Courtney E. Bakan

The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel monoclonal anti-PD-1 antibody.

T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg, PD-L1) on tumor cells; however, little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM), an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011, a novel anti-PD-1 antibody, enhances human NK-cell function against autologous, primary MM cells, seemingly through effects on NK-cell trafficking, immune complex formation with MM cells, and cytotoxicity specifically toward PD-L1(+) MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011s enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered.

A phase 1 trial of the anti-KIR antibody IPH2101 in patients with relapsed/refractory multiple myeloma.

Natural killer (NK) cells elicit cytotoxicity against multiple myeloma (MM); however, MM cells express HLA class I molecules as ligands to NK cell inhibitory killer immunoglobulin-like receptors (KIRs) as a means of immunoevasion. KIR-ligand mismatch may improve outcomes in allogeneic transplantation for MM. Extrapolating on this concept, we conducted a phase 1 trial of IPH2101, an anti-KIR antibody, in patients with relapsed/refractory MM. IPH2101 was administered intravenously every 28 days in 7 dose-escalated cohorts (0.0003-3 mg/kg) for up to 4 cycles. Pharmacokinetic, pharmacodynamic, and correlative immunologic studies were completed. A total of 32 patients were enrolled. The biologic endpoint of full KIR2D occupancy across the dosing cycle was achieved without dose-limiting toxicity or maximally tolerated dose. One severe adverse event was noted. Pharmacokinetic and pharmacodynamic findings approximated preclinical predictions, and IPH2101 enhanced ex vivo patient-derived NK cell cytotoxicity against MM. No objective responses were seen. No evidence of autoimmunity was observed. These findings suggest that IPH2101 is safe and tolerable at doses that achieve full inhibitory KIR saturation, and this approach warrants further development in MM. This trial was registered at www.clinicaltrials.gov as #NCT00552396.

Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC

Abstract Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1–CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.

IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect.

Multiple myeloma (MM) patients who receive killer cell Ig-like receptor (KIR) ligand-mismatched, T cell-depleted, allogeneic transplantation may have a reduced risk of relapse compared with patients who receive KIR ligand-matched grafts, suggesting the importance of this signaling axis in the natural killer (NK) cell-versus-MM effect. Expanding on this concept, IPH2101 (1-7F9), an anti-inhibitory KIR mAb, enhances NK-cell function against autologous MM cells by blocking the engagement of inhibitory KIR with cognate ligands, promoting immune complex formation and NK-cell cytotoxicity specifically against MM cell targets but not normal cells. IPH2101 prevents negative regulatory signals by inhibitory KIR, whereas lenalidomide augments NK-cell function and also appears to up-regulate ligands for activating NK-cell receptors on MM cells. Lenalidomide and a murine anti-inhibitory NK-cell receptor Ab mediate in vivo rejection of a lenalidomide-resistant tumor. These mechanistic, preclinical data support the use of a combination of IPH2101 and lenalidomide in a phase 2 trial for MM.

The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity

BackgroundWe 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.ResultsFLLL32 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-γ-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-γ, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-γ production when cultured with K562 targets as compared to vehicle (DMSO).ConclusionsThese 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.

Curcumin induces proapoptotic effects against human melanoma cells and modulates the cellular response to immunotherapeutic cytokines

Curcumin has potential as a chemopreventative and chemotherapeutic agent, but its interactions with clinically relevant cytokines are poorly characterized. Because cytokine immunotherapy is a mainstay of treatment for malignant melanoma, we hypothesized that curcumin could modulate the cellular responsiveness to interferons and interleukins. As a single agent, curcumin induced a dose-dependent increase in apoptosis of human melanoma cell lines, which was most prominent at doses >10 μmol/L. Immunoblot analysis confirmed that curcumin induced apoptosis and revealed caspase-3 processing, poly ADP ribose polymerase cleavage, reduced Bcl-2, and decreased basal phosphorylated signal transducers and activators of transcription 3 (STAT3). Despite its proapoptotic effects, curcumin pretreatment of human melanoma cell lines inhibited the phosphorylation of STAT1 protein and downstream gene transcription following IFN-α and IFN-γ as determined by immunoblot analysis and real time PCR, respectively. Pretreatment of peripheral blood mononuclear cells from healthy donors with curcumin also inhibited the ability of IFN-α, IFN-γ, and interleukin-2 to phosphorylate STAT proteins critical for their antitumor activity (STAT1 and STAT5, respectively) and their respective downstream gene expression as measured by real time PCR. Finally, stimulation of natural killer (NK) cells with curcumin reduced the level of interleukin-12–induced IFN-γ secretion, and production of granzyme b or IFN-γ upon coculture with A375 melanoma cells or NK-sensitive K562 cells as targets. These data show that although curcumin can induce apoptosis of melanoma cells, it can also adversely affect the responsiveness of immune effector cells to clinically relevant cytokines that possess antitumor properties. [Mol Cancer Ther 2009;8(9):2726–35]

A small molecule, LLL12 inhibits constitutive STAT3 and IL-6-induced STAT3 signaling and exhibits potent growth suppressive activity in human multiple myeloma cells.

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.

Recombinant interleukin-2 in patients aged younger than 60 years with acute myeloid leukemia in first complete remission: Results from Cancer and Leukemia Group B 19808

Recombinant interleukin‐2 (rIL‐2) induces cellular cytotoxicity against leukemia blasts. Patients with acute myeloid leukemia (AML) in first complete remission (CR) may harbor minimal residual disease that is susceptible to rIL‐2–activated effector cells.

Effects of induction with novel agents versus conventional chemotherapy on mobilization and autologous stem cell transplant outcomes in multiple myeloma.

Multiple myeloma (MM) is the top indication for high-dose chemotherapy (HDC) with autologous stem cell transplantation (SCT), a strategy which improves progression-free survival and potentially overall survival (OS). Novel induction regimens incorporating the immunomodulatory (IMID) agents, such as thalidomide and lenalidomide and the proteosome inhibitor bortezomib improve response rates and survival for newly diagnosed patients. Recent data temper enthusiasm for these treatments by illustrating difficulty in some circumstances with mobilizing CD34(+) hematopoietic stem cells for subsequent HDC/SCT. We compare conventional induction regimens with novel agent-based induction strategies and the associated effects on stem cell mobilization and HDC/SCT outcome in 224 patients. Although patients exposed to novel agent inductions collected generally fewer CD34(+) cells than patients induced with chemotherapy, these differences did not translate into adverse consequences with subsequent HDC/SCT. We show that an improvement in OS after HDC/SCT may be related to induction therapy with novel agents as opposed to chemotherapy. Our data extrapolate on prior work and expand on ongoing controversies about optimal induction regimens for patients with MM planned for subsequent HDC/SCT and optimal sequencing of therapies.