Coexpressed Catalase Protects Chimeric Antigen Receptor-Redirected T Cells as well as Bystander Cells from Oxidative Stress-Induced Loss of Antitumor Activity

Abstract

Treatment of cancer patients by adoptive T cell therapy has yielded promising results. In solid tumors, however, T cells encounter a hostile environment, in particular with increased inflammatory activity as a hallmark of the tumor milieu that goes along with abundant reactive oxygen species (ROS) that substantially impair antitumor activity. We present a strategy to render antitumor T cells more resilient toward ROS by coexpressing catalase along with a tumor specific chimeric Ag receptor (CAR) to increase their antioxidative capacity by metabolizing H2O2. In fact, T cells engineered with a bicistronic vector that concurrently expresses catalase, along with the CAR coexpressing catalase (CAR-CAT), performed superior over CAR T cells as they showed increased levels of intracellular catalase and had a reduced oxidative state with less ROS accumulation in both the basal state and upon activation while maintaining their antitumor activity despite high H2O2 levels. Moreover, CAR-CAT T cells exerted a substantial bystander protection of nontransfected immune effector cells as measured by CD3ζ chain expression in bystander T cells even in the presence of high H2O2 concentrations. Bystander NK cells, otherwise ROS sensitive, efficiently eliminate their K562 target cells under H2O2-induced oxidative stress when admixed with CAR-CAT T cells. This approach represents a novel means for protecting tumor-infiltrating cells from tumor-associated oxidative stress-mediated repression.

FIGURE 1.

Design of bicistronic expression vector for CARs and catalase expression. (A) Schematic diagram depicting the two sets of CARs used. (B) PBMCs from healthy donors were cultured for a 4 d and transduced using either bicistronic retroviral expression vectors for CARs and catalase or retroviral expression vector for CARs alone. Expression of CARs on transduced T cells was assessed by staining with PE conjugated F(ab′)₂ anti-human IgG that binds to the extracellular Fc region of the CAR and allophycocyanin-conjugated anti-CD3. PE-conjugated isotype Abs were used to confirm lack of nonspecific binding. CAR cells were gated on lymphocyte population in forward scatter and side scatter prior to gating CAR⁺ cells. (C) Protein lysates from MACS-sorted transduced T cells were analyzed by Western blot. Relative protein expression was determined by ImageJ analysis of the intensity of the bands from the Western blot. (D) MACS-sorted lysates were used to measure catalase activity. (E) Luminescence from 10⁵ transduced or nontransduced cells was measured after adding L-012 and H₂O₂. (F) Transduced T cells were permeabilized and rabbit polyclonal anti-human catalase Ab was used to stain for intracellular catalase. FITC-conjugated anti-rabbit IgG Ab was used to analyze the samples with flow cytometry. Data are presented as means ± SD. ***p < 0.005 by Student t test using GraphPad Prism 5.

FIGURE 2.

Oxidative state is reduced in CAR-CAT T cells compared with CAR T cells. (A) Directly after coculture with HEK293T cells, transduced T cells were labeled with CellROX (5 μM) in complete medium. (B) After short-term 1-h culture, or long-term 18-h culture, cells were labeled with PE anti-human IgG and Pacific Orange anti-human CD3 and samples were acquired on an LSR II. (C) To induce oxidative stress, freshly transduced T cells were loaded with CellROX and stimulated with PMA (3 μg/ml) and DHNQ (20 μM) for 2 h before acquiring samples by flow cytometry. Samples were analyzed using FlowJo.

FIGURE 3.

CAR-CAT T cells maintain viability and functionality under H₂O₂-induced oxidative stress. (A) After transduction with CAR or CAR-CAT, T cells were resuspended to 2 × 10⁵ in 200 μl complete medium RPMI 1640 containing 10% FCS and exposed to increasing concentrations of H₂O₂. After 24 h cells were stained with annexin V–FITC and 7-aminoactinomycin D and analyzed by FACS. (B) H₂O₂ (50 μM) was used to induce oxidative stress in 5 × 10³ engineered or nonmodified T cells being stimulated with CD3-CD28 proliferation beads for 4 d and cell proliferation was measured by [³H]thymidine incorporation for 4 h. (C) Transduced T cells were used as effectors for targeting Her2⁺ tumors at an E:T ratio of 1:2. Freshly transduced T cells were cultured overnight with increasing concentrations of H₂O₂ to induce oxidative stress. After 18 h, target cells were labeled with [⁵¹Cr] and transferred into effector cell–containing wells. Supernatant was collected after 24 h coculture and transferred to LumaPlates and read out on MicroBeta. The percentage specific lysis was calculated using the following formula: (CPM_sample − CPM_spontaneous)/(CPM_maximum − CPM_spontaneous). Data are presented as means ± SD. **p < 0.005 by two-way ANOVA using GraphPad Prism 5 for (B) and (C) between CAR and CAR-CAT.

FIGURE 4.

A bystander effect is mediated by CAR-CAT T cells toward nonmodified T and NK cells. (A) T cells were labeled with CellROX for both a short-term and long-term staining, and CellROX MFI on CAR⁻ T cells was determined after cells were acquired by FACS. (B) Healthy donor autologous T cells were CFSE labeled and admixed with transduced T cells followed by staining with Abs and CellROX and acquired by flow cytometry. (C) CAR-CAT and CAR⁻ cells were labeled with maleimide and surface thiols were evaluated. (D) Supernatants were collected from transduced and nontransduced T cells and tested for catalase activity. (E) H₂O₂ was used to induce oxidative stress in T cell culture for 2 h prior to staining. Cells (2 × 10⁵) were stained for CD3ζ using FITC-conjugated anti-CD3ζ after permeabilization with 0.25% PFA and digitonin. Cells were acquired by FACS, and change in CD3ζ MFI was calculated by: CD3ζ MFI_{(xμM H2O2)} − CD3ζ MFI_{(0μM H2O2)} gated on the CAR⁻ fraction. (F) NK cells were cocultured with engineered T cells at a ratio of 2:1 CAR T cell/NK cell overnight under oxidative stress induced by different concentrations H₂O₂. K562 target cells were loaded with [⁵¹Cr] and added to the NK/T cell mix after H₂O₂ coculture at a ratio of 1:1 NK cell/K562 cell. Supernatant (25 μl) was transferred to LumaPlates and read out on MicroBeta. Data are presented as means ± SD. *p < 0.05 by two-way ANOVA using GraphPad Prism 5 for (C) between CAR and CAR-CAT.