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PURPOSE To assess the efficacy of pembrolizumab in patients with advanced relapsed or refractory mycosis fungoides (MF) or Sézary syndrome (SS). PATIENTS AND METHODS CITN-10 is a single-arm, multicenter phase II trial of 24 patients with advanced MF or SS. Patients were treated with pembrolizumab 2 mg/kg every 3 weeks for up to 24 months. The primary end point was overall response rate by consensus global response criteria. RESULTS Patients had advanced-stage disease (23 of 24 with stage IIB to IV MF/SS) and were heavily pretreated with a median of four prior systemic therapies. The overall response rate was 38% with two complete responses and seven partial responses. Of the nine responding patients, six had 90% or more improvement in skin disease by modified Severity Weighted Assessment Tool, and eight had ongoing responses at last follow-up. The median duration of response was not reached, with a median response follow-up time of 58 weeks. Immune-related adverse events led to treatment discontinuation in four patients. A transient worsening of erythroderma and pruritus occurred in 53% of patients with SS. This cutaneous flare reaction did not result in treatment discontinuation for any patient. The flare reaction correlated with high PD-1 expression on Sézary cells but did not associate with subsequent clinical responses or lack of response. Treatment responses did not correlate with expression of PD-L1, total mutation burden, or an interferon-γ gene expression signature. CONCLUSION Pembrolizumab demonstrated significant antitumor activity with durable responses and a favorable safety profile in patients with advanced MF/SS.

Purpose: Preclinical data established IL15 as a homeostatic factor and powerful stimulator of NK and CD8+ T-cell function, the basis for clinical testing.Experimental Design: A first-in-human outpatient phase I dose escalation trial of subcutaneous (SC) rhIL15 was conducted in refractory solid tumor cancer patients. Therapy consisted of daily (Monday–Friday) subcutaneous injections of rhIL15 for two consecutive weeks (10 total doses/cycle). Clinical response was assessed by RECIST. Pharmacokinetics of rhIL15 and immune biomarkers were evaluated.Results: Nineteen patients were treated with rhIL15 at dose levels of 0.25, 0.5, 1, 2, and 3 mcg/kg/day. Fourteen patients completed ≥ 2 cycles of therapy that was well tolerated. One serious adverse event (SAE), grade 2 pancreatitis, required overnight hospitalization. Enrollment was halted after a patient receiving 3 mcg/kg/day developed a dose-limiting SAE of grade 3 cardiac chest pain associated with hypotension and increased troponin. No objective responses were observed; however, several patients had disease stabilization including a renal cell carcinoma patient who continued protocol treatment for 2 years. The treatment induced profound expansion of circulating NK cells, especially among the CD56bright subset. A proportional but less dramatic increase was found among circulating CD8+ T cells with maximal 3-fold expansion for the 2 and 3 mcg/kg patients.Conclusions: SC rhIL15 treatment was well tolerated, producing substantial increases in circulating NK and CD8+ T cells. This protocol establishes a safe outpatient SC rhIL15 regimen of 2 mcg/kg/day dosing amenable to self-injection and with potential as a combination immunotherapeutic agent. Clin Cancer Res; 24(7); 1525–35. ©2017 AACR.

Purpose: Human tumors consist of heterogeneous populations of cells with distinct marker expression and functional properties. In squamous cell carcinoma of the head and neck (SCCHN), CD44 is a well-characterized marker of a resilient subpopulation of cells associated with increased tumorigenesis, radioresistance, and chemoresistance. Evidence indicates that these cells have an immunosuppressive phenotype; however, mechanisms have been elusive. Experimental Design: Using primary human SCCHN tumor samples and patient-derived xenografts, we examined the phenotypes of subsets of tumor cells and investigated mechanisms regulating their immunogenicity. Results: CD44+ cells in primary human SCCHN were found to have an epithelial-to-mesenchymal (EMT) phenotype and were less immunogenic than CD44− cells when cultured with autologous CD8+ tumor-infiltrating T cells. Selective expression of the programmed death-ligand 1 (PD-L1) was observed on CD44+ cells compared with CD44− cells and was associated with constitutive phosphorylation of STAT3 on CD44+ cells. Importantly, inhibition of STAT3 decreased expression of PD-L1 on CD44+ cells. IFNγ treatment preferentially induced even further PD-L1 expression on CD44+ cells and was associated with enhanced IFNγ receptor expression and phosphorylation of STAT1. Finally, the decreased immunogenicity of CD44+ cells was partially reversed by antibody blockade of the programmed death 1 (PD-1) receptor, indicating that the differences in PD-L1 expression between CD44+ and CD44− cells are biologically and clinically relevant. Conclusions: Our findings provide a mechanism by which long-lived CD44+ tumor-initiating cells can selectively evade host immune responses and provide rationale for targeting the PD-1 pathway in the adjuvant therapy setting of SCCHN. Clin Cancer Res; 22(14); 3571–81. ©2016 AACR.

The recent success of checkpoint blockers to treat cancer has demonstrated that the immune system is a critical player in the war against cancer. Historically, anticancer therapeutics have been tested in syngeneic mouse models (with a fully murine immune system) or in immunodeficient mice that allow the engraftment of human xenografts. Animal models with functioning human immune systems are critically needed to more accurately recapitulate the complexity of the human tumor microenvironment. Such models are integral to better predict tumor responses to both immunomodulatory agents and directly antineoplastic therapies. In this regard, the development of humanized models is a promising, novel strategy that offers the possibility of testing checkpoint blockers' capacity and their combination with other antitumor drugs. In this review, we discuss the strengths and weaknesses of the available animal models regarding their capacity to evaluate checkpoint blockers and checkpoint blocker-based combination immunotherapy.

The dominant cancer treatment modalities such as chemotherapy, radiotherapy, and even targeted kinase inhibitors and mAbs are limited by low efficacy, toxicity, and treatment-resistant tumor subclones. Oncolytic viral therapy offers a novel therapeutic strategy that has the potential to dramatically improve clinical outcomes. Reovirus, a double-stranded benign human RNA virus, is a leading candidate for therapeutic development and currently in phase III trials. Reovirus selectively targets transformed cells with activated Ras signaling pathways; Ras genes are some of the most frequently mutated oncogenes in human cancer and it is estimated that at least 30% of all human tumors exhibit aberrant Ras signaling. By targeting Ras-activated cells, reovirus can directly lyse cancer cells, disrupt tumor immunosuppressive mechanisms, reestablish multicellular immune surveillance, and generate robust antitumor responses. Reovirus therapy is currently being tested in combination with radiotherapy, chemotherapy, immunotherapy, and surgery. In this review, we discuss the current successes of these combinatorial therapeutic strategies and emphasize the importance of prioritizing combination oncolytic viral therapy as reovirus-based treatments progress in clinical development. Mol Cancer Ther; 15(5); 767–73. ©2016 AACR.

Vaccination has had a tremendous impact on human health by harnessing the immune system to prevent and eradicate infectious diseases and this same approach might be used in cancer therapy. Cancer vaccine development has been slowed hindered by the paucity of universal tumor-associated antigens and the difficulty in isolating and preparing individualized vaccines ex vivo. Another approach has been to initiate or stimulate an immune response in situ (at the tumor site) and thus exploit the potentially numerous tumor-associated antigens there. Here, we review the many approaches that have attempted to accomplish effective in situ vaccination, using intratumoral administration of immunomodulators to increase the numbers or activation state of either antigen present cells or T cells within the tumor.

Introduction Blockade of the immune checkpoint programmed death receptor ligand-1 (PD-L1)/PD-1 pathway has well-established clinical activity across many tumor types. PD-L1 protein expression by immunohistochemistry is emerging as a predictive biomarker of response to these therapies. Here, we examine PD-L1 expression in a thymic epithelial tumor (TET) tissue microarray (TMA). Methods The TMA contained 69 TETs and 17 thymic controls, with each case represented by triplicate cores. The TMA was stained with rabbit monoclonal antibody (clone 15; Sino Biological, Beijing, China) to human PD-L1. PD-L1 staining was scored based on intensity as follows: 0=none, 1=equivocal/uninterpretable, 2=weak, and 3=intermediate-strong. Those cases with all cores scoring three in the epithelial component were categorized as PD-L1high and the remaining as PD-L1low. Results PD-L1high scores were more frequent in TETs than in controls (68.1% versus 17.6%; p = 0.0036). PD-L1 scores and histology were significantly correlated, with higher intensity staining in World Health Organization (WHO). B2/B3/C TETs. Only 14.8% of TETs had PD-L1 staining of associated lymphocytes. In an adjusted analysis (age/sex), PD-L1high TETs had a significantly worse overall survival (hazard ratio: 5.40, 95% confidence interval: 1.13–25.89; p = 0.035) and a trend for worse event-free survival (hazard ratio: 2.94, 95% confidence interval: 0.94–9.24; p = 0.064). Conclusions PD-L1 expression was present in all cases of TETs within the epithelial component but only in a minority in the lymphocytic component. TETs stained more intensely for PD-L1 than in controls, and PD-L1high TETs were associated with more aggressive histology and worse prognosis. This study lends rationale to a clinical trial with anti-PD-1/PD-L1 therapy in this rare tumor type.

The clinical successes of CTLA4 and PD-1 immune checkpoint blockade in aggressive malignancies such as metastatic melanoma and non-small cell lung carcinoma inaugurate a new era in oncology. Indeed, as opposed to tumor-targeted therapies, it is now clear that immune-targeted therapies designed to enhance the antitumor immune response are a relevant strategy to obtain long-term tumor responses. Interestingly, the study of tumor cell death biology has recently revealed that part of radiotherapy efficacy relies on its ability to trigger an immune response against tumor cells. This "immunogenic cell death" partly relies on the generation of damage-associated molecular patterns, which can stimulate immune sensors such as toll-like receptors. Tumor radiation therapy can therefore be envisioned as a strategy to perform an in situ immunization because it can initiate the release of tumor-associated antigens, deplete immune suppressors, and stimulate antigen-presenting cells via endogenous release of toll-like receptor agonists. Moreover, combinations of radiotherapy with immune checkpoint antibodies are synergistic in preclinical models. The translation of these observations in the clinic is ongoing in early phase I/II trials.

Preclinical evidence of successful combinations of ionizing radiation with immunotherapy has inspired testing the translation of these results to the clinic. Interestingly, the preclinical work has consistently predicted the responses encountered in clinical trials. The first example came from a proof-of-principle trial started in 2001 that tested the concept that growth factors acting on antigen-presenting cells improve presentation of tumor antigens released by radiation and induce an abscopal effect. Granulocyte-macrophage colony-stimulating factor was administered during radiotherapy to a metastatic site in patients with metastatic solid tumors to translate evidence obtained in a murine model of syngeneic mammary carcinoma treated with cytokine FLT-3L and radiation. Subsequent clinical availability of vaccines and immune checkpoint inhibitors has triggered a wave of enthusiasm for testing them in combination with radiotherapy. Examples of ongoing clinical trials are described in this report. Importantly, most of these trials include careful immune monitoring of the patients enrolled and will generate important data about the proimmunogenic effects of radiation in combination with a variety of immune modulators, in different disease settings. Results of these studies are building a platform of evidence for radiotherapy as an adjuvant to immunotherapy and encourage the growth of this novel field of radiation oncology.

Immune cell infiltration in the tumor microenvironment is of prognostic and therapeutic import. These immune cell subsets can be heterogeneous and are composed of mature antigen-presenting cells, helper and effector cytotoxic T cells, toleragenic dendritic cells, tumor-associated macrophages, and regulatory T cells, among other cell types. With the development of novel drugs that target the immune system rather than the cancer cells, the tumor immune microenvironment is not only prognostic for overall patient outcome, but also predictive for likelihood of response to these immune-targeted therapies. Such therapies aim to reverse the cancer immunotolerance and trigger an effective antitumor immune response. Two major families of immunostimulatory drugs are currently in clinical development: pattern recognition receptor agonists (PRRago) and immunostimulatory monoclonal antibodies (ISmAb). Despite their immune-targeted design, these agents have so far been developed clinically as if they were typical anticancer drugs. Here, we review the limitations of this conventional approach, specifically addressing the shortcomings of the usual schedules of intravenous infusions every 2 or 3 weeks. If the new modalities of immunotherapy target specific immune cells within the tumor microenvironment, it might be preferable to deliver them locally into the tumor rather than systemically. There is preclinical and clinical evidence that a therapeutic systemic antitumor immune response can be generated upon intratumoral immunomodulation. Moreover, preclinical results have shown that therapeutic synergy can be obtained by combining PRRagos and ISmAbs to the local tumor site. Clin Cancer Res; 20(7); 1747–56. ©2014 AACR.

A new era of cancer immunotherapy has brought not only successful cancer vaccines but also immunomodulators, such as those that target checkpoint blockade in order to induce endogenous host immune responses. However, the immune system of cancer patients can be compromised through multiple means, including immune suppression by the tumor and by prior therapies such as chemotherapy and radiation. Therefore, a comprehensive means of assessing patient immunocompetence would seem helpful for determining whether patients are ready to benefit from immunotherapy, and perhaps even which immunotherapy might be most appropriate for them. Unfortunately, there are no standardized tests for immune competence, nor is there agreement on what to measure and what will be predictive of outcome. In this review, we will discuss the technologies and assays that might be most useful for this purpose. We argue for a comprehensive approach that should maximize the chances of developing predictive biomarkers for eventual clinical use.

2500 Background: People with HIV have been excluded from immuno-oncology (IO) studies. Anti- PD-1/PD-L1 therapies are approved for a growing number of cancers. We evaluated pembrolizumab (pembro) in people with HIV and cancer. Methods: CITN-12 is a multicenter phase 1 trial. Key eligibility: advanced cancer; ECOG ≤1; CD4 ≥100 cells/μL; ≥4 weeks antiretroviral therapy (ART), HIV viral load (VL) < 200 copies/mL. Exclusion: uncontrolled HBV/HCV, autoimmune disease. Participants (pts) accrued into CD4 based cohorts (C): C1 100-199; C2 200-350; C3 > 350 CD4 cells/μL. Pembro 200 mg IV administered Q3W for up to 35 doses. Adverse events (AE) evaluated by CTCAE. Immune related AE ≥ grade (Gr) 2 were events of clinical interest (irECI). Clinical benefit (tumor shrinkage or stable disease [SD] ≥24 weeks) was estimated. Data were locked for safety analysis and publication once C2 and C3 completed accrual and all pts completed ≥2 cycles. Accrual continued for 6 C1 pts and a new phase 1b Kaposi sarcoma (KS) cohort (C4). Results: 30 pts, characteristics: C1 (6), C2 and 3 (12 each), median (med) age 57 years (range 39-77), 28 men, 2 women, 60% White, 30% Black, 10% Hispanic. Med CD4 285 cells/μL (132 - 966). Cancers: KS (6), non-Hodgkin lymphoma (NHL) (5), non-AIDS defining (19) – most common: anal (6) and squamous cell skin (3). Prior radiation (19), med prior systemic therapies 2 (0-8). Safety observed over 183 cycles, med 5 (1-32). Treatment emergent AE ≥ possibly attributed to pembro mostly Gr 1-2, with 20% of pts having Gr 3. irECI: hypothyroidism (6), elevated AST/ALT (1), pneumonitis (3), rash (2), musculoskeletal (1).1 KS pt developed KSHV-associated multicentric Castleman disease (KSHV-MCD) and died of the AE. HIV was controlled and increasing CD4 counts were observed. Best response: complete (lung, 1), partial (NHL, 2), SD ≥24 weeks (KS, 2), SD < 24 weeks (13), progressive disease (10), not evaluable (2). Conclusions: Pembro has acceptable safety in cancer pts with HIV on ART and > 100 CD4 cells/µL, similar to patients without HIV. Anti-PD-1 may unmask KSHV-MCD and such KSHV-viremic patients should be excluded. Clinical benefit was noted in several tumor types. Anti-PD1 is appropriate for FDA-approved indications in this population. Patients with HIV meeting appropriate eligibility criteria should be included in IO studies. Clinical trial information: NCT02595866.

Background: In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour's response to IR has not been addressed. Methods: We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro. Results: CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs. Conclusions: These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.

Background In the era of personalized cancer medicine, identifying techniques for effectively matching patients to efficacious treatments is a critical step in the treatment process. The advent of anti-cancer immunotherapies necessitates novel approaches to biomarker identification beyond traditional genomic profiling. One promising approach is incorporation of nomograms into treatment decisions. Nomograms are prediction tools, based on statistical modeling, designed to predict treatment outcomes. As a first step toward developing a nomogram, we conducted analyses to predict CD137 expression of natural killer cells after monoclonal antibody (mAb) treatment. Patients and methods Patient, tumor and immune characteristics were collected from 199 patients with breast cancer (N = 62), head/neck cancers (N = 46) and non-Hodgkin's lymphoma (NHL) (N = 91), who were receiving mAb therapy as part of clinical trials. The difference in CD137 expression before and after mAb therapy was assessed by flow cytometry. To evaluate those who respond to mAb therapy via increased CD137 expression, we applied classification and regression trees (CART), multivariable lasso regression tools and Random Forest. Results The CD137 expression was significantly different for each cancer type [mean (SD): Breast: 6.6 (6.5); Head/Neck: 11.0 (7.0); NHL: 7.5 (7.1), P

International audience; Checkpoint inhibitors (CPI), namely anti-CTLA4 and anti-PD1/PD-L1 antibodies, demonstrated efficacy across multiple types of cancer. However, only subgroups of patients respond to these therapies. Additionally, CPI can induce severe immune-related adverse events (irAE). Biomarkers that predict efficacy and toxicity may help define the patients who may benefit the most from these costly and potentially toxic therapies. In this study, we review the main biomarkers that have been associated with the efficacy (pharmacodynamics and clinical benefit) and the toxicity (irAE) of CPIs in patients.

Purpose: Urelumab is an agonist antibody to CD137 with potential application as an immuno-oncology therapeutic. Data were analyzed to assess safety, tolerability, and pharmacodynamic activity of urelumab, including the dose selected for ongoing development in patients with advanced solid tumors and lymphoma.Experimental Design: A total of 346 patients with advanced cancers who had progressed after standard treatment received at least one dose of urelumab in one of three dose-escalation, monotherapy studies. Urelumab was administered at doses ranging from 0.1 to 15 mg/kg. Safety analyses included treatment-related and serious adverse events (AEs), as well as treatment-related AEs leading to discontinuation and death, with a focus on liver function test abnormalities and hepatic AEs.Results: Urelumab doses between 1 and 15 mg/kg given every 3 weeks resulted in a higher frequency of treatment-related AEs than 0.1 or 0.3 mg/kg every 3 weeks. Dose was the single most important factor contributing to transaminitis development, which was more frequent and severe at doses ≥1 mg/kg. At the MTD of 0.1 mg/kg every 3 weeks, urelumab was relatively well tolerated, with fatigue (16%) and nausea (13%) being the most common treatment-related AEs, and was associated with immunologic and pharmacodynamic activity demonstrated by the induction of IFN-inducible genes and cytokines.Conclusions: Integrated evaluation of urelumab safety data showed significant transaminitis was strongly associated with doses of ≥1 mg/kg. However, urelumab 0.1 mg/kg every 3 weeks was demonstrated to be safe, with pharmacodynamic activity supporting continued clinical evaluation of this dose as monotherapy and in combination with other immuno-oncology agents. Clin Cancer Res; 23(8); 1929-36. ©2016 AACR. Purpose: Urelumab is an agonist antibody to CD137 with potential application as an immuno-oncology therapeutic. Data were analyzed to assess safety, tolerability, and pharmacodynamic activity of urelumab, including the dose selected for ongoing development in patients with advanced solid tumors and lymphoma.Experimental Design: A total of 346 patients with advanced cancers who had progressed after standard treatment received at least one dose of urelumab in one of three dose-escalation, monotherapy studies. Urelumab was administered at doses ranging from 0.1 to 15 mg/kg. Safety analyses included treatment-related and serious adverse events (AEs), as well as treatment-related AEs leading to discontinuation and death, with a focus on liver function test abnormalities and hepatic AEs.Results: Urelumab doses between 1 and 15 mg/kg given every 3 weeks resulted in a higher frequency of treatment-related AEs than 0.1 or 0.3 mg/kg every 3 weeks. Dose was the single most important factor contributing to transaminitis development, which was more frequent and severe at doses ≥1 mg/kg. At the MTD of 0.1 mg/kg every 3 weeks, urelumab was relatively well tolerated, with fatigue (16%) and nausea (13%) being the most common treatment-related AEs, and was associated with immunologic and pharmacodynamic activity demonstrated by the induction of IFN-inducible genes and cytokines.Conclusions: Integrated evaluation of urelumab safety data showed significant transaminitis was strongly associated with doses of ≥1 mg/kg. However, urelumab 0.1 mg/kg every 3 weeks was demonstrated to be safe, with pharmacodynamic activity supporting continued clinical evaluation of this dose as monotherapy and in combination with other immuno-oncology agents. Clin Cancer Res; 23(8); 1929-36. ©2016 AACR.

Studies of sequential anti-CD137/anti-CD20 therapy have previously shown that the efficacy of anti-CD20 was heavily reliant upon anti-CD137; however, the exact mechanism of the anti-B-cell lymphoma efficacy, and whether this correlates with enhanced adverse effects or toxicity, had not been elucidated. Here, we observed that sequential anti-CD137 administration with anti-CD20 resulted in a synergistic therapy, largely dependent upon Fc receptors (FcR), to prolong survival in an experimental B-cell lymphoma therapy model. Tumor suppression was accompanied by B cell depletion, which was not dependent on one activating FcR. Surprisingly, the B-cell activating factor (BAFF) was elevated in the plasma of mice receiving anti-CD137 alone or in combination with anti-CD20, while a selective increase in some plasma cytokines was also noted and triggered by anti-CD137. These effects were independent of activating FcR. Sustained treatment of advanced lymphoma revealed increased lymphocyte infiltrates into the liver and a significant decrease in the metabolic capability of the liver in mice receiving anti-CD137. Importantly, these effects were not exacerbated in mice receiving the anti-CD20/CD137 combination, and elevations in classical liver damage markers such as alanine aminotransferase (ALT) were less than that caused by the lymphoma itself. Thus, combined anti-CD20/anti-CD137 treatment increases the therapeutic index of anti-CD20 or anti-CD137 alone. These mouse data were corroborated by ongoing clinical development studies to assess safety, tolerability and pharmacodynamic activity of human patients treated by this approach. Together, these data support the use of this sequential antibody therapeutic strategy to improve the efficacy of rituximab in B-cell lymphoma patients.

International audience; The efficacy of PD-1/PD-L1 targeted therapies in addition to anti-CTLA-4 solidifies immunotherapy as a modality to add to the anticancer arsenal. Despite raising the bar of clinical efficacy, immunologically targeted agents raise new challenges to conventional drug development paradigms by highlighting the limited relevance of assessing standard pharmacokinetics (PK) and pharmacodynamics (PD). Specifically, systemic and intratumoral immune effects have not consistently correlated with standard relationships between systemic dose, toxicity, and efficacy for cytotoxic therapies. Hence, PK and PD paradigms remain inadequate to guide the selection of doses and schedules, both starting and recommended Phase 2 for immunotherapies. The promise of harnessing the immune response against cancer must also be considered in light of unique and potentially serious toxicities. Refining immune endpoints to better inform clinical trial design represents a high priority challenge. The Cancer Immunotherapy Trials Network investigators review the immunodynamic effects of specific classes of immunotherapeutic agents to focus immune assessment modalities and sites, both systemic and importantly intratumoral, which are critical to the success of the rapidly growing field of immuno-oncology.

The clinical efficacy of monoclonal antibodies as cancer therapeutics is largely dependent upon their ability to target the tumor and induce a functional antitumor immune response. This two-step process of ADCC utilizes the response of innate immune cells to provide antitumor cytotoxicity triggered by the interaction of the Fc portion of the antibody with the Fc receptor on the immune cell. Immunotherapeutics that target NK cells, γδ T cells, macrophages and dendritic cells can, by augmenting the function of the immune response, enhance the antitumor activity of the antibodies. Advantages of such combination strategies include: the application to multiple existing antibodies (even across multiple diseases), the feasibility (from a regulatory perspective) of combining with previously approved agents and the assurance (to physicians and trial participants) that one of the ingredients – the antitumor antibody – has proven efficacy on its own. Here we discuss current strategies, including biologic rationale and clinical results, which enhance ADCC in the following ways: strategies that increase total target–monoclonal antibody–effector binding, strategies that trigger effector cell ‘activating’ signals and strategies that block effector cell ‘inhibitory’ signals.

Adoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer. However, T-cell therapy may be limited by the cotransfer of regulatory T cells (Treg). Here, we explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while excluding Tregs. In a murine model of B-cell lymphoma, only CD137negCD44hi CD4 T cells infiltrated tumor sites and provided protection. Conversely, the population of CD137posCD44hi CD4 T cells consisted primarily of activated Tregs. Notably, this CD137pos Treg population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137pos cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137posCD44hi CD4 cells to CD137negCD44hi CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined a population of activated Tregs that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137pos CD4 T cells that were predominantly CD25posFoxP3pos Tregs. In conclusion, our findings identify 2 surface markers that can be used to facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory Treg population. Cancer Res; 72(5); 1239–47. ©2012 AACR.

Temozolomide sensitivity is determined by methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter. This study assessed whether the temozolomide dose can be tailored by MGMT promoter status and whether protracted, low-dose temozolomide can “prime” blasts in patients with unmethylated MGMT (unMGMT). Elderly patients with high-risk AML were stratified by MGMT methylation. Patients with methylated MGMT (mMGMT) received temozolomide 200 mg/m2 orally for 7 days every 4 weeks, while patients with unMGMT received temozolomide 100 mg/m2 orally for 14 days followed by 200 mg/m2 orally for 7 days every 6weeks. Of 36 patients (median age, 75 years), 31 (86%) had an unMGMT promoter. Overall response rate for the entire cohort was 36%. Patients with mMGMT and unMGMT had similar response rates (40% vs. 29%). Median duration of response and overall survival (OS) among responders were 29 and 35 weeks, respectively. Induction deaths (ID) occurred in 25% of patients, mostly caused by disease progression. Hematological toxicities were the most common adverse event. Toxicities were similar between patients on conventional versus protracted schedules. High HCT-CI scores were predictive of lower CR rate, higher ID, and shorter OS, while bone marrow blast count

Acute myeloid leukemia (AML) is a disease of the elderly. Poor outcomes with standard therapies necessitate novel approaches. Outpatient regimens sufficiently potent and well tolerated to induce remissions and enable continuation therapy may be beneficial. In this phase-1 study, we determined the maximum tolerated dose (MTD) and the efficacy for sequential azacitidine and lenalidomide as remission induction and continuation therapy in elderly, previously untreated patients. We investigated the impact on global DNA methylation and bone marrow cytokines, and sought biological predictors of response. Eighteen patients were enrolled. The MTD was not reached. Median follow-up was 8.2 months (10.3 months for survivors). Common adverse events included fatigue, injection site reactions, constipation, nausea, pruritus and febrile neutropenia. Ten patients responded (56%), and the rate of complete remissions (CRs) or CRs with incomplete recovery of blood counts for evaluable patients was 44% (7/16). The median response duration was 6.2 months. DNA demethylation and changes in bone marrow cytokines were observed; responders had a unique cytokine profile and a trend towards lower methylation levels. Sequential azacitidine and lenalidomide was well tolerated with encouraging clinical and biological activity in previously untreated elderly AML patients. This trial is registered at ClinicalTrials.gov (NCT00890929).

Immunophenotypic identification of myeloid specific antigens is an important diagnostic tool in the management of patients with acute myeloid leukemia (AML). These antigens allow determination of cell of origin and degree of differentiation of leukemia blasts. AML with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) is a relatively rare subtype of AML. The immunophenotypic characteristics of inv(3) AML patients are somewhat limited. We identified 14 new cases of hematological disorders with increased myeloid blasts carrying inv(3)(q21q26.2)/t(3;3)(q21;q26.2). Also, we identified another 13 cases previously published in the literature, where the immunophenotype of inv(3)(q21q26.2) was documented. As a group, patients with AML with inv(3)(q21q26.2) had high levels of early myeloid (CD13, CD33, CD117 and MPO) and uncommitted markers (CD34, HLA-DR and CD56) and a high rate of monosomy 7 in addition to the inv(3)(q21q26.2). Differential karyotype and expression of certain antigens were noted in patients with de novo AML with inv(3)(q21q26.2) vs. those with inv(3)(q21q26.2)-containing blasts.

The immunosuppressive effects of total lymphoid irradiation (TLI) for protection against graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation have been studied for years in animal models. In preclinical models of bone marrow transplantation non-myeloablative TLI conditioning protects against GvHD by skewing host T-cell subsets to favor regulatory natural killer T cells that suppress GvHD by polarizing donor T cells towards secretion of non-inflammatory cytokines such as IL-4. These preclinical models have recently been adapted to human transplantation.Patients receiving allogeneic hematopoietic cell transplantation for hematological malignancies conditioned with TLI and depletive T-cell antibodies showed sustained donor chimerism, a reduced incidence of acute GvHD yet retained anti-tumor activity. As in the preclinical models, the low incidence of GvHD is associated with increased IL-4 secretion by chimeric donor T cells.This review summarizes the evolution of the preclinical TLI protocols and their recent translation to clinical trials, and discusses the mechanisms involved in protection from GvHD and the induction of tolerance following mixed chimerism.

In almost half of patients diagnosed with Hodgkin's disease (HD), the malignant Reed-Sternberg (RS) cells express Epstein-Barr virus (EBV) antigens. Multiple translational efforts are actively investigating antitumor immune strategies by stimulating cytotoxic T lymphocytes (CTL) against tumor-associated EBV antigens. It has previously been believed that this therapeutic strategy and presence of EBV-specific CTLs are limited to EBV-positive HD. In an effort to explore the EBV-specific immune response, here we characterize EBV-specific CTL responses to lytic and latent EBV antigens in 12 consecutive EBV carriers with EBV-negative HD. Compared to healthy donors, we detected weak, baseline EBV-specific responses to both lytic and latent antigens by IFN-gamma ELISPOT in patients with EBV-negative HD at diagnosis. Chemoradiotherapy was associated temporally with a decrease EBV-specific responses. At final follow-up (24 months), recovery of EBV-specific CTL responses was observed with robustness of lytic-specific response equivalent to healthy controls. We confirm evidence of EBV-specific CTLs in patients with EBV-negative HD and provide the first report of dynamic variance in this population during treatment. Our observation challenges prior belief that patients with HD remain immunodeficient following therapy and argues that the clinical significance of the EBV-specific immune response in EBV-negative HD should be further investigated.

Natural killer/T-cell (NK/T) lymphomas represent a group of rare tumors of NK and NK-T cells. The World Health Organization classifies NK-cell tumors into three types, extranodal NK/T-cell lymphomas (ENKL, nasal and non-nasal), NK-cell leukemias, and a blastic variant (CD4-positive, CD56-positive hematodermic neoplasms). We focus our review to the current concepts in biology and treatment of ENKL. Though considerable advances have been made in our understanding of NK-cell biology, malignant transformation including the role of Epstein-Barr virus, and prognosis, the rare nature of ENKL and its heterogeneity limit the ability to standardize therapy. Radiotherapy is fundamental to treatment of early-stage disease with a role for chemoradiotherapy among high-risk patients. The clinical course of advanced disease is highly aggressive with frequent chemotherapy resistance and a poor prognosis. Therapeutic approaches to advanced-stage or relapsed and refractory disease, including the appropriate sequence of chemotherapy, combined modality therapy, and stem cell transplantation is not well-established. International and multicenter clinical trials are needed for this rare and aggressive disease.

We report here a case of a 57-year-old man with multiple myeloma and high-output cardiac failure (HOCF), which completely resolved upon treatment of his myeloma. Recurrence of high-output cardiac f...

International audience; Stage III metastatic melanomas require adequate adjuvant immunotherapy to prevent relapses. Prognostic factors are awaited to optimize the clinical management of these patients. The magnitude of metastatic lymph node invasion and the BRAF V600 activating mutation have clinical significance. Based on a comprehensive immunophenotyping of 252 parameters per patient in paired blood and metastatic lymph nodes performed in 39 metastatic melanomas, we found that blood markers were as contributive as tumor-infiltrated lymphocyte immunotypes, and parameters associated with lymphocyte exhaustion/suppression showed higher clinical significance than those related to activation or lineage. High frequencies of CD45RA þ CD4 þ and CD3 e CD56 e tumor-infiltrated lymphocytes appear to be independent prognostic factors of short progression-free survival. High NKG2D expression on CD8 þ tumor-infiltrated lymphocytes, low level of regulatory T-cell tumor-infiltrated lymphocytes, and low PD-L1 expression on circulating T cells were retained in the multivariate Cox analysis model to predict prolonged overall survival. Prospective studies are needed to determine whether such immunological markers may guide adjuvant therapies in stage III metastatic melanomas.

International audience; In November 2015, the CALYM Carnot Institute held a 2-d workshop to discuss the current and future development of immunomodulatory antibodies for the treatment of lymphoma. Highlights from the workshop are presented in this article. © 2016 Taylor & Francis Group, LLC.

Kenkre and colleagues report the absence of correlation between FcγRIIIa-V158F polymorphism and rituximab response in follicular lymphoma patients, a result which is in contrast with prior studies. This discrepancy recalls that many other factors (from the host and from the tumor) may influence the efficacy of rituximab in vivo. Clin Cancer Res; 22(4); 787–9. ©2015 AACR. See related article by Kenkre et al., p. 821

Despite the progress that has been made in other forms of cancer therapy, Provenge® (Sipuleucel-T) is the only FDA-approved vaccine for the treatment of cancer. To understand the current landscape of therapeutic oncology vaccines we performed a quantitative analysis of phase 2 and phase 3 therapeutic cancer vaccine trials. We highlight shifts in trends for the vaccine platforms examined, common adjuvant use, target indications, antibody or treatment combinations between past and recent trials as well as discuss the relationship between these trends and ratio between the number of phase 3: phase 2 for different vaccine platforms. Despite the poor success rate in vaccine approvals, registration of phase 3 trials between 2010 and 2014 were stable indicating continued investment and efforts towards development of immunotherapeutic vaccines. Electronic supplementary material The online version of this article (doi:10.1186/s40425-015-0093-x) contains supplementary material, which is available to authorized users.

Meeting abstracts The Cancer Immunotherapy Trials Network is conducting a Phase I, dose-escalation study of subcutaneous (SQ) rhIL-15 in advanced melanoma, renal cell, non-small cell lung (NSCLC) and squamous cell head and neck carcinoma patients. IL-15 has been identified as a high priority agent

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.

The consideration of the complex interplay between the tumour microenvironment (TME) and the immune response is the key for designing effective immunotherapies. Therapeutic strategies that harness co-stimulatory receptors have recently gained momentum in the clinic. One such strategy with promising clinical applications is the targeting of CD137, a member of the tumour necrosis factor receptor superfamily. Its expression on both innate and adaptive immune cells, coupled with its unique ability to potentiate antitumour responses through modulating the TME and to ameliorate autoimmune responses, has established it as an appealing target. In this review, we will discuss the various CD137-targeted immunotherapeutics that have reached clinical development, with a focus on recent advances and novel modalities such as CD137 chimeric antigen receptors and CD137 bispecific antibodies. We will also highlight the effect of CD137 targeting on the TME and discuss the importance of probing TME changes for predicting and testing the efficacy of CD137-mediated immunotherapy.

Purpose: The goals of the study were to elucidate the immune mechanisms that contribute to desirable complete remissions of murine colon tumors treated with single radiation dose of 30 Gy. This dose is at the upper end of the ablative range used clinically to treat advanced or metastatic colorectal, liver, and non–small cell lung tumors. Experimental Design: Changes in the tumor immune microenvironment of single tumor nodules exposed to radiation were studied using 21-day (>1 cm in diameter) CT26 and MC38 colon tumors. These are well-characterized weakly immunogenic tumors. Results: We found that the high-dose radiation transformed the immunosuppressive tumor microenvironment resulting in an intense CD8+ T-cell tumor infiltrate, and a loss of myeloid-derived suppressor cells (MDSC). The change was dependent on antigen cross-presenting CD8+ dendritic cells, secretion of IFNγ, and CD4+T cells expressing CD40L. Antitumor CD8+ T cells entered tumors shortly after radiotherapy, reversed MDSC infiltration, and mediated durable remissions in an IFNγ-dependent manner. Interestingly, extended fractionated radiation regimen did not result in robust CD8+ T-cell infiltration. Conclusions: For immunologically sensitive tumors, these results indicate that remissions induced by a short course of high-dose radiotherapy depend on the development of antitumor immunity that is reflected by the nature and kinetics of changes induced in the tumor cell microenvironment. These results suggest that systematic examination of the tumor immune microenvironment may help in optimizing the radiation regimen used to treat tumors by adding a robust immune response. Clin Cancer Res; 21(16); 3727–39. ©2015 AACR.

The 29 th annual meeting of the Society for Immunotherapy of Cancer (SITC) was held November 7-9, 2014 in National Harbor, MD and was organized by Dr. Arthur A. Hurwitz (National Cancer Institute), Dr. Kim A. Margolin (Stanford University), Dr. Daniel E. Speiser (Ludwig Center for Cancer Research, University of Lausanne) and Dr. Walter J. Urba (Earle A. Chiles Research Institute, Providence Cancer Center). This meeting included over 1,600 registered participants from 28 separate countries, making it the largest SITC meeting held to date. It highlighted significant worldwide progress in the development and application of cancer immunology to the practice of clinical oncology, including advances in diagnosis, prognosis and therapy, utilizing several immunological pathways and mechanisms for a variety of oncologic conditions. Presentations and posters demonstrated that many concepts that had been pursued preclinically in the past are now being translated into clinical practice, with clear benefits for patients.

In the past 5 years, immunomodulatory antibodies have revolutionized cancer immunotherapy. CD137, a member of the tumor necrosis factor receptor superfamily, represents a promising target for enhancing antitumor immune responses. CD137 helps regulate the activation of many immune cells, including CD4+ T cells, CD8+ T cells, dendritic cells, and natural killer cells. Recent studies indicate that the antitumor efficacy of therapeutic tumor-targeting antibodies can be augmented by the addition of agonistic antibodies targeting CD137. As ligation of CD137 provides a costimulatory signal in multiple immune cell subsets, combination therapy of CD137 antibody with therapeutic antibodies and/or vaccination has the potential to improve cancer treatment. Recently, clinical trials of combination therapies with agonistic anti-CD137 mAbs have been launched. In this review, we discuss the recent advances and clinical promise of agonistic anti-CD137 monoclonal antibody therapy. Clin Cancer Res; 21(14); 3113–20. ©2015 AACR.

Blockade of CTLA-4 by antibodies has potentiated antitumor T-cell responses in both preclinical models and clinical trials. However, treatment with CTLA-4 blocking antibodies is associated with autoimmune and inflammatory side effects. In this study, we propose a novel administration method for CTLA-4 blocking antibodies as monotherapy.We use different preclinical mouse models of cancer to investigate the local administration of CTLA-4 blocking antibody and its effect on cancer progression and the antitumor T-cell response.By injecting the antibodies in a subcutaneous slow-release delivery formulation in the tumor area, we show that an eight-fold lower dose of antibody is as effective in inducing tumor eradication as systemic delivery. A lower dose and slow release of the antibody results in thousand-fold decreased levels of antibody in the serum, reducing adverse events and the risk of autoimmunity. The main target and effector cells of the CTLA-4 blockade treatment in the studied tumor models are tumor-specific endogenous CD8(+) T cells that are capable of eradicating also distant tumors, whereas CD4(+) T cells do not play a prominent role in the antibody-mediated tumor eradication.Injecting CTLA-4 blocking antibody in a slow-release formulation close to the tumor is an effective way of activating the antitumor T-cell response. This administration method is associated with very low serum levels of antibody, which decreases the risk of treatment-induced side effects. These results call for exploration of a similar delivery principle in clinical settings.

Over the prior two decades, imiquimod, a toll-like receptor 7 agonist, has been applied to nearly 50 clinical settings. Because of its immunomodulatory role, the topical cream today, for the first time, is being applied to cutaneous breast cancer in preclinical models and in a phase II clinical trial. Clin Cancer Res; 18(24); 6571–3. ©2012 AACR.

The incidence pattern of adult acute leukemia (AL) in Hispanics is distinct, with increased B-cell acute lymphoblastic leukemia (ALL) and acute promyelocytic leukemia (APL) and decreased non-APL acute myeloid leukemia (AML). To better understand genetic versus environmental contributors, we assessed AL incidence rates in a population of adult California Hispanics according to birthplace. Using data from California AL patients ≥20 diagnosed between 2000-2009, incidence rate ratios (IRR) were employed to compare incidence rates of AL in foreign- versus United States (US)-born Hispanics. Compared to whites, Hispanics had increased incidence rates of B-cell ALL and APL, IRR2.13 (1.93-2.35) and 1.33 (1.12-1.57), respectively. No nativity differences in B-cell ALL were noted. Foreign-born Hispanics had a higher incidence rate of APL versus US-born Hispanics (IRR 1.79, 1.11-2.94). For adult Hispanics, increased B-cell ALL incidence rates may be due to heritable genetic factors; increased APL incidence rates may be due to as yet unknown environmental exposures.

An expanding panel of monoclonal antibodies (mAbs) that specifically target malignant cells or intercept trophic factors delivered by the tumor stroma is now available for cancer therapy. These mAbs can exert direct antiproliferative/cytotoxic effects as they inhibit pro-survival signal transduction cascades or activate lethal receptors at the plasma membrane of cancer cells, they can opsonize neoplastic cells to initiate a tumor-targeting immune response, or they can be harnessed to specifically deliver toxins or radionuclides to transformed cells. As an indication of the success of this immunotherapeutic paradigm, international regulatory agencies approve new tumor-targeting mAbs for use in cancer patients every year. Moreover, the list of indications for previously licensed molecules is frequently expanded to other neoplastic disorders as the results of large, randomized clinical trials become available. Here, we discuss recent advances in the preclinical and clinical development of tumor-targeting mAbs for oncological indications.

Background:PD-1 and PD-L1/PD-L2 are expressed by malignant T-cells in mycosis fungoides (MF) and Sézary syndrome (SS). PD-1 is additionally expressed by tumor-infiltrating cytotoxic T-cells and PD-L1 is expressed by macrophages and other stromal components of the tumor microenvironment in these diseases. Moreover, reports of 9p24.1/PD-L2 translocation and CTLA4-CD28 fusion events in MF/SS support a genomic basis for immune evasion. Here, we explore the clinical activity of pembrolizumab, an immune checkpoint inhibitor of the PD-1/PD-L1 axis, in MF/SS. Methods:Patients (pts) with MF/SS stages IB-IV treated with at least 1 prior systemic therapy were enrolled in this phase 2, single-arm study coordinated by the Cancer Immunotherapy Trials Network (CITN). A Simon two-stage design was applied where stage 2 is initiated if 1 of 9 pts had an objective response. An additional 15 pts were planned in stage 2. Pembrolizumab was administered at 2 mg/kg every 3 weeks and treatment was allowed up to 2 years. The primary endpoint was overall response rate (ORR) as determined by the consensus global response criteria. Secondary endpoints were safety/tolerability, time to response (TTR), duration of response (DOR) and progression-free survival (PFS). Correlative biomarker studies included immunohistochemistry (IHC) staining for PD-L1, PD-L2, and multiple immune subsets as well as serum analysis of 62 cytokines and chemokines. Phenotypic and functional profiling of malignant and non-malignant immune cells will be performed by flow cytometry and mass cytometry (CyTOF). Results: The study completed enrollment and all 24 patients received at least one dose of pembrolizumab. Median age was 67 (range 44-85); 18 were male. Patients were advanced stage with 23 patients (96%) stage IIB or higher, including 15 patients (63%) with stage IVA SS. Most pts were heavily treated with a median of 4 prior systemic therapies (range 1-11). The median follow-up time was 40 weeks (range 9-60 weeks). The objective response rate (ORR) was 38% with 1 complete response (CR) and 8 partial responses (PR). Of the responding pts, 6 pts had 90% or greater improvement in skin disease as measured by mSWAT. An additional 9 pts (38%) had stable disease (SD). The median TTR was 11 weeks (range 8-41 weeks). Responses were durable with 8 of 9 (89%) responses currently ongoing at a median of 32 weeks of duration (4-46). The median PFS has not yet been reached, and the one-year PFS was 69%. There was no significant association between response and clinical characteristics including stage, disease type (MF vs. SS), and number of prior therapies, nor with skin tissue expression of PD-1, PD-L1, PD-L2, or infiltrating CD8+ T-cells as determined by IHC. Planned additional correlatives including CyTOF profiling, gene expression profiling, T cell receptor high throughput sequencing, multiplexed ion beam imaging (MIBI), and whole exome sequencing will explore potential predictive biomarkers of response. Adverse events (AE) were consistent with those seen in prior studies of pembrolizumab with the exception of an immune-mediated skin flare reaction seen in 6 pts (2 grade 2 and 4 grade 3). Skin flares occurred exclusively in patients with SS (6/15; 40%) and were associated with lower serum levels of the cytokines IL-7 and SCF prior to pembrolizumab treatment (p=0.01 and p=0.02 respectively, n.s. by Bonferroni correction). Pts with the skin flare reaction experienced increases in serum IFN-gamma, IL-12p40, IL-15, LIF, G-CSF, and CCL4 following treatment. There were two treatment related serious adverse events (SAE), both immune related. One pt experienced grade 2 pneumonitis which resolved with systemic corticosteroids. Another patient experienced grade 3 diarrhea secondary to steroid-refractory duodenitis. Conclusions: Pembrolizumab has significant clinical activity in pts with previously treated MF/SS. Responses were durable and were not associated with any identifiable clinical or pathologic characteristics. Treatment was well tolerated with a toxicity profile consistent with prior pembrolizumab studies, though 40% of pts with SS developed a notable skin flare reaction. These findings support further study of PD-1 blockade in the treatment of MF and SS. A phase 2 trial of pembrolizumab in combination with interferon-gamma is being developed based on these results. Disclosures Porcu: Millenium: Other: investigator in a clinical trial; miRagen: Other: Investigator in a clinical trial; celgene: Other: Investigator in a clinical trial; Innate Pharma: Other: Investigator in a clinical trial. Foss:Celgene: Consultancy, Research Funding, Speakers Bureau; Eisai: Consultancy; Seattle Genetics: Consultancy, Speakers Bureau; Spectrum Pharmaceuticals: Consultancy. Moskowitz:Bristol Myers Squibb: Honoraria; Merck: Honoraria; Seattle Genetics: Honoraria, Research Funding. Sokol:Seattle Genetics: Consultancy; Spectrum: Consultancy. Yearley:Merck: Employment. Chartash:Merck: Employment. Townson:Merck: Employment. Horwitz:Spectrum: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Huya: Consultancy; Infinity: Consultancy, Research Funding; Kyowa Hakka Kirin: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Celgene: Consultancy; ADCT Therapeutics: Research Funding. Kim:Seattle Genetics: Consultancy, Other: Investigator in a clinical trial; Merck: Other: Investigator in a clinical trial; Neumedicine: Consultancy; Soligenix: Consultancy; Galderma: Consultancy; Genentech: Other: Investigator in a clinical trial; Innate Pharma: Other: Investigator in a clinical trial; Kyowa Hakko Kirin: Consultancy, Honoraria, Other, Research Funding; Millenium: Consultancy, Other: Investigator in a clinical trial; Eisai: Consultancy, Other: Investigator in a clinical trial; Actelion: Consultancy, Other: Investigator in a clinical trial; Celgene: Consultancy; MiRagen: Consultancy; Horizon: Consultancy.

Immunotherapeutic approaches targeting a single immune checkpoint, programmed death ligand 1, have resulted in unprecedented response rates and survival benefit across multiple cancer histologies. The efficacy of PD-1 blockade is augmented when delivered in combination with other immune checkpoints, specifically anti-CTLA-4, despite a significant increase in toxicity. The current challenge is identification of novel immunomodulatory targets with equivalent efficacy to PD1 blockade as monotherapy, and which can be applied in combination with anti-PD1 with a superior therapeutic index. As the majority of autoimmune toxicity occurs in healthy, non-malignant tissue, we developed a novel high throughput RNA screen to identify new immune checkpoint targets preferentially expressed in the tumor microenvironment. Across numerous tumor types including tumors targeted by monoclonal antibodies (Abs), ie rituximab, trastuzumab, among others, the screen reproducibly identified a list of novel targets for immunotherapy which through a secondary screen were refined for cell surface expression. By this approach we selected a novel type II transmembrane glycoprotein (TIITG) with dual function as an ectoenzyme and in signal transduction. Further experiments in-vitro and ex-vivo from tumor biopsies confirmed upregulation of the target on tumor-infiltrating, activated, immune cells including but not limited to innate immune effectors. In-vitro and in-vivo murine models using agonistic antibodies to the TIITG demonstrated (1) augmentation of natural killer cell spontaneous and antibody-dependent cytotoxicity, (2) inhibition of myeloid derived suppressor cell and regulatory T cell function, and (3) enhancement in depth and breadth of the adaptive response including post vaccination. Antagonistic Abs had minimal to no effect on the immune response, while agonistic Abs augmented type 1-cytokines (IFNg and TNFa) and cytotoxic markers, including CD107a, perforin, and granzyme. Surprisingly, in a murine autoimmune toxicity model established with prior immune checkpoint agonists, agonists of the TIITG elicited no apparent adverse effects, autoimmune or otherwise. Finally, human xenograft models support antitumor activity across over a dozen tumor histologies as monotherapy in addition to synergy with anti-PD1. In summary, using a novel screening technique we uncovered promising immune targets for cancer therapy. The first of which, TIITG, has completed lead selection and is being prepared to enter a first-in-human clinical trial. Citation Format: Narendiran Rajasekaran, Amani Makkouk, Russel K. Pachynski, Aruna Azameera, Cariad Chester, Xing Zhao, Atsushi Yonezawa, Holbrook E. Kohrt. Discovery and validation of a novel checkpoint target preferentially expressed in the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2318.

In 2015, an estimated 59,340 people will develop head and neck cancer and an estimated 12,290 deaths will occur. Recent evidence has demonstrated that the immune system plays a key role in the development, establishment, and progression of head and neck squamous cell carcinoma (HNSCC). Here, we conduct comprehensive immune profiling to better understand the immunophenotype of HNSCC patients, assess the applicability of immune-modulatory drugs, and identify prognostic biomarkers of response to cetuximab treatment. Our systems immunology approach is composed of three complimentary, high-dimensional technologies: time-of-flight mass cytometry (CyTOF), luminex, and the HIMChip microarray platform. Mass cytometry quantifies protein expression on a single-cell basis by utilizing transition element, isotope-tagged antibodies. The Luminex immunoassay measures plasma cytokine levels. The “HIMChip” microarray is a custom Agilent SurePrint HD 8×15k format array containing over 7,000 unique probes for over 4,274 human immune-related genes. We employed these technologies to study the global immune status of thirty HNSCC patients receiving treatment with cetuximab, an IgG1 monoclonal antibody (mAb) targeting the epidermal growth factor receptor (EGFR). Patients were consented to participate on the Phase 0 biomarker-focused clinical trial (NCT01114256) and peripheral blood was obtained before and after cetuximab treatment. Peripheral blood mononuclear cells and plasma were isolated from each time point and utilized in downstream analysis. Via manual gating, our CyTOF-based phenotyping measured 24 distinct populations and the expression of 5 activation markers and 5 checkpoint receptors. The results of manual gating were confirmed with an automated, unsupervised algorithmic analysis combining stochastic neighbor embedding and k-means clustering. The resulting 2D representation of our single-cell data preserved global geometries and facilitated exploration of unique subsets within the natural killer (NK) compartment. To assess the prognostic value of these NK cell subsets, we applied a lasso-regularized logistic regression model to stratify patients based on their clinical response to cetuximab therapy. We identified an NK cell subset that is associated with clinical benefit to therapy. Luminex analysis revealed a cetuximab-induced cytokine signature composed of VCAM1, IP-10, and VEGFD that may play a role in increasing NK cell trafficking to tumor (T-statistics of 1.77, 2.17, and 1.55 respectively). The HIMChip microarray results support our proteomic findings. These results provide the first known comprehensive immune profiling of HNSCC patients as they undergo treatment with cetuximab. Validation in a large cohort of patients is needed and future mechanistic studies will investigate the efficacy of our novel NK cell population in disease control. Citation Format: Cariad Chester, Ajay Fernandez, Atsushi Yonezawa, Xing Zhao, Naren Rajasekaran, Holbrook Kohrt. A systems immunology analysis to detect prognostic biomarkers in patients with squamous cell carcinomas of the head and neck. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1401.

AFM13 is an NK-cell engaging CD30/CD16A bi-specific tetravalent TandAb antibody currently in Phase 2 clinical development in Hodgkin Lymphoma (HL) and CD30+ malignancies. Immune checkpoints inhibitors have demonstrated clinical efficacy in a variety of cancers, including HL. NK-cells are also regulated by a number of check-points, prompting us to investigate the combination of AFM13 with several immuno-modulatory antibodies. In previous experiments we were able to demonstrate higher efficacy of AFM13 than several immuno-modulatory antibodies in monotherapy and strong synergy between AFM13 and an anti-PD-1 antibody in vitro, as well as in vivo PDX models with human CD30+ HL tumors. In order to investigate the underlying immunological mechanisms we employed the same PDX model by implanting tumor fragments derived from surgical specimens of HL patients in immuno-deficient mice. After establishing tumors, mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-PD-1 weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes, myeloid cells and intra-tumoral cytokines were evaluated on days 30, 44, and 58, i.e. 2, 16 and 30 days after treatment start. While monotherapy with AFM13 was reproducibly more potent than anti-PD-1, significant synergy was observed when both agents were combined. Analysis of the tumors on day 58 revealed a strong correlation between tumor growth inhibition and levels of tumor-infiltrating NK-cells, T-cells, myeloid cells and intra-tumoral cytokines such as IFNg. In contrast to anti-PD-1 monotherapy, which only induced T-cell infiltration, AFM13 monotherapy was able to induce infiltration of NK- and T-cells in the tumors, however the combination further enhanced infiltration of both, NK- and T-cells. AFM13 resulted in stronger infiltration of macrophages than anti-PD-1, which was also increased by the combination of both agents, therefore further supporting cross-talk between innate and adaptive immunity. Furthermore, tumor analyses at earlier time-points (days 30 and 44) showed that the initial immune response is characterized by NK-cell infiltration and activation, as well as infiltration of macrophages, whilst the adaptive immune response by T-cells and activated dendritic cells was more pronounced on day 58. Combining AFM13 and anti-PD-1 augments infiltration and activation of all immune subpopulations. In conclusion, our data support strong synergistic anti-tumor efficacy when AFM13 is combined with anti-PD-1 checkpoint blockade in HL PDX models, mediated by tumor-infiltrating lymphocytes, macrophages and dendritic cells, and provide strong evidence for cross-talk between innate and adaptive immunity induced by AFM13-recruited human NK-cells. Citation Format: Xing Zhao, Narendiran Rajasekaran, Uwe Reusch, Jens-Peter Marschner, Martin Treder, Holbrook E. Kohrt. Immune checkpoint inhibition by anti-PD-1 or CD137 co-stimulation enhances cytotoxicity towards CD30+ tumors mediated by the bispecific tetravalent CD30/CD16A TandAb AFM13. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2323.

Background: Merkel cell carcinoma (MCC) is an aggressive skin cancer linked to UV exposure and the Merkel cell polyomavirus (MCPyV). Cytotoxic chemotherapy is associated with high response rates in patients (pts) with metastatic MCC, although responses are seldom durable and median progression-free survival is only 13 weeks. MCC may be responsive to PD-1 pathway blockade as tumors often express PD-L1 and MCPyV-specific T cells express the inhibitory co-receptor PD-1. Methods: In this open-label, phase II trial by the Cancer Immunotherapy Trials Network (NCT02267603), adults with advanced/metastatic MCC and no prior systemic therapy were enrolled. Pembrolizumab, a humanized IgG4 anti-PD-1 monoclonal antibody, was given IV every 3 weeks (2 mg/kg). Response was assessed by RECIST 1.1 every 9-12 weeks. Presence of MCPyV was assessed by measuring serum antibody titers to the MCPyV oncoprotein and/or quantifying tumor oncoprotein expression by immunohistochemistry (IHC). Results: Twenty-six pts were treated (median duration 22 weeks, range 4 to 49 weeks as of 1/8/16 dataset). Twenty-three pts had at least one documented radiologic or clinical assessment of response. Fifteen of 23 evaluable pts (65%) responded (4 complete responses and 11 partial responses); 2 additional pts had stable disease and 6 had progressive disease as best response. Fourteen of 15 responses (93%) were ongoing at last follow-up. Ten responders had at least one follow-up evaluation with response duration ranging from 8+ to 27+ weeks beyond initial documented response. Treatment was generally well tolerated with mostly grade 1 or 2 adverse events. Two pts developed severe drug-related toxicities including grade 4 myocarditis (n = 1) and grade 4 transaminase elevation (n = 1), both of which improved with corticosteroid treatment. Both pts have ongoing tumor responses despite discontinuation of pembrolizumab. Sixteen of 26 pts (62%) were classified as having virus-positive MCC. Among the 23 evaluable pts, 9 of 14 (64%) of those with virus positive tumors responded, while 6 of 9 (67%) of pts with virus negative tumors responded. Analyses of T cells specific to MCPyV and titers of oncoprotein antibodies during therapy, as well as IHC characterization of tumor biopsies including PD-L1 expression are ongoing. Conclusions: First-line therapy with pembrolizumab in pts with advanced MCC is associated with frequent (65%) and durable responses. Notably, the response rate was similar among pts with virus-positive and virus-negative tumors. While virus-positive tumors are known to express highly antigenic MCPyV oncoproteins, virus-negative tumors are typically UV-induced and display strikingly elevated mutational burdens generating putative neo-antigens. Thus, potentially through distinct mechanisms, both virus-positive and virus-negative MCC tumors appear to be immunogenic and frequently responsive to anti-PD-1 therapy. Citation Format: Paul Nghiem, Shailender Bhatia, Evan Lipson, Ragini Kudchadkar, Natalie Miller, Adil Daud, Steven Fling, Philip Friedlander, Harriet Kluger, Holbrook Kohrt, Lisa Lundgren, Kim Margolin, Thomas Olencki, Sunil Reddy, Erica Shantha, William Sharfman, Elad Sharon, Michi Shinohara, John Thompson, Jennifer Yearley, Suzanne Topalian, Martin Cheever. Clinical activity, immune and viral correlates of PD-1 blockade with pembrolizumab as first systemic therapy in patients with advanced Merkel cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT096.

Prior studies have shown preliminary clinical efficacy in combining CPG-ODN with radiation therapy (XRT) to patients with indolent B-cell lymphoma. We report Phase 1 data of combination XRT and SD-101, a synthetic class C CPG-ODN, selected for strong induction of type I interferon. Methods: This dose-escalation Phase I trial (3+3 design) enrolled patients with untreated indolent B-cell lymphoma. The primary endpoints were safety and alpha-interferon-gene induction. Secondary endpoints included efficacy assessment using Cheson (1999) criteria and quantification of changes in tumor-infiltrating lymphocytes. Patients had a single lesion treated with XRT (2 Gy daily X 2 days) followed by weekly intratumoral SD-101 × 5 doses. Pharmacodynamic assessment included flow cytometry assessment of T-cell infiltrates in a FNA sample of the treated tumor and RT-PCR RNA assay of whole blood to assess induction of alpha-interferon genes. Efficacy assessment included imaging (CT at 3, 6, and every 6 months thereafter). Results: As of 23 Dec 2015, 13 patients total were enrolled and treated with SD-101 doses of 1, 2, 4 or 8 mg/dose. There were no dose limiting toxicities. Twelve patients received all planned doses of XRT and SD-101. One patient (8 mg) withdrew consent for treatment after receiving 1 SD-101 dose due to Grade 2 confusion and flu-like symptoms. The most common adverse events (AEs) (decreasing frequency) were chills, malaise, myalgia, fatigue, headache, pyrexia, and nausea, typically resolving within 48 hours. One serious AE occurred (Grade 3 Pulmonary Embolism (4 mg)) which was incidental and asymptomatic. There were 2 dose delays due to an AE (Grade 3 neutropenia) in the same patient (8 mg) that resolved without treatment. The other AE was Grade 3 malaise reported in 2 patients (8 mg). At study Day 90, a reduction of the product of diameters in treated tumors occurred in 12 patients (median -45.3%; range [-87, +100]) and in non-treated tumors occurred in 11 patients (median -8.1%; range [-48, +45]). An induction of alpha-interferon genes occurred at all dose levels with similar level of induction. At the treated site, regulatory T-cells (T Regs) were reduced in 8 of 10 patients (average decrease 22.3 + 9.5%) at Day 8. There was an average reduction of 83.3 + 9.9% in follicular T helper cells (Tfh) at Day 8 (n = 9 with baseline Tfh). Conclusions: SD-101 combined with radiation therapy showed preliminary abscopal anti-tumor activity and was well tolerated. A maximum tolerated dose has not yet been reached at the doses explored. Target engagement of the TLR-9 receptor (induction of interferon-alpha genes) was observed across all dose groups. A decrease in the proportion of T Regs and Tfh cells suggests modulation of their inhibitory effects and tumor growth promoting effects, respectively. The study is currently enrolling 2 expansion cohorts at 1 mg and 8 mg. Citation Format: Ronald Levy, Nancy Bartlett, Jonathan Friedberg, Patrick Reagan, Leo Gordon, Craig Bergman, Robert Coffman, Robert Janssen, Albert Candia, Michael Khodadoust, Matthew Frank, Steven Long, Debra Czerwinski, Michael Chu, Holbrook Kohrt. SD-101, a novel intratumoral class C CpG-ODN, given with low-dose radiation in patients with untreated low-grade B-cell lymphoma: interim results of a phase I trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT047.