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.

Antibody recruiting molecules (ARMs) represent an important class of “proximity-inducing” chemical tools with therapeutic potential. ARMs function by simultaneously binding to a hapten-specific serum antibody (Ab) (e.g., anti-dinitrophenyl (DNP)) and a cancer cell surface protein, enforcing their proximity. ARM anticancer efficacy depends on the formation of ARM:Ab complexes on the cancer cell surface, which activate immune cell recognition and elimination of the cancer cell. Problematically, ARM function in human patients may be limited by conditions that drive the dissociation of ARM:Ab complexes, namely, intrinsically low binding affinity and/or low concentrations of anti-hapten antibodies in human serum. To address this potential limitation, we previously developed a covalent ARM (cARM) chemical tool that eliminates the ARM:antibody equilibrium through a covalent linkage. In the current study, we set out to determine to what extent maximizing the stability of ARM:antibody complexes via cARMs enhances target immune recognition. We observe cARMs significantly increase target immune recognition relative to ARMs across a range of therapeutically relevant antibody concentrations. These results demonstrate that ARM therapeutic function can be dramatically enhanced by increasing the kinetic stability of ARM:antibody complexes localized on cancer cells. Our findings suggest that a) high titres/concentrations of target antibody in human serum are not neccessary and b) saturative antibody recruitment to cancer cells not sufficient, to achieve maximal ARM therapeutic function.

Covalent antibody recruiting molecules (cARMs) constitute a proximity-inducing chemical strategy to modulate the recognition and elimination of cancer cells by the immune system. Recognition is achieved through synthetic bifunctional molecules that use covalency to stably bridge endogenous hapten-specific antibodies like anti-dinitrophenyl (anti-DNP), with tumor antigens on cancer cell surfaces. To recruit these antibodies, cARMs are equipped with the native hapten-binding molecule. The majority of cancer-killing immune machinery, however, recognizes epitopes on protein ligands and not small molecule haptens (e.g., Fc receptors, pathogen-specific antibodies). To access this broader class of immune machinery for recruitment, we developed a covalent immune proximity-inducing strategy. This strategy uses synthetic bif unctional electrophilic peptides derived from the native protein ligand. These bifunctional peptides are engineered to contain both a tumor-targeting molecule and a sulfonyl (VI) fluoride exchange (SuFEx) electrophile. As a proof of concept, we synthesized bifunctional electrophilic peptides derived from glycoprotein D (gD) on herpes simplex virus (HSV), to recruit gD-specific serum anti-HSV antibodies to cancer cells expressing the prostate-specific membrane antigen (PSMA). We demonstrate that serum anti-HSV antibodies can be selectively and irreversibly targeted by these electrophilic peptides and that the reaction rate can be uniquely enhanced by tuning SuFEx chemistry without a loss in selectivity. In cellular assays, electrophilic peptides demonstrated enhanced anti-tumor immunotherapeutic efficacy compared to analogous peptides lacking electrophilic functionality. This enhanced efficacy was especially prominent in the context of (a) natural anti-HSV antibodies isolated from human serum and (b) harder to treat tumor cells associated with lower PSMA expression levels. Overall, we demonstrate a new covalent peptide-based approach to immune proximity induction and reveal the potential utility of anti-viral antibodies in synthetic tumor immunotherapy.

An accurate and precise ultra‐high performance liquid chromatography with tandem mass spectrometry (UHPLC‐MS/MS) method was validated for the analysis of glyphosate and its main transformation product (aminomethylphosphonic acid) in barley, malt, wheat, oats, and lentils. The validation data demonstrated good performance of the method. This UHPLC‐MS/MS method was also used to evaluate the performance of a commercially available enzyme‐linked immunosorbent assay (ELISA) test kit. For all of the grain matrices examined, the ELISA showed poor accuracy and precision at its stated lower working limit of 0.075 mg/kg; however, performance was acceptable at 0.30 mg/kg, as well as higher concentrations relevant to established maximum residue limits. At these relevant concentrations, the ELISA also produced results higher than the UHPLC‐MS/MS method. Although results from the two methods were linearly correlated, differences in the result values from the two methods differed among the grains studied and ranged from +1% for oats to +40% for glyphosate concentrations in barley. ELISA is a useful tool that is complemented by the comprehensive and sensitive UHPLC‐MS/MS method.

Chemical immunotherapeutic strategies including Antibody Recruiting Molecules (ARMs ‐ bivalent small molecules containing an antibody‐binding domain (ABD) and a target‐binding domain (TBD)) direct immune‐mediated clearance of diseased cells. Anti‐cancer ARM function relies on high tumor antigen valency, limiting function against lower antigen expressing tumors. To address this limitation, we report a tunable multivalent immune recruitment (MIR) platform to amplify/stabilize antibody recruitment to cells with lower antigen valencies. An initial set of polymeric ARMs (pARMs) were synthesized and screened to evaluate ABD/TBD copy number, ratio, and steric occlusion on specific immune induction. Most pARMs demonstrated simultaneous high avidity binding to anti‐dinitrophenyl antibodies and prostate‐specific membrane antigens on prostate cancer. Optimized pARMs mediated enhanced anti‐cancer immune function against lower antigen expressing target cells compared to an analogous ARM.