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// Anthony S. Malamas 1 , Scott A. Hammond 2 , Jeffrey Schlom 1 and James W. Hodge 1 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA 2 MedImmune LLC, Gaithersburg, Maryland, USA Correspondence to: James W. Hodge, email: // Keywords : OX40, cancer vaccines, combination immunotherapy, twist Received : July 22, 2017 Accepted : July 23, 2017 Published : August 05, 2017 Abstract OX40 is a costimulatory receptor that potentiates proliferation, survival, memory formation, and effector function of CD4 + and CD8 + T-cells, while overcoming the suppressive activity of regulatory T-cells (Tregs). Here, we explored the combination of an OX40L fusion protein (OX40L-FP) with a poxvirus-based cancer vaccine (MVA-Twist-TRICOM) to inhibit tumor metastasis in the 4T1 murine breast cancer model. Contrary to the single agent treatments, the combination therapy significantly decreased the number of metastatic colonies per lung and prolonged survival. Depletion studies demonstrated that these effects were mediated by both CD4 + and CD8 + T-cells. The combination therapy a) increased the total number of T-cells in the CD4 + Foxp3 - population and the CD4 + central and effector memory subsets within the lung, spleen, and draining lymph node, b) enhanced infiltration of CD4 + T-cells into metastatic areas of the lung, and (c) increased the number of functional CD8 + T-cells that produced IFNγ and TNFα. The combination therapy also promoted the development of KLRG1 - CD127 + memory precursor CD8 + T-cells, while reducing those with a KLRG1 + terminally differentiated phenotype. Moreover, the combination of OX40L-FP and vaccine induced greater CD4 + and CD8 + Twist-specific responses. In addition, Tregs isolated from mice receiving the combination were also less immunosuppressive in ex-vivo proliferation assays than those from the OX40L-FP and MVA-Twist-TRICOM monotherapy groups. Such results provide the rationale to combine co-stimulatory agonists with cancer vaccines for the treatment of tumor metastasis.

// Anthony S. Malamas 1 , Sofia R. Gameiro 1 , Karin M. Knudson 1 , James W. Hodge 1 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Correspondence to: James W. Hodge, email: jh241d@nih.gov Keywords: radium-223, alpha radiation, CTL-mediated lysis, immunogenic modulation, calreticulin Received: October 07, 2016 Accepted: November 07, 2016 Published: November 23, 2016 ABSTRACT Radium-223 dichloride (Xofigo ® ; 223 Ra) is an alpha-emitting radiopharmaceutical FDA-approved for the treatment of bone metastases in patients with advanced castration-resistant prostate cancer. It is also being examined clinically in patients with breast and lung carcinoma and patients with multiple myeloma. As with other forms of radiation, the aim of 223 Ra is to reduce tumor burden by directly killing tumor cells. External beam (photon) and proton radiation have been shown to augment tumor sensitivity to antigen-specific CD8 + cytotoxic T lymphocytes (CTLs). However, little is known about whether treatment with 223 Ra can also induce such immunogenic modulation in tumor cells that survive irradiation. We examined these effects in vitro by exposing human prostate, breast, and lung carcinoma cells to sublethal doses of 223 Ra. 223 Ra significantly enhanced T cell-mediated lysis of each tumor type by CD8 + CTLs specific for MUC-1, brachyury, and CEA tumor antigens. Immunofluorescence analysis revealed that the increase in CTL killing was accompanied by augmented protein expression of MHC-I and calreticulin in each tumor type, molecules that are essential for efficient antigen presentation. Enhanced tumor-cell lysis was facilitated by calreticulin surface translocation following 223 Ra exposure. The phenotypic changes observed after treatment appear to be mediated by induction of the endoplasmic reticulum stress response pathway. By rendering tumor cells more susceptible to T cell-mediated lysis, 223 Ra may potentially be effective in combination with various immunotherapies, particularly cancer vaccines that are designed to generate and expand patients’ endogenous antigen-specific T-cell populations against specific tumor antigens.

// Caroline Jochems 1, * , James W. Hodge 1, * , Massimo Fantini 1 , Rika Fujii 1 , Y. Maurice Morillon II 1 , John W. Greiner 1 , Michelle R. Padget 1 , Sarah R. Tritsch 1 , Kwong Yok Tsang 1 , Kerry S. Campbell 2 , Hans Klingemann 3 , Laurent Boissel 3 , Shahrooz Rabizadeh 4 , Patrick Soon-Shiong 3, 4 , Jeffrey Schlom 1 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 2 Immune Cell Development and Host Defense Program, Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA 3 NantKwest, Inc., Culver City, CA, USA 4 NantCell, LLC, Culver City, CA, USA * These authors contributed equally to this work Correspondence to: Jeffrey Schlom, email: js141c@nih.gov Keywords: ADCC, NK lysis, immunotherapy, high affinity CD16, cetuximab Received: October 07, 2016 Accepted: November 07, 2016 Published: November 16, 2016 ABSTRACT Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells. The studies reported here show high levels of granzyme in haNK cells, and demonstrate the effects of irradiation of haNK cells on multiple phenotypic markers, viability, IL-2 production, and lysis of a spectrum of human tumor cells. Studies also compare endogenous irradiated haNK lysis of tumor cells with that of irradiated haNK-mediated ADCC using cetuximab, trastuzumab and pertuzumab monoclonal antibodies. These studies thus provide the rationale for the potential use of irradiated haNK cells in adoptive transfer studies for a range of human tumor types. Moreover, since only approximately 10% of humans are homozygous for the high affinity V CD16 allele, these studies also provide the rationale for the use of irradiated haNK cells in combination with IgG1 anti-tumor monoclonal antibodies.

// Caroline Jochems 1 , Massimo Fantini 1 , Romaine I. Fernando 1 , Anna R. Kwilas 1 , Renee N. Donahue 1 , Lauren M. Lepone 1 , Italia Grenga 1 , Young-Seung Kim 2 , Martin W. Brechbiel 2 , James L. Gulley 3 , Ravi A. Madan 3 , Christopher R. Heery 1 , James W. Hodge 1 , Robert Newton 4 , Jeffrey Schlom 1, * , Kwong Y. Tsang 1, * 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2 Radioimmune Inorganic Chemistry Section, Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 3 Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 4 Incyte Corporation, Wilmington, DE, USA * These authors contributed equally to this work Correspondence to: Jeffrey Schlom, email: js141c@nih.gov Keywords: IDO inhibitor, dendritic cells, T cells, indoleamine-2,3-dioxygenase (IDO), Tregs Received: February 29, 2016 Accepted: April 26, 2016 Published: May 12, 2016 ABSTRACT Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro . Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83 + conventional DCs, and a decrease in immature activated Tim3 + NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 chordoma cell lines, first for expression of PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1 expression was markedly upregulated by IFN-γ in all 4 chordoma cell lines, which significantly increased sensitivity to ADCC. Brachyury is a transcription factor that is uniformly expressed in chordoma. Clinical trials are ongoing in which chordoma patients are treated with brachyury-specific vaccines. Co-incubating chordoma cells with brachyury-specific CD8+ T cells resulted in significant upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-γ production, and increased sensitivity of chordoma cells to avelumab-mediated ADCC. Residential cancer stem cell subpopulations of chordoma cells were also killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. These findings suggest that as a monotherapy for chordoma, avelumab may enable endogenous NK cells, while in combination with T-cell immunotherapy, such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via ADCC.

Among breast cancer types, triple-negative breast cancer (TNBC) has the fewest treatment options and the lowest 5-year survival rate. Androgen receptor (AR) inhibition has displayed efficacy against breast cancer preclinically and is currently being examined clinically in AR positive TNBC patients. Androgen deprivation has been shown to induce immunogenic modulation; the alteration of tumor cell phenotype resulting in increased sensitivity to immune-mediated killing. We evaluated the ability of AR inhibition to reduce the growth and improve the immune-mediated killing of breast cancer cells with differing expression of the estrogen receptor and AR. While AR expression was required for the growth inhibitory effects of enzalutamide on breast cancer cells, both enzalutamide and abiraterone improved the sensitivity of breast cancer cells to immune-mediated lysis independent of detectable AR expression. This increase in sensitivity was linked to an increase in cell surface tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression as well as a significant reduction in the expression of osteoprotegerin (OPG). The reduction in OPG was further examined and found to be critical for the increase in sensitivity of AR- TNBC cells to immune-mediated killing. The data presented herein further support the use of AR inhibition therapy in the AR+ TNBC setting. These data, however, also support the consideration of AR inhibition therapy for the treatment of AR- TNBC, especially in combination with cancer immunotherapy, providing a potential novel therapeutic option for select patients.

The clinical promise of cancer immunotherapy relies on the premise that the immune system can recognize and eliminate tumor cells identified as non-self. However, tumors can evade host immune surveillance through multiple mechanisms, including epigenetic silencing of genes involved in antigen processing and immune recognition. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can restore tumor immune recognition when combined with immunotherapy, such as immune checkpoint blockade and therapeutic cancer vaccines. We sought to examine the potential of clinically relevant exposure of prostate and breast human carcinoma cells to histone deacetylase (HDAC) inhibitors to reverse tumor immune escape to T-cell mediated lysis. Here we demonstrate that prostate (LNCAP) and breast (MDA-MB-231) carcinoma cells are more sensitive to T-cell mediated lysis in vitro after clinically relevant exposure to epigenetic therapy with either the pan-HDAC inhibitor vorinostat or the class I HDAC inhibitor entinostat. This pattern of immunogenic modulation was observed against a broad range of tumor-associated antigens, such as CEA, MUC1, PSA, and brachyury, and associated with augmented expression of multiple proteins involved in antigen processing and tumor immune recognition. Genetic and pharmacological inhibition studies identified HDAC1 as a key determinant in the reversal of carcinoma immune escape. Further, our findings suggest that the observed reversal of tumor immune evasion is driven by a response to cellular stress through activation of the unfolded protein response. This offers the rationale for combining HDAC inhibitors with immunotherapy, including therapeutic cancer vaccines.

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no “antigenic competition” in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.

Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Here we show for the first time that both enzalutamide and abiraterone render prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT.

// Duane H. Hamilton 1,* , Mary T. Litzinger 1,* , Alessandra Jales 1 , Bruce Huang 1 , Romaine I. Fernando 1 , James W. Hodge 1 , Andressa Ardiani 1 , David Apelian 2 , Jeffrey Schlom 1,* , and Claudia Palena 1,* 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 2 GlobeImmune Inc., Louisville, Colorado * These authors contributed equally to the work Correspondence: Claudia Palena, email: // Keywords : epithelial-mesenchymal transition, brachyury, T-box transcription factor, cancer vaccine, T-cell immunotherapy Received : August 16, 2013 Accepted : September 24, 2013 Published : September 26, 2013 Abstract The embryonic T-box transcription factor brachyury is aberrantly expressed in a range of human tumors. Previous studies have demonstrated that brachyury is a driver of the epithelial-mesenchymal transition (EMT), a process associated with cancer progression. Brachyury expression in human tumor cells enhances tumor invasiveness in vitro and metastasis in vivo , and induces resistance to various conventional therapeutics including chemotherapy and radiation. These characteristics, and the selective expression of brachyury for a range of human tumor types vs. normal adult tissues, make brachyury an attractive tumor target. Due to its intracellular localization and the “undruggable” character of transcription factors, available options to target brachyury are currently limited. Here we report on the development and characterization of an immunological platform for the efficient targeting of brachyury-positive tumors consisting of a heat-killed, recombinant Saccharomyces cerevisiae (yeast)–brachyury vector-based vaccine (designated as GI-6301) that expresses the full-length human brachyury protein. We demonstrate that human dendritic cells treated with recombinant yeast-brachyury can activate and expand brachyury-specific CD4 + and CD8 + T cells in vitro that, in turn, can effectively lyse human tumor cells expressing the brachyury protein. Vaccination of mice with recombinant yeast-brachyury is also shown here to elicit brachyury-specific CD4 + and CD8 + T-cell responses, and to induce anti-tumor immunity in the absence of toxicity. Based on these results, a Phase I clinical trial of GI-6301 is currently ongoing in patients with advanced tumors; to our knowledge, this is the first vaccine platform aimed at targeting a driver of tumor EMT that has successfully reached the clinical stage.

// Tadanobu Nagaya 1 , Yuko Nakamura 1 , Kazuhide Sato 1 , Toshiko Harada 1 , Peter L. Choyke 1 , James W. Hodge 2 , Jeffrey Schlom 2 , Hisataka Kobayashi 1 1 Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States of America 2 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States of America Correspondence to: Hisataka Kobayashi, email: kobayash@mail.nih.gov Keywords: near infrared photoimmunotherapy, PD-L1, lung cancer Received: July 30, 2016 Accepted: September 20, 2016 Published: October 03, 2016 ABSTRACT Near Infrared-Photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate (APC). Programmed cell death protein-1 ligand (PD-L1) is emerging as a molecular target. Here, we describe the efficacy of NIR-PIT, using fully human IgG 1 anti-PD-L1 monoclonal antibody (mAb), avelumab, conjugated to the photo-absorber, IR700DX, in a PD-L1 expressing H441 cell line, papillary adenocarcinoma of lung. Avelumab-IR700 showed specific binding and cell-specific killing was observed after exposure of the cells to NIR in vitro . In the in vivo study, avelumab-IR700 showed high tumor accumulation and high tumor-background ratio. Tumor-bearing mice were separated into 4 groups: (1) no treatment; (2) 100 μg of avelumab-IR700 i.v.; (3) NIR light exposure only, NIR light was administered; (4) 100 μg of avelumab-IR700 i.v., NIR light was administered. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other groups ( p < 0.001), and significantly prolonged survival was achieved ( p < 0.01 vs other groups). In conclusion, the anti-PD-L1 antibody, avelumab, is suitable as an APC for NIR-PIT. Furthermore, NIR-PIT with avelumab-IR700 is a promising candidate of the treatment of PD-L1-expressing tumors that could be readily translated to humans.

// Peter S. Kim 1 , Anna R. Kwilas 1 , Wenxin Xu 2 , Sarah Alter 2 , Emily K. Jeng 2 , Hing C. Wong 2 , Jeffrey Schlom 1, * , James W. Hodge 1, * 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2 Altor BioScience Corporation, Miramar, FL, USA * These authors have contributed equally to this work Correspondence to: James W. Hodge, e-mail: jh241d@nih.gov Keywords: IL-15, IL-15 superagonist, NK cells, immunotherapy Received: September 02, 2015 Accepted: February 11, 2016 Published: February 18, 2016 ABSTRACT Interleukin (IL)-15-N72D superagonist-complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT-803) has been reported to exhibit significant anti-tumor activity in murine myeloma, rat bladder cancer, and murine glioblastoma models. In this study, we examined the immunomodulatory and anti-tumor effects of IL-15SA/IL-15RαSu-Fc in tumor-free and highly metastatic tumor-bearing mice. Here, IL-15SA/IL-15RαSu-Fc significantly expanded natural killer (NK) and CD8 + T cells. In examining NK cell subsets, the greatest significant increase was in highly cytotoxic and migrating (CD11b + , CD27 hi ; high effector) NK cells, leading to enhanced function on a per-cell basis. CD8 + T cell subset analysis determined that IL-15SA/IL-15RαSu-Fc significantly increased IL-15 responding memory (CD122 + , CD44 + ) CD8 + T cells, in particular those having the innate (NKG2D + , PD1 − ) phenotype. In 4T1 breast tumor–bearing mice, IL-15SA/IL-15RαSu-Fc induced significant anti-tumor activity against spontaneous pulmonary metastases, depending on CD8 + T and NK cells, and resulting in prolonged survival. Similar anti-tumor activity was seen in the experimental pulmonary metastasis model of CT26 colon carcinoma cells, particularly when IL-15SA/IL-15RαSu-Fc was combined with a cocktail of checkpoint inhibitors, anti-CTLA-4 and anti-PD-L1. Altogether, these studies showed for the first time that IL-15SA/IL-15RαSu-Fc (1) promoted the development of high effector NK cells and CD8 + T cell responders of the innate phenotype, (2) enhanced function of NK cells, and (3) played a vital role in reducing tumor metastasis and ultimately survival, especially in combination with checkpoint inhibitors.

// Saddam M. Muthana 1, 2 , James L. Gulley 3 , James W. Hodge 4 , Jeffrey Schlom 4 , Jeffrey C. Gildersleeve 1 1 Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA 2 Chemistry Department, College of Science & General Studies, Alfaisal University, Riyadh, KSA 3 Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 4 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Correspondence to: Jeffrey C. Gildersleeve, e-mail: gildersj@mail.nih.gov Keywords: cancer vaccine, immunotherapy, PROSTVAC-FV, Glycan microarray, blood type Received: July 11, 2015 Accepted: August 04, 2015 Published: August 18, 2015 ABSTRACT Immunotherapies for cancer are transforming patient care, but clinical responses vary considerably from patient to patient. Simple, inexpensive strategies to target treatment to likely responders could substantially improve efficacy while simultaneously reducing health care costs, but identification of reliable biomarkers has proven challenging. Previously, we found that pre-treatment serum IgM to blood group A (BG-A) correlated with survival for patients treated with PROSTVAC-VF, a therapeutic cancer vaccine in phase III clinical trials for the treatment of prostate cancer. These results suggested that ABO blood type might influence efficacy. Unfortunately, blood types were not available in the clinical records for all but 8 patients and insufficient amounts of sera were left for standard blood typing methods. To test the hypothesis, therefore, we developed a new glycan microarray-based method for determining ABO blood type. The method requires only 4 μL of serum, provides 97% accuracy, and allows simultaneous profiling of many other serum anti-glycan antibodies. After validation with 220 healthy subjects of known blood type, the method was then applied to 74 PROSTVAC-VF patients and 37 control patients from a phase II trial. In this retrospective study, we found that type B and O PROSTVAC-VF patients demonstrated markedly improved clinical outcomes relative to A and AB patients, including longer median survival, longer median survival relative to Halabi predicted survival, and improved overall survival via Kaplan-Meier survival analysis ( p = 0.006). Consequently, blood type may provide an inexpensive screen to pre-select patients likely to benefit from PROSTVAC-VF therapy.

Several transcription factors play a role in the alteration of gene expression that occurs during cancer metastasis. Twist expression has been shown to be associated with the hallmarks of the metastatic process, as well as poor prognosis and drug resistance in many tumor types. However, primarily due to their location within the cell and the lack of a hydrophobic groove required for drug attachment, transcription factors such as Twist are difficult to target with conventional therapies. An alternative therapeutic strategy is a vaccine comprised of a Modified vaccinia Ankara (MVA), incorporating the Twist transgene and a TRIad of COstimulatory Molecules (B7-1, ICAM-1, LFA-3; TRICOM). Here we characterize an MVA-TWIST/TRICOM vaccine that induced both CD4+ and CD8+ Twist-specific T-cell responses in vivo. In addition, administration of this vaccine reduced both the primary tumor growth and metastasis in the 4T1 model of metastatic breast cancer. In the TRAMP transgenic model of spontaneous prostate cancer, MVA-TWIST/TRICOM alone significantly improved survival, and when combined with the androgen receptor antagonist enzalutamide, the vaccine further improved survival. These studies thus provide a rationale for the use of active immunotherapy targeting transcription factors involved in the metastatic process and for the combination of cancer vaccines with androgen deprivation.

// Sofia R. Gameiro 1 , Momodou L. Jammeh 1 , Max M. Wattenberg 1 , Kwong Y. Tsang 1 , Soldano Ferrone 2 , and James W. Hodge 1 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA 2 Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA Correspondence: James W. Hodge, email: // Keywords : radiation, immunogenic modulation, antigen-processing machinery, calreticulin, CTL, ER stress, PERK, TAP1, TAP2, calnexin, tapasin, LMP2, LMP7, LMP10 Received : December 18, 2013 Accepted : December 31, 2013 Published : December 31, 2013 Abstract Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response. This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone.