Preclinical Evaluation and Dosimetry of [111In]CHX-DTPA-scFv78-Fc Targeting Endosialin/Tumor Endothelial Marker 1 (TEM1)

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Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing

<div><p>Background</p><p>NY-ESO-1 belongs to the cancer/testis antigen (CTA) family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized <i>in vitro</i> treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC).</p><p>Methodology/Principal Findings</p><p>We demonstrated <i>de novo</i> induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(_157–165) peptide specific chimeric antigen receptor (CAR) CD8⁺ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels.</p><p>Conclusions/Significance</p><p>These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.</p></div

Optimization of DAC treatment for MCF7, U266, and ARK tumor cell lines.

<p>A. Variation of DAC concentration in cell culture medium as indicated (0–15 μM); white bars: isotype control, grey bars: detection with HLA-A2/NY-ESO-1p_157-165 specific Fab-tetramers. B. Variation of DAC-treatment intensity (0–4 times per day). C. Variation of DAC-treatment duration (1–3 days). All data are representative of at least three independent experiments performed in triplicate.</p

Effects of DAC treatment on NY-ESO-1 mRNA and protein expression in MCF7, U266, and ARK cell lines.

<p>A. NY-ESO-1 specific mRNA, quantified as copy numbers/μg RNA using qRT-PCR. All data are representative of three independent experiments performed in triplicate. B. NY-ESO-1 protein expression analyzed by Western blotting (n = 3). The first and second line show total cell lysate of the respective cell line +/- DAC treatment and detection via a NY-ESO-1 specific or α-Tubulin specific (loading control) antibody. The third line shows a Western blot of recombinant NY-ESO-1 protein as control.</p

Increased specific lysis of MCF7 and U266 tumor cells by CAR redirected CD8⁺ T cells after DAC treatment.

<p>Retrovirally transduced NY-ESO-1-specific and CEA-specific CAR redirected CD8⁺ T cells were cocultivated with U266 or MCF7 cells. NY-ESO-1 expression after DAC treatment statistically significantly enhanced the antigen specific killing of anti-NY-ESO-1 CAR redirected T cells in U266 (A), whereas the increased lysis of MCF7 was only detectable after DAC treatment (B). Antigen specific activation of anti-NY-ESO-1 CAR redirected CD8⁺ T cells was determined by IFN-gamma (C and D). All data are representative of three independent experiments performed in triplicate.</p

Quantification of HLA-A2/NY-ESO1p_157-165 complexes at the cell surface of MCF7, U266, and ARK tumor cell lines.

<p>A. Flow cytometry histogram after staining of the indicated tumor cell lines with HLA-A2/NY-ESO-1p_157-165 peptide specific Fab-T1 tetramer (blue/red) and isotype control (black/green). Cells were either untreated (black/blue) or DAC- treated (green/red). B. Quantification of the NY-ESO-1 peptides at the surface of the untreated (white bars) or DAC-treated (grey bars) tumor cell lines. Peptide numbers were calculated as described in materials and methods. C. Relative change in HLA-A2/NY-ESO-1p_157-165 peptide presentation on the tumor cell lines. Increase of NY-ESO-1p_157-165 peptide presentation is shown in relation to the total number of HLA-A2 molecules on the tumor cell lines following DAC treatment. ARK cells were used as a negative control. All data are representative of at least five independent experiments performed in triplicate.</p

Antigen-specific in vitro expansion of functional redirected NY-ESO-1-specific human CD8+ T-cells in a cell-free system

Background: Tumors can be targeted by the adoptive transfer of chimeric antigen receptor (CAR) redirected T-cells. Antigen-specific expansion protocols are needed to generate large quantities of redirected T-cells. We aimed to establish a protocol to expand functional active NY-ESO-1-specific redirected human CD8+ T-cells. Materials and Methods: The anti-idiotypic Fab antibody A4 with specificity for HLA-A*0201/NY-ESO-1157-165 was tested by competition assays using a HLA-A*0201/NY-ESO-1157-165 tetramer. HLA-A*0201/NY-ESO-1157-165 redirected T-cells were generated, expanded and tested for CAR expression, cytokine release, in vitro cytolysis and protection against xenografted HLA-A*0201/NY-ESO-1157-165–positive multiple myeloma cells. Results: A4 demonstrated antigen-specific binding to HLA-A*0201/NY-ESO-1157-165 redirected T-cells. Expansion with A4 resulted in 98% of HLA-A*0201/NY-ESO-1157-165 redirected T-cells. A4 induced strong proliferation, resulting in a 300-fold increase of redirected T-cells. After expansion protocols, redirected T-cells secreted Interleukin-2, (IL-2), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) and lysed target cells in vitro and were protective in vivo. Conclusion: A4 expanded HLA-A*0201/NY-ESO-1157-165 redirected T-cells with preservation of antigen-specific function