Design of an implantable active microport system for patient specific drug release.

We present a novel concept of an implantable active microport based on micro technology that incorporates a high-resolution volumetric dosing unit and a drug reservoir into the space of a conventional subcutaneous port. The controlled release of small drug volumes from such an "active microport" is crucial e.g. for innovative methods in cancer treatment or pain therapy. Our microport system delivers a flow rate in the range of 10-1,000 mul/h and enables a patient-specific release profile. The core of our device is a two-stage piezoelectric micropump. It features a backpressure-independent volumetric dosing capability i.e. a stable flow rate is ensured up to a backpressure of 30 kPa. The stroke volume and hence the resolution of the mircopump is voltage controlled and can be preset between 10 and 200 nl. A miniaturized high-performance electronic control unit enables freely programmable dosing profiles. This electronic circuit is optimized for both energy consumption and weight which are both essential for a portable device. The data of an implemented pressure sensor are used to permanently monitor the dosing process and to detect a potential catheter occlusion. A polyurethane soft lithography process is introduced for the fabrication of the prototype. Therewith, a compact multilayer system has been developed which measures only 50 x 35 x 25 mm(3).

A new liposomal formulation of Gemcitabine is active in an orthotopic mouse model of pancreatic cancer accessible to bioluminescence imaging.

Despite its rapid enzymatic inactivation and therefore limited activity in vivo, Gemcitabine is the standard drug for pancreatic cancer treatment. To protect the drug, and achieve passive tumor targeting, we developed a liposomal formulation of Gemcitabine, GemLip (Ø: 36 nm: 47% entrapment). Its anti-tumoral activity was tested on MIA PaCa-2 cells growing orthotopically in nude mice. Bioluminescence measurement mediated by the stable integration of the luciferase gene was employed to randomize the mice, and monitor tumor growth. GemLip (4 and 8 mg/kg), Gemcitabine (240 mg/kg), and empty liposomes (equivalent to 8 mg/kg GemLip) were injected intravenously once weekly for 5 weeks. GemLip (8 mg/kg) stopped tumor growth, as measured via in vivo bioluminescence, reducing the primary tumor size by 68% (SD +/- 8%; p < 0.02), whereas Gemcitabine hardly affected tumor size (-7%; +/- 1.5%). In 80% of animals, luciferase activity in the liver indicated the presence of metastases. All treatments, including the empty liposomes, reduced the metastatic burden. Thus, GemLip shows promising antitumoral activity in this model. Surprisingly, empty liposomes attenuate the spread of metastases similar to Gemcitabine and GemLip. Further, luciferase marked tumor cells are a powerful tool to observe tumor growth in vivo, and to detect and quantify metastases.

Cell fusion: an approach to generating constitutively proliferating human tumor antigen-presenting cells.

Somatic cell hybrids of HLA-A2(+) EBV-transformed B- or dendritic cells (DC) and allogeneic HLA-A2(-) melanoma cell line Me15 were obtained by in vitro electrofusion using an electroporator. Before fusion, melanoma cells were stably transfected with green fluorescent marker protein (GFP) and neomycin resistance gene (neo(+)). Stably growing hybrid antigen-presenting cells (HAPC) expressing HLA-DR and HLA-A2 (or HLA-A30/31), and melanoma-associated antigens (MART-1, gp100) were selected by a double strategy of immunomagnetic MACS and neomycin selection. Fusion efficiency ranged between 3% and 18% (mean: 8.0+/-4.7%) as defined by simultaneous GFP and HLA-A2 detection. Expression of melanoma-associated antigens (MART-1, gp100) in hybrid cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). HLA-restricted antigen-specific presentation of melanoma antigens was demonstrated by killing of semi-allogenic HAPC by HLA-A2-restricted MART-1 or gp100-specific cytotoxic T lymphocyte (CTL) clones. HLA restriction and antigen specificity were confirmed by inhibition of specific cytotoxicity by anti-HLA antibodies and cold target inhibition. During long-term (42-70 days) neomycin selection of HAPC, a drastic loss of antigen-presenting cell (APC)-derived determinants (e.g. HLA-DR, HLA-A2) was observed which, however, could be "reversed" by repeated MACSorting (days 10, 21 and 49). Our method allows the generation of semi-allogenic HAPC that constitutively proliferate in vitro. This opens the possibility of establishing a number of tumor-APC hybrids expressing defined HLA haplotypes and tumor antigens, of investigating their specific properties (e.g. antigen processing), and testing their diagnostic or therapeutic potential.

Phase I clinical trial with IL-2-transfected xenogeneic cells administered in subcutaneous metastatic tumours: clinical and immunological findings.

Various studies have emphasized an immunodepression state observed at the tumour site. To reverse this defect and based upon animal studies, we initiated a phase I clinical trial of gene therapy in which various doses of xenogeneic monkey fibroblasts (Vero cells) genetically engineered to produce human IL-2 were administered intratumorally in 8 patients with metastatic solid tumours. No severe adverse effect was observed in the 8 patients analysed during this clinical trial even in the highest dose (5 yen 107 cells) group. This absence of toxicity seems to be associated with rapid elimination of Vero-IL-2 cells from the organism. Indeed, exogenous IL-2 mRNA could no longer be detected in the peripheral whole blood 48 hours after Vero-IL-2 cell administration. In addition, we did not find any expression of exogenous IL-2 mRNA in post-therapeutic lesions removed 29 days after the start of therapy. A major finding of this trial concerns the two histological responses of two treated subcutaneous nodules not associated with an apparent clinical response. The relationship between local treatment and tumour regression was supported by replacement of tumour cells by inflammatory cells in regressing lesions and marked induction of T and natural killer cell derived cytokines (IL-2, IL-4, IFNg ...) in post-therapeutic lesions analysed 28 days after the start of Vero-IL-2 administration. Gene therapy using xenogeneic cells as vehicle may therefore present certain advantages over other vectors, such as its complete absence of toxicity. Furthermore, the in vivo biological effect of immunostimulatory genes, i.e IL-2-, may be potentiated by the xenogeneic rejection reaction.

Locally inducible CD66a (CEACAM1) as an amplifier of the human intestinal T cell response.

CD66a is an adhesion molecule member of the carcinoembryonic antigen immunoglobulin-like family present on the surface of epithelial cells, granulocytes and IL-2 activated T cells. We studied whether CD66a is expressed in vivo by T lymphocytes and whether it affects TCR-mediated activation. CD66a was detected by histochemistry, flow cytometry analysis, reverse transcription PCR and Western blot on fresh colon biopsies and T cell clones. A fraction of T cells in the lamina propria express CD66a, which is induced by IL-7 and IL-15 cytokines. T cells express four different CD66a splice variants and at least two forms of the protein are glycosylated in a cell type-specific manner. Triggering of CD66a on T cells with physiological ligands or with specific mAb increases TCR-mediated lymphokine release, in an antigen dose-independent manner. This effect requires the presence of the CD66a intracytoplasmic domain, which contains two immunoglobulin receptor family tyrosine-based inhibitory motif-like domains, as shown by stimulation of Jurkat cells transfected with different CD66a isoforms and is associated with increased induction of AP1 and NFkappaB transcription factors. These data indicate that CD66a amplifies T cell activation and thus could facilitate crosstalk between epithelial cells and T lymphocytes in intestinal immune response.

Synovial PMN show a coordinated up-regulation of CD66 molecules.

Changes in the expression of various activation-dependent surface markers have been reported for polymorphonuclear neutrophils (PMN) isolated from synovial fluid of patients with inflammatory joint diseases. We extend these findings to the expression of CD66 molecules and several other surface markers. Three members of the CD66 family, namely CD66a, CD66b, and CD66c, showed an up to fourfold up-regulation on synovial fluid PMN compared with peripheral blood PMN (PBG) of the same patients; CD59 was increased twofold, the expression of CD16 did not change, whereas CD62L was reduced by more than 50% on synovial fluid PMN. It is interesting that CD66a, CD66b, and CD66c showed a coordinated expression on PBG of patients and controls and a coordinated up-regulation on synovial neutrophils. In contrast, after in vitro stimulation of peripheral blood PMN with phorbol myristate acetate, CD66c was much less up-regulated compared with CD66a and CD66b. All samples of synovial fluid PMN exhibited an additional increase in the expression of CD66a, CD66b, and CD66c when stimulated with phorbol myristate acetate in vitro. Prostaglandins are known to inhibit various responses of neutrophils to inflammatory stimuli. We could show that prostaglandins inhibit N-formyl-methionyl-leucyl-phenylalanine-induced up-regulation of CD66 on peripheral blood PMN in a concentration-dependent manner.

Gene therapy with cytokine-transfected xenogenic cells (Vero-IL-2) in patients with metastatic solid tumors: mechanism(s) of elimination of the transgene-carrying cells.

Eleven patients with advanced cancer were treated in a clinical gene therapy trial by repeated intra- tumoral injections with different doses of xenogenic fibroblasts secreting high amounts of human interleukin-2 (Vero-IL2). Treatments in a total of 14 courses were well tolerated and resulted in clinical responses and measurable biological effects. Together with increases in serum interleukin-2 (IL-2), modifications of the V-beta T cell receptor repertoire and induction of intratumoral T-cell infiltration were observed. When the intratumoral expression of endogenous cytokine genes and the persistence of the IL-2 transgene at the application site and in peripheral blood were investigated, rapid disappearance of the transgene at the application site appeared to be the most prominent biological effect. Tests detecting a single Vero-IL2 cell against a background of 10(5) non-transfected cells were not able to demonstrate significant expression of exogenous IL-2 (i.e. the transgene or transgene-carrying cells) in tumor biopsies or blood at different times. Therefore, further studies were performed to evaluate the mechanism(s) involved in the rapid disappearance of xenogenic carrier cells in more detail. We show here that significant in vitro cytotoxicity against transgene-carrying Vero cells can be observed in peripheral blood of all the patients before treatment as well as in healthy controls. "Cold" target inhibition shows that significant killing of Vero-IL2 cells is mediated by natural killer (NK) cells. This was confirmed by showing that established CD3(-)/CD16(+)/CD56(+) peripheral blood NK cell clones kill both K562 and Vero-IL2 target cells. The failure of other mechanisms (complement, antibody-dependent cell cytotoxicity or cytotoxic T lymphocytes) to destroy xenogenic, histoincompatible Vero cells in vitro suggests that NK cells also might be responsible for the killing of Vero-IL2 in vivo and for the failure to detect the transgene at the application site. These results might also be of importance for some aspects of the current discussion of xenotransplantation.

Cancer gene and immunotherapy: recent developments.

Gene and immunotherapeutic approaches to treat human malignant tumors are reviewed. Special attention is given to the different strategies of cancer gene therapy and to recent aspects of cytokine-supported tumor immunotherapy or tumor-specific vaccination. The limitations of these therapy approaches are critically discussed especially with respect to immune escape mechanisms.

Gene therapy study of cytokine-transfected xenogeneic cells (Vero-interleukin-2) in patients with metastatic solid tumors.

On the basis of compelling preclinical data in cats and dogs, we initiated a clinical gene therapy study in nine patients with advanced solid tumors using xenogeneic fibroblasts secreting human interleukin (IL)-2 (Vero-IL-2 cells). Cohorts of three successive patients with tumors accessible to computed tomography- or ultrasound-guided injection were treated repeatedly with 5 x 10(5), 5 x 10(6), or 5 x 10(7) Vero-IL-2 cells. The endpoints of the study were feasibility, toxicity, and the clinical and biological effects of this novel approach to immunotherapy of cancer. Histopathological, immunological, and molecular analyses were performed on biopsy specimens of tumors and blood samples before, during, and after treatment. Treatment was well tolerated, and toxicity consisted of transient fever in one patient and short-lived, mild itching and erythema in two others. One patient with soft-tissue sarcoma showed a reduction of >90% and >50% of the volume of two distant, noninjected metastases, lasting for 29+ and 26 months, respectively. Four other patients showed stabilization of their disease for 3-9 months; of these patients, one with melanoma developed marked vitiligo. We conclude that repeated injections of < or =5 x 10(7) Vero-IL-2 cells are feasible and safe in heavily pretreated patients with advanced solid tumors. An additional evaluation of an intratumoral application of Vero-IL-2 seems warranted.

Gene therapy with cytokine-transfected xenogeneic cells in metastatic tumors.

On the basis of compelling preclinical data in cats and dogs we initiated a clinical gene therapy study in nine patients with advanced solid tumors using xenogeneic fibroblasts secreting human IL-2 (Vero-IL-2 cells). Cohorts of three successive patients with tumors accessible to CT- or ultrasound-guided injection were treated repeatedly with 5 x 10(5), 5 x 10(6), or 5 x 10(7) Vero-IL-2 cells. Endpoints of the study were feasibility, toxicity, and clinical and biological effects of this novel approach to immunotherapy of cancer. Histopathological, immunological and molecular analyses were performed on biopsy specimens of tumors and blood samples from before, during and after treatment. Low levels of serum antibodies to Vero cells developed in 2/9 patients. Analysis of tumor biopsies showed increased expression of CD3 mRNA and enhanced tumor infiltration with varying lymphocyte subpopulations after treatment. In addition, monoclonal alterations of the TCR repertoire of blood and tumor lymphocytes were observed. Treatment was well tolerated and toxicity consisted of transient fever in one patient and short-lived, mild itching and erythema in two others. One patient with soft tissue sarcoma showed a more than 90% and more than 50% reduction of the volume of two distant, non-injected metastases, respectively, lasting for 22+ months. Four other patients showed stabilization of their disease for three to nine months, among whom was a patient with melanoma who developed marked vitiligo. We conclude that repeated injection of up to 5 x 10(7) Vero-IL-2 cells was safe and showed biological and clinical activity in heavily pretreated patients with advanced solid tumors. Further evaluation of intratumoral application of Vero-IL-2 seems warranted.

A CD66a-specific, activation-dependent epitope detected by recombinant human single chain fragments (scFvs) on CHO transfectants and activated granulocytes.

Antibodies to CD66 recognize at least five members (CD66a-e) of the carcinoembryonic antigen (CEA) family. Recombinant human single-chain Fv fragments (scFvs) that bind specifically to CD66a (biliary glycoprotein) were obtained from a naive human scFv library. The scFvs bound to the N-domain of CD66a on Chinese hamster ovary (CHO) transfectants but did not bind to freshly isolated peripheral granulocytes or to dimethylsulfoxide-treated HL-60 cells. In contrast, scFvs bound well to granulocytes that were short-term activated with N-formyl-Met-Leu-Phe or phorbol 12-myristate 13-acetate and to human HL-60 cells that were treated with all-trans-retinoic acid to induce granulocytic differentiation. Quantification of antigenic sites showed that the activation-dependent CD66a epitopes were expressed on nearly all of the CD66a molecules on CHO-biliary glycoprotein transfectants, but they were detected only on a portion of the molecules on activated polymorphonuclear neutrophils and differentiated HL-60 cells. Binding of CD66a scFvs to their neoepitopes on prestimulated PMNs induced respiratory burst, suggesting that CD66a is capable of delivering transmembrane signals in these cells.

Glycoproteins of the carcinoembryonic antigen (CEA) family are expressed in sweat and sebaceous glands of human fetal and adult skin.

The carcinoembryonic antigen (CEA) family comprises a group of glycoproteins including the classical CEA, nonspecific cross-reacting antigens (NCA), and biliary glycoprotein (BGP). CEA glycoproteins have been identified in many glandular and mucosal tissues. In view of their putative role in cell adhesion, protein sorting, and signal transduction, CEA glycoproteins are thought to be involved in embryogenesis, architectual integrity, and secretory mechanisms of glandular epithelia. Since there are few data available on the expression of CEA-like proteins in human skin, the aim of this study was to immunohistochemically specify and localize the CEA glycoproteins in cutaneous adult and fetal glands using a panel of well-characterized antibodies. The secretory parts of eccrine sweat glands expressed CEA, NCA-90, and BGP, whereas apocrine glands remained unreactive for CEA glycoproteins. The ductal epithelia of both eccrine and apocrine glands contained CEA and NCA-90. Sebaceous glands were stained for BGP only. Electron microscopy of sweat glands showed CEA glycoprotein expression in cytoplasmic organelles and on microvilli lining the ductal surface. In sebaceous glands, BGP were demonstrated in small vesicles and along the cell membranes of differentiating sebocytes. Fetal development of cutaneous glands was associated with early expression of CEA glycoproteins. Additionally, mice transgenic for human CEA were shown to express CEA in sweat glands. The overall distribution of CEA glycoproteins in cutaneous glands was consistent with that in epithelia of other glandular tissues.