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Low immunogenicity anti-tnf-α humanized monoclonal antibody tcx063 and its application

A monoclonal antibody and humanized technology, applied in the fields of antibodies, applications, immunoglobulins, etc., can solve the problems of reducing the therapeutic benefit of patients, limiting antibody re-administration, and complicated drug delivery methods, achieving great application potential and value , reduce the amount of antibody medicine, and avoid the effect of reducing the efficacy of the medicine

Active Publication Date: 2022-03-25
北京天成新脉生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immune response may cause immune complex-mediated clearance of antibody or fragment from circulation and render repeated dosing inappropriate for therapy, reducing therapeutic benefit to patient and limiting re-administration of antibody
At the same time, due to the high immunogenicity, Infliximab needs to be administered through intravenous injection. Compared with subcutaneously administered monoclonal antibodies such as Adalim, the requirements for the operator and the administration environment are more stringent. Harsh and more complicated to administer, and drugs often have short half-lives when given intravenously
However, there are no reports of antibodies to TNF-α with low immunogenicity

Method used

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  • Low immunogenicity anti-tnf-α humanized monoclonal antibody tcx063 and its application
  • Low immunogenicity anti-tnf-α humanized monoclonal antibody tcx063 and its application
  • Low immunogenicity anti-tnf-α humanized monoclonal antibody tcx063 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Sequence analysis and design of humanized anti-TNF-α monoclonal antibody with reduced immunogenicity

[0081]The original sequence of Infliximab monoclonal antibody was analyzed and evaluated by commercial DNAStarTM software. The analysis results showed that the immunogenicity coefficient of Infliximab monoclonal antibody was 16.4, indicating high immunogenicity.

[0082] Find the FR segments of all light and heavy chains in the NCBI human antibody gene library, and use DNAStarTM software to perform immunogenicity analysis on the FR region sequences of human antibodies in the NCBI database, and screen out low immunogenicity human FR regions sequence, and construct a database of low immunogenicity human FR regions.

[0083] The non-antigen-binding fragments (ie, FR regions) in the variable region of the Infliximab antibody were evaluated for immunogenicity using DNAStarTM software, and highly immunogenic fragments were found. The highly immunogenic fragments w...

Embodiment 2

[0091] Embodiment 2 Construction of the expression vector of improved EF nigrosumab TCX063

[0092] According to the nucleotide sequences of the full-length heavy chain and light chain of the anti-TNF-α humanized monoclonal antibody obtained in Example 1, the restriction enzyme cleavage sites on both sides of the light chain sequence were designed as Hind III+EcoR I. The enzyme cleavage sites on both sides of the chain sequence are HindIII+EcoR I, and the full-length heavy chain and light chain full-length sequences carrying the cleavage sites were sent to Goldwisdom Corporation to synthesize the full gene sequence, and the ligation vector used for the synthesis was pUC57. Taking pEE12.4 (for heavy chain gene expression) and pEE6.4 (for light chain gene expression) as expression vectors, the above-mentioned expression vector and the synthesized gene sequence were respectively subjected to corresponding double digestion, and the obtained The target gene and expression vector we...

Embodiment 3

[0093] Example 3 Transient Expression and Purification of Improved EF Istizumab TCX063

[0094] The Escherichia coli DH5α strains carrying different heavy chain and light chain full-length gene expression vectors obtained in Example 2 were cultured, the culture was harvested, and the Qiagen UltraPure plasmid DNA purification kit was used to extract and purify the full-length heavy chain and light chain. gene expression vector. The above-purified plasmid DNA was used to transfect 293F cells using a liposome method kit from Invitrogen Company, and the transfection method was referred to the kit instructions.

[0095] 293F cells were transfected with a combination of expression plasmids carrying different light chain and heavy chain genes respectively, and the combination method is shown in Table 1. A total of 30 expression plasmid combinations were transiently expressed in 293F cells. After culturing the transfected 293F cells for 7 days, the culture supernatant was taken for a...

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Abstract

The invention relates to the field of biotechnology, in particular to TCX063, a humanized monoclonal antibody with low immunogenicity against TNF-α, and its application. The present invention provides anti-TNF-α humanized monoclonal antibody TCX063, which is an anti-TNF-α humanized monoclonal antibody with reduced immunogenicity obtained by modifying the FR region of Infliximab. The antibody provided by the present invention has the same TNF-α antigen-binding site as the Infliximab, and retains the antigen affinity and specificity of the Infliximab, but its immunogenicity is significantly lower than that of EF Liximaclonal antibody. Low immunogenicity TNF-α antibody will reduce the risk of drug side effects caused by the immune response caused by antibody immunogenicity in the human body; reduce the antibody drug ADA; prolong the half-life of the antibody in vivo; at the same time enable subcutaneous administration It becomes possible and has great application potential and value.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to low immunogenicity anti-TNF-α humanized monoclonal antibody TCX063 and application thereof. Background technique [0002] Tumor necrosis factor alpha (TNF-alpha) is a cytokine secreted by immune cells and naturally secreted in inflammatory and immune responses. Studies have shown that TNF-α tends to be up-regulated in patients with various chronic diseases (such as Crohn's disease, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, etc.) TNF-α levels are elevated in the synovial fluid of patients with rheumatoid arthritis (RA), and play an important role in processes such as pathological inflammation and joint destruction. The molecular weight of human TNF-α is 17kDa, and it can exist in the form of monomer or trimer in vivo, and its active form is trimer. TNF-α exerts its biological activity by interacting with the cell surface receptors p55 and p75. Currently,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/24C12N15/13A61K39/395A61P35/00A61P29/00A61P37/02G01N33/68G01N33/577
CPCC07K16/241A61P35/00A61P29/00A61P37/02G01N33/6863G01N33/577C07K2317/24C07K2317/567C07K2317/56C07K2317/73C07K2317/92A61K2039/505G01N2333/525A61K39/395C07K16/24G01N33/68A61P19/02
Inventor 孙乐任文林
Owner 北京天成新脉生物技术有限公司
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