Helen Sabzevari

Cutting Edge: Epigenetic Regulation of Foxp3 Defines a Stable Population of CD4+ Regulatory T Cells in Tumors from Mice and Humans

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.

Systemic Immunotherapy of Non-Muscle Invasive Mouse Bladder Cancer with Avelumab, an Anti-PD-L1 Immune Checkpoint Inhibitor

In an orthotopic model of non-muscle invasive bladder cancer, in which BCG had minimal activity, systemic administration of the anti–PD-L1 checkpoint inhibitor avelumab demonstrated durable antitumor responses and long-term survival mediated by CD4 and CD8 T cells. Bacillus Calmette–Guerin (BCG) is the standard of care for intravesical therapy for carcinoma in situ and non-muscle invasive, nonmetastatic human urothelial carcinoma. Although the responsiveness to this immunotherapeutic is believed to be linked with (i) a high number of somatic mutations and (ii) a large number of tumor-infiltrating lymphocytes, recent findings of the roles that inhibitory immune receptors and their ligands play in tumor evasion may provide insights into the limitations of the effectiveness of BCG and offer new targets for immune-based therapy. In this study, an aggressive, bioluminescent orthotopic bladder cancer model, MB49 tumor cells transfected with luciferase (MB49luc), was used to study the antitumor effects of avelumab, an antibody to PD-L1. MB49luc murine tumor cells form multifocal tumors on the mucosal wall of the bladder reminiscent of non-muscle invasive, nonmetastatic urothelial carcinomas. MB49luc bladder tumors are highly positive for the expression of PD-L1, and avelumab administration induced significant (P < 0.05) antitumor effects. These antitumor effects were more dependent on the presence of CD4 than CD8 T cells, as determined by in vivo immune cell depletions. The findings suggest that in this bladder tumor model, interruption of the immune-suppressive PD-1/PD-L1 complex releases a local adaptive immune response that, in turn, reduces tumor growth. This bladder tumor model can be used to further identify host antitumor immune mechanisms and evaluate combinations of immune-based therapies for carcinoma in situ and non-muscle invasive, nonmetastatic urothelial carcinoma, to provide the rationale for subsequent clinical studies. Cancer Immunol Res; 4(5); 452–62. ©2016 AACR.

Enhanced preclinical antitumor activity of M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β

Blockade of PD-L1 and TGF-β pathways with M7824 activates effective innate and adaptive antitumor immune responses. Doubling up against cancer Inhibitors of immune checkpoints are increasingly used for cancer immunotherapy because they decrease cancer-associated immunosuppression and improve patients’ own immune responses against the disease. However, this type of treatment is not always sufficient to ensure protective immunity. To reinforce the effects of immune checkpoint inhibition, Lan et al. designed a bifunctional protein called M7824, which simultaneously targets an immune checkpoint and a different immunosuppressive pathway. This bifunctional protein was more effective at restoring antitumor immunity than interventions targeting one pathway at a time, and it showed promising results in multiple mouse models, alone and in combination with other treatment modalities. 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.

Anti-tumor effects of anti-PD-L1 therapy in an orthotopic bladder tumor model

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).

Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion

A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi–a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1β), M2-associated (CCL13, CD206), Mreg-associated (IL-10, TGF-β), and angiogenesis associated (MMP9, VEGF) genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1β, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

Systemic immunotherapy of superficial mouse bladder cancer with Avelumab (MSB0010718C), an anti-PD-L1 immune checkpoint inhibitor

Bladder cancer is considered a malignancy that is responsive to immunotherapy because of the presence of a (a) high number of somatic mutations, (b) large number of tumor-infiltrating lymphocytes and (c) clinical response to the immune stimulant, Bacillus Calmette-Guerin (BCG). Recent findings of the roles that inhibitory immune receptors and their ligands play in tumor evasion provide some possible explanation as to the limitations of BCG and may offer new therapeutic approaches to some patients with bladder cancer. In this study, an aggressive, bioluminescent orthotopic bladder cancer model, MB49 tumor cells transfected with luciferase (MB49luc), was used to study the antitumor effects of an anti-PD-L1 antibody. MB49luc murine tumor cells form multifocal, superficial tumors on the mucosal wall of the bladder reminiscent of superficial human transitional cell bladder carcinomas. MB49luc bladder tumors were shown to constitutively express high PD-L1 levels and the administration of Avelumab, an anti-PD-L1 antibody, induced significant tumor reduction. Antitumor effects subsequently improved overall survival, and both were abrogated by selective in vivo depletion of CD4 or CD8 T cells, but not with NK cell depletion. These findings suggest that in this murine bladder tumor model, interruption of the immune suppressive PD-1/PD-L1 complex releases a local adaptive immune response that, in turn, significantly reduces tumor growth. This experimental bladder tumor model can be used to (a) identify host immune mechanisms responsible for tumor resistance or destruction and (b) evaluate combinations of immune-based therapy for bladder cancer which could provide rationale for future clinical studies.

Investigation of antibody dependent cellular cytotoxicity as a mechanism of action for a novel anti-PD-L1 monoclonal antibody

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

Abstract 2573: The immunocytokine NHS-IL12 as a potential cancer therapeutic

Targeted delivery of IL-12 might turn this cytokine into a safer, more effective cancer therapeutic. Here we describe a novel immunocytokine, NHS-IL12, consisting of two molecules of IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). The addition of the human IgG1 moiety resulted in a longer plasma half-life of NHS-IL12 than recombinant IL-12, and a selective targeting to murine tumors in vivo. Data from both in vitro assays using human PBMCs and in vivo primate studies showed that IFN-gamma production by immune cells is attenuated following treatment with the immunocytokine, suggesting an improved toxicity profile than seen with recombinant IL-12 alone. NHS-IL12 was superior to recombinant IL-12 when evaluated as an anti-tumor agent in three murine tumor models. Mechanistic studies utilizing immune cell subset-depleting antibodies, flow cytometric methods, and in vitro cytotoxicity and ELISA assays all indicated that the anti-tumor effects of NHS-IL12 were primarily CD8+ T cell-dependent and likely IL-12-mediated. Combining NHS-IL12 treatment with a cancer vaccine, radiation, or chemotherapy resulted in greater anti-tumor effects than each individual therapy alone. These preclinical findings provide a rationale for the clinical testing of this immunocytokine, both as a single agent and in combination with vaccines, radiation and chemotherapy.

Cyclophosphamide mechanism of action in preclinical tecemotide studies

Background Tecemotide is a MUC1 antigen-specific therapeutic cancer vaccine. In phase III clinical studies, delivery of tecemotide is preceded by a single low dose of cyclophosphamide (CPA) to inhibit regulatory T cells (Tregs) and enhance the response to the tumor-associated antigen. Here, we investigated effects of CPA on the immune environment and tumor growth in preclinical murine models. Methods The effect of CPA was investigated in tumor-free human MUC1 transgenic mice and anti-tumor responses were evaluated in the mice engrafted with syngeneic colorectal and ovarian cancer cells expressing human MUC1. The mice received either vehicle control, CPA (100 mg/ kg), tecemotide (100 μg) or CPA + tecemotide. The immune cell phenotype and function in the spleen were assessed on Days 1, 3 and 7 post-administration.

Abstract 1237: Mechanism of action of L-BLP25 in preclinical pancreatic tumor models.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnBackground. 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<0.02) and L-BLP25 + CPA (55.5%, p<0.02). In multiple studies, tumor volume was consistently lower in mice treated with L-BLP25 + CPA vs. L-BLP25 alone. L-BLP25 + CPA s.c. treatment was effective at day 49 with 44% greater inhibition of tumor growth and more pronounced extended survival compared to i.d. treatment, supporting the route of administration in the clinic. Various splenic immune cell populations were increased by s.c. L-BLP25 + CPA treatment compared to vehicle and L-BLP25 alone, including the CD8+ effector memory/Treg ratio. Mice treated with s.c. L-BLP25 + CPA after CD8+ T cell depletion had a greater tumor volume compared to T cell-sufficient mice, while CD4+ T cell depletion had no effect. In the orthotopic model of pancreatic cancer, median survival was improved with s.c. L-BLP25 + CPA treatment vs. vehicle (p<0.05) and vs. L-BLP25 alone (non-significant). When stimulated ex vivo with the BP25 peptide, the frequency of IFN-γ producing CD4+ T cells isolated from the spleens of mice treated with L-BLP25 + CPA was significantly higher than in all other treatment groups (p<0.001). CD8+ T cells were found to produce no IFN-γ after in vitro re-stimulation. Conclusions. When administered after a single, low-dose pre-treatment of CPA, L-BLP25 immunotherapy exhibits anti-tumor activity, immune activation and prolonged survival in murine pancreatic cancer models. T cell depletion data indicate a CD8+ T cell-mediated anti-tumor response, whilst IFN-γ production by CD4+ helper T cells may contribute to prolonged survival.nnCitation 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