Recombinant Anti-FAP antibody and use thereof

Abstract

The present invention relates to the field of antibody drugs, and in particular to a recombinant anti-fibroblast activation protein (FAP) antibody and a use thereof. Provided are an antibody protein that specifically binds to FAPα, a use of the antibody protein for diagnostic and therapeutic purposes, and a method for producing the antibody. Experiments show that the provided anti-FAP antibody has better efficacy and lower immunogenicity, and exhibits highly specific binding and fast and efficient internalization. The anti-FAP antibody has the potential to become therapeutic monoclonal antibodies due to a high affinity, high selectivity and a high biological activity level thereof.

Description

Recombinant anti-FAP antibodies and their applications

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on January 15, 2024, with application number 202410055515.5 and invention name “Recombinant anti-FAP antibodies and their applications”, the entire contents of which are incorporated by reference into this application. Technical Field

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

[0003] Malignant epithelial tumors are the leading cancer-related diseases that cause death in humans. These solid tumors often exhibit significant stromal reactions, such as those referred to as "desmoplastic stroma" or "reactive stroma," which account for 20-60% of the overall tumor mass and are 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 alpha (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 originally 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 cancer, the fibroblast response is characterized by the induction of a cell surface protein, fibroblast activation protein α (FAPα), a 95 kDa serine protease whose expression is highly restricted to developing organs, wound healing, and tissue remodeling.

[0005] FAP presents with the following features:

[0006] 1) Type II membrane glycoprotein with serine protease activity;

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

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

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

[0010] 5) have a faster internalization ability;

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

[0012] In concerted interactions with various components of the stroma, CAFs are able to promote neovascularization and tumor growth. CAFs have also been shown to be crucial for the development of aggressive tumors and tumor invasiveness during cancer progression. CAFs promote the spread and invasion of tumor cells into distant organs, thereby contributing to the formation of metastases. Importantly, stromal cells have also been implicated in the failure of systemic drug delivery to tumors and the emergence of drug resistance.

[0013] The identification of cellular and molecular targets that can abrogate stroma-tumor cell interactions and thereby attenuate tumorigenesis is currently a very important topic in translational oncology. Indeed, targeting the peritumoral stroma is a novel strategy for treating metastatic tumors, which account for over 90% of cancer patient mortality: only a few products have been approved for treatment to date, most of which are anti-angiogenic drugs. The identification and targeting of additional novel molecules within the tumor microenvironment is essential to increase the efficacy of conventional therapies in combination with stroma-based treatment approaches and represents a powerful approach for the treatment of cancer and metastasis.

[0014] To alter the non-human immunogenicity of the monoclonal antibody F19 (derived from the hybridoma cell line ATCC accession number HB 8269), antibody F19 was humanized with sibrotuzumab (BIBH1), a humanized antibody that specifically binds to FAP described in WO 99 / 57151. Phase I clinical studies targeting BIBH1, an anti-FAP monoclonal antibody radiolabeled with I31, showed good safety and tolerability (Scott et al., 2001; Hofheinz et al., 2003). However, in a Phase II clinical study in patients with advanced metastatic colorectal cancer, the majority of patients experienced persistent tumor progression, and the study was discontinued because minimal response was not achieved.

[0015] Boehringer-Ingelheim has developed anti-FAP MAb derivatives directed against both human and murine FAP proteins (US 2009 / 0304718 A1). They have been shown to specifically bind to FAP+ cells and be 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 intolerance-related effects.

[0016] hu36, an anti-FAP antibody developed by ONCOMATRYX BIOPHARMA, exhibits highly specific binding and rapid internalization. Furthermore, the inventors isolated the A chain of Nigrin b, which is produced in bacterial host cells, and conjugated it to an anti-FAP antibody for tumor treatment. It demonstrated tumor growth inhibition in multiple in vitro tumor models.

[0017] Despite these advances, there remains an unmet need for additional therapeutic strategies for the treatment of tumors including epithelial tumors, and for components for use in such therapeutic strategies. Summary of the Invention

[0018] In light of this, the present invention provides recombinant anti-FAP antibodies and their uses. The present invention provides antibody proteins that specifically bind to fibroblast activation protein alpha (FAPα), uses of such antibody proteins for diagnostic and therapeutic purposes, and methods for producing such antibodies. Experimental findings indicate that the humanized anti-FAP antibodies provided herein have reduced immunogenicity, exhibit highly specific binding, and can cross-bind proteins from different species, including human, mouse, and monkey. Their high affinity, selectivity, and biological activity make them suitable for therapeutic monoclonal antibodies.

[0019] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0020] The present invention provides a recombinant anti-FAP antibody, comprising a heavy chain and a light chain;

[0021] The heavy chain CDR region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0022] (I), the heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO. 2, 16, 29, 59, 67, 75 or 158; and

[0023] (II), the heavy chain CDR2 has an amino acid sequence as shown in SEQ ID NO. 4, 18, 30, 39, 49, 60, 68, 76, 85, 94 or 102; and

[0024] (III) the heavy chain CDR3 has an amino acid sequence as shown in SEQ ID NO. 6, 20, 32, 41, 51, 62, 70, 78, 87, 96, 104 or 161; or

[0025] (IV) an amino acid sequence as described in any one of (I) to (III) with one or more amino acids substituted, deleted or added; or

[0026] (V) an amino acid sequence having 80% or more identity with the amino acid sequence described in any one of (I) to (IV);

[0027] The light chain CDR region includes light chain CDR1, light chain CDR2 and light chain CDR3;

[0028] (VI), the light chain CDR1 has an amino acid sequence as shown in SEQ ID NO. 9, 23, 35, 44, 53, 64, 72, 80, 89, 98 or 105; and

[0029] (VII), the light chain CDR2 has an amino acid sequence as shown in SEQ ID NO. 11, 25, 36, 46, 55, 73, 91, 99 or 159; and

[0030] (VIII), the light chain CDR3 has the amino acid sequence shown in SEQ ID NO. 13, 27, 38, 48, 57, 66, 74, 83, 93, 100, 106 or 162; or

[0031] (IX) A sequence in which one or more amino acids are substituted, deleted or added to the amino acid sequence of any one of (VI) to (IX); or

[0032] (X) An amino acid sequence that is 80% or more identical to the amino acid sequence described in any one of (VI) to (X).

[0033] In some specific embodiments of the present invention, the recombinant anti-FAP antibody comprises one or more of a rabbit chimeric antibody, a humanized antibody, an affinity matured antibody, or a single-chain antibody;

[0034] The CDR region of the heavy chain of the rabbit chimeric antibody includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0035] (1) The heavy chain CDR1 has an amino acid sequence as shown in SEQ ID NO. 2, 16, 29, 59, 67 or 75; and

[0036] (2) the heavy chain CDR2 has an amino acid sequence as shown in SEQ ID NO. 4, 18, 30, 39, 49, 60, 68, 76, 85, 94 or 102; and

[0037] (3) The heavy chain CDR3 has an amino acid sequence as shown in SEQ ID NO. 6, 20, 32, 41, 51, 62, 70, 78, 87, 96 or 104; or

[0038] (4) an amino acid sequence as described in any one of (1) to (3) with one or more amino acids substituted, deleted or added; or

[0039] (5) an amino acid sequence having 80% or more identity with the amino acid sequence described in any one of (1) to (4);

[0040] The CDR region of the light chain of the rabbit chimeric antibody includes light chain CDR1, light chain CDR2 and light chain CDR3;

[0041] (6) the light chain CDR1 has an amino acid sequence as shown in SEQ ID NO. 9, 23, 35, 44, 53, 64, 72, 80, 89, 98 or 105; and

[0042] (7) the light chain CDR2 has an amino acid sequence as shown in SEQ ID NO. 11, 25, 36, 46, 55, 73, 91 or 99; and

[0043] (8) The light chain CDR3 has an amino acid sequence as shown in SEQ ID NO. 13, 27, 38, 48, 57, 66, 74, 83, 93, 100 or 106; or

[0044] (9) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in any one of (6) to (8), and having the same function as the amino acid sequence described in any one of (6) to (8); or

[0045] (10) An amino acid sequence having 80% or more identity with the amino acid sequence described in any one of (6) to (9).

[0046] In some specific embodiments of the present invention, the rabbit-derived chimeric antibody comprises a heavy chain and a light chain;

[0047] (11), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 2, 4 and 6, respectively; and

[0048] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 9, 11 and 13, respectively; or

[0049] (12) the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 16, 18 and 20, respectively; and

[0050] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 23, 25 and 27, respectively; or

[0051] (13) the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 29, 30 and 32, respectively; and

[0052] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 35, 36 and 38, respectively; or

[0053] (14) the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 16, 39 and 41, respectively; and

[0054] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 44, 46 and 48, respectively; or

[0055] (15) the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 16, 49 and 51, respectively; and

[0056] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 53, 55 and 57, respectively; or

[0057] (16) the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 59, 60 and 62, respectively; and

[0058] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 64, 25 and 66, respectively; or

[0059] (17) the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 67, 68 and 70, respectively; and

[0060] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 72, 73 and 74, respectively; or

[0061] (18), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 75, 76 and 78, respectively; and

[0062] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 80, 25 and 83, respectively; or

[0063] (19), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 29, 85 and 87, respectively; and

[0064] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 89, 91 and 93, respectively; or

[0065] (20), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 16, 94 and 96, respectively; and

[0066] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 98, 99 and 100, respectively; or

[0067] (21), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 75, 102 and 104, respectively; and

[0068] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 105, 25 and 106, respectively.

[0069] (22) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in any one of (11) to (21), and having the same function as the amino acid sequence described in any one of (11) to (21); or

[0070] (23) An amino acid sequence having 80% or more identity with the amino acid sequence described in any one of (11) to (22).

[0071] In some specific embodiments of the present invention, the CDR region of the heavy chain of the humanized antibody includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0072] <1> , the heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO. 2, 16, 59 or 67; and

[0073] <2> , the heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO. 4, 39, 60, 68 or 94; and

[0074] <3> , the heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO. 6, 41, 62, 70 or 96; or

[0075] <4> ,like <1> to <3> The amino acid sequence obtained by replacing, deleting or adding one or more amino acids in any one of the amino acid sequences, and <1> to <3> An amino acid sequence that is functionally equivalent to any one of the amino acid sequences; or

[0076] <5> 、and <1> to <4> An amino acid sequence having more than 80% identity with any of the amino acid sequences;

[0077] The CDR region of the light chain of the humanized antibody includes light chain CDR1, light chain CDR2 and light chain CDR3;

[0078] <6> , the light chain CDR1 has the amino acid sequence shown in SEQ ID NO.9, 44, 64, 72 or 147; and

[0079] <7> , the light chain CDR2 has the amino acid sequence shown in SEQ ID NO.11, 25, 46, 73 or 99; and

[0080] <8> , the light chain CDR3 has the amino acid sequence shown in SEQ ID NO.13, 48, 66, 74 or 100; or

[0081] <9> ,like <6> to <8> The amino acid sequence obtained by replacing, deleting or adding one or more amino acids in any one of the amino acid sequences, and <6> to <8> An amino acid sequence that is functionally equivalent to any one of the amino acid sequences; or

[0082] <10> 、and <6> to <9> The amino acid sequence of any one of the above has an amino acid sequence with an identity of 80% or more.

[0083] In some embodiments of the present invention, the humanized antibody comprises a heavy chain and a light chain;

[0084] <11> , the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 2, 4 and 6, respectively; and

[0085] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 9, 11 and 13, respectively; or

[0086] <12> , the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 16, 39 and 41, respectively; and

[0087] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 44, 46 and 48, respectively; or

[0088] <13> , the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 59, 60 and 62, respectively; and

[0089] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 64, 25 and 66, respectively; or

[0090] <14> , the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 67, 68 and 70, respectively; and

[0091] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 72, 73 and 74, respectively; or

[0092] <15> , the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 16, 94 and 96, respectively; and

[0093] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 147, 99 and 100, respectively; or

[0094] <16> ,like <11> to <15> The amino acid sequence obtained by replacing, deleting or adding one or more amino acids in any one of the amino acid sequences, and <11> to <15> An amino acid sequence that is functionally equivalent to any one of the amino acid sequences; or

[0095] <17> 、and <11> to <16> The amino acid sequence of any one of the above has an amino acid sequence with an identity of 80% or more.

[0096] In some specific embodiments of the present invention, the CDR region of the heavy chain of the affinity matured antibody includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0097] X1), the heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO.158 or 16; and

[0098] X2), the heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO.94; and

[0099] X3), the heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO.96 or 161; or

[0100] X4), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in any one of X1) to X3), and having the same function as the amino acid sequence described in any one of X1) to X3); or

[0101] X5) an amino acid sequence having 80% or more identity with the amino acid sequence described in any one of X1) to X4);

[0102] The CDR region of the light chain of the affinity matured antibody includes light chain CDR1, light chain CDR2 and light chain CDR3;

[0103] X6), the light chain CDR1 has the amino acid sequence shown in SEQ ID NO.9; and

[0104] X7), the light chain CDR2 has the amino acid sequence shown in SEQ ID NO.11 or 159; and

[0105] X8), the light chain CDR3 has the amino acid sequence shown in SEQ ID NO.13 or 162; or

[0106] X9) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in any one of X6) to X8), and having the same function as the amino acid sequence described in any one of X6) to X8); or

[0107] X10) An amino acid sequence that is more than 80% identical to the amino acid sequence described in any one of X6) to X9).

[0108] In some embodiments of the present invention, the affinity matured antibody comprises a heavy chain and a light chain;

[0109] X11), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 158, 94 and 96, respectively; and

[0110] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 9, 159 and 13, respectively; or

[0111] X12), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 16, 94 and 161, respectively; and

[0112] The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NOs. 9, 11 and 162, respectively; or

[0113] X13) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in X11) or X12), and having the same function as the amino acid sequence described in X11) or X12); or

[0114] X14) An amino acid sequence that is more than 80% identical to the amino acid sequence described in any one of X11) to X13).

[0115] In some specific embodiments of the present invention, the heavy chain FR region includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4;

[0116] I) the heavy chain FR1 has an amino acid sequence as shown in SEQ ID NO. 1, 15, 28, 58, 84, 101, 129, 136, or 142; and

[0117] The heavy chain FR2 has an amino acid sequence as shown in SEQ ID NO. 3, 17, 130, 137 or 160; and

[0118] The heavy chain FR3 has an amino acid sequence as shown in SEQ ID NO. 5, 19, 31, 40, 50, 61, 69, 77, 86, 95, 103, 131, 138 or 143; and

[0119] The heavy chain FR4 has an amino acid sequence as shown in SEQ ID NO. 7, 21, 33 or 42; or

[0120] II), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in I), and having the same function as the amino acid sequence described in I); or

[0121] III), an amino acid sequence having more than 80% identity with the amino acid sequence described in I) or II); or

[0122] The FR region of the light chain includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4;

[0123] IV), the light chain FR1 has an amino acid sequence as shown in SEQ ID NO. 8, 22, 34, 43, 52, 63, 71, 79, 88, 97, 132, 139, 144 or 146; and

[0124] The light chain FR2 has an amino acid sequence as shown in SEQ ID NO. 10, 24, 45, 54, 81, 90, 133 or 140; and

[0125] The light chain FR3 has an amino acid sequence as shown in SEQ ID NO. 12, 26, 37, 47, 56, 65, 82, 92, 134, 141 or 145; and

[0126] The light chain FR4 has the amino acid sequence shown in SEQ ID NO. 14 or 135; or

[0127] V) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in IV), and having the same function as the amino acid sequence described in IV); or

[0128] VI) an amino acid sequence that is more than 80% identical to the amino acid sequence described in IV) or V).

[0129] In some specific embodiments of the present invention, the recombinant anti-FAP antibody comprises one or more of a rabbit chimeric antibody, a humanized antibody, an affinity matured antibody, or a single-chain antibody;

[0130] The heavy chain FR region of the rabbit chimeric antibody includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4;

[0131] (A) the heavy chain FR1 has the amino acid sequence shown in SEQ ID NO. 1, 15, 28, 58, 84 or 101; and

[0132] The heavy chain FR2 has the amino acid sequence shown in SEQ ID NO. 3 or 17; and

[0133] The heavy chain FR3 has an amino acid sequence as shown in SEQ ID NO. 5, 19, 31, 40, 50, 61, 69, 77, 86, 95 or 103; and

[0134] The heavy chain FR4 has an amino acid sequence as shown in SEQ ID NO. 7, 21, 33 or 42; or

[0135] (B) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (A), and having the same function as the amino acid sequence described in (A); or

[0136] (C) an amino acid sequence having 80% or more identity with the amino acid sequence described in (A) or (B); or

[0137] The FR region of the rabbit-derived chimeric antibody light chain includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4;

[0138] (D) the light chain FR1 has an amino acid sequence as shown in SEQ ID NO. 8, 22, 34, 43, 52, 63, 71, 79, 88 or 97; and

[0139] The light chain FR2 has an amino acid sequence as shown in SEQ ID NO. 10, 24, 45, 54, 81 or 90; and

[0140] The light chain FR3 has an amino acid sequence as shown in SEQ ID NO. 12, 26, 37, 47, 56, 65, 82 or 92; and

[0141] The light chain FR4 has the amino acid sequence shown in SEQ ID NO.14; or

[0142] (E) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (D), and having the same function as the amino acid sequence described in (D); or

[0143] (F) An amino acid sequence having 80% or greater identity with the amino acid sequence described in (D) or (E).

[0144] In some specific embodiments of the present invention, the rabbit-derived chimeric antibody comprises a heavy chain and a light chain;

[0145] (G), FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 1, 3, 5 and 7, respectively; and

[0146] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 8, 10, 12 and 14, respectively; or

[0147] (H) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 15, 17, 19 and 21, respectively; and

[0148] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 22, 24, 26 and 14, respectively; or

[0149] (I) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 28, 17, 31 and 33, respectively; and

[0150] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 34, 24, 37 and 14, respectively; or

[0151] (J) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 28, 3, 40 and 42, respectively; and

[0152] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 43, 45, 47 and 14, respectively; or

[0153] (K) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 28, 3, 50 and 21, respectively; and

[0154] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 52, 54, 56 and 14, respectively; or

[0155] (L) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 58, 3, 61 and 7, respectively; and

[0156] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 63, 10, 65 and 14, respectively; or

[0157] (M) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 28, 3, 69 and 21, respectively; and

[0158] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 71, 24, 47 and 14, respectively; or

[0159] (N) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 28, 17, 77 and 21, respectively; and

[0160] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 79, 81, 82 and 14, respectively; or

[0161] (O) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 84, 3, 86 and 7, respectively; and

[0162] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 88, 90, 92 and 14, respectively; or

[0163] (P) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 28, 3, 95 and 7, respectively; and

[0164] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 97, 24, 12 and 14, respectively; or

[0165] (Q) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 101, 17, 103 and 21, respectively; and

[0166] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 22, 24, 26 and 14, respectively; or

[0167] (R), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in any one of (G) to (Q), and having the same function as the amino acid sequence described in any one of (G) to (Q); or

[0168] (S) An amino acid sequence that is 80% or more identical to the amino acid sequence described in any one of (G) to (R).

[0169] In some specific embodiments of the present invention, the heavy chain FR region of the humanized antibody includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4;

[0170] 1) the heavy chain FR1 has the amino acid sequence shown in SEQ ID NO. 129, 136 or 142; and

[0171] The heavy chain FR2 has the amino acid sequence shown in SEQ ID NO. 130 or 137; and

[0172] The heavy chain FR3 has an amino acid sequence as shown in SEQ ID NO. 131, 138 or 143; and

[0173] The heavy chain FR4 has the amino acid sequence shown in SEQ ID NO.7; or

[0174] 2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in 1), and having the same function as the amino acid sequence described in 1); or

[0175] 3) an amino acid sequence having 80% or more identity with the amino acid sequence described in 1) or 2); or

[0176] The FR region of the humanized antibody light chain includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4;

[0177] 4) the light chain FR1 has an amino acid sequence as shown in SEQ ID NO. 132, 139, 144 or 146; and

[0178] The light chain FR2 has the amino acid sequence shown in SEQ ID NO. 133 or 140; and

[0179] The light chain FR3 has an amino acid sequence as shown in SEQ ID NO. 134, 141 or 145; and

[0180] The light chain FR4 has the amino acid sequence shown in SEQ ID NO.135; or

[0181] 5) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in 4), and having the same function as the amino acid sequence described in 4); or

[0182] 6) An amino acid sequence that is 80% or more identical to the amino acid sequence described in 4) or 5).

[0183] In some embodiments of the present invention, the humanized antibody comprises a heavy chain and a light chain;

[0184] 7) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 129, 130, 131 and 7, respectively; and

[0185] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 132, 133, 134 and 135, respectively; or

[0186] 8) FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 136, 137, 138 and 7, respectively; and

[0187] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 139, 140, 141 and 135, respectively; or

[0188] 9), FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 142, 137, 143 and 7, respectively; and

[0189] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 144, 133, 145 and 135, respectively; or

[0190] 10), FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 142, 137, 143 and 7, respectively; and

[0191] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 146, 133, 134 and 135, respectively; or

[0192] 11), FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 136, 137, 138 and 7, respectively; and

[0193] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 144, 133, 145 and 135, respectively; or

[0194] 12) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in any one of 7) to 11), and having the same function as the amino acid sequence described in any one of 7) to 11); or

[0195] 13) An amino acid sequence that is 80% or more identical to the amino acid sequence described in any one of 7) to 12).

[0196] In some specific embodiments of the present invention, the heavy chain FR region of the affinity matured antibody includes heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4;

[0197] (X1) the heavy chain FR1 has the amino acid sequence shown in SEQ ID NO. 136; and

[0198] The heavy chain FR2 has the amino acid sequence shown in SEQ ID NO. 137 or 160; and

[0199] The heavy chain FR3 has the amino acid sequence shown in SEQ ID NO.138; and

[0200] The heavy chain FR4 has the amino acid sequence shown in SEQ ID NO.7; or

[0201] (X2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (X1), and having the same function as the amino acid sequence described in (X1); or

[0202] (X3), an amino acid sequence having 80% or greater identity with the amino acid sequence described in (X1) or (X2); or

[0203] The FR region of the affinity matured antibody light chain includes light chain FR1, light chain FR2, light chain FR3 and light chain FR4;

[0204] (X4), the light chain FR1 has the amino acid sequence shown in SEQ ID NO.132; and

[0205] The light chain FR2 has the amino acid sequence shown in SEQ ID NO.133; and

[0206] The light chain FR3 has the amino acid sequence shown in SEQ ID NO.134; and

[0207] The light chain FR4 has the amino acid sequence shown in SEQ ID NO.135; or

[0208] (X5) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (X4), and having the same function as the amino acid sequence described in (X4); or

[0209] (X6) An amino acid sequence that is 80% or more identical to the amino acid sequence described in (X4) or (X5).

[0210] In some embodiments of the present invention, the affinity matured antibody comprises a heavy chain and a light chain;

[0211] (X7), FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 136, 137, 138 and 7, respectively; and

[0212] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 132, 133, 134 and 135, respectively; or

[0213] (X8), FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NOs. 136, 160, 138 and 7, respectively; and

[0214] FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences shown in SEQ ID NOs. 132, 133, 134 and 135, respectively; or

[0215] (X9) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (X7) or (X8), and having the same function as the amino acid sequence described in (X7) or (X8); or

[0216] (X10) An amino acid sequence that is 80% or more identical to the amino acid sequence described in any one of (X7) to (X9).

[0217] In some specific embodiments of the present invention, the recombinant anti-FAP antibody comprises a heavy chain and a light chain;

[0218] (A1), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No. (2N+1) or SEQ ID No. (2X); and

[0219] (A2), the variable region of the light chain has the amino acid sequence shown in SEQ ID No. (2N+2) or SEQ ID No. (2X+1); or

[0220] (A3) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in (A1) or (A2); or

[0221] (A4) a sequence having an amino acid sequence homology of 80% or more to any one of (A1) to (A3);

[0222] N is selected from any integer between 53 and 63, 81 or 82;

[0223] The X is selected from any integer between 74 and 78.

[0224] In some specific embodiments of the present invention, the recombinant anti-FAP antibody comprises one or more of a rabbit chimeric antibody, a humanized antibody, an affinity matured antibody, or a single-chain antibody;

[0225] The rabbit-derived chimeric antibody comprises a heavy chain and a light chain;

[0226] (B1), the heavy chain variable region of the rabbit-derived chimeric antibody has the amino acid sequence shown in SEQ ID NO. (2N+1); and

[0227] (B2), the light chain variable region of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID NO. (2N+2); or

[0228] (B3) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (B1) or (B2); or

[0229] (B4) a sequence having an amino acid sequence homology of 80% or more to any one of (B1) to (B3);

[0230] The N is selected from any integer between 53 and 63.

[0231] In some specific embodiments of the present invention, the rabbit-derived chimeric antibody comprises a heavy chain and a light chain;

[0232] (B5), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.107; and

[0233] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.108; or

[0234] (B6), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.109; and

[0235] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.110; or

[0236] (B7), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.111; and

[0237] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.112; or

[0238] (B8), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.113; and

[0239] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.114; or

[0240] (B9), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.115; and

[0241] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.116; or

[0242] (B10), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.117; and

[0243] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.118; or

[0244] (B11), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.119; and

[0245] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.120; or

[0246] (B12), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.121; and

[0247] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.122; or

[0248] (B13), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.123; and

[0249] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.124; or

[0250] (B14), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.125; and

[0251] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.126; or

[0252] (B15), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.127; and

[0253] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.128; or

[0254] (B16) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (B5) to (B15); or

[0255] (B17) A sequence having 80% or more identity with the amino acid sequence shown in any one of (B5) to (B16).

[0256] In some specific embodiments of the present invention, the recombinant anti-FAP antibody, the humanized antibody comprises a heavy chain and a light chain;

[0257] (C1), the heavy chain variable region of the humanized antibody has the amino acid sequence shown in SEQ ID NO. (2X); and

[0258] (C2), the light chain variable region of the humanized antibody has the amino acid sequence shown in SEQ ID NO. (2X+1); or

[0259] (C3) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (C1) or (C2); or

[0260] (C4) a sequence having an amino acid sequence homology of more than 80% with any one of (C1) to (C3);

[0261] The X is selected from any integer between 74 and 78.

[0262] In some embodiments of the present invention, the humanized antibody comprises a heavy chain and a light chain;

[0263] (C5), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.148; and

[0264] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.149; or

[0265] (C6), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.150; and

[0266] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.151; or

[0267] (C7), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.152; and

[0268] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.153; or

[0269] (C8), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.154; and

[0270] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.155; or

[0271] (C9), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.156; and

[0272] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.157; or

[0273] (C10) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in any one of (C5) to (C9); or

[0274] (C11) A sequence having 80% or more identity with the amino acid sequence shown in any one of (C5) to (C10).

[0275] In some specific embodiments of the present invention, the recombinant anti-FAP antibody, the affinity matured antibody comprises a heavy chain and a light chain;

[0276] (D1), the heavy chain variable region of the affinity matured antibody has the amino acid sequence shown in SEQ ID No. (2N+1); and

[0277] (D2), the light chain variable region of the affinity matured antibody has the amino acid sequence shown in SEQ ID No. (2N+2); or

[0278] (D3) a sequence in which one or more amino acids are replaced by substitution, deletion, addition and / or replacement based on the amino acid sequence as shown in (D1) or (D2); or

[0279] (D4) a sequence having an amino acid sequence homology of 80% or more to any one of (D1) to (D3);

[0280] The N is selected from 81 or 82.

[0281] In some embodiments of the present invention, the affinity matured antibody comprises a heavy chain and a light chain;

[0282] (D5), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.163; and

[0283] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.164; or

[0284] (D6), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.165; and

[0285] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO.166; or

[0286] (D7) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (D5) or (D6); or

[0287] (D8) A sequence having 80% or more identity with the amino acid sequence shown in any one of (D5) to (D7).

[0288] In some specific embodiments of the present invention, the single-chain antibody has:

[0289] <e1>, any one or more of the amino acid sequences shown in SEQ ID NO.170 to SEQ ID NO.184; or

[0290] <e2>、In <e1>A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown; or

[0291] <e3>、and <e1>or <e2>The amino acid sequences shown have a homology of more than 80%.

[0292] In some specific embodiments of the present invention, the recombinant anti-FAP antibody further comprises a constant region;

[0293] 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 κ type.

[0294] In some embodiments of the invention, the FAP comprises human FAP, murine FAP and / or cynomolgus monkey FAP.

[0295] The present invention also provides a method for preparing the recombinant anti-FAP antibody, comprising taking the FAP immune receptor, isolating spleen cells of the receptor, and amplifying by PCR to obtain the light and heavy chain variable regions of the recombinant anti-FAP antibody;

[0296] Splicing the heavy chain variable region and the heavy chain constant region of the IgG1 subclass, constructing them into a mammalian cell expression vector to obtain a heavy chain vector; splicing the light chain variable region and the light chain constant region of the κ class antibody, constructing them into the mammalian cell expression vector to obtain a light chain vector;

[0297] The heavy chain vector and the light chain vector are taken, transfected into cells, cultured, and purified. The recombinant anti-FAP antibody is obtained by screening based on the affinity between the purified antibody and the FAP.

[0298] In some embodiments of the present invention, the subject comprises a New Zealand White rabbit.

[0299] In some specific embodiments of the present invention, the cells comprise HEK293 cells.

[0300] In some specific embodiments of the present invention, the preparation method further includes: combining the CDR region of the recombinant anti-FAP antibody with the framework region of a human antibody, splicing the heavy chain variable region and the heavy chain constant region of the IgG1 subclass, constructing them into a mammalian cell expression vector to obtain a heavy chain vector; splicing the light chain variable region and the light chain constant region of a κ class antibody, constructing them into the mammalian cell expression vector to obtain a light chain vector; taking the heavy chain vector and the light chain vector, transfecting cells, culturing, purifying, and screening the recombinant anti-FAP antibody based on the affinity of the purified antibody to the FAP.

[0301] In some specific embodiments of the present invention, the cells comprise HEK293 cells.

[0302] In some specific embodiments of the present invention, the preparation method further comprises: taking the recombinant anti-FAP antibody and / or the humanized antibody, randomly mutating the amino acids in the CDR region to obtain a mutated antibody, and screening the recombinant anti-FAP antibody based on the affinity between the mutated antibody and FAP.

[0303] In some specific embodiments of the present invention, the preparation method further comprises: sequentially connecting the pre-sequence, (G4S)3 and post-sequence to construct a eukaryotic expression vector upstream of the human IgG1 heavy chain constant region encoding gene to obtain a fusion protein expression plasmid, amplifying the fusion protein expression plasmid, isolating it, and co-transfecting it with PEI into cells, taking the supernatant, and purifying it to obtain the recombinant anti-FAP antibody:

[0304] ① The presequence has the amino acid sequence shown in SEQ ID NO.167, and the postsequence has the amino acid sequence shown in SEQ ID NO.169; or

[0305] ② The presequence has the amino acid sequence shown in SEQ ID NO.169, and the postsequence has the amino acid sequence shown in SEQ ID NO.167; or

[0306] ③ The presequence has the amino acid sequence shown in SEQ ID NO.156, and the postsequence has the amino acid sequence shown in SEQ ID NO.149; or

[0307] ④ The presequence has the amino acid sequence shown in SEQ ID NO.149, and the postsequence has the amino acid sequence shown in SEQ ID NO.156; or

[0308] ⑤ The presequence has the amino acid sequence shown in SEQ ID NO.163, and the postsequence has the amino acid sequence shown in SEQ ID NO.164; or

[0309] ⑥ The presequence has the amino acid sequence shown in SEQ ID NO.164, and the postsequence has the amino acid sequence shown in SEQ ID NO.163; or

[0310] 7. The preceding sequence has the amino acid sequence shown in SEQ ID NO.165, and the following sequence has the amino acid sequence shown in SEQ ID NO.166; or

[0311] ⑧ The presequence has the amino acid sequence shown in SEQ ID NO.166, and the postsequence has the amino acid sequence shown in SEQ ID NO.165; or

[0312] ⑨ The presequence has the amino acid sequence shown in SEQ ID NO.150, and the postsequence has the amino acid sequence shown in SEQ ID NO.151; or

[0313] ⑩ The preceding sequence has the amino acid sequence shown in SEQ ID NO.153, and the following sequence has the amino acid sequence shown in SEQ ID NO.152; or

[0314] The presequence has the amino acid sequence shown in SEQ ID NO.155, and the postsequence has the amino acid sequence shown in SEQ ID NO.154; or

[0315] The presequence has the amino acid sequence shown in SEQ ID NO.112, and the postsequence has the amino acid sequence shown in SEQ ID NO.111; or

[0316] The presequence has the amino acid sequence shown in SEQ ID NO.122, and the postsequence has the amino acid sequence shown in SEQ ID NO.121; or

[0317] The presequence has the amino acid sequence shown in SEQ ID NO.123, and the postsequence has the amino acid sequence shown in SEQ ID NO.124; or

[0318] The presequence has the amino acid sequence shown in SEQ ID NO.128, and the postsequence has the amino acid sequence shown in SEQ ID NO.127; or

[0319] As ① to The amino acid sequence obtained by replacing, deleting or adding one or more amino acids in any one of the amino acid sequences, and An amino acid sequence that is functionally equivalent to any one of the amino acid sequences; or

[0320] With Ru① to The amino acid sequence of any one of the above has an amino acid sequence with an identity of 80% or more.

[0321] The (G4S)3 has an amino acid sequence as shown in SEQ ID NO.168.

[0322] In some specific embodiments of the present invention, the cells comprise HEK293 cells.

[0323] The present invention also provides a biomaterial comprising any of the following:

[0324] a), a nucleic acid molecule encoding the recombinant anti-FAP antibody; and / or

[0325] b) an expression vector comprising a nucleic acid molecule encoding the recombinant anti-FAP antibody; and / or

[0326] c) a host that secretes the recombinant anti-FAP antibody; and / or

[0327] d) a recombinant anti-FAP antibody obtained by the preparation method; and / or

[0328] e) a chemically labeled or biologically labeled recombinant anti-FAP antibody and / or a combination of the recombinant anti-FAP antibody obtained by the preparation method.

[0329] The present invention also provides an immune cell, comprising: a nucleic acid molecule encoding the aforementioned recombinant anti-FAP antibody; and / or an expression vector comprising a nucleic acid molecule encoding the aforementioned recombinant anti-FAP antibody.

[0330] In some embodiments of the present invention, the immune cell is selected from cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβT cells and a γδT cell.

[0331] In some embodiments of the present invention, the immune cell expresses a chimeric antigen receptor (CAR), a T cell antigen coupling (TAC) receptor, or a T cell receptor (TCR).

[0332] Chimeric antigen receptor (CAR) combines many aspects of normal T cell activation into a single protein. They connect the extracellular antigen recognition domain to the intracellular signaling domain, activating T cells when antigen binds. CAR typically has the following regions: antigen binding domain, extracellular hinge region, transmembrane region and intracellular region. In some embodiments, the intracellular region includes an intracellular signaling domain or an intracellular signaling region.

[0333] The antigen binding domain is exposed to the outside of the cell and is located in the extracellular domain of the receptor. It interacts with potential target molecules and is responsible for targeting CAR-T cells to any cell that expresses a matching molecule. The antigen binding domain is typically derived from the variable region of a monoclonal antibody linked together as a single-chain variable fragment (scFv). scFv is a chimeric protein composed of a light chain (VL) and a heavy chain (VH) of an immunoglobulin and connected to a short linker peptide. In some embodiments, the heavy chain and the light chain have the aforementioned heavy chain variable region and light chain variable region sequences.

[0334] The present invention also provides the use of any of the following in the preparation of a drug targeting FAP:

[0335] A), the recombinant anti-FAP antibody; and / or

[0336] B) the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0337] C), the biological material; and / or

[0338] D) the immune cells.

[0339] The present invention also provides the use of any of the following in the preparation of a product for preventing and / or treating cancer:

[0340] A), the recombinant anti-FAP antibody; and / or

[0341] B) the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0342] C), the biological material; and / or

[0343] D) the immune cells.

[0344] In some specific embodiments of the present invention, the cancer comprises any one or more of ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, bladder cancer, colorectal cancer and brain metastasis cancer.

[0345] In some embodiments of the invention, the product comprises a drug and / or a vaccine.

[0346] The present invention also provides a drug comprising any of the following:

[0347] A), the recombinant anti-FAP antibody; and / or

[0348] B) the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0349] C), the biological material; and / or

[0350] D) the immune cells.

[0351] The present invention also provides a pharmaceutical combination comprising the drug and any other effective ingredients.

[0352] In some specific embodiments of the present invention, the other optional active ingredients include small molecule toxins.

[0353] The present invention also provides a vaccine comprising any of the following:

[0354] A), the recombinant anti-FAP antibody; and / or

[0355] B) the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0356] C), the biological material; and / or

[0357] D) the immune cells.

[0358] The present invention also provides the use of any of the following in the preparation of a reagent and / or kit for detecting FAP:

[0359] A), the recombinant anti-FAP antibody; and / or

[0360] B) the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0361] C), the biological material; and / or

[0362] D) the immune cells.

[0363] The present invention also provides reagents and / or kits, including any of the following:

[0364] A), the recombinant anti-FAP antibody; and / or

[0365] B) the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0366] C), the biological material; and / or

[0367] D) the immune cells.

[0368] The present invention also provides a method of treatment comprising administering to a subject any of the following:

[0369] A), the recombinant anti-FAP antibody; and / or

[0370] B) the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0371] C), the biological material; and / or

[0372] D), the immune cells; and / or

[0373] E), the drug; and / or

[0374] F), the drug combination.

[0375] The present invention also provides a prevention method comprising administering the vaccine to a subject.

[0376] The present invention also provides a detection method, wherein a sample to be tested is subjected to any of the following tests:

[0377] A), the recombinant anti-FAP antibody; and / or

[0378] The preparation method obtains recombinant anti-FAP antibodies; and / or

[0379] The biological material.

[0380] The present invention includes but is not limited to the following beneficial effects:

[0381] The anti-FAP antibodies provided by the present invention have better effects, lower immunogenicity, and exhibit highly specific binding as well as rapid and efficient internalization capabilities. Their high affinity, high selectivity, and high biological activity levels contribute to their potential as therapeutic monoclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0382] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0383] Figure 1 shows the affinity of the control antibody to the overexpressing cell line CHO-hFAP cells;

[0384] Figure 2 shows the affinity of the control antibody to the naturally expressing cell line U138MG cells;

[0385] FIG3 shows the binding test of anti-human FAP chimeric antibodies to CHO-hFAP cells;

[0386] FIG4 shows the binding test of anti-human FAP chimeric antibodies to CHO-hFAP cells;

[0387] FIG5 shows the binding test of anti-human FAP chimeric antibodies to CHO-hFAP cells;

[0388] FIG6 shows the binding test of anti-human FAP chimeric antibodies to CHO-mFAP cells;

[0389] FIG7 shows the binding test of anti-human FAP chimeric antibodies to CHO-mFAP cells;

[0390] FIG8 shows the binding test of anti-human FAP chimeric antibodies to CHO-mFAP cells;

[0391] FIG9 shows a binding test of anti-human FAP humanized antibodies to HT1080-hFAP cells;

[0392] FIG10 shows a binding test of anti-human FAP humanized antibodies to HT1080-hFAP cells;

[0393] FIG11 shows a binding test of anti-human FAP humanized antibodies to CHO-mFAP cells;

[0394] FIG12 shows a binding test of anti-human FAP humanized antibodies to CHO-mFAP cells;

[0395] FIG13 shows the binding test of affinity-matured combination molecules to HT1080-hFAP cells;

[0396] FIG14 shows the binding test of affinity-matured combination molecules to HT1080-hFAP cells;

[0397] FIG15 shows the binding test of affinity-matured combination molecules to CT26-mFAP cells;

[0398] FIG16 shows the binding test of affinity-matured combination molecules to CT26-mFAP cells;

[0399] FIG17 shows the binding test of affinity-matured combination molecules to 293-cynoFAP cells;

[0400] FIG18 shows the non-specific binding test of affinity-matured combination molecules to CHO-hDPP4 cells;

[0401] FIG19 shows the non-specific binding test of affinity-matured combination molecules to CHOK1 cells;

[0402] FIG20 shows the non-specific binding test of affinity-matured combination molecules to HEK293 cells;

[0403] Figure 21 shows a metabolic diagram in mice;

[0404] FIG22 shows a binding test of single-chain combination molecules to HT1080-hFAP cells;

[0405] FIG23 shows a binding test of single-chain combination molecules to HT1080-hFAP cells;

[0406] FIG24 shows a binding test of single-chain combination molecules to HT1080-hFAP cells;

[0407] FIG25 shows a binding test of single-chain combination molecules to HT1080-hFAP cells;

[0408] FIG26 shows a binding test of single-chain combination molecules to HT1080-hFAP cells;

[0409] FIG27 shows a binding test of single-chain combination molecules to HT1080-hFAP cells;

[0410] FIG28 shows a binding test of single-chain combination molecules to HT1080-hFAP cells;

[0411] FIG29 shows a binding test of single-chain combination molecules to CT26-mFAP cells;

[0412] FIG30 shows a binding test of single-chain combination molecules to CT26-mFAP cells;

[0413] FIG31 shows a binding test of single-chain combination molecules to CT26-mFAP cells;

[0414] FIG32 shows a binding test of single-chain combination molecules to CT26-mFAP cells;

[0415] FIG33 shows a binding test of single-chain combination molecules to CT26-mFAP cells;

[0416] FIG34 shows a binding test of single-chain combination molecules to CT26-mFAP cells;

[0417] FIG35 shows a binding test of single-chain combination molecules to 293-cynoFAP cells;

[0418] FIG36 shows a binding test of single-chain combination molecules to 293-cynoFAP cells;

[0419] FIG37 shows a binding test of single-chain combination molecules to 293-cynoFAP cells;

[0420] FIG38 shows a binding test of single-chain combination molecules to 293-cynoFAP cells;

[0421] FIG39 shows a non-specific binding assay of single-chain combination molecules to CHO-hDPP4 cells;

[0422] FIG40 shows a non-specific binding assay of single-chain combination molecules to CHO-hDPP4 cells;

[0423] FIG41 shows a non-specific binding assay of single-chain combination molecules to CHO-hDPP4 cells;

[0424] FIG42 shows a non-specific binding assay of single-chain combination molecules to CHO-hDPP4 cells;

[0425] FIG43 shows a non-specific binding assay of single-chain combination molecules to CHOK1 cells;

[0426] FIG44 shows a non-specific binding assay of single-chain combination molecules to CHOK1 cells;

[0427] FIG45 shows the non-specific binding test of single-chain combination molecules to CHOK1 cells. DETAILED DESCRIPTION

[0428] The present invention discloses recombinant anti-FAP antibodies and their applications. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine 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.

[0429] 1. Sequence synthesis and vector construction of tool antibodies

[0430] Table 1 Sequences of tool antibodies

[0431] 2. Purchase antigen and verify antigen binding activity

[0432] 2.1 Human FAP recombinant protein antigen information

[0433] The commercial reagents purchased are shown in Table 1:

[0434] Table 2 Commercial reagents

[0435] 2.2 Construction of Antigen Cell Lines of Different Species

[0436] Table 3 Sequences of antigens from different species

[0437] The hFAP nucleotide sequence was constructed into the commercially available pCDNA3.1 vector from Invitrogen. Specifically, the pCDNA3.1 vector was double-digested with KpnI and XhoI, and the hFAP nucleotide sequence was inserted. The vector sequence information was then confirmed by sequencing. After successful plasmid construction, it was transfected into human fibrosarcoma HT1080 cells using lipofectamine 3000. Subsequently, puromycin antibiotics were used to screen overexpressing monoclonal cells, and clones with slightly lower expression levels were selected. The successfully constructed cell line was named low-expressing HT1080-hFAP cells.

[0438] The hFAP nucleotide sequence was constructed into the commercially available pCDNA5 vector from Invitrogen. Specifically, the pCDNA5 vector was double-digested with NheI and PmeI, and the hFAP nucleotide sequence was inserted. The vector sequence information was then confirmed by sequencing. After successful plasmid construction, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary CHOK1 cells using lipofectamine 3000. Hygromycin was then used to select overexpressing cell lines. The successfully constructed cell lines were named CHO-hFAP and 293-hFAP cells, respectively.

[0439] The mFAP nucleotide sequence was constructed into the commercialized pCDNA5 vector from Invitrogen. Specifically, the pCDNA5 vector was double-digested with NheI and PmeI, and the mFAP nucleotide sequence was inserted. The vector sequence information was then confirmed by sequencing. After successful plasmid construction, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary CHOK1 cells using lipofectamine 3000. Hygromycin antibiotics were then used to select overexpressing cell lines. The successfully constructed cell lines were named CHO-mFAP and 293-mFAP cells, respectively.

[0440] The nucleotide sequence of the above-mentioned cynoFAP was constructed into the commercialized pCDNA5 vector of Invitrogen. Specifically, the pCDNA5 vector was double-digested with NheI and PmeI, and the nucleotide sequence of cynoFAP was inserted, and then the vector sequence information was confirmed by sequencing. After successful construction of the plasmid, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1 using lipofectamine3000, respectively. Subsequently, the overexpressing cell lines were selected using hygromycin antibiotics, and monoclonal overexpressing cell lines CHO-cynoFAP and 293-cynoFAP cells were finally obtained through monoclonal cloning.

[0441] 2.3 Construction of cell lines expressing proteins from the same family

[0442] Table 4 Protein sequences of the same family

[0443] The hDPP4 nucleotide sequence was constructed into the commercialized pCDNA5 vector from Invitrogen. Specifically, the pCDNA5 vector was double-digested with NheI and PmeI, and the hDPP4 nucleotide sequence was inserted. The vector sequence information was then confirmed by sequencing. After successful plasmid construction, it was transfected into Chinese hamster ovary (CHOK1) cells using lipofectamine 3000. Hygromycin was then used to select overexpressing cell lines, and monoclonal overexpressing cell lines (CHO-hDPP4) were finally obtained through monoclonal cloning.

[0444] 3. Purchase of antigen cell lines

[0445] Mouse FAP engineered cell line:

[0446] CT26-mouse-Fap-Cell-Line. Product number: KC-1284.

[0447] Natural tumor cells expressing human FAP:

[0448] U-138MG cells were purchased from ATCC, catalog number HTB-16.

[0449] Reagent: Z-Gly-Pro-AMC, brand BACHEM, product number 4002518.

[0450] The antibodies provided by the present invention have better effects and lower immunogenicity. The antibody sequences are shown in Tables 5 to 12.

[0451] Table 10 Variable region sequences of affinity matured optimized antibodies

[0452] Table 11 Composition sequence of single-chain antibody sequence

[0453] Table 12 Single-chain antibody sequences

[0454] Unless otherwise specified, the recombinant anti-FAP antibodies provided by the present invention and the raw materials and reagents used in their applications can be purchased from the market.

[0455] The present invention will be further described below in conjunction with the embodiments:

[0456] Example 1 FACS determination of affinity between antigen cells and tool antibodies

[0457] Experimental reagents and materials:

[0458] Table 13 Experimental reagents and materials

[0459] Experimental process:

[0460] 1) Cell collection and plating

[0461] a) Harvest cells in the logarithmic growth phase and ensure that the cell viability is above 90%.

[0462] b) Centrifuge at 1000 rpm for 5 minutes and discard the supernatant;

[0463] c) washing the cells once with PBS;

[0464] d) Resuspend the cells in FACS Buffer and count them;

[0465] e) Use FACS Buffer to prepare a density of 2×10 6 cells / mL cell suspension;

[0466] f) Add 50 μL of cell suspension to each well of a 96-well plate;

[0467] 2) Antibody incubation and detection

[0468] a) Add 50 μL of test sample of different concentrations to the experimental group, starting with a sample concentration of 20 μg / mL and setting a 3-fold dilution gradient with multiple points;

[0469] b) After mixing, incubate at 4°C in the dark overnight;

[0470] c) Wash the cells once with 200 μL of FACS Buffer, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0471] d) APC-labeled secondary antibody (1:1500 dilution) was added to the 96-well plate, and an equal volume of FACS buffer was added to the blank control group;

[0472] e) After mixing, incubate at 4°C in the dark for 40 minutes;

[0473] f) Wash the cells once with 200 μL of FACS Buffer each time, centrifuge at 1000 rpm for 5 minutes, and resuspend the cells in 100 μL of FACS Buffer.

[0474] g) RL-1 MFI readings were measured using Intellicyte plus flow cytometer (Excitation Laser: 488 nm Blue Laser).

[0475] 3) Data processing

[0476] FACS data were analyzed using Prism software.

[0477] Data analysis: Using the overexpression cell line CHO-hFAP cells and the native expression cell line U138MG cells, after in vitro cell biology combined with FACS testing, it can be concluded from the data that each control antibody has binding signals with both cell lines, with different high and low properties. Among them, the binding signal of 4B9 is higher, and the binding signal of hu36 is slightly lower (as shown in Table 14, Figures 1 and 2).

[0478] Table 14 EC50 and fluorescence values ​​of control antibody flow cytometry binding assay

[0479] Example 2: Production of Rabbit-derived Anti-Human FAP Monoclonal Antibodies (This work was commissioned to Shanghai Dinoxinan Biotechnology Co., Ltd.)

[0480] 1. Animal immunization

[0481] In order to obtain rabbit monoclonal antibodies that recognize human FAP antigens, the present invention chose to immunize New Zealand white rabbits. The first immunization was 200 μg. During the first immunization, Freund's complete adjuvant and an equal volume of antigen (human FAP-hFc protein, brand Acro Biosystems, product number FAP-H5263) were mixed and emulsified, and immunization was performed at multiple points on the back. Two weeks later, booster immunization was performed. The amount of antigen used for booster immunization was 100 μg. Freund's incomplete adjuvant and an equal volume of antigen were mixed and emulsified, and immunization was performed at multiple points on the back. After five immunizations, the titer of the immune serum was detected by conventional Elisa methods. Rabbits with high titers were selected, and a shock immunization was performed with 50 μg of protein intraperitoneally three days before screening antibodies. The antigen did not need to be emulsified with an adjuvant, and the buffer solution was PBS. The spleen was taken three days later.

[0482] 2. Spleen Cell Isolation

[0483] Rabbit spleens were surgically removed and placed in a sterile cell culture dish. The spleens were rinsed with DPBS containing 100 U / mL penicillin and 100 μg / mL streptomycin. The spleens were minced with surgical scissors and gently ground into single cells using a syringe plunger. The cell suspension was filtered through a 100 μm cell sieve, and the single-cell filtrate was collected. The cells were centrifuged at 1200 rpm for 3 minutes, the supernatant discarded, and the cells resuspended in RPMI-1640 supplemented with 5% fetal bovine serum.

[0484] 3. B cell culture and identification

[0485] Rabbit memory B cells were isolated by incubating biotin-labeled FAP protein with successfully immunized rabbit lymphocytes. Cultured in B cell culture medium in 96-well cell culture plates at 37°C and 5% CO2. After 10-14 days of culture, the supernatants of clones were assayed for protein binding activity by ELISA. Clones with binding activity exceeding 5-fold above background were considered positive. ELISA-positive supernatants were then assayed by FACS for binding to stably transfected CHO-hFAP and CHO-mFAP cells, and FACS-positive single clones were selected.

[0486] 4. Cloning of the gene encoding rabbit monoclonal antibody

[0487] Positive B cell clones were collected, and total RNA from selected clones was extracted using RNAiso Plus and reverse transcribed into cDNA. The light and heavy chain variable region sequences were amplified by PCR and constructed into a PTT5 expression vector containing the corresponding heavy and light chain constant regions. Sequencing was performed to obtain the correct sequence. The sequencing results were analyzed using VBASE2 (http: / / www.vbase2.org / vbscAb.php) to obtain the antibody light and heavy chain variable region sequences.

[0488] Example 3 Preparation of anti-human FAP chimeric antibodies

[0489] The heavy chain variable region sequence of a rabbit anti-human FAP monoclonal antibody was spliced ​​with the heavy chain constant region sequence of a publicly available human monoclonal antibody of the IgG1 subclass and constructed into a mammalian cell expression vector. The light chain variable region sequence of a rabbit anti-human FAP monoclonal antibody was spliced ​​with the light chain constant region sequence of a publicly available human monoclonal antibody of the κ subclass and constructed into a mammalian cell expression vector. The constructed heavy chain and light chain vectors of the anti-human FAP chimeric antibody were mixed and transfected into HEK293 cells using polyethyleneimine (PEI). Approximately 7 days later, the cell supernatant was collected and purified using MabSelect to obtain the anti-human FAP chimeric antibody protein.

[0490] Example 4 In vitro cell binding assay of anti-human FAP chimeric antibodies

[0491] The anti-human FAP chimeric antibody was diluted 2-fold starting from a starting concentration of 20 μg / mL for a total of 8 concentration points. 50 μL of the antibody at each concentration point was added to a 96-well plate. CHOK1 cells that highly expressed human FAP and mouse FAP on their cell surfaces were collected by centrifugation at 100g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA and centrifuged at 100g for 5 minutes at room temperature. The cells were resuspended at a density of approximately 2 × 10 6 50 μL of the culture medium was added to the wells of a 96-well plate containing the antibody. After incubation at 4°C for 1 hour, APC fluorescently labeled goat anti-human IgG secondary antibody was added. After another hour of incubation at 4°C, the mean fluorescence reading of the cell population was analyzed by flow cytometry, and a four-parameter curve fit was performed using Prism software.

[0492] Analysis of results: After in vitro cell biology binding tests, several antibodies obtained, such as chrD1, chrD7, and chrD25, can reach a plateau value at a lower concentration compared to the control cMFP5. In vitro binding experiments with CHO-mFAP cells also showed that several chimeric antibody molecules have good binding signals with mFAP (as shown in Figures 3 to 8).

[0493] Example 5 In vitro binding affinity and kinetics experiments of anti-human FAP chimeric antibodies

[0494] Antibody affinity was determined using the anti-human antibody capture method using a Fortebio instrument (BLITZ pro 1.1.0.28). For the assay, a capture antibody (AHC) bioprobe targeting the Fc region of an anti-human antibody was soaked in PBS for 10 minutes. 200 μL of diluted antibody sample (including the chimeric antibody of the present invention and a control antibody; the antibody working concentration was 15 μg / mL) was loaded onto the AHC bioprobe, which was then equilibrated in PBS for 100 seconds. The AHC probe was then subjected to a binding reaction with human FAP protein and mouse FAP protein (purchased from ACRO biosystem) for 600 seconds. The AHC probe was then transferred to PBS for a dissociation reaction for 600 seconds. After the experiment, the blank control response was subtracted, and a 1:1 Langmuir binding model was fitted using software to calculate the kinetic constants for antigen-antibody binding.

[0495] Result analysis: After in vitro kinetic binding activity analysis, the binding kinetic constant of the chimeric antibody to the recombinant hFAP protein was 10 -10 to 10 -12 In contrast, the binding kinetic constants of the benchmark antibodies cMFP5 and hu36 were between 10 -9 to 10 -11 The binding kinetic constant of the chimeric antibody to the recombinant mFAP protein was 10 -6 to 10 -12 In contrast, the binding kinetic constants of the benchmark antibodies cMFP5 and hu36 were between 10 -9 to 10 -11 The chimeric antibody molecules had comparable binding kinetics to the control antibody (as shown in Table 15 and Table 5).

[0496] Table 15 In vitro kinetic binding activity of antibodies to recombinant human FAP protein

[0497] Table 16 In vitro kinetic binding activity of antibodies to recombinant mouse FAP protein

[0498] Example 6 Humanization of Anti-Human FAP Rabbit Antibody

[0499] Combining the antibody encoding schemes of Kabat and Chothia, the amino acid sequence regions of the six antigen complementarity determining regions (CDRs) of the rabbit antibody's heavy and light chains, as well as the framework region that supports the antibody's conserved three-dimensional conformation, were determined. Subsequently, by analyzing and searching known human antibody sequences, the human heavy chain variable region sequence most similar to the rabbit antibody, such as IGHV1|IGHJ4*01, was selected. This framework region sequence was used as a template, and the rabbit antibody heavy chain CDRs were combined with the human antibody framework region to generate the humanized antibody heavy chain variable region sequence. The same process was used to generate the humanized antibody light chain variable region sequence. Antibodies with rabbit antibody CDRs directly grafted onto human framework regions often experience a sharp decrease in binding activity, necessitating the conversion of individual amino acids in the framework region from human to rabbit. To identify backmutation sites, the first step is to compare the designed humanized antibody sequence with the original rabbit antibody sequence to examine which amino acids differ and, secondly, to examine whether these amino acids play a significant role in supporting the antibody structure or in antigen binding. After humanization, the sequence needs to be checked for potential post-translational modification sites, such as N (asparagine) glycosylation sites, N deamidation sites, and D (aspartic acid) isomerization sites.

[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 and light chain vectors of the humanized anti-human FAP antibody were mixed and transfected into HEK293 cells using polyethyleneimine (PEI). After approximately 7 days, the cell supernatant was collected and purified using MabSelect to obtain the humanized anti-human FAP antibody protein.

[0501] Example 7 In vitro binding affinity and kinetics experiments of anti-human FAP humanized antibodies

[0502] Antibody affinity was determined using the anti-human antibody capture method using a Fortebio instrument (BLITZ pro 1.1.0.28). For the assay, a capture antibody (AHC) bioprobe targeting the Fc region of an anti-human antibody was soaked in PBS for 10 minutes. 200 μL of diluted antibody sample (including the chimeric antibody of the present invention and a control antibody; the antibody working concentration was 15 μg / mL) was loaded onto the AHC bioprobe, which was then equilibrated in PBS for 100 seconds. The AHC probe was then subjected to a binding reaction with human FAP protein and mouse FAP protein (purchased from ACRO biosystem) for 600 seconds. The AHC probe was then transferred to PBS for a dissociation reaction for 600 seconds. After the experiment, the blank control response was subtracted, and a 1:1 Langmuir binding model was fitted using software to calculate the kinetic constants for antigen-antibody binding.

[0503] Analysis of results: chrD1, chrD11, chrD16, chrD27, chrD5, and chrD18 were selected for humanized design and modification. Compared with chimeric antibodies, the modified humanized molecules maintained the binding activity with recombinant human and mouse proteins as much as possible at the in vitro kinetic binding activity level (as shown in Tables 17 to 22).

[0504] Table 17 In vitro kinetic binding activity of humanized antibodies to chrD1 and recombinant human and mouse FAP proteins

[0505] Table 18 In vitro kinetic binding activity of humanized antibodies against chrD11 and recombinant human and mouse FAP proteins

[0506] Table 19 In vitro kinetic binding activity of humanized antibodies against chrD16 and recombinant human and mouse FAP proteins

[0507] Table 20 In vitro kinetic binding activity of humanized antibodies against chrD27 and recombinant human and mouse FAP proteins

[0508] Table 21 In vitro kinetic binding activity of humanized antibodies to chrD5 and recombinant human and mouse FAP proteins

[0509] Table 22 In vitro kinetic binding activity of humanized antibodies against chrD18 and recombinant human and mouse FAP proteins

[0510] Example 8 In vitro cell binding assay of humanized anti-human FAP antibodies

[0511] The anti-human FAP humanized antibody was diluted 4-fold starting from a starting concentration of 20 μg / mL, for 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 expression of human FAP on the cell surface and CHOK1 cells with high expression of mouse FAP were collected by centrifugation at 100g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 minutes at room temperature, and resuspended at a density of approximately 2×10 6 50 μL of the culture medium was added to the wells of a 96-well plate containing the antibody. After incubation at 4°C for 1 hour, APC fluorescently labeled goat anti-human IgG secondary antibody was added. After another hour of incubation at 4°C, the mean fluorescence reading of the cell population was analyzed by flow cytometry. Prism software was used to perform a four-parameter curve fit and calculate the EC50 and top fluorescence intensity values.

[0512] Analysis of Results: Humanized antibodies were tested in vitro for binding to HT1080-hFAP cells, which underexpress hFAP, and CHO-mFAP cells, which overexpress mFAP. The results, shown in Figures 9 to 12 and Table 23, demonstrate that humanized molecules D1, D16, and D27 bind to HT1080-hFAP cells at the same level as the reference molecule 4B9. While all three antibodies bind to CHO-mFAP cells, the fluorescence signals are low.

[0513] Table 23 In vitro flow cytometry binding EC50 and top values ​​of fluorescence intensity of humanized antibodies to HT1080-hFAP and CHO-mFAP cells

[0514] Example 9 Affinity Maturation

[0515] This antibody modification uses yeast display technology to randomly mutate the amino acids in the six CDR regions of the heavy chain variable region and light chain variable region of the antibody molecule to build a library, and then screen out candidate antibody molecules with higher affinity.

[0516] 1. Design and construction of affinity matured antibody library

[0517] The heavy and light chain variable regions of affinity-matured parental antibody molecules, hzD1-H1L0 and hzD27-H1L0, were linked via a GS linker to construct single-chain variable fragments (scFvs). The CDR regions of these scFvs were the target of affinity maturation, while the framework sequences remained unchanged during the affinity maturation process. Separate antibody libraries were constructed from the six CDR regions of each parental antibody's heavy and light chains.

[0518] 2. Sorting of affinity matured antibody library

[0519] The resulting antibody library was subjected to magnetic bead sorting and flow cytometry: First, cells were incubated with 300 nM biotinylated antigen for 1 hour, followed by magnetic bead sorting (Invitrogen Cat: 11206D). The resulting antibody library was then subjected to flow cytometry. In the subsequent flow cytometry, the biotinylated antigen was incubated with the antibody library for 40 minutes at room temperature. Unbound biotinylated antigen was then washed away, and cells were harvested by centrifugation at 14,000 rpm for 1 minute at 4°C and washed twice with ice-cold PBS containing 1% BSA. Then, 100 μL of a 1:500 diluted fluorescent secondary antibody was added and incubated on ice for 30 minutes. Cells were harvested by centrifugation at 14,000 rpm for 1 minute at 4°C and washed twice with ice-cold PBS containing 1% BSA before flow cytometry. The antigen concentration used in each round is gradually reduced, and only 0.1-0.5% of the clones in the library are collected for culture in each round of sorting before the next round of sorting. Finally, after a total of 3-4 rounds of flow sorting, 96 yeasts are selected from each antibody library for sequencing analysis, and finally several unique mutant clones are obtained from the heavy chain antibody library and the light chain antibody library.

[0520] 3. Affinity maturation mutation sequence combination screening, identification and evaluation

[0521] 3.1. In vitro cell binding assay of affinity-matured combination molecules

[0522] The anti-human FAP affinity matured combination molecule antibody was diluted 4-fold starting from a starting concentration of 5 μg / mL, for a total of 7 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, CT26 cells overexpressing mouse FAP, and 293-cynoFAP cell lines overexpressing monkey FAP were collected by centrifugation at 100g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 minutes at room temperature, and resuspended at a density of approximately 2×10 6 50 μL of the culture medium was added to the wells of a 96-well plate containing the antibody. After incubation at 4°C for 1 hour, APC fluorescently labeled goat anti-human IgG secondary antibody was added. After another hour of incubation at 4°C, the mean fluorescence reading of the cell population was analyzed by flow cytometry, and a four-parameter curve fit was performed using Prism software.

[0523] Analysis of results: Affinity-matured combination molecules were subjected to in vitro cell binding assays with HT1080-hFAP cells, which underexpress hFAP; CT26-mFAP cells, which overexpress mFAP; and 293-cynoFAP cells, which overexpress monkey FAP. The results, shown in Figures 12 to 17, demonstrate that several affinity-matured combination molecules maintained high levels of binding to HT1080-hFAP cells, while also maintaining binding to 293-cynoFAP cells. Most importantly, the binding signal to CT26-mFAP cells was significantly enhanced.

[0524] 3.2 In vitro non-specific cell binding test of affinity maturation combination molecules

[0525] Anti-human FAP affinity-matured combination molecule antibodies were diluted to 20 μg / mL and 5 μg / mL, and 50 μL of the diluted antibodies were added to a 96-well plate. CHO-hDPP4 cells overexpressing human DPP4 on their 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 at a density of approximately 2 × 10 6 50 μL of the culture medium was added to the wells of a 96-well plate containing the antibody. After incubation at 4°C for 1 hour, APC fluorescently labeled goat anti-human IgG secondary antibody was added. After another hour of incubation at 4°C, the mean fluorescence reading of the cell population was analyzed by flow cytometry, and a four-parameter curve fit was performed using Prism software.

[0526] Analysis of results: Non-specific binding experiments were conducted on the affinity matured combination molecules with the cell line CHO-hDPP4 expressing the same family protein DPP4, as well as the Chinese hamster ovary cell line CHOK1 and the human embryonic kidney cell line HEK293, which are commonly used to express proteins. The results are shown in Figures 18 to 20. The results show that each affinity matured combination molecule has no non-specific binding signals with the same family protein and the empty cells used for expression.

[0527] 3.3. In vitro binding affinity and kinetics experiments of affinity-matured combination molecules

[0528] Antibody affinity was determined using the anti-human antibody capture method using a Fortebio instrument (BLITZ pro 1.1.0.28). For the assay, a capture antibody (AHC) bioprobe targeting the Fc region of an anti-human antibody was soaked in PBS for 10 minutes. 200 μL of diluted antibody sample (including the chimeric antibody of the present invention and a control antibody; the antibody working concentration was 15 μg / mL) was loaded onto the AHC bioprobe, which was then equilibrated in PBS for 100 seconds. The AHC probe was then subjected to a binding reaction with human FAP protein and mouse FAP protein (purchased from ACRO biosystem) for 600 seconds. The AHC probe was then transferred to PBS for a dissociation reaction for 600 seconds. After the experiment, the blank control response was subtracted, and a 1:1 Langmuir binding model was fitted using software to calculate the kinetic constants for antigen-antibody binding.

[0529] Result analysis: The affinity matured combination molecules were tested for in vitro kinetic binding. The results are shown in Table 24. The results show that the binding kinetic constant of each combination molecule to recombinant human FAP protein is maintained at 10 -12 level, and the binding kinetic constant of recombinant mouse FAP protein increased from 10 -9 Level increased to 10 -10 The level has been greatly improved.

[0530] Table 24 In vitro kinetic binding activity of affinity matured combination molecules

[0531] 3.4. Physical Characterization of Affinity Maturation Combination Molecular Monomer Ratio Analysis

[0532] Experimental instrument: UPLC CLASS ACQUITY H (WATERS).

[0533] Analytical column: TSKgel G3000SWXL 7.8*300 (TOSHI, Cat No 003C03326C.

[0534] Assay solution: 200 mM K2HPO4, 250 mM KCl, pH adjusted to 6.2 with HCl.

[0535] Analytical method: Inject 50 μL of 1 mg / mL antibody into a pre-equilibrated chromatography column. Flow at room temperature and a flow rate of 0.75 mL / min for 45 minutes. Simultaneously, monitor the absorbance of the A280 analyzer. The monomer content and ratio of the antibody are determined based on the peak elution time and volume.

[0536] Result analysis: The monomer rate analysis of affinity matured combination molecules showed that the monomer rate of each combination molecule was above 95%, and the monomer rate property was good (as shown in Table 25).

[0537] Table 25 Main peak retention time and monomer rate of antibodies

[0538] 3.5 Analysis of Antibody Hydrophobicity

[0539] Experimental instrument: ARC (Waters).

[0540] Analytical column used in the experiment: TSKgel Butyl-NPR (4.6 mm×3.5 cm, Cat No. 14947).

[0541] Assay solution: A. 20 mM Histidine, pH 6.0;

[0542] B. 20mM Histidine, 1.6M (NH4)2SO4.

[0543] Analysis method: Analyze the hydrophobic properties of the antibody according to the instructions for use of the hydrophobic chromatography column.

[0544] Result analysis: The hydrophobic properties of the affinity matured combination molecules were analyzed. The results showed that the hydrophobicity HIC values ​​of each combination molecule were greater than 0.7, indicating good hydrophobic properties (as shown in Table 26).

[0545] Table 26 Hydrophobic properties of antibodies

[0546] 3.6. Drug Metabolism Analysis in Antibody Mice

[0547] Experimental materials: antibody to be tested, serum collected from mice at different time points, antibody to be tested and antigen to be bound, anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML), HRP-labeled goat anti-human IgG secondary antibody (Jackson, code: 109-035-098).

[0548] Experimental methods:

[0549] Serum collection:

[0550] 1) Female Balb / C mice (3 mice / group) were administered 200 μg of 4B9, D27H1-D01L0, D27H4-D01L4, or D27H2-D01L1 via the tail vein.

[0551] 2) Blood was collected from the tail vein at the time points of the experimental design. The blood samples were placed at room temperature for at least 30 minutes. The serum was collected at 4000 rpm for 15 minutes and stored at -20°C. To prevent serum evaporation, the final serum collection volume should be greater than 20 μL.

[0552] 3) The final serum collection should be frozen at -20°C for at least 24 hours.

[0553] Detection method:

[0554] 1) PBS was used to coat the antigen and anti-IgG Fab monoclonal antibody in a 96-well enzyme-linked microtiter plate at 0.2 μg / mL, 100 μL per well, and incubated at 4°C overnight.

[0555] 2) Prepare the required reagents: Blocking solution 5% BSA + PBS Antibody diluent 5% BSA + PBS + 20% blank mouse serum ELISA plate washing solution 0.1% Tween + PBS

[0556] 3) Wash the coated ELISA plate three times with PBS, 300 μL / well;

[0557] 4) Add blocking solution, 200 μL / well, and block at 37°C for 1 h;

[0558] 5) Dilute the starting serum to an appropriate concentration using blocking buffer, and then dilute it to an appropriate concentration range using antibody diluent containing the same serum concentration. The specific dilution factor should be adjusted based on preliminary experiments. In principle, the final color value of the tested serum should be within the color value range of the standard curve.

[0559] 6) Dilute the antibody standard curve with antibody diluent. The dilution of the standard curve should still be adjusted according to the preliminary experiment so that the standard curve fits a linear curve (if suitable software is available, an S-shaped curve can also be fitted).

[0560] 7) Discard the blocking solution and add the diluted antibody and test serum to the ELISA plates for both coating methods, 100 μL / well, and incubate at 37°C for 1 hour.

[0561] 8) Wash the plate 3 times with PBST;

[0562] 9) Dilute the secondary antibody at 1:5000 and add it to the washed ELISA plate, 100 μL / well, and incubate at 37°C for 40 min.

[0563] 10) Wash the plate three times with PBST;

[0564] 11) TMB color development, 100 μL / well, protected from light for 10 min;

[0565] 12) Add 50 μL of 2 M HCl to stop the reaction and read the result at 450 nm.

[0566] Analysis of results: In vivo drug metabolism experiments were conducted on four samples, 4B9, D27H1-D01L0, D27H4-D01L4, and D27H2-D01L1. Female Balb / C mice were used, with three mice for each sample. Blood was collected at different time points after tail vein injection, and the antibody content in mouse serum was tested using a fully human antibody detection method. The results showed that the metabolic level of each molecule in mice was different. 4B9 and D27H1-D01L0 were slowly metabolized in vivo and had long half-lives of more than 100 hours. D27H4-D01L4 and D27H2-D01L1 were rapidly metabolized in vivo and had short half-lives of less than 100 hours (results are shown in Table 27 and Figure 20).

[0567] Table 27 Antibody drug metabolism analysis in mice

[0568] Example 10 Preparation and identification of single-chain antibodies

[0569] 1. Synthesis and expression of single-chain antibodies

[0570] The antibody's heavy and light chain variable regions, or the light and heavy chain variable regions, were linked via (G4S)3 and constructed into a eukaryotic expression vector upstream of the gene encoding the human IgG1 heavy chain constant region to generate a scFv and Fc fusion protein expression plasmid. The resulting plasmid was transformed into E. coli for amplification, and large quantities of the scFv and Fc fusion protein expression plasmid were isolated. The plasmids were mixed with PEI and co-transfected into HEK293 cells. Five to six days after cell transfection, the culture supernatant was collected and purified using a Mabselect affinity chromatography column to obtain the scFv and Fc fusion protein.

[0571] 2. In vitro cell binding test of single-chain antibody

[0572] The single-chain antibody was diluted 4-fold with buffer starting at a concentration of 120 nM, and 8 gradients were set. 50 μL of the diluted antibody was added to a 96-well plate. HT1080-hFAP cells overexpressing human FAP and CT26-mFAP cells overexpressing mouse FAP on the cell surface, as well as 293-cynoFAP cells overexpressing human embryonic kidney HEK293 cells, 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 at a density of approximately 2 × 10 6 50 μL of the culture medium was added to the wells of a 96-well plate containing the antibody. After incubation at 4°C for 1 hour, APC fluorescently labeled goat anti-human IgG secondary antibody was added. After another hour of incubation at 4°C, the mean fluorescence reading of the cell population was analyzed by flow cytometry, and a four-parameter curve fit was performed using Prism software.

[0573] Analysis of Results: In vitro cell binding assays were performed on each single-chain antibody with HT1080-hFAP cells, which underexpress hFAP; CT26-mFAP cells, which overexpress mFAP; and 293-cynoFAP cells, which overexpress monkey FAP. The results, shown in Figures 22 to 38, demonstrate that several single-chain antibody combinations maintained high levels of binding to HT1080-hFAP cells, while also maintaining binding to 293-cynoFAP cells and exhibiting varying degrees of binding signals to CT26-mFAP cells. Furthermore, the in vitro cell binding activity varied significantly depending on the position of the light and heavy chains within the same molecule. For example, the binding signals of D27H1-D01L0-LH to HT1080-hFAP cells and 293-cynoFAP cells were significantly stronger than those of D27H1-D01L0-HL, and the binding signals to CT26-mFAP cells were at a comparable level. It was also found that the properties of single-chain antibodies were different from those of the corresponding monoclonal antibodies. In the affinity maturation combination molecule identification mentioned above, the in vitro cell binding of D27H1-D01L0 was slightly lower than that of 4B9, but among the single-chain antibodies, the two variants of D27H1-D01L0 performed better than the single-chain antibody of 4B9.

[0574] 3. In vitro non-specific cell binding test of single-chain antibody

[0575] The single-chain antibody was diluted 4-fold with buffer starting at a concentration of 120 nM, and 4 gradients were set. 50 μL of the diluted antibody was added to a 96-well plate. CHO-hDPP4 cells overexpressing human DPP4 on the cell surface and Chinese hamster ovary cells CHOK1 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 at a density of approximately 2 × 10 6 50 μL of the culture medium was added to the wells of a 96-well plate containing the antibody. After incubation at 4°C for 1 hour, APC fluorescently labeled goat anti-human IgG secondary antibody was added. After another hour of incubation at 4°C, the mean fluorescence reading of the cell population was analyzed by flow cytometry, and a four-parameter curve fit was performed using Prism software.

[0576] Analysis of Results: In vitro cell binding assays were performed on each single-chain antibody with CHO-hDPP4 cells, which overexpress FAP homologous proteins, and CHOK1 cells, which express Chinese hamster ovary cells. The results, shown in Figures 39 to 45, demonstrate that no significant nonspecific binding signals were observed for any of the single-chain antibody combinations with either CHO-hDPP4 cells, which overexpress FAP homologous proteins, or CHOK1 cells, which express Chinese hamster ovary cells.

[0577] 4. In vitro binding affinity and kinetics experiments of single-chain antibody

[0578] Antibody affinity was determined using the anti-human antibody capture method using a Fortebio instrument (BLITZ pro 1.1.0.28). For the assay, a capture antibody (AHC) bioprobe targeting the Fc region of an anti-human antibody was soaked in PBS for 10 minutes. 200 μL of diluted antibody sample (including the chimeric antibody of the present invention and a control antibody; the antibody working concentration was 15 μg / mL) was loaded onto the AHC bioprobe, which was then equilibrated in PBS for 100 seconds. The AHC probe was then subjected to a binding reaction with human FAP protein and mouse FAP protein (purchased from ACRO biosystem) for 600 seconds. The AHC probe was then transferred to PBS for a dissociation reaction for 600 seconds. After the experiment, the blank control response was subtracted, and a 1:1 Langmuir binding model was fitted using software to calculate the kinetic constants for antigen-antibody binding.

[0579] Result analysis: The single-chain antibody molecules were tested for in vitro kinetic binding. The results are shown in Tables 28 to 30. The results show that the binding kinetic constants of each single-chain antibody molecule with recombinant human FAP protein are mostly maintained at 10 -12 The affinity of a few molecules, such as hzD11-H1L0-HL-scFv and chrD7-LH-scFv, was low. The binding kinetic constants with recombinant mouse FAP protein showed that the binding constant of the control antibody 4B9 was at the level of 10-9. Most of the single-chain antibody molecules of self-produced antibodies were at the same level. A few molecules, such as D27H2.D01L1-LH-scFv and chrD20-LH-scFv, had a higher affinity level, reaching 10 -12 level; the binding kinetic constants of the control molecule and the self-produced molecule with recombinant monkey FAP protein were all within 10 -12 levels, and a few molecules, such as hzD11-H1L0-HL-scFv and chrD7-LH-scFv, showed reduced affinity.

[0580] Table 28 In vitro kinetic binding activity of single-chain antibodies to recombinant human FAP protein

[0581] Table 29 In vitro kinetic binding activity of single-chain antibodies to recombinant mouse FAP protein

[0582] Table 30 In vitro kinetic binding activity of single-chain antibodies to recombinant monkey FAP protein

[0583] 5. Single-chain antibody physical characterization and monomer rate analysis

[0584] Experimental instrument: UPLC CLASS ACQUITY H (WATERS).

[0585] Analytical column: TSKgel G3000SWXL 7.8*300 (TOSHI, Cat No 003C03326C.

[0586] Assay solution: 200 mM K2HPO4, 250 mM KCl, pH adjusted to 6.2 with HCl.

[0587] Analytical method: Inject 50 μL of 1 mg / mL antibody into a pre-equilibrated chromatography column. Flow at room temperature and a flow rate of 0.75 mL / min for 45 minutes. Simultaneously, monitor the absorbance of the A280 analyzer. The monomer content and ratio of the antibody are determined based on the peak elution time and volume.

[0588] Analysis of results: The monomer rate of single-chain antibody molecules was analyzed. The results showed that the monomer rates of D27H1.D01L0-HL-scFv, D27H2.D01L1-LH-scFv and D27H5.D01L4-LH-scFv were relatively high, all above 95%; the monomer rates of D27H1.D01L0-LH-scFv, D27H5.D01L4-HL-scFv and D11-H1L0-LH-scFv were good, above 90%; the monomer rates of the remaining molecules were lower than 90% (as shown in Table 31).

[0589] Table 31 Main peak retention time and monomer rate of antibodies

[0590] The above describes in detail the recombinant anti-FAP antibodies and their applications provided by the present invention. This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above examples are intended only to facilitate understanding of the methods and core concepts of the present invention. It should be noted that those skilled in the art will be able to make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Recombinant anti-FAP antibody, characterized in that, comprising a heavy chain and a light chain; the CDR regions of the heavy chain include heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; (I), the heavy chain CDR1 has an amino acid sequence as shown in SEQ ID NO.2, 16, 29, 59, 67, 75, or 158; and (II), the heavy chain CDR2 has an amino acid sequence as shown in SEQ ID NO.4, 18, 30, 39, 49, 60, 68, 76, 85, 94, or 102; and (III), the heavy chain CDR3 has an amino acid sequence as shown in SEQ ID NO.6, 20, 32, 41, 51, 62, 70, 78, 87, 96, 104, or 161; or (IV), a sequence in which one or more amino acids are substituted, deleted, or added to the amino acid sequence described in any one of (I) to (III); or (V), an amino acid sequence having more than 80% identity with the amino acid sequence described in any one of (I) to (IV); the CDR regions of the light chain include light chain CDR1, light chain CDR2, and light chain CDR3; (VI), the light chain CDR1 has an amino acid sequence as shown in SEQ ID NO.9, 23, 35, 44, 53, 64, 72, 80, 89, 98, or 105; and (VII), the light chain CDR2 has an amino acid sequence as shown in SEQ ID NO.11, 25, 36, 46, 55, 73, 91, 99, or 159; and (VIII), the light chain CDR3 has an amino acid sequence as shown in SEQ ID NO.13, 27, 38, 48, 57, 66, 74, 83, 93, 100, 106, or 162; or (IX), a sequence in which one or more amino acids are substituted, deleted, or added to the amino acid sequence described in any one of (VI) to (IX); or (X), an amino acid sequence having more than 80% identity with the amino acid sequence described in any one of (VI) to (X).

2. The recombinant anti-FAP antibody according to claim 1, wherein The recombinant anti-FAP antibody includes one or more of a rabbit chimeric antibody, a humanized antibody, an affinity matured antibody, or a single-chain antibody; Optionally, the rabbit chimeric antibody includes a heavy chain and a light chain; (11), the heavy chain CDR1, CDR2, and CDR3 sequentially have amino acid sequences as shown in SEQ ID NO.2, 4, and 6; and the light chain CDR1, CDR2, and CDR3 sequentially have amino acid sequences as shown in SEQ ID NO.9, 11, and 13; or (12), the heavy chain CDR1, CDR2, and CDR3 sequentially have amino acid sequences as shown in SEQ ID NO.16, 18, and 20; and the light chain CDR1, CDR2, and CDR3 sequentially have amino acid sequences as shown in SEQ ID NO.23, 25, and 27; or (13), the heavy chain CDR1, CDR2, and CDR3 sequentially have amino acid sequences as shown in SEQ ID NO.29, 30, and 32; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 35, 36 and 38 in sequence; or (14), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 16, 39 and 41 in sequence; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 44, 46 and 48 in sequence; or (15), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 16, 49 and 51 in sequence; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 53, 55 and 57 in sequence; or (16), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 59, 60 and 62 in sequence; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 64, 25 and 66 in sequence; or (17), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 67, 68 and 70 in sequence; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 72, 73 and 74 in sequence; or (18), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 75, 76 and 78 in sequence; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 80, 25 and 83 in sequence; or (19), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 29, 85 and 87 in sequence; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 89, 91 and 93 in sequence; or (20), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 16, 94 and 96 in sequence; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 98, 99 and 100 in sequence; or (21), the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 75, 102 and 104 in sequence; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 105, 25 and 106 in sequence; or (22) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in any one of (11) to (21), and having the same function as the amino acid sequence described in any one of (11) to (21); or (23) An amino acid sequence having more than 80% identity with the amino acid sequence described in any one of (11) to (22).

3. The recombinant anti-FAP antibody according to claim 2, wherein, The humanized antibody comprises a heavy chain and a light chain; <11> The CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO.2, 4 and 6 respectively; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO.9, 11 and 13 respectively; or <12> The CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO.16, 39 and 41 respectively; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO.44, 46 and 48 respectively; or <13> The CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO.59, 60 and 62 respectively; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO.64, 25 and 66 respectively; or <14> The CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO.67, 68 and 70 respectively; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO.72, 73 and 74 respectively; or <15> The CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO.16, 94 and 96 respectively; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO.147, 99 and 100 respectively; or <16> An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in any one of <11> to <15>, and having the same function as the amino acid sequence described in any one of <11> to <15>; or <17> An amino acid sequence having more than 80% identity with the amino acid sequence described in any one of <11> to <16>.

4. The recombinant anti-FAP antibody according to claim 2, wherein The affinity matured antibody comprises a heavy chain and a light chain; X11) The CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO.158, 94 and 96 respectively; and The CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO.9, 159 and 13 respectively; or X12), the CDR1, CDR2, and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO.16, 94, and 161, respectively; and the CDR1, CDR2, and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO.9, 11, and 162, respectively; or X13), an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in X11) or X12), and having the same function as the amino acid sequence described in X11) or X12); or X14), an amino acid sequence having more than 80% identity with the amino acid sequence described in any one of X11) to X13).

5. The recombinant anti-FAP antibody according to any one of claims 1 to 4, characterized in that, comprising a heavy chain and a light chain; (A1), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No.(2N + 1) or SEQ ID No.(2X); and (A2), the variable region of the light chain has the amino acid sequence shown in SEQ ID No.(2N + 2) or SEQ ID No.(2X + 1); or (A3), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in (A1) or (A2); or (A4), a sequence having more than 80% homology with the amino acid sequence shown in any one of (A1) to (A3); wherein N is any integer selected from 53 to 63, 81, or 82; wherein X is any integer selected from 74 to 78.

6. The recombinant anti-FAP antibody according to any one of claims 1 to 5, characterized in that, The recombinant anti - FAP antibody includes one or more of a rabbit - derived chimeric antibody, a humanized antibody, an affinity - matured antibody, or a single - chain antibody; The rabbit - derived chimeric antibody includes a heavy chain and a light chain; (B1), the variable region of the heavy chain of the rabbit - derived chimeric antibody has the amino acid sequence shown in SEQ ID No.(2N + 1); and (B2), the variable region of the light chain of the rabbit - derived chimeric antibody has the amino acid sequence shown in SEQ ID No.(2N + 2); or (B3), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in (B1) or (B2); or (B4), a sequence having more than 80% homology with the amino acid sequence shown in any one of (B1) to (B3); wherein N is any integer selected from 53 to 63.

7. The recombinant anti-FAP antibody according to claim 6, wherein, The rabbit - derived chimeric antibody includes a heavy chain and a light chain; (B5), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.107; and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO.108; or (B6), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.109; and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO.110; or (B7), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.111; and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO.112; or (B8) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 113; and The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 114; or (B9) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 115; and The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 116; or (B10) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 117; and The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 118; or (B11) 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. 120; or (B12) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 121; and The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 122; or (B13) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 123; and The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 124; or (B14) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 125; and The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 126; or (B15) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 127; and The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 128; or (B16) 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 (B5) to (B15); or (B17) A sequence having a homology of 80% or more with the amino acid sequence shown in any one of (B5) to (B16).

8. The recombinant anti-FAP antibody according to claim 6, wherein The humanized antibody comprises a heavy chain and a light chain; (C1) The variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID NO(2X); and (C2) The variable region of the light chain of the humanized antibody has the amino acid sequence shown in SEQ ID NO.(2X + 1); or (C3) 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 (C1) or (C2); or (C4) A sequence having a homology of 80% or more with the amino acid sequence shown in any one of (C1) to (C3); X is any integer selected from 74 to 78.

9. The recombinant anti-FAP antibody according to claim 8, wherein, The humanized antibody comprises a heavy chain and a light chain; (C5) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 148; and The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 149; or (C6) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 150; and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 151; or (C7) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 152; and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 153; or (C8) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 154; and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 155; or (C9) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 156; and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 157; or (C10) 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 (C5) to (C9); or (C11) A sequence having a homology of 80% or more with the amino acid sequence shown in any one of (C5) to (C10).

10. The recombinant anti-FAP antibody according to claim 6, wherein The affinity matured antibody comprises a heavy chain and a light chain; (D1) The variable region of the heavy chain of the affinity matured antibody has the amino acid sequence shown in SEQ ID No. (2N + 1); and (D2) The variable region of the light chain of the affinity matured antibody has the amino acid sequence shown in SEQ ID No. (2N + 2); or (D3) 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 (D1) or (D2); or (D4) A sequence having a homology of 80% or more with the amino acid sequence shown in any one of (D1) to (D3); The N is selected from 81 or 82.

11. The recombinant anti-FAP antibody according to claim 10, wherein The affinity matured antibody comprises a heavy chain and a light chain; (D5) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 163; and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 164; or (D6) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 165; and the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 166; or (D7) 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 (D5) or (D6); or (D8) A sequence having a homology of 80% or more with the amino acid sequence shown in any one of (D5) to (D7).

12. The recombinant anti-FAP antibody according to claim 6, wherein The single-chain antibody has: <e1> any one or more of the amino acid sequences shown in SEQ ID NOs. 170 to 184; or <e2>, in such as <e1> a sequence in which one or more amino acids are substituted, deleted, added, and / or replaced on the basis of the shown amino acid sequence; or <e3>, and such as <e1>or <e2> a sequence having a homology of 80% or more with the shown amino acid sequence.

13. The recombinant anti-FAP antibody according to any one of claims 1 to 12, wherein It further comprises a constant region; 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 kappa type; Optionally, the FAP comprises human FAP, murine FAP, and / or cynomolgus monkey FAP.

14. The method for preparing the recombinant anti-FAP antibody according to any one of claims 1 to 13, characterized in that, It includes taking the FAP immunoreceptor, separating the spleen cells of the receptor, and obtaining the variable regions of the light and heavy chains of the recombinant anti-FAP antibody by PCR amplification; Splicing the variable region of the heavy chain and the heavy chain constant region of the IgG1 subclass, constructing it into a mammalian cell expression vector to obtain a heavy chain vector; splicing the variable region of the light chain and the light chain constant region of the kappa class antibody, constructing it into the mammalian cell expression vector to obtain a light chain vector; Taking the heavy chain vector and the light chain vector, transfecting cells, culturing, purifying, and screening to obtain the recombinant anti-FAP antibody according to the affinity between the purified antibody and the FAP.

15. A biological material, characterized in that, It includes any of the following: a), a nucleic acid molecule encoding the recombinant anti-FAP antibody according to any one of claims 1 to 13; and / or b), an expression vector containing the nucleic acid molecule encoding the recombinant anti-FAP antibody according to any one of claims 1 to 13; and / or c), a host secreting the recombinant anti-FAP antibody according to any one of claims 1 to 13; and / or d), the recombinant anti-FAP antibody obtained by the preparation method according to claim 14; and / or e), a conjugate of the recombinant anti-FAP antibody according to any one of claims 1 to 13 chemically or biologically labeled and / or the recombinant anti-FAP antibody obtained by the preparation method according to claim 14.

16. An immune cell, characterized in that, It includes: A nucleic acid molecule encoding the recombinant anti-FAP antibody according to any one of claims 1 to 13; and / or An expression vector containing the nucleic acid molecule encoding the recombinant anti-FAP antibody according to any one of claims 1 to 13.

17. The immune cell according to claim 16, wherein, The immune cells are selected from cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβT cells, and γδT cells.

18. The immune cell according to claim 17, wherein, The immune cells express a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC) receptor, or a T cell receptor (TCR).

19. Use of any of the following in the preparation of a drug targeting FAP: A), the recombinant anti-FAP antibody according to any one of claims 1 to 13; and / or B), the recombinant anti-FAP antibody obtained by the preparation method according to claim 14; and / or C), the biological material according to claim 15; and / or D), the immune cells according to any one of claims 16 to 18.

20. Use of any of the following in the preparation of a product for preventing and / or treating cancer: A), the recombinant anti-FAP antibody according to any one of claims 1 to 13; and / or B), the recombinant anti-FAP antibody obtained by the preparation method according to claim 14; and / or C), the biological material according to claim 15; and / or D), the immune cells according to any one of claims 16 to 18.

21. The application according to claim 20, wherein the cancer includes any one or more of ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, bladder cancer, colorectal cancer, and brain metastatic cancer; Optionally, the product includes a drug and / or a vaccine.

22. A drug, characterized in that, It includes any of the following: A), the recombinant anti-FAP antibody according to any one of claims 1 to 13; and / or B), the recombinant anti-FAP antibody obtained by the preparation method according to claim 14; and / or C), the biological material according to claim 15; and / or D), the immune cells according to any one of claims 16 to 18.

23. Pharmaceutical combination, characterized in that, It includes the drug according to claim 22, and any other active ingredient.

24. The pharmaceutical combination according to claim 23, wherein, The any other active ingredient includes a small molecule toxin.

25. A vaccine, characterized in that, It includes any of the following: A), the recombinant anti-FAP antibody according to any one of claims 1 to 13; and / or B), the recombinant anti-FAP antibody obtained by the preparation method according to claim 14; and / or C), the biological material according to claim 15; and / or D), the immune cells according to any one of claims 16 to 18.

26. A treatment method, characterized in that, It includes administering to a subject any of the following: A), the recombinant anti-FAP antibody according to any one of claims 1 to 13; and / or B), the recombinant anti-FAP antibody obtained by the preparation method according to claim 14; and / or C), the biological material according to claim 15; and / or D), the immune cells according to any one of claims 16 to 18; and / or E), the drug according to claim 22; and / or F), the drug combination according to claim 23 or 24.

27. A preventive method, characterized in that it includes administering to a subject the vaccine according to claim 25.

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