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 Donald.ORourke@uphs.upenn.edu

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

CART approaches to human GBM

Structural comparison for CARs used in GBM

Strategy to target heterogeneity

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

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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

EGFR mutational landscape in GBM: Penn Cohort

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

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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

Reversing anergy in tumor microenvironment

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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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

Platform of translational studies in canine spontaneous glioblastoma model

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

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Hu08HuBBz Hu08CaBBz UTD

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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

(Donald.ORourke@uphs.upenn.edu) • 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