Kuntz, Gabriele

Abstract Effective cancer therapy with a minimum of undesired side effects in multiple myeloma (MM) requires specifity of the therapeutic agent against the neoplastic cells. One approach to reach this aim is virotherapy using oncolytic measles virus. Attenuated replication-competent Edmonston lineage strains of measles virus (MV-Edm) have proven anti-tumor activity against xenograft models of human multiple myeloma, ovarian cancer, lymphoma and glioma. The virus is selectively oncolytic, causing extensive lethal cell to cell fusion via CD46, which is more highly expressed on tumor cells than on normal cells. However, MV-Edm retains the capacity to infect a variety of nontransformed cell types via its native receptors, CD46 and SLAM which are present on many different cell types. One approach to avoid this risk is to engineer the viral attachment protein to ablate its natural tropisms and at the same time, redirect its specificity to interact with alternative tumor specific receptors. The native measles Hemagglutinin (H) protein recognizes CD46 or SLAM resulting in membrane fusion and syncytia formation of cells. In this work the H protein was engineered to restrict and retarget membrane fusion through the display of a single-chain antibody (scFv) recognizing the Wue-1-antigen known to be highly specific for MM cells and abrogation of native measles binding domains for CD46 and SLAM by mutation. This modified H protein (chimeric H) was cloned in a full-length viral backbone including EGFP enabling to detect infected cells and syncytia formation under the UV light emitting green fluorescence (EGFP). On the basis of the parental measles virus expressing EGFP (MV-GFP) two different viruses were generated: non-ablated virus expressing the chimeric H protein including the scFv Wue-1 still competent to infect cells expressing CD46 and SLAM (MV-W1) with the chimeric H protein but ablated for the interactions with CD46 and SLAM (MV-W2). The genetically modified viruses propagated as recently described (Nat. Biotech., Vol.23, Nr.2, Feb.2005, pp.209–214.). To determine if the fully retargeted MV-Edm would be able to infect MM cell lines selectively a first array of infection assays was performed using the MM cell lines RPMI 8226 and ARH-77 expressing the Wue-1 antigen as expected targets and K562 and healthy CD40L activated CD19 positive B cells as controls. 24 to 96 hours after infection with MV-GFP, MV-W1 and MV-W2 we observed syncytia formation and expression of EGFP with MV-GFP and MV-W1 in all cells indicating that the modification of the virus with the scFv-Wue-1 doesn’t alter the potential to infect and kill cells compared to the parental virus. In contrast MV-W2 was able to form only EGFP positive plaques with the Wue-1 antigen positive cells RPMI 8226 and ARH-77 but did not infect K562 or CD19 positve B cells both negative for Wue-1 antigen. These results indicate that the measles virus vaccine strain Edmonston B can be modified to express a scFv recognizing the Wue-1 antigen. In addition the native H protein can be mutated resulting in ablation of the natural tropism towards CD46 and SLAM positive cells. Viruses with these modifications can be rescued and propagated in vitro and selectively infected Multiple Myeloma cells without causing damage to normal B-cell progenitors.

The applicability of cytoreductive treatment of malignant diseases using recombinant viruses strongly depends on specific recognition of surface receptors to target exclusively neoplastic cells. A recently generated monoclonal antibody (mAb), Wue-1, specifically detects CD138+ multiple myeloma (MM) cells. In this study, a haemagglutinin (H) protein that was receptor-blinded (i.e. did not bind to CD46 and CD150) was genetically re-engineered by fusing it to a single-chain antibody fragment (scFv) derived from the Wue-1 mAb open reading frame (scFv-Wue), resulting in the recombinant retargeted measles virus (MV)-Wue. MV-Wue efficiently targeted and fully replicated in primary MM cells, reaching titres similar to those seen with non-retargeted viruses. In agreement with its altered receptor specificity, infection of target cells was no longer dependent on CD150 or CD46, but was restricted to cells that had been labelled with Wue-1 mAb. Importantly, infection with MV-Wue rapidly induced apoptosis in CD138+ malignant plasma cell targets. MV-Wue is the first fully retargeted MV using the restricted interaction between Wue-1 mAb and primary MM cells specifically to infect, replicate in and deplete malignant plasma cells.