TCR gene therapy of spontaneous prostate carcinoma requires in vivo T cell activation

Abstract

Analogous to the clinical use of recombinant high-affinity Abs, transfer of TCR genes may be used to create a T cell compartment specific for self-Ags to which the endogenous T cell repertoire is immune tolerant. In this study, we show in a spontaneous prostate carcinoma model that the combination of vaccination with adoptive transfer of small numbers of T cells that are genetically modified with a tumor-specific TCR results in a marked suppression of tumor development, even though both treatments are by themselves without effect. These results demonstrate the value of TCR gene transfer to target otherwise nonimmunogenic tumor-associated self-Ags provided that adoptive transfer occurs under conditions that allow in vivo expansion of the TCR-modified T cells.

FIGURE 1. Detection of SV40 Large T expression in transformed cells

A-C, Large T expression in the prostate gland of a 9-week old TRAMP mouse. Prostate cells in areas undergoing atypical hyperplasia are large T immunostaining positive (B), whereas prostate cells with a normal morphology are large T immunostaining negative (C). Original magnifications: 10× (A) and 40× (B-C).

FIGURE 2. TRAMP mice are tolerant towards the tumor-associated SV40_IV epitope

A-B, TRAMP mice and as a control non-transgenic littermates received an intranasal flu-T infection. 10 days post infection, spleen cells were isolated and cultured in the presence of either SV40_404-411 or (as a control) the influenza A nucleoprotein derived PR_366-374 epitope (5×10^-4 μg/ml) for 14 days. Antigen specific T cells were measured by intracellular IFNγ staining after incubation with 100 ng/ml of the relevant peptide. Shown are dot-plots of 2 representative cultures (A) and cumulative data of all cultures (B). Numbers in upper right corner of dot-plots refer to percentage of IFNγ⁺ CD8⁺ cells of total CD8⁺ cells. Circles in graphs represent individual mice, bars indicate averages.

FIGURE 3. SV40_IV-TCR transduced T cells recognize antigen in vitro and in vivo

A, Flow cytometric analysis of SV40_IV-TCR (left panel) or mock (right panel) transduced T cells. The transduction efficiency of the SV40_IV-TCR was determined by calculating the percentage of Vβ9⁺ cells within the population of Vβ-pool⁺ cells (represented in right upper corner of dot plot). Average transduction percentage was 25-40%. B, Functional analysis of SV40_IV-TCR transduced splenocytes. Cells were incubated in the presence of SV40_404-411 (closed circles) or control OVA_257-264 peptide (open circles) at the indicated concentrations. Prior to incubation, a sample of the transduced T cells was stained with K^b-SV40_404-411 tetramers and PE-anti-CD8α antibodies to determine the percentage of SV40_IV-specific CD8⁺ cells. Post peptide stimulation, cells were stained with APC-anti-CD8α, permeabilized and stained with PE-anti-IFNγ. C, Flow cytometric analysis of blood cells of B6 (left panels) and TRAMP mice (right panels) that received 1×10⁵ SV40_IV-TCR transduced T cells (top panels) or no transfer of T cells (bottom panels) followed by an i.n. infection with 1000 p.f.u. of flu-T. Blood was sampled 3-14 days post infection. Circles represent TCR-transduced T cell responses in individual mice; bars indicate averages.

FIGURE 4. Homing and functional properties of SV40_IV-TCR modified T cells

10-week old TRAMP mice and control non-transgenic littermates received an adoptive transfer of 5×10⁵ SV40_IV-TCR transduced T cells, followed by vaccination by i.p. infection with 1×10⁵ p.f.u. of rVV-T. Control mice were solely vaccinated with rVV-T. 11 days post vaccination, the frequency of TCR transduced cells in spleen and prostate was assessed by analysing the percentage of Vβ9⁺Vβ-pool⁺ CD8⁺ cells of total Vβ-pool⁺ CD8⁺ cells. Functionality of SV40_IV-specific T cells was measured by intracellular IFN-γ staining after incubation for 4 hours with 100 ng/ml of the relevant peptide (SV40_404-411) or control peptide (OVA_257-264). Shown are dot-plots from a mouse in each of the treatment groups (A) and cumulative data from all mice (B). Numbers in upper right corner of dot-plots refer to percentage of Vβ9⁺Vβpool⁺ cells of total CD8⁺ cells or IFNγ⁺CD8⁺ cells of total CD8⁺ cells. Circles in graphs represent individual mice, bars indicate averages.

FIGURE 5. Adoptive transfer of SV40_IV-TCR transduced T cells results in a marked delay in tumor development

A, Outline of experiment. 10-week old TRAMP mice received an adoptive transfer of 2.5-5×10⁵ SV40_IV-TCR-transduced T cells, followed by vaccination by means of an i.p. infection with 1×10⁵ p.f.u. of rVV-T. 6 weeks later the same mice received a 2^nd infusion of 2.5-5 × 10⁵ SV40_IV-TCR transduced T cells, followed by i.n. infection with 1000 p.f.u. of flu-T. Control mice were either vaccinated with the 2 recombinant viruses, or were left untreated. Blood was sampled at various time points for 2 weeks post each infusion and analysed blindly by flow cytometry. At 28 weeks, mice were sacrificed and indicated sites were analysed for tumor development by histopathology. B, The percentage of Vβ9⁺Vβpool⁺ CD8⁺ cells of total Vβpool⁺ CD8⁺ cells in peripheral blood. Circles represent individual mice, bars indicate averages. Shown are results of 1 out of 2 experiments. C, Classification of tumor development in prostate gland, coagulation gland and seminal vesicles in treated and non-treated TRAMP mice. Bars depict the percentage of mice with the indicated lesion type within that cohort. In cases where multiple types of lesions were detected within one organ, the most severe type was used for scoring. Shown are the pooled results of two independent experiments, compared to pathology found in 9-weeks old TRAMP mice (top row). To test for treatment effects, Wilcoxon-Mann-Whitney tests were performed, adjusting for organ type. Vaccination only versus no treatment, p=0.9152; TCR gene transfer versus no treatment, p < 0.0001; TCR gene transfer versus vaccination only, p < 0.0001; TCR gene transfer versus the pathology found in 9 week old mice, p=0.3775. D, Macroscopic analysis of the male reproductive tract of a representative TRAMP mouse of each of the three treatment groups, compared to the male reproductive tract of a 28-week old non-transgenic littermate. E, SV40 large T expression in prostate and coagulation gland of a 28-weeks old TRAMP mouse after 2 transfers of SV40_IV-TCR transduced T cells in combination with viral vaccination. Dorsal prostate gland (D); lateral prostate gland (L); coagulation gland (C). Cells showing atypical hyperplasia are SV40 large T immunostaining positive (bottom panels), whereas cells with a normal morphology in the same coagulation gland are SV40 Large T immunostaining negative (left bottom panel). Original magnifications: 2.5x (top panel) and 40x (bottom panels).

FIGURE 6. Effect of vaccination and number of treatment cycles on anti-tumor efficacy of SV40_IV-TCR modified T cells

A & B, 10-week old TRAMP mice received an adoptive transfer of 2.5-5×10⁵ SV40_IV-TCR-transduced T cells, followed by vaccination by means of an i.p. infection with 1×10⁵ p.f.u. of rVV-T. 6 weeks later, mice did or did not receive a 2^nd infusion of 2.5-5 × 10⁵ SV40_IV-TCR transduced T cells, followed by i.n. infection with 1000 p.f.u. of flu-T. Control mice were vaccinated with the 2 recombinant viruses without ACT. C & D, 10-week old TRAMP mice received an adoptive transfer of 2.5-5×10⁵ SV40_IV-TCR-transduced T cells, followed by a second infusion 6 weeks later. Control mice were left untreated. A & C, Blood was sampled at the indicated time points to determine the percentage of Vβ9⁺Vβpool⁺ CD8⁺ cells of total Vβpool⁺ CD8⁺ cells. Circles represent individual mice, bars indicate averages. B & D, At 28 weeks, mice were sacrificed and indicated sites were analysed for tumor development by histopathology. Bars depict the percentage of mice with the indicated lesion type within that cohort. In cases where multiple types of lesions were detected within one organ, the most severe type was used for scoring. To test for treatment effects, Wilcoxon-Mann-Whitney tests were performed. B: Two cycles of ACT/ vaccination versus vaccination only, p < 0.001 ; One cycle of ACT/ vaccination versus vaccination only, p < 0.01; Two cycles of ACT/ vaccination versus one cycle of ACT/ vaccination, p =0.2166; D: TCR gene transfer without concomitant vaccination versus no treatment, p =0.5055.