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The delivery of immunomodulators directly into the tumor potentially harnesses the existing antigen, tumor-specific infiltrating lymphocytes, and antigen presenting cells. This can confer specificity and generate a potent systemic anti-tumor immune response with lower doses and less toxicity compared to systemic administration, in effect an in situ vaccine. Here, we test this concept using the novel combination of immunomodulators anti-CTLA4, -CD137, and -OX40. The triple combination administered intratumorally at low doses to one tumor of a dual tumor mouse model had dramatic local and systemic anti-tumor efficacy in lymphoma (A20) and solid tumor (MC38) models, consistent with an abscopal effect. The minimal effective dose was 10 μg each. The effect was dependent on CD8 T-cells. Intratumoral administration resulted in superior local and distant tumor control compared to systemic routes, supporting the in situ vaccine concept. In a single tumor A20 model, injection close to the tDLN resulted in similar efficacy as intratumoral and significantly better than targeting a non-tDLN, supporting the role of the tDLN as a viable immunotherapy target in addition to the tumor itself. Distribution studies confirmed expected concentration of antibodies in tumor and tDLN, in keeping with the anti-tumor results. Overall intratumoral or peri-tDLN administration of the novel combination of anti-CTLA4, anti-CD137, and anti-OX40, all agents in the clinic or clinical trials, demonstrates potent systemic anti-tumor effects. This immunotherapeutic combination is promising for future clinical development via both these safe and highly efficacious routes of administration.

The tyrosine kinase inhibitor, imatinib, has the potential to indirectly inhibit DNA repair. This mechanism of action has been shown to mediate sensitization to chlorambucil in chronic lymphocytic leukemia (CLL). To evaluate this effect in vivo, we performed a phase I study of chlorambucil combined with imatinib in relapsed CLL patients.The three dose levels studied included imatinib at 300, 400, or 600 mg/day. Imatinib was given on days 1-10, and chlorambucil (8 mg/m(2) daily) was given on days 3-7 of a 28-day cycle (up to 6 cycles).Eleven patients participated in this study. Low-grade gastrointestinal toxicities were observed in a dose-dependent manner. Forty-five percent of patients responded (two unconfirmed CRs and three PRs). Two responding patients were fludarabine refractory. The in vitro IC(50) of chlorambucil alone or in the presence of 5 μM imatinib in CLL lymphocytes correlated with the decrease in lymphocyte counts on day 15. Imatinib plasma concentrations achieved in patients were in the range of those effective in in vitro sensitization studies.The combination of chlorambucil and imatinib in patients with previously treated CLL was well tolerated and showed evidence of clinical efficacy. Based on our results, we recommend the 400 mg daily dose of imatinib on days 1-10 with 8 mg/m(3) chlorambucil on days 3-7 every 28 days as the phase II dose. This represents the first clinical trial examining the potential synergy between a tyrosine kinase inhibitor and a conventional alkylating agent for the treatment of CLL.

The peripheral T cell lymphomas (PTCLs) are a heterogeneous group of neoplasms for which standardized treatment approaches remain elusive. A number of new therapeutic agents have become available, of which monoclonal antibodies (MAbs) represent a powerful tool for targeted treatment of PTCLs. Therapeutic MAbs vary in their structure, targets, and mechanisms of action. Common mechanisms of action include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, direct apoptosis, blocking of receptors or signaling pathways, delivery of cytotoxic agents to tumor cells, and binding to and blocking biologically active molecules. This review will focus on recent published evidence for the various MAbs used in the treatment of PTCLs. The results overall have been very promising, and the future will see more trials with these antibodies alone and in various therapy combinations, as well as newer ones with novel modifications, conjugates, and targets.

International audience; Treatment with cetuximab, an EGFR-targeting IgG1 mAb, results in beneficial, yet limited, clinical improvement for patients with head and neck (HN) cancer as well as colorectal cancer (CRC) patients with WT KRAS tumors. Antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells contributes to the efficacy of cetuximab. The costimulatory molecule CD137 (4-1BB) is expressed following NK and memory T cell activation. We found that isolated human NK cells substantially increased expression of CD137 when exposed to cetuximab-coated, EGFR-expressing HN and CRC cell lines. Furthermore, activation of CD137 with an agonistic mAb enhanced NK cell degranulation and cytotoxicity. In multiple murine xenograft models, including EGFR-expressing cancer cells, HN cells, and KRAS-WT and KRAS-mutant CRC, combined cetuximab and anti-CD137 mAb administration was synergistic and led to complete tumor resolution and prolonged survival, which was dependent on the presence of NK cells. In patients receiving cetuximab, the level of CD137 on circulating and intratumoral NK cells was dependent on postcetuximab time and host FcyRIIIa polymorphism. Interestingly, the increase in CD137-expressing NK cells directly correlated to an increase in EGFR-specific CD8+ T cells. These results support development of a sequential antibody approach against EGFR-expressing malignancies that first targets the tumor and then the host immune system.

The mainstay of HER2+ breast cancer treatment has been monoclonal antibody therapy with Trastuzumab, a humanized antibody that targets HER-2. However, the efficacy of Trastuzumab monotherapy is only 10-15%. Therefore, strategies are being developed to enhance its therapeutic efficacy. Our previous study demonstrated that stimulation of NK cells with an anti-CD137 agonistic mAb enhanced Trastuzumab-mediated Antibody Dependent Cellular Cytotoxicity (ADCC). Anti-CD137 agonistic mAb enhanced anti-breast cancer activity of Trastuzumab in vivo in a xenotransplanted human breast cancer model (Kohrt et al. J Clin. Invest, 2012, 22:3). We have developed a treatment regimen consisting of sequential administration of Trastuzumab followed by CD137 antibody as three weekly injections. This regimen demonstrated more potent antitumor activity than administration of anti-CD137 mAb followed by Trastuzumab. Our combination therapy was superior to Traztuzumab alone in a primary HER2-overexpressing-breast tumor xenotransplant model but was ineffective in low HER-2 expressing breast cancer model. Next, we evaluated the therapeutic efficacy of FDA approved antibody drug conjugate Trastuzumab Emtansine (T-DM1) in combination with CD137 antibody. We used HER2 over-expressing HER18 xenograft model and an SU-258 primary breast cancer model in our studies. Following tumor inoculation, mice received either T-DM1 on day 3 (for HER18 xenograft), or day 30 (for SU-258) and anti-CD137 antibody on day 4(or HER18) or day 31(for SU-258) with each treatment repeated weekly for a total of three weeks. Sequential antibody strategy with T-DM1 and CD137 significantly improved survival compared to T-DM1 alone or with Trastuzumab and CD137 antibody. Our results demonstrate that T-DM1 retains its potency and demonstrates synergistic activity with anti-CD137 mAb therapy against HER2-overexpressing breast cancer models. Our results support a novel, sequential antibody approach against HER-2+breast cancer, by targeting first the tumor with an antibody drug conjugate and then the host immune system. Clinical investigations are now planned to determine the clinical outcome of our immunotherapy strategy. Citation Format: Suparna Dutt, Narendiran Rajasekaran, Aurelien Marabelle, Roch Houot, Mohith Sadaram, Jonathan Hebb, Idit Sagiv-Barfi, Sid Ambulkar, Amanda Rajapaksa, Cariad Chester, Erin Waller, Holbrook Kohrt. Sequential tumor and immune targeted immunotherapy: Anti-tumor activity of antibody drug conjugate Trastuzumab Emtansine (T-DM1) with CD137 stimulation in HER-2+ breast cancer therapy. [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 2466. doi:10.1158/1538-7445.AM2015-2466

Introduction: The BTK inhibitor, ibrutinib is FDA-approved in MCL and CLL, with activity in the majority of CD20+ B-cell malignancies. As rituximab-combination chemotherapy is today's standard of care in CD20+ B-cell malignancies, we previously investigated and determined ibrutinib antagonizes rituximab-dependent NK-cell mediated cytotoxicity (ADCC) due to ibrutinib's secondary irreversible binding to interleukin-2 inducible tyrosine kinase (ITK) which is required for FcR-stimulated NK cell function including calcium mobilization, granule release, and overall ADCC. We hypothesized that BTK inhibitors, BTK-InhA (ACP-196), BTK-InhB (BGB-3111) and BTK-InhC (undisclosed), each with lower ITK binding, may preserve NK cell function and therefore synergize rather than antagonize rituximab. Methods: Rituximab and trastuzumab-dependent NK-cell mediated cytotoxicity was assessed using lymphoma and HER2+ breast cancer cell lines as well as autologous CLL tumor cells. In vitro NK cell cytokine secretion, degranulation and cytotoxicity were assessed by IFN-g release, CD107a mobilization and chromium release. Results: FcR-stimulated NK cells following exposure to rituximab-coated lymphoma cells or trastuzumab-coated HER2+ breast cancer cells express high and moderate levels of ITK and BTK, respectively. Ibrutinib, in a dose-dependent manner (0.1 and 1uM), and not BTK-InhA, BTK-InhB or BTK-InhC (each at 1uM), inhibited both rituximab- and trastuzumab-induced NK cell cytokine secretion in vitro (Fig A *p=.018, **p=.002, ***p Conclusions: Ibrutinib is clinically effective as monotherapy and in combination with rituximab, despite inhibition of ADCC in vitro and in vivo murine models due to ibrutinib's secondary irreversible binding to ITK. Preclinically, the efficacy of therapeutics which do not inhibit NK cell function, including three novel BTK inhibitors, is superior to ibrutinib. Clinical investigation is needed to determine the impact of this finding on patients with lymphoma receiving rituximab. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Wang: BeiGene: Employment. Lannutti:Acerta Pharma: Employment. Johnson:Acerta Pharma: Employment.

Introduction: Kohrt et al., Blood, 2014 demonstrated that ibrutinib antagonizes ADCC function of rituximab in vitro in ADCC assays and in vivo in the DHL-4 xenograft model through inhibition of FcgammaR signaling in immune effector cells, possibly mediated by inhibition of ITK. Obinutuzumab (GA101) is a glycoengineered type II CD20 antibody that mediates higher direct cell death induction than rituximab, and by being glycoengineered mediates enhanced induction of ADCC and ADCP. Here we aimed to investigate the impact of ibrutinib on the immune effector function of obinutuzumab as compared to rituximab. Experimental methods: The impact of ibrutinib (dose range 30, 100, 300 ng/ml to cover Cmax and Ctrough in patients) on NK cell mediated ADCC induction by obinutuzumab and rituximab was investigated using SU-DHL4 and Z138 cells as targets in LDH and chromium release assays or measuring CD16 downmodulation and the degranulation marker CD107a. IFNg release as a surrogate for NK cell activation was investigated using DHL-4 target cells or an autologous in vitro system using leukemic cells derived from CLL/NHL patients. Depletion of CD19 positive B-cells was determined in whole blood from healthy volunteers in flow cytometry-based whole blood assay. In vivo the combination of obinutuzumab or rituximab (10 mg/kg once weekly for 3 weeks) with ibrutinib (25mg/kg BID days 14-28) was investigated in the DHL-4 xenograft model. Results: In ADCC assays, ibrutinib (dose range 30, 100, 300 ng/ml) resulted in a reduction of the ADCC potency of obinutuzumab and rituximab. However, at saturating antibody concentrations of 10 ug/ml, ADCC mediated by obinutuzumab was retained while ADCC mediated by rituximab was strongly reduced as measured by chromium release (Figure 1A). Interestingly, in the whole blood B cell depletion assay only little impact of ibrutinib on obinutuzumab-mediated B cell depletion in terms of EC50 and maximal killing was observed at clinically meaningful concentrations of ibrutinib (30, 100, 300 ng/ml), while the activity of rituximab could be completely abolished with 300 ng/ml ibrutinib (Figure 1B). Notably, control experiments using an effector dead version of obinutuzmab that cannot any longer mediate ADCC or ADCP demonstrate that the retained B cell depletion by obinutuzumab in presence of ibrutinib is not due to direct cell death induction, but also due to immune effector cell mediated function (ADCC and ADCP). In the DHL-4 xenograft model where ibrutinib as a single agent has no anti-tumoral efficacy, the combination resulted in a reduced anti-tumoral efficacy of rituximab, whereas efficacy of obinutuzumab was not affected (Figure 1C). Conclusions: Surprisingly, we found that the inhibitory effect of ibrutinib on the immune effector mediated activity of obinutuzumab is not observed when compared to rituximab. Most notably, ADCC at saturating antibody doses, whole blood B cell depletion and in vivo efficacy of obinutuzumab were retained in presence of clinically relevant concentrations of ibrutinib covering Cmax and Ctrough levels, whereas the activity of rituximab was almost completely abolished under these conditions. We hypothesize that the differential behavior of obinutuzumab and rituximab may be related to the enhanced FcgRIII affinity and stronger FcgRIII signaling activation mediated by obinutuzumab as a consequence of glycoengineering that may subsequently overwrite inhibitory effects of ibrutinib. While the clinical relevance of the observed preclinical antagonism for the combination of rituximab with ibrutinib still needs further clinical investigation, these preclinical data strongly support the clinical investigation of ibrutinib in combination with the glycoengineered Type II CD20 antibody obinutuzumab for the treatment of chronic lymphocytic leukemia and other B-cell malignancies. Figure 1 Figure 1. Disclosures Herter: Roche: Employment. Bacac:Roche: Employment. Umana:Roche: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Objective: Inhibition of indoleamine-2,3-dioxygenase-1 (IDO-1), indoleamine-2,3- dioxygenase-2 (IDO-2), and/or tryptophan-2,3-dioxygenase (TDO) represent novel opportunities for reversing the immunosuppressed microenvironment found in cancer patients. However, little is known about the relative expression of these enzymes in tumors and associated lymph nodes of mouse tumor models, including those previously utilized in the preclinical characterization of tool compounds or even clinical candidates that inhibit one of these enzymes. This report describes the systematic characterization of expression of these enzymes in several of the most commonly used mouse tumor models. Study Design: Tumor models evaluated included B16F10 melanoma, B16-GMCSF melanoma and PAN02 pancreatic in C57BL/6 mice, and 4T1 breast and CT26 colon in Balb/c mice. Cells were injected s.c. into the flanks of 8-week old female mice. In each model, tumors and draining lymph nodes were collected from separate cohorts of mice when the mean tumor volumes reached 100 and 1,000 mm3, respectively. Expression of the 3 enzymes of interest was assessed using a variety of techniques, including qRT-PCR, Western blot and FACS. Results: A highly complex picture arises from our study. Expression levels of each of the enzymes studied here were highly dependent on the choice of tumor cell, the tissue analyzed (tumor vs. lymph node), as well as the stage of growth of the tumor. In general, the most commonly expressed of the 3 enzymes was IDO-1, primarily on tumor cells and dendritic cells in the lymph nodes. Conclusion: It is critically important, in the preclinical evaluation of potential new drugs, to select mouse models that recapitulate specific and well-characterized elements of the signaling pathway targeted by those drugs. The results presented in this communication should be of particular interest to those investigators pursuing a therapeutic strategy of immunosuppression reversal. Citation Format: Rajkumar Noubade, Holbrook Kohrt, Lisa Marshall, Idit Sagiv-Barfi, Jonathan Hebb, Cariad Chester, Amanda Rajapaksa, Erin Waller, Steve Young, Jay Powers, Juan Jaen. Expression of tolerogenic enzymes IDO-1, IDO-2 and TDO in commonly used mouse tumor models and impact on model selection for evaluation of immunosuppression reversal by novel therapeutics. [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 3648. doi:10.1158/1538-7445.AM2014-3648

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Tumor irradiation induces innate and adaptive immune responses which, rarely, lead to tumor regression at distant sites, the abscopal effect. We have previously demonstrated that immunotherapy including Toll-like-receptor agonists (CpG) and checkpoint inhibitors (anti-CTLA4) both preclinically and clinically ([NCT00185965][1] & [NCT01769222][2]) can significantly increase the rate of systemic, abscopal responses (Kim, Blood 2012 & Brody, JCO 2010). Here we provide the first report of a preclinical murine model and patient case series following local radiation and systemic anti-PD-L1 ([NCT01375842][3]). Methods: Preclinical modeling was performed in a two-tumor, syngenic, A20, lymphoma BALB/c model combining fractionated single tumor radiation and systemic (i.p.) anti-PD-L1. Patients receiving MPDL3280A, a human mAb containing an engineered Fc-domain, as part of the phase 1 clinical trial with mixed responses or asymptomatic progression of disease were eligible for the addition of local radiation therapy. Murine and human immune responses including cell phenotype and function, specifically assessing expression of PD-L1 and production of IFNγ were determined by standard flow cytometry and time of flight mass cytometry (CyTOF). Results: Fractionated radiation delayed tumor growth at the treated site only, and systemic anti-PD-L1 reduced tumor growth rate at both sites, however despite prolonged survival all mice died by day 38 following either monotherapy (radiation or anti-PD-L1). In contrast, combination local fractionated radiation and systemic anti-PD-L1 flattened tumor growth at both the irradiated and un-irradiated site, and prolonged survival with 50% survival at day 48 post-tumor inoculation. Modulation of PD-L1 expression post-radiation and tumor-specific augmentation of IFNγ secretion correlated with the enhanced anti-tumor activity. Five patients including four with solid tumors received fractionated, non-definitive dose radiation with at least stabilization of systemic progression in all patients and a RECIST partial response at systemic sites in 1 patient, notably with a synovial sarcoma. Transient, grade 1-2 inflammatory adverse events (fevers, flu-like symptoms) occurred with no DLTs or serious immune-related toxicities. Modulation of PD-L1 expression, T cell phenotype and IFNγ secretion was observed and updated clinical and immune response will be presented. Conclusion: We provide the first report of combination local radiotherapy with anti-PD-L1 demonstrating synergy in a preclinical model and clinical activity in a limited case series. The magnitude of the immune response and abscopal response rate in mice and humans provides proof-of-concept that anti-PD-L1 may be an equally if not more potent combination immunotherapy with radiation compared to our experience with CpG and/or anti-CTLA4. Citation Format: Idit Sagiv-Barfi, Amanda Rajapaksa, Debra Czerwinski, Serena Chang, Jonathan Hebb, Cariad Chester, Erin Waller, Gregg Fine, Daniel Chen, Marcin Kowanetz, Bryan Irving, Ronald Levy, Holbrook Kohrt. Local tumor irradiation combined with α-PDL-1 immune checkpoint inhibition results in local and systemic anti-tumor responses: Successful translation of a mouse model to a human case series. [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 2941. doi:10.1158/1538-7445.AM2014-2941 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00185965&atom=%2Fcanres%2F74%2F19_Supplement%2F2941.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01769222&atom=%2Fcanres%2F74%2F19_Supplement%2F2941.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01375842&atom=%2Fcanres%2F74%2F19_Supplement%2F2941.atom

Objective: Very little is known about the cues parents use to assess pain in their children. This study has described the cues (verbal and nonverbal) parents reported using to determine how their children felt following surgery. Design and Subjects: The subjects were 176 parents of children undergoing short-stay or day surgery. Using pain diaries, parents were asked to provide written responses to the question Did your child give you any clues on how they were feeling? for the day of surgery and 2 days after their children's surgery. Parents also provided ratings of their children's pain five times per day using a visual analogue scale. Setting: The study was conducted at a tertiary care children's hospital. Results and Conclusions: Parents frequently cited using verbal report and appetite as cues to how their children were feeling. A variety of other cue types were also reported by parents, including activity level, sleep quality, visible/ audible discomfort, and physiological observations. Cue types were not significantly related to the child's gender, and only one cue type was significantly related to the child's age (appetite was used more often for older children than younger children). The presence or absence of illness behavior cues (e.g., protective behavior, visible/audible discomfort) as well as disruptions to normal behavior pattern cues (e.g., sleep, level of activity) was related, in the expected direction, to the pain intensity ratings. This study provides insights into the cues parents use to assess pain in their children and serves as a foundation for future studies on parents' assessment of children's pain.

Meeting abstracts To select maximally-efficacious, minimally-toxic regimens of combination tumor- and immune-targeted therapy, preclinical testing in immune-competent models is required. We previously demonstrated 4-1bb(CD137) stimulation augmented the innate immune response mediated by CD137+