Broadening a SARS-CoV-1-neutralizing antibody for potent SARS-CoV-2 neutralization through directed evolution.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing antibody that neutralizes one virus but not a related virus. Through a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1-neutralizing antibody, to bind to the receptor binding domain of SARS-CoV-2 with >1000-fold increased affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of engineered CR3022 elucidated the molecular mechanisms by which the antibody can accommodate sequence differences in the epitopes between SARS-CoV-1 and SARS-CoV-2. This workflow provides a blueprint for the rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.

Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins.

Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape pathways.

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we used a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduces the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity-matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for antiviral indications would benefit from affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

Spontaneous Glycan Reattachment Following N-Glycanase Treatment of Influenza and HIV Vaccine Antigens.

In cells, asparagine/N-linked glycans are added to glycoproteins cotranslationally, in an attachment process that supports proper folding of the nascent polypeptide. We found that following pruning of N-glycan by the amidase PNGase F, the principal influenza vaccine antigen and major viral spike protein hemagglutinin (HA) spontaneously reattached N-glycan to its de-N-glycosylated positions when the amidase was removed from solution. This reaction, which we term N-glycanation, was confirmed by site-specific analysis of HA glycoforms by mass spectrometry prior to PNGase F exposure, during exposure to PNGase F, and after amidase removal. Iterative rounds of de-N-glycosylation followed by N-glycanation could be repeated at least three times and were observed for other viral glycoproteins/vaccine antigens, including the envelope glycoprotein (Env) from HIV. Covalent N-glycan reattachment was nonenzymatic as it occurred in the presence of metal ions that inhibit PNGase F activity. Rather, N-glycanation relied on a noncovalent assembly between protein and glycan, formed in the presence of the amidase, where linearization of the glycoprotein prevented this retention and subsequent N-glycanation. This reaction suggests that under certain experimental conditions, some glycoproteins can organize self-glycan addition, highlighting a remarkable self-assembly principle that may prove useful for re-engineering therapeutic glycoproteins such as influenza HA or HIV Env, where glycan sequence and structure can markedly affect bioactivity and vaccine efficacy.

Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition.

Although mechanisms of acquired resistance of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here we observe that acquired resistance caused by the EGFR(T790M) gatekeeper mutation can occur either by selection of pre-existing EGFR(T790M)-positive clones or via genetic evolution of initially EGFR(T790M)-negative drug-tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug-tolerant cells had a diminished apoptotic response to third-generation EGFR inhibitors that target EGFR(T790M); treatment with navitoclax, an inhibitor of the anti-apoptotic factors BCL-xL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug-resistant cancer cells can both pre-exist and evolve from drug-tolerant cells, and they point to therapeutic opportunities to prevent or overcome resistance in the clinic.