Inquiry Basket
During the early development of antibody-drug conjugate (ADC), lysine is often selected as a well binding site with antibodies. As a leading service provider in antibody-drug conjugate research and discovery, BOC Sciences can perform advanced lysine conjugation technologies to develop antibody-drug conjugates. Based on a thorough understanding of customers' project requirements, our scientists are capable of inventing ADCs suitable for large-scale production to support commercial needs at a low cost.
One of the most common ADC conjugation strategies adapts lysine residues of antibodies, in which the nucleophilic NH2 group of lysine reacts with the electrophilic N-hydroxysuccinimide (NHS) group on the payload. Although this reaction is relatively simple, the high abundance of lysine residues has led to uneven ADCs formation under random distribution. In fact, the drug-to-antibody ratio (DAR) of lysine conjugation is controlled by the drug/antibody stoichiometric ratio and is widely used in various ADC development, including approved ADCs.
Fig. 1. Lysine-based conjugation strategies.
A humanized antibody contains 80 to 90 lysines, and partial modification of lysines does not significantly affect the natural disulfide bonds nor the stability, biophysical properties, or affinity of the overall antibody. However, each antibody can couple to 0-8 small-molecule drugs through lysine conjugations. Thus, coupling sites may occur at nearly 40 different lysine residues of light and heavy chains and generate more than a million ADCs. When a small molecule is anchored to a monoclonal antibody's lysine by a covalent bond, the ADC is able to control the number of small molecule drugs in the chain.
An example of a disulfide-containing ADC refers to the attachment of DM1 or DM4 to immunoglobulin, and it is transported to lysosomes by antigen endocytosis through vesicles, and monoclonal antibodies are degraded to provide lysine derivatives linked to small-molecule drugs. Further changes in the cell, including disulfide linkage, are broken by the exchange of disulfide bonds, and finally, the thiol may be methylated by methyltransferase to form a potent derivative of DM1 or DM4. Nevertheless, these methylated drugs can penetrate the cell membrane to exert a broader effect. Methylated drugs kill cells from not only the inside but also nearby cells that are not expressing antigens. According to the antibody's characteristics and the customer's specific requirements, BOC Sciences is capable of choosing the most appropriate coupling strategy for your interested ADCs.
One of the most common conjugation methods exploits the antibody's lysine residues, where the nucleophilic NH2-group of lysine reacts with an electrophilic N-hydroxysuccinimide (NHS) on the linker-payload. Due to differences in lysine position and microenvironment, protonated amino groups' pKa are varied, thus affecting the reaction rates. In a one-step binding, the lysine ε-amino group of the antibody reacts with the amine-reactive group of the drug to form an amide bond. Taking advantage of this property difference, BOC Sciences can offer multiple conjugation strategies for lysine residues and linkers.
Applying the two-step conjugation strategy, a chemical group is first introduced to modify the lysine residues. For example, sulfonyl acrylates are used as a reagent to modify a single lysine residue on native protein sequences. The regioselectivity of this reaction was due to the combination of designed sulfonyl acrylates and the unique local microenvironment surrounding each lysine. Moreover, it is predicted computationally that the lysine with the lowest pKa was prone to react preferentially at slightly basic pH in a site-specific manner. Thus, this conjugation technique retains the original secondary structure and protein function after conjugation. BOC Sciences is capable of coupling small molecule drugs to the peptide through the isomeric peptide bond, site-specifically in vitro, and to having the binding site apply only at the ε-amino site of lysine in amino acid sequence.
References
