Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell–mediated killing of HLA class I–expressing tumors. Lack of KIR-HLA class I interactions has ...been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell–mediated lysis of HLA-C–expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3–positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.
Historically, attempts at cancer immunotherapy have emphasized strategies designed to stimulate or augment the immune system into action. In the past decade, a complementary approach has developed, ...that of releasing immune cells from inhibitory restraint. Discoveries in the fundamental biology of how immunity is regulated, how the immune system interfaces with malignancy, and how cancer cells may exploit these processes to evade detection have all been translated into the rapidly growing field of therapeutic immune checkpoint inhibition for cancer. Myeloma is a malignancy associated with significant immune dysfunction imparted both by the disease itself as well as by many of the immunosuppressive therapies that have been used in the past. The growing body of preclinical data regarding immunoregulatory mechanisms that appear active in myeloma has begun to be translated to clinical trials targeting these signaling axes. This review will attempt to summarize the current understanding of the basic biology of several immune checkpoint pathways that may be important in myeloma and provide an up-to-date overview of recent and ongoing clinical trials of immune checkpoint inhibitors in myeloma. Finally, several current challenges and possible future directions of immune checkpoint blockade in myeloma will be reviewed.
We characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically ...inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells.
FLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-gamma-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-gamma, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-gamma production when cultured with K562 targets as compared to vehicle (DMSO).
These data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy.
Abstract
Multiple myeloma (MM) is an essentially incurable malignancy associated with profound immune dysregulation. Despite the advent of novel therapies and improvements in survival over the last ...10 years, death from progressive disease and infection remains a common outcome. Natural killer (NK) cells are CD56(+)CD3(−) large granular lymphocytes that constitute a key cellular subset of the innate immune system. For over 30 years, the relationship between NK cells and MM has been described in the clinical setting and characterized in the laboratory. Data suggest that NK cells may play a role in the immune response to MM; however, this effect is lost due to immunoevasive strategies utilized by MM. Nevertheless, progress in the understanding of the mechanisms perpetuating this effect have led to new opportunities to recover or augment NK cell function therapeutically in MM. In fact, the novel agents thalidomide, lenalidomide and bortezomib all confer anti-MM effects, in part, through enhancement of NK cell function. Currently, the development of therapies designed specifically to increase NK cell cytotoxicity against MM is under way. The present review summarizes the current understanding of the NK cell versus MM effect and characterizes therapeutic interventions that exert anti-MM efficacy via NK cell function against the disease.
We characterized the effects of a newly developed signal transducers and activators of transcription 3 (STAT3) inhibitor, LLL12 in multiple myeloma (MM) cells. LLL12 specifically inhibited STAT3 ...phosphorylation, nuclear localization, DNA binding activity, down‐regulated STAT3 downstream genes, and induced apoptosis in MM cells. Importantly, LLL12 significantly inhibited STAT3 phosphorylation, induced apoptosis in primary MM cells which came from patients that were clinically resistant to lenalidomide and bortezomib. LLL12 is a potent inhibitor of cell proliferation with IC50 values ranging between 0.26 and 1.96 μM in MM and primary MM cells. LLL12 also inhibited STAT3 phosphorylation induced by interleukin‐6 (IL‐6) and interferon‐α but not STAT1, STAT2, STAT4 and STAT6 phosphorylation induced by interferon‐α, interferon‐γ and IL‐4 indicating the selectivity of LLL12 for STAT3. The selectively of LLL12 on STAT3 was further demonstrated on 21 protein kinases, which LLL12 had IC50 values ≥73.92 μM. In addition, the pretreatment of LLL12 blocked the promotion of the cell proliferation and resistance to lenalidomide by IL‐6. Furthermore, LLL12 significantly blocked tumor growth of MM cells in mouse model. Our results indicate that LLL12 blocks constitutive STAT3 and IL‐6 induced STAT3 signaling and may be a potential therapeutic agent for MM.
In a phase 1 trial, idelalisib (GS-1101, CAL-101), a selective inhibitor of the lipid kinase PI3Kδ, was evaluated in 54 patients with relapsed/refractory chronic lymphocytic leukemia (CLL) with ...adverse characteristics including bulky lymphadenopathy (80%), extensive prior therapy (median 5 range 2-14 prior regimens), treatment-refractory disease (70%), unmutated IGHV (91%), and del17p and/or TP53 mutations (24%). Patients were treated at 6 dose levels of oral idelalisib (range 50-350 mg once or twice daily) and remained on continuous therapy while deriving clinical benefit. Idelalisib-mediated inhibition of PI3Kδ led to abrogation of Akt phosphorylation in patient CLL cells and significantly reduced serum levels of CLL-related chemokines. The most commonly observed grade ≥3 adverse events were pneumonia (20%), neutropenic fever (11%), and diarrhea (6%). Idelalisib treatment resulted in nodal responses in 81% of patients. The overall response rate was 72%, with 39% of patients meeting the criteria for partial response per IWCLL 2008 and 33% meeting the recently updated criteria of PR with treatment-induced lymphocytosis.1,2 The median progression-free survival for all patients was 15.8 months. This study demonstrates the clinical utility of inhibiting the PI3Kδ pathway with idelalisib. Our findings support the further development of idelalisib in patients with CLL. These trials were registered at clinicaltrials.gov as #NCT00710528 and #NCT01090414.
•Idelalisib was evaluated in 54 patients with heavily pretreated chronic lymphocytic leukemia, and target inhibition was documented in vivo.•Oral idelalisib therapy demonstrated a favorable safety profile and rapidly induced durable disease control in the majority of patients.
Multiple myeloma (MM) is an incurable hematologic malignancy of plasma cells, with an estimated 30,000 new cases diagnosed each year in the United States, signifying the need for new therapeutic ...approaches. We hypothesized that targeting MM using a bispecific antibody (biAb) to simultaneously engage both innate and adaptive cytolytic immune cells could present potent antitumor activity. We engineered a biAb by fusing an anti-CS1 single-chain variable fragment (scFv) and an anti-NKG2D scFv (CS1-NKG2D biAb). Although NKG2D is a potent activation receptor ubiquitously expressed on mostly cytolytic immune cells including NK cells, CD8
T cells, γδ T cells, and NKT cells, the CS1 tumor-associated antigen on MM represents a promising target. CS1-NKG2D biAb engaged human MM cell lines and NKG2D
immune cells, forming immune synapses. In effector cells, CS1-NKG2D biAb triggered the phosphorylation of AKT, a downstream protein kinase of the activated NKG2D-DAP10 complex. The EC
values of CS1-NKG2D biAb for CS1
and for CS1
MM cell lines with effector PBMCs were 10
and 10
mol/L, respectively. CS1-NKG2D biAb acted through multiple types of immune cells, and this induced cytotoxicity was both CS1- and NKG2D-specific.
, survival was significantly prolonged using CS1-NKG2D biAb in a xenograft NOD-SCID
(NSG) mouse model engrafted with both human PBMCs and MM cell lines. Collectively, we demonstrated that the CS1-NKG2D biAb facilitated an enhanced immune synapse between CS1
MM cells and NKG2D
cytolytic innate and antigen-specific effector cells, which, in turn, activated these immune cells for improved clearance of MM.
.
To evaluate the maximum-tolerated dose (MTD), safety, and efficacy of elotuzumab in combination with bortezomib in patients with relapsed or relapsed and refractory multiple myeloma (MM).
Elotuzumab ...(2.5, 5.0, 10, or 20 mg/kg intravenously IV) and bortezomib (1.3 mg/m(2) IV) were administered on days 1 and 11 and days 1, 4, 8, and 11, respectively, in 21-day cycles by using a 3 + 3 dose-escalation design. Patients with stable disease or better after four cycles could continue treatment until disease progression or unexpected toxicity. Responses were assessed during each cycle by using European Group for Blood and Marrow Transplantation (EBMT) criteria.
Twenty-eight patients with a median of two prior therapies were enrolled; three patients each received 2.5, 5.0, and 10 mg/kg of elotuzumab and 19 received 20 mg/kg (six during dose escalation and 13 during an expansion phase). No dose-limiting toxicities were observed during cycle 1 of the dose-escalation phase, and the MTD was not reached up to the maximum planned dose of 20 mg/kg. The most frequent grade 3 to 4 adverse events (AEs) were lymphopenia (25%) and fatigue (14%). Two elotuzumab-related serious AEs of chest pain and gastroenteritis occurred in one patient. An objective response (a partial response or better) was observed in 13 (48%) of 27 evaluable patients and in two (67%) of three patients refractory to bortezomib. Median time to progression was 9.46 months.
The combination of elotuzumab and bortezomib was generally well-tolerated and showed encouraging activity in patients with relapsed/refractory MM.
Accumulating evidence indicates that human natural killer (NK) cells develop in secondary lymphoid tissue (SLT) through a so-called “stage 3” developmental intermediate minimally characterized by a ...CD34−CD117+CD94− immunophenotype that lacks mature NK cell function. This stage 3 population is heterogeneous, potentially composed of functionally distinct innate lymphoid cell (ILC) types that include interleukin-1 receptor (IL-1R1)-positive, IL-22-producing ILC3s. Whether human ILC3s are developmentally related to NK cells is a subject of ongoing investigation. Here, we show that antagonism of the aryl hydrocarbon receptor (AHR) or silencing of AHR gene expression promotes the differentiation of tonsillar IL-22-producing IL-1R1hi human ILC3s to CD56brightCD94+ interferon (IFN)-γ-producing cytolytic mature NK cells expressing eomesodermin (EOMES) and T-Box Protein 21 (TBX21 or TBET). Hence, we demonstrate the lineage plasticity of human ILCs by identifying AHR as a transcription factor that prevents IL-1R1hi ILC3s from differentiating into NK cells.
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•IL-1β maintains a human population of IL-22+ IL-1R1hi ILC3s in SLT by regulating AHR•Inhibition of AHR in human IL-1R1hi ILC3s promotes NK cell differentiation in vitro•Expression of AHR prevents human IL-1R1hi ILC3 differentiation to NK cells
Natural killer (NK) cells are innate lymphoid cells (ILCs) and critical mediators of antitumor responses. The molecular mechanisms controlling NK cell development are largely unknown, and it is currently unclear whether human group 3 ILCs (ILC3s) are developmentally related to NK cells. In this study, Hughes et al. demonstrate that pharmacologic inhibition and genetic loss of the aryl hydrocarbon receptor promote in vitro differentiation of ILC3s to NK cells. These data provide new evidence for lineage plasticity among human ILCs.
The standard preparative regimen for autologous stem cell transplant (ASCT) in multiple myeloma (MM) is 200 mg/m
2
of intravenous melphalan; however, a dose of 140 mg/m
2
is often used when concerns ...exist related to patient age, performance status, organ function, and other factors. It is unclear whether a lower dose of melphalan impacts post-transplant survival outcomes. We performed a retrospective review of 930 patients with MM who underwent ASCT with 200 mg/m
2
versus 140 mg/m
2
melphalan. On univariable analysis, no difference in progression-free survival (PFS) was observed, however, an overall survival (OS) benefit was observed in patients receiving 200 mg/m
2
melphalan (p = 0.04). Multivariable analyses showed patients receiving 140 mg/m
2
faired no worse than those receiving 200 mg/m
2
. While a subset of younger patients with normal renal function may achieve superior OS with a standard dose of 200 mg/m
2
melphalan, these findings suggest an opportunity to individualize the ASCT preparative regimen to optimize outcomes.
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