Covalent Immune Recruiters: Tools to Gain Chemical Control Over Immune Recognition

Abstract

Unprecedented progress made in the treatment of cancer using the body’s own immune system has encouraged the development of synthetic molecule based immunotherapeutics. An emerging class of these compounds, called Antibody Recruiting Molecules (ARMs) or Antibody Engagers (AEs), functions by reversibly binding antibodies naturally present in human serum and recruiting these to cancer cells. The recruited antibodies then engage immune cells to form quaternary complexes that drive cancer erradication. Despite their promise, the requirement to form quaternary complexes governed by multiple equilibria complicates an understanding of their in vivo efficacy. Particularly problematic are low endogenous serum antibody concentrations and rapid clearance of AEs from circulation. Here we describe a new class of trifunctional chemical tools we call covalent immune recruiters (CIRs). CIRs covalently label specific serum antibodies in a selective manner with a target protein binding ligand. CIRs thereby exert well-defined control over antibody recruitment and simplify quaternary complex equilibium, enabling probing of the resultant effects on immune recognition. We demonstrate CIRs can selectively covalently label anti-DNP IgG, a natural human antibody, directly in human serum to drive efficient immune cell recognition of targets. We expect CIRs will be useful tools to probe how quaternary complex stability impacts the immune recognition of cancer in vivo, revealing new design principles to guide the development of future AEs.

Multivalent Antibody‐Recruiting Macromolecules: Linking Increased Binding Affinity with Enhanced Innate Immune Killing

“…[40,42] Despite this fact, of the three main haptens, DNP has received the most attention in the context of design antibody recruiting molecules, likely due to the commercial availability of affinity maturated anti-DNP antibodies, including mouse and human monoclonals that bind DNP with much higher affinity than endogenous anti-DNP in human serum. [43] However, it is also worth noting that DNP is also prone to nonspecific binding to albumin [44] and to aromatic residues such as tryptophan in the antigen binding site of antibodies. [45,46] By varying the ABT and TBT, many different ARMs systems have been developed, acting against a broad variety of disease relevant targets, including viruses and virally infected cells, [47,48] bacteria [36,49,50] and cancer cells.…”

Methods to Validate Binding and Kinetics of “Proximity‐Inducing” Covalent Immune‐Recruiting Molecules

“…As association time increases, more noncovalent complexes appear to form on the probe surface, which cannot be removed during the dissociation step. We hypothesize this represents slow higher-avidity binding of antibody to the surface or slow nonspecific antibody aggregation on the probe surface (see supporting information in Lake et al, 2020). To account for this we determined NCIR F at each time point and used this value to correct for the true extent of the covalent reaction.…”

“…Finally, we describe how to employ antibody-dependent cellular phagocytosis (ADCP) assays (Basic Protocol 3, Alternate Protocol 2) to define the CIR mechanism of action and demonstrate its anticancer function. All CIRs investigated Lake et al (2020) are shown. CIR1/2/3/4 and NCIR5 are referenced.…”

“…ABD, antibody-binding domain; ALD, antibody-labeling domain; CIR, covalent immune recruiters; NCIR, nonreactive CIR; TBD, target-binding domain. Adapted and reprinted with permission from Lake et al (2020). ACS Chemical Biology, 15, 1089-1095.…”

“…Synthetic methods affording compounds CIR1/CIR4, NCIR5, and DNP-glycine can be found in Lake et al (2020). NOTE: All compounds were produced according to previously published work (Lake et al, 2020). Compound samples are available by request to the authors (rulloa@ mcmaster.ca).…”