HER2 Antigen-Binding Construct Mutations for Reduced scFv Aggregation
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Solution Overview
Problem
Existing HER2-targeted antibody drugs face challenges such as antibody aggregation during expression and purification, leading to issues like immunogenicity, toxicity, degradation, impaired avidity, and loss of activity, especially in single-chain molecules like scFvs which form dimers, trimers, and tetramers.
Innovation Solution
Development of an antigen-binding construct with specific mutations in the heavy and light chain variable regions of scFv fragments, such as K30E and F53Y according to the Kabat numbering system, to reduce aggregation and enhance stability, while maintaining binding affinity to HER2.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If single-chain scFv molecules are used for HER2 targeting, then molecular flexibility and reduced immunogenicity are improved, but aggregation tendency increases leading to dimer, trimer and tetramer formation
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at defined positions (Kabat numbering positions 30, 31, 32, 53, 54, 55 in the variable regions) to alter the physical-chemical properties of the scFv molecule. These mutations change the aggregation propensity while maintaining binding function, directly resolving the contradiction between reduced immunogenicity and aggregation stability.
2Stability of the object's composition
If antibody aggregation is reduced through mutations, then stability and binding affinity are improved, but protein expression and purification complexity increases
Solution Approach 1:
The patent introduces specific amino acid substitutions (such as K30E, F53Y mutations) that modify the protein's physical-chemical parameters to reduce aggregation. These targeted parameter changes improve stability and binding affinity while the mutations are designed to be implementable in standard expression systems, balancing complexity improvements.
Data Source
AI summary
Disclosed are antigen binding constructs targeting HER2 and uses thereof. Specifically provided is an antibody comprising a monovalent antigen-binding fragment that specifically binds to the HER2 ECD4 antigen on HER2-expressing cells, with the heavy chain variable region containing a mutation at position 30 and the light chain variable region containing a mutation at position 53, both according to Kabat numbering, a nucleic acid that encodes the same, a vector that comprises said nucleic acid, a cell that comprises an isolated nucleic acid of said vector, and a pharmaceutical composition that comprises the foregoing. Further provided are uses thereof in aspects such as treating subjects who suffer from HER2-expressing tumors, killing HER2-expressing tumor cells, or inhibiting the growth of HER2-expressing tumor cells.