Chlorotoxin-directed CAR T cell therapy for recurrent glioblastoma: Interim clinical experience demonstrating feasibility and safety

Abstract

A challenge in treating glioblastoma (GBM) is its phenotypic heterogeneity between patients and within tumors. Chlorotoxin (CLTX), a peptide from scorpion venom, broadly binds glioma cells through a mechanism involving surface matrix metalloproteinase-2 (MMP-2). We previously developed chimeric antigen receptor (CAR) T cells incorporating CLTX as the GBM recognition domain. Here, we report interim clinical experience of a phase 1 trial evaluating intracavity/intratumoral (ICT) delivery of CLTX-CAR T cells in four patients with MMP-2-expressing recurrent GBM (NCT04214392), with the primary objectives of feasibility and safety. The therapy is well tolerated with no dose-limiting toxicities. Three of the four participants (75%) exhibit a best response of stable disease. CLTX-CAR T cells are detected in the tumor cavity fluid and at lower levels in the blood. Human anti-CAR antibody assays do not detect humoral immunogenicity against the CLTX-CAR. These observations support further clinical evaluation of CLTX-CAR therapy.

Keywords: T cells; chimeric antigen receptor; chlorotoxin; immunotherapy; phase 1 clinical trial.

Graphical abstract

Figure 1

Study overview (A) Schematic of patient treatment. Days shown are approximate. #, final imaging occurred between days 21 and 35. (B) Schema of the dose schedule, in which optional dosing cycles (four or more) were allowed after the initial three cycles of the DLT period. To be evaluable for dose escalation, disease response, and survival, patients had to receive at least 80% of the intended CLTX-CAR T cell dose in each cycle 1–3. (C) Tumor MMP-2 immunoreactivity on GBM cells as well as on tumor vasculature, and overlap of MMP-2 immunoreactivity with CLTX:biotin binding, for tissue specimens from treated patients. Left, immunohistochemistry (IHC) on archival tissue used for enrollment, and surgical tissue taken at time of catheter placement (pre-treatment). Right, immunofluorescence (IF) images at pre-treatment. MMP-2 H-scores as evaluated by a neuropathologist are indicated in the IHC images. Scale bars = 100 μm except for UPN 522 IF images (scale bar = 50 μm). See also Figures S2–S4. (D) CONSORT diagram of patient enrollment and treatment. ∗, UPN 479 CLTX-CAR T cell product failed the QC transgene copy-number test (woodchuck hepatitis virus posttranscriptional regulatory element [WPRE] analysis by qPCR); ∧, ineligibility due to complications from surgery. CAR, chimeric antigen receptor; CLTX, chlorotoxin; DLT, dose-limiting toxicity; ICT, intracavity/intratumoral; MRI, magnetic resonance imaging; PB, peripheral blood; PET, fluorodeoxyglucose-positron emission tomography; QC, quality control; TCF, tumor cavity fluid; UPN, unique patient number.

Figure 2

Treatment response to CLTX-CAR T cell therapy (A) Swimmer plot of treated participants and their clinical outcomes. Black lines indicate administered CAR T cell cycles. (B) MRI of UPN 487 showed absence of tumor recurrence following CLTX-CAR T cell infusions into the tumor resection cavity (yellow arrow), whereas the temporal tumor distant from the infusion site progressed (red arrow).

Figure 3

Persistence of CLTX-CAR T cells (A) Numbers of total CD3⁺ T cells and CD3⁺ CAR-expressing T cells in the TCF as determined by flow cytometry over all treatment cycles (C1–C8 as relevant; one sample per time point). Day 0 is immediately prior to the first CLTX-CAR T cell infusion. Representative histograms showing staining for CD19 (marking CAR positivity) and CD8 expression on the CD3-gated cells are depicted at the time when CLTX-CAR T cells reached their highest levels for each participant (i.e., C6, C4, and C2 for UPNs 479, 487, and 522, respectively). Percentages of immunoreactivity are indicated in each histogram. Note that TCF was not collected from UPN 502. (B) WPRE copy numbers per μg of whole-blood DNA over time. Averages of three technical replicates are depicted. Cycles of CAR T cell administration are indicated by dashed lines. UPN 487 had surgery at day 70 upon recurrence as indicated by the arrow and after the MRI shown in Figure 2A.

Figure 4

Assessment of participant cytokine profiles (A) Low-dimensional representations of cytokine measurements colored by compartment (serum [SER], CSF, or TCF; left), day of any given cycle (up to 8 cycles, with CxD0 being day 0 of cycles 2 or greater; center), or individual participants (right). These data are superimposed over those previously reported for our IL13Rα2-CAR T cell trial (small pale dots). (B) Heatmaps of median log10-fold changes in TCF cytokine levels pre-infusion (D0) and post-infusion (D1) across cycles (C1, C2, C3, etc.) relative to baseline values as indicated by the arrowheads at top. For UPN 487, the C2D0 sample was used as the baseline as a C1D0 pre-treatment sample was not available. All unavailable samples are indicated by slash marks. See also Figure S5 for graphical depiction of baseline values, Figure S6 for serum cytokine heatmaps, and Tables S1–S7 for all cytokine data (averages of two technical replicates).

Figure 5

HACA assay suggests non-immunogenicity of CLTX-CAR T cells (A) HACA assay schema. The different domains of a CLTX(EQ)28ζ CAR protein expressed on the cell surface are identified on the left. Human IgG1 molecules from participant serum or TCF that might possibly bind the extracellular domains of the CAR are depicted in gray. The fluorochrome-conjugated secondary antibody used to detect bound human IgG1 is depicted in red. A schematic of how the HACA assay was validated is depicted on the right, with the CD19t transduction marker that is also expressed as a component of the CLTX(EQ)28Z-T2A-CD19t_epHIV7 vector, the biosimilar humanized anti-CD19 IgG1 reagent (light gray), and the fluorochrome-conjugated secondary antibody (red). (B) HACA assay sensitivity was determined by titrating a biosimilar, humanized anti-CD19 antibody. The indicated concentrations of anti-CD19 (IgG1) antibody, followed by fluorochrome-conjugated anti-IgG1 secondary antibody (20 μg/mL), was used to stain CLTX(EQ)28ζ/CD19t+ (red lines) or mock-transduced (blue lines) Jurkat cells. Isotype (IgG1) control (3 μg/mL) followed by secondary antibody staining of CLTX(EQ)28ζ/CD19t+ Jurkat cells (gray lines) was also performed as a negative control. (C) Graphic representation of MFI ratios (MFI of CLTX-CAR Jurkat cells/MFI of mock Jurkat cells) from (B). Values of 1.3 or higher indicate a positive HACA response. (D) Participants’ serum (top) or TCF (bottom) samples were evaluated for IgG1 bound to CLTX(EQ)28ζ/CD19t+ (red lines) or mock-transduced (blue lines) Jurkat cells. Isotype control followed by secondary antibody staining of CLTX(EQ)28ζ/CD19t+ Jurkat cells (black lines) was again performed as a negative control. MFI ratios are indicated in each histogram. Each histogram is representative of three technical replicates.