Summary of recent advancesYeast surface display is being employed to engineer desirable properties into proteins for a broad variety of applications. Labeling with soluble ligands enables rapid and quantitative analysis of yeast-displayed libraries by flow cytometry, while libraries with insoluble or even as-yetuncharacterized binding targets can be screened through cell-surface selections. In parallel, the utilization of yeast surface display for protein characterization, including in particular the mapping of functional epitopes mediating protein-protein interactions, represents a significant recent advance.

Summary Cancer immunotherapies under development have generally focused on either stimulating T-cell immunity or driving antibody-directed effector functions of the innate immune system such as antibody-dependent cell-mediated cytotoxicity (ADCC). We find that a combination of an anti-tumor antigen antibody and an untargeted IL-2 fusion protein with delayed systemic clearance induces significant tumor control in aggressive isogenic tumor models via a concerted innate and adaptive response involving neutrophils, NK cells, macrophages, and CD8+ T-cells. This combination therapy induces an intratumoral “cytokine storm” and extensive lymphocyte infiltration. Adoptive transfer of anti-tumor T-cells together with this combination therapy leads to robust cures of established tumors and establishment of immunological memory.

Immunostimulatory agonists such as anti-CD137 and interleukin-2 (IL-2) have elicited potent anti-tumor immune responses in preclinical studies, but their clinical use is limited by inflammatory toxicities that result upon systemic administration. We hypothesized that by rigorously restricting the biodistribution of immunotherapeutic agents to a locally accessible lesion and draining lymph node(s), effective local and systemic anti-tumor immunity could be achieved in the absence of systemic toxicity. We anchored anti-CD137 and an engineered IL-2Fc fusion protein to the surfaces of PEGylated liposomes, whose physical size permitted dissemination in the tumor parenchyma and tumor-draining lymph nodes but blocked entry into the systemic circulation following intratumoral injection. In the B16F10 melanoma model, intratumoral liposome-coupled anti-CD137 + IL-2Fc therapy cured a majority of established primary tumors, while avoiding the lethal inflammatory toxicities caused by equivalent intratumoral doses of soluble immunotherapy. Immuno-liposome therapy induced protective anti-tumor memory and elicited systemic anti-tumor immunity that significantly inhibited the growth of simultaneously-established distal tumors. Tumor inhibition was CD8+ T-cell-dependent and was associated with increased CD8+ T-cell infiltration in both treated and distal tumors, enhanced activation of tumor-antigen-specific T-cells in draining lymph nodes, and a reduction in regulatory T-cells in treated tumors. These data suggest that local nanoparticle-anchored delivery of immuno-agonists represents a promising strategy to improve the therapeutic window and clinical applicability of highly potent but otherwise intolerable regimens of cancer immunotherapy.