Extracellular Matrix Scaffold-Assisted Tumor Vaccines Induce Tumor Regression and Long-Term Immune Memory.

Injectable scaffold delivery is a strategy to enhance the efficacy of cancer vaccine immunotherapy. The choice of scaffold biomaterial is crucial, impacting both vaccine release kinetics and immune stimulation via the host response. Extracellular matrix (ECM) scaffolds prepared from decellularized tissues facilitate a pro-healing inflammatory response that promotes local cancer immune surveillance. Here, an ECM scaffold-assisted therapeutic cancer vaccine that maintains an immune microenvironment consistent with tissue reconstruction is engineered. Several immune-stimulating adjuvants are screened to develop a cancer vaccine formulated with decellularized small intestinal submucosa (SIS) ECM scaffold co-delivery. It is found that the STING pathway agonist cyclic di-AMP most effectively induces cytotoxic immunity in an ECM scaffold vaccine, without compromising key interleukin 4 (IL-4) mediated immune pathways associated with healing. ECM scaffold delivery enhances therapeutic vaccine efficacy, curing 50-75% of established E.G-7OVA lymphoma tumors in mice, while none are cured with soluble vaccine. SIS-ECM scaffold-assisted vaccination prolonged antigen exposure is dependent on CD8+ cytotoxic T cells and generates long-term antigen-specific immune memory for at least 10 months post-vaccination. This study shows that an ECM scaffold is a promising delivery vehicle to enhance cancer vaccine efficacy while being orthogonal to characteristics of pro-healing immune hallmarks.

Tumor eradication by hetIL-15 locoregional therapy correlates with an induced intratumoral CD103intCD11b+ dendritic cell population.

Locoregional monotherapy with heterodimeric interleukin (IL)-15 (hetIL-15) in a triple-negative breast cancer (TNBC) orthotopic mouse model resulted in tumor eradication in 40% of treated mice, reduction of metastasis, and induction of immunological memory against breast cancer cells. hetIL-15 re-shaped the tumor microenvironment by promoting the intratumoral accumulation of cytotoxic lymphocytes, conventional type 1 dendritic cells (cDC1s), and a dendritic cell (DC) population expressing both CD103 and CD11b markers. These CD103intCD11b+DCs share phenotypic and gene expression characteristics with both cDC1s and cDC2s, have transcriptomic profiles more similar to monocyte-derived DCs (moDCs), and correlate with tumor regression. Therefore, hetIL-15, a cytokine directly affecting lymphocytes and inducing cytotoxic cells, also has an indirect rapid and significant effect on the recruitment of myeloid cells, initiating a cascade for tumor elimination through innate and adoptive immune mechanisms. The intratumoral CD103intCD11b+DC population induced by hetIL-15 may be targeted for the development of additional cancer immunotherapy approaches.

Regression and Eradication of Triple-Negative Breast Carcinoma in 4T1 Mouse Model by Combination Immunotherapies.

Triple-negative breast carcinoma (TNBC) is one of the most aggressive types of solid-organ cancers. While immune checkpoint blockade (ICB) therapy has significantly improved outcomes in certain types of solid-organ cancers, patients with immunologically cold TNBC are afforded only a modest gain in survival by the addition of ICB to systemic chemotherapy. Thus, it is urgently needed to develop novel effective therapeutic approaches for TNBC. Utilizing the 4T1 murine model of TNBC, we developed a novel combination immunotherapeutic regimen consisting of intratumoral delivery of high-mobility group nucleosome binding protein 1 (HMGN1), TLR2/6 ligand fibroblast-stimulating lipopeptide (FSL-1), TLR7/8 agonist (R848/resiquimod), and CTLA-4 blockade. We also investigated the effect of adding SX682, a small-molecule inhibitor of CXCR1/2 known to reduce MDSC trafficking to tumor microenvironment, to our therapeutic approach. 4T1-bearing mice responded with significant tumor regression and tumor elimination to our therapeutic combination regimen. Mice with complete tumor regressions did not recur and became long-term survivors. Treatment with HMGN1, FSL-1, R848, and anti-CTLA4 antibody increased the number of infiltrating CD4+ and CD8+ effector/memory T cells in both tumors and draining lymph nodes and triggered the generation of 4T1-specific cytotoxic T lymphocytes (CTLs) in the draining lymph nodes. Thus, we developed a potentially curative immunotherapeutic regimen consisting of HMGN1, FSL-1, R848, plus a checkpoint inhibitor for TNBC, which does not rely on the administration of chemotherapy, radiation, or exogenous tumor-associated antigen(s).

Heterodimeric IL-15 (hetIL-15) reduces circulating tumor cells and metastasis formation improving chemotherapy and surgery in 4T1 mouse model of TNBC.

Immunotherapy has emerged as a viable approach in cancer therapy, with cytokines being of great interest. Interleukin IL-15 (IL-15), a cytokine that supports cytotoxic immune cells, has been successfully tested as an anti-cancer and anti-metastatic agent, but combinations with conventional chemotherapy and surgery protocols have not been extensively studied. We have produced heterodimeric IL-15 (hetIL-15), which has shown anti-tumor efficacy in several murine cancer models and is being evaluated in clinical trials for metastatic cancers. In this study, we examined the therapeutic effects of hetIL-15 in combination with chemotherapy and surgery in the 4T1 mouse model of metastatic triple negative breast cancer (TNBC). hetIL-15 monotherapy exhibited potent anti-metastatic effects by diminishing the number of circulating tumor cells (CTCs) and by controlling tumor cells colonization of the lungs. hetIL-15 treatment in combination with doxorubicin resulted in enhanced anti-metastatic activity and extended animal survival. Systemic immune phenotype analysis showed that the chemoimmunotherapeutic regimen shifted the tumor-induced imbalance of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in favor of cytotoxic effector cells, by simultaneously decreasing PMN-MDSCs and increasing the frequency and activation of effector (CD8+T and NK) cells. Tumor resection supported by neoadjuvant and adjuvant administration of hetIL-15, either alone or in combination with doxorubicin, resulted in the cure of approximately half of the treated animals and the development of anti-4T1 tumor immunity. Our findings demonstrate a significant anti-metastatic potential of hetIL-15 in combination with chemotherapy and surgery and suggest exploring the use of this regimen for the treatment of TNBC.

Heterodimeric IL-15 delays tumor growth and promotes intratumoral CTL and dendritic cell accumulation by a cytokine network involving XCL1, IFN-γ, CXCL9 and CXCL10.

BACKGROUND: Interleukin-15 (IL-15) promotes growth and activation of cytotoxic CD8+ T and natural killer (NK) cells. Bioactive IL-15 is produced in the body as a heterodimeric cytokine, comprising the IL-15 and IL-15 receptor alpha chains (hetIL-15). Several preclinical models support the antitumor activity of hetIL-15 promoting its application in clinical trials. METHODS: The antitumor activity of hetIL-15 produced from mammalian cells was tested in mouse tumor models (MC38 colon carcinoma and TC-1 epithelial carcinoma). The functional diversity of the immune infiltrate and the cytokine/chemokine network within the tumor was evaluated by flow cytometry, multicolor immunohistochemistry (IHC), gene expression profiling by Nanostring Technologies, and protein analysis by electrochemiluminescence and ELISA assays. RESULTS: hetIL-15 treatment resulted in delayed primary tumor growth. Increased NK and CD8+ T cell tumoral infiltration with an increased CD8+/Treg ratio were found by flow cytometry and IHC in hetIL-15 treated animals. Intratumoral NK and CD8+ T cells showed activation features with enhanced interferon-γ (IFN-γ) production, proliferation (Ki67+), cytotoxic potential (Granzyme B+) and expression of the survival factor Bcl-2. Transcriptomics and proteomics analyses revealed complex effects on the tumor microenvironment triggered by hetIL-15 therapy, including increased levels of IFN-γ and XCL1 with intratumoral accumulation of XCR1+IRF8+CD103+ conventional type 1 dendritic cells (cDC1). Concomitantly, the production of the chemokines CXCL9 and CXCL10 by tumor-localized myeloid cells, including cDC1, was boosted by hetIL-15 in an IFN-γ-dependent manner. An increased frequency of circulating CXCR3+ NK and CD8+ T cells was found, suggesting their ability to migrate toward the tumors following the CXCL9 and CXCL10 chemokine gradient. CONCLUSIONS: Our results show that hetIL-15 administration enhances T cell entry into tumors, increasing the success rate of immunotherapy interventions. Our study further supports the incorporation of hetIL-15 in tumor immunotherapy approaches to promote the development of antitumor responses by favoring effector over regulatory cells and by promoting lymphocyte and DC localization into tumors through the modification of the tumor chemokine and cytokine milieu.

Heterodimeric IL15 Treatment Enhances Tumor Infiltration, Persistence, and Effector Functions of Adoptively Transferred Tumor-specific T Cells in the Absence of Lymphodepletion.

Purpose: Adoptive cell transfer (ACT) is a promising immunotherapeutic approach for cancer. Host lymphodepletion is associated with favorable ACT therapy outcomes, but it may cause detrimental effects in humans. We tested the hypothesis that IL15 administration enhances ACT in the absence of lymphodepletion. We previously showed that bioactive IL15 in vivo comprises a stable complex of the IL15 chain with the IL15 receptor alpha chain (IL15Rα), termed heterodimeric IL15 (hetIL15).Experimental Design: We evaluated the effects of the combination regimen ACT + hetIL15 in the absence of lymphodepletion by transferring melanoma-specific Pmel-1 T cells into B16 melanoma-bearing mice.Results: hetIL15 treatment delayed tumor growth by promoting infiltration and persistence of both adoptively transferred Pmel-1 cells and endogenous CD8+ T cells into the tumor. In contrast, persistence of Pmel-1 cells was severely reduced following irradiation in comparison with mice treated with hetIL15. Importantly, we found that hetIL15 treatment led to the preferential enrichment of Pmel-1 cells in B16 tumor sites in an antigen-dependent manner. Upon hetIL15 administration, tumor-infiltrating Pmel-1 cells showed a "nonexhausted" effector phenotype, characterized by increased IFNγ secretion, proliferation, and cytotoxic potential and low level of PD-1. hetIL15 treatment also resulted in an improved ratio of Pmel-1 to Treg in the tumor.Conclusions: hetIL15 administration improves the outcome of ACT in lymphoreplete hosts, a finding with significant implications for improving cell-based cancer immunotherapy strategies. Clin Cancer Res; 23(11); 2817-30. ©2016 AACR.