T cells redirected to interleukin-13Rα2 with interleukin-13 mutein--chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1

Abstract

Background aims: Outcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL) 13Rα2, human epidermal growth factor receptor 2, epidermal growth factor variant III or erythropoietin-producing hepatocellular carcinoma A2 has shown promise for the treatment of glioma in preclinical models. On the basis of IL13Rα2 immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARS with IL13 muteins as antigen-binding domains. Whereas the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells.

Methods: We constructed four second-generation CARs with IL13 muteins with one or two amino acid substitutions, and evaluated the effector function of IL13-mutein CAR T cells in vitro and in vivo.

Results: T cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by interferon-γ production. IL13 protein produced significantly more IL2, indicating that IL13 mutein-CAR T cells have a higher affinity to IL13Rα2 than to IL13Rα1. In cytotoxicity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. Although we observed no significant differences between IL13 mutein-CAR T cells in vitro, only T cells expressing IL13 mutein-CARs with an E13K amino acid substitution had anti-tumor activity in vivo that resulted in a survival advantage of treated animals.

Conclusions: Our study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit.

Keywords: CAR; GBM; IL13Rα1; IL13Rα2; T cells; cancer; immunotherapy.

Figure 1. Generation of IL13 mutein-CAR T cells

(A) Scheme of IL13 mutein-CARs. All four constructs consisted of an IgG1-CH2CH3 domain, a CD28 transmembrane (TM) domain, and costimulatory domains derived from CD28 and the CD3 ζ-chain. The binding domain contained different IL13 muteins with one (E13K or E13Y) or two amino acid substitution (E13K.K105R; E13Y.K105R). (C,B) CAR expression was confirmed using FACS analysis. Representative plots (B) and summary data (C) is shown (mean 80.2% – 85.6%, n=7-14 per CAR construct).

Figure 2. IL13 mutein-CAR T cells release cytokines after stimulation with recombinant IL13Rα1 and IL13Rα2 proteins

IL13 mutein-CAR or non-transduced (NT) T cells were stimulated with recombinant IL13Rα1, IL13Rα2, or IL4R proteins. After 24h IFNγ (A) or IL2 (B) was measured by ELISA (n=3). T cells expressing all four CAR constructs expressed significant levels of IFNγ (p<0.001) when stimulated with recombinant IL13Rα2 or IL13Rα1 proteins in comparison to IL4R stimulated T cells. Only stimulation with recombinant IL13Rα2 protein induced significant levels of IL2 (p<0.001).

Figure 3. Expression of IL13Rα1 and IL13Rα2 in cell lines

(A) qRT-PCR for IL13Rα1 or IL13Rα2. FACS analysis for IL13Rα1 (B) or IL13Rα2 (C).

Figure 4. IL13 mutein-CAR T cells recognize IL13Rα1- and IL13Rα2-positive cells

(A,B) IL13 mutein-CAR T cells (IL13Y-CAR, IL13K-CAR) were cocultured with genetically modified Raji cells expressing IL13Rα1, IL13Rα2 or GFP, or unmodified Raji cells (Co) at a 1:4 E:T ratio. NT and mhFAP-CAR T cells served as controls. After 24h IFNγ (A) or IL2 (B) was measured by ELISA (n=3). Only stimulation of IL13 mutein-CAR T cells with Raji-IL13Rα1 or Raji-IL13Rα2 cells resulted in significant production of IFNγ (p<0.001) and of IL2 (p<0.001). Raji-IL13Rα2 cells induced significant higher levels of IFNγ (p=0.0016) and IL2 (p=0.0018) in comparison to Raji-IL13Rα1 cells.

Figure 5. IL13 mutein-CAR T cells kill IL13Rα1- and IL13Rα2-positive cells

(A,B) IL13 mutein-CAR T cells (IL13Y-CAR, IL13K-CAR) killed Raji-IL13Rα1and Raji-IL13Rα2 cells in contrast to Raji and Raji-GFP cells in a standard 4h cytotoxicity assay. Controls (NT T cells or mhFAP-CAR T cells) exhibited no cytolytic activity (results for 2 donors are shown).

Figure 6. Treatment of glioma xenograft with T cells expressing IL13 mutein-CARs with E13K amino acid substitutions results in improved overall survival

U373 glioma bearing mice were treated on day 7 with IL13K- (n=11), IL13Y- (n=11), IL13KR- (n=11), or IL13YR-CAR (n=11) T cells. Untreated mice (n=10) or mice treated with NT T cells (n=9) served as controls. (A) Representative images for each group and quantitative bioluminescence (radiance = photons/sec/cm²/sr) imaging data for all mice are shown (dotted lines: individual mice; solid lines: median). (B) Kaplan-Meier survival analysis (NT-T cells vs IL13K-CAR T cells, p=0.009; NT-T cells vs IL13KR-CAR T cells, p<0.001; NT-T cells vs IL13Y-CAR T cells, p=0.322; NT-T cells vs IL13KR-CAR T cells, p=0.253).

Figure 6. Treatment of glioma xenograft with T cells expressing IL13 mutein-CARs with E13K amino acid substitutions results in improved overall survival

U373 glioma bearing mice were treated on day 7 with IL13K- (n=11), IL13Y- (n=11), IL13KR- (n=11), or IL13YR-CAR (n=11) T cells. Untreated mice (n=10) or mice treated with NT T cells (n=9) served as controls. (A) Representative images for each group and quantitative bioluminescence (radiance = photons/sec/cm²/sr) imaging data for all mice are shown (dotted lines: individual mice; solid lines: median). (B) Kaplan-Meier survival analysis (NT-T cells vs IL13K-CAR T cells, p=0.009; NT-T cells vs IL13KR-CAR T cells, p<0.001; NT-T cells vs IL13Y-CAR T cells, p=0.322; NT-T cells vs IL13KR-CAR T cells, p=0.253).