Site-Specific Antibody Conjugation to Engineered Double Cysteine Residues

Area of Science:

• Biotechnology
• Protein Engineering
• Antibody Engineering

Background:

• Site-specific antibody conjugation is crucial for developing homogeneous antibody-drug conjugates (ADCs) with improved therapeutic indices.
• Existing methods often face limitations in achieving high drug-to-antibody ratios (DAR > 2), particularly with engineered cysteines.

Purpose of the Study:

• To engineer novel antibody variants with free cysteine residues in the CH2 and CH3 regions to enable site-specific conjugation.
• To expand the capabilities of site-specific conjugation for achieving higher DAR values.

Main Methods:

• Antibody mutants with single and double cysteine residues were generated using site-directed mutagenesis.
• PEGylation was employed as a screening tool to assess conjugation efficiency and selectivity.
• Successful mutants were further characterized for expression, aggregation, and conjugation properties.

Main Results:

• Thirty-eight double cysteine mutants were generated, with most exhibiting comparable expression and low aggregation to wild-type antibodies.
• Seventeen double cysteine mutants demonstrated high conjugatability and selectivity via PEGylation screening.
• The study successfully demonstrated the feasibility of achieving site-specific antibody conjugates with DAR > 3.4.

Conclusions:

• Engineered cysteine residues in CH2 and CH3 regions offer a viable strategy for site-specific antibody conjugation.
• PEGylation serves as an effective method for rapid screening of antibody mutants for conjugation.
• The identified antibody mutants hold potential for next-generation ADC development with enhanced therapeutic properties.