Transduction of human renal carcinoma cells with human gamma-interferon gene via retroviral vector.

We used a retroviral vector containing a human gamma-interferon (gamma-IFN) gene to transduce 13 renal carcinoma cell lines. The transduction efficiencies ranged from 0% to 60%, as determined by using an analogous vector containing the LacZ marker gene. In addition, gene-transferred resistance to the antibiotic neomycin was used to select for transduced cells. Nine of 13 lines were successfully transduced. Transduction was associated with the morphologic change of elongation, and there was a marked decrease in cell growth rate. Transduced cells secreted varying amounts (20-1076 pg/10(6) cells/d) of gamma-IFN as measured by enzyme-linked immunosorbent assay for at least 2 to 3 weeks after transduction (including 1 day of transduction, 6-7 days of selection, and an additional 8-12 days before the first passage of the transduced cells). Human leukocyte antigen (HLA) class II expression was markedly increased in six of seven cell lines; HLA class I expression was significantly increased in two of eight lines. Transduced cells that were subjected to cryopreservation after irradiation still produced gamma-IFN and expressed HLA class I and II antigens, although generally at lower levels than before these manipulations. This study confirms that retroviral vector transduction of the human gamma-IFN gene into renal carcinoma cells is feasible and associated with persistent production of gamma-IFN and increased expression of HLA class I and II molecules, and these effects are retained after irradiation and cryopreservation. This suggests that an autologous tumor cell vaccine trial with irradiated gamma-IFN gene-transduced renal carcinoma cell is rationale and feasible.

Sustained cytokine production and immunophenotypic changes in human neuroblastoma cell lines transduced with a human gamma interferon vector.

The majority of human neuroblastomas express low to undetectable levels of major histocompatibility complex (MHC) class I and II antigens (MHC-I and -II). We studied the effects of gamma interferon (gamma-IFN) transduction on expression of these antigens in six human neuroblastoma cell lines with and without genomic amplification of the N-myc oncogene. All six were stably transduced with an MoMLV-based gamma-IFN retroviral vector (DAh gamma-IFN). G418-resistant cells were assayed for MHC-I, MHC-II, B7-1, and neuroblastoma-associated antigen expression, as well as for gamma-IFN levels in cell culture supernatants. Sustained gamma-IFN production, 2 to > 1000 units/10(6) cells/d, was attained for five of six transduced cell lines and persisted for up to 9 months. This resulted in marked upregulation of MHC-I and MHC-II expression in LA-N-1, LA-N-6, and CHLA-127 cells and moderate upregulation in SK-N-Fi and SK-N-AS cells. One cell line (LA-N-1) had marked induction of MHC-I and MHC-II despite marginal levels of gamma-IFN production. Expression of CD28 ligand B7-1 (as determined by BB1 antibody) remained unchanged in all gamma-IFN-transduced cell lines tested. Expression of several neuroblastoma-associated antigens (NKH1A, 126-4, HSAN 1.2, HNK, 459, and 390) was upregulated in some of the gamma-IFN-transduced cell lines. These results demonstrate that preparation of gamma-IFN expressing neuroblastoma cells for immunotherapeutic purposes is feasible and that gamma-IFN transduction results in phenotypic changes that may improve immunogenicity of human neuroblastoma cells.

Transduction of human melanoma cells with the gamma interferon gene enhances cellular immunity.

Human tumor cells transduced with the gamma interferon (gamma IFN) gene are currently used in specific active immunotherapy protocols to enhance the antitumor immune responses of cancer patients. This in vitro study was undertaken to examine the initial events in the cellular immune response that may occur following the administration of the gamma IFN-transduced cell vaccine. Human melanoma tumor cell lines were transduced with a MoMLV-based retroviral vector carrying the human gamma IFN gene. The transduced cells expressed the cytokine gene, secreted biologically active gamma IFN, and exhibited enhanced expression of MHC class I and class II (HLA-DR), and ICAM-1 surface antigens. The gamma IFN-transduced and corresponding parental melanoma cells were used for the induction of short-term lymphocyte cultures. Peripheral blood lymphocytes or lymph node cells from 20 melanoma patients were stimulated for 5 to 15 days with autologous or MHC class I-matched allogeneic parental or gamma IFN-transduced melanoma cells. Seven of the 20 lymphocyte cultures showed substantial increases in lytic activity following stimulation with the transduced melanoma cells in comparison to control lymphocyte cultures stimulated with unmodified parental melanoma. The cytolytic activity stimulated with gamma IFN-modified melanomas was mediated partly by MHC-restricted cytotoxic T lymphocytes and partly by NK cells. Lymphocyte cultures that displayed increases in cytotoxicity after stimulation with the gamma IFN-transduced melanoma cells also exhibited enhanced expression or induction of one or more of the following lymphokines: IL-4, IL-1 alpha, IL-1 beta, gamma IFN, and TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrovirus-mediated gene transfer of the human gamma-IFN gene: a therapy for cancer.

A retroviral vector-mediated gene transfer system was used to introduce m gamma-IFN and h gamma-IFN genes into mouse and human tumor cells, respectively. Murine tumor cell lines and primary human melanoma tumor cells were successfully transduced with gamma-IFN vector, and these transduced cells secreted measurable levels of biologically active m gamma-IFN and h gamma-IFN, respectively. Both murine and human tumor cell lines that expressed gamma-IFN exhibited increased surface expression of HLA class I antigens when tested by Western blot and FACS analysis. gamma-IFN--transduced human melanoma cells were more active in stimulating tumor-specific cytolytic activity of CTLs from melanoma patients in vitro. m gamma-IFN--transduced tumor cells were substantially less tumorigenic than the corresponding parent tumor cell lines in immune-competent mice. In addition, injection of m gamma-IFN--transduced tumor cells resulted in activation of tumor-specific CTL in vivo. We plan to use gamma-IFN--transduced autologous tumor cells to boost host immune responses as a potential therapy for human melanoma.

Human anti-mouse antibody response in cancer patients following single low-dose injections of radiolabeled murine monoclonal antibodies.

We examined the human anti-mouse antibody (HAMA) response in 61 cancer patients following a single, diagnostic injection of any one of ten 111In conjugated murine monoclonal antibodies. Between 1 and 22 mg of antibody containing 1-5 mCi 111In was administered. The populations studied included 30 patients with colorectal carcinoma (four different antibodies), 22 with malignant melanoma (four antibodies), and nine with prostate cancer (two antibodies). Forty-one percent of the patients developed HAMA within 14 days. Three patients (5%) developed an IgM response, five patients (8%) developed an IgG response, and 17 patients (28%) developed both IgM and IgG. Only 27% of the patients with colon cancer developed HAMA, compared to 55% of the melanoma patients and 56% of the prostate cancer patients. There were no correlations among injected dose, various clinical parameters, and HAMA response. There were variations in the HAMA response to different monoclonal antibodies, but population samples were too small to infer significance. Most of the HAMA responses had a significant proportion of idiotypic or isotypic specificity. Only 1/6 patients who were HAMA negative after the first infusion developed HAMA following subsequent infusions of the same monoclonal antibody. Our data demonstrate that a significant percent of cancer patients develop HAMA following a single, low-dose injection of a radiolabeled monoclonal antibody for diagnostic purposes. This may have important implications for the future therapeutic use of monoclonal antibodies in such patients.

Mechanisms of human CD5 modulation and capping induced by murine monoclonal antibody T101.

We have previously demonstrated that the murine monoclonal antibody T101 induces antigenic modulation when infused into patients with chronic lymphocytic leukemia and cutaneous T-cell lymphoma. In this paper, we extend our studies of T101-induced modulation and compare it to T101-induced capping. We found that, in contrast to antigenic modulation, capping occurred only in the presence of secondary anti-mouse IgG antisera and was altered by drugs that affect the cellular cytoskeleton or energy metabolism. F(ab')2 fragments of T101 induced antigenic modulation with kinetics similar to those of intact T101, but Fab-induced modulation proceeded more slowly and required the continual presence of Fab throughout the incubation. Experiments with radioiodinated T101 demonstrated that initial internalization of the antibody is followed by rapid efflux of intact, immunoreactive T101 from the cells. These data indicate important differences between capping and modulation and suggest that these two phenomena proceed by different mechanisms. More importantly, the data have implications for the potential therapeutic use of monoclonal antibody immunoconjugates.