Antitumor activity of cytotoxic T lymphocytes engineered to target vascular endothelial growth factor receptors - PubMed (original) (raw)

Antitumor activity of cytotoxic T lymphocytes engineered to target vascular endothelial growth factor receptors

Thomas M J Niederman et al. Proc Natl Acad Sci U S A. 2002.

Abstract

The demonstration that angiogenesis is required for the growth of solid tumors has fueled an intense interest in the development of new therapeutic strategies that target the tumor vasculature. Here we report the development of an immune-based antiangiogenic strategy that is based on the generation of T lymphocytes that possess a killing specificity for cells expressing vascular endothelial growth factor receptors (VEGFRs). To target VEGFR-expressing cells, recombinant retroviral vectors were generated that encoded a chimeric T cell receptor comprised of VEGF sequences linked to intracellular signaling sequences derived from the zeta chain of the T cell receptor. After transduction of primary murine CD8 lymphocytes by such vectors, the transduced cells were shown to possess an efficient killing specificity for cells expressing the VEGF receptor, Flk-1, as measured by in vitro cytotoxicity assays. After adoptive transfer into tumor-bearing mice, the genetically modified cytotoxic T lymphocytes strongly inhibited the growth of a variety of syngeneic murine tumors and human tumor xenografts. An increased effect on in vivo tumor growth inhibition was seen when this therapy was combined with the systemic administration of TNP-470, a conventional angiogenesis inhibitor. The utilization of the immune system to target angiogenic markers expressed on tumor vasculature may prove to be a powerful means for controlling tumor growth.

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Figures

Figure 1

Transduction of primary lymphocytes and cloned T cell lines by cTcR-expressing retroviral vectors. (A) Structure of retroviral vectors used in this study. VEGF-cTcR, VEGF chimeric TCR; MR-1-cTcR, MR-1 single-chain monoclonal antibody chimeric TCR; SD, splice donor; SA, splice acceptor; CMV IE, cytomegalovirus immediate early promoter; myc, human c-Myc epitope; CD8, CD8α hinge region; TCR, ζ chain of the TCR; LTR, long-terminal repeat. (B) FACS analysis of untransduced CTLs (dashed line) or CTLs transduced with VEGF-cTcR or MR-1-cTcR (solid line). Splenocytes transduced with VEGF-cTcR also were incubated with anti-CD8-FITC antibody (solid line) or an anti-rat IgG-FITC negative control antibody (dashed line). (C) FACS analysis of VEGF-cTcR clone 2 and MR-1-cTcR clone 8 cells. (D) Southern blot analysis of genomic DNA from transduced primary CD8+ splenocytes and CTL clones. Indicated is the expected 600-bp _Xba_I-_Bgl_II fragment containing the transgenic VEGF sequence and a background band that migrated as a 2.5-kb fragment. The one-copy control lane represents mock-transduced genomic DNA spiked with 12 pg of CMMP-VEGF-cTcR plasmid DNA, which correlates with an expected one copy of transgene per genome.

Figure 2

Assessment of binding of Flk-1 to cTcR-expressing cells. (A) FACS analysis demonstrating the binding of soluble Flk-Fc only to HeLa cells expressing VEGF-cTcR. CM, conditional medium. (B) Binding of soluble human KDR-Fc to a CTL clone expressing VEGF-cTcR (VEGF-cTcR clone 2). Cells were incubated with increasing concentrations of soluble KDR-Fc, washed, incubated with anti-human IgG-FITC, and subjected to FACS analysis. The data are plotted on a logarithmic scale. (Insert) Plot of the data on a linear scale to demonstrate saturable binding.

Figure 3

VEGF-cTcR T cells specifically lyse cells expressing Flk-1. (A) Primary VEGF-cTcR CTLs (squares) or MR-1-cTcR CTLs (circles) were incubated with either B16.F10 cells that either expressed (■ and ●, respectively) or did not express (□ and ○, respectively) Flk-1 at varying effector-to-target ratios, and cell lysis was determined by using a standard Cr51 release assay. (B) Primary VEGF-cTcR CTLs (■), VEGF-cTcR del Z CTLs (□), or MR-1-cTcR CTLs (●) were incubated with adherent MILE cells, and lysis was determined by using a standard dehydrogenase (LDH) release assay. (C) MILE cells were preincubated with no antibodies, anti-Flk-1 antibodies, or isotype control (IC) antibodies before incubation with CTLs expressing VEGF-cTcR (filled bars) or VEGF-cTcR delZ (open bars) in a 5-h cytotoxicity assay at an effector-to-target ratio of 15:1. Each data point reflects the mean of six independent determinations.

Figure 4

Adoptive immunotherapy using genetically modified CTLs. (A) CT26 cells (5 × 105, n = 6 per group) were implanted s.c. on BALB/c mice. (B) B16.F10 cells (7 × 105, n = 4 per group) were implanted on C57BL/6 mice. B16.F10 cells (5 × 105, n = 4 per group) (C) or LS174T cells (1 × 106, n = 7 per group) (D) were implanted on C57BL/6 nude mice. On the days indicated with an arrowhead, mice were treated with 5 × 106–9 × 106 VEGF-cTcR CTLs (□), MR-1-cTcR CTLs (♦), PBS (○), PBS with no exogenous IL-2 (▾), or VEGF-cTcR del Z (X). Daily i.p. injections of IL-2 started on the first day of CTL infusion (except for the group indicated with the inverted triangle in B). Tumor volume was calculated by using the formula width2 × length × 0.52, and the SEM is indicated with error bars. The ratio of the tumor volumes of the VEGF-cTcR CTL-treated mice to the PBS control mice (T/C) was determined for the last time point.

Figure 5

Effects of the combined treatment of genetically modified CTLs and TNP-470 on tumor growth. (A) CT26 adenocarcinoma cells (5 × 105, n = 4 per group) were implanted s.c. on BALB/c mice. B16.F10 melanoma cells (7 × 105, n = 4 per group) (B) or T241 fibrosarcoma cells (5 × 105, n = 3 per group) (C) were implanted on C57BL/6 mice. On the days indicated with an arrowhead, mice were treated with 5 × 106–10 × 106 VEGF-cTcR CTLs (□), VEGF-cTcR CTLs + TNP-470 (■), MR-1-cTcR CTLs + TNP-470 (▴), PBS + TNP-470 (●), or PBS (○). Mice were treated with TNP-470 every other day and IL-2 every day starting on the first day of CTL therapy. Tumor measurements and analyses were as described for Fig. 4. T/C, ratio of the tumor volumes of the VEGF-cTcR CTL-treated mice to the PBS control mice.

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Antitumor activity of cytotoxic T lymphocytes engineered to target vascular endothelial growth factor receptors - PubMed (original) (raw)