Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Here, we applied a structure-based computational approach to engineer anti-human epidermal growth factor receptor 2 (Her2) antibodies with selective binding in the acidic tumor microenvironment. We used an affinity maturation platform in which dual-pH histidine-scanning mutagenesis was implemented for pH selectivity optimization. Testing of a small set of designs for binding to the recombinant Her2 ectodomain led to the identification of antigen-binding fragment (Fab) variants with the desired pH-dependent binding behavior. Binding selectivity toward acidic pH was improved by as much as 25-fold relative to the parental bH1-Fab. In vitro experiments on cells expressing intact Her2 confirmed that designed variants formatted as IgG1/k full-size antibodies have high affinity and inhibit the growth of tumor spheroids at a level comparable to that of the benchmark anti-Her2 antibody trastuzumab (Herceptin®) at acidic pH, whereas these effects were significantly reduced at physiological pH. In contrast, both Herceptin and the parental bH1 antibody exhibited strong cell binding and growth inhibition irrespective of pH. This work demonstrates the feasibility of computational optimization of antibodies for selective targeting of the acidic environment such as that found in many solid tumors.

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Natriuretic peptide receptor A (NPRA) is a noncovalent homodimeric receptor, composed of an extracellular domain (ECD) with a ligand-binding site, a single transmembrane domain (TM), and an intracellular domain (ICD) exhibiting guanylyl cyclase activity. NPRA activation by atrial natriuretic peptide (ANP) leads to cGMP production, which plays important roles in cardiovascular homeostasis. Initial studies have shown that activation of NPRA involves a conformational change in the juxtamembrane domain (JM). However, crystallographic study of the soluble ECD of NPRA has failed to document JM structure, and the conformational change involved in transmembrane signal transduction is still unknown. To analyze this conformational change, we first sequentially substituted nine amino acids of the JM with a cysteine residue. By studying the mutant's capacity to form ANP-induced or constitutive covalent disulfide dimers, we evaluated the relative proximity of JM residues, before and after NPRA activation. These results obtained with the full-length receptor demonstrate a high proximity of specific JM residues and are in disagreement with crystallography data. We also tested the hypothesis that signal transduction involves a TM rotation mechanism leading to ICD activation. By introducing one to five alanine residues into the TM alpha-helix, we show that a TM rotation of 40 degrees leads to constitutive NPRA activation. We finally studied the role of the TM in NPRA dimerization. By using the ToxR system, we demonstrate that the last JM residues are required to stabilize the TM dimer. Using these experimental data, we generated a new molecular model illustrating the active conformation of NPRA, where the JM and TM are depicted.

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs.

The SARS coronavirus 2 (SARS-CoV-2) spike (S) protein binding to the human ACE2 receptor is the molecular event that initiates viral entry into host cells and leads to infection and virus replication. There is a need for agents blocking viral entry into host cells that are cross-reactive with emerging virus variants. VHH-72 is an anti-SARS-CoV-1 single-domain antibody that also exhibits cross-specificity with SARS-CoV-2 but with decreased binding affinity. Here we applied a structure-based approach to affinity-mature VHH-72 for the SARS-CoV-2 spike protein while retaining the original affinity for SARS-CoV-1. This was achieved by employing the computational platform ADAPT in a constrained dual-affinity optimization mode as a means of broadening specificity. Select mutants designed by ADAPT were formatted as fusions with a human IgG1-Fc fragment. These mutants demonstrated improved binding to the SARS-CoV-2 spike protein due to decreased dissociation rates. Functional testing for virus neutralization revealed improvements relative to the parental VHH72-Fc up to 10-fold using a SARS-CoV-2 pseudotyped lentivirus and 20-fold against the SARS-CoV-2 authentic live virus (Wuhan variant). Binding and neutralization improvements were maintained for some other SARS-CoV-2 variants currently in circulation. These improved VHH-72 mutants are predicted to establish novel interactions with the S antigen. They will be useful, alone or as fusions with other functional modules, in the global quest for treatments of COVID-19 infections.