WO2021129605A1 - Antibody against chemokine cx3cl1 and application thereof - Google Patents

Abstract

The present invention provides a humanized antibody against CX3CL1 or a fragment thereof, a coding nucleic acid of the antibody or a fragment thereof, a composition comprising the antibody or a fragment thereof, and a use thereof in treating a disease. The antibody or a fragment thereof provided by the present invention has a high affinity to CX3CL1, specifically blocks CX3CL1/CX3CR1 signaling pathway, and blocks the migration of CX3CR1-expressing cells.

Description

Antibodies against chemokine CX3CL1 and their applications

This patent application claims the priority rights of the Chinese invention patent application with the application number CN201911338538.2 filed on December 23, 2019, and the entire content of which is hereby incorporated by reference.

Technical field

The present invention relates to the field of biomedicine. Specifically, the present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to the chemokine CX3C subtype CX3CL1, and applications of the antibodies and antigen-binding fragments thereof.

Background technique

Chemokines are a class of functionally related small molecule secreted proteins. They are named "chemokines" because of their leukocyte chemotaxis and cytokine activity. In the human body, this family consists of about 50 related molecules, and there are similar homologs in other mammals. 20%-95% of chemokine sequences contain conserved cysteine residues. According to the number and spacing of cysteines, they are divided into four subtypes: CC, CXC, XC and CX3C subtypes (C is Cysteine, X is any amino acid).

The CX3C subtype has only one chemokine CX3CL1, also known as fractalkine (FKN) or neurotactin, which is the only membrane-bound chemokine. CX3CL1 is expressed in a variety of cells, and its only receptor is the chemokine receptor CX3CR1. CX3CR1 is a 7-pass transmembrane domain G protein-coupled receptor. Under normal circumstances, the body's natural killer cells (NK cells), monocytes, mast cells, platelets and effector T cell membranes all have the expression of CX3CR1.

CX3CL1/CX3CR1 plays a vital role in the process of inflammatory cells reaching the site of inflammation from the peripheral circulation. After the receptor CX3CR1 binds to the ligand CX3CL1, it triggers the influx of calcium ions to produce a chemotactic reaction of cells, thereby inducing cells to specific parts of the organism. CX3CR1 participates in some inflammatory processes mainly by inducing and recruiting NK cells, monocytes, macrophages, mast cells, and effector T cells to reach the site of inflammation. CX3CR1 not only has a chemotactic effect on these cells, but also because CX3CR1 has a special mucin stem structure, CX3CR1 also has a good adhesion to inflammatory cells, which makes CX3CR1 play a role in the pathological process of the occurrence and development of inflammation Important role. Studies have shown that the occurrence of pathological changes in many diseases is closely related to CX3CR1.

Japan Eisai successfully screened and obtained a high-affinity mouse monoclonal antibody 3A5-2 against human CX3CL1 through traditional hybridoma technology. The humanized antibody H3-2L4 still maintains a high affinity to human CX3CL1 (see CN102597003A), and is currently in clinical phase 2 or clinical phase 1/2 studies for rheumatoid and Crohn's disease. However, the H3-2L4 humanized antibody also retains more murine amino acids, which has a relatively high probability of causing rejection in the human body; in addition, the CDR region that binds to the target also retains several post-translational modification sites. Point, there is a risk of losing biological activity due to modification in the human body.

Summary of the invention

In order to minimize the rejection immune response caused by antibodies, human therapeutic antibodies should tend to contain as few murine amino acids as possible. In order to solve this technical problem, the present invention aims to minimize the number of murine amino acids of the target antibody through a more optimized humanized design, so that it is expected to have better in vivo safety.

In addition, the present invention also aims to use cell surface display technology to construct a library of the 6 CDR amino acids of humanized antibodies by random mutations, so as to screen out antibody molecules with higher affinity; The antibody was engineered for the second time to construct a library of random mutations with the full sequence of the variable region to screen antibodies that bind to mouse CX3CL1 with high affinity, and provide antibody options for studying the mechanism of CX3CL1 related diseases in mouse models.

In view of the above technical problems, the purpose of the present invention is to provide an anti-human chemokine CX3CL1 antibody or its fragment, especially a humanized antibody or its fragment, compared with the existing CX3CL1 humanized antibody, the present invention provides The antibody has fewer murine amino acids and has a higher affinity for CX3CL1. The purpose of the present invention is also to provide further obtained antibodies with high affinity to mouse CX3CL1. Based on the foregoing, the purpose of the present invention is also to provide applications of these antibodies.

Specifically, the present invention provides the following technical solutions.

The "fragment" of the antibody of the present invention encompasses various functional fragments of the antibody, for example, its antigen-binding portion, such as Fab, F(ab')2 or scFv fragments.

The CX3CL1 refers to fractalkine or neurotactin. Preferably, the CX3CL1 is a primate mammal CX3CL1, more preferably a human CX3CL1.

In one aspect, the present invention provides an antibody or fragment thereof, the antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL/VK), wherein the heavy chain variable region (VH ) Contains 3 CDRs (H-CDR1, H-CDR2, H-CDR3) from the heavy chain variable region shown in any of the following sequences:

SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; and/or

Wherein the light chain variable region (VL) comprises 3 CDRs (L-CDR1, L-CDR2, L-CDR3) from the light chain variable region shown in any of the following sequences:

SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31.

Preferably, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region (VH) comprises 3 CDRs (H-CDR1, H-CDR2 , H-CDR3):

SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; and/ or

Wherein the light chain variable region (VL) comprises 3 CDRs (L-CDR1, L-CDR2, L-CDR3) from the light chain variable region shown in any of the following sequences:

SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.

According to a specific embodiment of the present invention, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region (VH) and the light chain variable region (VL) comprise 6 CDRs from the following sequence combinations: (H-CDR1, H-CDR2, H-CDR3; L-CDR1, L-CDR2, L-CDR3):

(1) SEQ ID NO: 11+SEQ ID NO: 23;

(2) SEQ ID NO: 14+SEQ ID NO: 22;

(3) SEQ ID NO: 14+SEQ ID NO: 23;

(4) SEQ ID NO: 14+SEQ ID NO: 24;

(5) SEQ ID NO: 14+SEQ ID NO: 25;

(6) SEQ ID NO: 15+SEQ ID NO: 22;

(7) SEQ ID NO: 15+SEQ ID NO: 23;

(8) SEQ ID NO: 15+SEQ ID NO: 24;

(9) SEQ ID NO: 15+SEQ ID NO: 25;

(10) SEQ ID NO: 16+SEQ ID NO: 22;

(11) SEQ ID NO: 16+SEQ ID NO: 23;

(12) SEQ ID NO: 16+SEQ ID NO: 24;

(13) SEQ ID NO: 16+SEQ ID NO: 25;

(14) SEQ ID NO: 17+SEQ ID NO: 22;

(15) SEQ ID NO: 17+SEQ ID NO: 23;

(16) SEQ ID NO: 17+SEQ ID NO: 24;

(17) SEQ ID NO: 17+SEQ ID NO: 25;

(18) SEQ ID NO: 26+SEQ ID NO: 29;

(19) SEQ ID NO: 26+SEQ ID NO: 30;

(20) SEQ ID NO: 26+SEQ ID NO: 31;

(21) SEQ ID NO: 27+SEQ ID NO: 29;

(22) SEQ ID NO: 27+SEQ ID NO: 30;

(23) SEQ ID NO: 27+SEQ ID NO: 31;

(24) SEQ ID NO: 28+SEQ ID NO: 29;

(25) SEQ ID NO: 28 + SEQ ID NO: 30; or

(26) SEQ ID NO: 28 + SEQ ID NO: 31.

The combination of light and heavy chain CDRs provided by the present invention is derived from the above-mentioned light and heavy chain variable regions. Based on the amino acid sequence of a given light and heavy chain variable region, those skilled in the art can routinely determine the amino acid sequence of the CDR contained therein. . For example, according to a specific embodiment of the present invention, the Chothia coding method is used to divide the CDRs in the amino acid sequence of the variable region. The light and heavy chain CDRs and their combinations divided by methods known in the art are also encompassed within the scope of the present invention.

Preferably, the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of CDRs selected from:

(1) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPTQGDY) shown in SEQ ID NO: 37, 44, 42 in sequence; and, shown in SEQ ID NO: 49, 51, 48 L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(2) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 37, 41, 39 in sequence; and, shown in SEQ ID NO: 46, 51, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(3) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 37, 41, 39 in sequence; and, shown in SEQ ID NO: 49, 51, 48 L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(4) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 37, 41, 39 in sequence; and, shown in SEQ ID NO: 46, 52, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT);

(5) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 37, 41, 39 in sequence; and, shown in SEQ ID NO: 49, 52, 48 L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT);

(6) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 37, 44, 39 in sequence; and, shown in SEQ ID NO: 46, 51, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(7) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 37, 44, 39 in sequence; and, shown in SEQ ID NO: 49, 51, 48 L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(8) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 37, 44, 39 in sequence; and, shown in SEQ ID NO: 46, 52, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT);

(9) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 37, 44, 39 in sequence; and, shown in SEQ ID NO: 49, 52, 48 L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT);

(10) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NO: 37, 41, 40 in sequence; and, shown in SEQ ID NO: 46, 51, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(11) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NO: 37, 41, 40 in sequence; and, shown in SEQ ID NO: 49, 51, 48 L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(12) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NO: 37, 41, 40; and, shown in SEQ ID NO: 46, 52, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT);

(13) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NO: 37, 41, 40; and, shown in SEQ ID NO: 49, 52, 48 L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT);

(14) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NO: 37, 44, 40 in sequence; and, shown in SEQ ID NO: 46, 51, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(15) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NO: 37, 44, 40 in sequence; and, shown in SEQ ID NO: 49, 51, 48 L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(16) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NO: 37, 44, 40 in sequence; and, shown in SEQ ID NO: 46, 52, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT);

(17) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NO: 37, 44, 40 in sequence; and, shown in SEQ ID NO: 49, 52, 48 L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT);

(18) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSSKFNERFEG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 53, 54, 39; and, shown in SEQ ID NO: 46, 51, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(19) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSSKFNERFEG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 53, 54, 39; and, shown in SEQ ID NO: 46, 56, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNALAE), L-CDR3 (QQFWSTPYT);

(20) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 53, 55, 39 in sequence; and, shown in SEQ ID NO: 46, 51, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(21) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 53, 55, 39 in sequence; and, shown in SEQ ID NO: 46, 56, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNALAE), L-CDR3 (QQFWSTPYT);

(22) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 53, 55, 39; and, shown in SEQ ID NO: 46, 51, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT);

(23) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 53, 55, 39; and, shown in SEQ ID NO: 46, 56, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNALAE), L-CDR3 (QQFWSTPYT);

(24) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 53, 55, 39 in sequence; and, shown in SEQ ID NO: 46, 51, 48 L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT).

According to a specific embodiment of the present invention, in the antibody or fragment thereof, the heavy chain variable region comprises a sequence selected from:

Shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, The amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 or have at least 75% identity with the amino acid sequence The amino acid sequence of; and/or

The variable region of the light chain comprises a sequence selected from:

SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID The amino acid sequence of NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31 or an amino acid sequence having at least 75% identity with the amino acid sequence.

Preferably, according to a specific embodiment of the present invention, in the antibody or fragment thereof, the heavy chain variable region and the light chain variable region contained in the antibody or fragment thereof are selected from the following combinations:

(1) The amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

(2) The amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 22 or the amino acid sequence shown in SEQ ID NO: 22 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 22;

(3) The amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

(4) The amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 24;

(5) The amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence shown in SEQ ID NO: 25 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 25;

(6) The amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 22 or the amino acid sequence shown in SEQ ID NO: 22 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 22;

(7) The amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

(8) The amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 or The amino acid sequence shown in SEQ ID NO: 24 has an amino acid sequence with at least 75% identity;

(9) The amino acid sequence shown in SEQ ID NO: 15 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence shown in SEQ ID NO: 25 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 25;

(10) The amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 22 or the amino acid sequence shown in SEQ ID NO: 22 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 22;

(11) The amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

(12) The amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 24;

(13) The amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence shown in SEQ ID NO: 25 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 25;

(14) The amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 22 or the amino acid sequence shown in SEQ ID NO: 22 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 22;

(15) The amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

(16) The amino acid sequence shown in SEQ ID NO: 17 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 24;

(17) The amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence shown in SEQ ID NO: 25 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 25;

(18) The amino acid sequence shown in SEQ ID NO: 26 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 26; and, the amino acid sequence shown in SEQ ID NO: 29 or the amino acid sequence shown in SEQ ID NO: 29 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 29;

(19) The amino acid sequence shown in SEQ ID NO: 26 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 26; and, the amino acid sequence shown in SEQ ID NO: 30 or the amino acid sequence shown in SEQ ID NO: 30 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 30;

(20) The amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 26; and, the amino acid sequence shown in SEQ ID NO: 31 or the amino acid sequence shown in SEQ ID NO: 31 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 31;

(21) The amino acid sequence shown in SEQ ID NO: 27 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27; and, the amino acid sequence shown in SEQ ID NO: 29 or the amino acid sequence shown in SEQ ID NO: 29 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 29;

(22) The amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27; and, the amino acid sequence shown in SEQ ID NO: 30 or the amino acid sequence shown in SEQ ID NO: 30 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 30;

(23) The amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27; and, the amino acid sequence shown in SEQ ID NO: 31 or the amino acid sequence shown in SEQ ID NO: 31 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 31;

(24) The amino acid sequence shown in SEQ ID NO: 28 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28; and, the amino acid sequence shown in SEQ ID NO: 29 or the amino acid sequence shown in SEQ ID NO: 29 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 29;

(25) The amino acid sequence shown in SEQ ID NO: 28 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28; and, the amino acid sequence shown in SEQ ID NO: 30 or the amino acid sequence shown in SEQ ID NO: 30 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 30; or

(26) The amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28; and, the amino acid sequence shown in SEQ ID NO: 31 or the amino acid sequence shown in SEQ ID NO: 31 The amino acid sequence in SEQ ID NO: 31 has an amino acid sequence that is at least 75% identical.

Preferably, the antibody or fragment thereof is an antibody or an antigen-binding fragment thereof against chemokine CX3CL1, preferably mammalian CX3CL1, more preferably human CX3CL1, cynomolgus CX3CL1 or mouse CX3CL1;

Preferably, the antibody or antigen-binding fragment thereof comprises a VH framework region and a VL framework region;

The antibody is in any form such as monoclonal antibodies, single-chain antibodies, bifunctional antibodies, single domain antibodies, nanobodies, fully or partially humanized antibodies, or chimeric antibodies, or the antigen-binding fragment is a half antibody or Antigen-binding fragments of antibodies or halves, such as scFv, BsFv, dsFv, (dsFv) ₂ , Fab, Fab', F(ab') ₂ or Fv.

Further preferably, the antibody or fragment thereof further comprises a constant region of human or murine origin, preferably a heavy chain constant region (CH) and/or light chain constant region (CL) of human or murine origin; preferably, the The antibody or fragment thereof comprises a heavy chain and a light chain;

More preferably, the antibody or fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region.

Preferably, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is IgG4 subtype, and the light chain constant region is κ type;

Preferably, the heavy chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 35 or an amino acid sequence having at least 75% identity with the amino acid sequence; preferably, the monoclonal antibody The light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 75% identity with the amino acid sequence.

The at least 75% identity of the present invention is at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity, etc. Any percentage of identity ≥ 75%.

In particular, the antibody or fragment thereof of the present invention includes at least a heavy chain variable region and a light chain variable region, both of which include the above-mentioned CDRs and intervening framework regions (framework), and the arrangement of each domain is: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4. In this regard, at most 25% difference in amino acid sequence caused by the "at least 75% identity" may be present in any framework region in the variable region of the heavy chain or the variable region of the light chain, or in the present invention The antibody or fragment thereof in any domain or sequence other than the variable region of the heavy chain and the variable region of the light chain. The difference can be caused by amino acid deletion, addition or substitution at any position, wherein the substitution can be a conservative substitution or a non-conservative substitution.

According to specific embodiments of the present invention, particularly preferably, the present invention provides the following antibodies:

Humanized antibody AM85, the antibody comprising the heavy chain variable region shown in SEQ ID NO: 14, the light chain variable region shown in SEQ ID NO: 22, and the heavy chain constant region shown in SEQ ID NO: 35 , The light chain constant region shown in SEQ ID NO: 36;

Humanized antibody AM85-M3, the antibody comprising the heavy chain variable region shown in SEQ ID NO: 26, the light chain variable region shown in SEQ ID NO: 31, and the heavy chain shown in SEQ ID NO: 35 Constant region, the light chain constant region shown in SEQ ID NO: 36;

Humanized antibody AM85-M8, the antibody comprising the heavy chain variable region shown in SEQ ID NO: 28, the light chain variable region shown in SEQ ID NO: 30, and the heavy chain shown in SEQ ID NO: 35 Constant region, the light chain constant region shown in SEQ ID NO: 36.

On the other hand, based on the antibody or fragment thereof of the present invention, the present invention also provides a nucleic acid molecule that encodes any antibody or fragment thereof of the present invention or encodes the heavy chain CDR and light chain CDR contained in the antibody or fragment thereof. , Light chain variable region, heavy chain variable region, heavy chain or light chain.

In yet another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.

The vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter the host cell in any manner for the purpose of preservation or expression of antibodies. Therefore, in another aspect, the present invention provides a host cell comprising the nucleic acid molecule and/or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and/or vector of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

Based on the disclosure of the present invention, the antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention can be obtained by using any conventional technical methods known in the art. The antibodies or fragments thereof, nucleic acid molecules, vectors, and/or host cells may be included in a composition (for example, a pharmaceutical composition), more particularly in a pharmaceutical preparation, so as to be used for various purposes according to actual needs.

Therefore, in another aspect, the present invention also provides a composition comprising the antibody or fragment thereof, nucleic acid molecule, vector and/or host cell of the present invention. Preferably, the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients.

As antibodies or fragments thereof that bind to CX3CL1, especially human CX3CL1, the present invention also provides related applications of the above-mentioned topics.

Specifically, in another aspect, the invention provides the use of the antibody or fragments thereof, nucleic acid molecules, vectors, host cells or compositions in the preparation of medicines for the prevention, treatment and/or amelioration of inflammatory diseases ；

Preferably, the inflammatory disease is ulcerative colitis, Crohn’s disease, inflammatory bowel disease, rheumatoid arthritis, nephritis, glomerulonephritis, myositis, multiple sclerosis, optic neuromyelitis, atherosclerosis Like sclerosis, psoriasis, systemic lupus erythematosus (e.g. lupus or lupus nephritis of the central nervous system) or autoimmune hepatobiliary diseases.

Alternatively, the invention provides the use of the antibody or its fragment, nucleic acid molecule, vector, host cell or composition in the preparation of a medicament for blocking the CX3CL1/CX3CR1 signaling pathway and/or blocking CX3CR1 expressing cells migrate.

Alternatively, the invention provides the use of the antibody or its fragment, nucleic acid molecule, vector, host cell or composition as a medicament for studying the CX3CL1/CX3CR1 signaling pathway-related or CX3CL1 or CX3CR1-related disease mechanism in a mouse model.

In another aspect, the present invention provides a kit comprising the antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or composition of the present invention. The kit can be used for treatment, diagnosis or detection purposes. For example, the antibody or fragment thereof is contacted with the sample to be tested to detect the presence of CX3CL1 in the sample.

In yet another aspect, the present invention provides a method for preventing, treating and/or ameliorating inflammatory diseases, the method comprising administering to a subject in need the antibody or fragments thereof, nucleic acid molecules, vectors, The host cell or pharmaceutical composition, and optionally other drugs or means.

Preferably, the inflammatory disease is ulcerative colitis, Crohn’s disease, inflammatory bowel disease, rheumatoid arthritis, nephritis, glomerulonephritis, myositis, multiple sclerosis, optic neuromyelitis, atherosclerosis Like sclerosis, psoriasis, systemic lupus erythematosus (e.g. lupus or lupus nephritis of the central nervous system) or autoimmune hepatobiliary diseases.

Alternatively, the present invention provides a method for blocking the CX3CL1/CX3CR1 signaling pathway and/or blocking the migration of CX3CR1 expressing cells, the method comprising administering the antibody or fragment thereof of the present invention to a subject in need thereof, Nucleic acid molecule, vector, host cell or pharmaceutical composition, and optionally other drugs or means.

In the method of the present invention, the optional other drugs or means refer to other drugs or means that can be administered in combination with the antibody or fragments, nucleic acid molecules, vectors, host cells, or pharmaceutical compositions of the present invention, such as small molecule drugs. , Targeted drugs, antibodies and other recombinant protein drugs, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapy drugs and radiotherapy. The combined administration of the two can be carried out in any form, for example, simultaneously, continuously or at intervals.

In the method of the present invention, the "subject" may be a mammal, such as a primate or a rodent, and more preferably a human.

The present invention minimizes the number of murine amino acids of the target antibody (mouse monoclonal antibody 3A5-2) through a more optimized humanization design, so that the humanized antibody has better safety in vivo. Further, through the cell surface display technology, the present invention performs random mutations on the amino acids in the 6 CDR regions of the humanized antibody to construct a library, and through the flow sorting of the antibody library, the screening results are compared to 3A5-2. The original humanized antibody H3-2L4 has a higher affinity antibody molecule. Compared with H3-2L4, some of the affinity mature antibodies even have an order of magnitude increase in KD affinity, which hinders the CX3CL1/CX3CR1 signaling pathway. The maximum blocking rate is increased by 10%-20%.

Furthermore, the present invention has also carried out a second engineering modification on affinity matured antibodies, and also constructed a random mutation antibody library with the full sequence of the variable region. After the second engineering modification, the antibodies are compared with mice, cynomolgus monkeys and human CX3CL1. The binding kinetic parameters were screened to obtain an antibody that binds to mouse CX3CL1 with high affinity. This antibody retains its binding activity to human and cynomolgus CX3CL1, while its binding KD to mouse CX3CL1 is increased by 30-40 times. It reaches the nM level, so it can be used as a surrogate antibody to study the mechanism of CX3CL1 related diseases in mouse models.

In addition, the present invention has fully studied the physicochemical properties of the antibody, and found that the monomer ratio, hydrophilicity, and melting point (Tm) of the antibody are all at a normal level. In particular, the test found that the antibody of the present invention can block the migration activity of CX3CR1 expressing cells in vitro, and it is even 5-fold higher than that of H3-2L4.

In summary, compared with the existing CX3CL1 antibody, the antibody provided by the present invention is a more ideal clinical lead drug.

Description of the drawings

Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

Figure 1 shows the humanized design of the murine antibody 3A5-2.

Figure 2 shows the 6 CDRs of the humanized antibody 3A5-2_hz20 and the mutation site selection for affinity maturation modification.

Figure 3 is a schematic diagram of a two-step PCR method using mutant primers to prepare mutant single-chain antibody genes in affinity maturation modification.

Figure 4 shows the results of FACS binding analysis of mutant clones and antigens obtained by flow sorting.

Figure 5 shows the results of the cAMP experiment in which the affinity matured antibody blocks the downstream signaling pathway of the target.

Figure 6 shows the experimental results of affinity maturation antibody and engineered antibody blocking the migration activity of CX3CR1 expressing cells.

The best way to implement the invention

The present invention will be described below with reference to specific embodiments. Those skilled in the art can understand that these embodiments are only used to illustrate the present invention, and they do not limit the scope of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The medicinal materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified.

Human CX3CL1: The amino acid sequence is shown in SEQ ID NO: 32;

Mouse CX3CL1: The amino acid sequence is shown in SEQ ID NO: 33;

Cynomolgus monkey CX3CL1: the amino acid sequence is shown in SEQ ID NO: 34;

Heavy chain constant region: the amino acid sequence is shown in SEQ ID NO: 35;

Light chain constant region: the amino acid sequence is shown in SEQ ID NO: 36;

Human CX3CR1: The amino acid sequence is shown in SEQ ID NO: 58.

Example 1 Humanized design of mouse monoclonal antibody 3A5-2 and determination of in vitro binding activity of humanized antibody

1.1 Humanized design of mouse monoclonal antibody 3A5-2

Based on the antibody coding schemes of Kabat and Chothia, the amino acid sequence regions of the six antigen complementarity determinants (CDR) of the heavy and light chains of the murine antibody 3A5-2 and the framework regions (FR) supporting the conservative three-dimensional conformation of the antibody were determined. Then through analysis and search for known human antibody sequences, select the human antibody heavy chain variable region sequence that is most similar to the mouse antibody, such as IGHV1-69, select its antibody framework region sequence as a template, and use the mouse antibody heavy chain CDR Combine with human antibody FR to generate humanized antibody heavy chain variable region sequence. In the same process, the IGKV1-13 antibody framework region sequence was selected as the template to generate the humanized antibody light chain variable region sequence. In addition, determine the back mutation site: compare the designed humanized antibody sequence with the original murine antibody sequence, check which amino acids are different, and check whether these amino acids play an important role in supporting the structure of the antibody or binding to the antigen Important role. At the same time, check whether the humanized design sequence has potential post-translational modification sites, such as N (asparagine) glycosylation site, N deamidation site, D (aspartic acid) isomerization Sites, etc.

The humanized design of 3A5-2 is shown in Figure 1, in which the three humanized sequences (3A5-2_VH_hz0) of the heavy chain variable region (3A5-2_VH) and the light chain variable region (3A5-2_VK) of 3A5-2 are shown respectively. , 3A5-2_VH_hz1 and 3A5-2_VH_hz2; 3A5-2_VK_hz0, 3A5-2_VK_hz1 and 3A5-2_VK_hz2), the humanized sequence with the suffix "_hz0" indicates that the mouse antibody CDR is directly transplanted into the human framework region.

Synthesize the above-mentioned humanized heavy chain variable region gene and construct it into a mammalian cell expression vector containing the heavy chain constant region gene of the human monoclonal antibody IgG4 subclass; similarly, synthesize the light chain variable region gene, It was constructed into a mammalian cell expression vector containing the light chain constant region gene of human monoclonal antibody κ subclass. The constructed humanized antibody heavy chain vector and light chain vector were cross-paired (Table 1), and HEK293 cells were transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant was collected and purified by ProteinA to obtain the humanized antibody. Antibody protein.

The humanized antibody is named "murine antibody abbreviation_hz", and the antibody whose CDR of murine antibody is directly transplanted to the human framework region is named "murine antibody abbreviation_hz00", and the antibodies obtained by further modification are numbered according to different sequences .

Table 1. Humanized antibodies expressed in cross-pairing between heavy and light chains

1.2 In vitro binding activity determination of humanized antibody

The Fortebio (BLITZ pro1.1.0.28) instrument was used to analyze the binding kinetic parameters of the humanized antibody to the antigen human CX3CL1 (hCX3CL1). Before the measurement, the NTA biological probe was soaked in PBS for 10 minutes; then the probe was placed in PBS containing 100 nM of antigen for 300 seconds to capture the His-tagged antigen; the probe was further combined with 100 nM of antibody. The binding time is 400 seconds; after that, the probe is transferred to PBS for dissociation reaction, and the time is 600 seconds. After the experiment, the blank control response value was subtracted, and the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding. The results are shown in Table 2.

Table 2. Binding kinetic parameters of antibodies obtained after humanization of mouse anti-3A5-2

Ab ID	response	K D (M)	K on (1/Ms)	k off (s -1 )	K off /K off (H3-2L4)
H3-2L4	0.306	5.95E-09	4.42E+05	2.63E-03	1.00
3A5-2_hz00	0.252	9.05E-09	5.35E+05	4.84E-03	1.84
3A5-2_hz10	0.280	6.50E-09	5.16E+05	3.35E-03	1.28
3A5-2_hz20	0.217	1.49E-08	4.99E+05	7.45E-03	2.77
3A5-2_hz01	0.013	<1.0E-12	2.83E+04	<1.0E-07	NA
3A5-2_hz11	0.015	<1.0E-12	3.22E+04	<1.0E-07	NA
3A5-2_hz21	0.009	<1.0E-12	6.86E+04	<1.0E-07	NA
3A5-2_hz02	0.010	<1.0E-12	2.14E+04	<1.0E-07	NA
3A5-2_hz12	0.012	<1.0E-12	5.75E+04	<1.0E-07	NA
3A5-2_hz22	0.001	<1.0E-12	3.04E+04	<1.0E-07	NA

Example 2 Affinity maturation modification of humanized antibody

2.1 Design and construction of affinity maturation antibody library

The humanized antibody 3A5-2_hz20 was selected as the template for affinity maturation modification. The heavy chain variable region and light chain variable region of 3A5-2_hz20 are connected by GS linker to construct a single-chain variable fragment (scFv). The CDR of this single-chain antibody is the target of affinity maturation and transformation, and the skeleton The regions will remain unchanged during the affinity maturation modification, and a total of 6 CDRs in the heavy and light chains are constructed into separate antibody libraries. Based on experience and sequence characteristics, some amino acids in H-CDR3 remain unchanged during the modification of affinity maturation. As shown in Figure 2, the invariant amino acids are shown in bold italics.

A random mutation primer for CDR was designed and synthesized. Only 20% nucleotide mutations were introduced at each mutation position, and there were about 20 base pairs of unmutated bases on both sides of the primer. The mutation is integrated into the final single-chain antibody gene by two-step PCR with mutant primers and other conventional primers. The single-chain antibody gene has a 148bp overhang at the 5'end and a 222bp overhang at the 3'end, which is the same as the sequence on the display DNA vector pYD, which is the so-called homology region. The schematic diagram of this 2-step PCR method is shown in Figure 3.

The yeast strain EBY100 was co-transformed with 4μg linearized vector and 12μg single-chain antibody gene by electroporation, and then yeast used its homologous recombination mechanism to link the single-chain antibody gene to the display DNA vector. The capacity of each CDR region mutant antibody library is shown in Table 3.

Table 3. Storage capacity of 6 CDR mutant antibody libraries

#	Antibody Library	Number of amino acid mutations		Storage capacity
1	H1	10	1.0×10 8
2	H2	8	1.7×10 8
3	H3	7	1.6×10 8
4	L1	8	1.2×10 8
5	L2	8	1.1×10 8
6	L3	7	5×10 7

2.2 Flow sorting of affinity maturation antibody library

After 1 round of magnetic bead sorting for the 6 CDR mutant antibody libraries, 4 rounds of FACS sorting with successively decreasing antigen concentrations were performed. In the first round of sorting with 300nM biotinylated hCX3CL1, the antigen concentration in the second round was reduced to 100nM, the antigen concentration in the third round was reduced to 10nM, and the antigen concentration in the fourth round was reduced to 1nM. In each round of FACS sorting, 0.1% to 0.5% of the clones of the library are collected and cultured, and then the next round of sorting is performed, and finally mutant clones with improved affinity are isolated.

96 yeast colonies were selected for sequencing analysis, and finally 7 unique mutant clones were selected from the H2 library, 4 unique mutant clones were selected from the H3 library, and sorted according to affinity and mutations from L1 and Five unique mutant clones were selected from the L2 library, as shown in Table 4.

Table 4. Mutant clones selected by flow sorting

2.3 Affinity analysis of sorted clones

The mutant cloned yeast was inoculated into yeast culture medium to induce the expression of related single chain antibody. The yeast cells were collected by centrifugation at 14000 rpm for 1 minute, and washed once with PBS containing 1% BSA. The density of resuspended yeast cells is 5,000,000 cells/ml, and 100 μl of cells are added to each well of a 96-well U-shaped well plate. Add 100 μl of the biotinylated antigen solution in gradient dilutions, incubate with shaking at room temperature for 30 minutes, and place on ice for 10 minutes. The cells were collected by centrifugation at 14000 rpm at 4°C for 1 minute, and washed once with pre-cooled PBS containing 1% BSA. Add 100μl 1:500 diluted SA fluorescent secondary antibody, and incubate on ice for 30 minutes. The cells were collected by centrifugation at 14000 rpm at 4°C for 1 minute, and washed twice with pre-cooled PBS containing 1% BSA. Analyze the average fluorescence reading of the cell population with a flow cytometer. The results are shown in Figure 4, the EC50 values of the mutant clones after 5 rounds of sorting are higher than the H3-2L4 antibody.

2.4 Recombinant expression of affinity maturation antibody IgG

A comprehensive analysis of the above-mentioned affinity-enhancing cloned mutation sites is combined, and the related mutation sites of the 6 CDRs are combined. The antibody variable regions containing various combinations of mutation sites are shown in the 11 types of heavy chain variable regions (AM_vh1 to AM_vh11) and 8 types of light chain variable regions (AM_vl1 to AM_vl8) shown in the sequence listing and shown in Figure 1. 35A-2_VK-hz0.

Synthesize the heavy chain variable region gene and construct it into a mammalian cell expression vector containing the heavy chain constant region gene of the human monoclonal antibody IgG4 subclass; similarly, synthesize the light chain variable region gene and construct it into a mammalian cell expression vector containing the heavy chain constant region gene of the human monoclonal antibody IgG4 subclass; Human monoclonal antibody κ subclass light chain constant region gene in mammalian cell expression vector. As for the expression of humanized antibody in section 1.1 of Example 1, cross-pair the heavy chain vector and light chain vector of the constructed affinity mature antibody, transfect HEK293 cells with polyethyleneimine (PEI), and collect it after about 7 days The cell supernatant was purified with ProteinA to obtain the affinity mature antibody protein.

The affinity mature antibody is named "AMxy", where x is derived from the numerical number of the heavy chain variable region AM_vh, and y is derived from the numerical number of the light chain variable region AM_vl. For example, AM85 below includes AM_vh8 and AM_vl5.

2.5 Determination of kinetic parameters of affinity maturation antibody and human CX3CL1

Use GE's BIAcore instrument S200 to determine the antibody-antigen interaction force. Refer to the GE Human antibody capture kit (Cat. No. BR-1008-39, Lot10261753) operating instructions. First, the sensor chip CM5 analysis channel and the control sample channel are both saturated and coupled with the maximum amount of anti-human Fc antibody, and then flow through the analysis channel containing 7.5μg/ml anti-human CX3CL1 chimeric antibody buffer to make it evenly distributed, and finally flow through the gradient dilution antigen sample (initial concentration 20nM, 1:3 dilution 8 concentration points, and Set the 0.741nm concentration point to repeat), and measure the light response value that occurs after the antibody antigen binds. After fitting and analysis by the instrument software, the binding constant Kon and the dissociation constant Koff of the antibody, and the affinity constant KD are finally obtained.

The results show that most of the affinity mature antibodies that have undergone recombination of heavy and light chains have a certain degree of improvement in their binding activity to the antigen compared to H3-2L4. The binding activity of antibodies containing AM_vh8 or AM_vh9, AM_vl5 or AM_vl6 increased by an order of magnitude. See Table 5.

Table 5. Biacore analysis of the kinetic parameters of IgG antibodies after affinity maturation

2.6 Determination of the blocking activity of affinity maturation antibodies on the downstream signaling pathway of CX3CL1

The cAMP method is used for detection.

First, a stable cell line stably expressing human CX3CR1 was constructed on CHO-K1 cells, named CHOK1-CX3CR1 cells. The cells were cultured to 70% to 90% confluence, trypsinized and collected, centrifuged at 400g for 5 minutes, and the supernatant was discarded. Resuspend the cells in a medium containing IBMX to a density of 125,000 cells/ml, and add 4 μl of cells to each well in a 384-well plate. The antibody to be tested is dissolved in a medium containing 1% BSA, with an initial concentration of 30 μg/ml, 3 times dilution, 8 dilution concentrations (including 0 concentration), and 2 μl antibodies of each concentration are added to a 384-well plate. Use a medium containing 1% BSA to prepare CX3CL1 protein at a concentration of 0.2μg/ml, add 2μl to a 384-well plate, and place it at room temperature for 10 minutes. Use a medium containing 1% BSA to prepare Forskolin with a concentration of 5μM, add 2μl to a 384-well plate, and place it at 37°C for 30 minutes. Finally, add 5μl of anti-cAMP-Cryptate solution and 5ul of cAMP-d2 solution, read the plate for detection after 1 hour at room temperature.

The results show that compared with the H3-2L4 antibody, the affinity maturation antibody of the present invention has no significant difference in the IC50 value of blocking the signal pathway of CX3CL1 and its receptor, and the maximum blocking rate is increased by about 10%-20%. See Figure 5 and Table 6.

Table 6. cAMP experiment results of affinity maturation antibody blocking the downstream signaling pathway of the target

To	AM85	AM86	AM87	AM88	AM95	AM96	AM97	AM98	H3-2L4	hIgG
IC50(nM)	~2.253	1.69	1.734	1.901	1.186	1.746	1.677	~2.299	1.695	NA
Maximum blocking rate (%)	126	121	123	124	123	110	121	110	100	NA

Example 3 Engineering of a replacement antibody that binds to mouse CX3CL1

3.1 Construction and screening of engineered antibody library

The method is as described in the modification of antibody affinity maturation in Example 2. The affinity mature antibody AM85 was selected as the template for the secondary engineering, and an antibody library with random mutations in the full variable regions of the heavy and light chains was designed. After 5 rounds of FACS screening with successively decreasing mouse CX3CL1 antigen concentrations, clones with FACS binding antigen stronger than the template were obtained, which were re-expanded and the DNA was extracted and sequenced. Analyze the sequencing results, synthesize new antibody heavy and light chain variable region genes and construct them into eukaryotic expression vectors. The heavy and light chain expression vectors are cross-combined, transiently transfected into HEK293 cells for recombinant expression, and purified by ProteinA to obtain antibody proteins with SDS-PAGE purity of more than 95%.

The variable regions of various antibodies are shown in the three heavy chain variable regions (AM_vh8_m1 to AM_vh8_m3) and the three light chain variable regions (AM_vl5_m1 to AM_vl5_m3) shown in the sequence listing, respectively.

Table 7. Engineered antibodies expressed in cross-paired expression of heavy and light chains

3.2 Determination of the binding activity of engineered antibodies to CX3CL1 of different species

Methods As described above, the GE BIAcore instrument S200 was used to determine the binding kinetic parameters of the antibody to mice, cynomolgus monkeys and human CX3CL1 after the secondary engineering.

The results showed that the secondary engineered antibody, while retaining its binding activity to human and cynomolgus CX3CL1, its binding KD value to mouse CX3CL1 increased from the original level of about 50 nM by 30-40 times, reaching nM level. The re-engineered antibody can be used as a surrogate antibody to study the mechanism of CX3CL1 related diseases in mouse models. See Table 8.

Table 8. Species cross-binding activity of engineered antibodies

3.3 Determination of chemotactic activity of engineered antibodies to block CX3CL1 receptor expression cells

Culture the CHOK1-CX3CR1 cells stably expressing the CX3CR1 protein to a confluence of about 50%, wash the cells twice with PBS, and then add a serum-free medium for starvation and culture overnight. The cells were collected by trypsinization, centrifuged at 400g for 5 minutes, and the supernatant was discarded. Resuspend the cells in serum-free medium to ^{a density of 3×10 6} cells/ml. Add 600μl of culture medium to each well of the 24-well plate, add human CX3CL1 with a final concentration of 0.2μg/ml and different concentrations of the antibody to be tested; place the migration chamber in the 24-well plate, and add 50μl of cells to the upper migration chamber. After placing the 24-well plate in a 37°C incubator for 4 hours, transfer the upper migration chamber to another 24 empty plate containing 600μl of pancreatin, and place it in the 37°C incubator for further incubation for 10 minutes. Centrifuge at 500g for 1 minute. The migration chamber was discarded, and 50 μl of FBS was added to terminate the enzyme reaction. Combine the cells in the corresponding wells of the two 24-well plates, centrifuge at 500g for 5 minutes, discard the supernatant, add 120μl CellTiter-Glo, and check the luminescence intensity after standing at room temperature for 10 minutes.

The results showed that, compared with the H3-2L4 antibody, the first affinity maturation modified antibody AM85 and the second mouse CX3CL1 cross-binding activity modified antibody AM85-M3 and AM85-M8 blocked CX3CR1 expressing cells in vitro In terms of migration activity, the IC50 value has been significantly improved by about 5 times. See Figure 6 and Table 9.

Table 9. Experimental results of antibody blocking the migration activity of CX3CR1 expressing cells

Ab ID.	Maximum blocking rate (%)	IC50(nM)
H3-2L4	100	3.33
AM85	100	0.59
AM85-M3	100	0.68
AM85-M8	100	0.69

The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications according to the present invention, as long as they do not deviate from the spirit of the present invention, they shall fall within the scope of the appended claims of the present invention.

Claims (16)

1. An antibody or fragment thereof, said antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises any one of the following sequences The 3 CDRs (H-CDR1, H-CDR2, H-CDR3) of the variable region of the heavy chain shown:

   SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; and/or

   Wherein the light chain variable region (VL) comprises 3 CDRs (L-CDR1, L-CDR2, L-CDR3) from the light chain variable region shown in any of the following sequences:

   SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31.
2. The antibody or fragment thereof according to claim 1, wherein the heavy chain variable region (VH) comprises 3 CDRs (H-CDR1, H-CDR2, H-CDR3):

   SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; and/ or

   Wherein the light chain variable region (VL) comprises 3 CDRs (L-CDR1, L-CDR2, L-CDR3) from the light chain variable region shown in any of the following sequences:

   SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.
3. The antibody or fragment thereof according to claim 1 or 2, wherein the variable region of the heavy chain (VH) and the variable region of the light chain (VL) comprise 6 CDRs from the following sequence combinations (H-CDR1, H-CDR2, H-CDR3; L-CDR1, L-CDR2, L-CDR3):

   (1) SEQ ID NO: 11+SEQ ID NO: 23;

   (2) SEQ ID NO: 14+SEQ ID NO: 22;

   (3) SEQ ID NO: 14+SEQ ID NO: 23;

   (4) SEQ ID NO: 14+SEQ ID NO: 24;

   (5) SEQ ID NO: 14+SEQ ID NO: 25;

   (6) SEQ ID NO: 15+SEQ ID NO: 22;

   (7) SEQ ID NO: 15+SEQ ID NO: 23;

   (8) SEQ ID NO: 15+SEQ ID NO: 24;

   (9) SEQ ID NO: 15+SEQ ID NO: 25;

   (10) SEQ ID NO: 16+SEQ ID NO: 22;

   (11) SEQ ID NO: 16+SEQ ID NO: 23;

   (12) SEQ ID NO: 16+SEQ ID NO: 24;

   (13) SEQ ID NO: 16+SEQ ID NO: 25;

   (14) SEQ ID NO: 17+SEQ ID NO: 22;

   (15) SEQ ID NO: 17+SEQ ID NO: 23;

   (16) SEQ ID NO: 17+SEQ ID NO: 24;

   (17) SEQ ID NO: 17+SEQ ID NO: 25;

   (18) SEQ ID NO: 26+SEQ ID NO: 29;

   (19) SEQ ID NO: 26+SEQ ID NO: 30;

   (20) SEQ ID NO: 26+SEQ ID NO: 31;

   (21) SEQ ID NO: 27+SEQ ID NO: 29;

   (22) SEQ ID NO: 27+SEQ ID NO: 30;

   (23) SEQ ID NO: 27+SEQ ID NO: 31;

   (24) SEQ ID NO: 28+SEQ ID NO: 29;

   (25) SEQ ID NO: 28 + SEQ ID NO: 30; or

   (26) SEQ ID NO: 28+SEQ ID NO: 31;

   Preferably, the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of CDRs selected from:

   (1) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 44, 42; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 49, 51, 48 , L-CDR3;

   (2) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 41, 39; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 51, 48 , L-CDR3;

   (3) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 41, 39; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 49, 51, 48 , L-CDR3;

   (4) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 41, 39; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 52, 48 , L-CDR3;

   (5) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 41, 39; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 49, 52, 48 , L-CDR3;

   (6) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 44, 39; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 51, 48 , L-CDR3;

   (7) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 44, 39; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 49, 51, 48 , L-CDR3;

   (8) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 44, 39; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 52, 48 , L-CDR3;

   (9) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 44, 39; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 49, 52, 48 , L-CDR3;

   (10) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 41, 40; and L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 51, 48 , L-CDR3;

   (11) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 41, 40; and L-CDR1, L-CDR2 shown in SEQ ID NO: 49, 51, 48 , L-CDR3;

   (12) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 41, 40; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 52, 48 , L-CDR3;

   (13) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 41, 40; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 49, 52, 48 , L-CDR3;

   (14) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 44, 40; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 51, 48 , L-CDR3;

   (15) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 44, 40; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 49, 51, 48 , L-CDR3;

   (16) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 44, 40; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 52, 48 , L-CDR3;

   (17) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 37, 44, 40; and, L-CDR1, L-CDR2 shown in SEQ ID NO: 49, 52, 48 , L-CDR3;

   (18) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 53, 54 and 39 in sequence; and L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 51, and 48 in sequence , L-CDR3;

   (19) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 53, 54 and 39; and L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 56, 48 , L-CDR3;

   (20) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 53, 55, 39 in sequence; and L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 51, 48 in sequence , L-CDR3;

   (21) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 53, 55, 39; and L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 56, 48 , L-CDR3;

   (22) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 53, 55, 39; and L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 51, 48 , L-CDR3;

   (23) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 53, 55, 39; and L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 56, 48 , L-CDR3;

   (24) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO: 53, 55, 39; and L-CDR1, L-CDR2 shown in SEQ ID NO: 46, 51, 48 , L-CDR3.
4. The antibody or fragment thereof according to any one of claims 1 to 3, wherein the heavy chain variable region comprises a sequence selected from:

   Shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, The amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 or have at least 75% identity with the amino acid sequence The amino acid sequence of; and/or

   The variable region of the light chain comprises a sequence selected from:

   SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID The amino acid sequence of NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31 or an amino acid sequence having at least 75% identity with the amino acid sequence;

   Preferably, the heavy chain variable region and the light chain variable region contained in the antibody or fragment thereof are selected from the following combinations:

   (1) The amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

   (2) The amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 22 or the amino acid sequence shown in SEQ ID NO: 22 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 22;

   (3) The amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

   (4) The amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 24;

   (5) The amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence shown in SEQ ID NO: 25 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 25;

   (6) The amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 22 or the amino acid sequence shown in SEQ ID NO: 22 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 22;

   (7) The amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

   (8) The amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 or The amino acid sequence shown in SEQ ID NO: 24 has an amino acid sequence with at least 75% identity;

   (9) The amino acid sequence shown in SEQ ID NO: 15 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence shown in SEQ ID NO: 25 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 25;

   (10) The amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 22 or the amino acid sequence shown in SEQ ID NO: 22 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 22;

   (11) The amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

   (12) The amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 24;

   (13) The amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence shown in SEQ ID NO: 25 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 25;

   (14) The amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 22 or the amino acid sequence shown in SEQ ID NO: 22 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 22;

   (15) The amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 23;

   (16) The amino acid sequence shown in SEQ ID NO: 17 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 24;

   (17) The amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence shown in SEQ ID NO: 25 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 25;

   (18) The amino acid sequence shown in SEQ ID NO: 26 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 26; and, the amino acid sequence shown in SEQ ID NO: 29 or the amino acid sequence shown in SEQ ID NO: 29 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 29;

   (19) The amino acid sequence shown in SEQ ID NO: 26 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 26; and, the amino acid sequence shown in SEQ ID NO: 30 or the amino acid sequence shown in SEQ ID NO: 30 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 30;

   (20) The amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 26; and, the amino acid sequence shown in SEQ ID NO: 31 or the amino acid sequence shown in SEQ ID NO: 31 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 31;

   (21) The amino acid sequence shown in SEQ ID NO: 27 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27; and, the amino acid sequence shown in SEQ ID NO: 29 or the amino acid sequence shown in SEQ ID NO: 29 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 29;

   (22) The amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27; and, the amino acid sequence shown in SEQ ID NO: 30 or the amino acid sequence shown in SEQ ID NO: 30 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 30;

   (23) The amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27; and, the amino acid sequence shown in SEQ ID NO: 31 or the amino acid sequence shown in SEQ ID NO: 31 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 31;

   (24) The amino acid sequence shown in SEQ ID NO: 28 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28; and, the amino acid sequence shown in SEQ ID NO: 29 or the amino acid sequence shown in SEQ ID NO: 29 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 29;

   (25) The amino acid sequence shown in SEQ ID NO: 28 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28; and, the amino acid sequence shown in SEQ ID NO: 30 or the amino acid sequence shown in SEQ ID NO: 30 An amino acid sequence with at least 75% identity in the amino acid sequence of SEQ ID NO: 30; or

   (26) The amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28; and, the amino acid sequence shown in SEQ ID NO: 31 or the amino acid sequence shown in SEQ ID NO: 31 The amino acid sequence in SEQ ID NO: 31 has an amino acid sequence that is at least 75% identical.
5. The antibody or fragment thereof according to any one of claims 1 to 4, wherein the antibody or fragment thereof is directed against chemokine CX3CL1, preferably mammalian CX3CL1, more preferably human CX3CL1, cynomolgus CX3CL1, or Mouse CX3CL1 antibody or antigen-binding fragment thereof;

   Preferably, the antibody or antigen-binding fragment thereof comprises a VH framework region and a VL framework region;

   The antibody is in any form such as monoclonal antibodies, single-chain antibodies, bifunctional antibodies, single domain antibodies, nanobodies, fully or partially humanized antibodies, or chimeric antibodies, or the antigen-binding fragment is a half antibody or Antigen-binding fragments of antibodies or half-antibodies, such as scFv, BsFv, dsFv, (dsFv) ₂ , Fab, Fab', F(ab') ₂ or Fv;

   Preferably, the antibody or fragment thereof further comprises a constant region of human or murine origin, preferably a heavy chain constant region (CH) and/or light chain constant region (CL) of human or murine origin; preferably, the antibody Or its fragments include heavy chain and light chain;

   More preferably, the antibody or fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region.
6. The antibody or fragment thereof according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the The constant region of the heavy chain of the monoclonal antibody is of the IgG4 subtype, and the constant region of the light chain is of the κ type;

   Preferably, the heavy chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 35 or an amino acid sequence having at least 75% identity with the amino acid sequence;

   Preferably, the light chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 36 or an amino acid sequence having at least 75% identity with the amino acid sequence.
7. A nucleic acid molecule encoding the antibody or fragment thereof according to any one of claims 1 to 6, or encoding the heavy chain CDR, light chain CDR, light chain variable region, and heavy chain contained in the antibody or fragment thereof Variable region, heavy chain or light chain.
8. A vector comprising the nucleic acid molecule of claim 7.
9. A host cell comprising the nucleic acid molecule of claim 7 and/or the vector of claim 8, or the host cell is composed of the nucleic acid molecule of claim 7 and/or the vector of claim 8. The vector is transformed or transfected.
10. A composition comprising the antibody or fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the vector according to claim 8, or the host cell according to claim 9;

    Preferably, the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients.
11. The antibody or fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, or the combination of claim 10 Use of the drug in the preparation of a medicament for the prevention, treatment and/or amelioration of inflammatory diseases;

    Preferably, the inflammatory disease is ulcerative colitis, Crohn’s disease, inflammatory bowel disease, rheumatoid arthritis, nephritis, glomerulonephritis, myositis, multiple sclerosis, optic neuromyelitis, atherosclerosis Like sclerosis, psoriasis, systemic lupus erythematosus (e.g. lupus or lupus nephritis of the central nervous system) or autoimmune hepatobiliary diseases.
12. The antibody or fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, or the combination of claim 10 The use of the drug in the preparation of a medicament for blocking the CX3CL1/CX3CR1 signaling pathway and/or blocking the migration of CX3CR1 expressing cells.
13. The antibody or fragment thereof of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, or the combination of claim 10 The drug is used as a medicament to study the CX3CL1/CX3CR1 signaling pathway-related or the CX3CL1 or CX3CR1 related disease mechanism in a mouse model.
14. A kit comprising the antibody or fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the vector according to claim 8, and the antibody according to claim 9 A host cell or the composition of claim 10.
15. A method for preventing, treating and/or ameliorating inflammatory diseases, the method comprising administering the antibody or fragment thereof according to any one of claims 1 to 6, claim 7 to a subject in need thereof The nucleic acid molecule, the vector of claim 8, the host cell of claim 9, or the composition of claim 10, and optionally other drugs or means.
16. A method for blocking the CX3CL1/CX3CR1 signaling pathway and/or blocking the migration of CX3CR1 expressing cells, the method comprising administering the antibody of any one of claims 1 to 6 to a subject in need thereof Or a fragment thereof, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9, or the composition of claim 10, and optionally other drugs or means.

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