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. 2012 Mar 15;18(6):1672-83.
doi: 10.1158/1078-0432.CCR-11-3050. Epub 2012 Jan 30.

Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice

Dhanalakshmi Chinnasamy  1 , Zhiya Yu, Sid P Kerkar, Ling Zhang, Richard A Morgan, Nicholas P Restifo, Steven A Rosenberg
Affiliations

Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice

Dhanalakshmi Chinnasamy et al. Clin Cancer Res. .

Abstract

Purpose: We investigated the feasibility of delivering the proinflammatory cytokine interleukin (IL)-12 into tumor using T cells genetically engineered to express a chimeric antigen receptor (CAR) against the VEGF receptor-2 (VEGFR-2).

Experimental design: Two different strains of mice bearing five different established subcutaneous tumors were treated with syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressed single-chain murine IL-12 or an inducible IL-12 gene after host lymphodepletion. Tumor regression, survival of mice, and persistence of the transferred cells were evaluated.

Results: Adoptive transfer of syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressing single-chain IL-12 resulted in the regression of five different established tumors of different histologies without the need for IL-2 administration. T cells transduced with either anti-VEGFR-2 CAR or single-chain IL-12 alone did not alter the tumor growth indicating that both of them had to be expressed in the same cell to mediate tumor regression. Anti-VEGFR-2 CAR and IL-12-cotransduced T cells infiltrated the tumors, expanded, and persisted for prolonged periods. The antitumor effect did not require the presence of host T and B cells but was dependent on host IL-12R-expressing cells. The anti-VEGFR-2 CAR changed the immunosuppressive tumor environment by altering/reducing both the systemic and the intratumoral CD11b(+)Gr1(+) myeloid suppressor cell subsets that expressed VEGFR-2.

Conclusions: These results suggest that targeted delivery of IL-12 into the tumor environment with T cells redirected against VEGFR-2 is a promising approach for treating patients with a variety of solid tumor types.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1.
Figure 1.
Construction and characterization of recombinant retroviral vectors expressing CAR targeted against mouse VEGFR-2 and mouse single-chain IL-12. A, schematic representation of recombinant retroviral vectors used in this study. In DC101-CD828BBZ vector, the DC101 ScFv is made up of the variable regions of heavy (VH) and light chains (VL) of a rat IgG against mouse VEGFR-2 joined by a 218 linker that is linked to the hinge and transmembrane regions of the mouse CD8α chain, and the intracellular signaling sequences derived from mouse CD28, 4–1BB, and CD3Ϛ molecules. The Flexi-IL12 vector encodes the single-chain IL-12 comprised of p40 and p35 subunits of the murine IL-12 linked by (Gly4Ser)3 flexible linker. The NFAT-IL-12 vector expresses the murine single-chain Flexi-IL12 under the transcriptional control of the inducible minimal IL-2 promoter that contains 6 NFAT-binding motifs. SD, splice donor; SA, splice acceptor; PA2, polyadenylation signal. B, enriched splenic CD3þ T cells were stimulated for 2 days with ConA and IL-7 and then transduced with an empty or anti–VEGFR-2 CAR retroviral vector. The next day, cells were transduced with retroviral vector expressing Flexi-IL12 or NFAT-IL12 or left untransduced. Two days posttransduction, cells were analyzed for transgene expression by flow cytometry. Expression of IL-12 in transduced T cells was determined by intracellular FACS staining with or without PMA and ionomycin stimulation. Representative FACS data showing the percentage of T cells in each quadrant are presented. C, mouse T cells were transduced as described in Fig. 1B and 2 days later stimulated with PMA and ionomycin for 4 hours or cocultured with indicated target cell lines for 18 hours. Culture supernatants were assayed for secreted IL-12 by ELISA. Results are presented as the mean values ± SEM of triplicates. The data shown in Fig. B and C are representative of 3 independent experiments.
Figure 2.
Figure 2.
IL-12 cotransduction enhanced the antigen-specific immune responses of anti–VEGFR-2 CAR–transduced mouse T cells. Enriched CD3+ mouse T cells were transduced with the indicated retroviral vectors as described in Fig. 1. Two days later, cells were cocultured with VEGFR-2–negative MB49 tumor line or MB49 cells stably expressing mouse VEGFR-2 (referred as MB49-Flk1) for 18 to 24 hours. Culture supernatants were assayed for secreted IFN-γ (top) and TNF-α (bottom) by ELISA. Results are presented as the mean ± SEM of triplicates. The data shown are representative of 2 independent experiments.
Figure 3.
Figure 3.
Adoptively transferred anti–VEGFR-2 CAR and Flexi-IL12–engineered mouse T cells induced regression of multiple types of established syngeneic tumors in mice without exogenous IL-2 administration and increased the survival of tumor-bearing mice. Ten to 12 days old B16 (melanoma), MCA-205 (sarcoma), MC38 (colorectal adenocarcinoma), or MC17–51 (sarcoma) tumor-bearing C57BL/6 mice and 12 to 14 days old CT26 colon tumor-bearing BALB/c mice were sublethally irradiated at 5 Gy TBI and treated with 1 × 106 or 5 × 105 syngeneic T cells transduced with various retroviral vectors as indicated in the figure. Control group received no treatment. Each treatment group included a minimum 5 mice. Serial, blinded tumor measurements were obtained and the products of perpendicular diameters were plotted± SEM. P values are shown for anti–VEGFR-2 CAR and IL-12–transduced T cells versus no treatment (∗) or anti–VEGFR-2 CAR alone transduced T cells (∗∗). Mice treated with 1 × 106 anti–VEGFR-2 CAR and Flexi-IL12–transduced T cells died by day 14 postcell transfer irrespective of the tumo type and mouse strains used.
Figure 4.
Figure 4.
T cells engineered with anti–VEGFR-2 CAR and inducible IL-12 caused durable regressions of B16 melanoma without dose limiting toxicities, and host lymphodepletion is required for their enhanced antitumor effect. C57BL/6 mice bearing 12-day-old subcutaneous B16 melanoma were treated with indicated numbers of syngeneic T cells transduced with various retroviral vectors or left untreated as shown in the figures.Aand B, all mice received 5 Gy TBI before T-cell transfer. C, only some groups received TBI (+XRT) before T-cell transfer. Each treatment group included a minimum 5 mice. Serial, blinded tumor measurements were obtained and the products of perpendicular diameters were plotted ± SEM. P values shown in A and B: anti–VEGFR-2 CAR and IL-12–cotransduced T cells versus anti–VEGFR-2 CAR alone transduced T cells. P values shown in C: anti–VEGFR-2 CAR and NFAT-IL12–transduced T cells with 5 Gy TBI versus without TBI (–XRT). A, mice treated with 1 × 106 anti–VEGFR-2 CAR and Flexi-IL12–transduced T cells died by day 14 post-cell transfer (open square symbols).
Figure 5.
Figure 5.
Enhanced tumor infiltration of adoptively transferred anti–VEGFR-2 CAR and IL-12–cotransduced T cells in mice bearing established B16-F10 tumor. C57BL/6 mice–bearing B16 melanoma tumor were sublethally irradiated with 5 Gy TBI and treated with 1 × 106 Ly5.1+ syngeneic T cells transduced with anti–VEGFR-2 CAR or empty vector alone or either of these vectors cotransduced with NFAT-IL12 vector. Control group received no treatment. Each group included minimum of 14 mice. A, serial, blinded tumor measurements were obtained from 5 mice per group and the products of perpendicular diameters were plotted ± SEM. Tumors and spleens of 3 mice from each group were excised at different time points post-therapy and processed to obtain single-cell suspensions and analyzed by flow cytometry. Percentage of the Ly5.1+ lymphocytes was determined in total viable fraction of the cell preparations by flow cytometry. Absolute numbers of Ly5.1+ cells were determined by multiplying the percentage of Ly5.1+ cells by the total number of viable cells. B, representative FACS data from single-cell preparations of spleen and tumor tissues from 1 mouse in each group obtained on day 6 post-ACT showing the percentage Ly5.1+ cells gated in the total viable cell population. C, pooled data obtained from 3 mice from each group collected at indicated time points post-ACT showing the percentage and total number of Ly5.1+ cells in spleen and tumor tissues. Data represented as mean ± SEM.
Figure 6.
Figure 6.
Enhanced tumor regression mediated by anti–VEGFR-2 CAR and IL-12 T–cotransduced T cells was independent of host T and B cells or MHC class I expression of host cells but required a host response to transgenic IL-12. Mice that were genetically knocked out for indicated immune cell components/function in C57BL/6 background were subcutaneously implanted with B16 melanoma cells 12 days before therapy and treated with 1 × 106 T cells retrovirally modified to express anti–VEGFR-2 CAR or an empty vector alone or together with NFAT-IL12 vector. No treatment groups served as control. All mice received 5 Gy TBI before T-cell therapy. Serial, blinded tumor measurements were obtained from 5 mice per group and the products of perpendicular diameters obtained. All data are expressed as a mean ± SEM and is representative of at least 2 independent experiments. P values shown are compared with group treated with anti–VEGFR-2 CAR only transduced T cells.
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