A conjugate of an antibody against CCR5 and an antifusogenic peptide

A technology of -CCR5 and conjugates, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, non-active ingredients of polymer compounds, peptides, etc., can solve the problem that human health is not necessary

Inactive Publication Date: 2009-08-05
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Additionally, CCR5 function is not essential for human health

Method used

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  • A conjugate of an antibody against CCR5 and an antifusogenic peptide
  • A conjugate of an antibody against CCR5 and an antifusogenic peptide
  • A conjugate of an antibody against CCR5 and an antifusogenic peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0241] Preparation of anti-CCR5 antibody expression plasmid

[0242] will encode the anti-CCR5 antibody light chain variable domain (V L ) and human κ-light chain constant domain (C L ) gene fragment combination, will also be about the anti-CCR5 antibody heavy chain variable domain (V H ) and human γ1-heavy chain constant domain (C H 1-Hinge-C H 2-C H 3) The combination of gene fragments.

[0243] In the case of mAb CCR5 of SEQ ID NO: 63 / 64, the heavy and light chain variable domains were derived from mouse antibodies and the heavy and light chain constant domains were derived from human antibodies (C-κ and IgG1) .

[0244] Subsequently, the gene fragment encoding the complete anti-CCR5 antibody light chain is linked at the N-terminus and / or C-terminus to the nucleic acid encoding the anti-fusion peptide, including the linker sequence, and / or the encoding complete anti-CCR5 antibody The gene segment of the heavy chain is linked at the N-terminus and / or C-t...

Embodiment 2

[0300] Preparation of the final anti-CCR5 antibody expression plasmid

[0301] Using known recombination methods and techniques, by connecting the nucleic acid fragments, a fusion gene (heavy chain and / or light chain antibody fusion gene) is assembled, the fusion gene comprises mAb CCR5 gene fragment, optional linker gene fragment and antifusion peptide gene fragments. Nucleic acid sequences encoding the peptide linker and the anti-fusion polypeptide were separately synthesized by chemical synthesis, and then ligated into an E. coli plasmid for amplification. The nucleic acid sequence of the subclones was verified by DNA sequencing.

Embodiment 3

[0303] Transient expression of immunoglobulins and immunoglobulin variants in HEK293EBNA cells

[0304] Recombinant anti-CCR5 antibody and anti-CCR5 antibody-variants were passed through adherently grown HEK293-EBNA cells (human embryonic kidney cell line 293 expressing Epstein-Barr virus nuclear antigen; American Type Culture Collection ATCC # CRL-10852 ), the cells were grown in supplemented with 10% ultra-low IgG FCS (fetal calf serum, Gibco), 2 mM glutamine (Gibco), 1% volume / volume (v / v) non-essential amino acids (Gibco) and 250 μg / ml G418 (Roche Molecular Biochemicals) in DMEM (Dulbecco's modified Eagle medium, Gibco). For transfection, FuGENE TM 6 Transfection reagent (Roche Molecular Biochemicals) was used in a reagent (μl):DNA (μg) ratio of 3:1-6:1. The light and heavy chains comprising the anti-fusion peptide-anti-CCR5 antibody conjugate light and heavy chains were expressed from two different plasmids, respectively, using a 1:2-2:1 light chain-encoding plasm...

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Abstract

The current invention is related to a conjugate comprising one or more antifusogenic peptides and an antibody against an HIV gp120 binding cell surface receptor, characterized in that one to eight antifusogenic peptides are each conjugated to one terminus of the heavy and / or light chains of said antibody against an HIV gp120 binding cell surface receptor and to the pharmaceutical use of said conjugate.

Description

[0001] The present invention relates to a conjugate of an antibody against CCR5 and an anti-fusion peptide, wherein each of 1-8 anti-fusion peptides is conjugated to one end of the heavy and / or light chain of an anti-CCR5 antibody. The antifusogenic peptides may be different, similar or identical at the amino acid level. Background of the invention [0002] Infection of cells by human immunodeficiency virus (HIV) is achieved by a process in which the membrane of the cell to be infected fuses with the viral membrane. A general scheme of this process is proposed: the viral envelope glycoprotein complex (gp120 / gp41) interacts with cell surface receptors located on the membrane of the cell to be infected. Binding of gp120 to, for example, the CD4 receptor in combination with a co-receptor such as CCR-5 or CXCR-4 results in a conformational change of the gp120 / gp41 complex. As a result of the conformational change, the gp41 protein is able to insert into the membrane of the target...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61K47/42
CPCA61K47/48561C07K16/28C07K14/162C07K14/005A61K47/6811A61K47/6841C12N2740/16122
Inventor 迈克尔·勃兰特斯特凡·费希尔埃哈德·科佩茨基苏里亚娜拉亚娜·桑库拉特里拉尔夫·舒马赫
Owner F HOFFMANN LA ROCHE & CO AG
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