Monoclonal antibody against fibroblast activation protein and its application

Abstract

The present invention relates to FAP antibodies, antigen-binding fragments and uses thereof, and specifically provides antibodies or antigen-binding fragments thereof targeting FAP, or variants thereof having at least 85% sequence identity with the antibodies or antigen-binding fragments thereof and retaining their FAP binding activity.

Description

Technical Field

[0001] The present invention relates to the fields of biology and medicine; more specifically, the present invention relates to an antibody specifically binding to FAP, an antigen-binding fragment and uses thereof. Background Art

[0002] Fibroblast activation protein (FAP) is a serine hydrolase that belongs to the type II serine protease family. FAP is expressed on the surface of tumor-associated fibroblasts (TAF) in more than 90% of epithelial tumors, including colon cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, etc. It can also be expressed in gastric cancer stroma, some bone and soft tissue sarcoma cells, and is an important type of interstitial cell in the tumor microenvironment. In addition to tumors, FAP is also expressed on the surface of damaged stroma in idiopathic pulmonary fibrosis, hepatic parenchymal cells in patients with chronic hepatitis C, and myocardial interstitial cells in myocardial fibrosis. The patent of this invention screened 29 mouse FAP monoclonal antibodies with new variable region sequences through phage display mouse immune antibody library technology and verified their binding activity. Summary of the invention

[0003] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof targeting FAP, or a variant thereof having at least 85% sequence identity with the antibody or the antigen-binding fragment thereof and retaining its FAP binding activity.

[0004] In one or more embodiments, the antibody comprises: three HCDRs of the heavy chain variable region shown in any singular integer sequence in SEQ ID NO: 1-58, and / or three LCDRs of the light chain variable region shown in any even integer sequence in SEQ ID NO: 1-58.

[0005] In one or more embodiments, the HCDR1 of the antibody comprises any one selected from the group consisting of SEQ ID NOs: 59-87, or a sequence having at least 85% sequence identity thereto.

[0006] In one or more embodiments, the HCDR2 of the antibody comprises any one selected from the group consisting of SEQ ID NOs: 88-116, or a sequence having at least 85% sequence identity thereto.

[0007] In one or more embodiments, the HCDR3 of the antibody comprises any one selected from the group consisting of SEQ ID NOs: 117-145, or a sequence having at least 85% sequence identity thereto.

[0008] In one or more embodiments, the LCDR1 of the antibody comprises any one selected from the group consisting of SEQ ID NOs: 146-174, or a sequence having at least 85% sequence identity thereto.

[0009] In one or more embodiments, the LCDR2 of the antibody comprises any one selected from the group consisting of SEQ ID NOs: 175-203, or a sequence having at least 85% sequence identity thereto.

[0010] In one or more embodiments, the LCDR3 of the antibody comprises any one selected from the group consisting of SEQ ID NOs: 204-232, or a sequence having at least 85% sequence identity thereto.

[0011] In one or more embodiments, the antibody contains HCDR1, HCDR2, HCDR3 of an antibody shown in any row of Table 12, or a sequence having at least 85% sequence identity thereto.

[0012] In one or more embodiments, the antibody comprises LCDR1, LCDR2 and LCDR3 of an antibody shown in any row of Table 12, or a sequence having at least 85% sequence identity thereto.

[0013] In one or more embodiments, the antibody contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of an antibody shown in any row of Table 12, or a sequence having at least 85% sequence identity thereto.

[0014] In one or more embodiments, the light chain variable region of the antibody comprises a murine or human light chain FR region. In one or more embodiments, the heavy chain variable region of the antibody comprises a murine or human heavy chain FR region.

[0015] In one or more embodiments, FR1, FR2, FR3 and FR4 of the heavy chain variable region of the antibody are each independently selected from FR1, FR2, FR3 and FR4 of the heavy chain variable region represented by any singular integer in SEQ ID NOs: 1-58, and / or, FR1, FR2, FR3 and FR4 of the light chain variable region of the antibody are each independently selected from FR1, FR2, FR3 and FR4 of the light chain variable region represented by any even integer in SEQ ID NOs: 1-58.

[0016] In one or more embodiments, the antibody comprises a VH, a VL, or a VH and a VL as shown in any one of the antibodies in Table 11, or a sequence having at least 85% sequence identity thereto.

[0017] In one or more embodiments, the VH of the antibody has a sequence as shown in any single integer of SEQ ID NO: 1-58, or a sequence having at least 85% sequence identity therewith. Alternatively or in addition, the VL of the antibody has a sequence as shown in any double integer of SEQ ID NO: 1-58, or a sequence having at least 85% sequence identity therewith.

[0018] In one or more embodiments, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0019] In one or more embodiments, the heavy chain of the antibody comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, or a sequence having at least 85% sequence identity therewith. Alternatively or in addition, the light chain of the antibody comprises a light chain constant region of a human κ chain, λ chain, or a sequence having at least 85% sequence identity therewith.

[0020] In one or more embodiments, the heavy chain of the antibody comprises the heavy chain constant region of human IgG1 or a sequence having at least 85% sequence identity thereto, and the light chain comprises the light chain constant region of human κ chain or a sequence having at least 85% sequence identity thereto.

[0021] In one or more embodiments, the antibody is a multispecific antibody, preferably a bispecific antibody.

[0022] In one or more embodiments, the antibody is a monoclonal antibody.

[0023] In one or more embodiments, the antibody is a chimeric antibody or a murine antibody.

[0024] The present invention also provides a fusion protein or antibody conjugate, which comprises the antibody or antigen-binding fragment thereof described herein. Preferably, the antibody conjugate is an antibody-drug conjugate (ADC).

[0025] The present invention also provides a polynucleotide selected from:

[0026] (1) The coding sequence of the antibody or antigen-binding fragment thereof, fusion protein, or antibody conjugate described in any embodiment of the present invention;

[0027] (2) The complementary sequence of (1).

[0028] The present invention also provides a nucleic acid construct, which comprises the polynucleotide described in any embodiment herein.

[0029] In one or more embodiments, the nucleic acid construct is a vector, such as an integration vector, a cloning vector, or an expression vector.

[0030] The present invention also provides a phage comprising the antibody or antigen-binding fragment thereof described in any embodiment of the present invention or a library comprising the phage.

[0031] In one or more embodiments, the antibody or antigen-binding fragment thereof is displayed on the surface of the bacteriophage.

[0032] The present invention also provides a host cell, which:

[0033] (1) expressing and / or secreting the antibody or antigen-binding fragment thereof, fusion protein, or antibody conjugate described in any embodiment of the present invention;

[0034] (2) comprising a polynucleotide as described herein; and / or

[0035] (3) comprising the nucleic acid construct described herein.

[0036] In one or more embodiments, the host cell is selected from a prokaryotic cell or a eukaryotic cell.

[0037] In one or more embodiments, the host cell is a mammalian cell.

[0038] The present invention also provides a method for producing an antibody or an antigen-binding fragment thereof, comprising: culturing the host cell described herein under conditions suitable for producing the antibody or the antigen-binding fragment thereof, and optionally purifying the antibody or the antigen-binding fragment thereof from the culture.

[0039] The present invention also provides a kit for detecting FAP, which is used for evaluating the effect of drug treatment or diagnosing cancer, and the kit comprises the antibody or its antigen-binding fragment, fusion protein, antibody conjugate, polynucleotide, nucleic acid construct, phage or host cell described in any embodiment of the present invention.

[0040] In one or more embodiments, the kit further comprises a reagent for detecting the binding of FAP to an antibody or an antigen-binding fragment thereof, a fusion protein, or an antibody conjugate, for example, a reagent for detecting the binding by enzyme-linked immunosorbent assay.

[0041] In one or more embodiments, the reagent for detecting binding is a detectable label, such as biotin, that can be attached to an antibody or its antigen-binding fragment, a fusion protein, or an antibody conjugate. The detectable label is attached to the antibody or its antigen-binding fragment or is separately present in the kit.

[0042] The present invention also provides a non-diagnostic method for detecting the presence of FAP in a sample, the method comprising: incubating the sample with the antibody or antigen-binding fragment thereof, fusion protein, or antibody conjugate described in any embodiment of the present invention, and detecting the binding of FAP to the antibody or antigen-binding fragment thereof, fusion protein, or antibody conjugate, thereby determining the presence of FAP in the sample. The detection is enzyme-linked immunosorbent assay.

[0043] The present invention also provides use of the antibody or antigen-binding fragment thereof, fusion protein, or antibody conjugate described in any embodiment herein in the preparation of a kit for detecting FAP in a sample, evaluating the effect of drug treatment, or diagnosing cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Mouse serum ELISA test results.

[0045] Figure 2 : The ability of serum to block positive antibodies binding to NCI / 3T3-MFAP.

[0046] Figure 3 :Flowchart for construction of mouse immune library.

[0047] Figure 4 :Simplified diagram of Fab library plasmid.

[0048] Figure 5 : Non-reducing SDS-PAGE image.

[0049] Figure 6 : Restored SDS-PAGE image.

[0050] Figure 7 : SEC image of A008 antibody molecule.

[0051] Figure 8 : SEC image of A018 antibody molecule.

[0052] Fig. 9 :SEC image of A019 antibody molecule.

[0053] Fig.10 :SEC image of A034 antibody molecule.

[0054] Fig.11 :SEC image of A039 antibody molecule.

[0055] Fig.12 :SEC image of A045 antibody molecule.

[0056] Fig.13 :SEC image of A057 antibody molecule.

[0057] Fig.14:SEC image of A066 antibody molecule.

[0058] Fig.15 :SEC image of A080 antibody molecule.

[0059] Fig.16 :SEC image of A092 antibody molecule.

[0060] Fig.17 : SEC image of A122 antibody molecule.

[0061] Fig.18 :SEC image of A128 antibody molecule.

[0062] Fig.19 :SEC image of A139 antibody molecule.

[0063] Fig. 20 :ELISA was used to detect the binding activity of monoclonal antibodies to His-human FAP antigen protein.

[0064] Fig.21 : ELISA was used to detect the binding activity of monoclonal antibodies to His-cyno FAP antigen protein.

[0065] Fig. 22 :ELISA was used to detect the binding activity of monoclonal antibodies to His-mouse FAP antigen protein.

[0066] Fig.23 : FACS results of the selected 14 molecules and NIH / 3T3-HFAP cells.

[0067] Fig.24 : FACS results of the selected 14 molecules and NIH / 3T3-MFAP cells. DETAILED DESCRIPTION

[0068] Unless otherwise defined, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the technical scope of the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., ed., 1987 edition and periodic updates thereof); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0069] The present invention screened 29 mouse FAP monoclonal antibodies with novel variable region sequences through phage display mouse immune antibody library technology and verified their binding activity, which has clinical transformation value in the development of immunotherapy for fibroblasts.

[0070] As used herein, a "FAP antibody" is a protein that specifically binds to FAP, including but not limited to antibodies, antigen-binding fragments of antibodies, nanobodies, minibodies, affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

[0071] Herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2 and Fv. Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0072] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of 5 basic heterotetrameric units and an additional polypeptide called J chain, containing 10 antigen binding sites, while IgA antibodies contain 2-5 basic 4-chain units, which can be combined with J chains to form multivalent assemblies. In the case of IgG, the 4-chain unit is usually about 150,000 daltons. Each light chain is connected to the heavy chain by one covalent disulfide bond, while the two heavy chains are connected to each other by one or more disulfide bonds, the number of which depends on the isotype of the heavy chain. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH1, CH2 and CH3 for each α and γ chain) and four (CH1, CH2, CH3 and CH4 for μ and ε isotypes) constant domains (CH) and a hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. VL is aligned with VH, while CL is aligned with 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. Pairs of VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw, eds., Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be assigned to one of two distinct types, called 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 their heavy chain constant domains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes can be further divided into subclasses, for example humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as the participation of antibodies in antibody-dependent cell-mediated cytotoxicity.

[0073] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "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.

[0074] The term "variable" refers to the situation where certain segments in the variable domain vary widely in the antibody sequence. The variable domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, the variability is not evenly distributed across all the amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (in both the light chain and heavy chain variable domains), namely HCDR1, HCDR2, HCDR3 of the heavy chain variable domain and LCDR1, LCDR2 and LCDR3 of the light chain variable domain. 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 (FR1, FR2, FR3 and FR4), most of which adopt a β-sheet conformation and are connected by three HVRs that form a loop connection and in some cases form part of the β-sheet structure. The HVRs in each chain are kept together very closely by the FR regions and contribute to the formation of the antigen binding site of the antibody together with the HVRs of the other chain. Typically, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. For chimeric antibodies, the FR region can be non-human (e.g., mouse). Antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, comprehensive consideration of Kabat / Chothia, etc. These coding systems are known in the art, and can be specifically found, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence numbering of the antigen-binding protein can be based on the IMGT numbering scheme.

[0075] In the present invention, antibodies include murinized variants.

[0076] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, the CH2 domain and the CH3 domain from the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

[0077] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding region and / or variable region of an intact antibody. Antibody fragments are preferably antigen binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragments that should be able to increase half-life by chemical modification or by incorporation into liposomes. Digestion of antibodies with papain produces two identical antigen binding fragments called "Fab" fragments, and a residual "Fc" fragment, the name reflecting its ability to crystallize easily. Fab fragments consist of a complete light chain and heavy chain variable domain (VH) and one heavy chain first constant domain (CH1). Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of antibodies produces a larger F(ab')2 fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen binding activities and still able to cross-link antigen. Fab' fragments differ from Fab fragments by the addition of a few additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment contains the carboxyl terminal portions of the two heavy chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain types of cells.

[0078] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. From the folding of these two domains protrude six hypervariable loops (3 loops each from the heavy and light chains) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with less affinity than the complete binding site. "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the antibody VH and VL domains connected into one polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains so that the sFv forms the desired antigen binding structure.

[0079] Herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, except for possible natural mutations and / or post-translational modifications (such as isomerization, amidation) that may exist in a small amount, each antibody constituting the group is identical.Monoclonal antibodies are highly specific for a single antigenic site.Compared with polyclonal antibody preparations (which typically include different antibodies for different determinants (epitopes)), each monoclonal antibody is for a single determinant on the antigen.Except for their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture without being polluted by other immunoglobulins.The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody group and should not be interpreted as requiring the production of antibodies by any ad hoc method.For example, the monoclonal antibody used according to the present invention can be generated by a variety of techniques, including, for example, hybridoma method, phage display method, recombinant DNA method, and technology, single cell sequencing method for generating human or human-like antibodies from animals with part or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences.

[0080] Monoclonal antibodies herein also include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0081] A "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally contains sequences derived from non-human immunoglobulins. Thus, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions are exchanged with sequences found in human antibodies. Typically in a humanized antibody, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDRs). The CDRs (some or all of which are encoded by nucleic acids derived from non-human organisms) are transplanted into the β-folded framework of the variable region of a human antibody to produce an antibody whose specificity is determined by the transplanted CDRs. Methods for producing such antibodies are well known in the art, for example, using the humanization platform hu-mab (Claire Marks, Alissa M Hummer, Mark Chin, Charlotte M Deane. Humanization of antibodies using a machine learning approach on large-scale repertoire data, Bioinformatics (2021)) or BioPhi (David Prihoda, Jad Maamary, Andrew Waight, Veronica Juan, Laurence Fayadat-Dilman, Daniel Svozil & Danny A. Bitton (2022) BioPhi: A platform for antibody design, humanization, and humanness evaluation based on natural antibody repertoires and deep learning, mAbs, 14:1, DOI: https: / / doi.org / 10.1080 / 19420862.2021.2020203) to humanize existing antibodies, and using mice with genetically engineered immune systems to produce antibodies. In the present invention, antibodies include variants of each of the murine antibodies.

[0082] "Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibody explicitly excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries.

[0083] "Mouse antibodies" refer to spleen cells obtained from hyperimmune donors, i.e. antigen-immunized mice, which are then fused with myeloma cells and cloned to produce cloned cells that secrete monoclonal antibodies. The antibodies used are extracted from mice.

[0084] In one or more embodiments, the antibody described herein comprises: three HCDRs of the heavy chain variable region shown in any singular integer sequence in SEQ ID NO: 1-58, and / or three LCDRs of the light chain variable region shown in any even integer sequence in SEQ ID NO: 1-58.

[0085] In one or more embodiments, the HCDR1 of the antibody comprises any one selected from the group consisting of SEQ ID NO: 59-87, or a sequence having at least 85% sequence identity therewith. In one or more embodiments, the HCDR2 of the antibody comprises any one selected from the group consisting of SEQ ID NO: 88-116, or a sequence having at least 85% sequence identity therewith. In one or more embodiments, the HCDR3 of the antibody comprises any one selected from the group consisting of SEQ ID NO: 117-145, or a sequence having at least 85% sequence identity therewith. In one or more embodiments, the LCDR1 of the antibody comprises any one selected from the group consisting of SEQ ID NO: 146-174, or a sequence having at least 85% sequence identity therewith. In one or more embodiments, the LCDR2 of the antibody comprises any one selected from the group consisting of SEQ ID NO: 175-203, or a sequence having at least 85% sequence identity therewith. In one or more embodiments, the LCDR3 of the antibody comprises any one selected from the group consisting of SEQ ID NO: 204-232, or a sequence having at least 85% sequence identity therewith. In one or more embodiments, the antibody contains HCDR1, HCDR2, HCDR3 of an antibody shown in any row of Table 12, or a sequence having at least 85% sequence identity thereto. In one or more embodiments, the antibody contains LCDR1, LCDR2 and LCDR3 of an antibody shown in any row of Table 12, or a sequence having at least 85% sequence identity thereto.

[0086] In one or more embodiments, FR1, FR2, FR3 and FR4 of the heavy chain variable region of the antibody are each independently selected from FR1, FR2, FR3 and FR4 of the heavy chain variable region shown in any singular integer sequence of SEQ ID NO: 1-58, and / or, FR1, FR2, FR3 and FR4 of the light chain variable region of the antibody are each independently selected from FR1, FR2, FR3 and FR4 of the light chain variable region shown in any even integer sequence of SEQ ID NO: 1-58.

[0087] In one or more embodiments, the antibody comprises a VH, a VL, or a VH and a VL as shown in any one of the antibodies in Table 11, or a sequence having at least 85% sequence identity thereto.

[0088] In one or more embodiments, the VH of the antibody has a sequence as shown in any single integer of SEQ ID NO: 1-58, or a sequence having at least 85% sequence identity therewith. Alternatively or in addition, the VL of the antibody has a sequence as shown in any double integer of SEQ ID NO: 1-58, or a sequence having at least 85% sequence identity therewith.

[0089] The anti-FAP antibodies described herein may be monovalent or multivalent antibodies, multispecific antibodies comprising the anti-FAP antibody fragments described herein.

[0090] In one or more embodiments, the antibodies described herein further include a heavy chain constant region and / or a light chain constant region, the heavy chain constant region, such as a heavy chain constant region derived from IgA, IgD, IgE, IgG and IgM, or a heavy chain constant region derived from IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2, or a sequence thereof having at least 85% sequence identity, and a light chain constant region, such as a light chain constant region of a κ or λ chain, or a sequence thereof having at least 85% sequence identity.

[0091] The present invention also includes the antibody derivatives and analogs. "Derivatives" and "analogs" refer to polypeptides that substantially maintain the same biological function or activity as the antibodies of the present invention. The derivatives or analogs of the present invention may be (i) polypeptides having a substitution group in one or more amino acid residues, or (ii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these derivatives and analogs are within the scope known to those skilled in the art.

[0092] Without substantially affecting the activity of the antibody, a person skilled in the art may change one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein. For example, amino acids with similar properties are substituted in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For another example, adding one or more amino acids to the C-terminus and / or N-terminus will generally not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.

[0093] Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention.

[0094] In some embodiments, the sequence of the variant of the present invention may have at least 95%, 96%, 97%, 98% or 99% identity to its source sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters is used. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0095] The antibodies of the present invention can be prepared by conventional methods in the art, such as hybridoma technology well known in the art. The antibodies of the present invention can be prepared by conventional methods in the art, such as phage display technology well known in the art. Alternatively, the antibodies of the present invention or fragments thereof or antibody conjugates can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the present invention. Transformation can be carried out by any known method, for example, including packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial FAP binding properties.

[0096] Fusion Protein

[0097] The present application also provides a fusion protein, which comprises the antibody or antigen-binding fragment described in the present application as an expression target, an active molecule or a targeting molecule. Herein, "expression target" means that the fusion protein is an expression that needs to be prepared, such as a fusion protein formed by adding tags (such as His6 tags) at both ends of the antibody that facilitate protein expression or purification. The tag does not affect the function of the target protein and can be easily removed. Herein, "active molecule" refers to a molecule that exerts the final function of the fusion protein (such as neutralizing antigen). Herein, "targeting molecule" refers to a molecule that mediates the targeting, recognition, or binding of the fusion protein to the target.

[0098] Antibody Conjugates

[0099] The present application also provides an antibody conjugate, which comprises the antibody or antigen-binding fragment described in the present application. In the present application, the term "antibody conjugate" generally refers to a conjugate formed by conjugating the other agents (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors and cytotoxic agents) to the antibody or its antigen-binding fragment (e.g., covalently linked by a linker molecule), and the conjugate can be delivered to the target cell (e.g., tumor cell) by specifically binding the antibody or its antigen-binding fragment to the antigen on the target cell. The present invention also provides the use of the antibody described herein in the preparation of an immunoconjugate.

[0100] In certain embodiments, the antibody or antigen-binding fragment thereof described herein may be linked to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, a spectroscopic probe, etc. The link may be through one or more covalent bonds, or non-covalent interactions, and may include chelation. A variety of linkers (the linkers may be known in the art) may be used to form an antibody conjugate. In addition, the antibody conjugate may be provided in the form of a fusion protein, which may be expressed by a polynucleotide encoding the antibody conjugate. The antibody conjugate may also include, 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 anti-mitotic agents. In ADC, the antibody and therapeutic agent may be cross-linked through a linker that may be cleavable, such as a peptide linker, a disulfide linker, or a hydrazone linker.

[0101] Polynucleotide

[0102] The present invention also provides polynucleotides encoding the antibodies or antigen-binding fragments thereof, fusion proteins or antibody conjugates described herein. Polynucleotides encoding heavy chain variable regions, light chain variable regions, heavy chains, light chains and each CDR are provided herein. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded. The present invention also includes degenerate variants of polynucleotide sequences encoding antibodies or antigen-binding fragments thereof, fusion proteins or antibody conjugates, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

[0103] As those skilled in the art will understand, DNA is usually a double-stranded structure, with a sense strand and an antisense strand that complement each other. Among them, the sense strand, also known as the sense strand, refers to the strand with genetic information in the DNA double strand, which cannot be transcribed, and contains the same nucleotide sequence as the mRNA encoding the functional protein, with the only difference that the T in DNA is U in the mRNA. The antisense strand, also known as the template strand, refers to the DNA strand used by RNA polymerase during the transcription process, serving as a template for transcription, and the RNA polymerase moves along the template strand and transcribes it into mRNA, which is then translated into a polypeptide by the mRNA.

[0104] In a narrow sense, "polynucleotide sequence encoding..." or "coding sequence of..." refers to a sequence that is present on the antisense strand of DNA and can directly guide transcription, or a sequence that is present on mRNA and can directly guide translation. A polynucleotide may contain a coding sequence (or coding region) and a non-coding sequence (or non-coding region, such as introns). The coding sequence may be continuous or discontinuous, and discontinuous coding sequence fragments may be separated by non-coding sequences. The complete coding region obtained by sequentially connecting the coding sequence fragments is the coding sequence of the polypeptide.

[0105] According to the amino acid sequence and codons, those skilled in the art can easily obtain the coding sequence of the antibody or its fragment. The expression of the polypeptide in different species can also be changed by codon optimization in this field, and the codon preference of different species belongs to the routine technology in this field. Exemplarily, the sequences of the polynucleotides encoding the heavy chain variable region and the light chain variable region of the A004 antibody are shown in SEQ ID NO: 233 and SEQ ID NO: 234.

[0106] As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be produced, all of which encode the FAP antibodies of the present invention. Therefore, once a specific amino acid sequence has been identified, one skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence encoding the protein. Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0107] The full-length nucleotide sequence of the anti-FAP antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0108] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the host cells after proliferation by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0109] Therefore, the present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vector usually contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) usually include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, a replication origin, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multilinker region for inserting a nucleic acid encoding an anti-FAP antibody to be expressed, and a selectable marker element.

[0110] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0111] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0112] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0113] The polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0114] use

[0115] The inventors discovered, expressed and purified antibodies that can bind to FAP protein, and verified the ability of these antibodies to bind to antigens and cells through protein-level affinity testing.

[0116] Therefore, all aspects of the antibody conjugates described herein can be used to prepare drugs for preventing or treating various conditions and diseases described herein, especially diseases or conditions associated with cells expressing FAP. The antibody can be used as a targeting molecule that interacts with a target, or as an active molecule that kills cells. In some embodiments, the condition and disease are FAP-related diseases. Herein, FAP-related diseases include cancer, idiopathic pulmonary fibrosis, chronic hepatitis C, myocardial fibrosis, etc. Preferably, cancer includes colon cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, etc.

[0117] The present invention also provides the use of antibody conjugates as active ingredients for preparing pharmaceutical compositions, the pharmaceutical compositions comprising antibody conjugates and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants. Excipients are preferably non-toxic to the recipient at the dose and concentration used. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, the pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. These substances are known in the prior art. The optimal pharmaceutical composition can be determined based on the expected route of administration, delivery method, and required dosage.

[0118] Pharmaceutical compositions for in vivo administration are usually provided in the form of sterile preparations. Sterilization is achieved by filtering through a sterile filtration membrane. When the composition is freeze-dried, this method can be used for sterilization before or after freeze-drying and rehydration. The pharmaceutical composition of the present invention can be selected for parenteral delivery. The composition for parenteral administration can be stored in a freeze-dried form or in a solution. For example, it is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually placed in a container with a sterile access hole, such as an intravenous solution strip or a vial with a stopper that can be pierced by a hypodermic injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as oral). The preparation of the pharmaceutically acceptable composition is within the technology of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibody conjugates in sustained or controlled release delivery formulations. Technologies for preparing a variety of other sustained or controlled delivery methods (such as liposome carriers, bioerodible microparticles or porous beads and accumulated injections) are also known to those skilled in the art.

[0119] Once the pharmaceutical composition is formulated, it is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or in the form of a dehydrated or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form of rehydration before administration (e.g., lyophilization). The present invention also provides a kit for producing a single-dose administration unit. The kit of the present invention can each contain a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[0120] The present invention also provides a method for treating a patient (especially a FAP-related disease in a patient) by administering an antibody conjugate or a pharmaceutical composition thereof according to any embodiment of the present invention. As used herein, the terms "patient," "subject," "individual," and "subject" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., rats, mice, dogs, cats, rabbits, etc.), and most preferably a human. "Treatment" refers to the use of a treatment regimen described herein to a subject to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth). A treatment regimen that effectively treats a patient may vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the therapy to stimulate an anti-cancer response in the subject.

[0121] The therapeutically effective amount of the pharmaceutical composition containing the antibody conjugate of the present invention to be used will depend on, for example, the degree of treatment and the target. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will vary depending in part on the delivered molecule, indication, route of administration, and size (body weight, body surface or organ size) and / or condition (age and general health) of the patient. In certain embodiments, the clinician can titrate the dosage and change the route of administration to obtain the best therapeutic effect. For example, about 10 micrograms / kg body weight-about 50 milligrams / kg body weight per day.

[0122] The frequency of administration will depend on the pharmacokinetic parameters of the antibody conjugate in the formulation used. The clinician typically administers the composition until a dose is achieved to achieve the desired effect. The composition can therefore be administered as a single dose, or administered over time as two or more doses (which may or may not contain the same amount of the desired molecule), or administered by continuous infusion via an implantable device or catheter.

[0123] The route of administration of the pharmaceutical composition is according to known methods, for example, orally, by injection via intravenous, intraperitoneal, intracerebral (intracerebroparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional routes; by sustained release systems or by implant devices.

[0124] The present invention also provides the use of the anti-FAP antibodies described herein in the preparation of immune effector cells. The immune effector cells express anti-FAP antibodies or polypeptide molecules containing anti-FAP antibodies. In one or more embodiments, the anti-FAP antibodies are located on the surface of the immune effector cells. In one or more embodiments, the polypeptide molecule is a transmembrane molecule that transmits a signal targeting FAP to the cell, such as CAR or TCR.

[0125] Diagnostics, Tests and Kits

[0126] The antibodies of the present invention can be used in assays, such as binding assays, to detect and / or quantify FAP expressed in tissues or cells due to their high affinity for FAP. The antibodies can be used in studies to further study the role of FAP in disease. The method for detecting FAP is generally as follows: obtaining a cell and / or tissue sample; detecting the level of FAP in the sample.

[0127] The anti-FAP antibodies of the present invention can be used for diagnostic purposes to detect, diagnose or monitor diseases and / or conditions associated with FAP. The present invention provides for detecting the presence of FAP in a sample using classical immunohistological methods known to those skilled in the art. Detection of FAP can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of FAP include ELISA, FACS, RIA, etc.

[0128] For diagnostic applications, the antibody is typically labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: a radioisotope or radionuclide (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), a fluorescent group (e.g., FITC, rhodamine, lanthanide phosphors), an enzymatic group (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), a chemiluminescent group, a biotinyl group, or a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag), an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent. Various methods for labeling proteins are known in the art and can be used to perform the present invention.

[0129] Another aspect of the invention provides a method for detecting the presence of a test molecule that competes with the anti-FAP antibodies of the invention for binding to FAP. An example of such an assay would involve detecting the amount of free antibody in a solution containing a certain amount of FAP in the presence or absence of a test molecule. An increase in the amount of free anti-FAP antibody (i.e., antibody that is not bound to FAP) would indicate that the test molecule can compete with the antibody for binding to FAP. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of the antibody.

[0130] The present invention also provides a detection kit for detecting FAP levels, which includes an anti-FAP antibody, a lysis medium for dissolving a sample, and common reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0131] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, materials and methods conventional in the art.

[0132] Example

[0133] Example 1, mouse immunization

[0134] Through multiple immunizations with specific immunogens (proteins, cells or peptides, etc.), antibodies against specific immunogens are produced in mice. Mouse spleens are obtained for the construction of mouse immune libraries.

[0135] 1.1 Experimental Materials

[0136] 1.1.1 Reagents and consumables

[0137] Immune adjuvant (purchased from Sigma, F5881), phosphate buffered saline (20×PBS) (purchased from Biotechnology), FACS buffer (500ml PBS + 10ml FBS (2%) + 0.02% Na Azide), skimmed milk powder (purchased from Yili), 96 half-well plates (purchased from Corning), 96-well round-bottom culture plates (domestic), 2M stop solution (2 mol / L H2SO4), Tween-20 / Tween 20 (purchased from Sinopharm).

[0138] 1.1.2 Instruments and equipment

[0139] Amalgamator (purchased from Amalgamator), microplate reader (purchased from Molecular Devices, Max 190), cell flow cytometer (purchased from Backman, CytoFLEX), and processing software (FlowJo_V10).

[0140] 1.1.3 Experimental animals

[0141] Female Balb / c mice aged 6-8 weeks were purchased from Vital River and kept in an SPF animal room.

[0142] 1.1.4 Sample information

[0143]

[0144] 1.2 Experimental methods

[0145] 1.2.1 Mouse immunization method

[0146] Protein immunization: The first immunization antigen dose is 100 μg / mouse, with Freund's complete adjuvant; the subsequent immunization antigen dose is 50 μg / mouse with Freund's incomplete adjuvant, and the injection method is intraperitoneal and subcutaneous multiple injections. Cell immunization: The first immunization cell dose is 1×10 7 The subsequent immune cell volume was 5×10 6 The injection method was intraperitoneal injection. The mice were divided into 6 groups by different immunization methods. The immunization process of each group of mice is shown in Table 1.

[0147] Table 1: Mouse immunization process

[0148]

[0149] Note: hFAP protein indicates Human FAP-hFc protein, mFAP protein indicates Mouse FAP-His protein, hFAP cells indicate Human FAP-NIH / 3T3 stable cell line, and mFAP cells indicate Mouse FAP-NIH / 3T3 stable cell line.

[0150] 1.2.2 ELISA serum titer detection method

[0151] The plate was coated with His-Human FAP and His-Mouse FAP antigens and incubated overnight at 4°C; the blocking solution was used for blocking at room temperature for 2 hours; the serum was diluted 3 times and incubated at room temperature for 1 hour; the corresponding secondary antibody was added and incubated at room temperature for 1 hour, then the color was developed and terminated, and the OD value of each well was measured at a wavelength of 450 nm using an enzyme marker. The mouse serum titer was calibrated according to 1.65 times NC, and the titer was greater than 40,000, which was qualified.

[0152] 1.2.3 FACS detection method

[0153] Serum blocking antibody and cell binding experiment: NIH / 3T3-MFAP cells and positive control antibodies were incubated for 1 h; serum diluted 4 times in a gradient dilution was added and incubated at 4°C for 60 min; secondary antibody was added and incubated at 4°C for 30 min; MFI value was detected by flow cytometry. Whether the serum has the ability to block the binding of the positive control antibody to NIH / 3T3-MFAP was determined based on whether the serum has a blocking effect.

[0154] 1.3 Experimental Results

[0155] 1.3.1 Evaluation of mouse immune ELISA test results: The serum ELISA test results of the 18 immunized mice are shown in Figure 1-Figure 2 and Table 2.

[0156] Table 2: Titers of mouse five immune sera

[0157]

[0158] 1.3.2 Evaluation of mouse immune FACS test results

[0159] The sera of 18 immunized mice can specifically block the binding of the positive control antibody to NIH / 3T3-MFAP cells. The specific data are shown in Table 3.

[0160] Table 3: Ability of serum to block positive antibodies binding to NIH / 3T3-MFAP

[0161]

[0162] 1.4 Experimental Summary

[0163] A total of 18 Balb / C mice were immunized by protein single immunization, protein cross immunization, cell single immunization, and protein cell cross immunization. The results of ELISA and FACS serum titer tests showed that the mouse immune titer was qualified. The mice with qualified immunization can be used in the next step to construct a mouse immune antibody library.

[0164] Example 2: Construction of mouse immune library

[0165] Twelve mouse immune antibody libraries were constructed to screen monoclonal antibodies that bind to the target antigen. RNA from the spleen tissue of immunized mice was extracted and further reverse transcribed into cDNA to obtain all antibody nucleotide sequences. The antibody nucleotide sequences were then constructed into phage display vectors through molecular cloning technology, and the constructed vectors were further transformed into Escherichia coli by electroporation to obtain the mouse immune phage display library.

[0166] 2.1 Experimental Materials

[0167] 2.1.1 Reagents and consumables

[0168] Ultrapure water, anhydrous ethanol (purchased from Bio-Tech), competent cells, 2YT medium, 50×TAE, 1.5 mL centrifuge tubes (purchased from Axygen), and 96-well PCR plates (purchased from Bio-Tech).

[0169] 2.1.2 Instruments and equipment

[0170] Electrophoresis instrument (purchased from Liuyi Biotechnology, DYCP-32B), automatic gel image analyzer (purchased from Peiqing, JS-680D), electrotransfer instrument (purchased from BIO-RAD, MicroPulser), PCR instrument (purchased from Long Gene, A200).

[0171] 2.2 Experimental methods

[0172] The RNA of spleen cells of immunized mice was extracted and further reverse transcribed into cDNA to obtain all antibody nucleotide sequences. The light and heavy chain genes were amplified by molecular cloning technology. The antibody genes were recombined by in vitro ligation. The vector and Fab gene sequences were digested separately. The antibody nucleotide sequence was constructed into a phage display vector. The constructed vector was further transformed into Escherichia coli by electroporation to obtain a mouse immune phage display library. The flowchart of mouse immune library construction is shown in the figure. Figure 3 shown.

[0173] 2.3 Experimental Results

[0174] 2.3.1 Fab library capacity determination and sequence analysis

[0175] The 12 mouse immune antibody libraries were numbered L2230, L2231, L2232, L2233, L2234, L2235, L2236, L2237, L2238, L2239, L2240, and L2241, and the quality of the antibody libraries was identified. The library capacity of the 12 Fab libraries was determined by the dilution point plate method, and the correct insertion rate of the antibody gene was verified by monoclonal sequencing analysis. The measurement results of the Fab library constructed in this experiment are shown in Table 4. The library capacity is greater than the designed library capacity, and the correct insertion rate of the 12 libraries is greater than 80%, which meets the expected requirements.

[0176] Table 4: Fab library monoclonal sequencing analysis

[0177]

[0178] 2.3.2 Detection of antibody gene integrity in Fab antibody library

[0179] See the schematic diagram of Fab antibody library construction vector for Figure 4 After the library is built, the region between ApaL I and Not I is the full-length sequence of the antibody Fab.

[0180] 2.3.3 Analysis of the provenance of inserted antibody genes

[0181] The sequenced antibody sequences were further analyzed using the NCBI website to check whether the inserted antibody sequences were mouse antibodies, check the CDR3 of the light and heavy chains, and confirm the number of specific sequences. According to the sequence analysis results of the 12 antibody libraries, the inserted antibody sequences were all mouse antibodies.

[0182] 2.4 Experimental Summary

[0183] RNA was extracted from spleen cells of 18 mice and reverse transcribed into cDNA. The identification results were qualified and could be used for the next step of library construction. 12 mouse immune antibody libraries were constructed by grouped primer amplification method. The dilution spot plate method results showed that the library capacity of the 12 antibody libraries was greater than the designed library capacity. The sequencing analysis results showed that the correct insertion rate was greater than 80%, and the high richness could be reflected from CDR3. The quality of this library construction was qualified and could be used for subsequent screening.

[0184] Example 3: Screening of mouse immune library

[0185] The target antigen is used to screen mouse antibody molecules against the target from the mouse immune library. The sequence of the constructed mouse immune library antibody library is displayed on the phage surface through phage display technology, and then specific proteins, cells or peptides are used as antigens. After multiple rounds of screening, the phage-displayed antibodies that bind to the antigen are continuously enriched, and positive clones that specifically bind to the antigen are further screened through ELISA, and the positive clone antibody sequences are obtained through sequencing.

[0186] 3.1 Experimental Materials

[0187] 3.1.1 Reagents and consumables

[0188] Tween 20 (Sinopharm), glycerol (Sinopharm), 96-well half-well plate (Corning), 96-well round-bottom culture plate (domestic), 96-well square-hole deep-well plate (Biscellide), disposable sample loading chamber (Huaxiong), ELISA blocking solution (1% BSA), bovine serum albumin (Sinopharm), 2 M stop solution (2 mol / L H2SO4).

[0189] 3.1.2 Instruments and equipment

[0190] Ultra-clean workbench (purchased from Antai), plate washer (purchased from Molecular Devices, AquaMax2000), and microplate reader (purchased from Molecular Devices, SpectraMax190).

[0191] 3.3.3 Experimental samples

[0192]

[0193] 3.2 Experimental methods

[0194] 3.2.1 Phage preparation: The mouse immune library or the obtained output collection is subjected to a series of steps including inoculation, helper phage infection, phage expansion, phage precipitation and resuspension to prepare enriched phages for the next round of screening.

[0195] 3.2.2 Liquid phase screening: The biotin-labeled antigen is combined with the streptavidin-coupled magnetic beads, then incubated with the prepared phage, washed and eluted. After three rounds of screening, the specific monoclonal antibodies against the antigen are enriched.

[0196] 3.2.3 Solid phase screening: The antigen is coated on the surface of an immunotube with high adsorption capacity, and then the prepared phage is added to the immunotube for incubation, washing and elution. After three rounds of screening, the specific monoclonal antibodies against the antigen are enriched.

[0197] 3.2.4 Cell screening: The prepared negatively screened phages and antigen-overexpressing cells are incubated, washed and eluted. After three rounds of screening, the specific monoclonal antibodies against the antigen are enriched.

[0198] 3.2.5 Screening set detection: ELISA experiment is performed on the screening output set to detect the enrichment effect. After a series of steps such as coating, blocking, incubation, adding secondary antibody, color development, termination, and detection of OD value, a standard curve is generated by serial dilution of known standards to quantify the Fab expression supernatant, and the expression supernatant concentration is used as the horizontal axis and the OD value is used as the vertical axis to draw a graph, and the negative value is used as a reference to evaluate the enrichment degree of the pool.

[0199] 3.2.6 Monoclonal screening: The target antigen is coated on the plate, and a series of steps are followed, including coating, blocking, incubation, adding secondary antibodies, color development, termination, and OD value detection. Positive clones are identified according to a certain background value and sent for testing.

[0200] 3.3 Experimental Results

[0201] 3.3.1 Results of monoclonal screening

[0202] Monoclonal clones were selected from the enriched pool for primary screening, and the screening was performed using the His-Human FAP, His-Mouse FAP, and His-Cyno FAP antigen coating method. In the first primary screening, 604 clones from 7 plates were selected, and 147 positive clones binding to the His-Human FAP antigen, 148 positive clones binding to the His-Cyno FAP antigen, and 32 positive clones binding to the His-Mouse FAP antigen were obtained. 146 positive clones with cross-binding activity to human and monkey antigens were selected for sequencing analysis, and 32 molecules with unique sequences were obtained. In the second primary screening, 618 clones from 7 plates were selected, and 291 positive clones binding to the His-Human FAP antigen, 304 positive clones binding to the His-Cyno FAP antigen, and 101 positive clones binding to the His-Cyno FAP antigen were obtained. 98 positive clones with cross-binding activity to human, mouse, and monkey were selected and sequenced, and 5 new molecules with unique sequences were added.

[0203] 3.3.2 Full-length build information

[0204] According to the affinity ranking results of positive clones and sequence diversity analysis, a total of 37 molecules with unique sequences were obtained, and 29 molecules were selected for full-length construction. The unconstructed molecules contained post-translational modification sites or had low diversity, so no full-length construction was arranged.

[0205] 3.4 Experimental Summary

[0206] This experiment used solid phase, liquid phase crossover and cell screening to screen the constructed mouse immune library and obtain multiple sets of well-enriched output sets. A total of 1222 monoclonal clones from 14 plates were selected through ELISA screening, and 37 molecules with unique sequences were obtained. 29 of them were selected for full-length construction. Molecules containing post-translational modification sites or poor molecular diversity were not selected, and full-length construction was not performed. The number of molecules screened reached the expected goal, and full-length construction and eukaryotic level verification can be performed.

[0207] Example 4: Preparation and detection of monoclonal antibodies

[0208] Prepare target proteins that meet the requirements. Construct the target gene sequence into the pcDNA3.4 vector, use Expi CHO cells for transient expression, and perform concentration and activity tests after 2.5 days of expression in a small volume of 1 mL. Perform protein purification after 7 days of expression in 10 mL. Perform physical, chemical and biochemical tests on the purified proteins.

[0209] 4.1 Experimental Materials

[0210] 4.1.1 Reagents and consumables

[0211] Precast gel (purchased from GenScript), Xbridge BEH200Å (purchased from Waters), ProA probe (purchased from GATOR), Goat-Anti-Human-IgG-Fc-HRP (purchased from Abcam), TMB (purchased from SurModics), 96-well half-well plate (purchased from Corning), Anti-Human IgG, Fcγ(PE) (purchased from Jackson), Anti-Human IgG, Fcγ(PE) (purchased from Abcam).

[0212] 4.1.2 Instruments and equipment

[0213] Micro-spectrophotometer (purchased from Hangzhou Aosheng), eStain L1 protein stainer (purchased from GenScript), protein electrophoresis tank (purchased from BioRAD), HPLC (purchased from Agilent), label-free biomolecule analyzer (purchased from Gator Probe LIFE), microplate reader (purchased from Molecular Devices), flow cytometer (purchased from Beckman).

[0214] 4.2 Experimental methods

[0215] 4.2.1 PCR amplification of target gene

[0216] The PCR reaction system was prepared as follows:

[0217]

[0218] Place the PCR tube in the PCR instrument and perform the PCR reaction according to the following procedure:

[0219]

[0220] 4.2.2 DNA fragment recovery

[0221] The enzyme digestion products and PCR products were purified by agarose gel electrophoresis and gel recovery.

[0222] 4.2.3 Homologous recombination and transformation

[0223] The amount of vector used in a single reaction is 20-50 ng, and the molar ratio of vector to insert is 1:3. The required amount of insert is calculated. Take 5 µL of the recombinant product and add it to 50 µL of competent cells, flick the tube wall to mix, let it stand on ice for 5 min, heat shock in a 42℃ water bath for 60 s, immediately put it on ice for 3 min, and let it stand at room temperature for 3 min. In the clean bench, pour 6-8 sterilized coating beads on the surface of each plate solid culture medium, drop the competent cells after standing at room temperature on the coating beads, and oscillate horizontally so that the coating beads evenly coat the bacterial solution on the surface of the culture medium. After pouring out the coating beads, put the plate in a plastic bag without sealing, place it in a 37℃ incubator, and invert it for about 16 h. The positive clones are picked and identified and sequenced to verify, and the corresponding plasmid DNA is prepared for cell transfection. All 29 antibody molecules use the pcDNA3.4 vector, and the heavy chain type is IgG1. The sequences of the heavy chain constant region and the light chain constant region of the antibody are shown in SEQ ID NO:235 and SEQ ID NO:236.

[0224] 4.2.4 Cell transfection

[0225] Prepare well-grown Expi CHO cells. One day before transfection, add 3.00 × 10 6 cells / mL - 4.00×10 6 Cells were passaged at a density of 10 cells / mL and cultured overnight. Cell counts were performed on the day of transfection, and the cell density was controlled to be approximately 7.00×10 6 cells / mL - 10.00×10 6 cells / mL, and the viability was guaranteed to be >98%. Preheat Expi CHOExpression Medium at 37°C and adjust the cell density to 6.00×10 6 cells / mL, determine the cell amount based on the expression volume requirements.

[0226] Pipette 400 µL of OptiPRO SFM into a 1.5 ml EP tube, add 10 µg of plasmid DNA, and mix by inverting 10-15 times. Pipette 400 µL of OptiPRO SFM into a new 1.5 mL EP tube, add 32 µL of ExpressiFectamine CHO Reagent, and mix by inverting 4-5 times. Add the diluted transfection reagent to the diluted plasmid, and immediately mix by gently inverting 6-10 times. Incubate the transfection reagent / plasmid DNA mixture at room temperature for 1-5 min, then add it dropwise to the prepared cell suspension, gently shaking the cell suspension while adding. Incubate the transfected cells in a 37°C shaker with 7% CO2. Add feed on the first day after transfection (18 h-22 h), gently shake the cell suspension while adding, and transfer the shake flask to a shaker with 7% CO2 to continue incubation. On the fifth day after transfection, feed was added, and the cell suspension was gently shaken while adding, and the shake flask was returned to the shaker at 7% CO2 for continued culture. On the 2nd, 5th, and 7th day after transfection, different volumes of transfected cells were extracted in the order of expression numbers to measure cell density and viability. Samples were collected when the viable cell density was not less than 50% of the highest cell density and the cell viability was not less than 60%.

[0227] 4.2.5 Protein purification

[0228] Preparation: Fill an empty Thermo centrifugal column (Pierce Centrifuge Columns, 10 mL) with 2 mL of ProteinA / G filler, centrifuge at 100×g for 10 s or remove the preservation solution by gravity, and wash with more than 10 mL of deionized water and CIP buffer, respectively, and finally wash the CIP buffer with deionized water (CV=2 mL). Equilibration: Add 10-15 mL of Binding Buffer (20 mM Arg-Ac, pH 7.0), pipette to mix, add 10 mL of Binding Buffer again, and equilibrate again. Sample loading: Collect the cell expression supernatant by centrifugation at 15,000×g for 20 min at 4℃, add the supernatant after centrifugation to the centrifugal column, let it flow through the filler by gravity, and collect the flow-through. Washing: After loading, add more than 10 mL of Wash Buffer I (20 mM Arg-Ac, 500 mM NaCl, pH 7.0) and Wash Buffer II (20 mM Arg-Ac, pH 7.0) in sequence, and let it flow through the packing by gravity by standing, and remove the supernatant remaining in the packing and non-specifically bound proteins. Elution: Add 10 mL of Elution Buffer (100 mM Pro-Ac, pH3.3), let it flow through the packing by gravity by standing, and collect the eluate. Neutralization: After elution, adjust the pH of the sample to 5.0 with 0.5 M Ar. In-place cleaning: Rinse the column with 5-8 CV of deionized water, and then rinse the column with 5-8 CV of CIP buffer; after completion, rinse the residual buffer with 5-8 CV of deionized water. Storage: Rinse the column with 5-8 CV of storage solution, remove the residual deionized water, blow and mix, and store the column at 4°C. Concentration and replacement: Add the neutralized protein solution to the ultrafiltration tube, use a horizontal rotor, and centrifuge at 3500×g for 10 min (can be extended or shortened appropriately according to the concentration situation and needs). After the concentration is completed, discard the flow-through and record the volume of the retained solution; fill the ultrafiltration tube with 1×PBS, pH6.0, calculate the sample dilution factor, and centrifuge again several times until the sample's starting buffer is diluted more than 100 times. Quantitative packaging: Take 2 μL of the replaced sample and quantify it with Nano-300 (measure at least twice and take the average value), and calculate the theoretical concentration of the protein using the formula c=A280 / bε.

[0229] 4.3 Experimental Results

[0230] 4.3.1 SDS-PAGE identification

[0231] According to the concentration of the sample, 1 μg of the non-reduced sample was added to 4×SDS loading buffer, iodoacetamide (final concentration 40 mM), and heated in a 75°C dry bath for 10 min. 2 μg of the reduced sample was added to 4×SDS loading buffer, DTT (final concentration 5 mM), and heated in a 100°C dry bath for 10 min. SDS-PAGE was performed on the non-reduced sample and the reduced sample, respectively. Figure 5 and Figure 6 As shown; the purity of the reduced sample was calculated by Image J according to the peak area normalization method, and the purity of all molecules was greater than 90%.

[0232] 2.6.2 SEC Analysis

[0233] Mobile phase preparation: Prepare 0.15 M PBS and adjust the pH to 6.0. Sample treatment: Dilute the sample concentration to 0.5 mg / mL. Chromatographic column conditions: Use XBridge BEH 200Å, SEC 3.5 µm, 7.8×300 mm, set the column temperature to 25°C, and the detection baseline is stable. Parameter settings: Flow rate is set to 0.8 mL / min; sample injection volume is set to 20 μL; detection wavelength is 280 nm, bandwidth is 4 nm, reference wavelength is 360 nm, bandwidth is 100 nm, peak width (response time) is >0.1 min (2 s response time); slit is 4nm; negative absorbance baseline is 100 mAU. System suitability standards: The purity of the reference product Herceptin monomer is greater than 95%, the separation between BSA monomer and dimer is greater than 1.5, and the baseline is stable, then the system suitability is considered to have passed. SEC analysis was performed on the 13 expressed and purified molecules, and the results are as follows Figure 7-19 The retention time and purity results are summarized in Table 5.

[0234] Table 5: Monoclonal antibody SEC test results

[0235]

[0236] Example 5: Antibody affinity determination

[0237] 5.1 ELISA to detect the binding activity of monoclonal antibodies to His-human FAP, His-cyno FAP and His-mouse FAP antigen proteins

[0238] The antigen protein was diluted to a concentration of 2 μg / mL in carbonate buffer (pH 9.6) and coated on ELISA well plates (96-well plates), 30 μl per well, overnight at 4°C. The plate was washed 3 times with PBST (containing 0.1% Tween-20) solution, and blocked at room temperature for 2 h with 5% PBSM blocking solution (i.e., PBS solution containing 5% skim milk powder). The plate was washed 3 times with PBST, and the monoclonal antibody solution diluted in a gradient manner with 1% PBSM (i.e., PBS solution containing 1% skim milk powder) was added at 30 μL / well, and incubated at room temperature for 60 minutes. The plate was washed 3 times with PBST, and the Goat-Anti-human-IgG-Fc-HRP secondary antibody diluted 1:8000 was added, 30 μl per well, and incubated at room temperature for 50 minutes. The plate was washed 6 times with PBST, and 30 μL TMB was added to each well for color development. After reacting at room temperature for 30 minutes, 2 M stop solution was added to terminate the reaction, and OD450 was detected.

[0239] The results of ELISA binding activity test of 29 monoclonal molecules with His-human FAP, His-cyno FAP and His-mouse FAP antigen proteins are shown in the figure. Figure 20-22 Among them, the binding activity of A008, A018, A019, A020, A034, A045, A066, A080, A087, A098, A101, A122, and A128 molecules to His-human FAP antigen was better than that of the control antibody; the binding activity of A004, A010, A014, A036, and A124 molecules to His-human FAP antigen was equivalent to that of the control antibody. The binding activity of A008, A018, A019, A020, A034, A039, A045, A066, A080, A087, A122, and A128 molecules to His-mouse FAP antigen was better than that of the control antibody; the binding activity of A004, A010, A014, A036, A101, and A124 molecules to His-cyno FAP antigen was comparable to that of the control antibody. The EC50 values ​​of all monoclonal antibody molecules are shown in Table 6.

[0240] Table 6: EC50 values ​​of monoclonal antibody molecules detected by ELISA

[0241]

[0242] Note: NA means no binding.

[0243] 5.2 Binding activity of FAP-detected monoclonal antibodies to positive cell lines

[0244] Prepare cells: NIH / 3T3-HFAP cells and NIH / 3T3-MFAP cells with good growth status were digested and transferred to centrifuge tubes. The supernatant was removed by centrifugation, and the cell density was adjusted to 1×10 after resuspending and counting in culture medium. 6 cells / mL. Take a 96-well round-bottom plate and use a 100 μL pipette to add the cells to each well. Centrifuge at 300 g / min for 5 min and remove the supernatant.

[0245] Primary antibody: dilute the monoclonal antibody with FACS Buffer containing 2% FBS, with a total of 8 dilutions. Use a 100 μL 12-channel pipette to add the supernatant dilution to the 96-well cell plate, 100 μL per well, mix well, and incubate in a 4°C refrigerator for 1 h.

[0246] Secondary antibody: dilute the secondary antibody PE labelled anti-Human Fc (Jackson) 1:200 with FACS Buffer containing 2% FBS, centrifuge the cell culture plate to remove the supernatant, and use a 100 μL 12-channel pipette to add 100 μL of the secondary antibody dilution to the cell culture plate. Place the cell culture plate in a 4°C refrigerator and culture for 30 min. Centrifuge to remove the supernatant, wash the plate twice with FACS Buffer, and resuspend the cells in each well with 120 μL of FACS Buffer.

[0247] Data collection: Turn on the flow cytometer, and after the instrument is cleaned, perform FACS Binding detection. The binding data of 15 monoclonal antibody molecules to NIH / 3T3-HFAP cells and NIH / 3T3-MFAP cells are shown in Fig.23 and Fig.24 Among them, the binding ability of A008, A018, A039, A045, A057, A092, A128, and A139 molecules to NIH / 3T3-HFAP cells was stronger than that of the control antibody, and the ability of A080 and A122 molecules to bind to NIH / 3T3-HFAP cells was comparable to that of the control antibody. The EC50 values ​​of all monoclonal antibody molecules binding to cells are shown in Table 7 below.

[0248] Table 7: EC50 values ​​of monoclonal antibody molecules binding to target cells detected by FACS

[0249]

[0250] Note: NA means no binding.

[0251] 5.3 FAP antibody affinity determination

[0252] Preferably, 12 molecules are used for affinity determination of His-Human FAP, His-Cyno FAP, and His-Mouse FAP, respectively, and VH-VL is a positive control antibody.

[0253] Detection instrument: GATOR (ProbeLife)

[0254] Probe: ProA (LN 2212061 T5), Human antibody Capture

[0255] Conclusion: R2>0.95, the system adaptability passed, and the data is true. NA means no dissociation in 180s. N / A means no binding.

[0256] Table 8: Affinity determination with His-Human FAP protein

[0257]

[0258] Note: KD represents the binding dissociation constant; Ka represents the binding constant; Kd represents the dissociation constant; R2 represents the linear fitting constant; Rmax represents the maximum response value of the curve fitting.

[0259] Table 9: Affinity determination with His-Cyno FAP protein

[0260]

[0261] Note: KD represents the binding dissociation constant; Ka represents the binding constant; Kd represents the dissociation constant; R 2 represents the linear fitting constant; Rmax represents the maximum response value of the curve fitting.

[0262] Table 10: Affinity determination with His-Mouse FAP protein

[0263]

[0264] Note: KD represents the binding dissociation constant; Ka represents the binding constant; Kd represents the dissociation constant; R2 represents the linear fitting constant; Rmax represents the maximum response value of the curve fitting.

[0265] Table 11: Antibody heavy chain variable region (VH) and light chain variable region (VL) sequences and numbers

[0266]

[0267]

[0268]

[0269] Table 12: Antibody heavy chain complementary pairing region (HCDR) and light chain complementary pairing region (LCDR) sequences and numbers

[0270]

[0271]

[0272]

[0273]

[0274] Table 13. Nucleotide sequences and numbers of antibody heavy chain variable regions (VH) and light chain variable regions (VL)

[0275]

Claims

1. An antibody or antigen-binding fragment thereof targeting FAP, characterized in that: The antibody contains HCDR1 shown in SEQ ID NO:85, HCDR2 shown in SEQ ID NO:114, HCDR3 shown in SEQ ID NO:143, LCDR1 shown in SEQ ID NO:172, LCDR2 shown in SEQ ID NO:201 and LCDR3 shown in SEQ ID NO:

230.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: FR1, FR2, FR3 and FR4 of the heavy chain variable region of the antibody are each independently selected from FR1, FR2, FR3 and FR4 of the heavy chain variable region shown in SEQ ID NO:53, and / or, FR1, FR2, FR3 and FR4 of the light chain variable region of the antibody are each independently selected from FR1, FR2, FR3 and FR4 of the light chain variable region shown in SEQ ID NO:

54.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein: The antibody contains VH shown in SEQ ID NO:53 and VL shown in SEQ ID NO:

54.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein: The antibody further comprises a heavy chain constant region and / or a light chain constant region, and / or The antibody is a multispecific antibody, and / or The antibody is a chimeric antibody or a fully human antibody.

5. A fusion protein comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A polynucleotide selected from: (1) the coding sequence of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the fusion protein according to claim 5, (2) The complementary sequence of (1). A nucleic acid construct comprising the polynucleotide according to claim 6.

8. The nucleic acid construct according to claim 7, wherein The nucleic acid construct is a vector.

9. The nucleic acid construct according to claim 8, wherein The nucleic acid construct is an integration vector, a cloning vector or an expression vector.

10. A host cell, which: (1) containing, expressing and / or secreting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the fusion protein according to claim 5; (2) comprising the polynucleotide according to claim 6; and / or (3) A nucleic acid construct comprising any one of claims 7 to 9.

11. The host cell according to claim 10, characterized in that The host cell is selected from a prokaryotic cell or a eukaryotic cell.

12. The host cell according to claim 11, characterized in that The host cell is a mammalian cell.

13. A method for producing an antibody or an antigen-binding fragment thereof, comprising: Cultivating the host cell of any one of claims 10 to 12 under conditions suitable for producing the antibody or antigen-binding fragment thereof, and optionally purifying the antibody or antigen-binding fragment thereof from the culture.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the fusion protein according to claim 5 in the preparation of a kit for detecting FAP in a sample.

15. The use according to claim 14, characterized in that The detection is an enzyme-linked immunosorbent assay.

16. A kit for detecting FAP, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the fusion protein according to claim 5, the polynucleotide according to claim 6, the nucleic acid construct according to any one of claims 7 to 9, and the host cell according to any one of claims 10 to 12.

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