Bispecific Antibody Expression Tuning for Correct Chain Assembly
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Solution Overview
Problem
The production of bispecific antibodies in eukaryotic host cells is challenging due to promiscuous pairing of heavy and light chains, leading to low titer and insufficient product quality, which complicates manufacturing processes and increases costs.
Innovation Solution
A method involving the use of eukaryotic host cells with tailored translation initiation sequences to control the expression levels of polypeptide chains, where one subunit is expressed at a lower level than another, promoting correct assembly of multimeric polypeptides like bispecific antibodies, using knob-into-hole mutations in antibody Fc regions to enhance specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If co-expression of two antibodies in a single cell is used, then manufacturing process complexity is reduced, but chain mispairing increases leading to lower product quality
Solution Approach 1:
The patent introduces distinct sorting signals (different amino acid sequences) at specific locations (N- or C-termini) of different heavy chains. These local differences enable each heavy chain to be selectively recognized and paired with its corresponding light chain by cellular machinery, ensuring correct assembly without requiring separate expression systems.
Solution Approach 2:
The patent employs sorting signals as intermediary elements that mediate the specific interaction between heavy chains and their cognate light chains. These sorting signals act as molecular guides that facilitate correct pairing by being recognized by complementary binding partners in the assembly process, preventing promiscuous pairing while maintaining co-expression in a single cell line.
2Manufacturing precision
If separate expression of half-antibodies followed by in vitro assembly is used, then product quality is improved, but manufacturing time and cost increase
Solution Approach 1:
The patent enables the expression system to self-assemble the correct antibody structure in vivo within the host cell. The cellular machinery, guided by the sorting signals, automatically performs the pairing function that would otherwise require separate in vitro assembly steps. This self-assembly capability eliminates the need for complex downstream purification and assembly processes while maintaining high product quality.
3Ease of manufacture
If conventional translation initiation sequences are used for all polypeptide chains, then expression is simplified, but assembly efficiency decreases due to promiscuous pairing
Solution Approach 1:
The patent modifies translation initiation sequences (such as Kozak sequences) specifically for polypeptide chains that require enhanced or suppressed expression. By making these sequences different for different chains, the patent creates local expression differences that drive correct assembly kinetics without affecting the overall simplicity of the expression system. This allows tuning of expression levels to match assembly requirements.
Data Source
AI summary
The present disclosure relates to the production of multimeric polypeptides (e.g., comprising two or more subunits, each subunit comprising two or more polypeptide chains, such as a multispecific or bispecific antibody) in eukaryotic host cells. In some embodiments, provided herein are methods for producing a multimeric polypeptide in a eukaryotic host cell using a host cell that comprises a polynucleotide comprising a translation initiation sequence operably linked to an open-reading frame encoding each polypeptide of the multimeric polypeptide. Advantageously, the present disclosure demonstrates that tuning the strength of the translation initiation sequences linked to each open-reading frame allows for higher production of the multimeric polypeptide with fewer incorrectly assembled side products. The present disclosure further provides cells, methods of screening, and kits related thereto.