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Antibody of TNF (Tumor Necrosis Factor) alpha and application thereof

A technology of antibodies and antigens, applied in the direction of antibodies, applications, anti-inflammatory agents, etc., can solve the problems of insufficient affinity of monoclonal antibodies, cannot be used in clinical practice, and high production costs, achieve important economic value and social significance, and improve inhibition TNFα ability, effect of enhancing affinity

Inactive Publication Date: 2010-11-03
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Screening and identification of human antibody phage libraries to obtain therapeutic monoclonal antibodies is a common method, but its disadvantage is that the affinity of monoclonal antibodies identified by this method is not high enough to be used in clinical practice
Moreover, full-length therapeutic monoclonal antibodies must be expressed using mammalian cells, and the production cost is high

Method used

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  • Antibody of TNF (Tumor Necrosis Factor) alpha and application thereof
  • Antibody of TNF (Tumor Necrosis Factor) alpha and application thereof
  • Antibody of TNF (Tumor Necrosis Factor) alpha and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Materials and methods:

[0030] Recombinant human tumor necrosis factor TNFα was purchased from PeprotechAsia (Remcombinant Human TNF-α, Cat: 300-01A).

[0031] The phage display kit was purchased from Amersham Bioscience (Recombinant phage antibody system, Cat: 27-9401-01).

[0032] See SEQ ID NO:151 for the heavy chain sequence of D2E7. The D2E7 light chain sequence is shown in SEQ ID NO:152.

[0033] Linker sequence between heavy and light chains (SEQ ID NO: 138):

[0034] 5'-GGTGGAGGCGGTTCAGGTGGAGGCGGTTCAGGTGGAGGCGGTTCA-3', encoded amino acid sequence (SEQ ID NO: 135): 5'-(Gly 4 Ser) 3 -3', a total of 15 amino acids.

[0035] First, according to the heavy chain and light chain of D2E7 and the sequence of linker (linker), the complete nucleic acid sequence is artificially synthesized in the following order: (V H ) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker sequence-(V L )FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (D2E7scFv), see figure 1 .

[0036] Secondly, primers were synth...

Embodiment 2

[0228] Taking 1B7 as an example, the effect of each mutant amino acid on its affinity was further studied in depth. Compared with the wild-type D2E7, clone 1B7 has mutated one amino acid in each CDR region of the heavy chain and the light chain, and mutated 6 amino acids in total. Each mutated amino acid is back-mutated, that is, the mutated amino acid is restored. It is a wild-type sequence, and the results of phage Elisa show that the amino acid reversion (His to Ile) mutation in the heavy chain CDR2 region does not affect the affinity of the antibody, but the other 5 amino acid reversion mutations will affect the affinity of the antibody. In other words, the remaining 5 amino acid mutations can all improve the affinity of D2E7.

[0229] The mutation method and the phage Elisa method are described above.

Embodiment 3

[0230] Example 3: Expression and purification of full-length antibodies

[0231] Materials and methods:

[0232] Chinese hamster ovary cells CHO were purchased from ATCC, recombinant protein A rProtein A was purchased from GE Healthcare Bio-Sciences AB (rProtein A Sepharose Fast Flow, Cat: 17-1279-03), protein G (Protein G) was purchased from GE Healthcare Bio-Sciences AB (rProtein A Sepharose 4 FastFlow, Cat: 17-0618-06).

[0233] Full-length antibodies, including the variable and constant regions of the heavy chain (human gamma1, gamma2, gamma3, gamma4 regions), and the variable and constant regions of the light chain (human kappa or lambda region), usually need to be produced in lactation Animal cell expression has therapeutic activity. Commonly used mammalian cells include CHO, NSO, COS, SP2, 293 and other cells. The heavy chain and light chain of an antibody can be expressed separately on different vectors, or expressed on the same vector. Vectors usually require scre...

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Abstract

The invention relates to an antibody of TNF (Tumor Necrosis Factor) alpha and application thereof. Through carrying out mutation on a complementary determining region of the antibody of the TNF alpha, the complementary determining region and a full-length antibody of the antibody of the high-affinity TNF alpha are discovered. The antibody of the high-affinity TNF alpha can be used for preparing medicaments for treating diseases induced by the TNF alpha. The survival rate of a mouse is above 70 percent when the antibody is in 5 mu g of dosage level.

Description

technical field [0001] The present invention relates to inhibitors of TNFα (tumor necrosis factor alpha) and uses thereof. Specifically, the present invention relates to complementarity-determining regions of antibodies to TNFα, the antibodies and their use in the preparation of medicines for treating TNFα-mediated diseases. Background technique [0002] The body mainly expresses and secretes TNFα (tumor necrosis factor alpha) from monocytes and macrophages. The mature form of TNFα is a trimer composed of a single-chain molecular weight of 17kD polypeptide. TNFα induces inflammation, leading to tissue damage. Current studies have shown that TNFα mediates a variety of diseases, including autoimmune diseases, organ transplant rejection, infection, sepsis, tumors and so on. [0003] Due to the various harmful physiological effects of TNFα, screening and identification of TNFα inhibitors can be used to treat various diseases induced by TNFα, and monoclonal antibodies are curre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N15/13A61K39/395A61P1/00A61P1/04A61P17/06A61P19/02A61P19/04A61P19/08A61P29/00A61P31/00A61P35/00A61P37/02A61P37/06
Inventor 常永生方福德董学雨左瑾
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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