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Multi-specific antibody targeting HIV gp120 protein and human CD3 molecule and application thereof

A multi-specific antibody and specific technology, applied in the field of immunoglobulin, can solve the problem of inability to completely remove HIV, and achieve the effect of reducing the load of virus and virus pool

Active Publication Date: 2022-04-12
珠海臻谱基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, none of the programs used clinically to treat AIDS can completely eliminate HIV from the patient's body, and the patient needs to take medicine for life

Method used

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  • Multi-specific antibody targeting HIV gp120 protein and human CD3 molecule and application thereof
  • Multi-specific antibody targeting HIV gp120 protein and human CD3 molecule and application thereof
  • Multi-specific antibody targeting HIV gp120 protein and human CD3 molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1, Construction and expression of the recombinant vector of multispecific antibody HIV-DNS

[0078] 1. Structure of multispecific antibody HIV-DNS

[0079] (1) Schematic diagram of the multispecific antibody HIV-DNS structure as shown in figure 1 shown

[0080] The light chain variable region of the HIV-DNS multispecific antibody molecule adopts the HIV virus receptor-domain 1 of the human T cell surface glycoprotein CD4 molecule (https: / / www.uniprot.org / uniprot / P01730, Domain 1 of human T-cell surface glycoprotein CD4, CD4-D1), this domain is the main domain of HIV virus binding to human CD4+ cells (Primary receptor for human immunodeficiency virus-1 (HIV-1)), which can bind to gp120 protein of HIV .

[0081] The light chain constant region of HIV-DNS adopts the constant region of human immunoglobulin kappa light chain (humanImmunoglobulin kappa chain constant region, IGKC, https: / / www.uniprot.org / uniprot / P01834).

[0082] In the heavy chain of the anti-gp1...

Embodiment 2

[0123] Embodiment 2, ELISA method detects the binding force of HIV-DNS and HIV gp120 glycoprotein

[0124] Commercially purchased recombinant HIV gp120 glycoprotein (purchased from Abcam, product number 174070) was diluted to 1.0 ug / ml with coating solution, coated onto the microtiter plate at 100 ng / well, and kept overnight at 4 °C. Then add PBST solution containing 2% BSA to each well and incubate for blocking for 1.5-2 hours. During the incubation period, the blocking solution was diluted with PBST to a final BSA concentration of 0.5% BSA. As the antibody diluent, the multispecific antibody HIV-DNS was serially diluted, starting from 10 ug / ml to 1.25 pg / ml. Human IgG (Shanxi Kangbao Biological Products Co., Ltd.) was used as reference substance 1, and HIV-TS with the same target was used as reference substance 2. After the enzyme plate is sealed, it is fully washed. Add double-diluted antibodies, 100 ul / well, and incubate at 37°C for 1 hour. After washing thoroughly, add ...

Embodiment 3

[0126] Example 3. Surface plasmon resonance (SPR) method to detect the affinity of HIV-DNS to recombinant gp120

[0127] The measuring instrument adopts Biacore X100 of GE. Amine coupling kit, human antibody capture kit, CM5 chip, 10×HBS-EP pH 7.4 and other related reagents and consumables were purchased from GE Healthcare.

[0128] The affinity of multispecific antibody HIV-DNS was determined by capture method. When determining the affinity of the HIV-DNS multispecific antibody purified and prepared in step (3) of Example 1 to gp120, the anti-Fc antibody was coupled to the surface of the CM5 chip, and HIV-DNS was diluted to an appropriate concentration to capture the CM5 The surface of the chip is used as the stationary phase, and the recombinant gp120-His (purchased from Abcam, product number ab174070) is set to flow through the surface of the stationary phase in a series of concentration gradients using a single-cycle method. The flow rate is 30ul / min, and the affinity bet...

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Abstract

The invention discloses a multispecific antibody targeting HIV (human immunodeficiency virus) gp120 protein and human CD3 molecules and application of the multispecific antibody. The multi-specific antibody comprises an antibody specifically bound with gp120 and a single-chain antibody specifically bound with CD3 at the 471st-710th sites of which the amino acid sequence is sequence 1, and the antibody specifically bound with gp120 comprises a heavy chain variable region at the 1st-136th sites of which the amino acid sequence is sequence 1 and a light chain variable region at the 1st-125th sites of which the amino acid sequence is sequence 3, and the single-chain antibody is fused with the C terminal of the heavy chain of the antibody which is specifically combined with the gp120. The multispecific antibody targets different epitopes of gp120, the neutralizing effect of HIV viruses can be remarkably improved, the probability of generation of virus escape mutation is reduced, meanwhile, the viruses are better killed by targeting CD3 molecules, and the clinical curative effect of antibody treatment is improved.

Description

technical field [0001] The invention relates to the field of immunoglobulins, in particular to multispecific antibodies targeting HIV gp120 protein and human CD3 molecules and applications thereof. Background technique [0002] Acquired Immune Deficiency Syndrome (AIDS), also known as AIDS, is caused by infection with Human Immunodeficiency Virus (HIV). HIV can destroy the human immune system, and the most important CD4 in the human immune system + As the main target of attack, T lymphocytes destroy the cells in large numbers and make the human body lose its immune function. Therefore, the human body is susceptible to various diseases, and malignant tumors can occur, with a high mortality rate. HIV invades CD4 + For T cells, the CD4 molecule on the cell surface is the main receptor, and CCR5 or CXCR4 is the co-receptor. In the first step of infection, the virus approaches the cell through its outer membrane protein gp120 binding to the CD4 molecule on the cell, and furth...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13C12N15/85C12N5/10A61K39/42A61P31/18
Inventor 胡显文李磊焦长乐袁盛凌廖翔范泰漓杨立宪刘紫龙袁建军杜滨胡嘉琪王延明
Owner 珠海臻谱基因科技有限公司
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