Fc Variant Amino Acid Substitutions for Heterodimer Assembly
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Solution Overview
Problem
Current methods for producing heteromultimeric proteins, such as multispecific antibodies, face challenges including low yield, production of mixed products, and reduced effector function due to issues like mispairing and redox inefficiency, particularly in the formation of bispecific antibodies with a functional Fc domain.
Innovation Solution
The development of Fc variants with specific amino acid modifications, such as substitutions at residues 241 and 243, to disrupt homodimer formation and enhance yield, along with methods for producing these variants using expression vectors and host cells like CHO or E. coli, facilitating efficient production of heterodimeric proteins with improved mispairing and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods (chemical cross-linking, cell-fusion, recombinant DNA) are used to produce heteromultimeric proteins, then bispecific antibodies can be generated, but the yield is low and mixed products are produced due to mispairing and redox inefficiency
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (e.g., F241R/F243S, F241S/F243R, or F241R/F243S) into the Fc region of antibody heavy chains. These parameter changes in the protein sequence alter the dimerization interface properties to favor heterodimer formation over homodimer formation, thereby improving both yield and purity of bispecific antibodies without requiring complex purification processes.
2Ease of manufacture
If chemical cross-linking is used to assemble antibody chains, then heteromultimeric proteins can be formed, but the process is labor intensive and chemical modification steps alter protein integrity leading to poor stability
Solution Approach 1:
The patent replaces chemical cross-linking methods with a biologically-based solution by using Fc region amino acid substitutions to control dimerization. This substitution eliminates the need for chemical cross-linking agents and associated purification steps, simplifying the manufacturing process while maintaining protein stability through native-like disulfide bond formation at the dimer interface.
3Adaptability or versatility
If cell-fusion technology is used to express antibody chains, then multispecific molecules can be produced, but the desired heteromultimeric antibodies are only a small fraction requiring dramatic purification that reduces production yields
Solution Approach 1:
The patent applies local quality by introducing specific amino acid changes only at the Fc region dimerization interface (residues 241 and 243) while maintaining the rest of the antibody structure intact. This localized modification enables selective heterodimer formation and significantly increases the fraction of desired heteromultimeric antibodies, eliminating the need for extensive purification and improving overall production yield.
Data Source
AI summary
Described herein are Fc variants and methods for the efficient production of antibodies and other multimeric protein complexes (collectively referred to herein as heteromultimeric proteins). Heteromultimeric proteins may be capable of specifically binding to more than one target. The targets may be, for example, different epitopes on a single molecule or located on different molecules. The methods combine efficient, high gene expression level, appropriate assembly, and ease of purification for the heteromultimeric proteins. The invention also provides methods of using these heteromultimeric proteins, and compositions, kits and articles of manufacture comprising these antibodies.