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Multiple antibodies blocking PD-1 or PD-L1 have been approved for clinical use. However, not all patients successfully respond to monotherapy and the need for combinations has become more apparent. CTX-8371 combines PD-1/PD-L1 targeting in one bispecific, tetravalent molecule. CTX-8371 potently blocks PD-1/PD-L1 in vitro and in vivo. This robust activity may be explained by the unique mechanism of action (MOA) of CTX-8371 which involves cell surface PD-1 cleavage. Here we present further in vivo evidence of PD-1 cleavage by CTX-8371 detected during a dose range finding (DRF) study in cynomolgus macaques. Results of this study as well as in vitro receptor occupancy (RO) and the PK/PD profile of CTX-8371 in tumor-bearing mice are described. CTX-8371 was administered to female cynomolgus monkeys (n=3/group) in a single intravenous infusion at 2 mg/kg or weekly infusions at 10 or 50 mg/kg, for a total of 2 doses. PBMCs were isolated before, 24 hours after the first dose, or 24 hours after the second dose (for 10 and 50 mg/kg groups). Immunophenotyping, including PD-1 and PD-L1, was performed on PBMCs by flow cytometry. Mean serum concentration-time data was analyzed to derive PK parameters using noncompartmental and two-compartment models. PK was also determined in tumor-bearing mice using an MSD ELISA-based assay and non-compartmental analysis. In monkeys, CTX-8371 administration resulted in a marked decrease of PD-1+CD4+ and PD-1+CD8+ T cells in PBMCs on days 2 and 9 at all dose levels, indicating PD-1 cleavage from the cell surface. These results confirm in vitro, and in vivo MOA data obtained from mouse studies. Clearance and half-life of CTX-8371 were calculated to be ~6.87 mL/day/kg and 5.45 days, respectively, which fall within the expected ranges for a human IgG1 antibody in non-human primates (NHP) with linear PK. CTX-8371 infusion was well tolerated in cynomolgus monkeys. In tumor-bearing mice, the dose which results in 50% of maximum tumor growth inhibition was found to be 1-2 mg/kg. The minimum effective dose, at which at least 50% of animals exhibit greater than 50% tumor growth inhibition over two consecutive days, was 1mg/kg. It was observed that 11% (1/9), 78% (7/9) and 100% (9/9) of mice in 0.1 mg/kg, 1 mg/kg and 10 mg/kg dose groups, respectively, meet the above criteria. CTX-8371 also showed dose-dependent in vitro RO on T cells from peripheral blood of human, cynomolgus monkey, and transgenic hPD-1hPD-L1 mice. Data presented here confirm the unique MOA of CTX-8371, cleavage of PD-1 from the cell surface. The dual blockade of PD-1 and PD-L1, combined with PD-1 cleavage, may offer superior benefit compared to current checkpoint inhibitor therapies. RO data as well as murine and NHP PK/PD analyses will guide a projected human dose for future development. Citation Format: Diana I. Albu, Xianzhe Wang, Yan Qin, Vivian Li, Purushothama Nanjappa, Amy Daniel Ulumben, Ruturaj Jadhav, Jason Kong, Austin Ablicki, Neal Schilling, Thomas Schuetz, Susan Kalled, Bing Gong, Nelly Kuklin. Dose range finding study in non-human primates confirms the unique mechanism of action of CTX-8371, a novel bispecific antibody blocking PD-1 and PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3431.

e15056 Background: Checkpoint blockade therapies targeting PD-1 and PD-L1 have shown great success for the treatment of various malignancies. However, a substantial fraction of patients with PD-L1-positive tumors remain unresponsive to these therapies. Novel therapy with significantly greater activity than the leading PD-1/PD-L1 inhibitors is expected to bring additional clinical benefit to patients. Here we describe the preclinical evaluation of CTX-8371, which combines anti-PD-1 and anti-PD-L1 monoclonal antibodies in one bispecific tetravalent molecule. Methods: The immune-enhancing activity of CTX-8371 was tested in vitro in T cell activation assays and tumor cell killing assay. CTX-8371 anti-tumor efficacy in vivo was assessed using mouse tumor cells expressing human PD-L1 implanted in transgenic mice humanized at the PD-1 and PD-L1 loci. CTX-8371 anti-tumor activity was also tested in xenograft tumor models. The mechanism of action of CTX-8371 was investigated in vitro using Jurkat cells expressing PD-1 or PD-L1, human PBMCs, and in vivo in tumor-bearing, chimeric PD-1/PD-L1 transgenic mice. CTX-8371 PK was determined in mice using an MSD ELISA-based assay and in cynomolgus monkeys using a qualified ELISA method. Dose range finding and toxicokinetic studies were performed in cynomolgus monkeys. Results: CTX-8371 potently enhanced T cell activation and function in vitro and showed curative efficacy as monotherapy in multiple solid tumor models, isografts or xenografts. Furthermore, CTX-8371 demonstrated superior anti-tumor efficacy compared to Keytruda or atezolizumab in checkpoint inhibitors-sensitive and resistant syngeneic mouse tumor models. Mechanistically, in addition to blocking PD-1 interaction with PD-L1, CTX-8371 bispecific antibody facilitated cell to cell bridging between cells expressing PD-1 and cells expressing PD-L1. Furthermore, we show that simultaneous binding of CTX-8371 to both PD-1 and PD-L1 resulted in long term PD-1 shedding. This suggests that CTX-8371 may prevent or overcome T cell exhaustion within the tumor microenvironment, thus providing additional advantage over existing therapies. Lastly, excellent tolerability was observed in non-human primates given 2 weekly drug infusions at up to 50 mg/kg dose. Conclusions: CTX-8371 displays multiple mechanisms of action over monoclonal PD1/PD-L1 blockade. These unique pharmacological properties of CTX-8371 could explain the enhanced T cell responses to tumor antigens and superior efficacy over current monoclonal antibody therapies. With favorable PK/PD and toxicology profiles in mice and cynomolgus monkeys, CTX-8371 warrants further advancement to clinical testing.

Eribulin, a synthetic analog of the marine sponge natural product halichondrin B, is approved in the US for certain patients with advanced breast cancer or liposarcoma. Its mechanisms of action include both cytotoxic, antimitotic effects as well as non-cytotoxic effects on tumor vasculature, phenotype and microenvironment. Known aspects of eribulin's effects on tumor phenotype, including reversal of epithelial-mesenchymal transition (EMT) and induction of cellular differentiation in various preclinical models, led us to speculate that eribulin might also alter immune homeostasis in the in vivo setting, both in tumors themselves as well as supporting immune organs such as the spleen. Here, we report on eribulin's effects on both lymphoid and myeloid lineage cellular components in both spleen and tumors from immunocompetent mice bearing subcutaneous Lewis lung carcinoma (LLC) isografts. Surprisingly, spleens of vehicle treated, LLC tumor-bearing mice are approximately twice as large spleens from non-tumor-bearing mice, with an excellent correlation (r2 = 0.92) between individual tumor size and spleen weight arguing for a direct cause and effect relationship between the presence of tumor and splenomegaly. Tumor-induced splenomegaly is associated with increased proliferation of splenic non-lymphoid (CD4-/CD8-/CD19-) cells, including both CD11b+ mature myeloid lineage cells as well as CD11b- cells. Due to high levels of P-glycoprotein (PgP) characteristic of most murine tumors, LLC tumors respond poorly to eribulin, a known PgP substrate. Thus, 3 doses of MTD eribulin (1.6 mg/kg; Q4Dx3) led to only a 30% reduction in tumor volume relative to vehicle-treated mice. Unexpectedly, however, despite this reduced tumor volume, spleens from eribulin-treated mice were twice again larger than the already-enlarged spleens from vehicle-treated tumor-bearing mice, and the clear relationship between tumor size and spleen weight was abolished. This additional eribulin-induced splenomegaly was associated with enhanced proliferation of the same CD4-/CD8-/CD19- non-lymphoid cells, CD11b+ myeloid lineage cells and CD11b- cells as in vehicle-treated, tumor-bearing mice. Evaluation of immune cell components in the associated fixed tumors from eribulin- and vehicle-treated mice is currently ongoing to assess what effect, if any, such changes in the spleen will have on the tumor immune microenvironment; these results will be presented at the meeting. In conclusion, our results point to significant and unexpected effects of eribulin treatment on the immune cell landscape in spleens and tumors from tumor-bearing immunocompetent mice. Citation Format: Haochen Wu, Keyi Zhu, Xiaolong Tu, Diana Albu, Mary Woodall-Jappe, Bruce A. Littlefield. Evaluation of eribulin-induced alterations of the intact immune cell landscape in spleens and tumors from tumor-bearing immunocompetent mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5522.

Reprogramming of immunosuppressive tumor microenvironment (TME) by targeting alternatively activated tumor associated macrophages (M2TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Tregs), represents a promising strategy for developing novel cancer immunotherapy. Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite and mediator of chronic inflammation, has emerged as a powerful immunosuppressor in the TME through engagement with one or more of its 4 receptors (EP1-EP4). We have developed E7046, an orally bioavailable EP4-specific antagonist and show here that E7046 has specific and potent inhibitory activity on PGE2-mediated pro-tumor myeloid cell differentiation and activation. E7046 treatment reduced the growth or even rejected established tumors in vivo in a manner dependent on both myeloid and CD8+ T cells. Furthermore, co-administration of E7046 and E7777, an IL-2-diphtheria toxin fusion protein that preferentially kills Tregs, synergistically disrupted the myeloid and Treg immunosuppressive networks, resulting in effective and durable anti-tumor immune responses in mouse tumor models. In the TME, E7046 and E7777 markedly increased ratios of CD8+granzymeB+ cytotoxic T cells (CTLs)/live Tregs and of M1-like/M2TAM, and converted a chronic inflammation phenotype into acute inflammation, shown by substantial induction of STAT1/IRF-1 and IFNγ-controlled genes. Notably, E7046 also showed synergistic anti-tumor activity when combined with anti-CTLA-4 antibodies, which have been reported to diminish intratumoral Tregs. Our studies thus reveal a specific myeloid cell differentiation-modifying activity by EP4 blockade and a novel combination of E7046 and E7777 as a means to synergistically mitigate both myeloid and Treg-derived immunosuppression for cancer treatment in preclinical models.

Introduction: We report discovery and characterization of E7766, a structurally novel STING agonist, as a potential immunotherapy for solid cancers through intratumoral (IT) administration and for Bacillus Calmette-Guerin (BCG) unresponsive non-muscle invasive bladder cancer (NMIBC) through intravesical (VE) administration. Methods: E7766 was designed and synthesized to optimize the potency of binding to dimerized STING proteins of different genetic isoforms. The compound was extensively and comparatively characterized in a variety of biochemical, molecular and cellular, in vivo, ex vivo, and primary human tumor and cellular studies for potency, mechanisms and translational biomarkers. Novel preclinical models to mimic orthotopic NMIBC and deep lesion metastasis were developed, and co-crystalization with recombinant proteins of genetic variations was performed. Results: E7766, a novel Macrocycle-Bridged STING Agonist, showed highly specific and potent agonist activity in both human and mouse STING. In human PBMCs, E7766 demonstrated potent and consistent activity across seven tested human STING genotypes (IC50, 0.15-0.79 μM). By contrast, a reference cyclic dinucleotide STING agonist showed weaker potency and substantial variability across genotypes (IC50, 1.88 μM - >50 μM). Co-crystal structures indicated a structural basis for the superior interactions of E7766 with STING proteins compared with conventional cyclic dinucleotide STING agonists. Intravesical administration of E7766 to a preclinical orthotopic mouse bladder cancer model mimicking the BCG-unresponsive NMIBC demonstrated a dose-dependent and curative activity without serious adverse effects. The anti-tumoral activity was associated with a robust induction of IFNβ, CXCL10 and other downstream effectors of STING pathway inside the bladder cavity. In addition, single IT administration of E7766 to a subcutaneous (SC) tumor in mice bearing dual CT26 tumors in liver and SC lesion cured 90% of animals without recurrence for over 8 months. Those tumor-free animals rejected re-challenge of the same tumor cells in the absence of CD8+ T cells or NK cells, indicating the presence of a highly effective immune memory response following treatment with E7766 independent of either cell population alone. Conclusions: E7766 is a structurally novel and highly potent STING agonist with pan-genotypic activity, demonstrating curative anti-tumoral activity in murine models of BCG-unresponsive NMIBC and of metastatic tumors in deep lesions. Clinical investigation of E7766 is under discussion. Citation Format: Kuan-Chun Huang, Atsushi Endo, Shannon McGrath, Dinesh Chandra, Jiayi Wu, Dae-Shik Kim, Diana Albu, Christy Ingersoll, Karen Tendyke, Kara Loiacono, Thomas Noland, David Verbel, Chi Zhang, Ming-Hong Hao, Mark Matijevic, Vaishali Dixit, Renee R. Hukkanen, Janna Hutz, John Wang, Frank Fang, Xingfeng Bao, Donna Kolber-Simonds, Muzaffar Akram, Nadeem Sarwar. Discovery and characterization of E7766, a novel macrocycle-bridged STING agonist with pan-genotypic and potent antitumor activity through intravesical and intratumoral administration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3269.

Meeting abstracts Immunotherapies targeting the immune checkpoint receptors have shown great promise for a subset of cancer patients. However, robust and safe combination therapies are still needed to increase the benefit of cancer immunotherapy and bring it to broader patient populations. We have

CD8(+) T lymphocytes mediate the immune response to viruses, intracellular bacteria, protozoan parasites, and tumors. We provide evidence that the transcription factor Bcl11b/Ctip2 controls hallmark features of CD8(+) T cell immunity, specifically antigen (Ag)-dependent clonal expansion and cytolytic activity. The reduced clonal expansion in the absence of Bcl11b was caused by altered proliferation during the expansion phase, with survival remaining unaffected. Two genes with critical roles in TCR signaling were deregulated in Bcl11b-deficient CD8(+) T cells, CD8 coreceptor and Plcgamma1, both of which may contribute to the impaired responsiveness. Bcl11b was found to bind the E8I, E8IV, and E8V, but not E8II or E8III, enhancers. Thus, Bcl11b is one of the transcription factors implicated in the maintenance of optimal CD8 coreceptor expression in peripheral CD8(+) T cells through association with specific enhancers. Short-lived Klrg1(hi)CD127(lo) effector CD8(+) T cells were formed during the course of infection in the absence of Bcl11b, albeit in smaller numbers, and their Ag-specific cytolytic activity on a per-cell basis was altered, which was associated with reduced granzyme B and perforin.

Transcriptional control of gene expression in double-positive (DP) thymocytes remains poorly understood. We show that the transcription factor BCL11B plays a critical role in DP thymocytes by controlling positive selection of both CD4 and CD8 lineages. BCL11B-deficient DP thymocytes rearrange T cell receptor (TCR) α; however, they display impaired proximal TCR signaling and attenuated extracellular signal-regulated kinase phosphorylation and calcium flux, which are all required for initiation of positive selection. Further, provision of transgenic TCRs did not improve positive selection of BCL11B-deficient DP thymocytes. BCL11B-deficient DP thymocytes have altered expression of genes with a role in positive selection, TCR signaling, and other signaling pathways intersecting the TCR, which may account for the defect. BCL11B-deficient DP thymocytes also presented increased susceptibility to spontaneous apoptosis associated with high levels of cleaved caspase-3 and an altered balance of proapoptotic/prosurvival factors. This latter susceptibility was manifested even in the absence of TCR signaling and was only partially rescued by provision of the BCL2 transgene, indicating that control of DP thymocyte survival by BCL11B is nonredundant and, at least in part, independent of BCL2 prosurvival factors.

T regulatory (Treg) cells play an important role in maintaining immunological tolerance to self-antigens, thus limiting immune responses to tumor antigens. Therefore, depleting or suppressing Tregs is one strategy by which anti-tumor immunity can be restored. The immunotoxin ONTAK® is an IL-2-diphteria toxin fusion protein that has been shown to diminish Tregs in patients and animal models of cancer in peripheral blood using a systemic intravenous (i.v.) administration route. E7777 is a new version of ONTAK®. In this study we tested the hypothesis that locally-diminished Tregs by intratumoral (i.t.) administration of E7777 generate effective anti-tumor immune response at both local and systemic levels. First, we showed superior anti-tumor activity and safety of E7777 i.t. over E7777 i.v., where i.t. administration resulted in complete tumor regressions in both moderately immunogenic CT26 and non-immunogenic B16F10 tumors with minimal animal body weight loss. In contrast, only tumor growth delay was observed for E7777 i.v. with dose-limiting animal body weight reduction in the same models. Immune phenotyping showed a 4 fold reduction of intratumoral Tregs in treated CT-26 tumors without significant change of Tregs in the spleens of treated animals, confirming a local Treg-depleting effect of E7777 i.t. In contrast, intratumoral CD8+ T cells were not reduced. Second, E7777 i.t. enhanced overall anti-tumor immune response, manifested by significantly increased numbers of CD45+ hematopoietic cells, Granzyme B+ CD8+ cytotoxic T cells, and ratios of cytotoxic T cells/Tregs in the treated tumors. Importantly, E7777 i.t. also resulted in distant effects in the spleen characterized by increased ratio of T lymphocytes to myeloid cells and increased frequencies of both effector memory CD8+ T cells (CD8+CD62L-CD44+) and central memory CD8+ T cells (CD8+CD62L+CD44+) indicative of systemic immune activation. Consistent with the generation of immunological memory, 60% of the tumor-free animals treated with E7777 i.t. rejected completely and 40% displayed delayed tumor growth of B16F10 cell challenge while all naïve control animals grew tumors. Taken together, our results demonstrate that intratumoral Treg depletion by local administration of E7777 leads to an effective local and memory anti-tumor response in preclinical models and support further evaluation of local E7777 delivery as a cancer immunotherapy. Citation Format: Diana I. Albu, Christy Ingersoll, Kuan-Chun Huang, Mary Woodall-Jappe, Xingfeng Bao. Intratumoral Treg cell depletion by local administration of IL-2-Diphteria toxin fusion protein E7777 induces a therapeutic and memory anti-tumor immune response in preclinical models [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 4586. doi:10.1158/1538-7445.AM2017-4586

Purpose: Immunotherapies targeting immune checkpoint receptors have shown great promise for a subset of cancer patients; however, robust and safe combination therapies are still needed. In the tumor microenvironment, prostaglandin E2 receptor type 4 (EP4) signaling has been implicated in both protumoral myeloid cell differentiation and cytotoxic T cell exhaustion. We evaluated the combination of the EP4 antagonist E7046 (clinical trial NCT02540291) with anti-PD1 or anti-CTLA4 in preclinical tumor models, and also interrogated the relationship between PGE2 pathway activation and cancer patient survival. Materials/Methods: Mouse syngeneic tumor models CT-26 and 4T1 were used for pharmacological investigation. GMP grade E7046 was administered to tumor-bearing animals by oral gavage. Co-culture of EG7-OVA and OT1 cells in an antigen-specific cytotoxic T cell (CTL) activation assay provided mechanistic insights. For translational validation, transcripts of five major genes involved in PGE2 synthesis, transport and degradation were compared between malignant and normal tissues across all TCGA tumor types, and correlation of their expression with overall survival was assessed. Results: In the CT26 tumor model, the combination of E7046 and anti-PD1 resulted in significantly more tumor-free animals compared with either agent alone. In the 4T1 tumor model, the combination of E7046 and anti-CTLA4 was also more effective in suppressing tumor growth and tumor rejection compared with anti-CTLA4 alone, and was accompanied by a markedly increased accumulation of GZMB+CD8T+ CTLs in the treated tumors. Consistent with those findings, addition of anti-PD1 antibody promoted OVA-specific CTL activation in vitro while addition of PGE2 strongly inhibited it, as measured by IFNγ secretion. Inclusion of E7046 dose-dependently reversed the PGE2-induced suppressive activity in the presence of anti-PD1 antibody. Among major human PGE2 pathway genes, TCGA analysis showed that PTGES1 was upregulated and HPGD downregulated across a broad range of tumor types. In contrast, COX1, COX2 and PGT showed less difference between malignant and normal tissues. Importantly, these differences of one or multiple PGE2 pathway genes were strongly associated with patient survival in certain cancer types. Conclusions: A subset of human cancer types displays upregulated PGE2 pathway that is associated with a poorer prognosis. PGE2-EP4 signaling potently suppresses antigen-specific CTL activation in the presence of PD1 signaling blockade. The combination of EP4 antagonist E7046 with either anti-PD1 or anti-CTLA4 demonstrated superior anti-tumor activity compared with anti-PD1 or anti-CTLA4 alone. This increased activity was accompanied by increased CTL activation. Citation Format: Diana I. Albu, David Verbel, Yuan Huang, Donna Kolber-Simonds, Zichun Wang, Xulong Wang, Zoltan Dezso, Christy Ingersoll, Kuan-Chun Huang, Janna Hutz, Mary Woodall-Jappe, Xingfeng Bao. Specific inhibition of PGE2-EP4 signaling by E7046 promotes anti-tumor activity of checkpoint blockade agents through boosting cytotoxic T cell activity [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 4607. doi:10.1158/1538-7445.AM2017-4607

Purpose: To assess the combined activity of E7046 and E7777 against multiple murine syngeneic tumor models. Background: Both regulatory T cells (Tregs)and myeloid cells are significant constituents in the tumor microenvironment in many types of cancer, where they help to maintain an immunosuppressive milieu that inhibits cytotoxic T cell function, which favors tumor persistence. Prostaglandin E2 (PGE2) engages the EP4 receptor on monocytes to direct myeloid cell activities away from antigen presentation and toward immunosuppression (wound healing). PGE2-driven myeloid responses have been reported to enhance Treg immunosuppressive activity, while Tregs with high expression of the FoxP3 transcription factor have been reported to up-regulate cyclooxygenase expression and PGE2 secretion. E7046 is a highly selective small molecule that potently competes with PGE2 binding to EP4. Single agent E7046 slows the growth of multiple syngeneic murine tumor types, in a mechanism dependent upon the presence of T cells. Denileukin diftitox (ONTAK®), an IL-2/diphtheria toxin fusion protein, was originally approved by the US FDA for treating patients with relapsed/refractory cutaneous T cell lymphoma. E7777 is a new version of denileukin diftitox developed using an improved manufacturing process. We and others have shown that denileukin diftitox selectively depletes Tregs. Neither E7046 nor E7777 affects tumor cell viability in vitro. Here we assessed their combined activity against syngeneic tumor models and the mechanisms driving their activity. Methods: Tumors were implanted subcutaneously in groups of 5-10 female BALB/c mice. When tumors were > 50 mm3, mice were treated with E7046 (150 mg/kg, QD x 21, p.o.) and/or E7777 (2.5 or 3 μg/head, Q7D x 2 or 3, i.v). Tumors, spleens, and tumor-draining lymph nodes were excised and analyzed by flow cytometry for immune cell composition and function. Results: As single agents, both E7046 and E7777 delayed the growth of established tumors. When combined, their anti-tumor activities were significantly enhanced, with up to 20% of animals rendered stably tumor-free. Activity was significantly reduced if animals were treated with antibody to deplete CD8+ T cells. In the tumors, the combination treatment dramatically increased the ratio of CD8 T cells to CD4+CD25+Foxp3+ Tregs, and also decreased the frequency of immunosuppressive myeloid cells. Importantly, the CD8+ T cells in the treated tumors, but not in the control tumors, were found to express granzyme B. Spleens from the treated animals also showed an increased ratio of CD8+ T cells vs the highly active CD4+CD25+Foxp3+ ICOS+ Tregs. Conclusions: The combination of E7046 and E7777 showed promising preclinical anti-tumor activity via an immune-mediated mechanism. Citation Format: Diana I. Albu, Kuan-Chun Huang, Jiayi Wu, Xingfeng Bao, Kenichi Nomoto, Mary Woodall-Jappe. Preclinical immune antitumor activity of myeloid-targeting E7046 and Treg depleting E7777. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B034.

E7046 is a potent and selective small molecule antagonist of the type 4 prostaglandin E2 (PGE2) receptor EP4. The discovery and evaluation of this novel EP4 antagonist for cancer treatment was recently presented at the AACR 2015 annual conference (abstract # 275). Here we show new evidence of E7046 in vivo activity against a series of syngeneic tumor models as well as its pharmacological effect on APCMin/+ mice, which possess a mutation in the adenomatous polyposis coli (APC) tumor suppressor gene. We found that daily oral administration of E7046 was able to both slow down the growth of established subcutaneous tumors and significantly delay the recurrence of tumors after surgical resection. These activities of E7046 correlated with differences in the immune cell composition of the microenvironments of each type of tumor. In this context, cyclooxygenase 2 - positive (COX-2+) tumors that were rich in myeloid cells showed an enhanced response to E7046 regardless of their T lymphocyte infiltration status. In addition, E7046 was superior to the COX-2 inhibitor celecoxib against mutant APC-driven neoplastic polyp formation in the intestines of APCMin/+ mice exposed to dextran sodium sulfate. Using the APCMin/+ mouse model, we found that E7046 significantly reduced the combined colon polyp area and the size of individual polyps without influencing the total polyp number. For each of these parameters, E7046 activity against colon and small intestine tumors was greater than that of a comparable dose of celecoxib. Quantification of cyclin D1 staining in colon and small intestine polyps further indicated that both compounds led to a significant reduction in the proliferation of tumor cells, with a greater effect for E7046 compared to celecoxib. Overall, these preclinical results suggest that this agent should be further investigated in patients with COX-2+ tumors infiltrated with myeloid cells, including patients with APC-mutated colon cancer. First-In-Human study of E7046 is currently enrolling patients (IND 125272). Citation Format: Diana I. Albu, Jiayi Wu, Kuan-chung Huang, Renee Wright-Michaud, Shanqin Xu, Galina Kuznetsov, Xingfeng Bao, Mary Woodall-Jappe. Pharmacological profile of the PGE2 EP4 receptor antagonist E7046. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B198.

Bcl11b is required for optimal FoxP3 expression and suppressor function by regulatory T cells and for the generation of inducible regulatory T cells., Dysregulated CD4+ T cell responses and alterations in T regulatory cells (Treg cells) play a critical role in autoimmune diseases, including inflammatory bowel disease (IBD). The current study demonstrates that removal of Bcl11b at the double-positive stage of T cell development or only in Treg cells causes IBD because of proinflammatory cytokine-producing CD4+ T cells infiltrating the colon. Provision of WT Treg cells prevented IBD, demonstrating that alterations in Treg cells are responsible for the disease. Furthermore, Bcl11b-deficient Treg cells had reduced suppressor activity with altered gene expression profiles, including reduced expression of the genes encoding Foxp3 and IL-10, and up-regulation of genes encoding proinflammatory cytokines. Additionally, the absence of Bcl11b altered the induction of Foxp3 expression and reduced the generation of induced Treg cells (iTreg cells) after Tgf-β treatment of conventional CD4+ T cells. Bcl11b bound to Foxp3 and IL-10 promoters, as well as to critical conserved noncoding sequences within the Foxp3 and IL-10 loci, and mutating the Bcl11b binding site in the Foxp3 promoter reduced expression of a luciferase reporter gene. These experiments demonstrate that Bcl11b is indispensable for Treg suppressor function and for maintenance of optimal Foxp3 and IL-10 gene expression, as well as for the induction of Foxp3 expression in conventional CD4+ T cells in response to Tgf-β and generation of iTreg cells.

Invariant natural killer T cells (iNKT cells) are innate-like T cells important in immune regulation, antimicrobial protection, and anti-tumor responses. They express semi-invariant T cell receptors, which recognize glycolipid antigens. Their positive selection is mediated by double-positive (DP) thymocytes, which present glycolipid self-antigens through the noncanonical MHC class I-like molecule CD1d. Here we provide genetic and biochemical evidence that removal of the transcription factor Bcl11b in DP thymocytes leads to an early block in iNKT cell development, caused by both iNKT cell extrinsic and intrinsic defects. Specifically, Bcl11b -deficient DP thymocytes failed to support Bcl11b -sufficient iNKT precursor development due to defective glycolipid self-antigen presentation, and showed enlarged lysosomes and accumulation of glycosphingolipids. Expression of genes encoding lysosomal proteins with roles in sphingolipid metabolism and glycolipid presentation was found to be altered in Bcl11b- deficient DP thymocytes. These include cathepsins and Niemann–Pick disease type A, B, and C genes. Thus, Bcl11b plays a central role in presentation of glycolipid self-antigens by DP thymocytes, and regulates directly or indirectly expression of lysosomal genes, exerting a critical extrinsic role in development of iNKT lineage, in addition to the intrinsic role in iNKT precursors. These studies demonstrate a unique and previously undescribed role of Bcl11b in DP thymocytes, in addition to the critical function in positive selection of conventional CD4 and CD8 single-positive thymocytes.

One of the hallmarks of an immunosuppressive tumor microenvironment is the presence of myeloid-derived suppressor cells (MDSCs) and type 2 tumor-associated macrophages (TAMs). These myeloid cells derive from immature monocytes and their differentiation is highly regulated by the engagement of prostaglandin E2 (PGE2) receptor type 4 (EP4) with PGE2 in the tumor microenvironment. To understand the importance of EP4 for tumor support, we implanted mouse tumors in EP4 inducible knockout mice and found that these tumors grew significantly more slowly than in wild type mice, indicating an important role of host cell EP4 signaling for tumor progression. Here we report the development and evaluation of ER-886046, a novel and specific EP4 antagonist, for cancer treatment. Daily oral administration of ER-886046 inhibited the growth of multiple mouse syngeneic tumor models with an inhibitory activity up to 100%. The anti-tumor activity of ER-886046 was T cell-dependent since it was not observed in T cell deficient mice. Furthermore, we found in vitro and in vivo mechanistic evidence that ER-886046 interferes with tumor-induced monocyte differentiation into immunosuppressive type 2 macrophages and MDSCs, instead supporting monocyte differentiation into APCs and facilitating intratumoral T cell accumulation. Importantly, ER-886046 has a desirable pharmacokinetic and metabolism profile in mice, rats and dogs which qualifies it as a good candidate for clinical studies in humans. Thus, the preclinical data support further investigation of targeting EP4 signaling by ER-886046 as a novel immune therapy for cancer treatment in clinical setting. Citation Format: Diana I. Albu, Zichun Wang, Jiayi Wu, Kuan-chun Huang, Wei Li, Diana Liu, Galina Kuznetsov, Qian Chen, Xingfeng Bao, Mary Woodall-Jappe. ER-886046, an antagonist of PGE2 receptor type-4, induces an effective antitumor immune response in mice by attenuating intratumoral MDSCs and TAMs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 275. doi:10.1158/1538-7445.AM2015-275

BCL11A and BCL11B are transcriptional regulators important for lymphopoiesis and previously associated with hematopoietic malignancies. Ablation of the mouse Bcl11b locus results in failure to generate double-positive thymocytes, implicating a critical role of Bcl11b in T-cell development. However, BCL11B is also expressed in CD4+ T lymphocytes, both in resting and activated states. Here we show both in transformed and primary CD4+ T cells that BCL11B participates in the control of the interleukin-2 (IL2) gene expression following activation through T-cell receptor (TCR). BCL11B augments expression from the IL2 promoter through direct binding to the US1 site. In addition, BCL11B associates with the p300 coactivator in CD4+ T cells activated through TCR, which may account for its transcriptional activation function. These results provide the first evidence that BCL11B, originally described as a transcriptional repressor, activates transcription of a target gene in the context of T-cell activation.

We have investigated the induction of protective mucosal immunity to human immunodeficiency virus type 1 (HIV-1) isolate 89.6 by intranasal (i.n.) immunization of mice with gp120 and gp140 together with interleukin-12 (IL-12) and cholera toxin subunit B (CTB) as adjuvants. It was found that both IL-12 and CTB were required to elicit mucosal antibody responses and that i.n. immunization resulted in increased total, immunoglobulin G1 (IgG1), and IgG2a anti-HIV-1 antibody levels in serum; increased total, IgG1, IgG2a, and IgA antibody expression in bronchoalveolar lavage fluids; and increased IgA antibody levels in vaginal washes. Levels of anti-HIV-1 antibodies in both sera and secretions were higher in groups immunized with gp140 than in those immunized with gp120. However, only gp120-specific mucosal antibodies demonstrated neutralizing activity against HIV-1 89.6. Taken together, the results show that IL-12 and CTB act synergistically to enhance both systemic and local mucosal antibody responses to HIV-1 glycoproteins and that even though gp140 induces higher antibody titers than gp120, only gp120-specific mucosal antibodies interfere with virus infectivity.

Recent reports utilizing the MMTV-PyMT spontaneous model of breast cancer established that inhibition of macrophage recruitment to the tumors resulted in increased anti-cancer activity of paclitaxel. Eribulin mesylate (Halaven®) is a non-taxane microtubule-dynamics inhibitor recently approved for third line clinical use in patients with heavily pretreated metastatic breast cancer, based upon a statistically significant increase in median overall survival (OS) compared to treatment of physician's choice. In the present study we investigated whether systemic depletion of macrophages would enhance eribulin anti-tumor efficacy. In the subcutaneous Lewis lung carcinoma (LLC) syngeneic murine tumor model, we found that macrophage depletion via liposomal clodronate enhanced eribulin inhibition of tumor growth. Current studies are investigating the mechanism(s) of this activity. Citation Format: Diana I. Albu, Bruce A. Littlefield, Mary Woodall-Jappe. Macrophage depletion enhances the anticancer efficacy of eribulin mesylate in mice bearing syngeneic tumors. [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 1227. doi:10.1158/1538-7445.AM2013-1227

Numerous studies indicate that certain tumor-infiltrating macrophage subsets can support neoplastic growth. Recent reports showed that spleen can serve as an important reservoir for macrophages and neutrophils that replicate in situ, then migrate to support tumor growth. Eribulin mesylate (Halaven®) is a microtubule-dynamics inhibitor approved for third line clinical use in patients with heavily pretreated metastatic breast cancer based upon statistically significant increase in median overall survival (OS) compared to treatment of physician's choice. In an effort to understand whether eribulin has additional effects beyond its anti-mitotic activity, the current study addressed the effects of eribulin mesylate on immune cells. Using the subcutaneous syngeneic murine tumor model Lewis lung carcinoma (LLC), we found that the tumor shrinking effect of eribulin correlated with increased extramedullary hematopoiesis observed in the spleens of treated mice. Eribulin was also found to inhibit the proliferation of M2 tumor-associated macrophages (TAMs) in the spleens of LLC-bearing mice. Early effects of eribulin were studied using the CT-26 colon tumor isograft model and included increased differentiation and death of CD4+ and CD8+ effector T cells. Furthermore, eribulin caused increased CD25 expression on CD4+ Foxp3- T cells but not on CD4+ Foxp3+ T regulatory cells. Finally, frequencies of exhausted CD8+ T cells were reduced following eribulin treatment. Taken together, these results suggest that eribulin-mediated modulation of immune homeostasis especially in the spleen, may contribute to its anti-tumor effect. Citation Format: Diana I. Albu, Namita Kumar, Galina Kusnetzov, Shanqin Xu, Bruce Littlefield, Mary Woodall-Jappe. Eribulin mesylate alters immune homeostasis in mice bearing syngeneic tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B90.