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Low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and application thereof

A technology of monoclonal antibody and cloning antibody, which is applied in the fields of application, antibody, immunoglobulin, etc., and can solve problems such as short half-life, inapplicable treatment, and no antibody

Active Publication Date: 2020-11-10
ABMAX BIOTECHNOLOGY CO LTD
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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-alpha humanized monoclonal antibody TCX063 and application thereof
  • Low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and application thereof
  • Low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and application thereof

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]Using commercial DNAStarTM software to analyze and evaluate the original sequence of Infliximab, the analysis results show that the immunogenicity coefficient of Infliximab is 16.4, which is high in immunogenicity.

[0082] Search the FR regions of all light and heavy chains in the NCBI human antibody gene library, use DNAStarTM software to analyze the immunogenicity of the FR region sequences of human antibodies in the NCBI database, and screen out the human FR regions with low immunogenicity sequence, and construct a low immunogenic human FR region database.

[0083] DNAStarTM software was used to evaluate the immunogenicity of the non-antigen-binding fragment (ie, the FR region) in the variable region of the Infliximab antibody, and find highly immunogenic fragments. Perform BLAST on the highly immunogenic fragment and the sequence in the low immuno...

Embodiment 2

[0091] The construction of the expression vector of embodiment 2 improved EF interest monoclonal antibody TCX063

[0092] According to the nucleotide sequence of the heavy chain and the full-length light chain of the anti-TNF-α humanized monoclonal antibody obtained in Example 1, the enzyme cleavage sites on both sides of the light chain sequence were designed to be Hind III+EcoR I, and the heavy chain was designed. The enzyme cutting 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 enzyme cutting sites are sent to Jinweizhi Company to synthesize the whole gene sequence, and the connection vector used for the synthesis is pUC57. Using pEE12.4 (for heavy chain gene expression) and pEE6.4 (for light chain gene expression) as expression vectors, the above expression vectors and synthetic gene sequences were subjected to corresponding double enzyme digestion, and the obtained The target...

Embodiment 3

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

[0094] The Escherichia coli DH5α strains obtained in Example 2 carrying different heavy chain and light chain full-length gene expression vectors were cultured, the cultures were harvested, and the Qiagen UltraPure plasmid DNA purification kit was used to extract and purify the heavy chain and light chain full-length Gene expression vector. The above-mentioned purified plasmid DNA was transfected into 293F cells using the liposome method kit of 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, the combination methods are shown in Table 1, and a total of 30 combinations of expression plasmids were used for transient expression in 293F cells. After the transfected 293F cells were cultured for 7 days, the culture supernatant w...

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Abstract

The invention relates to the technical field of biology, in particular to a low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and an application thereof. The anti-TNF-alpha humanized monoclonal antibody TCX063 having reduced immunogenicity, provided by the invention is obtained by modifying an FR region of an Infliximab monoclonal antibody. The antibody provided by the invention has the same TNF-alpha antigen binding site as that of the Infliximab monoclonal antibody, and retains the antigen affinity and specificity of the Infliximab monoclonal antibody, but the immunogenicity is obviously lower than that of the Infliximab monoclonal antibody. The low-immunogenicity TNF-alpha antibody can reduce the risk of drug side effects caused by immune response caused by antibodyimmunogenicity original nature in a human body. The antibody drug ADA is reduced. The in-vivo half-life period of the antibody is prolonged. Besides, a subcutaneous administration mode becomes possible, and great application potential and value are achieved.

Description

technical field [0001] 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. Background technique [0002] Tumor necrosis factor alpha (TNF-α) is a cytokine secreted by immune cells and naturally secreted during inflammation and immune response. Studies have shown that the content of TNF-α in patients with various chronic diseases (such as Crohn's disease, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, etc.) tends to be up-regulated (for example, in rheumatoid TNF-α levels are elevated in the synovial fluid of patients with acute 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 biological activity by interacting with cell surface recep...

Claims

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Application Information

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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 ABMAX BIOTECHNOLOGY CO LTD
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