Covalent antibody recruiting molecules (cARMs) are synthetic bifunctional molecules (BMs) capable of inducing antibodies to recognise and eliminate cancer cells by the innate immune machinery. BMs have been developed to bridge endogenous immune receptors with tumour protein antigens to elicit a therapeutic immune response, however, the therapeutic effect of this three-component (e.g., tumour antigen/BM/antibody) “ternary” complex relies on its stability. If the binding affinity of BM to the immune receptor is too weak to form stable ternary complexes, it may fail to elicit the desired therapeutic function. Three factors may account for the weak BM affinity to immune receptors: endogenous immune receptor concentrations are below the KD governing binding, the BMs are being rapidly cleared, and/or the ternary complex cannot withstand the mechanical forces induced during cell activation. To circumvent these issues, these researchers sought to apply a bifunctional proximity-induction strategy that involves the development of protein-specific immune receptors without the dependency on ultra-high affinity binding peptides or multivalency. They hypothesised that modest-affinity peptides engineered with a strategically positioned electrophile could undergo a selective ligand-directed covalent linkage to a target immune receptor. When provided with a tumour-targeting molecule, the resultant bifunctional covalent peptide could irreversibly engage protein-specific immune receptors to form a highly stable ternary complex with tumour cells. To prove their concept, these researchers chose to target pre-existing viral-specific antibodies that recognise glycoproteins on the surface of the herpes simplex virus (HSV). These antibodies are also highly prevalent in the blood of cancer patients undergoing treatment with oncolytic virus (OV) tumour immunotherapy, thus the development of proximity-inducing molecules that redirect HSV-specific antibodies is a tactical plan, especially in combination with OV therapies. They theorised that small synthetic peptides derived from immunogenic epitopes on HSV glycoproteins may function as binding ligands to facilitate covalent proximity induction of anti-HSV antibodies with tumour cells. To investigate this, they designed a series of tumour-targeting bifunctional covalent peptides derived from glycoprotein D (gD) of HSV1/2. The peptides were subsequently modified with a tyrosine fluorosulfate (FSY) or aryl sulfonlyl fluoride (ASF) SuFEx handle, at different locations on the peptide. Next, the covalent peptides were equipped with a tumour antigen-binding molecule that targets the prostate-specific membrane antigen (PSMA), thereby creating the final bifunctional covalent peptide products. Kinetics assays revealed that bifunctional covalent peptides can selectively and covalently target specific anti-HSV antibodies in both human serum and the serum of mice treated with an OV therapeutic. Cell-based assays demonstrated that covalency significantly increases immunotherapeutic function against difficult-to-treat lower antigen-expressing tumour cells. This proof-of-concept research paper highlights the importance and potentially groundbreaking findings that a “covalent immune proximity-induction” strategy may enable the efficient recruitment of immune receptors via the incorporation of available modest affinity peptide ligands. Additionally, it revealed the potential value of anti-viral antibodies in synthetic tumour immunotherapy.
How was PEAKS used?
Bioinformatics Solution Inc.’s service department collaborated by performing antibody conjugate analysis. In-solution endoproteinase digestions of the monoclonal antibody (mAb), anti-HSV glycoprotein D antibody (clone LP14), Fluor-ASF-gD labelled mAb, and Fluor-FSY-gD labelled mAb were performed for mAb sequencing and conjugate analysis by LC-MS/MS. The antibodies were reduced with dithiothreitol and alkylated using iodoacetamide. The sample was equally divided into 6 aliquots for 6 individual enzyme digestions: LysC, Asp N, Chymotrypsin, Elastase, Trypsin, and Pepsin. After reversed phase cleanup of each digest, digests were analysed on an Orbitrap analyser (Orbitrap Fusion Lumos, Thermo Fisher Scientific). Both full MS scans and MS2 scans were acquired in the high resolution Orbitrap mass analyser. MS2 data were acquired using HCD and ETD followed by HCD (EThcD) fragmentation methods. All raw data files were used for de novo sequencing and conjugate analysis using the PEAKS AB 2.0 software with a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.02 Da. Carbamidomethylation was set as a fixed post translational modification (PTM) and deamidation (NQ), oxidation (M), and pyro-glu from Q were set as variable PTMs. Custom PTMs were applied to determine the location of the ASF-gD or FSY-gD peptides on the labelled LP14 antibodies.
McCann, Harrison M., et al. “Covalent Immune Proximity-Induction Strategy Using SuFEx-Engineered Bifunctional Viral Peptides.” ACS Chemical Biology (2022). PMID: 35522208. https://doi.org/10.1021/acschembio.2c00233
Abstract
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 bifunctional 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.