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Background Development of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. Recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to Ebola virus glycoprotein with only published sequences. Methods and Results A rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. A focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of EBOV GP from allosteric effectors reported from literature. Conclusions Critically, this workflow allows us to probe the epitope landscape of EBOV GP without any prior structural knowledge of the antigen or structural benchmark clones. Incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library’s full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. The use of high-throughput epitope binning during new outbreaks such as the current COVID-19 pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment., Starting with only published antibody sequences and soluble Ebola virus glycoprotein, high-throughput surface plasmon resonance is employed to rapidly screen and epitope bin hundreds of antibodies to fully characterize the epitope coverage of antibodies identified from a convalescent donor.

The Wnt/β-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.

Mouse tumor models have been successfully used to generate preclinical data for numerous clinical programs including immunotherapy. Preclinical in vivo studies are typically carried out using young mice (often less than 2 months old) to generate efficacy data, predictive biomarker and pharmacodynamic markers. In notable contrast, the majority of human cancer occur in adult and older patients. It has become increasingly clear that the immune system of young and old mice is quite different with regards to the relative abundance and functionality of different cell populations. Data generated using young mice could provide a distorted assessment of the potential activity of immuno-oncology drugs in the clinic. Therefore, we tested the activity of GITRL-Fc protein in both young and old mice (>9 months). Previously using extensive young mice experiments, we have shown that GITRL-Fc promoted a robust antitumor immune response and enhanced tumor-specific T-cell responses, particularly of the Th1 type, and also led to a reduction in Treg-mediated immunosuppressive activity. Compared to young mice, tumors grew faster in older mice, and peripheral blood of older tumor-bearing mice has fewer T cells and NK cells. The total MDSC population was increased in the blood and spleen of old tumor-bearing mice, with a significantly higher number of G-MDSCs in the blood. On the other hand, old mice had reduced “antigen-presenting cells” (macrophages/dendritic cells expressing MHCII) in the blood. Furthermore, splenocytes from old mice had impaired production of IL-2. GITRL-Fc significantly inhibited tumor growth in both older and younger mice. However, efficacy was more pronounced in young mice, which frequently exhibited complete tumor regression. There were fewer tumor-infiltrating immune cells with less CD8 T and NK cells in older mice compared to young mice, consistent with faster tumor growth. Interestingly, GITR expression in CD8 T cells in old mice was lower compared with young mice at the tumor site. In old mice, GITRL-Fc (mIgG2a) was still able to deplete Tregs in tumor and increase Tregs in the spleen as has been previously shown with GITRL-Fc in young mice. On the other hand, GITRL-Fc deficient in effector function (mIgG2a (N297A)) did not deplete Tregs in the tumor but did retain some antitumor growth activity, indicating a role for GITR signaling in the mechanism of efficacy by GITLR-Fc. In conclusion, the results demonstrate the potential for the aging of the immune system to impact the efficacy observed with immunotherapy agents and highlight the potential benefits of conducting efficacy studies with both young and older mice. Citation Format: Angie Inkyung Park, Minu K. Srivastava, Rui Yun, Jenny Pokorny, Janice Yu, Fumiko Axelrod, Austin Gurney. Effect of aging on the antitumor activity of GITRL-Fc [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 70.

Activation of the co-stimulatory receptor GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) by GITR-Ligand (GITRL) promotes proliferation and activation of effector T cells (T eff) and inhibits suppressive activity of regulatory T cells (Treg). Here, we have further characterized the mechanism of action of a single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc, 336B3) by examining pharmacodynamic (PD) biomarkers in time course studies. Mice bearing CT26.WT colon tumors were treated with weekly GITRL-Fc and sacrificed 24 hours, 7 and 14 days after the first dose. Immuno-phenotyping of tumor-associated immune cells revealed a reduction in Treg frequency in tumor by 24 hours post-dose that was maintained at 7 and 14 days. Furthermore, GITRL-Fc treatment increased activation markers on tumor-associated CD4+ and CD8+ T cells, suggesting an increased cytotoxic environment within the tumor. This was supported by significant and sustained increase in CD8+ T cell:Treg ratio in the tumor after GITRL-Fc treatment. To determine whether intratumoral (IT) injection of GITRL-Fc is an effective route of administration, we compared efficacy, pharmacodynamic (PD) markers and pharmacokinetics in IT- and intraperitoneal (IP)-injected mice bearing bilateral CT26.WT tumors. Both routes of administration showed similar tumor growth inhibition (TGI) and PD markers in both the treated and the abscopal tumors, but IT injection resulted in a significantly lower serum GITRL-Fc concentration, suggesting that IT administration may be an alternative route of administration to IP with similar efficacy. The GITRL-Fc molecule 336B3 is effector function competent and able to induce cell-mediated cytotoxicity upon binding. To determine whether this effector function is required for GITRL-Fc-induced TGI, we treated CT26.WT tumor-bearing mice with 336B3 and 336B22, a GITRL-Fc molecule deficient in effector function. The effector function-competent 336B3 induced significant TGI and a more robust activation of Teff cells and reduction in Treg frequency, when compared to 336B22, suggesting that effector function is important for efficacy. To identify biomarkers for GITRL-Fc, we performed microarray analyses on multiple syngeneic mouse models treated with GITRL-Fc and developed GITRL gene signatures from blood and from tumors. We also developed multiplexed immunohistochemistry panels designed to quantify frequency of GITR and GITRL expression (GITR+CD8, GITR+FOXP3) in tumors. In conclusion, we have examined the effects of GITRL-Fc on preclinical mouse models. Biomarker analysis showed that loss of Tregs, activation of T cells and Fc-mediated effector function are key elements in the mechanism of action of the molecule. We have identified potential biomarkers to be used for PD and potential predictive analysis in clinical trial patient samples. Citation Format: Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Reyhaneh Lahmy, Erwan Le Scolan, Rose Harris, Alayne Brunner, Min Wang, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Earth Light Lowe, Gilbert O'Young, Austin Gurney, Ann M. Kapoun. GITRL-Fc biomarker and mechanism study: GITRL-Fc reduces Treg frequency in tumors and requires effector function for inhibition of tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3826.

The immune checkpoint co-inhibitory receptor TIGIT (T cell immunoreceptor with Ig ITIM domain) is expressed on regulatory T cells (Tregs) and on activated CD4+ T, CD8+ T, and NK cells. We have reported that by blocking TIGIT activity with an IgG2a anti-TIGIT antibody (313R12), CD8+ and CD4+ Tcells and NK cells were activated, resulting in dose-dependent tumor growth inhibition (TGI) in multiple syngeneic mouse models. To explore the pharmacodynamics (PD) and mechanism of action of tumor growth inhibition (TGI) by anti-TIGIT antibodies, we examined the kinetics of immune cell frequency and activation in tumor by flow cytometry, qPCR and immunohistochemistry (IHC). We performed in vivo time course studies in the CT26.WT colon carcinoma model using weekly dosing at 0.1, 0.5 and 12.5 mg/kg anti-TGIT. Mice were sacrificed at 24 hours, 7 days and 14 days after the first dose for biomarker analysis. After 24 hours of treatment, Tregs in the tumor decreased and this reduction of Tregs was sustained at 7 and 14 days. Markers of immune cell activation and exhaustion such as CD69, PD1 and intracellular cytokines were modulated during the course of the study, suggesting a more cytotoxic intratumoral environment after 313R12 treatment. In addition, CD226, a binding partner of TIGIT, was significantly upregulated in T cells, Tregs and NK cells throughout the study, reflecting a feedback loop activated by inhibiting TIGIT activity. The anti-TIGIT antibody used in these studies, 313R12, is effector function competent and is able to induce cell-mediated cytotoxic effector functions upon binding. In order to determine whether effector function is necessary for anti-TIGIT antibody activity, we compared 313R12 with an effector function-deficient molecule, 313R13, in CT26.WT tumors. After 7 days, only 313R12 showed significant TGI compared to control-treated animals, suggesting that effector function is required for efficacy. While the effector function-deficient molecule 313R13 was able to similarly induce some changes in PD biomarkers, including immune cell activation, it required a higher dose than 313R12 to do so. To develop biomarkers for anti-TIGIT, we used microarray analyses to identify anti-TIGIT gene signatures in tumors and blood from multiple syngeneic models. In addition, we developed multiplexed IHC panels (e.g., TIGIT+CD8, TIGIT+FOXP3) to quantify expression of TIGIT and TIGIT ligand-positive immune cells in the tumor and surrounding stroma, and we profiled a panel of 80 human tumors with these panels. In summary, we examined the effects of anti-TIGIT antibodies on preclinical mouse models. Biomarker analysis demonstrated loss of Tregs and activation of T cells and NK cells, as well as effector function, as part of the mechanism of action of the molecule. We have also identified biomarkers that can be used for PD and potential predictive analysis in clinical trial samples. Citation Format: Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Erwan le Scolan, Rose Harris, Reyhaneh Lahmy, Alayne Brunner, Min Wang, Gilbert O'Young, Earth Light Lowe, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Austin Gurney, John Lewicki, Ann Kapoun. Anti-TIGIT biomarker study: Inhibition of TIGIT induces loss of Tregs from tumors and requires effector function for tumor growth inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5627.

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the TNF receptor superfamily. GITR, a co-stimulatory surface receptor, is activated upon binding to GITR Ligand (GITRL) and mediates co-stimulation of T-cell and NK responses and inhibits the suppressive activity of regulatory T-cells (Tregs). As such, GITR is an attractive immuno-oncology target for activation via GITR agonists. OMP-336B11 is a single-gene recombinant fusion protein consisting of two trimeric human GITRLs and a human immunoglobulin (IgG1) Fc domain. Murine preclinical studies using a surrogate GITRL-Fc fusion protein demonstrated robust antitumor efficacy. Functional characterization of OMP-336B11 in various human immune cell assays is presented here. In human peripheral blood mononuclear cells (PBMC), OMP-336B11 stimulated IL-2 cytokine release in a dose-dependent manner. In activated human T-cells, OMP-336B11 enhanced cell proliferation in a dose-responsive fashion. OMP-336B11 also augmented IL-2 induced IFNγ from human NK cells. To elicit NK mediated cytotoxicity of high GITR expressing cells (i.e., Tregs), OMP-336B11 is designed with an IgG1 Fc domain. Co-incubation of primary human NK cells (effector) and GITR expressing cells (target) resulted in an OMP-336B11 dependent dose-titratable increase in target cytotoxicity. Furthermore, OMP-336B11 agonistic activity was compared to anti-GITR agonist antibodies. By measuring cell proliferation and IFNγ from activated T-cells, OMP-336B11 demonstrated superior activity compared to the other agonist anti-GITR antibodies. In conclusion, OMP-336B11 is designed to induce effective GITR activation and also to mediate depletion of GITR-high cells. OMP-336B11 is currently undergoing phase I clinical study. Citation Format: Ivan H. Chan, Ming-Hong Xie, Andrew Lam, Fumiko T. Axelrod, Jennifer Elechko, Angie I. Park, Austin Gurney. In vitro functional activity of OMP-336B11, a GITRL-Fc fusion protein, on primary human immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2726.

Meeting abstracts Blocking DLL4, a Notch ligand, effectively inhibits tumor growth by increasing non-functional angiogenesis and decreasing the cancer stem cell (CSC) population. Preclinical studies have demonstrated inhibition of tumor growth by anti-DLL4 treatment and have led us to enter

Deregulation of the β-catenin signaling has long been associated with cancer. Intracellular components of this pathway, including axin, APC, and β-catenin, are frequently mutated in a range of human tumors, but the contribution of specific extracellular ligands that promote cancer development through this signaling axis remains unclear. We conducted a reporter-based screen in a panel of human tumors to identify secreted factors that stimulate β-catenin signaling. Through this screen and further molecular characterization, we found that R-spondin (RSPO) proteins collaborate with Wnt proteins to activate β-catenin. RSPO family members were expressed in several human tumors representing multiple malignancies, including ovarian, pancreatic, colon, breast, and lung cancer. We generated specific monoclonal antibody antagonists of RSPO family members and found that anti-RSPO treatment markedly inhibited tumor growth in human patient-derived tumor xenograft models, either as single agents or in combination with chemotherapy. Furthermore, blocking RSPO signaling reduced the tumorigenicity of cancer cells based on serial transplantation studies. Moreover, gene-expression analyses revealed that anti-RSPO treatment in responsive tumors strongly inhibited β-catenin target genes known to be associated with cancer and normal stem cells. Collectively, our results suggest that the RSPO family is an important stimulator of β-catenin activity in many human tumors and highlight a new effective approach for therapeutically modulating this fundamental signaling axis. Cancer Res; 76(3); 713–23. ©2015 AACR.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a co-inhibitory molecule containing an immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic tail, and is highly expressed on regulatory T cells and activated CD4+ T, CD8+ T, and NK cells. TIGIT competes with CD226, which contains an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic tail for ligands poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2), with higher affinity to PVR. The ligands are expressed on the surface of antigen presenting cells and at high levels on most tumors. Therefore, when TIGIT is present, the ligands preferentially engage TIGIT rather than CD226, leading to cell suppression. We have generated antibodies against TIGIT that blocks ligand binding and inhibits TIGIT signaling. The clinical candidate, OMP-313M32 binds human TIGIT but not rodent and non-human primate TIGIT. Therefore, a surrogate antibody was generated for pre-clinical assessments in mice. Antibody 313R12 is an anti-mouse TIGIT antibody that can block mouse PVR ligand binding and inhibit TIGIT signaling in a manner similar to the clinical candidate OMP-313M32. 313R12 inhibited the growth of syngeneic colon and kidney tumors in immune competent mice. In some cases, anti-TIGIT antibody 313R12 caused complete tumor regression and a potent anti-tumor immune memory response as demonstrated by the lack of tumor growth upon re-challenge of mice that remained tumor-free after prior anti-TIGIT treatment. Mechanistically, anti-TIGIT antibody 313R12 was shown to induce a Th1 response and increase cytotoxic T lymphocyte (CTL) activity. By in vivo depletion of T cell populations, we have shown that CD8 T cell depletion completely abrogated the anti-TIGIT therapeutic effect, whereas CD4 T cell depletion led to partial reversal of efficacy of anti-TIGIT. Therefore, both CD4+ and CD8+ T cells are critical for anti-TIGIT-mediated immune responses. Using mice reconstituted with human hematopoietic stem cells, we also demonstrated that the clinical candidate OMP-313M32 inhibits patient-derived melanoma tumor growth. Taken together, these data demonstrate that anti-TIGIT therapy suppresses tumor growth and generates long-term immunological memory against multiple tumors. Citation Format: Angie Inkyung Park, Minu Srivastava, Erin Mayes, Hyun-Bae Jie, Rui Yun, Christopher Murriel, Ming-hong Xie, Andrew Lam, May Ji, Fumiko Axelrod, Jorge Monteon, John Lewicki, Tim Hoey, Austin Gurney. Antibody against TIGIT (T cell immunoreceptor with Ig and ITIM domains) induces anti-tumor immune response and generates long-term immune memory [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 2003. doi:10.1158/1538-7445.AM2017-2003

TIGIT (T cell immunoreceptor with Ig and ITIM domains) has been recently described as an inhibitory receptor which blocks CD8 T cell-mediated anti-tumor immune responses. We have generated an anti-mouse TIGIT antibody (313R12) to evaluate drug efficacy and mechanism of action in pre-clinical tumor models. Anti-TIGIT as a single agent promoted an anti-tumor immune response in multiple syngeneic mouse tumor models. Anti-TIGIT enhanced tumor specific T cell responses, particularly of the Th1 type and reduced Th2 type responses and also increased the function of cytotoxic T cells. Furthermore, anti-TIGIT displayed combination activity with immune checkpoint inhibitors anti-PD1 and anti-PDL1 in inhibiting tumor growth, promoting complete tumor rejection and significantly increasing mouse survival in the murine CT26 colon carcinoma model as compared to controls and single agents alone. Mice “cured” with anti-TIGIT/anti-PDL1 or anti-TIGIT/anti-PD1 combination treatments did not form tumors upon subsequent re-challenges with increasing number of CT26 tumor cells, suggesting the existence of immunologic memory. IL2 and tumor-specific IFN-γ production by splenic T cells were increased in mice who responded to combination treatment compared to controls. Additionally, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population in responding mice. We also demonstrated a systemic increase in tumor-specific CD8 T cells after anti-TIGIT/anti-PDL1 combination treatment compared to controls. Therefore, these results suggest that co-targeting of TIGIT and PD1 or PDL1 may be an effective and durable cancer therapy by increasing T cell-mediated anti-tumor immune responses and promoting long-term immunological memory. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Janice Yu, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Jorge Monteon, Andrew Lam, May Ji, Yuwang Liu, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity [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 2612. doi:10.1158/1538-7445.AM2017-2612

Accumulating evidence has suggested that tumors may arise and grow as a result of the formation of a subset cell population termed cancer stem cells (CSC) or tumor initiating cells. Several research reports have indicated that CSCs are relatively resistant to conventional therapies. Thus, therapeutic strategies that specifically target cancer stem cells could have a major impact on cancer patient survival. Patient-derived tumor xenografts (PDX) have played a major role in the development of new cancer therapies. The advantage of PDX over standard cell-line xenograft models is that PDX retain much of the molecular, genetic, and histological heterogeneity of the original tumor and are minimally passed. In addition, self-renewal and lineage differentiation, the hallmarks of cancer stem cells, can be demonstrated through serial transplantation in immunodeficient mice. Our therapeutic approach in cancer stem cell drug discovery has been to target key developmental pathways that have been strongly implicated in cancer including the Notch pathway. We carried out tumorigenicity studies in three SCLC PDX models utilizing naïve cells positively enriched for either Notch2 or Notch3 expression. In two of the three models, Notch2+ and/or Notch3+ cell populations resulted in enhanced tumor formation. In a SCLC tumor that expressed low levels of Notch3, the Notch2+ population resulted in 70% tumor formation compared to CD44+ (10%) or CD133+ (45%) enriched populations. We have developed a monoclonal antibody, tarextumab (OMP-59R5), which selectively inhibits the function of both Notch2 and Notch3. Our preclinical data in PDX models demonstrate that tarextumab was efficacious in inhibiting the growth of various indications with minimal intestinal toxicity. Notably, the sensitivity of tarextumab in combination with gemcitabine or gemcitabine plus nab-paclitaxel in pancreatic tumors was associated with higher levels of Notch3 gene expression. Interference with Notch2/3 signaling by tarextumab delays tumor recurrence, decreases cancer stem cell frequency (as determined by in vivo LDA studies) and modulates the function of tumor vasculature. Our ALPINE Phase 1b clinical trial indicates that tarextumab is generally well-tolerated and shows signs of anti-tumor efficacy and modulation of Notch pathway signaling in the clinic. Furthermore, we observe a higher response rate and longer survival in patients with Notch3 high tumors receiving gemcitabine/nab-paclitaxel/tarextumab combination therapy. Analysis of pre- and post-treatment tumor biopsies showed an inhibition of Notch pathway and CSC gene signatures. Ongoing Phase 2 clinical trials evaluate first-line treatment with tarextumab in metastatic pancreatic cancer (ALPINE) and small cell lung cancer (PINNACLE). Collectively, these results demonstrate the utility of PDX models for discovery and development of anti-cancer stem cell therapeutics, identifying pharmacodynamic endpoints of drug actions, identifying predictive biomarkers for patient stratification, and translating these preclinical findings into clinical trials. Citation Format: Marcus M. Fischer, Wan-Ching Yen, Fumiko Axelrod, Christopher Bond, Jennifer Cain, Belinda Cancilla, Randall Henner, Rene Meisner, Aaron Sato, Jalpa Shaw, Tracy Tang, Breanna Wallace, Min Wang, Chun Zhang, Ann Kapoun, Lei Zhou, Jakob Dupont, John Lewicki, Austin Gurney, Tim Hoey. Use of patient-derived tumor xenografts (PDX) for discovery and development of an anti-Notch2/3 monoclonal antibody targeting cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A41.

GITRL (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the TNF superfamily and naturally exists as a membrane-anchored type II protein that self assembles as a trimer. GITRL activates the co-stimulatory receptor GITR. GITR is found primarily on activated T effector (Teff) cells and regulatory T (Treg) cells. Co-stimulation of GITR by agonist agents is hypothesized to promote anti-tumor immunity by enhancing Teff cell activity and inhibiting Treg suppression. We generated a novel single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc). GITRL-Fc activated GITR signaling more effectively than prototype GITR agonist antibody DTA-1. GITRL-Fc promoted a robust anti-tumor immune response in multiple syngeneic mouse tumor models. GITRL-Fc enhanced tumor specific T-cell responses, particularly of the Th1 type, and also led to reduction in Treg-mediated immunesuppressive activity. GITRL-Fc displayed single agent activity in inhibiting tumor growth and promoting complete tumor rejection in the murine CT26 colon carcinoma model and combination activity with anti-PDL1 as compared to anti-PDL1 and control IgG2a alone. Mice “cured” with GITRL or GITRL/anti-PDL1 combination treatments were protected from re-challenge with tumor cells, suggesting the existence of immunologic memory. More mice were protected from tumor re-challenge with the combination of GITRL-Fc and anti-PDL1, as compared to GITRL-Fc alone. Our results demonstrate that agonist GITRL-Fc induces potent T cell responses, overcomes Treg inhibition, and promotes anti-tumor activity in preclinical models as a single agent or in combination with anti PDL1. The mechanism of tumor eradication and induction of long-term immune memory response by the combination is under investigation and will be discussed at the presentation. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Hyun_Bae Jie, Fumiko Axelrod, Jorge Monteon, Ming-Hong Xie, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. GITR ligand fusion protein (GITRL-Fc) induces T cell mediated anti-tumor immune response and can combine with anti-PDL1 to enhance anti-tumor immunity and long-term immune memory. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2214.

Although the interaction between GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) and GITRL (its ligand) is important for the development of immune responses, the cellular mechanisms underlying anti-tumor immunity including both innate and adaptive immunity is not fully understood. We generated a novel single-gene linkerless GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc), and investigated its effect on controlling tumor growth and immune responses in preclinical tumor models. Treatment with GITRL-Fc significantly reduced tumor growth in several preclinical tumor models by inducing Th1 biased anti-tumor immunity and reducing Treg-mediated immune suppression. Immune cell depletion studies showed that anti-tumor immunity induced by GITRL-Fc depended on CD8+ T cells as well as NK cells. Furthermore, the combination of GITRL-Fc with PD-1 blockade significantly reduced the tumor growth in the Renca murine kidney adenocarcinoma tumor model. Taken together, these results suggest that GITRL-Fc can improve cancer treatment as a single agent or in combination therapy by enhancing innate and adaptive cellular immunity. Citation Format: Hyun-Bae Jie, Minu K. Srivastava, Erin Mayes, Rui Yun, Fumiko Axelrod, Jorge Monteon, Austin Gurney, Angie In-Kyung Park. GITRL-Fc can significantly reduce tumor growth by stimulating innate and adaptive immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3215.

GITRL, (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the TNF family of ligands and naturally exists as a membrane-anchored type II protein that self assembles as a trimer. GITRL activates the co-stimulatory receptor GITR. A novel single-gene linkerless GITRL trimer was shown to be functional when fused to either the N- or C-terminus of an immunoglobulin Fc domain, offering a flexible strategy that may also be amenable to the production of bispecific agents. GITRL-Fc activated GITR signaling more effectively than prototype GITR agonist antibody DTA-1. GITRL-Fc promoted a robust anti-tumor immune response in several murine tumor graft models, including the apparent total regression of some treated tumors. GITRL-Fc potentiated tumor specific T-cell responses, particularly of the Th1 type, increased antigen-specific CD8 response, and promoted a reduction in Treg-mediated immune-suppressive activity. Citation Format: Fumiko Axelrod, Hyun-Bae Jie, Erin Mayes, Jorge Monteon, Minu Srivastava, Rui Yun, Inkyung Angie Park, Austin Gurney. GITRL-Fc, an immunotherapeutic agent that stimulates T-cell-mediated antitumor immune response. [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 A058.

Blocking DLL4, a Notch ligand, effectively inhibits tumor growth by increasing non-functional angiogenesis and decreasing the cancer stem cells (CSC) population. We are currently testing an anti-DLL4 antibody, demcizumab, in Phase1B trials in NSCLC, pancreatic, and ovarian cancer. DLL4 is also known to modulate immune responses. In the current study we examine the impact of anti-DLL4 on anti-tumor immune responses as a single agent and in combination with the key immune checkpoint inhibitor Programmed Cell Death Protein 1 (PD1). While the recent clinical success of PD1 inhibitors represents a new and promising cancer immunotherapeutic approach, high initial response rates are often associated by a lack of long-term, durable effects in a significant number of patients. Therefore, we hypothesized that dual blockade of DLL4 and PD1 might further impact tumor growth by further enhancing anti-tumor immune immunity. Our data demonstrates that dual blockade of DLL4 and PD1 using antibodies not only reduces tumor growth, but also led to tumor rejection in ∼50% in CT26WT tumor-bearing mice, similar to those treated with anti-PD1 alone (no tumor rejection was observed with anti-DLL4 alone). Anti-PD1 increased specific CD8+ T cell-mediated IFN-γ production while decreasing IL6. Anti-DLL4 treatment reduced IL17 production. Interestingly, only the dual blockage led to increased production of IL2 by splenocytes. Since IL2 is required for secondary population expansion of CD8+ memory T cells, increased IL2 in the combination group suggests potential for increased T cell activation, maintenance and memory T cell function, as compared to single agent anti-DLL4 and anti-PD1. While anti-PD1 reduced inhibition of CD4+ T cell proliferation by Tregs, the dual blockade significantly reduced Treg-mediated CD8+ T cell suppression. Furthermore, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population. Interestingly, anti-PD1 decreased granulocytic MDSCs, while anti-DLL4 reduced monocytic MDSCs. Mice cured with single-agent anti-PD1 and anti-DLL4/anti-PD1 combination treatments were protected from series of re-challenge with tumor cells, suggesting the existence of immunologic memory. Interestingly, more mice were protected from tumor re-challenge when both DLL4 and PD1 were blocked, as compared to PD1 alone. Surprisingly, mice previously treated with the anti-DLL4/anti-PD1 combination produced more IL2, clearly indicating the role of DLL4 blockade in enhancing anti-tumor immunity. Therefore, these results show that dual targeting of DLL4 and PD1 may be an effective and durable cancer therapy by increasing anti-tumor immune response and promoting long-term immunological memory. Citation Format: Minu Srivastava, Christopher L. Murriel, Julie Roda, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Rui Yun, Erin Mayes, Trevor Bentley, Belinda Cancilla, Raymond Tam, Tracy Tang, Ann Kapoun, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Dual targeting of Delta-like ligand 4 (DLL4) and programmed death 1(PD1) inhibits tumor growth and generates enhanced long-term immunological memory. [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 255. doi:10.1158/1538-7445.AM2015-255

One of the fundamental principles of pharmacology is that most drugs have side effects. Although considerable attention is paid to detrimental side effects, drugs can also have beneficial side effects. Given the time and expense of drug development, it would be particularly exciting if a systematic method could be applied to reveal all of the activities, including the unappreciated actions, of a potential drug. The present study takes the first step along this path. An activity-based proteomics strategy was used to simultaneously identify targets and screen for their inhibitors in prostate cancer. Orlistat, a Food and Drug Administration-approved drug used for treating obesity, was included in this screen. Surprisingly, we find a new molecular target and a potential new application for Orlistat. Orlistat is a novel inhibitor of the thioesterase domain of fatty acid synthase, an enzyme strongly linked to tumor progression. By virtue of its ability to inhibit fatty acid synthase, Orlistat halts tumor cell proliferation, induces tumor cell apoptosis, and inhibits the growth of PC-3 tumors in nude mice.

Both Notch/Delta-like ligand 4 (DLL4) and vascular endothelial growth factor (VEGF) pathways play a critical role in angiogenesis and tumor growth. Due to differential regulatory effects of VEGF and DLL4 on the vasculature, inhibition of DLL4 or VEGF inhibits tumor growth by distinct mechanisms: anti-DLL4 treatment induces an abnormal increase of poorly perfused blood vessels, which results in a nonproductive angiogenesis unable to support tumor growth, whereas the anti-VEGF therapy significantly decreases vasculature reducing the blood supply to tumors. We have recently developed a high binding affinity bispecific monoclonal antibody that targets both human DLL4 and human VEGF. In vitro, this antibody exhibited nanomolar affinity to hVEGF and hDLL4, and reduced HUVEC proliferation induced by VEGF. The bispecific antibody demonstrated significant in vivo anti-tumor efficacy in various solid tumors, delayed tumor recurrence following termination of chemotherapy, and decreased the frequency of tumor initiating cells. Analysis of tumor vasculature after treatment with anti-DLL4/VEGF revealed inhibition of vascular gene expression and endothelial cell proliferation, indicating that the anti-VEGF effect on the vasculature is dominant over the anti-DLL4 effect. Notably, at doses where both anti-DLL4 and anti-VEGF alone produced suboptimal anti-tumor effect, dual targeting resulted in additive tumor growth inhibition. These results indicate that our bispecific anti-DLL4/VEGF is broadly efficacious and may be useful for treatment of a variety of solid tumors. Citation Format: Wan-Ching Yen, Fumiko Axelrod, Chris Bond, Jennifer Cain, Cecile Chartier, Marcus Fischer, Shirley Ma, Rene Meisner, Janak Raval, Jalpa Shah, Austin Gurney, John Lewicki, Timothy Hoey. Dual targeting of DLL4 and VEGF signaling by a novel bispecific antibody inhibits tumor growth and reduces cancer stem cell frequency. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 207. doi:10.1158/1538-7445.AM2014-207

Deregulation of the canonical Wnt/beta-catenin signaling pathway has long been associated with cancer. Intracellular components of this pathway, including Axin, APC, and beta-catenin are frequently mutated in a range of human tumors. The identity of specific extracellular ligands that contribute to human cancer development through this signaling axis has remained unclear. Molecular characterization of the secreted beta-catenin signaling activities produced by minimally passaged human tumor xenograft models identified RSPO family members produced by multiple tumor types including ovarian, pancreatic, colon, breast and non-small cell lung cancer. In human tumor xenograft models that had RSPO overexpression, in some instances due to genomic translocation, anti-RSPO treatment markedly inhibited tumor growth. In addition, striking combination activity with standard of care chemotherapy agents resulted in regression of established tumors. These results highlight the potential for therapeutic intervention with this newly appreciated signaling axis. Citation Format: Austin Gurney, Fumiko Axelrod, Chris Bond, Jennifer Cain, Cecile Chartier, Marcus Fischer, May Ji, Chris Murriel, Janak Raval, Jalpa Shah, Min Wang, Wan-Ching Yen, Ann Kapoun, John Lewicki, Timothy Hoey. Inhibition of R-spondin (RSPO) signaling reduces the growth of multiple human tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1764. doi:10.1158/1538-7445.AM2014-1764

Maspin, a member of the serine protease inhibitor (serpin) family, is a tumor suppressor in breast and prostate cancer. To address molecular mechanisms underlying maspin's activity, we restored its expression in invasive carcinoma cells and analyzed the resulting changes by shotgun proteomics. Using a mass spectrometry-based multidimensional proteomic method, we observed changes to the expression of approximately 27% of the detectable proteome. In particular, we noted changes to the expression of proteins that regulate cytoskeletal architecture, cell death, and protein turnover. In each case, changes in protein expression were accompanied by measurable changes in tumor cell phenotype. Thus, maspin-expressing cells exhibit a more prominent actin cytoskeleton, a reduced invasive capacity, an increased rate of spontaneous apoptosis, and an altered proteasome function. These observations reveal for the first time the far reaching effects of maspin on multiple protein networks and a new hypothesis of maspin function based on the regulation of proteasome function.

Both Notch/Delta-like ligand 4 (DLL4) and vascular endothelial growth factor (VEGF) pathways play a critical role in angiogenesis and tumor growth. Due to differential regulatory effects of VEGF and DLL4 on the vasculature, blockage of DLL4 or VEGF inhibits tumor growth by distinct mechanisms: anti-DLL4 treatment induces an abnormal increase of poorly perfused blood vessels, which results in a nonproductive angiogenesis unable to support tumor growth, whereas the anti-VEGF therapy significantly decreases vasculature reducing the blood supply to tumors. In our study, we have developed a bispecific monoclonal antibody that targets both human DLL4 (hDLL4) and human VEGF (hVEGF). In vitro, this antibody demonstrated nanomolar affinity to hVEGF and hDLL4, and reduced HUVEC proliferation induced by VEGF (EC50 0.86 ug/ml). To test the activity of this bispecific antibody in vivo, we developed a human skin graft model in NOD/SCID mice, and implanted human tumor specimen-derived colon cancer cells intradermally into these skin transplants. The tumor model was selected based on its sensitivity to both the anti-human DLL4 antibody, OMP-21M18, and to the human VEGF inhibitor bevacizumab. The skin graft model provides a suitable human microenvironment for evaluating anti-tumor efficacy and anti-angiogenesis of the bispecific antibody directed against the human component of DLL4 and VEGF and allows a comparison to OMP-21M18 and also to bevacizumab. Each of these treatments was administered to mice intraperitoneally at a dose of 25 mg/Kg weekly. The bispecific antibody caused a significant inhibition of tumor growth (87% TGI) compared to control antibody (p=0.00001), and this effect was superior to either OMP-21M18 (45% TGI) or bevacizumab (70%TGI). The inhibition of tumor growth by the bispecific antibody was consistently associated with increased blood vessels, up-regulated VEGFA and VEGFR2, and enhanced hypoxia and these effects were more pronounced compared to OMP-21M18. As expected, in this model bevacizumab caused a significant decrease of blood vessels, down-regulated VEGFR2, and increased hypoxia. In separate experiments with mice bearing subcutaneous human colon tumors, the bispecific antibody delayed tumor recurrence following termination of chemotherapy and impacted tumorigenicity by decreasing the frequency of tumor initiating cells. These results suggest that our bispecific anti-DLL4 and anti-VEGF antibody is a potential candidate in the treatment of tumors driven by both VEGF and Notch/DLL4 signaling pathways. Citation Format: Lucia Beviglia, Pete Yeung, Wang-Ching Yen, Belinda Cancilla, Sato Aaron, Chris Bond, Janak Raval, Fumiko Axelrod, Cecile Chartier, Shirley Ma, Austin Gurney, John Lewicki, Ann M. Kapoun, Timothy Hoey. In vivo evaluation of anti-tumor activity by an anti-VEGF and anti-DLL4 bispecific antibody in a humanized model of skin graft. [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 4330. doi:10.1158/1538-7445.AM2013-4330

The R-spondin-LGR signaling axis is a recently appreciated pathway that promotes beta-catenin signaling in normal stem cell populations. Beta-catenin signaling had long been considered synonymous with Wnt signaling. We conducted a reporter-based screen for secreted beta-catenin signaling activities produced by a panel of human tumors. This exercise and further molecular characterization efforts discovered RSPO activity produced by numerous human tumors of multiple tumor types including ovarian, pancreatic, colon, breast and non-small cell lung cancer. Specific antibody antagonists of RSPO family members were developed. In minimally passaged human tumor xenograft models, anti-RSPO treatment markedly inhibited tumor growth in several tumor types. Moreover, RPSO blockade promoted tumor differentiation and reduced the frequency of tumor initiating cells. These data highlight the potential for therapeutic intervention with this newly appreciated signaling axis. Citation Format: Austin Gurney, Cecile Chartier, Fumiko Axelrod, Jennifer Cain, Wan-Ching Yen, Christopher Murriel, Janak Raval, Marcus Fischer, Jalpa Shah, May Ji, Christopher Bond, Ann Kapoun, John Lewicki, Timothy Hoey. R-Spondin (RSPO) signaling drives the growth of multiple human tumor types. [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 218. doi:10.1158/1538-7445.AM2013-218

The Wnt/beta-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several co-receptors, is known to play a critical role in several major cancer types. Mutations in the signalling pathway occur in most cases of human colon cancer, and activation of Wnt signalling through various mechanisms has been reported in multiple major tumor types. We have developed a novel Wnt pathway antagonist antibody, OMP-18R5 which was initially identified by binding to Frizzled7, and subsequently found to also bind several other human FZDs through a conserved epitope within the extracellular domain. OMP-18R5 blocks Wnt binding and canonical signalling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, OMP-18R5 inhibited the growth of a broad range of tumor types and has been found to be particularly active in breast cancer. OMP-18R5 treatment reduced tumor growth and the frequency of tumor initiating cells in combination with pacilitaxel. Furthermore, we found that OMP-18R5 treatment can restore chemosensitivity in drug resistant breast tumors. Molecular analyses indicated OMP-18R5 inhibits the expression of EMT markers in breast tumors, thus providing additional evidence linking EMT with resistance to chemotherapy and cancer stem cells. In addition to combination activity with paclitaxel, OMP-18R5 treatment also resulted in increased anti-tumor activity in combination with trastuzumab in a Her2+ breast cancer model. These data suggest that OMP-18R5, which has recently entered Phase 1 clinical testing, may be useful in the treatment of various types of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3356. doi:1538-7445.AM2012-3356