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Human anti-human alpha interferon antibody and its application

A kind of antibody and antibody fragment technology, applied in human anti-human alpha interferon antibody and its application field

Active Publication Date: 2017-11-17
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, a large number of studies have shown that a variety of autoimmune diseases may be related to the high expression of type I interferon

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Screening and identification of specific human-derived anti-human interferon alpha antibody

[0057] 1. Materials and methods:

[0058]1. Materials: A large-capacity fully synthetic phage single-chain antibody library was constructed by the Academy of Military Medical Sciences of the Chinese People's Liberation Army (ZL200910091261.8), with a library capacity of 1.35×10 10 . Antigens for screening the antibody library were human IFNα_2b recombinantly expressed in Escherichia coli (ProSpec-Tany, Israel) and HSA-IFNα_1b and HSA-IFNα_2b fused with human serum albumin (HSA) (Antibody Online Company). The bacterial strain is XL1-Blue (Stratagene, USA); the phage used is M13KO7 (Invitrogene, USA). The eukaryotic expression vectors pABG1, pABK and pABL are preserved in our laboratory. The vector structure map is shown in Figure 1. Mammalian cells FreeStyle TM HEK293-T was purchased from Invitrogene. The variable region nucleic acid sequence synthesis service of M...

Embodiment 2

[0082] Example 2 Analysis of AIA22 affinity and neutralizing activity in vitro

[0083] 1. Materials and methods:

[0084] 1. Materials: CM5 chips were purchased from GE Healthcare Life Sciences. IFNα_1, IFNα_2, IFNα_4, IFNα_5, IFNα_6, IFNα_8, IFNα_10, IFNα_14, IFNα_16, IFNα_17, IFNα_21 and other subtypes were purchased from PBL Company (product number 11002-1). Daudi cells (human Burkitt's lymphoma cells) were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences. CCK-8 was purchased from Japan Dojin Chemical Co., Ltd. Neonatal bovine serum NCS was purchased from PAA Company. RPMI1640 cell culture medium was purchased from Gibco. Other related materials are the same as in Example 1.

[0085] 2. Method

[0086] 2.1 BIAcore3000 System Determination of Antibody Affinity

[0087] In this example, the BIAcore3000 system was used to detect the affinity of AIA22 to IFNα_2b and HSA-IFNα_1b. The specific steps are as follows: ...

Embodiment 3

[0116] Example 3 AIA22 light and heavy chain CDR region alanine scanning and mutant evaluation

[0117] 1. Materials and methods:

[0118] 1. Materials: Fortebio Octet system and Anti-Human IgG Fc Capture were purchased from iResearch Biotechnology (Shanghai) Co., Ltd. Other relates to material with embodiment 1, embodiment 2.

[0119] 2. Method

[0120] 2.1 Alanine scanning

[0121] Site-directed mutagenesis primers were designed to scan the 6 CDR regions of the AIA22 antibody for alanine, and if the position was alanine, it was mutated to Gly and Ser respectively. Methods The plasmid site-directed mutagenesis method was used, and the specific implementation method can refer to the literature [Wang Ronghao, Chen Ruichuan, Liu Runzhong. An optimized method for fast point mutations. Journal of Xiamen University (Natural Science Edition), 2008, Vol47, sup2, 282-285].

[0122] 2.2. ELISA detection of relative affinity

[0123] Antigens (HSA-IFNα_1b and HSA-IFNα_2b) were di...

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PUM

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Abstract

The invention provides a human anti-human alpha interferon antibody and its coding gene and application. Through the combination of genetic engineering and phage surface display technology, genetically engineered single-chain antibodies against multiple subtypes of human IFNα were screened from the fully synthetic single-chain human antibody library, and the variable region gene sequences of the antibodies were obtained. Here Based on the construction of fully human monoclonal Fab and IgG antibody recombinant vectors, high-purity antibody molecules can be obtained through mammalian cell expression and purification. The binding affinity of the Fab of the antibody obtained in the present invention to human IFNα-2 is not greater than 0.5nM, and the affinity to IFNα-1 is not greater than 5nM. The antibody of the invention can effectively block the biological activity of IFNα-2 and other subtypes of IFNα at the cellular level, and the invention provides a specific antibody drug for the treatment of interferon-related diseases, such as systemic lupus erythematosus and other autoimmune diseases .

Description

technical field [0001] The present invention relates to the preparation and application of human genetically engineered antibodies for treatment, mainly targeting various subtypes of human α interferon (interferon alpha, IFNα), and inhibiting the biological function of IFNα by blocking the combination of IFNα and IFNα receptors. function, so as to achieve the treatment of systemic lupus erythematosus and other autoimmune diseases related to IFNα with genetically engineered antibodies. Background technique [0002] Interferon (interferon, IFN) is a kind of glycoprotein produced by viruses or other kinds of interferon inducers to stimulate the reticuloendothelial system, macrophages, lymphocytes and somatic cells. Interferon is a broad-spectrum antiviral agent, which does not directly kill or inhibit viruses, but mainly makes cells produce antiviral proteins through the action of cell surface receptors, thereby inhibiting virus replication; at the same time, it can also enhanc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/24C12N15/13C12N15/85C07K19/00A61K39/395A61K47/68A61K51/10A61P37/02A61P19/02A61P17/06A61P17/00A61P3/10A61P21/00
Inventor 孙志伟王双杜鹏仇玮祎余云舟
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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