Tricistronic viral vectors co-expressing interleukin-12 (1L-12) and CD80 (B7-1) for the immunotherapy of cancer: Preclinical studies in myeloma

Abstract

Synergy between interleukin -12 ( IL -12 ) and B7 -1 ( CD80 ) for cancer immunotherapy has previously been demonstrated in animal models of breast cancer, lymphoma, and multiple myeloma. With a view to human clinical application, tricistronic retroviral and adenovirus vectors co -expressing IL -12 ( IL -12p40 plus IL -12p35 ) and CD80 were constructed by utilizing two internal ribosome entry site ( IRES ) sequences to link the three cDNAs. A murine stem cell virus ( MSCV ) -based retroviral vector ( MSCV -hIL12.B7 ) utilized distinct IRES sequences from the encephalomyocarditis virus ( EMCV ) and the foot -and -mouth disease virus ( FMCV ), whereas Ad5 -based adenovirus vectors contained transcriptional units with two EMCV IRES sequences under the control of murine ( AdMh12.B7 ) or human ( AdHh12.B7 ) cytomegalovirus promoters. AdMh12.B7 was found to consistently direct higher levels of IL -12 and CD80 expression than AdHh12.B7 following infection of a number of human tumor cell lines. In preclinical studies, the human myeloma cell line U266 was infected with MSCV -hIL12.B7 and a resulting clonal cell line, U / MSCV -h12.B7, was generated with stable expression of CD80 and secreting IL -12 at 1 ng / 24 h / 10 6 cells. By comparison, following AdMh12.B7 infection, 81% of infected U266 cells ( U / AdMh12.B7 ) expressed CD80 and secreted IL -12 at 25 ± 50 ng / 24 h / 10 6 cells. Both engineered myeloma cell lines stimulated enhanced allogeneic mixed lymphocyte proliferation and provoked increases in cytotoxic T -lymphocyte responses and -interferon release from normal donor lymphocytes exposed to parental U266 cells. These results suggest potential clinical utility of AdMh12.B7 in immunotherapy strategies for the treatment of multiple myeloma and other cancers.

Highly suppressible expression of single‐chain interleukin‐12 by doxycycline following adenoviral infection with a single‐vector Tet‐regulatory system

“…Different pathways of secretion and glycosylation capacities might explain differences in transgene expression by U266. The hCMV-promoter we used provided IL12 expression levels in U266 cells similar to previous findings [41]. However, further studies are necessary to fully explore this phenomenon of transgene-dependent promoter performance.…”

Circumventing Recombination Events Encountered with Production of a Clinical-Grade Adenoviral Vector with a Double-Expression Cassette

“…However, this approach cannot guarantee uniform infection of all cells with both vectors because of the relative inefficiency of gene transfer procedures. Another approach involves the use of bicistronic vectors coexpressing the genes linked by an internal ribosomal entry site (Wen et al, 2001;Wang et al, 2005). Use of this method results in valid correlations in expression, but the absolute level of expression of the second gene is generally reduced.…”

Decreased tumorigenic potential of EphA2-overexpressing breast cancer cells following treatment with adenoviral vectors that express EphrinA1

“…[17][18][19][20] The results of animal studies and clinical trials in humans for cancer therapy have further increased interest in adenoviral-based therapy. [21][22][23][24][25][26] Based on the differential ligand binding that EphA2 demonstrates in normal versus malignant cells, we describe herein the engineering of HAd type 5 (HAd5) vectors that express secreted forms of EphrinA1 and show that these vectors can negatively regulate tumor cell growth in vitro and in vivo.…”