Volume 15, Issue 1, Pages 114-122 (January 2007) Infected Cell Carriers: A New Strategy for Systemic Delivery of Oncolytic Measles Viruses in Cancer Virotherapy  Ianko D Iankov, Boris Blechacz, Chunsheng Liu, Jeffrey D Schmeckpeper, James E Tarara, Mark J Federspiel, Noel Caplice, Stephen J Russell  Molecular Therapy  Volume 15, Issue 1, Pages 114-122 (January 2007) DOI: 10.1038/sj.mt.6300020 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Heterofusion of measles-infected carrier cells is resistant to antibody neutralization. Transfer of MV-GFP infection (MOI=0.2) from Ug-C811 (U-937 green clone) cells to RFP+ovarian cancer cell clones SR-B2 (a) and SR-A3 (b) in the presence of 1:100 diluted highly neutralizing human serum. (c and d) Similar results obtained with infected human OECs (confocal microscopy). As in the previous experiments, cell carriers were treated with antibody and complement to prevent infection by cell-free virions or cell-surface virions. Number of GFP+syncitia per well was counted on days 2 and 3 using fluorescent and confocal microscopy. The samples were run in 4–6 wells and the experiment was repeated twice. In (e) infected U-937 were overlaid on SR-B2 cells and cultured in the presence of different dilutions of highly neutralizing serum. Heterofusion was 16-32 times more resistant to neutralization than cell-free virions as determined by the VN test. MV-GFP-infected (MOI=0.5) U-937 (103) were overlaid on SR-B2 cells and cultured in the presence of different dilutions of highly neutralizing human serum. Control samples of infected delivery cells were cultured in the presence of fusion inhibitory peptide to calculate the number of infected cells by flow cytometry. The efficiency of infection in U-937 was 68%. The VN test with 103 PFU of cell-free MV-GFP was used as control. At a dilution of 1:20 the same serum antibodies had no effect on the size of syncytia in the target ovarian cancer cells overlaid with (f) MV-GFP-infected U-937 (fluorescent microscopy). MV-GFP absorbed on the surface of J774A.1 mouse macrophages was able to infect Vero cells after overlaying (left) but was completely neutralized (right) by (g) MAb CL48 and complement treatment. Molecular Therapy 2007 15, 114-122DOI: (10.1038/sj.mt.6300020) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 In vivo heterofusion in the peritoneal cavity. MV-NIS-infected UR-D7 cells (RFP+clone of U-937 monocytes) were administered i.p. to SCID mice bearing i.p. GFP-expressing KAS6/1 myeloma xenografts. The results show cells isolated from three representative animals (in rows) by peritoneal lavage 2 days after injection of MV delivery cells. The percentage of double-positive (GFP/RFP) cells varied between 0.31 and 5.87% in individual animals (five per group). Molecular Therapy 2007 15, 114-122DOI: (10.1038/sj.mt.6300020) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Systemic and i.p. in vivo delivery of MV-GFP infection using different cell carriers. MV-GFP-infected cells (U-937 or PBMCs) were administered i.v. to mice bearing systemic RR-E63 (RFP+Raji cells) lymphoma xenografts. GFP-positive syncytia were seen in meningeal infiltrates 3 days after i.v. injection of infected (a) U-937 cells or (b) PBMCs demonstrated by confocal microscopy. (c) Large multinucleated syncytia were seen in an i.p. SR-B2 (RFP+SKOV3ip.1 clone) ovarian cancer tumor model 3 days after injection of 2 × 106 MV-GFP-infected U-937 cells. Massive MV infection in the tumors was observed in all mice engrafted i.p. with HUH-7 hepatocellular carcinoma cells 3 days after i.p. injection of the carrier cells. Green fluorescent lesions (white arrows) in representative animals from groups treated with MV-GFP-infected (d) U-937 cells or (e) OEC. GFP-positive tumor lesions were also observed at (f) the liver in all mice and large syncytia were demonstrated by (g) confocal microscopy. Samples from the GFP-positive tumors were overlaid on Vero monolayers and MV-GFP was isolated. Molecular Therapy 2007 15, 114-122DOI: (10.1038/sj.mt.6300020) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Therapeutic potency of MV-GFP-infected carrier cells in an ovarian cancer model. Nude mice were engrafted i.p. with SKOV3ip.1 cells and treatment was started 10 days later using five repeated i.p. injections of (a) MV-GFP-infected U-937 (RFP+clone UR-D7) cells. Median survival was significantly (P<0.001) prolonged for both groups of carrier-cell-treated animals (UR-D7>70 days) compared with the controls (median survival, 24 days). (b) The efficacy of a single injection of cell carriers was compared with multiple applications in SCID mice engrafted i.p. with SKOV3ip.1 tumors. The therapy was started on day 7 using infected UR-D7 monocytic cell carriers. Control mice received non-infected delivery cells on day 7. A single therapeutic injection significantly (P<0.001) improved animal survival more than twice (median survival, 60 days) compared with the control group (median survival, 26 days). All mice treated with repeated injections of MV-infected carrier cells survived without symptoms or site injection tumors for more than 80 days. Ten mice per group were used in both experiments. Molecular Therapy 2007 15, 114-122DOI: (10.1038/sj.mt.6300020) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 MV-infected cells, but not naked MVs, can bypass measles neutralizing antibodies in i.v. and i.p. delivery models. Using the systemic Raji (RFP+RR-E63 cells) tumor model, (a) MV-GFP-infected PBMCs or (b) U-937 successfully delivered the virus to RR-E63 lesions in the central nerve system in the presence of 1:4–1:8 neutralizing human antibodies in the plasma. In the presence of neutralizing antibodies, (c) MV-GFP-infected OEC, or (d) U-937 cells successfully delivered infection to i.p. HUH-7 tumors. In contrast, (e) cell-free MV was completely neutralized and no GFP+ was observed compared with (f) control, cell-free MV without antibodies. (g) Large GFP-positive tumor lesions and syncytia were observed in all animals treated with cell-associated MV. (h) MV-GFP could be recovered from all GFP-positive lesions after overlaying the tumor samples on Vero monolayers. Molecular Therapy 2007 15, 114-122DOI: (10.1038/sj.mt.6300020) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions