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Anti-TNF-α humanized monoclonal antibody tcx060 with low immunogenicity and low ADCC/CDC function and its application

A monoclonal antibody, humanized technology, applied in the direction of DNA/RNA fragments, antibodies, applications, etc., can solve the problems of reducing the therapeutic benefit of patients, increasing the risk of side effects, limiting the re-administration of antibodies, etc., to achieve great application potential and Value, low immunogenicity, effect of improving drug safety

Active Publication Date: 2022-04-19
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, the original research of Cytol beads needs to be used in combination with immunosuppressants, which greatly increases the risk of side effects
However, there are no reports of antibodies to TNF-α with low immunogenicity

Method used

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  • Anti-TNF-α humanized monoclonal antibody tcx060 with low immunogenicity and low ADCC/CDC function and its application
  • Anti-TNF-α humanized monoclonal antibody tcx060 with low immunogenicity and low ADCC/CDC function and its application
  • Anti-TNF-α humanized monoclonal antibody tcx060 with low immunogenicity and low ADCC/CDC function and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Analysis and Design of Certolizumab Monoclonal Antibody with Low ADCC / CDC Function and Reduced Immunogenicity

[0054] The present invention analyzes and evaluates the original sequence of the certoliz monoclonal antibody by using the commercial DNAStarTM software, and the analysis result shows that the immunogenicity of the original certoliz monoclonal antibody sequence is very low. It is speculated that after the PEGylation of the original certolizumab monoclonal antibody was performed to remove the ADCC / CDC function, the multimer of the antibody increased, which in turn caused the ADA of the drug to be as high as 23%. Therefore, it is necessary to remove the PEGylation modification of the original certolizumab monoclonal antibody, and at the same time reduce its ADCC / CDC function. The amino acid sequences of the full length of the light chain and the variable region of the heavy chain of the original certo beads are shown in SEQ ID NO.1 and SEQ ID NO.2, res...

Embodiment 2

[0056] Example 2 Construction of the expression vector of anti-TNF-α humanized monoclonal antibody TCX060

[0057] According to the amino acid sequence of the full-length heavy chain and light chain of the anti-TNF-α fully humanized monoclonal antibody H2L0 obtained in Example 1 and the codon preference of the host, the nucleotides encoding the heavy chain H2 and the light chain L0 were designed sequence (wherein the nucleotide sequence of L0 is shown in SEQ ID NO.6, and the nucleotide sequence of H2 is shown in SEQ ID NO.5), and the enzyme cutting sites on both sides of the light chain sequence are designed to be Hind III+EcoR I. Design the enzyme cutting sites on both sides of the heavy chain sequence as Hind III+EcoR I, send the full-length heavy chain and light chain full-length sequences carrying the enzyme cutting sites to Jinweizhi Company to synthesize the whole gene sequence, and synthesize the used The connection vector is pUC57. Using pEE12.4 (for heavy chain expre...

Embodiment 3

[0059] Example 3 Transient Expression of Anti-TNF-α Humanized Monoclonal Antibody TCX060

[0060] The Escherichia coli DH5α strains obtained in Example 2 carrying the expression vectors of the full-length genes of the heavy chain H2, H0 and light chain L0 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.

[0061] The positive cells obtained by transfection were used for antibody expression, and the expression level of the monoclonal antibody TCX060 whose combination of heavy chain and light chain is H2L0 is shown in Table 2.

[0062] Table 2 The expression level detection result of monoclonal antibody TCX060 (mg / L)

[0063]

[0064]The ant...

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Abstract

The invention relates to the field of biotechnology, in particular to TCX060, a humanized monoclonal antibody against TNF-α with low immunogenicity and low ADCC / CDC function, and its application. The present invention provides an anti-TNF-α humanized monoclonal antibody TCX060, which is obtained by modifying the certolizumab monoclonal antibody as follows: (1) removing PEG modification; (2) transforming it into a full-length antibody of human IgG1 subtype; (3) A flexible amino acid stretch is inserted between the CDR3 and CH2 regions. The binding affinity of TCX060 to human TNF‑α is similar to that of certolizumab, and it can specifically block the binding of TNF‑α to TNF receptors on the cell surface. The originality is significantly lower than that of certolizumab monoclonal antibody, which reduces the risk of immunogenicity of the antibody in vivo while removing the ADCC / CDC function, and has excellent application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to TCX060, an anti-TNF-α humanized monoclonal antibody with low immunogenicity and low ADCC / CDC function, 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/24C12N15/13A61K39/395A61P35/00A61P29/00A61P37/02
CPCC07K16/241A61P35/00A61P29/00A61P37/02C07K2317/24C07K2317/76C07K2317/92C07K2317/33C07K2317/732C07K2317/734A61K2039/505A61P37/04C12N15/62C07K2317/52C07K2317/524C07K2317/565
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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