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Combination chemotherapy regimen incorporating CD20 antibodies are commonly used in the treatment of CD20-positive non–Hodgkin's lymphoma (NHL). Fludarabine phosphate (F-araAMP), cyclophosphamide, and CD20 antibodies (Rituximab) constitute the FCR regimen for treating selected NHL, including aggressive mantle cell lymphoma (MCL). As an alternative to the CD20 antibody, we generated a CD20-targeted measles virus (MV)–based vector. This vector was also armed with the prodrug convertase purine nucleoside phosphorylase (PNP) that locally converts the active metabolite of F-araAMP to a highly diffusible substance capable of efficiently killing bystander cells. We showed in infected cells that early prodrug administration controls vector spread, whereas late administration enhances cell killing. Control of spread by early prodrug administration was also shown in an animal model: F-araAMP protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Enhanced oncolytic potency after extensive infection was shown in a Burkitt's lymphoma xenograft model (Raji cells): After systemic vector inoculation, prodrug administration enhanced the therapeutic effect synergistically. In a MCL xenograft model (Granta 519 cells), intratumoral (i.t.) vector administration alone had high oncolytic efficacy: All mice experienced complete but temporary tumor regression, and survival was two to four times longer than that of untreated mice. Cells from MCL patients were shown to be sensitive to infection. Thus, synergy of F-araAMP with a PNP-armed and CD20-targeted MV was shown in one lymphoma therapy model after systemic vector inoculation. [Cancer Res 2007;67(22):10939–47]

An immunocompetent model is required to test therapeutic regimens for clinical trials with the oncolytic measles virus (MV). Toward developing this model, a retargeted MV that enters murine colon adenocarcinoma cells forming tumors in syngeneic C57BL/6 mice was generated. Since MV infection tends to be less efficient in murine than in human cells, the targeted virus was also armed with the prodrug convertase, purine nucleoside phosphorylase (PNP), and named MV-PNP-antiCEA. We have shown before that in cultured cells, infection with this virus activated the prodrug, 6-methylpurine-2'-deoxyriboside (MeP-dR), causing extensive cytotoxicity. When injected intratumorally (IT), MV-PNP-antiCEA inhibited subcutaneous tumor growth marginally, but subsequent administration of the prodrug enhanced the oncolytic effect. Systemic delivery of MV-PNP-antiCEA alone had no substantial oncolytic effects, but in combination with the prodrug it was therapeutic, revealing synergistic effects between virus and prodrug. Immunosuppression with cyclophosphamide (CPA) retarded the appearance of MV neutralizing antibodies and enhanced oncolytic efficacy: survival was 100%, with 9 out of 10 animals going into complete remission. This immunocompetent murine model facilitates the testing of therapeutic regimens for clinical trials.

The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Our study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein.

VEGFR-2 is expressed on tumor vasculature and a target for anti-angiogenic intervention. VXM01 is a first in kind orally applied tumor vaccine based on live, attenuated Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2. We here studied the safety, tolerability, T effector (Teff), T regulatory (Treg) and humoral responses to VEGFR2 and anti-angiogenic effects in advanced pancreatic cancer patients in a randomized, dose escalation phase I clinical trial. Results of the first 3 mo observation period are reported. Locally advanced or metastatic, pancreatic cancer patients were enrolled. In five escalating dose groups, 30 patients received VXM01 and 15 placebo on days 1, 3, 5, and 7. Treatment was well tolerated at all dose levels. No dose-limiting toxicities were observed. Salmonella excretion and salmonella-specific humoral immune responses occurred in the two highest dose groups. VEGFR2 specific Teff, but not Treg responses were overall increased in vaccinated patients. We furthermore observed a significant reduction of tumor perfusion after 38 d in vaccinated patients together with increased levels of serum biomarkers indicative of anti-angiogenic activity, VEGF-A, and collagen IV. Vaccine specific Teff responses significantly correlated with reductions of tumor perfusion and high levels of preexisting VEGFR2-specific Teff while those showing no antiangiogenic activity had low levels of preexisting VEGFR2 specific Teff, showed a transient early increase of VEGFR2-specific Treg and reduced levels of VEGFR2-specific Teff at later time points - pointing to the possibility that early anti-angiogenic activity might be based at least in part on specific reactivation of preexisting memory T cells.

Cancer cells secrete matrix metalloproteinases (MMP) that degrade the extracellular matrix and are responsible for some hallmarks of malignant cancer. Many viruses, including a few currently used in oncolytic virotherapy clinical trials, depend on intracellular proteases to process their proteins and activate their particles. We show here for measles virus (MV) that particle activation can be made dependent of proteases secreted by cancer cells. The MV depends on the intracellular protease furin to process and activate its envelope fusion (F) protein. To make F protein activation cancer cell specific, we introduced hexameric sequences recognized by an MMP and identified the mutant proteins most effective in fusing MMP-expressing human fibrosarcoma cells (HT1080). We showed that an MMP inhibitor interferes with syncytia formation elicited by mutant F proteins and confirmed MMP-dependent cleavage by Edman degradation sequence analysis. We generated recombinant MVs expressing the modified F proteins in place of furin-activated F. These viruses spread only in cells secreting MMP. In nude mice, an MMP-activated MV retarded HT1080 xenograft growth as efficiently as the furin-activated MV vaccine strain. In MV-susceptible mice, the furin-activated virus caused lethal encephalitis upon intracerebral inoculation, whereas the MMP-activated did not. Thus, MV particle activation can be made dependent of proteases secreted by cancer cells, enhancing safety. This study opens the perspective of combining targeting at the particle activation, receptor recognition, and selective replication levels to improve the therapeutic index of MV and other viruses in ongoing clinical trials of oncolysis. (Cancer Res 2006; 66(15): 7694-700)

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus , Chandipura virus , has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae , the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses , Ephemeroviruses , and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

Background: Advanced pancreatic adenocarcinoma still remains associated with a desperate prognosis. Nevertheless, treatment options for patients with metastasized disease have improved considerably over the last few years. Recently, cytotoxic combination therapies such as the FOLFIRINOX regimen and combined nab-paclitaxel/gemcitabine have shown improved overall survival compared to gemcitabine alone. There is still no standard of care in second-line therapy for patients with disease progression. Case Report: We report the case of a 47-year-old patient who dramatically responded to second-line treatment with nab-paclitaxel and gemcitabine after primary progression to the FOLFIRINOX protocol. Conclusion: Second-line treatment after FOLFIRINOX is feasible for patients with good performance status. Our case report supports preclinical findings that suggest that pancreatic cancer is a heterogeneous disease. Further studies that characterize possible subgroups and identify predictive molecular markers to guide therapy are warranted.

Canine distemper virus (CDV) infects many carnivores, including ferrets and dogs, and is the member of the Morbillivirus genus most easily amenable to experimentation in a homologous small-animal system. To gain insights into the determinants of CDV pathogenesis, we isolated a strain highly virulent for ferrets by repeated passaging in these animals. Sequence comparison of the genome of this strain with that of its highly attenuated precursor revealed 19 mutations distributed almost evenly in the six genes. We then recovered a virus from a cDNA copy of the virulent CDV strain's consensus sequence by using a modified reverse genetics system based on B cells. We infected ferrets with this virus and showed that it fully retained virulence as measured by the timing of rash appearance, disease onset, and death. Body temperature, leukocyte number, lymphocyte proliferation activity, and cell-associated viremia also had similar kinetics. We then addressed the question of the relative importance of the envelope and other viral constituents for virulence. Viruses in which the envelope genes (matrix, fusion, and hemagglutinin) of the virulent strain were combined with the other genes of the attenuated strain caused severe rash and fever even if the disease onset was delayed. Viruses in which the nucleocapsid, polymerase, and phosphoprotein genes (coding also for the V and C proteins) of the virulent strain were combined with the envelope genes of the attenuated strain caused milder signs of disease. Thus, virulence-inducing mutations have accumulated throughout the genome.

Background Methylentetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism and consequently could be an important factor for the efficacy of a treatment with 5-fluorouracil. Our aim was to evaluate the prognostic and predictive value of two well characterized constitutional MTHFR gene polymorphisms for primarily resected and neoadjuvantly treated esophagogastric adenocarcinomas. Methods 569 patients from two centers were analyzed (gastric cancer: 218, carcinoma of the esophagogastric junction (AEG II, III): 208 and esophagus (AEG I): 143). 369 patients received neoadjuvant chemotherapy followed by surgery, 200 patients were resected without preoperative treatment. The MTHFR C677T and A1298C polymorphisms were determined in DNA from peripheral blood lymphozytes. Associations with prognosis, response and clinicopathological factors were analyzed retrospectively within a prospective database (chi-square, log-rank, cox regression). Results Only the MTHFR A1298C polymorphisms had prognostic relevance in neoadjuvantly treated patients but it was not a predictor for response to neoadjuvant chemotherapy. The AC genotype of the MTHFR A1298C polymorphisms was significantly associated with worse outcome (p = 0.02, HR 1.47 (1.06-2.04). If neoadjuvantly treated patients were analyzed based on their tumor localization, the AC genotype of the MTHFR A1298C polymorphisms was a significant negative prognostic factor in patients with gastric cancer according to UICC 6th edition (gastric cancer including AEG type II, III: HR 2.0, 95% CI 1.3-2.0, p = 0.001) and 7th edition (gastric cancer without AEG II, III: HR 2.8, 95% CI 1.5-5.7, p = 0.003), not for AEG I. For both definitions of gastric cancer the AC genotype was confirmed as an independent negative prognostic factor in cox regression analysis. In primarily resected patients neither the MTHFR A1298C nor the MTHFR C677T polymorphisms had prognostic impact. Conclusions The MTHFR A1298C polymorphisms was an independent prognostic factor in patients with neoadjuvantly treated gastric adenocarcinomas (according to both UICC 6th or 7th definitions for gastric cancer) but not in AEG I nor in primarily resected patients, which confirms the impact of this enzyme on chemotherapy associated outcome.

In 1997 TTV was detected using representational difference analysis (RDA) in serum of a patient with posttransfusion hepatitis unrelated to known hepatitis viruses. The genome of TTV is a circular single-stranded DNA molecule of 3852 nt with negative polarity. TTV possibly can be grouped either into the existing family Circoviridae or into a recently established virus family "Circinoviridae". Analysis of the complete DNA nucleotide sequence of TTV identified three partially overlapping open reading frames (ORFs). Neither DNA nucleotide nor corresponding amino acid sequences of TTV do show significant homologies to known sequences. TTV DNA nucleotide sequences amplified by PCR from sera of different patients show considerable sequence variations. Although the natural route of transmission of TTV is still unknown, there is clear evidence for a transmission of TTV through blood and blood products. TTV DNA can be detected in the feces of infected individuals suggesting that it may be possible to attract TTV infection from environmental sources. Since the discovery of TTV, numerous studies have investigated the prevalence of TTV infections in different human population groups all over the world. All these studies are based on PCR detection systems, but the technical aspects of the PCR systems vary significantly between the different investigators. The results of the epidemiological studies do not show a clear picture. The discovery of TTV as a viral agent and particularly the identification of a high percentage of infected carriers in the healthy human population raises the following questions: Firstly, what is the origin and molecular relatedness of TT virus. Secondly, what is the significance of TTV as a human pathogen. And thirdly, what are the exact molecular mechanisms of viral replication. To answer these questions it will be necessary to determine the primary structure and the coding capacity of several TTV patient isolates.

Tupaia herpesviruses (THVs) have been isolated from malignant lymphomas and from degenerating lung or spleen cell cultures of tree shrews (Tupoia spp.), but because of a lack of genetic information the final classification of THVs is still open. In the present work the viral DNA polymerase (DPOL) gene was mapped within the genome of the different THV strains using PCR and degenerate oligonucleotide primers. Nucleotide sequences of the DPOL genes of THV strains 1 to 5 were determined and used for comparative analyses. The transcriptional activity of the THV-2 DPOL gene was confirmed by RT-PCR. It was found that the different THV strains are very closely related to each other. When compared to other herpesviruses the highest amino acid sequence identities detected were with DPOLs of the murine and human cytomegaloviruses. These results justify the conclusion that THVs are members of the subfamily Betaherpes-virinae.

BACKGROUND Recent data suggest primary resection as the preferable approach in patients with signet ring cell gastric cancer (SRC). The aim of our retrospective exploratory study was to evaluate the influence of SRC on prognosis and response in esophagogastric adenocarcinoma treated with neoadjuvant chemotherapy. METHODS A total of 723 locally advanced esophagogastric adenocarcinomas (cT3/4 N any) documented in a prospective database from two academic centers were classified according to the WHO definition for SRC (more than 50 % SRC) and analyzed for their association with response and prognosis after neoadjuvant treatment. RESULTS A total of 235 tumors (32.5 %) contained SRC. Median survival of SRC was 26.3 compared with 46.6 months (p < 0.001) for non-SRC. SRC were significantly associated with female gender, gastric localization, advanced ypT and R1/2 categories, and lower risk of surgical complications and anastomotic leakage (each p < 0.001). Clinical (21.1 vs. 33.7 %, p = 0.001) and histopathological response (less than 10 % residual tumor: 16.3 vs. 28.9 %, p < 0.001) were significantly less frequent in SRC. Clinical response (p = 0.003) and complete histopathological response (pCR) (3.4 %) (p = 0.003) were associated with improved prognosis in SRC. Clinical response, surgical complications, ypTN categories, but not SRC were independent prognostic factors in forward Cox regression analysis in R0 resected patients. Risk of peritoneal carcinomatosis was increased (p < 0.001), while local (p = 0.015) and distant metastases (p = 0.02) were less frequent than in non-SRC. CONCLUSIONS Prognosis of SRC is unfavorable. Although response to neoadjuvant chemotherapy is rare in SRC, it is associated with improved outcome. Thus, chemotherapy might not generally be abandoned in SRC. A stratification based on SRC should be included in clinical trials.

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased antitumor activity, and are currently under investigation in clinical phase 1 trials. Approaches with other viral vectors have shown that insertion of immunomodulatory transgenes enhances the therapeutic potency. In this study, we engineered MV for expression of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). For the first time, therapeutic efficacy and adaptive immune response in the context of MV oncolysis could be evaluated in the previously established immunocompetent murine colon adenocarcinoma model MC38cea. MC38cea cells express the human carcinoembryonic antigen (CEA), allowing for infection with retargeted MV. Intratumoral application of MV-GMCSF significantly delayed tumor progression and prolonged median overall survival compared with control virus-treated mice. Importantly, more than one-third of mice treated with MV-GMCSF showed complete tumor remission and rejected successive tumor reengraftment, demonstrating robust long-term protection. An enhanced cell-mediated tumor-specific immune response could be detected by lactate dehydrogenase assay and interferon-γ enzyme-linked immunospot assay. Furthermore, MV-GMCSF treatment correlated with increased abundance of tumor-infiltrating CD3(+) lymphocytes analyzed by quantitative microscopy of tumor sections. These findings underline the potential of oncolytic, GM-CSF-expressing MV as an effective therapeutic cancer vaccine actively recruiting adaptive immune responses for enhanced therapeutic impact and tumor elimination. Thus, the treatment benefit of this combined immunovirotherapy approach has direct implications for future clinical trials.

Pancreatic ductal adenocarcinoma is one of the most aggressive cancers, and surgical resection is a requirement for a potential cure. However, the majority of patients are diagnosed with advanced-stage disease, either metastatic (50%) or locally advanced cancer (30%). Although palliative chemotherapy is the standard of care for patients with metastatic disease, management of locally advanced adenocarcinoma is controversial. Several treatment options, including extended surgical resections, neoadjuvant therapy with subsequent resections, as well as palliative radiotherapy and/or chemotherapy, should be considered. However, there is little evidence available to support treatment options for locally advanced disease. As valid predictive biomarkers for stratification of therapy are not available today, future trials need to define the role of the different treatment options. This Review summarizes the current evidence and discusses available treatment options for both locally advanced and metastatic pancreatic adenocarcinoma.

The influence of the local immune response on the clinical course of colorectal cancer (CRC) has been analyzed extensively. Analyzing the invasive margin of human CRC liver metastases, we identified a protumoral mechanism of T-cell-derived CCL5 that leads to immune cell exploitation by tumor cells. Two distinct subsets of myeloid cells produce CXCL9/CXCL10, which induce an influx of T cells into the invasive margin. CCL5 is produced by these exhausted T cells and stimulates tumor cell proliferation and invasive behavior via CCR5 on tumor cells and macrophages. CCR5 inhibition in patient-derived functional in vitro organotypic culture models induced macrophage repolarization with anti-tumoral effects. These immunomodulatory and anti-tumoral effects of CCR5 blockade then could be confirmed in a phase I trial with a CCR5 antagonist in advanced refractory CRC patients with liver metastases. Amelioration of tumor-promoting inflammation on the tumor tissue level and objective tumor responses in advanced metastatic CRC patients were observed. Citation Format: Niels Halama, Inka Zoernig, Anna Berthel, Christoph Kahlert, Fee Klupp, Meggy Suarez-Carmona, Juergen Krauss, Karsten Brand, Felix Lasitschka, Tina Lerchl, Claudia Luckner-Minden, Alexis Ulrich, Juergen Weitz, Martin Schneider, Markus W. Buechler, Laurence Zitvogel, Thomas Herrmann, Axel Benner, Christina Kunz, Stephan Luecke, Christoph Springfeld, Christine Falk, Dirk Jaeger. Macrophage repolarization therapy in metastatic colorectal cancer: CCR5 inhibition [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B037.

Introduction: An underlying basis of cancer immunotherapy is the activation of the patient's own immune effector cells to specifically eradicate tumor cells. Different strategies of cancer immunotherapy have shown outstanding success in a number of patients across various tumor types. However, currently only a fraction of patients respond to immunotherapy, and therapy works only in a subset of tumors. To understand why, an integrated analysis of host and tumor factors is required. Fundamental issues in the analysis of key prognostic and predictive components include elucidating the complex interaction between tumors and their microenvironment, determining the type and densities of tumor-infiltrating immune cells, and understanding how the mutational load influences therapy outcome. Sophisticated analysis is required for patients with refractory or rare cancers to explore new therapeutic strategies. We established a systems medicine approach to obtain detailed data about tumor-host interactions, which may provide information on clinical outcome and help identify patients who are most likely to benefit from immunotherapy. Methods and Results: Whole-exome sequencing of tumor and control samples is performed from freshly obtained patient samples. In addition, whole-transcriptome sequencing of tumor tissue is performed if possible. Single nucleotide variations (SNVs) are detected in each tumor. Sequence-based HLA typing and computational epitope prediction methods are used to assess the immunogenicity of each SNV. The grade of immune cell infiltration and the expression of selected immunomodulatory proteins, e.g. PD-1 and PD-L1, is assessed by immunohistochemical stainings of tissue samples. Additionally, cytokine and chemokine profiles are determined in the tumor microenvironment. The resulting data can aid in treatment decision, e.g. PD-1 blocking antibodies can be administered when elevated PD-1 expression is detected. Alternatively, peptide vaccination is considered if a sufficient number of immunogenic mutations is detected. Based on the mutational profiles, mutation specific 29mer peptides and corresponding wild type peptides are produced for ELIspot assays with patient PBMCs to assess mutation-specific T cell reactivity. To assess the changes over time, blood and tissue are obtained during therapy to monitor therapy-induced changes in the periphery and the tumor microenvironment. Conclusion: Here we present our established workflow to systematically analyze tumor-host interactions in cancer patients and show preliminary data of different facets and clinical implications of these complex interactions. To evaluate and validate immunological and pathological characteristics of a tumor and the methods to assess its immunogenicity as well as its complex interplay with the microenvironment, comprehensive translational studies have to be conducted. Eventually, the obtained information will help to identify important subgroups and patients who are most likely to benefit from a certain type of immunotherapy. Citation Format: Zeynep Kosaloglu, Inka Zoernig, Niels Halama, Bruno Koehler, Anne Katrin Berger, Christoph Springfeld, Alexis Ulrich, Martin Schneider, Felix Lasitschka, Hanno Glimm, Stefan Froehling, Benedikt Brors, Dirk Jaeger. Combining immunomics and genomics for immunotherapy of refractory and rare cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-287.

e15739Background: Hyperbilirubinaemia is a common disease effect in patients (pts) with metastatic pancreatic cancer (mPC). As clinical trials often exclude them, data on management of these pts ar...

3091Background: VXM01 is an orally applied tumor vaccine based on live, attenuated salmonella bacteria carrying a eukaryotic expression plasmid, which encodes VEGFR-2. A recent randomized and place...

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.

Lentiviral vectors (LVs) are vectors of choice for many gene therapy applications since they mediate long term gene expression and can transduce dividing and non-dividing cells. Recently, efficient targeting of LVs pseudotyped with the measles virus (MV) glycoproteins has been reported. However, MV antibodies in patients might limit the clinical use of these vectors. Thus, aim of this study was the development of targeted LVs pseudotyped with the glycoproteins of Tupaia paramyxovirus (TPMV). Since this animal paramyxovirus does not infect humans, no TPMV antibodies in patients are expected. For efficient incorporation in LVs, the TPMV glycoproteins, the hemagglutinin (H) and fusion (F) protein, were modified by truncation of their cytoplasmic tails. Targeting was achieved by displaying a single-chain antibody against the B cell surface marker CD20 on the H protein. The modified proteins were biochemically characterized and tested for their functionality. Unexpectedly, it was observed that an additional proteolytic cleavage of the F protein occurs during activation, resulting in the fragments F1a, F1b and F2. The newly identified fragment F1a was detected in virions and in supernatant of transfected cells. The F1a/F1b cleavage site was mapped and a cysteine protease was identified as likely activating protease. The data indicate that F protein processing is more complex than expected. After characterization, the modified TPMV glycoproteins were screened in all combinations for their ability to form functional pseudotyped LVs. Most efficient pseudotype formation was achieved with CT truncations of 80 amino acids (aa) for H (HΔ80αCD20) and 32 aa for F (FΔ32) (titers ~ 106 t.u./ml). The resulting vectors selectively transduced CD20-positive cells in a mixed cell population. Furthermore, they mediated efficient gene transfer into activated and quiescent primary human B cells. Neutralization assays showed that TPMV-pseudotyped vectors were not neutralized by human sera containing MV antibodies. In conclusion, it was demonstrated that targeted LVs pseudotyped with TPMV glycoproteins can be generated and escape neutralization by MV antibodies. Remarkably, the vectors are able to efficiently transduce even quiescent B cells. Hence, they might be a valuable vector choice when systemic application of targeted lentiviral vectors in humans is required.

First-line treatment of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC) is based on platinum, 5-fluorouracil (5-FU) and the monoclonal antiEGFR antibody cetuximab. However, in most cases this chemoimmunotherapy does not cure the disease, and more than 50% of HNSCC patients are dying because of local recurrence of the tumors. In the majority of cases, HNSCC overexpress the epidermal growth factor receptor (EGFR), and its presence is associated with a poor outcome. In this study, we engineered an EGFR-targeted oncolytic measles virus (MV), armed with the bifunctional enzyme cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT). CD/UPRT converts 5-fluorocytosine (5-FC) into the chemotherapeutic 5-FU, a mainstay of HNSCC chemotherapy. This virus efficiently replicates in and lyses primary HNSCC cells in vitro. Arming with CD/UPRT mediates efficient prodrug activation with high bystander killing of non-infected tumor cells. In mice bearing primary HNSCC xenografts, intratumoral administration of MV-antiEGFR resulted in statistically significant tumor growth delay and prolongation of survival. Importantly, combination with 5-FC is superior to virus-only treatment leading to significant tumor growth inhibition. Thus, chemovirotherapy with EGFR-targeted and CD/UPRT-armed MV is highly efficacious in preclinical settings with direct translational implications for a planned Phase I clinical trial of MV for locoregional treatment of HNSCC.

Top of pageAbstract Infection with wild-type measles virus (MV), a negative-stranded RNA virus of the family Paramyxoviridae, has been associated with spontaneous remissions in patients with lymphoma and leukaemia. In addition, studies with lymphoma, myeloma, glioma and ovarian cancer xenografts have documented oncolytic effects of an Edmonston lineage virus (MV-Edm) on a wide array of cancer types. Recombinant MV with targeted cell entry have been produced: specifity domains displayed on the viral attachment protein H mediate entry into normally non-permissive cells via designated receptors. Moreover, viruses that are |[ldquo]|blind|[rdquo]| for their natural receptors have been generated by mutating residues responsible for entry through CD46 and SLAM. To engineer the most efficient and safe vectors for clinical trials we pursue three independent approaches to be eventually combined: i) Vector retargeting at the cell entry level. ii) Vector arming with suicide genes. iii) Production of vectors with a stealth envelope that is not recognized by measles-specific antibodies. RESULTS. We have produced viruses expressing suicide genes: thymidine kinase (TK), cytosine deaminase (CD) and the E. coli purine nucleoside phosphorylase (PNP), that kills infected and bystander cells very effectively and have combined arming with receptor-targeting. We have begun to analyse these new viruses. First, the ability of retargeted MV to retard the growth of tumor xenografts was demonstrated in an immunocompetent mouse model based on murine MC38cea cells expressing the designated receptor human CEA. Second, the efficacy of recombinant viruses with combined oncolytic specificities in entering and killing target cells was demonstrated in vitro. The efficacy of these viruses is currently under investigation in a CD20/lymphoma xenograft model. Third, we are producing vectors with the glycoproteins F and H from the Tupaia paramyxovirus (TPMV) in place of the homologous MV proteins. We have previously shown that the TPMV H protein can be retargeted to support fusion of cells expressing a designated receptor. Now we show that a chimeric virus with the MV proteins N, P, L, M and the TPMV F and H protein can be rescued but does not spread efficiently. DISCUSSION. The success of oncolytic virotherapy depends on the specificity of viral replication in cancer cells, the efficacy of the oncolytic agent in eliminating those cells, and their ability of spreading without being neutralized by antibodies. We have obtained targeted and armed viruses, and have made good progress toward the production of stealth particles.

To engineer a targeting envelope for gene and oncolytic vector delivery, we characterized and modified the envelope proteins of Tupaia paramyxovirus (TPMV), a relative of the morbilli- and henipaviruses that neither infects humans nor has cross-reactive relatives that infect humans. We completed the TPMV genomic sequence and noted that the predicted fusion (F) protein cleavage-activation site is not preceded by a canonical furin cleavage sequence. Coexpression of the TPMV F and hemagglutinin (H) proteins induced fusion of Tupaia baby fibroblasts but not of human cells, a finding consistent with the restricted TPMV host range. To identify the factors restricting fusion of non- Tupaia cells, we initially analyzed F protein cleavage. Even without an oligo- or monobasic protease cleavage sequence, TPMV F was cleaved in F1 and F2 subunits in human cells. Edman degradation of the F1 subunit yielded the sequence IFWGAIIA, placing the conserved phenylalanine in position 2, a novelty for paramyxoviruses but not the cause of fusion restriction. We then verified whether the lack of a TPMV H receptor limits fusion. Toward this end, we displayed a single-chain antibody (scFv) specific for the designated receptor human carcinoembryonic antigen on the TPMV H ectodomain. The H-scFv hybrid protein coexpressed with TPMV F mediated fusion of cells expressing the designated receptor, proving that the lack of a receptor limits fusion and that TPMV H can be retargeted. Targeting competence and the absence of antibodies in humans define the TPMV envelope as a module to be adapted for ferrying ribonucleocapsids of oncolytic viruses and gene delivery vectors.

The Tupaia herpesviruses (THVs) have been isolated from malignant lymphoma tissue cultures and from degenerating lung and spleen cell cultures of tree shrews (Tupaia spp.). Recently we succeeded in the localization of the gene locus of the THV DNA polymerase (DPOL) gene within the viral genome. Based on these results the highly conserved gene cluster of herpesviruses encoding the DPOL, the glycoprotein B (gB), a probable processing and transport protein (PRTP), and the major DNA binding protein (DNBI) was characterized in the genome of THV strain 2 (THV-2) in its entirety. The complete nucleotide sequence of the gene cluster was determined and it was discovered that the THV-2 gene products are most closely related to the corresponding proteins of mammalian cytomegaloviruses. The transcriptional activity of the four genes was confirmed by amplification of a part of the corresponding mRNAs obtained from infected cell RNA by RT-PCR. The homology values and the overall structure of the gene cluster, that shows specific colinearity with the corresponding clusters of the mammalian cytomegaloviruses, is further evidence that THV-2 is a member of the subfamily Betaherpesvirinae.

Measles virus (MV) is oncolytic: wild type MV infection occasionally induces lymphoma regression in humans, and the MV vaccine strain has been used to eliminate different types of human cancer xenografts in mice. Towards enhancing the MV specificity for different tumor types we are operating at different levels, including selective viral activation by tumor-specific matrix metalloproteinases (MMPs). We report here the engineering of a recombinant MV which fusion (F) protein is selectively cleaved and activated by a MMP. The F protein of wild type and vaccine MV is produced as an inative precursor F0 that is cleaved into an amino-terminal F2 subunit and a membrane-anchored F1 subunit bearing a fusion peptide. MV F0 is activated intracellularly by furin, but we have previously engineered a F protein activated by the extracellular protease trypsin (J. Gen. Virol. 81, 441-9, 2000).

Wild type MV infection occasionally induces lymphoma regression in humans, and recombinant viruses based on the vaccine MV strain are currently being developed as therapeutics for use in clinical trials for different types of cancer. Towards producing recombinant MV that target different tumor types we are operating at multiple levels: first, we have altered cell entry specificity by re-engineering the attachment protein (hemagglutinin, H) to bind to cancer-specific glycoproteins (J. Virol. 75:2087-96, 2001; J. Virol. 78, 302-13, 2004; Nat. Biotechnol. 22:331-6, 2004). Moreover, we have produced fusion (F) proteins that are selectively activated by matrix metalloproteases (Springfeld et al., this Abstract book), altered the viral host control evasion proteins (Devaux et al., this Abstract book; von Messling and Cattaneo, this Abstract book), and expressed suicide genes from an additional transcription unit (Mol. Ther. 9:S224, 2004). However a standing issue with the use of recombinant MV in human clinical trials is the frequent presence of neutralizing antibodies in human sera. Therefore, the possibility of exchanging the viral envelope with that of an animal virus that is not MV cross-reactive is being explored.

Infection with wild-type measles virus (MV), a negative-stranded RNA virus of the family Paramyxoviridae, has been associated with spontaneous remissions in some patients with Hodgkins lymphoma and leukemia. Subsequent studies in vitro and in mouse models of lymphoma, myeloma, glioma, and ovarian cancer with an attenuated MV strain of the Edmonston lineage (MV-Edm) have demonstrated a strong oncolytic effect. However, in these mouse models, the implanted tumors could not always be eradicated by the treatment with MV-Edm. Therefore, MV with enhanced and inducible oncolytic properties are highly desirable.