Yoshikazu Koshita

In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate

To target disseminated tumors in vivo, transgenes [ β‐galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV‐TK)] were conjugated to transferrin (Tf) by a biotin‐streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf‐R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf‐β ‐galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf‐R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK‐1) whereas in normal diploid cells (HEL), which express low levels of Tf‐R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf‐HSV‐TK gene conjugate for massively metastasized k562 tumors in severe combined immunedeficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tumors.—Sato, Y., Yamauchi, N., Takahashi, M., Sasaki, K., Fukaura, F., Neda, H., Fujii, S., Hirayama, M., Itoh, Y., Koshita, Y., Kogawa, K., Kato, J., Sakamaki, S., Niitsu, Y. In vivo gene delivery to tumor cells by transferrin‐streptavidin‐DNA conjugate. FASEB J. 14, 2108–2118 (2000)

Characterization of tumor-necrosis-factor-gene-transduced tumor-infiltrating lymphocytes from ascitic fluid of cancer patients : analysis of cytolytic activity, growth rate, adhesion molecule expression and cytokine production

We characterized tumor-infiltrating lymphocytes (TIL) from ascites of patients with ovarian or pancreatic cancer in which the human tumor necrosis factor (TNF) gene was successfully transduced with retrovirus vector. The TNF-gene-transduced TIL (TNF-TIL) from these patients showed a higher level of TNF production and higher cytotoxic activity against K562 and Daudi cells than did neomycin-phosphotransferase-gene-transduced TIL (neo-TIL). Of these TIL preparations, only that from pancreatic cancer was further characterized since it was collected in a relatively large amount. In spite of the fact that the autologous tumor cells showed resistance to soluble TNF, the TNF-TIL clearly demonstrated enhanced cytotoxicity against them as compared with neo-TIL. The enhanced cytotoxicity was ascribed to autocrine effects of secreted TNF on TIL, which included augmentation of adhesion molecule (CD2 and CD11a) and interleukin-2 receptor expression, and elevation of production of interferon γ, lymphotoxin and granulocyte/macrophage-colonystimulating factor and its paracrine effect on target cells to facilitate them to be more susceptible to TIL.

Augmented systemic immunity in mice implanted with tumor necrosis factor-α, gene-transduced Meth-A cells

Syngeneic BALB/c mice bearing methylcholanthrene‐induced fibrosarcoma (Meth‐A) cells transduced with a tumor necrosis factor (TNF) gene showed a longer life span and tumor regression as compared with mice carrying TNF‐non‐producing Meth‐A cells. To elucidate the mechanism of the reduced tumorigenicity of TNF‐producing Meth‐A, we compared systemic immune responses between mice bearing high TNF producer (C5) and unmodified Meth‐A cells (M0). The results indicated that the cytotoxic activity of lymphokine‐activated killer cells (LAK) induced from spleen cells of mice bearing C5 was higher against both M0 and C5 than that of LAK from mice bearing M0. Also, C5 was more sensitive to LAK induced from spleen cells of C5‐ and M0‐ bearing mice than M0. We also found that cytotoxic T lymphocyte from spleen cells of mice transplanted with C5 was more cytotoxic to M0 than that from mice with M0. In addition, the population of Lyt2 (CD8)‐positive T cells was higher in freshly isolated spleen cells of mice transplanted with C5 than from mice with M0. Finally, we observed a higher expression of MHC class 1 antigen on C5 than on M0. These observations suggest that the augmented host systemic immunity in mice carrying TNF gene‐modified Meth‐A cells is one of the mechanisms of the reduced tumorigenicity of C5 and that the increased systemic immunity can be ascribed to the increased immunogenicity of the tumor cells. Thus, the use of TNF gene‐modified tumor cells as vaccines appears to be promising for therapeutic and/or prophylactic application.

Synergistic Suppressive Effect of Double Transfection of Tumor Necrosis Factor‐α and Interleukin 12 Genes on Tumorigenicity of Meth‐A Cells

Tumor necrosis factor‐α(TNF‐α) and interleukin 12 (IL‐12), both potent antitumor cytokines, are known to be involved in the hosts antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer/lymphocyteactivated killer (NK/LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1‐type cellular immunity and enhances the activities of natural killer T (NKT), NK/LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the hosts immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK/LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor‐specific immunity was confirmed by rechallenge with parental Meth‐A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF‐αand IL‐12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.

Mechanisms of tumor regression of TNF gene-transduced Meth-A cells transplanted in mice.

We transduced a tumor necrosis factor (TNF) gene into methylcholanthreninduced mouse fibrosarcoma (Meth-A) cells retrovirally to establish a high TNF producer clone (C5). C5 was subcutaneously transplanted into syngeneic mice, resulting in remarkable tumor regression and a much longer life span as compared with subjects transplanted with the parent Meth-A (MO) or NeoR genetransduced Meth-A (ML). Since C5 was resistant to exogenous TNF in vim, it seems unlikely that the observed tumor regression was due to any direct antitumor effect of TNF produced from C5. In this presentation, we studied mechanisms of regression of the C5 tumor. First, we investigated whether augmented systemic immunity was induced in C5-bearing mice. Cytotoxic effects of interleukin-2 (IL-2) activated splenocytes (LAK) from MOand C5-transplanted mice were studied against MO and C5, respectively, by 51Cr release assay; LAK from C5-bearing mice showed higher cytotoxic activity, the level of which was higher in targeting C5 than MO. Cytotoxic effects of CTL induced from splenocytes of MOand C5-bearing mice were also studied against MO, showing increased cytotoxic activity of CTL from mice with C5. SurFace antigens of freshly isolated splenocytes from C5and MO-bearing mice were also analyzed by flow cytometry, resulting in higher expression of T cell (particularly CD8+ cells) in splenocytes from C5-bearing mice than that from MO-bearing ones. Second, we studied whether C5 accompanied any alterations compared with MO. Sensitivities of MO and C5 to IL-2-activated splenocytes from allogenic normal mouse (allogenic LAK) were studied; C5 showed a higher sensitivity to the LAK than MO. Sensitivities of MO and C5 to CTL induced from splenocytes of C5-bearing mice were also studied; C5 showed a higher sensitivity to the CTL. Major histocompatibility complex (MHC) antigen and adhesion molecule expression of MO and C5 were investigated; class 1 expression was higher in C5 than in MO. Third, we analyzed C5 and MO tumor tissue with immunohistochemical staining; C5 tumor revealed higher expression of CD4, CD8, ICAM-1, and VCAM1.