1. Volume 17, Issue 10, Pages 1779-1787 (October 2009)
Immunotherapy for Osteosarcoma: Genetic Modification of T cells Overcomes Low Levels of Tumor Antigen Expression 
Nabil Ahmed, Vita S Salsman, Eric Yvon, Chrystal U Louis, Laszlo Perlaky, Winfried S Wels, Meghan K Dishop, Eugenie E Kleinerman, Martin Pule, Cliona M Rooney, Helen E Heslop, Stephen Gottschalk 
Molecular Therapy 
Volume 17, Issue 10, Pages (October 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Primary osteosarcomas and osteosarcoma cell lines express human epidermal growth factor receptor 2 (HER2) at low levels. (a) Using the HER2-specific mouse monoclonal antibody NCL-L-CB11 (Novocastra, Newcastle Upon Tyne, UK), seven of eight primary osteosarcoma samples representing different histological subtypes showed detectable HER2 expression. One of eight samples did not express HER2 (bottom right panel). (b) Fluorescence-activated cell sorter analysis: osteosarcoma cells lines (U2-OS, Sa-OS, HOS, SK-ES-1, MNNG, Hs 894, Hs 899, LM7, MG-63, SJSA-1, 143b, and ) and breast cancer cell lines (HER2-negative: MDA-MB-468, HER2-low: MCF-7, HER2-high: SK-BR-3) were stained for HER2 expression (isotype control: open curves; HER2: solid curves. Osteosarcoma cell lines expressed HER2 at low levels comparable to MCF-7.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Trastuzumab fails to inhibit the proliferation of human epidermal growth factor receptor 2 (HER2)-positive osteosarcoma cell lines. (a) Tumor cells were cultured for 4 days in the presence of increasing concentrations of trastuzumab prior to performing a proliferation assay. Trastuzumab efficiently inhibited the proliferation of the HER2-high expressing cell line SK-BR-3, where as no inhibition was observed for the HER2-low expressing cell lines Sa-OS, MG-63, HOS, MNNG, LM7 or the HER2-negative cell line MDA-MB-468. Results from two experiments; done in triplicates are shown. (b) To test for complement mediated cytotoxicity, 51Cr-labeled tumor cells incubated for 30–60 minutes with baby-rabbit complement in the presence of increasing concentrations of trastuzumab. While trastuzumab efficiently lysed the HER2-high expressing cell line SK-BR-3, no lysis was observed for the HER2-low expressing cell lines Sa-OS, U2-OS, MG-63, HOS, MNNG, LM7, or the HER2-negative cell line MDA-MB-468. Results from two experiments done in triplicates are shown.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Characterization of human epidermal growth factor receptor 2 (HER2) CAR.CD28.ζ T cells. (a) Scheme of SFG retroviral vector encoding HER2.CD28.ζ CAR. (b) Depending on the donor, a median of 72% (range 45–92%) of T cells transduced with the HER2.CD28.ζ CAR retrovirus were positive for the transgene as judged by fluorescence-activated cell sorter analysis. CAR, chimeric antigen receptor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Human epidermal growth factor receptor 2 (HER2)-specific T cells secrete immunostimulatory cytokines, proliferate and kill HER2-expressing osteosarcoma cells in coculture. HER2-specific T cells were stimulated with HER2-positive (U2-OS, Sa-OS, SK-ES-1, MNNG, MG-63, HOS, Hs 894, Hs 899, and LM7) or HER2-negative (MDA-MB-468) cells. 24–48-hours poststimulation the (a) IFN-γ and (b) IL-2 concentration was determined. (c) T-cell proliferation was determined by counting viable cells (Trypan blue exclusion) 3 days poststimulation. Only HER2-specific T cells produced IFN-γ and IL-2 and proliferated after exposure to HER2-positive cells in comparison to nontransduced T cells. Results from three experiments from two donors done in duplicates are shown. (d) Only HER2-specific T cells killed HER2 positive osteosarcoma cells, in a 4 hour 51Cr-release cytotoxicity assay; nontransduced (NT) T cells did not. The HER2 negative cell line MDA-MB-468, and HER2-negative fibroblasts, lymphoblastoid B-cell lines (LCL) and T-cell blasts were not killed by HER2-specific or NT T cells. SK-BR-3 was used as a positive control. Results from three experiments done in triplicates are shown.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Adoptively transferred human epidermal growth factor receptor 2 (HER2)-specific T cells induce regression of intraperitoneal osteosarcoma xenografts. 2 × 106 LM7.eGFP.FFLuc cells were injected intraperitoneally in 9- to 12-week-old nonobese diabetic–severe combined immunodeficient mice (day 0) followed by intraperitoneal injection of 10 × 106 HER2-specific T cells or nontransduced T cells (NT T cells) on days 2, 3 and 4 or days 8, 9 and 10 after tumor inoculation. (a) Although tumors regressed over a period of 24–72 hours in response to injection of HER2-specific T cells (two lower rows), tumors grew progressively in untreated mice as shown for four representative animals and in mice receiving nontransduced T cells (upper two rows). (b) Quantitative bioluminescence imaging: the pretreatment bioluminescence values on day 2 were comparable between animals treated with HER2-specific T cells and those treated with NT T cells (P = 0.94). As early as 24 hours after initiation of treatment, the median photon emission from animals receiving HER2-specific T cells was significantly lower than those receiving NT T cells and for untreated tumors (P < and P < for animals treated on day 2 and those treated on day 8, respectively). Values are corrected to background bioluminescence. Two-tailed P value, Mann–Whitney U test are reported. Solid arrows: time of T-cell injection. (c) Survival analysis was performed at day 160. Mice from the untreated and NT T-cell control groups had a median survival of 63 days (range 29–66 days) and 65 days (range 49–104 days), respectively. Injection of HER2-specific T cells into mice bearing small or large tumors resulted in a significant survival advantage; mice bearing small tumors had a median survival of greater 160 days (P < 0.001) where as mice bearing large tumors had a median survival of 88 days (range 62 to >160 days; P < 0.01).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Adoptive transfer of human epidermal growth factor receptor 2 (HER2)-specific T cells induces regression of established osteosarcoma lung metastasis. 2 × 106 LM7.eGFP.FFLuc cells were injected systemically into the tail vein in 9- to 12-week-old Nu/Nu mice (day 0) followed by tail-vein injection of 10 × 106 HER2-specific T cells on days 2, 3, and 4 after tumor inoculation. (a) While tumors regressed in response to injection of HER2-specific T cells, tumors grew progressively in untreated mice. (b) Kaplan–Meier survival curve: mice treated with HER2-specific T cells had a significantly longer survival probability (P < 0.001) in comparison to untreated mice.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions