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The prognosis for cats with fibrosarcoma is still poor as the treatment options existing to date do not lead to satisfying results. After sole surgical removal of the tumor, up to 70 % of the cats develop local recurrences. Even with adjuvant radiation and/or chemotherapy, the recurrence rate can just be reduced to at most 40 %. In the present work an alternative treatment method in terms of neoadjuvant immunostimulatory gene therapy should be established. Via plasmids, three feline cytokine genes were transferred into the tumor. The plasmids coded for feIL-2, feIFN-γ and feGM-CSF. Using magnetofection, gene transfer should be optimized. The aim of this clinical dose escalation study was to define a well tolerated dose, which can be tested for its efficacy in a subsequent phase-II trial. Therefore the cats were examined at defined time points for treatment-related toxicity up to 180 days after surgery. Two more check-ups on day 270 and 360 after surgery were performed to elongate the observation period for local recurrences. Prerequisite for a cat’s admission to the study was the localization of the fibrosarcoma (primary tumor or recurrence) on the trunk. The tumor had to be excised in one setting without limb amputation. Affected cats were neither allowed to be pretreated with chemo-, radiation- or gene therapy nor with corticosteroids within the past six weeks. Further exclusion criteria were hints for metastases or other severe illnesses which reduce life expectancy to less than one year. Due to the potential teratogenic effect of the expressed cytokines, pregnancy had to be ruled out. Only cats with histopathologically confirmed fibrosarcoma continued the study after surgery. As this was a scientific study with a drug not registered yet, written informed consent from the owners was a prerequisite for the participation of each cat. Four treatment groups with defined dose escalation were prospectively fixed. The dose of the feline cytokines was 15, 50, 150 and 450 µg per plasmid in group I, II, III and IV. The initial dose (3 x 15 µg) was oriented to the total dose for small oral melanomas in dogs (400 µg) established by DOW et al. and is 1/10 of it. Plasmids as non-viral vectors were chosen for several reasons. Their handling is not liable to such strict regulations as the potentially more dangerous viral vectors. Their production is simple and affordable. They induce fewer side effects than viral vectors. Therefore plasmids are more suitable for application in veterinary clinical practice. Equal amounts of the plasmids were brought into 0.9 % saline. The positively charged magnetic nanoparticles were brought into solution with aqua for injections and were mixed with the plasmid formulation in a 1:1 ratio. This formulation had a total volume of 500 µl and was injected twice intratumorally in weekly intervals. Transfection was enhanced and targeted to the tumor area by the application of a neodymium-iron-boron magnet for the duration of 60 minutes. One week after the second application, wide en-bloc resection of the tumor was performed. Four cats were assigned to each treatment group. As questionable toxicity occured in group IV, four more cats were added to this group. A control group also consisting of four cats received surgery without neoadjuvant therapy. For ethical reasons, the application of empty plasmids was avoided so that in this group surgery could be performed without delay. Medical care of all the cats was carried out by the same team of internists, anesthetists and surgeons. Clinical and laboratory parameters were evaluated according to the VCOG-CTCAE system. All adverse events were registered, classified with severity grades and correlated to treatment. Plasma samples of all cats up to 14 days after surgery were examined for the existence of feGM-CSF and feIFN-γ with commercially available ELISA kits. Statistical analyses were performed comparing the treatment groups themselves as well as treatment groups and the control group regarding body weight, white blood cells and differential blood counts. Only one cat out of group IV showed adverse events during the neoadjuvant treatment period, which were classified as grade 3 and which were probably correlated to treatment (correlation grade 4). For this reason four more cats were treated with the highest dose. None of these cats showed side effects that could be correlate to treatment. The occurrence of early recurrences in four cats of group IV was outstanding, but of course, the expressiveness of this statement is low regarding the small group sizes. However it is known that biological drugs, especially IL-2, often do not have a linear dose-response profile. Therefore the optimal effective dose lies within a strictly defined area and is probably lower than the maximal tolerated dose. The highest applied dose which is 450 µg per plasmid was defined as a well tolerated dose. It can be safely tested for its efficacy in a subsequent phase-II trial. Because of the reflections mentioned above it would undoubtedly be convenient to test the third dose (150 µg per plasmid) in parallel for its effectiveness.

The feline fibrosarcoma is a spontaneous malignant soft tissue sarcoma. Due to the high recurrence rate of approximatelly 70 % even after radical surgical excision the prognosis is very poor. There have been carried out different gene-therapy protocolls adjuvant to tumour excision, yet. In the following study a local, nonviral gene-transfer with the feline cytokine-genes Interleukin 2, Granulocyte-macrophage colony-stimulating factor and Interferon γ is conducted. As local gene delivery system serves a collagen-sponge loaded with a plasmid-DNA-PEI-PEG-formulation. The immunomodulating cytokines are expected to improve the antigen presentation, to activate immune effector cells and to generate memory cells against specific tumour antigens. The goal is to enhance tumour cell killing and anti cancer immune response to extend the tumour free survival time or even to decrease the recurrence rate in fibrosarcoma bearing cats. First a phase I dose escalation study is carried out using these vector-loaded collagen sponge. The adjuvant immunotherapy is combined with the surgical standard treatment of the feline fibrosarcoma. Blood was collected from healthy cats. Feline peripheral mononuclear blood cells were separated, cultured and stimulated in vitro by Concanavalin A. mRNA was isolated and reverse transcribed in cDNA. The cDNA served as template for the PCR amplification. Specific primers are based on already published sequences and introduced restriction endonuclease sequences in the amplified product. PCR products and expression vector were cut with these restriction enzymes. After ligation of PCR products and expression vector p55pCMV_ivs_luc+ transformation in DH 10 B bacteria was performed. Further analysed inserts were sequenced. Before therapeutic use the cytokines had to show biological activity. Recombinant proteins are expressed in the mammalian cell line COS-7. The feIL-2 and feGM-CSF bioactivity is demonstrated in proliferation assays, using the IL-2 and GM-CSF dependent cell lines CTLL-2 and TF-1. The biological acitivty of feIFNγ is measured by FACS analysis. A MHC I and II induction assay was performed on feline fibrosarcoma cells. After plasmid DNA preparation with polykation polyethylenimine and protective copolymers (polyethylene glycol) the collagen sponge is loaded with these gene vectors under sterile conditions. The vector loaded sponge is stored at 4 °C till implanted in the tumour bed of fibrosarcoma bearing cats.

Das feline Fibrosarkom hat mit einer Rezidivneigung von bis zu 70 % nach alleiniger chirurgischer Entfernung eine ungünstige Heilungsprognose. Auch adjuvante Chemo- oder Strahlentherapien führen nur in 42 % der Patienten zu einer rezidivfreien Zeit von über einem Jahr. In der vorliegenden Arbeit soll ein nonvirales Gentransfersystem etabliert werden, bei dem erstmalig eine Kombination aus drei felinen Zytokin-Genen zur adjuvanten Immunstimulation nach Tumorexstirpation beim Fibrosarkom der Katze eingesetzt wird. Ziel dieser Studie ist die Festlegung einer maximal tolerierten Dosis. Mit Hilfe eines Nebenwirkungskataloges, der auf der Common-Toxicity-Criteria-Tabelle des National Cancer Instiute basiert, werden klinische wie auch labordiagnostische Parameter objektiv erfasst und in Relation zur Therapie gestellt. Nach definierten Aufnahmekriterien werden Katzen mit Fibrosarkomen von prak-tischen Tierärzten an die Medizinische Kleintierklinik überwiesen. Es werden nur Tiere in die Studie aufgenommen, deren Tumoren (Primärtumor oder Rezidiv) am Rumpf lokalisiert sind. Die Tumorexstirpation muss in einer Sitzung möglich sein und darf dabei weder zu einer Gliedmaßenamputation noch zur Eröffnung einer Körperhöhle führen. Katzen, bei denen Hinweise auf Metastasen oder eine andere schwere Krankheit vorliegen, sowie Tiere, die bereits zuvor mit einer Chemo-, Strahlen- oder Gentherapie behandelt wurden, können nicht in die Studie aufge-nommen werden. Nach Tumorexstirpation wird den Tieren ein Kollagenschwamm in das Tumorbett implantiert. Dieser Kollagenschwamm trägt Plasmide, die jeweils für feIL-2, feIFNγ und feGM-CSF kodieren. Die Plasmid-DNA ist zusätzlich an Polyethylenimin (PEI) assoziiert und mit einem Hüllpolymer (P6YE5C) ummantelt. Insgesamt werden 15 Tiere in vier verschiedenen Dosisgruppen therapiert. Gruppe I (n=3) mit 75 μg je Plasmid, Gruppe II (n=3) mit 150 μg je Plasmid, Gruppe III (n=6) mit 300 μg je Plasmid und in Dosisgruppe IV (n=3) mit 600 μg je Plasmid. Als Ver-gleichsgruppe gelten vier Katzen, die unter gleichen Bedingungen nur mit einer en bloc Resektion behandelt werden. In die Studie werden 15 Katzen (mk=9, wk=6) im Alter zwischen drei und 15 Jahren aufgenommen. Die Fibrosarkome sind häufiger im Interscapularbereich lokalisiert. Bei keinem Patienten kommt es zu therapiebedingten Beeinträchtigungen des Allgemeinbefindens. Drei Katzen entwickeln postoperativ ein Serom, das ohne thera-peutisches Eingreifen resorbiert wird. Systemische Veränderungen, die auf die Therapie zurückgeführt werden, manifestieren sich erst in Dosisgruppe IV mit einem Abfall der Lymphozytenpopulation. Dabei fallen in einem Zeitraum bis 45 Tage nach Implantation des Kollagenschwammes die Lymphozyten bis zu 70 % des Ausgangs-wertes ab. Bei dieser Phase I-Studie handelt es sich um eine Dosisfindungsstudie. Diese wird durch das Auftreten erster Nebenwirkungen, die auf das Transgen oder auf dessen Expressionsprodukt zurückgeführt werden können, beendet. Die maximal tolerierte Dosis wird somit in vorliegender Studie auf 600 μg je Zytokin-Plasmid festgesetzt.

Feline herpesvirus-1 (FHV-1)-infection, also known as feline viral rhinotracheitis (FVR) is distributed world wide in the cat population, with a high incidence in colony cats (>70%). FHV-1 typically infects and replicates in epithelial tissue of the upper respiratory tract and conjunctiva causing cytopathic lesions. The virus is recognized as one of the most important pathogens of feline upper respiratory tract infections, conjunctivitis and keratitis in cats. Following primary infection over 80% of cats are unable to eliminate the virus and develop a carrier state, with intermittent episodes of virus shedding. During latency, the virus persists most often in the trigeminal ganglion and the disease can be reactivated following stress and corticosteroid therapy. Successful treatment, particularly of ocular manifestations associated with recrudescent infection such as dendritic ulcers remains difficult. Available antiviral drugs are virostatic and in particular, the systemic application is associated with severe toxic side effects. Human natural and recombinant interferons and feline interferons have been described in their use against a selection of feline and canine viruses (JAMESON and ESSEX, 1983; FULTON and BURGE, 1985; WEISS and TOIVIO-KINNUCAN, 1988; WEISS, 1989; WEISS and OOSTROM-RAM, 1990; TRUYEN et al., 2001). To date, only two in vitro studies indicate a potent antiviral activity of the recombinant FeIFN-ω against FHV-1 (MOCHIZUKI et al., 1994, TRUYEN et al., 2001). The purpose of this study was to evaluate the antiviral efficacy of the recently available rFeIFN-ω (Virbagen®-ω) and the human rHuIFN-α2b (Intron®-A) on the replication of FHV-1 in vitro. A wider range of concentrations (100 U/ml – 500,000 U/ml) was studied than used in the previous studies (WEISS , 1989; MOCHIZUKI et al., 1994, TRUYEN et al., 2001) in order to repeat previously tested concentration and further, to determine whether the drugs have a dose-dependant response of antiviral activity and which concentration would be the most effective treatment. The species-specificity of type I interferons is well known and therefore, it was suggested that rFeIFN-ω would result in a more profound effect compared to the rHuIFN-α2b due to its homologous nature. In addition to cell and virus culture techniques, a methodology for the plaque reduction assay was established. Furthermore, for the first time antiviral efficacy for interferon was additionally measured by plaque size reductions and is reported in this study. An in vitro MTT-Assay was integrated in the experiment to exclude possible cytotoxic effects that could, in principle, contribute to the antiviral effects observed with either of the interferon treatments. For the plaque reduction assay, confluent monolayers of Crandell feline kidney (CRFK)-cells were grown in 24-well cell culture plates. Cells were treated with either rFeIFN-ω (Virbagen® Omega) or rHuIFN-α2b (Intron®-A) across a set of serial dilutions (100 U/ml - 500,000 U/ml). Cells were treated six hours before addition of FHV-1, concurrent with addition of FHV-1 and each drug concentration was added to the overlay medium (2X DMEM 1:1 with 1.6% Agarose). The treatments were performed in duplicates including virus controls, which received PBS instead of either one of the interferons and assays were performed on six occasions. Following incubation of 72 hrs, the cells were fixed with formalin, overlay plugs were discarded and the remaining cell layers were stained with crystal violet. Plaque numbers were counted under an inverted microscope and Plaque diameters were measured using a reticule. For the MTT-assay, 96-well culture plates were seeded with CRFK-cells. In each of the test wells (n=16) growth medium was supplemented with either rHuIFN-α2b or with rFeIFN-ω. Control wells (n=72) received growth medium with PBS instead of either one of the Interferons. Cell-death controls were treated with ethanol to obtain 100% cell death. After incubation, the medium was aspirated and replaced with medium containing the MTT-solution. Following another incubation period, the medium of each well was removed and replaced with a solubilization solution (0.1N HCL/Isopropanol). The plates were incubated for an additional five minutes to dissolve the crystals and the plates were read using a plate reader. The average optical densities were calculated for each dilution and compared with that of the positive control wells. A one-way ANOVA and Dunnett’s test were used for the statistical analysis of all experiments. A significant reduction of plaque numbers was observed for rFeIFN-ω at 100,000 U/ml with a plaque reduction of 54.7 % and at 500,000 U/ml with a plaque reduction of 59.8 %. Plaque sizes were significantly reduced by 47.5 % at 100,000 U/ml and by 81 % at 250,000 U/ml and 70.5% at 500,000 U/ml. Recombinant HuIFN-α2b treatment did not succeed to produce any significant plaque number reduction. However, significant plaque size reductions were observed following treatment with 100,000 U/ml, 250,000 U/ml and 500,000 U/ml with reductions of 56 %, 75.7% and 69% respectively. None of the high-dose treatments of either rHuIFN-α2b or rFeIFN-ω caused significant cellular toxicity in the MTT-Assay. Therefore, the antiviral activity demonstrated by both interferons is not attributable to an in vitro effect on the cellular viability of CRFK-cells. In agreement with previous authors, this study was able to demonstrated that rFeIFN-ω and rHuIFN-α2b have inhibitory effects on the replication of FHV-1. For rFeIFN-ω the antiviral effect is dose-dependent and could be reliably detected at high concentrations (> 50,000 U/ml) using both the plaque number and plaque size measurements. Treatment with high concentrations of rHuIFN-α2b also resulted in an antiviral effect, which was only detected at using the plaque size measure; there was no statistical evidence for a reduction in the plaque number measurement. The significantly smaller plaque sizes in drug–treated cell cultures indicate that high-dose treatment with rFeIFN-ω or rHuIFN-α2b may have potential efficacy on reducing the dimensions of FHV-1 induced cytopathic lesions. Treatment with rFeIFN-ω has shown more profound effects in antiviral activity compared to rHuIFN-α2b and, in contrast to rHuIFN-α2b, it also offers the advantage of a homologous compound. This is consistent with recently published results obtained on in vivo activity, which have demonstrated that high-dose treatment of rFeIFN-ω shows good antiviral efficacy and clinical improvement (VERNEUIL, 2004; BRAECKLEIN et al., 2003). Therefore, there are indications that rFeIFN-ω may provide effective prophylactic and therapeutic treatment for FHV-1 infected cats.

SUMMARY Immunological monitoring in the context of gene therapy of feline fibrosarcomas Cytokine kinetics and humoral response to vector and transgene In the context of an adenoviral gene therapy of the fibrosarcoma of the cat we introduced the human interleukin 2 (huIL 2)-gene and the feline Interferon g (feIFN g)-gene, one or the other or a combination of both. The aim of these studies is to evaluate the toxic risk and the therapeutic potential of multiple injections of two adenoviral vectors (type 5) expressing immunostimulating genes as adjuvant therapy in cats with spontaneous cancers. Several immunological parameters such as cytokine concentration, antibodies of different classes against vector and transgene and their neutralizing capacity were examined in the thesis at hand in connexion with the questions of adenoviruses in cats, pre-existing immunity and influence of inflammatory context and way of application. The kinetic of the production of IL 2 in different therapeutic approaches was evaluated. A dose-dependency was shown. The levels found in the group treated with a combination of both genes were lower than in the group treated with just AdhuIL 2. Prospects of explications are the shutdown of the viral CMV-Promoter due to the presence of interferon, the rivalry of the two transgenes to transduce a cell and the antiproliferative and antiviral effect of IFN g which directly reduces vectors and infected cells and indirectly lowers the IL 2-production by this means. More than that the application mode played an important role: The plasmic cytokine concentration was more than doubled after intratumoral injection compared to a postoperative use to the tumour’s bed. The serum peak 24 hours after vector application, which quickly redescended, is no decisive factor for the course of cytokine concentration observed locally at the tumor site. Literature shows rather a consistent local secretion for a period of nine days with intensity ten times higher than measured in blood circulation. There are indices that the serum level of IL 2 the day after the first gene transfer may be predictive for the appearance of recidivism within the first year. The tendency of infected cells in?vitro to express more IL 2 without the presence of IFN g is the same. The possibility to dose feIFN g per ELISA is presently not given. Furthermore the development of anti-huIL 2-antibodies was surveyed for a period of one year following the gene-carrying vector application. The immunoglobulins appeared early in the group of treatment with IL 2 alone (day 30), and a tardily rise of Ig G was detected in the therapeutic group with association to IFN g (day 90 to 180). By measuring the existing antibodies capable to detect or to neutralize as well the used adenovirus in ELISAs and biological assays the influence of the transgene on the antiadenoviral immune response could be proofed as well as the influence of the lieu of administration in which the vector is applied. Antiviral Ig M showed up day 14 and Ig G day 30, but their occurrence was much higher in animals treated with interferon (solitary or in association). The neutralizing capacity reached high percentages even in elevated dilutions after just one week. Moreover the immunological response to the vector depends on the application mode in terms of considerably reduced neutralizing antibodies after intratumoral injection compared to the postoperative inoculation to the tumor-surrounding tissues. The parallel-lessened defence to the transgene in this case may be assumed. Altogether the described methods are convenient to ensure an immunological monitoring of veterinary patients during an adenoviral gene therapy and to document the reactions to the applied viral vector and the expressed transgene. RESUME Surveillance immunologique du chat lors de la thérapie génique du fibrosarcome félin Cinétique des cytokines et réponse humorale contre vecteur et transgène Dans le contexte d´une thérapie génique adenovirale du fibrosarcome du chat nous avons introduit les gènes de l´interleukine 2 humaine (huIL 2) ou de l´interféron g félin (feIFN g), seuls ou en combinaison. Le but de ces études est d’évaluer le risque toxique et le potentiel thérapeutique d´injections multiples de deux vecteurs adénoviraux (type 5), contenant des gènes immunostimulateurs, comme thérapie adjuvante dans des cancers spontanés des chats. Plusieurs paramètres immunologiques comme la concentration des cytokines, des anticorps de classes différentes contre le vecteur ou le transgène et leur capacité de neutralisation ont été examinés dans la thèse présentée en rapport avec la question d´adénovirus chez le chat pour déterminer l’impact de l´immunité anti-adénovirale préexistante, l´influence d´un contexte inflammatoire et le mode d´administration. La cinétique de la production d´IL 2 dans différentes approches thérapeutiques a été évaluée. Une relation dose-réponse a été démontrée. Les niveaux trouvés dans le groupe traité avec une combinaison de deux gènes étaient plus bas que dans le groupe traité avec AdhuIL 2 seul. Ces résultats peuvent être expliqués par la coupure du promoteur viral CMV due à la présence de l´interféron dans le tissu, la compétition des deux vecteurs à transgènes différents à transduire une cellule et l´effet antiproliferatif et antiviral de l´IFN g qui limite la persistance de l’infection et engendre de ce fait une baisse de la production d´IL 2. De plus, le mode d´administration joue un rôle important: La concentration de cytokines dans le sérum a plus que doublé après une injection intratumorale, contrairement à une application postopératoire dans le lit tumoral. Le pic de concentration de cytokines dans le sérum, qui apparaît 24 heures après l´administration du vecteur et qui disparaît très vite, n´est pas un facteur décisif pour l’évolution de la concentration de cytokine observée localement au site de la tumeur. La littérature montre plutôt une sécrétion locale continue pour une période de neuf jours, avec une intensité dix fois plus élevée que celle mesurée dans la circulation sanguine. Certaines indications montrent que le niveau d´IL 2 dans le sérum, un jour après le premier transfert génétique, peut refléter les risques d’une récidive au cours de la première année. KeywordsBitte tragen Sie hLa tendance des cellules infectées in?vitro d´exprimer plus d´IL 2 sans la présence d´IFN g est la même. La possibilité de doser feIFN g par ELISA n´est actuellement pas donnée. En outre l´ apparition d´anticorps anti-huIL 2 a été surveillée pendant un an, après administration du vecteur porteur des gènes. Ces immunoglobulines se produisent tôt dans le groupe de traitement avec l’IL 2 seul (jour 30), et une hausse tardive des Ig G a été détectée dans le groupe thérapeutique avec l´association d´IFN g (jour 90 à 180). En mesurant la quantité d’anticorps capables de détecter ou de neutraliser même l´adénovirus utilisé, par des tests d´ELISA et biologiques, l´influence du transgène sur la réponse immunitaire anti-adénovirale et aussi l´influence de l´endroit de l´application dans lesquels les vecteurs sont injectés ont été démontrées. Les Ig M antiviraux se manifestaient jour 14 et les Ig G jour 30, mais étaient, en l´occurrence, beaucoup plus forte chez des animaux traités avec l´interféron (seul ou en association). La capacité de neutralisation atteint des pourcentages élevés à des dilutions importantes après une semaine de traitement. De plus, la réponse immunologique contre le vecteur dépend de la voie d´ administration: la quantité d’anticorps neutralisants est nettement réduite après injection intratumorale, contrairement à une inoculation postopératoire dans les tissus autour de la tumeur. La défense contre le transgène peut être soupçonnée d´être diminuée en parallèle. En tout, les méthodes décrites sont adéquates pour garantir une surveillance immunologique des patients vétérinaires lors d´une thérapie génique adénovirale et pour témoigner les réactions au vecteur viral appliqué et au transgène exprimé.