Multispecific Antibody Heterodimerization via CH1-CL Charge Engineering
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Solution Overview
Problem
Current methods for producing multispecific antibodies face challenges such as immune responses against linkers, poor stability due to proteolytic cleavage, and high levels of undesired side products, which affect yield and therapeutic efficacy.
Innovation Solution
Development of multispecific antibodies with specific amino acid substitutions in the CH1 and CL domains, such as replacing amino acids at positions 124, 147, and 213, and using VH/VL domain replacement to enhance heterodimerization and reduce Bence-Jones-type side products, thereby improving yield and stability.
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 proteolytic cleavage increase
Solution Approach 1:
The invention removes linkers from the antibody structure entirely, using direct fusion of heavy and light chains without intervening linker sequences. This extraction of the harmful linker component eliminates the source of immunogenicity and proteolytic vulnerability while maintaining the multispecific functionality through alternative structural arrangements.
Solution Approach 2:
The invention creates composite antibody structures by fusing heavy and light chains directly to form integrated heterodimeric or heterotetrameric molecules. This composite approach combines different antibody specificities into a single unified structure without requiring external linker components, achieving both structural integrity and functional versatility.
2Stability of the object's composition
If quadroma technology is used to produce bispecific antibodies, then natural antibody structure is retained, but mispaired byproducts and production yield increase
Solution Approach 1:
The invention introduces asymmetric charge distributions at the heavy-light chain interface through specific amino acid substitutions. By creating asymmetric electrostatic interactions (positive charges on one chain paired with negative charges on the other), the design ensures directional and specific pairing between heavy and light chains, preventing mispairing while maintaining natural antibody structure.
Solution Approach 2:
The invention modifies the electrostatic parameters at the heavy-light chain interface by substituting specific amino acids with charged residues. This parameter change creates complementary charge patterns that thermodynamically favor correct heterodimer formation over homodimer or mispaired structures, thereby improving both structural fidelity and production yield.
3Manufacturing precision
If knobs-into-holes technology is used to force heavy chain pairing, then heterodimer formation is improved, but light chain identity constraints increase
Solution Approach 1:
The invention copies the successful heterodimerization strategy from the knobs-into-holes approach (which modifies CH3 domains) and applies it to the CH1/CL interface instead. By creating complementary charged residues at this earlier interface, the invention achieves heterodimer specificity without requiring identical light chains, as the charge-based recognition occurs before light chain pairing constraints would apply.
Data Source
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AI summary
The present invention relates to multispecific antibodies, their manufacture and use.