CH3 Domain Heterodimer Design for Bispecific Antibody Production
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Solution Overview
Problem
Existing methods for producing bispecific antibodies face challenges such as heterogeneity, instability, and low yield due to the formation of homodimers, making it difficult to achieve stable and efficient production of multifunctional antibodies.
Innovation Solution
A heterodimer molecule is developed by optimizing the CH3 domain of antibody heavy chains with specific amino acid mutations, such as Y349C/T366W, D356C/T366S, L368A/Y407V/F405K, to enhance the formation of heterodimers over homodimers, improving yield and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If chemical crosslinking method is used to prepare bispecific antibodies, then the preparation process is simple, but the products exhibit heterogeneity and instability with low yield
Solution Approach 1:
The patent applies parameter changes by modifying amino acid sequences at specific positions in the Fc region (e.g., position 366 in the CH3 domain) to alter the binding characteristics. By changing the chemical parameters of the protein structure, the invention achieves stable heterodimer formation while maintaining simple production processes, resolving the contradiction between manufacturing simplicity and product reliability.
2Reliability
If hybridizing F(ab')2 molecules method is used, then bispecific antibodies can be produced, but the preparation process is time-consuming and labor-consuming with very low yield
Solution Approach 1:
The patent implements preliminary action by pre-modifying the Fc region amino acid sequences before the actual bispecific antibody formation process. By preparing the modified Fc regions in advance (e.g., introducing specific amino acids at positions 366, 409, 411), the subsequent assembly process becomes more efficient and yields higher productivity, eliminating the need for time-consuming post-processing steps.
3Reliability
If murine hybridoma method is used, then a reliable source can be obtained, but randomly pairing between light chains and heavy chains produces multiple possible antibody forms making production and purification very difficult
Solution Approach 1:
The patent applies local quality by making specific localized modifications to the Fc region amino acid sequences rather than random modifications. By introducing specific amino acids at predetermined positions (such as position 366 in the CH3 domain), the invention creates localized heterogeneity that guides specific pairing between light and heavy chains, thereby simplifying production and purification while maintaining source reliability.
4Strength
If 'knob into hole' model is used to enhance heterodimer formation, then the binding capacity is strengthened, but homodimer formation still occurs reducing heterodimer proportion
Solution Approach 1:
The patent applies asymmetry by introducing asymmetric amino acid modifications at specific positions in the Fc region. By creating asymmetric binding interfaces through targeted mutations (e.g., different amino acids at corresponding positions in different chains), the invention strongly promotes heterodimer formation while preventing homodimer formation, thereby achieving high heterodimer proportion while maintaining strong binding capacity.
Data Source
AI summary
A heterodimer molecule based on CH3, and a preparation method therefor and a use thereof. By comprehensively considering various interactions between molecules, for example, an ionic action, a hydrophobic interaction and a spatial action, a preferred Fc mutant sequence being more inclined to form a heterodimer rather than a homodimer is screened, and accordingly, the yield of the heterodimer molecule is greatly improved, thereby creating conditions for the preparation of bispecific molecules and the like.