doi: 10.1158/0008-5472.CAN-12-3913.
Epub 2013 Apr 30.
Simultaneous targeting of tumor antigens and the tumor vasculature using T lymphocyte transfer synergize to induce regression of established tumors in mice
Affiliations
- PMID: 23633494
- PMCID: PMC3686092
- DOI: 10.1158/0008-5472.CAN-12-3913
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Simultaneous targeting of tumor antigens and the tumor vasculature using T lymphocyte transfer synergize to induce regression of established tumors in mice
Cancer Res.
.
Abstract
Most systemic cancer therapies target tumor cells directly, although there is increasing interest in targeting the tumor stroma that can comprise a substantial portion of the tumor mass. We report here a synergy between two T-cell therapies, one directed against the stromal tumor vasculature and the other directed against antigens expressed on the tumor cell. Simultaneous transfer of genetically engineered syngeneic T cells expressing a chimeric antigen receptor targeting the VEGF receptor-2 (VEGFR2; KDR) that is overexpressed on tumor vasculature and T-cells specific for the tumor antigens gp100 (PMEL), TRP-1 (TYRP1), or TRP-2 (DCT) synergistically eradicated established B16 melanoma tumors in mice and dramatically increased the tumor-free survival of mice compared with treatment with either cell type alone or T cells coexpressing these two targeting molecules. Host lymphodepletion before cell transfer was required to mediate the antitumor effect. The synergistic antitumor response was accompanied by a significant increase in the infiltration and expansion and/or persistence of the adoptively transferred tumor antigen-specific T cells in the tumor microenvironment and thus enhanced their antitumor potency. The data presented here emphasize the possible beneficial effects of combining antiangiogenic with tumor-specific immunotherapeutic approaches for the treatment of patients with cancer.
©2013 AACR.
Figures
A, CD3+ T cells from splenocytes of Wt or transgenic Pmel mice were stimulated with ConA and IL-7 or 1 μM hgp10025–33 peptide respectively for 2 days in T cell media containing 30 IU /mL rhIL-2 and transduced with retroviral vectors expressing an anti-VEGFR2 CAR (DC101 CAR) or an empty vector. Two days later T cells were analyzed for expression of the DC101 CAR and Pmel TCR (measured by Vβ13 staining) by FACS. Cells were also costained for CD3ε expression using allo phycocyanin (APC) conjugated rat anti-mouse CD3ε. CD3+ viable T cells were gated. Representative FACS data from 3 experiments showing the percentage of cells in each quadrant are shown. B, Two days after transduction, 105 effector mouse T cells were cocultured with indicated target cells at 1:1 ratio for 18 hours. Where indicated 2 effector T cell types were mixed in equal numbers (each 5 × 104) and cocultured with 105 target cells. Target cells were pulsed with indicated concentrations of either hgp10025-33 peptide or irrelevant control peptide prior to coculture. Culture supernatants were assayed for secreted IFN-γ by ELISA. The data shown are representative of two independent experiments.
A, groups of 5 C57BL/6 mice bearing B16 tumors were sublethally irradiated with 5 Gy TBI and treated with 105, 106, or 107 Tg-Pmel T cells, open repertoire T cells from Wt mice transduced with an empty vector or an anti-VEGFR CAR, or Tg-Pmel T cells transduced with an anti-VEGFR2 CAR. Some groups received a combination of Tg-Pmel T cells and anti-VEGFR2 CAR transduced open repertoire T cells. Control groups received neither T cells nor vaccine nor rhIL-2. All treatment groups received a single dose of 2 × 107 pfu vaccinia virus expressing hgp100 antigen and 2 daily doses of 2.2 × 105 IU rhIL-2 per dose for 3 consecutive days. Serial, blinded tumor measurements were obtained and the products of perpendicular diameters were plotted ± SEM. The data shown are representative of three independent experiments. B, groups of 5 C57BL/6 mice bearing B16 tumors were sublethally irradiated with 5 Gy TBI and treated with different numbers and combinations of T cells as indicated in the figure. Tumor area (left) and survival (right) in mice receiving various treatments compared with untreated controls are shown. All treatment groups received rhIL-2 for 3 days. Where indicated some groups received hgp100 expressing vaccinia virus vaccine in conjunction with cell transfer. The data shown are representative of 2 independent experiments.
A, schematic representation of tumor antigen-specific TCRs expressing recombinant retroviral vectors used in this study. In the MSGV1 retroviral vectors expressing the codon optimized Pmel TCR (Vα1, Vβ13), TRP-1 TCR (Vα3.2, Vβ14), or TRP-2 TCR (Vα12.1, Vβ3) recognizing the murine gp100, tyrosinase-related-protein-1 (TRP-1), or tyrosinase-related-protein-2 (TRP-2) antigens respectively, the corresponding Vα and Vβ cDNAs were fused together using the picornavirus foot-and-mouth disease virus 2A sequence. SD, splice donor; SA, splice acceptor; LTR, long terminal repeat; PA2, polyadenylation signal. B, enriched splenic CD3+ T cells from C57BL/6 mice were stimulated for 2 days with ConA and IL-7 and then transduced with an empty or anti-VEGFR2 CAR retroviral vector. The next day, cells were transduced with retroviral vector expressing one of the TCRs or left untransduced and analyzed by FACS tw days later. Cells were also costained for CD3ε expression using allo phycocyanin (APC) conjugated rat anti-mouse CD3ε. FACS data showing the percentage of cells in gated CD3+ viable T cells are presented. Data are representative of 2 independent experiments.
Enriched splenic CD3+ T cells obtained from C57BL/6 mice were stimulated for 2 days with ConA and IL-7 and then transduced with an empty or anti-VEGFR2 CAR retroviral vector. The next day, cells were transduced with retroviral vector expressing one of the TCRs or left untransduced. Two days later transduced T cells were adoptively transferred into B16 tumor bearing C57BL/6 mice (5 mice per group) as indicated in the figure. Animals received 5 Gy TBI prior to T cell transfer and concurrently received vaccine and rhIL-2 as described in legend for Figure 2A. Serial, blinded tumor measurements were obtained and the products of perpendicular diameters were plotted ± SEM. The data shown are representative of 2 independent experiments.
C57BL/6 mice bearing B16 melanoma tumor were sublethally irradiated with 5 Gy TBI and treated with 106 Thy1.1+ Wt T cells or 106 Thy1.1+ Tg-Pmel T cells mixed with 5 × 106 Ly5.1+ syngeneic open repertoire Wt T cells transduced with a retroviral vector expressing the control CAR (SP6 CAR) or an anti-VEGFR2 CAR (DC101 CAR). Animals concurrently received a single dose of 2 × 107 pfu vaccinia virus expressing hgp100 and twice daily rhIL-2 administration for 3 days. Control group received no treatment. Tumors and spleens of 3 mice from each group were excised at different time points post therapy and single cell suspensions were made as described in materials and methods. Cell preparations were stained with FITC-labeled anti-rat Thy1.1 and PE-labeled mouse anti-mouse Ly5.1 antibodies and analyzed by flow cytometry. Representative flow cytometry data from single cell preparations of spleen and tumor tissues from one mouse in each group obtained on day 3, 6, and 9 post T cell treatment indicating the percentage Thy1.1+ and Ly5.1+ cells gated in the total viable cell population are shown.
C57BL/6 mice bearing B16 melanoma tumor were sublethally irradiated with 5 Gy TBI. Mice received T cell treatments as described in Figure 5. Single cell preparations from tumors and spleens of 3 mice from each group were prepared and analyzed by flow cytometry as described in Figure 5. The absolute numbers of Thy1.1+ and Ly5.1+ cells were determined by multiplying the % Thy1.1+ and Ly5.1+ cells obtained by FACS by the total number of viable cells. A, pooled data obtained from three mice from each group collected at indicated time points post ACT showing the absolute numbers of Thy1.1+ and Ly5.1+ cells in spleen and tumor tissues. B, fold changes in the absolute numbers of Thy1.1+ in the spleen and tumor tissues of mice treated with a mixture of 106 Tg-Pmel and 5 × 106 anti-VEGFR2 CAR (DC101 CAR) transduced Wt T cells compared to that of mice treated with a mixture of 106 Tg-Pmel and 5 × 106 SP6 CAR (control CAR) transduced Wt T cells. The data shown are representative of 2 independent experiments.
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