Variable heavy-variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light...

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Bispecific antibodies offer therapeutic advantages but face production challenges.
• Correct heavy and light chain assembly are critical for functional bispecific antibodies.
• Existing methods like knobs-into-holes and CH3 charge interactions address heavy chain assembly, while light chain assembly requires further optimization.

Purpose of the Study:

• To develop an improved CrossMab design principle for efficient bispecific antibody production.
• To enable correct light chain assembly in CrossMabVH-VL and CrossMabFab formats.
• To overcome limitations of previous immunoglobulin domain crossover approaches.

Main Methods:

• Engineering of CrossMab formats, specifically CrossMabVH-VL and CrossMabFab.
• Application of site-specific positional exchanges of charged amino acid pairs in the constant domain.
• Analysis of side product formation in different CrossMab designs.

Main Results:

• The CrossMabCH1-CL exchange initially showed a favorable profile without significant side products.
• Novel CrossMab design principle enables correct light chain assembly in CrossMabVH-VL format.
• Improvements were achieved for the CrossMabFab design, reducing unwanted side products.

Conclusions:

• The improved CrossMab design principle offers a more efficient strategy for producing bispecific antibodies.
• This advancement facilitates the development of therapeutic antibodies with enhanced specificity and efficacy.
• The study provides a valuable tool for antibody engineering and biopharmaceutical development.