Checkpoint blockade with antibodies against CTLA-4 or PD-1 elicits durable tumor regressions in metastatic cancer, but these dramatic responses are confined to a minority of patients1–3. This suboptimal outcome is likely due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint4–8. Here we demonstrate a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse melanoma model; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal anti-tumor efficacy required four components: a tumor antigen targeting antibody, an extended half-life recombinant IL-29, anti-PD-1, and a powerful T-cell vaccine10. Depletion experiments revealed that CD8+ T-cells, cross-presenting DCs, and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake, and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies capable of curing a majority of tumors in experimental settings typically viewed as intractable.

Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47-SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophagemediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.immunotherapy | protein engineering | cancer | macrophage | T cell

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.

Ras is at the hub of signal transduction pathways controlling cell proliferation and survival. Its mutants, present in about 30% of human cancers, are major drivers of oncogenesis and render tumors unresponsive to standard therapies. Here we report the engineering of a protein scaffold for preferential binding to K-Ras G12D. This is the first reported inhibitor to achieve nanomolar affinity while exhibiting specificity for mutant over wild type (WT) K-Ras. Crystal structures of the protein R11.1.6 in complex with K-Ras WT and K-Ras G12D offer insight into the structural basis for specificity, highlighting differences in the switch I conformation as the major defining element in the higher affinity interaction. R11.1.6 directly blocks interaction with Raf and reduces signaling through the Raf/MEK/ERK pathway. Our results support greater consideration of the state of switch I and provide a novel tool to study Ras biology. Most importantly, this work makes an unprecedented contribution to Ras research in inhibitor development strategy by revealing details of a targetable binding surface. Unlike the polar interfaces found for Ras/effector interactions, the K-Ras/R11.1.6 complex reveals an extensive hydrophobic interface that can serve as a template to advance the development of high affinity, non-covalent inhibitors of K-Ras oncogenic mutants.

Summary Numerous synergistic cancer immunotherapy combinations have been identified, but the effects of relative dose timing are rarely considered. In established syngeneic mouse tumor models, we found that staggering IFNα administration after, rather than before or simultaneously with, serum-persistent IL-2 and tumor-specific antibody significantly increased long-term survival. Successful combination therapy required IFNα-induced activation of cross-presenting CD8α+ DCs following release of antigenic tumor debris by the IL-2-and-antibody-mediated immune response. Due to decreased phagocytic ability post-maturation, DCs activated too early captured less antigen and could not effectively prime CD8+ T cells. Temporally programming DC activation to occur after tumoricidal activity enhanced tumor control by multiple distinct combination immunotherapies, highlighting dose schedule as an underappreciated factor that can profoundly affect the success of multi-component immunotherapies.