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

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 co-stimulatory 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-γ (IFN-γ) 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-12/10⁶ 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.

Overview summary

Preclinical studies have shown that interleukin-12 (IL-12) may be effective in the treatment of cancer. Studies have shown tumor regression when IL-12 has been administered by injection directly into or close to the site of a tumor. However, the best results have been seen when tumors have been modified to secrete IL-12. Development of vectors containing IL-12 is difficult because bioactive IL-12 is comprised of two polypeptides, each derived from separate genes. Therefore, we used a linker sequence to create a single cDNA yielding a single-chain polypeptide, which gives bioactive IL-12. This fusion gene, called Flexi-12, has been packaged into an adeno-associated viral vector and then used to deliver IL-12 to cryopreserved acute myeloid leukemic (AML) blasts. These AML blasts express high levels of bioactive IL-12 and will be used to generate cancer vaccines.