Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2 Fusion cDNA for Cancer Gene Immunotherapy

Abstract

Genetic engineering of tumor cells to express both granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-2 can induce synergistic immune antitumor effects. Paradoxically, the combination has also been reported to down-regulate certain immune functions, highlighting the unpredictability of dual cytokine use. We hypothesized that a GM-CSF and IL-2 fusion transgene (GIFT) could circumvent such limitations yet preserve synergistic features. We designed a fusion cDNA of murine GM-CSF and IL-2. Protein structure computer modeling of GIFT protein predicted for intact ligand binding domains for both cytokines. B16 mouse melanoma cells were gene modified to express GIFT (B16GIFT), and these cells were unable to form tumors in C57bl/6 mice. Irradiated B16GIFT whole-cell tumor vaccine could also induce absolute protective immunity against challenge by live B16 cells. In mice with established melanoma, B16GIFT therapeutic cellular vaccine significantly improved tumor-free survival when compared with B16 expressing both IL-2 and GM-CSF. We show that GIFT induced a significantly greater tumor site recruitment of macrophages than combined GM-CSF and IL-2 and that macrophage recruitment arises from novel chemotactic feature of GIFT. In contrast to suppression by GM-CSF of natural killer (NK) cell recruitment despite coexpression of IL-2, GIFT leads to significant functional NK cell infiltration as confirmed in NK-defective beige mice. In conclusion, we demonstrated that a fusion between GM-CSF and IL-2 can invoke greater antitumor effect than both cytokines in combination, and novel immunobiological properties can arise from such chimeric constructs.

An Engineered GM-CSF-CCL2 Fusokine Is a Potent Inhibitor of CCR2-Driven Inflammation As Demonstrated in a Murine Model of Inflammatory Arthritis

“…1C). Furthermore, we have previously demonstrated that the N-terminal GM-CSF component of two distinct fusokines (GIFT2 and GIFT15) will bind to GM-CSF receptor and will act as a mitogen for GM-CSF-dependent JAWSII cells and will also lead to normal downstream signaling (as represented by STAT5 phosphorylation) akin to monomeric GM-CSF (10,11). We were unable to make similar analysis of GMME1 due to its pro-apoptotic properties on JAWSII cells in vitro (data not shown).…”

“…GM-CSF was selected as the N-terminal partner because it is permissive for high fusokine secretion by transfected cells (10,11). Furthermore, GM-CSF has a fairly long plasma half-life reported to be ϳ6 h (12,13), which may markedly extend the in vivo bioavailability of C-terminal domains such as truncated CCL2.…”

GM-CSF–Based Fusion Cytokines as Ligands for Immune Modulation

“…To evaluate whether the distinct GM-CSF and IL-2 domains of GIFT-2 were functional, GIFT-2 was applied to the JAWSII and CTLL-2 cell lines to evaluate whether the fusokine could replicate GM-CSF's and IL-2's respective abilities to induce their proliferation. IL-2 was as effective as its unfused counterparts at maintaining these cells lines in culture; however, contrary to pIXY 321, GIFT-2 did not significantly enhance their proliferation (41). In vitro investigations into GIFT-2's ability to mediate tumor rejection in mice found that GIFT-2-stimulated NK cells adopted a novel proinflammatory phenotype (42); these NK cells produced significantly more IFN-g than did cells treated with GM-CSF, IL-2, or the combination of the two, and they were highly cytolytic and resistant to GM-CSF's regulatory effects (43).…”

“…Despite the overlapping functions of their source material, the GIFTs differed remarkably from one another in terms of their signaling and their net effects. GIFT-2 retained an IL-2-like phenotype, but with the hyperinduction of STAT1, STAT3, and STAT5 (41). GIFT-15 completely altered IL-15R signaling because it induced a hyperphosphorylation of STAT3 and a hypophosphorylation of STAT5 (44) We speculate that GIFT-15's higher affinity for the IL-15Ra-chain and the GM-CSF moiety have altered receptor-binding properties in terms of how GIFT-15 fits into the IL-15-binding cleft (44), but it is not obvious how this alters the phosphorylation of Jaks and STATs further downstream.…”

Protective Immunity against Bordetella pertussis by a Recombinant DNA Vaccine and the Effect of Coinjection with a Granulocyte‐Macrophage Colony Stimulating Factor Gene

“…GM-CSF is a hematopoietic growth factor that stimulates neutrophil, monocyte/macrophage, and eosinophil colony formation, and has been applied in a variety of clinical and research settings (14,16). GM-CSF can elicit high cytotoxic T cell immune response in DNA vaccine against hepatitis C virus (HCV, 23), tuberculosis (TB, 33) and tumors (29).…”