Construction and Biological Characterization of an Interleukin-12 Fusion Protein (Flexi-12): Delivery to Acute Myeloid Leukemic Blasts Using Adeno-Associated Virus

Abstract

Interleukin-12 (IL-12) is a cytokine that exhibits pleiotropic effects on lymphocytes and natural killer cells and has been shown to have promise for the immunotherapy of cancer. The combination of the immune costimulatory molecule B7.1 and IL-12 has been shown to be synergistic for T cell activation. By transfecting tumor cells with both IL-12 and B7.1 cDNAs, it may be possible to use these modified targets as vaccines. A major obstacle in designing a vector to deliver these genes results from the structure of IL-12. Functional IL-12 is a heterodimer composed of two distinct subunits that are encoded by separate genes on different chromosomes. Production of functional IL-12 requires the coordinated expression of both genes. This presents several problems in vectors, particularly those in which additional genes, either a co-stimulatory gene or a selectable marker, are inserted. Therefore, we have constructed a single cDNA that encodes a single-chain protein, called Flexi-12, which retains all of the biological characteristics of recombinant IL-12 (rIL-12). The monomeric polypeptide Flexi-12 is able to induce the proliferation of phytohemagglutinin (PHA) blasts, induce PHA blasts to secrete interferon-gamma (IFN-gamma) and additionally, by preincubation, enhance the killing of K562 targets by PBLs. These phenomena are in a dose-dependent manner comparable to that seen with rIL-12. We have also shown that tyrosine phosphorylation of the STAT 4 transcription factor, which has been shown to be unique to the IL-12 signaling pathway, occurs with Flexi-12 at levels similar to those seen with rIL-12. We have packaged Flexi-12 into a recombinant adeno-associated virus (AAV) and used this vector to infect acute myeloid leukemic (AML) blasts. Infected AML blasts produced between 2 and 6 ng of IL-12/10(6) cells per ml per 48 hr. These studies also confirm that AAV is an efficient delivery vehicle for cytokines to leukemic cells. Direct analysis of these modified cells acting as tumor vaccines is underway.

A strategy for treatment of Epstein–Barr virus-positive Hodgkin's disease by targeting interleukin 12 to the tumor environment using tumor antigen-specific T cells

“…36,21 Our data confirm that this approach leads to production of high levels of biologically functional IL12 with little evident impairment of T-cell behavior.…”

“…Flexi-IL12, first described by Anderson et al 21 consists of the p35 and p40 subunits of IL12 connected by a flexible linker. The linker sequence enables both subunits to fold in their natural three-dimensional structure and the single polypeptide has the biological activity of human IL12 (p70).…”

Improved antitumour immunity in murine neuroblastoma using a combination of IL-2 and IL-12

“…The plasmid pCMV-Flexi12 encodes the cDNA for both human IL-12 genes (p35 and p40) as a fusion protein (Anderson et al, 1997). pCI-IL2 plasmid carrying the human IL-2 cDNA was constructed by incorporation of the hIL2 cDNA into the EcoRI-NotI restriction sites of the pCI plasmid (Promega, Southampton, UK).…”

Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12)

“…57 AAV vectors have several potential advantages over retroviral and adenoviral gene transfer vectors and recently, an rAAV vector expressing a human IL-12 fusion protein has been reported and characterized. 51 At 48 hours following infection of human acute myelocytic leukemia blasts with this vector, which did not contain a selectable marker, 2-6 ng of biologically functional human IL-12/mL/10 6 cells/48 hours was produced. Unlike mIL-12 which maintains activity on both mouse and human lymphocytes, human IL-12 is biologically inactive on murine cells.…”

“…Recently, an rAAV vector expressing human IL-12 has been reported. 51 Because human IL-12 is nonfunctional in mice, this vector cannot be used to model the effects of the AAV vector-mediated expression of IL-12 in vivo against murine tumor models.…”