Bispecific Antibody Format with Segmented Light Chains
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Solution Overview
Problem
Existing bispecific antibody formats face challenges in maintaining high binding affinity to two different antigens, particularly when the antigen binding affinity of the inner variable region decreases, and the production process is complicated by the need for linker cleavage, which affects the stability and yield of the antibody.
Innovation Solution
A novel bispecific antibody format is developed, comprising two heavy chains and two light chains with specific variable and constant regions, where the amino terminus of one light chain is linked to the carboxy terminus of another via a peptide linker containing a protease recognition sequence, allowing for intracellular cleavage during production, thereby maintaining affinity and simplifying the production process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a linker is used to connect variable regions in existing bispecific antibody formats, then the antibody can recognize two different antigens, but the antigen binding affinity of the inner variable region decreases
Solution Approach 1:
The patent divides the light chain into two separate entities: a first light chain that pairs with the first heavy chain variable region, and a second light chain that pairs with the second heavy chain variable region. This segmentation eliminates the need for linkers connecting variable regions, thereby maintaining high binding affinity for both antigens without the affinity loss associated with linked structures.
Solution Approach 2:
The patent employs asymmetric chain pairing where the first heavy chain variable region is paired with the first light chain, and the second heavy chain variable region is paired with the second light chain. This asymmetric configuration allows each variable region to maintain its native pairing and high binding affinity, avoiding the structural constraints that cause affinity loss in symmetric linked formats.
2Reliability
If enzymatic cleavage of the linker is performed to restore affinity, then the antigen binding affinity of the inner variable region is improved, but the structure stability between heavy chain variable region and light chain variable region on the outer side decreases
Solution Approach 1:
By segmenting the light chain into two separate light chains, the patent eliminates the need for enzymatic cleavage of linkers. Each light chain remains stably paired with its corresponding heavy chain variable region throughout the antibody structure, maintaining structural stability without requiring post-purification cleavage steps.
3Reliability
If enzymatic cleavage of the linker is performed after purification, then the antigen binding affinity is restored, but the production process becomes complicated and the amount of produced antibody decreases
Solution Approach 1:
The segmented light chain design eliminates the need for post-purification enzymatic cleavage steps. The antibody can be directly purified in its functional form with both light chains properly paired, simplifying the production process and increasing the yield of functional antibody.
Solution Approach 2:
The correct pairing of light chains with heavy chain variable regions is established during the antibody assembly process itself, rather than requiring subsequent cleavage and reconfiguration steps. This preliminary correct configuration eliminates complex post-purification processing.
4Reliability
If a long or flexible linker is used to restore affinity, then the antigen binding affinity of the inner variable region is improved, but the linker cannot be universally used and requires screening
Solution Approach 1:
The patent eliminates the need for linkers entirely by segmenting the light chain into two separate light chains. This approach provides universal applicability across different antibody combinations without requiring linker screening or optimization for each specific pair of antigens.
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
The novel format retains high binding affinity to both antigens and simplifies the production process by enabling efficient cleavage and separation of antibody fragments, improving stability and yield compared to traditional methods.
Implementation Method 1
the amino terminus of one light chain is linked to the carboxy terminus of another via a peptide linker containing a protease recognition sequence, allowing for intracellular cleavage during production
Data Source
AI summary
[Problem] Provided is a bispecific antibody with a novel format that retains high binding affinity to both antigens, and can be efficiently produced in a commercial production process.[Means for Solution] A bispecific antibody comprising two heavy chains, two first light chains, and two second light chains, in which the heavy chains each comprise a first heavy chain variable region, a CH1 region, a first linker, a second heavy chain variable region, and a heavy chain constant region in order from the amino terminus side; the first light chains comprise a first light chain variable region and a first light chain constant region; the second light chains comprise a second light chain variable region and a second light chain constant region; the first heavy chain variable region and the first light chain variable region form a first antigen binding site; the second heavy chain variable region and the second light chain variable region form a second antigen binding site; and the first antigen binding site and the second antigen binding site recognize different antigens each other.