Phase II trial of Modified Vaccinia Ankara (MVA) virus expressing 5T4 and high dose Interleukin-2 (IL-2) in patients with metastatic renal cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Interleukin-2 (IL-2) induces durable objective responses in a small cohort of patients with metastatic renal cell carcinoma (RCC) but the antigen(s) responsible for tumor rejection are not known. 5T4 is a non-secreted membrane glycoprotein expressed on clear cell and papillary RCCs. A modified vaccinia virus Ankara (MVA) encoding 5T4 was tested in combination with high-dose IL-2 to determine the safety, objective response rate and effect on humoral and cell-mediated immunity.</p> <p>Methods</p> <p>25 patients with metastatic RCC who qualified for IL-2 were eligible and received three immunizations every three weeks followed by IL-2 (600,000 IU/kg) after the second and third vaccinations. Blood was collected for analysis of humoral, effector and regulatory T cell responses.</p> <p>Results</p> <p>There were no serious vaccine-related adverse events. While no objective responses were observed, three patients (12%) were rendered disease-free after nephrectomy or resection of residual metastatic disease. Twelve patients (48%) had stable disease which was associated with improved median overall survival compared to patients with progressive disease (not reached vs. 28 months, p = 0.0261). All patients developed 5T4-specific antibody responses and 13 patients had an increase in 5T4-specific T cell responses. Although the baseline frequency of Tregs was elevated in all patients, those with stable disease showed a trend toward increased effector CD8+ T cells and a decrease in Tregs.</p> <p>Conclusion</p> <p><b>V</b>accination with MVA-5T4 did not improve objective response rates of IL-2 therapy but did result in stable disease associated with an increase in the ratio of 5T4-specific effector to regulatory T cells in selected patients.</p> <p>Trial registration number</p> <p>ISRCTN83977250</p

Targeting the local tumor microenvironment with vaccinia virus expressing B7.1 for the treatment of melanoma

Immunotherapy for the treatment of metastatic melanoma remains a major clinical challenge. The melanoma microenvironment may lead to local T cell tolerance in part through downregulation of costimulatory molecules, such as B7.1 (CD80). We report the results from the first clinical trial, to our knowledge, using a recombinant vaccinia virus expressing B7.1 (rV-B7.1) for monthly intralesional vaccination of accessible melanoma lesions. A standard 2-dose–escalation phase I clinical trial was conducted with 12 patients. The approach was well tolerated with only low-grade fever, myalgias, and fatigue reported and 2 patients experiencing vitiligo. An objective partial response was observed in 1 patient and disease stabilization in 2 patients, 1 of whom is alive without disease 59 months following vaccination. All patients demonstrated an increase in postvaccination antibody and T cell responses against vaccinia virus. Systemic immunity was tested in HLA-A*0201 patients who demonstrated an increased frequency of gp100 and T cells specific to melanoma antigen recognized by T cells 1 (MART-1), also known as Melan-A, by ELISPOT assay following local rV-B7.1 vaccination. Local immunity was evaluated by quantitative real-time RT-PCR, which suggested that tumor regression was associated with increased expression of CD8 and IFN-γ. The local delivery of vaccinia virus expressing B7.1 was well tolerated and represents an innovative strategy for altering the local tumor microenvironment in patients with melanoma

Isogenic human trophectoderm cells demonstrate the role of NDUFA4 and associated variants in ZIKV infection

Summary: Population-based genome-wide association studies (GWAS) normally require a large sample size, which can be labor intensive and costly. Recently, we reported a human induced pluripotent stem cell (hiPSC) array-based GWAS method, identifying NDUFA4 as a host factor for Zika virus (ZIKV) infection. In this study, we extended our analysis to trophectoderm cells, which constitute one of the major routes of mother-to-fetus transmission of ZIKV during pregnancy. We differentiated hiPSCs from various donors into trophectoderm cells. We then infected cells carrying loss of function mutations in NDUFA4, harboring risk versus non-risk alleles of SNPs (rs917172 and rs12386620) or having deletions in the NDUFA4 cis-regulatory region with ZIKV. We found that loss/reduction of NDUFA4 suppressed ZIKV infection in trophectoderm cells. This study validated our published hiPSC array-based system as a useful platform for GWAS and confirmed the role of NDUFA4 as a susceptibility locus for ZIKV in disease-relevant trophectoderm cells

Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting

<div><p>Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13^NEGSSEA4^POSTra-1-60^POS on day 7–10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and “contaminating” partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential <i>in vitro</i> and <i>in vivo</i>. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.</p> </div

Enhanced derivation and maintenance of virally reprogrammed fibroblasts using Fluorescence Activated Cell Sorting.

<p>(<b>A</b>) CD13^NEGSSEA4^POSTra-1-60^NEG and CD13^NEGSSEA4^POSTra-1-60^POS populations from the manually derived 1018 clone were sorted onto MEF feeder layers and expanded for 20 days prior to reanalysis by flow cytometry to assess retention of sorted surface markers. dpi = days post infection. dps = days post sort. (<b>B</b>) CD13^NEGSSEA4^POS and CD13^NEGSSEA4^POSTra-1-60^POS populations were sorted onto MEF layers at seven days post infection and imaged at 3 and 17 dps to assess colony formation. (<b>C</b>) Colony counts arising from the sorted cell populations shown in Panel B at 17 dps (25 dpi). (<b>D</b>) Gating structure used in the analysis of CD13^POS cells present within the SSEA4^POSTra-1-60^POS population at 7 dpi. (<b>E</b>) Fluorescence microscopy demonstrating NANOG expression in CD13^POS cell at 7 dpi. 40× magnification. CD13 shown in red. Nanog in shown Green. Values designated %T indicates proportion of total cells within the culture positive for the indicated combinations of surface markers. Values without T designation indicate the proportion of CD13^NEGSSEA4^POS cells that are Tra-1-60^POS or Tra-1-60^NEG in Panel A and D.</p

Stability of Fluorescence Activated Cell Sorted and Manually Derived IPSC Lines.

<p>Three individual clones were selected from foreskin (0819) fibroblasts lines which previously underwent four factor retroviral reprogramming and were derived by either FACS (<b>A, C₁–C₃</b>) or manual (<b>B, C₄–C₆</b>) techniques were analyzed by flow cytometry for pluripotent surface marker expression following expansion on murine embryonic fibroblasts for 12–14 passages. Clones C3 and C6 were adapted to Matrigel and mTSER media around passage11 and expanded for several passages prior to surface marker analysis by flow cytometry to demonstrate stability following changes in culture conditions. Events displayed in the 2D scatter plots are “live” cells as defined by forward and side scatter properties expressing indicated surface markers.</p