A Phase I/II Trial of Cetuximab in Combination with Interleukin-12 Administered to Patients with Unresectable Primary or Recurrent Head and Neck Squamous Cell Carcinoma.

PURPOSE: mAbs including cetuximab can induce antibody-dependent cellular cytotoxicity (ADCC) and cytokine production mediated via innate immune cells with the ability to recognize mAb-coated tumors. Preclinical modeling has shown that costimulation of natural killer (NK) cells via the Fc receptor and the IL12 receptor promotes NK-cell-mediated ADCC and production of cytokines. PATIENTS AND METHODS: This phase I/II trial evaluated the combination of cetuximab with IL12 for the treatment of EGFR-expressing head and neck cancer. Treatment consisted of cetuximab 500 mg/m2 i.v. every 2 weeks with either 0.2 mcg/kg or 0.3 mcg/kg IL12 s.c. on days 2 and 5 of the 2-week cycle, beginning with cycle 2. Correlative studies from blood draws obtained prior to treatment and during therapy included measurement of ADCC, serum cytokine, and chemokine analysis, determination of NK cell FcγRIIIa polymorphisms, and an analysis of myeloid-derived suppressor cell (MDSC) frequency in peripheral blood. RESULTS: The combination of cetuximab and IL12 was well tolerated. No clinical responses were observed, however, 48% of patients exhibited prolonged progression-free survival (PFS; average of 6.5 months). Compared with patients that did not exhibit clinical benefit, patients with PFS >100 days exhibited increased ADCC as therapy continued compared with baseline, greater production of IFNγ, IP-10, and TNFα at the beginning of cycle 8 compared with baseline values and had a predominance of monocytic MDSCs versus granulocytic MDSCs prior to therapy. CONCLUSIONS: Further investigation of IL12 as an immunomodulatory agent in combination with cetuximab in head and neck squamous cell carcinoma is warranted.

A phase I study of recombinant (r) vaccinia-CEA(6D)-TRICOM and rFowlpox-CEA(6D)-TRICOM vaccines with GM-CSF and IFN-α-2b in patients with CEA-expressing carcinomas.

Prime-boost vaccination with recombinant (r) vaccinia(V)-CEA(6D)-TRICOM (triad of co-stimulatory molecules B7.1, ICAM-1 and LFA-3) and rFowlpox(F)-CEA(6D)-TRICOM infect antigen-presenting cells and direct expression of co-stimulatory molecules. We hypothesized that co-administration of vaccine with GM-CSF and interferon alpha (IFN-α) would have efficacy in CEA-expressing cancers. Patients with CEA-expressing cancers received the rV-CEA(6D)-TRICOM vaccine subcutaneously (s.c.) on day 1 followed by GM-CSF s.c. to the injection site on days 1-4. In Cycle 1, patients received thrice weekly s.c. injections of IFN-α-2b the week after rV-CEA(6D)-TRICOM. In Cycles 2-4, patients received thrice weekly s.c. injections of IFN-α-2b the same week that rF-CEA(6D)-TRICOM was given. The first cohort received no IFN followed by dose escalation of IFN-α in subsequent cohorts. Thirty-three patients were accrued (mean 59.8 years). Grade 3 toxicities included fatigue and hyperglycemia. Grade 4-5 adverse events (unrelated to treatment) were confusion (1), elevated aspartate transaminase (AST)/alanine transaminase (ALT) (1), and sudden death (1). No patients had a partial response, and eight patients exhibited stable disease of ≥3 months. Median progression-free survival and overall survival (OS) were 1.8 and 6.3 months, respectively. Significantly higher serum CD27 levels were observed after vaccine therapy (p = 0.006 post 1-2 cycles, p = 0.003 post 3 cycles, p = 0.03 post 4-7 cycles) and 42 % of patients assayed developed CEA-specific T cell responses. Pre-treatment levels of myeloid-derived suppressor cells correlated with overall survival (p = 0.04). Administration of IFN-α led to significantly increased OS (p = 0.02) compared to vaccine alone. While the vaccine regimen produced no clinical responses, IFN-α administration was associated with improved survival.

A Phase I Trial to Evaluate Antibody-Dependent Cellular Cytotoxicity of Cetuximab and Lenalidomide in Advanced Colorectal and Head and Neck Cancer.

mAbs can induce antibody-dependent cellular cytotoxicity (ADCC) via the innate immune system's ability to recognize mAb-coated cancer cells and activate immune effector cells. Lenalidomide is an immunomodulatory agent with the capacity to stimulate immune cell cytokine production and ADCC activity. This phase I trial evaluated the combination of cetuximab with lenalidomide for the treatment of advanced colorectal and head and neck squamous cell cancers (HNSCC). This trial included patients with advanced colorectal cancer or HNSCC. Treatment consisted of cetuximab 500 mg/m(2) i.v. every two weeks with lenalidomide given orally days 1-21 on a 28-day cycle. Three dose levels of lenalidomide were evaluated (15, 20, 25 mg). Correlative studies included measurement of ADCC, FcγRIIIA polymorphism genotyping, measurement of serum cytokine levels, and flow cytometric analysis of immune cell subtypes. Twenty-two patients were enrolled (19 colorectal cancer, 3 HNSCC). Fatigue was the only dose-limiting toxicity. One partial response was observed and 8 patients had stable disease at least 12 weeks. The recommended phase II dose is cetuximab 500 mg/m(2) with lenalidomide 25 mg daily, days 1-21. Correlative studies demonstrated a dose-dependent increase in natural killer cytotoxic activity with increasing doses of lenalidomide. Cetuximab and lenalidomide were well tolerated. There was a lenalidomide dose-dependent increase in ADCC with higher activity in patients enrolled in cohort 3 than those enrolled in cohorts 1/2. Although response was not a primary endpoint, there was evidence of antitumor activity for the combination therapy. Further investigation of lenalidomide as an immunomodulator in solid tumors is warranted. Mol Cancer Ther; 15(9); 2244-50. ©2016 AACR.

The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells.

Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 µM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 µM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells.

Patients with pancreatic adenocarcinoma exhibit elevated levels of myeloid-derived suppressor cells upon progression of disease.

Elevated levels of myeloid-derived suppressor cells (MDSCs) induced by tumor-derived factors are associated with inhibition of immune responses in patients with gastrointestinal malignancies. We hypothesized that pro-MDSC cytokines and levels of MDSC in the peripheral blood would be elevated in pancreatic adenocarcinoma patients with progressive disease. Peripheral blood mononuclear cells (PBMCs) were isolated from 16 pancreatic cancer patients undergoing chemotherapy and phenotyped for MDSC using a five antigen panel (CD33, HLA-DR, CD11b, CD14, CD15). Patients with stable disease had significantly lower MDSC levels in the peripheral blood than those with progressive disease (1.41 ± 1.12 vs. 5.14 ± 4.58 %, p = 0.013, Wilcoxon test). A cutoff of 2.5 % MDSC identified patients with progressive disease. Patients with ECOG performance status ≥2 had a weaker association with increased levels of MDSC. Plasma was obtained from 15 chemonaive patients, 13 patients undergoing chemotherapy and 9 normal donors. Increases in the levels of pro-MDSC cytokines were observed for pancreatic cancer patients versus controls, and the pro-MDSC cytokine IL-6 was increased in those patients undergoing chemotherapy. This study suggests that MDSC in peripheral blood may be a predictive biomarker of chemotherapy failure in pancreatic cancer patients.

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-α (IFN-α) 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-α-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/m(2) IFN-α subcutaneously thrice weekly. During weeks 2-4 of cycle 1, bortezomib was administered intravenously weekly along with IFN-α thrice weekly. There was a treatment break during week 5. After cycle 1, bortezomib was administered in combination with IFN-α. Bortezomib was administered in escalating doses (1.0, 1.3, or 1.6 mg/m) 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/m(2). 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-α 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-α can be safely administered to melanoma patients.

Control of pH during plasmid preparation by alkaline lysis of Escherichia coli.

Alkaline lysis of Escherichia coli is usually the method of choice for plasmid preparation, but ''ghost bands" of denatured supercoiled DNA can result if the pH is too high or the period of lysis is too long. By replacing the usual sodium hydroxide lysis solution with an arginine buffer prepared in the range of pH 11.4 to 12.0, we were able to stabilize the pH during lysis and obtain plasmid that is suitably pure for restriction digestion and DNA sequencing.

A nonalkaline method for isolating sequencing-ready plasmids.

We describe a simple method of isolating plasmid DNA directly from Escherichia coli culture medium by addition of lithium acetate and Sodium dodecyl sulphate, followed by centrifugation and alcohol precipitation. The plasmid is sufficiently pure that it can be used in many enzyme-based reactions, including DNA sequencing and restriction analysis. Chromosomal DNA contamination is significantly reduced by pretreatment of the culture with DNase I, suggesting that much of the contaminant is associated with permeable dead cells. Chromosomal DNA contaminant can also be selectively denatured without damage to the supercoiled plasmid by alkaline denaturation in an arginine buffer or heat treatment in the presence of urea or N,N-dimethylformamide.