Combination immunotherapy has recently emerged as a powerful cancer treatment strategy. A promising treatment approach utilizes coadministration of antagonistic antibodies to block checkpoint inhibitor receptors, such as antiprogrammed cell death-1 (aPD1), alongside agonistic antibodies to activate costimulatory receptors, such as antitumor necrosis factor receptor superfamily member 4 (aOX40). Optimal T-cell activation is achieved when both immunomodulatory agents simultaneously engage T-cells and promote synergistic proactivation signaling. However, standard administration of these therapeutics as free antibodies results in suboptimal T-cell binding events, with only a subset of the T-cells binding to both aPD1 and aOX40. Here, it is shown that precise spatiotemporal codelivery of aPD1 and aOX40 using nanoparticles (NP) (dual immunotherapy nanoparticles, DINP) results in improved T-cell activation, enhanced therapeutic efficacy, and increased immunological memory. It is demonstrated that DINP elicits higher rates of T-cell activation in vitro than free antibodies. Importantly, it is demonstrated in two tumor models that combination immunotherapy administered in the form of DINP is more effective than the same regimen administered as free antibodies. This work demonstrates a novel strategy to improve combination immunotherapy using nanotechnology.

Figure 1 IL22 induces an ER stress/unfolded protein response transcriptional module in colonic epithelial cells. (A) Heat map demonstrating pathway specific transcript expression in murine colonoids treated with (+IL22, n=3) or without (control, n=3) recombinant IL22. Mouse gene 2.0 ST array platform (affymetrix). (B) GSEA evaluating enrichment of ER stress response transcriptional module in IL22 treated colonoids. A core set of colonic epithelial-specific ER stress genes was defined by analysing significantly differentially expressed (p<0.05 and absolute value of the log2 fold change >±2) transcripts in colonoids treated with tunicamycin (n=3) or medium alone (n=3). (C) Expression of ER stress response transcripts in IL22 treated WT and Il22ra1 −/− colonoids (RNA-seq dataset ERR247358-ERR247389, Pham et al, 2014). 18 (D) Enrichment analysis for ER stress-related functional annotation groups (GO biological processes) in IL22-treated colonoids from dataset ERR247358-ERR247389. (E) Microarray analysis of core ER stress response transcripts in colonoids treated with tunicamycin (n=3), tunicamycin+IL22 (n=3) or untreated (control, n=3). (F) Real-time PCR quantification of ER stress transcripts in colonoids treated with IL22 (n=11), IL17A (n=6) and IL22+IL17A (n=6) and unexposed controls. *P<0.01. (G) Immunoblot and densitometry quantification (H) detecting GRP78 protein expression in colonoids treated with different cytokines. *P<0.026, one tailed t test. ER, endoplasmic reticulum; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; IL22, interleukin-22.on July 6, 2020 by guest. Protected by copyright.

Macrophages exhibit phenotypic heterogeneity under both physiological and pathological conditions. Applications targeting M2-like tumor-associated macrophages (TAMs) improve outcome in solid tumors. Considerable differences are detected between leukemia-associated macrophages (LAMs) and TAMs. However, application to induce M1 characteristics in heterogeneous LAMs has not been established. Here we analyzed clinical relevance of macrophage phenotypes in human acute myeloid leukemia (AML), studied phenotypic evolution of bone marrow (BM) and spleen (SP) LAMs in mouse AML and T cell acute lymphoblastic leukemia (T-ALL) models, explored mechanism leading to different LAM phenotypes and tried to eliminate pro-leukemic effects by inducing M1 characteristics. The results showed that more M2-like LAMs but not total LAMs correlated with worse prognosis in AML patients. Heterogeneity of LAM activation in tissue-specific leukemic microenvironments was observed in both AML and ALL models, SP LAMs evolved with more M2 characteristics while BM LAMs with more M1 characteristics. Furthermore, IRF7 contributed to M1 characteristics through the activation of SAPK/JNK pathway. Moreover, targeting IRF7-SAPK/JNK pathway to induce M1 characteristics in LAMs contributed to prolonged survival in leukemia mice. Our study provides the potential target for macrophage based immuno-therapy strategy against leukemia.