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An antigen-binding molecule, a pharmaceutical composition, and a method

A technology of antigen-binding molecules and binding capabilities, applied in the fields of botanical equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc.

Pending Publication Date: 2022-02-25
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limitation is that one antibody molecule typically has two binding sites and therefore only neutralizes two targets after administration (one antigen per binding site)

Method used

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  • An antigen-binding molecule, a pharmaceutical composition, and a method
  • An antigen-binding molecule, a pharmaceutical composition, and a method
  • An antigen-binding molecule, a pharmaceutical composition, and a method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0654] Preparation of recombinant C1r2s2

[0655] 1.1. Expression and purification of cynomolgus monkey C1r2s2 His / Flag tetramer

[0656] Sequences used for expression and purification were: Cynomolgus Cls (SEQ ID NO: 1 ) with a C-terminal GGGGS linker and FLAG tag and Cynomolgus Clr with a C-terminal GGGGS linker and 8×histidine tag. The cynomolgus Clr sequence has the R463QS654A mutation (SEQ ID NO: 2). To express recombinant cynomolgus C1r2s2 His / Flag tetramer, FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA) was used to transiently co-express cynomolgus C1s-Flag and cynomolgus C1r-His. Conditioned medium expressing recombinant cynomolgus C1r2s2 His / Flag tetramer was applied to anti-FlagM2 affinity resin (Sigma) and eluted with Flag peptide (Sigma). Fractions containing recombinant cynomolgus C1r2s2 His / Flag tetramer were passed through an IMAC column (GE Healthcare) and eluted with an imidazole gradient. The eluted fractions containing the recombinant C1r2s2 ...

Embodiment 2

[0660] 2.1 Preparation of cynomolgus Fcγ receptor (cynomolgus FcγR)

[0661] Synthesis of extracellular domain genes for cynomolgus FcγRs was constructed by cloning the cDNA of each FcγR from cynomolgus monkeys using methods known to those skilled in the art. The amino acid sequence of the constructed FcγR extracellular domain is shown in the sequence list: (for cynomolgus FcγRIa, SEQ ID NO: 5; for cynomolgus FcγRIIa1, SEQ ID NO: 6; for cynomolgus FcγRIIa2, SEQ ID NO: 7; for cynomolgus FcγRIIa3, SEQ ID NO: 8; for cynomolgus FcγRIIb, SEQ ID NO: 9; for cynomolgus FcγRIIIa (R), SEQ ID NO: 10; for cynomolgus FcγRIIIa (S), SEQ ID NO: 11). His-tags were then added to their C-terminals and each of the obtained genes was inserted into expression vectors designed for expression in mammalian cells. The expression vector was introduced into FreeStyle293 cells (Invitrogen) derived from human embryonic kidney cells to express the target protein. After culturing, the resulting culture su...

Embodiment 3

[0663] Preparation of anti-C1s antibody

[0664] 3.1. Production of COS0098pHv1 and preparation of COS0098pHv1-SG1077 and COS0098pHv1-SG1

[0665] The variable regions of some anti-C1s antibodies COS0098bb (VH, SEQ ID NO: 12; VL, SEQ ID NO: 13, as described in PCT / JP2018 / 042054) were humanized to reduce the potential immunogenicity of the antibodies. The complementarity determining regions (CDRs) of the anti-C1s rabbit antibody were grafted onto the cognate human antibody framework (FR) using the conventional CDR grafting method (Nature 321:522-525 (1986)). The gene encoding the humanized VH was synthesized and combined with human IgG1 CH (SG1, SEQ ID NO: 14), modified human IgG1 CH such as SG1077 (SEQ ID NO: 15) and SG1148 (SEQ ID NO: 16). The gene encoding the humanized VL was synthesized and combined with human CL (SK1, SEQ ID NO: 26) and modified human CL (KOMC, SEQ ID NO: 17). Those combined sequences were cloned into expression vectors.

[0666] A number of mutations ...

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Abstract

The present invention provides antigen-binding molecules that bind to C1s and comprise a heavy chain variable region, a light chain variable region and an Fc region, wherein (i) the heavy chain variable region and / or the light chain variable region comprise at least one amino acid that can increase the ratio of KD value of the antigen-binding molecule to C1s in acidic pH range to KD value of the antigen-binding molecule to C1s in neutral pH range, KD (acidic pH range) / KD (neutral pH range), compared to that of the first reference antibody; (ii) the Fc region comprises at least one amino acid that can increase binding ability of the antigen-binding molecule to FcRn in acidic pH range compared to that of the second reference antibody; and (iii) the Fc region comprises at least one amino acid that can increase binding ability of the antigen-binding molecule to Fc[gamma] receptor in neutral pH range compared to that of the second reference antibody.

Description

technical field [0001] The present invention relates to antigen binding molecules, pharmaceutical compositions and methods. Background technique [0002] The complement system consists of approximately 25-30 complement proteins that play key roles in host defense against pathogens, foreign antigens and tumor cells. The complement system is also involved in maintaining homeostasis by clearing the body of immune complexes and apoptotic cells. Complement components exert these functions by interacting in a cascade of enzymatic processes and membrane-bound events. The net result of these processes is the production of products with lytic, immunomodulatory, and opsonic functions. [0003] It is well known that the complement system can be divided into three distinct pathways: the classical pathway, the lectin pathway and the alternative pathway. Although each pathway begins differently, all three pathways converge and share the same terminal complement components (C5 to C9), w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13
CPCC07K16/18C07K2317/92C07K2317/72C07K2317/24C07K2317/94C07K2317/56
Inventor 古贺光
Owner CHUGAI PHARMA CO LTD