Cysteine Based Conjugation

As an industrial leader in antibody-drug conjugate (ADC) development and with our experienced science teams, Creative Biolabs provides the most comprehensive cysteine-based conjugation approaches to generate highly customized ADCs in a timely and cost efficient way. With a deep understanding of thiol conjugation chemistry, scientists at Creative Biolabs will help design and prepare thiol-based ADCs using optimized linkers and strategies.

Cysteine residues serve essential roles in protein structure and function due to their highly reactive thiol side chains that form inter or intra molecular di-sulfide bonds to enable correct protein folding. Thiol reactivity also provides an appealing route for conjugating toxic agents to antibodies. A full IgG scaffold contains 16 cysteine pairs, forming 12 intra- and 4 inter-chain disulfide bonds. Due to higher solvent accessibility, the four inter-chain disulfides forming Cys residues are the main targets for conjugation, but occasions where conjugation occurred on intra-chain cysteines have also been reported. What’s more, strategies using engineered Cys residues for site specific conjugation without the partial reduction of the endogenous disulfide bonds have been well-established and termed EnCys-mAb.

Conjugation based on inter-chain cysteines

For conventional ADC production, 8 nucleophilic cysteine residues are first liberated from the reduced inter-chain disulfide bonds via reducing agents and they are later conjugated with drug-linker complexes. This approach generates ADCs with heterogeneous conjugation sites and a different number of drugs attached, resulting in a drug to antibody ratio (DAR) ranging from 0~8. As an example, Adcetris, one of the FDA approved ADCs, is prepared via non-specific cysteine conjugation.

Conjugation through reduced inter-chain cysteine produced an uneven distribution of DAR profiles (mAbs, 2014).

Selective reduction of certain inter-chain disulfide bonds to develop relatively site-specific ADCs can be achieved taking advantage of the varied susceptibility of different disulfide bonds in an antibody towards reduction agents. Typically, partial reduction with either dithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP) results in the disruption of the heavy-light inter-chain disulfides to release free Cys for drug conjugation, while treatment using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) yields drug conjugates connect to the Cys residues usually involved in heavy-heavy inter-chain disulfides.

Another method to develop cysteine–based site specific antibody conjugation involves the mutation of some hinge region cysteine residues to serine. Several studies have shown that these cysteine-serine mutations in an antibody have no significant influence on antibody binding affinity and in vitro conjugate stability. These mutations have also been demonstrated not to induce antibody aggregation.

Reduction and alkylation of inter-chain cysteine residues on antibodies containing several cysteine-to-serine mutations (Bioconjugate Chem, 2015).

What’s more, using novel dibromomaleimide and bis-sulfone linkers, bridging alkylation of the reduced inter-chain cysteine residues can be readily achieved. These linkers generate a stable covalent linkage between the antibody heavy and light chains, keep the structure of the antibody intact, and in the meantime, tightly control the ligand-to-antibody ratio of the final ADC products.

Reduction and bridging alkylation of inter-chain cysteine residues with bis-sulfone linkers (Bioconjugate Chem, 2015).

Conjugation based on engineered cysteines

Cysteine residues can be engineered into specific sites on the backbone of an antibody. This approach, designated as EnCys-mAb, is a valuable strategy for the generation of antibody-drug conjugates with improved homogeneity.

Conjugation through engineered cysteine residues (EnCys-mAb). Cys residues were engineered into specific sites antibody and result in a narrow DAR profiles (mAbs, 2014).

Advantages of cysteine based conjugation:

• Cysteine-based conjugation offers flexible choices for generating either non-specific or site-specific ADCs.
• Conventional cysteine-based conjugation is easy to perform without the need for antibody engineering.
• Site specific conjugation can be achieved through partial reduction or EnCys-mAb and generates homogeneous ADCs with controlled DAR and drug-attachment sites.
• This strategy is suitable for drug conjugation to both Fab region and full length IgG.

Creative Biolabs provides the most comprehensive antibody modification and conjugation strategies in a timely and cost efficient manner. We are dedicated to providing our clients with high-quality services and contribute to your ADC development projects. Please contact us for more information and a detailed quote.

References:

1. Behrens, C.R.; et al. Methods for site-specific drug conjugation to antibodies. mAbs. 2014, 6 (1): 46–53.
2. Dennler, P.; et al. Antibody conjugates: from heterogeneous populations to defined reagents. Antibodies. 2015, 4: 197-224.
3. Agarwal, P.; et al. Site-specific antibody−drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjugate Chem. 2015, 26: 176−192.