Abstract:
The development of effective gene therapies using cytokines requires an understanding of the differential effects of individual cytokines as paracrine agents during the course of an immune response. We have created retroviral vectors based on the modified Maloney retroviral vector, MFG expressing a single, double (DFG), or triple (TFG) tandem array of genes as a single polycistronic transcript, utilizing the EMCV internal ribosomal entry sequence (IRES). High level production of the p40 and p35 chains of mIL-12, B7.1, vIL-10 or mIL-10 along with NeoR in murine fibroblasts or tumors was used to dissect the biology of these interesting cytokines. Systemic administration of rmIL-12 or rhIL-10 was effective in the eradication of murine tumors at doses of 0.1 ug/d and 40 ug/d, respectively, and required the presence of a radiation sensitive host effector. For IL-12, either CD4+ or CD8+ cells were required; asialo-GM1+ cells appeared to play no role and IFN gamma but not TNF alpha production was required using in vivo antibody neutralization studies. Using paracrine delivery by transfected fibroblasts or tumors, mIL-12 production (150 ng/10(6) cells/24 hr) was sufficient to prevent outgrowth of the immunogenic MCA-207 and poorly immunogenic MCA-102. Depletion of CD4+/CD8+ cells prevented acquisition of long term protective immunity but not the delay in tumor growth observed with local IL-12 production. Depletion of NK cells delayed the ultimate rejection of tumor and with CD4+/CD8+ depletion, all antitumor effects. B7.1 expression enhanced the antitumor effects of IL-12. vIL-10 is the EBV gene now believed to be expressed early following B-cell infection and during latency. Transfection of highly immunogenic tumors with vIL-10 allowed rapid growth and prevented effective eradication. mIL-10 expression conversely was associated with uniform development of tumors but late eradication. Systemic delivery of IL-2 or IL-12 could overcome the local immunosuppressive effects of vIL-10 production. Development of vIL-10, perhaps in association with the HSV-1 ICP47 gene product which prevents peptide assembly with Class I MHC molecules, has promise for the gene therapy of transplantation and that of hIL-12 for cancer gene therapy.
Keywords: Animal Cytokines/*GENETICS CD4-CD8 Ratio *Gene Therapy Mice Neoplasms, Experimental/*THERAPY Receptors, Antigen, T-Cell, gamma-delta/*GENETICS *Transplantation JOURNAL ARTICLE
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