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.
Subscribe to Journal
Get full journal access for 1 year
We are sorry, but there is no personal subscription option available for your country.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Data generated or analyzed during this study and all unique materials can be made available by the corresponding author.
Imperato, J. P., Paleologos, N. A. & Vick, N. A. Effects of treatment on long-term survivors with malignant astrocytomas. Ann. Neurol. 28, 818–822 (1990).
Mullard, A. FDA approves first CAR T therapy. Nat. Rev. Drug Discov. 16, 669 (2017).
O’Rourke, D. M. et al. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci. Transl. Med. 9, 586 (2017).
Ahmed, N. et al. HER2-specific chimeric antigen receptor-modified virus-specific T cells for progressive glioblastoma: a phase 1 dose-escalation trial. JAMA Oncol. 3, 1094–1101 (2017).
Brown, C. E. et al. Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N. Engl. J. Med. 375, 2561–2569 (2016).
Choi, B. D. et al. Bispecific antibodies engage T cells for antitumor immunotherapy. Expert Opin. Biol. Ther. 11, 843–853 (2011).
Verhaak, R. G. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17, 98–110 (2010).
Wikstrand, C. J., McLendon, R. E., Friedman, A. H. & Bigner, D. D. Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor, EGFRvIII. Cancer Res. 57, 4130–4140 (1997).
Felsberg, J. et al. Epidermal growth factor receptor variant III (EGFRvIII) positivity in EGFR-amplified glioblastomas: prognostic role and comparison between primary and recurrent tumors. Clin. Cancer Res. 23, 6846–6855 (2017).
Uhlen, M. et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol. Cell. Proteomics 4, 1920–1932 (2005).
Choi, B. D. et al. Systemic administration of a bispecific antibody targeting EGFRvIII successfully treats intracerebral glioma. Proc. Natl Acad. Sci. USA 110, 270–275 (2013).
Priceman, S. J. et al. Regional delivery of chimeric antigen receptor-engineered T cells effectively targets HER2(+) breast cancer metastasis to the brain. Clin. Cancer Res. 24, 95–105 (2018).
Choi, B. D. et al. Intracerebral delivery of a third generation EGFRvIII-specific chimeric antigen receptor is efficacious against human glioma. J. Clin. Neurosci. 21, 189–190 (2014).
Fajardo, C. A. et al. Oncolytic adenoviral delivery of an EGFR-targeting T-cell engager improves antitumor efficacy. Cancer Res. 77, 2052–2063 (2017).
Bargou, R. et al. Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science 321, 974–977 (2008).
Sadelain, M., Riviere, I. & Riddell, S. Therapeutic T cell engineering. Nature 545, 423–431 (2017).
Long, A. H. et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat. Med. 21, 581–590 (2015).
Pandita, A., Aldape, K. D., Zadeh, G., Guha, A. & James, C. D. Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR. Genes Chromosomes Cancer 39, 29–36 (2004).
Radaelli, E. et al. Immunohistopathological and neuroimaging characterization of murine orthotopic xenograft models of glioblastoma multiforme recapitulating the most salient features of human disease. Histol. Histopathol. 24, 879–891 (2009).
Johnson, L. A. et al. Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma. Sci. Transl. Med. 7, 275ra222 (2015).
Agero, A. L. et al. Dermatologic side effects associated with the epidermal growth factor receptor inhibitors. J. Am. Acad. Dermatol. 55, 657–670 (2006).
Nanney, L. B., Magid, M., Stoscheck, C. M. & King, L. E. Jr. Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J. Invest. Dermatol. 83, 385–393 (1984).
Majzner, R. G. & Mackall, C. L. Tumor antigen escape from CAR T-cell therapy. Cancer Discov. 8, 1219–1226 (2018).
Moscatello, D. K. et al. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res. 55, 5536–5539 (1995).
Kloss, C. C., Condomines, M., Cartellieri, M., Bachmann, M. & Sadelain, M. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat. Biotechnol. 31, 71–75 (2013).
Cho, J. H., Collins, J. J. & Wong, W. W. Universal chimeric antigen receptors for multiplexed and logical control of T cell responses. Cell 173, 1426–1438 e1411 (2018).
Roybal, K. T. et al. Precision tumor recognition by T cells with combinatorial antigen-sensing circuits. Cell 164, 770–779 (2016).
Caruso, H. G. et al. Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity. Cancer Res. 75, 3505–3518 (2015).
Choi, B. D. et al. Human regulatory T cells kill tumor cells through granzyme-dependent cytotoxicity upon retargeting with a bispecific antibody. Cancer Immunol. Res. 1, 163 (2013).
Rafiq, S. et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat. Biotechnol. 36, 847–856 (2018).
Reardon, D. A. et al. OS10.3 randomized phase 3 study evaluating the efficacy and safety of nivolumab vs bevacizumab in patients with recurrent glioblastoma: CheckMate 143. Neuro Oncol. 19, iii21–iii21 (2017).
Wherry, E. J. et al. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4, 225–234 (2003).
Kaech, S. M. & Wherry, E. J. Heterogeneity and cell-fate decisions in effector and memory CD8+ T cell differentiation during viral infection. Immunity 27, 393–405 (2007).
Santomasso, B. D. et al. Clinical and biological correlates of neurotoxicity associated with CAR T-cell therapy in patients with B-cell acute lymphoblastic leukemia. Cancer Discov. 8, 958–971 (2018).
Day, C. P., Merlino, G. & Van Dyke, T. Preclinical mouse cancer models: a maze of opportunities and challenges. Cell 163, 39–53 (2015).
Westphal, M., Maire, C. L. & Lamszus, K. EGFR as a target for glioblastoma treatment: an unfulfilled promise. CNS Drugs 31, 723–735 (2017).
Choi, B. D., Maus, M. V., June, C. H. & Sampson, J. H. Immunotherapy for glioblastoma: adoptive T-cell strategies. Clin. Cancer Res. 0432.CCR-18-1625 (2018).
Choi, B. D., Curry, W. T., Carter, B. S. & Maus, M. V. Chimeric antigen receptor T-cell immunotherapy for glioblastoma: practical insights for neurosurgeons. Neurosurg. Focus 44, E13 (2018).
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).
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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).
About this article
Cite this article
Choi, B.D., Yu, X., Castano, A.P. et al. CAR-T cells secreting BiTEs circumvent antigen escape without detectable toxicity. Nat Biotechnol 37, 1049–1058 (2019). https://doi.org/10.1038/s41587-019-0192-1
ESMO Open (2021)
British Journal of Cancer (2021)
Bcl-2 Enhances Chimeric Antigen Receptor T Cell Persistence by Reducing Activation-Induced Apoptosis