CN117946265A - Anti-CX 3CL1 antibody and application thereof - Google Patents

Anti-CX 3CL1 antibody and application thereof Download PDF

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Publication number
CN117946265A
CN117946265A CN202410062177.8A CN202410062177A CN117946265A CN 117946265 A CN117946265 A CN 117946265A CN 202410062177 A CN202410062177 A CN 202410062177A CN 117946265 A CN117946265 A CN 117946265A
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seq
sequence
variable region
antibody
chain variable
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周雅琼
聂磊
柳颖
吴振华
陈刚
王海彬
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Zhejiang Borui Biopharmaceutical Co ltd
Haizheng Biopharmaceutical Co ltd
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Zhejiang Borui Biopharmaceutical Co ltd
Haizheng Biopharmaceutical Co ltd
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Abstract

The present invention belongs to the field of biology, and provides an antibody or an antigen binding fragment thereof which specifically binds to CX3CL1, and the antibody comprises a heavy chain variable region and a light chain variable region. The anti-CX 3CL1 antibody or the antigen binding fragment thereof has stronger binding property with human or monkey CX3CL1, can inhibit migration of CX3CR 1-expressing cells, blocks the binding of CX3CL1 with a receptor thereof, inhibits cAMP activity, and has good patent medicine prospect.

Description

Anti-CX 3CL1 antibody and application thereof
Technical Field
The invention belongs to the field of biology, and relates to an anti-CX 3CL1 antibody.
Background
Chemokines are a class of functionally related small molecule secreted proteins, termed "chemokines" for their leukocyte chemotaxis and cytokine activity. Chemokines can be divided into 4 subfamilies, depending on the number and spacing of the first two cysteines of the N-terminal conserved cysteine structural motif: c-, CC-, CXC-, and CX 3C-like chemokines (wherein C refers to cysteine and X can be any amino acid). Fractalkine (Fkn, CX3CL 1) was found in 1997 to be the only member of the current CX 3C-class of chemokines. Fractalkine has chemotactic function, and is involved in migration and activation of leukocytes, especially phagocytes and lymphocytes, while exhibiting adhesion, mediating intercellular adhesion.
Inflammatory factors such as Tumor Necrosis Factor (TNF) - α, interleukin (IL) -1, interferon (IFN) - γ, etc. can induce the expression of membrane-bound FRACTLKINE on endothelial cells. The secreted Fractalkine only contains a chemotactic protein functional region and a part of mucin-like structural region, has strong chemotactic activity on monocytes, NK cells and T cells, and can enhance the adhesion of leukocytes by the expression in activated endothelial cells. CX3CL1 can be induced in endothelial cells by pro-inflammatory cytokines (TNF. Alpha., IFN. Gamma., IL-1. Beta.) and LPS. CX3CR1 is a specific receptor for Fractalkine, is mainly expressed on cytotoxic effector lymphocytes (including NK cells, cytotoxic T lymphocytes and macrophages), and is a highly selective chemokine receptor and surface marker on cytotoxic effector lymphocytes. Soluble Fractalkine can induce NK cell transmembrane transfer and release particles, thereby improving cell lysis. Like NK cells, CD8T cells and CD4T cells expressing CX3CR1 can terminally differentiate into effector cells with cytotoxic particles, and excessive cytotoxic lymphocyte activation will lead to vascular and tissue damage.
CX3CL1 and Rheumatoid Arthritis (RA), crohn's disease, rheumatoid Vasculitis (RV),Development of various diseases such as Sjogren's Syndrome (SS), systemic Lupus Erythematosus (SLE), scleroderma, multiple sclerosis and arteriosclerosis is involved. Soluble Fractalkine was found in cerebrospinal fluid of patients with neuropsychiatric lupus, at/>In the serum of patients with the syndrome, rheumatic arthritis and type 2 diabetes, the soluble Fractalkine is higher than that of the patients in the control group. The development of anti-CX 3CL1 drugs can directly block related channels, thereby achieving the aim of inhibiting inflammation.
Disclosure of Invention
It is an object of the present invention to provide anti-CX 3CL1 antibodies that provide selectivity for the treatment of related diseases.
In a first aspect, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to CX3CL1, comprising a heavy chain variable region comprising three Complementarity Determining Regions (CDRs): HCDR1, HCDR2 and HCDR3, the light chain variable region comprising three Complementarity Determining Regions (CDRs): LCDR1, LCDR2, and LCDR3; wherein,
The amino acid sequence of HCDR1 is shown in SEQ ID NO. 11;
The amino acid sequence of HCDR2 is shown in SEQ ID NO. 12, 19, 21, 23 or 43;
The amino acid sequence of HCDR3 is shown in SEQ ID NO. 13;
The amino acid sequence of LCDR1 is shown as SEQ ID NO. 15;
the amino acid sequence of LCDR2 is shown as SEQ ID NO. 16;
the amino acid sequence of LCDR3 is shown as SEQ ID NO. 17; or alternatively
The amino acid sequence of HCDR1 is shown in SEQ ID NO. 3;
The amino acid sequence of HCDR2 is shown in SEQ ID NO. 4;
the amino acid sequence of HCDR3 is shown in SEQ ID NO. 5;
The amino acid sequence of LCDR1 is shown in SEQ ID NO. 7;
the amino acid sequence of LCDR2 is shown in SEQ ID NO. 8;
the amino acid sequence of LCDR3 is shown in SEQ ID NO. 9.
The CDRs of the present invention are defined according to the Kabat numbering convention. Those skilled in the art will readily identify CDRs defined by each numbering system and the correspondence between the different numbering systems is well known to those skilled in the art. In some embodiments, the CDRs are defined according to Kabat, IMGT, chothia, abM or Contact numbering rules.
In some embodiments, the invention provides an anti-CX 3CL1 antibody or antigen binding fragment thereof comprising a variable heavy chain region and a variable light chain region, wherein:
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is as shown in SEQ ID NO. 10, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO. 10; the sequence of the light chain variable region is shown as SEQ ID NO. 14 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 14; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is as shown in SEQ ID NO. 18 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO. 18; the sequence of the light chain variable region is shown as SEQ ID NO. 20 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 20; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is as shown in SEQ ID NO. 22 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO. 22; the sequence of the light chain variable region is shown as SEQ ID NO. 20 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 20; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is as shown in SEQ ID NO. 24 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO. 24; the sequence of the light chain variable region is shown as SEQ ID NO. 20 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 20; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is shown as any one of SEQ ID NO 27-35 or SEQ ID NO 39, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO 27-35 or SEQ ID NO 39; the sequence of the light chain variable region is shown in any one of SEQ ID NOs 36-38 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in any one of SEQ ID NOs 36-38; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is as shown in SEQ ID NO. 2, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO. 2; the sequence of the light chain variable region is shown as SEQ ID NO. 6 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 6.
In some preferred embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 10, and the light chain variable region sequence is shown as SEQ ID NO. 14; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 18, and the light chain variable region sequence is shown as SEQ ID NO. 20; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 22, and the light chain variable region sequence is shown as SEQ ID NO. 20; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 24, and the light chain variable region sequence is shown as SEQ ID NO. 20; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 39, and the sequence of the light chain variable region is shown as any one of SEQ ID NO. 36-38; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is shown in any one of SEQ ID NO. 27-35, and the light chain variable region sequence is shown in any one of SEQ ID NO. 36-38; or alternatively
The heavy chain variable region sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 2, and the light chain variable region sequence is shown as SEQ ID NO. 6.
In some embodiments, the heavy chain variable region of the antibody or antigen binding fragment thereof further comprises a framework region of human or murine origin, and/or the light chain variable region of the antibody or antigen binding fragment thereof further comprises a framework region of human or murine origin.
In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and/or a light chain constant region; the heavy chain constant region and/or the light chain constant region is of human or murine origin.
In a preferred embodiment, the heavy chain constant region and/or the light chain constant region is of human origin, wherein the sequence of the heavy chain constant region is shown in SEQ ID NO. 25 and the sequence of the light chain constant region is shown in SEQ ID NO. 26.
In preferred embodiments, the antibody or antigen binding fragment thereof is an animal-derived antibody, chimeric antibody, humanized antibody, or a combination thereof.
In some embodiments, the anti-CX 3CL1 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein,
The amino acid sequence of the heavy chain is shown as SEQ ID NO. 40 or 42, or comprises the amino acid sequence similar to SEQ ID NO:40 or 42, and the amino acid sequence of the light chain has an amino acid sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:41 or comprises a sequence identical to SEQ ID NO:41 has an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
In some embodiments, the anti-CX 3CL1 antibody or antigen binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence set forth in SEQ ID No. 40, and the light chain has the amino acid sequence set forth in SEQ ID NO: shown at 41.
In some embodiments, the anti-CX 3CL1 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence as shown in SEQ ID NO. 42 and the light chain has an amino acid sequence as shown in SEQ ID NO: shown at 41.
In some embodiments, the antibody or antigen-binding fragment thereof is an antibody full-length protein, or an antigen-binding fragment.
In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, fab ', (Fab') 2, fd, fv, disulfide-linked Fv, scFv, and dAb.
In a second aspect, there is provided a biomaterial, which may be:
(1) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention; preferably, the nucleic acid molecule is a DNA molecule or an RNA molecule; based on the amino acid sequences of the antibodies disclosed herein, one skilled in the art can infer the DNA or RNA sequences encoding the amino acid sequences and arrange suitable expression elements for them to enable the DNA or RNA molecules to express the antibodies of the invention;
(2) A vector comprising a nucleic acid molecule as described above; preferably, the vector is a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector;
(3) A host cell containing the nucleic acid molecule or the vector described above, or a culture such as a culture solution or a bacterial suspension obtained by culturing the host cell.
In a third aspect, there is provided a method of preparing an antibody or antigen binding fragment thereof of the invention, comprising:
1) Chemical synthesis, based on the amino acid sequence of the antibody or antigen binding fragment thereof; or alternatively
2) Biological preparation comprising culturing the cells of the second aspect above and harvesting the antibody or antigen-binding fragment thereof.
In a fourth aspect, there is provided a composition comprising an anti-CX 3CL1 antibody or antigen binding fragment thereof, or biological material, of the invention.
In some embodiments, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.
In a fifth aspect, there is provided a kit comprising an antibody or antigen-binding fragment thereof, a biological material, or an antibody composition of the invention; the kit is useful for diagnosing or detecting CX3CL1 protein or CX3CL1 protein-expressing cells in an isolated sample.
In a sixth aspect, there is provided the use of an antibody or antigen-binding fragment, biomaterial or antibody composition of the invention for the manufacture of a medicament for the prophylaxis and/or treatment of a disease associated with expression or dysfunction of CX3CL 1.
In some embodiments, the disease associated with CX3CL1 expression or dysfunction is selected from: rheumatic Arthritis (RA), crohn's disease, ulcerative colitis, diabetes, rheumatoid Vasculitis (RV), and,The symptoms of the disease include Sjogren's Syndrome (SS), systemic Lupus Erythematosus (SLE), neuropsychiatric lupus, scleroderma, multiple sclerosis and arteriosclerosis.
In a seventh aspect, the present invention provides a method of preventing and/or treating a disease associated with CX3CL1 expression or dysfunction, the method comprising administering to the human individual an effective amount of an anti-CX 3CL1 antibody or antigen binding fragment thereof of any one of the preceding claims, or a biological material as described above, or a composition as described above.
The anti-CX 3CL1 antibody or the antigen binding fragment thereof disclosed by the invention has stronger binding property with human or monkey CX3CL1, can inhibit migration of CX3CR 1-expressing cells, block the binding of CX3CL1 with a receptor thereof and inhibit cAMP activity. Therefore, the antibody and the antigen binding fragment thereof have good patent medicine prospect.
Detailed Description
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Definition of the definition
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of cell culture, biochemistry, nucleic acid chemistry, immunology laboratories and the like as used herein are all conventional procedures widely used in the corresponding fields.
Throughout the specification and claims, the words "comprise," "have," "include," and the like are to be construed as having an inclusive, rather than an exclusive or exhaustive, meaning unless the context clearly requires otherwise; that is, the meaning of "including but not limited to". Unless otherwise indicated, "comprising" includes "consisting of … …". For example, for a polypeptide comprising SEQ ID NO:2, which specifically covers HCDR1 having an amino acid sequence as set forth in SEQ ID NO:2, and HCDR1.
The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in j.biol. Chem,243, p3558 (1968).
As used herein, a natural "antibody" refers to an immune protein capable of binding to an antigen, which is produced by the body as a result of stimulation by the antigen, and which has a protective effect, and is secreted by plasma cells (effector B cells); "immunoglobulin" refers to an immunoglobulin having antibody (Ab) activity or chemical structure similar to an antibody molecule. Antibodies are typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as k (kappa) and (lambda) light chains. Heavy chains are classified into five classes, μ, γ, α, δ and ε, according to their constant region antigenicity, and the corresponding immunoglobulins are IgM, igG, igA, igD and IgE, respectively. And the isotypes of antibodies were defined as IgM, igD, igG, igA and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is composed of 3 CDRs and 4 FRs arranged from amino-terminus to carboxyl-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The assignment of amino acids in various regions or domains can be followed by Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991)), or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883.
As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, such as may be defined in accordance with the Kabat numbering system (Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md,1991)、Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (LEFRANC ET al., dev. Comparat. Immunol.27:55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to the person skilled in the art (see, for example, LEFRANC ET al. Dev. Comparat. Immunol.27:55-77, 2003). Unless otherwise indicated, the variable region and CDR sequences in the examples of the present invention apply the "Kabat" numbering convention. Although in particular embodiments one numbering system (e.g., kabat) is used to define amino acid residues, the corresponding schemes for other numbering systems are to be considered equivalent.
As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.
As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. Non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') 2, fd, fv, complementarity Determining Region (CDR) fragments, scFv, and the like (technologies available from Domatis), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide.
"Fab" consists of a light chain and a heavy chain CH1 and variable domains.
"Fab'" is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain.
"F (ab ') 2", which is a bivalent fragment formed by the ligation of two Fab' fragments through the disulfide bridge of the hinge region.
"Fd" having VH and CH1 domains; (iv) Fd' fragments having a VH and CH1 domain and having one or more cysteine residues at the C-terminal end of the CH1 domain.
"Fv" having the VL and VH domains of one arm of an antibody.
"DAb" consisting of a VH domain.
"Single chain antibody molecule (scFv)" means a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) linked by a linker, such scFv molecules may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH.
The term "IgG-type antibody" refers to an antibody having "heavy" and "light" chains crosslinked via intra-and inter-chain disulfide bonds, with a typical "Y" structure. Each heavy chain consists of an N-terminal HCVR and a heavy chain constant region ("HCCR"). Each light chain consists of LCVR and a light chain constant region ("LCCR").
As used herein, the term "full length antibody" means an antibody consisting of two "full length heavy chains" and two "full length light chains". Wherein "full length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to C-terminal direction; and, when the full length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-to C-terminal direction. A "full length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to C-terminal direction. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. The full length antibodies of the invention may be from a single species, e.g., human; chimeric or humanized antibodies are also possible. The full length antibodies of the invention comprise two antigen binding sites formed by VH and VL pairs, respectively, which specifically recognize/bind the same antigen.
The term "capable of specifically binding", "specifically binding" or "binding" refers to an antibody that is capable of binding to a certain antigen or epitope with a higher affinity than other antigens or epitopes. Typically, an antibody binds an antigen or epitope with an equilibrium dissociation constant (KD) of about 1 x 10 -7 M or less (e.g., about 1 x 10 -8 M or less). In some embodiments, the antibody binds to an antigen with a KD of 10% or less (e.g., 1%) of the KD of the antibody to a non-specific antigen (e.g., BSA, casein). KD can be measured using known methods, for example by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or epitope thereof may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, e.g., cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno), chimpanzee (Pan troglodes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset).
As used herein, the terms "monoclonal antibody," "mAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" merely indicates the character of the antibody as being obtained from a population of highly homologous antibodies, and is not to be construed as requiring preparation of the antibody by any particular method.
Monoclonal antibodies of the invention may be prepared by a variety of techniques, such as hybridoma techniques (see, e.g., kohler et al, nature,256:495, 1975), recombinant DNA techniques (see, e.g., U.S. patent application 4,816,567), or phage antibody library techniques (see, e.g., clackson et al Nature352:624-628,1991, or Marks et al J.mol.biol.222:581-597, 1991).
Antibodies can be purified by well-known techniques, such as affinity chromatography using protein a or protein G. Subsequently or alternatively, a specific antigen (the target molecule recognized by the antibody) or an epitope thereof may be immobilized on the column, and the immunospecific antibody may be purified by immunoaffinity chromatography. Purification of immunoglobulins can be referred to, for example D.Wilkinson(The Scientist,published by The Scientist,Inc,Philadelphia Pa.,Vo1.14,No.8(Apr.17,2000),pp.25--28).
As used herein, the term "chimeric antibody (Chimeric antibody)" refers to an antibody in which a portion of the light chain or/and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular class or subclass of antibody) and another portion of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different class or subclass of antibody), but which nevertheless retains binding activity for the antigen of interest (u.s.p 4,816,567to Cabilly et al.; morrison et al, proc.Natl.Acad.Sci.USA,81:6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies (e.g., human murine combination antibodies) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., human antibody).
As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Typically, at least one or two, but typically all three, acceptor CDRs (of the heavy and/or light immunoglobulin chains) of the humanized antibody are replaced by donor CDRs. Immunoglobulins that provide CDRs are referred to as "donors" and immunoglobulins that provide frameworks are referred to as "acceptors". In one embodiment, the donor immunoglobulin is a non-human (e.g., murine) antibody, and the acceptor framework may be a naturally occurring human framework, or a sequence having about 85%, 90%, 95%, 99% or more identity thereto. Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).
The term "sequence identity" refers to the degree (percent) to which the amino acids/nucleic acids of two sequences are identical at equivalent positions when optimally aligned. During the alignment, gaps may be allowed to be introduced as necessary to obtain the maximum percent sequence identity, but any conservative substitutions are not considered to form part of the sequence identity. To determine percent sequence identity, alignment may be accomplished by techniques known in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences compared.
The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid encoding the antigen binding molecule refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present at one or more positions in a host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with degenerate bases and/or deoxyinosine residues, as described in detail below.
The term "pharmaceutical composition" means a mixture comprising one or more antigen binding molecules or antibodies described herein and other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients.
The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.
As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK293 cells or human cells.
The term "subject" or "individual" includes both human and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein unless specifically indicated. As used herein, the term "cynomolgus monkey (cyno)" or "cynomolgus monkey (cynomolgus)" refers to cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.
"Administering" or "administering," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous pharmaceutical, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.
The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in the body of a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cyst fluid, tears, fecal matter, sputum, mucous secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneal cavity and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluid, and the like, tissue biopsy samples, fine needle punctures, surgically excised tissues, organ cultures, or cell cultures.
"Treatment" and "treatment" (and grammatical variations thereof) refer to a clinical intervention intended to be administered to an individual being treated, and may be performed for prophylactic purposes, or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis. In some embodiments, the molecules of the present disclosure are used to delay the formation of a disease or to slow the progression of a disease.
An "effective amount" is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate such symptoms and/or underlying etiology, prevent the appearance of symptoms and/or underlying etiology, and/or ameliorate or improve the damage caused by or associated with a disease state. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or condition, particularly a state or condition associated with the disease state, or otherwise prevent, hinder, delay or reverse the progression of the disease state or any other undesirable condition associated with the disease in any way. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. The complete therapeutic or prophylactic effect does not necessarily occur after administration of one dose, but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations. The "therapeutically effective amount" and "prophylactically effective amount" may vary depending on a variety of factors: such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of effective therapeutic agents or combinations of therapeutic agents include, for example, improved health of a patient.
The present invention will be further illustrated by the following specific examples, but the scope of the present invention is not limited thereto.
Unless otherwise specified, the reagents and apparatus used in the present invention are all conventional reagents and apparatus and are commercially available; the methods used are all conventional techniques, and a person skilled in the art can undoubtedly complete the experiments and obtain the corresponding results according to the description.
Example 1: screening of anti-CX 3CL1 murine monoclonal antibody
The present invention uses hybridoma technology to produce monoclonal antibodies (mabs) with specificity and high affinity for human and monkey CX3CL 1.
This example describes a method for preparing a murine anti-human CX3CL1 monoclonal antibody. The extracellular CX3CL1 protein (UniProt No.: P78423, SEQ ID NO: 1) was expressed first as an immunogen, the C-terminus of the extracellular CX3CL1 protein amino acid sequence (Q25-Q341) was added with a 6XHis (CX 3CL1 (Q25-Q341) -6 XHis) or human IgG1 Fc (CX 3CL1 (Q25-Q341) -IgG1 Fc) tag, then the encoding nucleic acid was cloned into an expression vector, 293 cells were transiently transfected with the expression vector, and after 7 days the cell culture broth was collected and purified.
To prepare murine anti-human CX3CL1 monoclonal antibodies, SJL mice (purchased from Ming's chemistry) were immunized in two groups with two tagged CX3CL1 proteins, each group being immunized 3 times in total. Serum immunotiter detection was performed on mice after immunization by ELISA. The two tagged CX3CL1 proteins were coated on the microplate at 1. Mu.g/mL overnight at 4℃and then blocked with blocking solution at 37℃for 1h, after washing the plate, the mouse serum was added to the plate and incubated at 37℃for 1 h. Wash plates were added HRP-labeled goat anti-mouse IgG (purchased from Thermofisher) and reacted at 37 ℃ for 1 hour. After washing the plate, TMB solution was added, the reaction was stopped by 5 minutes at room temperature in the dark, and then 2N H 2SO4 was added. The absorbance was measured at a wavelength of 450nm on a microplate reader.
Mice with high immune titers are selected, spleen cells are fused with myeloma cells, and two rounds of screening of cell culture supernatants are performed by ELISA. Positive hybridoma clones were transferred to 24-well plates for culture after primary screening, followed by secondary screening. And (3) subcloning the positive clone obtained by screening, detecting the subclone again by adopting a primary screening and secondary screening mode, and selecting a positive monoclonal to produce antibodies and freeze the stock. Hybridoma cell lines with monoclonal numbers of 36C7-1G2, 15F7B2 and 18H6F1 are selected for amplification culture, and the supernatant is purified to produce murine monoclonal antibodies mAb017, mAb022 and mAb032.
Human CX3CL1:
QHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAAGGMDESVVLEPEATGESSSLEPTPSSQEAQRALGTSPELPTGVTGSSGTRLPPTPKAQDGGPVGTELFRVPPVSTAATWQSSAPHQPGPSLWAEAKTSEAPSTQDPSTQASTASSPAPEENAPSEGQRVWGQGQSPRPENSLEREEMGPVPAHTDAFQDWGPGSMAHVSVVPVSSEGTPSREPVASGSWTPKAEEPIHATMDPQRLGVLITPVPDAQAATRRQ(SEQ ID NO:1).
example 2: sequencing of murine monoclonal antibodies and production of chimeric antibodies
Three hybridoma cell lines of 36C7-1G2, 15F7B2 and 18H6F1 corresponding to mAb017, mAb022 and mAb032 are subjected to antibody sequence determination, and 3 monoclonal antibodies with specific sequences are obtained after sequencing, wherein the specific sequences are respectively shown as follows:
>mAb017-VH
QIQLVQSGPELKKPGETVKISCKASVYTFTEYAMHWVKQAPGKGFKWMGWINTNSGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCGRGRVVGSFDYWGQGTTLTVSS(SEQ ID NO:2); Wherein,
HCDR1：EYAMH(SEQ ID NO:3)；
HCDR2：WINTNSGEPTYADDFKG(SEQ ID NO:4)；
HCDR3：GRVVGSFDY(SEQ ID NO:5)；
>mAb017-VL
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQFYSYPLTFGAGTKLELK(SEQ ID NO:6);
LCDR1：KSSQSLLYSSNQKNYLA(SEQ ID NO:7)；
LCDR2：WASTRES(SEQ ID NO:8)；
LCDR3：QQFYSYPLT(SEQ ID NO:9)；
>mAb022-VH
EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVFYCASSGNSYVDYWGQGTTLTVSS(SEQ ID NO:10); Wherein,
HCDR1：DYYMN(SEQ ID NO:11)；
HCDR2：DINPNNGGTNYNQKFKG(SEQ ID NO:12)；
HCDR3：SGNSYVDY(SEQ ID NO:13)；
>mAb022-VL
DIVMTQSHKFMSTSVGDRVNITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSSPYTFGGGTKLEIK(SEQ ID NO:14); Wherein,
LCDR1：KASQDVSTAVA(SEQ ID NO:15)；
LCDR2：WASTRHT(SEQ ID NO:16)；
LCDR3：QQHYSSPYT(SEQ ID NO:17)；
>mAb032-VH
EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASSGNSYVDYWGQGTTLTVSS(SEQ ID NO:18); Wherein,
HCDR1：DYYMN(SEQ ID NO:11)；
HCDR2：DINPNNGGTSYNQKFKG(SEQ ID NO:19)；
HCDR3：SGNSYVDY(SEQ ID NO:13)；
>mAb032-VL
DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSSPYTFGGGTKLEIK(SEQ ID NO:20); Wherein,
LCDR1：KASQDVSTAVA(SEQ ID NO:15)；
LCDR2：WASTRHT(SEQ ID NO:16)；
LCDR3：QQHYSSPYT(SEQ ID NO:17)。
The variable regions of the light chains of the mAb022 and mAb032 monoclonal antibodies obtained are connected with the constant region of the human IgG2 antibody by utilizing a DNA recombination technology to construct chimeric antibodies C022 and C032. Wherein, the CDR2 of mAb032 has NG hot spot, so 2 different mutants, C032NA and C032QG, are prepared simultaneously.
The sequence of the antibody fragment containing the mutation site is as follows:
C032NA-HCDR2：DINPNNGTSYNQKFKG(SEQ ID NO:21)；
C032NA-VH：
EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNAGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASSGNSYVDYWGQGTTLTVSS(SEQ ID NO:22);
C032QG-HCDR2：DINPNGGTSYNQKFKG(SEQ ID NO:23)；
C032QG-VH：
EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNQGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASSGNSYVDYWGQGTTLTVSS(SEQ ID NO:24).
the chimeric antibody is expressed by connecting the light and heavy chain variable region genes of the chimeric antibody with the constant region genes of the human IgG2 antibody, connecting the genes into an expression vector pcDNA3.4 by adopting the technology of total gene synthesis, and transfecting a mammalian cell CHOK1 (purchased from ATCC). Purification the anti-human CX3CL1 human murine chimeric antibody was isolated from the serum-free culture supernatant containing the antibody of interest using protein A affinity chromatography. Wherein the heavy chain constant region amino acid sequence of the chimeric antibody is shown in SEQ ID NO:25, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: shown at 26.
CH:
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:25);
CL:
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:26).
The obtained chimeric antibody was sequenced and the results were identical.
Example 3: humanization and expression of anti-CX 3CL1 monoclonal antibodies
3.1 Humanization of C032QG mab
And selecting the C032QG monoclonal antibody for humanization, transplanting the CDR region of the C032QG antibody to a matched human variable region gene framework sequence in a CDR transplanting mode, and designing back mutation in the framework region to finally obtain the heavy chain variable region and the light chain variable region of the humanized antibody of the C032 QG. The C032QG monoclonal antibody was humanized using a heavy chain variable region template of 2 human antibodies and a template of 1 light chain variable region, and the sequences of the heavy chain variable regions of the HumAb QG humanized antibodies obtained were as follows:
HumAb032 VH G1-0
EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMNWVQQAPGKGLEWMGDINPNQGGTSYNQKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCATSGNSYVDYWGQGTTVTVSS(SEQ ID NO:27);
HumAb032 VH G1-1
EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMNWVQQAPGKSLEWMGDINPNQGGTSYNQKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTTVTVSS(SEQ ID NO:28);
HumAb032 VH G1-2
EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWMGDINPNQGGTSYNQKFKGRVTLTVDTSTDTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTTVTVSS(SEQ ID NO:29);
HumAb032 VH G1-3
EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWMGDINPNQGGTSYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTTVTVSS(SEQ ID NO:30);
HumAb032 VH G1-4
EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWIGDINPNQGGTSYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTTVTVSS(SEQ ID NO:31);
HumAb032 VH G1-5
EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWIGDINPNQGGTSYNQKFKGKATLTVDKSTSTAYMELRSLRSEDTAVYYCASSGNSYVDYWGQGTTVTVSS(SEQ ID NO:32);
HumAb032 VH G2-0
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGDINPNQGGTSYNQKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASSGNSYVDYWGQGTTVTVSS(SEQ ID NO:33);
HumAb032 VH G2-1
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVKQAPGQSLEWMGDINPNQGGTSYNQKFKGKVTMTVDKSISTAYMELSRLRSDDTAVYYCASSGNSYVDYWGQGTTVTVSS(SEQ ID NO:34);
HumAb032 VH G2-2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVKQAPGQSLEWIGDINPNQGGTSYNQKFKGKATLTVDKSISTAYMELSRLRSDDTAVYYCASSGNSYVDYWGQGTTVTVSS(SEQ ID NO:35).
the sequence of the light chain variable region is shown below:
HumAb032 VL g0
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLLYWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSSPYTFGGGTKVEIK(SEQ ID NO:36);
HumAb032 VL g1
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLLYWASTRHTGVPDRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSSPYTFGGGTKVEIK(SEQ ID NO:37);
HumAb032 VL g2
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYWASTRHTGVPDRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSSPYTFGGGTKVEIK(SEQ ID NO:38).
The variable region of the obtained humanized antibody was combined with the heavy chain constant region of a human antibody (heavy chain constant region of IgG2 shown in SEQ ID NO: 25) and the light chain constant region of a light chain constant region of kappa shown in SEQ ID NO:26 to obtain 27 full-length humanized antibodies, as shown in Table 1.
TABLE 1 variable region combinations of humanized antibodies
3.2 Humanization of C022 mab
Since only 1 amino acid difference exists between C022 and C032, the difference amino acid between C022 and C032 in the heavy chain variable region CDR2 of HumAb-4 is directly mutated into the amino acid of C022, and HumAb022-4 is obtained. The CDR2, heavy chain variable region and light chain variable region sequences of HumAb022-4 are shown below (wherein the light chain variable region is identical to the light chain variable region of HumAb 032-4).
HCDR2：DINPNQGGTNYNQKFKG(SEQ ID NO:43)；
HumAb022-4VH
EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWMGDINPN QGGTNYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTT VTVSS(SEQ ID NO:39);
HumAb022-4VL: (same HumAb VL g 0)
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLLYWASTRHTG VPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSSPYTFGGGTKVEIK(SEQ ID NO:36).
The full length sequence of an exemplary humanized antibody is as follows:
1、HumAb022-4
Heavy chain:
EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWMGDINPNQGGTNYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:40);
light chain:
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLLYWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSSPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:41)
2、HumAb032-4
Heavy chain:
EVQLVQSGAEVKKPGATVKISCKASGYTFTDYYMNWVQQAPGKSLEWMGDINPNQGGTSYNQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCASSGNSYVDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:42);
Light chain (same HumAb022-4 light chain):
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLLYWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSSPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:41).
All humanized antibodies were expressed using the production method of the chimeric antibody of example 2, and the sequence of the humanized antibody was detected, resulting in coincidence.
The sequence numbers of the murine, chimeric and humanized antibodies and fragments thereof related to the above examples are shown in Table 2.
Table 2: sequence numbers of antibodies and fragments thereof
Example 4: preparation of positive control antibodies
CX3CL1 positive control antibody was produced according to the antibody sequence published by Tabs database Quetmolimab and designated Tab#1.
Tab#1 heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVKQAPGQGLEWIGWIYPGDGSPKFNERFKGRTTLTADKSTNTAYMLLSSLRSEDTAVYFCATGPTDGDYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:44);
Tab#1 light chain
DIQMTQSPSSLSASVGDRVTITCRASGNIHNFLAWYQQKPGKAPKLLIYNEKTLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQFWSTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:45).
Test example 1: ELISA binding of antibodies to human CX3CL1, monkey CX3CL1
Two antigen proteins were diluted with PBS: human CX3CL1 extracellular region protein (huCX CL1-His, i.e., CX3CL1 (Q25-Q341) -6 XHis) and monkey CX3CL1 extracellular region protein (cynoCX CL1-His, available from Baiying organism B23523001) proteins were brought to 1. Mu.g/mL, ELISA microwell plates were added at 100. Mu.L/well and incubated overnight at 4 ℃. Blocking was performed for two hours at 37℃with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH7.4, the percentages being mass percentages). The antibody was added in a gradient of 8 concentration points (initial concentration 100nM, 10-fold gradient dilution, including 0 point). 100. Mu.L/well, incubated at 37℃for 1 hour. Plates were washed 3 times and secondary antibody (purchased from Thermofisher) was added: anti-mouse IgG-HRP (1:5000 dilution), 100. Mu.L/well, incubated at 37℃for 1 hour. The plate was washed 3 times, TMB chromogenic solution was added thereto, 100. Mu.L/well, and after incubation at 37℃for 10 minutes, the chromogenic reaction was terminated by adding 2N H 2SO4 thereto. OD450nm readings were taken with a microplate reader.
As shown in tables 3-1 to 3-4, the obtained monoclonal antibodies were well bound to extracellular proteins of human CX3CL1 and monkey CX3CL 1.
TABLE 3-1 binding of murine antibody to human CX3CL1 and monkey CX3CL1
TABLE 3-2 binding of chimeric antibodies to human CX3CL1 and monkey CX3CL1
TABLE 3-3 binding of humanized antibodies to human CX3CL1 and monkey CX3CL1
TABLE 3-4 binding of humanized antibodies to human CX3CL1 and monkey CX3CL1
Test example 2: detection of binding Activity of monoclonal antibody blocking human CX3CL1 and receptor
Antigen huCX CL1-His was diluted to 0.2. Mu.g/mL with PBS, and the mab was diluted in a gradient (initial concentration 15. Mu.g/mL, 2-fold gradient dilution, 7 concentration points). 60. Mu.L of antigen was mixed with 60. Mu.L of mab and incubated at 4℃for 30 min. Human CX3CR1 overexpressing cells (CHOK 1-hCX CR1, human CX3CR1, uniprot number P49238, were transformed into CHOK1 cells by lentiviral means to obtain a stably transformed cell line) were digested with TrypLE (available from Gibco), collected by centrifugation and resuspended to a cell density of 2E6 cells/ml in PBS buffer. The cell suspension was added to a 96-well cell plate at 100. Mu.L per well, and the supernatant was discarded after centrifugation. Cells were resuspended by adding antigen and mab mixture at 100 μl/well and incubated for 1 hour at 4deg.C. Cells were washed 2 times with PBS, 100. Mu.l of 0.5. Mu.g/mL (1:1000) anti-His-647 fluorescent secondary antibody (cat: A01802-100) was added to each well, cells were resuspended, incubated at 4℃for 1 hour in the absence of light, cells were washed 2 times with PBS, and FACS analysis was performed on PBS resuspended cells.
The results are shown in tables 4-1 to 4-4 below, and the monoclonal antibodies tested can better block the binding of human CX3CL1 to the receptor.
TABLE 4-1 blocking Activity of murine antibodies
Antibodies to IC50(nM)
mAb017 6.1
mAb022 7.6
mAb032 11.5
TABLE 4-2 blocking Activity of chimeric antibodies
Antibodies to IC50(nM) Antibodies to IC50(nM)
C022 0.885 C032NA 0.865
C032 0.731 C032QG 0.811
TABLE 4-3 blocking Activity of humanized antibodies
Antibodies to IC50(nM) Antibodies to IC50(nM)
HumAb032-1 2.396 HumAb032-15 1.366
HumAb032-2 1.525 HumAb032-16 1.303
HumAb032-3 1.283 HumAb032-17 1.396
HumAb032-4 1.223 HumAb032-18 1.324
HumAb032-5 1.086 HumAb032-19 1.267
HumAb032-6 1.251 HumAb032-20 1.068
HumAb032-7 2.665 HumAb032-21 1.159
HumAb032-8 1.495 HumAb032-22 2.779
HumAb032-9 1.281 HumAb032-23 1.5
HumAb032-10 1.267 HumAb032-24 1.366
HumAb032-11 1.068 HumAb032-25 1.303
HumAb032-12 1.159 HumAb032-26 1.369
HumAb032-13 2.779 HumAb032-27 1.324
HumAb032-14 1.5 C032QG 1.492
TABLE 4-4 blocking Activity of humanized antibodies
Antibodies to IC50(nM)
HumAb022-4 1.275
C022 1.702
Tab#1 1.957
Test example 3: detection of inhibition of cell secretion cAMP Activity by monoclonal antibodies
The CHOK1-hCX CR1 cells were digested, centrifuged, collected according to standard procedures, resuspended in F12K (Hyclone, cat#SH 30526.01) and incubated at room temperature for 30-40 minutes. mu.L of 0.4. Mu.g/mL huCX CL1-His (antigen concentration 0.2. Mu.g/mL in murine mAb experiments) was mixed with 10. Mu.L of mAb (initial concentration 15. Mu.g/mL, 2-fold gradient dilution, 8 gradient) and incubated for 30 min at room temperature. Cells were collected by centrifugation, resuspended in F12K containing 0.5mM IBMX (Sigma) to a cell concentration of 2.5E5/ml (halving the cell concentration in murine monoclonal antibody experiments) and added to a new assay plate (Corning # 3365) at 10. Mu.L/well. The antigen-antibody mixture was then added to the assay plate at 10. Mu.L/well and the cells were incubated at room temperature for 10 minutes. 9.9. Mu.M Forskolin (MCE, CAS# 66575-29-9) was added to the assay plate at 5. Mu.L/well and incubated for 30 minutes at 37 ℃. Subsequently, 10. Mu.l of the mixture was transferred to the assay plate (OptiPlate-384), 5. Mu.l of cAMP-Cryptate working solution (Cisbio, 62AM9PEC Lot#02E) was added and mixed well, and 5. Mu.l of anti-cAMP-d 2 working solution (Cisbio, 62AM9PEC Lot#02E) was added. Incubate for 1 hour at room temperature. Fluorescence values at two different wavelengths (665 nm and 620 nm) were read on Envision.
The results are shown in tables 5-1 to 5-3 below, and the monoclonal antibodies tested are better able to block the binding of human CX3CL1 to the receptor, thereby inhibiting cellular cAMP production and being superior to the control antibody Tab#1.
TABLE 5-1 inhibition of cAMP Activity of murine antibodies
Antibodies to IC50(nM) MAX
mAb017 0.70 100.0
mAb022 0.80 105.2
mAb032 1.07 114.9
TABLE 5-2 inhibition of cAMP Activity of chimeric antibodies
Antibodies to IC50(nM) MAX
C022 3.284 92.88
C032 3.404 95.82
C032NA 3.633 92.51
C032QG 3.963 96.95
Tab#1 5.686 94.04
TABLE 5-3 inhibition of cAMP Activity of antibodies
Antibodies to IC50(nM) MAX
HumAb022-4 2.69 100.5
C022 2.41 100.1
Tab#1 4.07 92.5
Test example 4: detection of migration Activity of monoclonal antibodies
The CHOK1-CX3CR1 cells were washed with PBS and then starved overnight with F12K medium alone. The CHOK1-hCX CR1 cells were digested, centrifuged and collected following standard procedures. Cells were resuspended with F12K and adjusted to a concentration of 3E6/ml (halving the cell concentration in chimeric antibody experiments). mu.L of cells (3E 6/ml) and 60. Mu.L of gradient diluted monoclonal antibody (initial concentration 30. Mu.g/ml, 2-fold gradient dilution, 5 concentration spots) were mixed. The chamber (Corning, 3422) was transferred to a 24-well plate, 100 μl of the cell and antibody mixture was added, and 300 μl of 1 μg/ml concentration huCX CL1-His and 300 μl of the diluted murine monoclonal antibody described above were added to the lower well of the 24-well plate while several antigen-free wells were set. The culture plate is not moved during the cell migration process after the migration for 4 hours at 37 ℃. The migrated cells were then collected and the chamber was gently transferred to a new 24-well plate containing 600 μl PBS. Cells under the chamber membrane and cells under the original 24-well plate were collected and transferred to a U-bottom 96-well plate (Corning, # 3799), and the supernatant was discarded from the centrifuge plate. Mu. L CELLTITER-Glo was added to each well and 120. Mu.L of cell lysate was transferred to the assay plate (Corning, # 3917), left to stand for 10 minutes and luminescence (1 sec/well) was recorded by Thermo VarioSkan Lux.
The results are shown in tables 6-1 to 6-3 below, and the monoclonal antibodies tested can better block the binding of human CX3CL1 to the receptor, thereby inhibiting cell migration and being superior to the control Tab#1.
TABLE 6-1 inhibition of cell migration Activity of murine antibodies
Antibodies to IC50(nM)
mAb017 7.55
mAb022 7.19
mAb032 8.72
TABLE 6-2 inhibition of cell migration Activity of chimeric antibodies
Antibodies to IC50(nM) Antibodies to IC50(nM)
C022 2.892 C032QG 2.265
C032 2.491 Tab#1 19.010
C032NA 4.005
TABLE 6-3 inhibition of cell migration Activity of antibodies
Antibodies to IC50(nM)
HumAb022-4 0.299
C022 0.579

Claims (10)

1. An antibody or antigen-binding fragment thereof that specifically binds CX3CL1, comprising a heavy chain variable region comprising three Complementarity Determining Regions (CDRs): HCDR1, HCDR2 and HCDR3, the light chain variable region comprising three Complementarity Determining Regions (CDRs): LCDR1, LCDR2, and LCDR3; wherein,
The amino acid sequence of HCDR1 is shown in SEQ ID NO. 11;
The amino acid sequence of HCDR2 is shown in SEQ ID NO. 12, 19, 21, 23 or 43;
The amino acid sequence of HCDR3 is shown in SEQ ID NO. 13;
The amino acid sequence of LCDR1 is shown as SEQ ID NO. 15;
the amino acid sequence of LCDR2 is shown as SEQ ID NO. 16;
the amino acid sequence of LCDR3 is shown as SEQ ID NO. 17; or alternatively
The amino acid sequence of HCDR1 is shown in SEQ ID NO. 3;
The amino acid sequence of HCDR2 is shown in SEQ ID NO. 4;
the amino acid sequence of HCDR3 is shown in SEQ ID NO. 5;
The amino acid sequence of LCDR1 is shown in SEQ ID NO. 7;
the amino acid sequence of LCDR2 is shown in SEQ ID NO. 8;
the amino acid sequence of LCDR3 is shown in SEQ ID NO. 9.
2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein:
The heavy chain variable region sequence is shown as SEQ ID NO. 10, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 10; the sequence of the light chain variable region is shown as SEQ ID NO. 14 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 14; or alternatively
The heavy chain variable region sequence is shown as SEQ ID NO. 18 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown as SEQ ID NO. 18; the sequence of the light chain variable region is shown as SEQ ID NO. 20 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 20; or alternatively
The heavy chain variable region sequence is as shown in SEQ ID NO. 22 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO. 22; the sequence of the light chain variable region is shown as SEQ ID NO. 20 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 20; or alternatively
The heavy chain variable region sequence is as shown in SEQ ID NO. 24, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO. 24; the sequence of the light chain variable region is shown as SEQ ID NO. 20 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 20; or alternatively
The heavy chain variable region sequence is shown as any one of SEQ ID NO 27-35 or SEQ ID NO 39, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO 27-35 or SEQ ID NO 39; the sequence of the light chain variable region is shown in any one of SEQ ID NOs 36-38 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in any one of SEQ ID NOs 36-38; or alternatively
The heavy chain variable region sequence is shown as SEQ ID NO. 2, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 2; the sequence of the light chain variable region is shown as SEQ ID NO. 6 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NO. 6;
Preferably, the method comprises the steps of,
The sequence of the heavy chain variable region is shown as SEQ ID NO. 10, and the sequence of the light chain variable region is shown as SEQ ID NO. 14; or alternatively
The sequence of the heavy chain variable region is shown as SEQ ID NO. 18, and the sequence of the light chain variable region is shown as SEQ ID NO. 20; or alternatively
The sequence of the heavy chain variable region is shown as SEQ ID NO. 22, and the sequence of the light chain variable region is shown as SEQ ID NO. 20; or alternatively
The sequence of the heavy chain variable region is shown as SEQ ID NO. 24, and the sequence of the light chain variable region is shown as SEQ ID NO. 20; or alternatively
The sequence of the heavy chain variable region is shown as SEQ ID NO. 39, and the sequence of the light chain variable region is shown as any one of SEQ ID NO. 36-38; or alternatively
The sequence of the heavy chain variable region is shown in any one of SEQ ID NO 27-35, and the sequence of the light chain variable region is shown in any one of SEQ ID NO 36-38; or alternatively
The sequence of the heavy chain variable region is shown as SEQ ID NO.2, and the sequence of the light chain variable region is shown as SEQ ID NO. 6.
3. The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising a heavy chain constant region and/or a light chain constant region; the heavy chain constant region and/or the light chain constant region is of human or mouse origin; preferably, the heavy chain constant region and/or the light chain constant region are human, wherein the sequence of the heavy chain constant region is shown as SEQ ID NO. 25, and the sequence of the light chain constant region is shown as SEQ ID NO. 26.
4. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence as set forth in SEQ ID No. 40 or 42, or comprises an amino acid sequence that is identical to SEQ ID NO:40 or 42, and the amino acid sequence of the light chain has an amino acid sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:41 or comprises a sequence identical to SEQ ID NO:41 has an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity;
Preferably, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 40, and the amino acid sequence of the light chain is shown as SEQ ID NO: 41; or alternatively
The amino acid sequence of the heavy chain is shown as SEQ ID NO. 42, and the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 41.
5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', (Fab') 2, fd, fv, disulfide-linked Fv, scFv, and dAb.
6. A biomaterial, which may be:
(1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-5; preferably, the nucleic acid molecule is a DNA molecule or an RNA molecule;
(2) A vector comprising the nucleic acid molecule of (1); preferably, the vector is a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector;
(3) A host cell comprising the nucleic acid molecule according to (1) or the vector according to (2), or a culture such as a culture solution or a bacterial suspension obtained by culturing the above host cell.
7. The method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which comprises:
1) Chemical synthesis, based on the amino acid sequence of the antibody or antigen binding fragment thereof; or alternatively
2) Biological preparation comprising culturing the host cell of claim 6 and harvesting the antibody or antigen-binding fragment thereof.
8. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5, or the biological material of claim 6; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.
9. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5, the biological material of claim 6, or the antibody composition of claim 8; preferably, the kit is used for diagnosing or detecting CX3CL1 protein or CX3CL1 protein expressing cells in an ex vivo sample.
10. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5, the biological material of claim 6, or the antibody composition of claim 8 for the preparation of a medicament for the prevention and/or treatment of a disease associated with CX3CL1 expression or dysfunction;
preferably, the disease associated with CX3CL1 expression or dysfunction is selected from: rheumatic Arthritis (RA), crohn's disease, ulcerative colitis, diabetes, rheumatoid Vasculitis (RV), and, The symptoms of the disease include Sjogren's Syndrome (SS), systemic Lupus Erythematosus (SLE), neuropsychiatric lupus, scleroderma, multiple sclerosis and arteriosclerosis.
CN202410062177.8A 2024-01-16 2024-01-16 Anti-CX 3CL1 antibody and application thereof Pending CN117946265A (en)

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