www.sciencemag.org/cgi/content/full/311/5768/1780/DC1

Supporting Online Material for

Synergistic Antitumor Effects of Immune Cell-Viral Biotherapy

Steve H. Thorne, Robert S Negrin, Christopher H Contag*

*To whom correspondence should be addressed, E-mail: ccontag@cmgm.stanford.edu

Published 24 March 2006, Science 311, 1780 (2006).

DOI: 10.1126/science.1121411

This PDF file includes

Materials and Methods Figs. S1 to S7 Reference

www.sciencemag.org/cgi/content/full/311/5768/1780/DC1

Supporting Online Material for

Synergistic Antitumor Effects of Immune Cell-Viral Biotherapy

Steve H. Thorne, Robert S Negrin, Christopher H Contag*

*To whom correspondence should be addressed, E-mail: ccontag@cmgm.stanford.edu

Published 24 March 2006, Science 311, 1780 (2006).

DOI: 10.1126/science.1121411

This PDF file includes

Materials and Methods Figs. S1 to S7 Reference

Science Supplementary Online Material Materials and Methods Cells and viruses. Human ovarian cancer cell lines were SKOV-3 (obtained from ATCC) and UCI-101 (obtained from Drs. P. DiSaia and A. Manetta, University of California). Murine mammary adenocarcinoma cell lines were 4T1 and JC (obtained from ATCC). Stably transduced versions of all cell lines were produced by retroviral transfection in order to produce cells expressing click beetle red luciferase and puromycin (for selection). Growth characteristics of the transfected cells were compared to the parental strains in vitro to verify no effects due to the retroviral insertion. The human B-cell lymphoma cell line (OCI-ly8) has been previously characterized. The expansion of CIK cells has been described previously (S1). The strains of vaccinia virus used were (i) wild type strain Western Reserve (obtained from ATCC); (ii) Western Reserve containing a single deletion in the viral thymidine kinase (TK) gene, and expressing luciferase (provided by D. Bartlett, University of Pittsburg); (iii) Western Reserve containing a single deletion in the viral growth factor (VGF) gene and expressing beta-galactosidase (provided by D Bartlett, originally constructed by B Moss, NIH); and (iv) the double deleted virus (vvDD) containing deletions in both the TK and VGF genes and expressing either green fluorescent protein (GFP) from within the site of the TK gene (provided by D Bartlett) or expressing luciferase from the TK gene (constructed by homologous recombination of the plasmid pSC-65 containing the firefly luciferase gene from pGL3 (Promega, Madison) into the TK gene of the VGF deleted virus). Viral replication and plaque assays. Cell lines to be assayed were infected by addition of virus at the multiplicity of infection (MOI) (plaque forming units (PFUs)/cell) indicated. After 2 hours of infection, the medium was changed and at various times after this samples were taken of either (i) media alone and (ii) cells resuspended in PBS, or else media and cells were sampled together. Cells were lysed by three cycles of freeze/thaw to release intracellular virus. Plaque assay was then performed following serial dilution in 6-well plates on the BS-C-1 cell line (obtained from ATCC). Cell viability assays. Pre-infection of CIK cells was achieved by addition of virus to CIK cells at an MOI of 1.0, waiting 2 hours before centrifuging CIK cells to remove excess virus. Cell lines to be assayed (expressing luciferase) were treated in triplicate with virus, CIK cells or pre-infected CIK cells at different MOI’s (CIK cells or viral PFUs per target cell) and left for the indicated times. Luciferin substrate was then added (10 µl of 30 mg/ml luciferin (Xenogen Corp., Alameda) to 1 ml media), and cell viability measured as light output relative to untreated control plates using an IVIS 50 imaging system (Xenogen). In addition, virus expressing GFP or CIK cells pre-infected with virus expressing GFP were added to unlabelled monolayers of target cells at MOI of 0.05. Viral infection (GFP expression) was then monitored at different time points using a Zeiss Axiovert fluorescence microscope with GFP filters. Cellular cytotoxicity assay. Tumor cell lysis by effector cells was assessed by measurement of the luciferase activity of surviving target cells. Target cells expressing luciferase were plated into black-walled 96-well plates at 1 x 104 cells/well. Effector cells were then added at specified effector-to-target ratios; wells representing all ratios of cells (as well as target only wells) were plated in triplicate and incubated for 4 hours at 37°C, 5%CO2 (N.B. incubation was too short for viral-mediated cytopathic effect to occur). In

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some experiments CIK cells were mixed with NKG2D blocking antibody (20 µg/ml for 30 mins)(R&D Systems, Minneapolis) prior to addition to target cells. Luciferin was then added to each well [2 µl/well of 30 mg/ml luciferin (Xenogen)] and light output (photons·second-1/well) measured on an IVIS 50 imaging system (Xenogen). Percent cytotoxicity was then determined relative to control wells (target only or target only pre-treated with 70% ethanol). Flow cytometry assays. Flow cytometry assays were performed on cells alone or on cells pre-infected with vvDD expressing GFP or vvDD. Cells were then stained with cell surface markers including anti-MHC Class I polypeptide-related sequence A/B (MICA/B) antibody conjugated to PE at a dilution of 1:200, anti-mouse CD3-FITC at 1:50, anti-mouse DX5-PE at 1:50, anti-human CD3-PerCPCy5.5 at 1:20, anti-human CD56-APC at 1:100, anti-human CD16-PE at 1:100, anti-vaccinia polyclonal-FITC at 1:200, anti-mouse NKG2D-PE at 1:50 and anti-human NKG2D-APC at 1:100 (all antibodies from BD Pharmingen, San Diego; Abcam, Cambridge; or eBioscience, San Diego). Intracellular cytokine staining for mouse interferon-gamma was performed after application of golgi stop for 24 hours and permeabilization of cells with cytofix/cytoperm (BD Pharmingen). Staining was with anti-IFNγ XMG1.2-APC (eBioscience). Samples and isotype controls were run on a FACScaliber (Becton Dickinson, San Jose). Mouse tumor models. CD1 athymic nu/nu mice were used for xenograft studies and BALB/c mice were used for syngeneic models. All mice were female, aged 8 to 10 weeks and obtained from Charles River Co. (Wilmington). Mice received subcutaneous (fatpad) or intraperitoneal injections of 1 x 106 tumor cells. Those mice injected subcutaneously received treatment by a single tail vein injection once palpable tumors of 50-100 mm3 were formed (tumor size was determined by caliper measurement or bioluminescence imaging as indicated). Animals injected intraperitoneally received luciferase-labeled tumor cells only and were imaged regularly by bioluminescence imaging. These mice were treated with a single intravenous tail vein injection of therapeutic as described 3 days after tumor implantation. Bioluminescence imaging was performed on days 1 and 3 after tumor implantation to confirm tumor cell signal was increasing prior to treatment. Non-invasive imaging assays. Both bioluminescence and fluorescence imaging modalities were utilized to image tumor cells, virus or CIK cell biodistribution. Animals were anesthetized and injected with luciferin (150 mg/kg) 5 min prior to bioluminescence imaging. Animals were placed on a warmed stage (37°C) and imaged using an IVIS 200 system (Xenogen) for bioluminescence or Cy5.5 fluorescence imaging and a Maestro (Cambridge Research & Instrumentation, Inc., Woburn) for GFP imaging. Whenever fluorescence imaging was used, an initial background image was first taken for background subtraction immediately before the fluorescence image, and no luciferin was injected. Appropriate filter sets were used for Cy5.5 and GFP imaging. Labeling of CIK cells for imaging. CIK cells were either labeled with click beetle red luciferase by retroviral transduction, or with Cy5.5 NHS ester (GE Healthcare, Amersham Biosciences, Pittsburg). Immunofluorescence microscopy. Tumors from mice treated with Cy5.5 labeled CIK cells and/or vvDD-GFP were frozen in optimal cutting temperature compound (OCT) and sectioned (8-10 microns). Tissue slices were fixed in acetone, blocked with 2% fetal calf serum (FCS) and stained using primary antibodies to CD-31 or GFP (Invitrogen, Molecular Probes, Carlsbad) as indicated. Secondary staining was with AlexaFluor

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conjugated antibodies (Invitrogen, Molecular Probes) and nuclear staining with Sytox Blue (Invitrogen, Molecular Probes). Sections were then examined using a Zeiss Axiovert or a Leica Confocal microscope. Reference S1. 1. P. H. Lu, R. S. Negrin, J. Immunol. 153, 1687 (1994).

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Supplementary Figures;

Fig. S1. (A) Replication kinetics of vvDD in human CIK cells. CIK cells were infected with vvDD at an MOI of 1.0 PFU/cell. At the indicated times, samples were taken and the cells were separated from the media by centrifugation. Viral infectious units were titered in both the media (open squares) and cellular (open triangles) fractions. At the same time points we counted total CIK cell numbers in both the vvDD infected cultures (closed squares) and in control uninfected CIK cultures (closed triangles). (B) Immunofluorescence of a UCI-101 human ovarian cancer cell monolayer at the times indicated following addition of vvDD-GFP, alone or within pre-infected CIK cells. (Scale bar, 200 µm)

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Fig. S2. Effect of vaccinia (vvDD) infection on expression of cell surface and functional markers in human and mouse CIK cells. (A) Mouse CIK cells (top panels) labeled with mouse T-cell (CD3) and NK cell (DX5) markers prior to infection with vvDD (0 hour) and at 48 hours after infection (at a multiplicity of infection of 2.0). Human CIK cells (bottom panels) labeled with mouse T-cell (CD3) and NK cell (CD56 and CD16) markers prior to infection with vvDD (0 hour) and at 48 hours after infection (at a multiplicity of infection of 2.0). Expression of CD56 and CD16 on CD3-positive, gated populations are shown. (B) Staining of human CIK cells and human ovarian tumor SKOV-3 cells with a polyclonal anti-vaccinia antibody after infection with vvDD. Human CIK cells are stained 48 hours after infection and SKOV-3 are stained 8 hours after infection. Unshaded graphs are uninfected cells treated with the same antibody (C) Staining with anti-NKG2D antibody. Human CIK cells either uninfected (blue graph) or previously infected with vvDD for 48 hours (red graph) were stained with anti-NKG2D antibody. Unshaded graph is isotype control. (D) Intracellular staining for interferon-gamma. Mouse CIK cells, uninfected (blue graph) or previously infected with vvDD for 48 hours (red graph), were treated with golgi stop for 24 hours before permeabilization and staining with anti-interferon-gamma antibody. Unshaded graph is isotype control

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Fig. S3. UCI-101 or SKOV-3 cells alone or infected with vvDD-GFP for 24 hours were stained with anti-MHC Class I polypeptide-related sequence A/B (MICA/MICB) antibody and the numbers of cells expressing these cell surface markers determined by flow cytometry. Unshaded graphs are isotype controls and results are representative of three experiments.

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Fig. S4. Fluorescence and bioluminescence imaging to monitor trafficking of uninfected and vvDD-infected CIK cells. (A) Immunocompetent BALB/c mice bearing subcutaneous 4T1 tumors (50-100 mm3) were treated with a single tail vein injection of 1 x 107 CIK cells or 1 x 107 CIK cells pre-infected with vvDD. The CIK cells had previously been labeled by covalent conjugation to Cy5.5 dye. Subsequent fluorescence imaging with an IVIS 200 system was used to quantify Cy5.5 signal from within the tumors of these animals (n = 3 mice per group). (B) Immunodeficient (athymic nu/nu) mice carrying subcutaneous SKOV-3 (top panel) or UCI-101 (bottom panel) tumors (arrowed) were treated (day 0) with a single intravenous injection of either 1 x 107 CIK cells labeled with Cy5.5 (left animal) or 1 x 107 Cy5.5 labeled CIK cells infected with vvDD (right animal). Cy5.5 fluorescence was imaged using an IVIS200 system (Xenogen). A PBS control mouse was included for comparison on day 2 (large arrow). (C) In an equivalent experiment, a mouse bearing an UCI-101 tumor was treated with 1 x 107 CIK cells transfected with retrovirus to express luciferase and infected with vvDD (MOI 1.0) on day 0. Bioluminescence was imaged using an IVIS200 system. Small arrows indicate locations of tumors.

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Fig. S5. Immunofluorescence microscopy of MICA/B expression in a treated SKOV-3 tumor. A mouse bearing a subcutaneous SKOV-3 tumor was treated with a tail vein injection of 1 x 107 CIK cells labeled with Cy5.5 and infected with vvDD-GFP (MOI 1.0). Tumors were recovered 48 hours after treatment and frozen sections were stained with (A) a nuclear dye (Sytox Blue, Invitrogen Molecular Probes); (B) anti-GFP antibody: or (C) anti-MICA/B. Fluorescence was imaged, along with Cy5.5 fluorescence (D) using a Leica confocal microscope. An overlay image is also shown (E). Scale bar 100 µm.

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Fig. S6. Immunofluorescence microscopy showing CIK-mediated delivery of vvDD to UCI-101 human ovarian tumor xenografts. A mouse bearing a subcutaneous UCI-101 tumor was treated with a tail vein injection of 1 x 107 human CIK cells infected with vvDD-GFP (MOI 1.0) or 1 x 107 PFU vvDD-GFP. Tumors were recovered and frozen sections were stained, (A) Tumors from animals receiving vvDD-GFP or CIK pre-infected with vvDD-GFP via tail vein injection. Sections (48 hours after treatment) were stained with anti-CD31 (endothelial cell marker) (magnification 400x)(scale bar 50 µm). (B) Mice receiving human CIK cells labeled with cy5.5 fluorescent dye and pre-infected with vvDD-GFP, and tumors recovered after 24 hours. Sections were labeled with a nuclear dye (Sytox Blue, Invitrogen, Molecular Probes) and anti-GFP antibody (green). Fluorescence was imaged, along with Cy5.5 fluorescence (red) using a Leica confocal microscope. An overlay image is shown. (C) as in (B), only tumors recovered 72 hours after treatment. Arrows indicate area of overlapping green and red, indicating infected CIK cells within the tumor (scale bar 100 µm).

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Fig. S7. Inhibition of 4T1 tumor growth by combination therapies. (A) BALB/c mice received an intraperitoneal injection of 1 x 106 4T1 cells labeled with luciferase. Three days after tumor cell injection, mice were treated with a single tail vein injection of PBS alone; or 1 x 107 CIK cells and 1 x 107 vvDD PFU delivered separately; or 1 x 107 CIK cells pre-infected with vvDD. Luciferase expression was measured 3 days after treatment. (B) Quantification of average tumor signal for animals in each group shown in (A). Pre-infected CIK cell treatment significantly reduced tumor burden relative to both control treated animals (**P = 0.0104) and to animals treated with CIK and vvDD separately (*P = 0.0464)(Student’s t test)(Error bars are SEM)