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Purpose:The success of checkpoint blockade has led to a significant increase in the development of a broad range of immunomodulatory molecules for the treatment of cancer, including agonists against T-cell costimulatory receptors, such as OX40. Unlike checkpoint blockade, where complete and sustained receptor saturation may be required for maximal activity, the optimal dosing regimen and receptor occupancy for agonist agents is less well understood and requires further study.Experimental Design:We integrated both preclinical and clinical biomarker data sets centered on dose, exposure, receptor occupancy, receptor engagement, and downstream pharmacodynamic changes to model the optimal dose and schedule for the OX40 agonist antibody BMS-986178 alone and in combination with checkpoint blockade.Results:Administration of the ligand-blocking anti-mouse surrogate antibody OX40.23 or BMS-986178 as monotherapy or in combination with checkpoint blockade led to increased peripheral CD4+ and CD8+ T-cell activation in tumor-bearing mice and patients with solid tumors, respectively. OX40 receptor occupancy between 20% and 50% both in vitro and in vivo was associated with maximal enhancement of T-cell effector function by anti-OX40 treatment, whereas a receptor occupancy > 40% led to a profound loss in OX40 receptor expression, with clear implications for availability for repeat dosing.Conclusions:Our results highlight the value of an integrated translational approach applied during early clinical development to aggregate preclinical and clinical data in an effort to define the optimal dose and schedule for T-cell agonists in the clinic.

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Purpose: The success of checkpoint blockade has led to a significant increase in the development of a broad range of immunomodulatory molecules for the treatment of cancer, including agonists against T-cell costimulatory receptors, such as OX40. Unlike checkpoint blockade, where complete and sustained receptor saturation may be required for maximal activity, the optimal dosing regimen and receptor occupancy for agonist agents is less well understood and requires further study. Experimental Design: We integrated both preclinical and clinical biomarker data sets centered on dose, exposure, receptor occupancy, receptor engagement, and downstream pharmacodynamic changes to model the optimal dose and schedule for the OX40 agonist antibody BMS-986178 alone and in combination with checkpoint blockade. Results: Administration of the ligand-blocking anti-mouse surrogate antibody OX40.23 or BMS-986178 as monotherapy or in combination with checkpoint blockade led to increased peripheral CD4+ and CD8+ T-cell activation in tumor-bearing mice and patients with solid tumors, respectively. OX40 receptor occupancy between 20% and 50% both in vitro and in vivo was associated with maximal enhancement of T-cell effector function by anti-OX40 treatment, whereas a receptor occupancy > 40% led to a profound loss in OX40 receptor expression, with clear implications for availability for repeat dosing. Conclusions: Our results highlight the value of an integrated translational approach applied during early clinical development to aggregate preclinical and clinical data in an effort to define the optimal dose and schedule for T-cell agonists in the clinic.

Recent advances in cancer treatment with checkpoint blockade of receptors such as CTLA-4 and PD-1 have demonstrated that combinations of agents with complementary immunomodulatory effects have the potential to enhance antitumor activity as compared to single agents. We investigated the efficacy of immune-modulatory interleukin-21 (IL-21) combined with checkpoint blockade in several syngeneic mouse tumor models. After tumor establishment, mice were administered recombinant mouse IL-21 (mIL-21) alone or in combination with blocking monoclonal antibodies against mouse PD-1 or CTLA-4. In contrast to monotherapy, IL-21 enhanced antitumor activity of mCTLA-4 mAb in four models and anti-PD-1 mAb in two models, with evidence of synergy for one or both of the combination treatments in the EMT-6 and MC38 models. The enhanced efficacy was associated with increased intratumoral CD8+ T cell infiltrates, CD8+ T cell proliferation, and increased effector memory T cells, along with decreased frequency of central memory CD8+ T cells. In vivo depletion of CD8+ T cells abolished the antitumor activities observed for both combination and monotherapy treatments, further supporting a beneficial role for CD8+ T cells. In all studies, the combination therapies were well tolerated. These results support the hypothesis that the combination of recombinant human IL-21 with CTLA-4 or PD-1 monoclonal antibodies could lead to improved outcomes in cancer patients.

Background: Multicolor flow cytometry has provided deep characterization of tumor-infiltrating lymphocytes (TILs). Routine fluorescence-activated cell sorting (FACS) methods discard cells after counting, precluding transcriptome analysis, which is key to understanding the activation states of immune cells. FACS-sorted cells pose 3 problems for transcriptomics: low cell count, cell rupture from high-pressure sort process, and inefficient centrifugation and recovery of cells from large collection volumes. SMART-Seq (TaKaRa/Clontech) is a highly sensitive, reverse transcriptase-based method to amplify low-input RNA that is commonly used to generate RNA-Seq libraries. After FACS, cell RNA can be purified by Qiagen RNeasy or nucleic acid binding solid-phase reversible immobilization (SPRI) magnetic beads. Alternatively, cells can be spun down after FACS then directly lysed as input to the SMART-Seq method. We sought to compare different methods and design a high-throughput protocol to capture the transcriptome from FACS-sorted, rare immune cell types. Methods: A dilution series of 50-5000 human T cells was generated to test efficiency and sensitivity of RNA extraction. We compared RNA extraction yields by column (Qiagen RNeasy Low Input Micro Kit) or SPRI bead (Agencourt RNA-Advanced Cell v2, Beckman Coulter). Direct lysis of cells in SMART-Seq buffer was tested as an alternative to extraction. RNA yields were quantified by High Sensitivity Agilent Bioanalyzer or digital droplet PCR (BioRad). Extracted RNA was reverse transcribed and amplified using SMART-Seq v2 followed by library preparation (Nextera XT DNA kit) and sequencing (NovaSeq Illumina). To obtain TILs, mice were implanted subcutaneously with MC38 tumor cells, then tumors were harvested, disassociated, and FACS sorted to isolate T cells (CD4+, CD8+) and macrophages (yields ranged from 1000-8000 cells). Results: Direct lysis followed by SMART-Seq was found to work well with diluted T cells; however, the method was sensitive to large buffer volumes and was unreliable for small numbers of pelleted, FACS-sorted cells ( Conclusions: Use of SPRI beads to extract RNA from FACS-sorted immune cells was sensitive and reproducible, enabling transcriptomic characterization of Citation Format: Aiqing He, Manling Ma-Edmonds, Chan Gao, Vishal Patel, David Galbraith, Yan Zhang, Qi Guo, Heshani DeSilva, Gennaro Dito, Amy Truong, Sunil Kuppasani, Kandasamy Ravi, Omar Jabado. Accessing the transcriptome of low-abundance tumor-infiltrating lymphocytes with SPRI beads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2807.

Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) binding agent, has proven to be an effective monotherapy for metastatic melanoma and has shown antitumor activity in trials when administered with other therapeutic agents. We hypothesized that the combination of ipilimumab with chemotherapeutic agents, such as ixabepilone, paclitaxel, etoposide, and gemcitabine, may produce therapeutic synergy based on distinct but complementary mechanisms of action for each drug and unique cellular targets. This concept was investigated using a mouse homolog of ipilimumab in preclinical murine tumor models, including SA1N fibrosarcoma, EMT-6 mammary carcinoma, M109 lung carcinoma, and CT-26 colon carcinoma. Results of CTLA-4 blockade in combination with one of various chemotherapeutic agents demonstrate that synergy occurs in settings where either agent alone was not effective in inducing tumor regression. Furthermore, when combined with CTLA-4 blockade, ixabepilone, etoposide, and gemcitabine elicited prolonged antitumor effects in some murine models with induction of a memory immune response. Future investigations are warranted to determine which specific chemo-immunotherapy combinations, if any, will produce synergistic antitumor effects in the clinical setting.

Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report methodology using primers for a portion of the H2-kb murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 &mgr;M primer, a 20-fold increase in the median manufacturer’s recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 &mgr;g of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection limit half as sensitive as that found when no carrier DNA was present.

Elotuzumab is a humanized monoclonal antibody (mAb) that binds specifically to Signaling Lymphocytic Activation Molecule F7 (SLAMF7, also known as CS1), a glycoprotein expressed on the surface of multiple myeloma (MM) tumor cells. SLAMF7 has not been detected on hematopoietic stem cells or on other normal tissues, but is expressed on some normal leukocytes, including natural killer (NK) cells. Elotuzumab enhances NK cell activation directly via SLAMF7 and also enhances NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) of SLAMF7-expressing MM cells. NK cell-mediated ADCC can be enhanced by stimulating activating NK receptors as well as by blocking inhibitory NK receptors. IL-21 is a pleiotropic cytokine that has been shown to augment the ability of NK cells to mediate ADCC. CD137 (4-1BB) agonism has been shown to enhance both NK cell ADCC in vitro and the efficacy of a tumor-specific monoclonal antibody in a syngeneic mouse tumor model. NK cell-mediated ADCC can be reduced by inhibitory signaling via killer immunoglobulin-like receptors (KIRs) on NK cells, which recognize MHC class I molecules as ligands. Lirilumab is a human IgG4 mAb that binds inhibitory KIR2DL1/2/3, blocking interaction with HLA-C ligands and promoting NK cell activity. We investigated the ability of IL-21, agonistic CD137 mAb, and lirilumab to augment elotuzumab activity in vitro and in mouse models to support and guide clinical evaluation. Standard cytotoxicity assays were used to assess in vitro ADCC with human peripheral blood mononuclear cells or purified NK cells as effectors and SLAMF7-expressing tumor cells as targets. Flow cytometry was used to evaluate CD107a expression on NK cells. Efficacy was assessed in immunodeficient mice using a SLAMF7-expressing OPM2 human MM subcutaneous xenograft model. IL-21 was able to increase elotuzumab-mediated ADCC in vitro, but showed little or no enhancement of elotuzumab activity in the OPM2 tumor model. CD137 agonism showed minimal enhancement of elotuzumab ADCC in vitro, but was able to synergize with elotuzumab in vivo to mediate potent anti-tumor activity in the OPM2 xenograft tumor model. Tumors progressed in all eight of the mice treated with either buffer control or with anti-CD137 mAb alone. Of the eight mice treated with elotuzumab alone, one achieved a complete regression by the end of the study. In contrast, seven of eight mice in the group treated with both elotuzumab and anti-CD137 mAb achieved complete regression. Blocking the inhibitory KIR pathway with lirilumab augmented elotuzumab-mediated ADCC in vitro and synergized with elotuzumab to mediate potent anti-tumor activity using a mouse model expressing a KIR2DL3 transgene on a RAG-deficient background. Tumors progressed in all ten mice treated with either isotype control antibody or with lirilumab alone. Of the ten mice treated with elotuzumab alone, two achieved partial regressions and another mouse achieved a complete regression by the end of the study. Six of the ten mice in the group treated with both elotuzumab and lirilumab achieved complete regression and one mouse achieved a partial regression. Together, these approaches demonstrate that targeting activating and inhibitory receptors on NK cells can enhance or synergize with elotuzumab to increase NK cell ADCC towards myeloma cells. This study is being funded by Bristol-Myers Squibb. Editorial assistance was provided by Caudex Medical and was funded by Bristol-Myers Squibb. Disclosures Robbins: Bristol-Myers Squibb: Employment; Bristol-Myers Squibb: Equity Ownership. Jure-Kunkel:Bristol-Myers Squibb: Employment; Bristol-Myers Squibb: Equity Ownership. Dito:Bristol-Myers Squibb: Employment. Andre:Innate Pharma: Employment. Zhang:Bristol-Myers Squibb: Employment. Bezman:Bristol-Myers Squibb: Employment. Graziano:Bristol-Myers Squibb: Employment; Bristol-Myers Squibb: Equity Ownership.

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Nivolumab (BMS-936558; MDX-1106; ONO-4538) is a fully human IgG4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), inhibiting the downregulation of antitumor T-cell functions. Nivolumab has shown activity in advanced solid tumors, including renal cell carcinoma (RCC), melanoma, and non-small cell lung cancer (Topalian SL, et al. NEJM 2012;366:2443-54). Sunitinib and sorafenib are anti-angiogenic tyrosine kinase inhibitors (TKIs) used for the treatment of RCC. We investigated the activity of nivolumab in combination with TKIs in a preclinical RCC murine model. Methods: The murine RCC (Renca) tumor cell line was maintained in vitro and implanted subcutaneously into 8-12 week old female Balb/c mice. When mean tumor volume reached approximately 90-100 mm3, mice were randomized into groups of eight. Vehicle control, sunitinib 120 mg/kg, or sorafenib 200 mg/kg were administered orally once daily for 14 days. The nivolumab surrogate antibody, an IgG1 anti-mouse PD-1 monoclonal antibody (clone 4H2), was administered at 10 mg/kg by intraperitoneal injection every four days for four cycles. Immunohistochemistry and flow cytometry analyses were used to assess immune cell infiltration of tumors. Results: Sunitinib monotherapy showed activity in the Renca murine RCC model producing tumor growth inhibition of 84% by the end of treatment; however, tumors grew progressively after cessation of therapy. Conversely, while the anti-PD-1 monoclonal antibody was inactive in this model, addition of anti-PD-1 to sunitinib produced significant antitumor activity, resulting in complete tumor regressions or marked delay in tumor growth. Combination anti-PD-1 plus sunitinib therapy also had no effect on the body weight of the mice. Immunohistochemical analysis demonstrated that sunitinib monotherapy led to an influx of immune cells predominantly into the tumor periphery, whereas greater infiltration of immune cells throughout the tumor was observed with combination anti-PD-1 and sunitinib therapy. In contrast, combination treatment with anti-PD-1 antibody and sorafenib did not enhance antitumor activity in this murine RCC model. Further exploration of the mechanisms contributing to this synergy will be presented. Conclusions: Combination therapy with sunitinib and anti-PD-1 antibody demonstrated an enhanced effect against murine RCC. Safety and response to nivolumab plus sunitinib, pazopanib, or ipilimumab in patients with metastatic RCC are being assessed in an ongoing phase 1 study ([NCT01472081][1]). Citation Format: Gregg Masters, Gennaro Dito, Becky Penhallow, Anne Lewin, Henry Kao, Maria N. Jure-Kunkel. Antitumor activity of anti-PD-1 in combination with tyrosine kinase inhibitors in a preclinical renal cell carcinoma model. [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 5016. doi:10.1158/1538-7445.AM2014-5016 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01472081&atom=%2Fcanres%2F74%2F19_Supplement%2F5016.atom

3019 Background: Interleukin 21 (IL-21), a γc chain family cytokine, is produced primarily by CD4+ T cells and has many effects on immune cells, including enhancing CD8+ T cell and NK cell proliferation and cytotoxicity. Recombinant IL-21 (rIL-21) therapy resulted in objective responses in ~20% of melanoma and renal cell carcinoma patients. In mouse models, monoclonal antibody (mAb) blockade of CTLA-4 prolongs antigen-specific T cell responses, while blockade of programmed death 1 (PD-1) reverses tumor induced T cell suppression. Ipilimumab, a CTLA-4 blocking mAb, significantly improved overall survival in patients with metastatic melanoma in 2 phase III trials, and in phase I studies a PD-1 blocking mAb (nivolumab) has antitumor activity in various cancers. Side effect profiles for each mAb have been related to their mechanism and are generally manageable. It was hypothesized that combination of IL-21 plus CTLA-4 or PD-1 blockade may enhance antitumor responses, potentially leading to improved clinical activity. Methods: Preclinical studies were conducted to test the antitumor activity of mouse IL-21 (mIL-21) in combination with an anti-mouse PD-1 (mPD-1) mAb (4H2-IgG1) or with an anti-mCTLA-4 blocking mAb (9D9-IgG2b) in syngeneic mouse tumor models, including MC38, CT-26, EMT-6, and B16F10. mIL-21 was tested at doses ranging from 50-200 μg/dose, administered up to 3d/wk. mCTLA-4 mAb or mPD-1 mAb were administered 3-4x total at 200-300 μg/dose. Results: Combination treatments produced enhanced antitumor activity vs. monotherapy. In the MC38 model, mIL-21 treatment led to 30% median tumor growth inhibition (TGI) by d29, while mPD-1 mAb produced 60% median TGI and 1/10 tumor-free mice. Combination of both agents led to synergistic antitumor activity, with complete regressions (CR) in 7/10 mice and 99.9% median TGI (p=0.046). CTLA-4 mAb + mIL-21 also produced synergistic activity in the MC38 model. By d21, mIL-21 monotherapy induced 34% TGI while CTLA-4 mAb resulted in 28% TGI, with no CR in either group. Combination resulted in 6/8 mice with CR and 86% TGI (p

e13055 Background: The antitumor activity and immunomodulatory effect of an ipilimumab homolog antibody in combination with BMS-908662, a selective RAF inhibitor, were assessed in preclinical model...

The development and progression of prostate cancer is known to be dependent on androgens and their signaling mediated by the androgen receptor (AR). The primary therapeutic intervention involves using agents that lower serum testosterone (e.g., LHRH agonists), often in concert with an AR antagonist, such as bicalutamide. Despite a favorable initial anti-tumor response, most patients progress to the advanced hormone-refractory disease. The development of resistance to anti-androgen therapy has been shown to be associated with an increase in the levels of both AR mRNA and protein. This observation supports the concept that an AR antagonist with a significant improvement in potency as compared to bicalutamide and a broader spectrum of in vivo anti-tumor activity, including the bicalutamide-refractory human prostate tumor xenografts, may provide a significant clinical advantage in the treatment of advanced prostate cancer. This presentation will describe structure-activity relationships in a novel tetracyclic series of androgen receptor antagonists leading up to the identification of BMS-779333. It is a potent AR full antagonist, which exhibited broad spectrum efficacy in four human prostate tumor xenograft models. BMS-779333 did not exhibit agonist activity for AR mutant isoforms. Tumors that failed bicalutamide treatment were shown to retain their sensitivity to respond to BMS-779333. Transcriptomic changes in LuCaP-35 tumors treated with BMS-779333 were closer to castration than with other drug treatments. Based on its overall profile, BMS-779333 was selected for further preclinical evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5782.

3048 Background: The antitumor activity of a homolog of ipilimumab, a CTLA-4 blocking agent, was investigated in combination with ixabepilone, the first in a new class of microtubule-stabilizing dr...