Antibodies against chemokine CX3CL1 and their applications

Abstract

The present invention provides a humanized antibody against CX3CL1 or a fragment thereof, a coding nucleic acid of the antibody or the fragment, a composition comprising the antibody or the fragment, etc., and their uses in treating diseases. The antibody or the fragment provided by the present invention has a high affinity for CX3CL1, specifically blocks the CX3CL1 / CX3CR1 signaling pathway, and blocks the migratory activity of CX3CR1-expressing cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to antibodies and antigen-binding fragments thereof that specifically bind to the chemokine CX3C subtype CX3CL1, as well as applications of the antibodies and antigen-binding fragments thereof. Background Art

[0002] Chemokines are a class of functionally related small secreted proteins, named "chemokines" for their leukocyte chemotaxis and cytokine activity. In humans, this family consists of approximately 50 related molecules, with close homologs also found in other mammals. Chemokine sequences contain conserved cysteine ​​residues in 20% to 95% of their sequences. Chemokines are classified into four subtypes based on the number and spacing of cysteines: CC, CXC, XC, and CX3C (C represents cysteine ​​and X represents any amino acid).

[0003] The CX3C subtype has only one chemokine, CX3CL1, also known as fractalkine (FKN) or neurotactin, and is the only membrane-bound chemokine. CX3CL1 is expressed in a variety of cells, and its sole receptor is the chemokine receptor CX3CR1. CX3CR1 is a seven-transmembrane domain G protein-coupled receptor. Under normal circumstances, CX3CR1 is expressed on the membranes of natural killer cells (NK cells), monocytes, mast cells, platelets, and effector T cells.

[0004] CX3CL1 / CX3CR1 plays a crucial role in the arrival of inflammatory cells from the peripheral circulation to sites of inflammation. Binding of the receptor CX3CR1 to its ligand CX3CL1 triggers calcium influx, generating a chemotactic response that directs cells to specific sites in the body. CX3CR1 participates in some inflammatory processes primarily by inducing and recruiting natural killer (NK) cells, monocytes, macrophages, mast cells, and effector T cells to sites of inflammation. CX3CR1 not only acts as a chemoattractant for these cells but also, due to its unique mucin stalk structure, allows it to adhere to inflammatory cells. This makes CX3CR1 a crucial player in the pathogenesis and progression of inflammation. Research has shown that the pathological changes of many diseases are inextricably linked to CX3CR1.

[0005] Using traditional hybridoma technology, Japan's Eisai Co., Ltd. successfully screened and obtained a high-affinity mouse monoclonal antibody 3A5-2 targeting human CX3CL1. The humanized antibody H3-2L4 still maintains a high affinity for human CX3CL1 (see CN102597003A) and is currently in Phase 2 or Phase 1 / 2 clinical trials for rheumatoid arthritis and Crohn's disease. However, the humanized H3-2L4 antibody retains a large number of mouse-derived amino acids, making it relatively likely to cause an immune rejection response in humans. In addition, the CDR region that binds to the target also retains several post-translational modification sites, which poses a risk of loss of biological activity in the human body due to modification. Summary of the Invention

[0006] To minimize antibody-induced rejection immune responses, therapeutic antibodies for human use should be designed to contain as few mouse-derived amino acids as possible. To address this technical issue, the present invention aims to minimize the number of mouse-derived amino acids in the target antibody through a more optimized humanized design, resulting in an expected improved in vivo safety profile.

[0007] In addition, the present invention also aims to construct a library by randomly mutating the six CDR amino acids of the humanized antibody through cell surface display technology to screen for antibody molecules with higher affinity; and, the affinity-matured antibody can also be subjected to a second engineering transformation to construct an antibody library with full-sequence random mutations in the variable region to screen for antibodies that bind to mouse CX3CL1 with high affinity, providing antibody selection for studying the mechanism of action of CX3CL1-related diseases in mouse models.

[0008] In response to the above technical problems, the present invention aims to provide an antibody or fragment thereof, particularly a humanized antibody or fragment thereof, against the human chemokine CX3CL1. Compared to existing humanized CX3CL1 antibodies, the antibodies provided by the present invention have fewer mouse amino acids and a higher affinity for CX3CL1. A further object of the present invention is to provide further antibodies with high affinity for mouse CX3CL1. Based on the foregoing, the present invention also aims to provide applications of these antibodies.

[0009] Specifically, the present invention provides the following technical solutions.

[0010] The "fragments" of the antibodies of the present invention encompass various functional fragments of antibodies, such as antigen-binding portions thereof, such as Fab, F(ab')2 or scFv fragments.

[0011] The CX3CL1 is a fractalkine or a neuron chemokine. Preferably, the CX3CL1 is a primate mammalian CX3CL1, more preferably a human CX3CL1.

[0012] In one aspect, the present invention provides an 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) comprises three CDRs (H-CDR1, H-CDR2, H-CDR3) from a heavy chain variable region shown in any one of the following sequences:

[0013] and / or

[0014] wherein the light chain variable region (VL) comprises three CDRs (L-CDR1, L-CDR2, L-CDR3) from the light chain variable region shown in any one of the following sequences:

[0015] 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.

[0016] Preferably, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region (VH) comprises three CDRs (H-CDR1, H-CDR2, H-CDR3) from the heavy chain variable region shown in any one of the following sequences:

[0017] 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

[0018] wherein the light chain variable region (VL) comprises three CDRs (L-CDR1, L-CDR2, L-CDR3) from the light chain variable region shown in any one of the following sequences:

[0019] 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.

[0020] 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 six CDRs (H-CDR1, H-CDR2, H-CDR3; L-CDR1, L-CDR2, L-CDR3) from the following sequence combinations:

[0021] (1)SEQ ID NO:11+SEQ ID NO:23;

[0022] (2)SEQ ID NO:14+SEQ ID NO:22;

[0023] (3) SEQ ID NO:14+SEQ ID NO:23;

[0024] (4)SEQ ID NO:14+SEQ ID NO:24;

[0025] (5)SEQ ID NO:14+SEQ ID NO:25;

[0026] (6) SEQ ID NO:15+SEQ ID NO:22;

[0027] (7) SEQ ID NO:15+SEQ ID NO:23;

[0028] (8) SEQ ID NO:15+SEQ ID NO:24;

[0029] (9) SEQ ID NO:15+SEQ ID NO:25;

[0030] (10)SEQ ID NO:16+SEQ ID NO:22;

[0031] (11)SEQ ID NO:16+SEQ ID NO:23;

[0032] (12)SEQ ID NO:16+SEQ ID NO:24;

[0033] (13) SEQ ID NO:16+SEQ ID NO:25;

[0034] (14)SEQ ID NO:17+SEQ ID NO:22;

[0035] (15)SEQ ID NO:17+SEQ ID NO:23;

[0036] (16)SEQ ID NO:17+SEQ ID NO:24;

[0037] (17)SEQ ID NO:17+SEQ ID NO:25;

[0038] (18) SEQ ID NO:26+SEQ ID NO:29;

[0039] (19) SEQ ID NO:26+SEQ ID NO:30;

[0040] (20)SEQ ID NO:26+SEQ ID NO:31;

[0041] (21)SEQ ID NO:27+SEQ ID NO:29;

[0042] (22)SEQ ID NO:27+SEQ ID NO:30;

[0043] (23) SEQ ID NO:27+SEQ ID NO:31;

[0044] (24)SEQ ID NO:28+SEQ ID NO:29;

[0045] (25) SEQ ID NO: 28 + SEQ ID NO: 30; or

[0046] (26) SEQ ID NO:28+SEQ ID NO:31.

[0047] The combination of light and heavy chain CDRs provided herein is derived from the light and heavy chain variable regions described above. Based on the amino acid sequences of the given light and heavy chain variable regions, those skilled in the art can routinely determine the amino acid sequences of the CDRs contained therein. For example, according to a specific embodiment of the present invention, the Chothia numbering method is used to delineate the CDRs in the variable region amino acid sequence. Light and heavy chain CDRs and combinations thereof obtained by delineation using methods known in the art are also encompassed within the scope of the present invention.

[0048] Preferably, the heavy chain variable region (VH) and the light chain variable region (VL) comprise a CDR combination selected from the following:

[0049] (1) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPTQGDY) shown in SEQ ID NOs: 37, 44, and 42, respectively; and L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 49, 51, and 48, respectively;

[0050] (2) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 37, 41, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 51, and 48, respectively;

[0051] (3) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 37, 41, and 39, respectively; and L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 49, 51, and 48, respectively;

[0052] (4) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 37, 41, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 52, and 48, respectively;

[0053] (5) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 37, 41, and 39, respectively; and L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 49, 52, and 48, respectively;

[0054] (6) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 37, 44, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 51, and 48, respectively;

[0055] (7) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 37, 44, and 39, respectively; and L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 49, 51, and 48, respectively;

[0056] (8) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 37, 44, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 52, and 48, respectively;

[0057] (9) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 37, 44, and 39, respectively; and L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 49, 52, and 48, respectively;

[0058] (10) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NOs: 37, 41, and 40, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 51, and 48, respectively;

[0059] (11) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NOs: 37, 41, and 40, respectively; and L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 49, 51, and 48, respectively;

[0060] (12) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NOs: 37, 41, and 40, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 52, and 48, respectively;

[0061] (13) H-CDR1 (NYYIH), H-CDR2 (WYLPGDDSPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NOs: 37, 41, and 40, respectively; and L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 49, 52, and 48, respectively;

[0062] (14) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NOs: 37, 44, and 40, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 51, and 48, respectively;

[0063] (15) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NOs: 37, 44, and 40, respectively; and L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 49, 51, and 48, respectively;

[0064] (16) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NOs: 37, 44, and 40, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 52, and 48, respectively;

[0065] (17) H-CDR1 (NYYIH), H-CDR2 (WFIPGSDPPKFNERFKG), H-CDR3 (GPAPAEGDY) shown in SEQ ID NOs: 37, 44, and 40, respectively; and L-CDR1 (RASGRIHGFLA), L-CDR2 (TDNTLSG), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 49, 52, and 48, respectively;

[0066] (18) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSSKFNERFEG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 53, 54, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 51, and 48, respectively;

[0067] (19) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSSKFNERFEG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 53, 54, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNALAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 56, and 48, respectively;

[0068] (20) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 53, 55, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 51, and 48, respectively;

[0069] (21) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 53, 55, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNALAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 56, and 48, respectively;

[0070] (22) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 53, 55, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 51, and 48, respectively;

[0071] (23) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NOs: 53, 55, and 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNALAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NOs: 46, 56, and 48, respectively;

[0072] (24) H-CDR1 (NYNIH), H-CDR2 (WYLPGDDSPRFNERFKG), H-CDR3 (SPTDETQGDY) shown in SEQ ID NO: 53, 55, 39, respectively; and L-CDR1 (RASGRIHDFLA), L-CDR2 (TDNTLAE), L-CDR3 (QQFWSTPYT) shown in SEQ ID NO: 46, 51, 48, respectively.

[0073] 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 the following:

[0074] the amino acid sequence 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, 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 an amino acid sequence that is at least 75% identical to said amino acid sequence; and / or

[0075] The light chain variable region comprises a sequence selected from the group consisting of:

[0076] The amino acid sequence of 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, or an amino acid sequence that is at least 75% identical to said amino acid sequence.

[0077] Preferably, according to a specific embodiment of the present invention, in the antibody or fragment thereof, the heavy chain variable region and light chain variable region comprised by the antibody or fragment thereof are selected from the following combinations:

[0078] (1) the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 11; and, the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 23;

[0079] (2) the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 22;

[0080] (3) the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 23;

[0081] (4) the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 24;

[0082] (5) the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 14; and, the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 25;

[0083] (6) the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 22;

[0084] (7) the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 23;

[0085] (8) the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 24;

[0086] (9) the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 25;

[0087] (10) the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 22;

[0088] (11) the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 23;

[0089] (12) the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 24;

[0090] (13) the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 16; and, the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 25;

[0091] (14) the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 22;

[0092] (15) the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 23;

[0093] (16) the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 24;

[0094] (17) the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 17; and, the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 25;

[0095] (18) the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 26; and, the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 29;

[0096] (19) the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 26; and, the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 30;

[0097] (20) the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 26; and, the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 31;

[0098] (21) the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 27; and, the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 29;

[0099] (22) the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 27; and, the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 30;

[0100] (23) the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 27; and, the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 31;

[0101] (24) the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 28; and, the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 29;

[0102] (25) the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 28; and, the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 30; or

[0103] (26) the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 28; and, the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 31.

[0104] Preferably, the antibody or fragment thereof is an antibody or antigen-binding fragment thereof against chemokine CX3CL1, preferably mammalian CX3CL1, more preferably human CX3CL1, cynomolgus monkey CX3CL1 or mouse CX3CL1;

[0105] Preferably, the antibody or antigen-binding fragment thereof comprises a VH framework region and a VL framework region;

[0106] The antibody is in any form such as a monoclonal antibody, a single-chain antibody, a bifunctional antibody, a single-domain antibody, a nanobody, a fully or partially humanized antibody or a chimeric antibody, or the antigen-binding fragment is a half antibody or an antigen-binding fragment of an antibody or a half antibody, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

[0107] Further preferably, the antibody or fragment thereof further comprises a human or murine constant region, preferably a human or murine heavy chain constant region (CH) and / or a light chain constant region (CL); preferably, the antibody or fragment thereof comprises a heavy chain and a light chain;

[0108] More preferably, the antibody or fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a light chain constant region of kappa or lambda type.

[0109] 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 of the IgG4 subtype, and the light chain constant region is of the κ type;

[0110] Preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence as shown in SEQ ID NO: 35, or an amino acid sequence that is at least 75% identical to the amino acid sequence; preferably, the light chain constant region of the monoclonal antibody comprises the amino acid sequence as shown in SEQ ID NO: 36, or an amino acid sequence that is at least 75% identical to the amino acid sequence.

[0111] 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 identity ≥ 75%.

[0112] In particular, the antibodies or fragments thereof of the present invention comprise at least a heavy chain variable region and a light chain variable region, both of which include the above-mentioned CDRs and an intervening framework region, and the arrangement of the various domains is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this regard, the "at least 75% identity" resulting in a maximum of 25% difference in amino acid sequence may exist in any framework region in the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibodies or fragments thereof of the present invention. The difference may be caused by deletion, addition or substitution of amino acids at any position, wherein the substitution may be a conservative substitution or a non-conservative substitution.

[0113] According to a specific embodiment of the present invention, particularly preferably, the present invention provides the following antibodies:

[0114] The humanized antibody AM85 comprises a heavy chain variable region set forth in SEQ ID NO: 14, a light chain variable region set forth in SEQ ID NO: 22, a heavy chain constant region set forth in SEQ ID NO: 35, and a light chain constant region set forth in SEQ ID NO: 36;

[0115] The humanized antibody AM85-M3 comprises a heavy chain variable region set forth in SEQ ID NO: 26, a light chain variable region set forth in SEQ ID NO: 31, a heavy chain constant region set forth in SEQ ID NO: 35, and a light chain constant region set forth in SEQ ID NO: 36;

[0116] The humanized antibody AM85-M8 comprises a heavy chain variable region shown in SEQ ID NO: 28, a light chain variable region shown in SEQ ID NO: 30, a heavy chain constant region shown in SEQ ID NO: 35, and a light chain constant region shown in SEQ ID NO: 36.

[0117] 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, light chain CDR, light chain variable region, heavy chain variable region, heavy chain or light chain contained in the antibody or fragment thereof.

[0118] In another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phage vector.

[0119] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect a host cell or enter a host cell in any manner for purposes such as storage or expression of antibodies. Therefore, in another aspect, the present invention provides a host cell comprising a nucleic acid molecule and / or vector of the present invention, or transformed or transfected by a 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.

[0120] 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 techniques known in the art. The antibodies or fragments thereof, nucleic acid molecules, vectors and / or host cells can be included in compositions (e.g., pharmaceutical compositions), more particularly in pharmaceutical preparations, and thus used for various purposes according to actual needs.

[0121] 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 a pharmaceutically acceptable excipient.

[0122] As antibodies or fragments thereof that bind to CX3CL1, especially human CX3CL1, the present invention also provides related applications of the above subject matter.

[0123] Specifically, in another aspect, the invention provides use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell or composition in the preparation of a medicament for preventing, treating and / or ameliorating inflammatory diseases;

[0124] Preferably, the inflammatory disease is ulcerative colitis, Crohn's disease, inflammatory bowel disease, rheumatoid arthritis, nephritis, glomerulonephritis, myositis, multiple sclerosis, neuromyelitis optica, atherosclerosis, psoriasis, systemic lupus erythematosus (e.g., lupus of the central nervous system or lupus nephritis), or autoimmune hepatobiliary disease.

[0125] Alternatively, the invention provides use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell or composition in the preparation of a drug for blocking the CX3CL1 / CX3CR1 signaling pathway and / or blocking the migration of CX3CR1-expressing cells.

[0126] Alternatively, the invention provides the use of the antibody or fragment thereof, nucleic acid molecule, vector, host cell or composition as a medicament for studying the mechanism of action of CX3CL1 / CX3CR1 signaling pathway-related or CX3CL1 or CX3CR1-related diseases in a mouse model.

[0127] In another aspect, the present invention provides a kit comprising the antibody or fragment thereof, nucleic acid molecule, vector, host cell, and / or composition described herein. The kit can be used for therapeutic, diagnostic, or detection purposes. For example, the antibody or fragment thereof can be contacted with a sample to detect the presence of CX3CL1.

[0128] The present invention minimizes the number of murine amino acids in the target antibody (mouse monoclonal antibody 3A5-2) through a more optimized humanized design, thereby improving the in vivo safety of the humanized antibody. Furthermore, using cell surface display technology, the present invention randomly mutates the amino acids in the six CDR regions of the humanized antibody to construct a library. Through flow cytometry sorting of the antibody library, antibody molecules with higher affinity than the original humanized antibody H3-2L4 of 3A5-2 were screened. Compared with H3-2L4, some of the affinity-matured antibodies even have an affinity increase of one order of magnitude in KD, and block the CX3CL1 / CX3CR1 signaling pathway with comparable blocking efficiency and a 10%-20% increase in maximum blocking rate.

[0129] Furthermore, the present invention also performed a second engineering modification on the affinity matured antibody, and similarly constructed an antibody library with full-sequence random mutations in the variable region. By measuring the binding kinetic parameters of the secondary engineered antibodies to mouse, cynomolgus macaque, and human CX3CL1, antibodies with high-affinity binding to mouse CX3CL1 were screened. While retaining their binding activity to human and cynomolgus macaque CX3CL1, the antibody's KD for binding to mouse CX3CL1 was increased by 30-40 times to the nanomolar level. Therefore, it can be used as a surrogate antibody to study the mechanism of action of CX3CL1-related diseases in mouse models.

[0130] Furthermore, the present invention thoroughly studied the physicochemical properties of the antibody and found that its monomer rate, hydrophilicity, and melting point (Tm) were all within normal levels. In particular, testing revealed that the antibody of the present invention can block the migration activity of CX3CR1-expressing cells in vitro, with a five-fold improvement compared to H3-2L4.

[0131] In summary, compared with existing CX3CL1 antibodies, the antibodies provided by the present invention are more ideal clinical lead drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0133] Figure 1 The humanized design of the murine antibody 3A5-2 is shown.

[0134] Figure 2 Shown are the six CDRs of the humanized antibody 3A5-2_hz20 and the mutation site selection for affinity maturation engineering.

[0135] Figure 3 Schematic diagram of the two-step PCR method using mutant primers to prepare mutant single-chain antibody genes in affinity maturation modification.

[0136] Figure 4The results of FACS binding analysis of mutant clones obtained by flow cytometry sorting and antigen are shown.

[0137] Figure 5 The results of a cAMP experiment showing that affinity-matured antibodies blocked the target downstream signaling pathway.

[0138] Figure 6 The results of the experiment showing that affinity matured antibodies and engineered antibodies blocked the migration activity of CX3CR1-expressing cells. DETAILED DESCRIPTION

[0139] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0140] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0141] Human CX3CL1: amino acid sequence shown in SEQ ID NO: 32;

[0142] Mouse CX3CL1: amino acid sequence shown in SEQ ID NO: 33;

[0143] Cynomolgus monkey CX3CL1: amino acid sequence shown in SEQ ID NO: 34;

[0144] Heavy chain constant region: amino acid sequence shown in SEQ ID NO: 35;

[0145] Light chain constant region: amino acid sequence shown in SEQ ID NO: 36;

[0146] Human CX3CR1: The amino acid sequence is shown in SEQ ID NO: 58.

[0147] Example 1 Humanized design of mouse monoclonal antibody 3A5-2 and in vitro binding activity determination of the humanized antibody

[0148] 1.1 Humanized design of mouse monoclonal antibody 3A5-2

[0149] Combining the antibody coding schemes of Kabat and Chothia, the amino acid sequence regions of the six antigen complementarity determining clusters (CDRs) of the heavy and light chains of the murine antibody 3A5-2 and the framework region (FR) that supports the conserved three-dimensional conformation of the antibody were determined. Subsequently, by analyzing and searching known human antibody sequences, the human antibody heavy chain variable region sequence that is most similar to the murine antibody, such as IGHV1-69, was selected. Its antibody framework region sequence was selected as a template, and the murine antibody heavy chain CDR was combined with the human antibody FR to generate a humanized antibody heavy chain variable region sequence. In the same process, the IGKV1-13 antibody framework region sequence was selected as a template to generate a humanized antibody light chain variable region sequence. In addition, the back mutation site was determined: the designed humanized antibody sequence and the original murine antibody sequence were compared to check which amino acids were different and whether these amino acids played an important role in supporting the antibody structure or in binding to the antigen. At the same time, check whether the humanized sequence has potential post-translational modification sites, such as N (asparagine) glycosylation site, N deamidation site, D (aspartic acid) isomerization site, etc.

[0150] The humanized design of 3A5-2 is shown in Figure 1 , which shows three humanized sequences (3A5-2_VH_hz0, 3A5-2_VH_hz1 and 3A5-2_VH_hz2; 3A5-2_VK_hz0, 3A5-2_VK_hz1 and 3A5-2_VK_hz2) of the 3A5-2 heavy chain variable region (3A5-2_VH) ​​and light chain variable region (3A5-2_VK), respectively. The humanized sequence with the suffix "_hz0" indicates that the murine antibody CDR is directly transplanted into the human framework region.

[0151] The humanized heavy chain variable region gene was synthesized and constructed into a mammalian cell expression vector containing the heavy chain constant region gene of the human monoclonal antibody IgG4 subclass. Similarly, the light chain variable region gene was synthesized and constructed into a mammalian cell expression vector containing the light chain constant region gene of the human monoclonal antibody κ subclass. The constructed humanized antibody heavy chain vector and light chain vector were cross-paired (Table 1) and transfected into HEK293 cells using polyethyleneimine (PEI). After approximately 7 days, the cell supernatant was collected and purified using Protein A to obtain the humanized antibody protein.

[0152] The humanized antibodies were named "mouse antibody abbreviation_hz", among which the antibodies in which the mouse antibody CDR was directly transplanted to the human framework region were named "mouse antibody abbreviation_hz00", and the antibodies obtained by further modification were numbered according to different sequences.

[0153] Table 1. Humanized antibodies expressed by cross-pairing of heavy and light chains

[0154]

[0155] 1.2 In vitro binding activity assay of humanized antibodies

[0156] The binding kinetics of the humanized antibody to the antigen human CX3CL1 (hCX3CL1) were analyzed using a Fortebio (BLITZ pro1.1.0.28) instrument. Before the assay, the NTA bioprobe was soaked in PBS for 10 minutes. The probe was then placed in PBS containing 100 nM antigen for 300 seconds to capture the His-tagged antigen. The probe then bound to 100 nM antibody for 400 seconds. The probe was then transferred to PBS for 600 seconds of dissociation. After the experiment, the blank control response was subtracted, and a 1:1 Langmuir binding model fit was performed using the software to calculate the kinetic constants for antigen-antibody binding. The results are shown in Table 2.

[0157] Table 2. Binding kinetic parameters of antibodies obtained after humanization of mouse anti-3A5-2

[0158] Ab ID response <![CDATA[K D (M)]]> <![CDATA[K on (1 / Ms)]]> <![CDATA[k off (s -1 )]]> <h2 style=";text-align:left;direction:ltr"><![CDATA[K <h2 style=";text-align:left;direction:ltr"> off <h2 style=";text-align:left;direction:ltr"> / K<h2 style=";text-align:left;direction:ltr"> off <h2 style=";text-align:left;direction:ltr"> (H3-2L4)]]><h2 style=";text-align:left;direction:ltr"> 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

[0159] Example 2 Affinity maturation of humanized antibodies

[0160] 2.1 Design and construction of affinity matured antibody library

[0161] The humanized antibody 3A5-2_hz20 was selected as the template for affinity maturation. The heavy chain variable region and light chain variable region of 3A5-2_hz20 were 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, while the framework region will remain unchanged during affinity maturation. A total of 6 CDRs of the heavy and light chains were constructed into separate antibody libraries. Based on experience and sequence characteristics, some amino acids in H-CDR3 remain unchanged during affinity maturation, such as Figure 2 Invariant amino acids are shown in bold italics.

[0162] Random mutagenesis primers for CDRs were designed and synthesized, introducing only 20% nucleotide mutations at each mutation position, with approximately 20 base pairs of unmutated bases on both sides of the primers. The mutations were incorporated into the final single-chain antibody gene through two-step PCR using the mutagenesis primers and other conventional primers. The single-chain antibody gene has a 148 bp overhang at the 5' end and a 222 bp overhang at the 3' end, which are identical to the sequences on the display DNA vector pYD, the so-called homology region. A schematic diagram of this two-step PCR method is shown in Figure 3 .

[0163] 4 μg of linearized vector and 12 μg of single-chain antibody gene were co-transformed into yeast strain EBY100 by electroporation. The yeast then utilized its homologous recombination machinery to ligate the single-chain antibody gene into the display DNA vector. The library capacity of each CDR region mutant antibody library is shown in Table 3.

[0164] Table 3. Library capacity of 6 CDR mutant antibody libraries

[0165] # Antibody Library Number of amino acid mutations Storage capacity 1 H1 10 <![CDATA[1.0×10 8 ]]> 2 H2 8 <![CDATA[1.7×10 8 ]]> 3 H3 7 <![CDATA[1.6×10 8 ]]> 4 L1 8 <![CDATA[1.2×10 8 ]]> 5 L2 8 <![CDATA[1.1×10 8 ]]> 6 L3 7 <![CDATA[5×10 7 ]]>

[0166] 2.2 Flow cytometry sorting of affinity matured antibody library

[0167] After one round of magnetic bead sorting, the six CDR mutant antibody libraries underwent four rounds of FACS sorting at decreasing antigen concentrations. The first round used 300 nM biotinylated hCX3CL1, the second round reduced to 100 nM, the third to 10 nM, and the fourth to 1 nM. After each round of FACS sorting, 0.1% to 0.5% of the clones in the library were collected for culture and then subjected to the next round of sorting, ultimately isolating mutant clones with improved affinity.

[0168] 96 yeast colonies were selected for sequencing analysis, and ultimately 7 unique mutant clones were selected from the H2 library, 4 unique mutant clones were selected from the H3 library, and 5 unique mutant clones were selected from the L1 and L2 libraries based on affinity ranking and mutation, respectively, as shown in Table 4.

[0169] Table 4. Mutant clones selected by flow cytometry

[0170]

[0171] 2.3 Affinity analysis of sorted clones

[0172] The yeast of mutant clones was inoculated into yeast culture medium to induce the expression of relevant single-chain antibodies. Yeast cells were collected by centrifugation at 14000rpm for 1 minute and washed once with PBS containing 1% BSA. The density of resuspended yeast cells was 5000000 / ml, and 100μl of cells were added to each well of a 96-well U-shaped plate. 100μl of gradient diluted biotinylated antigen solution was added, incubated with shaking at room temperature for 30 minutes, and then placed on ice for 10 minutes. Centrifuge at 14000rpm for 1 minute at 4°C to collect cells, and wash once with pre-cooled PBS containing 1% BSA. Add 100μl of 1:500 diluted SA fluorescent secondary antibody and incubate on ice for 30 minutes. Centrifuge at 14000rpm for 1 minute at 4°C to collect cells, and wash twice with pre-cooled PBS containing 1% BSA. The average fluorescence reading of the cell population was analyzed by flow cytometry. The results are as follows. Figure 4As shown, the EC50 values ​​of the mutant clones after 5 rounds of sorting for binding to the antigen were higher than that of the H3-2L4 antibody.

[0173] 2.4 Recombinant Expression of Affinity Matured Antibody IgG

[0174] After comprehensive analysis of the mutation sites of the affinity-enhancing clones above, the relevant mutation sites of the six CDRs were combined. The antibody variable regions containing various combined mutation sites are shown in the sequence table as 11 heavy chain variable regions (AM_vh1 to AM_vh11) and 8 light chain variable regions (AM_vl1 to AM_vl8), as well as Figure 1 35A-2_VK-hz0 shown.

[0175] The heavy chain variable region gene was synthesized and constructed into a mammalian cell expression vector containing a heavy chain constant region gene of the human monoclonal antibody IgG4 subclass. Similarly, the light chain variable region gene was synthesized and constructed into a mammalian cell expression vector containing a light chain constant region gene of the human monoclonal antibody κ subclass. As described in Section 1.1 of Example 1 for humanized antibody expression, the constructed heavy chain vector and light chain vector of the affinity matured antibody were cross-paired and transfected into HEK293 cells using polyethyleneimine (PEI). After approximately 7 days, the cell supernatant was collected and purified using Protein A to obtain the affinity matured antibody protein.

[0176] The affinity matured antibodies were 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, e.g., AM85 below comprises AM_vh8 and AM_vl5.

[0177] 2.5 Determination of kinetic parameters of affinity-matured antibodies binding to human CX3CL1

[0178] Antibody-antigen interaction was measured using a GE BIAcore S200 instrument. Following the instructions for the GE Human Antibody Capture Kit (Cat. No. BR-1008-39, Lot 10261753), the CM5 sensor chip was first saturated with the maximum amount of anti-human Fc antibody in both the analysis channel and the control sample channel. A buffer containing 7.5 μg / ml anti-human CX3CL1 chimeric antibody was then passed through the analysis channel to ensure uniform distribution. Finally, a gradient dilution of the antigen sample (starting at 20 nM, diluted 1:3 over eight concentrations, with a repeat at 0.741 nm) was passed through both the analysis channel and the control sample channel. The light response generated by antibody-antigen binding was measured. The binding constant, Kon, and dissociation constant, Koff, as well as the affinity constant, KD, were obtained using instrument software fitting and analysis.

[0179] The results showed that most affinity-matured antibodies, which underwent heavy-light chain recombination, showed improved antigen binding activity compared to H3-2L4. Antibodies containing AM_vh8, AM_vh9, AM_vl5, or AM_vl6 showed an improvement in binding activity by as much as an order of magnitude. See Table 5.

[0180] Table 5. Kinetic parameters of IgG antibodies after affinity maturation analyzed by Biacore

[0181]

[0182]

[0183] 2.6 Determination of the blocking activity of affinity-matured antibodies on CX3CL1 downstream signaling pathways

[0184] The cAMP assay was used for detection.

[0185] First, a stable cell line stably expressing human CX3CR1 was established in CHO-K1 cells and named CHOK1-CX3CR1 cells. The cells were cultured to 70% to 90% confluence, harvested by trypsinization, centrifuged at 400g for 5 minutes, and the supernatant discarded. The cells were resuspended in medium containing IBMX to a density of 125,000 cells / ml, and 4μl of cells were added to each well of a 384-well plate. The antibody to be tested was dissolved in medium containing 1% BSA at a starting concentration of 30μg / ml. 8 dilutions (including 0) were prepared 3-fold, and 2μl of each concentration of antibody was added to a 384-well plate. CX3CL1 protein was prepared at a concentration of 0.2μg / ml in medium containing 1% BSA, 2μl of which was added to a 384-well plate and incubated at room temperature for 10 minutes. Forskolin was prepared at a concentration of 5μM in medium containing 1% BSA, 2μl of which was added to a 384-well plate and incubated at 37°C for 30 minutes. Finally, add 5 μl of anti-cAMP-Cryptate solution and 5 μl of cAMP-d2 solution, and let it stand at room temperature for 1 hour before reading the plate for detection.

[0186] The results showed that compared with the H3-2L4 antibody, the affinity matured antibody of the present invention had no significant difference in IC50 value for blocking the signaling pathway between CX3CL1 and its receptor, while the maximum blocking rate was increased by about 10%-20%. Figure 5 and Table 6.

[0187] Table 6. cAMP assay results of affinity matured antibodies blocking target downstream signaling pathways

[0188] 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

[0189] Example 3Engineering of surrogate antibodies that bind mouse CX3CL1

[0190] 3.1 Construction and screening of engineered antibody libraries

[0191] The method is as described in the antibody affinity maturation modification of Example 2. The affinity matured antibody AM85 was selected as the template for secondary engineering modification, and an antibody library with random mutations in the entire variable region of the heavy and light chains was designed. After 5 rounds of FACS screening with decreasing concentrations of mouse CX3CL1 antigen, clones with FACS binding antigen ability stronger than the template were obtained, which were re-expanded and cultured, and DNA was extracted for sequencing. The sequencing results were analyzed, and new antibody heavy and light chain variable region genes were synthesized and constructed into a eukaryotic expression vector. The heavy and light chain expression vectors were cross-combined, transiently transfected into HEK293 cells for recombinant expression, and purified by Protein A in one step to obtain an antibody protein with an SDS-PAGE purity of more than 95%.

[0192] The variable regions of various antibodies are shown in the sequence table as three heavy chain variable regions (AM_vh8_m1 to AM_vh8_m3) and three light chain variable regions (AM_vl5_m1 to AM_vl5_m3).

[0193] Table 7. Engineered antibodies expressed by heavy and light chain cross-pairing

[0194]

[0195] 3.2 Determination of the binding activity of engineered antibodies to CX3CL1 from different species

[0196] Methods As described above, the binding kinetic parameters of the secondary engineered antibody to mouse, cynomolgus monkey and human CX3CL1 were determined using the GE BIAcore instrument S200.

[0197] Results showed that the re-engineered antibody retained its binding activity to human and cynomolgus monkey CX3CL1, while also increasing its KD value for mouse CX3CL1 by 30-40 times from the original level of approximately 50 nM to the nanomolar range. This re-engineered antibody can be used as a surrogate antibody to study the mechanisms of action of CX3CL1-related diseases in mouse models. See Table 8.

[0198] Table 8. Species cross-binding activity of engineered antibodies

[0199]

[0200] 3.3 Determination of chemotactic activity of engineered antibodies blocking CX3CL1 receptor-expressing cells

[0201] CHOK1-CX3CR1 cells stably expressing CX3CR1 protein were cultured to approximately 50% confluence. The cells were washed twice with PBS and then starved overnight in serum-free medium. The cells were harvested by trypsinization and centrifuged at 400 g for 5 minutes. The supernatant was discarded. The cells were resuspended in serum-free medium to a concentration of 3 × 10 6 Cells were cultured at a density of 100 cells / ml. 600 μl of culture medium was added to each well of a 24-well plate, along with human CX3CL1 at a final concentration of 0.2 μg / ml and various concentrations of the test antibody. A migration chamber was placed in the 24-well plate, and 50 μl of cells were added to the upper migration chamber. After incubating the 24-well plate in a 37°C incubator for 4 hours, the upper migration chamber was transferred to another empty 24-well plate containing 600 μl of trypsin and incubated in a 37°C incubator for another 10 minutes. After centrifugation at 500 g for 1 minute, the migration chamber was discarded and 50 μl of FBS was added to terminate the enzyme reaction. Cells from corresponding wells of two 24-well plates were pooled and centrifuged at 500 g for 5 minutes. The supernatant was discarded and 120 μl of CellTiter-Glo was added. The cells were incubated at room temperature for 10 minutes, and the luminescence intensity was measured.

[0202] The results showed that compared with the H3-2L4 antibody, the antibody AM85 after the first affinity maturation and the antibodies AM85-M3 and AM85-M8 after the second mouse CX3CL1 cross-binding activity modification had an IC50 value significantly increased by about 5 times in terms of in vitro blocking the migration activity of CX3CR1-expressing cells. Figure 6 and Table 9.

[0203] Table 9. Results of the antibody-blocking experiment on the migration activity of CX3CR1-expressing cells

[0204] 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

[0205] 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 based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention. Sequence Listing <110> Maiwei (Shanghai) Biotechnology Co., Ltd. Shanghai Puming Biotechnology Co., Ltd. <120> Antibodies targeting chemokine CX3CL1 and their applications <130> LC19110082 <160> 58 <170> SIPOSequenceListing 1.0 <210> 1 <211> 119 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> 3A5-2_VH_hz0 <400> 1 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Thr Asp Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 2 <211> 119 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> 3A5-2_VH_hz1 <400> 2 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Thr Asp Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115[[ID=​​​​​​​​​​​​<222> ()..() <223> 3A5-2_VH_hz2 <400> 3 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Gln Gly Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95<0OO0670>[[ID=ao]]Ala Thr Gly Pro Thr Gln Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 了` <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() It should be noted that there seems to be an error in the original text where <210> 了 is incorrect. It is likely supposed to be something else. This might cause some inaccuracies in the translation if the context is not fully understood.<223> 3A5-2_VK_hz0 <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Asn Glu Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> 3A5-2_VK_hz1 <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Leu 35 40 45 Tyr Asn Glu Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 6 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> 3A5-2_VK_hz2 <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Leu 35 40 45 Tyr Asn Glu Lys Thr Leu Ala Gln Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 122 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh1 <400> 7 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Gln Gly Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Pro Thr Asp Glu Thr Gln Gly Asp Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 8 <211> 121 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh2 <400> 8 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45<Gly Trp Ile Tyr Pro Gly Gln Gly Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Ala Pro Ala Glu Gly Asp Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 9 <211> 119 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh3 <400> 9 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met[[ID=​​Gly Trp Tyr Leu Pro Gly Asp Asp Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Thr Gln Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 10 <211> 119 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh4 <400> 10 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Val Asp Pro Gly Tyr Gly Pro His Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Thr Gln Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 11 <211> 119 <212> PRT <213> Artificial <220> <​​​​​​​​​​​​​​​​​​​Gly Trp Phe Ile Pro Gly Ser Asp Pro Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Thr Gln Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 12 <211> 119 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh6 <400> 12 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Leu Leu Pro Gly Glu Asp Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Thr Gln Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 13 <211> 119 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh7 <​​​​​​​​​​​​​Gly Trp Thr Tyr Pro Gly Gln Gly Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Thr Gln Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 14 <211> 122 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh8 <400> 14 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Tyr Leu Pro Gly Asp Asp Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Pro Thr Asp Glu Thr Gln Gly Asp Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 15 <211> 122 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh9 <400> 15 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Phe Ile Pro Gly Ser Asp Pro Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Pro Thr Asp Glu Thr Gln Gly Asp Tyr Phe Asp Tyr Trp 100 105 110[[ID=I5]] Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 16 <211> 121 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh10 <400> 16 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Tyr Leu Pro Gly Asp Asp Ser Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Ala Pro Ala Glu Gly Asp Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 17 <211> 121 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh11 <400> 17 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Phe Ile Pro Gly Ser Asp Pro Pro Lys Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Pro Ala Pro Ala Glu Gly Asp Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 18 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl1 <(400)> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Arg Ile His Asp Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Asn Glu Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl2 <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Arg Ile His Gly Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Asn Glu Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 20 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl3 <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Thr Asp Asn Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 21 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl4 <400> 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Thr Asp Asn Thr Leu Ser Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 22 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl5 <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Arg Ile His Asp Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Thr Asp Asn Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 [[ID=]]<211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl6 <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Arg Ile His Gly Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Thr Asp Asn Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 24 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl7 <400> 24 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly<--Removed duplicate line break here--> 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Arg Ile His Asp Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Thr Asp Asn Thr Leu Ser Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 25 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl8 <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Arg Ile His Gly Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Thr Asp Asn Thr Leu Ser Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 26 <211> 122 <212> PRT <213> Artificial <220> <221> PEPTIDE <222>()..() <223> AM_vh8_m1 <400> 26 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 三十 Asn Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Tyr Leu Pro Gly Asp Asp Ser Ser Lys Phe Asn Glu Arg Phe 50 55 60 Glu Gly Arg Val Thr Ile Thr Ala Asp Arg Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Pro Thr Asp Glu Thr Gln Gly Asp Tyr Phe Asp Tyr Trp 100 105 110<**0001225**>Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 27 <211> 122 <212> PRT <213> Artificial[[ID=3**1**]] <220> <221> PEPTIDE <222> ()..() <223> AM_vh8_m2 <400> 27 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Met Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Asn Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Tyr Leu Pro Gly Asp Asp Ser Pro Arg Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Pro Thr Asp Glu Thr Gln Gly Asp Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 28 <211> 122 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vh8_m3 <400> 28 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Asn Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Tyr Leu Pro Gly Asp Asp Ser Pro Arg Phe Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Pro Thr Asp Glu Thr Gln Gly Asp Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120​​​​​​​​​​​​​​​​​​​​​​​​​​​Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Phe Thr Asp Asn Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 30 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl5_m2 <400> 30 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Arg Ile His Asp Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu<00​​Tyr Thr Asp Asn Ala Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Thr Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 31 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> AM_vl5_m3 <400> 31 Asp Ile Arg Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Arg Ile His Asp Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Thr Asp Asn Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 32 <211> 397 <212> PRT <213> human CX3CL1 <400> 32 Met Ala Pro Ile Ser Leu Ser Trp Leu Leu Arg Leu Ala Thr Phe Cys 1 5 10 15 His Leu Thr Val Leu Leu Ala Gly Gln His His Gly Val Thr Lys Cys 20 25 30 Asn Ile Thr Cys Ser Lys Met Thr Ser Lys Ile Pro Val Ala Leu Leu 35 40 45 Ile His Tyr Gln Gln Asn Gln Ala Ser Cys Gly Lys Arg Ala Ile Ile 50 55 60 Leu Glu Thr Arg Gln His Arg Leu Phe Cys Ala Asp Pro Lys Glu Gln 65 70 75 80 Trp Val Lys Asp Ala Met Gln His Leu Asp Arg Gln Ala Ala Ala Leu 85 90 95 Thr Arg Asn Gly Gly Thr Phe Glu Lys Gln Ile Gly Glu Val Lys Pro 100 105 110 Arg Thr Thr Pro Ala Ala Gly Gly Met Asp Glu Ser Val Val Leu Glu 115 120 125 Pro Glu Ala Thr Gly Glu Ser Ser Ser Leu Glu Pro Thr Pro Ser Ser 130 135 140 Gln Glu Ala Gln Arg Ala Leu Gly Thr Ser Pro Glu Leu Pro Thr Gly 145 150 155 160 Val Thr Gly Ser Ser Gly Thr Arg Leu Pro Pro Thr Pro Lys Ala Gln 165 170 175 Asp Gly Gly Pro Val Gly Thr Glu Leu Phe Arg Val Pro Pro Val Ser 180 185 190 Thr Ala Ala Thr Trp Gln Ser Ser Ala Pro His Gln Pro Gly Pro Ser 195 200 205 Leu Trp Ala Glu Ala Lys Thr Ser Glu Ala Pro Ser Thr Gln Asp Pro 210 215 220 Ser Thr Gln Ala Ser Thr Ala Ser Ser Pro Ala Pro Glu Glu Asn Ala 225 230 235 240 Pro Ser Glu Gly Gln Arg Val Trp Gly Gln Gly Gln Ser Pro Arg Pro 245 250 255 Glu Asn Ser Leu Glu Arg Glu Glu Met Gly Pro Val Pro Ala His Thr 260 265 270 Asp Ala Phe Gln Asp Trp Gly Pro Gly Ser Met Ala His Val Ser Val 275 280 285 Val Pro Val Ser Ser Glu Gly Thr Pro Ser Arg Glu Pro Val Ala Ser 290 295 300 Gly Ser Trp Thr Pro Lys Ala Glu Glu Pro Ile His Ala Thr Met Asp 305 310 315 320 Pro Gln Arg Leu Gly Val Leu Ile Thr Pro Val Pro Asp Ala Gln Ala 325 330 335 Ala Thr Arg Arg Gln Ala Val Gly Leu Leu Ala Phe Leu Gly Leu Leu 340 345 350 Phe Cys Leu Gly Val Ala Met Phe Thr Tyr Gln Ser Leu Gln Gly Cys 355 360 365 Pro Arg Lys Met Ala Gly Glu Met Ala Glu Gly Leu Arg Tyr Ile Pro 370 375 380 Arg Ser Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val 385 390 395 <210> 33 <211> 395 <212> PRT <213> mouse CX3CL1 <400> 33 Met Ala Pro Ser Pro Leu Ala Trp Leu Leu Arg Leu Ala Ala Phe Phe 1 5 10 15 His Leu Cys Thr Leu Leu Pro Gly Gln His Leu Gly Met Thr Lys Cys 20 25 30 Glu Ile Met Cys Asp Lys Met Thr Ser Arg Ile Pro Val Ala Leu Leu 35 40 45 Ile Arg Tyr Gln Leu Asn Gln Glu Ser Cys Gly Lys Arg Ala Ile Val 50 55 60 Leu Glu Thr Thr Gln His Arg Arg Phe Cys Ala Asp Pro Lys Glu Lys 65 70 75 80 Trp Val Gln Asp Ala Met Lys His Leu Asp His Gln Ala Ala Ala Leu 85 90 95 Thr Lys Asn Gly Gly Lys Phe Glu Lys Arg Val Asp Asn Val Thr Pro 100 105 110 Gly Ile Thr Leu Ala Thr Arg Gly Leu Ser Pro Ser Ala Leu Thr Lys 115 120 125 Pro Glu Ser Ala Thr Leu Glu Asp Leu Ala Leu Glu Leu Thr Thr Ile 130 135 140 Ser Gln Glu Ala Arg Gly Thr Met Gly Thr Ser Gln Glu Pro Pro Ala 145 150 155 160 Ala Val Thr Gly Ser Ser Leu Ser Thr Ser Glu Ala Gln Asp Ala Gly 165 170 175 Leu Thr Ala Lys Pro Gln Ser Ile Gly Ser Phe Glu Ala Ala Asp Ile 180 185 190 Ser Thr Thr Val Trp Pro Ser Pro Ala Val Tyr Gln Ser Gly Ser Ser 195 200 205 Ser Trp Ala Glu Glu Lys Ala Thr Glu Ser Pro Ser Thr Thr Ala Pro 210 215 220 Ser Pro Gln Val Ser Thr Thr Ser Pro Ser Thr Pro Glu Glu Asn Val 225 230 235 240 Gly Ser Glu Gly Gln Pro Pro Trp Val Gln Gly Gln Asp Leu Ser Pro 245 250 255 Glu Lys Ser Leu Gly Ser Glu Glu Ile Asn Pro Val His Thr Asp Asn 260 265 270 Phe Gln Glu Arg Gly Pro Gly Asn Thr Val His Pro Ser Val Ala Pro 275 280 285 Ile Ser Ser Glu Glu Thr Pro Ser Pro Glu Leu Val Ala Ser Gly Ser 290 295 300 Gln Ala Pro Lys Ile Glu Glu Pro Ile His Ala Thr Ala Asp Pro Gln 305 310 315 320 Lys Leu Ser Val Leu Ile Thr Pro Val Pro Asp Thr Gln Ala Ala Thr 325 330 335 Arg Arg Gln Ala Val Gly Leu Leu Ala Phe Leu Gly Leu Leu Phe Cys 340 345 350 Leu Gly Val Ala Met Phe Ala Tyr Gln Ser Leu Gln Gly Cys Pro Arg 355 360 365 Lys Met Ala Gly Glu Met Val Glu Gly Leu Arg Tyr Val Pro Arg Ser 370 375 380 Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val 385 390 395 <210> 34 <211> 408 <212> PRT <213> cyno CX3CL1 <400> 34 Met Ala Pro Ile Ser Leu Ser Trp Leu Leu His Leu Ala Thr Leu Cys 1 5 10 15 His Leu Thr Val Leu Leu Ala Gly Gln His His Gly Val Thr Lys Cys 20 25 30 Asn Ile Thr Cys Ser Lys Met Thr Ser Lys Ile Pro Val Ala Leu Leu 35 40 45 Ile His Tyr Gln Gln Asn Gln Glu Ser Cys Gly Lys Arg Ala Ile Val 50 55 60 Leu Glu Thr Arg Gln His Arg Leu Phe Cys Ala Asp Pro Lys Glu Gln 65 70 75 80 Trp Val Lys Asp Ala Met Gln His Leu Asp Arg Gln Ala Ala Ala Leu 85 90 95 Thr Arg Asn Gly Gly Thr Phe Glu Lys Gln Val Gly Leu Val Lys Pro 100 105 110 Arg Thr Thr Leu Ala Ala Arg Gly Met Glu Glu Ser Ala Val Pro Glu 115 120 125 Pro Glu Ala Thr Gly Glu Ser Ser Ser Leu Lys Pro Thr Pro Ser Ser 130 135 140 Arg Glu Ala Gln Thr Ala Leu Gly Thr Ser Pro Glu Gln Ser Thr Gly 145 150 155 160 Val Thr Gly Ser Ser Gly Thr Gly Leu Pro Leu Thr Pro Lys Ala Gln 165 170 175 Asp Gly Gly Pro Val Gly Thr Glu Leu Phe Arg Gly Pro Pro Val Ser 180 185 190 Thr Ala Ala Ala Trp Gln Ser Ser Ala Pro His Gln Pro Gly Pro Gly 195 200 205 Leu Trp Ala Glu Gly Lys Thr Ser Glu Ala Pro Ser Thr Gln Asp Pro 210 215 220 Ser Thr Gln Ala Ser Ser Asn Pro Arg Ala Ser Ser Thr Gln Ala Ser 225 230 235 240 Thr Thr Ser Ser Pro Ala Pro Glu Glu Asn Thr Pro Ser Glu Gly Gln 245 250 255 Pro Val Trp Gly Gln Gly Gln Ser Pro Arg Pro Glu Asn Ser Leu Glu 260 265 270 Arg Glu Glu Met Gly Pro Val Pro Ala His Thr Asp Ala Phe Gln Asp 275 280 285 Trp Gly Pro Gly Ser Met Ala His Val Ser Val Val Pro Val Ser Ser 290 295 300 Glu Gly Thr Pro Ser Arg Glu Pro Val Val Ser Gly Ser Trp Thr Pro 305 310 315 320 Lys Ala Glu Glu Pro Ile His Ala Thr Met Asp Pro Gln Arg Leu Gly 325 330 335 Val Leu Ile Thr Pro Val Pro Asp Ser Gln Ala Ala Thr Arg Arg Gln 340 345 350 Ala Val Gly Leu Leu Ala Phe Leu Gly Leu Leu Phe Cys Leu Gly Val 355 360 365 Ala Met Phe Ala Tyr Gln Ser Leu Gln Gly Cys Pro Arg Lys Met Ala 370 375 380 Gly Glu Met Val Glu Gly Leu Arg Tyr Ile Pro Arg Ser Cys Gly Ser 385 390 395 400 Asn Ser Tyr Val Leu Val Pro Val 405 <210> 35 <211> 327 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain constant region <400> 35 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15<00015Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 36 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain constant region <400> 36 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 37 <211> 5 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR1 <400> 37 Asn Tyr Tyr Ile His 1 5 <210> 38<​​​​​​​​​​​​​​​​​​​​​​​​​​<212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR3 <400> 39 Ser Pro Thr Asp Glu Thr Gln Gly Asp Tyr 1 5 10 <210> 40 <211> 9 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR3 <400> 40 Gly Pro Ala Pro Ala Glu Gly Asp Tyr 1 5 <210> 41 <211> 17 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR2 <400> 41 Trp Tyr Leu Pro Gly Asp Asp Ser Pro Lys Phe Asn Glu Arg Phe Lys 1 5 10 15 Gly <210> 42 <211> 7 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR3 <400> 42 Gly Pro Thr Gln Gly Asp Tyr 1 5 <210> 43 <211> 17 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR2 <400> 43 Trp Val Asp Pro Gly Tyr Gly Pro His Lys Phe Asn Glu Arg Phe Lys 1 5 10 15 Gly <210> 44 <211> 17 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR2 <400> 44 Trp Phe Ile Pro Gly Ser Asp Pro Pro Lys Phe Asn Glu Arg Phe Lys 1 5 10 15 Gly <210> 45 <211> 17 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR2 <400> 45 Trp Leu Leu Pro Gly Glu Asp Ser Pro Lys Phe Asn Glu Arg Phe Lys 1 5 10 15 Gly <210> 46 <211> 11 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> L-CDR1 <400> 46 Arg Ala Ser Gly Arg Ile His Asp Phe Leu Ala 1 5 10 <210> 47 <211> 7 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> L-CDR2 <400> 47 Asn Glu Lys Thr Leu Ala Asp 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> L-CDR3 <400> 48 Gln Gln Phe Trp Ser Thr Pro Tyr Thr 1 5 <210> 49 <211> 11 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> L-CDR1 <400> 49 Arg Ala Ser Gly Arg Ile His Gly Phe Leu Ala 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> L-CDR1 <400> 50 Arg Ala Ser Gly Asn Ile His Asn Phe Leu Ala 1​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Thr Asp Asn Thr Leu Ser Gly 1 5 <210> 53 <211> 5 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR1 <400> 53 Asn Tyr Asn Ile His 1 5 <210> 54 <211> 17 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR2 <400> 54 Trp Tyr Leu Pro Gly Asp Asp Ser Ser Lys Phe Asn Glu Arg Phe Glu 1 5 10 15 Gly <210> 55 <211> 17 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> H-CDR2 <400> 55 Trp Tyr Leu Pro Gly Asp Asp Ser Pro Arg Phe Asn Glu Arg Phe Lys 1 5 10 15 Gly <210> 56 <211> 7 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> L-CDR2 <400> 56 Thr Asp Asn Ala Leu Ala Glu 1 5 <210> 57 <211> 17 <212> PRT​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Asn Leu Leu Val Val Phe Ala Leu Thr Asn Ser Lys Lys Pro Lys Ser 50 55 60 Val Thr Asp Ile Tyr Leu Leu Asn Leu Ala Leu Ser Asp Leu Leu Phe 65 70 75 80 Val Ala Thr Leu Pro Phe Trp Thr His Tyr Leu Ile Asn Glu Lys Gly 85 90 95 Leu His Asn Ala Met Cys Lys Phe Thr Thr Ala Phe Phe Phe Ile Gly 100 105 110 Phe Phe Gly Ser Ile Phe Phe Ile Thr Val Ile Ser Ile Asp Arg Tyr 115 120 125 Leu Ala Ile Val Leu Ala Ala Asn Ser Met Asn Asn Arg Thr Val Gln 130 135 140 His Gly Val Thr Ile Ser Leu Gly Val Trp Ala Ala Ala Ile Leu Val 145 150 155 160 Ala Ala Pro Gln Phe Met Phe Thr Lys Gln Lys Glu Asn Glu Cys Leu 165 170 175 Gly Asp Tyr Pro Glu Val Leu Gln Glu Ile Trp Pro Val Leu Arg Asn 180 185 190 Val Glu Thr Asn Phe Leu Gly Phe Leu Leu Pro Leu Leu Ile Met Ser 195 200 205 Tyr Cys Tyr Phe Arg Ile Ile Gln Thr Leu Phe Ser Cys Lys Asn His 210 215 220 Lys Lys Ala Lys Ala Ile Lys Leu Ile Leu Leu Val Val Ile Val Phe 225 230 235 240 Phe Leu Phe Trp Thr Pro Tyr Asn Val Met Ile Phe Leu Glu Thr Leu 245 250 255 Lys Leu Tyr Asp Phe Phe Pro Ser Cys Asp Met Arg Lys Asp Leu Arg 260 265 270 Leu Ala Leu Ser Val Thr Glu Thr Val Ala Phe Ser His Cys Cys Leu 275 280 285 Asn Pro Leu Ile Tyr Ala Phe Ala Gly Glu Lys Phe Arg Arg Tyr Leu 290,295,300 Tyr His Leu Tyr Gly Lys Cys Leu Ala Val Leu Cys Gly Arg Ser Val 305 310 315 320 His Val Asp Phe Ser Ser Glu Ser Gln Arg Ser Arg His Gly Ser 325 330 335 Val Leu Ser Ser Asn Phe Thr Tyr His Thr Ser Asp Gly Asp Ala Leu 340 345 350 Many Many Many 355

Claims

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) and the light chain variable region (VL) comprise the amino acid sequences of 6 CDRs (H-CDR1, H-CDR2, H-CDR3; L-CDR1, L-CDR2, L-CDR3) from the following sequence combinations, wherein the sequences in the sequence combinations are the amino acid sequences of the heavy chain variable region and the light chain variable region of an anti-CX3CL1 antibody respectively: (1) SEQ ID NO:14 and SEQ ID NO:22; (2) SEQ ID NO:26 and SEQ ID NO:31; or (3) SEQ ID NO:28 and SEQ ID NO:

30.

2. The antibody or fragment thereof according to claim 1, wherein, the heavy chain variable region (VH) and the light chain variable region (VL) comprise a CDR combination selected from the following: (1) H-CDR1, H-CDR2, H-CDR3 shown sequentially in SEQ ID NO:37, 41, 39; and, L-CDR1, L-CDR2, L-CDR3 shown sequentially in SEQ ID NO:46, 51, 48; (2) H-CDR1, H-CDR2, H-CDR3 shown sequentially in SEQ ID NO:53, 54, 39; and, L-CDR1, L-CDR2, L-CDR3 shown sequentially in SEQ ID NO:46, 51, 48; or (3) H-CDR1, H-CDR2, H-CDR3 shown sequentially in SEQ ID NO:53, 55, 39; and, L-CDR1, L-CDR2, L-CDR3 shown sequentially in SEQ ID NO:46, 56, 48.

3. The antibody or fragment thereof according to claim 1 or 2, wherein, the heavy chain variable region and the light chain variable region comprised by the antibody or fragment thereof are selected from the following combinations: (1) The amino acid sequence shown in SEQ ID NO:14; and, the amino acid sequence shown in SEQ ID NO:22; (2) The amino acid sequence shown in SEQ ID NO:26; and, the amino acid sequence shown in SEQ ID NO:31; or (3) The amino acid sequence shown in SEQ ID NO:28; and, the amino acid sequence shown in SEQ ID NO:

30.

4. The antibody or fragment thereof according to any one of claims 1 to 3, wherein, the antibody or fragment thereof is an antibody or antigen-binding fragment thereof against chemokine CX3CL1.

5. The antibody or fragment thereof according to claim 4, wherein, the chemokine CX3CL1 is mammalian CX3CL1.

6. The antibody or fragment thereof according to claim 4, wherein, the chemokine CX3CL1 is human CX3CL1.

7. The antibody or fragment thereof according to claim 4, wherein, The chemokine CX3CL1 is cynomolgus monkey CX3CL1.

8. The antibody or fragment thereof according to claim 4, wherein, the chemokine CX3CL1 is murine CX3CL1.

9. The antibody or fragment thereof according to claim 4, wherein, The antibody is a monoclonal antibody, single-chain antibody, bifunctional antibody, fully or partially humanized antibody or chimeric antibody, or the antigen-binding fragment is scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv.

10. The antibody or fragment thereof according to claim 4, wherein, the antibody or its antigen-binding fragment further comprises a human or murine constant region.

11. The antibody or fragment thereof according to claim 4, wherein, the antibody or its antigen-binding fragment further comprises a human or murine heavy chain constant region (CH) and / or light chain constant region (CL).

12. The antibody or fragment thereof according to claim 4, wherein, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain.

13. The antibody or fragment thereof according to claim 4, wherein, the antibody or its antigen-binding fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a light chain constant region of κ or λ type.

14. The antibody or fragment thereof according to claim 4, wherein, the antibody is a monoclonal antibody.

15. The antibody or fragment thereof according to claim 14, wherein, the antibody is a murine, chimeric or humanized monoclonal antibody.

16. The antibody or fragment thereof according to claim 14, wherein, the heavy chain constant region of the monoclonal antibody is of IgG4 subtype and the light chain constant region is of κ type.

17. The antibody or fragment thereof according to claim 14, wherein, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 35 or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the monoclonal antibody 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.

18. A nucleic acid molecule encoding the antibody or fragment thereof according to any one of claims 1 to 17.

19. A vector comprising the nucleic acid molecule according to claim 18.

20. A host cell, which comprises the nucleic acid molecule according to claim 18 or the vector according to claim 19, or the host cell is transformed or transfected with the nucleic acid molecule according to claim 18 or the vector according to claim 19.

21. A composition comprising the antibody or fragment thereof according to any one of claims 1 to 17, the nucleic acid molecule according to claim 18, the vector according to claim 19 or the host cell according to claim 20.

22. The composition according to claim 21, wherein, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable excipient.

23. A kit, which comprises the antibody or fragment thereof according to any one of claims 1 to 17, the nucleic acid molecule according to claim 18, the vector according to claim 19, the host cell according to claim 20 or the composition according to claim 21 or 22.

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