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. 2012 Nov 1;18(21):5949-60.
doi: 10.1158/1078-0432.CCR-12-0319. Epub 2012 Sep 10.

Suppression of human glioma xenografts with second-generation IL13R-specific chimeric antigen receptor-modified T cells

Seogkyoung Kong  1 , Sadhak Sengupta, Betty Tyler, Anthony J Bais, Qiangzhong Ma, Saryn Doucette, Jinyuan Zhou, Ayguen Sahin, Bob S Carter, Henry Brem, Richard P Junghans, Prakash Sampath
Affiliations

Suppression of human glioma xenografts with second-generation IL13R-specific chimeric antigen receptor-modified T cells

Seogkyoung Kong et al. Clin Cancer Res. .

Abstract

Purpose: Glioblastoma multiforme (GBM) remains highly incurable, with frequent recurrences after standard therapies of maximal surgical resection, radiation, and chemotherapy. To address the need for new treatments, we have undertaken a chimeric antigen receptor (CAR) "designer T cell" (dTc) immunotherapeutic strategy by exploiting interleukin (IL)13 receptor α-2 (IL13Rα2) as a GBM-selective target.

Experimental design: We tested a second-generation IL13 "zetakine" CAR composed of a mutated IL13 extracellular domain linked to intracellular signaling elements of the CD28 costimulatory molecule and CD3ζ. The aim of the mutation (IL13.E13K.R109K) was to enhance selectivity of the CAR for recognition and killing of IL13Rα2(+) GBMs while sparing normal cells bearing the composite IL13Rα1/IL4Rα receptor.

Results: Our aim was partially realized with improved recognition of tumor and reduced but persisting activity against normal tissue IL13Rα1(+) cells by the IL13.E13K.R109K CAR. We show that these IL13 dTcs were efficient in killing IL13Rα2(+) glioma cell targets with abundant secretion of cytokines IL2 and IFNγ, and they displayed enhanced tumor-induced expansion versus control unmodified T cells in vitro. In an in vivo test with a human glioma xenograft model, single intracranial injections of IL13 dTc into tumor sites resulted in marked increases in animal survivals.

Conclusions: These data raise the possibility of immune targeting of diffusely invasive GBM cells either via dTc infusion into resection cavities to prevent GBM recurrence or via direct stereotactic injection of dTcs to suppress inoperable or recurrent tumors. Systemic administration of these IL13 dTc could be complicated by reaction against normal tissues expressing IL13Ra1.

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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 of IL13 CAR. A, plasmid map of IL13-CAR.pMFG. The IL13.CD28.ζ CAR expression cassette was constructed and cloned into BamHI and NotI sites of the MFG retroviral expression vector plasmid. B, CARs with different signal sequences (IL13, immunoglobulin heavy chain), with and without CD8a hinge, were constructed. Efficacy of CAR expression on the transduced T cells was tested by flow cytometry, with percent positive listed in each panel.
Figure 2
Figure 2
Expression of CAR on transduced T cells. A, graphic representation of the IL13 CAR showing monomeric and dimeric forms. B, Western blotting of CAR expression on membranes of transduced T cells, confirming monomer and dimer size bands versus control UnTd T cells. C, flow cytometric profiles of IL13 CAR expression on transduced T cells, with percent positive listed. Mean fluorescence intensity (MFI) of positive fractions was similarly high among all constructs (>10,000).
Figure 3
Figure 3
T-cell signaling via different CAR formats assessed by IL2 production by transduced T cells. A, flow cytometric profile of IL13Rα1 and IL13Rα2 expression on U251 glioma cells and Thp-1 cells. B, bar graph representing specific IL2 secretion by IL13 CAR transduced T cells upon coculturing with either IL13R (a1+a2−) Thp-1 cells (white bars) or IL13R (a1−a2+) U251 glioma cells (black bars) after background subtractions (Materials and Methods). IL2 levels are normalized to the percent of modified cells (range, 20%–24%) and expressed in pg/106 modified T cells/24 hours. Error bars represent SD from the mean of triplicate results.
Figure 4
Figure 4
Functional characterization of IL13 CAR T cells. A, cytotoxic effects of IL13 CAR transduced versus untransduced T cells on U251 glioma cells (black bars) or control HUVEC cells (white bars). Left, cytotoxicity after 5 hours of co-culture with an E:T ratio of 5:1. Right, cytotoxicity after 22 hours of coculture with E:T ratio of 0.5:1. Error bars represent SEM from the mean of triplicate results. B, cytokine secretion by IL13 CAR transduced or untransduced T cells upon coculture with U251 glioma cells (black bars) or control HUVEC cells (white bars). Left, IL2; right, IFNγ. Cytokine levels expressed as pg/106 total T cells/24 hours, uncorrected for modified fraction (30%). Error bars represent SEM from the mean of triplicate results. C, dTc cell proliferation upon coculturing 1:1 with control Daudi cells (left) and U251 glioma cells (right) on days 0, 3, 9, and 14 and cell counts assayed (♦; UnTd T cells; Ж, IL13-CAR T cells). D, enrichment of IL13 CAR T cells upon coculture with stimulators. T-cell expansions in C with either U251 glioma cells or control Daudi cells were assayed by flow on day 0 and day 14. Top, untransduced (UnTd) T cells; bottom, IL13 CAR transduced T cells. The x-axis represents IL13 CAR expression, whereas y-axis represents cell count in the histogram plots.
Figure 5
Figure 5
Successful suppression of human glioma xenografts by IL13 CAR+ T cells. Rats with 6-day established U251 glioblastoma tumors were randomized into 3 groups for treatment on day 0: (i) CAR+ T cells, (ii) UnTd T cells, and (iii) no treatment. A, representative brain histology. At day 7, after T-cell injection, progressing and regressing tumors were assessed by H&E (a and b) and anti-CD3 mAb staining (c–e); (a) UnTd: A 4-mm tumor is shown; (b) dTc: An indistinct band of tumor with a 5-mm zone of associated inflammation. Immunostaining: Anti-CD3 immunostaining showed increased T cells in the dTc treated tumors (d and e) versus tumors treated with UnTd T cells (c). B, T2-weighted MRI of tumor-bearing animals at indicated times following treatment. For each rat, 3 acquired slices are displayed at each time point to span the tumor zone. No visual tumor (open arrow) was observed in animals treated with CAR+ T cells. In contrast, large tumors (solid arrow) were observed in control group animals that grew progressively. C, Kaplan–Meier progression-free survival.
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