Eric Luedke

Cetuximab therapy in head and neck cancer: Immune modulation with interleukin-12 and other natural killer cell–activating cytokines

BACKGROUND Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide. Greater than 90% of SCCHN of the oropharynx overexpress the epidermal growth factor receptor (EGFR or HER1). Cetuximab (Erbitux-TM) is a humanized anti-HER1 monoclonal antibody (mAb) that binds to HER1 overexpressing tumor cells. Cetuximab has a direct effect on HER1-positive cancer cells, but it also can activate immune cells that bear receptors for the Fc (constant portion) of IgG such as natural killer (NK) cells. NK cells have an activating Fc receptor for IgG (FcγRIIIa), which mediates Ab dependent cellular cytotoxicity (ADCC) and enhances production of interferon-γ (IFN-γ) in response to Ab-coated targets. Interleukin-12 (IL-12) is a cytokine produced by antigen-presenting cells that stimulates IFN-γ production from NK cells. We hypothesized that IL-12 would enhance the anti-tumor activity of cetuximab by activating the FcR effector mechanisms of NK cells. METHODS Expression of HER1 was measured on human papilloma virus (HPV)-positive (UD-SCC2, UM-SCC47) and HPV-negative (Cal27, UM-SCC74B) SCCHN cell lines by immunoblot analysis and flow cytometry. NK cells from normal donors were treated overnight with IL-2 (100 U), IL-12, IL-15, or IL-21 (all 10 ng/mL) and tested for ADCC versus cetuximab-coated cancer cells in a 4 hr (51)Cr assay. Release of cytokines by NK cells in response to cetuximab-coated cells was measured by ELISA. Phosphorylation of the ERK transcription factor in NK cells was measured by flow cytometry. The efficacy of combination therapy with cetuximab plus IL-12 was evaluated in a murine tumor model of head and neck cancer. RESULTS All cell lines showed >99% expression of HER1 by flow cytometry and immunoblot analysis except UM-SCC74B (73%). Normal NK cells mediated 49.4% lysis of cetuximab-coated SCCHN cell lines as compared to 7.6% lysis of cells treated with control IgG (P = .0002). NK cell lysis of cetuximab-coated SCCHN cells was markedly enhanced by 12 hr pre-treatment of NK cells with IL-12 (71.6% lysis, P = .005 vs cetuximab alone). As a control, IL-12-activated NK cells were tested against IgG-treated cells. ADCC under these conditions was just 21.7%. Similar levels of lysis were noted for both HPV-positive and HPV-negative and cell lines. Other NK cell activating factors such as IL-2, IL-15, and IL-21 were also able to enhance NK cell ADCC. The stimulus of IL-12 and cetuximab-coated tumor cells induced the synergistic production of nanogram levels of IFN-γ (>6-fold increase over controls) (P < .001). A similar effect was seen for NK cell production of the chemokines RANTES, MIP-1α, and IL-8. Phosphorylation of ERK (which is critical for FcR-mediated ADCC and cytokine production) was enhanced in NK cells exposed to IL-12 and IgG as compared to control conditions. The combination of cetuximab plus IL-12 resulted in a reduction in tumor burden when compared to either agent alone in a murine xenograft model of SCCHN. CONCLUSION Cytokine stimulation of NK cells in the presence of cetuximab-coated head and neck cancer cells leads to enhanced NK cell mediated ADCC and cytokine secretion independent of tumor cell HPV-status. Cytokine administration could be a useful adjuvant in the cetuximab treatment of HER1-positive head and neck cancer.

Monoclonal antibody therapy of pancreatic cancer with cetuximab: potential for immune modulation.

Pancreatic cancer is a devastating disease, with a median survival of around 6 months for patients with stage IV disease. The epidermal growth factor receptor (EGFR, or HER1) belongs to the erbB receptor tyrosine kinase family. HER1-mediated cell signaling has been shown to play a major role in promoting tumor proliferation, angiogenesis, metastasis, and evasion of apoptosis. Over-expression of HER1 is observed in multiple human malignancies, including colorectal, lung, breast and pancreatic cancers. In pancreatic carcinoma, over-expression of HER1 is observed in greater than 70% of patients and is associated with a poor prognosis and a significant decrease in survival. Cetuximab (Erbitux) is a chimeric monoclonal antibody (mAb) that binds to the extracellular domain of the HER1 molecule preventing ligand binding and promoting internalization and subsequent degradation of the HER1 receptor. Cetuximab has shown anti-tumor activity either alone or in combination with other agents and is currently FDA approved for use in both squamous cell carcinoma of the head and neck (SCCHN) and colorectal carcinoma. Research efforts continue to elucidate a possible role for cetuximab in the treatment of pancreatic cancer. Despite promising preclinical work, phase II and phase III clinical trials have failed to consistently show efficacy of cetuximab treatment in advanced pancreatic cancer either alone or in combination with cytotoxic agents. Alternative approaches to HER1 blockade and mAbs including immune modulation with cytokines might be necessary in order to improve the efficacy of mAbs in pancreatic cancer therapy.

IL-21 Enhances Natural Killer Cell Response to Cetuximab-Coated Pancreatic Tumor Cells.

Purpose: Alternative strategies to EGFR blockage by mAbs is necessary to improve the efficacy of therapy in patients with locally advanced or metastatic pancreatic cancer. One such strategy includes the use of NK cells to clear cetuximab-coated tumor cells, as need for novel therapeutic approaches to enhance the efficacy of cetuximab is evident. We show that IL-21 enhances NK cell-mediated effector functions against cetuximab-coated pancreatic tumor cells irrespective of KRAS mutation status. Experimental Design: NK cells from normal donors or donors with pancreatic cancer were used to assess ADCC, IFN-γ release, and T-cell chemotaxis toward human pancreatic cancer cell lines. The in vivo efficacy of IL-21 in combination with cetuximab was evaluated in a subcutaneous and intraperitoneal model of pancreatic cancer. Results: NK cell lysis of cetuximab-coated wild-type and mutant kras pancreatic cancer cell lines were significantly higher following NK cell IL-21 treatment. In response to cetuximab-coated pancreatic tumor cells, IL-21–treated NK cells secreted significantly higher levels of IFN-γ and chemokines, increased chemotaxis of T cells, and enhanced NK cell signal transduction via activation of ERK and STAT1. Treatment of mice bearing subcutaneous or intraperitoneal EGFR-positive pancreatic tumor xenografts with mIL-21 and cetuximab led to significant inhibition of tumor growth, a result further enhanced by the addition of gemcitabine. Conclusions: These results suggest that cetuximab treatment in combination with IL-21 adjuvant therapy in patients with EGFR-positive pancreatic cancer results in significant NK cell activation, irrespective of KRAS mutation status, and may be a potential therapeutic strategy. Clin Cancer Res; 23(2); 489–502. ©2016 AACR.

A naonoporous cell-therapy device with controllable biodegradation for long-term drug release.

Herein we describe the development and implementation of a nanoporous cell-therapy device with controllable biodegradation. Dopamine-secreting PC12 cells were housed within newly formulated alginate-glutamine degradable polylysine (A-GD-PLL) microcapsules. The A-GD-PLL microcapsules provided a 3-D microenvironment for good spatial cell growth, viability and proliferation. The microcapsules were subsequently placed within a poly(ethylene glycol) (PEG)-coated poly(ε-caprolactone) (PCL) chamber covered with a PEG-grafted PCL nanoporous membrane formed by phase inversion. To enhance PC12 cell growth and to assist in controlled degradation of both the PC12 cells and the device construct, small PCL capsules containing neural growth factor (PCL-NGF) and a poly(lactic-co-glycolic acid) pellet containing glutamine (PLGA-GLN) were also placed within the PCL chamber. Release of NGF from the PCL-NGF capsules facilitated cell proliferation and viability, while the controlled release of GLN from the PLGA-GLN pellet resulted in A-GD-PLL microcapsule degradation and eventual PC12 cell death following a pre-specified period of time (4 weeks in this study). In vivo, our device was found to be well tolerated and we successfully demonstrated the controlled release of dopamine over a period of four weeks. This integrated biodegradable device holds great promise for the future treatment of a variety of diseases.

MICA-Expressing Monocytes Enhance Natural Killer Cell Fc Receptor-Mediated Antitumor Functions

Natural killer (NK) cells secrete immunostimulatory factors like IFNγ in response to tumors. Engagement of monocyte MICA and NK cell NKG2D promoted and enhanced the NK response to HER2+ breast tumors treated with mAb to HER2 in a murine model. Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA–NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell–monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778–89. ©2017 AACR.

A phase I trial of bortezomib and interferon-α-2b in metastatic melanoma.

The possibility that cytokine administration could enhance the antitumor effects of proteasome inhibition was explored. It was found that coadministration of bortezomib and interferon-&agr; (IFN-&agr;) induced synergistic apoptosis in human melanoma cell lines and prolonged survival in a murine model of melanoma. A phase I study was conducted to determine the tolerability and the maximum tolerated dose of bortezomib when administered in combination with IFN-&agr;-2b to patients with metastatic melanoma. Patients were treated on a 5-week cycle. In week 1 of cycle 1, patients received 5 million U/m2 IFN-&agr; subcutaneously thrice weekly. During weeks 2–4 of cycle 1, bortezomib was administered intravenously weekly along with IFN-&agr; thrice weekly. There was a treatment break during week 5. After cycle 1, bortezomib was administered in combination with IFN-&agr;. Bortezomib was administered in escalating doses (1.0, 1.3, or 1.6 mg/m2) to cohorts of 3 patients. Sixteen patients were treated (8 women, 8 men; median age 59 y). Common grade 3 toxicities included fatigue (5), vomiting (3), and diarrhea (3). Grade 4 toxicities included fatigue (3) and lymphopenia (1). The maximum tolerated dose for bortezomib was 1.3 mg/m2. One patient had a partial response, and 7 had stable disease. Progression-free survival was 2.5 months, and overall survival was 10.3 months. Bortezomib administration did not augment the ability of IFN-&agr; to induce phosphorylation of STAT1 in circulating immune cells; however, it did lead to reduced plasma levels of proangiogenic cytokines. The combination of bortezomib and IFN-&agr; can be safely administered to melanoma patients.

The combination of MLN2238 (ixazomib) with interferon-alpha results in enhanced cell death in melanoma

The ubiquitin-proteasome signaling pathway is critical for cell cycle regulation and neoplastic growth. Proteasome inhibition can activate apoptotic pathways. Bortezomib, a selective proteasome inhibitor, has anti-melanoma activity. MLN2238 (ixazomib), an oral proteasome inhibitor, has improved pharmacotherapeutic parameters compared to bortezomib. Interferon-alpha (IFN-α), an immune boosting agent, is FDA-approved for treatment of melanoma. In this study in vitro and in vivo evaluation of the antitumor potential of ixazomib and combination treatments with ixazomib and IFN-α were performed. Apoptosis induced by ixazomib was first observed at 12 hours and was maximal at 48 hours with similar levels of cell death compared to bortezomib. IFN-α alone had little effect on cell viability in vitro. However, the combination of ixazomib with IFN-α significantly enhanced ixazomibs ability to induce apoptotic cell death in BRAF V600E mutant and BRAF wild-type human melanoma tumor cells. The combination of ixazomib and IFN-α also enhanced inhibition of cell proliferation in BRAF V600E mutant melanoma tumor cells; however, this was not seen in BRAF wild-type cells. Ixazomib-induced apoptosis was associated with processing of the pro-apoptotic proteins procaspase-3, -7, -8, and -9, and cleavage of poly-ADP-ribose polymerase (PARP). In an in vivo xenograft model of human melanoma, combination treatment with IFN-α-2b and ixazomib demonstrated a significant reduction in tumor volume when compared to vehicle (p = 0.005) and single therapy ixazomib (p = 0.017) and IFN-α-2b (p = 0.036). These pre-clinical results support further evaluation of combination treatment with ixazomib and IFN-α for the treatment of advanced BRAF V600E mutant melanoma.

NK Cell–Mediated Antitumor Effects of a Folate-Conjugated Immunoglobulin Are Enhanced by Cytokines

Many tumors express receptors for the vitamin folate (FRs). Folate-conjugated IgG, which can bind both FR+ tumors and NK-cell receptors for IgG, induced potent NK-cell antitumor responses that were further augmented by cytokine therapy. Optimally effective antitumor therapies would not only activate immune effector cells but also engage them at the tumor. Folate conjugated to immunoglobulin (F-IgG) could direct innate immune cells with Fc receptors to folate receptor–expressing cancer cells. F-IgG bound to human KB and HeLa cells, as well as murine L1210JF, a folate receptor (FR)–overexpressing cancer cell line, as determined by flow cytometry. Recognition of F-IgG by natural killer (NK) cell Fc receptors led to phosphorylation of the ERK transcription factor and increased NK cell expression of CD69. Lysis of KB tumor cells by NK cells increased by about 5-fold after treatment with F-IgG, an effect synergistically enhanced by treatment with IL2, IL12, IL15, or IL21 (P < 0.001). F-IgG also enhanced the lysis of chronic lymphocytic leukemia cells by autologous NK cells. NK cells significantly increased production of IFNγ, MIP-1α, and RANTES in response to F-IgG–coated KB target cells in the presence of the NK cell–activating cytokine IL12, and these coculture supernatants induced significant T-cell chemotaxis (P < 0.001). F-IgG-coated targets also stimulated FcR-mediated monocyte effector functions. Studies in a murine leukemia model confirmed the intratumoral localization and antitumor activity of F-IgG, as well as enhancement of its effects by IL12 (P = 0.05). The antitumor effect of this combination was dependent on NK cells and led to decreased tumor cell proliferation in vivo. Thus, F-IgG can induce an immune response against FR-positive tumor cells that is mediated by NK cells and can be augmented by cytokine therapy. Cancer Immunol Res; 4(4); 323–36. ©2016 AACR.

Interleukin-21 activates natural killer cell activity against cetuximab-coated pancreatic tumor cells

Natural killer (NK) cells are large granular lymphocytes that play a critical role in anti-cancer mechanisms. These effector cells mediate antibody-dependent cellular cytotoxicity (ADCC) and produce IFN-γ in response to antibody-coated targets. The monoclonal antibody cetuximab targets the epidermal growth factor receptor (EGFR), which is over expressed in the majority of pancreatic cancers, but fails to effectively block MAP kinase activity when KRAS is concurrently mutated. Interleukin-21 (IL-21) is an immunomodulatory cytokine that stimulates the anti-tumor response of NK cells. We hypothesized that IL-21 treatment of NK cells would enhance their response to cetuximab-coated EGFR-positive pancreatic tumor cells, regardless of KRAS mutational status and thereby generate an effective anti-tumor response. Expression of EGFR was measured on six human pancreatic cancer cell lines. All six cell lines showed >85% expression of EGFR by flow cytometry, which was confirmed by immunoblot assay. NK cells from normal healthy donors and pancreatic cancer patients were treated overnight with IL-21 (10 ng/ml) and tested for their ability to lyse cetuximab-coated tumor cells in a standard 4-hour 51Cr release assay. NK cell killing of cetuximab-coated wild-type and mutant KRAS cell lines was significantly higher following NK cell IL-21 treatment, as compared to controls (p < 0.001). In response to cetuximab-coated pancreatic tumor cells, IL-21 treated NK cells secreted significantly higher levels of IFN-γ (p < 0.001), released higher levels of the chemokines IL-8, MIP1α/β, and RANTES (p < 0.001), induced greater chemotaxis of T cells (p < 0.01), and enhanced NK cell signal transduction via activation of ERK and STAT1. Treatment of mice bearing subcutaneous EGFR-positive/KRAS mutant pancreatic tumor xenographs with mIL-21 and cetuximab led to significant inhibition of tumor growth as compared to control conditions (p < 0.05). This result was confirmed in a second intraperitoneal model of pancreatic cancer. Treatment of tumor bearing mice with gemcitabine and cetuximab in combination led to only a modest reduction in tumor burden in vivo (as is seen in humans), but this effect was markedly enhanced by the addition of IL-21 (p < 0.05). Overall, this data suggests that cetuximab treatment in combination with IL-21 can greatly enhance NK cell effector functions and mediate a significant anti-tumor response in EGFR-positive pancreatic cancers in the presence of mutated KRAS.

Activation of the FcgammaReceptorIIIa on human natural killer cells leads to increased expression of functional interleukin-21 receptor

ABSTRACT Natural killer (NK) cells are innate immune effector cells that play a crucial role in immune surveillance and the destruction of cancer cells. NK cells express a low-affinity receptor for the Fc or constant region of immunoglobulin G (FcγRIIIa) and multiple cytokine receptors that respond to antibody-coated targets and cytokines in the tumor microenvironment. In the present work, microarray gene expression analysis revealed that the IL-21 receptor (IL-21R) was strongly upregulated following FcR stimulation. The IL-21R was found to be upregulated on FcR-stimulated NK cells at the transcript level as determined by reverse transcription polymerase chain reaction (RT-PCR). Immunoblot analysis revealed that protein expression of the IL-21R peaked at 8 h post-stimulation of the FcR. Inhibition of the mitogen-activated protein kinase (MAPK) pathway downstream of the FcR blocked the induction of IL-21R expression. Increased expression of the IL-21R sensitized NK cells to IL-21 stimulation, as treatment of FcR-stimulated NK cells led to significantly increased phosphorylation of STAT1 and STAT3, as measured by intracellular flow cytometry and immunoblot analysis. Following FcR-stimulation, IL-21-activated NK cells were better able to mediate the lysis of trastuzumab-coated human epidermal growth factor receptor 2 (HER2+) SK-BR-3 tumor cells as compared to control-treated cells. Likewise, IL-21-induced NK cell secretion of IFNγ following exposure to antibody-coated tumor cells was enhanced following FcR-stimulation. The analysis of NK cells from patients receiving trastuzumab therapy for HER2+ cancer exhibited increased levels of the IL-21R following the administration of antibody suggesting that the presence of monoclonal antibody-coated tumor cells in vivo can stimulate the increased expression of IL-21R on NK cells.