Construction and characterization of retroviral vectors expressing biologically active human interleukin-12.
Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hIL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neoR) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/10(6) cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/10(6) cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-gamma (IFN-gamma) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer.
Zitvogel
L
L
Department of Surgery, University of Pittsburgh School of Medicine, PA 15261.
Tahara
H
H
Cai
Q
Q
Storkus
W J
WJ
Muller
G
G
Wolf
S F
SF
Gately
M
M
Robbins
P D
PD
Lotze
M T
MT
D000818:Animals; D001704:Biopolymers; D018076:DNA, Complementary / Q000096:biosynthesis; D004686:Encephalomyocarditis virus / Q000235:genetics*; D005814:Genes, Viral; D005815:Genetic Code; D005819:Genetic Markers; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D018664:Interleukin-12 / Q000096:biosynthesis*; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics
D001704:Biopolymers; D018076:DNA, Complementary; D005819:Genetic Markers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D018664:Interleukin-12; D007371:Interferon-gamma