Volume 7, Issue 1, Pages 62-72 (January 2003) An oncolytic measles virus engineered to enter cells through the CD20 antigen  Amanda D Bucheit, Shaji Kumar, Deanna M Grote, Yukang Lin, Veronika von Messling, Roberto B Cattaneo, Adele K Fielding  Molecular Therapy  Volume 7, Issue 1, Pages 62-72 (January 2003) DOI: 10.1016/S1525-0016(02)00033-3 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Schematic representation of the MV genome, a cloning intermediate plasmid, and the amino acid sequences of the H protein–αCD20 sequence junction. (A) A schematic representation of the full-length MV cDNA. The genes are denoted by open boxes and annotated by the letters N (nucleocapsid), P (phosphoprotein), M (matrix), F (fusion), H (hemagglutinin), and L (polymerase). The intergenic regions are shown in black. (B) An expanded schematic of the H-fusion proteins. H is shown by the open box, the linker region by the hatched box, and the scFV anti-CD20 antibody by the shaded box. (C) The amino acid sequences of the C terminus of MV-H followed by the SfiI restriction site, the linker sequence, and the sequence of the N terminus of the scFV anti-CD20 antibody. Molecular Therapy 2003 7, 62-72DOI: (10.1016/S1525-0016(02)00033-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 HαCD20 expression plasmids efficiently induce syncytium formation in CHOCD20 but not in unmodified CHO cells. (A) Flow-cytometric analysis of CHO (gray line) and CHOCD20 cells (black line) after incubation with an anti-CD20 antibody. (B) The number of syncytia formed and the distribution of syncytial size after co-transfection of plasmids encoding F protein with either H or the CD20- targeted H proteins into Vero, CHO, and CHOCD20 cells. The total number of syncytia is shown on the y-axis. The dotted bars represent syncytia with < 5 nuclei; dark gray bars 6–20 nuclei; hatched bars, 21–50 nuclei; black bars, > 50 nuclei. The pairs of plasmids transfected in each case are shown beneath the bars. (C) Photomicrograph of representative fields of view. The cell lines are named on the left and the plasmid used are denoted along the top. Molecular Therapy 2003 7, 62-72DOI: (10.1016/S1525-0016(02)00033-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 The chimeric H-anti-CD20 proteins are efficiently incorporated into the envelope of MV virions, and the Factor Xa protease cleavage site in MVHXαCD20 is efficiently cleaved by Factor Xa protease. (A) Immunoblot of sucrose gradient–purified MVNSe and MVHXαCD20 revealed with an antibody against H glycoprotein. The purified viruses were treated with varying concentrations of Factor Xa protease before loading. The concentration of Factor Xa in μg/μl is shown at the top of the blot. The numbers on the left refer to the molecular weight in kDA. Lanes 1–4, MVNSe; lanes 5–8, MVHXαCD20. (B) Immunoblot of sucrose gradient–purified MVNSe, MVHαCD20, and MVHXαCD20 revealed with an antibody against MV-H glycoprotein. The purified viruses were either treated with 50 μg/μl of Factor Xa protease or left untreated. The presence or absence of Factor Xa is denoted by a − or + sign above the lane. Molecular Therapy 2003 7, 62-72DOI: (10.1016/S1525-0016(02)00033-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 MVHαCD20 binds specifically to CHOCD20 cells. A flow-cytometric virus-binding assay was carried out on CHO and CHOCD20 cells. Histograms representing fluorescence intensity are shown for (A) CHO and (B) CHOCD20. The shaded histogram represents the no-virus control (primary and secondary antibodies only), and the line histogram represents the binding of MVHαCD20. Molecular Therapy 2003 7, 62-72DOI: (10.1016/S1525-0016(02)00033-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 MVHαCD20 and MVHXαCD20 enter CHO cells through human CD20. (A) Photomicrographs of CHO and CHOCD20 after infection with MVNSe and MVαCD20. (B) Titers of virus obtained 48 hours after infection of Vero cells and CHOCD20 cells with MVNSe, MV HαCD20, or MVHXαCD20 after treatment with 0 (dotted bars), 5 (black bars), or 50 (hatched bars) μg/μl of Factor. Xa protease. Virus titer at 48 hours after infection is shown on the y-axis. Each set of bars is labeled below with the input virus. Molecular Therapy 2003 7, 62-72DOI: (10.1016/S1525-0016(02)00033-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 MVHαCD20 has the same growth kinetics as the nontargeted MVNSe on a human cell line expressing high amounts of human CD20. (A) Flow-cytometric analysis of HT1080 (gray line) and HT1080CD20 cells (black line) after incubation with an anti-CD20 antibody. (B) Growth curves of MVNSe and MVHαCD20 on HT1080 and HT1080CD20 cells. The y-axis shows virus titer, the x-axis hours after infection. The black line represents the growth curve for MVNSe and the gray line that for MVHαCD20. Molecular Therapy 2003 7, 62-72DOI: (10.1016/S1525-0016(02)00033-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 7 MVHαCD20 suppresses the growth of human CD20+ tumors more efficiently than does a nontargeted MV. (A) Kaplan–Meier analysis of time taken to reach a tumor volume of 0.6 cm3. Bilateral human HT1080 xenografts were established, one side expressing CD20 and the other being CD20−. Mice were injected i.p. with either MVNSe or MVHαCD20. The curves are labeled a–d, defined in the figure, represent the four different experimental situations. (B) Graph showing the mean (± SEM) ratio of tumor volumes for each of the HT1080 and HT1080CD20 tumors, normalized for MVNSe injection. (C) Bar chart showing titers of replicating MV (pfu / g of tumor tissue) recovered from excised CD20+ and CD20− HT1080 tumor xenografts. Each set of bars is labeled beneath with the input virus and the tumor type. Molecular Therapy 2003 7, 62-72DOI: (10.1016/S1525-0016(02)00033-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions