scholarly journals Augmentation of antitumor immunity by tumor cells transduced with a retroviral vector carrying the interleukin-2 and interferon-gamma cDNAs

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1289-1298 ◽  
Author(s):  
FM Rosenthal ◽  
K Cronin ◽  
R Bannerji ◽  
DW Golde ◽  
B Gansbacher
Keyword(s):  
Gene Transfer ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Antitumor Immunity ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Ifn Gamma ◽  
Gm Csf

Abstract Therapeutic models using gene transfer into tumor cells have pursued three objectives: (1) to induce rejection of the tumor transduced with therapeutic genes, (2) to induce immune-mediated regression of metastatic disease, and (3) to induce long-lasting immunity to protect against challenge with tumor cells or clinical regrowth of micrometastatic disease. Because in vivo therapy for patients with cancer using gene transfer would, as a first step, attempt to eliminate the existing tumor, we have investigated whether antitumor immunity induced by tumor cells secreting a single cytokine could be increased by cotransfer of a second cytokine gene. To test this approach, CMS-5, a murine fibrosarcoma, was transduced with retroviral vectors carrying interleukin-2 (IL-2), interferon-gamma (IFN-gamma), or granulocyte- macrophage-colony-stimulating factor (GM-CSF) cDNA alone or IL-2 cDNA in combination with IFN-gamma or GM-CSF cDNA. Single cytokine-secreting clones were selected to match levels of cytokine production by double cytokine-secreting clones so that similar amounts of cytokine were secreted. IFN-gamma- and IL-2/IFN-gamma-secreting CMS-5 cells showed increased levels of major histocompatability complex class I expression compared with IL-2- and GM-CSF-secreting or parental CMS-5 cells, IL- 2/IFN-gamma-secreting CMS-5 cells were always rejected by syngeneic mice, whereas the same number of CMS-5 cells secreting only one of these cytokines or mixtures of single cytokine-secreting CMS-5 cells were not rejected. In vivo depletion of CD4+, CD8+, or natural-killer effector cell subpopulations showed that CD8+ cytotoxic T cells were primarily responsible for rejection of IL-2/IFN-gamma-transduced tumor cells. Our data show the successful use of a single retroviral vector to stably transduce two cytokine genes into the same tumor cell, leading to an increased effect on the in vivo induction of antitumor immunity.

scholarly journals Augmentation of antitumor immunity by tumor cells transduced with a retroviral vector carrying the interleukin-2 and interferon-gamma cDNAs

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1289-1298
Author(s):  
FM Rosenthal ◽  
K Cronin ◽  
R Bannerji ◽  
DW Golde ◽  
B Gansbacher
Keyword(s):  
Gene Transfer ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Antitumor Immunity ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Ifn Gamma ◽  
Gm Csf

Therapeutic models using gene transfer into tumor cells have pursued three objectives: (1) to induce rejection of the tumor transduced with therapeutic genes, (2) to induce immune-mediated regression of metastatic disease, and (3) to induce long-lasting immunity to protect against challenge with tumor cells or clinical regrowth of micrometastatic disease. Because in vivo therapy for patients with cancer using gene transfer would, as a first step, attempt to eliminate the existing tumor, we have investigated whether antitumor immunity induced by tumor cells secreting a single cytokine could be increased by cotransfer of a second cytokine gene. To test this approach, CMS-5, a murine fibrosarcoma, was transduced with retroviral vectors carrying interleukin-2 (IL-2), interferon-gamma (IFN-gamma), or granulocyte- macrophage-colony-stimulating factor (GM-CSF) cDNA alone or IL-2 cDNA in combination with IFN-gamma or GM-CSF cDNA. Single cytokine-secreting clones were selected to match levels of cytokine production by double cytokine-secreting clones so that similar amounts of cytokine were secreted. IFN-gamma- and IL-2/IFN-gamma-secreting CMS-5 cells showed increased levels of major histocompatability complex class I expression compared with IL-2- and GM-CSF-secreting or parental CMS-5 cells, IL- 2/IFN-gamma-secreting CMS-5 cells were always rejected by syngeneic mice, whereas the same number of CMS-5 cells secreting only one of these cytokines or mixtures of single cytokine-secreting CMS-5 cells were not rejected. In vivo depletion of CD4+, CD8+, or natural-killer effector cell subpopulations showed that CD8+ cytotoxic T cells were primarily responsible for rejection of IL-2/IFN-gamma-transduced tumor cells. Our data show the successful use of a single retroviral vector to stably transduce two cytokine genes into the same tumor cell, leading to an increased effect on the in vivo induction of antitumor immunity.

scholarly journals Retroviral gene transfer induced constitutive expression of interleukin- 2 or interferon-gamma in irradiated human melanoma cells

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2817-2825 ◽  
Author(s):  
B Gansbacher ◽  
K Zier ◽  
K Cronin ◽  
PA Hantzopoulos ◽  
B Bouchard ◽  
...  
Keyword(s):  
Gamma Irradiation ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Ifn Gamma ◽  
Melanoma Cell Lines

Cytokines are important modulators of host antitumor responses. Two of these cytokines, interleukin-2 (IL-2) and interferon gamma (IFN-gamma), are produced after antigen-induced activation of helper lymphocytes. The cytokines are released into the immediate vicinity where they either interact with the appropriate receptors on effector cell populations or are rapidly degraded. To mimic this physiologic release of cytokines at the effector-target site, we used retroviral vectors to transduce melanoma cells with the IL-2 or IFN-gamma cDNA. Five melanoma cell lines were transduced with IL-2- or IFN-gamma-containing vectors and secreted IL-2 at 1 to 40 U/mL/10(6) cells/24 h or IFN-gamma 1 to 8 U/mL/10(6) cells/24 h, respectively. After gamma irradiation, these cells continued to secrete cytokines for about 3 to 4 weeks. Secretion of IFN-gamma induced upregulation of major histocompatibility complex class I molecules in a subset of melanoma cell lines. IL-2 production by human melanoma xenografts induced tumor rejection in BALB/c nu/nu mice, showing the in vivo effect of this cytokine. This study shows that (1) human melanoma cells can be stably transduced with cytokine- containing retroviral vectors; (2) cytokines are secreted constitutively by the transduced tumor cells and have the expected biologic effects in vitro and in vivo; and (3) after gamma irradiation, cytokines continue to be secreted for several weeks. These data suggest that irradiated cytokine-secreting allogenic or autologous tumor cells can be used in vaccination protocols for cancer patients.

Retroviral gene transfer induced constitutive expression of interleukin- 2 or interferon-gamma in irradiated human melanoma cells

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2817-2825 ◽  
Author(s):  
B Gansbacher ◽  
K Zier ◽  
K Cronin ◽  
PA Hantzopoulos ◽  
B Bouchard ◽  
...  
Keyword(s):  
Gamma Irradiation ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Ifn Gamma ◽  
Melanoma Cell Lines

Abstract Cytokines are important modulators of host antitumor responses. Two of these cytokines, interleukin-2 (IL-2) and interferon gamma (IFN-gamma), are produced after antigen-induced activation of helper lymphocytes. The cytokines are released into the immediate vicinity where they either interact with the appropriate receptors on effector cell populations or are rapidly degraded. To mimic this physiologic release of cytokines at the effector-target site, we used retroviral vectors to transduce melanoma cells with the IL-2 or IFN-gamma cDNA. Five melanoma cell lines were transduced with IL-2- or IFN-gamma-containing vectors and secreted IL-2 at 1 to 40 U/mL/10(6) cells/24 h or IFN-gamma 1 to 8 U/mL/10(6) cells/24 h, respectively. After gamma irradiation, these cells continued to secrete cytokines for about 3 to 4 weeks. Secretion of IFN-gamma induced upregulation of major histocompatibility complex class I molecules in a subset of melanoma cell lines. IL-2 production by human melanoma xenografts induced tumor rejection in BALB/c nu/nu mice, showing the in vivo effect of this cytokine. This study shows that (1) human melanoma cells can be stably transduced with cytokine- containing retroviral vectors; (2) cytokines are secreted constitutively by the transduced tumor cells and have the expected biologic effects in vitro and in vivo; and (3) after gamma irradiation, cytokines continue to be secreted for several weeks. These data suggest that irradiated cytokine-secreting allogenic or autologous tumor cells can be used in vaccination protocols for cancer patients.

scholarly journals Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity.

1990 ◽  
Vol 172 (4) ◽  
pp. 1217-1224 ◽  
Author(s):  
B Gansbacher ◽  
K Zier ◽  
B Daniels ◽  
K Cronin ◽  
R Bannerji ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Tumor Cells ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Cytolytic Activity ◽  
Tumor Formation ◽  
Tumor Bearing ◽  
Low Levels ◽  
Parental Tumor ◽  
Observation Period

To study the effects of localized secretion of cytokines on tumor progression, the gene for human interleukin 2 (IL-2) was introduced via retroviral vectors into CMS-5 cells, a weakly immunogenic mouse fibrosarcoma cell line of BALB/c origin. Secretion of low levels of IL-2 from the tumor cells abrogated their tumorigenicity and induced a long-lasting protective immune response against a challenge with a tumorigenic dose of parental CMS-5 cells. Co-injection of IL-2-producing CMS-5 cells with unmodified tumor cells inhibited tumor formation even when highly tumorigenic doses of CMS-5 cells were used. Cytolytic activity in mice injected with parental CMS-5 cells was transient and was greatly diminished 3 wk after injection, as commonly observed in tumor-bearing animals. However, in mice injected with IL-2-producing cells, tumor-specific cytolytic activity persisted at high levels for the duration of the observation period (at least 75 d). High levels of tumor-specific cytolytic activity could also be detected in parental CMS-5 tumor-bearing animals 18 d after inoculation with tumor cells, if IL-2-producing CMS-5 cells but not unmodified parental tumor cells were used as targets. These studies highlight the potential advantages of localized secretion of cytokines mediated via gene transfer to induce potent anti-tumor immune responses.

Vaccination With Irradiated, Autologous Melanoma Cells Engineered to Secrete Granulocyte-Macrophage Colony-Stimulating Factor by Adenoviral-Mediated Gene Transfer Augments Antitumor Immunity in Patients With Metastatic Melanoma

2003 ◽  
Vol 21 (17) ◽  
pp. 3343-3350 ◽  
Author(s):  
Robert Soiffer ◽  
F. Stephen Hodi ◽  
Frank Haluska ◽  
Ken Jung ◽  
Silke Gillessen ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Metastatic Melanoma ◽  
Tumor Cells ◽  
Antitumor Immunity ◽  
Melanoma Cells ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Purpose: Vaccination with irradiated, autologous melanoma cells engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) by retroviral-mediated gene transfer generates potent antitumor immunity in patients with metastatic melanoma. Further clinical development of this immunization scheme requires simplification of vaccine manufacture. We conducted a phase I clinical trial testing the biologic activity of vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF by adenoviral-mediated gene transfer.Patients and Methods: Excised metastases were processed to single cells, transduced with a replication-defective adenoviral vector encoding GM-CSF, irradiated, and cryopreserved. Individual vaccines were composed of 1 × 106, 4 × 106, or 1 × 107tumor cells, depending on overall yield, and were injected intradermally and subcutaneously at weekly and biweekly intervals.Results: Vaccines were successfully manufactured for 34 (97%) of 35 patients. The average GM-CSF secretion was 745 ng/106cells/24 hours. Toxicities were restricted to grade 1 to 2 local skin reactions. Eight patients were withdrawn early because of rapid disease progression. Vaccination elicited dense dendritic cell, macrophage, granulocyte, and lymphocyte infiltrates at injection sites in 19 of 26 assessable patients. Immunization stimulated the development of delayed-type hypersensitivity reactions to irradiated, dissociated, autologous, nontransduced tumor cells in 17 of 25 patients. Metastatic lesions that were resected after vaccination showed brisk or focal T-lymphocyte and plasma cell infiltrates with tumor necrosis in 10 of 16 patients. One complete, one partial, and one mixed response were noted. Ten patients (29%) are alive, with a minimum follow-up of 36 months; four of these patients have no evidence of disease.Conclusion: Vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF by adenoviral-mediated gene transfer augments antitumor immunity in patients with metastatic melanoma.

scholarly journals Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 670-677 ◽  
Author(s):  
WJ Murphy ◽  
JR Keller ◽  
CL Harrison ◽  
HA Young ◽  
DL Longo
Keyword(s):  
Growth Factors ◽  
Nk Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Hematopoietic Growth Factors ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Growth Promoting

Abstract Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon- gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G- CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.

scholarly journals Rapid activation of proteins that interact with the interferon gamma activation site in response to multiple cytokines

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2063-2071 ◽  
Author(s):  
P Lamb ◽  
LV Kessler ◽  
C Suto ◽  
DE Levy ◽  
HM Seidel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Interferon Gamma ◽  
Regulation Of Gene Expression ◽  
Interleukin 2 ◽  
Sequence Element ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Rapid Changes ◽  
Rapid Activation

Many cytokines and growth factors trigger rapid changes in gene expression upon binding to their receptors. In many cases, the mechanism by which these changes are affected is unknown. In this report, we show that interleukin-2 (IL-2), IL-3, IL-4, IL-6, leukemia inhibitory factor (LIF), erythropoietin (Epo), and granulocyte- macrophage colony-stimulating factor (GM-CSF) treatment of cells causes rapid activation of DNA-binding activities that recognize a DNA sequence element previously implicated in regulation of gene expression by interferon gamma (IFN gamma). The IL-4-, IL-6-, and GM-CSF-induced complexes can be distinguished from the recently characterized IFN gamma-activated protein p91 on the basis of mobility in polyacrylamide gels, sequence preferences, and lack of reactivity with an anti-p91 antiserum. The IL-4- and GM-CSF-induced complexes react with antiphosphotyrosine antibodies, demonstrating the presence of phosphotyrosine-containing proteins in these DNA-binding complexes. Transcriptional activation of a reporter gene linked to a synthetic IFN gamma-responsive promoter is observed in response to IFN gamma, IL-6, and LIF. These data suggest a pathway by which cytokines induce rapid changes in gene expression.

scholarly journals Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 670-677 ◽  
Author(s):  
WJ Murphy ◽  
JR Keller ◽  
CL Harrison ◽  
HA Young ◽  
DL Longo
Keyword(s):  
Growth Factors ◽  
Nk Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Hematopoietic Growth Factors ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Growth Promoting

Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon- gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G- CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.

scholarly journals IP-10, a -C-X-C- chemokine, elicits a potent thymus-dependent antitumor response in vivo.

1993 ◽  
Vol 178 (3) ◽  
pp. 1057-1065 ◽  
Author(s):  
A D Luster ◽  
P Leder
Keyword(s):  
Tumor Cells ◽  
Interferon Gamma ◽  
T Lymphocyte ◽  
Inflammatory Infiltrate ◽  
Antitumor Effect ◽  
Genetically Engineered ◽  
Antitumor Response ◽  
Ifn Gamma ◽  
Biologic Property

IP-10 is a member of the -C-X-C-chemokine superfamily of proinflammatory cytokines whose secretion is induced by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). To date no function has been described for IP-10. We have genetically engineered tumor cells to secrete high levels of murine IP-10 and demonstrate that while IP-10 has no effect on the growth of these tumor cells in culture, it elicits a powerful host-mediated antitumor effect in vivo. The IP-10 antitumor response is T lymphocyte dependent, non-cell autonomous, and appears to be mediated by the recruitment of an inflammatory infiltrate composed of lymphocytes, neutrophils, and monocytes. These results document an important biologic property of IP-10 and raise the possibility that some of the T cell-directed effects of IFN-gamma and LPS may be mediated by this chemokine.

Synthetic Vascular Grafts Seeded with Genetically Modified Endothelium in the Dog: Evaluation of the Effect of Seeding Technique and Retroviral Vector on Cell Persistence in Vivo

1995 ◽  
Vol 4 (2) ◽  
pp. 219-235 ◽  
Author(s):  
Jill E. Sackman ◽  
Michael B. Freeman ◽  
Mark G. Petersen ◽  
Zuhair Allebban ◽  
Glenn P. Niemeyer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Gene Sequence ◽  
Genetically Modified ◽  
Vascular Grafts ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Marker Genes ◽  
Growth Hormone Gene

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial NeoR gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) pre-clotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 × 103 to 1.5 × 105 cells/cm2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 μg/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the NeoR gene sequence at 2 and 3 wk. In contrast to transduced EC's, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.

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