CN120365430A

CN120365430A - Recombinant anti-FAP antibody and application thereof

Info

Publication number: CN120365430A
Application number: CN202411965980.9A
Authority: CN
Inventors: 徐晓红; 吴建; 桂勋
Original assignee: Maiwei Shanghai Biotechnology Co ltd; Jiangsu T Mab Biopharma
Current assignee: Maiwei Shanghai Biotechnology Co ltd; Jiangsu T Mab Biopharma
Priority date: 2024-01-05
Filing date: 2024-12-30
Publication date: 2025-07-25

Abstract

本发明涉及抗体药物领域，具体涉及重组抗FAP抗体及其应用。本发明提供了重组抗FAP抗体及其应用。本发明提供了重组抗FAP抗体及其应用。本发明提供的重组抗FAP抗体，展现高度特异性结合以及快速和高效的内化能力，其高选择性，高生物学活性的水平，有助于成为治疗用单克隆抗体或偶联小分子毒素的抗体偶联药物。The present invention relates to the field of antibody drugs, and in particular to recombinant anti-FAP antibodies and their applications. The present invention provides recombinant anti-FAP antibodies and their applications. The present invention provides recombinant anti-FAP antibodies and their applications. The recombinant anti-FAP antibodies provided by the present invention exhibit highly specific binding and rapid and efficient internalization capabilities, and their high selectivity and high biological activity levels contribute to becoming therapeutic monoclonal antibodies or antibody-drug conjugates conjugated to small molecule toxins.

Description

Recombinant anti-FAP antibody and application thereof

The present application claims priority from the national intellectual property agency, application number 202410018607.6, chinese patent application entitled "recombinant anti-FAP antibody and use thereof," filed on month 05 2024, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the field of antibody medicines, in particular to a recombinant anti-FAP antibody and application thereof.

Background

Malignant epithelial tumors are the major cancer-related diseases in human death. These solid tumors often exhibit a significant interstitial response, such as what is known as "desmoplastic interstitial" or "reactive interstitial", which accounts for 20-60% of the overall tumor mass and are characterized by the presence of large numbers of interstitial cells and dense extracellular matrix (ECM). A highly consistent molecular characteristic of the reactive matrix of many types of epithelial cancers is the induction of fibroblast activation protein alpha (hereinafter FAP). In particular, an appreciable number of cancer-associated fibroblasts (CAF) are often observed in the tumor-associated stroma of various human cancers including breast, lung, colon and pancreas cancers.

FAP alpha is a cell surface molecule of reactive stromal fibroblasts originally identified by monoclonal antibody F19, is a cell surface glycoprotein of reactive stromal fibroblasts, and can be used as a potential antibody target in human epithelial cancers. In many types of human cancers, the fibroblast response is characterized by the induction of a cell surface protein, fibroblast activation protein α (fapα), a 95kDa serine protease whose expression is highly localized to developmental organs, wound healing and tissue remodeling.

FAP exhibits the following features:

1) Type II membrane glycoprotein with serine protease activity

2) 89% Human-murine protein homology, 99% human-monkey protein homology

3) Tumor stroma is expressed in >90% of cancers (breast, pancreatic, lung, bladder and colon cancers)

4) Transient and highly localized expression in normal adult tissues and developing organs during wound healing

5) Has faster internalization capability

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

CAF is capable of promoting neovascularization and tumor growth in the case of coordinated interactions with the different components of the stroma, and has also been shown to be critical for the development of invasive tumors and tumor invasiveness during cancer progression, and to promote the spread and infiltration of tumor cells in distant organs, thereby causing the formation of metastases. Importantly, the correlation of stromal cells with systemic drug delivery failure to tumors and emergence of drug resistance has also been indicated.

Identification of cellular and molecular targets that eliminate the stromal-tumor cell interaction and thereby attenuate tumorigenesis is currently an important topic in transformed oncology. Indeed, targeting the peri-tumor stroma is a novel strategy for treating metastatic tumors representing mortality in more than 90% of cancer patients, with only a few products to date being approved for treatment, most of them being anti-angiogenic drugs. The identification and targeting of other novel molecules within the tumor microenvironment is necessary to increase the efficacy of conventional therapies combined with interstitial-based therapies and represents an effective approach for cancer and metastasis therapy.

To alter the non-human immunogenicity of the monoclonal antibody of F19 (by hybridoma cell line ATCC accession number HB 8269), antibody F19 is humanized sibrotuzumab (BIBH 1), a humanized antibody that specifically binds FAP is described in WO 99/57151. Phase I clinical studies on BIBH1, i.e., I31 radiolabeled anti-FAP mab, showed good safety and tolerability (Scott et al, 2001; hofheinz et al, 2003), but in phase II clinical studies with patients with advanced metastatic colorectal cancer, most patients found sustained tumor progression, and the study was aborted due to the failure to meet minimum requirements.

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

2009/0304718 Al). They showed specific binding to fap+ cells in vitro and were internalized. In pancreatic and lung cancer xenograft models, treatment with murine MAb MFP5-DM1 immunotoxins induced long-lasting inhibition and complete regression of tumor growth without any intolerance-related effects.

ONCOMATRYX BIOPHARMA the anti-FAP antibody hu36 developed exhibited highly specific binding and rapid internalization. Furthermore, the inventors isolated the a-chain of melanin (Nigrin) b produced in bacterial host cells and conjugated to anti-FAP antibodies for use in the treatment of tumors. It exhibits an effect of inhibiting tumor growth in various tumor models in vitro.

Despite these advances, there remains an unmet need for other therapeutic strategies for treating tumors, including epithelial tumors, as well as for components used in such therapeutic strategies.

Disclosure of Invention

In view of this, the present invention provides recombinant anti-FAP antibodies and uses thereof.

The invention provides recombinant anti-FAP antibodies and uses thereof. The recombinant anti-FAP antibody provided by the invention has high specific binding and rapid and efficient internalization capability, has high selectivity and high biological activity level, and is favorable for becoming a monoclonal antibody for treatment or an antibody coupling drug coupled with small molecule toxin.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides recombinant anti-FAP antibodies comprising a heavy chain and a light chain:

the CDR regions of the heavy chain include CDR1, CDR2, and CDR3:

(I) CDR1 of said heavy chain having an amino acid sequence as shown in SEQ ID No.2, 16, 27, 38, 48, 56, 67 or 107, and

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

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

(IV) a sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as shown in any one of (I) to (III), or

(V) a sequence having 80% or more homology with the amino acid sequence as defined in any one of (I) to (IV);

the CDR regions of the light chain include CDR1, CDR2, and CDR3:

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

(VII), CDR2 of said light chain having an amino acid sequence as shown in SEQ ID No.11, 35, 45, 63 or 74, and

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

(IX) a sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as shown in any one of (VI) to (VIII), or

(X) a sequence having 80% or more homology with the amino acid sequence as defined in any one of (VI) to (IX).

In some embodiments of the invention, the recombinant anti-FAP antibodies include rabbit chimeric antibodies and humanized antibodies;

In some embodiments of the invention, the CDR regions of the heavy chain of the rabbit chimeric antibody comprise CDR1, CDR2 and CDR3:

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

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

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

IV) substitution, deletion, addition and/or substitution of a sequence of 1 or more amino acids based on the amino acid sequence as shown in any one of I) to III), or

V) a sequence having 80% or more homology with the amino acid sequence shown in any one of I) to IV);

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

VI), the CDR1 of the light chain of said rabbit chimeric antibody having the amino acid sequence as shown in SEQ ID No.9, 21, 34, 44, 52, 62 or 73, and

VII) the CDR2 of the light chain of said rabbit chimeric antibody has the amino acid sequence as shown in SEQ ID No.11, 35, 45, 63 or 74, and

VIII), the CDR3 of the light chain of said rabbit chimeric antibody has the amino acid sequence as shown in SEQ ID No.13, 24, 37, 47, 55, 65 or 76, or

IX), a sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as shown in any one of VI) to VIII), or

X) a sequence having 80% or more homology with the amino acid sequence shown in any one of VI) to IX).

In some embodiments of the invention, the rabbit chimeric antibody comprises chrX1, chrX6, chrX8, chrX9, chrA, chrA, or chrA18;

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

The CDR1, CDR2 and CDR3 of the light chain of the chrysX 1 have the amino acid sequences shown in SEQ ID No.9, 11 and 13 in sequence, or

B) CDR1, CDR2 and CDR3 of the heavy chain of said chrX6 have in sequence the amino acid sequences shown in SEQ ID No.16, 18 and 19, and

The CDR1, CDR2 and CDR3 of the light chain of the chrysX 6 have the amino acid sequences shown in SEQ ID No.21, 11 and 24 in sequence, or

C) CDR1, CDR2 and CDR3 of the heavy chain of said chrX8 have in sequence the amino acid sequences shown in SEQ ID No.27, 29 and 31, and

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

D) CDR1, CDR2 and CDR3 of the heavy chain of said chrX9 have in sequence the amino acid sequences shown in SEQ ID Nos. 38, 40 and 42, and

The CDR1, CDR2 and CDR3 of the light chain of chrX9 have the amino acid sequences shown in SEQ ID Nos. 44, 45 and 47 in sequence, or

E) CDR1, CDR2 and CDR3 of the heavy chain of chrA have the amino acid sequences shown in SEQ ID Nos. 48, 49 and 6 in this order, and

The CDR1, CDR2 and CDR3 of the light chain of chrA have the amino acid sequences shown in SEQ ID Nos. 52, 11 and 55 in order, or

F) CDR1, CDR2 and CDR3 of the heavy chain of chrA have the amino acid sequences shown in SEQ ID Nos. 56, 57 and 59 in this order, and

CDR1, CDR2 and CDR3 of the light chain of chrA have SEQ ID Nos. 62, 63 and 65 in this order, or

G) CDR1, CDR2 and CDR3 of the heavy chain of chrA have the amino acid sequences shown in SEQ ID Nos. 67, 69 and 71 in this order, and

The CDR1, CDR2 and CDR3 of the light chain of chrA have the amino acid sequences shown in SEQ ID Nos. 73, 74 and 76 in order, or

H) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of a) to g), or

J) A sequence having 80% or more homology with the amino acid sequence shown in any one of a) to h).

In some embodiments of the invention, the CDR regions of the heavy chain of the humanized antibody comprise CDR1, CDR2 and CDR3:

(A) CDR1 of the heavy chain of the humanized antibody has an amino acid sequence as shown in SEQ ID No.107, and

(B) CDR2 of the heavy chain of the humanized antibody has an amino acid sequence as shown in SEQ ID No.18, and

(C) CDR3 of the heavy chain of the humanized antibody has an amino acid sequence as shown in SEQ ID No.108, or

(D) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of (A) to (C), or

(E) A sequence having 80% or more homology with the amino acid sequence as set forth in any one of (A) to (D);

the CDR regions of the light chain of the humanized antibody include CDR1, CDR2, and CDR3:

(F) CDR1 of the light chain of the humanized antibody has an amino acid sequence as shown in SEQ ID No.112, and

(G) CDR2 of the light chain of the humanized antibody has an amino acid sequence as shown in SEQ ID No.11, and

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

(J) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of (F) to (G), or

(K) A sequence having 80% or more homology with the amino acid sequence as defined in any one of (F) to (J).

In some embodiments of the invention, the humanized antibody comprises hzX6-H29L3, hzX6-H29L10, hzX6-H29L12, hzX-H32L 3, hzX6-H32L10, hzX6-H32L12, hzX6-H33L3, hzX6-H33L10, or hzX6-H33L12;

k) CDR1, CDR2 and CDR3 of the heavy chain of hzX-H29L 3 have the amino acid sequences shown in SEQ ID Nos. 107, 18 and 108 in that order, and

The CDR1, CDR2 and CDR3 of the light chain of hzX-H29L 3 have the amino acid sequences shown in SEQ ID No.112, 11 and 24 in sequence, or

L), CDR1, CDR2 and CDR3 of the heavy chain of hzX-H29L 10 having in sequence the amino acid sequences shown in SEQ ID No.107, 18 and 108, and

The CDR1, CDR2 and CDR3 of the light chain of hzX-H29L 10 have the amino acid sequences shown in SEQ ID No.112, 11 and 116 in order, or

M), CDR1, CDR2 and CDR3 of the heavy chain of hzX-H29L 12 having in sequence the amino acid sequences shown in SEQ ID Nos. 107, 18 and 108, and

The CDR1, CDR2 and CDR3 of the light chain of hzX-H29L 12 have the amino acid sequences shown in SEQ ID Nos. 112, 11 and 117 in order, or

N), CDR1, CDR2 and CDR3 of the heavy chain of hzX-H32L 3 having in sequence the amino acid sequences shown in SEQ ID No.107, 18 and 108, and

The CDR1, CDR2 and CDR3 of the light chain hzX-H32L 3 have the amino acid sequences shown in SEQ ID No.112, 11 and 24 in sequence, or

O), CDR1, CDR2 and CDR3 of the heavy chain of hzX-H32L 10 having in sequence the amino acid sequences shown in SEQ ID No.107, 18 and 108, and

The CDR1, CDR2 and CDR3 of the light chain of hzX-H32L 10 have the amino acid sequences shown in SEQ ID No.112, 11 and 116 in order, or

P), CDR1, CDR2 and CDR3 of the heavy chain of hzX-H32L 12 having in sequence the amino acid sequences shown in SEQ ID No.107, 18 and 108, and

The CDR1, CDR2 and CDR3 of the light chain of hzX-H32L 12 have the amino acid sequences shown in SEQ ID No.112, 11 and 117 in order, or

Q), CDR1, CDR2 and CDR3 of the heavy chain of hzX-H33L 3 having the amino acid sequences shown in SEQ ID Nos. 107, 18 and 108 in that order, and

The CDR1, CDR2 and CDR3 of the light chain hzX-H33L 3 have the amino acid sequences shown in SEQ ID No.112, 11 and 24 in sequence, or

R), CDR1, CDR2 and CDR3 of the heavy chain of hzX-H33L 10 having the amino acid sequences shown in SEQ ID Nos. 107, 18 and 108 in that order, and

The CDR1, CDR2 and CDR3 of the light chain of hzX-H33L 10 have the amino acid sequences shown in SEQ ID Nos. 112, 11 and 116 in order, or

S), CDR1, CDR2 and CDR3 of the heavy chain of hzX-H33L 12 having the amino acid sequences shown in SEQ ID No.107, 18 and 108 in that order, and

The CDR1, CDR2 and CDR3 of the light chain of hzX-H33L 12 have the amino acid sequences shown in SEQ ID Nos. 112, 11 and 117 in order, or

T) a sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as shown in any one of k) to s), or

U) a sequence having 80% or more homology with the amino acid sequence shown in any one of k) to t).

In some embodiments of the invention, the FR region of the heavy chain comprises FR1, FR2, FR3 and FR4:

(1) FR1 of said heavy chain has the amino acid sequence shown as SEQ ID No.1, 15, 26, 66 or 106, and

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

(3) FR3 of said heavy chain has an amino acid sequence as shown in SEQ ID No.5, 30, 41, 50, 58, 70, 150, 109 or 110, and

(4) FR4 of said heavy chain has the amino acid sequence shown as SEQ ID No.7, 32 or 60, or

(5) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of (1) to (4), or

(6) A sequence having 80% or more homology with the amino acid sequence as set forth in any one of (1) to (5);

the FR regions of the light chain include FR1, FR2, FR3 and FR4:

(7) FR1 of said light chain has the amino acid sequence shown as SEQ ID No.8, 20, 33, 43, 51, 61, 72 or 111, and

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

(9) FR3 of said light chain has the amino acid sequence shown as SEQ ID No.12, 23, 36, 46, 54, 64, 75 or 114, and

(10) FR4 of said light chain has the amino acid sequence shown as SEQ ID No.14, 25, 77 or 115, or

(11) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of (7) to (10), or

(12) A sequence having 80% or more homology with the amino acid sequence as set forth in any one of (7) to (11).

in some embodiments of the invention, the FR region of the heavy chain of the rabbit chimeric antibody comprises FR1, FR2, FR3 and FR4:

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

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

(15) FR3 of the heavy chain of said rabbit chimeric antibody has the amino acid sequence shown as SEQ ID No.5, 30, 41, 50, 58 or 70, and

(16) FR4 of the heavy chain of said rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No.7, 32 or 60, or

(17) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of (13) to (16), or

(18) A sequence having 80% or more homology with the amino acid sequence as set forth in any one of (13) to (17);

The FR regions of the light chain of the rabbit chimeric antibody include FR1, FR2, FR3 and FR4:

(19) FR1 of the light chain of said rabbit chimeric antibody has the amino acid sequence shown as SEQ ID No.8, 20, 33, 43, 51, 61 or 72, and

(20) FR2 of the light chain of said rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No.10, 22 or 53, and

(21) FR3 of the light chain of said rabbit chimeric antibody has the amino acid sequence shown as SEQ ID No.12, 23, 36, 46, 54, 64 or 75, and

(22) FR4 of the light chain of said rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No.14, 25 or 77, or

(23) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of (19) to (22), or

(24) A sequence having 80% or more homology with the amino acid sequence as set forth in any one of (19) to (23).

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

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

2) FR1, FR2, FR3 and FR4 of said heavy chain of chrX6 have in sequence the amino acid sequences shown in SEQ ID No.15, 17, 5 and 7, and

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

3) FR1, FR2, FR3 and FR4 of said heavy chain of chrX8 have in sequence the amino acid sequences shown in SEQ ID No.26, 28, 30 and 32, and

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

4) FR1, FR2, FR3 and FR4 of said heavy chain of chrX9 have in sequence the amino acid sequences shown in SEQ ID No.26, 39, 41 and 7, and

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

5) FR1, FR2, FR3 and FR4 of said chrA heavy chain have the amino acid sequences shown in SEQ ID No.1, 39, 50 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of chrA have the amino acid sequences shown in SEQ ID No.51, 53, 54 and 25 in order, or

6) FR1, FR2, FR3 and FR4 of said chrA heavy chain have the amino acid sequences shown in SEQ ID No.26, 3, 58 and 60 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of chrA have the amino acid sequences shown in SEQ ID No.61, 22, 64 and 25 in order, or

7) FR1, FR2, FR3 and FR4 of said chrA heavy chain have the amino acid sequences shown in SEQ ID No.66, 68, 70 and 7 in this order, and

The FR1, FR2, FR3 and FR4 of the light chain of chrA have the amino acid sequences shown in SEQ ID No.72, 22, 75 and 77 in order, or

8) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of 1) to 7), or

9) A sequence having 80% or more homology with the amino acid sequence shown in any one of 1) to 8).

In some embodiments of the invention, the FR regions of the heavy chain of the humanized antibody comprise FR1, FR2, FR3 and FR4:

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

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

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

D) FR4 of the heavy chain of said humanized antibody has the amino acid sequence shown in SEQ ID No.7 or

E) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as shown in any one of A) to D), or

F) A sequence having 80% or more homology with the amino acid sequence shown in any one of A) to E);

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

g) FR1 of the light chain of said humanized antibody has the amino acid sequence shown in SEQ ID No.111, and

H) FR2 of the light chain of said humanized antibody has the amino acid sequence shown as SEQ ID No.113, and

J) FR3 of the light chain of said humanized antibody has the amino acid sequence shown as SEQ ID No.114, and

K) FR4 of the light chain of said humanized antibody has the amino acid sequence shown as SEQ ID No.115, or

L), a sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of (19) to (22), or

M) a sequence having 80% or more homology with the amino acid sequence as set forth in any one of (19) to (23).

1) FR1, FR2, FR3 and FR4 of said hzX heavy chain of H29L3 have the amino acid sequences shown in SEQ ID No.106, 28, 150 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of hzX-H29L 3 have the amino acid sequences shown in SEQ ID No.111, 113, 114 and 115 in order, or

2) FR1, FR2, FR3 and FR4 of said hzX heavy chain of H29L10 have the amino acid sequences shown in SEQ ID No.106, 28, 150 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of hzX-H29L 10 have the amino acid sequences shown in SEQ ID No.111, 113, 114 and 115 in order, or

3) FR1, FR2, FR3 and FR4 of said hzX heavy chain of H29L12 have the amino acid sequences shown in SEQ ID No.106, 28, 150 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of hzX-H29L 12 have the amino acid sequences shown in SEQ ID No.111, 113, 114 and 115 in order, or

4) FR1, FR2, FR3 and FR4 of said hzX heavy chain of H32L3 have the amino acid sequences shown in SEQ ID No.106, 28, 109 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of hzX-H32L 3 have the amino acid sequences shown in SEQ ID No.111, 113, 114 and 115 in order, or

5) FR1, FR2, FR3 and FR4 of said hzX heavy chain of hzX-H32L 10 have the amino acid sequences shown in SEQ ID No.106, 28, 109 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of hzX-H32L 10 have the amino acid sequences shown in SEQ ID No.111, 113, 114 and 115 in order, or

6) FR1, FR2, FR3 and FR4 of said hzX heavy chain of hzX-H32L 12 have the amino acid sequences shown in SEQ ID No.106, 28, 109 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of hzX-H32L 12 have the amino acid sequences shown in SEQ ID No.111, 113, 114 and 115 in order, or

7) FR1, FR2, FR3 and FR4 of said hzX heavy chain of H33L3 have the amino acid sequences shown in SEQ ID No.106, 28, 110 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of hzX-H33L 3 have the amino acid sequences shown in SEQ ID No.111, 113, 114 and 115 in order, or

8) FR1, FR2, FR3 and FR4 of said hzX heavy chain of hzX-H33L 10 have the amino acid sequences shown in SEQ ID No.106, 28, 110 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of hzX-H33L 10 have the amino acid sequences shown in SEQ ID No.111, 113, 114 and 115 in order, or

9) FR1, FR2, FR3 and FR4 of said hzX heavy chain of hzX-H33L 12 have the amino acid sequences shown in SEQ ID No.106, 28, 110 and 7 in order, and

The FR1, FR2, FR3 and FR4 of the light chain of hzX-H33L 12 have the amino acid sequences shown in SEQ ID No.111, 113, 114 and 115 in order, or

10 Substituted, deleted, added and/or substituted with a sequence of 1 or more amino acids based on the amino acid sequence as set forth in any one of 1) to 9), or

11 A sequence having 80% or more homology with the amino acid sequence shown in any one of 1) to 10).

In some embodiments of the present invention,

(I) The variable region of the heavy chain having an amino acid sequence as shown in SEQ ID No.78, 80, 82, 84, 86, 88, 90, 118, 119 or 120, and

(Ii) The variable region of the light chain has an amino acid sequence as shown in SEQ ID No.79, 81, 83, 85, 87, 89, 91, 121, 122 or 123, or

(Iii) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence shown in (i) or (ii), or

(Iv) A sequence having 80% or more homology with the amino acid sequence as defined in any one of (i) to (iii).

In some embodiments of the present invention,

I) The variable region of the heavy chain having the amino acid sequence shown as SEQ ID No.78, and

The variable region of the light chain has an amino acid sequence shown as SEQ ID No. 79;

Or (b)

Ii) the variable region of the heavy chain has the amino acid sequence shown as SEQ ID No.80, and

The variable region of the light chain has an amino acid sequence shown as SEQ ID No. 81;

Or (b)

Iii) The variable region of the heavy chain having the amino acid sequence shown as SEQ ID No.82, and

The variable region of the light chain has an amino acid sequence shown as SEQ ID No. 83;

Or (b)

Iv) the variable region of the heavy chain has the amino acid sequence as shown in SEQ ID No.84, and

The variable region of the light chain has an amino acid sequence shown as SEQ ID No. 85;

Or (b)

V) the variable region of the heavy chain has the amino acid sequence shown as SEQ ID No.86, and

The variable region of the light chain has an amino acid sequence shown as SEQ ID No. 87;

Or (b)

Vi) the variable region of the heavy chain has the amino acid sequence shown as SEQ ID No.88, and

The variable region of the light chain has an amino acid sequence shown as SEQ ID No. 89;

Or (b)

Vii) the variable region of said heavy chain has the amino acid sequence shown as SEQ ID No.90, and

The variable region of the light chain has an amino acid sequence shown as SEQ ID No. 91;

Or (b)

Viii) the variable region of the heavy chain has the amino acid sequence shown as SEQ ID No.118, and

The variable region of the light chain has an amino acid sequence shown as SEQ ID No. 121;

Or (b)

Ix) the variable region of said heavy chain has the amino acid sequence as shown in SEQ ID No.119, and

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

Or (b)

X) the variable region of the heavy chain has the amino acid sequence shown as SEQ ID No.120, and

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

xi), a sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as shown in any one of i) to x), or

Xii) a sequence having 80% or more homology with the amino acid sequence as set forth in any one of i) to xi).

In some embodiments of the present invention,

I) A nucleic acid molecule encoding the variable region of the heavy chain having a nucleotide sequence as set forth in SEQ ID No.92, 94, 96, 98, 100, 102, 104, 124, 125 or 126, and

Ii) the nucleic acid molecule encoding the variable region of the light chain has the nucleotide sequence as shown in SEQ ID No.93, 95, 97, 99, 101, 103, 105, 127, 128 or 129, or

Iii) A nucleotide sequence which encodes the same protein as the nucleotide sequence set forth in i) or ii) but differs from the nucleotide sequence set forth in i) or ii) by the degeneracy of the genetic code, or

Iv) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in any one of i) to iii) and functionally identical or similar to the nucleotide sequence shown in any one of i) to iii), or

V) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence of any one of i) to iv).

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

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

(Vi) The variable region of the heavy chain of the humanized antibody has an amino acid sequence as shown in 118, 119 or 120, and

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

(Vii) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence shown in (v) or (vi), or

(Ix) A sequence having 80% or more homology with the amino acid sequence as defined in any one of (v) to (vii).

In some embodiments of the invention, the rabbit chimeric antibody comprises:

1) The variable region of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No.78, and

The variable region of the light chain of the rabbit chimeric antibody has an amino acid sequence shown as SEQ ID No. 79;

Or (b)

2) The variable region of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No.80, and

The variable region of the light chain of the rabbit chimeric antibody has an amino acid sequence shown as SEQ ID No. 81;

Or (b)

3) The variable region of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No.82, and

The variable region of the light chain of the rabbit chimeric antibody has an amino acid sequence shown as SEQ ID No. 83;

Or (b)

4) The variable region of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No.84, and

The variable region of the light chain of the rabbit chimeric antibody has an amino acid sequence shown as SEQ ID No. 85;

Or (b)

5) The variable region of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No.86, and

The variable region of the light chain of the rabbit chimeric antibody has an amino acid sequence shown as SEQ ID No. 87;

Or (b)

6) The variable region of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No.88, and

The variable region of the light chain of the rabbit chimeric antibody has an amino acid sequence shown as SEQ ID No. 89;

Or (b)

7) The variable region of the heavy chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No.90, and

The variable region of the light chain of the rabbit chimeric antibody has an amino acid sequence shown as SEQ ID No. 91;

Or (b)

In some embodiments of the invention, the humanized antibody:

1) The variable region of the heavy chain of the humanized antibody has an amino acid sequence as shown in 118, and

The variable region of the light chain of the humanized antibody has an amino acid sequence as shown at 121;

Or (b)

2) The variable region of the heavy chain of the humanized antibody has an amino acid sequence as shown in 119, and

The variable region of the light chain of the humanized antibody has an amino acid sequence as shown in 122;

Or (b)

3) The variable region of the heavy chain of the humanized antibody has an amino acid sequence as shown in 120, and

The variable region of the light chain of the humanized antibody has an amino acid sequence as shown in 123;

Or (b)

4) A sequence in which 1 or more amino acids are substituted, deleted, added and/or substituted on the basis of the amino acid sequence as set forth in any one of 1) to 3), or

5) A sequence having 80% or more homology with the amino acid sequence shown in any one of 1) to 4).

In some embodiments of the invention vi) a nucleic acid molecule encoding the heavy chain variable region of said rabbit chimeric antibody has the nucleotide sequence shown as SEQ ID No.92, 94, 96, 98, 100, 102 or 104, and

The nucleic acid molecule encoding the light chain variable region of the rabbit chimeric antibody has a nucleotide sequence as shown in SEQ ID No.93, 95, 97, 99, 101, 103 or 105, or

Vii), a nucleic acid molecule encoding the heavy chain variable region of said humanized antibody has a nucleotide sequence as set forth in SEQ ID No.124, 125 or 126, and

The nucleic acid molecule encoding the light chain variable region of the humanized antibody has a nucleotide sequence as shown in SEQ ID No.127, 128 or 129, or

Ix), a nucleotide sequence which encodes the same protein as the nucleotide sequence set forth in vi) or vii), but which differs from the nucleotide sequence set forth in vi) or vii) by the degeneracy of the genetic code, or

X), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in any one of vi) to ix), and functionally identical or similar to the nucleotide sequence shown in any one of vi) to ix), or

Xi) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence of any one of vi) to x).

In some embodiments of the invention, the recombinant anti-FAP antibodies further comprise a constant region;

The heavy chain constant region of the recombinant anti-FAP antibody comprises human IgG1, and the light chain constant region of the recombinant anti-FAP antibody comprises human kappa type.

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

The invention also provides a preparation method of the recombinant anti-FAP antibody, which comprises the following steps:

step 1, taking FAP antigen immune receptor, separating spleen cells of the receptor, and carrying out PCR amplification to obtain a light chain variable region of the recombinant anti-FAP antibody;

Step 2, splicing the heavy chain variable region and the heavy chain constant region, constructing the heavy chain variable region and the heavy chain constant region into an expression vector to obtain a heavy chain vector;

and 3, taking the heavy chain vector and the light chain vector, transfecting, culturing, purifying and screening to obtain the recombinant anti-FAP antibody.

In some embodiments of the invention, the receptor comprises a New Zealand white rabbit;

the heavy chain constant region is a heavy chain constant region of a human IgG1 subclass;

the light chain constant region is the light chain constant region of a human kappa antibody;

The constructed expression vector is a mammalian cell expression vector.

In some embodiments of the invention, the preparation method comprises the steps of:

Step A, combining a CDR region of the recombinant anti-FAP antibody with a human antibody framework region, splicing the heavy chain variable region and the heavy chain constant region, constructing an expression vector to obtain a heavy chain vector, splicing the light chain variable region and the light chain constant region, constructing the expression vector to obtain a light chain vector;

and B, taking the heavy chain vector and the light chain vector, carrying out transfection, culturing, purifying and screening to obtain the humanized antibody of the recombinant anti-FAP antibody.

In some embodiments of the invention, the heavy chain constant region is a heavy chain constant region of the human IgG1 subclass;

The constructed expression vector is a mammalian cell expression vector.

Based on the above study, the present invention also provides a biomaterial comprising any of the following:

(a) Nucleic acid encoding said recombinant anti-FAP antibody or said recombinant anti-FAP antibody produced by said production method, and/or

(B) Nucleic acid encoding said recombinant anti-FAP antibody, and acceptable carrier therefor, or

The recombinant anti-FAP antibody prepared by the preparation method and an acceptable carrier, and/or

(C) A host secreting said recombinant anti-FAP antibody or said recombinant anti-FAP antibody produced by said production method, and/or

(D) Said recombinant anti-FAP antibody, chemically-labeled or biomarker, or said recombinant anti-FAP antibody produced by said production method, and/or

(E) The recombinant anti-FAP antibody coupled with a carrier or the recombinant anti-FAP antibody prepared by the preparation method;

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

The invention also provides application of any of the following in preparing FAP-targeted drugs:

① . The recombinant anti-FAP antibody, and/or

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

③ . The biological material.

The invention also provides the use of any of the following for the preparation of a product for the prevention and/or treatment of a disease:

① . The recombinant anti-FAP antibody, and/or

③ . The biological material.

In some embodiments of the invention, the disease comprises any one or more of human epithelial cancer, breast cancer, pancreatic cancer, lung cancer, bladder cancer, or colon cancer.

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

The invention also provides a medicament, which comprises any of the following auxiliary materials and pharmaceutically acceptable auxiliary materials:

① . The recombinant anti-FAP antibody, and/or

③ . The biological material.

The invention also provides a pharmaceutical combination comprising the medicament and any other active ingredients.

In some embodiments of the invention, the other optional active ingredient comprises a small molecule toxin.

The invention also provides a vaccine comprising any of the following:

① . The recombinant anti-FAP antibody, and/or

③ . The biological material.

The invention also provides application of any of the following in preparation of a reagent and/or a kit for detecting FAP:

① . The recombinant anti-FAP antibody, and/or

③ . The biological material.

The invention also provides reagents and/or kits comprising any of the following:

① . The recombinant anti-FAP antibody, and/or

③ . The biological material.

The invention also provides a method of treatment comprising administering to a subject any of:

① . The recombinant anti-FAP antibody, and/or

③ . The biological material.

The invention provides recombinant anti-FAP antibodies and uses thereof. The invention provides recombinant anti-FAP antibodies and uses thereof. The recombinant anti-FAP antibody provided by the invention has high specific binding and rapid and efficient internalization capability, has high selectivity and high biological activity level, and is favorable for becoming a monoclonal antibody for treatment or an antibody coupling drug coupled with small molecule toxin. The antibody provided by the invention has better effect and lower immunogenicity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1a shows a control antibody flow-through binding assay to CHO-hFAP cells in the experimental example;

FIG. 1b shows a control antibody flow-through binding assay to U138MG cells in the experimental example;

FIG. 2a shows a cell-stream binding assay for anti-human FAP chimeric antibodies chrysX 1, chrysX 6, chrysX 8, chrysX 9 and HT1080-hFAP of example 1;

FIG. 2b shows a flow-through assay of anti-human FAP chimeric antibodies chrA, chrA, chrA18, hu36 and HT1080-hFAP cells of example 1;

FIG. 3a shows the use of cells of the low expressing cell lines HT1080-hFAP in example 3 for testing the endocytic activity of anti-human FAP chimeric antibodies chrysX 1, chrysX 6, chrysX 8, chrysX 9;

FIG. 3b shows the use of cells of the low expressing cell lines HT1080-hFAP in example 3 to test endocytosis of anti-human FAP chimeric antibodies chrA7, chrA, chrA18, hu 36;

FIG. 4a shows the binding assay of humanized antibodies to HT1080-hFAP cells of example 6;

FIG. 4b shows the binding assay of humanized antibodies to CT26-mFAP cells in example 6;

FIG. 4c shows the binding assay of humanized antibodies to CHO-cynoFAP cells in example 6;

FIG. 5a shows the binding assay of anti-human FAP humanized antibodies to CHO-hDPP4 cells in example 7;

FIG. 5b shows an experiment of the binding of anti-human FAP humanized antibodies to CHOK1 cells in example 7;

FIG. 5c shows an experiment of binding of anti-human FAP humanized antibodies to HEK293 cells in example 7;

FIG. 6 shows the use of HT1080-hFAP cells in example 8 to test endocytosis activity of anti-human FAP humanized antibodies;

FIG. 7a shows a killing assay for testing human FAP humanized antibodies hzX6-H29L3, hzX6-H29L10 and hzX6-H29L12 using HT1080-hFAP cells in example 9;

FIG. 7b shows a killing assay for testing human FAP humanized antibodies hzX6-H32L3, hzX6-H32L10 and hzX6-H32L12 using HT1080-hFAP cells in example 9;

FIG. 7c shows a killing assay for testing human FAP humanized antibodies hzX6-H33L3, hzX6-H33L10 and hzX6-H33L12 using HT1080-hFAP cells in example 9;

FIG. 7d shows a killing experiment using 293-hFAP cells to test human FAP humanized antibodies hzX-H29L 3, hzX6-H29L10 and hzX6-H29L12 in example 9;

FIG. 7e shows a killing experiment using 293-hFAP cells to test human FAP humanized antibodies hzX-H32L 3, hzX6-H32L10 and hzX6-H32L12 in example 9;

FIG. 7f shows a killing experiment using 293-hFAP cells to test human FAP humanized antibodies hzX-H33L 3, hzX6-H33L10, and hzX6-H33L12 in example 9.

Detailed Description

The invention discloses a recombinant anti-FAP antibody and application thereof, and a person skilled in the art can refer to the content of the invention to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

TABLE 1 amino acid sequence of chimeric antibody and CDR partitioning thereof

The variable region sequences of the rabbit monoclonal antibodies are shown below.

1. Rabbit-derived antibody

TABLE 2

Note that the underlined CDRs are labeled in the same manner as the subsequent sequences according to the Kabat antibody coding scheme.

TABLE 3 chimeric antibody nucleotide sequences

TABLE 4 humanized molecular light heavy chain variable region amino acid CDR partitioning

2. Humanized engineered antibodies

TABLE 5

Note that the underlined CDRs are labeled in the same manner as the subsequent sequences according to the Kabat antibody coding scheme. PTM loci and corresponding variants thereof are marked by character thickening inclination, and the subsequent sequence marking modes are the same.

TABLE 6 nucleotide sequence of humanized molecules

1. Sequence synthesis and vector construction of tool antibodies

1.1Boehringer-Ingelheim anti-human FAP humanized monoclonal antibody sibrotuzumab (BIBH 1)

BIBH1 heavy chain variable region amino acid sequence (SEQ ID No. 130):

QVQLVQSGAEVKKPGASVKVSCKTSRYTFTEYTIHWVRQAPGQRLEWIGGINPNNGIPNYNQKF KGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRRIAYGYDEGHAMDYWGQGTLVTVSS

BIBH1 light chain variable region amino acid sequence (SEQ ID No.131:

DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVP DRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK

1.2Boehringer-Ingelheim anti-human FAP murine monoclonal antibody cMFP5

MFP5 heavy chain variable region amino acid sequence (SEQ ID No. 132):

QVQLQQSGAELARPGASVNLSCKASGYTFTNNGINWLKQRTGQGLEWIGEIYPRSTNTLYNEKF KGKATLTADRSSNTAYMELRSLTSEDSAVYFCARTLTAPFAFWGQGTLVTVSA

MFP5 light chain variable region amino acid sequence (SEQ ID No. 133):

QIVLTQSPAIMSASPGEKVTMTCSASSGVNFMHWYQQKSGTSPKRWIFDTSKLASGVPARFSGSG SGTSYSLTISSMEAEDAATYYCQQWSFNPPTFGGGTKLEIK

1.3ONCOMATRYX BIOPHARMA anti-FAP antibody hu36 developed

Hu36 heavy chain variable region amino acid sequence (SEQ ID No. 134):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYNEK FKDRVTMTADTSTSTVYMELSSLRSEDTAVYYCARHGGTGRGAMDYWGQGTLVTVSS

hu36 light chain variable region amino acid sequence (SEQ ID No. 135):

DIQMTQSPSSLSASVGDRVTITCRASKSVSTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKLEIK

1.4 Michaelson biological self-production Isotype control (abbreviated as SH-NC)

SH-NC heavy chain variable region amino acid sequence (SEQ ID No. 136):

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPIFGSSNYAQKFQ GRVTITADESTSTAYMELSSLRSEDTAVYYCAESPLGGGSGYSVSWFDPWGQGTLVTVSS

SH-NC light chain variable region amino acid sequence (SEQ ID No. 137):

EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSG SGTEFTLTISSLQSEDFAVYYCQQYSNWPPWTFGQGTKVEIK

1.5 protein expression System IgG1 heavy chain constant region G1m3 amino acid sequence of PTT5 vector (SEQ ID No. 138):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

IgG1 heavy chain constant region G1m3 nucleotide sequence of PTT5 vector used by protein expression System (SEQ ID No. 139):

gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa

1.6 protein expression System IgG1 light chain constant region Km3 amino acid sequence of PTT5 vector (SEQ ID No. 140):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

IgG1 light chain constant region Km3 nucleotide sequence of PTT5 vector used by protein expression System (SEQ ID No. 141):

cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatccccgcgaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaaccgcggagagtgt

2. purchasing antigens and validating antigen binding Activity

2.1. Human FAP recombinant protein antigen information

Commercial reagents were purchased as follows:

TABLE 7

Name of the name	Manufacturer' s	Model number
			Human FAP-His proteins	Acro	FAP-H5244
Human FAP-hFc protein	Acro	FAP-H5263
			Murine FAP-His protein	Acro	FAP-M52H3
Monkey FAP-His protein	Acro	FAP-C52H3

2.2. Construction of antigen cell lines of different species:

the amino acid sequence of human FAP protein (hFAP for short) is as follows:

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

MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD

the nucleotide sequence of human FAP protein (hFAP for short) is as follows (SEQ ID No. 143):

atgaagacttgggtaaaaatcgtatttggagttgccacctctgctgtgcttgccttattggtgatgtgcattgtcttacgcccttcaagagttcataactctgaagaaaatacaatgagagcactcacactgaaggatattttaaatggaacattttcttataaaacattttttccaaactggatttcaggacaagaatatcttcatcaatctgcagataacaatatagtactttataatattgaaacaggacaatcatataccattttgagtaatagaaccatgaaaagtgtgaatgcttcaaattacggcttatcacctgatcggcaatttgtatatctagaaagtgattattcaaagctttggagatactcttacacagcaacatattacatctatgaccttagcaatggagaatttgtaagaggaaatgagcttcctcgtccaattcagtatttatgctggtcgcctgttgggagtaaattagcatatgtctatcaaaacaatatctatttgaaacaaagaccaggagatccaccttttcaaataacatttaatggaagagaaaataaaatatttaatggaatcccagactgggtttatgaagaggaaatgcttgctacaaaatatgctctctggtggtctcctaatggaaaatttttggcatatgcggaatttaatgatacggatataccagttattgcctattcctattatggcgatgaacaatatcctagaacaataaatattccatacccaaaggctggagctaagaatcccgttgttcggatatttattatcgataccacttaccctgcgtatgtaggtccccaggaagtgcctgttccagcaatgatagcctcaagtgattattatttcagttggctcacgtgggttactgatgaacgagtatgtttgcagtggctaaaaagagtccagaatgtttcggtcctgtctatatgtgacttcagggaagactggcagacatgggattgtccaaagacccaggagcatatagaagaaagcagaactggatgggctggtggattctttgtttcaacaccagttttcagctatgatgccatttcgtactacaaaatatttagtgacaaggatggctacaaacatattcactatatcaaagacactgtggaaaatgctattcaaattacaagtggcaagtgggaggccataaatatattcagagtaacacaggattcactgttttattctagcaatgaatttgaagaataccctggaagaagaaacatctacagaattagcattggaagctatcctccaagcaagaagtgtgttacttgccatctaaggaaagaaaggtgccaatattacacagcaagtttcagcgactacgccaagtactatgcacttgtctgctacggcccaggcatccccatttccacccttcatgatggacgcactgatcaagaaattaaaatcctggaagaaaacaaggaattggaaaatgctttgaaaaatatccagctgcctaaagaggaaattaagaaacttgaagtagatgaaattactttatggtacaagatgattcttcctcctcaatttgacagatcaaagaagtatcccttgctaattcaagtgtatggtggtccctgcagtcagagtgtaaggtctgtatttgctgttaattggatatcttatcttgcaagtaaggaagggatggtcattgccttggtggatggtcgaggaacagctttccaaggtgacaaactcctctatgcagtgtatcgaaagctgggtgtttatgaagttgaagaccagattacagctgtcagaaaattcatagaaatgggtttcattgatgaaaaaagaatagccatatggggctggtcctatggaggatacgtttcatcactggcccttgcatctggaactggtcttttcaaatgtggtatagcagtggctccagtctccagctgggaatattacgcgtctgtctacacagagagattcatgggtctcccaacaaaggatgataatcttgagcactataagaattcaactgtgatggcaagagcagaatatttcagaaatgtagactatcttctcatccacggaacagcagatgataatgtgcactttcaaaactcagcacagattgctaaagctctggttaatgcacaagtggatttccaggcaatgtggtactctgaccagaaccacggcttatccggcctgtccacgaaccacttatacacccacatgacccacttcctaaagcagtgtttctctttgtcagactaa

The amino acid sequence of the murine FAP protein (mFAP for short) is as follows:

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

MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD

The nucleotide sequence of the murine FAP protein (mFAP for short) is shown in (SEQ ID No. 145)

Atgaagacatggctgaaaactgtctttggagttaccaccctggctgcgcttgctttagtggtgatatgcattgtcttacgtccctcaagagtttacaaacctgaaggaaacacaaagagagctcttaccttgaaggatattttaaatggaacattctcatataaaacatattttcccaactggatttcagaacaagaatatcttcatcaatctgaggatgataacatagtattttataatattgaaacaagagaatcatatatcattttgagtaatagcaccatgaaaagtgtgaatgctacagattatggtttgtcacctgatcggcaatttgtgtatctagaaagtgattattcaaagctctggcgatattcatacacagcgacatactacatctacgaccttcagaatggggaatttgtaagaggatacgagctccctcgtccaattcagtatctatgctggtcgcctgttgggagtaaattagcatatgtatatcaaaacaatatttatttgaaacaaagaccaggagatccaccttttcaaataacttatactggaagagaaaatagaatatttaatggaataccagactgggtttatgaagaggaaatgcttgccacaaaatatgctctttggtggtctccagatggaaaatttttggcatatgtagaatttaatgattcagatataccaattattgcctattcttattatggtgatggacagtatcctagaactataaatattccatatccaaaggctggggctaagaatccggttgttcgtgtttttattgttgacaccacctaccctcaccacgtgggcccaatggaagtgccagttccagaaatgatagcctcaagtgactattatttcagctggctcacatgggtgtccagtgaacgagtatgcttgcagtggctaaaaagagtgcagaatgtctcagtcctgtctatatgtgatttcagggaagactggcatgcatgggaatgtccaaagaaccaggagcatgtagaagaaagcagaacaggatgggctggtggattctttgtttcgacaccagcttttagccaggatgccacttcttactacaaaatatttagcgacaaggatggttacaaacatattcactacatcaaagacactgtggaaaatgctattcaaattacaagtggcaagtgggaggccatatatatattccgcgtaacacaggattcactgttttattctagcaatgaatttgaaggttaccctggaagaagaaacatctacagaattagcattggaaactctcctccgagcaagaagtgtgttacttgccatctaaggaaagaaaggtgccaatattacacagcaagtttcagctacaaagccaagtactatgcactcgtctgctatggccctggcctccccatttccaccctccatgatggccgcacagaccaagaaatacaagtattagaagaaaacaaagaactggaaaattctctgagaaatatccagctgcctaaagtggagattaagaagctcaaagacgggggactgactttctggtacaagatgattctgcctcctcagtttgacagatcaaagaagtaccctttgctaattcaagtgtatggtggtccttgcagccagagtgttaagtctgtgtttgctgttaattggataacttatctcgcaagtaaggaggggatagtcattgccctggtagatggtcggggcactgctttccaaggtgacaaattcctgcatgccgtgtatcgaaaactgggtgtatatgaagttgaggaccagctcacagctgtcagaaaattcatagaaatgggtttcattgatgaagaaagaatagccatatggggctggtcctacggaggttatgtttcatccctggcccttgcatctggaactggtcttttcaaatgtggcatagcagtggctccagtctccagctgggaatattacgcatctatctactcagagagattcatgggcctcccaacaaaggacgacaatctcgaacactataaaaattcaactgtgatggcaagagcagaatatttcagaaatgtagactatcttctcatccacggaacagcagatgataatgtgcactttcagaactcagcacagattgctaaagctttggttaatgcacaagtggatttccaggcgatgtggtactctgaccagaaccatggtatatcatctgggcgctcccagaatcatttatatacccacatgacgcacttcctcaagcaatgcttttctttatcagactga

The amino acid sequence of the cynomolgus monkey FAP protein (cynoFAP for short) is as follows:

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

MKTWVKIVFGVATSAVLALLVMCIVLRPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD

The cynomolgus monkey FAP protein (cynoFAP for short) has the nucleotide sequence as follows (SEQ ID No. 147):

atgaagacttgggtaaaaatcgtatttggagttgccacctctgctgtgcttgccttattggtgatgtgcattgtcttacgccctccaagagttcataactctgaagaaaatacaatgagagcactcacactgaaggatattttaaatgggacattttcttataaaacattttttccaaactggatttcaggacaagaatatcttcatcaatctgcagataacaatatagtactttataatattgaaacaggacaatcatataccattttgagtaacagaaccatgaaaagtgtgaatgcttcaaattatggcttatcacctgatcggcaatttgtatatctagaaagtgattattcaaagctttggagatactcttacacagcaacatattacatctatgaccttagcaatggagaatttgtaagaggaaatgagcttcctcgtccaattcagtatttatgctggtcgcctgttgggagtaaattagcatatgtctatcaaaacaatatctatttgaaacaaagaccaggagatccaccttttcaaataacatttaatggaagagaaaataaaatatttaatggaatcccagactgggtttatgaagaggaaatgcttgctacaaaatatgctctctggtggtctcctaatggaaaatttttggcatatgcggaatttaatgatacagatataccagttattgcctattcctattatggcgatgaacaatatcccagaacaataaatattccatacccaaaggccggagctaagaatccttttgttcggatatttattatcgataccacttaccctgcgtatgtaggtccccaggaagtgcctgttccagcaatgatagcctcaagtgattattatttcagttggctcacgtgggttactgatgaacgagtatgtttgcagtggctaaaaagagtccagaatgtttcggtcttgtctatatgtgatttcagggaagactggcagacatgggattgtccaaagacccaggagcatatagaagaaagcagaactggatgggctggtggattctttgtttcaacaccagttttcagctatgatgccatttcatactacaaaatatttagtgacaaggatggctacaaacatattcactatatcaaagacactgtggaaaatgctattcaaattacaagtggcaagtgggaggccataaatatattcagagtaacacaggattcactgttttattctagcaatgaatttgaagattaccctggaagaagaaacatctacagaattagcattggaagctatcctccaagcaagaagtgtgttacttgccatctaaggaaagaaaggtgccaatattacacagcaagtttcagcgactacgccaagtactatgcacttgtctgctatggcccaggcatccccatttccacccttcatgacggacgcactgatcaagaaattaaaatcctggaagaaaacaaggaattggaaaatgctttgaaaaatatccagctgcctaaagaggaaattaagaaacttgaagtagatgaaattactttatggtacaagatgattcttcctcctcaatttgacagatcaaagaagtatcccttgctaattcaagtgtatggtggtccctgcagtcagagtgtaaggtctgtatttgctgttaattggatatcttatcttgcaagtaaggaagggatggtcattgccttggtggatggtcgaggaacagctttccaaggtgacaaactcctgtatgcagtgtatcgaaagctgggtgtttatgaagttgaagaccagattacagctgtcagaaaattcatagaaatgggtttcattgatgaaaaaagaatagccatatggggctggtcctatggaggatatgtttcatcactggcccttgcatctggaactggtcttttcaaatgtgggatagcagtggctccagtctccagctgggaatattacgcgtctgtctacacagaaagattcatgggtcttcccacaaaggatgataatcttgagcactacaaaaattcaactgtgatggcaagagcagaatatttcagaaatgtagactatcttctcatccacggaacagcagatgataatgtgcactttcaaaactcagcacagattgctaaagctctggttaatgcacaagtggatttccaggcaatgtggtactctgaccagaaccacggcttatccggcctgtccacgaaccacttatacacccacatgacccactttctaaagcagtgtttttctttgtcggactaa

The nucleotide sequence of hFAP is constructed on a pCDNA3.1 vector commercialized by Invitrogen, specifically, the pCDNA3.1 vector is subjected to double digestion by using KpnI and XhoI, the nucleotide sequence of hFAP is inserted, then sequencing is carried out to confirm the sequence information of the vector, after the plasmid is constructed successfully, lipofectamine3000 is used for transfecting human fibrosarcoma HT1080 cells, then puromycin antibiotics are used for screening over-expression monoclonal cells, clones with slightly lower expression level are selected, and a successful cell strain is constructed and named as low-expression HT1080-hFAP cells.

The nucleotide sequence hFAP was constructed on a pCDNA5 vector commercialized by Invitrogen, specifically, the pCDNA5 vector was double digested with NheI and PmeI, the nucleotide sequence hFAP was inserted and then sequenced to confirm the vector sequence information, and after the plasmid construction was successful, lipofectamine3000 was used to transfect human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1, and then Hygromycin antibiotics were used to screen the overexpressed cell lines, and successful cell lines were constructed, designated CHO-hFAP and 293-hFAP cells, respectively.

The nucleotide sequence of cynoFAP is constructed on a pCDNA5 vector commercialized by Invitrogen, specifically, after the pCDNA5 vector is subjected to double digestion by using NheI and PmeI, the nucleotide sequence of cynoFAP is inserted, and then sequencing is carried out to confirm the sequence information of the vector, after the plasmid is constructed successfully, the plasmid is respectively transfected into human embryo kidney HEK293 cells and Chinese hamster ovary cells CHOK1 by using lipofectamine3000, and then an overexpressing cell strain is screened by using Hygromycin antibiotics, and monoclonal overexpressing cell strains CHO-cynoFAP and 293-cynoFAP cells are finally obtained by monoclonalization.

2.3. Construction of cell lines of proteins of the same family

The amino acid sequence of human DPP4 protein (abbreviated as hDPP 4) is as follows (SEQ ID No. 148):

MKTPWKVLLGLLGAAALVTIITVPVVLLNKGTDDATADSRKTYTLTDYLKNTYRLKLYSLRWISDHEYLYKQENNILVFNAEYGNSSVFLENSTFDEFGHSINDYSISPDGQFILLEYNYVKQWRHSYTASYDIYDLNKRQLITEERIPNNTQWVTWSPVGHKLAYVWNNDIYVKIEPNLPSYRITWTGKEDIIYNGITDWVYEEEVFSAYSALWWSPNGTFLAYAQFNDTEVPLIEYSFYSDESLQYPKTVRVPYPKAGAVNPTVKFFVVNTDSLSSVTNATSIQITAPASMLIGDHYLCDVTWATQERISLQWLRRIQNYSVMDICDYDESSGRWNCLVARQHIEMSTTGWVGRFRPSEPHFTLDGNSFYKIISNEEGYRHICYFQIDKKDCTFITKGTWEVIGIEALTSDYLYYISNEYKGMPGGRNLYKIQLSDYTKVTCLSCELNPERCQYYSVSFSKEAKYYQLRCSGPGLPLYTLHSSVNDKGLRVLEDNSALDKMLQNVQMPSKKLDFIILNETKFWYQMILPPHFDKSKKYPLLLDVYAGPCSQKADTVFRLNWATYLASTENIIVASFDGRGSGYQGDKIMHAINRRLGTFEVEDQIEAARQFSKMGFVDNKRIAIWGWSYGGYVTSMVLGSGSGVFKCGIAVAPVSRWEYYDSVYTERYMGLPTPEDNLDHYRNSTVMSRAENFKQVEYLLIHGTADDNVHFQQSAQISKALVDVGVDFQAMWYTDEDHGIASSTAHQHIYTHMSHFIKQCFSLP

the nucleotide sequence of the human DPP4 protein is as follows (SEQ ID No. 149):

atgaagacaccgtggaaggttcttctgggactgctgggtgctgctgcgcttgtcaccatcatcaccgtgcccgtggttctgctgaacaaaggcacagatgatgctacagctgacagtcgcaaaacttacactctaactgattacttaaaaaatacttatagactgaagttatactccttaagatggatttcagatcatgaatatctctacaaacaagaaaataatatcttggtattcaatgctgaatatggaaacagctcagttttcttggagaacagtacatttgatgagtttggacattctatcaatgattattcaatatctcctgatgggcagtttattctcttagaatacaactacgtgaagcaatggaggcattcctacacagcttcatatgacatttatgatttaaataaaaggcagctgattacagaagagaggattccaaacaacacacagtgggtcacatggtcaccagtgggtcataaattggcatatgtttggaacaatgacatttatgttaaaattgaaccaaatttaccaagttacagaatcacatggacggggaaagaagatataatatataatggaataactgactgggtttatgaagaggaagtcttcagtgcctactctgctctgtggtggtctccaaacggcacttttttagcatatgcccaatttaacgacacagaagtcccacttattgaatactccttctactctgatgagtcactgcagtacccaaagactgtacgggttccatatccaaaggcaggagctgtgaatccaactgtaaagttctttgttgtaaatacagactctctcagctcagtcaccaatgcaacttccatacaaatcactgctcctgcttctatgttgataggggatcactacttgtgtgatgtgacatgggcaacacaagaaagaatttctttgcagtggctcaggaggattcagaactattcggtcatggatatttgtgactatgatgaatccagtggaagatggaactgcttagtggcacggcaacacattgaaatgagtactactggctgggttggaagatttaggccttcagaacctcattttacccttgatggtaatagcttctacaagatcatcagcaatgaagaaggttacagacacatttgctatttccaaatagataaaaaagactgcacatttattacaaaaggcacctgggaagtcatcgggatagaagctctaaccagtgattatctatactacattagtaatgaatataaaggaatgccaggaggaaggaatctttataaaatccaacttagtgactatacaaaagtgacatgcctcagttgtgagctgaatccggaaaggtgtcagtactattctgtgtcattcagtaaagaggcgaagtattatcagctgagatgttccggtcctggtctgcccctctatactctacacagcagcgtgaatgataaagggctgagagtcctggaagacaattcagctttggataaaatgctgcagaatgtccagatgccctccaaaaaactggacttcattattttgaatgaaacaaaattttggtatcagatgatcttgcctcctcattttgataaatccaagaaatatcctctactattagatgtgtatgcaggcccatgtagtcaaaaagcagacactgtcttcagactgaactgggccacttaccttgcaagcacagaaaacattatagtagctagctttgatggcagaggaagtggttaccaaggagataagatcatgcatgcaatcaacagaagactgggaacatttgaagttgaagatcaaattgaagcagccagacaattttcaaaaatgggatttgtggacaacaaacgaattgcaatttggggctggtcatatggagggtacgtaacctcaatggtcctgggatcgggaagtggcgtgttcaagtgtggaatagccgtggcgcctgtatcccggtgggagtactatgactcagtgtacacagaacgttacatgggtctcccaactccagaagacaaccttgaccattacagaaattcaacagtcatgagcagagctgaaaattttaaacaagttgagtacctccttattcatggaacagcagatgataacgttcactttcagcagtcagctcagatctccaaagccctggtcgatgttggagtggatttccaggcaatgtggtatactgatgaagaccatggaatagctagcagcacagcacaccaacatatatatacccacatgagccacttcataaaacaatgtttctctttaccttag

the nucleotide sequence of the hDPP4 is constructed on a pCDNA5 vector commercialized by Invitrogen, specifically, the pCDNA5 vector is subjected to double digestion by using NheI and PmeI, the nucleotide sequence of the hDPP4 is inserted and then sequenced to confirm the sequence information of the vector, after the plasmid is constructed successfully, the plasmid is transfected into Chinese hamster ovary cells CHOK1 by using lipofectamine3000, and then an overexpressed cell strain is screened by using Hygromycin antibiotics, and finally a monoclonal overexpressed cell strain CHO-hDPP4 is obtained by monoclonalization.

3. Purchase of antigen cell lines

Murine FAP engineered cell line:

CT26-mouse-Fap-Cell-Line Kang Yuanbo, product number of Biotechnology (Beijing) Co., ltd., KC-1284, hereinafter referred to as CT26-mFAP Cell Line

Natural tumor cells expressing human FAP:

u-138MG cell was purchased from ATCC, cat# HTB-16

The recombinant anti-FAP antibody and the raw materials and reagents used in the application of the recombinant anti-FAP antibody provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples:

Test examples

Facs determination of affinity of antigen cells to tool antibodies

1. Experimental reagents and materials:

TABLE 8

2. The experimental procedure is as follows:

1) Cell collection and plating

A) Harvesting cells in logarithmic growth phase to ensure cell viability above 90%.

B) Centrifuging at 1000r/min for 5min, and discarding supernatant;

c) Cells were washed once with PBS;

d) Cells were resuspended using FACS Buffer (i.e., 1% bsa, dpbs) and counted;

e) Cell suspensions with a density of 2X 10 ⁶ cells/mL were prepared using FACS Buffer;

f) 50. Mu.L of the cell suspension was added to each 96-well plate;

2) Antibody incubation and detection

A) Adding 50 mu L of test samples with different concentrations into the experimental group, and setting 3-fold dilution multiple gradient points at the beginning of the sample concentration of 20 mu g/ml;

b) After mixing, placing the mixture at a temperature of 4 ℃ and incubating overnight in a dark place;

c) Cells were washed 1 time with FACS Buffer, centrifuged at 200. Mu.L each time at 1000r/min for 5min, and the supernatant was discarded;

d) APC-labeled secondary antibodies (1:1500-fold dilution) were added to 96-well plates, and the blank group was added to an equal volume of FACS Buffer;

e) After being evenly mixed, the mixture is placed at 4 ℃ and incubated for 40min in dark;

f) Cells were washed 1 time with FACS Buffer, centrifuged at 200. Mu.L each time at 1000r/min for 5min, and finally resuspended with 100. Mu.LFACS Buffer;

g) RL-1MFI readings (accounting Laser: 640: 640nmBlue Laser) were measured with INTELLICYTE PLUS flow cytometer.

3) Data processing

FACS data were analyzed with Prism software.

Analysis of the data using the over-expressed cell line CHO-hFAP cells and the naturally expressed cell line U138MG cells, the in vitro cell biological binding FACS test showed that the three control antibodies had binding signals to both cell lines, with the BIBH and cMFP5 binding signals being higher and the hu36 binding signal being slightly lower, as shown in fig. 1a, 1 b:

TABLE 9 EC50 and fluorescence values for control antibody flow binding experiments

Preparation example

1. Generation of rabbit anti-human FAP monoclonal antibody (this working line entrusted to development of Shandor Kadsura Biotech Co., ltd.)

1) Immunization of animals

In order to obtain the rabbit monoclonal antibody which can recognize the human FAP antigen, the invention selects and immunizes New Zealand white rabbits. The first immunization was performed using 200. Mu.g of human FAP-hFc protein (purchased from Acro Biosystems under the trade designation FAP-H5263) and was performed by mixing Freund's complete adjuvant with an equal volume of antigen for emulsification at the time of priming and back multipoint injection immunization. Two weeks apart, booster immunization was performed with antigen dose of 100 μg, and Freund's incomplete adjuvant and equal volume of antigen were mixed and emulsified for back multipoint injection immunization. The immunization was boosted 1 time every two weeks, and after five times of immunization, the immune serum titers were detected by the Elisa conventional method. Rabbits with high titers were selected and were immunized by one impact with 50 μg of protein by intraperitoneal injection 3 days prior to antibody screening, the antigen being emulsified without adjuvant, and the buffer being PBS. Spleen was taken three days later.

2) Spleen cell separation

Spleens from rabbits were surgically removed and placed in sterile cell culture dishes and the spleens were rinsed with DPBS containing 100U/ml penicillin and 100. Mu.g/ml streptomycin. The spleen was minced with surgical scissors, gently ground into single cells with a syringe core, and finally the cell suspension was filtered with a 100 μm cell sieve, and the single cell filtrate was collected. Centrifugation at 1200rpm for 3min, the supernatant was discarded and the cells were resuspended in RPMI-1640 containing 5% fetal bovine serum.

3) B cell culture and identification

And incubating the FAP protein marked by biotin with successfully immunized rabbit lymphocytes, and sorting to obtain the memory B cells of the rabbits. The culture was performed in 96-well cell culture plates using B cell medium at 37 ℃ and 5% co 2. After 10-14 days of culture, the binding activity of the clone supernatant was detected at the protein level by ELISA, and clones 5-fold or more above background were judged to be positive. ELISA positive supernatants were tested for binding to stable cells CHO-hFAP and CHO-mFAP by FACS method and FACS positive monoclonal was finally selected.

4) Cloning of genes encoding Rabbit monoclonal antibodies

B cell positive clones are collected, part of positive clones are selected, total RNA is extracted by RNAiso Plus and is reversely transcribed into cDNA, the light chain variable region and heavy chain variable region sequences are amplified by a PCR method, and the sequences are constructed on a PTT5 expression vector containing corresponding heavy chain constant region and light chain constant region for sequencing, so that the correct sequences are obtained. The sequencing result is analyzed by VBASE2 (http:// www.vbase2.org/vbscAb. Php) to obtain the light and heavy chain variable region sequence of the antibody.

2. Preparation of chimeric anti-human FAP antibodies

Splicing together the variable region sequence of the heavy chain of the rabbit anti-human FAP monoclonal antibody and the published variable region sequence of the heavy chain of the human monoclonal antibody IgG1 subclass to construct into a mammalian cell expression vector, and splicing together the variable region sequence of the light chain of the rabbit anti-human FAP monoclonal antibody and the published variable region sequence of the light chain of the human monoclonal antibody kappa subclass to construct into a mammalian cell expression vector. The constructed heavy chain vector and light chain vector of the anti-human FAP chimeric antibody are paired and mixed, HEK293 cells are transfected by Polyethyleneimine (PEI), cell supernatants are collected after about 7 days, and the anti-human FAP chimeric antibody protein is obtained by purifying by using MabSelect.

Example 1 in vitro cell binding assay for chimeric antibodies against human FAP

The anti-human FAP chimeric antibody was diluted 4-fold in gradient ratio from the initial concentration of 20. Mu.g/mL, and the total concentration was 8, and 50. Mu.L of the antibody at each concentration was added to a 96-well plate. HT1080-hFAP cells with low expression of human FAP on the cell surface were collected by centrifugation at 100g for 5 min, washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 min, resuspended at a density of about 2X 10 ⁶ cells per ml, and 50. Mu.L was added to wells of a 96-well plate to which antibodies were added. After incubation at 4℃for 1 hour, an APC fluorescent-labeled goat anti-human IgG secondary antibody was added. After further incubation at 4 degrees for 1 hour, the average fluorescence readings of the cell populations were analyzed by flow cytometry and 4-parameter fitting curves were performed using prism software, as shown in fig. 2a, 2b.

Results analysis several chimeric antibodies were obtained that were superior to the control in both EC50 and top value of fluorescence MFI compared to control hu36, through in vitro cell biological binding assays performed with HT1080-hFAP cells expressing human FAP.

Table 10

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

Example 2 in vitro binding affinity and kinetic experiments of chimeric anti-human FAP antibodies

Antibody affinity was determined using an anti-human antibody capture method using a Fortebio (BLITZ pro 1.1.0.28) instrument. In the measurement, a capture Antibody (AHC) biological probe (purchased from SARTORIUS, cat# 18-5060) of the Fc fragment of an anti-human antibody was immersed in PBS for 10min, 200. Mu.l of diluted antibody sample (comprising the chimeric antibody of the present invention and a control antibody; working concentration of the antibody is 15. Mu.g/mL) was loaded on the AHC biological probe, and then equilibrated in PBS for 100s, and further the AHC probe was subjected to a binding reaction with human FAP protein and murine FAP protein (purchased from ACRO biosystem) for 600s, and then the AHC probe was transferred into PBS for a dissociation reaction for 600s. After the experiment, the blank control response value is deducted, and a 1:1Langmuir binding mode fitting is performed by software to calculate the kinetic constant of antigen-antibody binding.

The results analysis shows that the binding kinetics constant of the chimeric antibody and the recombinant human FAP protein is 10 ^-10 -10 ^-12, the binding kinetics constant of the target antibody hu36 is 10 ^-9, and the binding kinetics constant of the chimeric antibody and the recombinant mouse FAP protein is 10 ^-9 -10 ^-12, and the binding kinetics constant of the target antibody hu36 is 10 ^-9. Each chimeric antibody had a better kinetic binding signal to both hFAP and mFAP.

Table 11 in vitro kinetic binding Activity of chimeric antibodies and recombinant human FAP proteins

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

Table 12 in vitro kinetic binding Activity of chimeric antibodies and recombinant murine FAP proteins

EXAMPLE 3 endocytosis assay of chimeric anti-human FAP antibodies

Firstly, HT1080-hFAP cells are prepared, a pancreatiless digestive solution (brand GIBCO, product number 13151014) is used for digestion counting, a cell culture medium (brand GIBCO, product number 22400097) is used for resuspension, the cell density is adjusted to 2E6/mL, an endocytic reagent (purchased from Sartorius, product number 90565) is reconstituted with sterile water to a final concentration of 100 mug/mL, the molecular weight of the endocytic reagent is about 1/3 of the molecular weight of the antibody, so that the mass ratio of the reagent antibody is 3:1 when the reagent antibody is incubated, the mass ratio of the antibody and the endocytic reagent is 1:1 when the reagent antibody is incubated, the cell culture medium (brand GIBCO, product number 22400097) is used for diluting the antibody to 2 mug/mL, the labeled reagent is incubated in a 37 ℃ incubator for 15 minutes, the labeled antibody reagent marker is diluted by a gradient of 7 times, the diluted antibody reagent marker is added into the prepared cells, the prepared cells are placed in the 37 ℃ incubator for endocytosis for two hours, the culture plate is taken out, and a flow meter is directly used for reading the sample and the fluorescent curve is analyzed by a curve fitting mode shown in figure 35 b and 3% and 35 b.

Analysis of the data the endocytosis of anti-human FAP chimeric antibodies was tested using the low expressing cell line HT1080-hFAP cells, and from the results, the chimeric antibodies chrX1, chrX6, chrX9 and chrA, chrA12 all underwent endocytosis faster, comparable or better than the control antibody hu 36.

TABLE 13 statistical tables of EC50 of anti-human FAP chimeric antibody endocytosis experiments

Antibody name	EC50
		chrX1	0.04774
chrX6	0.0633
		chrX8	0.3673
chrX9	0.06337
		chrA7	0.07154
chrA12	0.08361
		chrA18	0.2441
hu36	0.1405

EXAMPLE 4 humanization of anti-human FAP Rabbit antibody

The amino acid sequence regions of the 6 Complementarity Determining Regions (CDRs) of the heavy and light chains of the rabbit antibody were determined by combining the antibody coding schemes of Kabat, chothia, and the framework regions supporting the conserved three-dimensional conformation of the antibody (frameworkregion). Then, by analyzing and searching the known human antibody sequence, the human antibody heavy chain variable region sequence which is most similar to the rabbit antibody is selected, such as IGHV 1|IGHJ4.times.01, the antibody framework region sequence is selected as a template, and the rabbit antibody heavy chain CDR is combined with the human antibody framework region, so that the humanized antibody heavy chain variable region sequence is finally generated. The same procedure was followed to generate humanized antibody light chain variable region sequences. Antibodies in which CDRs of a rabbit antibody are directly grafted to human framework regions often exhibit a dramatic decrease in binding activity, thus requiring the replacement of individual amino acids of the framework regions from human to rabbit. The site of the back mutation is determined, firstly by checking which amino acids differ against the designed humanized antibody sequence and the original rabbit antibody sequence, and secondly by checking whether these amino acids play an important role in supporting the antibody structure or in binding to the antigen. The sequence after the design is humanized while checking for potential post-translational modification sites such as N (asparagine) glycosylation sites, N deamidation sites, D (aspartic acid) isomerization sites, etc.

The humanized antibody variable region heavy chain gene is constructed into a mammalian cell expression vector containing the heavy chain constant region gene of the human monoclonal antibody IgG1 subclass, and the light chain gene is constructed into a mammalian cell expression vector containing the light chain constant region gene of the human monoclonal antibody kappa subclass. And (3) pairing and mixing the constructed heavy chain vector and the light chain vector of the anti-human FAP humanized antibody, transfecting HEK293 cells by using Polyethyleneimine (PEI), collecting cell supernatants after about 7 days, and purifying by using MabSelect to obtain the anti-human FAP humanized antibody protein.

Example 5 in vitro binding affinity and kinetic experiments of humanized antibodies against human FAP

Antibody affinity was determined using an anti-human antibody capture method using a Fortebio (BLITZ pro 1.1.0.28) instrument. In the measurement, a capture Antibody (AHC) biological probe (purchased from SARTORIUS, cat# 18-5060) of the Fc fragment of an anti-human antibody is soaked in PBS for 10min, 200 μl of diluted antibody sample (comprising the chimeric antibody and a control antibody of the invention; working concentration of the antibody is 15 μg/mL) is loaded on the AHC biological probe, and then the mixture is equilibrated in PBS for 100s, and the AHC probe is further subjected to a binding reaction with a series of gradients (purchased from ACRO biosystem, cat# FAP-M52H 3) of human FAP protein, monkey FAP protein and mouse FAP protein for 600s, and then the AHC probe is transferred into PBS for dissociation reaction. After the experiment, the blank control response value is deducted, a 1:1Langmuir binding mode fitting is carried out by software, the kinetic constant of antigen-antibody binding is calculated, and the curve treatment and fitting are carried out by using the self-contained software of the instrument.

And (3) data analysis, namely, through in-vitro dynamic binding activity analysis, the binding dynamic constants of the anti-human FAP humanized antibody, the recombinant human FAP protein, the recombinant mouse FAP protein and the recombinant monkey FAP protein are all kept at 10 ^-12 level, and compared with the binding dynamic constants of the target antibody hu36 and the human, mouse and monkey FAP proteins are kept at 10 ^-10 -10 ^-12 level. Each humanized antibody has a better kinetic binding signal.

TABLE 14 in vitro kinetic binding data for humanized antibodies and recombinant human FAP proteins

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

TABLE 15 in vitro kinetic binding data for humanized antibodies and recombinant monkey FAP proteins

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

TABLE 16 in vitro kinetic binding data for humanized antibodies and recombinant murine FAP proteins

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

Example 6 in vitro cell binding experiments with humanized antibodies against human FAP

The humanized antibody against human FAP was diluted 4-fold in gradient ratio from an initial concentration of 20. Mu.g/mL, for a total of 8 concentration spots, and 50. Mu.L of the antibody at each concentration spot was added to a 96-well plate. 100g of cells were collected by centrifugation at room temperature for 5 minutes, HT1080-hFAP cells expressing human FAP at low cell surface, 293-cynoFAP cells expressing monkey FAP at high level and CT26-mFAP cells overexpressing mouse FAP were washed once with PBS containing 0.5% BSA, centrifuged at room temperature for 5 minutes at 100g, resuspended cells at a density of about 2X106 cells per ml, and 50. Mu.L were added to wells of 96-well plates to which antibodies were added. After incubation at 4℃for 1 hour, an APC fluorescent-labeled goat anti-human IgG secondary antibody was added. After further incubation at 4 degrees for 1 hour, the average fluorescence readings of the cell populations were analyzed using a flow cytometer, 4-parameter fitting curves were performed using prism software and EC50 and top values of fluorescence intensity were calculated as shown in fig. 4a, 4b, and 4 c.

Analysis of data humanized antibodies were tested for cell binding in vitro with cells HT1080-hFAP and CT26-mFAP, which are cells under hFAP and cells CT26-mFAP, which are cells over-expressed mFAP, respectively, and CHO-cynoFAP, which are cells over-expressed cynoFAP protein. The results show that the humanized molecules of each light and heavy chain combination can mostly maintain the binding capacity with HT1080-hFAP cells, CT26-mFAP cells and CHO-cynoFAP cells, and the binding signal is not lower than the signal value of the high concentration of the antibody hu 36.

Table 17 Top values of in vitro flow binding EC50 and fluorescence intensity of humanized antibodies to HT1080-hFAP, 293-cynoFAP cells and CT26-mFAP cells

Example 7 in vitro cell non-specific binding assay of anti-human FAP humanized antibodies

Anti-human FAP affinity maturation combinatorial antibodies were diluted 4-fold at 20. Mu.g/mL in 4 gradients and 50. Mu.L of diluted antibodies were added to 96-well plates. The cells were collected by centrifugation at 100g at room temperature for 5 minutes, the CHO-hDPP4 cells over-expressing human DPP4 on the cell surface, chinese hamster ovary cells CHOK1, human embryonic kidney cells HEK293, washed once with PBS containing 0.5% bsa, centrifuged at 100g at room temperature for 5 minutes, resuspended at a density of about 2x106 cells per ml, and 50 μl was added to wells of a 96-well plate to which antibodies were added. After incubation at 4℃for 1 hour, an APC fluorescent-labeled goat anti-human IgG secondary antibody was added. After further incubation at 4 degrees for 1 hour, the average fluorescent readings of the cell populations were analyzed by flow cytometry and shown in bar graphs using prism software as shown in fig. 5a, 5b, 5c.

The data analysis shows that the non-specific binding experiment of the anti-human FAP humanized antibody and cell strain CHO-hDPP4 expressing the same family protein DPP4 and cell strain Chinese hamster ovary cell CHOK1 and human embryo kidney cell HEK293 which are commonly used for expressing the protein is carried out, and the result shows that each anti-human FAP humanized antibody molecule has no non-specific binding signal with the same family protein and the empty cell for expression.

TABLE 18

Example 8 detection of endocytosis Activity of anti-human FAP humanized antibody molecules

Firstly, HT1080-hFAP cells are prepared, a pancreatiless digestive solution (brand GIBCO, product number 13151014) is used for digestion counting, a cell culture medium (brand GIBCO, product number 22400097) is used for resuspension, cell density is adjusted to 2E6/mL, an endocytic reagent (purchased from Sartorius, product number 90565) is reconstituted with sterile water to a final concentration of 100 mug/mL, the molecular weight of the endocytic reagent is about 1/3 of the molecular weight of an antibody, so that the mass ratio of the reagent antibody is 3:1 when the reagent antibody is labeled, the antibody and the endocytic reagent are diluted to 2 mug/mL according to the mass ratio of 1:1 by using the cell culture medium, the labeled antibody is incubated in a 37 ℃ incubator for 15 minutes, the labeled antibody reagent marker is diluted by a gradient of 7 times, the diluted antibody reagent marker is added into the prepared cells, the prepared cells are placed in the incubator for two hours at 37 ℃, a culture plate is taken out, a flow analyzer is directly used for analyzing RL-1 channel, fluorescence and the mass ratio of the endocytosis reagent is read, and the mass ratio of the endocytosis reagent is embezzle% is calculated by a curve fitting mode shown in a graph 6.

The results analysis shows that the endocytosis activity of the anti-human FAP humanized antibodies is tested by using the HT1080-hFAP cell low expression cell line, and according to the results, each humanized antibody can be endocytosed faster.

Example 9 in vitro killing experiments with anti-human FAP humanized antibodies

In vitro killing experiments use two low expression cell strains HT1080-hFAP and 293-hFAP, cell digestion counts, use corresponding culture media (HT 1080-hFAP cells use RPMI1640 culture media, 293-hFAP cells use DMEM culture media) to adjust cell density to 5E4/mL,50 μl/well was plated in 96 Kong Baiban, a small molecule toxin coupling reagent 20ADC alpha HFc-CL-MMAE (purchased from moradec, cat# AH-102 AE-50) was diluted to 2 μg/mL using cell corresponding culture media, the diluted small molecule toxin coupling reagent was used to dilute humanized antibody to 0.4 μg/mL, then a 3-fold dilution was set to 10 gradients, the diluted serial antibodies 50 μl/well were added to a white plate plated with cells, placed in a 37 ℃ incubator for 4 days, titer-Glo (purchased from promega, cat# G7573) was prepared according to the reagent instructions, 100 μl/well was added to the cell plate, a shaking instrument was used for 5 minutes, and the light emitting curve was fitted with a four-dimensional graph, such as that of 7-7 sm curve graph was read.

The data analysis shows that each anti-human FAP humanized antibody molecule has in-vitro cell killing activity by using low expression cell strains HT1080-hFAP and over expression cell strains 293-hFAP, wherein the killing activity of H29L12, H32L12 and H33L12 is better and similar to that of a control antibody hu 36.

Table 19 anti-human FAP humanized antibody in vitro killing experimental data

Example 10 physical characterization of anti-human FAP humanized antibody molecules Single body Rate analysis

Experimental instrument UPLC CLASS ACQUITYH (WATERS)

Analytical column TSKgel G3000SWXL 7.8 x 300 (TOSHI, catNo 003C 03326C)

Analytical solution 200mM K2HPO4,250Mm KCl, pH was adjusted to 6.2 using HCl

The analysis method comprises the steps of injecting 50 μl of antibody with concentration of 1mg/ml into a pre-equilibrated chromatographic column, flowing at room temperature for 45min at a flow rate of 0.75ml/min, detecting the absorption value of machine A280, and judging the monomer content and proportion of the antibody according to the peak-out time and the peak-out volume.

The result analysis shows that the monomer ratio of the anti-human FAP humanized antibody molecule is over 95 percent, and the monomer ratio has good performance.

TABLE 20 main peak retention time and monomer Rate of antibodies

EXAMPLE 11 humanized antibody hydrophobic Property analysis

Experimental apparatus ARC (Waters)

Analytical column for experiments TSKgel Butyl-NPR (4.6mmX3.5cm,CatNo 14947)

The analytical solution A.20mM HISTIDINE, pH6.0;

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

Analytical methods the hydrophobic properties of antibodies were analyzed according to the instructions of the hydrophobic chromatography column.

The results of the analysis of the hydrophobicity of the humanized antibody molecules against human FAP show that the hydrophobicity HIC value of each combined molecule is more than 0.7, and the hydrophobicity is good.

TABLE 21 hydrophobic Properties of humanized antibodies

Antibodies to	Retention time of main peak (min)	HIC
			Hu36	13.579	1.08
hzX6-H29L3	13.409	1.09
			hzX6-H29L10	13.358	1.10
hzX6-H32L10	13.362	1.10
			hzX6-H33L10	13.368	1.10

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A recombinant anti-FAP antibody, characterized in that it comprises a heavy chain and a light chain:

The CDR region of the heavy chain includes CDR1, CDR2 and CDR3:

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

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

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

(IV) a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence as shown in any one of (I) to (III); or

(V) a sequence having an amino acid sequence homology of more than 80% with any one of (I) to (IV);

The CDR region of the light chain includes CDR1, CDR2 and CDR3:

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

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

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

(IX) a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence of any one of (VI) to (VIII); or

(X) A sequence having 80% or more homology with the amino acid sequence shown in any one of (VI) to (IX).

2. The recombinant anti-FAP antibody according to claim 1, characterized in that the recombinant anti-FAP antibody comprises a rabbit chimeric antibody and a humanized antibody;

Optionally, the rabbit-derived chimeric antibody comprises chrX1, chrX6, chrX8, chrX9, chrA7, chrA12 or chrA18;

a), CDR1, CDR2 and CDR3 of the heavy chain of chrX1 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 of the chrX1 have the amino acid sequences shown in SEQ ID No.9, 11 and 13 respectively; or

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

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

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

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

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

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

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

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

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

The CDR1, CDR2 and CDR3 of the light chain of chrA12 have SEQ ID Nos. 62, 63 and 65, respectively; or

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

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

h) a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence as shown in any one of a) to g); or

j) A sequence having a homology of more than 80% with the amino acid sequence shown in any one of a) to h).

3. The recombinant anti-FAP antibody of claim 2, wherein the humanized antibody comprises hzX6-H29L3, hzX6-H29L10, hzX6-H29L12, hzX6-H32L3, hzX6-H32L10, hzX6-H32L12, hzX6-H33L3, hzX6-H33L10 or hzX6-H33L12;

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

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

1), the CDR1, CDR2 and CDR3 of the heavy chain of hzX6-H29L10 have the amino acid sequences shown in SEQ ID No.107, 18 and 108 respectively; and

The CDR1, CDR2 and CDR3 of the light chain of hzX6-H29L10 have the amino acid sequences shown in SEQ ID Nos. 112, 11 and 116, respectively; or

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

The CDR1, CDR2 and CDR3 of the light chain of hzX6-H29L12 have the amino acid sequences shown in SEQ ID Nos. 112, 11 and 117, respectively; or

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

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

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

The CDR1, CDR2 and CDR3 of the light chain of hzX6-H32L10 have the amino acid sequences shown in SEQ ID Nos. 112, 11 and 116, respectively; or

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

The CDR1, CDR2 and CDR3 of the light chain of hzX6-H32L12 have the amino acid sequences shown in SEQ ID Nos. 112, 11 and 117, respectively; or

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

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

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

The CDR1, CDR2 and CDR3 of the light chain of hzX6-H33L10 have the amino acid sequences shown in SEQ ID Nos. 112, 11 and 116, respectively; or

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

The CDR1, CDR2 and CDR3 of the light chain of hzX6-H33L12 have the amino acid sequences shown in SEQ ID Nos. 112, 11 and 117, respectively; or

t), a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in any one of k) to s); or

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

4. The recombinant anti-FAP antibody according to any one of claims 1 to 3, characterized in that

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

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

or

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

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

or

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

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

or

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

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

or

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

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

or

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

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

or

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

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

or

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

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

or

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

The variable region of the light chain has the amino acid sequence shown in SEQ ID No.122;

or

x), the variable region of the heavy chain has the amino acid sequence shown in SEQ ID No.120; and

The variable region of the light chain has the amino acid sequence shown in SEQ ID No.123;

xi), a sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence as shown in any one of i) to x); or

xii) A sequence having at least 80% homology with the amino acid sequence shown in any one of i) to xi).

5. The recombinant anti-FAP antibody according to any one of claims 2 to 4, wherein the rabbit chimeric antibody comprises:

1) The variable region of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No. 78; and

The variable region of the light chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No.79;

or

2) The variable region of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No. 80; and

The variable region of the light chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No.81;

or

3) The variable region of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No.82; and

The variable region of the light chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No.83;

or

4) The variable region of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No. 84; and

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

or

5) The variable region of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No. 86; and

The variable region of the light chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No.87;

or

6) The variable region of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No. 88; and

The variable region of the light chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No.89;

or

7) The variable region of the heavy chain of the rabbit chimeric antibody has the amino acid sequence shown in SEQ ID No. 90; and

The variable region of the light chain of the rabbit chimeric antibody has an amino acid sequence as shown in SEQ ID No.91;

or

8) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in any one of 1) to 7); or

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

6. The recombinant anti-FAP antibody according to any one of claims 2 to 5, wherein the humanized antibody:

1) The variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in 118; and

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

or

2) The variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in 119; and

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

or

3) The variable region of the heavy chain of the humanized antibody has the amino acid sequence shown in 120; and

The variable region of the light chain of the humanized antibody has the amino acid sequence shown in 123;

or

4) A sequence in which one or more amino acids are substituted, deleted, added and/or replaced based on the amino acid sequence shown in any one of 1) to 3); or

5) A sequence having a homology of 80% or more to the amino acid sequence shown in any one of 1) to 4).

7. The recombinant anti-FAP antibody according to any one of claims 1 to 6, further comprising a constant region;

The heavy chain constant region of the recombinant anti-FAP antibody includes human IgG1; the light chain constant region of the recombinant anti-FAP antibody includes human κ type;

Optionally, the FAP comprises human FAP, mouse 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 comprises the following steps:

Step 1: taking FAP antigen immune receptors, isolating spleen cells of the receptors, and amplifying by PCR to obtain the light and heavy chain variable regions of the recombinant anti-FAP antibody;

Step 2: splicing the heavy chain variable region and the heavy chain constant region, constructing them into an expression vector, and obtaining a heavy chain vector; splicing the light chain variable region and the light chain constant region, constructing them into the expression vector, and obtaining a light chain vector;

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

9. The preparation method according to claim 8, characterized in that the recipient comprises New Zealand white rabbits;

The heavy chain constant region is a heavy chain constant region of human IgG1 subclass;

The light chain constant region is a light chain constant region of a human κ antibody;

The constructed expression vector is a mammalian cell expression vector.

10. Biomaterial, characterized in that it comprises any of the following:

(a) a nucleic acid encoding the recombinant anti-FAP antibody according to any one of claims 1 to 7 or the recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9; and/or

(b), a nucleic acid encoding the recombinant anti-FAP antibody according to any one of claims 1 to 7 and an acceptable vector; or

A recombinant anti-FAP antibody prepared by the preparation method of claim 8 or 9 and an acceptable carrier; and/or

(c) a host that secretes the recombinant anti-FAP antibody according to any one of claims 1 to 7 or the recombinant anti-FAP antibody produced by the preparation method according to claim 8 or 9; and/or

(d) a chemically labeled or biologically labeled recombinant anti-FAP antibody according to any one of claims 1 to 7 or a recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9; and/or

(e) a recombinant anti-FAP antibody according to any one of claims 1 to 7, or a recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9, coupled to a carrier;

(f) An antibody-drug conjugate obtained by covalently coupling a drug with the recombinant anti-FAP antibody according to any one of claims 1 to 7 or the recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9 through a linker.

11. Use of any of the following in the preparation of a drug targeting FAP:

①. The recombinant anti-FAP antibody according to any one of claims 1 to 7; and/or

②. The recombinant anti-FAP antibody prepared by the preparation method according to claim 8 or 9 and/or

③. The biomaterial as described in claim 10.

12. Use of any of the following in the preparation of products for preventing and/or treating diseases:

③. The biomaterial as described in claim 10.

13. The use according to claim 12, characterized in that the disease comprises any one or more of human epithelial cancer, breast cancer, pancreatic cancer, lung cancer, bladder cancer or colon cancer.

14. The use according to claim 12 or 13, characterized in that the product comprises a medicine and/or a vaccine.

15. A drug, characterized in that it comprises any of the following items and pharmaceutically acceptable excipients:

③. The biomaterial as described in claim 10.

16. A drug combination, characterized in that it comprises the drug according to claim 15 and any other active ingredients.

17. The pharmaceutical combination of claim 16, wherein the other arbitrary active ingredients include small molecule toxins.