Lysine Based Conjugation

As a professional service provider in antibody-drug conjugates (ADCs) design and preparation, Creative Biolabs offers lysine-based conjugation strategy suitable for cost-effective and multi-scale preparation of various ADC products. We are committed to customarily design and conduct each project according to your budget, timeline, and specific requirements.

Lysine-based conjugation strategy

In ADC development, lysine-based conjugation is one of the most widely used non-specific conjugation strategies. This conjugation occurs on reactive amine side chains of lysine residues due to their good nucleophilicity. The immunoglobulin (IgG) scaffold contains over 80 lysine residues, most of which are exposed on the surface of the antibody. Among the surface lysine residues, more than 20 have been shown as highly solvent-accessible and can serve as potential ADC conjugation sites. Lys conjugation follows two main strategies that result in the formation of a stable amide or amidine bond between the protein and the drug-linker complex. Generally speaking, activated esters on the drug-linker complexes, often O-succinimide reagents such as N-hydroxysuccinimidyl (NHS) or sulfo-NHS esters, react with the antibody lysine residues and achieve the conjugation via amide bonds. On the other hand, stable amidine bonds can be generated on an antibody by the reaction of imido ester compounds, such as Traut’s reagent, with antibody lysine residues.

Many ADCs have been developed using lysine-based conjugation strategies. The FDA-approved gemtuzumab ozogamicin (trade name, Mylotarg) and trastuzumab emtansine (trade name, Kadcyla) are both generated by modifying the accessible lysine residues on the surface of the corresponding antibodies. Meanwhile, dozens of emerging ADCs under clinical evaluation are developed using lysine conjugation strategies, despite the potential of highly heterogeneous products with a relatively wide drug to antibody ratio (DAR) distribution.

Lysine-based conjugation and the expected DAR profiles. Due to the uneven distribution and different solvent accessibility of antibody surface lysine residues, lysine-based conjugation can yield ADCs with a wide DAR profile (BioProcess International, 2014).

With a full understanding of lysine side chain conjugation chemistry and the principle of ADC development, Creative Biolabs provides two different strategies for clients who prefer lysine-based conjugations, namely the one-step conjugation approach and the two-step conjugation approach:

(1) One-step conjugation— a straightforward direct conjugation of a drug-linker complex containing an amine-reactive group to the antibody via amide bonds. This approach has been widely used to introduce non-cleavable linkers with different cytotoxic agents, such as maytansine derivatives and PBD dimers, onto antibodies. Occasional reports of lysine conjugation based ADCs bearing cleavable linkers are also available.

(2) Two-step conjugation— this approach is carried out in a two-step fashion. In the first step, a small bi-functional reagent containing both an amine- and a thiol-reactive functional groups is reacted with the available lysine ε-amino groups to serve as a chemical adaptor, leaving free thiol-reactive groups on the antibody. In the second step, the payload drugs or drug-linker complexes are attached to the thiol-reactive groups introduced previously to form the ADC. The two-step approach is often used when the drug/drug-linker complex contains a thiol-reactive module or as an alternative route when introducing an amine-reactive module into the drug or drug-linker complex is proven to be difficult. Typically, there are four small adaptors commonly used in the two-step conjugation: SPDB disulfide, MCC (maleimidomethyl cyclohexane-1-carboxylate), sulfo-SPDB, and Hydrazine.

Structures of chemical adaptors involved in the two-step Lysine-based conjugation approaches (Pharm Res, 2015).

Advantages of lysine based conjugation:

• Suitable for the conjugation to both the Fab region and full length IgG.
• A simple process does not require any form of antibody engineering or modification.
• Multiple conjugation sites enabling the sufficient loading of payload drugs.
• One of the most commonly used approaches.
• High chemical versatility to incorporate a variety of cytotoxic agents into antibodies.

Creative Biolabs is dedicated to help customized preparation of high-quality Lys-based ADCs in a timely and cost efficient manner. Please contact us for more information and a detailed quote.

References:

1. Walker, J.M.; et al. Antibody-drug conjugates. Humana Press. 2013.
2. Bhat, A.S.; et al. The next step in homogenous bioconjugate development: optimizing payload placement and conjugate composition. BioProcess International. 2014.
3. Jain, N.; et al. Current ADC linker chemistry. Pharm Res. 2015, 32: 3526–3540.

For Research Use Only. NOT FOR CLINICAL USE.