Using clinically approved cyclophosphamide regimens to control the humoral immune response to oncolytic viruses.

Oncolytic viruses can be neutralized in the bloodstream by antiviral antibodies whose titers increase progressively with each exposure, resulting in faster virus inactivation and further reductions in efficacy with each successive dose. A single dose of cyclophosphamide (CPA) at 370 mg m(-2) was not sufficient to control the primary antiviral immune responses in mice, squirrel monkeys and humans. We therefore tested clinically approved multidose CPA regimens, which are known to kill proliferating lymphocytes, to determine if more intensive CPA therapy can more effectively suppress antiviral antibody responses during virotherapy. In virus-susceptible mice, primary antibody responses to intravenously (i.v.) administered oncolytic measles virus (MV) or vesicular stomatitis virus (VSV) were partially or completely suppressed, respectively, by oral (1 mg × 8 days) or systemic (3 mg × 4 days) CPA regimens initiated 1 day before virus. When MV- or VSV-immune mice were re-challenged with the respective viruses and concurrently treated with four daily systemic doses of CPA, their anamnestic antibody responses were completely suppressed and antiviral antibody titers fell significantly below pre-booster levels. We conclude that the CPA regimen of four daily doses at 370 mg m(-2) should be evaluated clinically with i.v. virotherapy to control the antiviral antibody response and facilitate effective repeat dosing.

Oncolytic measles virus strains have significant antitumor activity against glioma stem cells.

Glioblastoma (GBM) is the most common primary brain tumor in adults and has a dismal prognosis despite multimodality treatment. Given the resistance of glioma stem cells (GSC) to chemotherapy and radiation therapy, their eradication could prevent tumor recurrence. We sought to evaluate the antitumor activity of measles virus (MV) derivatives against GSC. We generated neurosphere cultures from patient-derived primary tumor GBM xenografts, and we characterized them for the GSC markers CD133, SOX2, Nestin, ATF5 and OLIG2. Using the MV-strains MV-GFP, MV-CEA and MV-NIS we demonstrated infection, viral replication and significant cytopathic effect in vitro against GSC lines. In tumorigenicity experiments, GBM44 GSC were infected with MV in vitro and subsequently implanted into the right caudate nucleus of nude mice: significant prolongation of survival in mice implanted with infected GSC was observed, compared with mock-infected controls (P=0.0483). In therapy experiments in GBM6 and GBM12 GSC xenograft models, there was significant prolongation of survival in MV-GFP-treated animals compared with inactivated virus-treated controls (GBM6 P=0.0021, GBM12 P=0.0416). Abundant syncytia and viral replication was demonstrated in tumors of MV-treated mice. Measles virus derivatives have significant antitumor activity against glioma-derived stem cells in vitro and in vivo.

Pinhole micro-SPECT/CT for noninvasive monitoring and quantitation of oncolytic virus dispersion and percent infection in solid tumors.

The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to: 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). Sodium iodide symporter (NIS) RNA level and dispersion pattern were determined in control and MV-NIS-infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with (123)I or (99)TcO(4) micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r(2)=0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS-infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from non-infected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection, and can replace more time-consuming and expensive analyses (for example, autoradiography and IHC) that require animal killing.

Phase I trial of systemic administration of Edmonston strain of measles virus genetically engineered to express the sodium iodide symporter in patients with recurrent or refractory multiple myeloma.

MV-NIS is an Edmonston lineage oncolytic measles virus expressing the human sodium iodide symporter-a means for monitoring by non-invasive imaging of radioiodine. Patients with relapsed, refractory myeloma who had explored all other treatment options were eligible for this Phase I trial. Cohort 1 was treated with intravenous MV-NIS, and Cohort 2 received cyclophosphamide 2 days prior to MV-NIS. Thirty-two patients were treated. Cohort 1 initially enrolled to four dose levels without reaching maximum tolerated dose (MTD) and subsequently to two higher dose levels when improved virus manufacture technology made it possible. MTD was not reached in Cohort 1, and TCID50 1011 is the dose being used in a Phase II trial of single agent MV-NIS. Grade 3-4 adverse events in both cohorts at all dose levels were: neutropenia (n=9); leukocyte count decreased (n=5); thrombocytopenia (n=2); and CD4 lymphocytes decreased, anemia and lymphopenia (each n=1). MV-N RNA sequences were amplified from gargle specimens, blood and urine. 123I scans were positive in eight patients. One patient achieved a complete response; transient drops in serum free light chains were seen in other patients. MV-NIS is capable of replicating before being cleared by the immune system. Oncolytic viruses offer a promising new modality for the targeted infection and destruction of disseminated myeloma.

Pharmacokinetics of oncolytic measles virotherapy: eventual equilibrium between virus and tumor in an ovarian cancer xenograft model.

Because of their ability to replicate, the dose-response relationships of oncolytic viruses cannot easily be predicted. To better understand the pharmacokinetics of virotherapy in relation to viral dose and schedule, we administered MV-CEA intraperitoneally in an orthotopic mouse model of ovarian cancer. MV-CEA is an attenuated oncolytic measles virus engineered to express soluble human carcinoembryonic antigen (CEA), and the virus is currently undergoing phase I clinical testing in patients with ovarian cancer. Plasma CEA levels correlate with numbers of virus-infected tumor cells at a given time, and were used as a surrogate to monitor the profiles of viral gene expression over time. The antineoplastic activity of single- or multiple-dose MV-CEA was apparent over a wide range of virus doses (10(3)-10(8) TCID(50)), with little reduction in observed antitumor efficacy, even at the lowest tested dose. However, analysis of CEA profiles of treated mice was highly informative, illustrating the variability in virus kinetics at different dose levels. The highest doses of virus were associated with higher initial levels of tumor cell killing, but the final outcome of MV-CEA therapy at all dose levels was a partial equilibrium between virus and tumor, resulting in significant slowing of tumor growth and enhanced survival of the mice.

Enzymatic sulfation of steroids. XV. Studies differentiating between rat liver androgen, estrogen, bile acid, glucocorticoid and phenol sulfotransferases.

Earlier reports left the number of enzymes that catalyzed phenol, androgen, estrogen, bile acid and glucocorticoid sulfation obscure. Here, we have utilized chromatographic, immunochemical and endocrinologic methods to compare and differentiate these enzymes in rat liver. Sulfotransferases I, II, and III--which sulfate glucocorticoids--were used in this comparison. We found that phenols were sulfated by phenol sulfotransferases 1 and 2, which were unrelated to the other enzymes studied here. Large amounts of phenol sulfotransferase 1 were found in both sexes. Large amounts of phenol sulfotransferase 2 were restricted to males. By contrast, the small amount of androgen sulfation found in both sexes appeared to be mediated by sulfotransferase II, which preferred 3 beta-hydroxysteroids, but also sulfated estrogens and glucocorticoids to lesser extents. The sulfation of estrogens presented a more complex picture. Most estradiol sulfotransferase activity in both sexes was due to an enzyme that sulfated estrone poorly, and did not sulfate the other steroids tested. This specific estradiol sulfotransferase was unrelated to the other sulfotransferases described here. Smaller amounts of estrogen sulfotransferase activity that sulfated estradiol and estrone equally well were present at concentrations dependent on the sex of test animals. This enzyme activity appeared to be due to sulfotransferases I, II and III. Most bile acid sulfotransferase activity eluted from DEAE-Sephadex A-50 columns with sulfotransferases I and II. However, data with males suggested that these enzymes were not responsible. Thus, phenols, androgens, estrogens and glucocorticoids were sulfated by six enzymes of differing substrate specificity: phenol sulfotransferases 1 and 2, specific estradiol sulfotransferase, and sulfotransferases I, II, and III. Unique bile acid sulfotransferases also appeared probable.

Inhibition of the Aurora A kinase augments the anti-tumor efficacy of oncolytic measles virotherapy.

Oncolytic measles virus (MV) strains have demonstrated broad spectrum preclinical anti-tumor efficacy, including breast cancer. Aurora A kinase controls mitotic spindle formation and has a critical role in malignant transformation. We hypothesized that the Aurora A kinase inhibitor MLN8237 (alisertib) can increase MV oncolytic effect and efficacy by causing mitotic arrest. Alisertib enhanced MV oncolysis in vitro and significantly improved outcome in vivo against breast cancer xenografts. In a disseminated MDA-231-lu-P4 lung metastatic model, the MV/alisertib combination treatment markedly increased median survival to 82.5 days with 20% of the animals being long-term survivors versus 48 days median survival for the control animals. Similarly, in a pleural effusion model of advanced breast cancer, the MV/alisertib combination significantly improved outcome with a 74.5 day median survival versus the single agent groups (57 and 40 days, respectively). Increased viral gene expression and IL-24 upregulation were demonstrated, representing possible mechanisms for the observed increase in anti-tumor effect. Inhibiting Aurora A kinase with alisertib represents a novel approach to enhance MV-mediated oncolysis and antitumor effect. Both oncolytic MV strains and alisertib are currently tested in clinical trials, this study therefore provides the basis for translational applications of this combinatorial strategy in the treatment of patients with advanced breast cancer.

Characterization of two HDL subfractions and LpA-I, LpA-I:A-II distribution profiles and clinical characteristics of hyperalphalipoproteinemic subjects without cholesterol ester transfer protein deficiency.

The aims of the present study were (i) to characterize the HDL2, HDL3 and the LpA-I, LpA-I:A-II distribution, (ii) to investigate the prevalence of atherosclerotic lesions and (iii) to assess the activity of cholesteryl ester transfer protein (CETP) in 29 hyperalphalipoproteinemic (HALP) patients (HDL-C=90+/-11 mg/dl) with combined hypercholesterolemia (LDL-C=180+/-16 mg/dl). According to the HDL2/HDL3 and LpA-I/LpA-I:A-II ratios, two HALP profiles (A and B) were defined: in 22 patients (HALP profile A) these ratios were increased compared to the normolipidemic control subjects (1.19+/-0.11 versus 0.53+/-0.19, P < 0.001 and 1.01+/-0.2 versus 0.51+/-0.25, P < 0.001, respectively) and in seven patients (HALP profile B) these ratios were within the normal range (0.64+/-0.20 and 0.69+/-0.2, respectively). The atherosclerotic lesions were assessed by ultrasonography of the carotid arteries. Amongst patients with HALP profile A, 17 were free from lesions, five had intimal wall thickening and none displayed plaques, whereas for patients within the HALP profile B, only one was free from lesions, two had intimal wall thickening and four displayed plaques. CETP activities (348+/-116 versus 371+/-75%/ml/h) and CETP concentrations (2.4+/-0.5 versus 2.5+/-0.6 microg/ml) were similar in HALP profiles A and B, however these values were both higher than in control subjects (190+/-40%/ml/h, P < 0.001 and 1.8+/-0.3 microg/ml, P < 0.001, respectively). Hence the hyperalphalipoproteinemic profiles (A and B) described here were not related to CETP deficiency. In conclusion, the HALP profile A was characterized by both increased HDL2/HDL3 and LpA-I/LpA-I:A-II ratios and was associated with a low prevalence of atherosclerosis, whereas the HALP profile B, characterized by HDL2/HDL3 and LpA-I/LpA-I:A-II ratios within the normal range, was less cardioprotective.

Interrelationships between postprandial lipoprotein B:CIII particle changes and high-density lipoprotein subpopulation profiles in mixed hyperlipoproteinemia.

We studied the relationships postprandially between triglyceride-rich lipoprotein (TRL) and high-density lipoprotein (HDL) in 11 mixed hyperlipoproteinemia (MHL) and 11 hypercholesterolemia (HCL) patients. The high and prolonged postprandial triglyceridemia response observed in MHL but not HCL patients was essentially dependent on very-low-density lipoprotein (VLDL) changes. This abnormal response was related to decreased lipoprotein lipase (LPL) activity (-48.7%, P<.01) in MHL compared with HCL subjects. Cholesteryl ester transfer protein (CETP) activity was postprandially enhanced only in MHL patients, and this elevation persisted in the late period (+19% at 12 hours, P<.05), sustaining the delayed enrichment of VLDL with cholesteryl ester (CE). The late postprandial period in MHL patients was also characterized by high levels of apolipoprotein B (apoB)-containing lipoproteins with apoCIII ([LpB:CIII] +36% at 12 hours, P<.01) and decreased levels of apoCIII contained in HDL ([LpCIII-HDL] -34% at 12 hours, P<.01), reflecting probably a defective return of apoCIII from TRL toward HDL. In MHL compared with HCL patients, decreased HDL2 levels were related to both HDL2b and HDL2a subpopulations (-57% and -49%, respectively, P<.01 for both) and decreased apoA-I levels (-53%, P<.01) were equally linked to decreased HDL2 with apoA-I only (LpA-I) and HDL2 with both apoA-I and apoA-II ([LpA-I:A-II] -55% and -52%, respectively, P<.01 for both). The significant inverse correlations between the postprandial magnitude of LpB:CIII and HDL2-LpA-I and HDL2b levels in MHL patients underline the close TRL-HDL interrelationships. Our findings indicate that TRL and HDL abnormalities evidenced at fasting were postprandially amplified, tightly interrelated, and persistent during the late fed period in mixed hyperlipidemia. Thus, these fasting abnormalities are likely postprandially originated and may constitute proatherogenic lipoprotein disorders additional to the HCL in MHL patients.

Direct isolation of labeled low density lipoproteins for the determination of cholesteryl ester transfer protein activity.

The measurement of the activity of cholesteryl ester transfer protein (CETP), is of high clinical interest and this study reports the use of a direct LDL isolation (d-LDL) technique to determine in one step the amount of radiolabeled cholesteryls esters ([3H]-CE) transferred from exogenous HDL3 to LDL, avoiding the conveniences of the usually used ultracentrifugation or precipitation of apo-B containing lipoproteins in the CETP methodologies. The d-LDL technique providing a specific immunoprecipitation of VLDL, IDL and HDL allowed to directly determine the [3H]-CE transferred on LDL (d-[3H]-CE-LDL). Two methodologies were assayed for the CETP activity using either exogenous or endogenous lipoproteins, and the results with the d-LDL technique were compared with those obtained using the ultracentrifugation (u-[3H]-CE-LDL) considered as the reference method. The intra- and inter-assays were similar in both techniques for the two CETP activity assays. Strong positive correlations were established between values obtained with d-[3H]-CE-LDL and u-[3H]-CE-LDL isolation procedures for CETP activities with exogenous or endogenous lipoproteins (r = 0.972; p = 0.0001 and r = 0.965; p = 0.0001 respectively). In conclusion, the d-LDL technique represents an easy and accurate procedure to measure directly, in normotriglyceridemic plasmas, the amount of [3H]-CE transferred from HDL to LDL by the CETP.

Meningeal myeloma deposits adversely impact the therapeutic index of an oncolytic VSV.

Vesicular stomatitis virus (VSV) is neuropathogenic in rodents but can be attenuated 50-fold by engineering the mouse interferon-beta (IFN-ß) gene into its genome. Intravenously administered VSVs encoding IFN-ß have potent activity against subcutaneous tumors in the 5TGM1 mouse myeloma model, without attendant neurotoxicity. However, when 5TGM1 tumor cells were seeded intravenously, virus-treated mice with advanced myeloma developed clinical signs suggestive of meningoencephalitis. Co-administration of a known active antimyeloma agent did not prolong survival, further suggesting that deaths were due to viral toxicity, not tumor burden. Histological analysis revealed that systemically administered 5TGM1 cells seed to the CNS, forming meningeal tumor deposits, and that VSV infects and destroys these tumors. Death is presumably a consequence of meningeal damage and/or direct transmission of virus to adjacent neural tissue. In light of these studies, extreme caution is warranted in clinical testing of attenuated VSVs, particularly in patients with CNS tumor deposits.

Polyinosinic acid decreases sequestration and improves systemic therapy of measles virus.

Off-target binding or vector sequestration can significantly limit the efficiency of systemic virotherapy. We report here that systemically administered oncolytic measles virus (MV) was rapidly sequestered by the mononuclear phagocytic system (MPS) of the liver and spleen in measles receptor CD46-positive and CD46-negative mice. Since scavenger receptors on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated here if MV uptake was mediated by scavenger receptors on Kupffer cells. Pretreatment of cells with poly(I), a scavenger receptor ligand, reduced MV expression by 99% in murine (J774A.1) macrophages and by 50% in human (THP-1) macrophages. Pre-dosing of mice with poly(I) reduced MPS sequestration of MV and increased circulating levels of MV by 4 to 15-folds at 2 min post virus administration. Circulating virus was still detectable 30 min post infusion in mice pre-dosed with poly(I) whereas no detectable MV was found at 5-10 min post infusion if mice did not receive poly(I). MPS blockade by poly(I) enhanced virus delivery to human ovarian SKOV3ip.1 and myeloma KAS6/1 xenografts in mice. Higher gene expression and improved control of tumor growth was noted early post therapy. Based on these results, incorporation of MPS blockade into MV treatment regimens is warranted.

Curative one-shot systemic virotherapy in murine myeloma.

Current therapy for multiple myeloma is complex and prolonged. Antimyeloma drugs are combined in induction, consolidation and/or maintenance protocols to destroy bulky disease, then suppress or eradicate residual disease. Oncolytic viruses have the potential to mediate both tumor debulking and residual disease elimination, but this curative paradigm remains unproven. Here, we engineered an oncolytic vesicular stomatitis virus to minimize its neurotoxicity, enhance induction of antimyeloma immunity and facilitate noninvasive monitoring of its intratumoral spread. Using high-resolution imaging, autoradiography and immunohistochemistry, we demonstrate that the intravenously administered virus extravasates from tumor blood vessels in immunocompetent myeloma-bearing mice, nucleating multiple intratumoral infectious centers that expand rapidly and necrose at their centers, ultimately coalescing to cause extensive tumor destruction. This oncolytic tumor debulking phase lasts only for 72 h after virus administration, and is completed before antiviral antibodies become detectable in the bloodstream. Antimyeloma T cells, cross-primed as the virus-infected cells provoke an antiviral immune response, then eliminate residual uninfected myeloma cells. The study establishes a curative oncolytic paradigm for multiple myeloma where direct tumor debulking and immune eradication of minimal disease are mediated by a single intravenous dose of a single therapeutic agent. Clinical translation is underway.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Preclinical pharmacology and toxicology of intravenous MV-NIS, an oncolytic measles virus administered with or without cyclophosphamide.

MV-NIS is an oncolytic measles virus encoding the human thyroidal sodium iodide symporter (NIS). Here, we report the results of preclinical pharmacology and toxicology studies conducted in support of our clinical protocol "Phase I Trial of Systemic Administration of Edmonston Strain of Measles Virus, Genetically Engineered to Express NIS, with or without Cyclophosphamide, in Patients with Recurrent or Refractory Multiple Myeloma." Dose-response studies in the KAS-6/1 myeloma xenograft model demonstrated a minimum effective dose of 4 x 10(6) TCID50 (tissue culture infectious dose 50)/kg. Toxicity studies in measles-naive squirrel monkeys and measles-susceptible transgenic mice were negative at intravenous doses up to 10(8) and 4 x 10(8) TCID50/kg, respectively. Abundant viral mRNA, maximal on day 8, was detected in cheek swabs of squirrel monkeys, more so after pretreatment with cyclophosphamide. On the basis of these data, the safe starting dose of MV-NIS for our clinical protocol was set at 1-2 x 10(4) TCID50/kg (10(6) TCID50 per patient).

Effects of the gag region on genome stability: avian retroviral vectors that contain sequences from the Bryan strain of Rous sarcoma virus.

We have previously described replication-competent Schmidt Ruppin-A Rous sarcoma virus (RSV)-based retroviral vectors that can be used to deliver and express genes in avian cells. We have continued to modify the prototype vectors to develop a more versatile and efficient system. Substitution of the polymerase (pol) region from the Bryan high-titer RSV (BH-RSV) for the SR-A RSV pol region of these retroviral vectors causes these viruses to replicate more efficiently. We cloned the gag regions from two independent BH-RSV-transformed cell lines and tested whether substituting either of these gag regions would improve the replication and/or gene expression of the vectors. Chimeric vectors were constructed in which the gag region of the prototype vector (SR-A RSV) was replaced with the corresponding segment of BH-RSV gag in vectors that had either the original SR-A RSV pol or the BH-RSV pol region. All vectors contained the bacterial chloramphenicol acetyltransferase gene (CAT). The results indicate that different SR-A RSV and BH-RSV gag-pol chimeras can significantly affect the level of viral and CAT gene expression. The insertion of one of the BH-RSV gag regions, but not the other, gave rise to viruses with unstable genomes.

Selection of a subgroup A avian leukosis virus [ALV(A)] envelope resistant to soluble ALV(A) surface glycoprotein.

The host developing resistance to retroviral infection is believed to be a major force in the evolution of multiple receptor usage by retroviruses. The avian leukosis-sarcoma virus (ALV) group of retroviruses provides a powerful system for studying the envelope-receptor interactions involved in retrovirus entry; different members of this group of closely related viruses use distinct cellular receptors. Analysis of the ALV envelope subgroups suggests that the different ALVs evolved from a common ancestor by mutations in the env gene. Cells and animals that express subgroup A ALV envelope glycoproteins are highly resistant to ALV(A) infection due to receptor interference. In this study, we tested whether expression of a soluble form of subgroup A surface glycoprotein (SU) would result in receptor interference and whether this interference would select for resistant viruses with altered receptor usage. Chicken cells expressing the secreted ALV(A) SU immunoadhesin SU(A)-rIgG, which contains the subgroup A SU domain fused to the constant region of a rabbit immunoglobulin (IgG) heavy chain, showed significant receptor interference. A variant virus resistant to SU(A)-rIgG receptor interference was obtained. This virus had a six-amino-acid deletion in the subgroup A hr1 that altered receptor usage. This approach may identify regions of SU that play a critical role in receptor specificity.