Antibody Fusion Protein Preparation via Split Intein Trans-Splicing
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Solution Overview
Problem
Current methods for producing bispecific antibodies face challenges such as mispairing of heavy and light chains, immune response, and stability issues due to structural differences from natural antibodies.
Innovation Solution
A method involving the split of a bispecific antibody into antigen A and antigen B binding portions, expressed separately, and then ligated into an intact antibody using protein trans-splicing mediated by a split intein, ensuring correct pairing and structural integrity similar to natural antibodies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If bispecific antibodies are produced using conventional methods (cell fusion, chemical modification, genetic recombination), then bispecific antibody molecules can be obtained, but mispairing of heavy and light chains occurs and structural stability is compromised
Solution Approach 1:
The invention divides the bispecific antibody production into separate heavy chain and light chain expression components. Each chain is expressed independently with optimized coding sequences, and they assemble correctly through natural antibody assembly mechanisms in the host cell, eliminating mispairing issues associated with conventional co-expression methods
Solution Approach 2:
The invention optimizes the coding sequences of heavy and light chains by adjusting nucleotide parameters to enhance expression levels and reduce mispairing. Specific codon optimizations and sequence modifications are applied to ensure correct pairing while maintaining structural integrity and immunogenicity
2Adaptability or versatility
If structural differences from natural antibodies are introduced to create bispecific antibodies, then dual antigen binding capability is achieved, but immunogenicity and stability are reduced
Solution Approach 1:
The invention optimizes coding sequences through codon optimization and nucleotide parameter adjustment to enhance expression of the bispecific antibody while maintaining natural antibody structure. This approach preserves immunogenicity and stability despite the dual-specificity modifications
Solution Approach 2:
The invention uses gene synthesis techniques to create optimized copies of the heavy and light chain coding sequences that maintain the natural antibody structure while enabling dual antigen binding. The synthesized genes are designed to express proteins with correct folding and natural immunogenicity properties
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach eliminates mispairing of heavy and light chains, enhances stability and immunogenicity, and allows for the production of humanized or fully human bispecific antibodies with retained effector functions and extended half-life.
Implementation Method 1
ligated into an intact antibody using protein trans-splicing mediated by a split intein
Data Source
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Figure 5(A)
AI summary
The present disclosure relates to the field of biological technologies, and discloses a method for expressing and preparing a polyvalent multi-specific antibody and an immune hybrid protein. In the present disclosure, by using the characteristics of protein Intein, a novel method for preparing a polyvalent specific antibody, a hybrid immune protein having an antibody fused to a cytokine, an immunotoxin having an antibody fused to a toxin, or an immune hybrid protein having an antibody fused to other active proteins is designed and developed. In the present disclosure, each portion of a hybrid protein is respectively expressed in a suitable prokaryotic or eukaryotic cell system, separated and purified by high-performance affinity chromatography, and then spliced in vitro by trans-splicing mediated by the intein, to prepare a polyvalent specific antibody and an immune hybrid protein. The method has high production efficiency, and wide scope of application, and facilitates the separation and purification of the products. The present disclosure provides a novel method for preparing biological drugs for directionally attacking cancers or other diseases.