A decoy receptor for SARS-CoV-2: For severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to enter human cells, the spike protein on the surface of the virus must bind to the host receptor protein, angiotensin-converting enzyme 2 (ACE2). A soluble version of the receptor is being explored as a therapeutic. Chan et al. used deep mutagenesis to identify ACE2 mutants that bind more tightly to the spike protein and combined mutations to further increase binding affinity (see the Perspective by DeKosky). A promising variant was engineered to be a stable dimer that has a binding affinity for the spike protein; it is comparable with neutralizing antibodies and neutralized both SARS-CoV-2 and SARS-CoV-1 in a cell-based assay. In addition, the similarity to the natural receptor may limit the possibility for viral escape. Science , this issue p. 1261 ; see also p. 1167

Cytokines are a class of potent immunoregulatory proteins that are secreted in response to various stimuli and act locally to regulate many aspects of human physiology and disease. Cytokines play important roles in cancer initiation, progression, and elimination, and thus, there is a long clinical history associated with the use of recombinant cytokines to treat cancer. However, the use of cytokines as therapeutics has been limited by cytokine pleiotropy, complex biology, poor drug-like properties, and severe dose-limiting toxicities. Nevertheless, cytokines are crucial mediators of innate and adaptive antitumor immunity and have the potential to enhance immunotherapeutic approaches to treat cancer. Development of immune checkpoint inhibitors and combination immunotherapies has reinvigorated interest in cytokines as therapeutics, and a variety of engineering approaches are emerging to improve the safety and effectiveness of cytokine immunotherapy. In this review we highlight recent advances in cytokine biology and engineering for cancer immunotherapy.

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors. Significance CAR T cells have shown significant promise in treating hematopoietic cancers but challenges remain, including antigen loss and identification of targets in solid tumors. Aberrant O-linked glycosylation in solid cancers is common, often resulting in expression of cell-surface neoantigens. We used directed evolution to engineer the binding site of an antibody so variants reacted more broadly with tumor-specific glycoprotein epitopes containing Tn (GalNAc-O-S/T). As CARs, these variants showed improved broader activity against mouse and human cancer cells with dysregulated O-glycosylation. Even cancer cells without MUC1, the most studied cancer-associated glycoantigen, were recognized by the engineered CARs. Cancer-specific recognition was retained due to Tn-antigen requirement, but broader activity against the glycoprotein backbone provides opportunities for potent use with minimal antigen-loss escape.

Most affinity-maturation campaigns for antibodies and T cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by group at the α:β interface, at a significant distance from the TCR/pepMHC binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART- 1/HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single codon libraries of both CDR and non- CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1/HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (α and β) and surface-exposed framework residues. Mutational analyses showed that both α:β interface and CDR residues were important in maintaining binding to MART- 1/HLA-A2, likely due to either structural requirements for proper α:β association or direct contact with the ligand. More surprisingly, many α:β interface substitutions yielded improved binding to MART-1/HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (β) was most prevalent. Further analysis of β showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the α:β interface, at a significant distance from the TCR/pepMHC binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART- 1/HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.

The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.