Dual Variable Domain Antibody-Drug Conjugation Platform for Site-Specific Conjugation Chemistry

Highly Functional Antibody-Agent Conjugates with Dual Variable Domain for Conjugation to a Second Moiety

These modified 38C2 catalytic antibodies enable improved antibody-drug conjugation. Antibody-agent conjugates depict a powerful tool in basic research and the diagnosis and therapy of diseases, leveraging the high specificity of monoclonal antibodies. Currently, 11 FDA-approved antibody-drug conjugates (ADCs) are on the market. Common strategies for using antibody-agent conjugates involve using natural or uniquely engineered reactive amino acids or carbohydrates to assemble antibodies and small molecules in site-specific manners. The catalytic antibody 38C2, and its humanized version, h38C2, contain a uniquely reactive lysine residue (Lys99) in its active site. Exploiting this feature enables the development of a variety of highly homogenous antibody-small molecule conjugates, including dual variable domain-based antibody-drug conjugates for the rapid, precise, efficient, and stable conjugation of highly cytotoxic payloads under mild conditions. However, there is an unmet need for alternative and better ways of antibody-agent conjugation.

Researchers at the University of Florida have developed a highly functional dual variable domain (DVD) platform with two variable binding sites in tandem on each of the two arms of the antibody molecule. With h38C2 being the second moiety, these dual variable domains allow site-specific agent conjugation. Replacing Lys99 in h38C2 with Arg99 and Cys99 creates novel orthogonal antibody-agent conjugation opportunities, enabling the simultaneous delivery of two agents with an additive or synergistic activity.

Application

Modified catalytic antibodies 38C2 and h38C2 for the development of site-specific dual variable domain (DVD)- antibody-drug conjugations and other antibody-agent conjugates

Advantages

• Fast and efficient site-specific antibody-agent conjugation under physiological conditions not requiring reduction, oxidation, unnatural amino acids, or enzymes. Can create an independent second conjugation site through specific mutations, increasing the number of potential applications, including delivering two synergistic therapeutic drugs together, attaching a therapeutic drug and targeting molecules on the same antibody
• Can accommodate different therapeutic agents, such as cytotoxic agents, peptides, nucleic acids (including siRNA), or detectable labels, enabling a variety of therapeutic and diagnostic applications

Technology

These functionalized h38C2 catalytic antibodies entail orthogonal site-specific conjugation sites, generating a highly functional dual variable domain (DVD platform. The heavy chain variable region of the antibody includes a uniquely reactive lysine (Lys99), arginine (Arg99), or cysteine (Cys99) residue capable of reacting with a linker, providing an attachment motif for conjugation with a drug moiety. Adding an alternative conjugation chemistry, researchers at the University of Florida modified the Lys99 residue via arylation to functionalize the h38C2 antibody. This enables the development of ADCs with compounds conjugating to Lys99, the reactive residue, via a heteroaryl methylsulfonyl linker. In providing alternative conjugation chemistry, the modified h38C2 antibody compounds produce alternative means for bioconjugation. The resulting functionalized antibody-agent conjugates can include antibody-drug conjugates (ADCs); antibody-siRNA conjugates; and chemically programmed antibodies, bispecific antibodies, and chimeric antigen receptors. To further broaden the utility of the dual variable domain (DVD) platform, orthogonal conjugation chemistries for alternative reactive residues, Arg99 and Cys99, which can replace Lys99, were developed.