Antibody Construct Expression With 2A Linkers for Correct Assembly
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Solution Overview
Problem
Existing methods for producing bispecific antibodies suffer from incorrect assembly, mispairing of polypeptide chains, and imbalanced expression, leading to reduced yield, purity, and increased production costs, with a risk of anti-drug antibodies.
Innovation Solution
Encoding at least two different polypeptide chains of an antibody construct within the same open reading frame using a self-cleaving 2A peptide linker, ensuring balanced expression and correct assembly by translating chains in close proximity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If polypeptide chains are expressed from separate expression cassettes, then each chain can be independently optimized, but incorrect assembly and mispairing occur due to imbalanced mRNA levels
Solution Approach 1:
The patent merges multiple polypeptide chain encoding sequences into a single open reading frame separated by 2A self-cleaving peptide linkers. This ensures that all polypeptide chains are translated from the same mRNA molecule, guaranteeing equimolar expression levels and preventing mispairing, while the 2A peptides enable ribosomal skipping to generate separate functional chains.
Solution Approach 2:
The patent segments the encoded polypeptide chains using 2A self-cleaving peptide linkers positioned between consecutive chains. These linkers cause ribosomal skipping during translation, resulting in physical separation of the polypeptide chains while maintaining their co-expression from a single mRNA, thus preventing incorrect assembly.
2Ease of manufacture
If conventional expression methods are used, then production cost is reduced, but yield and purity of correctly assembled antibody constructs decrease
Solution Approach 1:
The patent changes the expression parameter by using a single open reading frame with 2A peptide linkers instead of separate expression cassettes for each polypeptide chain. This parameter change ensures equimolar translation of all chains from one mRNA, dramatically increasing the yield of correctly assembled antibody constructs while maintaining cost-effectiveness through simplified production.
3Manufacturing precision
If protein chains are modified to improve assembly, then correct pairing is enhanced, but risk of inducing anti-drug antibodies increases
Solution Approach 1:
The patent introduces 2A self-cleaving peptide linkers as intermediaries between polypeptide chains. These linkers facilitate correct chain separation and assembly without requiring modifications to the antibody chains themselves, thereby preventing the induction of anti-drug antibodies while ensuring proper pairing through controlled expression stoichiometry.
Data Source
AI summary
The present invention pertains to expression vector designs for antibody constructs. Different polypeptide chains of an antibody construct are encoded within the same open reading frame, connected to each other by a 2A peptide linker. This expression vector design leads to homogeneous expression and correct assembly of the antibody construct.