Anti-TNFalpha fully human monoclonal antibodies and application thereof

A fully human antibody and antibody technology, applied in the fields of application, antibody, anti-animal/human immunoglobulin, etc., can solve the problem of infeasible immunity

Active Publication Date: 2015-09-09
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-TNFalpha fully human monoclonal antibodies and application thereof
  • Anti-TNFalpha fully human monoclonal antibodies and application thereof
  • Anti-TNFalpha fully human monoclonal antibodies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Preparation of hybridoma and vector

[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 Baiyiqiao Shenzhou, diluted with PBS, filtered with a 0.22μm needle-type sterile filter, and packed into 1.5ml cell cryopreservation tubes in- Store at 80°C.

[0074] 1.2 Human lymphocyte preparation

[0075] Aseptically harvest fresh tissue samples of chronic tonsillitis (in cooperation with the Department of Otolaryngology, Affiliated Hospital of Xuzhou Medical College, tonsils were taken from patients undergoing tonsillectomy, 10 patients, aged 5 to 10 years), and lymphocyte separator (GE) Isolate the lymphocytes and take 30×10 6 A; aseptically extract human fresh peripheral blood cells, separate the lymphocytes with lymphocyte separation solution (GE), and take 30×10 6 One.

[0076] 1.3 In vitro lymphocyte immunity

[0077...

Embodiment 2

[0109] Example 2: Preparation and identification of monoclonal antibodies

[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, 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, DMEM medium containing 15% fetal bovine serum 3.76.109 cells, gradually reduce the serum to serum-free or serum-free medium containing only a small amount of serum (Invitrogen, 12338-026), put it in 225cm 2 Square bottle, according to inoculation 1×10 5 Cells / ml, inoculate 100ml, inoculate 4 bottles for each cell line.

[0112] 2. Antibody purification

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

Embodiment 3

[0114] 3. Example 3: Affinity maturation

[0115] In order to improve the affinity of the antibody, the constructed single-chain antibody scFv is used as a template to perform random mutations, and the mutant single-chain antibody is phage-displayed, and then screened by ELISA to obtain antibodies with higher affinity.

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

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

[0118] Sequence analysis of antibody gene heavy and light chains. The total RNA of anti-TNFα monoclonal antibody cells uses Invitrogen's reagent kit (15596-026) for extraction; then use the random primers in Takara's 5'RACE FULL kit (D315), using total RNA as template, reverse transcription into the first strand of eDNA, and follow the kit Operating steps in the i...

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Abstract

Belonging to the technical field of antibody drugs, the invention provides anti-TNFalpha fully human monoclonal antibodies and application thereof. The antibodies are derived from human hybrid tumors and are obtained by cytotoxin functional test screening and affinity screening by phage display technology, and have excellent affinity and specificity to TNFalpha. In pharmacodynamic experiments of cell models, the antibodies can effectively neutralize NFalpha and the cytological effect generated thereby. In animal experiments, the antibodies also can effectively neutralize TNFalpha and inhibit the phenotype brought about by TNFalpha in animal models. The effect is not lower than or is better than Humira.

Description

Technical field [0001] The present invention relates to the technical field of antibody drugs. Specifically, it relates to a new set of fully human monoclonal antibody molecules, which can be used to treat diseases caused by TNFα. Background technique [0002] It is well known that the tumor necrosis factor (TNF) family is involved in the regulation of various immune and inflammatory responses, and is a key cytokine for 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 are: rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, sjogren's syndrome, scleroderma, polyarteritis nodosa, and Wegener granulomatosis. TNF participates in the complex cell network response of rheumatoid arthritis (RA) and regulates the inflammatory response, so it is theoretically effective for TNF to treat RA and other autoimmune diseases. In fa...

Claims

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Application Information

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Patent Type & Authority Applications(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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