Chimeric antigen receptor (CAR)-T-cell therapy for solid tumors is limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR-T cells directed against single antigens. Here, we developed a bicistronic construct to drive expression of a CAR specific for EGFRvIII, a glioblastoma-specific tumor antigen, and a bispecific T-cell engager (BiTE) against EGFR, an antigen frequently overexpressed in glioblastoma but also expressed in normal tissues. CART.BiTE cells secreted EGFR-specific BiTEs that redirect CAR-T cells and recruit untransduced bystander T cells against wild-type EGFR. EGFRvIII-specific CAR-T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive glioblastoma. However, CART.BiTE cells eliminated heterogenous tumors in mouse models of glioblastoma. BiTE-EGFR was locally effective but was not detected systemically after intracranial delivery of CART.BiTE cells. Unlike EGFR-specific CAR-T cells, CART.BiTE cells did not result in toxicity against human skin grafts in vivo.
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We thank M. Gianatasio, C. Lu, J. Messerschmidt, D. Millar, L. Riley, M. Cabral-Rodriguez and E. Schiferle for their expert technical assistance. This work was supported by grants from the National Institutes of Health (grant no. R25NS065743; B.D.C.), the Neurosurgery Research & Education Foundation and B*Cured Research Fellowship Grant (B.D.C.), the Society for Immunotherapy of Cancer–AstraZeneca Postdoctoral Cancer Immunotherapy in Combination Therapies Clinical Fellowship Award (B.D.C.) and The Jenny Fund (W.T.C.). This work was also supported by the Damon Runyon-Rachleff Innovation Award (M.V.M.) and Stand Up to Cancer (M.V.M.). We thank the following core facilities of the MGH Cancer Center: MGH Blood Bank, Flow Cytometry, Confocal Microscopy and Histopathology.
B.D.C. and M.V.M. are inventors on patents related to the use of engineered cell therapies and bispecific T-cell engagers for GBM and other cancers. B.D.C. received commercial research grants from ACEA Biosciences. M.V.M. received commercial research grants from Kite Pharma, TCR2, Agentus and CRISPR Therapeutics (unrelated to this work), and is a consultant or advisory board member for Adaptimmune, Agentus, Cellectis, CRISPR Therapeutics, Kite Pharma, Novartis, TCR2 and Windmil (unrelated to this work).
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Integrated supplementary information
(a) Tissue microarray showing EGFR expression by immunohistochemistry across several normal healthy human CNS tissues (top) and glioblastoma specimens (bottom). Details regarding each specimen may be found in Supplementary Table 1. (b) IHC for EGFR in human skin harvested from engrafted mice. (c) Expression of EGFR on HaCat (keratinocyte), U87 and U251 cell lines relative to isotype staining by flow cytometery. (d) Western blot analysis for EGFR expression in U87, U251 and HaCat cell lines. Lanes were loaded with 30 μg of total protein and subjected to SDS-PAGE and blotting with anti-EGFR antibody and anti-β-actin antibody. Experiments were repeated independently with similar results. Representative data are shown.
Supplementary Figure 2 EGFRvIII-negative tumor antigen escape following treatment with EGFRvIII CAR T cells.
(a) A heterogeneous population (30% EGFRvIII-positive, 70% wild-type) of U87 glioma cells was implanted in the flanks of NSG mice. Mice were treated intravenously (IV) by tail vein on day 2 post-implantation with untransduced (UTD) T cells or CART-EGFRvIII. Flank implantation allowed for concomitant caliper measurements of tumor growth once EGFRvIII-positive cells were eliminated. (b) Bioluminescence analysis of EGFRvIII-expressing tumor growth over time. (c) Caliper measurements of overall tumor growth in mice treated with UTD cells alone (black) versus CART-EGFRvIII (green), n = 5 biologically independent animals (mean + SD is depicted).
Supplementary Figure 3 Systemic versus intraventricular delivery of CAR T cells against tumors in the brain.
(a) Schematic representation of experimental design in which 5 × 103 U87vIII cells were implanted orthotopically into the brains (intracebral, IC) of NSG mice and treated with either intravenous (IV) or intraventricular (IVT) CAR T cells (1 × 106 transduced cells). (b) Survival plot of mice treated by CART-EGFRvIII, grouped by route-of-delivery, compared to treatment with UTD cells, n = 5 animals per group.
Supplementary Figure 4 Quantification of tumor-infiltrating and circulating CAR T cells in mice with brain tumors.
(a) H&E and (b) Human CD3 IHC of brain tumors (U87vIII) from mice treated with CART-EGFRvIII.BiTE-EGFR cells. (c) U87KO (EGFR-negative), U87 or U87vIII gliomas were implanted intracranially in NSG mice. Mice were infused intraventricularly on day 14 post-implantation with CART-EGFRvIII.BiTE-EGFR cells. Brain tumors were isolated on day 7 post-treatment and assessd for CAR-transduced cells (mCherry), (d) ddPCR for the 4-1BB-CD3ζ transgene and (e) human CD3+ events. (f) Circulating blood was also assessed by flow cytometry and (g) ddPCR. (h) Gating strategy for infiltrating and (i) circulating CAR T cells. The experiment was repeated with similar results. Representative data are shown, n = 3 biologically independent animals (mean + SEM is depicted; unpaired, one-tailed t-test, * = p < 0.05, ** = p < 0.01, *** = p < 0.001).
Supplementary Figure 5 Sorted Tregs can be redirected to kill GBM by BiTE secreted from CAR T cells.
Bioluminescence-based cytotoxicity assay measuring activity of bystander Tregs against U87, using a transwell system. T cells transduced with either CART-EGFRvIII.BiTE-CD19 (purple) or CART-EGFRvIII.BiTE-EGFR (orange) were cultured in top wells while sorted primary human Tregs (CD4+CD25+CD127dim/-) and U87 targets were placed in bottom wells. The assay was performed in triplicate, n = 3 biologically independent wells (mean + SEM is depicted).
Cytotoxicity of UTD cells (black) or CART-EGFRvIII.BiTE-EGFR cells (orange) against U87 by bioluminescence-based assay at indicated E:T ratios after 18 h. The assay was performed in triplicate, n = 3 biologically independent wells (mean + SEM is depicted).
(a) U87vIII glioma cells were implanted intracranially in NSG mice. Mice were treated intraventricularly on day 7 post-implantation with CART-EGFRvIII.BiTE-EGFR cells. On day 7 post-treatment, skin grafts from tumor-bearing mice were harvested and subjected to IHC for CD3. (b) Positive control skin graft harvested from a mouse treated with systemic EGFR CAR T cells. The experiment was repeated independently twice with similar results. Representative data are shown.
(a) U87KO (EGFR-negative), U87, or U87vIII glioma cells were implanted intracranially in NSG mice. Mice were treated intraventricularly on day 14 post-implantation with CART-EGFRvIII.BiTE-EGFR cells. On day 7 post-treatment, whole brain and blood were isolated from the treated mice. Proteins were extracted from brain tissue and blood. Each lane was loaded with 30 μg total protein and subjeced to SDS-PAGE and blotting with anti-His-tag antibody for BiTE and anti-β-actin antibody. Lanes 1 and 3 included 5 ng of added purified BiTE. (b) Protein samples from blood or brain (c) were analyzed by His Tag ELISA. 10 ng of BiTE was loaded for blood positive controls and 5 ng of BiTE was loaded for brain positive controls. The experiments were repeated independently three times with similar results. Representative data are shown. ELISA was performed in experimental groups as indicated, n = 3 biologically independent animals (mean + SEM is depicted, statistical significance determined by unpaired, two-tailed t-test).