Glenn Dranoff

Melanoma inhibitor of apoptosis protein (ML-IAP) is a target for immune-mediated tumor destruction

The identification of antigens associated with tumor destruction is a major goal of cancer immunology. Vaccination with irradiated tumor cells engineered to secrete granulocyte–macrophage colony stimulating factor generates potent, specific, and long-lasting antitumor immunity through improved tumor antigen presentation by dendritic cells and macrophages. A phase I clinical trial of this immunization strategy in patients with disseminated melanoma revealed the consistent induction in distant metastases of dense T and B cell infiltrates that effectuated substantial tumor necrosis and fibrosis. To delineate the target antigens of this vaccine-stimulated tumor destruction, we screened a melanoma cDNA expression library with postimmunization sera from a long-term responding patient (K030). High-titer IgG antibodies recognized melanoma inhibitor of apoptosis protein (ML-IAP), a caspase antagonist containing a single baculoviral IAP repeat and a COOH-terminal RING domain. Although K030 harbored antibodies to ML-IAP at the time of study entry, multiple courses of vaccination over 4 years increased antibody titers and elicited isotype switching. Moreover, lymphocyte infiltrates in necrotic metastases included CD4+ and CD8+ T cells specific for ML-IAP, as revealed by proliferation, tetramer, enzyme-linked immunospot, and cytotoxicity analysis. Whereas melanoma cells in densely infiltrated lesions showed strong ML-IAP expression by immunohistochemistry, lethal disease progression was associated with the loss of ML-IAP staining and the absence of lymphocyte infiltrates. These findings demonstrate that ML-IAP can serve as a target for immune-mediated tumor destruction, but that antigen-loss variants can accomplish immune escape.

Characterization of a spontaneous murine astrocytoma and abrogation of its tumorigenicity by cytokine secretion.

OBJECTIVEnThe promise of immunotherapies developed against brain tumors in animal models has not been realized in human clinical trials. This may be because of the routine use of rodent tumors artificially induced by chemicals or viruses that do not accurately portray the intrinsic qualities of spontaneously arising human tumors and that often fail to incorporate the role of immunosuppressants, such as transforming growth factor-beta, that are secreted by human gliomas. From an astrocytoma that arose spontaneously in inbred VM/Dk mice, we have characterized a highly tumorigenic spontaneous murine astrocytoma cell line (SMA-560) that retains features of glial differentiation and naturally produces high levels of biologically active transforming growth factor-beta. We have used this model to determine whether cytokine production by tumor cells will inhibit intracerebral astrocytoma growth.nnnMETHODSnPackaging cell lines producing replication-incompetent retroviral vectors were used to transfect the SMA-560 cell line in vitro with the genes encoding the murine cytokines interleukin (IL)-2, IL-3, IL-4, IL-6, tumor necrosis factor-alpha, gamma-interferon, or granulocyte-macrophage colony-stimulating factor or the costimulatory molecule B7.1 (CD80).nnnRESULTSnMice challenged intracerebrally with 5000 untransfected SMA-560 cells all succumbed to tumor within 30 days, with a median survival of 25 days. In contrast, mice challenged with SMA-560 cells producing IL-2, IL-4, or tumor necrosis factor-alpha each had a more than 400% increase in median survival (P < 0.0001). In these groups, 78.3% (18 of 23 mice), 66.7% (10 of 15 mice), and 60% (6 of 10 mice) of the mice, respectively, remained alive without evidence of tumor for longer than 100 days after the initial tumor challenge. All other cytokines tested and the expression of B7.1 failed to result in an increase in median survival.nnnCONCLUSIONnUsing a spontaneous astrocytoma model in an inbred mouse strain, we have shown that cytokine production by glial tumors can abrogate their tumorigenicity in vivo despite production of transforming growth factor-beta. These results predict that approaches directed at cytokine production within intracerebral astrocytomas may be efficacious in human trials and that the immunological privilege of the brain may not be absolute under such conditions.

STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment

STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether the inactivation of tumor suppressor genes, such as STK11/LKB1, exerts similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T-cell-suppressive effects, along with a corresponding increase in the expression of T-cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/LKB1-inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1-targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL6-neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1-mutated tumors with PD-1-targeting antibody therapies.

The Human Vaccines Project: A roadmap for cancer vaccine development

A concerted international effort is necessary to achieve clinically effective cancer vaccines. Cancer vaccine development has been vigorously pursued for 40 years. Immunity to tumor antigens can be elicited by most vaccines tested, but their clinical efficacy remains modest. We argue that a concerted international effort is necessary to understand the human antitumor immune response and achieve clinically effective cancer vaccines.

Vaccines Combined with Immune Checkpoint Antibodies Promote Cytotoxic T-cell Activity and Tumor Eradication

Despite dramatic clinical successes for cancer vaccines and immune checkpoint blockade, disease usually progresses. In a mouse model that combined vaccines with checkpoint blockade, significant CTL activation, tumor eradication, and long-term survival was achieved. We demonstrate that a poly(lactide-co-glycolide) (PLG) cancer vaccine can be used in combination with immune checkpoint antibodies, anti–CTLA-4 or anti–PD-1, to enhance cytotoxic T-cell (CTL) activity and induce the regression of solid B16 tumors in mice. Combination therapy obviated the need for vaccine boosting and significantly skewed intratumoral reactions toward CTL activity, resulting in the regression of B16 tumors up to 50 mm2 in size and 75% survival rates. These data suggest that combining material-based cancer vaccines with checkpoint antibodies has the potential to mediate tumor regression in humans. Cancer Immunol Res; 4(2); 95–100. ©2015 AACR.

Soluble PD-L1 as a Biomarker in Malignant Melanoma Treated with Checkpoint Blockade.

Melanoma cells could secrete several splice variants of PD-L1. Secretion differed among patients, and was affected by checkpoint therapy, with some changes associated with progressive disease, and others with favorable outcomes, suggesting circulating PD-L1 as a dynamic biomarker. Blockade of the pathway including programmed death-ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) has produced clinical benefits in patients with a variety of cancers. Elevated levels of soluble PD-L1 (sPD-L1) have been associated with worse prognosis in renal cell carcinoma and multiple myeloma. However, the regulatory roles and function of sPD-L1 particularly in connection with immune checkpoint blockade treatment are not fully understood. We identified four splice variants of PD-L1 in melanoma cells, and all of them are secreted. Secretion of sPD-L1 resulted from alternate splicing activities, cytokine induction, cell stress, cell injury, and cell death in melanoma cells. Pretreatment levels of sPD-L1 were elevated in stage IV melanoma patient sera compared with healthy donors. High pretreatment levels of sPD-L1 were associated with increased likelihood of progressive disease in patients treated by CTLA-4 or PD-1 blockade. Although changes in circulating sPD-L1 early after treatment could not distinguish responders from those with progressive disease, after five months of treatment by CTLA-4 or PD-1 blockade patients who had increased circulating sPD-L1 had greater likelihood of developing a partial response. Induction of sPD-L1 was associated with increased circulating cytokines after CTLA-4 blockade but not following PD-1 blockade. Circulating sPD-L1 is a prognostic biomarker that may predict outcomes for subgroups of patients receiving checkpoint inhibitors. Cancer Immunol Res; 5(6); 480–92. ©2017 AACR.

Advances in Therapeutic Cancer Vaccines.

Therapeutic cancer vaccines aim to induce durable antitumor immunity that is capable of systemic protection against tumor recurrence or metastatic disease. Many approaches to therapeutic cancer vaccines have been explored, with varying levels of success. However, with the exception of Sipuleucel T, an ex vivo dendritic cell vaccine for prostate cancer, no therapeutic cancer vaccine has yet shown clinical efficacy in phase 3 randomized trials. Though disappointing, lessons learned from these studies have suggested new strategies to improve cancer vaccines. The clinical success of checkpoint blockade has underscored the role of peripheral tolerance mechanisms in limiting vaccine responses and highlighted the potential for combination therapies. Recent advances in transcriptome sequencing, computational modeling, and material engineering further suggest new opportunities to intensify cancer vaccines. This review will discuss the major approaches to therapeutic cancer vaccination and explore recent advances that inform the design of the next generation of cancer vaccines.

Immunotherapy at Large: Balancing tumor immunity and inflammatory pathology

Gertrude Elion and George Hitchings, pioneers of cancer chemotherapy, taught that the search for selectivity in drug discovery often involved ‘enlightened empiricism’ together with a healthy dose of serendipity1. Two recently published clinical trials that report the remarkable antitumor effects of combination therapy using antibodies targeting programmed death-1 (PD-1) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4)2,3, two negative immune regulatory molecules expressed on T cells, illustrate how these principles of drug development apply to cancer immunotherapy. n nThe passive transfer of tumor-reactive T cells via bone marrow transplantation and the use of tumor-selective antibodies are well-established cancer therapies, but the crafting of effective strategies to stimulate endogenous antitumor immunity has long proved elusive4. As many attempts at amplifying positive immune signaling networks failed to elicit clinically significant tumor destruction, an alternative approach aims to do the opposite—interfering with immune checkpoints that attenuate the function of activated T cells in the tumor microenvironment5. Inspired by promising findings in experimental tumor models, two large randomized studies showing that a fully human antibody to CTLA-4 (ipilimumab) prolonged survival in patients with advanced melanoma, with about 20% of treated subjects achieving durable clinical benefits (which in some cases exceeded ten years), finally took place. However, the treatment also caused a diversity of inflammatory pathologies, most commonly affecting the intestinal tract, skin and endocrine tissues, consistent with the phenotype of mice lacking CTLA-4. Serious toxicity was observed in about 20% of treated subjects, but this could be dissociated from the antitumor effects, indicating that tumor immunity and inflammatory pathology are closely related but, nonetheless, separable. n nMotivated in large measure by the success of ipilimumab in patients, clinical exploration of blocking antibodies to PD-1 also began. Although many studies had delineated a key role for PD-1 in limiting T cell responses in diverse settings, including experimental tumors6, they did not anticipate the robust antitumor activities of anti–PD-1 antibodies in patients with cancer. The recent work with lambrolizumab2 and an earlier trial using nivolumab7 have convincingly established the ability of anti–PD-1 antibodies to trigger clinically significant tumor destruction in a high proportion of patients with advanced melanoma. The impressive durable regressions revealed in these two investigations exceed expectations gleaned from preclinical systems, highlighting some of the challenges in using murine models as a guide to designing new therapeutics for humans8 and illustrating the continuing role of serendipity in drug development. n nAbout 38% of 135 individuals with metastatic melanoma treated at any dose-level of lambrolizumab and 52% at an apparently optimal level displayed a tumor response; remarkably, 77% of all patients showed some reduction in tumor burden2. Even though small-molecule inhibitors of BRAF and MEK, key oncogenic drivers in melanoma, may also accomplish tumor regressions in genetically defined subsets, these typically are of short duration because of the rapid emergence of drug-resistant tumor cells9. In contrast, most responses to PD-1 blockade were sustained, although longer follow-up is required to clarify the durability of tumor control more precisely. n nAnti–PD-1 antibodies may have a more favorable safety profile compared to ipilimumab, although severe pneumonitis developed in a small number of patients7. The reduction in toxicity is in accordance with the milder inflammatory pathology in PD-1– compared to CTLA-4-deficient mice, which probably reflects the more restricted role of PD-1 in constraining ongoing immune responses in contrast to the requirement for CTLA-4 in immune homeostasis6. Encouraging anti-tumor effects and safety profiles have also been described with blocking antibodies to PD ligand-1 (PD-L1)10, underscoring the therapeutic potential of interfering with this pathway in different ways. Moreover, anti–PD-1 and anti–PD-L1 antibodies seem to be broadly active against cancer, provoking tumor regressions in patients with advanced non–small-cell lung carcinoma, renal cell carcinoma and other tumor types11. n nGiven the clinical efficacy of infusing anti–CTLA-4 or anti–PD-1 antibodies as monotherapy and the related but distinct functions of the molecules in the immune system6, combinations of the two agents might prove to be more potent than either alone. Anti–PD-1 antibodies elicited similar response rates in patients who were previously treated with ipilimumab (and presumably derived minimal or no clinical benefit) and in those who were not2. This suggests PD-1–mediated immune inhibition may act, at least in some cases, to restrict the impact of CTLA-4 inhibition, which can increase amounts of PD-L1 in the tumor microenvironment (owing to an unleashed interferon-γ production by T cells) that can then engage PD-1 on activated T cells to dampen proliferation and cytotoxicity. n nTo explore this idea further, Wolchok et al.3 administered nivolumab and ipilimumab concurrently to 53 patients with metastatic melanoma. Antitumor effects were evident in 65% of subjects, including objective responses in 40%. In contrast to sequential treatment, the concurrent infusion of anti–CTLA-4 and anti–PD-1 antibodies led to a high rate of serious inflammatory toxicity (53% of patients), which often required immunosuppressive therapy for management. These results imply that CTLA-4 and PD-1 have nonredundant roles in the maintenance of immune tolerance in humans, an insight that should inform our understanding of the pathogenesis of some autoimmune disorders12. Randomized clinical trials to delineate more thoroughly the potential advantages and risks of concurrent or sequential combination therapy versus individual blockade of CTLA-4 or PD-1 are underway (Fig. 1). n n n nFigure 1 n nThe need to balance the antitumor activity and toxicity of single and combinatorial immunotherapies. Blocking the immune inhibitory molecules CTLA-4 and PD-1, alone or in combination, results in variable levels of antitumor activity and toxicity. Further ... n n n nThe elevated incidence of toxicity with simultaneous CTLA-4 and PD-1 inhibition emphasizes the need to craft complementary strategies that afford a more favorable therapeutic index for antibody treatment. This might be accomplished through preferential targeting of antitumor T cells rather than homeostatic immune networks that preserve tissue integrity. Because CTLA-4 and PD-1 are both expressed upon T cell activation, combinatorial schemes should be directed toward enhancing the numbers of stimulated tumor-reactive T cells, which may allow less intense levels of immune checkpoint blockade to amplify tumor-specific immunity more selectively. Cancer vaccines that deliver tumor antigens in an optimal spatial and temporal fashion while promoting maturation of dendritic cells, which amplify antitumor T cell activation, constitute one promising approach to expand the effector T cell pool. Treatment strategies that may evoke immunogenic tumor cell death in situ, such as oncolytic viruses, focal radiation, tumor-directed antibodies (and drug conjugates) and some small-molecule inhibitors or cytotoxic agents, are also attractive options9. Antibodies targeting other immunoregulatory molecules expressed on T cells or natural killer cells, an innate cell population with potent antitumor cytotoxicity potential13, are additional compelling possibilities. n nAn alternative scheme to improve therapeutic index might aim to restore homeostatic networks that are compromised with immune checkpoint blockade. A recent clinical trial in patients with advanced melanoma suggests that systemic administration of the cytokine granulocyte-macrophage colony–stimulating factor (GM-CSF) may reduce the incidence of severe colitis and pneumonitis caused by anti–CTLA-4 antibodies yet increase overall survival14. Although the precise basis for these intriguing findings remains to be clarified, the roles of GM-CSF in both supporting regulatory T cells that attenuate inflammation in the intestinal and pulmonary mucosa and augmenting antitumor immunity might be involved15. n nThe clinical success of CTLA-4 and PD-1 blockade alone and together may come as a surprise to the greater cancer research and cancer medicine communities. Earlier uncertainty regarding a role for immunity in tumor pathogenesis reinforced the investigation of tumor cell–autonomous alterations and encouraged the evaluation of therapeutics using human tumor cell xenografts propagated in immune-deficient mice. Nonetheless, the demonstration that two molecules expressed on T cells may be the difference between survival and death in a significant proportion of patients with advanced cancer should prompt a reexamination of these research strategies and focus attention toward illuminating the ways in which specific steps of transformation are directly coupled to immune escape16. A deeper understanding of the links between oncogenesis and host evasion should accelerate the crafting of new combinatorial treatments that intensify tumor immunity but temper inflammatory pathology.

Interleukin-17A Promotes Lung Tumor Progression through Neutrophil Attraction to Tumor Sites and Mediating Resistance to PD-1 Blockade

Introduction Proinflammatory cytokine interleukin‐17A (IL‐17A) is overexpressed in a subset of patients with lung cancer. We hypothesized that IL‐17A promotes a protumorigenic inflammatory phenotype and inhibits antitumor immune responses. Methods We generated bitransgenic mice expressing a conditional IL‐17A allele along with conditional KrasG12D and performed immune phenotyping of mouse lungs, a survival analysis, and treatment studies with antibodies either blocking programmed cell death 1 (PD‐1) or IL‐6 or depleting neutrophils. To support the preclinical findings, we analyzed human gene expression data sets and immune profiled patient lung tumors. Results Tumors in IL‐17:KrasG12D mice grew more rapidly, resulting in a significantly shorter survival as compared with that of KrasG12D mice. IL‐6, granulocyte colony‐stimulating factor (G‐CSF), milk fat globule‐EGF factor 8 protein, and C‐X‐C motif chemokine ligand 1 were increased in the lungs of IL17:Kras mice. Time course analysis revealed that levels of tumor‐associated neutrophils were significantly increased, and lymphocyte recruitment was significantly reduced in IL17:KrasG12D mice as compared with in KrasG12D mice. In therapeutic studies PD‐1 blockade was not effective in treating IL‐17:KrasG12D tumors. In contrast, blocking IL‐6 or depleting neutrophils with an anti–Ly‐6G antibody in the IL17:KrasG12D tumors resulted in a clinical response associated with T‐cell activation. In tumors from patients with lung cancer with KRAS mutation we found a correlation between higher levels of IL‐17A and colony‐ stimulating factor 3 and a significant correlation among high neutrophil and lower T‐cell numbers. Conclusions Here we have shown that an increase in a single cytokine, IL‐17A, without additional mutations can promote lung cancer growth by promoting inflammation, which contributes to resistance to PD‐1 blockade and sensitizes tumors to cytokine and neutrophil depletion.

T-Cell Exhaustion Signatures Vary with Tumor Type and Are Severe in Glioblastoma

Purpose: T-cell dysfunction is a hallmark of glioblastoma (GBM). Although anergy and tolerance have been well characterized, T-cell exhaustion remains relatively unexplored. Exhaustion, characterized in part by the upregulation of multiple immune checkpoints, is a known contributor to failures amid immune checkpoint blockade, a strategy that has lacked success thus far in GBM. This study is among the first to examine, and credential as bona fide, exhaustion among T cells infiltrating human and murine GBM. Experimental Design: Tumor-infiltrating and peripheral blood lymphocytes (TILs and PBLs) were isolated from patients with GBM. Levels of exhaustion-associated inhibitory receptors and poststimulation levels of the cytokines IFNγ, TNFα, and IL2 were assessed by flow cytometry. T-cell receptor Vβ chain expansion was also assessed in TILs and PBLs. Similar analysis was extended to TILs isolated from intracranial and subcutaneous immunocompetent murine models of glioma, breast, lung, and melanoma cancers. Results: Our data reveal that GBM elicits a particularly severe T-cell exhaustion signature among infiltrating T cells characterized by: (1) prominent upregulation of multiple immune checkpoints; (2) stereotyped T-cell transcriptional programs matching classical virus-induced exhaustion; and (3) notable T-cell hyporesponsiveness in tumor-specific T cells. Exhaustion signatures differ predictably with tumor identity, but remain stable across manipulated tumor locations. Conclusions: Distinct cancers possess similarly distinct mechanisms for exhausting T cells. The poor TIL function and severe exhaustion observed in GBM highlight the need to better understand this tumor-imposed mode of T-cell dysfunction in order to formulate effective immunotherapeutic strategies targeting GBM. Clin Cancer Res; 24(17); 4175–86. ©2018 AACR. See related commentary by Jackson and Lim, p. 4059