Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression

Abstract

Human epidermal growth factor receptor 2 (HER2) is expressed by the majority of human osteosarcomas and is a risk factor for poor outcome. Unlike breast cancer, osteosarcoma cells express HER2 at too low, a level for patients to benefit from HER2 monoclonal antibodies. We reasoned that this limitation might be overcome by genetically modifying T cells with HER2-specific chimeric antigen receptors (CARs), because even a low frequency of receptor engagement could be sufficient to induce effector cell killing of the tumor. HER2-specific T cells were generated by retroviral transduction with a HER2-specific CAR containing a CD28.zeta signaling domain. HER2-specific T cells recognized HER2-positive osteosarcoma cells as judged by their ability to proliferate, produce immunostimulatory T helper 1 cytokines, and kill HER2-positive osteosarcoma cell lines in vitro. The adoptive transfer of HER2-specific T cells caused regression of established osteosarcoma xenografts in locoregional as well as metastatic mouse models. In contrast, delivery of nontransduced (NT) T cells did not change the tumor growth pattern. Genetic modification of T cells with CARs specific for target antigens, expressed at too low a level to be effectively recognized by monoclonal antibodies, may allow immunotherapy to be more broadly applicable for human cancer therapy.

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 143.98.2) 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.

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.

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.

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.

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 < 0.002 and P < 0.008 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).

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.