Protease-Cleavable Linkers for Correct Multispecific Antibody Pairing
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Solution Overview
Problem
The challenge in generating multispecific antibodies lies in achieving correct light chain association without light chain mispairing, which is difficult due to the need for extensive modifications at the Fab fragment interface and ensuring optimal ratios of light and heavy chains from different vectors/plasmids.
Innovation Solution
A protease-cleavable linker with the sequence HRRX1X2RSVDE is introduced to facilitate light chain pairing, allowing for intracellular protease-cleavage and correct association of heavy and light chains, forming active antigen binding sites upon protease-cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If design-derived multispecific formats with extensive modifications at the Fab fragment interface are used to achieve correct light chain pairing, then light chain association accuracy is improved, but device complexity and manufacturing difficulty increase significantly
Solution Approach 1:
The invention divides the light chain into two separate expression vectors: one expressing the N-terminal portion (including variable domain) and another expressing the C-terminal portion (including constant domain). This segmentation allows independent optimization of each fragment's expression and simplifies the pairing mechanism, as the protease-cleavable linker naturally facilitates correct reassembly without requiring extensive interface modifications.
Solution Approach 2:
The invention introduces a protease-cleavable linker as an intermediary element between the N-terminal and C-terminal light chain portions. This linker serves as a temporary connector that enables correct light chain association during expression and can be subsequently removed by protease treatment, achieving correct pairing without permanent structural modifications to the antibody fragments.
2Productivity
If multiple plasmids encoding light chains and heavy chains are co-expressed to produce multispecific antibodies, then productivity is improved, but expression ratio control becomes difficult leading to mispairing
Solution Approach 1:
The invention merges the N-terminal and C-terminal light chain portions into a single expression construct connected by a protease-cleavable linker. This ensures that both portions are expressed in stoichiometric equivalence from a single plasmid, eliminating the ratio control problems associated with co-expressing multiple plasmids while maintaining high productivity through efficient single-plasmid expression.
3Reliability
If extensive modifications are made at the Fab fragment interface to enforce correct light chain association, then light chain mispairing is reduced, but ease of manufacture deteriorates due to intensive design requirements
Solution Approach 1:
The invention employs a self-service mechanism where the protease-cleavable linker automatically ensures correct light chain association through its cleavage properties. The linker is designed to be cleaved by specific proteases, which naturally occurs during or after expression, eliminating the need for manual intervention or complex design strategies to enforce correct pairing. The system self-corrects any potential mispairing through the protease cleavage event.
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 enables efficient production of multispecific antibodies with correct light chain pairing and enhanced antigen binding activity, reducing the need for sequence modifications and improving expression ratios.
Implementation Method 1
PCL is a protease-cleavable linker comprising at least one protease-cleavage site; allowing for intracellular protease-cleavage and correct association of heavy and light chains
Data Source
Figure 1A~1B
Figure 2A~2C
Figure 2D
AI summary
The present invention relates to protein molecules comprising at least one protease-cleavable linker. It also relates to protein molecules obtainable by protease-cleavage of such protein molecules and their use in therapy.