Tumor cell transduction with the herpes simplex virus (HSV) thymidine kinase (tk) gene and treatment with ganciclovir (GCV) is a widely studied cancer gene therapy. Connexin (Cx)-dependent gap junctions between cells facilitate the intercellular spread of TK-activated GCV, thereby creating a bystander effect that improves tumor cell killing. However, tumor cells often have reduced connexin expression, thus thwarting bystander killing and the effectiveness of TK/GCV gene therapy. To improve the effectiveness of this therapy, we compared an HSV vector (TOCX) expressing Cx43 in addition to TK with an isogenic tk vector (TOZ.1) for their abilities to induce bystander killing of Cx-positive U-87 MG human glioblastoma cells and Cx-negative L929 fibrosarcoma cells in vitro and in vivo. The results showed that low-multiplicity infection of U-87 MG cells with TOCX only minimally increased GCV-mediated cell death compared with infection by TOZ.1, consistent with the endogenous level of Cx in these cells. In contrast, bystander killing of L929 cells was markedly enhanced by vector-mediated expression of Cx. In vivo experiments in which U-87 MG cells were preinfected at low multiplicity and injected into the flanks of nude mice showed complete cures of all animals in the TOCX group following GCV treatment, whereas untreated animals uniformly formed fatal tumors. TOCX injection into U-87 MG intradermal and intracranial tumors resulted in prolonged survival of the host animals in a GCV-dependent manner. Together, these results suggest that the combination of TK and Cx may be beneficial for the treatment of human glioblastoma.

To obtain high-titer recombinant retroviruses, we constructed plasmid pDL+, which carries the extended Psi region and the polyomavirus early region, including the replication origin and the early gene. Although pDL+ is useful for obtaining high-titer recombinant retroviruses, this vector plasmid is difficult to modify further for tissue-specific expression of foreign genes. To overcome this problem, the coding region of the polyomavirus early gene was expressed in the packaging cell lines. We modified the packaging cell lines, psi2 and PA317, by stably introducing the polyomavirus early gene, and established psiMP34 and psiMP37 from psi2, and PAMP51 from PA317. In the transient expression system using plasmids with and without the polyomavirus replication origin, the titers of recombinant retrovirus produced by these cell lines were 10-100 times higher than produced by the parent cell line and reached levels of 0.5-1.5 x 106 cfu/ml. Expression of the polyomavirus early gene in the packaging cell lines did not stimulate the production of replication-competent retrovirus. We also routinely established stable clones producing retrovirus titers over 1 x 10(7) cfu/ml from psiMP34 and PAMP51. We found that the activity of the long terminal repeat (LTR) promoter is stimulated by the polyomavirus early region protein(s) in these cell lines. Therefore, increases titer can be expected to occur in all the retroviral vectors in which LTR promoter is used to transcribe the retroviral genome.

Experiments were carried out in a nude mouse model of human glioblastoma to determine whether gamma-knife radiosurgery combined with herpes simplex virus thymidine kinase (tk) suicide gene therapy and tumor necrosis factor alpha (TNFalpha) gene transfer provided an improved multimodality treatment of this disease. Animals were inoculated intracerebrally with 2 x 10(5) U-87MG human glioblastoma cells to establish brain tumors. At 3 days postinoculation, the tumor region was injected with 2 x 10(6) infectious particles of highly defective herpes simplex viral vectors expressing the viral tk gene with the kinetics of a viral immediate early gene either alone (T.1) or together with TNF alpha (TH:TNF). Subgroups of animals were given daily intraperitoneal injections of ganciclovir (GCV) for 10 days and/or subjected to gamma-knife radiosurgery on the fifth day post tumor-cell implantation. Comparisons of animal survival showed that the TH:TNF vector in combination with radiosurgery and GCV administration provided the most effective therapy; eight of nine animals survived for 75 days compared to four of eight using the next best protocol. These findings suggest that gene therapy in combination with more conventional therapeutic methods may provide an improved strategy for extending the life expectancy of patients afflicted with this ultimately fatal disease.

Herpes simplex virus thymidine kinase (HSV-TK) and

l‐Type amino acid transporter 1 (LAT1) disulfide linked to CD98 heavy chain (hc) is highly expressed in most cancer cells, but weakly expressed in normal cells. In the present study, we developed novel anti‐LAT1 mAbs and showed internalization activity, inhibitory effects of amino acid uptake and cell growth and antibody‐dependent cellular cytotoxicity, as well as in vivo antitumor effects in athymic mice. Furthermore, we examined the reactivity of mAbs with LAT1 of Macaca fascicularis to evaluate possible side‐effects of antihuman LAT1 mAbs in clinical trials. Antihuman LAT1 mAbs reacted with ACHN human and MK.P3 macaca kidney‐derived cells, and this reactivity was significantly decreased by siRNAs against LAT1. Macaca LAT1 cDNA was cloned from MK.P3, and only two amino acid differences between human and macaca LAT1 were seen. RH7777 rat hepatoma and HEK293 human embryonic kidney cells expressing macaca LAT1 were established as stable transfectants, and antihuman LAT1 mAbs were equivalently reactive against transfectants expressing human or macaca LAT1. Dual (high and low) avidity modes were detected in transfectants expressing macaca LAT1, MK.P3, ACHN and HCT116 human colon cancer cells, and KA values were increased by anti‐CD98hc mAb, suggesting anti‐LAT1 mAbs detect an epitope on LAT1‐CD98hc complexes on the cell surface. Based on these results, LAT1 may be a promising anticancer target and Macaca fascicularis can be used in preclinical studies with antihuman LAT1 mAbs.