Cancer vaccines genetically engineered to produce interleukin 2 have been investigated intensively in a series of animal models and are at the point of entering into clinical trials. In this study we demonstrate a strong correlation between the rate of interleukin 2 production and the protection efficiency of murine S91 melanoma cell (clone M-3) vaccines. Best immunization is achieved with vaccines producing medium interleukin 2 levels of 1000-3000 units per

Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells,(ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction.

Hemophilia A is caused by defects in the factor VIII gene. This results in life-threatening hemorrhages and severe arthropathies. Today, hemophiliacs are treated with human blood-derived factor VIII. In the future, it may be possible to use gene therapy to avoid long-term complications of conventional therapy and to improve the quality of life. However, initial gene therapy models using retroviral vectors and nonviral gene transfer tcnques to introduce factor VIII gene constructs have been hampered by low expression levels of factor VIII. We show here that high expression levels of the B-domain-deleted human factor VIII in primar mouse fibroblasts and myoblasts are obtained by using receptor-m ted, adenovirus-augmented gene delivery (transferrinfection). We demonstrate that, presumably owing to the high molecular weight of factor VIII or its metabolic instability, secretion into the blood and attainment of therapeutic in vvo levels of factor VIII is achieved only if trnected autologous primary fibroblasts or myoblasts are delivered to the liver or spleen, but not if myoblasts are implanted into muscle, a strategy known to be successful for factor IX delivery.In hemophilia A there is a genetic defect of the coagulation factor VIII, a trace plasma glycoprotein, which acts as a cofactor for factor IX in the activation of factor X (reviewed in refs. 1 and 2). The factor VIII gene encodes a protein of 2351 amino acids containing six domains (3-5). The central, B domain of 980 amino acids, to which high-mannose oligosaccharides are added in the endoplasmic reticulum, is discarded during proteolytic activation of factor VIII (1,3,5). The B-domain-deleted form of the factor VIII gene has been chosen for use in gene transfer experiments with recombinant retrovirus because (i) the retroviral vectors do not readily accommodate the full-length cDNA (8.8 kb) and (ii) the B-domain-deleted protein is more easily processed in transduced cells than the full-length protein (6-9). However, the expression of factor VIII obtained with these retroviral vectors was too low for in vivo generation of detectable plasma levels of factor VIII (10). The insufficient synthesis rates after retrovirus-mediated transduction have been explained, at least in part, by the observation that sequences of the factor VIII gene interfere with its own transcription, resulting in low titers of the retroviral vectors and in low factor VIII production rates in the transduced cells (ref. 11 and R. C. Hoeben, personal communication). With "transferrinfection," neither the problem of interference of factor VIII sequences with vector production nor such size restriction for the delivery of DNA molecules exists, because the gene, highly condensed by polylysine-modified transferrin, is carried on the surface of the adenovirus (refs. 12-15 and unpublished results). Moreover, transferrinfection has been shown to introduce %20 copies of genes into cells, which correlates with high expression rates. To make our data directly comparable with published work, ...

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