid sequence of the extracellular domain of human FAP (i.e., residues 26 to 760 in Q12884) was obtained;

[0117] S1.2. According to the amino acid sequence of the constant region of human immunoglobulin gamma1 (IgG1) (P01857) on the protein database Uniprot, the domain amino acid sequence of human IgG1-Fc (hFc) (i.e., residues 104 to 330 in P01857) was obtained;

[0118] S1.3. The DNA fragments in steps S1.1 to S1.2 were obtained by artificial synthesis. The synthesized gene sequences were double-digested with HindIII and EcoRI from Fermentas and subcloned into the commercial vector pcDNA4 / myc-HisA (Invitrogen, V863-20). The accuracy of the constructed plasmid was verified by sequencing to obtain the recombinant plasmid DNA, namely: pcDNA4-hFAP-hFc.

[0119] S1.4. The amino acid sequence of enhanced green fluorescent protein EGFP (C5MKY7), the amino acid sequence of human FAP (Q12884), the amino acid sequence of mouse FAP (P97321), and the amino acid sequence of monkey FAP (A0A7N9D0S5) were obtained according to the information on the protein database Uniprot.

[0120] S1.5. The DNA fragment in step S1.4 was obtained by artificial synthesis. The synthesized gene sequences were double-digested with HindIII and EcoRI of Fermentas and subcloned into the commercial vector pcDNA4 / myc-HisA (Invitrogen, V863-20). The accuracy of the constructed plasmid was verified by sequencing to obtain recombinant plasmid DNA, namely: pcDNA4-hFAP-EGFP, pcDNA4-mFAP-EGFP, and pcDNA4-cynoFAP-EGFP.

[0121] S1.6, transfect the EGFP recombinant plasmid in step S1.5 into HEK293 (ATCC, CRL-1573 TM ) cells, and the expression of hFAP, mFAP, and cynoFAP was confirmed by fluorescence activated ACS 48 h after transfection.

[0122] S1.7, transient transfection of pcDNA4-hFAP-Fc into HEK293 cells for protein production. The recombinant expression plasmid was diluted with Freestyle293 medium and the PEI (Polyethylenimine) solution required for transformation was added. Each group of plasmid / PEI mixture was added to the cell suspension and cultured at 37°C, 10% CO2, 90rpm; after 5-6 days of culture, the transient expression culture supernatant was collected and preliminarily purified by Protein A affinity chromatography to obtain hFAP-Fc protein samples for use in the following examples.

[0123] Example 2 Screening of anti-FAP antibodies

[0124] hFAP-Fc was used to immunize alpacas once every two weeks, with 0.25 mg each time, for a total of four immunizations. In the last two times, peripheral blood was collected from the alpacas, and peripheral blood lymphocytes were separated and total RNA was extracted. The extracted RNA was reverse transcribed into cDNA, and the variable region (VHH) gene of the alpaca heavy chain antibody was amplified by RT-PCR. The gene was cloned into a phage vector; the competent E. coli cells were electrotransformed to construct a phage display nanoantibody library against hFAP-Fc. The anti-hFAP antibody, 329-VHH, was finally obtained by panning and screening the library with hFAP-Fc.

[0125] Example 3 Humanization of anti-FAP antibody

[0126] The NG and NHS on CDR2 and CDR3 of 329-VHH are deamidation and glycosylation sites, which were first mutated to NA and QHS, respectively, to obtain 329-VHH-NA-QHS, and then humanized on this sequence to obtain VHH1-5;

[0127] The affinity was detected by biomembrane interferometry (BLI). The antibody was immobilized on AHCbiosensor (Sartorius, Lot: 2201027311) at a final concentration of 1ug / ml, and the antigen (FAP-his Acro, Cat: FAP-H5244) was diluted to different concentrations (100nM, 2-fold dilution in 5 gradients) for detection. The results are shown in Figure 1A and Figure 1B .

[0128] Example 4 Binding of humanized anti-FAP antibodies to human, mouse, and cyno FAP-EGFP cells

[0129] HEK293 cells expressing hFAP-EGFP, mFAP-EGFP and cynoFAP-EGFP constructed in Example 1 were resuspended in 0.5% PBS-BSA Buffer, anti-hFAP mAb protein was added, and incubated on ice for 20 min. After washing, eBioscience secondary antibody anti-hIg-PE was added and incubated on ice for 20 min. After washing, the cells were resuspended in 500 μl 0.5% PBS-BSA Buffer and detected by flow cytometry. The results are shown in Figure 2. Figure 2 As shown, the three antibodies (VHH-1, VHH-1, and VHH-1) can bind to hFAP-EGFP, mFAP-EGFP, and cynoFAP-EGFP cells.

Claims

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 according to 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.

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