A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma

Abstract

We conducted a first-in-human study of intravenous delivery of a single dose of autologous T cells redirected to the epidermal growth factor receptor variant III (EGFRvIII) mutation by a chimeric antigen receptor (CAR). We report our findings on the first 10 recurrent glioblastoma (GBM) patients treated. We found that manufacturing and infusion of CAR-modified T cell (CART)-EGFRvIII cells are feasible and safe, without evidence of off-tumor toxicity or cytokine release syndrome. One patient has had residual stable disease for over 18 months of follow-up. All patients demonstrated detectable transient expansion of CART-EGFRvIII cells in peripheral blood. Seven patients had post-CART-EGFRvIII surgical intervention, which allowed for tissue-specific analysis of CART-EGFRvIII trafficking to the tumor, phenotyping of tumor-infiltrating T cells and the tumor microenvironment in situ, and analysis of post-therapy EGFRvIII target antigen expression. Imaging findings after CART immunotherapy were complex to interpret, further reinforcing the need for pathologic sampling in infused patients. We found trafficking of CART-EGFRvIII cells to regions of active GBM, with antigen decrease in five of these seven patients. In situ evaluation of the tumor environment demonstrated increased and robust expression of inhibitory molecules and infiltration by regulatory T cells after CART-EGFRvIII infusion, compared to pre-CART-EGFRvIII infusion tumor specimens. Our initial experience with CAR T cells in recurrent GBM suggests that although intravenous infusion results in on-target activity in the brain, overcoming the adaptive changes in the local tumor microenvironment and addressing the antigen heterogeneity may improve the efficacy of EGFRvIII-directed strategies in GBM.

Conflict of interest statement

Competing interests: M.V.M., B.L.L., J.B., J.J.M., C.H.J., and H.O. are inventors on patent application #20170008963 and granted U.S. patent # 9394368 held by the University of Pennsylvania and Novartis that cover the use of CAR T cells targeting EGFRvIII. K.M., R.I., and J.L.B. are employees of Novartis; A.S. is a former employee of Novartis. All other authors declare that they have no competing interests.

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Fig. 1. Protocol design, consort diagram, and clinical outcomes in subjects infused with CART-EGFRvIII

(A) Protocol schema for EGFRvIII testing, leukapheresis, and manufacturing, treatment with CAR T cells directed to EGFRvIII, and follow-up. (B) Consort diagram indicating the number of subjects screened and enrolled on the study. (C) Swimmer’s plot describing time on study for each subject (black), duration of follow-up off study (that is, survival beyond progression or initiation of other therapy) (gray), and present status. Arrows indicate ongoing survival. Asterisks indicate surgical intervention. Dashed vertical line indicates 1-year time point. (D) OS plotted as Kaplan-Meier estimate for all subjects. X axis is shown in days. Tick marks indicate each censored subject (that is, subjects who are alive at the data cutoff point).

Fig. 2. Engraftment of CART-EGFRvIII and cytokine modulation in the peripheral blood

(A) CART-EGFRvIII engraftment and persistence in the peripheral blood by flow cytometry detecting the CAR on CD3+ T cells (left y axis, blue curve) and by qPCR detecting CAR sequences (right y axis, red curve) in PBMC genomic DNA. Pharmacokinetics over the first 30 days for each subject are shown. (B) Fold change in IL-6 levels in the peripheral blood of each subject over time (black squares, plotted on the left y axis). Baseline IL-6 levels for subjects 201, 202, 204, 205, 207, 209, 211, 213, 216, and 217 were 2.57, 7.5, 8.58, 4.90, 1.78, 4.40, 0.35, 3.11, 8.89, and 33.85 pg/ml, respectively. Baseline level of IL-10 in subject 207 was 2.03 pg/ml. Data for all other cytokines are shown in table S6. C-reactive protein (CRP) levels are plotted in the subjects in whom it was measured over time (green triangles, right y axis). Significant clinical events such as seizures, surgery, or administration of siltuximab are noted on the x axis.

Fig. 3. Effects of CART-EGFRvIII on radiological and/or pathological assessments in two subjects

(A) MRI performed before and after administration of gadolinium (gad) in subject 205. T1 post-contrast and FLAIR images are shown for the indicated time points. (B) Histological analysis of surgical specimens obtained from subject 205, 2 months after CART-EGFRvIII infusion. Hematoxylin and eosin (H&E)–stained sections and immunohistochemistry for CD3 to demonstrate T cells are shown. Scale bar, 200 μm. (C) MRI (T1 post-contrast images) shown at the indicated time points for subject 209. This subject underwent surgical resection of one portion of the tumor after the 3-month scan.

Fig. 4. T cell trafficking and effect on EGFR mutations in brain tumors after intravenous infusion of CART-EGFRvIII

(A) Comparison of CART-EGFRvIII quantification in brain tumor specimens compared to peripheral blood in each of the seven patients who underwent surgical resection at various time points (subject # and day # indicated on the x axis) after CART-EGFRvIII infusion. Ratio is calculated on the basis of copies per microgram of genomic DNA in cells. (B) Levels of expression of EGFRvIII as determined by NGS of purified genomic DNA in brain tumor specimens obtained before or after CART-EGFRvIII infusion. Subject numbers are indicated along the right, and each subject is color-coded for clarity. Where more than one sample was obtained and tested separately, points show the mean. Bar indicates P value between pre- and post-CART levels in paired specimens by the Wilcoxon matched-pairs signed-rank test. (C) EGFR amplification in brain tumor specimens obtained pre– and post–CART-EGFRvIII infusion from all tissue samples tested. Bar indicates P value between pre- and post-CART levels in paired specimens by the Wilcoxon matched-pairs signed-rank test. Subject numbers are indicated along the right, and each subject is color-coded for clarity. (D) TCRβ CDR3 deep sequencing analysis of T cells infiltrating brain tumor specimens obtained before and after CART-EGFRvIII and (E) in the matching infusion product and post-CART brain tumor–infiltrating lymphocytes in three subjects (205, 209, and 211). Unique TCR sequences in the pre-infusion tumor biopsy are shown in the green box along the x axis in (D) and in the infusion product in the green box along the x axis in (E). In (D) and (E), these are compared with post-infusion tumor specimen clonotype repertoire shown in red along the y axis. Shared clonotypes are displayed in a blue box.

Fig. 5. Immunohistochemistry of the tumor microenvironment in GBM specimens before and after CART-EGFRvIII infusion

(A) T cell infiltration and phenotyping in pre– and post–CART-EGFRvIII infusion specimens from subject 216; pre-infusion specimens are from day −81, whereas post-infusion specimens are from day +13, relative to CART-EGFRvIII infusion at day 0. Top row shows low-power magnification of CD3 immunohistochemical stain, with high-power magnification as inset; ISH specifically for CAR sequences. T cell phenotyping is shown with ISH for IFN-γ and with immunohistochemistry for CD8, granzyme B (GRZMB), and the IL-2 receptor α chain (CD25). Scale bars, 4 mm (low-power graphs) and 200 μm (high-power graphs). (B) In situ assessment of immunosuppressive molecules in the tumor microenvironment is shown before and after CART-EGFRvIII infusion in patient 216, including IDO1, PD-L1, FoxP3, TDO, IL-10, and TGFβ. (C) Summary table with heat map of T cell infiltration, CART-EGFRvIII trafficking, and tumor microenvironment in seven subjects before and after treatment with CART-EGFRvIII. NP, not performed; 0, not detectable. Date indicates day of specimen relative to CART-EGFRvIII infusion, which was designated as day 0.

Fig. 6. Immunohistochemical colocalization of CD3/FoxP3 and CD8/Ki67

(A and B) Analysis of brain tumor samples performed pre– and post–CART-EGFRvIII infusion. Representative analysis of CD3/FoxP3 from patient 216 is shown in (A), with quantitative analysis of percent FoxP3+ CD3 cells shown for five subjects’ brain tumor samples in (B). Two-tailed paired t test analysis did not demonstrate statistical significance (P = 0.10). Hem, hematoxylin. (C) Representative analysis of CD8/Ki67 staining in patient 213, and (D) quantitative analysis of percent Ki67+ CD8 cells for five subjects’ brain tumor samples. Two-tailed paired t test analysis did not demonstrate statistical significance (P = 0.07).