Joseph F. Grosso

Safety and Activity of Anti–PD-L1 Antibody in Patients with Advanced Cancer

BACKGROUND Programmed death 1 (PD-1) protein, a T-cell coinhibitory receptor, and one of its ligands, PD-L1, play a pivotal role in the ability of tumor cells to evade the hosts immune system. Blockade of interactions between PD-1 and PD-L1 enhances immune function in vitro and mediates antitumor activity in preclinical models. METHODS In this multicenter phase 1 trial, we administered intravenous anti-PD-L1 antibody (at escalating doses ranging from 0.3 to 10 mg per kilogram of body weight) to patients with selected advanced cancers. Anti-PD-L1 antibody was administered every 14 days in 6-week cycles for up to 16 cycles or until the patient had a complete response or confirmed disease progression. RESULTS As of February 24, 2012, a total of 207 patients--75 with non-small-cell lung cancer, 55 with melanoma, 18 with colorectal cancer, 17 with renal-cell cancer, 17 with ovarian cancer, 14 with pancreatic cancer, 7 with gastric cancer, and 4 with breast cancer--had received anti-PD-L1 antibody. The median duration of therapy was 12 weeks (range, 2 to 111). Grade 3 or 4 toxic effects that investigators considered to be related to treatment occurred in 9% of patients. Among patients with a response that could be evaluated, an objective response (a complete or partial response) was observed in 9 of 52 patients with melanoma, 2 of 17 with renal-cell cancer, 5 of 49 with non-small-cell lung cancer, and 1 of 17 with ovarian cancer. Responses lasted for 1 year or more in 8 of 16 patients with at least 1 year of follow-up. CONCLUSIONS Antibody-mediated blockade of PD-L1 induced durable tumor regression (objective response rate of 6 to 17%) and prolonged stabilization of disease (rates of 12 to 41% at 24 weeks) in patients with advanced cancers, including non-small-cell lung cancer, melanoma, and renal-cell cancer. (Funded by Bristol-Myers Squibb and others; ClinicalTrials.gov number, NCT00729664.).

PD-1 Blockade with Nivolumab in Relapsed or Refractory Hodgkin's Lymphoma

BACKGROUND Preclinical studies suggest that Reed-Sternberg cells exploit the programmed death 1 (PD-1) pathway to evade immune detection. In classic Hodgkins lymphoma, alterations in chromosome 9p24.1 increase the abundance of the PD-1 ligands, PD-L1 and PD-L2, and promote their induction through Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. We hypothesized that nivolumab, a PD-1-blocking antibody, could inhibit tumor immune evasion in patients with relapsed or refractory Hodgkins lymphoma. METHODS In this ongoing study, 23 patients with relapsed or refractory Hodgkins lymphoma that had already been heavily treated received nivolumab (at a dose of 3 mg per kilogram of body weight) every 2 weeks until they had a complete response, tumor progression, or excessive toxic effects. Study objectives were measurement of safety and efficacy and assessment of the PDL1 and PDL2 (also called CD274 and PDCD1LG2, respectively) loci and PD-L1 and PD-L2 protein expression. RESULTS Of the 23 study patients, 78% were enrolled in the study after a relapse following autologous stem-cell transplantation and 78% after a relapse following the receipt of brentuximab vedotin. Drug-related adverse events of any grade and of grade 3 occurred in 78% and 22% of patients, respectively. An objective response was reported in 20 patients (87%), including 17% with a complete response and 70% with a partial response; the remaining 3 patients (13%) had stable disease. The rate of progression-free survival at 24 weeks was 86%; 11 patients were continuing to participate in the study. Reasons for discontinuation included stem-cell transplantation (in 6 patients), disease progression (in 4 patients), and drug toxicity (in 2 patients). Analyses of pretreatment tumor specimens from 10 patients revealed copy-number gains in PDL1 and PDL2 and increased expression of these ligands. Reed-Sternberg cells showed nuclear positivity of phosphorylated STAT3, indicative of active JAK-STAT signaling. CONCLUSIONS Nivolumab had substantial therapeutic activity and an acceptable safety profile in patients with previously heavily treated relapsed or refractory Hodgkins lymphoma. (Funded by Bristol-Myers Squibb and others; ClinicalTrials.gov number, NCT01592370.).

Immune Inhibitory Molecules LAG-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4(+) and CD8(+) T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3(-/-)Pdcd1(-/-) mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

Cutting Edge: An In Vivo Requirement for STAT3 Signaling in TH17 Development and TH17-Dependent Autoimmunity

STAT3 activation has been observed in several autoimmune diseases, suggesting that STAT3-mediated pathways promote pathologic immune responses. We provide in vivo evidence that the fundamental role of STAT3 signaling in autoimmunity relates to its absolute requirement for generating TH17 T cell responses. We show that STAT3 is a master regulator of this pathogenic T cell subtype, acting at multiple levels in vivo, including TH17 T cell differentiation and cytokine production, as well as induction of RORγt and the IL-23R. Neither naturally occurring TH17 cells nor TH17-dependent autoimmunity occurs when STAT3 is ablated in CD4 cells. Furthermore, ablation of STAT3 signaling in CD4 cells results in increased TH1 responses, indicating that STAT3 signaling skews TH responses away from the TH1 pathway and toward the TH17 pathway. Thus, STAT3 is a candidate target for TH17-dependent autoimmune disease immunotherapy that could selectively inhibit pathogenic immune pathways.

LAG-3 regulates CD8+ T cell accumulation and effector function in murine self- and tumor-tolerance systems

Lymphocyte activation gene-3 (LAG-3) is a cell-surface molecule with diverse biologic effects on T cell function. We recently showed that LAG-3 signaling is important in CD4+ regulatory T cell suppression of autoimmune responses. Here, we demonstrate that LAG-3 maintains tolerance to self and tumor antigens via direct effects on CD8+ T cells using 2 murine systems. Naive CD8+ T cells express low levels of LAG-3, and expression increases upon antigen stimulation. Our data show increased levels of LAG-3 protein on antigen-specific CD8+ T cells within antigen-expressing organs or tumors. In vivo antibody blockade of LAG-3 or genetic ablation of the Lag-3 gene resulted in increased accumulation and effector function of antigen-specific CD8+ T cells within organs and tumors that express their cognate antigen. Most notably, combining LAG-3 blockade with specific antitumor vaccination resulted in a significant increase in activated CD8+ T cells in the tumor and disruption of the tumor parenchyma. A major component of this effect was CD4 independent and required LAG-3 expression by CD8+ T cells. Taken together, these data demonstrate a direct role for LAG-3 on CD8+ T cells and suggest that LAG-3 blockade may be a potential cancer treatment.

Nivolumab in Patients With Relapsed or Refractory Hematologic Malignancy: Preliminary Results of a Phase Ib Study

PURPOSE Cancer cells can exploit the programmed death-1 (PD-1) immune checkpoint pathway to avoid immune surveillance by modulating T-lymphocyte activity. In part, this may occur through overexpression of PD-1 and PD-1 pathway ligands (PD-L1 and PD-L2) in the tumor microenvironment. PD-1 blockade has produced significant antitumor activity in solid tumors, and similar evidence has emerged in hematologic malignancies. METHODS In this phase I, open-label, dose-escalation, cohort-expansion study, patients with relapsed or refractory B-cell lymphoma, T-cell lymphoma, and multiple myeloma received the anti-PD-1 monoclonal antibody nivolumab at doses of 1 or 3 mg/kg every 2 weeks. This study aimed to evaluate the safety and efficacy of nivolumab and to assess PD-L1/PD-L2 locus integrity and protein expression. RESULTS Eighty-one patients were treated (follicular lymphoma, n = 10; diffuse large B-cell lymphoma, n = 11; other B-cell lymphomas, n = 10; mycosis fungoides, n = 13; peripheral T-cell lymphoma, n = 5; other T-cell lymphomas, n = 5; multiple myeloma, n = 27). Patients had received a median of three (range, one to 12) prior systemic treatments. Drug-related adverse events occurred in 51 (63%) patients, and most were grade 1 or 2. Objective response rates were 40%, 36%, 15%, and 40% among patients with follicular lymphoma, diffuse large B-cell lymphoma, mycosis fungoides, and peripheral T-cell lymphoma, respectively. Median time of follow-up observation was 66.6 weeks (range, 1.6 to 132.0+ weeks). Durations of response in individual patients ranged from 6.0 to 81.6+ weeks. CONCLUSION Nivolumab was well tolerated and exhibited antitumor activity in extensively pretreated patients with relapsed or refractory B- and T-cell lymphomas. Additional studies of nivolumab in these diseases are ongoing.

Eos Mediates Foxp3-Dependent Gene Silencing in CD4+ Regulatory T Cells

Treg Responses to Eos CD4+ regulatory T cells (Tregs) are critical for keeping our immune system in check: They prevent immune responses from getting out of hand and keep autoimmunity at bay. By activating the expression of some genes and turning off expression of others, the master regulatory transcription factor of Tregs, Foxp3, endows these cells with the appropriate gene expression program to mediate their suppressive effects. Pan et al. (p. 1142, published online 20 August) now demonstrate that the transcription factor Eos is selectively required for Foxp3-mediated gene suppression in mice. Genes normally suppressed by Foxp3 in Tregs remained “on” when Eos expression was suppressed, whereas genes activated by Foxp3 were unaffected. Treg function was also affected by Eos suppression. With half their genetic program disrupted, these cells resembled an intermediate between Tregs and conventional CD4+ T cells—unable to suppress immune responses properly and partially responsive to T cell–activating stimulation. A transcription factor required for gene suppression in regulatory T cells is identified. CD4+ regulatory T cells (Tregs) maintain immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses. The core genetic program of Tregs and their ability to suppress pathologic immune responses depends on the transcription factor Foxp3. Despite progress in understanding mechanisms of Foxp3-dependent gene activation, the molecular mechanism of Foxp3-dependent gene repression remains largely unknown. We identified Eos, a zinc-finger transcription factor of the Ikaros family, as a critical mediator of Foxp3-dependent gene silencing in Tregs. Eos interacts directly with Foxp3 and induces chromatin modifications that result in gene silencing in Tregs. Silencing of Eos in Tregs abrogates their ability to suppress immune responses and endows them with partial effector function, thus demonstrating the critical role that Eos plays in Treg programming.

A role for the transcription factor Helios in human CD4+CD25+ regulatory T cells

Relative upregulation of the Ikaros family transcription factor Helios in natural regulatory T cells (Tregs) has been reported by several groups. However, a role for Helios in regulatory T cells has not yet been described. Here, we show that Helios is upregulated in CD4(+)CD25(+) regulatory T cells. Chromatin-immunoprecipitation (ChIP) experiments indicated that Helios binds to the FoxP3 promoter. These data were further corroborated by experiments showing that knocking-down Helios with siRNA oligonucleotides results in down-regulation of FoxP3. Functionally, we found that suppression of Helios message in CD4(+)CD25(+) T cells significantly attenuates their suppressive function. Taken together, these data suggest that Helios may play an important role in regulatory T cell function and support the concept that Helios may be a novel target to manipulate Treg activity in a clinical setting.

Tc17 CD8 T Cells: Functional Plasticity and Subset Diversity

IL-17-secreting CD8 T cells (Tc17) have been described in several settings, but little is known regarding their functional characteristics. While Tc1 cells produced IFN-γ and efficiently killed targets, Tc17 cells lacked lytic function in vitro. Interestingly, the small numbers of IFN-γ-positive or IL-17/IFN-γ-double-positive cells generated under Tc17 conditions also lacked lytic activity and expressed a similar pattern of cell surface proteins to IL-17-producing cells. As is the case for Th17 (CD4) cells, STAT3 is important for Tc17 polarization, both in vitro and in vivo. Adoptive transfer of highly purified, Ag-specific IL-17-secreting Tc17 cells into Ag-bearing hosts resulted in near complete conversion to an IFN-γ-secreting phenotype and substantial pulmonary pathology, demonstrating functional plasticity. Tc17 also accumulated to a greater extent than did Tc1 cells, suggesting that adoptive transfer of CD8 T cells cultured in Tc17 conditions may have therapeutic potential for diseases in which IFN-γ-producing cells are desired.

Cyclophosphamide Augments Antitumor Immunity: Studies in an Autochthonous Prostate Cancer Model

To study the immune response to prostate cancer, we developed an autochthonous animal model based on the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse in which spontaneously developing tumors express influenza hemagglutinin as a unique, tumor-associated antigen. Our prior studies in these animals showed immunologic tolerance to hemagglutinin, mirroring the clinical situation in patients with cancer who are generally nonresponsive to their disease. We used this physiologically relevant animal model to assess the immunomodulatory effects of cyclophosphamide when administered in combination with an allogeneic, cell-based granulocyte-macrophage colony-stimulating factor-secreting cancer immunotherapy. Through adoptive transfer of prostate/prostate cancer-specific CD8 T cells as well as through studies of the endogenous T-cell repertoire, we found that cyclophosphamide induced a marked augmentation of the antitumor immune response. This effect was strongly dependent on both the dose and the timing of cyclophosphamide administration. Mechanistic studies showed that immune augmentation by cyclophosphamide was associated with a transient depletion of regulatory T cells in the tumor draining lymph nodes but not in the peripheral circulation. Interestingly, we also noted effects on dendritic cell phenotype; low-dose cyclophosphamide was associated with increased expression of dendritic cell maturation markers. Taken together, these data clarify the dose, timing, and mechanism of action by which immunomodulatory cyclophosphamide can be translated to a clinical setting in a combinatorial cancer treatment strategy.