Recombinant anti-FAP antibody and application thereof

Abstract

The invention relates to the field of antibody drugs, in particular to a recombinant anti-FAP antibody and application thereof. The present invention provides antibody proteins that specifically bind to fibrocyte-activated protein alpha (FAP alpha), the use of said antibody proteins for diagnostic and therapeutic purposes and methods of producing the above antibodies. Experiments find that the anti-FAP antibody provided by the invention is better in effect and lower in immunogenicity, shows high specific binding and rapid and efficient internalization ability, and is helpful to become a monoclonal antibody for treatment due to high affinity, high selectivity and high biological activity level.

Description

[0001] This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on January 15, 2024, with the application number 2024100555155 and the invention title "Recombinant Anti-FAP Antibody and Its Application", the entire content of which is incorporated herein by reference. Technical Field

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

[0003] FAPα is a cell surface molecule of reactive stromal fibroblasts initially identified by the monoclonal antibody F19, a cell surface glycoprotein of reactive stromal fibroblasts, and can serve as a potential antibody target in human epithelial cancers. In many types of human cancers, fibroblast responses are characterized by the induction of cell surface proteins, namely fibroblast activation protein α (FAPα), which is a 95 kDa serine protease with highly restricted expression in developing organs, wound healing, and tissue remodeling.

[0004] The anti-FAP antibody hu36 developed by ONCOMATRYX BIOPHARMA exhibits highly specific binding and rapid internalization. In addition, the inventors isolated the A chain of nigrin b produced in bacterial host cells and conjugated it to the anti-FAP antibody for tumor treatment. It shows the effect of inhibiting tumor growth in various in vitro tumor models.

[0005] Despite these advancements, there remains an unmet need for other treatment strategies for treating, including epithelial tumors, and for the components used in such treatment strategies. Summary of the Invention

[0006] In view of this, the present invention provides recombinant anti-FAP antibodies and their applications. The present invention provides an antibody protein that specifically binds to fibroblast activation protein α (FAPα), the application of the antibody protein for diagnostic and therapeutic purposes, and a method for producing the above antibody. The present invention discovers through experiments that the humanized anti-FAP antibody provided by the present invention has lower immunogenicity, exhibits highly specific binding, can cross-react with proteins of different species such as humans, mice, and monkeys, and its high affinity, high selectivity, and high biological activity levels contribute to becoming a therapeutic monoclonal antibody.

[0007] To achieve the above invention objectives, the present invention provides the following technical solutions:

[0008] The present invention provides a recombinant anti-FAP antibody, comprising a heavy chain and a light chain;

[0009] The CDR regions of the heavy chain include heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3;

[0010] (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

[0011] (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

[0012] (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

[0013] (IV), a sequence of the amino acid sequence as described in any one of (I) to (III) with one or more amino acids substituted, deleted or added; or

[0014] (V), an amino acid sequence having more than 80% identity with the amino acid sequence as described in any one of (I) to (IV);

[0015] The CDR regions of the light chain include light chain CDR1, light chain CDR2, and light chain CDR3;

[0016] (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

[0017] (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

[0018] (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

[0019] (IX), a sequence of the amino acid sequence as described in any one of (VI) to (IX) with one or more amino acids substituted, deleted or added; or

[0020] (X), an amino acid sequence having more than 80% identity with the amino acid sequence as described in any one of (VI) to (X).

[0021] In some specific embodiments of the present invention, 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;

[0022] The CDR regions of the heavy chain of the rabbit chimeric antibody include heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3;

[0023] (1), the heavy chain CDR1 has an amino acid sequence as shown in SEQ ID NO.2, 16, 29, 59, 67, or 75; and

[0024] (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

[0025] (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

[0026] (4), the amino acid sequence as described in any one of (1) to (3) with one or more amino acids substituted, deleted, or added; or

[0027] (5), an amino acid sequence having more than 80% identity with the amino acid sequence as described in any one of (1) to (4);

[0028] The CDR regions of the light chain of the rabbit chimeric antibody include light chain CDR1, light chain CDR2, and light chain CDR3;

[0029] (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

[0030] (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

[0031] (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

[0032] (9), an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence as described in any one of (6) to (8), and having the same function as the amino acid sequence as described in any one of (6) to (8); or

[0033] (10) An amino acid sequence having more than 80% identity with the amino acid sequence described in any one of (6) to (9).

[0034] In some specific embodiments of the present invention, the rabbit-derived chimeric antibody comprises a heavy chain and a light chain;

[0035] (11) The CDR1, CDR2, and CDR3 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.2, 4, and 6; and

[0036] The CDR1, CDR2, and CDR3 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.9, 11, and 13; or

[0037] (12) The CDR1, CDR2, and CDR3 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.16, 18, and 20; and

[0038] The CDR1, CDR2, and CDR3 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.23, 25, and 27; or

[0039] (13) The CDR1, CDR2, and CDR3 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.29, 30, and 32; and

[0040] The CDR1, CDR2, and CDR3 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.35, 36, and 38; or

[0041] (14) The CDR1, CDR2, and CDR3 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.16, 39, and 41; and

[0042] The CDR1, CDR2, and CDR3 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.44, 46, and 48; or

[0043] (15) The CDR1, CDR2, and CDR3 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.16, 49, and 51; and

[0044] The CDR1, CDR2, and CDR3 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.53, 55, and 57; or

[0045] (16) The CDR1, CDR2, and CDR3 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.59, 60, and 62; and

[0046] 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

[0047] (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

[0048] 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

[0049] (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

[0050] 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

[0051] (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

[0052] 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

[0053] (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

[0054] 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

[0055] (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

[0056] 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

[0057] (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 an amino acid sequence having the same function as the amino acid sequence described in any one of (11) to (21); or

[0058] (23) An amino acid sequence having more than 80% identity with the amino acid sequence described in any one of (11) to (22).

[0059] In some specific embodiments of the present invention, the CDR regions of the heavy chain of the humanized antibody include heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3;

[0060] <1>. The heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO.2, 16, 59, or 67; and

[0061] <2>. The heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO.4, 39, 60, 68, or 94; and

[0062] <3>. The heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO.6, 41, 62, 70, or 96; or

[0063] <4>. 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 <1> to <3>, and having the same function as the amino acid sequence described in any one of <1> to <3>; or

[0064] <5>. An amino acid sequence having more than 80% identity with the amino acid sequence described in any one of <1> to <4>;

[0065] The CDR regions of the light chain of the humanized antibody include light chain CDR1, light chain CDR2, and light chain CDR3;

[0066] <6>. The light chain CDR1 has the amino acid sequence shown in SEQ ID NO.9, 44, 64, 72, or 147; and

[0067] <7>. The light chain CDR2 has the amino acid sequence shown in SEQ ID NO.11, 25, 46, 73, or 99; and

[0068] <8>. The light chain CDR3 has the amino acid sequence shown in SEQ ID NO.13, 48, 66, 74, or 100; or

[0069] <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

[0070] <10>. An amino acid sequence having more than 80% identity with the amino acid sequence described in any one of <6> to <9>.

[0071] In some specific embodiments of the present invention, the humanized antibody comprises a heavy chain and a light chain;

[0072] <11>, the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO.2, 4 and 6 in sequence; and

[0073] the CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO.9, 11 and 13 in sequence; or

[0074] <12>, 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

[0075] 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

[0076] <13>, 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

[0077] 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

[0078] <14>, 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

[0079] 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

[0080] <15>, 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

[0081] the CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO.147, 99 and 100 in sequence; or

[0082] <16>, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence according to any one of <11> to <15>, and an amino acid sequence having the same function as the amino acid sequence according to any one of <11> to <15>; or

[0083] <17> An amino acid sequence having more than 80% identity with the amino acid sequence described in any one of <11> to <16>.

[0084] In some specific embodiments of the present invention, the CDR regions of the heavy chain of the affinity matured antibody include heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3;

[0085] X1), the heavy chain CDR1 has the amino acid sequence as shown in SEQ ID NO. 158 or 16; and

[0086] X2), the heavy chain CDR2 has the amino acid sequence as shown in SEQ ID NO. 94; and

[0087] X3), the heavy chain CDR3 has the amino acid sequence as shown in SEQ ID NO. 96 or 161; or

[0088] 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

[0089] X5), an amino acid sequence having more than 80% identity with the amino acid sequence described in any one of X1) to X4);

[0090] The CDR regions of the light chain of the affinity matured antibody include light chain CDR1, light chain CDR2, and light chain CDR3;

[0091] X6), the light chain CDR1 has the amino acid sequence as shown in SEQ ID NO. 9; and

[0092] X7), the light chain CDR2 has the amino acid sequence as shown in SEQ ID NO. 11 or 159; and

[0093] X8), the light chain CDR3 has the amino acid sequence as shown in SEQ ID NO. 13 or 162; or

[0094] 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

[0095] X10), an amino acid sequence having more than 80% identity with the amino acid sequence described in any one of X6) to X9);

[0096] In some specific embodiments of the present invention, the affinity matured antibody comprises a heavy chain and a light chain;

[0097] X11), the CDR1, CDR2, and CDR3 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO. 158, 94, and 96; and

[0098] the CDR1, CDR2, and CDR3 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO. 9, 159, and 13; or

[0099] X12), the CDR1, CDR2, and CDR3 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO. 16, 94, and 161; and

[0100] the CDR1, CDR2, and CDR3 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO. 9, 11, and 162; or

[0101] X13), an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence as described in X11) or X12), and having the same function as the amino acid sequence as described in X11) or X12); or

[0102] X14), an amino acid sequence having more than 80% identity with the amino acid sequence as described in any one of X11) to X13).

[0103] In some specific embodiments of the present invention, the FR regions of the heavy chain include heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4;

[0104] I) The heavy chain FR1 has the amino acid sequence shown in SEQ ID NO. 1, 15, 28, 58, 84, 101, 129, 136, or 142; and

[0105] the heavy chain FR2 has the amino acid sequence shown in SEQ ID NO. 3, 17, 130, 137, or 160; and

[0106] the heavy chain FR3 has the amino acid sequence shown in SEQ ID NO. 5, 19, 31, 40, 50, 61, 69, 77, 86, 95, 103, 131, 138, or 143; and

[0107] the heavy chain FR4 has the amino acid sequence shown in SEQ ID NO. 7, 21, 33, or 42; or

[0108] II), an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence as described in I), and having the same function as the amino acid sequence as described in I); or

[0109] III), an amino acid sequence having more than 80% identity with the amino acid sequence described in I) or II); or

[0110] The FR regions of the light chain include light chain FR1, light chain FR2, light chain FR3, and light chain FR4;

[0111] IV), the light chain FR1 has the amino acid sequence shown in SEQ ID NO. 8, 22, 34, 43, 52, 63, 71, 79, 88, 97, 132, 139, 144, or 146; and

[0112] The light chain FR2 has the amino acid sequence shown in SEQ ID NO. 10, 24, 45, 54, 81, 90, 133, or 140; and

[0113] The light chain FR3 has the amino acid sequence shown in SEQ ID NO. 12, 26, 37, 47, 56, 65, 82, 92, 134, 141, or 145; and

[0114] The light chain FR4 has the amino acid sequence shown in SEQ ID NO. 14 or 135; or

[0115] 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

[0116] VI), an amino acid sequence having more than 80% identity with the amino acid sequence described in IV) or V).

[0117] In some specific embodiments of the present invention, 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;

[0118] The FR regions of the heavy chain of the rabbit chimeric antibody include heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4;

[0119] (A) The heavy chain FR1 has the amino acid sequence shown in SEQ ID NO. 1, 15, 28, 58, 84, or 101; and

[0120] The heavy chain FR2 has the amino acid sequence shown in SEQ ID NO. 3 or 17; and

[0121] The heavy chain FR3 has the amino acid sequence shown in SEQ ID NO. 5, 19, 31, 40, 50, 61, 69, 77, 86, 95, or 103; and

[0122] The heavy chain FR4 has an amino acid sequence as shown in SEQ ID NO.7, 21, 33 or 42; or

[0123] (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

[0124] (C), an amino acid sequence having more than 80% identity with the amino acid sequence described in (A) or (B); or

[0125] The FR regions of the light chain of the rabbit-derived chimeric antibody include light chain FR1, light chain FR2, light chain FR3 and light chain FR4;

[0126] (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

[0127] the light chain FR2 has an amino acid sequence as shown in SEQ ID NO.10, 24, 45, 54, 81 or 90; and

[0128] 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

[0129] the light chain FR4 has an amino acid sequence as shown in SEQ ID NO.14; or

[0130] (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

[0131] (F), an amino acid sequence having more than 80% identity with the amino acid sequence described in (D) or (E).

[0132] In some specific embodiments of the present invention, the rabbit-derived chimeric antibody comprises a heavy chain and a light chain;

[0133] (G), the FR1, FE2, FR3 and FR4 of the heavy chain have amino acid sequences as shown in SEQ ID NO.1, 3, 5 and 7 in sequence; and

[0134] the FR1, FE2, FR3 and FR4 of the light chain have amino acid sequences as shown in SEQ ID NO.8, 10, 12 and 14 in sequence; or

[0135] (H) The FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 15, 17, 19, and 21, respectively; and

[0136] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 22, 24, 26, and 14, respectively; or

[0137] (I) The FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 28, 17, 31, and 33, respectively; and

[0138] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 34, 24, 37, and 14, respectively; or

[0139] (J) The FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 28, 3, 40, and 42, respectively; and

[0140] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 43, 45, 47, and 14, respectively; or

[0141] (K) The FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 28, 3, 50, and 21, respectively; and

[0142] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 52, 54, 56, and 14, respectively; or

[0143] (L) The FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 58, 3, 61, and 7, respectively; and

[0144] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 63, 10, 65, and 14, respectively; or

[0145] (M) The FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 28, 3, 69, and 21, respectively; and

[0146] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 71, 24, 47, and 14, respectively; or

[0147] (N) The FR1, FE2, FR3, and FR4 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.28, 17, 77, and 21; and

[0148] The FR1, FE2, FR3, and FR4 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.79, 81, 82, and 14; or

[0149] (O) The FR1, FE2, FR3, and FR4 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.84, 3, 86, and 7; and

[0150] The FR1, FE2, FR3, and FR4 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.88, 90, 92, and 14; or

[0151] (P) The FR1, FE2, FR3, and FR4 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.28, 3, 95, and 7; and

[0152] The FR1, FE2, FR3, and FR4 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.97, 24, 12, and 14; or

[0153] (Q) The FR1, FE2, FR3, and FR4 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.101, 17, 103, and 21; and

[0154] The FR1, FE2, FR3, and FR4 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.22, 24, 26, and 14; or

[0155] (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

[0156] (S) An amino acid sequence having more than 80% identity with the amino acid sequence described in any one of (G) to (R).

[0157] 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;

[0158] 1) The heavy chain FR1 has the amino acid sequence shown in SEQ ID NO.129, 136, or 142; and

[0159] The heavy chain FR2 has an amino acid sequence as shown in SEQ ID NO. 130 or 137; and

[0160] The heavy chain FR3 has an amino acid sequence as shown in SEQ ID NO. 131, 138 or 143; and

[0161] The heavy chain FR4 has an amino acid sequence as shown in SEQ ID NO. 7; or

[0162] 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

[0163] 3), an amino acid sequence having more than 80% identity with the amino acid sequence described in 1) or 2); or

[0164] The FR regions of the humanized antibody light chain include light chain FR1, light chain FR2, light chain FR3 and light chain FR4;

[0165] 4), the light chain FR1 has an amino acid sequence as shown in SEQ ID NO. 132, 139, 144 or 146; and

[0166] The light chain FR2 has an amino acid sequence as shown in SEQ ID NO. 133 or 140; and

[0167] The light chain FR3 has an amino acid sequence as shown in SEQ ID NO. 134, 141 or 145; and

[0168] The light chain FR4 has an amino acid sequence as shown in SEQ ID NO. 135; or

[0169] 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

[0170] 6), an amino acid sequence having more than 80% identity with the amino acid sequence described in 4) or 5).

[0171] In some specific embodiments of the present invention, the humanized antibody comprises a heavy chain and a light chain;

[0172] 7), the FR1, FE2, FR3 and FR4 of the heavy chain have amino acid sequences as shown in SEQ ID NO. 129, 130, 131 and 7 in sequence; and

[0173] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 132, 133, 134, and 135, respectively; or

[0174] 8), the FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 136, 137, 138, and 7, respectively; and

[0175] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 139, 140, 141, and 135, respectively; or

[0176] 9), the FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 142, 137, 143, and 7, respectively; and

[0177] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 144, 133, 145, and 135, respectively; or

[0178] 10), the FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 142, 137, 143, and 7, respectively; and

[0179] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 146, 133, 134, and 135, respectively; or

[0180] 11), the FR1, FE2, FR3, and FR4 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 136, 137, 138, and 7, respectively; and

[0181] The FR1, FE2, FR3, and FR4 of the light chain have the amino acid sequences shown in SEQ ID NO. 144, 133, 145, and 135, respectively; or

[0182] 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

[0183] 13), an amino acid sequence having more than 80% identity with the amino acid sequence described in any one of 7) to 12).

[0184] In some specific embodiments of the present invention, the FR regions of the heavy chain of the affinity matured antibody include heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4;

[0185] (X1) The heavy chain FR1 has the amino acid sequence shown in SEQ ID NO. 136; and

[0186] The heavy chain FR2 has the amino acid sequence shown in SEQ ID NO. 137 or 160; and

[0187] The heavy chain FR3 has the amino acid sequence shown in SEQ ID NO. 138; and

[0188] The heavy chain FR4 has the amino acid sequence shown in SEQ ID NO. 7; or

[0189] (X2), an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence as described in (X1), and having the same function as the amino acid sequence described in (X1); or

[0190] (X3), an amino acid sequence having more than 80% identity with the amino acid sequence as described in (X1) or (X2); or

[0191] The FR regions of the light chain of the affinity matured antibody include light chain FR1, light chain FR2, light chain FR3, and light chain FR4;

[0192] (X4), the light chain FR1 has the amino acid sequence shown in SEQ ID NO. 132; and

[0193] The light chain FR2 has the amino acid sequence shown in SEQ ID NO. 133; and

[0194] The light chain FR3 has the amino acid sequence shown in SEQ ID NO. 134; and

[0195] The light chain FR4 has the amino acid sequence shown in SEQ ID NO. 135; or

[0196] (X5), an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence as described in (X4), and having the same function as the amino acid sequence described in (X4); or

[0197] (X6), an amino acid sequence having more than 80% identity with the amino acid sequence as described in (X4) or (X5).

[0198] In some specific embodiments of the present invention, the affinity matured antibody includes a heavy chain and a light chain;

[0199] (X7), the FR1, FE2, FR3, and FR4 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.136, 137, 138, and 7; and

[0200] the FR1, FE2, FR3, and FR4 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.132, 133, 134, and 135; or

[0201] (X8), the FR1, FE2, FR3, and FR4 of the heavy chain sequentially have the amino acid sequences shown in SEQ ID NO.136, 160, 138, and 7; and

[0202] the FR1, FE2, FR3, and FR4 of the light chain sequentially have the amino acid sequences shown in SEQ ID NO.132, 133, 134, and 135; or

[0203] (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

[0204] (X10), an amino acid sequence having more than 80% identity with the amino acid sequence described in any one of (X7) to (X9).

[0205] In some specific embodiments of the present invention, the recombinant anti-FAP antibody comprises a heavy chain and a light chain;

[0206] (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

[0207] (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

[0208] (A3), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids on the basis of the amino acid sequence shown in (A1) or (A2); or

[0209] (A4), a sequence having more than 80% homology with the amino acid sequence shown in any one of (A1) to (A3);

[0210] The N is selected from any integer in 53 to 63, 81, or 82;

[0211] The X is selected from any integer in 74 to 78.

[0212] In some specific embodiments of the present invention, 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;

[0213] The rabbit chimeric antibody includes a heavy chain and a light chain;

[0214] (B1), the heavy chain variable region of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID NO. (2N + 1); and

[0215] (B2), the light chain variable region of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID NO. (2N + 2); or

[0216] (B3), a sequence with substitution, deletion, addition, and / or replacement of one or more amino acids based on the amino acid sequence shown in (B1) or (B2); or

[0217] (B4), a sequence with a homology of more than 80% to the amino acid sequence shown in any one of (B1) to (B3);

[0218] The N is selected from any integer between 53 and 63.

[0219] In some specific embodiments of the present invention, the rabbit chimeric antibody includes a heavy chain and a light chain;

[0220] (B5), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 107; and

[0221] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 108; or

[0222] (B6), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 109; and

[0223] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 110; or

[0224] (B7), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 111; and

[0225] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 112; or

[0226] (B8), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 113; and

[0227] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 114; or

[0228] (B9), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 115; and

[0229] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 116; or

[0230] (B10), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 117; and

[0231] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 118; or

[0232] (B11), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 119; and

[0233] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 120; or

[0234] (B12), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 121; and

[0235] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 122; or

[0236] (B13), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 123; and

[0237] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 124; or

[0238] (B14), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 125; and

[0239] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 126; or

[0240] (B15), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 127; and

[0241] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 128; or

[0242] (B16), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown in any one of (B5) to (B15); or

[0243] (B17) A sequence with more than 80% homology to the amino acid sequence shown in any one of (B5) to (B16).

[0244] In some specific embodiments of the present invention, the recombinant anti-FAP antibody, the humanized antibody comprises a heavy chain and a light chain;

[0245] (C1) The variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in SEQ ID NO. (2X); and

[0246] (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

[0247] (C3) A sequence with one or more amino acids substituted, deleted, added and / or replaced on the basis of the amino acid sequence shown in (C1) or (C2); or

[0248] (C4) A sequence with more than 80% homology to the amino acid sequence shown in any one of (C1) to (C3);

[0249] X is selected from any integer between 74 and 78.

[0250] In some specific embodiments of the present invention, the humanized antibody comprises a heavy chain and a light chain;

[0251] (C5) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 148; and

[0252] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 149; or

[0253] (C6) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 150; and

[0254] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 151; or

[0255] (C7) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 152; and

[0256] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 153; or

[0257] (C8) The variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 154; and

[0258] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 155; or

[0259] (C9), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 156; and

[0260] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 157; or

[0261] (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

[0262] (C11), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of (C5) to (C10).

[0263] 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;

[0264] (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

[0265] (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

[0266] (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

[0267] (D4), a sequence having a homology of 80% or more with the amino acid sequence shown in any one of (D1) to (D3);

[0268] The N is selected from 81 or 82.

[0269] In some specific embodiments of the present invention, the affinity matured antibody comprises a heavy chain and a light chain;

[0270] (D5), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 163; and

[0271] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 164; or

[0272] (D6), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 165; and

[0273] The variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 166; or

[0274] (D7), a sequence with substitution, deletion, addition and / or replacement of one or more amino acids based on the amino acid sequence shown in (D5) or (D6); or

[0275] (D8), a sequence with a homology of more than 80% to the amino acid sequence shown in any one of (D5) to (D7).

[0276] In some specific embodiments of the present invention, the single-chain antibody has:

[0277] <e1>, any one or more of the amino acid sequences shown in SEQ ID NO. 170 to SEQ ID NO. 184; or

[0278] <e2>, in such as <e1>A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids based on the amino acid sequence shown; or

[0279] <e3>, such as <e1>Or <e2>Sequences with more than 80% amino acid sequence homology as shown.

[0280] In some specific embodiments of the present invention, the recombinant anti-FAP antibody further comprises a constant region;

[0281] 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 the kappa type.

[0282] In some specific embodiments of the present invention, the FAP comprises human FAP, murine FAP, and / or cynomolgus monkey FAP.

[0283] The present invention also provides a method for preparing the recombinant anti-FAP antibody, comprising immunizing a receptor with the FAP, isolating spleen cells of the receptor, and PCR amplifying to obtain the light and heavy chain variable regions of the recombinant anti-FAP antibody;

[0284] Splicing the heavy chain variable region 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 light chain variable region 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;

[0285] 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.

[0286] In some specific embodiments of the present invention, the receptor comprises New Zealand white rabbits.

[0287] In some specific embodiments of the present invention, the cells comprise HEK293 cells.

[0288] In some specific embodiments of the present invention, the preparation method further comprises: combining the CDR region of the recombinant anti-FAP antibody with the human antibody framework region, splicing the heavy chain variable region 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 light chain variable region 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.

[0289] In some specific embodiments of the present invention, the cells comprise HEK293 cells.

[0290] In some specific embodiments of the present invention, the preparation method further includes: 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 according to the affinity between the mutated antibody and FAP.

[0291] In some specific embodiments of the present invention, the preparation method further includes: successively connecting the pre-sequence, (G4S)3 and the post-sequence, constructing them into 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, separating it and co-transfecting it into cells with PEI, taking the supernatant and purifying it to obtain the recombinant anti-FAP antibody:

[0292] ① The pre-sequence has the amino acid sequence shown in SEQ ID NO.167, and the post-sequence has the amino acid sequence shown in SEQ ID NO.169; or

[0293] ② The pre-sequence has the amino acid sequence shown in SEQ ID NO.169, and the post-sequence has the amino acid sequence shown in SEQ ID NO.167; or

[0294] ③ The pre-sequence has the amino acid sequence shown in SEQ ID NO.156, and the post-sequence has the amino acid sequence shown in SEQ ID NO.149; or

[0295] ④ The pre-sequence has the amino acid sequence shown in SEQ ID NO.149, and the post-sequence has the amino acid sequence shown in SEQ ID NO.156; or

[0296] ⑤ The pre-sequence has the amino acid sequence shown in SEQ ID NO.163, and the post-sequence has the amino acid sequence shown in SEQ ID NO.164; or

[0297] ⑥ The pre-sequence has the amino acid sequence shown in SEQ ID NO.164, and the post-sequence has the amino acid sequence shown in SEQ ID NO.163; or

[0298] ⑦ The pre-sequence has the amino acid sequence shown in SEQ ID NO.165, and the post-sequence has the amino acid sequence shown in SEQ ID NO.166; or

[0299] ⑧ The pre-sequence has the amino acid sequence shown in SEQ ID NO.166, and the post-sequence has the amino acid sequence shown in SEQ ID NO.165; or

[0300] ⑨ The foregoing sequence has the amino acid sequence shown in SEQ ID NO. 150, and the following sequence has the amino acid sequence shown in SEQ ID NO. 151; or

[0301] ⑩ The foregoing 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

[0302] The foregoing sequence has the amino acid sequence shown in SEQ ID NO. 155, and the following sequence has the amino acid sequence shown in SEQ ID NO. 154; or

[0303] The foregoing sequence has the amino acid sequence shown in SEQ ID NO. 112, and the following sequence has the amino acid sequence shown in SEQ ID NO. 111; or

[0304] The foregoing sequence has the amino acid sequence shown in SEQ ID NO. 122, and the following sequence has the amino acid sequence shown in SEQ ID NO. 121; or

[0305] The foregoing sequence has the amino acid sequence shown in SEQ ID NO. 123, and the following sequence has the amino acid sequence shown in SEQ ID NO. 124; or

[0306] The foregoing sequence has the amino acid sequence shown in SEQ ID NO. 128, and the following sequence has the amino acid sequence shown in SEQ ID NO. 127; or

[0307] Such as ① to 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 the above, and having the same function as the amino acid sequence described in any one of ① to An amino acid sequence having the same function as the amino acid sequence described in any one of the above; or

[0308] Having 80% or more identity with the amino acid sequence described in any one of ① to An amino acid sequence having 80% or more identity with the amino acid sequence described in any one of the above.

[0309] (G4S)3 has the amino acid sequence shown in SEQ ID NO. 168.

[0310] In some specific embodiments of the present invention, the cells include HEK293 cells.

[0311] The present invention also provides biomaterials, including any of the following:

[0312] a), a nucleic acid molecule encoding the recombinant anti-FAP antibody; and / or

[0313] b), an expression vector containing the nucleic acid molecule encoding the recombinant anti-FAP antibody; and / or

[0314] c), a host secreting the recombinant anti-FAP antibody; and / or

[0315] d), the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0316] e), a conjugate of the chemically labeled or biologically labeled recombinant anti-FAP antibody and / or the recombinant anti-FAP antibody obtained by the preparation method.

[0317] The present invention also provides an immune cell, including: a nucleic acid molecule encoding the aforementioned recombinant anti-FAP antibody; and / or an expression vector containing the nucleic acid molecule encoding the aforementioned recombinant anti-FAP antibody.

[0318] In some specific 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, αβ T cells, and a γδ T cell.

[0319] In some specific embodiments of the present invention, the immune cell expresses a chimeric antigen receptor (CAR), a T cell antigen coupler (TAC) receptor, or a T cell receptor (TCR).

[0320] Chimeric antigen receptors (CARs) combine many aspects of normal T cell activation into a single protein. They link an extracellular antigen recognition domain to an intracellular signaling domain that activates the T cell when the antigen binds. CARs typically have the following regions: an antigen-binding domain, an extracellular hinge region, a transmembrane region, and an intracellular region. In some embodiments, the intracellular region contains an intracellular signaling domain or an intracellular signaling region.

[0321] The antigen-binding domain is exposed to the outside of the cell and is part of the extracellular domain of the receptor. It interacts with potential target molecules and is responsible for targeting CAR-T cells to any cell expressing a matching molecule. The antigen-binding domain typically derives from the variable regions of monoclonal antibodies linked together as a single-chain variable fragment (scFv). The scFv is a chimeric protein composed of the light chain (VL) and heavy chain (VH) of an immunoglobulin and is linked to a short linker peptide. In some embodiments, the heavy chain and light chain have the aforementioned heavy chain variable region and light chain variable region sequences.

[0322] The present invention also provides the use of any of the following in the preparation of a drug targeting FAP:

[0323] A), the recombinant anti-FAP antibody; and / or

[0324] B), the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0325] C), the biological material; and / or

[0326] D), the immune cells.

[0327] The present invention also provides the use of any of the following in the preparation of a product for preventing and / or treating cancer:

[0328] A), the recombinant anti-FAP antibody; and / or

[0329] B), the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0330] C), the biological material; and / or

[0331] D), the immune cells.

[0332] In some specific embodiments of the present invention, 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.

[0333] In some specific embodiments of the present invention, the product includes a drug and / or a vaccine.

[0334] The present invention also provides a drug, including any of the following:

[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 a drug combination, including the drug and any other active ingredient.

[0340] In some specific embodiments of the present invention, the other active ingredient includes a small molecule toxin.

[0341] The present invention also provides a vaccine, including any of the following:

[0342] A), the recombinant anti-FAP antibody; and / or

[0343] B), the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0344] C), the biological material described above; and / or

[0345] D), the immune cells described above.

[0346] The present invention also provides the use of any of the following in the preparation of reagents and / or kits for detecting FAP:

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

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

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

[0350] D), the immune cells described above.

[0351] The present invention also provides reagents and / or kits, including any of the following:

[0352] A), the recombinant anti-FAP antibody described above; and / or

[0353] B), the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0354] C), the biological material described above; and / or

[0355] D), the immune cells described above.

[0356] The present invention also provides a treatment method, including administering to a subject any of the following:

[0357] A), the recombinant anti-FAP antibody described above; and / or

[0358] B), the recombinant anti-FAP antibody obtained by the preparation method; and / or

[0359] C), the biological material described above; and / or

[0360] D), the immune cells described above; and / or

[0361] E), the drug described above; and / or

[0362] F), the drug combination described above; and / or

[0363] G), the vaccine described above.

[0364] The present invention includes, but is not limited to, achieving the following beneficial effects:

[0365] The anti-FAP antibody provided by the present invention has better efficacy, lower immunogenicity, exhibits highly specific binding as well as rapid and efficient internalization ability, and its high affinity, high selectivity, and high level of biological activity contribute to becoming a monoclonal antibody for therapeutic use. BRIEF DESCRIPTION OF THE DRAWINGS

[0366] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0367] Figure 1 Showing the affinity of the control antibody for the overexpressing cell line CHO-hFAP cells;

[0368] Figure 2 Showing the affinity of the control antibody for the naturally expressing cell line U138MG cells;

[0369] Figure 3 Showing the binding test of the anti-human FAP chimeric antibody to CHO-hFAP cells;

[0370] Figure 4 Showing the binding test of the anti-human FAP chimeric antibody to CHO-hFAP cells;

[0371] Figure 5 Showing the binding test of the anti-human FAP chimeric antibody to CHO-hFAP cells;

[0372] Figure 6 Showing the binding test of the anti-human FAP chimeric antibody to CHO-mFAP cells;

[0373] Figure 7 Showing the binding test of the anti-human FAP chimeric antibody to CHO-mFAP cells;

[0374] Figure 8 Showing the binding test of the anti-human FAP chimeric antibody to CHO-mFAP cells;

[0375] Figure 9 Showing the binding test of the anti-human FAP humanized antibody to HT1080-hFAP cells;

[0376] Figure 10 Showing the binding test of the anti-human FAP humanized antibody to HT1080-hFAP cells;

[0377] Figure 11 Showing the binding test of the anti-human FAP humanized antibody to CHO-mFAP cells;

[0378] Figure 12 Showing the binding test of the anti-human FAP humanized antibody to CHO-mFAP cells;

[0379] Figure 13 Show the binding test of the affinity matured combinatorial molecule with HT1080-hFAP cells;

[0380] Figure 14 Show the binding test of the affinity matured combinatorial molecule with HT1080-hFAP cells;

[0381] Figure 15 Show the binding test of the affinity matured combinatorial molecule with CT26-mFAP cells;

[0382] Figure 16 Show the binding test of the affinity matured combinatorial molecule with CT26-mFAP cells;

[0383] Figure 17 Show the binding test of the affinity matured combinatorial molecule with 293-cynoFAP cells;

[0384] Figure 18 Show the non-specific binding test of the affinity matured combinatorial molecule with CHO-hDPP4 cells;

[0385] Figure 19 Show the non-specific binding test of the affinity matured combinatorial molecule with CHOK1 cells;

[0386] Figure 20 Show the non-specific binding test of the affinity matured combinatorial molecule with HEK293 cells;

[0387] Figure 21 Show the metabolic map in mice;

[0388] Figure 22 Show the binding test of the single-chain combinatorial molecule with HT1080-hFAP cells;

[0389] Figure 23 Show the binding test of the single-chain combinatorial molecule with HT1080-hFAP cells;

[0390] Figure 24 Show the binding test of the single-chain combinatorial molecule with HT1080-hFAP cells;

[0391] Figure 25 Show the binding test of the single-chain combinatorial molecule with HT1080-hFAP cells;

[0392] Figure 26 Show the binding test of the single-chain combinatorial molecule with HT1080-hFAP cells;

[0393] Figure 27 Show the binding test of the single-chain combinatorial molecule with HT1080-hFAP cells;

[0394] Figure 28 Show the binding test of the single-chain combinatorial molecule with HT1080-hFAP cells;

[0395] Figure 29 Show the binding test of the single-chain combinatorial molecule with CT26-mFAP cells;

[0396] Figure 30 Show the binding test of the single-chain combinatorial molecule with CT26-mFAP cells;

[0397] Figure 31 Show the binding test of the single-chain combinatorial molecule with CT26-mFAP cells;

[0398] Figure 32 Show the binding test of the single-chain combinatorial molecule with CT26-mFAP cells;

[0399] Figure 33 Show the binding test of the single-chain combinatorial molecule with CT26-mFAP cells;

[0400] Figure 34 Show the binding test of the single-chain combinatorial molecule with CT26-mFAP cells;

[0401] Figure 35 Show the binding test of the single-chain combinatorial molecule with 293-cynoFAP cells;

[0402] Figure 36 Show the binding test of the single-chain combinatorial molecule with 293-cynoFAP cells;

[0403] Figure 37 Show the binding test of the single-chain combinatorial molecule with 293-cynoFAP cells;

[0404] Figure 38 Show the binding test of the single-chain combinatorial molecule with 293-cynoFAP cells;

[0405] Figure 39 Show the non-specific binding test of the single-chain combinatorial molecule with CHO-hDPP4 cells;

[0406] Figure 40 Show the non-specific binding test of the single-chain combinatorial molecule with CHO-hDPP4 cells;

[0407] Figure 41 Show the non-specific binding test of the single-chain combinatorial molecule with CHO-hDPP4 cells;

[0408] Figure 42 Show the non-specific binding test of the single-chain combinatorial molecule with CHO-hDPP4 cells;

[0409] Figure 43 Show the non-specific binding test of the single-chain combined molecule with CHOK1 cells;

[0410] Figure 44 Show the non-specific binding test of the single-chain combined molecule with CHOK1 cells;

[0411] Figure 45 Show the non-specific binding test of the single-chain combined molecule with CHOK1 cells. Detailed implementation mode

[0412] The present invention discloses a recombinant anti-FAP antibody and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0413] 1. Sequence synthesis and vector construction of the tool antibody

[0414] Table 1 Sequences of the tool antibody

[0415]

[0416]

[0417]

[0418] 2. Purchase of antigens and verification of antigen binding activity

[0419] 2.1 Information on the human FAP recombinant protein antigen

[0420] Purchase commercial reagents as shown in Table 1:

[0421] Table 2 Commercial reagents

[0422] Name Brand Model Human FAP-His Protein AcroBiosystems FAP-H5244 Human FAP-hFc Protein AcroBiosystems FAP-H5263 Human FAP-Biotin Protein AcroBiosystems FAP-H82Q6 Mouse FAP-His Protein AcroBiosystems FAP-M52H3 Cynomolgus FAP-His Protein AcroBiosystems FAP-C52H3

[0423] 2.2 Construction of antigen cell lines of different species

[0424] Table 3 Sequences of antigens of different species

[0425]

[0426]

[0427]

[0428] The nucleotide sequence of hFAP described above was constructed into the commercially available pCDNA3.1 vector from Invitrogen. Specifically, after double digestion of the pCDNA3.1 vector with KpnI and XhoI, the nucleotide sequence of hFAP was inserted, and then the vector sequence information was confirmed by sequencing. After successful plasmid construction, it was transfected into human fibrosarcoma HT1080 cells using lipofectamine3000. Subsequently, puromycin antibiotic was used to screen for overexpressing monoclonal cells, and a clone with slightly lower expression was selected. The successfully constructed cell line was named low-expression HT1080-hFAP cells.

[0429] The nucleotide sequence of hFAP described above was constructed into the commercially available pCDNA5 vector from Invitrogen. Specifically, after double digestion of the pCDNA5 vector with NheI and PmeI, the nucleotide sequence of hFAP was inserted, and then the vector sequence information was confirmed by sequencing. After successful plasmid construction, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1 using lipofectamine3000. Subsequently, Hygromycin antibiotic was used to screen for overexpressing cell lines, and the successfully constructed cell lines were named CHO-hFAP and 293-hFAP cells, respectively.

[0430] The nucleotide sequence of mFAP described above was constructed into the commercially available pCDNA5 vector from Invitrogen. Specifically, after double digestion of the pCDNA5 vector with NheI and PmeI, the nucleotide sequence of mFAP was inserted, and then the vector sequence information was confirmed by sequencing. After successful plasmid construction, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1 using lipofectamine3000. Subsequently, Hygromycin antibiotic was used to screen for overexpressing cell lines, and the successfully constructed cell lines were named CHO-mFAP and 293-mFAP cells, respectively.

[0431] The nucleotide sequence of cynoFAP described above was constructed into the commercially available pCDNA5 vector from Invitrogen. Specifically, after double digestion of the pCDNA5 vector with NheI and PmeI, the nucleotide sequence of cynoFAP was inserted, and then the vector sequence information was confirmed by sequencing. After successful plasmid construction, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1 respectively using lipofectamine3000. Subsequently, Hygromycin antibiotic was used to screen for overexpressing cell lines, and through monoclonalization, monoclonal overexpressing cell lines CHO-cynoFAP and 293-cynoFAP cells were finally obtained.

[0432] 2.3 Construction of cell lines of proteins from the same family

[0433] Table 4 Homologous protein sequences

[0434]

[0435]

[0436] The nucleotide sequence of the above-mentioned hDPP4 was constructed onto the commercially available pCDNA5 vector from Invitrogen. Specifically, after double digestion of the pCDNA5 vector with NheI and PmeI, the nucleotide sequence of hDPP4 was inserted, and then the vector sequence information was confirmed by sequencing. After successful plasmid construction, it was transfected into Chinese hamster ovary cells CHOK1 using lipofectamine3000. Subsequently, an overexpressing cell line was screened using Hygromycin antibiotic, and through monoclonalization, a monoclonal overexpressing cell line, CHO-hDPP4 cells, was finally obtained.

[0437] 3. Purchase of antigen cell lines

[0438] Mouse FAP engineered cell line:

[0439] CT26-mouse-Fap-Cell-Line, product number: KC-1284, from Kangyuan Botech (Beijing) Co., Ltd.

[0440] Natural tumor cells expressing human FAP:

[0441] U-138MG cell was purchased from ATCC, catalog number HTB-16.

[0442] Reagent: Z-Gly-Pro-AMC, brand BACHEM, catalog number 4002518.

[0443] The antibodies provided by the present invention have better effects and lower immunogenicity. The antibody sequences are shown in Tables 5 to 12.

[0444]

[0445]

[0446]

[0447] Table 6 Variable region sequences of rabbit-derived chimeric antibodies

[0448]

[0449]

[0450] Note: Underlined are CDRs, and the CDR annotation method is based on the Kabat antibody coding scheme.

[0451]

[0452]

[0453] Variable region sequences of humanized antibodies in Table 8

[0454]

[0455]

[0456] Variable region sequences of affinity matured and optimized antibodies in Table 10

[0457]

[0458] Comprising sequences of single-chain antibody sequences in Table 11

[0459]

[0460]

[0461]

[0462] Single-chain antibody sequences in Table 12

[0463]

[0464]

[0465]

[0466] Unless otherwise specified, the raw materials and reagents used in the recombinant anti-FAP antibodies and their applications provided by the present invention are commercially available.

[0467] The present invention will be further described below in conjunction with examples:

[0468] Example 1 Determination of the affinity between antigen cells and the tool antibody by Facs

[0469] Experimental reagents and materials:

[0470] Experimental reagents and materials in Table 13

[0471]

[0472] Experimental procedure:

[0473] 1) Cell collection and seeding

[0474] a) Harvest cells in the logarithmic growth phase to ensure that the cell viability is above 90%.

[0475] b) Centrifuge at 1000 r / min for 5 min, then discard the supernatant;

[0476] c) Wash the cells once with PBS;

[0477] d) Resuspend the cells with FACS Buffer and count them;

[0478] e) Prepare a cell suspension with a density of 2×10 6 cells / mL using FACS Buffer;

[0479] f) Add 50 μL of the cell suspension to each well of a 96-well plate;

[0480] 2) Antibody Incubation and Detection

[0481] a) Add 50 μL of test samples with different concentrations to the experimental group. The sample concentration starts from 20 μg / mL and is set with multiple gradients of 3-fold dilution;

[0482] b) After mixing, incubate overnight at 4°C in the dark;

[0483] c) Wash the cells once with FACS Buffer, 200 μL each time, centrifuge at 1000 r / min for 5 min, and discard the supernatant;

[0484] d) Add APC-labeled secondary antibody (diluted 1:1500) to the 96-well plate, and add an equal volume of FACS Buffer to the blank control group;

[0485] e) After mixing, incubate at 4°C in the dark for 40 min;

[0486] f) Wash the cells once with FACS Buffer, 200 μL each time, centrifuge at 1000 r / min for 5 min, and finally resuspend the cells with 100 μL of FACS Buffer;

[0487] g) Detect the RL-1 MFI value using an Intellicyte plus flow cytometer (Excitation Laser: 488 nm Blue Laser).

[0488] 3) Data Processing

[0489] Analyze the FACS data using Prism software.

[0490] Data analysis: Using the overexpressing cell line CHO-hFAP cells and the naturally expressing cell line U138MG cells, through 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 relatively high, and the binding signal of hu36 is slightly lower (as shown in Tables 14, Figure 1 , Figure 2 ).

[0491] Table 14 EC50 and fluorescence values of the control antibody flow cytometry binding experiment

[0492]

[0493] Example 2 Generation of rabbit anti-human FAP monoclonal antibody (this work was entrusted to Shanghai DinoXinAn Biotechnology Co., Ltd. for development)

[0494] 1. Animal immunization

[0495] In order to obtain rabbit monoclonal antibodies that recognize human FAP antigen, the present invention selects to immunize New Zealand white rabbits. The first immunization is 200 μg. At the first immunization, Freund's complete adjuvant and an equal volume of antigen (human FAP-hFc protein, brand Acro Biosystems, catalog number FAP-H5263) are mixed and emulsified, and immunized by multiple-point injection on the back. After a two-week interval, booster immunization is carried out. The antigen dosage for booster immunization is 100 μg. Freund's incomplete adjuvant and an equal volume of antigen are mixed and emulsified, and immunized by multiple-point injection on the back. After five immunizations, the titer of the immune serum is detected by the conventional Elisa method. Select rabbits with high titers, and perform a boost immunization 3 days before screening the antibodies by intraperitoneal injection of 50 μg of protein. This antigen does not need to be emulsified with adjuvant, and the buffer is PBS. Take the spleen three days later.

[0496] 2. Isolation of spleen cells

[0497] The rabbit spleen is surgically removed and placed in a sterile cell culture dish. The spleen is rinsed with DPBS containing 100 U / mL penicillin and 100 μg / mL streptomycin. The spleen is cut into pieces with surgical scissors, and the spleen is gently ground into single cells with a syringe core. Finally, the cell suspension is filtered through a 100 μm cell sieve, and the single-cell filtrate is collected. Centrifuge at 1200 rpm for 3 minutes, discard the supernatant, and resuspend the cells with RPMI-1640 containing 5% fetal bovine serum.

[0498] 3. B cell culture and identification

[0499] Co-incubate biotin-labeled FAP protein with successfully immunized rabbit lymphocytes, and sort out memory B cells of rabbits. Culture them in a 96-well cell culture plate at 37 °C under 5% CO2 using B cell medium. After 10 - 14 days of culture, detect the binding activity of the clone supernatant at the protein level by ELISA, and clones with a binding activity greater than 5 times the background are determined to be positive. The supernatants positive by ELISA are detected for their binding to stably transfected cells CHO-hFAP and CHO-mFAP by FACS method, and finally FACS-positive monoclonal clones are selected.

[0500] 4. Cloning of genes encoding rabbit monoclonal antibodies

[0501] Collect B cell positive clones, select some positive clones to extract total RNA with RNAiso Plus and reverse transcribe it into cDNA. Amplify the light chain variable region and heavy chain variable region sequences by PCR method, and construct them into the PTT5 expression vector containing the corresponding heavy chain constant region and light chain constant region for sequencing to obtain the correct sequences. Analyze the sequencing results with VBASE2 (http: / / www.vbase2.org / vbscAb.php) to obtain the light and heavy chain variable region sequences of the antibody.

[0502] Example 3 Preparation of anti-human FAP chimeric antibody

[0503] Splice the heavy chain variable region sequence of the rabbit-derived anti-human FAP monoclonal antibody with the heavy chain constant region sequence of the publicly reported human monoclonal antibody IgG1 subclass, and construct it into a mammalian cell expression vector; splice the light chain variable region sequence of the rabbit-derived anti-human FAP monoclonal antibody with the light chain constant region sequence of the publicly reported human monoclonal antibody κ subclass, and construct it into a mammalian cell expression vector. Pair and mix the constructed heavy chain vector and light chain vector of the anti-human FAP chimeric antibody, transfect HEK293 cells with polyethyleneimine (PEI). Collect the cell supernatant about 7 days later, and purify the anti-human FAP chimeric antibody protein using Mabselect.

[0504] Example 4 In vitro cell binding experiment of anti-human FAP chimeric antibody

[0505] Perform 2-fold serial dilutions of the anti-human FAP chimeric antibody starting from an initial concentration of 20 μg / mL, with a total of 8 concentration points. Take 50 μL of the antibody at each concentration point and add it to a 96-well plate. Centrifuge CHOK1 cells with high surface expression of human FAP and mouse FAP at 100 g for 5 minutes at room temperature, wash the cells once with PBS containing 0.5% BSA, centrifuge at 100 g for 5 minutes at room temperature, and resuspend the cells to a density of approximately 2×10 6 Cells per milliliter, take 50 μL and add it to the wells of a 96-well plate with antibodies added. After incubating at 4°C for 1 hour, add the APC-fluorescently labeled goat anti-human IgG secondary antibody. After continuing to incubate at 4°C for 1 hour, analyze the average fluorescence reading of the cell population using a flow cytometer, and perform a 4-parameter fitting curve using prism software.

[0506] Result analysis: After in vitro cell biology binding tests, several antibodies obtained can reach the plateau value at lower concentrations compared with the control cMFP5, such as chrD1, chrD7, chrD25, etc.; The in vitro binding experiment with CHO-mFAP cells also shows that several chimeric antibody molecules have good binding signals with mFAP (as Figures 3 - 8 shown).

[0507] Example 5 In vitro binding affinity and kinetics experiments of anti-human FAP chimeric antibodies

[0508] Using a Fortebio (BLITZ pro1.1.0.28) instrument, the antibody affinity was determined by the anti-human antibody capture method. During the determination, the capture antibody (AHC) biosensor of the Fc segment of the anti-human antibody was soaked in PBS for 10 min; 200 μL of the diluted antibody sample (including the chimeric antibody of the present invention and the control antibody; the working concentration of the antibody was 15 μg / mL) was loaded onto the AHC biosensor, then equilibrated in PBS for 100 s, and further the AHC biosensor was subjected to a binding reaction with human FAP protein and mouse FAP protein (purchased from ACRObiosystem) for 600 s. After that, the AHC biosensor was transferred to PBS for a dissociation reaction for 600 s. After the experiment, the blank control response value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constants of antigen-antibody binding.

[0509] Result analysis: After in vitro kinetic binding activity analysis, the binding kinetic constants of the chimeric antibody with recombinant hFAP protein were at the level of 10 -10 to 10 -12 level. In contrast, the binding kinetic constants of the benchmark antibodies cMFP5 and hu36 were at the level of 10 -9 to 10 -11 level; The binding kinetic constants of the chimeric antibody with recombinant mFAP protein were at the level of 10 -6 to 10 -12 level. In contrast, the binding kinetic constants of the benchmark antibodies cMFP5 and hu36 were at the level of 10 -9 to 10 -11 level. The binding kinetic levels of the chimeric antibody molecules were comparable to those of the control antibodies (as shown in Table 15 and Table 5).

[0510] Table 15 In vitro kinetic binding activity of antibodies and recombinant human FAP protein

[0511] Antibody Concentration (nM) Response Value KD (M) kon (1 / Ms) kdis (1 / s) FullR^2 cMFP5 100 0.9513 1.60E-11 1.66E+05 2.65E-06 0.9985 hu36 100 0.8344 3.21E-09 9.96E+05 3.19E-03 0.9161 chrD1 100 0.7325 8.87E-12 3.33E+05 2.96E-06 0.9865 chrD4 100 0.7111 <1.0E-12 1.37E+05 <1.0E-07 0.997 chrD7 100 0.8452 3.19E-10 3.09E+05 9.87E-05 0.9865 chrD16 100 1.0203 <1.0E-12 4.53E+05 <1.0E-07 0.9414 chrD20 100 1.0123 <1.0E-12 3.18E+05 <1.0E-07 0.9731

[0512] Table 16 In vitro kinetic binding activity of antibodies and recombinant mouse FAP protein

[0513] Antibody Concentration (nM) Response Value KD (M) kon (1 / Ms) kdis (1 / s) FullR^2 cMFP5 100 0.7412 7.04E-11 1.19E+05 8.38E-06 0.9997 hu36 100 0.7302 3.22E-09 5.74E+05 1.85E-03 0.9214 chrD1 100 0.2769 1.65E-08 3.58E+05 5.91E-03 0.9955 chrD4 100 0.4925 <1.0E-12 6.94E+04 <1.0E-07 0.9981 chrD7 100 0.5009 5.30E-09 2.87E+05 1.52E-03 0.9742 chrD16 100 0.5841 7.99E-09 7.51E+05 6.00E-03 0.9823 chrD20 100 0.7656 9.24E-11 1.57E+05 1.45E-05 0.9978

[0514] Example 6 Humanization of rabbit-derived anti-human FAP antibody

[0515] Based on the antibody coding schemes of Kabat and Chothia, the amino acid sequence regions of the six antigen - complementary determining regions (CDRs) of the heavy and light chains of the rabbit - derived antibody and the framework region that supports the conserved three - dimensional conformation of the antibody were determined. Subsequently, by analyzing and searching known human antibody sequences, the human heavy - chain variable region sequence most similar to the rabbit - derived antibody was selected, such as IGHV1|IGHJ4*01, and its antibody framework region sequence was used as a template. The rabbit - derived heavy - chain CDRs were combined with the human antibody framework region to finally generate the humanized heavy - chain variable region sequence. In the same process, the humanized light - chain variable region sequence was generated. When the CDRs of the rabbit - derived antibody are directly transplanted into the human framework region, the binding activity of the antibody often drops sharply. Therefore, individual amino acids in the framework region need to be changed back from human to rabbit - derived. To determine the sites of back - mutation, first, compare the designed humanized antibody sequence with the original rabbit - derived antibody sequence to check which amino acids are different; second, check whether these amino acids play an important role in supporting the antibody structure or in binding to the antigen. At the same time, when checking the sequence after humanization design, it is necessary to check whether there are some potential post - translational modification sites, such as N (asparagine) glycosylation sites, N - deamidation sites, D (aspartic acid) isomerization sites, etc.

[0516] The humanized antibody heavy - chain variable region gene was constructed into a mammalian cell expression vector containing the heavy - chain constant region gene of the human monoclonal antibody IgG1 subclass; the light - chain gene was constructed into a mammalian cell expression vector containing the light - chain constant region gene of the human monoclonal antibody κ subclass. The constructed heavy - chain vector and light - chain vector of the humanized anti - human FAP antibody were paired and mixed, and HEK293 cells were transfected using polyethyleneimine (PEI). About 7 days later, the cell supernatant was collected, and the humanized anti - human FAP antibody protein was purified using Mabselect.

[0517] Example 7 In vitro binding affinity and kinetic experiments of humanized anti - human FAP antibody

[0518] The antibody affinity was determined by using the Fortebio (BLITZ pro 1.1.0.28) instrument and the anti-human antibody capture method. During the determination, the capture antibody (AHC) biosensor of the Fc segment of the anti-human antibody was immersed in PBS for 10 min; 200 μL of the diluted antibody sample (including the chimeric antibody of the present invention and the control antibody; the working concentration of the antibody was 15 μg / mL) was loaded onto the AHC biosensor, and then equilibrated in PBS for 100 s. Further, the AHC biosensor was subjected to a binding reaction with human FAP protein and murine FAP protein (purchased from ACRObiosystem) for 600 s. After that, the AHC biosensor was transferred to PBS for a dissociation reaction for 600 s. After the experiment, the blank control response value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constants of antigen-antibody binding.

[0519] Result analysis: chrD1, chrD11, chrD16, chrD27, chrD5, and chrD18 were selected for humanization design and modification. Compared with the chimeric antibody, the humanized molecule after modification maintained the binding activity with recombinant human and murine proteins at the in vitro kinetic binding activity level as much as possible (as shown in Tables 17 - 22).

[0520] Table 17 In vitro kinetic binding activity of the humanized antibody of chrD1 with recombinant human and murine FAP proteins

[0521]

[0522] Table 18 In vitro kinetic binding activity of the humanized antibody of chrD11 with recombinant human and murine FAP proteins

[0523]

[0524] Table 19 In vitro kinetic binding activity of the humanized antibody of chrD16 with recombinant human and murine FAP proteins

[0525]

[0526]

[0527] Table 20 In vitro kinetic binding activity of the humanized antibody of chrD27 with recombinant human and murine FAP proteins

[0528]

[0529] Table 21 In vitro kinetic binding activity of the humanized antibody of chrD5 with recombinant human and murine FAP proteins

[0530]

[0531] In vitro kinetic binding activity of humanized antibody against chrD18 and recombinant human and murine FAP proteins

[0532]

[0533] Example 8 In vitro cell binding experiment of anti-human FAP humanized antibody

[0534] The anti-human FAP humanized antibody was serially diluted 4-fold starting from an initial concentration of 20 μg / mL, with a total of 8 concentration points. 50 μL of the antibody at each concentration point was added to a 96-well plate. HT1080-hFAP cells with low surface expression of human FAP and CHOK1 cells with high expression of murine FAP were collected by centrifugation at 100 g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100 g for 5 minutes at room temperature, and resuspended to a density of approximately 2×10 6 cells per milliliter. 50 μL of the cell suspension was added to the wells of the 96-well plate that already contained the antibody. After incubation at 4°C for 1 hour, APC-fluorescently labeled goat anti-human IgG secondary antibody was added. After continued incubation at 4°C for 1 hour, the mean fluorescence readings of the cell population were analyzed using a flow cytometer. Prism software was used to perform a four-parameter fitting curve and calculate the EC50 and the top value of the fluorescence intensity.

[0535] Result analysis: In vitro cell binding experiments were carried out with the humanized antibody against low-expressing hFAP cells (HT1080-hFAP cells) and overexpressing mFAP cells (CHO-mFAP cells). The results are as Figures 9 - 12 shown in Table 23. The results indicate that the humanized molecules of D1, D16, and D27 have binding to HT1080-hFAP cells at the same level as the reference molecule 4B9. All three have binding to CHO-mFAP cells, but the fluorescence signal values are relatively low.

[0536] Table 23 In vitro flow cytometry binding EC50 and top values of fluorescence intensity of humanized antibody with HT1080-hFAP and CHO-mFAP cells

[0537]

[0538] Example 9 Affinity maturation

[0539] In this antibody modification, yeast display technology was used to randomly mutate the amino acids in the 6 CDR regions of the heavy chain variable region and the light chain variable region of the antibody molecule to construct a library, and then candidate antibody molecules with higher affinity were screened from it.

[0540] 1. Design and construction of the affinity-matured antibody library

[0541] The affinity-matured parental antibody molecules hzD1-H1L0 and hzD27-H1L0 were used to construct single-chain antibodies (single-chain variable fragment, scFv) by connecting their heavy-chain variable regions and light-chain variable regions through GS linkers, respectively. The CDR regions of the single-chain antibodies were the targets for affinity maturation modification, while the framework region sequences remained unchanged during the affinity maturation modification. Six CDR regions, including the heavy and light chains of each parental antibody, were constructed into separate antibody libraries respectively.

[0542] 2. Sorting of the affinity-matured antibody libraries

[0543] The obtained antibody libraries were sorted by magnetic beads and flow cytometry: First, incubate with 300 nM biotinylated antigen for 1 hour for magnetic bead sorting (Invitrogen Cat: 11206D), and then sort the antibody library obtained after magnetic bead sorting by flow cytometry. In the subsequent flow cytometry sorting, incubate the biotinylated antigen with the antibody library at room temperature for 40 min, then wash away the unbound biotinylated antigen, centrifuge at 14,000 rpm for 1 minute at 4 °C to collect cells, and wash twice with pre-cooled PBS containing 1% BSA. Add 100 μL of 1:500 diluted fluorescent secondary antibody and incubate on ice for 30 minutes. Centrifuge at 14,000 rpm for 1 minute at 4 °C to collect cells, wash twice with pre-cooled PBS containing 1% BSA, and then perform flow cytometry sorting. The antigen concentration used in each round was gradually decreased, and only 0.1-0.5% of the clones in the library were collected for culture in each round of sorting, and then the next round of sorting was carried out. Finally, after a total of 3-4 rounds of flow cytometry sorting, 96 yeast cells were selected from each antibody library for sequencing analysis, and several unique mutant clones were finally obtained from the heavy-chain antibody library and the light-chain antibody library.

[0544] 3. Screening, identification and evaluation of affinity-matured mutant sequence combinations

[0545] 3.1 In vitro cell binding test of affinity-matured combined molecules

[0546] The affinity-matured combined molecule antibody against human FAP was serially diluted 4-fold starting from an initial concentration of 5 μg / mL, with a total of 7 concentration points. 50 μL of the antibody at each concentration point was added to a 96-well plate. Centrifuge the HT1080-hFAP cells with low surface expression of human FAP, CT26 cells overexpressing murine FAP, and 293-cynoFAP cell line overexpressing cynomolgus FAP at 100 g for 5 minutes at room temperature to collect cells. Wash the cells once with PBS containing 0.5% BSA, centrifuge at 100 g for 5 minutes at room temperature, and resuspend the cells to a density of approximately 2×10 6 Cells per milliliter, take 50 μL and add it to the wells of a 96-well plate that has already been added with antibodies. After incubating at 4°C for 1 hour, add the APC-fluorescently labeled goat anti-human IgG secondary antibody. After continuing to incubate at 4°C for 1 hour, analyze the average fluorescence reading of the cell population using a flow cytometer, and perform a 4-parameter fitting curve using Prism software.

[0547] Result analysis: The combinatorial molecules after affinity maturation were respectively carried out in vitro cell binding experiments with cells HT1080-hFAP with low expression of hFAP, cells CT26-mFAP with overexpression of mFAP, and 293-cynoFAP with overexpression of cynomolgus FAP. The results are as Figures 12 - 17 shown. The results indicate that several combinatorial molecules after affinity maturation maintained a relatively high level of binding to HT1080-hFAP cells, while maintaining binding to 293-cynoFAP cells. Most importantly, the binding signal to CT26-mFAP cells was greatly improved.

[0548] 3.2 In vitro cell non-specific binding test of combinatorial molecules after affinity maturation

[0549] Dilute the anti-human FAP combinatorial molecule antibody after affinity maturation to concentrations of 20 μg / mL and 5 μg / mL. Take 50 μL of the diluted antibody and add it to a 96-well plate. Centrifuge at 100 g at room temperature for 5 minutes to collect CHO-hDPP4 cells with overexpression of human DPP4 on the cell surface, Chinese hamster ovary cells CHOK1, and human embryonic kidney cells HEK293. Wash the cells once with PBS containing 0.5% BSA, centrifuge at 100 g at room temperature for 5 minutes, and resuspend the cells to a density of approximately 2×10 6 cells per milliliter. Take 50 μL and add it to the wells of the 96-well plate that has already been added with the antibody. After incubating at 4°C for 1 hour, add the APC-fluorescently labeled goat anti-human IgG secondary antibody. After continuing to incubate at 4°C for 1 hour, analyze the average fluorescence reading of the cell population using a flow cytometer, and perform a 4-parameter fitting curve using Prism software.

[0550] Result analysis: Conduct non-specific binding experiments on the combinatorial molecules after affinity maturation with the cell line CHO-hDPP4 expressing the same family protein DPP4 and the commonly used cell lines for protein expression, Chinese hamster ovary cells CHOK1 and human embryonic kidney cells HEK293. The results are as Figures 18 - 20 shown. The results indicate that there is no non-specific binding signal between each combinatorial molecule after affinity maturation and the same family protein and the empty cells used for expression.

[0551] 3.3 In vitro binding affinity and kinetics experiments of combinatorial molecules after affinity maturation

[0552] The antibody affinity was determined using a Fortebio (BLITZ pro 1.1.0.28) instrument by the anti-human antibody capture method. During the determination, the capture antibody (AHC) biosensor of the Fc segment of the anti-human antibody was immersed in PBS for 10 min; 200 μL of the diluted antibody sample (including the chimeric antibody of the present invention and the control antibody; the working concentration of the antibody was 15 μg / mL) was loaded onto the AHC biosensor, then equilibrated in PBS for 100 s, and further the AHC biosensor was subjected to a binding reaction with human FAP protein and mouse FAP protein (purchased from ACRObiosystem) for 600 s. After that, the AHC biosensor was transferred to PBS for a dissociation reaction for 600 s. After the experiment, the blank control response value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constants of antigen-antibody binding.

[0553] Result analysis: The in vitro kinetic binding of the affinity matured combinatorial molecules was tested, and the results are shown in Table 24. The results indicate that the binding kinetic constants of each combinatorial molecule with the recombinant human FAP protein remained at the 10 -12 level, and the binding kinetic constants of the combinatorial molecules with the recombinant mouse FAP protein increased from the 10 -9 level to the 10 -10 level, showing a significant improvement.

[0554] Table 24 In vitro kinetic binding activities of the affinity matured combinatorial molecules

[0555]

[0556]

[0557] 3.4. Analysis of monomer ratio in the physical characterization of the affinity matured combinatorial molecules

[0558] Experimental instrument: UPLC CLASS ACQUITY H (WATERS).

[0559] Analytical column: TSKgel G3000SWXL 7.8*300 (TOSHI, CatNo 003C03326C.

[0560] Analytical solution: 200 mM K2HPO4, 250 mM KCl, and the pH was adjusted to 6.2 using HCl.

[0561] Analysis method: 50 μL of the antibody with a concentration of 1 mg / mL was injected into the pre-equilibrated chromatography column, and the flow rate was 0.75 mL / min at room temperature for 45 min, and the absorbance value of the machine A280 was detected simultaneously. Then, the monomer content and ratio of the antibody were judged according to the peak elution time and peak elution volume.

[0562] Result analysis: Monomer ratio analysis was performed on the affinity matured combinatorial molecules. The results showed that the monomer ratios of all combinatorial molecules were above 95%, indicating good monomer ratio properties (as shown in Table 25).

[0563] Table 25 Main peak retention time and monomer ratio of antibodies

[0564] Antibody Retention Time of Main Peak (min) Monomer Ratio SEC (%) D27H1-D01L0 13.542 98.28 D27-AM26 13.449 97.99 D27-AM46 13.723 98.16 D27H2-D01K1 14.044 97.49 D27H4-D01K1 14.376 98.06 D27H4-D01K4 13.795 98.37 D27H5-D01K4 13.574 98.41

[0565] 3.5. Hydrophobic property analysis of antibodies

[0566] Experimental instrument: ARC (Waters).

[0567] Analytical column for experiment: TSKgel Butyl-NPR (4.6 mm × 3.5 cm, CatNo 14947).

[0568] Analytical solution: A. 20 mM Histidine, pH 6.0;

[0569] B. 20 mM Histidine, 1.6 M (NH4)2SO4.

[0570] Analysis method: According to the usage instructions of the hydrophobic chromatography column, the hydrophobic properties of the antibodies were analyzed.

[0571] Result analysis: Hydrophobic property analysis was performed on the affinity matured combinatorial molecules. The results showed that the hydrophobic HIC values of all combinatorial molecules were greater than 0.7, indicating good hydrophobic properties (as shown in Table 26).

[0572] Table 26 Hydrophobic properties of antibodies

[0573]

[0574]

[0575] 3.6. Analysis of drug metabolism of antibodies in mice

[0576] Experimental materials: Antibodies to be tested, sera collected from mice at different time points, antibodies to be tested binding to antigens, anti-huIgGFab monoclonal antibody (Sigma, I5260-1ML), HRP-labeled goat anti-human IgG secondary antibody (Jackson, code: 109-035-098).

[0577] Experimental methods:

[0578] Serum collection:

[0579] 1) Female Balb / C mice, 3 mice per group, were given samples such as 4B9, D27H1-D01L0, D27H4-D01L4, D27H2-D01L1, etc. via the tail vein at a dose of 200 μg / mouse;

[0580] 2) Blood samples were collected from the tail vein at the time points designed in the experiment, and the blood samples were collected. After standing at room temperature for more than 30 min, the serum was collected at 4000 rpm for 15 min and stored at -20°C. To prevent serum evaporation, the final volume of collected serum should be preferably greater than 20 μL;

[0581] 3) The last collected serum was frozen at -20°C for at least 24 h.

[0582] Detection method:

[0583] 1) The binding antigen and anti-IgG Fab monoclonal antibody were coated on a 96-well ELISA plate with PBS at a concentration of 0.2 μg / mL, 100 μL per well, and incubated overnight at 4°C;

[0584] 2) Prepare the required reagents:

[0585] Blocking solution: 5% BSA + PBS

[0586] Antibody diluent: 5% BSA + PBS + 20% blank mouse serum

[0587] ELISA plate washing solution: 0.1% Tween + PBS

[0588] 3) The coated ELISA plate was washed three times with PBS, 300 μL per well;

[0589] 4) Add the blocking solution, 200 μL per well, and block at 37°C for 1 h;

[0590] 5) The initial serum was diluted to an appropriate concentration with the blocking solution and then diluted to an appropriate concentration range with the antibody diluent containing the same serum concentration. The dilution factor of the specific concentration needs to be adjusted according to the preliminary experiment. In principle, the final color development value of the detected serum should be within the color development value range of the standard curve;

[0591] 6) The antibody standard curve was diluted with the antibody diluent. The dilution of the standard curve was still adjusted according to the preliminary experiment to fit a linear curve (if there is appropriate software, an S-shaped curve can also be fitted).

[0592] 7) Pour out the blocking solution, and add the diluted antibody and the serum to be detected to the ELISA plates with the two coating methods, 100 μL per well, and incubate at 37°C for 1 h;

[0593] 8) Wash the plate 3 times with PBST;

[0594] 9) Dilute the secondary antibody at 1:5000 and add it to the well-washed ELISA plate, 100 μL per well, incubate at 37 °C for 40 min;

[0595] 10) Wash the plate 3 times with PBST;

[0596] 11) Develop color with TMB, 100 μL per well, protect from light for 10 min;

[0597] 12) Add 50 μL of 2M HCl to terminate the reaction and read the absorbance at 450 nm.

[0598] Result analysis: Mouse in vivo drug metabolism experiments were carried out on four samples, namely 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 measured using the fully human anti-detection method. The results showed that the in vivo metabolism levels of each molecule were different. The metabolism of 4B9 and D27H1-D01L0 in vivo was slow, with a long half-life, greater than 100 hours; the metabolism of D27H4-D01L4 and D27H2-D01L1 in vivo was fast, with a short half-life, less than 100 hours (the results are shown in Table 27, Figure 20 as shown).

[0599] Table 27 Analysis of Antibody In Vivo Drug Metabolism in Mice

[0600]

[0601]

[0602] Example 10 Preparation and Identification of Single-Chain Antibody

[0603] 1. Synthesis and Expression of Single-Chain Antibody

[0604] Link the heavy chain variable region and light chain variable region of the antibody with (G4S)3, or link the light chain variable region and heavy chain variable region with (G4S)3, and construct them into a eukaryotic expression vector upstream of the human IgG1 heavy chain constant region coding gene to obtain a fusion protein expression plasmid of scFv and Fc. Amplify the obtained plasmid in Escherichia coli, isolate a large number of fusion protein expression plasmids containing scFv and Fc, mix the plasmid with PEI and co-transfect it into HEK293 cells. 5 - 6 days after cell transfection, take the culture supernatant, purify the expression supernatant using a Mabselect affinity chromatography column to obtain a fusion protein of scFv and Fc.

[0605] 2. In Vitro Cell Binding Test of Single-Chain Antibody

[0606] Start with a concentration of 120 nM for the single-chain antibody, dilute it 4-fold with buffer, set up 8 gradients, and add 50 μL of the diluted antibody to a 96-well plate. Centrifuge the HT1080-hFAP cells overexpressing human FAP on the cell surface, CT26-mFAP cells overexpressing murine FAP, and 293-cynoFAP cells overexpressing cynomolgus FAP in human embryonic kidney cells HEK293 at 100 g for 5 minutes at room temperature. Wash the cells once with PBS containing 0.5% BSA, centrifuge at 100 g for 5 minutes at room temperature, and resuspend the cells to a density of approximately 2×10 6 cells per milliliter. Take 50 μL and add it to the wells of the 96-well plate already containing the antibody. After incubating at 4 °C for 1 hour, add the secondary antibody, APC-labeled goat anti-human IgG. Continue to incubate at 4 °C for 1 hour, and then analyze the average fluorescence readings of the cell population using a flow cytometer. Perform a 4-parameter fitting curve using Prism software.

[0607] Result analysis: Conduct in vitro cell binding experiments for each single-chain antibody with HT1080-hFAP cells with low expression of hFAP, CT26-mFAP cells overexpressing mFAP, and 293-cynoFAP cells overexpressing cynomolgus FAP. The results are as Figures 22 - 38 shown. The results indicate that several single-chain combination molecules maintain a relatively high level of binding to HT1080-hFAP cells, while also maintaining binding to 293-cynoFAP cells, and there are varying degrees of binding signals to CT26-mFAP cells. Moreover, for different positions of the same heavy and light chain molecules, there are significant differences in their in vitro cell binding activities. For example, the binding signals of D27H1-D01L0-LH to HT1080-hFAP cells and 293-cynoFAP cells are significantly stronger than those of D27H1-D01L0-HL, and their binding signals to CT26-mFAP cells are at a comparable level. Additionally, it was also found that the properties of the single-chain antibody are different from those of the corresponding monoclonal antibody. In the identification of the affinity maturation combination molecule D27H1-D01L0 mentioned above, its in vitro cell binding 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.

[0608] 3. In vitro cell non-specific binding test of single-chain antibody

[0609] Start with a concentration of 120 nM for the single-chain antibody, dilute it 4-fold with buffer, set up 4 gradients, and add 50 μL of the diluted antibody to a 96-well plate. Centrifuge the CHO-hDPP4 cells overexpressing human DPP4 on the cell surface and Chinese hamster ovary cells CHOK1 at 100 g for 5 minutes at room temperature. Wash the cells once with PBS containing 0.5% BSA, centrifuge at 100 g for 5 minutes at room temperature, and resuspend the cells to a density of approximately 2×10 6 Cells per milliliter, take 50 μL and add it to the wells of a 96-well plate with antibodies added. After incubating at 4°C for 1 hour, add the APC-fluorescently labeled goat anti-human IgG secondary antibody. After continuing to incubate at 4°C for 1 hour, analyze the average fluorescence reading of the cell population using a flow cytometer, and perform a 4-parameter fitting curve using Prism software.

[0610] Result analysis: In vitro cell binding experiments were carried out on each single-chain antibody with human DPP4 cells (CHO-hDPP4 cells) overexpressing FAP homologous proteins and Chinese hamster ovary cells (CHOK1) expressing empty cells. The results are as Figures 39 - 45 shown. The results indicate that no obvious non-specific binding signals were observed between all single-chain combination molecules and human DPP4 cells (CHO-hDPP4 cells) overexpressing homologous proteins and Chinese hamster ovary cells (CHOK1) expressing empty cells.

[0611] 4. In vitro binding affinity and kinetics experiments of single-chain antibodies

[0612] Using a Fortebio (BLITZ pro1.1.0.28) instrument, the antibody affinity was measured by the anti-human antibody capture method. During the measurement, the capture antibody (AHC) biological probe of the Fc segment of the anti-human antibody was immersed in PBS for 10 min; 200 μL of the diluted antibody sample (including the chimeric antibody of the present invention and the control antibody; the working concentration of the antibody was 15 μg / mL) was loaded onto the AHC biological probe, then equilibrated in PBS for 100 s, and further the AHC probe was reacted with human FAP protein and mouse FAP protein (purchased from ACRObiosystem) for 600 s. After that, the AHC probe was transferred to PBS for dissociation reaction for 600 s. After the experiment, the blank control response value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constants of antigen-antibody binding.

[0613] Result analysis: The in vitro kinetic binding of single-chain antibody molecules was tested. The results are shown in Tables 28 to 30. The results indicate that the binding kinetic constants of most single-chain antibody molecules with recombinant human FAP protein remained at 10 -12 level. For a small number of molecules, such as hzD11-H1L0-HL-scFv and chrD7-LH-scFv, the affinity level was relatively low; the binding kinetic constants with recombinant mouse FAP protein showed that the binding constant of the control antibody 4B9 was at the 10-9 level, and most of the single-chain antibody molecules of self-produced antibodies were at the same level. For a small number of molecules, such as D27H2.D01L1-LH-scFv and chrD20-LH-scFv, the affinity level was higher, reaching 10 -12 level; for the binding kinetic constants with recombinant monkey FAP protein, both the control molecule and the self-produced molecule were at 10 -12 At the horizontal level, the affinity levels of a few molecules, such as hzD11-H1L0-HL-scFv and chrD7-LH-scFv, are reduced.

[0614] Table 28 In vitro kinetic binding activities of single-chain antibodies and recombinant human FAP protein

[0615] Single-chain Antibody Protein Concentration (nM) Response Value KD (M) kon (1 / Ms) kdis (1 / s) FullR^2 4B9-HL-scFv 100 0.6186 <1.0E-12 2.73E+05 <1.0E-07 0.9799 4B9-LH-scFv 100 0.6399 1.51E-10 2.98E+05 4.49E-05 0.9856 D27H1.D01L0-HL-scFv 100 1.1869 <1.0E-12 4.32E+05 <1.0E-07 0.9776 D27H1.D01L0-LH-scFv 100 1.0699 <1.0E-12 4.43E+05 <1.0E-07 0.975 D27H2.D01L1-HL-scFv 100 1.2121 <1.0E-12 3.61E+05 <1.0E-07 0.9843 D27H2.D01L1-LH-scFv 100 1.0627 <1.0E-12 3.86E+05 <1.0E-07 0.9766 D27H5.D01L4-HL-scFv 100 1.1894 <1.0E-12 4.33E+05 <1.0E-07 0.9748 D27H5.D01L4-LH-scFv 100 1.0339 <1.0E-12 4.39E+05 <1.0E-07 0.9697 hzD11-H1L0-HL-scFv 100 0.7406 1.95E-10 2.91E+05 5.68E-05 0.9831 hzD11-H1L0-LH-scFv 100 0.7763 <1.0E-12 3.38E+05 <1.0E-07 0.9845 hzD18-H0L0-HL-scFv 100 0.7511 <1.0E-12 1.92E+05 <1.0E-07 0.9945 hzD18-H0L0-LH-scFv 100 0.7825 <1.0E-12 1.87E+05 <1.0E-07 0.9942 chrD7-HL-scFv 100 0.6157 <1.0E-12 2.32E+05 <1.0E-07 0.9952 chrD7-LH-scFv 100 0.6275 2.08E-09 2.41E+05 5.00E-04 0.9961 chrD20-HL-scFv 100 0.558 <1.0E-12 1.28E+05 <1.0E-07 0.9899 chrD20-LH-scFv 100 0.6671 <1.0E-12 1.38E+05 <1.0E-07 0.9938 chrD25-HL-scFv 100 0.8418 <1.0E-12 4.40E+05 <1.0E-07 0.9683 chrD25-LH-scFv 100 0.8768 <1.0E-12 4.29E+05 <1.0E-07 0.965 chrD30-HL-scFv 100 0.3589 <1.0E-12 8.38E+04 <1.0E-07 0.997 chrD30-LH-scFv 100 0.4986 <1.0E-12 1.07E+05 <1.0E-07 0.9985 chrD4-HL-scFv 100 0.2639 <1.0E-12 5.92E+04 <1.0E-07 0.9985 chrD4-LH-scFv 100 0.5667 <1.0E-12 1.32E+05 <1.0E-07 0.9991 hzD16H0L0-HL-scFv 100 0.5852 <1.0E-12 3.47E+05 <1.0E-07 0.978 hzD16-H0L0-LH-scFv 100 0.8989 <1.0E-12 3.78E+05 <1.0E-07 0.9826

[0616] Table 29 In vitro kinetic binding activities of single-chain antibodies and recombinant murine FAP protein

[0617]

[0618]

[0619] Table 30 In vitro kinetic binding activities of single-chain antibodies and recombinant simian FAP protein

[0620]

[0621]

[0622] 5. Physical characterization of single-chain antibodies - monomer ratio analysis

[0623] Experimental instrument: UPLC CLASS ACQUITYH (WATERS).

[0624] Analysis column: TSKgel G3000SWXL 7.8*300 (TOSHI, CatNo 003C03326C).

[0625] Analysis solution: 200 mM K2HPO4, 250 mM KCl, adjusted to pH 6.2 with HCl.

[0626] Analysis method: Inject 50 μL of the antibody at a concentration of 1 mg / mL into the pre-equilibrated chromatography column, at room temperature, with a flow rate of 0.75 mL / min for 45 min, and simultaneously detect the absorbance value at A280 of the machine. Then, determine the monomer content and ratio of the antibody based on the peak elution time and peak volume.

[0627] Result analysis: Monomer ratio analysis was carried out on single-chain antibody molecules. The results showed that the monomer ratios of D27H1.D01L0-HL-scFv, D27H2.D01L1-LH-scFv, and D27H5.D01L4-LH-scFv were relatively high, all above 95%; the monomer ratios of D27H1.D01L0-LH-scFv, D27H5.D01L4-HL-scFv, and D11-H1L0-LH-scFv were in good properties, above 90%; the monomer ratios of the remaining molecules were below 90% (as shown in Table 31).

[0628] Table 31 Retention time of main peak and monomer ratio of antibodies

[0629] antibody Retention time of main peak (min) Monomer ratio SEC (%) 4B9-HL-scFv 11.357 99.05 4B9-LH-scFv 11.506 98.40 D27H1.D01L0-HL-scFv 11.242 94.88 D27H1.D01L0-LH-scFv 11.403 91.67 D27H2.D01L1-HL-scFv 11.592 89.49 D27H2.D01L1-LH-scFv 12.063 97.24 D27H5.D01L4-HL-scFv 11.330 92.30 D27H5.D01L4-LH-scFv 11.41 95.35 hzD11-H1L0-HL-scFv 11.015 89.95 hzD11-H1L0-LH-scFv 11.01 93.30 hzD18-H0L0-HL-scFv 11.068 69.36 hzD18-H0L0-LH-scFv 11.184 54.33 chrD7-HL-scFv 11.046 61.91 chrD7-LH-scFv 11.113 51.65 chrD20-HL-scFv 11.293 62.06 chrD20-LH-scFv 11.54 62.00 chrD25-HL-scFv 11.049 88.59 chrD25-LH-scFv 11.255 60.14 chrD30-HL-scFv 9.998 87.42 chrD30-LH-scFv 10.28 80.30 hzD16-H0L0-HL-scFv 11.397 87.75 hzD16-H0L0-LH-scFv 11.19 76.30

[0630] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope 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), the amino acid sequence as described in any one of (I) to (III) with one or more amino acids substituted, deleted, or added; or (V), an amino acid sequence having more than 80% identity with the amino acid sequence as 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), the amino acid sequence as described in any one of (VI) to (IX) with one or more amino acids substituted, deleted, or added; or (X), an amino acid sequence having more than 80% identity with the amino acid sequence as described in any one of (VI) to (X).

2. The recombinant anti-FAP antibody according to claim 1, characterized in that, 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 CDR1, CDR2, and CDR3 of the heavy chain sequentially have amino acid sequences as shown in SEQ ID NO.2, 4, and 6; and the CDR1, CDR2, and CDR3 of the light chain sequentially have amino acid sequences as shown in SEQ ID NO.9, 11, and 13; or (12), the CDR1, CDR2, and CDR3 of the heavy chain sequentially have amino acid sequences as shown in SEQ ID NO.16, 18, and 20; and the CDR1, CDR2, and CDR3 of the light chain sequentially have amino acid sequences as shown in SEQ ID NO.23, 25, and 27; or (13), the CDR1, CDR2, and CDR3 of the heavy chain 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); Optionally, 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>; Optionally, 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 as described in X11) or X12), and having the same function as the amino acid sequence as described in X11) or X12); or X14), an amino acid sequence having more than 80% identity with the amino acid sequence as described in any one of X11) to X13).

3. The recombinant anti-FAP antibody according to claim 1 or 2, 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 on the basis of the amino acid sequence as shown in (A1) or (A2); or (A4), a sequence having more than 80% homology with the amino acid sequence as shown in any one of (A1) to (A3); N is selected from any integer in the range of 53 to 63, 81 or 82; X is selected from any integer in the range of 74 to 78.

4. The recombinant anti-FAP antibody according to claim 2, wherein The rabbit-derived chimeric antibody comprises 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 with substitution, deletion, addition and / or replacement of one or more amino acids based on the amino acid sequence shown in any one of (B5) to (B15); or (B17), a sequence with a homology of more than 80% to the amino acid sequence shown in any one of (B5) to (B16); Optionally, 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 with substitution, deletion, addition and / or replacement of one or more amino acids based on the amino acid sequence shown in any one of (C5) to (C9); or (C11), a sequence with a homology of more than 80% to the amino acid sequence shown in any one of (C5) to (C10); Optionally, 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).

5. The recombinant anti-FAP antibody according to any one of claims 2 to 4, characterized in that The single-chain antibody has: <e1>Any one or more of the amino acid sequences shown in SEQ ID NO. 170 to SEQ ID NO. 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.

6. The recombinant anti-FAP antibody according to any one of claims 1 to 5, characterized in that, It also includes a constant region; The heavy chain constant region of the recombinant anti-FAP antibody includes human IgG1; the light chain constant region of the recombinant anti-FAP antibody includes kappa type.

7. The recombinant anti-FAP antibody according to any one of claims 1 to 6, characterized in that, The FAP includes human FAP, murine FAP, and / or cynomolgus monkey FAP.

8. The method for preparing a recombinant anti-FAP antibody according to any one of claims 1 to 7, characterized in that, It includes taking the FAP immunized receptor, isolating the spleen cells of the receptor, and PCR amplifying to obtain the light and heavy chain variable regions of the recombinant anti-FAP antibody; Splicing the heavy chain variable region 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 light chain variable region 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 according to the affinity between the purified antibody and the FAP to obtain the recombinant anti-FAP antibody.

9. 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 7; 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 7; and / or c) A host secreting the recombinant anti-FAP antibody according to any one of claims 1 to 7; and / or d) The recombinant anti-FAP antibody obtained by the preparation method according to claim 8; and / or e) A conjugate of the recombinant anti-FAP antibody according to any one of claims 1 to 7 chemically or biologically labeled and / or the recombinant anti-FAP antibody obtained by the preparation method according to claim 8.

10. 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 7; and / or An expression vector containing the nucleic acid molecule encoding the recombinant anti-FAP antibody according to any one of claims 1 to 7.

11. The immune cell according to claim 10, 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.

12. The immune cell according to claim 10, wherein, The immune cells express a chimeric antigen receptor (CAR), a T cell antigen conjugate (TAC) receptor, or a T cell receptor (TCR).

13. 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 7; and / or B), the recombinant anti-FAP antibody obtained by the preparation method according to claim 8; and / or C), the biological material according to claim 9; and / or D), the immune cells according to any one of claims 10 to 12.

14. 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 7; and / or B), the recombinant anti-FAP antibody obtained by the preparation method according to claim 8; and / or C), the biological material according to claim 9; and / or D), the immune cells according to any one of claims 10 to 12.

15. The use according to claim 14, 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.

16. The application according to claim 14 or 15, characterized in that, The product includes a drug and / or a vaccine.

17. A drug, characterized in that, Includes any of the following: A), the recombinant anti-FAP antibody according to any one of claims 1 to 7; and / or B), the recombinant anti-FAP antibody obtained by the preparation method according to claim 8; and / or C), the biological material according to claim 9; and / or D), the immune cells according to any one of claims 10 to 12.

18. A pharmaceutical combination, characterized in that, Includes the drug according to claim 17, and any other active ingredient, Optionally, the any other active ingredient includes a small molecule toxin.

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