Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4

Abstract

Polyclonal T-cells can be directed against cancer using transmembrane fusion molecules known as chimeric antigen receptors (CARs). Although preclinical studies have provided encouragement, pioneering clinical trials using CAR-based immunotherapy have been disappointing. Key obstacles are the need for robust expansion ex vivo followed by sustained survival of infused T-cells in patients. To address this, we have developed a system to achieve selective proliferation of CAR(+) T-cells using IL-4, a cytokine with several pathophysiologic and therapeutic links to cancer. A chimeric cytokine receptor (4alphabeta) was engineered by fusion of the IL-4 receptor alpha (IL-4Ralpha) ectodomain to the beta(c) subunit, used by IL-2 and IL-15. Addition of IL-4 to T-cells that express 4alphabeta resulted in STAT3/STAT5/ERK phosphorylation and exponential proliferation, mimicking the actions of IL-2. Using receptor-selective IL-4 muteins, partnering of 4alphabeta with gamma(c) was implicated in signal delivery. Next, human T-cells were engineered to co-express 4alphabeta with a CAR specific for tumor-associated MUC1. These T-cells exhibited an unprecedented capacity to elicit repeated destruction of MUC1-expressing tumor cultures and expanded through several logs in vitro. Despite prolonged culture in IL-4, T-cells retained specificity for target antigen, type 1 polarity, and cytokine dependence. Similar findings were observed using CARs directed against two additional tumor-associated targets, demonstrating generality of application. Furthermore, this system allows rapid ex vivo expansion and enrichment of engineered T-cells from small blood volumes, under GMP-compliant conditions. Together, these findings provide proof of principle for the development of IL-4-enhanced T-cell immunotherapy of cancer.

Figures

FIGURE 1.

Co-expression of 4αβ and a CAR in human T-cells. A, in 4αβ, the ectodomain of IL-4Rα was fused to the transmembrane and endodomain of βc. IL-4-mediated pairing of 4αβ with γc is shown. Stoichiometric co-expression of 4αβ and a MUC1-specific CAR (HOX), or matched truncated control, HTr, was achieved using the SFG retroviral vector (B) containing an intervening Thosea Asigna (T2A) peptide, placed downstream of a furin cleavage site.

FIGURE 2.

IL-4 delivers an IL-2-like signal via 4αβ. A, CTLL-2 cells were engineered to express HOX, 4αβ-HOX, or 4αβ-HTr. Cell surface expression of 4αβ (open histograms) was demonstrated by flow cytometry (shaded histogram, background staining of HOX+ cells). B, engineered CTLL-2 cells were stimulated with human IL-2 (100 units/ml) or human IL-4 (30 ng/ml) for 15 min. Phosphorylation of STAT5 and p42/44 ERKs was assayed by Western blotting. C, triplicate cultures were established at 5 × 103/ml in human IL-2, human IL-4, or the derived muteins IL-4DD or IL-4 DE (concentrations as specified). Cell numbers were reevaluated after 6 days. Similar results were obtained in two to five independent experiments.

FIGURE 3.

Expression and signaling by 4αβ in human T-cells. Human T-cells were engineered to express HOX, 4αβ-HOX, or 4αβ-HTr. A, co-expression of the MUC1 CAR (HOX or HTr) and 4αβ was demonstrated by flow cytometry. B, Western blotting of lysates from engineered T-cells, probed with anti-CD3ζ antiserum. Endogenous CD3ζ serves as loading control (arrowed). C, engineered T-cells were stimulated with the indicated ILs for 15 min and analyzed for STAT phosphorylation by Western blotting. Total STAT3 and STAT5 levels (from the same lysates, blotted separately) were used as loading controls. Similar results were obtained in three independent experiments.

FIGURE 4.

Tumor cell killing by IL-4-stimulated T-cells. A, human T-cells were engineered to express 4αβ-HOX, 4αβ-HTr, or HOX and then cultured for 1 week in IL-2 (100 units/ml) or IL-4 (30 ng/ml). T-cells were then established in (B) triplicate 4-h cytotoxicity assays using the indicated targets or (C) overnight cytotoxicity assays using MUC1-expressing MDA-MB-435 target cells. In C, T-cells and targets were cultured at a 1:1 ratio. After staining for the MUC1 CAR, cultures were analyzed by flow cytometry to distinguish between tumor cells (large side scatter; SSC, orange) and T-cells. Expression of the MUC1 CAR is shown on the x axis (blue).

FIGURE 5.

Proliferation of engineered T-cells when stimulated via chimeric antigen and chimeric cytokine receptors. T-cells were engineered to express 4αβ-HOX and were periodically stimulated with MUC1+ tumor cells: BT20 (A), T47D (B), or MDA-MB-435 MUC1 (C). Cultures were maintained in IL-2 (100 units/ml), IL-4 (30 ng/ml), or no cytokine. Control T-cells were untrans(duced) or expressed HTr, 4αβ-HTr, or DOX (a matched CAR than lacks an scFv). Each cycle of stimulation with fresh tumor cells is indicated by overhead arrows. The cell number was evaluated periodically in triplicate. Cultures were terminated if tumor cells/monolayers were not destroyed within 1 week. Data are presented as mean ± S.D. (error bars) and are representative of at least two separate experiments with each tumor target. D, T-cells expressing 4αβ-HOX were propagated in IL-4 and stimulated six times with MDA-MB-435 MUC1 tumor cells (C) and then established in a secondary 4-h cytotoxicity assay with the indicated MUC1POS (T47D, ZR75, RPMI 8226, MCF7, or MDA-MB-435 MUC1) or MUC1NEG (MDA-MB-435) tumor cell targets. *, p < 0.05 compared with control T-cell population(s).

FIGURE 6.

Ex vivo expansion of T-cells that co-express 4αβ with an ErbB-specific CAR under closed and GMP compliant conditions. A, two replicate experiments in which engineered T-cells were expanded in clinical grade culture bags using IL-4. B, cultures expanded in IL-4 were periodically analyzed by flow cytometry for CAR expression. Markers were set using an isotype control (<1% staining). C, T-cells shown in A were analyzed by flow cytometry on days 4 and 10. Parallel microcultures were maintained in IL-2 and analyzed by flow cytometry on day 10. D, T-cells shown in A were harvested on day 10, stimulated with PMA and ionomycin, and analyzed for production of the indicated cytokines by intracellular staining and flow cytometry.