Off-the-shelf, steroid-resistant, IL13Rα2-specific CAR T cells for treatment of glioblastoma

Abstract

Background: Wide-spread application of chimeric antigen receptor (CAR) T cell therapy for cancer is limited by the current use of autologous CAR T cells necessitating the manufacture of individualized therapeutic products for each patient. To address this challenge, we have generated an off-the-shelf, allogeneic CAR T cell product for the treatment of glioblastoma (GBM), and present here the feasibility, safety, and therapeutic potential of this approach.

Methods: We generated for clinical use a healthy-donor derived IL13Rα2-targeted CAR+ (IL13-zetakine+) cytolytic T-lymphocyte (CTL) product genetically engineered using zinc finger nucleases (ZFNs) to permanently disrupt the glucocorticoid receptor (GR) (GRm13Z40-2) and endow resistance to glucocorticoid treatment. In a phase I safety and feasibility trial we evaluated these allogeneic GRm13Z40-2 T cells in combination with intracranial administration of recombinant human IL-2 (rhIL-2; aldesleukin) in six patients with unresectable recurrent GBM that were maintained on systemic dexamethasone (4-12 mg/day).

Results: The GRm13Z40-2 product displayed dexamethasone-resistant effector activity without evidence for in vitro alloreactivity. Intracranial administration of GRm13Z40-2 in four doses of 108 cells over a two-week period with aldesleukin (9 infusions ranging from 2500-5000 IU) was well tolerated, with indications of transient tumor reduction and/or tumor necrosis at the site of T cell infusion in four of the six treated research subjects. Antibody reactivity against GRm13Z40-2 cells was detected in the serum of only one of the four tested subjects.

Conclusions: This first-in-human experience establishes a foundation for future adoptive therapy studies using off-the-shelf, zinc-finger modified, and/or glucocorticoid resistant CAR T cells.

Keywords: IL13Rα2-CAR T cells; allogeneic; glioblastoma; glucocorticoid receptor; off-the-shelf.

Fig. 1

Characterization of GRm13Z40-2 cell product. (A) Schematic of the IL13-zetakine plasmid containing the hygromycin phosphotransferase-HSV thymidine kinase selection/PET reporter fusion gene (HyTK) with poly-adenosine tail (pA) under the control of the cytomegalovirus immediate-early promoter (CMVp), and the IL13-zetakine receptor gene under the control of the human elongation factor 1α promoter (EF1p). (B) Illustration of the IL13-zetakine receptor, where the IL13Rα2-specific ligand IL13(E13Y), IgG4Fc linker (huγ ₄F_c), CD4 transmembrane domain (huCD4 tm), and CD3ζ cytoplasmic signaling domain (huCD3ζ cyt) are depicted. (C) Schematic of the GR-ZFN-containing Ad5/35 vector SB-313, where the inverted terminal repeats (ITR), the psi packaging sequence (ψ), the zinc finger nuclease (ZFN) expression cassette containing the CMV promoter (CMVp), the two GR-locus targeting ZFN open reading frames (ZFN9674, ZFN9666) separated by the 2A ribosomal skip sequence, and the chimeric fiber protein sequence containing the shaft and knob domain from adenovirus serotype 35 (35K) in place of that for adenovirus serotype 5 are indicated. (D) Schematic of the human glucocorticoid receptor (hGR) protein sequence, where the N-terminal domain (NTD), DNA binding domain (DBD), ligand binding domain (LBD), and SB-313 target site at amino acid 412 are indicated. (E) Diagram of the manufacturing process, with day of each step indicated. PBMC, peripheral blood mononuclear cells; OKT3, a CD3 agonistic antibody used to activate T cells; REM, rapid expansion method involving 14-day cycles of stimulation with OKT3, rhIL-2 and irradiated feeders as previously described. (F) GRm13Z40-2 cells were stained with fluorochrome-conjugated reagents specific for the indicated markers (shaded histograms), with IL13-specific staining used to detect the CAR, and the IOTest® Beta Mark TCR V-beta Repertoire Kit used to determine TCR Vβ9 predominance. Percentages of positive cells above isotype control staining (open histograms) are indicated. Isotype control staining was used to set the quadrants in right-most histogram. (G) Sequence diversity of the GR locus as determined with PCR products incubated with the Cel-1 mismatch specific nuclease, with band analysis showing a 36% frequency of GR locus modification. Depicted are parental IL13Z40 cells (nontransduced with SB-313), and GRm13Z40-2 clinical product cells. M, marker lane. (H) Same cells as in (G) were either treated with 10^-6 M dexamethasone (DEX) or left untreated (no DEX) for 20 hours and message RNA levels of glucocortoid induced gene FKBP5 (left) and glucocorticoid-repressed gene IFN-γ (right) were analyzed by RT-PCR. Mean ± S.D. of 4 RT-PCR runs are depicted. *, P ≤ .0089 using a paired Student’s t-test; #, P = .0067 when compared to untreated cells using a paired Student’s t-test. (I) GRm13Z40-2 cells or control IL13Z03 (nontransduced with SB-313) cells were used as effectors in a 4-hour ⁵¹Cr release assay using the indicated E:T ratios. The IL13Rα2-positive tumor targets were U87 and U251T cells, and Daudi cells engineered to express IL13Rα2 (Daudi-Rα2); the negative tumor target controls were the LCL and Daudi parental cells. Mean ± S.E.M. of triplicate wells are depicted.

Fig. 2

GRm13Z40-2 cells lack alloreactivity. Alloreactivity was tested using a 4-hour ⁵¹Cr-release assay with GRm13Z40-2 effectors against EBV-transformed lymphoblastic cell lines (LCL) generated from donors of different allogeneic HLA types (indicated in Table at right). TM-LCL-OKT3 targets expressed the CD3 agonist OKT3 and acted as a positive control.

Fig. 3

Treatment schema and target antigen expression of excised tumors. (A) Intratumoral treatment overview for the 4 cycles of 10⁸ GRm13Z40-2 cells administered twice a week, with IL-2 administered by convection-enhanced delivery (CED) on days 2-5 of week one, and days 1-5 of week 2. (B) Recurrent tumors of each UPN resected at time of Rickham placement underwent immunohistochemical (IHC) staining using IL13Rα2-specific DAB with hematoxylin counterstain. Scale bars indicate 100 µm. H scores are indicated in parenthases, and were obtained by the formula: (3 x percentage of strongly staining cells) + (2 x percentage of moderately staining cells) + percentage of weakly staining cells, giving a range of 0 to 300 (Modified from); plus signs indicate that membranous staining was observed.

Fig. 4

Imaging demonstrates increased necrosis near site of CAR T cell injection. Axial T1 with contrast MRI images show preoperative recurrent tumors extending into left splenium of UPN 100 (A) and into the corpus callosum of UPN 104 (B), with PET imaging obtained after catheter placement demonstrating hypermatabolic tumors at each site. One month after treatment initiation (“Post Therapy” panels at right), local necrosis is noted in areas of previous MRI enhancement (white arrow heads); and PET imaging shows hypometabolic tumors, consistent with necrosis, surrounding each catheter site (white arrow heads). Mean standardized uptake values (SUVs) of tumor site normalized to background SUVs are indicated in each PET panel.

Fig. 5

Comparison of injection and distal tumor sites post-therapy. (A) Axial T1 with contrast MRI images show recurrent tumor of UPN 103, with no significant radiological changes noted adjacent to catheter (red arrow head) at 3 weeks after initiation of therapy, but more heterogenous enhancement of the tumor bulk surrounding the catheter at 10 weeks after initiation of therapy (top panels). PET imaging obtained after catheter insertion (red arrow head) and at 3 and 7 weeks after initiation of therapy shows hypermetabolic tumor (bottom panels). Biopsy sites collected at 10 weeks are indicated with red outlines. Mean SUVs of tumor site normalized to background SUVs are indicated in each PET panel. (B) IL13Rα2 IHC and hematoxylin and eosin (HE) staining of biopsies collected at 10 weeks after initiation of therapy. H scores for IL13Rα2 staining are indicated in parentheses, and were obtained as described in Figure 3 legend. Injection site shows 40% necrosis, which is chiefly represented by large areas of confluent coagulative necrosis (dashed oval, blue box magnified in far-right panel). Distal site shows predominantly viable tissue with foci of pseudo-palisading necrosis representing 10% of the resected tissue. Red boxes outline areas of viable tumor as magnified in the subsequent panels. Scale bars indicate 2.5 mm (images at left) and 100 µm (magnified images at right). (C) Tumor biopsies of injection and distal sites collected at 10 weeks after initiation of therapy were examined for the presence of CD3+ and CD8+ T cells by IHC. Scale bars indicate 50 µm. Enumeration of CD3+ cells in the 2.5 x 10⁸ μm² area of each biopsy IHC section is depicted at the right. (D) Detection of male CAR T cells in biopsies collected at 10 weeks after initiation of therapy. Upon alignment of FISH and CD8 IHC brightfield images, two CD8+ male cells (Target 52 depicted, white arrowhead; Target 110 not shown) were identified in the injection site biopsy section, and one CD8+ male cell (Target 112 depicted, white arrowhead) was identified in the distal site biopsy section. Scale bars indicate 50 µm.