Targeting the Respiratory Syncytial Virus N0-P Complex with Constrained α-Helical Peptides in Cells and Mice.

Respiratory syncytial virus (RSV) is the main cause of severe respiratory infection in young children worldwide, and no therapies have been approved for the treatment of RSV infection. Data from recent clinical trials of fusion or L polymerase inhibitors for the treatment of RSV-infected patients revealed the emergence of escape mutants, highlighting the need for the discovery of inhibitors with novel mechanisms of action. Here we describe stapled peptides derived from the N terminus of the phosphoprotein (P) that act as replication inhibitors. We demonstrate that these peptides inhibit RSV replication in vitro and in vivo by preventing the formation of the N0-P complex. The present strategy provides a novel means of targeting RSV replication with constrained macrocyclic peptides or small molecules and is broadly applicable to other viruses of the Mononegavirales order.

Site-selective template-directed synthesis of antibody Fc conjugates with concomitant ligand release.

Template-directed methods are emerging as some of the most effective means to conjugate payloads at selective sites of monoclonal antibodies (mAbs). We have previously reported a method based on an engineered Fc-III reactive peptide to conjugate a radionuclide chelator to K317 of antibodies with the concomitant release of the Fc-III peptide ligand. Here, our method was redesigned to target two lysines proximal to the Fc-III binding site, K248 and K439. Using energy minimization predictions and a semi-combinatorial synthesis approach, we sampled multiple Fc-III amino acid substituents of A3, H5, L6 and E8, which were then converted into Fc-III reactive conjugates. Middle-down MS/MS subunit analysis of the resulting trastuzumab conjugates revealed that K248 and K439 can be selectively targeted using the Fc-III reactive variants L6Dap, L6Orn, L6Y and A3K or A3hK, respectively. Across all variants tested, L6Orn-carbonate appeared to be the best candidate, yielding a degree and yield of conjugation of almost 2 and 100% for a broad array of payloads including radionuclide chelators, fluorescent dyes, click-chemistry reagents, pre-targeted imaging reagents, and some cytotoxic small molecules. Furthermore, L6Orn carbonate appeared to yield similar conjugation results across multiple IgG subtypes. In vivo proof of concept was achieved by conjugation of NODAGA to the PD1/PD-L1 immune checkpoint inhibitor antibody atezolizumab, followed by PET imaging of PD-L1 expression in mice bearing PD-L1 expressing tumor xenograft using radiolabeled [64Cu]Cu-atezolizumab.

Template directed synthesis of antibody Fc conjugates with concomitant ligand release.

Antibodies are an attractive therapeutic modality for cancer treatment as they allow the increase of the treatment response rate and avoid the severe side effects of chemotherapy. Notwithstanding the strong benefit of antibodies, the efficacy of anti-cancer antibodies can dramatically vary among patients and ultimately result in no response to the treatment. Here, we have developed a novel means to regioselectively label the Fc domain of any therapeutic antibody with a radionuclide chelator in a single step chemistry, with the aim to study by SPECT/CT imaging if the radiolabeled antibody is capable of targeting cancer cells in vivo. A Fc-III peptide was used as bait to bring a carbonate electrophilic site linked to a metal chelator and to a carboxyphenyl leaving group in close proximity with an antibody Fc nucleophile amino acid (K317), thereby triggering the covalent linkage of the chelator to the antibody lysine, with the concomitant release of the carboxyphenyl Fc-III ligand. Using CHX-A''-DTPA, we radiolabeled trastuzumab with indium-111 and showed in biodistribution and imaging experiments that the antibody accumulated successfully in the SK-OV-3 xenograft tumour implanted in mice. We found that our methodology leads to homogeneous conjugation of CHX-A''-DTPA to the antibody, and confirmed that the Fc domain can be selectively labeled at K317, with a minor level of unspecific labeling on the Fab domain. The present method can be developed as a clinical diagnostic tool to predict the success of the therapy. Furthermore, our Fc-III one step chemistry concept paves the way to a broad array of other applications in antibody bioengineering.