Volume 21, Issue 2, Pages 291-299 (February 2013) Transient Removal of CD46 Is Safe and Increases B-cell Depletion by Rituximab in CD46 Transgenic Mice and Macaques  Ines Beyer, Hua Cao, Jonas Persson, Hongjie Wang, Ying Liu, Roma Yumul, Zongyi Li, Douglas Woodle, Ronald Manger, Michael Gough, Diane Rocha, Jaclyn Bogue, Audrey Baldessari, Ronald Berenson, Darrick Carter, André Lieber  Molecular Therapy  Volume 21, Issue 2, Pages 291-299 (February 2013) DOI: 10.1038/mt.2012.212 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Studies with human cell lines. (a–c) Ad35K++ enhances complement-dependent cytotoxicity triggered by (b) alemtuzumab and (c) trastuzumab in vitro. Tumor cells expressing the corresponding monoclonal antibodies (mAb) targets were treated with mAb (15 µg/ml) and/or Ad35K++ (25 µg/ml). In previous studies, Ad35K++ -mediated decrease of CD46 from the cell surface was highest at 8–10 hours after addition of Ad35K++.13 On the basis of this, the interval between Ad35K++ and mAb addition was selected. Normal human serum (NHS) was added as a source of complement in the indicated groups. Cell viability of phosphate-buffered saline-treated cells was adjusted to 100%. The right most bars show the effect of the combination therapy and can be compared with the third bar from the left as the mAb alone (+NHS). N = 3. The difference between mAb/NHS vs. Ad35K++ plus mAb/NHS is significant (P < 0.01). (d,e) Ad35K++ improves trastuzumab therapy in vivo. Her2/neu-positive breast cancer cells BT474-M1 were injected into the mammary fat pad. Mice with established tumors were then injected intravenously with Ad35K++ (2 mg/kg) followed by trastuzumab (2 mg/kg) 10 hours later. Treatment was repeated 7 days later. N = 10. The difference between the two groups was significant (P < 0.01) from day 100 on. Molecular Therapy 2013 21, 291-299DOI: (10.1038/mt.2012.212) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Studies with hCD46/hCD20 transgenic mice. (a) Flow cytometry analysis of human CD46 and CD20-expressing mouse lymphoma cell line 38C13-hCD20/CD46. (b) Representative images of tumor localization in transgenic mice. hCD46/CD20 transgenic mice were intravenously injected with syngeneic 38C13-hCD20/CD46C lymphoma cells. Tissues were analyzed 1 week later; the upper image shows the presence of human CD46 positive tumor cells in brown in the bone marrow. The lower image shows human CD20-positive liver metastases (green). The scale bars are 20 µm. (c) Survival of hCD46/CD20 transgenic mice carrying 38C13-hCD46/CD20 lymphoma cells. Ad35K++ (2 mg/kg) or phosphate-buffered saline (PBS) was injected at day 14, followed 10 hours later by rituximab (2 mg/kg). In one group of mice, a second round of treatment was given 1 week after the first treatment (Ad35K++ plus rituximab) 2x. Onset of hind leg paralysis was used as the end point in Kaplan–Meier survival studies. N = 5. Control vs. rituximab: P = 0.08; rituximab vs. Ad35K++ plus rituximab: P = 0.02. Note that from day 28 on, antibodies against Ad35K++ were detectable by ELISA in serum of Ad35K++ injected mice (see arrow). (d) Anti-Ad35K++ serum antibodies in the group that received two rounds of Ad35K++/rituximab injection. The days of Ad35K++/rituximab injection are indicated on the horizontal axis by arrows. Serum was diluted 1:10 and anti-Ad35K++ antibodies were measured by ELISA. Shown are optical density450 absorption values. N = 5. (e) Immune responses against the Hepatitis B virus E antigen (HBeAg) in Ad35K++ or PBS-treated animals. The double transgenic mice were intravenously injected with PBS and Ad35K++ (2 mg/kg). Three days later, intramuscular immunizations with an adenoviral HBe vaccine were performed. Twelve days later, spleens were harvested and splenocytes were stimulated either with soluble HBeAg or control antigen (HBsAg). Twenty-four hours after stimulation, interferon-γ enzyme linked immunosorbent spots were performed. The number of spots was expressed as the average +/− the SD. N = 5 animals per group. PBMC, peripheral blood mononuclear cells. Molecular Therapy 2013 21, 291-299DOI: (10.1038/mt.2012.212) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 In vitro studies with monkey cells. (a) Ad35K++ hemagglutination assay. Serial dilutions of Ad35K++ were incubated with 1% erythrocytes and hemagglutination was assessed 1 hour later. (b) Downregulation of CD46 on Macaca fascicularis peripheral blood mononuclear cells (PBMC). PBMC cells were incubated with 25 µg/ml Ad35K++ for 12 hours. CD46 levels were analyzed using phycoerythrin (PE)-conjugated antibodies. Shown is a representative sample. The CD46 mean fluorescence intensity (MFI) in phosphate-buffered saline (PBS)-treated cells was 280(+/−40) (N = 3). The MFI in Ad35K++ treated cells was 175(+/−22). P < 0.01. (c) B-cell depletion in vitro. PBMCs were purified from M. fascicularis, and cultured for 1 day. CD20-positive cells were sorted via flow cytometry using a (crossreacting) anti-human CD20-PE antibody. For complement-dependent cytotoxicity assays, CD20- positive cells were incubated with 25 µg/ml Ad35K++ or PBS for 10 hours. Rituximab (15 µg/ml) or PBS was added, followed by autologous serum (20% final concentration, as a source of complement) 30 minutes later. Cell viability was measured after 3 hours of incubation based on trypan blue exclusion. N = 8. Rituximab vs. Ad35K++ plus rituximab: P < 0.01. Molecular Therapy 2013 21, 291-299DOI: (10.1038/mt.2012.212) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Analysis of Ad35K++ serum clearance, CD46 on peripheral blood mononuclear cells (PBMCs), and serum cytokine and chemokine levels in animals that received intravenous Ad35K++ (4 mg/kg). (a) Serum concentrations of Ad35K++ after intravenous injection of 4 mg/kg of Ad35K++. (b) CD46 on PBMCs measured by flow cytometry. The percentage of CD46+ cells before injection was taken as 100%. (c) Pretreatment levels were taken as 100%. Interleukin (IL)-1β, IFN-γ, IL-10, IL12/23 were also measured but not detected or unchanged and therefore not graphed. IFN, interferon; MCP, monocyte chemotactic protein. Molecular Therapy 2013 21, 291-299DOI: (10.1038/mt.2012.212) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Rituximab-mediated depletion of peripheral blood CD20-positive cells. (a) Percentage of peripheral blood CD20-positive cells. Pretreatment levels were taken as 100%. The animals were euthanized at day 3 and CD20-positive cells were measured in the spleen and bone marrow. (b) A rituximab dose of 0.01 mg/kg did not significantly reduce the percentage of peripheral CD20-positive cells. The animals were followed for 7 days. (c–f) Ad35K++ enhances rituximab-mediated depletion of peripheral CD20-positive cells. Animals A11020 and A11319 received rituximab (0.01 mg/kg) 2 days after Ad35K++ injection (4 mg/kg). For animals A11314 and A11294 the time interval between the two drugs was 3 days. (c) Peripheral blood CD20-positive cells. Pretreatment levels were taken as 100%. (d) CD20-positive cells were subgated using flow cytometry for CD46high cells. (e,f) Experimental controls: (e) CD20-positive cells in animals that received Ad35K++ only. (f) CD21-positive cells in animals that received the combination Ad35K++ and rituximab. Molecular Therapy 2013 21, 291-299DOI: (10.1038/mt.2012.212) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Ad35K++ and anti-Ad35K++ antibody serum levels in all animals that received intravenous Ad35K++ injection. Note that animal A11319 had antibodies that reacted with Ad35K++. In spite of this, Ad35K++ was functional (see Figure 5c,d). (a) anti-Ad35K++ serum antibodies. Serum was diluted 1:10 and anti-Ad35K++ antibodies were measured by ELISA. Shown are optical density450 absorption values. (b) Ad35K++ concentrations. Molecular Therapy 2013 21, 291-299DOI: (10.1038/mt.2012.212) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions