[35] Microencapsulation of genetically engineered cells for cancer therapy

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generate highly purified vector for large-scale studies in large animals or clinical trials. In time this drawback will be rectified and amplicon vectors will move into the mainstream of the gene therapy repertoire. Acknowledgment This work was supported by NIH CA69246 and NIH CA99004.

[35] Microencapsulation of Genetically Engineered Cells for Cancer Therapy B y J . - M A T F H I A S LOHR, ROBERT SALLER, BRIAN SALMONS, a n d W A L T E R H . GUNZBURG

Introduction Gene therapy is usually regarded as the transfer of new genetic information into target cells with resulting therapeutic potential. However, in practice, this conventional definition is often widened to include forms of therapeutic vaccination and cell therapy in which nonautologous genetically modified cells are transplanted into a patient, rather than attempting to modify the patient's own cells. On a conceptual level, such an approach could offer major advantages over conventional gene therapy since it would allow the controlled placement of the production site of therapeutic molecules (viral vectors, antibodies, cytokines, other proteins) and prohibit spillover of the transgene into the general system (systemic circulation) of the host.1 However gene therapy in general and cancer gene therapy in particular are hampered by several obstacles including (1) the controlled expression of the therapeutic gene at the right time and place and (2), in the case of transplantation of genetically modified cells, the maintainance of the integrity of the transplanted cells and their protection from the host immune system. The first problem is generally tackled by searching for tissue or cell type-specific promoters and engineering them to control the therapeutic gene, usually in the context of a vector.2-6 The second problem has not yet been satisfyingly tackled and, depending I A. M. Sun, Ann. N.Y. Acad. Sci. 831, 271-279 (1997). 2 W. H. Gtinzburg and B. Salmons, Mol. Med. Today 1, 410 (1995). 3 K. W. Peng and S. J. Russell, Curr. Opin. Biotechnol. 10, 454 (1999). 4 R. J. Yanez and A. C. Porter, Gene Ther. 5, 149 (1998). 5 D. M. Nettelbeck, V. Jerome, and R. MUller, Trends Genet. 16, 174 (2000). 6 W. H. Gtinzburg and B. Salmons, J. Mol. Med. 74, 171 (1996).

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on the origin of the transplanted cells, they have recently been found to harbor additional risks such as the production of potentially pathogenic species-jumping viruses. 7 One solution to the problem of maintaining transplanted cells could be to gather the transfected cells in a distinct containment and, by the same token, shield the genetically engineered cells from the host immune system. Microencapsulation was envisioned more than 40 years ago, 8,9 however, because of the lack of biocompatible procedures for preparing microcapsules capable of containing viable cells, it took another 30 years before living cells (islets of Langerhans) could be encapsulated. 10 We have established a system to encapsulate genetically engineered cells expressing the 2B 1 isomer of the rat cytochrome p450 gene (CYP2B 1), which is capable of converting ifosfamide into its active compounds, phosporamide mustard and acrolein. 11 In contrast to the widely used alginate/L-polylysine system, 27 we utilized sodium cellulose sulfate (NaCS) as polyanion and poly(diallyldimethylammonium chloride) (PDADMAC) as polycation. 12 We have exploited this system for cancer gene therapy, 13'14 but it represents a basic technology for a whole variety of other clinical aplications. Pancreatic cancer served as our model system since it is a devastating disease 15 in which death is usually caused by the primary tumor, and even emerging therapeutic concepts 16'17have not yet contributed to a significant improvement of patient survival or quality of life. Chemotherapy is limited in its effectivity by (1) access to the tumor, (2) the degree of drug sensitivity of the tumor, and (3) local and systemic toxicity of the chemotherapeutic agent. In pancreatic carcinoma, chemotherapy is mainly delivered systemically, TM although recent developments include the placement of intraarterial catheters delivering the cytotoxic drugs into the celiac

7 W. H. Giinzburgand B. Salmons, Mol. Med. Today 6, 199 (2000). 8 T. M. S. Chang, "Hemoglobin Corpuscles." Research report for Honours Physiology, Medical Library, McGiil University,McGill UniversityPress, Montreal (1957). 9 T. M. S. Chang,Ann. N.Y. Acad. Sci. 831, 249 (1997). l0 E Lim and A. M. Sun, Science 210, 908 (1980). 11H. A. A. M. Dirven, B. van Ommen, and E J. van Blaederen, Chem. Res. Toxicol. 9, 351 (1996). 12n. Dautzenberg, U. Schuldt, G. Grasnik, P. M. E Karle, M. LShr, M. Pelegrin, M. Piechacyk, K. v. Rombs, W. H. Gtinzburg,B. Salmons, and R. M. Sailer, Ann. N.Y. Acad. Sci. 875, 46 (1999). 13M. L6hr, P. Mtiller, E Karle, J. Stange, S. Mitzner, H. Nizze, S. Liebe, B. Salmons, and W. H. Giinzburg, Gene Ther. 5, 1070 (1998). 14T. Kammert6ns,W. Gelbmann,P. Karle, B. Salmons, W. H. Gtinzburg,and W. Uckert, Cancer Gene Ther 7, 629 (2000). 15S. Rosewiczand B. Wiedenmann,Lancet 349, 485 (1997). 16W. H. Gtinzburgand B. Salmons, Trends Mol. Med. 7, 30 (2000). 17L. Rosenberg,Drugs 59, 1071 (2000). 18j. D. Ahlgren, Cancer 78, 654 (1996).

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trunk. 19-22 On the other hand, instillation of solid microspheres for chemoembolization is a routine procedure for hepatic masses not suitable for surgery. 23-25 With this background, we reasoned that local application of microencapsulated cells capable of locally converting ifosfamide to its active, antitumor metabolites might result in good antitumor efficacy. Since much of the systemically applied ifosfamide should be locally converted at the tumorsite, rather than relying, as in conventional chemotherapy, on conversion at the natural site of expression of the CYP2B 1 enzyme, the liver, followed by systemic distribution of the relatively short-lived antitumor metabolites, we expected to be able to reduce the dose of ifosfamide used, resulting in reduced systemic side effects, without losing antitumor efficacy. 29 For application in humans, a supraselective intraarterial instillation in tumor-vascularizing arteries seemed to provide a minimally invasive approach for the exact placement of encapsulated cells. Methodology and Results Vector Construct

The cDNA coding for the CYP2B 1 enzyme 26 was cloned into the plasmid pcDNA3 and the resulting expression plasmid pC3/2B1, in which the 2B 1 cytochrome isoform is expressed from a linked cytomegalovirus promoter, 31 is amplified in Escherichia coli. Preparation was performed using the Maxi Preparation Kit and the Pyrogen Extraction Kit (both Qiagen, Germany). Sequencing confirmed the integrity of the insertion and of the plasmid vector DNA. Subsequent preparations were quantified by photometric and agarose gel analysis, and identity was confirmed by restriction enzyme digestion. Plasmid pC3/2B 1, which carries a neomycin (G418) resistance gene, was transferred into qualified (tested free of a panel of viruses and other adventitious agents,

19R. Kawasaki, S. Morita, Y. Noda, and A. Tsuji, Cardiovasc. lnterventional Radiol. 21, 152 (1998). 2oO. Ishikawa, H. Ohigashi, Y. Sasaki, K. Masao, T. Kabuto, H. Furukawa, and S. Imaoka, Hepatogastroenterology 45, 644 (1998). 21 C. A. Maurer, M. M. Bomer, J. Lauffer, H. Friess, K. Z'graggen, J. Triller, and M. W. Buchler, Int. J. Pancreatol. 23, 181 (1998). 22E K. Wacker, J. Boese-Landgraf,A. Wagner, D. Albrecht, K. J. Wolf, and F. Fobbe, Cardiovasc. lntervent. Radiol. 20, 128 (1997). 23p. Berghammer, E Pfeffel, E Winkelbauer, C. Wiltschke, T. Sehenk, J. Lammer, C. Muller, and C. Zielinski, Cardiovasc. Intervent. Radiol. 21, 214 (1998). 24E Florio, M. Nardella, S. Balzano, E. Caturelli, D. Siena, and M. Cammisa, Cardiovasc. Intervent. Radiol. 20, 23 (1997). 25Grouped'Etude et de Traitement du CarcinomeHepatocellulaire,N. Engl. J. Med. 332, 1256(1995). 26K. M. Kedzie, C. A. Balfour, G. Y. Escobar, S. W. Grimm, Y. A. He, D. J. Pepped, J. W. Regan, J. C. Stevens, and J. R. Halpert, J. Biol. Chem. 266, 22515 (1991).

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Q-One, Glasgow, UK) 293 cells (human embryonic kidney cell line; ATCC) by transfection using a Pharmacia Transfection Kit according to the manufacturer's instructions. Subsequently the cells were grown for 2 days and then selected for G418 resistance. Resistant clones were isolated and tested for the presence and activity of the vector. The expression of biologically active CYP2B 1 in the transfectants was determined using a biochemical assay, which is specific for the cytochrome P450 isoforms 1A1 and 2B 1.27 In this assay 7-pentoxy resorufin is converted by CYP2B 1 into resorufin. The amount of produced resorufin was measured with a fluorometer at 530 nm excitation and 590 nm emission.13 A standard curve was produced using different amounts of purified resorufin (Sigma). The cell clone 22P1G, 28 which showed the highest enzymatic activity, was chosen for further experimentation. Encapsulation

For encapsulation, CYP2B 1 transfected 22P1G cells are suspended in sodium cellulose sulfate (NaCS) or phosphate-buffered saline (PBS, pH 7) containing 2-5% NaCS (Fig. 1A) depending on the degree of sulfation (polyanionic solution). The suspension is passed through an adjustable droplet generation system (Inotech AG, Switzerland). The cell-containing droplets eventually pass into a precipitation bath containing 3-4% polydiallyldimethylammonium (PDADMAC; Fig. 1B) in NaC1 depending on the concentration of the NaCS (polycationic solution). On contact of the polyanion with the polycationic solution, a polyelectrolyte complex starts forming producing a capsule membrane, which forms from the outside toward the center (Fig. 2A). Histological sectioning of the capsules followed by microscopic analysis reveal that the cells are distributed throughout the capsule, however they are more concentrated in the center, while the polymerized cellulose sulfate is concentrated toward the surface of the capsule (Fig 2B). The capsules are then washed twice with saline before they are stored at 4°. 13 Survival of cells is measured according to the manufacturer's recommendations with the two-color LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes), which shows a green fluorescence for living cells and a red fluorescence for dead cells in confocal laser microscopy (Fig. 2C). The capsules had an average diameter of 0.7-0.85. They can withstand considerable physical pressure and are able to withstand delivery through an angiography catheter. In vitro and in vivo monitoring revealed that the capsules are flexible enough to take different shapes while remaining intact. Preparations of capsules were tested in several ways. One experiment consisted of forcing capsules through different diameters of needles attached to a syringe. The capsules 27V. Kurowskiand T. Wagner,Cancer Chemother. PharmacoL 33, 36 (1993). 28W.H. Gtinzburg,P. Karle,R. Renz,B. Salmons,and M. Renner,Ann. N. Y Acad. Sci. 880,326 (1999).

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(A) H 13

HOCHz

.

\

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(pD Aliquote and store at 4°C FIG. 1. Production of microcapsules. (A) Chemical structure of the polyelectrolytes. Sodium cellulose sulfate (NaCS; upper structure) and poly(diallyldimethylammonium chloride) (PDADMAC; lower structure). (B) Schematic of the encapsulation process. A suspension of a minimum of 3 x 108 cells is mixed with NaCs in a loading vessel and injected into the vibrating nozzle of an encapsulation machine. The resultant droplets pass into a polymerization solution of PDADMAC where they solidify and then are washed and aliquotted.

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(B) (c)

FIG. 2. Capsules and encapsulated cells in vitro. (A) Phase contrast microscopy of capsules lacking cells (empty) with a diameter of ~0.7 mm. (B) H&E staining of a section of encapsulated cells. (C) LIVE/DEAD assay of encapsulated cells in confocal laser microscopy 24 hr after encapsulation demonstrating that most of the cells retain viability (white cells).

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could pass through needle diameters down to 15-gauge without demonstrating leakage of cells in subsequent tissue culture. Furthermore, capsules were instilled into angiography catheters in isotonic contrast medium (Visipaque) to test the pressure that resulted after loading up to 1000 capsules into a standard 2.3F/140 cm angiography catheter (Cordis). Capsules could be easily pushed by hand through the catheter. The capsules were returned to tissue culture after this experiment. They retained integrity, i.e., no outgrowth of cells could be observed. The cells remained viable (LIVE/DEAD assay) at the same level as prior to the experiment. The same was true for the enzyme activity (resorufin assay). Animal Models

Initial studies were aimed at detection of immediate or delayed toxic effects in rodents. In order to assess acute toxicity, ground capsule material was injected at a concentration of 2000 mg/kg body weight into 10 immunocompetent mice. Animals were followed for 14 days; however, no toxicity was observed in any of the animals. Chronic toxicity studies were carried out in rabbits, which were injected intraperitoneally with up to 10 times the planned clinical dose. The animals were monitored for weight loss, fever, or any abnormal behavior, but no toxicity could be detected, even at the histological level, after a period of 3 months, indicating that the capsule material is well tolerated over the long term. To ascertain the tolerance to the capsule material at the planned site of clinical application, empty capsules were injected orthotopically into the pancreas of both nude and immunocompetent mice. 29 A mild foreign body reaction was seen surrounding the capsules; however, no systemic reaction or granuloma formation could be observed3° (Fig. 3A). The antitumor effect of the administration of microencapsulated CYP2B1producing cells followed by systemic ifosfamide application was investigated using encapsulated CYP2B l-expressing feline kidney cells and has been reported previously.31 Briefly, a suspension of 1 x 106 PANC-1 cells was injected subcutaneously into athymic nude mice.31 Once tumors reached a volume of 1 cm 3, capsule implantation took place. The capsules were carefully aspirated from a container with normal medium without supplements or fetal calf serum into a 1 ml syringe (insulin syringe; Braun, Melsungen, Germany) without a needle attached to it. Twenty to 40 capsules were injected into the preformed tumor through a 21-gauge needle (Microlance 3, inner diameter 0.6 mm) that was shortened to a 1 cm needle length, resharpened, and sterilized. Animals were then treated with low-dose ifosfamide and MESNA (both at 100 mg/kg) i.p. every 3 days for 2 weeks. 13 This 29 X. Fu, E Guadagni, and R. M. Hoffman, Proe. Natl. Acad. Sci. U.S.A. 89, 5645 (1992). 30 p. Miiller, R. Jesnowski, P. Karle, R. Renz, R. Sailer, H. Stein, K. Ptischel, K. von Rombs, H. Nizze, S. Liebe, T. Wagner, W. Gtinzburg, B. Salmons, and M. LOhr, Ann. N.Y. Acad. Sci. 880, 337 (1999). 31 M. IdShr, B. Trautmann, M. G/Sttler, S. Peters, I. Zauner, B. Maillet, and G. K18ppel, Br. J. Cancer 69, 144 (1994).

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FIG. 3. Capsules and encapsulated cells in vivo. (A) Mouse pancreas 1 week after implantation of an empty capsule (H&E stain). (B) Gross appearanceof xenotransplanted humanpancreatic carcinoma on nude mousewithouttreatment (left), partial remission(middle), and completeremission (right) after instillation of CYP2B1-expressing, microencapsulated cells and ifosfamide treatment for 3 weeks. resulted in a complete remission of the established tumors in almost 20% of the animals and partial remission in another 50% 13 (Fig. 3B). In order to quantitate the added value of the local conversion of ifosfamide, the former experiment was repeated, but this time 40 capsules were injected on one side of the tumor only, which was marked. Animals were then treated with a single dose of ifosfamide (100 mg/kg). After 30 min, i.e., reaching the plateau phase in blood plasma (Fig. 4A), animals were anesthetized and tumor tissue was removed in three parts: side of the capsule implantation, middle portion, and the side opposite to the capsule implantation. Ifosfamide and its active metabolites were measured in the snap-frozen tissue as well as in blood plasma. Although the levels of ifosfamide were the same at all three sites (Fig. 4B), 4-hydroxyifosfamide was at least two times higher where the capsules were implanted (Fig. 4C).

[35]

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Plasma levels after i.p, application of Ifosfamlde

45O 400 35O 3OO

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FIG. 4. Pharmacokinetics of ifosfamide in xenografted human pancreatic carcinomas on the nude mouse. (A) Plasma levels of ifosfamide in three groups of animals carrying tumors and having received ifosfamide only (ifo alone II), naked CYP2B 1-expressing cells (CYP2B 1+ifo A), or microencapsulated CYP2B 1-expressing cells (CapCells +ifo 0). (B) Tissue levels of ifosfamide for the three groups described in panel A. In all groups, the levels are about the same. (C) Tissue levels of the metabolized, activated ifosfamide, i.e., 4-hydroxyifosfarnide, in the three groups. The levels are almost three times higher in the group treated with the encapsulated CYP2B1 cells and 50% higher at the site of the capsule implantation.

612

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(c)

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Adolescent pigs (mean age 90 days, mean weight 46 kg) were kept fasting overnight. Capsules were produced as described above. 13'32'33After encapsulation, the capsules were washed, aliquotted at 100 capsules per vial, and kept at 4 ° until further use. A parallel sample was assessed for viability and enzyme activity as described. 32 For angiography, animals were sedated and placed in a supine position on the angiography table. Animals were intubated and ventilated. The femoral artery was punctured after open preparation with a 20-gauge angiography needle. A 4F introducer system (Terumo) was placed in by the Seldinger technique. 34 Under fluoroscopy, the celiac trunc was catheterized with a 4F Cobra 2 guiding catheter (Cordis). Supraselective cannulation was achieved by further advancement of the guidewire and a coaxial 2.3F microcatheter system (Cordis). In general, the splenic lobe was cannulated (Fig. 5A). Initial angiography was performed with Visipaque 270 (Nycomed). After positive placement of the catheter in the main vessel leading into the splenic lobe of the pancreas, 100 capsules were instilled slowly. At the end, control angiography was performed. The introductory set was removed, the wound closed, and the animals were allowed to 32E Karle, E M~iller, R. Sailer, K. von Rombs, R. Renz, H. Nizze, S. Liebe, W. H. Gtinzburg, B. Salmons, and M. Lfhr, Adv. Exp. Med. Biol. 451, 97 (1998). 33j. Stange, S. Mitzner, H. Dautzenberg, W. Ramlow,M. Knippel, M. Steiner, B. Ernst, R. Schmidt, and H. Klinkmann, Biomat. Art. Cells. lmmob. Biotech. 21, 343 (1993). 34j. C. Kr6ger, H. Bergmeister, A. Hoffmeyer,M. Ceijna, E Katie, R. Sailer, I. Schwendenwein, K. von Rombs, S. Liebe, W. H. Giinzburg,B. Salmons, K. Hauenstein, U. Losert, and M. Ltihr,Ann. N.Y. Acad. Sci. 880, 374 (1999).

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(A)

(B)

FIG. 5. Preclinical feasibility studies for angiographic access in the pig. (A) Supraselective angiography of the splenic lobe of the pig pancreas. (B) Histological appearance of the intra-arterially instilled microcapsules (H&E stain).

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wake. Animals were monitored clinically and by lab tests for a week. During the first 24 hr, the pigs were kept NPO (nothing per os) but received saline infusions (500 ml/12 hr). A total of 18 animals were investigated. In all animals, it was possible to cannulate both the splenic and the duodenal lobe arteries selectively. In all animals, instillation of capsules was successful. Manipulation with the guidewire, injection of contrast dye, and instillation of capsules resulted in a significant and prolonged vascular spasm that resolved spontaneously within 6 to 15 minutes. None of the animals developed pancreatic symptoms, but the first animal experienced slight tenderness of the abdomen. The amylase levels in all animals remained within normal limits. At the end of the observation period, another control angiography was performed. In addition, ifosfamide was administered systemically at low dosage (1 g/m2). Finally, the animals were sacrificed and the pancreas and other inner organs were removed. There was no visible macroscopic or microscopic damage to the pancreas. The capsules were found in capillaries, partly in conjunction with thrombotic material but never occluding the entire vessel (Fig. 5B). Studies in Humans

A nonrandomized, phase I/II study was designed for patients with advancedstage pancreatic carcinoma not suitable for curative surgery.29 The study was approved by the state Ethics Committee, the Somatic Gene Therapy Commission of the German Federal Medical Association (KSG-BAK), the Working Party for Oncology (AGO) of the German Society for Gastroenterology (DGVS), and the German Working Party for Gene Therapy (DAG-GT). For the clinical study, the entire process of culturing the cells carrying the therapeutic gene and the encapsulation process was transferred to a contract research organization to ensure compliance with GCP-GCH guidelines. The cells were cultured under constant GMP conditions. After passing the requested quality assurance tests, the capsules were released for clinical use (Fig. 6A). The selected patients underwent angiography after giving informed consent. Angiography for celiac trunc visualization was performed in a standard manner via the femoral route. A 4F introducer system (Terumo) was placed by the Seldinger technique. Under fluoroscopy, the celiac trunk and splenic and common hepatic arteries, as well as the superior mesenteric artery (SMA), were visualized with Visipaque 270 (Nycomed). Relating the localization of the tumor in the CAT scan to the vessel anatomy, the most appropriate access to the tumor was determined. Supraselective catheterization of the transversal artery was performed with a coaxial 2.3F microcatheter system (Cordis). 34 After documentation of the correct placement, 300 microcapsules were slowly instilled through the catheter one by one. For this procedure, a translucent Luer-lock connector on the catheter is mandatory (Fig. 6B). To have best control over the capsule instillation, 1-ml insulin syringes were utilized. The capsule instillation was followed by another control

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angiography and documentation of the correct catheter placement (Fig. 6C). Finally, the catheters and the introducer set were withdrawn. Patients rested for 48 hr. During this period, the patient was monitored clinically by follow-up lab tests and abdominal ultrasound in order to detect any abnormality in the upper abdomen, e.g., pancreatitis or ischemia. Patients received ifosfamide at 1 g/m z body surface for three consecutive days (day 2-4). This regimen was repeated starting at day 22. The application

(A)

FIG.6. Clinical study in humans. (A) Aspirationof capsules with an insulin syringe from a container from a clinical batch. Capsules freely float in the syringe. (B) Instillation of the capsules into a 2.3F microcatheter. Note the translucent adaptor piece of the catheter allowing the visual control of the capsule instillation (arrow). (C) Supraselective angiography of the arteria transversalis (branch of the A. pancreatoduodenalis).

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(B)

(c)

FIG. 6. (continued)

of ifosfamide was accompanied by monitoring of the drug and its metabolites. Routine laboratory monitoring according to oncological standards was performed during the entire period. Patients were followed up for a total o f 5 months at least. A total of 14 patients were treated according to protocol. The procedure was well tolerated. None of the patientsdeveloped any signs o f allergic reaction or pancreatitis. The treatment resulted in two partial remissions and stabilization in the remainder of the patients. The survival time was extended from 25 months (recent control group at study center) to 44 months. 35 35M. LOhr,A. Hoffmeyer,J. C. Kr/Jger,M. Freund, J. Hain, A. Holle, E Karle, W. T. Knffel, S. Liebe, E MUller,H. Nizze, M. Renner, R. M. Sailer, B. Salmons, T. Wagner, K. Hanenstein, and W. H. Gtinzburg,Lancet 357, 1591 (2001).

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Discussion With the series of experiments described, we could demonstrate positive proof for the use of genetically engineered, microencapsulated cells in cancer therapy with pancreatic carcinoma as a model system. Although several gene therapy approaches have been described in experimental pancreatic c a n c e r , 36-4° this is one of the very few gene/cell therapy protocols that reached clinical trial. 16 In animal models, encapsulation using biodegradable polylactic acid microspheres releasing recombinant interleukin-12 (IL-12) has been used for in situ tumor immunotherapy leading to tumor reduction. 41 However, this was a cell-free system relying on a relatively short-term release of IL-12. The microcapsules described in this paper have also been shown to be capable of secreting antibodies produced by encapsulated hybridoma cells42 even resulting in protection from a virally induced disease 43 and could be used to deliver other biomolecules. Another new and promising delivery technique encompasses the application of gene activated matrix (GAM) directly to target tissue. 44 Although this is feasable for superficial sites such as wounds, it does not allow delivery to inner parts of the body. The general route of in vivo delivery of therapeutic agents, including gene therapy reagents, is direct injection into a body compartment (e.g., abdominal cavity) or into a peripheral vein. In addition, catheter-directed gene delivery has been investigated with application of adenoviral vectors to swine lung arteries demonstrating reporter gene for several weeks. 45 The vascular delivery of genetic vectors for prevention of restenosis and coronary angiogenesis has used a wide range of devices and m e t h o d s . 46-48 Although gene transfer has been demonstrated

36 K. Makinen, S. Loimas, J. Wahlfors, E. Alhava, and J. Janne, J. Gene. Med. 2, 361 (2000). 37 K. Makinen, S. Loimas, V. M. Kosma, J. Wahlfors, S. Yla-Herttuala, E. Alhava, and J. Janne, Ann. Chir. Gynaecol. 89, 99 (2000). 38 H. Kijima and K. J. Scanlon, Mol. Biotechnol. 14, 59 (2000). 39 D. Evoy, E. A. Hirschowitz, H. A. Naama, X. K. Li, R. G. Crystal, J. M. Daly, and M. D. Lieberman, J. Surg. Res. 69, 226 (1997). 4o j. M. DiMaio, B. M. Clary, D. E Via, E. Coveney, T. N. Pappas, and H. K. Lyerly, Surgery 116, 205 (1994). 41 N. K. Egilmez, Y. S. Jong, M. S. Sabel, J. S. Jacob, E. Mathiowitz, and R. B. Bankert, Cancer Res. 60, 3832 (2000). 42 M. Pelegrin, M. Matin, D. Noel, M. Del Rio, R. Sailer, S. Stange, S. Mitzner, W. H. Giinzburg, and M. Piechaczyk, Gene Ther. 5, 828 (1998). 43 M. Pelegrin, M. Matin, A. Oates, D. Noel, R. Sailer, B. Salmons, and M. Piechaczyk, Hum. Gene Ther. 11, 1407 (2000). 44 j. Bonadio, J. Mol. Med. 78, 303 (2000). 45 S. Badran, S. K. Schachtner, H. S. Baldwin, and J. J. Rome, Hum. Gene Ther. 11, 1113 (2000). 46 M. Simons, R. O. Bonow, J. Chrono, H. K. Hammond, R. J. Laham, W. Li, M. Pike, E W. Sellke, T. J. Stegmann, J. E. Udelson, and T. K. Rosengart, Circulation 102, e73 (2000). 47 S. Nikol, T. Huehns, E. Kransz, S. Esin, M. G. Engelmann, D. Winder, B. Salmons, and B. HSfling, Gene Ther. 6, 737 (1999).

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for each device,49 most studies of catheter-based gene transfer have revealed low efficiency except in the context of prolonged vessel occlusion with ligated branches or a double-balloon catheter. 5° However, this concept is not applicable to anything else but a blood vessel, certainly not for cancer gene therapy of tumors in solid organs. Here, the placement of cells contained in a microcapsule represents a definite advantage. Outlook The capsule technology employed here, though already mature, has still considerable room for optimization. Furthermore, application of capsules containing CYP2B1 expressing cells in conjunction with ifosfamide, as used in our experiments, may prove effective for other cancers, e.g., peritoneal metastasis/malignant ascites, soft tissue sarcoma, and hepatic masses (primary and metastatic). Acknowledgments This work was supported by the Danish Government (V~estfond), a project Grant by Bavarian Nordic A/S, Copenhagen, Denmark, the Minister of Science and Education, Berlin, Germany (bmbf BOE 21--03113673), and EC Grant BIO4-CT-0100. This work is dedicated to our patients with pancreatic carcinoma who joined our inaugural study, and to Professor Horst Dauzenberg, the entrepreneur of capsule technology, who died much too early. The work with the capsules owes much to many investigators who contributed substantially to the many aspects of this project. Their input is gratefully acknowledged: Zoltan Bago, Stephan Benz, Mathias Freund, Johannes Hain, Karlheinz Hauenstein, Anne Hoffmeyer, Ralf Jesnowski, Jens Krtger, Karle, Stefan Liebe, Udo Losert, Petra Miiller, Steffen Mitzner, Horst Nizze, Alexander Probst, Matthias Renner, Regina Renz, Jan Stange, Hartmut Stein, Kerstin yon Rombs, Thomas Wagner, and Inge Walter. We also thank Asger Amund and Peter Wulff for their encouragement and continuous support.

48 T. Huehns, E. Krausz, S. Mrochen, M. Schmid, S. Esin, M. G. Engelmann, E K. Schrittenloher, B. HOfling, W. H. Giinzburg, and S. Nikol, Atherosclerosis 144, 135 (1999). 49 S. Baek and K. L. March, Circ. Res. 82, 295 (1998). 50 j. j. Rome, V. Shayani, K. D. Newman, S. Farrell, S. W. Lee, R. Virmani, and D. A. Dichek, Hum. Gene Ther. 5, 1249 (1994).