Fm Rosenthal

A phase‐I clinical study of autologous tumor cells plus interleukin‐2‐gene‐transfected allogeneic fibroblasts as a vaccine in patients with cancer

Tumor cells transfected to express immunostimulatory cytokines, or admixed with similarly modified bystander cells, are able to induce immune responses against unmodified tumor cells in animal models. For treatment of human patients, a vaccine composed of autologous tumor cells and IL‐2‐secreting allogeneic fibroblasts was developed. Autologous tumor cells were isolated from biopsy specimens. A clone (KMST6.14) of an immortalized human fibroblast line that stably secreted 5290 IU IL‐2 per 106 cells and per 24 hr was obtained by cationic lipofection with an expression construct for human IL‐2 and Neor. Fifteen patients with refractory malignant tumors received 3–4 injections of irradiated KMST6.14 and autologous tumor cells in a phase‐I clinical trial. Increasing transient inflammatory responses without systemic toxicity developed at vaccination sites and after injections with irradiated tumor cells only (p < 0.05). These sites contained a dense infiltrate of CD3+ T cells with numbers of CD4+ helper cells exceeding those of CD8+ cytotoxic T cells (CTL). CD8+ T‐cell lines isolated from vaccination sites of 2 malignant melanoma patients but not of renal‐cell carcinoma patients exhibited a dominant lytic activity against autologous tumor cells in vitro. CD8+ T‐cell clones established from the vaccination site of 1 of 2 renal‐cell carcinoma patients preferentially lysed autologous and partially matched allogeneic renal‐cell carcinoma cells. In conclusion, a vaccine composed of IL‐2 gene‐transfected allogeneic fibroblasts and autologous tumor cells is able to enhance specific anti‐tumor T‐cell responses in vivo without major side‐effects. Malignant melanoma and renal‐cell carcinoma appear to be promising entities for testing of similar approaches in future therapeutic trials. Int. J. Cancer, 70:269–277, 1997.

Transplanted Bone Marrow Cells Preferentially Home To The Vessels Of In Situ Generated Murine Tumors Rather Than Of Normal Organs

Transplanted bone marrow‐derived (BM) cells have been shown to home into the tumor vessels of s.c. implanted tumor models and to functionally contribute to tumor neoangiogenesis and tumor growth. However, whether BM cells contribute to the vessels of in situ developing tumors remains unknown. We have taken advantage of the in situ generation of mammary tumors in transgenic mice carrying the polyoma virus middle T oncogene (MMTV‐PyVT) to determine whether transplanted BM cells home to and incorporate into the intratumoral vessels. Unfractionated BM from lacZ+ROSA 26 mice was used to rescue irradiated MMTV‐PyVT transgenic mice or their wild‐type congenics. All transgenic mice were sacrificed when they developed easily palpable mammary tumors. BM cells recruited and incorporated into the vasculature were identified by coexpression of lacZ and CD31, evidence that these cells had a distinctive, elongated appearance and that they lined the vessel structures. We found that BM cells home to and incorporate into 1.3% of the vessels of all in situ generated mammary adenocarcinomas examined (n = 8). In contrast, BM cells did not recruit into the vessels of colon or liver of the tumor‐bearing mice. Whether these cells contribute to new vessel formation via vasculogenesis or angiogenesis or simply attach to, and integrate into, the growing tips or shafts of pre‐existing vessels has to be determined. BM could be used as a vehicle for the specific transport of antiangiogenic signals into the tumor vascular bed.

Pilot study for the evaluation of T-cell-mediated tumor immunotherapy by cytokine gene transfer in patients with malignant tumors

Induction of T-cell-mediated cytotoxicity and immunity against specific tumor antigens by vaccination with devitalized autologous tumor tissue and (a) autologous or (b) allogeneic fibroblasts as a constitutive paracrine source of interleukin-2 (IL-2). benefit for solid tumors, notably malignant melanoma and renal cell carcinoma. However, objective responses occur only in a minority of patients and are usually transient, and treatment is often associated with significant toxicity. Therefore a more selective delivery of immunostimulatory cytokines may improve treatment results. Recent data indicate the presence of MHC-restricted, tumor-specific cytotoxic T cells (CTL) in patients with malignant melanoma and renal cell carcinoma that may be capable of efficient tumor rejection. These CTL recognize tumor-associated antigens, for example, mutated proto-oncogenes, genes with expression restricted to the tumor cell lineage, and genes that are normally expressed only in the fetal period. Current theory holds that these CTL may not be activated properly due to the lack of costimulatory signals in the immediate context of their target cells and are therefore unable to lyze tumor cells in vivo. Those costimulatory signals appear to be provided physiologically by accessory molecules (e.g., B7) or soluble cytokines. In accordance with this theory, animal studies have demonstrated the therapeutic efficacy of tumor vaccines that have been genetically modified to secrete T-cell stimulating cytokines as a second signal for CTL activation. Based on these studies, this clinical trial attempts to induce antitumor immunity by stimulating tumor-specific CTL with a genetically engineered vaccine. The vaccine is composed of autologous tumor cells, presumably carrying tumor-associated antigens, mixed with IL-2 secreting fibroblasts as a paracrine source of IL-2 to provide an efficient costimulatory signal for activation of CTL.

Cloning and sequence analysis of the immediate promoter region and cDNA of porcine granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells committed to the neutrophil/granulocyte lineage. Recombinant G-CSF (rG-CSF) is routinely used in the prevention of chemotherapy-induced neutropenia and in the setting of bone marrow transplantation. Chronic idiopathic and congenital neutropenic disorders also show improvement after rG-CSF injections. Applications of either rG-CSF or G-CSF gene transfected cells into mice give rise to leukocytosis, which can be measured easily. This makes G-CSF a versatile tool for studying systemic effects of therapeutic proteins delivered by genetically modified cells in vivo. Although the biological activity of G-CSF is not species-specific, studies on long-term expression would require the use of species-identical proteins in order to avoid host immune reactions against the foreign gene product. Because of the physiological and immunological similarity of pigs and human, the pig has become an important large-animal model for biomedical research. We have therefore cloned porcine G-CSF cDNA from RNA isolated from pig PBLs. Pig G-CSF is a 195-amino-acid polypeptide that shares a high degree of homology to human (78%), murine (71%) as well as rat (68%) G-CSF. In contrast to human and murine, but not to rat G-CSF, a different ATG translation start codon is used, resulting in a shorter, but still functional signal sequence.

Guidelines for the design and implementation of clinical studies in somatic cell therapy and gene therapy

According to Sects. 2 and 3 of the German drug statute, (Arzneimittelgesetz, AMG) gene technology and gene therapy products as well as somatic cell therapy products are regarded as drugs and are therefore subject to Sects. 40 and 41, stipulating the requirements for clinical studies designed to test drugs. Major aspects of drug production are similarly defined in this statute (e.g., product license, Sect. 13). Below, we summarize the guidelines and recommendations for conducting clinical studies in somatic-cell therapy and gene therapy. In addition to laboratory licenses, a number of other aspects of drug production and rules regarding the design of clinical protocols must be considered. For example, all clinical protocols must be submitted to the government agency of the state (Land). This agency has a cer-

Particular Treatment Procedures

Hematopoetic stem cells are a small and predominantly dormant population of undifferentiated cells. They are characterized by the ability to self renew by continuous cell division and to differentiate into lymphoid, myeloid, erythroid, or megakaryocytic cells (7 Chap. 1.3). Hematopoietic stem cells transplanted after radiotherapy radiotherapy (total body irradiation) and/or high-dose chemotherapy from autologous or allogeneic sources are subjected to intense proliferation and differentiation inside the recipient. The contribution of individual cell types to shortand longterm bone marrow recovery after transplantation has not been fully elucidated, however, stem cells are responsible for maintaining continuous hematopoiesis.