Tri- or Tetraspecific Antibody Engineering via Knobs-into-Holes
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Solution Overview
Problem
Current methods for developing bispecific antibodies face challenges such as immune responses, stability issues, and production inefficiencies due to the use of linkers and mispaired by-products, which affect their therapeutic efficacy and production yields.
Innovation Solution
A trispecific or tetraspecific antibody is designed by replacing variable and constant domains of antibody pairs and fusing antigen binding peptides via peptide connectors to the C- or N-terminus of the antibody chains, improving the ratio of desired to undesired products and stabilizing the heterodimer formation through modifications like the 'knobs-into-holes' technology.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If linkers are used to fuse antibody core to binding proteins, then antibody engineering flexibility is improved, but immunogenicity and stability deteriorate due to foreign peptides eliciting immune responses and being prone to proteolytic cleavage
Solution Approach 1:
The patent removes the linker component entirely from the antibody structure. Instead of using linkers to connect antigen-binding domains to the Fc region, the invention employs direct fusion or alternative coupling methods that eliminate foreign peptide sequences, thereby preventing immunogenicity and proteolytic cleavage while maintaining engineering flexibility through modular domain design
Solution Approach 2:
The patent creates composite antibody structures by fusing antigen-binding proteins (such as scFv or Fab fragments) directly to the Fc region of IgG antibodies. This composite approach combines the specificity of antigen-binding domains with the stability and effector functions of the Fc region, eliminating the need for linker peptides while maintaining structural integrity and functional versatility
2Stability of the object's composition
If quadroma technology is used to produce bispecific antibodies similar to natural antibodies, then structural similarity to natural antibodies is improved, but production yield deteriorates due to random pairing generating up to ten different antibody species
Solution Approach 1:
The patent introduces asymmetric modifications to the antibody heavy chains, specifically using 'knobs-into-holes' technology where one heavy chain contains a protruding 'knob' and the other contains a complementary 'hole'. This asymmetric design creates steric complementarity that forces heterodimer formation between different heavy chains while preventing homodimer formation, thereby achieving high yields of desired bispecific antibodies with natural-like structure
Solution Approach 2:
The patent modifies physical and chemical parameters of the antibody structure, specifically introducing disulfide bridges between heavy chains and altering the charge distribution at the interface. These parameter changes stabilize the heterodimer configuration and shift the equilibrium toward desired bispecific product formation, dramatically improving production yield while maintaining structural similarity to natural antibodies
3Productivity
If knobs-into-holes technology is used to force pairing of different antibody heavy chains, then heterodimer formation yield is improved, but light chain compatibility requirements worsen as identical light chains are required for both heavy chains
Solution Approach 1:
The patent segments the antibody structure into independent modular domains, separating the light chain pairing requirements from the heavy chain heterodimerization mechanism. By designing the knobs-into-holes interface specifically on heavy chains and using flexible linker regions or alternative light chain pairing strategies, the invention allows different light chain combinations to be paired with the heterodimeric heavy chains, thereby maintaining high heterodimer yield while enabling versatility in antigen specificity combinations
Data Source
AI summary
The present invention relates to tri- or tetraspecific antibodies, their manufacture and use.