Integrative Development of a TLR8 Agonist for Ovarian Cancer Chemoimmunotherapy Bradley J

Published OnlineFirst October 4, 2016; DOI: 10.1158/1078-0432.CCR-16-1453

Cancer Therapy: Clinical Clinical Cancer Research Integrative Development of a TLR8 Agonist for Ovarian Cancer Chemoimmunotherapy Bradley J. Monk1, Andrea Facciabene2, William E. Brady3, Carol A. Aghajanian4, Paula M. Fracasso5, Joan L. Walker6, Heather A. Lankes3, Kristi L. Manjarrez7, Gwenn-ael€ H. Danet-Desnoyers2, Katherine M. Bell-McGuinn4, Carolyn K. McCourt8, Alexander Malykhin2, Robert M. Hershberg7, and George Coukos2

Abstract

Purpose: Immunotherapy is an emerging paradigm for the non-human primates and healthy human subjects, whereas treatment of cancer, but the potential efficacy of many drugs the effects of the motolimod/PLD combination in tumor- cannot be sufficiently tested in the mouse. We sought to develop bearing NSG-HIS mice closely mimicked those in patients a rational combination of motolimod—a novel Toll-like receptor with ovarian cancer treated in a phase Ib trial (NCT01294293). 8 (TLR8) agonist that stimulates robust innate immune responses The NSG-HIS mouse helped elucidate the mechanism of action in humans but diminished responses in mice—with pegylated of the combination and revealed a positive interaction liposomal doxorubicin (PLD), a chemotherapeutic that induces between the two drugs in vivo. The combination produced no immunogenic cell death. dose-limiting toxicities in patients with ovarian cancer. Two Experimental Design: We followed an integrative pharmaco- subjects (15%) had complete responses and 7 subjects (53%) logic approach including healthy human volunteers, non-human had disease stabilization. A phase II study was consequently primates, NSG-HIS ("humanized immune system") mice recon- initiated. þ stituted with human CD34 cells, and patients with cancer to test Conclusions: These results are the first to demonstrate the value the effects of motolimod and to assess the combination of of pharmacologic approaches integrating the NSG-HIS mouse, motolimod with PLD for the treatment of ovarian cancer. non-human primates, and patients with cancer for the develop- Results: The pharmacodynamic effects of motolimod ment of novel immunomodulatory anticancer agents with monotherapy in NSG-HIS mice closely mimicked those in human specificity. Clin Cancer Res; 1–12. 2016 AACR.

Introduction immunosuppressive functions. However, select chemotherapeutic agents such as anthracyclines may promote tumor cell death Most chemotherapeutic agents are believed to work indepen- through mechanisms that activate antitumor immune response dently of the host's immune system and some even appear to exert (1), thereby converting the tumor microenvironment (TME) from an immunologic sanctuary to a setting characterized by immunogenic tumor cell death. This transition is associated with the 1Arizona Oncology (US Oncology Network), University of Arizona College of Medicine–Phoenix, Creighton University School of Medicine at St. Joseph's Hospital, surface expression of stress-associated markers such as calreticulin Phoenix, Arizona. 2University of Pennsylvania, Philadelphia, Pennsylvania. 3NRG on injured cells, which induce active phagocytosis by myeloid Oncology Statistics and Data Management Center, Roswell Park Cancer Institute, dendritic cells (mDC) and promote antigen processing and cross- Buffalo, New York. 4Memorial Sloan Kettering Cancer Center, New York, New York. presentation (2). Furthermore, the release into the TME of high- 5 6 University of Virginia, Charlottesville, Virginia. University of Oklahoma Health motility group box 1 (HMGB1) protein, a danger-associated 7 Sciences Center, Oklahoma City, Oklahoma. VentiRx Pharmaceuticals, Seattle, molecular pattern (DAMP) molecule, triggers the innate immune Washington. 8Washington University School of Medicine, St. Louis, Missouri. response (3, 4). Despite these immune-activating processes, Note: Supplementary data for this article are available at Clinical Cancer tumor-inﬁltrating mDCs are likely to be suppressed by factors Research Online (http://clincancerres.aacrjournals.org/). within the TME (5), and phagocytosis of necrotic cells may not be B.J. Monk, A. Facciabene, and W.E. Brady contributed equally to this article. a sufﬁcient stimulus for optimal cross-presentation of tumor Current address for G. Coukos: Ludwig Institute for Cancer Research and antigens by tumor DCs to trigger protective cytotoxic T-cell Department of Oncology, University of Lausanne, Lausanne 1007, Switzerland. responses. B.J. Monk, W.E. Brady, C.A. Aghajanian, P.M. Fracasso, J.L. Walker, H.A. Lankes, Toll-like receptors (TLR) comprise a family of 13 surface or and K.M. Bell-McGuinn are study members of NRG Oncology and Gynecologic endosomal receptors that recognize ligands that are broadly Oncology Group. shared by pathogens, collectively referred to as pathogen-associ- Corresponding Author: Bradley J. Monk, Arizona Oncology (US Oncology Net- ated molecular pattern (PAMP) molecules. PAMPs activate DCs work), University of Arizona College of Medicine–Phoenix, Creighton University and stimulate the release of cytokines and chemokines, thus School of Medicine at St. Joseph Hospital, 2222 E. Highland Avenue, Suite 400, facilitating the development of adaptive immune responses Phoenix, AZ 85016. Phone: 888-972-2873; Fax: 602-283-3040; E-mail: (6, 7). In humans, TLR8 is distinguished from other endosomally [email protected] expressed TLRs by its unique appearance on mDCs, monocytes, doi: 10.1158/1078-0432.CCR-16-1453 and natural killer (NK) cells. The natural ligand of TLR8 is foreign 2016 American Association for Cancer Research. single-stranded RNA; engagement triggers cellular activation and

www.aacrjournals.org OF1

Monk et al.

phase Ib clinical study in women with recurrent ovarian cancer. Translational Relevance Motolimod plus PLD induced potent immune activation, which An integrative pharmacologic approach was used to identify was similar to that observed in the NSG-HIS mouse and in healthy a rational treatment approach for ovarian cancer by combining human volunteers, and was well tolerated without unexpected or motolimod—a Toll-like receptor 8 (TLR8) agonist that stimu- synergistic toxicities. lates human (but not mouse) innate immune responses— with pegylated liposomal doxorubicin (PLD), a chemothera- Materials and Methods peutic drug that induces immunogenic cell death. Multispecies experiments were performed using human volunteers, non- Drugs human primates, NSG-HIS ("humanized immune system") Motolimod (VTX-2337) was supplied by VentiRx Pharmaceu- þ mice reconstituted with human CD34 cells, and patients with ticals, Inc. For preclinical studies, PLD (Ben Venue Laboratories ovarian cancer. Study results demonstrated the pharmacody- Inc.) was purchased from the University of Pennsylvania Hospital namic effects of motolimod and PLD as well as providing (Philadelphia, PA) pharmacy. For the GOG-9925 study, PLD was insight into the mechanism of action of the combination and supplied by each clinical site's pharmacy. the impact on tumor growth. This integrative approach offers a new innovation to the rapidly expanding domain of immuno- Human volunteers oncology by providing additional methods to enhance clinical Five men and 5 women healthy volunteers enrolled in a development of immunomodulatory agents with human randomized, open-label, phase I study of motolimod (Study specificity. The findings also have broad implications for the VRXP-A105) received 2 doses of motolimod 2.5 mg/m2 subcu- utility of NSG-HIS mice in clinical and translational human taneously separated by 1 week (17). Plasma samples were col- immunology. lected predose and 6 hours after dosing and analyzed for mediator levels as described above. This clinical study was conducted in accordance with Good Clinical Practice guidelines and the ethical principles on the basis of the Declaration of Helsinki. Approval for study procedures was obtained from the institutional review maturation, including the release of helper T (TH)1-polarizing boards of each study site, and all subjects provided written cytokines. On the basis of a small genetic deletion in the murine informed consent upon study enrollment. TLR8 gene (8), the activity of TLR8 agonists in mice is significantly attenuated compared with that in humans and other species. Primate studies Consequently, the in vivo biologic activity of TLR8 agonists has not Studies in cynomolgus monkeys were conducted at Charles been extensively studied to date. Nonetheless, the specific expres- River Laboratories, Preclinical Services, in accordance with the sion of TLR8 within human myeloid cells offers unique oppor- recommendations in the Guide for the Care and Use of Laboratory tunities for anticancer therapy. Animals of the NIH (Bethesda, MD). Study protocols were We have developed a synthetic, small-molecule agonist of TLR8 reviewed and approved by the Charles River Institutional Animal (motolimod; previously identified as VTX-2337) comprising a Care and Use Committee. Three male cynomolgus monkeys were 2-aminobenzazepine core (9). Motolimod is selective for TLR8 2 þ þ þ administered a single dose of motolimod 3.6 mg/m . Plasma and potently stimulates CD14 monocytes and HLA-DR CD11c þ þ samples were collected predose and 6 hours postdose and ana- mDCs but not HLADR CD11c CD123 plasmacytoid DCs (9). lyzed as described above. For in vitro studies, 7 mL of peripheral In mDCs, motolimod activated NF-kB and induced expression of blood was collected from 3 males and 3 females and added to IL12 and TNFa. A completed phase I clinical study has demon- TruCulture tubes (Myriad-RBM) containing 2 mL of culture media strated the tolerability and biologic activity of motolimod mono- plus 300 nmol/L motolimod. Tubes were incubated for 24 hours; therapy and revealed predictable pharmacokinetic and pharma- supernatants were stored at 35 C until analysis for mediator codynamic profiles (10). levels as described above. Given the stimulatory effects of motolimod on mDCs and monocytes, we hypothesized that motolimod would be an optimal partner for immunomodulatory chemotherapies such NSG-HIS mice and immune analyses null as anthracyclines, as simultaneous activation of PAMP and We used NOD-scid IL2rg (NSG) mice reconstituted with DAMP pathways during tumor antigen exposure could produce human immune system (NSG-HIS) to test the biologic effects of positive interactions. To test this hypothesis, we chose pegy- motolimod and the combination with PLD. To generate human lated liposomal doxorubicin (PLD) because of favorable phar- ovarian tumors, we injected mice with OVCAR5 cells. Details of fl macokinetics and pharmacodynamics, limited hematopoietic mouse experiments as well as ow cytometric analysis of human toxicity, and preferential accumulation in tumors (11). We leukocytes harvested from NSG-HIS mice, and testing of reactive fi chose ovarian cancer because of the implicated role of immune human tumor-in ltrating lymphocytes (TIL) from mouse tumors mechanisms (12–14) and because PLD is standard of care in are provided in Supplementary Material. relapsed disease (15). Because the activity of TLR8 agonists in mice varies from that in Measurement of cytokines in human, primate, and murine humans (16), we pursued an integrative pharmacologic approach blood to study the combination of PLD and motolimod in parallel Human and non-human primate cytokines in biologic samples models including in vitro human, in vivo non-human primate, and (TruCulture supernatants and plasma samples) were measured in vivo humanized mouse to acquire knowledge on the mecha- using Luminex-based technology (HumanMAP panel; Myriad nism of action of the combination, which was then evaluated in a RBM). Plasma samples from CD-1 mice were pooled at each time

OF2 Clin Cancer Res; 2016 Clinical Cancer Research

TLR8 Agonist for Ovarian Cancer Chemoimmunotherapy

point, and cytokines were measured using the Myriad-RBM donor hematopoietic stem cells (HSC), develops all lineages of RodentMAP panel. the human immune system (NSG-HIS; ref. 20). NSG mice were given 2.75-Gy whole-body irradiation and then intravenously þ In vitro tumor cell viability assessment and Western blot injected with about 1 105 human donor cord blood CD34 analysis cells. Consistent with published data (21), a high level of human OVCAR5 cells were treated with PLD (0.1, 1, and 10 mg/mL) hematopoietic cell engraftment was seen in NSG-HIS mice within and/or recombinant human TNFa (20 ng/mL). Cell viability was 12 weeks, giving rise to human immune T- and B-lymphocytes as assessed by Annexin V staining (Pharmingen #559763). Protein well as monocytes and dendritic cells repopulating peripheral extraction and Western blot analysis were performed as described blood, spleen, and lymph nodes (Supplementary Fig. S2). elsewhere (18). Primary antibody against c-FLIP (Cell Signaling We next tested whether human immune cells in NSG-HIS #3210 rabbit) and secondary antibody (BioRad 172-1019) were mice were sensitive to the effects of motolimod. Compared used at room temperature. Protein bands were visualized using with untreated controls, mice treated with motolimod at 1.5 or ECL (Amersham #RPN2132) with X-films (Bioexpress # F-9023). 15 mg/m2 showed a dose-dependent increase in plasma levels of human cytokines and chemokines, including IL6, IL12p70, Clinical study design TNFa,MCP-1,andMIP-1b 6 hours following treatment that GOG-9925 was a phase Ib, open-label, dose-escalation study of declined to baseline by 24 hours (Fig. 1A). Although no motolimod in combination with either PLD or paclitaxel in individual cytokines were significantly increased (due to the subjects with advanced ovarian cancer. The trial utilized a tradi- small sample size), a mixed-effect linear model combining all tional 3 þ 3 dose-escalation design to identify the maximum analytes showed a significant treatment effect at the highest tolerated dose (MTD) of each combination (19). Primary objec- dose (P ¼ 0.01). Consistent with activation of human mono- tives were to assess safety and tolerability, secondary objectives cytes in vivo, motolimod upregulated the co-stimulatory mar- þ þ were to evaluate pharmacokinetics and pharmacodynamics, and kers CD86 in human CD14 and CD11c cells harvested from exploratory objectives included assessment of antitumor activity. NSG-HIS spleens (Fig. 1B and C). The study was conducted at 4 Gynecologic Oncology Group To further assess the suitability of the NSG-HIS mouse model, centers in the United States (June 2011 to September 2013) in we conducted comparative pharmacodynamic studies assessing accordance with Good Clinical Practice guidelines and the Decla- the in vivo plasma biomarker response to motolimod in NSG-HIS ration of Helsinki. Approval was obtained from each site's Insti- mice, non-human primates and healthy human volunteers. Doses tutional Review Board. All subjects provided written informed that were proven to be safe and biologically active for each species consent. For each subject in cohort 1, PLD (40 mg/m2)was were used (10, 17). A dose-dependent increase in cytokines and administered on day 1, followed by 2.5 mg/m2 motolimod on chemokines indicative of TLR8-induced monocyte activation— day 3; each subject was assessed over 24 hours for safety. In the including IL6, MCP-1, and MIP-1b—were observed in all species absence of overt toxicity, the next subject in the cohort was (Fig. 2). Collectively, these data demonstrate that NSG-HIS mice enrolled. Enrollment of subsequent cohorts proceeded without exhibit sensitivity to TLR8 stimulation similar to that of non- such staggering. All enrolled subjects were assessed for dose-lim- human primates and humans. iting toxicities (DLT), defined as treatment-related adverse events (AE) that occurred during the first cycle of treatment and met PLD and motolimod achieve potent immunomodulation in standard predefined toxicity criteria. DLTs and other toxicities were NSG-HIS mice the basis for decisions regarding dose escalation, de-escalation, and Given the immunomodulatory effects of doxorubicin (22, cohort expansion. Details on eligibility criteria and study assess- 23) and prior evidence of positive interactions with innate ment are provided in Supplementary Material. immunity activation (24), we next tested whether concomitant administration of PLD would attenuate motolimod-induced Statistical analyses TLR8 activation in vivo. NSG-HIS mice were administered Details on statistical analyses are provided in Supplementary intraperitoneally PLD once followed by subcutaneous motoli- Material. mod on the same day or 2 to 5 days later. Whether a concurrent or sequential dose schedule was utilized, the acute response in plasma cytokines and chemokines and in human monocytes Results that was previously observed with motolimod monotherapy Motolimod induces immune activation in NSG-HIS mice was not significantly attenuated by the addition of PLD (Fig. 3A similar to non-human primates and humans and Supplementary Fig. S3). Given the slow release of doxo- To screen for suitable preclinical models, we compared the rubicin in the TME and to maximize the timing of drug inter- response to motolimod in vitro of peripheral blood mononuclear actions, we adopted a staggered schedule for all subsequent cells (PBMC) obtained from human healthy volunteers, non- murine experiments, with administration of PLD (50 mg/m2) human primates, and wild-type CD1 mice. Confirming the on day 1, followed by motolimod at either 1.5 or 15 mg/m2 on reported phylogenetic differences in TLR8, leukocytes from days 5, 7, and 9 of each 14-day cycle. This combination was wild-type mice were minimally responsive to motolimod relative administered for 3 cycles. In pilot experiments, both dose levels to non-human primate and human leukocytes (Supplementary of motolimod were biologically active, eliciting cytokine and Fig. S1). We thus sought an immunocompetent murine model chemokine responses comparable to those shown in Figs. 1 that would allow us to explore the therapeutic potential of and2,withnoacutetoxicities.The1.5mg/m2 dose level was motolimod for tumor immunotherapy in vivo at clinically relevant selected for use in our subsequent studies, as it more closely doses. We selected the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD- approximated the relevant human dose levels identified in a scid-IL2rgnull NSG) mouse which, when reconstituted with human previous phase I study of motolimod (10).

www.aacrjournals.org Clin Cancer Res; 2016 OF3

Monk et al.

Figure 1. Motolimod induces human immune cell activation in the NSG-HIS mouse. A, NSG-HIS mice (n ¼ 3 mice/group) were administered vehicle (control) or motolimod at 1.5 or 15 mg/m2. Plasma samples were obtained 6 hours after dosing and analyzed by HumanMAP assay. A mixed-effect linear model combining all analytes showed a significant treatment effect at the highest dose (P 0.01). Representative cytokines and chemokines are displayed. Increase(s) that approached but did not achieve statistical significance with respect to the control (P 0.1; one-sided Wilcoxon rank-sum test) are identified with (#). B, Representative images of flow cytometric analysis of human immune cells collected from NSG-HIS spleen 6 hours after administration of vehicle (control) or motolimod 1.5 or 15 mg/m2 show upregulation of the costimulatory marker hCD86 in hCD14þ spleen cells following treatment with motolimod. C, Significant upregulation of hCD86 in hCD14þ and hCD11cþ cells in NSG-HIS mouse spleens 6 hours after dosing with motolimod 15 mg/m2 (n ¼ 3 mice/group). Significant increase(s) with respect to the control (P 0.05; one-sided Wilcoxon rank-sum test) are identified with an asterisk ().

PLD plus motolimod effectively control tumors in NSG-HIS To test whether the drugs mobilized immunity against tumors, þ mice we examined tumors for overall human CD45 leukocyte infil- þ The potential antitumor effects of PLD plus motolimod in tration. Rare human CD45 cells were detected in OVCAR5 ovarian cancer were next evaluated using NSG-HIS mice bearing tumors of untreated mice by immunohistochemical (IHC) anal- HLA-matched human ovarian tumor/human bone marrow chi- ysis, indicating a baseline low-level tumor inflammation. Mice þ meric xenografts. NSG-HIS mice reconstituted with CD34 cord treated with either PLD or motolimod monotherapy or with the þ þ blood cells from HLA-A2 donor neonates (NSG-HIS-A2) were combination exhibited a marked increase in CD45 cells (Fig. þ inoculated with subcutaneous HLA-A2 OVCAR5 tumors. 3C). Consistent with activation of human tumor macrophages Tumor grafts were accepted at a rate >95% and treatment began and DCs, there was an increase in the frequency of CD40-expres- þ þ when tumors were approximately 50 mm3 (day 28). As sing human CD11b and CD11c cells in tumors following expected, mice treated with PLD alone showed reduced tumor treatment with either drug alone or the combination relative to þ growth compared with untreated animals. Motolimod alone controls (Fig. 3D, top). An increase in human CD8 TILs was also did not significantly affect tumor growth relative to untreated seen in mice receiving motolimod alone or motolimod in com- þ mice. However, the combination of PLD and motolimod bination with PLD, whereas activation of CD8 T lymphocytes, markedly reduced tumor growth compared with either agent judged by the upregulation of CD69, was noted following treat- alone, including 2 of 10 mice who experienced complete ment with motolimod in combination with PLD (Fig. 3D, bot- disappearance of their tumors (Fig. 3B). tom). Thus, the addition of PLD did not attenuate the effect of

OF4 Clin Cancer Res; 2016 Clinical Cancer Research

TLR8 Agonist for Ovarian Cancer Chemoimmunotherapy

Figure 2. The NSG-HIS mouse model is suitable for the preclinical assessments of motolimod. A, In vivo plasma biomarker response to motolimod, administered at the respective doses shown, measured at 6 hours, is compared across NSG-HIS mice (n ¼ 3/group), non-human primates (n ¼ 3/group), and healthy human volunteers (n ¼ 10/group; 5 males and 5 females). Multiple cytokines and chemokines indicative of TLR8 stimulation and immune activation were evaluated using HumanMAP. Representative analytes showing fold increase changes are shown. B, In healthy volunteers (n ¼ 10), signiﬁcant upregulation in several analytes is shown 6 hours after dosing with motolimod (P 0.01, one-sided Wilcoxon signed-rank test) as denoted by asterisks ().

motolimod, and together the two drugs activated tumor-infiltrat- inhibitory protein (c-FLIP), an inhibitor of procaspase-8 recruiting monocytes and T cells, which was associated with significant ment, which prevents apoptosis induced by TNF family death tumor control. ligands (Fig. 4B; ref. 28). þ In view of the observed increase in activated human CD8 TILs PLD plus motolimod cooperate in antitumor innate and following PLD plus motolimod, we also asked whether TILs were adaptive immunity mechanisms in part responsible for the tumor growth restriction in this model. Activated macrophages secrete high amounts of TNFa, which Human TILs were expanded ex vivo from each treatment group can induce apoptosis in susceptible cells expressing the TNF using IL2 and tested against OVCAR5 cells, using TILs from the receptor 1 (TNFR1; ref. 25). We confirmed by Western blotting same human donor in each experiment. Consistent with the that OVCAR5 cells express TNFR1 (Supplementary Fig. S4). Given significant CD69 upregulation seen in TILs following combina- the potent activation of tumor macrophages by PLD plus moto- tion treatment, TILs from mice treated with PLD plus motolimod limod, we hypothesized that the therapeutic interaction could be exhibited high levels of cytotoxicity against OVCAR cells ex vivo, partly explained by PLD rendering tumor cells susceptible to whereas TILs from mice treated with monotherapy exhibited death induced by TNFa. In agreement with prior observations modest cytotoxicity by chromium release assay ex vivo (Supple- (26), OVCAR5 cells were initially resistant to TNFa even at high mentary Fig. S5). There was no cytotoxicity from TILs expanded levels (Fig. 4A). However, cells pre-exposed to PLD became from control mice. These results were consistent among different þ sensitive to TNFa killing in vitro. Consistent with their increased human CD34 cell donors. To further test the contribution of TILs susceptibility to TNFa-induced cell death, and in agreement with to restrict tumor growth, TILs were harvested from tumors at the prior evidence (27), treatment of OVCAR5 cells with PLD signif- end of treatment from each experimental group and from control icantly reduced cellular FADD-like IL1b-converting enzyme- mice, expanded ex vivo using IL2, and adoptively transferred to

www.aacrjournals.org Clin Cancer Res; 2016 OF5

Monk et al.

Figure 3. Preclinical studies of motolimod (Moto) plus PLD in NSG-HIS mice. A, NSG-HIS mice were given PLD alone (intraperitoneally, 50 mg/m2), motolimod alone (subcutaneously 2 days after PLD dosing, 1.5 mg/m2), the combination, or vehicle control. Plasma was collected 6 hours after dosing, and levels of cytokines and chemokines were assessed using the HumanMAP panel. Select biomarkers are shown. Symbols denote P values compared with control (, P 0.05; #, P 0.1; 1-sided Wilcoxon rank-sum test). B, Tumor-bearing NSG-HIS mice inoculated with the human ovarian cancer cell line OVCAR5 were randomized to receive vehicle control, PLD, Moto, or the combination. Tumor growth was assessed at multiple time points after inoculation. The combination of PLD þ Moto showed highly significant improvement compared with control, PLD, and Moto. Two of the 10 mice in the PLD þ Moto arm had no measurable tumors at the last 2 time points. Two asterisks () denote P 0.01; 2-sided log-linear mixed-effect model. C, IHC was used to assess the presence of human (h)CD45þ tumor-infiltrating leukocytes. D, Combination of PLD plus motolimod resulted in significant increases in hCD11bþ and hCD11cþ cells expressing hCD40 (top). The combination also yielded increases in the frequency of hCD8þ T cells expressing hCD69 (bottom). Treatment group(s) (n ¼ 5/group) with significant magnitude increase(s) with respect to the control (P 0.05; 1-sided Wilcoxon rank-sum test) are identified with an asterisk ().

treatment-na€ve NSG-HIS-A2 mice bearing established OVCAR5 (Study GOG-9925). A standard dose-escalation design was used to tumors. TILs from animals treated with PLD plus motolimod evaluate the combination of 40 mg/m2 PLD with 3 dose levels of conferred a highly significant antitumor effect (Fig. 4C), suggest- motolimod (2.5, 3.0, and 3.5 mg/m2), which were selected on the ing that the increased efficacy of the PLD plus motolimod com- basis of the results of a previous phase I dose-seeking study of bination was, in part, also due to the cytolytic activity of TILs. single-agent motolimod (doses of 0.1 to 3.9 mg/m2)inadultswith advanced cancer (10). PLD was administered intravenously on day Translation of the PLD plus motolimod combination into the 1 of a 28-day cycle, followed by subcutaneous motolimod on days clinic 3, 10, and 17. Cycles of therapy were repeated until disease On the basis of the above preclinical data, a phase Ib study of progression or unacceptable toxicity. PLD plus motolimod was initiated in women with recurrent or Following enrollment of the first and second cohorts [PLD þ persistent ovarian cancer following platinum-based chemotherapy 2.5 mg/m2 motolimod (n ¼ 3) and PLD þ 3.0 mg/m2 motolimod

OF6 Clin Cancer Res; 2016 Clinical Cancer Research

TLR8 Agonist for Ovarian Cancer Chemoimmunotherapy

Figure 4. Innate and adaptive immune-mediated interactions underlie the effect of the motolimod plus PLD combination. A, OVCAR5 cells were evaluated in vitro for sensitivity to TNFa. Cells were left untreated (control) or were exposed to TNFa or PLD alone or were exposed to TNFa after pre-exposure to PLD. After treatment, cell death was measured using a flow cytometry–based Annexin V/7-AAD staining protocol. For each treatment, representative flow cytometric plots are shown with corresponding quantitative measurements of the percentage of apoptotic and necrotic cells. B, Western blot analysis of c-FLIP expression in isolated OVCAR5 tumor cells at baseline and after treatment with PLD at 1 or 10 mg/mL. C, To test the ability of TILs to restrict tumor growth in NSG-HIS-A2 mice, ex vivo expanded TILs from mice previously treated with PLD alone (PLD TIL), PLD þ motolimod (PLD þ Moto TIL), or from untreated mice (Untreated TIL) were transferred adoptively to treatment-na€ve NSG-HIS-A2 mice bearing established OVCAR5 tumors (n ¼ 10/group). NSG-HIS-A2 mice that received no TILs served as the control. Tumor volume was assessed periodically after transfer; animals receiving PLD þ motolimod TIL had highly significant reduction in tumor volume compared with control, PLD alone, and untreated TIL. Two asterisks () denote P 0.01; 2-sided log-linear mixed-effect model.

(n ¼ 3), respectively], study accrual was suspended because of a no serious, unexpected drug-related AEs. There was no evi- global shortage in the commercial supply of PLD. After a 6-month dence of toxicity interactions between motolimod and PLD or disruption, PLD availability returned and enrollment proceeded motolimod and paclitaxel. No signiﬁcant drug-related gastro- [cohort 3; PLD þ 3.5 mg/m2 motolimod (n ¼ 7)]. During the intestinal, neurologic, or cardiac toxicities were observed. interim absence of PLD, there was a need to identify alternate Clinical tolerability of motolimod was better at the 2.5 and combinations wherein motolimod could augment the activity of 3.0 mg/m2 dose levels compared with 3.5 mg/m2.Themost existing chemotherapeutics. Because paclitaxel is an alternate common AEs on each treatment regimen are provided in Table standard-of-care chemotherapy for this patient population, the 1. Eleven of the 20 subjects experienced at least 1 AE grade protocol was amended to include a second treatment arm eval- III in severity; AEs in 7 of these subjects were considered uating motolimod plus paclitaxel. In this regimen, a single-dose related to combination therapy. Treatment-related events level of motolimod (3.0 mg/m2) was combined with paclitaxel grade III observed in subjects receiving PLD plus motolimod (80 mg/m2). Both agents were administered on days 1, 8, and 15 included neutropenia (n ¼ 3); anemia (n ¼ 2); mucositis oral of a 28-day cycle. Cycles were repeated until disease progression or (n ¼ 2); lymphopenia, palmar–plantar erythrodysesthesia unacceptable toxicity. syndrome, urinary tract infection, and leukopenia (n ¼ 1 Twenty subjects were evaluated on Study GOG-9925: 13 trea- each); and chills, fever, and vomiting (all in one subject). ted with PLD plus motolimod and 7 treated with paclitaxel plus All of these events were attributed to PLD except for the chills, motolimod. The majority of participants (n ¼ 16, 80%) were fever, and vomiting. Treatment-related events grade III Caucasian and aged between 50 and 69 years (80%) (Supple- observed in subjects receiving paclitaxel plus motolimod mentary Table S1). included bladder infection, fatigue, febrile neutropenia, neu- Motolimod in combination with PLD or paclitaxel was tropenia, and leukopenia (all reported in 1 subject and attrib- generally safe and well tolerated. There were no DLTs and uted to paclitaxel).

www.aacrjournals.org Clin Cancer Res; 2016 OF7

Monk et al.

Table 1. AE and laboratory abnormalities reported in 20% of subjects PLD þ motolimod (N ¼ 13) Paclitaxel þ motolimod (N ¼ 7) All grades Grade III/IV All grades Grade III/IV AE n (%) n (%) n (%) n (%) Abdominal pain 7 (54) 2 (15) 3 (49) 0 (0) Alopecia 3 (23) 0 (0) 2 (29) 0 (0) Anemia 10 (77) 2 (15) 7 (100) 0 (0) Anorexia 6 (46) 0 (0) 1 (14) 0 (0) Anxiety 3 (23) 0 (0) 1 (14) 0 (0) Arthralgia 6 (46) 0 (0) 3 (43) 0 (0) Back pain 5 (39) 0 (0) 0 (0) 0 (0) Chills 9 (69) 1 (8) 3 (49) 0 (0) Constipation 7 (54) 0 (0) 2 (29) 0 (0) Cough 3 (23) 1 (8) 2 (29) 0 (0) Diarrhea 5 (39) 0 (0) 4 (57) 1 (14) Dyspnea 4 (31) 1 (8) 3 (43) 0 (0) Fatigue 13 (100) 0 (0) 7 (100) 1 (14) Fever 11 (85) 1 (8) 3 (43) 0 (0) Headache 9 (69) 0 (0) 1 (14) 0 (0) Hypertension 4 (31) 1 (8) 1 (14) 1 (14) Injection site reaction 12 (92) 0 (0) 5 (71) 0 (0) Mucositis oral 7 (54) 2 (15) 0 (0) 0 (0) Myalgia 2 (15) 0 (0) 2 (29) 0 (0) Nausea 10 (77) 1 (8) 4 (57) 0 (0) Palmar–plantar erythrodysesthesia syndrome 4 (31) 1 (8) 0 (0) 0 (0) Paresthesia 4 (31) 0 (0) 0 (0.0) 0 (0) Peripheral sensory neuropathy 6 (46) 0 (0) 4 (57) 0 (0) Vomiting 10 (77) 2 (15) 5 (71) 0 (0) Weight loss 2 (15) 0 (0) 2 (29) 0 (0) Laboratory abnormality Hypoalbuminemia 3 (23) 2 (15) 2 (29) 0 (0) Hypokalemia 2 (15) 1 (8) 2 (29) 0 (0) Hyponatremia 2 (15) 1 (8) 2 (29) 1 (14) Platelet count decreased 4 (31) 1 (8) 0 (0) 0 (0) Neutrophil count decreased 8 (62) 3 (23) 1 (14) 1 (14) White blood cell decreased 8 (62) 1 (8) 1 (14) 1 (14) NOTE: AEs were graded according to the Common Terminology Criteria for Adverse Events (CTCAE).

The biologic effects of PLD plus motolimod are similar in limod did not attenuate the immunostimulatory effects of patients with ovarian cancer and NSG-HIS mice motolimod in humans. Standard pharmacokinetic evaluations of each drug were per- Next, we conducted comparative pharmacodynamic assess- formed to rule out any alternations in the metabolism and ments in women enrolled in the GOG-9925 study and in NSG- elimination due to drug–drug interactions. In both regimens, HIS mice bearing ovarian tumor xenografts treated with PLD plus pharmacokinetic parameters associated with motolimod were motolimod. In both clinical study participants and NSG-HIS within the range of previously reported values (Supplementary mice, plasma levels of IL6, MCP-1, and MIP-1b increased in a Fig. S6A). Exposure to PLD was consistent with historical data dose-related manner in response to motolimod (Fig. 5C). The (Supplementary Fig. S6B). Plasma levels of paclitaxel were also magnitude of the pharmacodynamic effects seen in NSG-HIS mice within the expected range. dosed with 15 mg/m2 motolimod plus PLD was comparable with The pharmacodynamic effect of motolimod in combination the levels seen in study participants who received 3.5 mg/m2 with PLD or paclitaxel was evaluated in ovarian cancer patients motolimod plus PLD. by quantitative assessment of more than 90 cytokines, chemokines, and other immune inflammatory mediators. These data Clinical results with the combination of PLD plus motolimod confirmed that motolimod was biologically active when com- The antitumor activity of the PLD/motolimod combination bined with either PLD or paclitaxel, with the induction of was assessed as an exploratory objective of the GOG-9925 study. multiple inflammatory biomarkers indicative of TLR8 stimu- At the time of study entry, subjects had evidence of recurrent lation and immune activation. Motolimod was biologically disease either by biochemical criteria (elevated CA-125) or by active at all 3 dose levels tested (Fig. 5A). Specifically, moto- imaging (measurable or nonmeasurable disease on CT or MRI). limod plus PLD increased plasma levels of G-CSF, IL6, MCP-1, Of the 13 subjects treated with PLD plus motolimod, one subject and MIP-1b at one or more of the motolimod dose levels. with nonmeasurable disease treated with 2.5 mg/m2 of motoli- Importantly, the magnitude of biomarker response was main- mod achieved a complete response (CR; Fig. 5D). Interestingly, tained over multiple treatments over 2 cycles, with no evidence this subject received PLD plus 2.5 mg/m2 motolimod for 2 cycles of augmentation or attenuation (Fig. 5B). Results with the on study, with stable disease (SD) and interval improvement on paclitaxel regimen were similar (Supplementary Fig. S7). Taken interval imaging. Because of the shortage of PLD, she was unable together, these results indicated that, as seen in the NSG-HIS to receive additional study drugs and elected to forego additional mouse, the combination of cytotoxic chemotherapy plus moto- anticancer therapy. A CA125 level within normal range by cycle 4

OF8 Clin Cancer Res; 2016 Clinical Cancer Research

TLR8 Agonist for Ovarian Cancer Chemoimmunotherapy

AD40 mg/m2 PLD + Motolimod 2.5 mg/m2 3.0 mg/m2 3.5 mg/m2 MMP9 MIP-1β Log fold MCP-1 increase Insulin 0.25 IL8 2 IL6 Baseline: Day 0 End of cycle 6: Day 311

Analyte IL1RA 16

IL10 128 GH G-CSF

Cycle 1 Day 3 Cycle 1 Day 17 Cycle 2 Day 3 B 1,000

100

10 G-CSF

1,000

10 IL6 Time: Before 10,000 After

1,000 Plasma level (pg/mL) Plasma MCP-1

1,000 MIP-1 β 1,00

2.5 3.0 3.5 2.5 3.0 3.5 2.5 3.0 3.5 Motolimod dose (mg/m2) C IL6 MCP-1 MIP-1β E Subject 256.0 1 2 32.0 3 4 4.0 5 6

Fold increase 7 0.5 8 9 10 NSG-HIS Mice Ovarian cancer patients 11 Dose: Low Medium High 12 13 0 4812 16 20 24 28 32 36 40 44 48 52 Weeks Subject with biochemical disease only Start of response Complete response Progressive disease Stable disease Discontinued treatment due to Unevaluable PLD shortage Discontinued treatment due to CR

Figure 5. Biologic effects of motolimod in humans. The in vivo cytokine response to motolimod was assessed in women with advanced ovarian cancer who were treated with 40 mg/m2 of PLD in combination with motolimod at 2.5 (n ¼ 3), 3.0 (n ¼ 3), or 3.5 mg/m2 (n ¼ 7). Plasma was collected before the initial dose of motolimod and

8 hours after the dose, and levels of selected cytokines and chemokines were assessed using the HumanMAP panel. A, Immune mediator concentrations were log2- transformed, and fold changes were calculated for each analyte level by dividing the postdose value by the corresponding value from the predose sample. The corresponding heatmap for mediators exhibiting a mean log fold increase of 1.3 over baseline is shown. B, Mediators that broadly demonstrated a dose-related increase of 1.3-fold at 1 or more of the motolimod dose levels included G-CSF, IL6, MIP-1b, and MCP-1, and this response was maintained over 2 treatment cycles. C, To demonstrate the translational relevance of the antitumor response seen in NSG-HIS mice treated with the combination of PLD þ motolimod, the murine plasma mediator response was compared with that observed in clinical participants. Many of the mediators induced in the plasma of women with ovarian cancer (n ¼ 13) were also induced in NSG-HIS mice (n ¼ 6) following motolimod administration. Overall, the magnitude of the mediator response (fold increase with respect to baseline/control) in mice dosed with 15 mg/m2 motolimod was comparable to what was seen in ovarian subjects at the 3.5 mg/m2 dose. Response following PLD þ motolimod in recurrent stage 3c ovarian cancer. D, This subject with recurrent nontarget (nonmeasurable) disease was enrolled on study GOG-9925 and received PLD 40 mg/m2 (day 1), followed by 2.5 mg/m2 of motolimod (days 3, 10, and 17) for 2 cycles without major incident. Her disease remained stable during a 5-month treatment-free interval necessitated by a shortage in the global supply of PLD. Thereafter, she received 4 additional cycles of PLD plus motolimod and achieved a CR (no evidence of disease). E, Swim plot illustrating the course of the 13 subjects who received the PLD plus motolimod combination. and a repeat PET/CT conﬁrming ongoing stabilization of disease 5 additional cycles of PLD plus motolimod (6 cycles total) and months later in the absence of therapy suggest a durable beneﬁt achieved a CR, then elected to discontinue study treatment. Post- from the 2 cycles of PLD/motolimod. She subsequently received 4 study follow-up showed the CR was maintained for 10 weeks

www.aacrjournals.org Clin Cancer Res; 2016 OF9

Monk et al.

without additional therapy. (Fig. 5D). Another subject enrolled which resulted in significant activation of tumor-infiltrating þ þ on the basis of biochemical evidence of disease (see Materials and CD11b monocytes and CD11c DCs, which in turn were asso- þ Methods) and treated at the 3.0 mg/m2 dose level achieved a ciated with activation of alloreactive CD8 T cells. A concomitant biochemical CR after 3 cycles. She continued treatment for 3 elevation of human TH1-polarizing and other inflammatory cyto- additional cycles, for a total of 6 cycles, and then elected to go off- kines in the plasma documented effective systemic activation of treatment. The CR endured for 16 additional weeks without immune cells with the PLD/motolimod combination, which anticancer therapy. Moreover, SD was seen in 8 subjects, including preserved the potency of single-agent motolimod therapy by 1 subject with biochemical disease only; 3 additional subjects had eliciting IL12 and TNFa responses. Additional distinct favorable progressive disease (PD; Fig. 5E). Overall, the response rate of features of the combination included enhanced IFNg and lower subjects treated with PLD plus motolimod was 15% (n ¼ 2) and levels of IL10 relative to motolimod monotherapy. the disease control rate (DCR) was 69% (n ¼ 9; Supplementary Activated TILs were, in part, responsible for the observed Table S2). Of the 7 subjects who received paclitaxel plus moto- therapeutic effect of the PLD/motolimod combination in this limod, one achieved a partial response (PR), 2 experienced SD, mouse model, which shows that PLD plus motolimod signifi- and 4 had PD, with a DCR of 43% (n ¼ 3; Supplementary Table cantly reprogrammed the TME through activation of monocytes S2). Notably, the 2 subjects who achieved CR with PLD plus to overcome local T-cell tolerance. It is also possible that tumor motolimod and the subject who achieved a PR with paclitaxel and restriction was mediated, in part, by activated macrophages. Our motolimod achieved their best response after receiving 3 cycles in vitro experiments demonstrated a synergistic interaction of therapy. between TNFa, a cytokine released by activated macrophages (and effector T cells), and PLD. Pretreatment of tumor cells with PLD was required to induce susceptibility to TNFa-mediated Discussion death, which was explained by downregulation of c-FLIP induced Cancer immunotherapy has resulted in major successes in a in tumor cells stressed by PLD. An additional interaction could variety of solid tumors. Durable objective responses and long- entail the upregulation of the proapoptotic molecule Bfk by both term survival benefits have been achieved with T-cell–based TNFa and cytotoxic chemotherapy (30). These results prove the adoptive therapy as well as using antibodies that activate T cells principle of a positive interaction of the combination due to in vivo by blocking key lymphocyte-inhibitory receptors (29). independent effects of PLD and motolimod on tumor cells and Although the field of T-cell activation has significantly advanced, immune effector cells respectively. It is likely that in tumors TNFa activation of innate immunity still remains an elusive yet key goal is derived from both activated tumor monocytes as well as of immunotherapy. activated effector TILs. Additional and complementary positive TLR8 is an innate sensor that may facilitate the transition from immunomodulatory mechanisms may be envisioned in human innate to adaptive immune responses owing to its ability to tumors in vivo, including the induction of immunogenic cell activate monocytes and mDC. Because of phylogenetic differences death induced by PLD, which could further contribute to proper in TLR8 and the relative lack of activity in mice, TLR8 agonists have antigen presentation by DCs, the production of T-cell recruiting not been evaluated as cancer therapies to date. We therefore chemokines by activated tumor macrophages, and a possible sought to assess for the first time, the application of a novel reprogramming of additional immunosuppressive monocyte small-molecule TLR8 agonist, motolimod, in combination with populations such as myeloid-derived suppressive cells (MDSC), immunogenic chemotherapy using a humanized mouse model. which should be further investigated if the combination proves Comparative pharmacodynamic studies confirmed that the effective in the clinic. human immune system in non–tumor-bearing NSG-HIS mice On the basis of the promising preclinical results, we developed responds to motolimod in a similar manner to non-human the GOG-9925 phase I clinical study, adopting a phased admin- primates and normal human volunteers, with clear dose-depen- istration schedule with motolimod initiated 2 days after PLD in dent activation of plasma and immune cell biomarkers. Having each cycle. This schedule was selected to maximize the PLD- identified an active dose range that closely recapitulated the associated antigen-shedding effects on the tumor. Despite the optimal dose identified in a phase 1 study in patients with cancer absence of DLTs, the clinical tolerability of motolimod was (10), we examined the combination of motolimod with PLD, a considered better at 3.0 mg/m2 compared with 3.5 mg/m2.As cytotoxic drug with favorable pharmacokinetic and immuno- all dose levels were associated with robust pharmacodynamic modulatory profiles. The NSG-HIS model proved a clinically responses, and evidence of antitumor activity was observed at the relevant system for evaluating human immunotherapies such as 2.5 and 3.0 mg/m2 dose levels, 3.0 mg/m2 was selected as the motolimod and helped determine the pharmacodynamic effects recommended phase II dose. Similar to observations in the and test the antitumor efficacy of the combination. mouse, PLD did not attenuate the immune signature of motoli- Clinical development of immunomodulatory agents that rec- mod in patients. Similar biomarker responses were observed in ognize human targets but not their murine counterparts is chal- ovarian cancer subjects as in the NSG-HIS mice, indicating the lenging. Although the NSG-HIS-A2 tumor model has limitations accuracy of pharmacodynamics predictions in the NSG-HIS related to the partial HLA mismatch and multiple minor H mouse. Notable AEs including thrombocytopenia, palmar–plan- mismatching between bone marrow and tumor allograft, it offers tar erythrodysesthesia syndrome, and peripheral sensory neurop- an acceptable immunocompetent surrogate mouse model with athy were typically attributed to PLD and/or prior therapy rather which to examine the effects of therapy on the TME. Despite the than to motolimod and were generally grade I or II in severity. potential allorecognition, tumors engrafted successfully and grew Furthermore, the incidence of these events was similar to those in NSG-HIS-A2 mice, indicating local tolerance mechanisms reported in other trials of PLD in this setting (31–33). Finally, the enacted by the tumor. These tolerance mechanisms were effec- clinical benefit observed with PLD plus motolimod suggests that tively disrupted by the combination of PLD plus motolimod, the combination may improve clinical outcomes. The time to best

OF10 Clin Cancer Res; 2016 Clinical Cancer Research

TLR8 Agonist for Ovarian Cancer Chemoimmunotherapy

response following treatment with motolimod plus chemother- Gradalis, ImmunoGen, Insys, Merck, NuCana, Oxigene, Pfizer, Roche/Genen- apy was consistent with reports of delayed clinical response that tech, TESARO, Inc., and Vermillion. R.M. Hershberg holds ownership interest have been observed with other immunotherapies such as ipili- (including patents) in VentiRx Pharmaceuticals. G. Coukos reports receiving commercial research grants from Boehringer-Ingelheim and Celgene; reports mumab (34, 35). receiving speakers' bureau honoraria from Genentech and Roche; and is a Although the phase I safety data are encouraging, it is important consultant/advisory board member for Genetech, Novartis, Roche, and Sanofi- to note that PLD shortage during the study likely limited the Aventis. No potential conflicts of interest were disclosed by the other authors. . understanding of the clinical effect of the chemoimmunotherapy combination and may have tainted the toxicity endpoints, as PLD Authors' Contributions is known to have delayed toxicity as well as efficacy. On the basis Conception and design: B.J. Monk, A. Facciabene, W.E. Brady, C.A. Aghajanian, of these preclinical and initial clinical data, a randomized, P.M. Fracasso, J.L. Walker, H.A. Lankes, K.L. Manjarrez, R.M. Hershberg, placebo-controlled trial comparing PLD to PLD plus motoli- G. Coukos mod in platinum-resistant ovarian cancer was initiated (GOG- Development of methodology: B.J. Monk, A. Facciabene, C.A. Aghajanian, 3003, NCT01666444). This trial enrolled nearly 300 women; P.M. Fracasso, J.L. Walker, H.A. Lankes, G.H. Danet-Desnoyers, R.M. Hershberg, G. Coukos data for the primary endpoint of overall survival are expected Acquisition of data (provided animals, acquired and managed patients, before the end of 2016. In addition, the immune effects elicited provided facilities, etc.): B.J. Monk, A. Facciabene, C.A. Aghajanian, J.L. Walker, by the combination suggest that PLD/motolimod chemoim- H.A. Lankes, G.H. Danet-Desnoyers, K.M. Bell-McGuinn, C.K. McCourt, munotherapy could be effectively combined with approaches A. Malykhin further enhancing T-cell function, such as checkpoint blockade Analysis and interpretation of data (e.g., statistical analysis, biostatistics, immunotherapy. This hypothesis is being tested in a phase I/IIa computational analysis): B.J. Monk, W.E. Brady, C.A. Aghajanian, P.M. Fra- – casso, J.L. Walker, H.A. Lankes, K.L. Manjarrez, G.H. Danet-Desnoyers, study assessing the PLD/motolimod combination with an anti A. Malykhin, R.M. Hershberg, G. Coukos PD-L1 neutralizing antibody in the same patient population Writing, review, and/or revision of the manuscript: B.J. Monk, A. Facciabene, (NCT02431559). W.E. Brady, C.A. Aghajanian, P.M. Fracasso, J.L. Walker, H.A. Lankes, In summary, we followed an integrative pharmacologic K.L. Manjarrez, G.H. Danet-Desnoyers, K.M. Bell-McGuinn, C.K. McCourt, approach that used traditional approaches, as well as a human- R.M. Hershberg, G. Coukos ized mouse model to advance the clinical development of moto- Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): B.J. Monk, W.E. Brady, J.L. Walker, H.A. Lankes, limod for cancer therapy. The NSG-HIS model mimicked human K.L. Manjarrez, G.H. Danet-Desnoyers donors and non-human primates and proved useful in exploring Study supervision: B.J. Monk, W.E. Brady, C.A. Aghajanian, P.M. Fracasso, combinations of motolimod with chemotherapy drugs, allowing J.L. Walker, H.A. Lankes, G. Coukos identification of appropriate combination schedules and dissect- ing interactions at the level of the TME. Despite its limitations, this Acknowledgments model offers important advantages including experimental We thank Gregory N. Dietsch, PhD, for providing the analysis of pharma- throughput and cost containment relative to non-human pri- cokinetic and pharmacodynamic samples; Raphael Gottardo, PhD, for provid- mates. In addition, this model solves a major limitation in cancer ing statistical analyses of the preclinical data; and Eilidh Williamson, PhD, for drug development by allowing assessment of the effects of drug providing medical writing assistance. combinations on the human peripheral immune system and in the TME. Further optimization of mice reconstituted with a Grant Support human immune system and bearing human tumor grafts can be This study was supported by National Cancer Institutegrants: GOG Tissue envisioned, including the use of autologous tumor and peripheral Bank (U10 CA27469, U24 CA114793, and NRG Oncology Operations grant number U10 CA180868) as well as NRG SDMCgrant U10 CA180822,) and the HSCs to obviate current limitations from alloreactive graft GOG Statistical and Data Center (CA37517). GOG-9925 was an NRG Oncology models. and Gynecologic Oncology Group study. Support was also provided by VentiRx Pharmaceuticals. Disclosure of Potential Conflicts of Interest The costs of publication of this article were defrayed in part by the payment of B.J. Monk reports receiving commercial research grants from Amgen, Array, page charges. This article must therefore be hereby marked advertisement in Eli Lilly, Genentech, Janssen/Johnson & Johnson, Morphotek, Novartis, and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. TESARO, Inc.; reports receiving speakers' bureau honoraria from AstraZeneca, Janssen/Johnson & Johnson, Myriad, and Roche/Genentech; and is a consul- Received June 7, 2016; revised August 19, 2016; accepted September 8, 2016; tant/advisory board member for Amgen, AstraZeneca, Bayer, Cerulean, Clovis, published OnlineFirst October 4, 2016.

References 1. Casares N, Pequignot MO, Tesniere A, Ghiringhelli F, Roux S, Chaput N, 5. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor myeloid cells by tumours. Nat Rev Immunol 2012;12:253–68. cell death. J Exp Med 2005;202:1691–701. 6. O'Neill LA, Golenbock D, Bowie AG. The history of Toll-like recep- 2. Zitvogel L, Kepp O, Senovilla L, Menger L, Chaput N, Kroemer G. Immu- tors - redefining innate immunity. Nat Rev Immunol 2013;13: nogenic tumor cell death for optimal anticancer therapy: the calreticulin 453–60. exposure pathway. Clin Cancer Res 2010;16:3100–4. 7. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune 3. Aguilar-Cazares D, Meneses-Flores M, Prado-Garcia H, Islas-Vazquez L, responses. Nat Immunol 2004;5:987–95. Rojo-Leon V, Romero-Garcia S, et al. Relationship of dendritic cell density, 8. Liu J, Xu C, Hsu LC, Luo Y, Xiang R, Chuang TH. A five-amino-acid motif in HMGB1 expression, and tumor-infiltrating lymphocytes in non-small cell the undefined region of the TLR8 ectodomain is required for species- lung carcinomas. Appl Immunohistochem Mol Morphol 2014;22:105–13. specific ligand recognition. Mol Immunol 2010;47:1083–90. 4. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll- 9. Lu H, Dietsch GN, Matthews MA, Yang Y, Ghanekar S, Inokuma M, et al. like receptor 4-dependent contribution of the immune system to anticancer VTX-2337 is a novel TLR8 agonist that activates NK cells and augments chemotherapy and radiotherapy. Nat Med 2007;13:1050–9. ADCC. Clin Cancer Res 2012;18:499–509.

www.aacrjournals.org Clin Cancer Res; 2016 OF11

Monk et al.

10. Northfelt DW, Ramanathan RK, Cohen PA, Von Hoff DD, Weiss GJ, 24. Alagkiozidis I, Facciabene A, Carpenito C, Benencia F, Jonak Z, Adams S, Dietsch GN, et al. A phase I dose-finding study of the novel Toll-like et al. Increased immunogenicity of surviving tumor cells enables coop- receptor 8 agonist VTX-2337 in adult subjects with advanced solid tumors eration between liposomal doxorubicin and IL-18. J Transl Med or lymphoma. Clin Cancer Res 2014;20:3683–91. 2009;7:104. 11. Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated 25. Gaur U, Aggarwal BB. Regulation of proliferation, survival and apoptosis liposomal doxorubicin: review of animal and human studies. Clin Phar- by members of the TNF superfamily. Biochem Pharmacol 2003;66: macokinet 2003;42:419–36. 1403–8. 12. Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani 26. Xiao CW, Yan X, Li Y, Reddy SA, Tsang BK. Resistance of human ovarian G, et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer cells to tumor necrosis factor alpha is a consequence of nuclear cancer. N Engl J Med 2003;348:203–13. factor kappaB-mediated induction of Fas-associated death domain-like 13. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, et al. Intraepithelial interleukin-1beta-converting enzyme-like inhibitory protein. Endocrinol- CD8þ tumor-infiltrating lymphocytes and a high CD8þ/regulatory T cell ogy 2003;144:623–30. ratio are associated with favorable prognosis in ovarian cancer. Proc Natl 27. Kelly MM, Hoel BD, Voelkel-Johnson C. Doxorubicin pretreatment Acad Sci U S A 2005;102:18538–43. sensitizes prostate cancer cell lines to TRAIL induced apoptosis which 14. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, et al. Specific correlates with the loss of c-FLIP expression. Cancer Biol Ther 2002; recruitment of regulatory T cells in ovarian carcinoma fosters immune 1:520–7. privilege and predicts reduced survival. Nat Med 2004;10:942–9. 28. Piao X, Komazawa-Sakon S, Nishina T, Koike M, Piao JH, Ehlken H, et al. c- 15. Coleman RL, Monk BJ, Sood AK, Herzog TJ. Latest research and treatment FLIP maintains tissue homeostasis by preventing apoptosis and pro- of advanced-stage epithelial ovarian cancer. Nat Rev Clin Oncol grammed necrosis. Sci Signal 2012;5:ra93. 2013;10:211–24. 29. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. 16. Govindaraj RG, Manavalan B, Basith S, Choi S. Comparative analysis of Nature 2011;480:480–9. species-specific ligand recognition in Toll-like receptor 8 signaling: a 30. van der Sluis TC, van Duikeren S, Huppelschoten S, Jordanova ES, Beyr- hypothesis. PLoS One 2011;6:e25118. anvand Nejad E, Sloots A, et al. Vaccine-induced tumor necrosis factor- 17. Dietsch GN, Randall TD, Gottardo R, Northfelt DW, Ramanathan RK, producing T cells synergize with cisplatin to promote tumor cell death. Clin Cohen PA, et al. Late-stage cancer patients remain highly responsive to Cancer Res 2015;21:781–94. immune activation by the selective TLR8 agonist motolimod (VTX-2337). 31. Monk BJ, Herzog TJ, Kaye SB, Krasner CN, Vermorken JB, Muggia FM, et al. Clin Cancer Res 2015;21:5445–52. Trabectedin plus pegylated liposomal doxorubicin in recurrent ovarian 18. Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, et al. cancer. J Clin Oncol 2010;28:3107–14. Inhibition of death receptor signals by cellular FLIP. Nature 1997;388: 32. Colombo N, Kutarska E, Dimopoulos M, Bae DS, Rzepka-Gorska I, 190–5. Bidzinski M, et al. Randomized, open-label, phase III study comparing 19. Le Tourneau C, Lee JJ, Siu LL. Dose escalation methods in phase I cancer patupilone (EPO906) with pegylated liposomal doxorubicin in plati- clinical trials. J Natl Cancer Inst 2009;101:708–20. num-refractory or -resistant patients with recurrent epithelial ovarian, 20. Gorden KB, Gorski KS, Gibson SJ, Kedl RM, Kieper WC, Qiu X, et al. primary fallopian tube, or primary peritoneal cancer. J Clin Oncol Synthetic TLR agonists reveal functional differences between human TLR7 2012;30:3841–7. and TLR8. J Immunol 2005;174:1259–68. 33. Vergote I, Finkler NJ, Hall JB, Melnyk O, Edwards RP, Jones M, et al. 21. Tanaka S, Saito Y, Kunisawa J, Kurashima Y, Wake T, Suzuki N, et al. Randomized phase III study of canfosfamide in combination with pegy- Development of mature and functional human myeloid subsets in lated liposomal doxorubicin compared with pegylated liposomal doxo- hematopoietic stem cell-engrafted NOD/SCID/IL2rgammaKO mice. J rubicin alone in platinum-resistant ovarian cancer. Int J Gynecol Cancer Immunol 2012;188:6145–55. 2010;20:772–80. 22. Alagkiozidis I, Facciabene A, Tsiatas M, Carpenito C, Benencia F, Adams S, 34. McDermott D, Lebbe C, Hodi FS, Maio M, Weber JS, Wolchok JD, et al. et al. Time-dependent cytotoxic drugs selectively cooperate with IL-18 for Durable benefit and the potential for long-term survival with immu- cancer chemo-immunotherapy. J Transl Med 2011;9:77. notherapy in advanced melanoma. Cancer Treat Rev 2014;40: 23. Simpkins F, Flores A, Chu C, Berek JS, Lucci J III, Murray S, et al. Che- 1056–64. moimmunotherapy using pegylated liposomal Doxorubicin and interleu- 35. Hoos A, Eggermont AM, Janetzki S, Hodi FS, Ibrahim R, Anderson A, et al. kin-18 in recurrent ovarian cancer: a phase I dose-escalation study. Cancer Improved endpoints for cancer immunotherapy trials. J Natl Cancer Inst Immunol Res 2013;1:168–78. 2010;102:1388–97.

OF12 Clin Cancer Res; 2016 Clinical Cancer Research

Integrative Development of a TLR8 Agonist for Ovarian Cancer Chemoimmunotherapy

Bradley J. Monk, Andrea Facciabene, William E. Brady, et al.

Clin Cancer Res Published OnlineFirst October 4, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-1453

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2016/10/04/1078-0432.CCR-16-1453.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2017/02/01/1078-0432.CCR-16-1453. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.