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Antibody aiming at chemokine CX3CL1 and application thereof

An antibody and L-CDR3 technology, applied in the field of biomedicine, can solve the problems of loss of biological activity and high probability of immune rejection, and achieve good in vivo safety

Pending Publication Date: 2021-07-09
SHANGHAI PUREMAB BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the H3-2L4 humanized antibody still retains more mouse-derived amino acids, and the probability of causing rejection immune reactions in humans is relatively high; in addition, its CDR region that binds to the target also retains several post-translational modification positions point, there is a risk of loss of biological activity in the human body due to modification

Method used

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  • Antibody aiming at chemokine CX3CL1 and application thereof
  • Antibody aiming at chemokine CX3CL1 and application thereof
  • Antibody aiming at chemokine CX3CL1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[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 complementarity determinants (CDRs) of the heavy and light chains of the murine antibody 3A5-2 and the framework regions (FRs) supporting the conservative three-dimensional conformation of the antibody were determined. Then search for known human antibody sequences through analysis, select the human antibody heavy chain variable region sequence that is most similar to the mouse antibody, such as IGHV1-69, select its antibody framework region sequence as a template, and convert the mouse antibody heavy chain CDR Combining with human antibody FRs, humanized antibody heavy chain variable region sequences are generated. In the same process, select the IGKV1-13 antibody...

Embodiment 2

[0159] Example 2 Affinity Maturation Engineering of Humanized Antibodies

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

[0161] The humanized antibody 3A5-2_hz20 was selected as a template for affinity maturation engineering. The heavy chain variable region and light chain variable region of 3A5-2_hz20 were connected by GS linker to form a single-chain variable fragment (scFv). The region will remain unchanged in the affinity maturation transformation, and a total of 6 CDRs of the heavy and light chains will be constructed into separate antibody libraries. Based on experience and sequence properties, some amino acids in H-CDR3 are kept unchanged in the modification of affinity maturation, such as figure 2 Invariant amino acids are shown in bold italics.

[0162] Random mutation primers for CDR were designed and synthesized. Only 20% nucleotide mutations were introduced into each mutation position, and there were about 20 base pairs of unmut...

Embodiment 3

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

[0190] 3.1 Construction and screening of engineered antibody library

[0191] The method was as described in the modification of antibody affinity maturation in Example 2. Affinity maturation antibody AM85 was selected as a template for secondary engineering, and an antibody library with random mutations in the full variable region of the heavy and light chains was designed. After 5 rounds of FACS screening with successively decreasing concentrations of mouse CX3CL1 antigen, clones whose FACS binding ability to antigen was stronger than template were obtained, which were re-expanded and cultured, and DNA was extracted and sequenced. Analyze the sequencing results, synthesize new antibody heavy and light chain variable region genes, and construct them into eukaryotic expression vectors. The heavy and light chain expression vectors were cross-combined, transiently transfected into HEK293 cells for ...

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PUM

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Abstract

The invention provides a humanized antibody aiming at CX3CL1 or a fragment thereof, a coding nucleic acid of the antibody or the fragment thereof, a composition containing the antibody or the fragment thereof and the like, and an application of the humanized antibody or the fragment thereof to treatment of diseases. The antibody or the fragment thereof provided by the invention has high affinity to CX3CL1, specifically blocks a CX3CL1 / CX3CR1 signal channel, and blocks the migration activity of CX3CR1 expression cells.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular, the present invention relates to an antibody specifically binding to chemokine CX3C subtype CX3CL1 and its antigen-binding fragment, as well as the application of the antibody and its antigen-binding fragment. Background technique [0002] Chemokines are a class of functionally related small molecule secreted proteins, which are named "chemokines" because of their leukocyte chemotaxis and cytokine activities. In humans, this family consists of about 50 related molecules and has close homologues in other mammals. 20% to 95% of chemokine sequences contain conserved cysteine ​​residues, which are divided into four subtypes according to the number and spacing of cysteines: CC, CXC, XC and CX3C subtypes (C is Cysteine, X is any amino acid). [0003] CX3C subtype has only one chemokine, CX3CL1, also known as fractalkine (FKN) or neuron chemokine (neurotactin), which is the only membr...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/13A61K39/395A61P1/00A61P19/02A61P29/00A61P13/12A61P25/00A61P17/06A61P9/10A61P37/02A61P1/16A61P17/00
CPCA61K2039/505A61P1/00A61P1/16A61P9/10A61P13/12A61P17/00A61P17/06A61P19/02A61P25/00A61P29/00A61P37/02C07K16/24C07K2317/24C07K2317/56C07K2317/565C07K2317/92A61K39/395A61P37/06C07K16/00C12N5/10C12N15/63
Inventor 林鉴蔺利娟朱戬王晋吴建王骊淳任红媛毕建军
Owner SHANGHAI PUREMAB BIO TECH CO LTD
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