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Development of CAR T Cell Platform for Therapeutic Applications to Glioblastoma
Yibo Yin, Zev A. Binder, Radhika Thokala, Donald M. O’Rourke Department of Neurosurgery
Center for Cellular Immunotherapies Abramson Cancer Center
University of Pennsylvania [email protected]
Outline
Strategy to target Glioblastoma heterogeneity Reversing anergy in tumor microenvironment (TME)
Platform of translational studies in canine
spontaneous glioblastoma model
Current malignant glioma CART clinical trials
• EGFRvIII NCT02209376 UPENN NCT01454596 NCI NCT02664363 Duke University
• HER2 NCT02442297 Baylor
• EphA2 NCT02575261 China
• IL13Rα2 NCT02208362 City of Hope
Target NCT# Location/Sponsor
(zetakine)
Johnson LA, et al. Cell Res 27(1), 38-58, 2017
EGFRvIII-targeting CAR T cells
O’Rourke 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. 2017 Jul 19;9(399). O’Rourke 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. 2017 Jul 19;9(399). No subjects experienced evidence of EGFR-directed toxicity or systemic cytokine release syndrome.
The aim of this Phase Ⅰ study was not survival but rather to demonstrate T cell entry and safety.
Target heterogeneity-specific target editing
EGFRvIII expression post-infusion EGFR amplification post-infusion
O’Rourke 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. 2017 Jul 19;9(399).
Decrease in target antigen (EGFRvIII) was not mirrored by a decrease in EGFR amplification.
Patient
205
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Pre-CART EGFRvIII 21% EGFR p.A289V EGFR amp 9-fold
EGFRvIII 95% EGFR amp 10-fold
EGFRvIII 60% EGFR p.R108K EGFR amp 8-fold
EGFRvIII 42% EGFR p.G598V EGFR amp 7-fold PIK3CA p.E542K
EGFRvIII 70% EGFR p.R108K EGFR amp 12-fold
Post-CART EGFRvIII negative EGFR amp 5-fold
EGFRvIII 72-95% (multiple areas tested) EGFR amp 16-fold
EGFRvIII 13% EGFR amp 5-fold
EGFRvIII negative EGFR amp 10-fold PIK3CA p.E542K
EGFRvIII 0, 9, 57, 95% EGFR p.R108K EGFR amp 12-fold
Specificity of Antigen Editing after EGFRvIII CART Infusion EGFR alterations post-infusion
EGFR alterations co-occurring with EGFRvIII did not change with 2173 EGFRvIII-CAR T treatment.
Dr. Zev A. Binder Senior Research Investigator
Dr. Radhika Thokala Postdoctoral fellow
Heterogeneity: EGFR Extracellular Domain Mutations confer oncogenic phenotype: EGFR A289V
Binder et al. Cancer Cell, In Press
Phage Display to identify EGFR ECD* novel mAbs (Radhika Thokala, Mike Milone, Don Siegel)
Heterogeneity: IL13 Receptor α2
• There are two receptors for IL13 (IL13Rα1 & IL13Rα2).
• The signaling pathway of IL13Rα1 associates with apoptosis.
• IL13Rα2 is a decoy receptor and tumor associated antigen.
Thaci B, et al. Neuro-Oncology 16(10), 1304–1312, 2014
IL13 zetakine CAR T cells
Penn BTTB: Examination of primary glioma stem cell cultures reveals mixed expression of IL13Rα2 (Zhang, Yin, Binder)
Logan Zhang
Zetakine CAR not only demonstrates binding to IL13Rα2 but also binds to IL13Rα1
Kong S, et al. Clin Cancer Res 18(21), 5949-5960, 2012
zetakine CAR
scFv CAR
mutant IL13 protein
scFv
IL13 Receptor α2 is a promising target for CAR T therapy, but needs to be further explored
• There are two receptors for IL13 (IL13Rα1 & IL13Rα2).
• The signaling pathway of IL13Rα1 associates with apoptosis.
• IL13Rα2 is a decoy receptor and tumor associated antigen.
Thaci B, et al. Neuro-Oncology 16(10), 1304–1312, 2014 Brown CE, et al. N Engl J Med 375(26), 2561–2569, 2016
Intr
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Del
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Intr
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Further evaluation of routes of delivery should be conducted prior to additional human trials.
Humanized IL13Rα2 targeting CAR T cells (Hu07,08 scFv BBz) were specifically stimulated by IL13Rα2, but not by
IL13Rα1 Hu07BBz Hu08BBz IL13Rα1 IL13Rα2
A549
D
270
IFNγ
CD8 Fully humanized scFv based IL13Rα2 targeting CAR T cells with high specificity and affinity.
Yibo Yin, M.D. Postdoctoral fellow
IL13Rα2 specific CAR T cells inhibit tumor growth of NSG mice with D270 intracranial gliomas.
Yibo Yin, Alina Boesteanu, Kristin Blouch, Laura Johnson
Pico de Coana Y, et al. Trends Mol Med 21(8), 482–491, 2015
Immune Checkpoint Receptors
• Crucial molecules for fine-tuning immune responses
• Involved in the mechanism of resistance of tumor immunotherapy
• Blocking immune checkpoint receptors might be able to enhance the function of CAR-T cells.
O’Rourke 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. 2017 Jul 19;9(399).
TME response
O’Rourke 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. 2017 Jul 19;9(399).
The tissue microenvironment demonstrated a responsive increase in immunosuppressive markers post-CART infusion.
TME response
Differential immune checkpoint expression
Co-culture experiments: CAR T cell populations expressed different levels of immune checkpoint molecules.
NSG mice
5e5 D270 s.c.
anti-EGFRvIII BBz
anti-IL13Rα2 BBz
Bioluminescent imaging Tumor calipering
every 3-4 days
CAR T cells combined checkpoint blockade treat NSG mice with D270 s.c.
Endpoint: 2cm in any direction
2e6 CAR+ i.v.
anti-PD-1
PBS
anti-CTLA-4
anti-TIM-3
200ug/mouse/4days i.p. N=5 per group
(2173BBz)
(Hu08BBz)
Combination therapy of CAR T cells and immune checkpoint blockade can be optimized based on the CAR target.
Anti-PD-1 Anti-CTLA-4 Anti-TIM-3
Yibo Yin, Alina Boesteanu, Kristin Blouch, Laura Johnson
Checkpoint blockade enhanced CAR T cells function in the NSG mice with D270 s.c. model.
STUDY SCHEMA: New Trial Cohort 2018 Phase I Study of EGFRvIII (2173)-directed CAR T cells in combination with dual PD-1 and IDO inhibition in patients with newly diagnosed, MGMT-unmethylated glioblastoma
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Short course radiation (40 Gy in 15 fx) Concurrent TMZ 75 mg/m2
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PD
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Maintenance PD-1/IDO Inhibition Q2W Cycles Until Progression or Intolerance
Surgical resection if clinically indicated
Off Study
PROGRESSIVE DISEASE PER RANO CRITERIA
2W 2W 2W 2W
3 weeks
Stephen Bagley, Arati Desai
Canine GBM – a spontaneous, immunocompetent model • Retrospective study of necropsy dogs
• 2% brain tumors; 70% gliomas; astrocytomas 17-28% of all primary CNS tumors • Boxers and Boston terriers have a higher frequency
• Clinical signs: mentation changes, pacing, seizures, vestibular disturbance, vision loss
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Canine GBM Treatment and Prognosis Palliative care:
Steroids at anti-inflammatory dosing, AEDs (phenobarbital), Omeprazole
Chemotherapy: CCNU 60 mg/m2 every 4-6 weeks Cost prohibitive and few trials: Temozolomide at 65-100 mg/m2 for 5 days, repeated every 2-3 weeks
Radiotherapy: Conventional: 16 fractions x 3 Gy, total 48 Gy Stereotactic Radiosurgery: 1-3 fractions of total 12-25 Gy
Surgery: Cost prohibitive, owner perceived QOL concerns Based on location, offered as a debulking/biopsy Often an en bloc approach, however, due to consistency of glial tumors often not possible. Post-op effects: amaurosis, seizures, change in behavior
Prognosis: Case studies: 3-12 months with chemo, surgical debulking, or radiation (alone or in combination) Systematic review: 127 cases of intra-axial tumor MST was 226 (unrelated to treatment modality)
Comparative Oncology Program (COP) at NIH and the Comparative Oncology Trials Consortium
https://ccr.cancer.gov/comparative-oncology-program/research/cbtc
https://www.washingtonpost.com/national/health-science/new-tricks-in-canine-cancer-research-may-improve-treatments-for-humans-too/2016/11/26/837dcd52-a4fc-11e6-8042-f4d111c862d1_story.html?utm_term=.851c973bbd2f
Harley, a 4-year-old boxer who has leukemia, receives an infusion of his own T cells at the University of Pennsylvania’s Ryan Veterinary Hospital. The cells have been genetically modified to help combat his cancer. (Katherine Frey/The Washington Post)
Available model for IL13Rα2 targeted Therapy
Spontaneous canine gliomas can be used as an immunocompetent model system.
caIF
Nγ
caCD4
Hu08HuBBz Hu08CaBBz UTD
CAR
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Canine IL13Rα2 targeting CAR T cells respond to canine glioblastoma cells (J3T)
Yibo Yin, Nicola Mason, M. Kazim Panjwani, Avery Posey Jr.
Canine IL13Rα2 targeting CAR T cells inhibit canine tumor growth of NSG mice with J3T intracranial gliomas.
Canine Glioma Trial IL13Rα2 homology ~70%
Intact tumor microenvironment (TME)
Canine CAR T cells and universal human CAR T cells
CAR T cells +/- PD1 blockade (canine PD1 mAb available)
Platform of translational studies in canine spontaneous
glioblastoma model human trial design
Nicola Mason and team, Penn Vet School
Conclusions
• Target heterogeneity
Multi-targeting e.g. EGFRvIII, IL13Rα2, EphA2 and HER2
• Tumor microenvironment
Selected ICBs e.g. anti-PD-1, anti-CTLA-4 and anti-TIM3
• Canine spontaneous glioblastoma
Reliable platform IL13Rα2 targeting CAR therapy
Acknowledgments and Funding • Donald M. O’Rourke
([email protected]) • Zev A. Binder • Radhika Thokala • Logan Zhang • Yibo Yin
• Zhiguo Lin • Carl H. June • MacLean Nasrallah • Nicola J. Mason
• M. Kazim Panjwani • Avery D. Posey Jr. • Michael C. Milone • Donald L. Siegel
• Laura A. Johnson (GSK) • Alina C. Boesteanu • Chong Xu • Jesse L. Rodriguez • Danielle R. Cook • Kristin Blouch • Bevin McGettigan-Croce
Funding • The Templeton Family Initiative in
Neuro-Oncology • The Maria and Gabriele Troiano
Brain Cancer Immunotherapy Fund • NIH DP2 CA174502 • NIH NINDS R01NS042645 • NNSFC 8177101833, 81571646