Recombinant interleukin-21 plus sorafenib for metastatic renal cell carcinoma: a phase 1/2 study.

BACKGROUND: Despite the positive impact of targeted therapies on metastatic renal cell carcinoma (mRCC), durable responses are infrequent and an unmet need exists for novel therapies with distinct mechanisms of action. We investigated the combination of recombinant Interleukin 21 (IL-21), a cytokine with unique immunostimulatory properties, plus sorafenib, a VEGFR tyrosine kinase inhibitor. METHODS: In this phase 1/2 study, 52 mRCC patients received outpatient treatment with oral sorafenib 400 mg twice daily plus intravenous IL-21 (10-50 mcg/kg) on days 1-5 and 15-19 of each 7-week treatment course. The safety, antitumor activity, pharmacokinetic and pharmacodynamic effects of the combination were evaluated. RESULTS: In phase 1 (n = 19), the maximum tolerated dose for IL-21 with the standard dose of sorafenib was determined to be 30 mcg/kg/day; grade 3 skin rash was the only dose-limiting toxicity. In phase 2, 33 previously-treated patients tolerated the combination therapy well with appropriate dose reductions; toxicities were mostly grade 1 or 2. The objective response rate was 21% and disease control rate was 82%. Two patients have durable responses that are ongoing, despite cessation of both IL-21 and sorafenib, at 41+ and 30+ months, respectively. The median progression-free survival in phase 2 was 5.6 months. The pharmacokinetic and pharmacodynamic properties of IL-21 appeared to be preserved in the presence of sorafenib. CONCLUSION: IL-21 plus sorafenib has antitumor activity and acceptable safety in previously treated mRCC patients. IL-21 may represent a suitable immunotherapy in further exploration of combination strategies in mRCC. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00389285.

Pharmacokinetics and pharmacodynamics of pegylated interferon lambda-1 in cynomolgus monkeys.

Type III lambda interferons (IFNs) have activity similar to type I IFNs, but a more restricted receptor distribution. A pegylated human IFN lambda-1 (pegIFNλ) is under development for chronic hepatitis C. Induction of receptor signaling (STAT1 phosphorylation) and expression of interferon-stimulated genes (ISGs) by pegIFNλ were assessed in, respectively, cynomolgus monkey leukocyte subsets and hepatocytes stimulated in vitro. ISG induction by pegIFNλ or IFNα was also assessed in peripheral leukocytes and liver biopsies after single and repeat (x3) dosing of pegIFNλ (0.03, 0.3, 3.0 mg/kg) or unpegylated IFNα-2b (10(7) IU/kg). Single-dose pharmacokinetics of pegIFNλ were evaluated. Strong ISG induction occurred in cultured hepatocytes and liver biopsies with both pegIFNλ and IFNα. However, STAT1 phosphorylation, MHC class 1 upregulation, and ISG induction in leukocytes only occurred with IFNα. Serum neopterin was unaffected by pegIFNλ; however, ß-2-microglobulin was elevated at all doses. The terminal half-life of pegIFNλ was 23 h with a 59 mL/kg volume of distribution, consistent with other pegylated IFNs. Serum exposure was dose-proportional across the dosing range. These data demonstrate the suitability of cynomolgus monkeys for the preclinical evaluation of pegIFNλ. Additionally, the absence of pegIFNλ pharmacologic activity in leukocytes is consistent with its low receptor expression in blood.

Interferon lambda as a potential new therapeutic for hepatitis C.

Interferon lambdas (IFN-lambda) are Type III interferons with biological activity, including induction of antiviral genes, similar to Type I IFNs, but signal through a distinct receptor complex. The expression pattern for the IFN-lambda receptor is more cell specific than the widely distributed IFN-alpha receptor, suggesting in vivo, IFN-lambda may have fewer side effects than IFN-alpha, such as less hematologic toxicities. A PEGylated form of IFN-lambda (PEG-rIL-29) was well tolerated in animals and did not result in hematologic toxicity. Clinical data from initial studies of PEG-rIL-29 has demonstrated antiviral effects in patients with hepatitis C without producing hematologic toxicity. These preclinical and early clinical data support PEG-rIL-29 as a potential new therapeutic agent for treatment of patients with hepatitis C.

IL-21 induces in vivo immune activation of NK cells and CD8(+) T cells in patients with metastatic melanoma and renal cell carcinoma.

PURPOSE: Human interleukin-21 (IL-21) is a class I cytokine previously reported in clinical studies on immune responsive cancers. Here we report the effects of systemic IL-21 therapy on the immune system in two phase 1 trials with this novel cytokine. EXPERIMENTAL DESIGN: Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 microg/kg using two planned treatment regimens: thrice weekly for 6 weeks (3/week); or once daily for five consecutive days followed by nine dose-free days (5 + 9). The following biomarkers were studied in peripheral blood mononuclear cells (PBMC) during treatment: phosphorylation of STAT3, alterations in the composition of leukocyte subsets, ex vivo cytotoxicity, expression of effector molecules in enriched CD8(+) T cells and CD56(+) NK cells by quantitative RT-PCR, and gene array profiling of CD8(+) T cells. RESULTS: Effects of IL-21 were observed at all dose levels. In the 5 + 9 regimen IL-21 induced a dose dependent decrease in circulating NK cells and T cells followed by a return to baseline in resting periods. In both CD8(+) T cells and CD56(+) NK cells we found up-regulation of perforin and granzyme B mRNA. In addition, full transcriptome analysis of CD8(+) T cells displayed changes in several transcripts associated with increased cell cycle progression, cellular motility, and immune activation. Finally, cytotoxicity assays showed that IL-21 enhanced the ability of NK cells to kill sensitive targets ex vivo. CONCLUSIONS: IL-21 was biologically active at all dose levels administered with evidence of in vivo NK cell and CD8(+) T cell activation.

The Salmonella enterica serovar typhimurium translocated effectors SseJ and SifB are targeted to the Salmonella-containing vacuole.

The Salmonella enterica serovar Typhimurium type III secretion system (TTSS) encoded in Salmonella pathogenicity island 2 (SPI-2) promotes replication within host cells and systemic infection of mice. The SPI-2 TTSS is expressed following Salmonella internalization into host cells and translocates effectors across the membrane of the Salmonella-containing vacuole (SCV). Two effectors with similar amino-terminal domains, SseJ and SifB, localize to the SCV membrane in infected HEp-2 cells and subsequently traffic away from the SCV along Salmonella-induced-filaments (Sifs). Following infection of RAW cells, SseJ and SifB localize to the SCV as well as LAMP-1-positive, vesicular-appearing structures distant from the SCV. Trafficking of SseJ and SifB away from the SCV requires the SPI-2 effector SifA. Deletion of sseJ, but not sifB, leads to attenuation of Salmonella replication in mice following intraperitoneal inoculation. The contribution of SseJ to in vivo replication is identical in wild-type and sifA deletion backgrounds, suggesting that SseJ trafficking away from the SCV along Sifs is unnecessary for its virulence function.

SpiC is required for translocation of Salmonella pathogenicity island 2 effectors and secretion of translocon proteins SseB and SseC.

The Salmonella pathogenicity island 2 (SPI2) type III secretion system (TTSS) promotes Salmonella enterica serovar Typhimurium virulence for mice and increased survival and replication within eukaryotic cells. After phagocytosis, Salmonella serovar Typhimurium assembles the SPI2 TTSS to translocate over a dozen effector proteins across the phagosome membrane. SpiC has been previously shown to be a translocated effector with a large contribution to virulence (K. Uchiya, M. A. Barbieri, K. Funato, A. H. Shah, P. D. Stahl, and E. A. Groisman, EMBO J. 18:3924-3933, 1999). This report demonstrates by competitive index that the virulence phenotype of a spiC mutant is equivalent to that of a secretion component mutant. In addition, translocation of SPI2 effector proteins was shown to require SpiC. Thus, the severe virulence phenotype resulting from deletion of spiC is likely due to the inability to translocate all SPI2 effectors. SpiC was also required to secrete translocon proteins SseB and SseC but not translocated effector SseJ, indicating that lack of assembly of the translocon explains the spiC mutant phenotype.

Transcription of the SsrAB regulon is repressed by alkaline pH and is independent of PhoPQ and magnesium concentration.

The Salmonella pathogenicity island 2 (SPI-2) type III secretion system is expressed by intracellular bacteria and translocates effector proteins across the vacuolar membrane. Signals sensed by Salmonella enterica serovar Typhimurium in the intracellular compartment activate SPI-2 gene expression through the two-component regulatory system SsrAB. The effects of environmental and genetic signals on expression of the SsrAB-regulated gene sspH2 were examined. SsrAB-dependent activation of sspH2 was detected in the presence of both low and moderate concentrations of magnesium or calcium and at acidic and neutral pHs. The levels of expression were comparable to those detected in bacteria recovered from cultured macrophages. The induction in media at alkaline pHs (pH 7.5 and 8.0) was greatly reduced compared to the induction observed at pH 7.0 or at a lower pH, suggesting that alkaline pH represses SsrAB activation. In addition, the PhoPQ two-component system, which is also activated intracellularly, was not required for activation of SsrAB.