RFUSIN2 - a Clinical Grade Lentiviral Vector Co-Expressing CD80/IL-2 Manufactured Under GMP for a Phase I Clinical Trial Study of Immune Gene Therapy for Poor Prognosis Acute Myeloid Leukaemia

Patient derived primary AML blasts cells can be efficiently modified (40–100%) to coexpress CD80 and IL-2 following a single round of transduction using a self-inactivating lentiviral vector. This modification produces significant immune stimulation as measured by proliferation, cytokine release and NK and T-cell cytolytic activity against AML in both autologous and allogeneic settings in vitro. In mouse models of leukaemia, CD80/IL-2 modified leukaemic cells result in rejection of previously established leukaemia and the longer survival of the vaccinated animals. Following regulatory approval in the UK, we have initiated a Phase I clinical study using Lentivirus Transduced AML cells expressing CD80 and IL-2 for the potential enhancement of Graft versus Leukaemia effect in poor prognosis AML. The UK regulatory approved vector, RFUSIN2, is a self-inactivating lentivirus vector, which becomes immobile following vector integration. RFUSIN2 encodes CD80 and IL-2 as a fusion protein, which is endogenously cleaved to generate biologically active CD80 that is membrane anchored and IL-2 which is secreted. Here we describe our in-house manufacturing of RFUSIN2 under EU compliant GMP conditions, which resulted in the production of >3x10¹⁰ concentrated infectious particles. Extensive safety characterisation of this vector has been carried out. The vector supernatant batch is free of viral contaminants including: Porcine and bovine viruses, other retroviruses, HepA/B/C, HIV-I/II, EBV, and CMV; it is also free of other contaminants including mycoplasma, yeast, fungus and bacteria. RFUSIN2 supernatant is absent of any replication competent lentivirus (RCL) or partial recombinants and is unable to mobilise or to achieve secondary transfer.We present a summary of the protocol for the generation of the autologous AML cell vaccine. The clinical protocol, encompassing the treatment schedule, cohorts, study end points and analytical assays are described.