Engaging the Immune Response to Normalize the Tumor Microenvironment

Solid tumors exist as heterogeneous populations comprised not only of malignant cells, but various other cell types, including cells that make up the vasculature, that can strongly influence tumorgenicity. Many forms of solid cancers are highly vascularized due to dysregulated angiogenesis. The tumor vasculature is classified by leaky, chaotic blood vessels consisting of several components including vascular endothelial cells and pericytes, as well vascular progenitors, resulting in vascular permeability and high interstitial pressure. As a result, the tumor vasculature limits the access of immune effector cells to the tumor, and may in part be responsible for the modest success observed in many current anti-cancer immunotherapies. Current first-line therapeutics in the advanced stage disease setting include anti-angiogenic small molecule drugs that have yielded high objective clinical response rates, however these responses tend to be transient in nature, with most patients becoming drug-refractory. Anti-tumor vasculature vaccines may promote the reconditioning of the tumor microenvironment by coordinately promoting a pro-inflammatory environment and the specific immune targeting of tumor-associated stromal cell populations that contribute to vasculature destabilization. Implementing a vaccine with these therapeutic effects is a promising treatment option that may extend disease-free intervals and overall patient survival. I show that vaccines specifically targeting tumor vasculature populations can “normalize” the tumor microenvironment, as shown by upregulation of proinflammatory molecules within the tumor as well as vascular remodeling promoting enhanced recruitment of CD8+ T cells, resulting in superior anti-tumor efficacy

Antagonism of regulatory ISGs enhances the anti-melanoma efficacy of STING agonists

BackgroundStimulator of Interferon Genes (STING) is a dsDNA sensor that triggers type I inflammatory responses. Recent data from our group and others support the therapeutic efficacy of STING agonists applied intratumorally or systemically in a range of murine tumor models, with treatment benefits associated with tumor vascular normalization and improved immune cell recruitment and function within the tumor microenvironment (TME). However, such interventions are rarely curative and STING agonism coordinately upregulates expression of immunoregulatory interferon-stimulated genes (ISGs) including Arg2, Cox2, Isg15, Nos2, and Pdl1 that may limit treatment benefits. We hypothesized that combined treatment of melanoma-bearing mice with STING agonist ADU-S100 together with antagonists of regulatory ISGs would result in improved control of tumor growth vs. treatment with ADU-S100 alone.MethodsMice bearing either B16 (BRAFWTPTENWT) or BPR20 (BRAFV600EPTEN-/-) melanomas were treated with STING agonist ADU-S100 plus various inhibitors of ARG2, COX2, NOS2, PD-L1, or ISG15. Tumor growth control and changes in the TME were evaluated for combination treatment vs ADU-S100 monotherapy by tumor area measurements and flow cytometry/transcriptional profiling, respectively.ResultsIn the B16 melanoma model, we noted improved antitumor efficacy only when ADU-S100 was combined with neutralizing/blocking antibodies against PD-L1 or ISG15, but not inhibitors of ARG2, COX2, or NOS2. Conversely, in the BPR20 melanoma model, improved tumor growth control vs. ADU-S100 monotherapy was only observed when combining ADU-S100 with ARG2i, COX2i, and NOS2i, but not anti-PD-L1 or anti-ISG15. Immune changes in the TME associated with improved treatment outcomes were subtle but included increases in proinflammatory innate immune cells and activated CD8+CD69+ T cells and varied between the two tumor models.ConclusionsThese data suggest contextual differences in the relative contributions of individual regulatory ISGs that serve to operationally limit the anti-tumor efficacy of STING agonists which should be considered in future design of novel combination protocols for optimal treatment benefit

UPDATE ON VACCINE DEVELOPMENT FOR RENAL CELL CANCER.

Renal cell carcinoma (RCC) remains a significant health concern that frequently presents as metastatic disease at the time of initial diagnosis. Current first-line therapeutics in the advanced stage setting include anti-angiogenic drugs that have yielded high rates of objective clinical response, however these tend to be transient in nature, with many patients becoming refractory to chronic treatment with these agents. Adjuvant immunotherapies remain viable candidates to sustain disease-free and overall patient survival. In particular, vaccines designed to optimize the activation, maintenance and recruitment of specific immunity within/into the tumor site continue to evolve. Based on the integration of increasingly refined immunomonitoring systems in both translational models and clinical trials, allowing for the improved understanding of treatment mechanism(s) of action, further refined (combinational) vaccine protocols are currently being developed and evaluated. This review will provide a brief history of RCC vaccine development, discussing the successes and deficiencies in such approaches, before providing a rationale for developing combinational vaccine approaches that may provide improved clinical benefits to patients with RCC

Immunotherapeutic targeting of HSP90 client proteins in BRAF-inhibitor resistant melanoma

Therapy options for patients with advanced stage melanoma have improved steadily over the past decade, with recent clinical successes noted for BRAF inhibitors (BRAFi). BRAFi such as dabrafenib are highly specific for BRAF mutant, BRAFV600E, which is expressed in approximately 50% of melanomas. Although treatment with BRAFi is highly efficacious with substantial tumor regression and increased patient survival, the response is short-lived, with treatment-refractory progressive disease developing as early as six months. Refractory progressive disease develops in association with tumor cell adoption of alternate signaling pathways (linked to tumor cell (over)expressed BRAFi-resistance associated molecules (BRAFi-RAM) such as FGFR3, MEK, PDGFRb, SRC, and STAT3, among others) supporting their continued survival, growth, and metastatic potential. It is worthwhile noting that each of these molecules represents a “client” protein of HSP90, a molecular chaperone commonly overexpressed in human melanomas where it serves to post-translationally stabilize a broad range of clients. Treatment of melanoma cells in vitro or in vivo with HSP90 inhibitor (HSP90i), leads to the rapid proteasome-dependent degradation of HSP90 client proteins, with the resulting peptides used to load MHC class I complexes on the tumor cell surface, thereby conditionally-enhancing specific CD8+ T cell recognition. In this study, we hypothesize that specific vaccination against BRAFi-RAM in combination with systemic HSP90i treatment will promote tumor regression and increase overall survival in a BRAFi-resistant melanoma mouse model. By treating with increasing concentrations of dabrafenib, we have selected for and maintained a dabrafenib-resistant BP (BRAF V600E ; PTEN-/-) mouse melanoma cell line (BPR20) that demonstrates upregulation of BRAFi-RAM and increased activation of receptor tyrosine kinase signaling pathways. Upregulated BRAFi-RAM in BPR20 underwent proteasome-dependent degradation when treated with ganetespib, an HSP90i. Furthermore, a proportion of the upregulated BRAFi-RAM were immunogenic. Specific CD8+ T cells were generated when wild-type C57Bl/6 mice were vaccinated with IL-12-expressing dendritic cells pulsed with a BRAFi-RAM peptide pool, and those CD8+ T cells demonstrated enhanced specific reactivity to syngeneic cells presenting relevant antigen as revealed by IFNg release assays. These results suggest that a polyepitope vaccine based on BRAFi-resistance associated HSP90 client proteins could define a novel immunotherapeutic strategy for the (co)treatment of patients with advanced-stage melanomas

Vaccination with EphA2-derived T cell-epitopes promotes immunity against both EphA2-expressing and EphA2-negative tumors

BACKGROUND: A novel tyrosine kinase receptor EphA2 is expressed at high levels in advanced and metastatic cancers. We examined whether vaccinations with synthetic mouse EphA2 (mEphA2)-derived peptides that serve as T cell epitopes could induce protective and therapeutic anti-tumor immunity. METHODS: C57BL/6 mice received subcutaneous (s.c.) vaccinations with bone marrow-derived dendritic cells (DCs) pulsed with synthetic peptides recognized by CD8+ (mEphA2(671–679), mEphA2(682–689)) and CD4+ (mEphA2(30–44)) T cells. Splenocytes (SPCs) were harvested from primed mice to assess the induction of cytotoxic T lymphocyte (CTL) responses against syngeneic glioma, sarcoma and melanoma cell lines. The ability of these vaccines to prevent or treat tumor (s.c. injected MCA205 sarcoma or B16 melanoma; i.v. injected B16-BL6) establishment/progression was then assessed. RESULTS: Immunization of C57BL/6 mice with mEphA2-derived peptides induced specific CTL responses in SPCs. Vaccination with mEPhA2 peptides, but not control ovalbumin (OVA) peptides, prevented the establishment or prevented the growth of EphA2+ or EphA2-negative syngeneic tumors in both s.c. and lung metastasis models. CONCLUSIONS: These data indicate that mEphA2 can serve as an attractive target against which to direct anti-tumor immunity. The ability of mEphA2 vaccines to impact EphA2-negative tumors such as the B16 melanoma may suggest that such beneficial immunity may be directed against alternative EphA2+ target cells, such as the tumor-associated vascular endothelial cells

Complementary Dendritic Cell–activating Function of CD8+ and CD4+ T Cells: Helper Role of CD8+ T Cells in the Development of T Helper Type 1 Responses

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of “T helper (Th)” signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood– or peripheral blood–isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-γ at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I–presented epitopes by antigen-specific CD8+ T cells results in the TNF-α– and IFN-γ–dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I–restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I–presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections

Bi- and Tri-Specific T Cell Engager-Armed Oncolytic Viruses: Next-Generation Cancer Immunotherapy

Oncolytic viruses (OVs) are potent anti-cancer biologics with a bright future, having substantial evidence of efficacy in patients with cancer. Bi- and tri-specific antibodies targeting tumor antigens and capable of activating T cell receptor signaling have also shown great promise in cancer immunotherapy. In a cutting-edge strategy, investigators have incorporated the two independent anti-cancer modalities, transforming them into bi- or tri-specific T cell engager (BiTE or TriTE)-armed OVs for targeted immunotherapy. Since 2014, multiple research teams have studied this combinatorial strategy, and it showed substantial efficacy in various tumor models. Here, we first provide a brief overview of the current status of oncolytic virotherapy and the use of multi-specific antibodies for cancer immunotherapy. We then summarize progress on BiTE and TriTE antibodies as a novel class of cancer therapeutics in preclinical and clinical studies, followed by a discussion of BiTE- or TriTE-armed OVs for cancer therapy in translational models. In addition, T cell receptor mimics (TCRm) have been developed into BiTEs and are expected to greatly expand the application of BiTEs and BiTE-armed OVs for the effective targeting of intracellular tumor antigens. Future applications of such innovative combination strategies are emerging as precision cancer immunotherapies

Toll like receptor-3 ligand poly-ICLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models

BACKGROUND: Toll-like receptor (TLR)3 ligands serve as natural inducers of pro-inflammatory cytokines capable of promoting Type-1 adaptive immunity, and TLR3 is abundantly expressed by cells within the central nervous system (CNS). To improve the efficacy of vaccine strategies directed against CNS tumors, we evaluated whether administration of a TLR3 ligand, polyinosinic-polycytidylic (poly-IC) stabilized with poly-lysine and carboxymethylcellulose (poly-ICLC) would enhance the anti-CNS tumor effectiveness of tumor peptide-based vaccinations. METHODS: C57BL/6 mice bearing syngeneic CNS GL261 glioma or M05 melanoma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes- mEphA2 (671–679), hgp100 (25–33) and mTRP-2 (180–188) for GL261, or ovalbumin (OVA: 257–264) for M05. The mice also received intramuscular (i.m.) injections with poly-ICLC. RESULTS: The combination of subcutaneous (s.c.) peptide-based vaccination and i.m. poly-ICLC administration promoted systemic induction of antigen (Ag)-specific Type-1 CTLs expressing very late activation antigen (VLA)-4, which confers efficient CNS-tumor homing of vaccine-induced CTLs based on experiments with monoclonal antibody (mAb)-mediated blockade of VLA-4. In addition, the combination treatment allowed expression of IFN-γ by CNS tumor-infiltrating CTLs, and improved the survival of tumor bearing mice in the absence of detectable autoimmunity. CONCLUSION: These data suggest that poly-ICLC, which has been previously evaluated in clinical trials, can be effectively combined with tumor Ag-specific vaccine strategies, thereby providing a greater index of therapeutic efficacy

JAK3/STAT5/6 Pathway Alterations Are Associated with Immune Deviation in CD8+ T Cells in Renal Cell Carcinoma Patients

To investigate the molecular mechanisms underlying altered T cell response in renal cell carcinoma (RCC) patients, we compared autologous and allogeneic CD8+ T cell responses against RCC line from RCC patients and their HLA-matched donors, using mixed lymphocyte/tumor cell cultures (MLTCs). In addition, we analyzed the expression of molecules associated with cell cycle regulation. Autologous MLTC responder CD8+ T cells showed cytotoxic activity against RCC cell lines; however the analysis of the distribution of CD8+ T-cell subsets revealed that allogenic counterparts mediate superior antitumor efficacy. In RCC patients, a decreased proliferative response to tumor, associated with defects in JAK3/STAT5/6 expression that led to increased p27KIP1 expression and alterations in the cell cycle, was observed. These data define a molecular pathway involved in cell cycle regulation that is associated with the dysfunction of tumor-specific CD8+ effector cells. If validated, this may define a therapeutic target in the setting of patients with RCC