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Anti-tnfα fully human monoclonal antibody and its application

A fully human antibody, antibody technology, applied in applications, antibodies, anti-animal/human immunoglobulins, etc., can solve problems such as infeasible immunity

Active Publication Date: 2018-04-17
BEIJING ANBAOKANG BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When murine MAb production became mature, researchers expected to produce human MAb through conventional hybridoma technology, but because human hybridoma cell lines cannot stably produce high-yield antibodies, and for many antigens, human In vivo immunity is not feasible

Method used

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  • Anti-tnfα fully human monoclonal antibody and its application
  • Anti-tnfα fully human monoclonal antibody and its application
  • Anti-tnfα fully human monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: the preparation of hybridoma and carrier

[0071] 1. Antigen and lymphocyte preparation and immunization

[0072] 1.1 Antigen TNFα protein preparation

[0073] Human TNFα protein (drug target) and monkey TNFα (drug target) protein were purchased from Yiqiao Shenzhou, diluted with PBS, filtered with a 0.22 μm needle sterile filter, and distributed in 1.5ml cell cryopreservation tubes in- Store at 80°C.

[0074] 1.2 Human lymphocyte preparation

[0075]Fresh tissue samples of chronic tonsillitis were collected aseptically (in cooperation with the Department of Otolaryngology, Affiliated Hospital of Xuzhou Medical College, tonsils were taken from patients undergoing tonsillectomy, 10 patients, aged 5-10 years), and lymphocyte separation medium (GE) Lymphocytes were separated, and 30×10 6 pcs; Aseptically extract human fresh peripheral blood cells, separate the lymphocytes in them with lymphocyte separation medium (GE), take 30×10 6 indivual.

[0076] ...

Embodiment 2

[0109] Example 2: Preparation and Identification of Monoclonal Antibody

[0110] 1. Preparation of antibodies by in vitro culture method

[0111] The 1.1.71, 1.5.58, 1.6.17, 1.24.2, 2.2.108, 2.39.45, 3.85.3, 3.4.32, 3.12.78, 3.76.109 cells, gradually reduce serum to serum-free or serum-free medium containing only a small amount of serum (Invitrogen, 12338-026), put in 225cm 2 Square bottle, according to inoculation 1×10 5 cells / ml, inoculate 100ml, and inoculate 4 bottles of each strain of cells.

[0112] 2. Antibody purification

[0113] Take out at 225cm 2 The cell supernatant of the square flask cultured for about 10 days was centrifuged to remove cell debris, and the antibody was purified by rProteinA affinity chromatography. Cell culture supernatant at pH 7.4 was filtered through a 0.22 μm filter membrane and loaded directly, then washed with pH 7.4 PBS to 10 times the volume of the column bed, and then eluted with pH 3.0 glycine solution, collected and eluted in...

Embodiment 3

[0114] 3. Example 3: Affinity maturation

[0115] In order to improve the affinity of the antibody, the constructed single-chain antibody scFv was used as a template for random mutation, and the mutated single-chain antibody was subjected to phage display, and then screened by ELISA to obtain an antibody with higher affinity.

[0116] 3.1 Sequence the heavy and light chains of Anti-TNFα

[0117] After immunization, fusion, and monoclonalization, anti-TNFα monoclonal antibody cell lines were selected as templates for antibody affinity maturation experiments based on the results of affinity and cell function experiments.

[0118] Sequence analysis of heavy and light chains of antibody genes. The total RNA of the anti-TNFα monoclonal antibody cells was used by Invitrogen Reagent kit (15596-026) was used for extraction; then random primers in the 5'RACE FULL kit (D315) of Takara Company were used, and total RNA was used as a template to reverse transcribe into first-strand eDN...

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Abstract

The invention belongs to the technical field of antibody drugs, and provides anti-TNFα fully human monoclonal antibodies and applications thereof. These antibodies are derived from human hybridomas and obtained through cytotoxic function test screening and affinity screening by phage display technology. Excellent affinity and specificity for TNFα. In the pharmacodynamic experiments of cell models, these antibodies can effectively neutralize TNFα and its cellular effects. In animal experiments, these antibodies can also effectively neutralize TNFα and inhibit the phenotype brought about by TNFα in animal models. Its effect shows no less or better than Humira.

Description

technical field [0001] The invention relates to the technical field of antibody drugs. Specifically, it relates to a new set of fully human monoclonal antibody molecules that can be used in the treatment of diseases caused by TNFα. Background technique [0002] It is well known that the tumor necrosis factor (tumor necrosis factor, TNF) family is involved in the regulation of various immune and inflammatory responses, and is a key cytokine in autoimmune diseases. Autoimmune diseases refer to diseases caused by the body's immune response to self-antigens, resulting in damage to its own tissues. Common systemic autoimmune diseases include: rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polyarteritis nodosa, Wegener's granulomatosis. TNF participates in the complex cell network response of rheumatoid arthritis (RA) and regulates the inflammatory response. Therefore, targeting TNF to treat RA and other autoimmune di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/24C12N15/13C12N15/63A61K39/395A61P19/02A61P29/00A61P1/00A61P17/06
Inventor 冯晓张洁涵李福军王涛李新灵
Owner BEIJING ANBAOKANG BIO PHARMA CO LTD
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