David C. Binder

Relapse or eradication of cancer is predicted by peptide-major histocompatibility complex affinity.

Cancers often relapse after adoptive therapy, even though specific T cells kill cells from the same cancer efficiently in vitro. We found that tumor eradication by T cells required high affinities of the targeted peptides for major histocompatibility complex (MHC) class I. Affinities of at least 10 nM were required for relapse-free regression. Only high-affinity peptide-MHC interactions led to efficient cross-presentation of antigen, thereby stimulating cognate T cells to secrete cytokines. These findings highlight the importance of targeting peptides with high affinity for MHC class I when designing T cell-based immunotherapy.

Molecular Pathways: Targeting IDO1 and Other Tryptophan Dioxygenases for Cancer Immunotherapy

Indoleamine 2, 3-dioxygenase 1 (IDO1), IDO2, and tryptophan 2, 3-dioxygenase (TDO) comprise a family of enzymes that catalyze the first- and rate-limiting step associated with the catabolic conversion of tryptophan (Trp) into kynurenine (Kyn). Through subsequent enzymatic and spontaneous reactions, Kyn is further converted into the energetic substrates, NAD+ and ATP, to fuel cellular metabolic functions. Coincidently, the depletion of Trp and accumulation of Kyn has been demonstrated to induce effector T-cell apoptosis/dysfunction and immunosuppressive regulatory T-cell induction, respectively. Similar to other immune checkpoints, IDO1 and TDO are suggested to be important targets for immunotherapeutic intervention. This is represented by the recent growth of efforts to inhibit the Trp-to-Kyn pathway as a means to control immunosuppression. Inhibitors currently in clinical trials, INCB024360, GDC-0919, indoximod, and an IDO1 peptide-based vaccine, are being evaluated for their efficacy against a wide range of cancers including melanoma, glioblastoma, non–small cell lung, pancreatic, and/or breast cancer, as well as metastatic disease. Despite the rapid development of potent clinical grade inhibitors, strategic questions remain. Here, we review the state of the literature with respect to current therapeutic inhibitors of tryptophan catabolism, evaluation of those efforts preclinically and clinically, compensatory changes that occur with therapeutic targeting, as well as newly recognized signaling features that raise critical questions to the field. Given the rapidly evolving interest in determining how IDO1/TDO, and to an unknown extent, IDO2, can be targeted for increasing cancer immunotherapeutic efficacy, we present a brief but comprehensive analysis that addresses critical questions, while highlighting the mechanics that remain to be explored. Clin Cancer Res; 21(24); 5427–33. ©2015 AACR.

Antigen-Specific Bacterial Vaccine Combined with Anti-PD-L1 Rescues Dysfunctional Endogenous T Cells to Reject Long-Established Cancer

Mobilizing the latent pool of tumor-specific T cells in patients is a goal for immunotherapy. Using Salmonella typhimurium to deliver tumor-specific antigens into the tumor, Binder and colleagues found that when combined with αPD-L1 blocking antibodies, this regimen rescued endogenous dysfunctional tumor-specific CD8 T cells and eradicated the established tumors. Immunogenic tumors grow progressively even when heavily infiltrated by CD8+ T cells. We investigated how to rescue CD8+ T-cell function in long-established immunogenic melanomas that contained a high percentage of endogenous PD-1+ tumor-specific CD8+ T cells that were dysfunctional. Treatment with αPD-L1– and αCTLA-4–blocking antibodies did not prevent tumors from progressing rapidly. We then tested exogenous tumor-specific antigen delivery into tumors using Salmonella Typhimurium A1-R (A1-R) to increase antigen levels and generate a proinflammatory tumor microenvironment. Antigen-producing A1-R rescued the endogenous tumor-specific CD8+ T-cell response: Proliferation was induced in the lymphoid organs and effector function was recovered in the tumor. Treatment with antigen-producing A1-R led to improved mouse survival and resulted in 32% rejection of long-established immunogenic melanomas. Following treatment with antigen-producing A1-R, the majority of tumor-specific CD8+ T cells still expressed a high level of PD-1 in the tumor. Combining antigen-producing A1-R with αPD-L1-blocking antibody enhanced the expansion of tumor-specific CD8+ T cells and resulted in 80% tumor rejection. Collectively, these data show a powerful new therapeutic approach to rescue dysfunctional endogenous tumor-specific CD8+ T cells and eradicate advanced immunogenic tumors. Cancer Immunol Res; 1(2); 123–33. ©2013 AACR.

Densely Granulated Murine NK Cells Eradicate Large Solid Tumors

Natural killer (NK) cells inhibit early stages of tumor formation, recurrence, and metastasis. Here, we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL-15 receptor α on cancer cells was needed to efficiently induce granulated NK cells, and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors.

Immunotherapy for cancer in the central nervous system: Current and future directions

ABSTRACT Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and still remains incurable. Although immunotherapeutic vaccination against GBM has demonstrated immune-stimulating activity with some promising survival benefits, tumor relapse is common, highlighting the need for additional and/or combinatorial approaches. Recently, antibodies targeting immune checkpoints were demonstrated to generate impressive clinical responses against advanced melanoma and other malignancies, in addition to showing potential for enhancing vaccination and radiotherapy (RT). Here, we summarize the current knowledge of central nervous system (CNS) immunosuppression, evaluate past and current immunotherapeutic trials and discuss promising future immunotherapeutic directions to treat CNS-localized malignancies.

Microvessel density is not increased in prostate cancer: digital imaging of routine sections and tissue microarrays.

Angiogenesis is considered a prognostic factor and therapy target in many tumors but remains controversial in prostate cancer. This study compares the microvessel density of normal prostate and prostate cancer of different grades using an automated approach to determine its clinical utility. Neoplastic and normal prostatic tissues from 60 prostatectomies were examined by routine histological sections (group I); 136 prostatectomies were used to create tissue microarrays (group II). Microvessel density was calculated using CD31 immunostaining. Automated Cellular Image System (ChromaVision, San Juan Capistrano, CA) and Aperio automated systems were used to digitally analyze microvessel density in Groups I and II respectively. Microvessel density was not significantly increased in tumor versus normal prostate in Group I (P = .303). Both the mean vessel count and vessel area were significantly higher in normal tissue than in tumor either by Automated Cellular Image System or Aperio analysis (P < .05). Aperio analysis in group II additionally showed significantly higher values in normal tissue for vessel lumen (P < .001), whereas vessel perimeter, wall thickness, vessel compactness, and shape were not significantly different (P > .05). Aperio comparison of low- versus high-grade prostate cancer demonstrated that only mean vessel count was increased in high-grade tumors (P = .047); no other automated parameter in either group showed significant association with Gleason scores. Irrespective of methodology, microvessel density was not increased in prostate cancer compared to normal prostate. The bias of using vascular hot spots that possibly contributed to previous contradictory results has been mitigated by automated microvessel density quantitation here. Similar microvessel density of low- and high-grade tumors indicate that microvessel density is neither an important nor reliable prognostic marker for prostate cancer.

Adoptively Transferred Immune T Cells Eradicate Established Tumors despite Cancer-Induced Immune Suppression

Myeloid-derived CD11b+Gr1+ suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are considered a major obstacle for effective adoptive T cell therapy. Myeloid cells suppress naive T cell proliferation ex vivo and can prevent the generation of T cell responses in vivo. We find, however, that adoptively transferred immune T cells eradicate well-established tumors in the presence of MDSCs and TAMs, which are strongly immunosuppressive ex vivo. These MDSCs and TAMs were comparable in numbers and immunosuppressive capacity among different tumor models. Longitudinal microscopy of tumors in vivo revealed that after T cell transfer, tumor vasculature and cancer cells disappeared simultaneously. During T cell–mediated tumor destruction, the tumor stroma contained abundant myeloid cells (mainly TAMs) that retained their suppressive properties. Preimmunized but not naive mice resisted immune suppression caused by an unrelated tumor burden, supporting the idea that in vivo, myeloid immunosuppressive cells can suppress naive but not memory T cell responses.

Radiotherapy and immune checkpoint blockade: potential interactions and future directions

Combining radiation and immune checkpoint blockade provides synergistic antitumor responses in animal models and a small subset of patients. New preclinical data have provided mechanistic insight into this treatment interaction and identification of therapeutic targets to optimize this approach.

Cytosolic group IVa phospholipase A2 mediates IL-8/CXCL8-induced transmigration of human polymorphonuclear leukocytes in vitro

BackgroundCytosolic gIVaPLA2 is a critical enzyme in the generation of arachidonate metabolites and in induction of β2-integrin adhesion in granulocytes. We hypothesized that gIVaPLA2 activation also is an essential downstream step for post adhesive migration of PMN in vitro.MethodsMigration of PMNs caused by IL-8/CXCL8 was assessed using a transwell migration chamber. PMNs were pretreated with two structurally unrelated inhibitors of gIVaPLA2, arachidonyl trifluoromethylketone (TFMK) or pyrrophenone, prior to IL-8/CXCL8 exposure. The fraction of migrated PMNs present in the lower chamber was measured as total myeloperoxidase content. GIVaPLA2 enzyme activity was analyzed using [14C-PAPC] as specific substrate F-actin polymerization and cell structure were examined after rhodamine-phalloidin staining.ResultsIL-8/CXCL8-induced migration of PMNs was elicited in concentration- and time-dependent manner. Time-related phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus was observed for PMNs stimulated with IL-8/CXCL8 in concentration sufficient to cause upstream phosphorylation of MAPKs (ERK-1/2 and p38) and Akt/PKB. Inhibition of gIVaPLA2 corresponded to the magnitude of blockade of PMN migration. Neither AA nor LTB4 secretion was elicited following IL-8/CXCL8 activation. In unstimulated PMNs, F-actin was located diffusely in the cytosol; however, a clear polarized morphology with F-actin-rich ruffles around the edges of the cell was observed after activation with IL-8/CXCL8. Inhibition of gIVaPLA2 blocked change in cell shape and migration caused by IL-8/CXCL8 but did not cause F-actin polymerization or translocation of cytosolic F-actin to inner leaflet of the PMN membrane.ConclusionWe demonstrate that IL-8/CXCL8 causes a) phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus, b) change in cell shape, c) polymerization of F-actin, and d) chemoattractant/migration of PMN in vitro. Inhibition of gIVaPLA2 blocks the deformability and subsequent migration of PMNs caused by IL-8/CXCL8. Our data suggest that activation of gIVaPLA2 is an essential step in PMN migration in vitro.

Non-tumor cell IDO1 predominantly contributes to enzyme activity and response to CTLA-4/PD-L1 inhibition in mouse glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor in adults with a median survival of 14.6months. A contributing factor to GBM aggressiveness is the intratumoral expression of the potently immunosuppressive enzyme, indoleamine 2,3 dioxygenase 1 (IDO1). The enzymatic activity of IDO1 is associated with the conversion of tryptophan into downstream kynurenine (Kyn), which has previously been hypothesized to contribute toward the suppression of tumor immunity. Utilizing the syngeneic, immunocompetent, intracranial GL261 cell GBM model, we previously demonstrated that tumor cell, but not non-tumor cell IDO1, suppresses T cell-mediated brain tumor regression in mice. Paradoxically, we also showed that the survival advantage mediated by immune checkpoint blockade is abrogated by non-tumor cell IDO1 deficiency. Here, we have built on our past observations and confirm the maladaptive role of tumor cell IDO1 in a novel mouse GBM model. We also demonstrate that, non-tumor cells, rather than mouse GBM cells, are the dominant contributor to IDO1-mediated enzyme activity. Finally, we show the novel associations between maximally-effective immune-checkpoint blockade-mediated survival, non-tumor cell IDO1 and intra-GBM Kyn levels. These data suggest for the first time that, GBM cell-mediated immunosuppression is IDO1 enzyme independent, while the survival benefits of immune checkpoint blockade require non-tumor cell IDO1 enzyme activity. Given that current clinical inhibitors vary in their mechanism of action, in terms of targeting IDO1 enzyme activity versus enzyme-independent effects, this work suggests that choosing an appropriate IDO1 pharmacologic will maximize the effectiveness of future immune checkpoint blockade approaches.