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Purpose: IFN-α is a pleiotropic cytokine possessing immunomodulatory properties that may improve the efficacy of therapeutic cancer vaccines. The aim of this study was to evaluate the effectiveness and compatibility of combining recombinant IFN-α with poxvirus vaccines targeting the human carcinoembryonic antigen (CEA) in murine models of colorectal and pancreatic adenocarcinomas, where CEA is a self-antigen.Experimental Design: The phenotypic and functional effects of IFN-α were evaluated in the draining inguinal lymph nodes of tumor-free mice. We studied the effect of the site of IFN-α administration (local versus distal) on antigen-specific immune responses to poxvirus vaccination. Mechanistic studies were conducted to assess the efficacy of IFN-α and CEA-directed poxvirus vaccines in tumor-bearing CEA transgenic mice.Results: We identified a dose and schedule of IFN-α that induced a locoregional expansion of the draining inguinal lymph nodes and improved cellular cytotoxicity (natural killer and CD8+) and antigen presentation. Suppression of the vaccinia virus was avoided by administering IFN-α distal to the site of vaccination. The combination of IFN-α and vaccine inhibited tumor growth, improved survival, and elicited CEA-specific CTL responses in mice with CEA+ adenocarcinomas. In mice with pancreatic tumors, IFN-α slowed tumor growth, induced CTL activity, and increased CD8+ tumor-infiltrating lymphocytes.Conclusions: These data suggest that IFN-α can be used as a biological response modifier with antigen-directed poxvirus vaccines to yield significant therapeutic antitumor immune responses. This study provides the rationale and mechanistic insights to support a clinical trial of this immunotherapeutic strategy in patients with CEA-expressing carcinomas.

Supplementary Figures S1-S7 from The Antitumor and Immunoadjuvant Effects of IFN-α in Combination with Recombinant Poxvirus Vaccines

Supplementary Figure 1 from Intravesical Immunotherapy of Superficial Bladder Cancer with Chitosan/Interleukin-12

Supplementary Figure Legends 1-2 from Intravesical Immunotherapy of Superficial Bladder Cancer with Chitosan/Interleukin-12

Intravesical BCG has been used successfully to treat superficial bladder cancer for three decades. However, 20% to 30% of patients will fail initial BCG therapy and 30% to 50% of patients will develop recurrent tumors within 5 years. Alternative or complementary strategies for the management of superficial bladder cancer are needed. Interleukin-12 (IL-12) is a potent TH1 cytokine with robust antitumor activity and the ability to potentiate immunologic memory. Unfortunately, intravesical IL-12 did not show antitumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that coformulation of IL-12 with chitosan, a biocompatible, mucoadhesive polysaccharide, could improve intravesical IL-12 delivery and provide an effective and durable alternative for the treatment of superficial bladder cancer. In antitumor studies, 88% to 100% of mice bearing orthotopic bladder tumors were cured after four intravesical treatments with chitosan/IL-12. In contrast, only 38% to 60% of mice treated with IL-12 alone and 0% treated with BCG were cured. Antitumor responses following chitosan/IL-12 treatments were durable and provided complete protection from intravesical tumor rechallenge. Urinary cytokine analysis showed that chitosan/IL-12 induced multiple TH1 cytokines at levels significantly higher than either IL-12 alone or BCG. Immunohistochemistry revealed moderate to intense tumor infiltration by T cells and macrophages following chitosan/IL-12 treatments. Bladder submucosa from cured mice contained residual populations of immune cells that returned to baseline levels after several months. Intravesical chitosan/IL-12 is a well-tolerated, effective immunotherapy that deserves further consideration for testing in humans for the management of superficial bladder cancer. [Cancer Res 2009;69(15):6192–9]

BackgroundIn recent years, a regulatory network involving nectin/nectin-like immune receptors has emerged as a potential point of manipulation for cancer immunotherapy. Central to this axis, CD226 (DNAM-1) is a T and NK cell co-stimulatory receptor that competes for ligand (CD155 and CD112) binding with multiple inhibitory receptors (TIGIT, CD96, and PVRIG [CD112R]). Despite a large body of literature for TIGIT, detailed cellular characterization of the entire axis is still lacking. Therefore, we used mass cytometry (CyTOF) to systematically evaluate expression of the CD226 axis in tumors from a range of indications.MethodsTo thoroughly characterize the CD226 axis in the tumor microenvironment, we immunophenotyped approximately 100 tumor samples derived from a variety of cancer types using a bespoke 46-parameter CyTOF panel. Human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol. Using a suite of high-dimensional analytical tools, including FlowSOM, UMAP, and tSNE, we revealed distinct expression profiles for each receptor; a finding that was previously obscured due to a lack of sufficient resolution.ResultsWe observed a notable divergence in expression profiles between the CD226 axis members across tumor indications. For example, TIGIT expression was found to be highest on activated CD4+ regulatory T (Treg) cells, where its expression correlated strongly with ICOS, FoxP3, CD25, and CCR8. By contrast, CD96 and PVRIG exhibited broad expression across intratumoral T and NK cell populations. Other receptors (e.g., CD226) demonstrated variegated expression profiles across T and NK cell subsets. Finally, despite relatively consistent expression profiles of certain CD226 axis (i.e., TIGIT on Treg cells) across tumors, we also found several cell subsets/clusters unique to specific indications.ConclusionsUsing high-parameter CyTOF analysis, we were able to thoroughly characterize the CD226 axis (CD226, TIGIT, CD96, PVRIG) and related immune receptors across a range of tumor indications. These analyses revealed divergent expression profiles for each CD226 axis member, suggesting distinct/contextual biological role(s) for each receptor. However, future studies will need to dissect the importance of the distinct cellular representation for each CD226 axis member.Ethics ApprovalAll samples were purchased from Discovery Life Sciences (DLS). DLS represents and warrants that it has ownership of all Products available for sale and has properly obtained, where required under HHS/OHRP 45 CFR 46.102 (d) (f), IRB approval (or appropriate research approval for institutions outside the U.S.) for study protocols and informed consent documents for all human subject derived biological materials.

Background Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. Methods We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. Results Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. Conclusions Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors. Fil: Torres, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Regge, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Secchiari, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Friedrich, Adrián David. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Spallanzani, Raúl Germán. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Raffo Iraolagoitia, Ximena Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Nuñez, Sol Yanel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Sierra, Jessica Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ziblat, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Santilli, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Gilio, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Almada, Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Lauché, Constanza Elena. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Pardo, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Inmunova S.A; Argentina Fil: Domaica, Carolina Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Fuertes, Mercedes Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Madauss, Kevin Patrick. Glaxo Smith Kline; Estados Unidos Fil: Hance, Kenneth W.. Glaxo Smith Kline; Estados Unidos Fil: Gloger, Israel S.. Glaxo Smith Kline; Estados Unidos Fil: Zylberman, Vanesa. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Goldbaum, Fernando Alberto. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Zwirner, Norberto Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina

In recent years, antibody-based immunotherapies targeting immune checkpoint proteins cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death receptor-1 (PD-1), have revolutionized the treatment of cancer. Despite durable clinical responses in many indications, a significant number of patients fail to yield meaningful benefit, emphasizing the need to identify novel and/or complementary therapeutic interventions (e.g., alternative immune checkpoints, cytotoxic agents, etc.). Inducible T cell Co-Stimulator (ICOS/CD278) is a receptor belonging to the CD28/CTLA-4 superfamily that is predominantly expressed on T cells shortly after T cell receptor (TCR) activation. A growing body of non-clinical and clinical evidence supports the concept of antibody-mediated induction of ICOS signaling as a means to promote durable antitumor responses, particularly in collaboration with other checkpoint immunotherapies. To support ongoing and future clinical studies for feladilimab (GSK3359609), a non-depleting IgG4 ICOS agonist antibody currently being evaluated in pivotal clinical trials, we conducted a series of in vivo studies using a rodent surrogate (7E.17G9 mouse [m]IgG1; analogous non-depleting Fc) in ICOS-sensitive (EMT6 breast carcinoma) and ICOS-insensitive (CT26 colon carcinoma) tumor models. Female BALB/c mice with pre-established EMT6 tumors, exhibited partial responses to GSK'609 surrogate/tool antibody (7E.17G9 mIgG1), with ~20% (range 0-40%) of mice demonstrating tumor clearance. Concurrent administration of anti-PD-1 (RMP1-14 rat [r]IgG2a) and CTLA-4 (9D9 mIgG2b) dramatically improved tumor growth inhibition and survival. Notably, a triplet of TIM-3 blockade (RMT3-23 rIgG2a) in concert with ICOS and PD-1 mAbs further enhanced the survival of EMT6 tumor-bearing mice, suggesting complementary therapeutic mechanisms. In addition to combination with immune checkpoint blockade, we also explored the potential of ICOS co-stimulation with different focal irradiation (FRT) doses and frequencies in both EMT6 and CT26 tumor models. Intriguingly, we observed dramatic differences in response to concurrent FRT and ICOS mAb depending on the tumor model and associated FRT dose and frequency. Further, we identified a FRT dose threshold required for ICOS agonist appreciable combination responses, enabling exploration of additional immunotherapeutic combinations on top of ICOS/FRT. Collectively, these data support the combination of ICOS co-stimulation with different checkpoint immunotherapies (doublets and triplets), several of which are currently being evaluated in clinical studies. Moreover, these data demonstrate potential of ICOS co-stimulation with cytotoxic strategies, such as FRT - providing various dosing considerations and a framework future therapeutic intervention strategies. Citation Format: Jeremy D. Waight, Meixia Bi, Chris Hopson, Sapna Yadavilli, Kenneth W. Hance, Marc Ballas, Axel Hoos. ICOS co-stimulation in combination immune checkpoint blockade and/or dose-optimized focal irradiation results in enhanced tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1849.

Immune cell functions are regulated by co-inhibitory and co-stimulatory receptors. The first two generations of cancer immunotherapy agents consist primarily of antagonist antibodies that block negative immune checkpoints, such as programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte protein 4 (CTLA4). Looking ahead, there is substantial promise in targeting co-stimulatory receptors with agonist antibodies, and a growing number of these agents are making their way through various stages of development. This Review discusses the key considerations and potential pitfalls of immune agonist antibody design and development, their differentiating features from antagonist antibodies and the landscape of agonist antibodies in clinical development for cancer treatment.

Antibodies targeting immune checkpoints are emerging as potent and viable cancer therapies, but not all patients respond to these as single agents. Concurrently targeting additional immunosuppressive pathways is a promising approach to enhance immune checkpoint blockade, and bifunctional molecules designed to target two pathways simultaneously may provide a strategic advantage over the combination of two single agents. M7824 (MSB0011359C) is a bifunctional fusion protein composed of a monoclonal antibody against programmed death ligand 1 (PD-L1) fused to the extracellular domain of human transforming growth factor-β (TGF-β) receptor II, which functions as a "trap" for all three TGF-β isoforms. We demonstrate that M7824 efficiently, specifically, and simultaneously binds PD-L1 and TGF-β. In syngeneic mouse models, M7824 suppressed tumor growth and metastasis more effectively than treatment with either an anti-PD-L1 antibody or TGF-β trap alone; furthermore, M7824 extended survival and conferred long-term protective antitumor immunity. Mechanistically, the dual anti-immunosuppressive function of M7824 resulted in activation of both the innate and adaptive immune systems, which contributed to M7824's antitumor activity. Finally, M7824 was an effective combination partner for radiotherapy or chemotherapy in mouse models. Collectively, our preclinical data demonstrate that simultaneous blockade of the PD-L1 and TGF-β pathways by M7824 elicits potent and superior antitumor activity relative to monotherapies.

CD4+ regulatory T cells (Tregs) are critical for maintaining self-tolerance and function to prevent autoimmune disease. High densities of intratumoral Tregs are generally associated with poor patient prognosis, a correlation attributed to their broad immune-suppressive features. Two major populations of Tregs have been defined, thymically derived natural Tregs (nTregs) and peripherally induced Tregs (iTregs). However, the relative contribution of nTregs versus iTregs to the intratumoral Treg compartment remains controversial. Demarcating the proportion of nTregs versus iTregs has important implications in the design of therapeutic strategies to overcome their antagonistic effects on antitumor immune responses. We used epigenetic, phenotypic, and functional parameters to evaluate the composition of nTregs versus iTregs isolated from mouse tumor models and primary human tumors. Our findings failed to find evidence for extensive intratumoral iTreg induction. Rather, we identified a population of Foxp3-stable nTregs in tumors from mice and humans.

Background: Serum adiponectin, leptin, C-peptide, and homocysteine are indicators for obesity, hyperinsulinemia, and chronic inflammation, which have all been associated with colorectal cancer. Aims: To determine whether serum adiponectin, leptin, C-peptide, and homocysteine are associated with fat, fiber, fruit and vegetable, flavonol, or dry bean intake and colorectal adenoma recurrence. Methods: Using logistic regression, we estimated odds ratios (OR) and 95% confidence intervals (95% CI) for adenoma recurrence in 627 participants from the control arm of the Polyp Prevention Trial, a 4-year trial that examined the effectiveness of a low-fat, high-fiber, high-fruit and vegetable diet on adenoma recurrence. Results: Serum concentrations of C-peptide and homocysteine were inversely related to fiber, fruit and vegetable, and flavonol intake and positively related to percentage of calories from fat (all Ptrend ≤0.01). High homocysteine concentrations were associated with any (4th versus 1st quartile: OR, 2.26; 95% CI, 1.30-3.94) and more than one adenoma recurrence (OR, 2.11; 95% CI, 1.01-4.40). Individuals in the highest, versus lowest, tertile of serum leptin concentration had a decreased risk of advanced adenoma recurrence (OR, 0.22; 95% CI, 0.06-0.79). Conclusion: Our results suggest that serum homocysteine may serve as an indicator of dietary exposure, including a low-fat and high-fiber, high-fruit and vegetable, and high-flavonol diet, as well as colorectal adenoma recurrence. Impact: Discovering biomarkers that are both modifiable and can predict cancer risk is critical. We identified serum homocysteine as a novel indicator that is modified by diet and predicts risk of adenoma recurrence. Cancer Epidemiol Biomarkers Prev; 19(6); 1441–52. ©2010 AACR.

The prevalence of obesity, an established risk factor for several chronic diseases, including cancer, has risen dramatically over the past 4 decades. Dietary change and/or increased physical activity are the most commonly recommended lifestyle-based strategies for preventing or reversing obesity. One of several physiological systems that may be enhanced by dietary change and exercise is the immune system. In this study, we examined the effects of energy restriction (ER; 30% reduction relative to control energy intake) and/or exercise (EX; voluntary wheel running) on systemic and mucosal immune function. Female C57BL/6 mice were randomized into 4 treatment conditions: 1) controls consumed ad libitum (AL); 2) AL with access to running wheels (AL + EX); 3) 30% ER; and 4) 30% ER with access to running wheels (ER + EX). Both ER and EX reduced spleen weight and the number of splenic T and B lymphocytes (P < 0.05). ER enhanced natural killer (NK) cell function, but reduced concanavalin A (Con A)-induced T-cell proliferation (P < 0.05). In contrast, EX enhanced Con A-induced proliferation and cytokine production from Peyer's patch cells (P < 0.05). These data suggest that ER and EX enhance some, but not all, components of the immune system and are likely working via different biological mechanisms to regulate NK and T-cell function.

Sustained, local delivery of immunomodulatory cytokines is under investigation for its ability to enhance vaccine and anti-tumor responses both clinically and preclinically. This study evaluates the ability of chitosan, a biocompatible polysaccharide, to (1) control the dissemination of a cytokine, GM-CSF, and (2) enhance the immunoadjuvant properties of GM-CSF. While cytokines have previously been delivered in lipid-based adjuvants and other vehicles, these do not have the clinical safety profile or unique properties of chitosan. We found that chitosan solution maintained a measurable depot of recombinant GM-CSF (rGM-CSF) at a subcutaneous injection site for up to 9 days. In contrast, when delivered in a saline vehicle, rGM-CSF was undetectable in 12-24h. Furthermore, a single s.c. injection of 20 microg rGM-CSF in chitosan solution (chitosan/rGM-CSF(20 microg)) transiently expanded lymph nodes up to 4.6-fold and increased the number of MHC class II expressing cells and dendritic cells by 7.4-fold and 6.8-fold, respectively. These increases were significantly greater than those measured when rGM-CSF was administered in saline at the standard preclinical dose and schedule, i.e. 4 daily s.c. injections of 20 microg. Furthermore, lymph node cells from mice injected with chitosan/rGM-CSF(20 microg) induced greater allogeneic T cell proliferation, indicating enhanced antigen presenting capability, than lymph node cells from mice injected with rGM-CSF alone. Finally, in vaccination experiments, chitosan/rGM-CSF was superior to either chitosan or rGM-CSF alone in enhancing the induction of antigen-specific CD4(+) proliferation, peptide-specific CD8(+) pentamer staining and cytotoxic T cell lysis. Altogether, chitosan/rGM-CSF outperformed standard rGM-CSF administrations in dendritic cell recruitment, antigen presentation and vaccine enhancement. We conclude that chitosan solution is a promising delivery platform for the sustained, local delivery of rGM-CSF.

Intestinal calcium absorption is a critical step in the maintenance of calcium homeostasis [Fleet, 2006]. It occurs by both paracellular and transcellular pathways [Bronner, 2003; Hoenderop et al., 2005]. The transcellular calcium absorption pathway is an active process that is regulated by the active form of vitamin D, 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). 1,25 (OH)2D3 increases efficiency of transcellular Calcium absorption by increasing the abundance of critical proteins: calbindin D9k (CaBPD9k), the transient receptor vanelloid family member 6 (TRPV6), and the plasma membrane calcium ATPase 1b (PMCA1b) [Fleet et al., 2002; Song et al., 2003b; Fleet, 2006]. The actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR), a transcription factor that belongs to nuclear hormone receptor superfamily [Haussler et al., 1998]. The deletion of VDR in the enterocyte results in a 70% reduction in intestinal calcium absorption efficiency that is coincident with a significant reduction in CaBPD9k, TRPV6, and PMCA1b gene expression [Van Cromphaut et al., 2001; Song et al., 2003a].Previous research shows that binding of 1,25(OH)2D3 to VDR in the cytoplasm of cells stimulates heterodimerization of VDR with RXR and the redistribution of the VDR-RXR- hormone complex to the nucleus [Barsony et al., 1990; Michigami et al., 1999; Racz and Barsony, 1999; Sunn et al., 2001]. The nuclear import of the VDR-RXR-hormone complex is active [Racz and Barsony, 1999;Miyauchi et al., 2005], enhanced by 1,25(OH)2D3 treatment [Michigami et al., 1999; Racz and Barsony, 1999; Sunn et al., 2001], dependent upon the presence of intact nuclear localization sequences (NLSs) in both VDR and RXR [Prufer et al., 2000], and requires various importins [Miyauchi et al., 2005; Yasmin et al., 2005]. Most of the studies examining the mechanism of VDR nuclear import have been conducted in COS-7 kidney cells (characterized by low expression of endogenous VDR) and some characteristics of VDR import have been confirmed in cells from bone, skin, or kidney. In contrast the impact of 1,25(OH)2D3 treatment on VDR distribution has not been examined in absorptive enterocytes, a primary target cell of 1,25(OH)2D3 action. In this article we have examined the nucleocytoplasmic trafficking of VDR receptor in proliferating and differentiated Caco-2 cells. Caco-2 cells are an intestinal cell line that spontaneously differentiates and recapitulates many of the features of the absorptive epithelial cell of the small intestine, including vitamin D regulated intestinal calcium absorption [Giuliano and Wood, 1991; Fleet et al., 2002]. We find that VDR nuclear import occurs under basal conditions and that import is accelerated by 1,25(OH)2D3. This process is not influenced by the state of cellular differentiation. Our data also suggest that 1,25(OH)2D3-induced nuclear import is balanced by nuclear export in Caco-2 cells. In addition, our data are consistent with an essential role for nuclear import of VDR in the genomic responses of enterocytes to 1,25(OH)2D3.

The parental cell line (P) of Caco-2 cells and two clones, BBe and TC7, were studied at 11 days postconfluence to test the facilitated diffusion model of vitamin D-mediated intestinal calcium absorption (CaTx). Nuclear vitamin D receptor (nVDR) and calbindin D9k(CaBP) were measured by Western blot; 1,25-hydroxyvitamin D324-hydroxylase (CYP24), CaBP, plasma membrane Ca-ATPase (PMCA), and Ca transport channel (CaT1) mRNA levels were examined by RT-PCR; and net apical-to-basolateral CaTx was examined after treating cells with vehicle or 10 nM calcitriol for 8 (mRNA levels) or 48 h (protein, CaBP mRNA, CaTx). nVDR level was lowest in BBe (38% P value) and directly related to CYP24 induction (TC7 = P, which were 1.56 times greater than BBe). nVDR was inversely related to the vitamin D-induced levels of CaT1 mRNA, CaBP mRNA, PMCA mRNA, and net CaTx, with the highest induction seen in BBe. Basal CaBP mRNA (86 times greater than P) and protein levels were highest in TC7 cells and were not associated with higher net CaTx, suggesting CaBP may not be rate limiting for CaTx in these cells.

Cyclic dinucleotide (CDN) analogs and small molecule agonists of STING (STimulator of INterferon Genes protein) are currently being developed as immuno-oncology agents based on their ability to stimulate potent anti-tumoral responses in the tumor micro-environment (TME) (1,2). STING agonists act primarily on APCs such as dendritic cells (DCs), resulting in enhanced type I interferon signalling and resulting cytotoxic T cell (CTL) responses (3). Germline gain of function mutations in STING (gene name TMEM173) exist and cause the rare disease SAVI (4). Patients with SAVI present as infants with symptoms such as chronic rash, lesions, and systemic inflammation and eventually succumb to more serious complications such as lung disease. At the functional level, they display elevated levels of interferon responsive genes indicative of constitutive STING activation. Thus, while chronic agonism of this pathway leads to an autoimmune/autoinflammatory phenotype, acute dosing has benefit in a tumor setting as an immunological stimulant. We designed an in silico approach to identify other genes with the same potential disease/phenotype profile as STING. We combined knowledge of Mendelian disease genetics with pathway phenotype associations such as involvement in immune cell anergy, activation, costimulation, chemotaxis, and migration. In addition, we determined a putative set of genes associated with immune privileged sites by combining textmining of biomedical literature with mRNA gene expression analysis. Taken together, these approaches provided a list of potential immuno-oncology targets that also included several well-known targets such as CTLA4, ICOS, and OX40 which are either approved or in clinical trials as immuno-oncology agents. This list was then manually curated and prioritized based on literature, and will be functionally validated in ongoing experimental systems. References: 1) Corrales, L., et. al. “Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity” 2015, Cell Reports, v.11, 1018-1030. 2) Chandra, D., et. al. “STING Ligand c-di-GMP Improves Cancer Vaccination against Metastatic Breast Cancer” 2014, Cancer Immunol. Res., v.2(9), 901-910. 3) Woo, S.-R., et. al. “STING-Dependent Cytosolic DNA Sensing Mediates Innate Immune Recognition of Immunogenic Tumors” 2014, Immunity, v.41, 830-842. 4) Liu, Y., et. al. “Activated STING in a Vascular and Pulmonary Syndrome” 2014, N. Engl. J. Med., v.371(6), 507-518. Citation Format: Benjamin Schwartz, Kenneth Hance, Johannes Freudenberg, Christopher Shelton, Junping Jing, Jingsong Yang. Monogenic autoimmune diseases as a starting point for new immunooncology targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-114. doi:10.1158/1538-7445.AM2017-LB-114

Background: Macrophage inhibitory cytokine-1 (MIC-1/GDF15) mediates nonsteroidal anti-inflammatory drug (NSAID) protection from colonic polyps in mice and is linked to the development of colorectal carcinoma in humans. Therefore, changes in serum MIC-1/GDF15 levels could predict the presence of premalignant colonic polyposis and assist in population screening strategies. Methods: Serum MIC-1/GDF15 levels were measured in subjects in the Polyp Prevention Trial, in which NSAID use and colon cancer risk factors were defined. Subjects had an initial adenoma removed, a repeat colonoscopy removing previously unidentified polyps, and serum MIC-1/GDF15 estimation. Three years later recurrent adenomas were identified and serum MIC-1/GDF15 levels reestimated. The relationship between serum MIC-1/GDF15 levels and adenoma presence or recurrence was examined. Results: Serum MIC-1/GDF15 levels differed by adenoma status and were significantly related to colon cancer risk factors. In addition, mean serum MIC-1/GDF15 levels rose with increasing numbers of adenomas present and high-risk adenoma recurrence. NSAID users had higher serum MIC-1/GDF15 concentrations, which were related to protection from adenoma recurrence. Furthermore, adjusted serum MIC-1/GDF15 levels at final follow-up were related to adenoma recurrence (highest quartile MIC-1/GDF15; OR = 14.7, 95% CI: 3.0–73). Conclusions: These data suggest that MIC-1/GDF15 mediates at least some of the protection afforded by NSAIDs against human colonic polyposis. Furthermore, serum MIC-1/GDF15 levels vary with the development of adnenomatous colonic polyps. Impact: Serum MIC-1/GDF15 determination may hold promise as the first serum screening test to assist the detection of premalignant adenomatous colonic polyposis. Cancer Epidemiol Biomarkers Prev; 21(2); 337–46. ©2011 AACR.

We have previously shown that the suppressive function of regulatory T cells (Tregs) from peripheral blood mononuclear cells (PBMCs) is enhanced in patients with prostate cancer when compared with healthy individuals. Two phase II studies using the PSA-TRICOM vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC) showed evidence of patient benefit in terms of enhanced survival. The Halabi nomogram has been used to predict survival (HPS) of patients with mCRPC treated with conventional chemotherapy or second-line hormonal therapy. Tregs from PBMCs of patients (n = 23) with mCRPC were obtained pre- and post-three monthly vaccinations, and analyzed for number, phenotype, and suppressive function. Changes post- versus pre-vaccination in these parameters were compared with 3-year survival and HPS. No differences in Treg numbers were observed post- versus pre-vaccination. Trends (P = 0.029) were observed between overall survival (OS) and a decrease in Treg suppressive function post- versus pre-vaccination. Trends were also observed in analyzing effector:Treg (CD4(+)CD25(+)CD127(-)FoxP3(+)CTLA4(+)) ratio post- versus pre-vaccination with OS versus HPS. These data provide preliminary evidence for a possible association between improved OS and a decrease in Treg function when PBMCs are analyzed after three monthly vaccinations. Patients with an OS HPS were more likely to have decreased Treg function following vaccine. Larger studies to confirm and extend these findings are warranted.

Intravesical BCG has been used successfully to treat superficial bladder cancer for three decades. However, 20% to 30% of patients will fail initial BCG therapy and 30% to 50% of patients will develop recurrent tumors within 5 years. Alternative or complementary strategies for the management of superficial bladder cancer are needed. Interleukin-12 (IL-12) is a potent TH1 cytokine with robust antitumor activity and the ability to potentiate immunologic memory. Unfortunately, intravesical IL-12 did not show antitumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that coformulation of IL-12 with chitosan, a biocompatible, mucoadhesive polysaccharide, could improve intravesical IL-12 delivery and provide an effective and durable alternative for the treatment of superficial bladder cancer. In antitumor studies, 88% to 100% of mice bearing orthotopic bladder tumors were cured after four intravesical treatments with chitosan/IL-12. In contrast, only 38% to 60% of mice treated with IL-12 alone and 0% treated with BCG were cured. Antitumor responses following chitosan/IL-12 treatments were durable and provided complete protection from intravesical tumor rechallenge. Urinary cytokine analysis showed that chitosan/IL-12 induced multiple TH1 cytokines at levels significantly higher than either IL-12 alone or BCG. Immunohistochemistry revealed moderate to intense tumor infiltration by T cells and macrophages following chitosan/IL-12 treatments. Bladder submucosa from cured mice contained residual populations of immune cells that returned to baseline levels after several months. Intravesical chitosan/IL-12 is a well-tolerated, effective immunotherapy that deserves further consideration for testing in humans for the management of superficial bladder cancer. [Cancer Res 2009;69(15):6192–9]

We examined the role of the extracellular signal regulated kinases (ERK) in 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3))-induced gene expression in the differentiated Caco-2 cells. 1,25(OH)(2)D(3)-regulated expression of the 25-hydroxyvitamin D, 24-hydroxylase (CYP24) gene (both natural gene and promoter construct) was strongly modulated by altering ERK activity (i.e., reduced by MEK inhibitors and dominant negative (dn) ERK1 and ERK2, activated by epidermal growth factor) but ERK inhibition had no effect on 1,25(OH)(2)D(3)-regulated expression of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6). ERK5-mediated phosphorylation of the transcription factor Ets-1 enhanced 1,25(OH)(2)D(3)-mediated CYP24 gene transcription in proliferating but not differentiated Caco-2 cells due to reduced levels of ERK5 and Ets-1 (total and phosphoprotein levels) in differentiated cells. MEK inhibition reduced 1,25(OH)(2)D(3)-induced 3X-VDRE promoter activity but had no impact on the association of vitamin D receptor (VDR) with chromatin suggesting a role for co-activator recruitment in ERK-modulation of vitamin D-regulated CYP24 gene activation. Chromatin immunoprecipitation assays revealed that the ERK1/2 target, mediator 1 (MED1), is recruited to the CYP24, but not the TRPV6, promoter following 1,25(OH)(2)D(3) treatment. MED1 phosphorylation was sensitive to activators and inhibitors of the ERK1/2 signaling and MED1 siRNA reduced 1,25(OH)(2)D(3)-regulated human CYP24 promoter activity. This suggests ERK1/2 signaling enhances 1,25(OH)(2)D(3) effects on the CYP24 promoter by MED1-mediated events. Our data show that there are both promoter-specific and cell stage-specific roles for the ERK signaling pathway on 1,25(OH)(2)D(3)-mediated gene induction in enterocyte-like Caco-2 cells.

Purpose: IFN-α is a pleiotropic cytokine possessing immunomodulatory properties that may improve the efficacy of therapeutic cancer vaccines. The aim of this study was to evaluate the effectiveness and compatibility of combining recombinant IFN-α with poxvirus vaccines targeting the human carcinoembryonic antigen (CEA) in murine models of colorectal and pancreatic adenocarcinomas, where CEA is a self-antigen.Experimental Design: The phenotypic and functional effects of IFN-α were evaluated in the draining inguinal lymph nodes of tumor-free mice. We studied the effect of the site of IFN-α administration (local versus distal) on antigen-specific immune responses to poxvirus vaccination. Mechanistic studies were conducted to assess the efficacy of IFN-α and CEA-directed poxvirus vaccines in tumor-bearing CEA transgenic mice.Results: We identified a dose and schedule of IFN-α that induced a locoregional expansion of the draining inguinal lymph nodes and improved cellular cytotoxicity (natural killer and CD8+) and antigen presentation. Suppression of the vaccinia virus was avoided by administering IFN-α distal to the site of vaccination. The combination of IFN-α and vaccine inhibited tumor growth, improved survival, and elicited CEA-specific CTL responses in mice with CEA+ adenocarcinomas. In mice with pancreatic tumors, IFN-α slowed tumor growth, induced CTL activity, and increased CD8+ tumor-infiltrating lymphocytes.Conclusions: These data suggest that IFN-α can be used as a biological response modifier with antigen-directed poxvirus vaccines to yield significant therapeutic antitumor immune responses. This study provides the rationale and mechanistic insights to support a clinical trial of this immunotherapeutic strategy in patients with CEA-expressing carcinomas.

Purpose: Saccharomyces cerevisiae, a nonpathogenic yeast, has been used previously as a vehicle to elicit immune responses to foreign antigens, and tumor-associated antigens, and has been shown to reduce tumor burden in mice. Studies were designed to determine if vaccination of human carcinoembryonic antigen (CEA)-transgenic (CEA-Tg) mice (where CEA is a self-antigen) with a recombinant S. cerevisiae construct expressing human CEA (yeast-CEA) elicits CEA-specific T-cell responses and antitumor activity. Experimental Design: CEA-Tg mice were vaccinated with yeast-CEA, and CD4+ and CD8+ T-cell responses were assessed after one and multiple administrations or vaccinations at multiple sites per administration. Antitumor activity was determined by tumor growth and overall survival in both pulmonary metastasis and s.c. pancreatic tumor models. Results: These studies demonstrate that recombinant yeast can break tolerance and that (a) yeast-CEA constructs elicit both CEA-specific CD4+ and CD8+ T-cell responses; (b) repeated yeast-CEA administration causes increased antigen-specific T-cell responses after each vaccination; (c) vaccination with yeast-CEA at multiple sites induces a greater T-cell response than the same dose given at a single site; and (d) tumor-bearing mice vaccinated with yeast-CEA show a reduction in tumor burden and increased overall survival compared to mock-treated or control yeast-vaccinated mice in both pulmonary metastasis and s.c. pancreatic tumor models. Conclusions: Vaccination with a heat-killed recombinant yeast expressing the tumor-associated antigen CEA induces CEA-specific immune responses, reduces tumor burden, and extends overall survival in CEA-Tg mice. These studies thus form the rationale for the incorporation of recombinant yeast-CEA and other recombinant yeast constructs in cancer immunotherapy protocols.

Regular moderate exercise has been proposed to enhance immune function, but its effects on immunity and their consequences have not been well studied. Mice without (AL) or with access (AL + EX) to voluntary running wheels were vaccinated with a model antigen (ovalbumin (OVA)) via intranasal or subcutaneous routes to target the mucosal and systemic immune compartments, respectively. EX enhanced OVA-specific CD4 + T cell cytokine production and proliferation in all lymphoid organs examined without changes in cell distribution in any organ. These results suggest that coupling moderate exercise with vaccination may enhance vaccine efficacy for the prevention and/or therapy of numerous diseases.

Inflammatory breast carcinoma (IBC) appears to be a relatively rare form of breast cancer with distinct clinicopathologic features (1-4). It is characterized by an early age at diagnosis, poor nuclear grade, negative hormone receptor status, and poor survival outcome. Although IBC is readily distinguishable from most forms of breast cancer, it can be confused with noninflammatory locally advanced breast cancer (LABC). Consistent with Haagensen’s original description of IBC (3), the American Joint Committee on Cancer (AJCC) provides the current gold standard definition for this form of breast cancer, describing it as a clinicopathologic entity that is characterized by diffuse erythema and edema (peau d’orange), often without an underlying palpable mass (5). The clinical presentation of IBC is due to tumor emboli within dermal lymphatic vessels, which may or may not be present on skin biopsy. Therefore, the AJCC relies on the clinical features of IBC and considers the pathologic features to be supportive of, but not necessary for, diagnosis. Some investigators (6-8) have classified IBC into three separate groups: 1) IBC cases with clinical features only, 2) IBC cases with clinical and pathologic features, and 3) IBC cases with pathologic features only. However, none of these definitions appears to capture all of the unique clinical and/or pathologic characteristics of the disease. Possibly because of varying case definitions, population-based estimates for IBC incidence range widely, from

Background. L-BLP25 is an investigational antigen-specific cancer immunotherapeutic agent targeting human mucin 1 (MUC1). A single low dose of cyclophosphamide (CPA) is known to be immunostimulatory and may enhance the anti-tumor effects of L-BLP25 when given 3 days before treatment initiation. Here we report the immune and anti-tumor efficacy of L-BLP25 with CPA pretreatment in subcutaneous (s.c.) and orthotopic models of murine pancreatic cancer. These models allow for reproduction of the clinical scenario, where 8 weekly doses of L-BLP25 are administered. Methods. Murine pancreatic tumor cells Panc02 transfected with human MUC1 were injected for comparison s.c. or orthotopically into human MUC1 transgenic mice. Mice received either vehicle control (PBS), CPA (100 mg/kg), L-BLP25 (100 μg), or L-BLP25 (100 μg) + CPA (100 mg/kg). CPA was administered intravenously (i.v.) 3 days prior to initiation of 8 weekly intradermal (i.d.) or s.c. L-BLP25 injections. Tumor volume was monitored and splenic cell populations were quantified by FACS. In the s.c. tumor model, the contribution of CD4+ and CD8+ T cell subsets to the inhibition of tumor growth was studied. In the orthotopic model, antigen-specific IFN-γ secretion was assessed via ex vivo T cell re-stimulation with antigen alone. Results. In the s.c. model of pancreatic cancer, tumor volume was reduced compared to vehicle at day 49 after i.d. administration of L-BLP25 (41.4%, p Citation Format: Kenneth Hance, Bo Marelli, Jin Qi, Guozhong Qin, Huakui Yu, Hong Wang, Xiaomei Xu, Robert Tighe, Beatrice Brunkhorst, Yan Lan, Robert Hofmeister, Helen Sabzevari, Michael Wolf. Mechanism of action of L-BLP25 in preclinical pancreatic tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1237. doi:10.1158/1538-7445.AM2013-1237

B144 Obesity increases the risk of several solid tumors, including breast, prostate, pancreatic, and colon. One of several mechanisms that may contribute to the relationship between obesity and tumor risk is an obesity-induced impairment in immunity. We have previously shown that numerous innate and adaptive immune responses, including NK cell cytotoxicity, antigen-specific CD4+ T cell proliferation, and CD8+ T cell cytotoxicity, are impaired in diet-induced obese (DIO) mice. The current study was designed to determine if the obesity-induced decrements in immunity were reversible with weight loss achieved via the most common obesity interventions used in humans: exercise (EX), energy restriction (ER), or a combination of EX+ER. Female C57BL/6 mice consumed a 60 kcal % fat diet, with 20 kcal % protein and carbohydrate, ad libitum for 15 weeks to induce obesity. After achieving a mean (± SD) body weight of 48.3 ± 1.9 grams, DIO mice were randomized to one of the following intervention groups (n=7-9/group): 1) DIO controls, which continued on 60 kcal % fat diet ad libitum; 2) DIO mice with access to running wheels (DIO+EX); 3) DIO mice placed on an energy-restricted diet consisting of a 30% reduction in kcal from fat (42 kcal % fat diet, with 29 kcal% protein & carbohydrate) (DIO+ER); or 4) DIO mice placed on an ER diet with access to running wheels (DIO+ER+EX). Mice on the aforementioned interventions consumed 20.9 ± 0.6 kcal/d, 20.6 ± 0.3 kcal/d, 15.6 ± 0.4 kcal/d, and 15.4 ±0.3 kcal/d, respectively. Mice were on one of the four interventions for 8 weeks prior to receiving a primary vaccination with a subcutaneous pox virus-based vaccine, followed by 2 booster vaccinations 2 and 4 weeks following the prime. Animals were sacrificed 3 weeks following the last boost, and spleens were collected for assessment of immune function. Following 15 weeks on the interventions, DIO, DIO+EX, DIO+ER, and DIO+ER+EX mice weighed 55.3 ± 1.3 g, 50.2 ± 1.3 g, 39.3 ± 1.7 g, 28.5 ± 0.8 g, respectively. Only mice on the ER diets (DIO+ER and DIO+ER+EX mice) weighed significantly less than the DIO control mice (P Citation Information: Cancer Prev Res 2008;1(7 Suppl):B144.

Purpose Expression of the immune checkpoint protein PD-L1 constitutes a major mechanism of tumor immune evasion. Multiple clinical trials in solid tumors have demonstrated that inhibition of tumor PD-L1 or immune effector PD-1 via monoclonal antibodies (mAbs) can produce dramatic clinical responses in many cancer patients. The main function of these mAbs is to inhibit signaling induced by ligation of PD-L1 on tumor cells with PD-1 on tumor infiltrating immune effectors. Antibody-dependent cellular cytotoxicity (ADCC) represents an additional mechanism of action for mAbs of the IgG1 isotype. In the current study, we describe investigations of a novel anti-PD-L1 mAb of the IgG1 isotype (MSB0010718). This mAb is currently in Phase I clinical trials for patients with metastatic or locally advanced solid tumors at the NCI, and is the first such mAb with the capacity to induce ADCC of PD-L1 positive tumor cells. We sought to investigate MSB0010718’s ability to induce ADCC and to determine factors affecting tumor cell sensitivity to this mechanism. Results

Antibodies that block PD-1/PD-L1 interactions have shown efficacy against both lung and skin cancers in early-stage clinical trials, and may also be effective in other tumor types, particularly bladder tumors. PD-L1 expression has been previously reported to correlate with high-grade tumors, a high recurrence rate, and reduced survival rate in patients with bladder cancer. These findings and the high frequency of somatic mutations found in bladder tumors indicate that bladder cancer patients may respond well to anti-PD-L1 therapy. Both murine (MB49, MBT-2) and human (J82, T24, TCCSUP) bladder cancer cell lines constitutively express PD-L1 as determined by flow cytometry. As expected, in vitro IFN-γ addition up-regulated PD-L1 expression levels on each of those tumor cells. A human IgG1 anti-PD-L1 antibody, MSB0010718C, induced ADCC activity in vitro against all three human bladder cancer cell lines following treatment with IFN-γ. In initial in vivo murine studies, the growth of s.c. MB49 tumors in syngeneic mice was significantly delayed following three i.p. injections of 400 µg of the MSB0010718C anti-PD-L1 antibody. An orthotopic bladder model consisting of the MB49 cells tagged with luciferase (MB49-luc) was also used to evaluate the antitumor efficacy of the anti-PD-L1 antibody. MB49-luc cells were instilled intravesically (bladder) in B6 mice. Beginning at 7 or 10 days post-instillation, three i.p. injections of the anti-PD-L1 antibody substantially reduced tumor volumes, as determined by intravital imaging, leading to long-term tumor-free survival for 40-60% of the treated mice. While initial immune cell subset depletion studies implicated both CD4+ and CD8+ T cells, continuing efforts will further define the cellular mechanisms responsible for the antitumor effects of the anti-PD-L1 antibody. Taken together, these results suggest that MSB0010718C therapy might be used to activate both innate and adaptive immune mechanisms to treat PD-L1-expressing bladder tumors. Furthermore, the MB49 tumor model can be used to evaluate the combined effects of anti-PD-L1 and other therapeutic agents, particularly ones that induce IFN-γ production and tumor PD-L1 up-regulation. MSB0010718C is currently being evaluated in a Phase I clinical trial (NCT01772004).