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Our previous studies showed that treatment of mice with total body irradiation (TBI) or total lymphoid tissue irradiation markedly changes the balance of residual T cell subsets to favor CD4+CD44hi NKT cells because of the differential resistance of the latter subset to cell death. The object of the current study was to further elucidate the changed balance and mechanisms of differential radioresistance of T cell subsets after graded doses of TBI. The experimental results showed that CD4+ T cells were markedly more resistant than CD8+ T cells, and CD44hi T cells, including NKT cells and memory T cells, were markedly more resistant than CD44lo (naive) T cells. The memory T cells immunized to alloantigens persisted even after myeloablative (1000 cGy) TBI and were able to prevent engraftment of bone marrow transplants. Although T cell death after 1000 cGy was prevented in p53−/− mice, there was progressive T cell death in p53−/− mice at higher doses. Although p53-dependent T cell death changed the balance of subsets, p53-independent T cell death did not. In conclusion, resistance of CD44hi T cells to p53-dependent cell death results in the persistence of immunological memory after TBI and can explain the immune-mediated rejection of marrow transplants in sensitized recipients.

The majority of patients with acute myeloid leukaemia (AML) are elderly. Advancements in supportive care and regimen intensification have resulted in improvements in clinical outcomes for younger AML patients, but analogous improvements in older patients have not been realized. While outcomes are compromised by increased comorbidities and susceptibility to toxicity from therapy, it is now recognized that elderly AML represents a biologically distinct disease that is more aggressive and less responsive to therapy. Some patients tolerate and benefit from intensive remission-induction approaches, while others are best managed with less aggressive strategies. The challenge is to differentiate these groups based on host-related and biological features, in order to maximize the therapeutic benefit and minimize toxicity. As more is understood about the complicated pathogenesis and molecular basis of AML, there are more opportunities to develop and test targeted therapies. Elderly patients, with their narrow therapeutic window, are well positioned to derive a benefit from these novel agents, and therefore, despite a difficult past, there are reasons to be optimistic about the future of elderly AML.

We designed a whole tumor cell vaccine by “loading” lymphoma tumor cells with CG-enriched oligodeoxynucleotide (CpG), a ligand for the Toll-like receptor 9 (TLR9). CpG-loaded tumor cells were phagocytosed, delivering both tumor antigen(s) and the immunostimulatory CpG molecule to antigen-presenting cells (APCs). These APCs then expressed increased levels of costimulatory molecules and induced T-cell immunity. TLR9 was required in the APCs but not in the CpG-loaded tumor cell. We demonstrate that T cells induced by this vaccine are effective in adoptive cellular therapy for lymphoma. T cells from vaccinated mice transferred into irradiated, syngeneic recipients protected against subsequent lymphoma challenge and, remarkably, led to regression of large and established tumors. This therapeutic effect could be transferred by CD4+ but not by CD8+ T cells. A CpG-loaded whole-cell vaccination is practical and has strong potential for translation to the clinical setting. It is currently being tested in a clinical trial of adoptive immunotherapy for mantle-cell lymphoma.

Background: Current treatment of advanced stage Mycosis fungoides (MF) and Sézary syndrome (SS) remains unsatisfactory. Complete responses (CR) are typically Methods: This single-arm, multicenter study by the Cancer Immunotherapy Trials Network (CITN) enrolled 24 patients with MF/SS stages IB-IV, with at least one prior systemic therapy. Pembrolizumab was administered at 2 mg/kg every 3 weeks for up to two years. The primary endpoint was overall response rate (ORR) using global response criteria according to the ISCL/EORTC consensus guidelines. Skin responses were measured by mSWAT. Secondary endpoints were safety/tolerability, time to response (TTR), duration of response (DOR) and progression-free survival (PFS). Correlative studies included immunohistochemistry (IHC), mass cytometry, whole exome sequencing, gene expression profiling, and serum cytokine analysis. Results: Patients had advanced stage disease (23/24 with stage IIB-IV MF/SS), and were heavily pretreated (median of 4 prior systemic therapies). The ORR was 38% with 2 CR and 7 PR. Of the 9 responding patients, 6 had ≥90% improvement in skin disease by mSWAT. The median TTR was 11 weeks. Responses were durable, with 8 of 9 responses ongoing at last follow up (median DOR 64 weeks, range 32-153 weeks). One responding patient progressed 2 months after discontinuing treatment due to an adverse event (AE). The median PFS was not reached. Overall, the toxicity profile was similar to prior studies of pembrolizumab. Four patients discontinued treatment due to treatment related serious AEs of duodenitis, pneumonitis, hepatitis, and corneal ulcer. Skin flare reactions were observed early in the treatment course in 40% of patients with SS, but none with MF. The skin flare reactions did not result in any treatment discontinuation, and did not correlate with subsequent response to treatment. There was no significant association between response and clinical characteristics including stage, disease type (MF vs. SS), or number of prior therapies. IHC assessment of PD-1, PD-L1, and PD-L2 did not predict response. Treatment resulted in an increase of PD-L1 expression by both IHC and nanoString analysis. A nanoString18 gene signature of tumor inflammation that is predictive of response to pembrolizumab in other tumor types was not predictive in this cohort. High dimensional mass cytometry enabled precise identification and phenotyping of malignant T cells. There was a positive correlation between PD-1 expression on the malignant T cells and development of the skin flare reaction. Whole exome sequencing revealed genomic disruptions of PD-1 signaling including copy loss of PD-1. No associations were found between outcomes and genomic events involving the PD1/PD-L1 axis, total mutation number, or neoantigen numbers. Conclusions: The 38% ORR rate in this heavily pretreated population was 38% with a highly encouraging DOR of 64 weeks. No predictive biomarkers have emerged thus far, but additional studies are ongoing. Additional combination studies with pembrolizumab are warranted to improve response rates. Figure. Figure. Disclosures Khodadoust: Innate Pharma: Research Funding. Porcu:Innate Pharma: Consultancy. Foss:Miragen: Consultancy, Speakers Bureau; Seattle genetics: Consultancy; Spectrum: Consultancy; Mallinkrodt: Consultancy. Moskowitz:Bristol Myers-Squibb: Consultancy, Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Merck: Research Funding; Takeda: Honoraria; ADC Therapeutics: Research Funding. Shustov:Seattle Genetics: Research Funding. Sokol:Seattle Genetics: Consultancy; Mallinckrodt Pharmaceuticals: Consultancy; Spectrum Pharmaceuticals: Consultancy. Yearley:Merck: Employment. Horwitz:Mundipharma: Consultancy; Celgene: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Research Funding; Kyowa-Hakka-Kirin: Consultancy, Research Funding; Forty Seven: Consultancy, Research Funding; Millennium/Takeda: Consultancy, Research Funding; Infinity/Verastem: Consultancy, Research Funding; Aileron Therapeutics: Consultancy, Research Funding; Portola: Consultancy; Trillium: Consultancy; Innate Pharma: Consultancy; Spectrum: Research Funding; Corvus: Consultancy. Kim:miRagen: Research Funding; Horizon Pharma: Consultancy, Research Funding; Neumedicine: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Galderma: Research Funding; Soligenix: Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tetralogic: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Portola: Research Funding; Merck: Research Funding; Medivir: Membership on an entity's Board of Directors or advisory committees; Kyowa-Kirin-Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Forty Seven Inc: Research Funding.

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.

Purpose of review The immune modulatory effects of total lymphoid irradiation (TLI) for graft-versus-host disease (GVHD) protection and transplantation tolerance following allogeneic bone marrow and organ transplantation have been studied for years in animal models. In preclinical models nonmyeloablative TLI conditioning alters residual host T cell subsets to favor regulatory natural killer T cells that suppress GVHD and prevent organ allograft rejection. These preclinical models have been recently adapted to human transplantation. Recent findings 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 graft antitumor activity. As in the preclinical models, nonmyeloablative TLI conditioning significantly altered residual host T cell subsets favoring natural killer T cells, and the low incidence of GVHD was associated with increased IL-4 secretion by chimeric donor T cells. The TLI regimen used in cancer patients was modified to determine conditions for stable mixed chimerism and tolerance induction following combined hematopoietic cell and kidney transplantation. Summary 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.

Despite the success of passive immunotherapy with monoclonal antibodies (mAbs), many lymphoma patients eventually relapse. Induction of an adaptive immune response may elicit active and long-lasting antitumor immunity, thereby preventing or delaying recurrence. Immunomodulating mAbs directed against immune cell targets can be used to enhance the immune response to achieve efficient antitumor immunity. Anti-CD137 agonistic mAb has demonstrated antitumor efficacy in various tumor models and has now entered clinical trials for the treatment of solid tumors. Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma. We found that human primary lymphoma tumors are infiltrated with CD137+ T cells. We therefore hypothesized that lymphoma would be susceptible to treatment with anti-CD137 agonistic mAb. Using a mouse model, we demonstrate that anti-CD137 therapy has potent antilymphoma activity in vivo. The antitumor effect of anti-CD137 therapy was mediated by both natural killer (NK) and CD8 T cells and induced long-lasting immunity. Moreover, the antitumor activity of anti-CD137 mAb could be further enhanced by depletion of regulatory T cell (Tregs). These results support the evaluation of anti-CD137 therapy in clinical trials for patients with lymphoma.

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.

Tumor immunotherapy has had demonstrable efficacy in patients with cancer. The most promising results have been with T-cell–based therapies. These include adoptive cell transfer of tumor-infiltrating lymphocytes, genetically engineered T cells, and immune checkpoint inhibitor antibodies. In this review, we describe the different T-cell–based strategies currently in clinical trials and put their applications, present and future, into perspective. Cancer Immunol Res; 3(10); 1115–22. ©2015 AACR.

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.

SummaryInterleukin-2 (IL-2) regulates lymphocyte function by signaling through heterodimerization of the IL-2Rβ and γc receptor subunits. IL-2 is of considerable therapeutic interest, but harnessing its actions in a controllable manner remains a challenge. Previously, we have engineered an IL-2 “superkine” with enhanced affinity for IL-2Rβ. Here, we describe next-generation IL-2 variants that function as “receptor signaling clamps.” They retained high affinity for IL-2Rβ, inhibiting binding of endogenous IL-2, but their interaction with γc was weakened, attenuating IL-2Rβ-γc heterodimerization. These IL-2 analogs acted as partial agonists and differentially affected lymphocytes poised at distinct activation thresholds. Moreover, one variant, H9-RETR, antagonized IL-2 and IL-15 better than blocking antibodies against IL-2Rα or IL-2Rβ. Furthermore, this mutein prolonged survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation.

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.

The authors have declared that no conflict of interest exists.; International audience; Cancer immunotherapy is a rapidly evolving field that offers a novel paradigm for cancer treatment: therapies focus on enhancing the immune system's innate and adaptive anti-tumor response. Early immunotherapeutics have achieved impressive clinical outcomes and monoclonal antibodies are now integral to therapeutic strategies in a variety of cancers. However, only recently have antibodies targeting innate immune cells entered clinical development. Innate immune effector cells play important roles in generating and maintaining antitumor immunity. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important innate immune mechanisms for tumor eradication. These cytolytic processes are initiated by the detection of a tumor-targeting antibody and can be augmented by activating co-stimulatory pathways or blocking inhibitory signals on innate immune cells. The combination of FDA-approved monoclonal antibodies with innate effector-targeting antibodies has demonstrated potent preclinical therapeutic synergy and early-phase combinatorial clinical trials are ongoing.

We have designed a novel therapeutic approach for lymphoma that combines targeted kinase inhibition with in situ vaccination. Intratumoral injection of an unmethylated cytosine guanine dinucleotide (CpG)-enriched oligodeoxynucleotide, an agonist for the toll-like receptor 9 (TLR9), induces the activation of natural killer cells, macrophages, and antigen presenting cells that control tumor growth at the local site. Ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase, a key enzyme in the signaling pathway downstream of B-cell receptor, is an effective treatment against many types of B-cell lymphomas. The combination of intratumoral injection of CpG with systemic treatment by ibrutinib resulted in eradication of the tumors not only in the injected site, but also at distant sites. Surprisingly, this combinatorial antitumor effect required an intact T-cell immune system since it did not occur in nude, severe combined immunodeficiency, or T-cell depleted mice. Moreover, T cells from animals treated with intratumoral CpG and ibrutinib prevented the outgrowth of newly injected tumors. This result suggests that ibrutinib can induce immunogenic cell death of lymphoma cells and that concomitant stimulation of antigen-presenting cells in the tumor microenvironment by toll-like receptor ligands can lead to a powerful systemic antitumor immune response.

Monoclonal antibodies can block cellular interactions that negatively regulate T-cell immune responses, such as CD80/CTLA-4 and PD-1/PD1-L, amplifying preexisting immunity and thereby evoking antitumor immune responses. Ibrutinib, an approved therapy for B-cell malignancies, is a covalent inhibitor of BTK, a member of the B-cell receptor (BCR) signaling pathway, which is critical to the survival of malignant B cells. Interestingly this drug also inhibits ITK, an essential enzyme in Th2 T cells and by doing so it can shift the balance between Th1 and Th2 T cells and potentially enhance antitumor immune responses. Here we report that the combination of anti–PD-L1 antibody and ibrutinib suppresses tumor growth in mouse models of lymphoma that are intrinsically insensitive to ibrutinib. The combined effect of these two agents was also documented for models of solid tumors, such as triple negative breast cancer and colon cancer. The enhanced therapeutic activity of PD-L1 blockade by ibrutinib was accompanied by enhanced antitumor T-cell immune responses. These preclinical results suggest that the combination of PD1/PD1-L blockade and ibrutinib should be tested in the clinic for the therapy not only of lymphoma but also in other hematologic malignancies and solid tumors that do not even express BTK.

It is increasingly recognized that cells within an antigen-specific CD8 T-cell population may be diverse in recognition efficiency for target, which may significantly affect the overall efficacy of the response in clinical settings such as viral infections and cancer. CD8 T cells with seemingly identical antigen specificity, particularly those elicited by cancer vaccines, may be heterogeneous for sensitivity and recognition efficiency for the cognate peptide and functional state in vivo. Analysis of individual T-cell clones derived from an antigen-specific T-cell population would provide an accurate assessment of the overall response; however, this is time- and labor-intensive, preventing rapid and routine assessment of patient samples from clinical trials. By stimulating antigen-specific T cells that otherwise appear homogeneous on tetramer staining with graded amounts of cognate peptides, the authors show that individual cells downmodulate surface T-cell receptors (TCR) and thus lose tetramer reactivity with variable dynamics within the T-cell population. The dynamics of TCR downregulation represent an accurate assessment of an individual cell's antigen sensitivity, recognition efficiency, and relative functional state within an antigen-specific population and have direct correlation to killing capacity by chromium release as well as degranulation by CD107 mobilization. Furthermore, despite correlation of average T-cell function by all three techniques, TCR downregulation uncovered heterogeneity in T-cell responses after vaccination among patient samples directly ex vivo. When examined using this novel technique, antigen-specific T cells elicited by vaccination with heteroclitic peptides exhibited significantly different recognition efficiencies for the heteroclitic versus native peptides, translating into differences in functional responses. With advancing cancer vaccine trials, the capacity to detect and functionally characterize antigen-specific T-cell responses in detail is critical. Techniques, as presented here, that rapidly assess the overall antigen sensitivity, recognition efficiency, and functional status of patients' T-cell responses will guide future vaccine trials and immunotherapies.

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.

The present study examined significant risk factors, including child maltreatment, for child psychopathology in a cross-cultural setting. Ninety-nine Mongolian boys, ages 3-10 years, were assessed. Primary caregivers (PCG) completed structured interviews including the Emory Combined Rating Scale (ECRS) and the Mood and Feelings Questionnaire (MFQ). Structural equation modeling identifies eight risk factors affecting child psychopathology: Three with direct effects (severity of physical punishment, PCG's MFQ score, and PCG's education), three with indirect effects (cultural acceptance of violence as discipline, presence of community violence, and contact with extended family), and two with direct and indirect effects (quality of marriage/presence of spousal abuse, and household size). Results support the ecological-transactional theory of developmental psychopathology in a cross-cultural setting. Structural equation modeling provides a useful technique to isolate specific sites for intervention, while maintaining a comprehensive perspective of risk factor interaction.

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.

Presentation of novel antigenic peptides (neoantigens) that distinguish malignant from normal cells by major histocompatibility complexes (MHC) can serve as potent substrates for specific anti-tumor immune responses. We sought to identify mantle cell lymphoma (MCL) neoantigens by taking an integrated genomic and proteomic strategy that interrogates antigen peptides presented by MHC-class I and class II. Peptides bound to MHC were purified via immunoprecipitation followed by identification using mass spectrometry. Mass spectra were searched against patient-specific proteome databases generated by whole exome sequencing and targeted immunoglobulin gene sequencing. This approach was applied to systematically characterize over 36,000 immunopeptides from 17 patients’ tumor specimens. Interestingly, 52 neoantigenic peptides were derived from the lymphoma immunoglobulin (Ig) heavy or light chain variable regions. Although we identified MHC presentation of private germline polymorphic alleles, no mutated peptides were recovered from non-Ig somatically mutated genes. Furthermore, somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC-II. T-cells specific for Ig-derived neoantigens were found in two patients. Following ex vivo activation and expansion, the T-cells were remarkably able to mediate killing of autologous lymphoma cells. These results demonstrate that combining MHC isolation, peptide identification and exome sequencing is an effective platform to uncover tumor neoantigens. Application of this strategy to MCL implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

The goal of this study was to determine whether a combination of local tumor irradiation and autologous T-cell transplantation can effectively treat metastatic 4T1 breast cancer in mice. BALB/c mice were injected subcutaneously with luciferase-labeled 4T1 breast tumor cells and allowed to grow for 21 days, at which time metastases appeared in the lungs. Primary tumors were treated at that time with 3 daily fractions of 20 Gy of radiation each. Although this approach could eradicate primary tumors, tumors in the lungs grew progressively. We attempted to improve efficacy of the radiation by adding autologous T-cell infusions. Accordingly, T cells were purified from the spleens of tumor-bearing mice after completion of irradiation and cryopreserved. Cyclophosphamide was administered thereafter to induce lympho-depletion, followed by T-cell infusion. Although the addition of cyclophosphamide to irradiation did not improve survival or reduce tumor progression, the combination of radiation, cyclophosphamide and autologous T-cell infusion induced durable remissions and markedly improved survival. We conclude that the combination of radiation and autologous T-cell infusion is an effective treatment for metastatic 4T1 breast cancer.

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 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.

Total lymphoid irradiation (TLI) with antithymocyte globulin (ATG) is a unique regimen that prepares recipients for allogeneic hematopoietic cell transplantation by targeting lymph nodes, while sparing large areas of the bone marrow. TLI is reported to increase the frequency of CD4(+)CD25(+)FoxP3(+) T-regulatory cells (Treg) relative to conventional T cells. In this study, barriers to hematopoietic stem cell (HSC) engraftment following this nonmyeloablative conditioning were evaluated. TLI/ATG resulted in profound lymphoablation but endogenous host HSC remained. Initial donor HSC engraftment occurred only in radiation exposed marrow sites, but gradually distributed to bone marrow outside the radiation field. Sustained donor engraftment required host lymphoid cells insofar as lymphocyte deficient Rag2γc(-/-) recipients had unstable engraftment compared with wild-type. TLI/ATG treated wild-type recipients had increased proportions of Treg that were associated with increased HSC frequency and proliferation. In contrast, Rag2γc(-/-) recipients who lacked Treg did not. Adoptive transfer of Treg into Rag2γc(-/-) recipients resulted in increased cell cycling of endogenous HSC. Thus, we hypothesize that Treg influence donor engraftment post-TLI/ATG by increasing HSC cell cycling, thereby promoting the exit of host HSC from the marrow niche. Our study highlights the unique dynamics of donor hematopoiesis following TLI/ATG, and the effect of Treg on HSC activity.

The aryl hydrocarbon receptor (AhR) has become increasingly recognized for its role in the differentiation and activity of immune cell subsets; however, its role in regulating the activity of natural killer (NK) cells has not been described. Here, we show that AhR expression is induced in murine NK cells upon cytokine stimulation. We show that in the absence of AhR, NK cells have reduced cytolytic activity and reduced capacity to control RMA-S tumor formation in vivo, despite having normal development and maturation markers. Although AhR was first identified to bind the xenobiotic compound dioxin, AhR is now known to bind a variety of natural exogenous (e.g., dietary) and endogenous ligands. We show that activation of AhR with an endogenous tryptophan derivative, 6-formylindolo[3,2- b ]carbazole, potentiates NK cell IFN-γ production and cytolytic activity. Further, administration of 6-formylindolo[3,2- b ]carbazole in vivo enhances NK cell control of tumors in an NK cell- and AhR-dependent manner. Finally, similar effects on NK cell potency occur with AhR dietary ligands, potentially explaining the numerous associations that have been observed in the past between diet and NK cell function. Our studies introduce AhR as another regulator of NK cell activity in vivo.

Chronic graft-versus-host disease (GVHD) results in significant morbidity and mortality, limiting the benefit of allogeneic hematopoietic cell transplantation (HCT). Peripheral blood gene expression profiling of the donor immune repertoire following HCT may provide associated genes and pathways thereby improving the pathophysiologic understanding of chronic GVHD. We profiled 70 patients and identified candidate genes that provided mechanistic insight in the biologic pathways that underlie chronic GVHD. Our data revealed that the dominant gene signature in patients with chronic GVHD represented compensatory responses that control inflammation and included the interleukin-1 decoy receptor, IL-1 receptor type II, and genes that were profibrotic and associated with the IL-4, IL-6 and IL-10 signaling pathways. In addition, we identified three genes that were important regulators of extracellular matrix. Validation of this discovery phase study will determine if the identified genes have diagnostic, prognostic or therapeutic implications.

Defects in T-cell function in patients with cancer might influence their capacity to mount efficient antitumor immune responses. Here, we identified highly reduced IL-4-, IL-10-, and IL-21-induced phosphorylation of STAT6 and STAT3 in tumor-infiltrating T cells (TILs) in follicular lymphoma (FL) tumors, contrasting other non-Hodgkin lymphoma TILs. By combining phospho-protein-specific flow cytometry with several T-cell markers, we identified that CD4(+)CD45RO(+)CD62L(-) FL TILs were largely nonresponsive to cytokines, in contrast to the corresponding autologous peripheral blood subset. We observed differential expression of the inhibitory receptor PD-1 in FL TILs and peripheral blood T cells. Furthermore, CD4(+)PD-1(hi) FL TILs, containing T(FH) and non-T(FH) cells, had lost their cytokine responsiveness, whereas PD-1 TILs had normal cytokine signaling. However, this phenomenon was not tumor specific, because tonsil T cells were similar to FL TILs. FL tumor cells were negative for PD-1 ligands, but PD-L1(+) histiocytes were found within the T cell-rich zone of the neoplastic follicles. Disruption of the microenvironment and in vitro culture of FL TILs could restore cytokine signaling in the PD-1(hi) subset. Because FL TILs in vivo probably receive suppressive signals through PD-1, this provides a rationale for testing PD-1 Ab in combination with immunotherapy in patients with FL.

Hypothalamic-pituitary-adrenal (HPA) axis activity is related to childhood disruptive behavior disorders and to exposure to abuse and neglect. This study explores the relationship of diurnal salivary cortisol levels with oppositional defiant disorder (ODD) and caregiver attitudes toward physical punishment among boys in Mongolia.Salivary cortisol was collected in the home or institution 4 times daily for 4 days from 46 boys, aged 4-10 years, in Ulaanbaatar, Mongolia. Caregivers rated child disruptive behavior symptoms, attitudes toward physical punishment, and community violence exposures. Mixed effects models were used to estimate the association of psychopathology and caregiver attitudes with salivary cortisol levels.Boys meeting criteria for ODD displayed consistently lower diurnal salivary cortisol levels compared to boys without ODD diagnoses. Controlling for ODD diagnosis, boys with depression showed higher cortisol levels throughout the day. No other diagnosis was associated with cortisol levels. Psychiatric diagnosis accounted for 17% of between individual variations in cortisol levels unexplained by the covariates. In a separate model, caregivers' beliefs regarding physical punishment accounted for 11% of between individual differences: boys with caregivers who stated physical punishment was necessary for discipline displayed hypocortisolism. Institutionalization did not associate with cortisol levels.Salivary cortisol data from a non-Western naturalistic setting support an association of reduced basal HPA activity with disruptive behavior disorders and caregiver attitudes toward discipline. These findings suggest HPA functioning may be a reflection of or mediate disruptive behavior disorders in children across ethnic and cultural settings.

Background: Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), plays an emerging role in targeted therapy of several lymphoid malignancies. Due to its activity in B-cell receptor (BCR) dependent human B-cell cancers, Ibrutinib is currently approved for the treatment of patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In addition, Ibrutinib responses can be seen in patients with follicular lymphoma (FL) and in a subset of patients with ABC- (activated B-cell-) like diffuse large B-cell lymphoma (DLBCL) (Wilson et al., Nat Med 2015; Fowler et al., ASH Annual Meeting, 2015). However, resistance mutations in BTK and other genes (e.g., PLCγ2)are a major cause of treatment failure and early identification of those aberrations remains challenging. Circulating tumor DNA (ctDNA) is an emerging biomarker with potential for disease monitoring as well as biopsy-free detection of evolving somatic aberrations. In this case study, we show that noninvasive genotyping of blood plasma allows early detection of resistance mutations duringIbrutinib therapy in a patient with relapsed/refractory follicular lymphoma associated with histological transformation. Methods: We profiled two tumor lymph node (LN) biopsy specimens and 8 plasma samples before, during, and after Ibrutinib therapy of a 67-year-old man with relapsed/refractory FL with evidence of transformation. Preceding Ibrutinib, this patient received 6 lines of lymphoma treatment over a ~3 year disease course, including Rituximab monotherapy, Benadmustine/Rituximab, and R-CHOP. Despite achieving stable disease after 12 weeks, the patient experienced significant disease progression after additional 4 months on Ibrutinib (Figure 1). We profiled these 10 serial samples using CAPP-Seq, a capture-based targeted high-throughput sequencing (HTS) method (Newman et al., Nat Med, 2014), which allows the sensitive identification of emerging somatic aberrations in circulating tumor DNA. Results: We detected the emergence of two independent BTK C481S resistance mutations in the blood plasma during Ibrutinib therapy, which were undetectable immediately before treatment. The major resistance clone harboring the BTK C481S A>T mutation (Figure 1, orange) was identified 2 months after the start of Ibrutinib, with allele frequencies as low as 0.03%. The second resistant subclone emerged 63 days later, carrying a BTK C481S somatic mutation with a substitution of C to G (Figure 1, green). Importantly, despite encoding an identical amino acid mutation, these two adjacent variants were never observed within the samectDNA molecule, demonstrating convergent evolution of independent resistantsubclones. We confirmed the existence of the two resistance mutations by tumor genotyping of a LN biopsy after clinical diagnosis of disease progression. Notably, while the minorsubclone completely disappeared under subsequentimmunochemotherapy with Rituximab/Bendamustine/Lenalidomide (RBL), the major resistance clone reappeared at the time of disease progression and the patient ultimately succumbed to his disease. Conclusions: Resistance mutations in BTK C481S have been described in tumor cells of Ibrutinib-refractory patients with CLL and MCL, with in vitro evidence of responsiveness to BH3-mimetics and SYK inhibitors. This study, to our knowledge, is the first report of its noninvasive detection in the plasma of a patient with Non-Hodgkin lymphoma, with the subsequent partial eradication of a resistant subclone. Therefore, we envision that biopsy-free genotyping will allow early detection of lymphoma patients developing Ibrutinib resistance and will be incorporated into future clinical trials involving targeted therapies. Figure 1 Figure 1. Disclosures Newman: Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Varian Medical Systems: Research Funding; Novartis: Consultancy; Roche: Consultancy; Quanticel Pharmaceuticals: Consultancy.

Neoantigens arising through somatic mutations are increasingly recognized as key tumor antigens driving clinical immune responses. We sought to identify human lymphoma neoantigens through a genomic and proteomic characterization of peptide ligands of major histocompatibility complex class I (MHC-I) and class II (MHC-II) of a cohort of patients with untreated mantle cell lymphoma. Peptide identification was performed through analysis of MHC-I and MHC-II peptide ligands using liquid chromatography and tandem mass spectrometry (LC/MS-MS), and augmented by generating patient-specific proteome databases through tumor:normal whole exome sequencing and targeted immunoglobulin gene sequencing in 17 patients. A total of 66 neoantigen peptides from 9 patients were found to be presented by MHC, and surprisingly all neoantigenic peptides were derived from either the lymphoma immunoglobulin heavy or light chain genes. There was no detectable MHC presentation of hundreds of candidate somatic neoantigenic peptides from other genes despite widespread antigen presentation of the proteome including proteins encoded by germline heterozygous single nucleotide polymorphisms. Clonotypic lymphoma immunoglobulin sequences were among the most commonly presented proteins by both MHC-I and MHC-II, yet MHC-I presentation of variable regions was nearly absent and included only a single neoantigen derived from IGVH. In contrast, the variable region was frequently presented by MHC-II and included neoantigen peptides created through somatic hypermutation or VDJ recombination. Autologous circulating T-cells specific for immunoglobulin-derived neoantigens could be detected using MHC-II tetramers and their frequency dynamically changed with vaccination and therapy. These findings suggest that immunoglobulin derived neoantigens are frequently presented by lymphoma, and represent potential targets for CD4, but not CD8, T-cell mediated therapies. Figure. MHC-I and MHC-II presentation of lymphoma immunoglobulin. (A) MHC-I (left) and MHC-II (right) presented peptides derived from patients' immunoglobulin were mapped to the immunoglobulin domains. Peptides recovered from all seventeen patients are mapped to a schematic of the IgM molecule with a heat map indicating the number of recovered peptides covering each position. (B) Expanded view of the immunoglobulin heavy chain variable region. The heatmap represents the total number of MHC-presented peptides that span that position, including non-neoantigens for MHC-I (above) and MHC-II (below). Neoantigen peptides created by either somatic hypermutation or VDJ rearrangement are mapped above or below the heat map. Red, positions within the recovered peptides which are altered from germline variable genes thus creating neoantigens. Boxes, peptides derived from a single patient. Figure. MHC-I and MHC-II presentation of lymphoma immunoglobulin. (A) MHC-I (left) and MHC-II (right) presented peptides derived from patients' immunoglobulin were mapped to the immunoglobulin domains. Peptides recovered from all seventeen patients are mapped to a schematic of the IgM molecule with a heat map indicating the number of recovered peptides covering each position. (B) Expanded view of the immunoglobulin heavy chain variable region. The heatmap represents the total number of MHC-presented peptides that span that position, including non-neoantigens for MHC-I (above) and MHC-II (below). Neoantigen peptides created by either somatic hypermutation or VDJ rearrangement are mapped above or below the heat map. Red, positions within the recovered peptides which are altered from germline variable genes thus creating neoantigens. Boxes, peptides derived from a single patient. Disclosures Newman: Roche: Consultancy. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies: Employment. Faham:Adaptive Biotechnologies Corp: Employment.

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.