Recombinant anti-FAP antibody and application thereof

Abstract

The invention relates to the field of antibody drugs, in particular to a recombinant anti-FAP antibody and application thereof. The invention provides a recombinant anti-FAP antibody and application thereof. The invention provides a recombinant anti-FAP antibody and application thereof. The recombinant anti-FAP antibody provided by the invention shows high specific binding and rapid and efficient internalization ability, and the high selectivity and high biological activity level of the recombinant anti-FAP antibody are helpful to become a monoclonal antibody for treatment or an antibody coupling drug for coupling small molecule toxins.

Description

[0001] This application claims the priority of a Chinese patent application with the application number 202410018607.6 and the invention title "Recombinant Anti-FAP Antibody and Its Application" filed with the National Intellectual Property Administration on January 5, 2024, the entire content of which is incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of antibody drugs, and specifically to recombinant anti-FAP antibodies and their applications. Background Art

[0003] Malignant epithelial tumors are the main cancer-related diseases causing human death. These solid tumors often exhibit a significant stromal response, such as what is called "desmoplastic stroma" or "reactive stroma", which accounts for 20 - 60% of the total tumor mass and is characterized by the presence of a large number of stromal cells and a dense extracellular matrix (ECM). A highly consistent molecular feature of the reactive stroma of many types of epithelial cancers is the induction of fibroblast activation protein α (hereinafter referred to as FAP). Specifically, a considerable number of cancer-associated fibroblasts (CAFs) are often observed in the tumor-associated stroma of various human cancers including breast cancer, lung cancer, colon cancer, and pancreatic cancer.

[0004] FAPα is a cell surface molecule of reactive stromal fibroblasts initially identified by the monoclonal antibody F19. It is a cell surface glycoprotein of reactive stromal fibroblasts and can serve as a potential antibody target in human epithelial cancers. In many types of human cancers, the fibroblast response is characterized by the induction of a cell surface protein, namely fibroblast activation protein α (FAPα), which is a 95 kDa serine protease whose expression is highly restricted to developing organs, wound healing, and tissue remodeling.

[0005] FAP exhibits the following characteristics:

[0006] 1) A type II membrane glycoprotein with serine protease activity

[0007] 2) 89% human-mouse protein homology, 99% human-monkey protein homology

[0008] 3) The tumor stroma is expressed in >90% of cancers (breast cancer, pancreatic cancer, lung cancer, bladder cancer, and colon cancer)

[0009] 4) Transient and highly restricted expression in normal adult tissues during wound healing and in developing organs

[0010] 5) Having a relatively fast internalization ability

[0011] 6) Involved in extracellular matrix remodeling, tumor growth, and metastasis.

[0012] In concert with distinct components of the stroma, CAFs can promote neo-angiogenesis and tumor growth; it has also been shown that CAFs are crucial for the development of aggressive tumors and tumor invasiveness during cancer progression; CAFs promote the spread and infiltration of tumor cells in distant organs, thereby contributing to the formation of metastases. Importantly, the correlation of stromal cells with the failure of systemic drug delivery to tumors and the emergence of drug resistance has also been indicated.

[0013] The identification of cellular and molecular targets that abrogate stroma-tumor cell interactions and thereby attenuate tumorigenesis is currently a major theme in translational oncology. Indeed, targeting the tumor stroma is a novel strategy for treating metastatic tumors that account for more than 90% of cancer patient mortality: to date, only a few products have received therapeutic approval, and most of them are anti-angiogenic drugs. The identification and targeting of other novel molecules within the tumor microenvironment are necessary to increase the efficacy of conventional therapies in combination with stroma-based treatment approaches and represent a promising approach for the treatment of cancer and metastases.

[0014] To alter the non-human immunogenicity of the monoclonal antibody F19 (from hybridoma cell line ATCC accession number HB 8269), the antibody F19 was humanized to sibrotuzumab (BIBH1), a humanized antibody that specifically binds FAP and is described in WO 99 / 57151. A phase I clinical study of BIBH1, i.e., I31 radiolabeled anti-FAP monoclonal antibody, demonstrated good safety and tolerability (Scott et al., 2001; Hofheinz et al., 2003), but in a phase II clinical study in patients with advanced metastatic colorectal cancer, most patients showed persistent tumor progression, and the study was aborted due to failure to meet the minimum requirements.

[0015] Boehringer-Ingelheim has developed anti-FAP MAb derivatives against both human and murine FAP proteins (US

[0016] 2009 / 0304718A1). They showed specific binding to FAP+ cells and were internalized in vitro. In pancreatic and lung cancer xenograft models, treatment with the murine MAb MFP5-DM1 immunotoxin induced long-lasting inhibition of tumor growth and complete regression without any associated effects of intolerance.

[0017] The anti-FAP antibody hu36 developed by ONCOMATRYX BIOPHARMA exhibits high specific binding and rapid internalization. In addition, the inventors isolated the A chain of nigrin b, which is produced in bacterial host cells, and conjugated it to the anti-FAP antibody for tumor treatment. It showed an effect of inhibiting tumor growth in various in vitro tumor models.

[0018] Despite these advancements, there remains an unmet need for other treatment strategies for treating, including epithelial tumors, and for components used in such treatment strategies. SUMMARY OF THE INVENTION

[0019] In view of this, the present invention provides a recombinant anti-FAP antibody and its applications.

[0020] The present invention provides a recombinant anti-FAP antibody and its applications. The recombinant anti-FAP antibody provided by the present invention exhibits a high degree of specific binding and rapid and efficient internalization ability, and its high selectivity and high level of biological activity contribute to its becoming a monoclonal antibody for treatment or an antibody-drug conjugate conjugated with a small molecule toxin.

[0021] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0022] The present invention provides a recombinant anti-FAP antibody, which comprises a heavy chain and a light chain:

[0023] The CDR regions of the heavy chain include CDR1, CDR2, and CDR3:

[0024] (I), the CDR1 of the heavy chain has an amino acid sequence as shown in SEQ ID No. 2, 16, 27, 38, 48, 56, 67, or 107; and

[0025] (II), the CDR2 of the heavy chain has an amino acid sequence as shown in SEQ ID No. 4, 18, 29, 40, 49, 57, or 69; and

[0026] (III), the CDR3 of the heavy chain has an amino acid sequence as shown in SEQ ID No. 6, 19, 31, 42, 59, 71, or 108; or

[0027] (IV), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of (I) to (III); or

[0028] (V), a sequence having a homology of more than 80% with the amino acid sequence shown in any one of (I) to (IV);

[0029] The CDR regions of the light chain include CDR1, CDR2, and CDR3:

[0030] (VI), the CDR1 of the light chain has an amino acid sequence as shown in SEQ ID No. 9, 21, 34, 44, 52, 62, 73, or 112; and

[0031] (VII), the CDR2 of the light chain has an amino acid sequence as shown in SEQ ID No. 11, 35, 45, 63 or 74; and

[0032] (VIII), the CDR3 of the light chain has an amino acid sequence as shown in SEQ ID No. 13, 24, 37, 47, 55, 65, 76, 116 or 117; or

[0033] (IX), a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids based on the amino acid sequence shown in any one of (VI) to (VIII); or

[0034] (X), a sequence having a homology of more than 80% with the amino acid sequence shown in any one of (VI) to (IX).

[0035] In some specific embodiments of the present invention, the recombinant anti-FAP antibody includes rabbit chimeric antibody and humanized antibody;

[0036] In some specific embodiments of the present invention, the CDR regions of the heavy chain of the rabbit chimeric antibody include CDR1, CDR2 and CDR3:

[0037] I), the CDR1 of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No. 2, 16, 27, 38, 48, 56 or 67; and

[0038] II), the CDR2 of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No. 4, 18, 29, 40, 49, 57 or 69; and

[0039] III), the CDR3 of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No. 6, 19, 31, 42, 59 or 71; or

[0040] IV), a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids based on the amino acid sequence shown in any one of I) to III); or

[0041] V), a sequence having a homology of more than 80% with the amino acid sequence shown in any one of I) to IV);

[0042] The CDR regions of the light chain of the rabbit chimeric antibody include CDR1, CDR2 and CDR3:

[0043] VI), the CDR1 of the light chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No. 9, 21, 34, 44, 52, 62 or 73; and

[0044] VII), the CDR2 of the light chain of the rabbit-derived chimeric antibody has an amino acid sequence shown in SEQ ID No. 11, 35, 45, 63 or 74; and

[0045] VIII), the CDR3 of the light chain of the rabbit-derived chimeric antibody has an amino acid sequence shown in SEQ ID No. 13, 24, 37, 47, 55, 65 or 76; or

[0046] IX), a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids based on the amino acid sequence shown in any one of VI) - VIII); or

[0047] X), a sequence having a homology of more than 80% with the amino acid sequence shown in any one of VI) - IX).

[0048] In some specific embodiments of the present invention, the rabbit-derived chimeric antibody includes chrX1, chrX6, chrX8, chrX9, chrA7, chrA12 or chrA18;

[0049] a), the CDR1, CDR2 and CDR3 of the heavy chain of chrX1 have the amino acid sequences shown in SEQ ID No. 2, 4 and 6 in sequence; and

[0050] the CDR1, CDR2 and CDR3 of the light chain of chrX1 have the amino acid sequences shown in SEQ ID No. 9, 11 and 13 in sequence; or

[0051] b), the CDR1, CDR2 and CDR3 of the heavy chain of chrX6 have the amino acid sequences shown in SEQ ID No. 16, 18 and 19 in sequence; and

[0052] the CDR1, CDR2 and CDR3 of the light chain of chrX6 have the amino acid sequences shown in SEQ ID No. 21, 11 and 24 in sequence; or

[0053] c), the CDR1, CDR2 and CDR3 of the heavy chain of chrX8 have the amino acid sequences shown in SEQ ID No. 27, 29 and 31 in sequence; and

[0054] the CDR1, CDR2 and CDR3 of the light chain of chrX8 have the amino acid sequences shown in SEQ ID No. 34, 35 and 37 in sequence; or

[0055] d), the CDR1, CDR2 and CDR3 of the heavy chain of chrX9 have the amino acid sequences shown in SEQ ID No. 38, 40 and 42 in sequence; and

[0056] The CDR1, CDR2, and CDR3 of the light chain of chrX9 have the amino acid sequences shown in SEQ ID No. 44, 45, and 47, respectively; or

[0057] e), the CDR1, CDR2, and CDR3 of the heavy chain of chrA7 have the amino acid sequences shown in SEQ ID No. 48, 49, and 6, respectively; and

[0058] The CDR1, CDR2, and CDR3 of the light chain of chrA7 have the amino acid sequences shown in SEQ ID No. 52, 11, and 55, respectively; or

[0059] f), the CDR1, CDR2, and CDR3 of the heavy chain of chrA12 have the amino acid sequences shown in SEQ ID No. 56, 57, and 59, respectively; and

[0060] The CDR1, CDR2, and CDR3 of the light chain of chrA12 have SEQ ID No. 62, 63, and 65, respectively; or

[0061] g), the CDR1, CDR2, and CDR3 of the heavy chain of chrA18 have the amino acid sequences shown in SEQ ID No. 67, 69, and 71, respectively; and

[0062] The CDR1, CDR2, and CDR3 of the light chain of chrA18 have the amino acid sequences shown in SEQ ID No. 73, 74, and 76, respectively; or

[0063] h), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids on the basis of the amino acid sequence shown in any one of a) to g); or

[0064] j), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of a) to h).

[0065] In some specific embodiments of the present invention, the CDR regions of the heavy chain of the humanized antibody include CDR1, CDR2, and CDR3:

[0066] (A), the CDR1 of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 107; and

[0067] (B), the CDR2 of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 18; and

[0068] (C), the CDR3 of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 108; or

[0069] (D), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of (A) to (C); or

[0070] (E), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of (A) to (D);

[0071] The CDR regions of the light chain of the humanized antibody include CDR1, CDR2, and CDR3:

[0072] (F), the CDR1 of the light chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 112; and

[0073] (G), the CDR2 of the light chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 11; and

[0074] (H), the CDR3 of the light chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 24, 116, or 117; or

[0075] (J), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of (F) to (G); or

[0076] (K), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of (F) to (J).

[0077] In some specific embodiments of the present invention, the humanized antibody includes hzX6-H29L3, hzX6-H29L10, hzX6-H29L12, hzX6-H32L3, hzX6-H32L10, hzX6-H32L12, hzX6-H33L3, hzX6-H33L10, or hzX6-H33L12;

[0078] k), the CDR1, CDR2, and CDR3 of the heavy chain of hzX6-H29L3 have the amino acid sequences shown in SEQ ID No. 107, 18, and 108 in sequence; and

[0079] the CDR1, CDR2, and CDR3 of the light chain of hzX6-H29L3 have the amino acid sequences shown in SEQ ID No. 112, 11, and 24 in sequence; or

[0080] l), the CDR1, CDR2, and CDR3 of the heavy chain of hzX6-H29L10 have the amino acid sequences shown in SEQ ID No. 107, 18, and 108 in sequence; and

[0081] The CDR1, CDR2 and CDR3 of the light chain of hzX6-H29L10 have the amino acid sequences shown in SEQ ID No. 112, 11 and 116 respectively; or

[0082] m), the CDR1, CDR2 and CDR3 of the heavy chain of hzX6-H29L12 have the amino acid sequences shown in SEQ ID No. 107, 18 and 108 respectively; and

[0083] The CDR1, CDR2 and CDR3 of the light chain of hzX6-H29L12 have the amino acid sequences shown in SEQ ID No. 112, 11 and 117 respectively; or

[0084] n), the CDR1, CDR2 and CDR3 of the heavy chain of hzX6-H32L3 have the amino acid sequences shown in SEQ ID No. 107, 18 and 108 respectively; and

[0085] The CDR1, CDR2 and CDR3 of the light chain of hzX6-H32L3 have the amino acid sequences shown in SEQ ID No. 112, 11 and 24 respectively; or

[0086] o), the CDR1, CDR2 and CDR3 of the heavy chain of hzX6-H32L10 have the amino acid sequences shown in SEQ ID No. 107, 18 and 108 respectively; and

[0087] The CDR1, CDR2 and CDR3 of the light chain of hzX6-H32L10 have the amino acid sequences shown in SEQ ID No. 112, 11 and 116 respectively; or

[0088] p), the CDR1, CDR2 and CDR3 of the heavy chain of hzX6-H32L12 have the amino acid sequences shown in SEQ ID No. 107, 18 and 108 respectively; and

[0089] The CDR1, CDR2 and CDR3 of the light chain of hzX6-H32L12 have the amino acid sequences shown in SEQ ID No. 112, 11 and 117 respectively; or

[0090] q), the CDR1, CDR2 and CDR3 of the heavy chain of hzX6-H33L3 have the amino acid sequences shown in SEQ ID No. 107, 18 and 108 respectively; and

[0091] The CDR1, CDR2 and CDR3 of the light chain of hzX6-H33L3 have the amino acid sequences shown in SEQ ID No. 112, 11 and 24 respectively; or

[0092] r), the CDR1, CDR2 and CDR3 of the heavy chain of hzX6-H33L10 have the amino acid sequences shown in SEQ ID No. 107, 18 and 108 respectively; and

[0093] the CDR1, CDR2 and CDR3 of the light chain of hzX6-H33L10 have the amino acid sequences shown in SEQ ID No. 112, 11 and 116 respectively; or

[0094] s), the CDR1, CDR2 and CDR3 of the heavy chain of hzX6-H33L12 have the amino acid sequences shown in SEQ ID No. 107, 18 and 108 respectively; and

[0095] the CDR1, CDR2 and CDR3 of the light chain of hzX6-H33L12 have the amino acid sequences shown in SEQ ID No. 112, 11 and 117 respectively; or

[0096] t), a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in any one of k) to s); or

[0097] u), a sequence having a homology of more than 80% with the amino acid sequence shown in any one of k) to t).

[0098] In some specific embodiments of the present invention, the FR regions of the heavy chain include FR1, FR2, FR3 and FR4:

[0099] (1), the FR1 of the heavy chain has the amino acid sequence shown in SEQ ID No. 1, 15, 26, 66 or 106; and

[0100] (2), the FR2 of the heavy chain has the amino acid sequence shown in SEQ ID No. 3, 17, 28, 39 or 68; and

[0101] (3), the FR3 of the heavy chain has the amino acid sequence shown in SEQ ID No. 5, 30, 41, 50, 58, 70, 150, 109 or 110; and

[0102] (4), the FR4 of the heavy chain has the amino acid sequence shown in SEQ ID No. 7, 32 or 60; or

[0103] (5), a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in any one of (1) to (4); or

[0104] (6) A sequence with a homology of more than 80% to the amino acid sequence shown in any one of (1) to (5);

[0105] The FR regions of the light chain include FR1, FR2, FR3 and FR4:

[0106] (7) The FR1 of the light chain has the amino acid sequence shown in SEQ ID No. 8, 20, 33, 43, 51, 61, 72 or 111; and

[0107] (8) The FR2 of the light chain has the amino acid sequence shown in SEQ ID No. 10, 22, 53 or 113; and

[0108] (9) The FR3 of the light chain has the amino acid sequence shown in SEQ ID No. 12, 23, 36, 46, 54, 64, 75 or 114; and

[0109] (10) The FR4 of the light chain has the amino acid sequence shown in SEQ ID No. 14, 25, 77 or 115; or

[0110] (11) A sequence with one or more amino acids substituted, deleted, added and / or replaced based on the amino acid sequence shown in any one of (7) to (10); or

[0111] (12) A sequence with a homology of more than 80% to the amino acid sequence shown in any one of (7) to (11).

[0112] In some specific embodiments of the present invention, the recombinant anti-FAP antibody includes a rabbit chimeric antibody and a humanized antibody;

[0113] In some specific embodiments of the present invention, the FR regions of the heavy chain of the rabbit chimeric antibody include FR1, FR2, FR3 and FR4:

[0114] (13) The FR1 of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No. 1, 15, 26 or 66; and

[0115] (14) The FR2 of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No. 3, 17, 28, 39 or 68; and

[0116] (15) The FR3 of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No. 5, 30, 41, 50, 58 or 70; and

[0117] (16) The FR4 of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 7, 32 or 60; or

[0118] (17) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequence shown in any one of (13) to (16); or

[0119] (18) A sequence having a homology of more than 80% with the amino acid sequence shown in any one of (13) to (17);

[0120] The FR regions of the light chain of the rabbit-derived chimeric antibody include FR1, FR2, FR3 and FR4:

[0121] (19) The FR1 of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 8, 20, 33, 43, 51, 61 or 72; and

[0122] (20) The FR2 of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 10, 22 or 53; and

[0123] (21) The FR3 of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 12, 23, 36, 46, 54, 64 or 75; and

[0124] (22) The FR4 of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 14, 25 or 77; or

[0125] (23) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequence shown in any one of (19) to (22); or

[0126] (24) A sequence having a homology of more than 80% with the amino acid sequence shown in any one of (19) to (23).

[0127] In some specific embodiments of the present invention, the rabbit-derived chimeric antibody includes chrX1, chrX6, chrX8, chrX9, chrA7, chrA12 or chrA18;

[0128] 1) The FR1, FR2, FR3 and FR4 of the heavy chain of the chrX1 have the amino acid sequences shown in SEQ ID No. 1, 3, 5 and 7 in sequence; and

[0129] The FR1, FR2, FR3, and FR4 of the light chain of chrX1 have the amino acid sequences shown in SEQ ID No. 8, 10, 12, and 14, respectively; or

[0130] 2), the FR1, FR2, FR3, and FR4 of the heavy chain of chrX6 have the amino acid sequences shown in SEQ ID No. 15, 17, 5, and 7, respectively; and

[0131] The FR1, FR2, FR3, and FR4 of the light chain of chrX6 have the amino acid sequences shown in SEQ ID No. 20, 22, 23, and 25, respectively; or

[0132] 3), the FR1, FR2, FR3, and FR4 of the heavy chain of chrX8 have the amino acid sequences shown in SEQ ID No. 26, 28, 30, and 32, respectively; and

[0133] The FR1, FR2, FR3, and FR4 of the light chain of chrX8 have the amino acid sequences shown in SEQ ID No. 33, 22, 36, and 25, respectively; or

[0134] 4), the FR1, FR2, FR3, and FR4 of the heavy chain of chrX9 have the amino acid sequences shown in SEQ ID No. 26, 39, 41, and 7, respectively; and

[0135] The FR1, FR2, FR3, and FR4 of the light chain of chrX9 have the amino acid sequences shown in SEQ ID No. 43, 22, 46, and 25, respectively; or

[0136] 5), the FR1, FR2, FR3, and FR4 of the heavy chain of chrA7 have the amino acid sequences shown in SEQ ID No. 1, 39, 50, and 7, respectively; and

[0137] The FR1, FR2, FR3, and FR4 of the light chain of chrA7 have the amino acid sequences shown in SEQ ID No. 51, 53, 54, and 25, respectively; or

[0138] 6), the FR1, FR2, FR3, and FR4 of the heavy chain of chrA12 have the amino acid sequences shown in SEQ ID No. 26, 3, 58, and 60, respectively; and

[0139] The FR1, FR2, FR3, and FR4 of the light chain of chrA12 have the amino acid sequences shown in SEQ ID No. 61, 22, 64, and 25, respectively; or

[0140] 7), the FR1, FR2, FR3, and FR4 of the heavy chain of said chrA18 have the amino acid sequences shown in SEQ ID No. 66, 68, 70, and 7 respectively; and

[0141] the FR1, FR2, FR3, and FR4 of the light chain of said chrA18 have the amino acid sequences shown in SEQ ID No. 72, 22, 75, and 77 respectively; or

[0142] 8), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of 1) to 7); or

[0143] 9), a sequence having a homology of more than 80% with the amino acid sequence shown in any one of 1) to 8).

[0144] In some specific embodiments of the present invention, the FR regions of the heavy chain of the humanized antibody include FR1, FR2, FR3, and FR4:

[0145] A), the FR1 of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 106; and

[0146] B), the FR2 of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 28; and

[0147] C), the FR3 of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 150, 109, or 110; and

[0148] D), the FR4 of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 7; or

[0149] E), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of A) to D); or

[0150] F), a sequence having a homology of more than 80% with the amino acid sequence shown in any one of A) to E);

[0151] The FR regions of the light chain of the humanized antibody include FR1, FR2, FR3, and FR4:

[0152] G), the FR1 of the light chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 111; and

[0153] H), the FR2 of the light chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 113; and

[0154] J), the FR3 of the light chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 114; and

[0155] K), the FR4 of the light chain of the humanized antibody has the amino acid sequence shown in SEQ ID No. 115; or

[0156] L), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of (19) to (22); or

[0157] M), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of (19) to (23).

[0158] In some specific embodiments of the present invention, the humanized antibody includes hzX6-H29L3, hzX6-H29L10, hzX6-H29L12, hzX6-H32L3, hzX6-H32L10, hzX6-H32L12, hzX6-H33L3, hzX6-H33L10 or hzX6-H33L12;

[0159] 1), the FR1, FR2, FR3, and FR4 of the heavy chain of hzX6-H29L3 have the amino acid sequences shown in SEQ ID No. 106, 28, 150, and 7, respectively; and

[0160] the FR1, FR2, FR3, and FR4 of the light chain of hzX6-H29L3 have the amino acid sequences shown in SEQ ID No. 111, 113, 114, and 115, respectively; or

[0161] 2), the FR1, FR2, FR3, and FR4 of the heavy chain of hzX6-H29L10 have the amino acid sequences shown in SEQ ID No. 106, 28, 150, and 7, respectively; and

[0162] the FR1, FR2, FR3, and FR4 of the light chain of hzX6-H29L10 have the amino acid sequences shown in SEQ ID No. 111, 113, 114, and 115, respectively; or

[0163] 3), the FR1, FR2, FR3, and FR4 of the heavy chain of hzX6-H29L12 have the amino acid sequences shown in SEQ ID No. 106, 28, 150, and 7, respectively; and

[0164] the FR1, FR2, FR3, and FR4 of the light chain of hzX6-H29L12 have the amino acid sequences shown in SEQ ID No. 111, 113, 114, and 115, respectively; or

[0165] 4), the FR1, FR2, FR3, and FR4 of the heavy chain of hzX6-H32L3 have the amino acid sequences shown in SEQ ID No. 106, 28, 109, and 7, respectively; and

[0166] the FR1, FR2, FR3, and FR4 of the light chain of hzX6-H32L3 have the amino acid sequences shown in SEQ ID No. 111, 113, 114, and 115, respectively; or

[0167] 5), the FR1, FR2, FR3, and FR4 of the heavy chain of hzX6-H32L10 have the amino acid sequences shown in SEQ ID No. 106, 28, 109, and 7, respectively; and

[0168] the FR1, FR2, FR3, and FR4 of the light chain of hzX6-H32L10 have the amino acid sequences shown in SEQ ID No. 111, 113, 114, and 115, respectively; or

[0169] 6), the FR1, FR2, FR3, and FR4 of the heavy chain of hzX6-H32L12 have the amino acid sequences shown in SEQ ID No. 106, 28, 109, and 7, respectively; and

[0170] the FR1, FR2, FR3, and FR4 of the light chain of hzX6-H32L12 have the amino acid sequences shown in SEQ ID No. 111, 113, 114, and 115, respectively; or

[0171] 7), the FR1, FR2, FR3, and FR4 of the heavy chain of hzX6-H33L3 have the amino acid sequences shown in SEQ ID No. 106, 28, 110, and 7, respectively; and

[0172] the FR1, FR2, FR3, and FR4 of the light chain of hzX6-H33L3 have the amino acid sequences shown in SEQ ID No. 111, 113, 114, and 115, respectively; or

[0173] 8), the FR1, FR2, FR3, and FR4 of the heavy chain of hzX6-H33L10 have the amino acid sequences shown in SEQ ID No. 106, 28, 110, and 7, respectively; and

[0174] the FR1, FR2, FR3, and FR4 of the light chain of hzX6-H33L10 have the amino acid sequences shown in SEQ ID No. 111, 113, 114, and 115, respectively; or

[0175] 9), the FR1, FR2, FR3, and FR4 of the heavy chain of hzX6-H33L12 have the amino acid sequences shown in SEQ ID No. 106, 28, 110, and 7, respectively; and

[0176] the FR1, FR2, FR3, and FR4 of the light chain of hzX6-H33L12 have the amino acid sequences shown in SEQ ID No. 111, 113, 114, and 115, respectively; or

[0177] 10), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of 1) to 9); or

[0178] 11), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of 1) to 10).

[0179] In some specific embodiments of the present invention,

[0180] (i), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 78, 80, 82, 84, 86, 88, 90, 118, 119, or 120; and

[0181] (ii), the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 79, 81, 83, 85, 87, 89, 91, 121, 122, or 123; or

[0182] (iii), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in (i) or (ii); or

[0183] (iv), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of (i) to (iii).

[0184] In some specific embodiments of the present invention,

[0185] i), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 78; and

[0186] the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 79;

[0187] or

[0188] ii), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 80; and

[0189] the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 81;

[0190] or

[0191] iii), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 82; and

[0192] the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 83;

[0193] or

[0194] iv), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 84; and

[0195] the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 85;

[0196] or

[0197] v), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 86; and

[0198] the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 87;

[0199] or

[0200] vi), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 88; and

[0201] the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 89;

[0202] or

[0203] vii), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 90; and

[0204] the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 91;

[0205] or

[0206] viii), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 118; and

[0207] the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 121;

[0208] or

[0209] ix), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 119; and

[0210] The variable region of the light chain has an amino acid sequence as shown in SEQ ID No. 122;

[0211] or

[0212] (x) The variable region of the heavy chain has an amino acid sequence as shown in SEQ ID No. 120; and

[0213] The variable region of the light chain has an amino acid sequence as shown in SEQ ID No. 123;

[0214] (xi) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids on the basis of the amino acid sequence shown in any one of (i) to (x); or

[0215] (xii) A sequence having a homology of 80% or more with the amino acid sequence shown in any one of (i) to (xi).

[0216] In some specific embodiments of the present invention,

[0217] (i) The nucleic acid molecule encoding the variable region of the heavy chain has a nucleotide sequence as shown in SEQ ID No. 92, 94, 96, 98, 100, 102, 104, 124, 125, or 126; and

[0218] (ii) The nucleic acid molecule encoding the variable region of the light chain has a nucleotide sequence as shown in SEQ ID No. 93, 95, 97, 99, 101, 103, 105, 127, 128, or 129; or

[0219] (iii) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (i) or (ii), but is different from the nucleotide sequence shown in (i) or (ii) due to the degeneracy of the genetic code; or

[0220] (iv) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in any one of (i) to (iii), and having the same or similar function as the nucleotide sequence shown in any one of (i) to (iii); or

[0221] (v) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (i) to (iv).

[0222] In some specific embodiments of the present invention, the recombinant anti-FAP antibody includes a rabbit chimeric antibody and a humanized antibody;

[0223] (v) The variable region of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No. 78, 80, 82, 84, 86, 88, or 90; and

[0224] The variable region of the light chain of the rabbit-derived chimeric antibody has an amino acid sequence as shown in SEQ ID No. 79, 81, 83, 85, 87, 89 or 91; or

[0225] (vi), the variable region of the heavy chain of the humanized antibody has an amino acid sequence as shown in 118, 119 or 120; and

[0226] the variable region of the light chain of the humanized antibody has an amino acid sequence as shown in 121, 122 or 123; or

[0227] (vii), a sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequence as shown in (v) or (vi); or

[0228] (ix), a sequence having a homology of more than 80% with the amino acid sequence as shown in any one of (v) to (vii).

[0229] In some specific embodiments of the present invention, the rabbit-derived chimeric antibody includes:

[0230] 1), the variable region of the heavy chain of the rabbit-derived chimeric antibody has an amino acid sequence as shown in SEQ ID No. 78; and

[0231] the variable region of the light chain of the rabbit-derived chimeric antibody has an amino acid sequence as shown in SEQ ID No. 79;

[0232] or

[0233] 2), the variable region of the heavy chain of the rabbit-derived chimeric antibody has an amino acid sequence as shown in SEQ ID No. 80; and

[0234] the variable region of the light chain of the rabbit-derived chimeric antibody has an amino acid sequence as shown in SEQ ID No. 81;

[0235] or

[0236] 3), the variable region of the heavy chain of the rabbit-derived chimeric antibody has an amino acid sequence as shown in SEQ ID No. 82; and

[0237] the variable region of the light chain of the rabbit-derived chimeric antibody has an amino acid sequence as shown in SEQ ID No. 83;

[0238] or

[0239] 4), the variable region of the heavy chain of the rabbit-derived chimeric antibody has an amino acid sequence as shown in SEQ ID No. 84; and

[0240] The variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 85;

[0241] or

[0242] 5), the variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 86; and

[0243] the variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 87;

[0244] or

[0245] 6), the variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 88; and

[0246] the variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 89;

[0247] or

[0248] 7), the variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 90; and

[0249] the variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 91;

[0250] or

[0251] 8), a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in any one of 1) to 7); or

[0252] 9), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of 1) to 8).

[0253] In some specific embodiments of the present invention, the humanized antibody:

[0254] 1), the variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in 118; and

[0255] the variable region of the light chain of the humanized antibody has the amino acid sequence shown in 121;

[0256] or

[0257] 2), the variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in 119; and

[0258] the variable region of the light chain of the humanized antibody has the amino acid sequence shown in 122;

[0259] or

[0260] 3), the variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID NO: 120; and

[0261] the variable region of the light chain of the humanized antibody has the amino acid sequence shown in SEQ ID NO: 123;

[0262] or

[0263] 4), a sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in any one of 1) to 3); or

[0264] 5), a sequence having a homology of more than 80% with the amino acid sequence shown in any one of 1) to 4).

[0265] In some specific embodiments of the present invention, vi), the nucleic acid molecule encoding the variable region of the heavy chain of the rabbit chimeric antibody has the nucleotide sequence shown in SEQ ID NO: 92, 94, 96, 98, 100, 102 or 104; and

[0266] the nucleic acid molecule encoding the variable region of the light chain of the rabbit chimeric antibody has the nucleotide sequence shown in SEQ ID NO: 93, 95, 97, 99, 101, 103 or 105; or

[0267] vii), the nucleic acid molecule encoding the variable region of the heavy chain of the humanized antibody has the nucleotide sequence shown in SEQ ID NO: 124, 125 or 126; and

[0268] the nucleic acid molecule encoding the variable region of the light chain of the humanized antibody has the nucleotide sequence shown in SEQ ID NO: 127, 128 or 129; or

[0269] ix), a nucleotide sequence encoding the same protein as that shown in vi) or vii), but different from the nucleotide sequence shown in vi) or vii) due to the degeneracy of the genetic code; or

[0270] x), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in any one of vi) to ix), and having the same or similar function as the nucleotide sequence shown in any one of vi) to ix); or

[0271] xi) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of vi) to x).

[0272] In some specific embodiments of the present invention, the recombinant anti-FAP antibody further comprises a constant region;

[0273] The heavy chain constant region of the recombinant anti-FAP antibody comprises human IgG1; the light chain constant region of the recombinant anti-FAP antibody comprises human kappa type.

[0274] In some specific embodiments of the present invention, the FAP includes human FAP, murine FAP, and / or cynomolgus monkey FAP.

[0275] The present invention also provides a method for preparing the recombinant anti-FAP antibody, comprising the following steps:

[0276] Step 1: Immunize a receptor with the FAP antigen, isolate the spleen cells of the receptor, and perform PCR amplification to obtain the variable regions of the light and heavy chains of the recombinant anti-FAP antibody;

[0277] Step 2: Splice the variable region of the heavy chain and the constant region of the heavy chain, construct them into an expression vector to obtain a heavy chain vector; splice the variable region of the light chain and the constant region of the light chain, construct them into the expression vector to obtain a light chain vector;

[0278] Step 3: Take the heavy chain vector and the light chain vector, transfect, culture, purify, and screen to obtain the recombinant anti-FAP antibody.

[0279] In some specific embodiments of the present invention, the receptor includes New Zealand white rabbits;

[0280] The heavy chain constant region is the heavy chain constant region of the human IgG1 subclass;

[0281] The light chain constant region is the light chain constant region of human kappa class antibodies;

[0282] The constructed expression vector is a mammalian cell expression vector.

[0283] In some specific embodiments of the present invention, the preparation method comprises the following steps:

[0284] Step A: Combine the CDR regions of the recombinant anti-FAP antibody with the framework regions of human antibodies, splice the variable region of the heavy chain and the constant region of the heavy chain, construct them into an expression vector to obtain a heavy chain vector; splice the variable region of the light chain and the constant region of the light chain, construct them into the expression vector to obtain a light chain vector;

[0285] Step B: Take the heavy chain vector and the light chain vector, transfect, culture, purify, and screen to obtain a humanized antibody of the recombinant anti-FAP antibody.

[0286] In some specific embodiments of the present invention, the heavy chain constant region is the heavy chain constant region of the human IgG1 subclass;

[0287] The light chain constant region is the light chain constant region of human kappa class antibodies;

[0288] The constructed expression vector is a mammalian cell expression vector.

[0289] On the basis of the above research, the present invention also provides biological materials, including any of the following:

[0290] (a) Nucleic acids encoding the recombinant anti-FAP antibody or the recombinant anti-FAP antibody prepared by the preparation method; and / or

[0291] (b) Nucleic acids encoding the recombinant anti-FAP antibody and an acceptable vector; or

[0292] The recombinant anti-FAP antibody prepared by the preparation method and an acceptable vector; and / or

[0293] (c) Hosts secreting the recombinant anti-FAP antibody or the recombinant anti-FAP antibody prepared by the preparation method; and / or

[0294] (d) Chemically or biologically labeled recombinant anti-FAP antibody or the recombinant anti-FAP antibody prepared by the preparation method; and / or

[0295] (e) The recombinant anti-FAP antibody or the recombinant anti-FAP antibody prepared by the preparation method coupled with a vector;

[0296] (f) Antibody-drug conjugate obtained by covalently coupling the recombinant anti-FAP antibody or the recombinant anti-FAP antibody prepared by the preparation method with a drug through a linker.

[0297] The present invention also provides the use of any of the following in the preparation of a drug targeting FAP:

[0298] ①. The recombinant anti-FAP antibody; and / or

[0299] ②. The recombinant anti-FAP antibody prepared by the preparation method and / or

[0300] ③. The biological material.

[0301] The present invention also provides the use of any of the following in the preparation of a product for preventing and / or treating diseases:

[0302] ①. The recombinant anti-FAP antibody; and / or

[0303] ②. The recombinant anti-FAP antibody prepared by the preparation method and / or

[0304] ③. The biological material.

[0305] In some specific embodiments of the present invention, the diseases include any one or more of human epithelial cancer, breast cancer, pancreatic cancer, lung cancer, bladder cancer or colon cancer.

[0306] In some specific embodiments of the present invention, the product includes a drug and / or a vaccine.

[0307] The present invention also provides a drug, including any one of the following and a pharmaceutically acceptable excipient:

[0308] ①. The recombinant anti-FAP antibody; and / or

[0309] ②. The recombinant anti-FAP antibody prepared by the preparation method and / or

[0310] ③. The biological material.

[0311] The present invention also provides a drug combination, including the drug and any other active ingredient.

[0312] In some specific embodiments of the present invention, the other active ingredient includes a small molecule toxin.

[0313] The present invention also provides a vaccine, including any one of the following:

[0314] ①. The recombinant anti-FAP antibody; and / or

[0315] ②. The recombinant anti-FAP antibody prepared by the preparation method and / or

[0316] ③. The biological material.

[0317] The present invention also provides the use of any one of the following in the preparation of a reagent and / or kit for detecting FAP:

[0318] ①. The recombinant anti-FAP antibody; and / or

[0319] ②. The recombinant anti-FAP antibody prepared by the preparation method and / or

[0320] ③. The biological material.

[0321] The present invention also provides a reagent and / or kit, including any one of the following:

[0322] ①. The recombinant anti-FAP antibody; and / or

[0323] ②. The recombinant anti-FAP antibody prepared by the preparation method and / or

[0324] ③. The biological material.

[0325] The present invention also provides a treatment method, including administering any one of the following to a subject:

[0326] ①. The recombinant anti-FAP antibody; and / or

[0327] ②. The recombinant anti-FAP antibody prepared by the preparation method and / or

[0328] ③. The biomaterial.

[0329] The present invention provides a recombinant anti-FAP antibody and its applications. The recombinant anti-FAP antibody provided by the present invention exhibits a high degree of specific binding as well as rapid and efficient internalization ability. Its high selectivity and high level of biological activity contribute to its potential as a therapeutic monoclonal antibody or an antibody-drug conjugate conjugated with a small molecule toxin. The antibody provided by the present invention has better effects and lower immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0330] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0331] Figure 1a Showing the flow cytometry binding experiment of the control antibody and CHO-hFAP cells in the test example;

[0332] Figure 1b Showing the flow cytometry binding experiment of the control antibody and U138MG cells in the test example;

[0333] Figure 2a Showing the flow cytometry binding experiment of the anti-human FAP chimeric antibodies chrX1, chrX6, chrX8, chrX9 and HT1080-hFAP cells in Example 1;

[0334] Figure 2b Showing the flow cytometry binding experiment of the anti-human FAP chimeric antibodies chrA7, chrA12, chrA18, hu36 and HT1080-hFAP cells in Example 1;

[0335] Figure 3a Showing the endocytosis activity test of the anti-human FAP chimeric antibodies chrX1, chrX6, chrX8, chrX9 using the low-expression cell line HT1080-hFAP cells in Example 3;

[0336] Figure 3b Showing the endocytosis activity test of the anti-human FAP chimeric antibodies chrA7, chrA12, chrA18, hu36 using the low-expression cell line HT1080-hFAP cells in Example 3;

[0337] Figure 4a Showing the binding experiment of the humanized antibody and HT1080-hFAP cells in Example 6;

[0338] Figure 4b Showing the binding experiment of the humanized antibody and CT26-mFAP cells in Example 6;

[0339] Figure 4c Experiment on the binding of the humanized antibody in Example 6 to CHO-cynoFAP cells;

[0340] Figure 5a Experiment on the binding of the anti-human FAP humanized antibody in Example 7 to CHO-hDPP4 cells;

[0341] Figure 5b Experiment on the binding of the anti-human FAP humanized antibody in Example 7 to CHOK1 cells;

[0342] Figure 5c Experiment on the binding of the anti-human FAP humanized antibody in Example 7 to HEK293 cells;

[0343] Figure 6 Experiment on the endocytosis activity of the anti-human FAP humanized antibody tested using HT1080-hFAP cells in Example 8;

[0344] Figure 7a Experiment on the killing assay of the human FAP humanized antibodies hzX6-H29L3, hzX6-H29L10, and hzX6-H29L12 tested using HT1080-hFAP cells in Example 9;

[0345] Figure 7b Experiment on the killing assay of the human FAP humanized antibodies hzX6-H32L3, hzX6-H32L10, and hzX6-H32L12 tested using HT1080-hFAP cells in Example 9;

[0346] Figure 7c Experiment on the killing assay of the human FAP humanized antibodies hzX6-H33L3, hzX6-H33L10, and hzX6-H33L12 tested using HT1080-hFAP cells in Example 9;

[0347] Figure 7d Experiment on the killing assay of the human FAP humanized antibodies hzX6-H29L3, hzX6-H29L10, and hzX6-H29L12 tested using 293-hFAP cells in Example 9;

[0348] Figure 7e Experiment on the killing assay of the human FAP humanized antibodies hzX6-H32L3, hzX6-H32L10, and hzX6-H32L12 tested using 293-hFAP cells in Example 9;

[0349] Figure 7fThe killing experiment of human FAP humanized antibodies hzX6-H33L3, hzX6-H33L10, and hzX6-H33L12 was tested using 293-hFAP cells in Example 9. Detailed implementation mode

[0350] The present invention discloses recombinant anti-FAP antibodies and their applications. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve them. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0351] Table 1: Amino acid sequences of chimeric antibodies and their CDR divisions

[0352]

[0353]

[0354]

[0355] The variable region sequences of rabbit monoclonal antibodies are shown as follows.

[0356] 1. Rabbit antibody

[0357] Table 2

[0358]

[0359]

[0360] Note: The underlined part is the CDR. The CDR annotation method is based on the Kabat antibody coding scheme, and the subsequent sequence coding method is the same.

[0361] Table 3: Nucleotide sequences of chimeric antibodies

[0362]

[0363]

[0364]

[0365] Table 4 Amino acid CDR divisions of the variable regions of the heavy and light chains of humanized molecules

[0366]

[0367]

[0368] 2. Humanized Antibodies

[0369] Table 5

[0370]

[0371]

[0372] Note: The underlined sequences are CDRs, and the CDR annotation method is based on the Kabat antibody coding scheme. The subsequent sequence coding method is the same. The PTM sites and their corresponding variants are marked in bold and italic characters. The subsequent sequence annotation method is the same.

[0373] Table 6 Nucleotide Sequences of Humanized Molecules

[0374]

[0375]

[0376] 1. Sequence Synthesis and Vector Construction of the Tool Antibody

[0377] 1.1 Humanized Monoclonal Antibody sibrotuzumab (BIBH1) against Human FAP from Boehringer-Ingelheim

[0378] Amino Acid Sequence of the Heavy Chain Variable Region of BIBH1 (SEQ ID No. 130):

[0379] QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKF KGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSS

[0380] Amino Acid Sequence of the Light Chain Variable Region of BIBH1 (SEQ ID No. 131):

[0381] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVP DRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK

[0382] 1.2 Murine Monoclonal Antibody cMFP5 against Human FAP from Boehringer-Ingelheim

[0383] Amino Acid Sequence of the Heavy Chain Variable Region of MFP5 (SEQ ID No. 132):

[0384] QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKF KGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSA

[0385] Amino acid sequence of the variable region of the MFP5 light chain (SEQ ID No. 133):

[0386] QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSG SGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIK

[0387] 1.3 Anti-FAP antibody hu36 developed by ONCOMATRYX BIOPHARMA

[0388] Amino acid sequence of the variable region of the Hu36 heavy chain (SEQ ID No. 134):

[0389] QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYNEK FKDRVTMTADTSTSTVYMELSSLRSEDTAVYYCARHGGTGRGAMDYWGQGTLVTVSS

[0390] Amino acid sequence of the variable region of the Hu36 light chain (SEQ ID No. 135):

[0391] DIQMTQSPSSLSASVGDRVTITCRASKSVSTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKLEIK

[0392] 1.4 Isotype control (abbreviation: SH-NC) self-produced by Mabwell Biologics

[0393] Amino acid sequence of the variable region of the SH-NC heavy chain (SEQ ID No. 136):

[0394] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPIFGSSNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAESPLGGGSGYSVSWFDPWGQGTLVTVSS

[0395] Amino acid sequence of the IgG1 heavy chain constant region G1m3 of the PTT5 vector used in the 1.5 protein expression system (SEQ ID No. 137):

[0396] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSNWPPWTFGQGTKVEIK

[0397] Amino acid sequence of the IgG1 heavy chain constant region G1m3 of the PTT5 vector used in the 1.5 protein expression system (SEQ ID No. 138):

[0398] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0399] Nucleotide sequence of the IgG1 heavy chain constant region G1m3 of the PTT5 vector used in the protein expression system (SEQ ID No. 139):

[0400] gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa

[0401] 1. The amino acid sequence (SEQ ID No. 140) of the Km3 of the IgG1 light chain constant region of the PTT5 vector used in the protein expression system:

[0402] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0403] The nucleotide sequence (SEQ ID No. 141) of the Km3 of the IgG1 light chain constant region of the PTT5 vector used in the protein expression system:

[0404] cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatccccgcgaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaaccgcggagagtgt

[0405] 2. Purchase of antigens and verification of antigen-binding activity

[0406] 2.1. Information on the recombinant human FAP protein antigen

[0407] The following commercial reagents were purchased:

[0408] Table 7

[0409] Name Manufacturer Model Human FAP-His Protein Acro FAP-H5244 Human FAP-hFc Protein Acro FAP-H5263 Mouse FAP-His Protein Acro FAP-M52H3 Monkey FAP-His Protein Acro FAP-C52H3

[0410] 2.2. Construction of antigen cell lines of different species:

[0411] The amino acid sequence of human FAP protein (abbreviation: hFAP) is as follows:

[0412] >FAP Homo sapiens (Human) Q12884 (SEQ ID No. 142)

[0413] MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD

[0414] The nucleotide sequence of human FAP protein (abbreviated as hFAP) is as follows (SEQ ID No. 143):

[0415]

[0416] The amino acid sequence of mouse FAP protein (abbreviated as mFAP) is as follows:

[0417] >FAP Mus musculus (Mouse) P97321 (SEQ ID No.144)

[0418] MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD

[0419] The nucleotide sequence of mouse FAP protein (abbreviated as mFAP) is as follows: (SEQ ID No.145)

[0420]

[0421] The amino acid sequence of cynomolgus FAP protein (abbreviated as cynoFAP) is as follows:

[0422] >FAP Macaca fascicularis(Crab-eatingmacaque)(Cynomolgus monkey)A0A2K5VGF4(SEQ IDNo.146)

[0423] MKTWVKIVFGVATSAVLALLVMCIVLRPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD

[0424] The nucleotide sequence of cynomolgus macaque FAP protein (abbreviated as cynoFAP) is as follows (SEQ ID No.147):

[0425]

[0426] The nucleotide sequence of the above-mentioned hFAP was constructed into the commercially available pCDNA3.1 vector of Invitrogen. Specifically, after the pCDNA3.1 vector was digested with KpnI and XhoI, the nucleotide sequence of hFAP was inserted, and then the vector sequence information was confirmed by sequencing. After the plasmid construction was successful, it was transfected into human fibrosarcoma HT1080 cells using lipofectamine3000. Subsequently, puromycin antibiotic was used to screen for overexpressing monoclonal cells, and the clone with slightly lower expression was selected. The successfully constructed cell line was named low-expression HT1080-hFAP cells.

[0427] The nucleotide sequence of the above-mentioned hFAP was constructed into the commercially available pCDNA5 vector of Invitrogen. Specifically, after the pCDNA5 vector was digested with NheI and PmeI, the nucleotide sequence of hFAP was inserted, and then the vector sequence information was confirmed by sequencing. After the plasmid construction was successful, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1 using lipofectamine3000. Subsequently, Hygromycin antibiotic was used to screen for overexpressing cell lines, and the successfully constructed cell lines were named CHO-hFAP and 293-hFAP cells respectively.

[0428] The nucleotide sequence of the above-mentioned cynoFAP was constructed into the commercially available pCDNA5 vector of Invitrogen. Specifically, after the pCDNA5 vector was digested with NheI and PmeI, the nucleotide sequence of cynoFAP was inserted, and then the vector sequence information was confirmed by sequencing. After the plasmid construction was successful, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1 respectively using lipofectamine3000. Subsequently, Hygromycin antibiotic was used to screen for overexpressing cell lines, and through monoclonalization, the monoclonal overexpressing cell lines CHO-cynoFAP and 293-cynoFAP cells were finally obtained.

[0429] 2.3. Construction of cell lines of proteins in the same family

[0430] The amino acid sequence of human DPP4 protein (abbreviation hDPP4) is as follows (SEQ ID No.148):

[0431] MKTPWKVLLGLLGAAALVTIITVPVVLLNKGTDDATADSRKTYTLTDYLKNTYRLKLYSLRWISDHEYLYKQENNILVFNAEYGNSSVFLENSTFDEFGHSINDYSISPDGQFILLEYNYVKQWRHSYTASYDIYDLNKRQLITEERIPNNTQWVTWSPVGHKLAYVWNNDIYVKIEPNLPSYRITWTGKEDIIYNGITDWVYEEEVFSAYSALWWSPNGTFLAYAQFNDTEVPLIEYSFYSDESLQYPKTVRVPYPKAGAVNPTVKFFVVNTDSLSSVTNATSIQITAPASMLIGDHYLCDVTWATQERISLQWLRRIQNYSVMDICDYDESSGRWNCLVARQHIEMSTTGWVGRFRPSEPHFTLDGNSFYKIISNEEGYRHICYFQIDKKDCTFITKGTWEVIGIEALTSDYLYYISNEYKGMPGGRNLYKIQLSDYTKVTCLSCELNPERCQYYSVSFSKEAKYYQLRCSGPGLPLYTLHSSVNDKGLRVLEDNSALDKMLQNVQMPSKKLDFIILNETKFWYQMILPPHFDKSKKYPLLLDVYAGPCSQKADTVFRLNWATYLASTENIIVASFDGRGSGYQGDKIMHAINRRLGTFEVEDQIEAARQFSKMGFVDNKRIAIWGWSYGGYVTSMVLGSGSGVFKCGIAVAPVSRWEYYDSVYTERYMGLPTPEDNLDHYRNSTVMSRAENFKQVEYLLIHGTADDNVHFQQSAQISKALVDVGVDFQAMWYTDEDHGIASSTAHQHIYTHMSHFIKQCFSLP

[0432] The nucleotide sequence of human DPP4 protein is as follows (SEQ ID No.149):

[0433]

[0434] The nucleotide sequence of the above-mentioned hDPP4 was constructed onto the commercially available pCDNA5 vector from Invitrogen. Specifically, after double digestion of the pCDNA5 vector with NheI and PmeI, the nucleotide sequence of hDPP4 was inserted, and then the vector sequence information was confirmed by sequencing. After successful plasmid construction, it was transfected into Chinese hamster ovary cells CHOK1 using lipofectamine3000. Subsequently, an overexpressing cell line was screened using Hygromycin antibiotic, and through monoclonalization, a monoclonal overexpressing cell line, CHO-hDPP4 cells, was finally obtained.

[0435] 3. Purchase of antigen cell lines

[0436] Mouse FAP engineering cell line:

[0437] CT26-mouse-Fap-Cell-Line, product number KC-1284 of Kangyuanbochuang Biotechnology (Beijing) Co., Ltd., hereinafter simply referred to as CT26-mFAP cell line

[0438] Natural tumor cells expressing human FAP:

[0439] U-138MG cells were purchased from ATCC, catalog number HTB-16

[0440] The recombinant anti-FAP antibody provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0441] The present invention will be further illustrated below in conjunction with examples:

[0442] Test examples

[0443] Facs determination of the affinity between antigen cells and tool antibodies

[0444] 1. Experimental reagents and materials:

[0445] Table 8

[0446]

[0447] 2. Experimental procedure:

[0448] 1) Cell collection and seeding

[0449] a) Harvest cells in the logarithmic growth phase to ensure that the cell viability is above 90%.

[0450] b) Centrifuge at 1000 r / min for 5 min, then discard the supernatant;

[0451] c) Wash the cells once with PBS;

[0452] d) Resuspend the cells with FACS Buffer (i.e., 1% BSA, DPBS) and count them;

[0453] e) Prepare a cell suspension with a density of 2×10 6 cells / mL using FACS Buffer;

[0454] f) Add 50 μL of the cell suspension to each well of a 96-well plate;

[0455] 2) Antibody Incubation and Detection

[0456] a) Add 50 μL of test samples at different concentrations to the experimental group. The sample concentration starts from 20 μg / ml and is serially diluted 3-fold with multiple gradient points;

[0457] b) After mixing, incubate overnight at 4°C in the dark;

[0458] c) Wash the cells once with 200 μL of FACS Buffer each time, centrifuge at 1000 r / min for 5 min, and discard the supernatant;

[0459] d) Add APC-labeled secondary antibody (diluted 1:1500) to the 96-well plate, and add an equal volume of FACS Buffer to the blank control group;

[0460] e) After mixing, incubate at 4°C in the dark for 40 min;

[0461] f) Wash the cells once with 200 μL of FACS Buffer each time, centrifuge at 1000 r / min for 5 min, and finally resuspend the cells with 100 μL of FACS Buffer;

[0462] g) Detect the RL-1 MFI value using an Intellicyte plus flow cytometer (Excitation Laser: 640 nm Blue Laser).

[0463] 3) Data Processing

[0464] Analyze the FACS data using Prism software.

[0465] Data Analysis: Using the overexpressing cell line CHO-hFAP cells and the native expressing cell line U138MG cells, through in vitro cell biology combined with FACS testing, it can be concluded from the data that the three control antibodies have binding signals with both cell lines. Among them, the binding signals of BIBH1 and cMFP5 are relatively high, and the binding signal of hu36 is slightly lower, as Figure 1a 、 Figure 1b shown:

[0466] Table 9 EC50 and fluorescence values of the control antibody flow cytometry binding experiment

[0467]

[0468] Preparation Example

[0469] 1. Generation of rabbit anti-human FAP monoclonal antibody (this work was entrusted to Shanghai Dinocean Biotech Co., Ltd. for development)

[0470] 1). Animal immunization

[0471] In order to obtain rabbit monoclonal antibodies that recognize human FAP antigen, the present invention selects New Zealand white rabbits for immunization. For the first immunization, 200 μg of human FAP-hFc protein (purchased from Acro Biosystems, product number FAP-H5263) was used. At the first immunization, Freund's complete adjuvant and an equal volume of antigen were mixed and emulsified, and then injected intradermally at multiple points on the back for immunization. After a two-week interval, booster immunization was carried out. The antigen dosage for booster immunization was 100 μg. Freund's incomplete adjuvant and an equal volume of antigen were mixed and emulsified, and then injected intradermally at multiple points on the back for immunization. Booster immunization was carried out once every two weeks. After five immunizations, the titer of the immune serum was detected by the conventional Elisa method. Rabbits with high titers were selected and given a boost injection of 50 μg of protein intraperitoneally three days before antibody screening. No adjuvant emulsification was required for this antigen, and the buffer was PBS. Three days later, the spleen was taken.

[0472] 2). Isolation of spleen cells

[0473] The rabbit spleen was surgically removed and placed in a sterile cell culture dish. The spleen was rinsed with DPBS containing 100 U / ml penicillin and 100 μg / ml streptomycin. The spleen was minced with surgical scissors and gently ground into single cells with a syringe plunger. Finally, the cell suspension was filtered through a 100 μm cell sieve, and the single cell filtrate was collected. Centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended with RPMI-1640 containing 5% fetal bovine serum.

[0474] 3). B cell culture and identification

[0475] Biotin-labeled FAP protein was co-incubated with successfully immunized rabbit lymphocytes to sort out rabbit memory B cells. In a 96-well cell culture plate, the cells were cultured using B cell medium at 37 °C and 5% CO2. After 10-14 days of culture, the binding activity of the clone supernatant was detected at the protein level by ELISA. Clones with a binding activity greater than 5 times the background were determined to be positive. The supernatant of ELISA-positive clones was detected by FACS for its binding to stably transfected cells CHO-hFAP and CHO-mFAP, and finally FACS-positive monoclonal clones were selected.

[0476] 4). Cloning of the gene encoding the rabbit monoclonal antibody

[0477] Collect B cell positive clones, select some positive clones, extract total RNA using RNAiso Plus and reverse transcribe it into cDNA. By PCR method, amplify the light chain variable region and heavy chain variable region sequences, and construct them onto the PTT5 expression vector containing the corresponding heavy chain constant region and light chain constant region for sequencing to obtain the correct sequence. Analyze the sequencing results using VBASE2 (http: / / www.vbase2.org / vbscAb.php) to obtain the light and heavy chain variable region sequences of the antibody.

[0478] 2. Preparation of anti-human FAP chimeric antibody

[0479] Ligate the heavy chain variable region sequence of the rabbit anti-human FAP monoclonal antibody with the heavy chain constant region sequence of the publicly available human monoclonal antibody IgG1 subclass and construct it into a mammalian cell expression vector; ligate the light chain variable region sequence of the rabbit anti-human FAP monoclonal antibody with the light chain constant region sequence of the publicly available human monoclonal antibody κ subclass and construct it into a mammalian cell expression vector. Mix the constructed heavy chain vector and light chain vector of the anti-human FAP chimeric antibody in pairs, transfect HEK293 cells using polyethyleneimine (PEI), collect the cell supernatant about 7 days later, and purify to obtain the anti-human FAP chimeric antibody protein using Mabselect.

[0480] Example 1 In vitro cell binding experiment of anti-human FAP chimeric antibody

[0481] Perform 4-fold serial dilution of the anti-human FAP chimeric antibody starting from an initial concentration of 20 μg / mL, with a total of 8 concentration points. Take 50 μL of the antibody at each concentration point and add it to a 96-well plate. Centrifuge the HT1080-hFAP cells with low surface expression of human FAP at 100 g for 5 minutes at room temperature, wash the cells once with PBS containing 0.5% BSA, centrifuge at 100 g for 5 minutes at room temperature, and resuspend the cells to a density of approximately 2×10 6 cells per milliliter. Take 50 μL and add it to the wells of the 96-well plate already containing the antibody. Incubate at 4°C for 1 hour, then add the APC-fluorescently labeled goat anti-human IgG secondary antibody. Continue to incubate at 4°C for 1 hour, and then analyze the average fluorescence reading of the cell population using a flow cytometer. Use prism software to perform a 4-parameter fitting curve, as Figure 2a 、 Figure 2b 。

[0482] Result analysis: Through in vitro cell biology binding tests with HT1080-hFAP cells expressing human FAP, several chimeric antibodies were obtained that were superior to the control hu36 in terms of both the EC50 and the top value of the fluorescence intensity MFI.

[0483] Table 10

[0484] Antibody Name EC50 MFI(Top) chrX1 0.1054 404752 chrX6 0.3469 436774 chrX8 0.8334 372077 chrX9 0.2238 350370 chrA7 0.1157 467802 chrA12 0.6685 629040 chrA18 1.036 372469 hu36 1.328 310733

[0485] Example 2 In vitro binding affinity and kinetics experiments of anti-human FAP chimeric antibodies

[0486] Using a Fortebio (BLITZ pro1.1.0.28) instrument, the antibody affinity was measured by the anti-human antibody capture method. During the measurement, the capture antibody (AHC) biosensor for the Fc segment of the anti-human antibody (purchased from SARTORIUS, product number 18-5060) was immersed in PBS for 10 min; 200 μl of the diluted antibody sample (including the chimeric antibody of the present invention and the control antibody; the working concentration of the antibody was 15 μg / mL) was loaded onto the AHC biosensor, and then equilibrated in PBS for 100 s. Further, the AHC biosensor was subjected to a binding reaction with human FAP protein and mouse FAP protein (purchased from ACRO biosystem) for 600 s. After that, the AHC biosensor was transferred to PBS for a dissociation reaction for 600 s. After the experiment, the blank control response value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constants of antigen-antibody binding.

[0487] Result analysis: Through the analysis of the in vitro kinetic binding activity, the binding kinetic constant of the chimeric antibody with recombinant human FAP protein was at the level of 10 -10 to 10 -12 level. In contrast, the binding kinetic constant of the reference antibody hu36 was at the level of 10 -9 level; the binding kinetic constant of the chimeric antibody with recombinant mouse FAP protein was at the level of 10 -9 to 10 -12 level. In contrast, the binding kinetic constant of the reference antibody hu36 was at the level of 10 -9 level. Each chimeric antibody had good kinetic binding signals with hFAP and mFAP.

[0488] Table 11 In vitro kinetic binding activity of chimeric antibodies with recombinant human FAP protein

[0489] Antibody Response Value KD(M) kon(1 / Ms) kdis(1 / s) chrX1 1.0048 <1.0E-12 5.16E+05 <1.0E-07 chrX6 0.8739 5.61E-11 4.15E+05 2.33E-05 chrX8 0.7973 <1.0E-12 2.54E+05 <1.0E-07 chrX9 0.9728 5.75E-10 4.07E+05 2.34E-04 chrA7 0.8483 5.26E-10 4.85E+05 2.55E-04 chrA12 0.5886 <1.0E-12 4.93E+05 <1.0E-07 chrA18 0.7021 6.66E-10 2.63E+05 1.75E-04 Hu36 0.8476 1.236E-09 6.60E+05 8.15E-04

[0490] Table 12 In vitro kinetic binding activity of chimeric antibodies with recombinant mouse FAP protein

[0491]

[0492]

[0493] Example 3 Intracellular endocytosis experiment of anti-human FAP chimeric antibody

[0494] Method steps: First, prepare HT1080-hFAP cells, digest and count them using a non-trypsin digestion solution (brand GIBCO, catalog number 13151014), resuspend them using a cell culture medium (brand GIBCO, catalog number 22400097), and adjust the cell density to 2E6 / mL; Resuspend the endocytosis reagent (purchased from Sartorius, catalog number 90565) with sterile water to a final concentration of 100 μg / mL; The molecular weight of the endocytosis reagent is approximately 1 / 3 of the antibody molecular weight. Therefore, when incubating the reagent and antibody at the same mass ratio during labeling, the molar ratio is 3:1. Dilute both the antibody and the endocytosis reagent to 2 μg / mL at a mass ratio of 1:1 using a cell culture medium (brand GIBCO, catalog number 22400097), and incubate and label them in a 37 °C incubator for 15 minutes; Dilute the labeled antibody reagent label in a 2-fold gradient for 7 points; Add the diluted antibody reagent label to the prepared cells, and place them in a 37 °C incubator for endocytosis for two hours; Take out the culture plate, directly use a flow analyzer to analyze and read the fluorescence and endocytosis percentage in the RL-1 channel, and fit the obtained %gate with a curve by the four-parameter method using prism software, as Figure 3a 、 Figure 3b shown.

[0495] Data analysis: Use the low-expression cell line HT1080-hFAP cells to test the endocytosis activity of the anti-human FAP chimeric antibody. According to the results, it can be seen that the chimeric antibodies chrX1, chrX6, chrX9, as well as chrA7 and chrA12, can all be endocytosed relatively quickly, equivalent to or better than the control antibody hu36.

[0496] Table 13 Statistical table of EC50 for the endocytosis experiment of anti-human FAP chimeric antibodies

[0497] Antibody Name EC50 chrX1 0.04774 chrX6 0.0633 chrX8 0.3673 chrX9 0.06337 chrA7 0.07154 chrA12 0.08361 chrA18 0.2441 hu36 0.1405

[0498] Example 4 Humanization of a rabbit-derived anti-human FAP antibody

[0499] Based on the antibody coding schemes of Kabat and Chothia, the amino acid sequence regions of six antigen - complementary determining regions (CDRs) of the heavy and light chains of rabbit - derived antibodies and the framework regions that support the conserved three - dimensional conformation of the antibody were determined. Subsequently, by analyzing and searching known human antibody sequences, the human heavy - chain variable - region sequence most similar to the rabbit - derived antibody was selected, such as IGHV1|IGHJ4*01, and its antibody framework - region sequence was used as a template. The rabbit - derived heavy - chain CDRs were combined with the human antibody framework region to finally generate the humanized heavy - chain variable - region sequence. Through the same process, the humanized light - chain variable - region sequence was generated. When the CDRs of rabbit - derived antibodies are directly transplanted into the human framework region, the binding activity of the antibody often drops sharply. Therefore, individual amino acids in the framework region need to be changed back from human to rabbit - derived. To determine the sites of back - mutation, first, compare the designed humanized antibody sequence with the original rabbit - derived antibody sequence to check which amino acids are different; second, check whether these amino acids play an important role in supporting the antibody structure or in binding to the antigen. At the same time, when checking the sequence after humanization design, it is necessary to check whether there are some potential post - translational modification sites, such as N (asparagine) glycosylation sites, N - deamidation sites, D (aspartic acid) isomerization sites, etc.

[0500] The humanized antibody variable - region heavy - chain gene was constructed into a mammalian - cell expression vector containing the heavy - chain constant - region gene of the human monoclonal antibody IgG1 subclass; the light - chain gene was constructed into a mammalian - cell expression vector containing the light - chain constant - region gene of the human monoclonal antibody κ subclass. The constructed heavy - chain vector and light - chain vector of the anti - human FAP humanized antibody were paired and mixed, and HEK293 cells were transfected using polyethyleneimine (PEI). About 7 days later, the cell supernatant was collected, and the anti - human FAP humanized antibody protein was purified using Mabselect.

[0501] Example 5 In vitro binding affinity and kinetics experiments of the anti - human FAP humanized antibody

[0502] The antibody affinity was determined by the anti-human antibody capture method using the Fortebio (BLITZ pro1.1.0.28) instrument. During the determination, the capture antibody (AHC) bioprobe of the anti-human antibody Fc segment (purchased from SARTORIUS, item number 18-5060) was soaked in PBS for 10 min; 200 μl of the diluted antibody sample (including the chimeric antibody of the present invention and the control antibody; the antibody working concentration was 15 μg / mL) was loaded onto the AHC bioprobe, and then equilibrated in PBS for 100 s, and the AHC probe was further combined with a series of gradients of human FAP protein, monkey FAP protein and mouse FAP protein (purchased from ACRO biosystem, item number FAP-M52H3) for 600 s. After that, the AHC probe was transferred to PBS for a dissociation reaction for 600 s. After the experiment was completed, the blank control response value was deducted, and the 1:1 Langmuir binding mode fitting was performed using the software, the kinetic constants of antigen-antibody binding were calculated, and the curve processing and fitting were performed using the instrument's own software.

[0503] Data analysis: After in vitro kinetic binding activity analysis, the binding kinetic constants of anti-human FAP humanized antibody to recombinant human FAP protein, recombinant mouse FAP protein and recombinant monkey FAP protein were all maintained at 10 -12 In contrast, the binding kinetic constants of the benchmark antibody hu36 to human, mouse, and monkey FAP proteins were between 10 -10 Up to 10 -12 Each humanized antibody has a good kinetic binding signal.

[0504] Table 14 In vitro kinetic binding data of humanized antibodies and recombinant human FAP protein

[0505] Antibody Name Response Value KD(M) kon(1 / Ms) kdis(1 / s) hu36 0.6156 <1.0E-12 6.29E+05 <1.0E-07 hzX6-H29L3 0.5686 <1.0E-12 4.60E+05 <1.0E-07 hzX6-H29L10 0.5886 <1.0E-12 4.61E+05 <1.0E-07 hzX6-H29L12 0.4717 <1.0E-12 5.42E+05 <1.0E-07 hzX6-H32L3 0.4859 <1.0E-12 5.41E+05 <1.0E-07 hzX6-H32L10 0.6159 <1.0E-12 4.71E+05 <1.0E-07 hzX6-H32L12 0.5106 <1.0E-12 5.46E+05 <1.0E-07 hzX6-H33L3 0.5203 <1.0E-12 5.55E+05 <1.0E-07 hzX6-H33L10 0.6188 <1.0E-12 4.85E+05 <1.0E-07 hzX6-H33L12 0.4287 <1.0E-12 4.28E+05 <1.0E-07

[0506] Table 15 In vitro kinetic binding data of humanized antibodies and recombinant monkey FAP protein

[0507] Antibody Name Response Value KD(M) kon(1 / Ms) kdis(1 / s) hu36 0.6399 9.89E-10 9.68E+05 9.57E-04 hzX6-H29L3 0.6586 <1.0E-12 4.70E+05 <1.0E-07 hzX6-H29L10 0.6652 <1.0E-12 4.81E+05 <1.0E-07 hzX6-H29L12 0.5585 <1.0E-12 5.36E+05 <1.0E-07 hzX6-H32L3 0.5651 <1.0E-12 5.47E+05 <1.0E-07 hzX6-H32L10 0.6912 <1.0E-12 4.86E+05 <1.0E-07 hzX6-H32L12 0.5847 <1.0E-12 5.58E+05 <1.0E-07 hzX6-H33L3 0.5639 <1.0E-12 6.66E+05 <1.0E-07 hzX6-H33L10 0.6893 <1.0E-12 5.01E+05 <1.0E-07 hzX6-H33L12 0.503 <1.0E-12 4.44E+05 <1.0E-07

[0508] Table 16 In vitro kinetic binding data of humanized antibodies and recombinant mouse FAP protein

[0509] Antibody Name Response Value KD(M) kon(1 / Ms) kdis(1 / s) hu36 0.6427 4.95E-10 8.69E+05 4.30E-04 hzX6-H29L3 0.6239 <1.0E-12 5.87E+05 <1.0E-07 hzX6-H29L10 0.6287 <1.0E-12 6.03E+05 <1.0E-07 hzX6-H29L12 0.5394 <1.0E-12 6.47E+05 <1.0E-07 hzX6-H32L3 0.5479 <1.0E-12 6.62E+05 <1.0E-07 hzX6-H32L10 0.6512 <1.0E-12 5.82E+05 <1.0E-07 hzX6-H32L12 0.5609 <1.0E-12 6.41E+05 <1.0E-07 hzX6-H33L3 0.5639 <1.0E-12 6.66E+05 <1.0E-07 hzX6-H33L10 0.6509 <1.0E-12 6.01E+05 <1.0E-07 hzX6-H33L12 0.4982 <1.0E-12 5.32E+05 <1.0E-07

[0510] Example 6 In vitro cell binding experiment of anti-human FAP humanized antibody

[0511] The anti-human FAP humanized antibody was serially diluted 4-fold starting from an initial concentration of 20 μg / mL, with a total of 8 concentration points. 50 μL of the antibody at each concentration point was added to a 96-well plate. HT1080-hFAP cells with low surface expression of human FAP, 293-cynoFAP cells with high expression of cynomolgus FAP, and CT26-mFAP cells overexpressing murine FAP were collected by centrifugation at 100 g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100 g for 5 minutes at room temperature, and resuspended to a density of approximately 2 x 10^6 cells per milliliter. 50 μL of the cell suspension was added to the wells of the 96-well plate containing the antibody. After incubation at 4°C for 1 hour, APC-conjugated goat anti-human IgG secondary antibody was added. After continued incubation at 4°C for 1 hour, the mean fluorescence readings of the cell population were analyzed using a flow cytometer. Prism software was used to perform a four-parameter fitting curve and calculate the EC50 and the top value of the fluorescence intensity, as Figure 4a and Figure 4b 、 Figure 4c shown.

[0512] Data analysis: In vitro cell binding assays were performed with the humanized antibody against low-expressing hFAP cells (HT1080-hFAP cells), overexpressing mFAP cells (CT26-mFAP cells), and CHO-cynoFAP cells overexpressing cynoFAP protein. The results showed that most of the humanized molecules with different heavy and light chain combinations could maintain the binding ability to HT1080-hFAP cells, CT26-mFAP cells, and CHO-cynoFAP cells, and the binding signal was not lower than the signal value of the reference antibody hu36 at high concentrations.

[0513] Table 17 In vitro flow cytometry binding EC50 and top values of fluorescence intensity of the humanized antibody to HT1080-hFAP, 293-cynoFAP cells, and CT26-mFAP cells

[0514]

[0515] Example 7 In vitro cell non-specific binding test of the anti-human FAP humanized antibody

[0516] The anti-human FAP affinity-matured combinatorial molecular antibody was serially diluted 4-fold starting from a concentration of 20 μg / mL for 4 gradients. 50 μL of the diluted antibody was added to a 96-well plate. CHO-hDPP4 cells overexpressing human DPP4 on the cell surface, Chinese hamster ovary cells CHOK1, and human embryonic kidney cells HEK293 were collected by centrifugation at 100 g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100 g for 5 minutes at room temperature, and resuspended to a density of approximately 2 x 10^6 cells per milliliter. 50 μL of the cell suspension was added to the wells of the 96-well plate containing the antibody. After incubation at 4°C for 1 hour, APC-conjugated goat anti-human IgG secondary antibody was added. After continued incubation at 4°C for 1 hour, the mean fluorescence readings of the cell population were analyzed using a flow cytometer, and a histogram was presented using Prism software, as shown in Figure 5a and 5b 5c.

[0517] Data analysis: A non-specific binding experiment of the anti-human FAP humanized antibody was carried out with the cell line CHO-hDPP4 expressing the same family protein DPP4 and the commonly used protein-expressing cell lines Chinese hamster ovary cells CHOK1 and human embryonic kidney cells HEK293. The results showed that there was no non-specific binding signal between each anti-human FAP humanized antibody molecule and the same family protein or the empty cells used for expression.

[0518] Table 18

[0519]

[0520] Example 8 Detection of the endocytic activity of the anti-human FAP humanized antibody molecule

[0521] Method steps: First, HT1080-hFAP cells were prepared, digested and counted using a non-trypsin digestion solution (brand GIBCO, product number 13151014), resuspended using a cell culture medium (brand GIBCO, product number 22400097), and the cell density was adjusted to 2E6 / mL; an endocytic reagent (purchased from Sartorius, product number 90565) was reconstituted with sterile water to a final concentration of 100 μg / mL; the molecular weight of the endocytic reagent was approximately 1 / 3 of the antibody molecular weight, so when incubating with the same mass ratio of the reagent and the antibody, the molar ratio was 3:1. The antibody and the endocytic reagent were both diluted to 2 μg / mL in a 1:1 mass ratio using the cell culture medium and incubated for labeling at 37°C in an incubator for 15 minutes; the labeled antibody reagent label was serially diluted 2-fold for 7 points; the diluted antibody reagent label was added to the prepared cells and placed in a 37°C incubator for endocytosis for two hours; the culture plate was taken out, and the fluorescence and endocytosis percentage were directly analyzed using a flow analyzer in the RL-1 channel. The obtained %gate was curve-fitted by the four-parameter method using Prism software, as shown in Figure 6 as shown.

[0522] Result analysis: The endocytosis activity of the anti-human FAP humanized antibody was tested using the low-expression cell line HT1080-hFAP cells. According to the results, it can be seen that each humanized antibody can be endocytosed relatively quickly.

[0523] Example 9 In vitro killing experiment of anti-human FAP humanized antibody

[0524] For the in vitro killing experiment, two low-expression cell lines, HT1080-hFAP and 293-hFAP, were used. The cells were digested and counted, and the cell density was adjusted to 5E4 / mL using the corresponding culture medium (RPMI1640 medium for HT1080-hFAP cells and DMEM medium for 293-hFAP cells), and 50 μl / well was plated in a 96-well white plate; the small molecule toxin coupling reagent 20ADCαHFc-CL-MMAE (purchased from moradec, product number AH-102AE-50) was diluted to 2 μg / mL using the corresponding cell culture medium; the humanized antibody was diluted to 0.4 μg / mL using the diluted small molecule toxin coupling reagent, and then serially diluted 3-fold to set 10 gradients; 50 μl / well of the diluted series of antibodies was added to the white plate seeded with cells; it was placed in an incubator at 37 °C for 4 days; CellTiter-Glo (purchased from promega, product number G7573) was prepared according to the reagent instruction manual, and 100 μl / well was added to the cell plate. After shaking and mixing for 5 minutes, the chemiluminescence was read using a microplate reader; the killing curve was fitted with four parameters using prism software, as Figure 7a - 7f .

[0525] Data analysis: The in vitro killing experiment was carried out using the low-expression cell line HT1080-hFAP and the over-expression cell line 293-hFAP. The results showed that each anti-human FAP humanized antibody molecule had in vitro cell killing activity, and among them, H29L12, H32L12, and H33L12 had better killing activity, similar to the control antibody hu36.

[0526] Table 19 Data of in vitro killing experiment of anti-human FAP humanized antibody

[0527]

[0528] Example 10 Analysis of monomer ratio of physical characterization of anti-human FAP humanized antibody molecule

[0529] Experimental instrument: UPLC CLASS ACQUITYH (WATERS)

[0530] Analysis column: TSKgel G3000SWXL 7.8*300 (TOSHI, CatNo 003C03326C

[0531] Analysis solution: 200 mM K2HPO4, 250 mM KCl, pH adjusted to 6.2 with HCl

[0532] Analysis method: Inject 50 μl of an antibody solution with a concentration of 1 mg / ml into a pre-equilibrated chromatography column. At room temperature, flow at a rate of 0.75 ml / min for 45 min, and simultaneously detect the absorbance at A280 of the machine. Determine the monomer content and ratio of the antibody based on the peak elution time and peak volume.

[0533] Result analysis: Conduct monomer ratio analysis on the anti-human FAP humanized antibody molecule. The results show that the monomer ratios of the molecules are all above 95%, and the monomer ratio properties are good.

[0534] Table 20 Main peak retention time and monomer ratio of the antibody

[0535]

[0536]

[0537] Example 11 Hydrophobic property analysis of humanized antibody

[0538] Experimental instrument: ARC (Waters)

[0539] Analytical column for experiment: TSKgel Butyl-NPR (4.6 mm X 3.5 cm, CatNo 14947)

[0540] Analysis solution: A. 20 mM Histidine, pH 6.0;

[0541] B. 20 mM Histidine, 1.6 M (NH4)2SO4

[0542] Analysis method: Analyze the hydrophobic properties of the antibody according to the instructions for use of the hydrophobic chromatography column.

[0543] Result analysis: Conduct hydrophobic property analysis on the anti-human FAP humanized antibody molecule. The results show that the hydrophobic HIC values of each combined molecule are all greater than 0.7, and the hydrophobic properties are good.

[0544] Table 21 Hydrophobic properties of humanized antibody

[0545] Antibody Retention Time of Main Peak (min) HIC Hu36 13.579 1.08 hzX6-H29L3 13.409 1.09 hzX6-H29L10 13.358 1.10 hzX6-H32L10 13.362 1.10 hzX6-H33L10 13.368 1.10

[0546] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Recombinant anti-FAP antibody, characterized in that, It includes a heavy chain and a light chain: The CDR regions of the heavy chain include CDR1, CDR2, and CDR3: (Ⅰ) The CDR1 of the heavy chain has an amino acid sequence as shown in SEQ ID No.2, 16, 27, 38, 48, 56, 67, or 107; and (Ⅱ) The CDR2 of the heavy chain has an amino acid sequence as shown in SEQ ID No.4, 18, 29, 40, 49, 57, or 69; and (Ⅲ) The CDR3 of the heavy chain has an amino acid sequence as shown in SEQ ID No.6, 19, 31, 42, 59, 71, or 108; or (Ⅳ) A sequence with substitution, deletion, addition, and / or replacement of one or more amino acids based on the amino acid sequence shown in any one of (Ⅰ) to (Ⅲ); or (V) A sequence with a homology of more than 80% to the amino acid sequence shown in any one of (Ⅰ) to (Ⅳ); The CDR regions of the light chain include CDR1, CDR2, and CDR3: (Ⅵ) The CDR1 of the light chain has an amino acid sequence as shown in SEQ ID No.9, 21, 34, 44, 52, 62, 73, or 112; and (Ⅶ) The CDR2 of the light chain has an amino acid sequence as shown in SEQ ID No.11, 35, 45, 63, or 74; and (Ⅷ) The CDR3 of the light chain has an amino acid sequence as shown in SEQ ID No.13, 24, 37, 47, 55, 65, 76, 116, or 117; or (Ⅸ) A sequence with substitution, deletion, addition, and / or replacement of one or more amino acids based on the amino acid sequence shown in any one of (Ⅵ) to (Ⅷ); or (Ⅹ) A sequence with a homology of more than 80% to the amino acid sequence shown in any one of (Ⅵ) to (Ⅸ).

2. The recombinant anti-FAP antibody according to claim 1, wherein The recombinant anti-FAP antibody includes a rabbit-derived chimeric antibody and a humanized antibody; Optionally, the rabbit-derived chimeric antibody includes chrX1, chrX6, chrX8, chrX9, chrA7, chrA12, or chrA18; a) The CDR1, CDR2, and CDR3 of the heavy chain of chrX1 have amino acid sequences shown in SEQ ID No.2, 4, and 6 in sequence; and The CDR1, CDR2, and CDR3 of the light chain of chrX1 have amino acid sequences shown in SEQ ID No.9, 11, and 13 in sequence; or b) The CDR1, CDR2, and CDR3 of the heavy chain of chrX6 have amino acid sequences shown in SEQ ID No.16, 18, and 19 in sequence; and The CDR1, CDR2, and CDR3 of the light chain of chrX6 have amino acid sequences shown in SEQ ID No.21, 11, and 24 in sequence; or c) The CDR1, CDR2, and CDR3 of the heavy chain of chrX8 have amino acid sequences shown in SEQ ID No.27, 29, and 31 in sequence; and The CDR1, CDR2 and CDR3 of the light chain of the said chrX8 have the amino acid sequences shown in SEQ ID No.34, 35 and 37 respectively; or d), The CDR1, CDR2 and CDR3 of the heavy chain of the said chrX9 have the amino acid sequences shown in SEQ ID No.38, 40 and 42 respectively; and The CDR1, CDR2 and CDR3 of the light chain of the said chrX9 have the amino acid sequences shown in SEQ ID No.44, 45 and 47 respectively; or e), The CDR1, CDR2 and CDR3 of the heavy chain of the said chrA7 have the amino acid sequences shown in SEQ ID No.48, 49 and 6 respectively; and The CDR1, CDR2 and CDR3 of the light chain of the said chrA7 have the amino acid sequences shown in SEQ ID No.52, 11 and 55 respectively; or f), The CDR1, CDR2 and CDR3 of the heavy chain of the said chrA12 have the amino acid sequences shown in SEQ ID No.56, 57 and 59 respectively; and The CDR1, CDR2 and CDR3 of the light chain of the said chrA12 have SEQ ID No.62, 63 and 65 respectively; or g), The CDR1, CDR2 and CDR3 of the heavy chain of the said chrA18 have the amino acid sequences shown in SEQ ID No.67, 69 and 71 respectively; and The CDR1, CDR2 and CDR3 of the light chain of the said chrA18 have the amino acid sequences shown in SEQ ID No.73, 74 and 76 respectively; or h), A sequence obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequence shown in any one of a) - g); or j), A sequence having a homology of more than 80% with the amino acid sequence shown in any one of a) - h).

3. The recombinant anti-FAP antibody according to claim 2, wherein The said humanized antibody includes hzX6 - H29L3, hzX6 - H29L10, hzX6 - H29L12, hzX6 - H32L3, hzX6 - H32L10, hzX6 - H32L12, hzX6 - H33L3, hzX6 - H33L10 or hzX6 - H33L12; k), The CDR1, CDR2 and CDR3 of the heavy chain of the said hzX6 - H29L3 have the amino acid sequences shown in SEQ ID No.107, 18 and 108 respectively; and The CDR1, CDR2 and CDR3 of the light chain of the said hzX6 - H29L3 have the amino acid sequences shown in SEQ ID No.112, 11 and 24 respectively; or l), The CDR1, CDR2 and CDR3 of the heavy chain of the said hzX6 - H29L10 have the amino acid sequences shown in SEQ ID No.107, 18 and 108 respectively; and The CDR1, CDR2 and CDR3 of the light chain of the said hzX6 - H29L10 have the amino acid sequences shown in SEQ ID No.112, 11 and 116 respectively; or m) The CDR1, CDR2, and CDR3 of the heavy chain of hzX6-H29L12 have the amino acid sequences shown in SEQ ID No. 107, 18, and 108, respectively; and the CDR1, CDR2, and CDR3 of the light chain of hzX6-H29L12 have the amino acid sequences shown in SEQ ID No. 112, 11, and 117, respectively; or n) The CDR1, CDR2, and CDR3 of the heavy chain of hzX6-H32L3 have the amino acid sequences shown in SEQ ID No. 107, 18, and 108, respectively; and the CDR1, CDR2, and CDR3 of the light chain of hzX6-H32L3 have the amino acid sequences shown in SEQ ID No. 112, 11, and 24, respectively; or o) The CDR1, CDR2, and CDR3 of the heavy chain of hzX6-H32L10 have the amino acid sequences shown in SEQ ID No. 107, 18, and 108, respectively; and the CDR1, CDR2, and CDR3 of the light chain of hzX6-H32L10 have the amino acid sequences shown in SEQ ID No. 112, 11, and 116, respectively; or p) The CDR1, CDR2, and CDR3 of the heavy chain of hzX6-H32L12 have the amino acid sequences shown in SEQ ID No. 107, 18, and 108, respectively; and the CDR1, CDR2, and CDR3 of the light chain of hzX6-H32L12 have the amino acid sequences shown in SEQ ID No. 112, 11, and 117, respectively; or q) The CDR1, CDR2, and CDR3 of the heavy chain of hzX6-H33L3 have the amino acid sequences shown in SEQ ID No. 107, 18, and 108, respectively; and the CDR1, CDR2, and CDR3 of the light chain of hzX6-H33L3 have the amino acid sequences shown in SEQ ID No. 112, 11, and 24, respectively; or r) The CDR1, CDR2, and CDR3 of the heavy chain of hzX6-H33L10 have the amino acid sequences shown in SEQ ID No. 107, 18, and 108, respectively; and the CDR1, CDR2, and CDR3 of the light chain of hzX6-H33L10 have the amino acid sequences shown in SEQ ID No. 112, 11, and 116, respectively; or s) The CDR1, CDR2, and CDR3 of the heavy chain of hzX6-H33L12 have the amino acid sequences shown in SEQ ID No. 107, 18, and 108, respectively; and the CDR1, CDR2, and CDR3 of the light chain of hzX6-H33L12 have the amino acid sequences shown in SEQ ID No. 112, 11, and 117, respectively; or t) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of k) to s); or u) A sequence with a homology of more than 80% to the amino acid sequence shown in any one of k) to t).

4. The recombinant anti-FAP antibody according to any one of claims 1 to 3, characterized in that i) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 78; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 79; or ii) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 80; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 81; or iii) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 82; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 83; or iv) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 84; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 85; or v) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 86; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 87; or vi) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 88; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 89; or vii) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 90; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 91; or viii) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 118; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 121; or ix) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 119; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 122; or x) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. 120; and The variable region of the light chain has the amino acid sequence shown in SEQ ID No. 123; xi) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequence shown in any one of i) to x); or xii) A sequence with a homology of more than 80% to the amino acid sequence shown in any one of i) to xi).

5. The recombinant anti-FAP antibody according to any one of claims 2 to 4, characterized in that, The rabbit-derived chimeric antibody comprises: 1) The variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 78; and The variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 79; or 2) The variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 80; and The variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 81; or 3), the variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 82; and the variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 83; or 4), the variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 84; and the variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 85; or 5), the variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 86; and the variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 87; or 6), the variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 88; and the variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 89; or 7), the variable region of the heavy chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 90; and the variable region of the light chain of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID No. 91; or 8), a sequence in which one or more amino acids are substituted, deleted, added, and / or replaced on the basis of the amino acid sequence shown in any one of 1) to 7); or 9), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of 1) to 8).

6. The recombinant anti-FAP antibody according to any one of claims 2 to 5, characterized in that, The humanized antibody: 1), the variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in 118; and the variable region of the light chain of the humanized antibody has the amino acid sequence shown in 121; or 2), the variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in 119; and the variable region of the light chain of the humanized antibody has the amino acid sequence shown in 122; or 3), the variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in 120; and the variable region of the light chain of the humanized antibody has the amino acid sequence shown in 123; or 4), a sequence in which one or more amino acids are substituted, deleted, added, and / or replaced on the basis of the amino acid sequence shown in any one of 1) to 3); or 5), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of 1) to 4).

7. The recombinant anti-FAP antibody according to any one of claims 1 to 6, characterized in that, It also includes a constant region; The heavy chain constant region of the recombinant anti-FAP antibody includes human IgG1; the light chain constant region of the recombinant anti-FAP antibody includes human kappa type; Optionally, the FAP includes human FAP, mouse FAP, and / or cynomolgus monkey FAP.

8. The preparation method of the recombinant anti-FAP antibody according to any one of claims 1 to 7, characterized in that, It includes the following steps: Step 1: Immunize a receptor with FAP antigen, isolate the spleen cells of the receptor, and PCR amplify the light and heavy chain variable regions of the recombinant anti-FAP antibody; Step 2: Splice the heavy chain variable region and the heavy chain constant region, construct them into an expression vector to obtain a heavy chain vector; splice the light chain variable region and the light chain constant region, construct them into the expression vector to obtain a light chain vector. Step 3: Take the heavy chain vector and the light chain vector, transfect, culture, purify, and screen to obtain the recombinant anti-FAP antibody.

9. The preparation method according to claim 8, wherein The receptor includes New Zealand white rabbits. The heavy chain constant region is the heavy chain constant region of human IgG1 subclass. The light chain constant region is the light chain constant region of human κ class antibody. The constructed expression vector is a mammalian cell expression vector.

10. A biological material, characterized in that, It includes any of the following: (a) Nucleic acid encoding the recombinant anti-FAP antibody according to any one of claims 1 to 7 or the recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9; and / or (b) Nucleic acid encoding the recombinant anti-FAP antibody according to any one of claims 1 to 7 and an acceptable vector; or The recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9 and an acceptable vector; and / or (c) A host secreting the recombinant anti-FAP antibody according to any one of claims 1 to 7 or the recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9; and / or (d) Chemically or biologically labeled recombinant anti-FAP antibody according to any one of claims 1 to 7 or the recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9; and / or (e) The recombinant anti-FAP antibody according to any one of claims 1 to 7 or the recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9 coupled with a vector; (f) An antibody-drug conjugate obtained by covalently coupling the recombinant anti-FAP antibody according to any one of claims 1 to 7 or the recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9 with a drug through a linker.

11. Use of any of the following in the preparation of a drug targeting FAP: ① The recombinant anti-FAP antibody according to any one of claims 1 to 7; and / or ② The recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9; and / or ③ The biological material according to claim 10.

12. Use of any of the following in the preparation of a product for preventing and / or treating diseases: ① The recombinant anti-FAP antibody according to any one of claims 1 to 7; and / or ② The recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9; and / or ③ The biological material according to claim 10.

13. The application according to claim 12, characterized in that, The diseases include any one or more of human epithelial cancer, breast cancer, pancreatic cancer, lung cancer, bladder cancer or colon cancer.

14. The application according to claim 12 or 13, characterized in that, The product includes drugs and / or vaccines.

15. A drug, characterized in that, It includes any of the following and pharmaceutically acceptable excipients: ① The recombinant anti-FAP antibody according to any one of claims 1 to 7; and / or ② The recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9; and / or ③ The biological material according to claim 10.

16. A pharmaceutical combination, characterized in that, It includes the drug according to claim 15 and any other active ingredient.

17. The pharmaceutical combination according to claim 16, wherein, The any other active ingredient includes small molecule toxins.

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