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

A technology of antibodies and sequences, applied in the direction of antibodies, applications, anti-inflammatory agents, etc., can solve the problems of high production costs, inability to be used in clinics, and insufficient affinity of monoclonal antibodies, so as to achieve enhanced affinity, important economic value and social Significance, the effect of improving the ability to inhibit TNFα

Inactive Publication Date: 2014-04-02
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 Method:

[0030] Recombinant human tumor necrosis factor TNFα was purchased from PeprotechAsia (Remcombinant HumanTNF-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 sequence of the D2E7 heavy chain. See SEQ ID NO:152 for the sequence of the D2E7 light chain.

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

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

[0035] First, according to the D2E7 heavy chain, light chain and linker sequence, the complete nucleic acid sequence was 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, the primers were synthesized, and the sequence of e...

Embodiment 2

[0228] Taking 1B7 as an example, we further studied the influence of each mutant amino acid on its affinity. Compared with wild-type D2E7, clone 1B7 has one amino acid mutation in each CDR region of the heavy chain and light chain, and a total of 6 amino acids are mutated. Each mutated amino acid is back-mutated, that is, the mutated amino acid is restored. For the wild-type sequence, the results of phage Elisa showed that the amino acid recovery (His mutation to Ile) mutation in the heavy chain CDR2 region did not affect the affinity of the antibody, but the other 5 amino acid back mutations would affect the affinity of the antibody. In other words, the remaining 5 amino acid mutations can all increase the affinity of D2E7.

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

Embodiment 3

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

[0231] Materials and Method:

[0232] Chinese hamster ovary cell CHO was 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), and protein G (Protein G) was purchased from GE Healthcare Bio-Sciences AB (rProtein A Sepharose 4FastFlow, Cat: 17-0618-06).

[0233] Full-length antibodies, including the variable and constant regions of the heavy chain (human gamma1, gamma2, gamma3, and gamma4 regions), and the variable and constant regions of the light chain (human kappa or lambda regions), usually need to be breast-feeding Animal cell expression has therapeutic activity. Commonly used mammalian cells include CHO, NS0, COS, SP2, 293 and other cells. The heavy chain and light chain of an antibody can be expressed separately on different vectors or on the same vector. Carriers usually require screening marker genes....

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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 the complementarity determining regions of TNFα antibodies, the antibodies and their use in the preparation of drugs for treating TNFα-mediated diseases. Background technique [0002] The body mainly expresses and secretes TNFα (tumor necrosis factor alpha) by monocytes and macrophages. The mature form of TNFα is a trimer composed of a single-chain polypeptide with a molecular weight of 17kD. TNFα induces inflammation and causes 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 identifying inhibitors of TNFα can be used to treat various diseases induced by TNFα, and monoclonal antibodies are currently very...

Claims

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Application Information

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Patent Type & Authority Patents(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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