Looking at the Future of CAR T-Cell Therapy: Combinations and Beyond David G. Maloney, MD, PhD Novel Approaches to CAR T-Cell Therapy: Combinations and Beyond Fred Hutch Cancer Research Center

David G. Maloney, MD, PhD Member-FHCRC, Professor-University of Washington Medical Director Bezos Family Immunotherapy Clinic Norma and Leonard Klorfine Endowed Chair for Clinical Research Disclosures

• Juno Therapeutics/Celgene: research funding • Advisory board participation – Celgene/Juno – Roche/Genentech – Kite Pharma/Gilead – Novartis CD19 Directed CAR T-Cell Therapy: Current Paradigm

• Collection of cells by resting state leukapheresis • T cells from patients often compromised in number or subsets • Cell manufacture (2-4 weeks) • May include isolation of T-cell subsets • T-cell stimulation • Transduction with viral vector (gamma or lentivirus containing the CAR) • Cell growth to target numbers (generally 1-2 million/kg) • Pass release criteria for safety and FDA specifications • Lymphodepletion chemotherapy • Given to deplete endogenous T cells and increase engraftment of CAR-T • Generally with cyclophosphamide and fludarabine • CAR T-cell infusion (usually cryopreserved and contain low level DMSO) • Monitoring for CRS and neurotoxicity Current CD19 CAR-T Toxicity: CRS and Neurotoxicity

Cytokine release syndrome (CRS) • Early hemodynamic changes associated with capillary leak • High fever, hypotension, tachycardia, decrease in protein/albumin, weight gain • Coagulopathy and increased transfusion requirements • Increased risk of hepatic and renal dysfunction • Elevated AST, ALT, bilirubin, alkaline phosphatase, creatinine • Cardiac arrhythmias • Elevated serum cytokines (IL-6 and many others) • Elevated serum CRP and ferritin • Usually responds to tocilizumab ± dexamethasone Neurotoxicity • Often presents with word finding difficulties and can progress to coma • May be associated with cerebral edema or seizures • Often onset 2-4 days after CRS • Generally reversible, but may be fatal • Not clear if treatments are effective (usually treated with steroids) How to Improve Outcome?

• Smarter, controllable CAR-T cells • Current CAR-T cells + additional agents • Checkpoint blockade with PD-1 or PD-L1 antibodies • Ibrutinib • Universal CAR-T cells “off the shelf” • UCART19 • Other CAR constructs • NK-T CAR Smarter, Controllable CAR-T cells Chimeric Antigen Receptor Design and Evolution

Fesnak AD, June CH, Levine BL, Nature Reviews, 2016 New Chimeric Antigen Receptor Models and Concepts

Fesnak AD, June CH, Levine BL, Nature Reviews, 2016 New Chimeric Antigen Receptor Models and Concepts, cont.

Fesnak AD, June CH, Levine BL, Nature Reviews, 2016 Controlling CARs by Boolean Logic Circuits

Chang ZL and YY Chen, Trends in Mol Med, 2018 Controllable CAR: SUPRA CAR

Cho, JH et al, Cell, 2018 Controllable CAR: SUPRA CAR, cont.

Cho, JH et al, Cell, 2018 Controllable CAR: SUPRA CAR, cont.

Cho, JH et al, Cell, 2018 Controllable CAR: SUPRA CAR, cont.

Cho, JH et al, Cell, 2018 Controllable CAR: SUPRA CAR, cont.

Cho, JH et al, Cell, 2018 CAR-T cell Combinations ZUMA-1: Preliminary Analysis of B-Cell and Immune-Related Molecules at Progression by Central Review

Progression Biopsies N=21

CD19+ CD19- CD19 (n=21) 14/21 (67) 7/21 (33)

PD-L1+ PD-L1 PD-L1 N/E PD-L1+ PD-L1- PD-L1 N/E PD-L1 (n=19) 9/14 (64) 4/14 (29) 1/14 (7) 4/7 (57) 2/7 (29) 1/7 (14)

• Post-progression tumor biopsies (21 evaluable patients) - 33% were CD19- by central review - 62% were PD-L1+ by central review Baseline and post-progression samples were not obtained from the same lesions. CD19 H-score of 0 was determined negativity. H-scores ≥1 were considered positive. H-score was calculated as a product of IHC intensity (scale 1-3) multiplied by the percentage of tumor cells at a given intensity (0%-100%). PD-L1 status was determined by the percentage of tumor cells with any membrane staining above background or by the percentage of tumor-associated immune cells with staining at any intensity above background. IHC, immunohistochemistry; N/E, not evaluable; PD-L1, programmed cell death-ligand 1. Neelapu et al, ASH 2017, Abstract 578 ZUMA-1: Representative Immunohistochemistry of B-Cell and Immune-Related Molecules Patient Summary CD19 at Baseline CD19 at PD PD-L1 at PD

BOR: PR BL: CD19+ PD: CD19– PD-L1+

BOR: PR BL: CD19+ PD: CD19+ PD-L1+

All images at 40× magnification. Scale bar indicates 60 µm. BL, baseline; BOR, best objective response; PD, progressive disease; PD-L1, programmed cell death-ligand 1. Neelapu et al, ASH 2017, Abstract 578 JCAR017 Re-expansion and Tumor Regression Following Incisional Tumor Biopsy

Case Report 68-yo woman with refractory DLBCL Flu/Cy lymphodepletion JCAR017 CD19 specific CAR-T cell infusion no CRS or NT 1 month PET negative CR, including brain mass 3 month extra cranial PD Incisional biopsy Re-expansion of CAR-T and remission

Pre CAR-T 1 mo post CAR-T, CR 3 mo post CAR-T, PD Post-incisional Bx, CR Abramson J et al, NEJM, 2017 Improving CD19 CAR T-Cell Therapy Decrease tumor escape mechanisms • CD19 negative relapse • Target additional tumor associated antigens • Multiple CAR-T products • Cells with multiple CAR-receptors

• Block PD-1/PD-L1 signaling • Combine CAR-T with PD-1 or PD-L1 mAbs • Knock out PD-1 in the CAR-T cells (armored CARS) CAR-T Cell Combinations: Checkpoint Inhibitor mAbs PD-L1 tumor expression Case Report 35-yo man with refractory PMBCL Cy x 6 doses for lymphodepletion CART19 5.3 x 106/kg Day 26 PD with symptoms, increased mass Rx: Pembrolizumab 2 mg/kg Activation of CAR-T cells and tumor reduction

Chong EA, Schuster SJ, Blood, 2017 Ongoing Clinical Trials of Anti CD19 CAR-T and Checkpoint mAbs

Multiple ongoing clinical trials (few highlighted here) • Axicabtagene ciloleucel and atezolizumab (anti-PD-L1) • Full dose mAb evaluated from day 21 to day 1 post CAR-T in phase 1 (3 x 3 design, n=9) • mAb q 3 weeks x 4 doses • Well tolerated, no obvious increase CRS or NT (ASH 2017) • Phase 2 enrolling • JCAR014 and durvalumab (FHCRC #9457) • Evaluating post-treatment durvalumab (q month) • Evaluating pre-treatment (d -1) durvalumab followed by post- treatment durvalumab for up to 1 year • JCAR017 and durvalumab (enrolling) Ibrutinib May Improve CAR-T Cell Function and Decrease CRS • Pts with CLL have defective T cells from CLL exposure • Ibrutinib treated patients have • Improved CAR-T generation and expansion • Better T-cell function • Less CRS • Better clinical activity from CAR-T?

Ibrutinib improves CAR-T expansion

Fraietta JA et al. Blood 2016;127:1117-1127 Ibrutinib Potentiates CTL019 Cell Expansion and Engraftment, Which Correlates With Clinical Response

Fraietta JA et al. Blood 2016;127:1117-1127 Universal CAR-T?

Autologous CAR-T Approach Universal CAR-T ➢ Patient specific therapy ➢ ”Off the shelf” universal donor/cell line ➢ Pts have compromised T cells ➢ Cells with no prior exposure to cytotoxins ➢ Autologous leukaphereses ➢ “Off the shelf” ➢ Requires 3-4 weeks for production ➢ “Off the shelf,” could be pre-shipped to center

➢ Less immune rejection ➢ Immune rejection requires additional engineering ➢ No risk of GVHD ➢ Risk of GVHD requires additional engineering

➢ Cells may persist a long time ➢ Shorter persistence Engineered “Off the Shelf” CAR-T Cells

CS1CS1 o r

UCART Attributes

• CAR expression to redirect T-cells to tumor antigens • Suicide gene (RQR8) for safety • Inactivation of the TCRa constant gene (TRAC) eliminates the TCR and minimizes risk of GvHD TALEN

• Additional gene editing to Donor blood cells Product • Facilitate continued lymphodepletion (CD52,dCK) • Prevent CAR-T cell cross-reactivity (CS1/SLAMF7) TALEN mediated gene editing to • To checkpoint pathways inhibition enable an allogenic approach (PD1,Lag3,…) • To delay rejection by T-cells (MCH-I inactivation) • … UCART19* Initial Proof of Concept in ALL

 1st patient dosed in June 2015 (compassionate)

 Phase 1 trials (US/EU) started in June 2016 Tumor cell

 4 recruiting centers (EU and US)

 14 patients treated disclosed (7 adults and 7 pediatric)**

 Results in line with early autologous CAR-T UCART19 phase 1 results published in past years

 Patients failed >5 lines of treatment, including autologous CAR-T

 Ph1b expansion at U Penn and MD Anderson

* UCART19 is exclusively licensed to Servier and under a joint clinical development program between Servier and Pfizer ** Including compassionate UCART19 Clinical Trial Results

Phase 1 pediatric B-cell ALL trial • Used as a bridge to allogeneic HCT (MRD negative remission) • N=6, age 3.75 y (range 0.8-16.4 y) • 5 patients had < 10% BM blasts • Flu/Cy/alemtuzumab lymphodepletion • Toxicity • CRS in all patients, ( 1 grade 1, 4 grade 2, 1 grade 3) • NT, 2 grade 1, 1 grade 2 • 3 prolonged cytopenia • 1 grade 1 cutaneous GVHD • 5 had viral infections (adeno, CMV, metapneumo, BK) • 5/6 MRD negative (flow), 3/6 PCR (d28-42) and 5 received allogenic HCT (d49-62) • Conditioning Flu/TBI ± Cy/ATG and all received rituximab • 3/5 alive (2 PD and 1 TRM [infections]) • 2 in MRD negative remission (10 and 11 months) Veys P, abstract/presentation at EBMT, 2018 Universal CD19 CAR-T: FT819

Manufactured from an induced pluripotent stem cell • Unlimited self renewal CAR construct introduced into the TRAK locus in single pluripotent cell • Lacks endogenous TCR • Uniform CAR expression • Better elimination of tumor (mouse) c/w conventional CAR-T Second targeting receptor containing CD16 Fc • Binds to mAbs coating tumor cells providing additional activity through ADCC • May help prevent antigen negative escape

Valamehr B et al, AACR, 2018 Cord Blood Derived CD19 Specific NK-T CAR

NK-T cells derived from cord blood • Universal donor, no HLA matching • Multiple products per cord, “off the shelf”

Genetically engineered to express • CD19 CAR with CD28 costimulatory domain • Secrete IL-15 (promotes NK cell proliferation and survival) • iCasp9

Initial clinical trial results encouraging • NK-T cell engraftment and expansion in vivo • CRS • Tumor responses in NHL and ALL Rezvani K and Shpall EJ, MD Anderson CAR NK Cells in Patients With Relapsed/Refractory B-lymphoid Malignancies (CLL, NHL, ALL) PI: Katy Rezvani MD, PhD; MD Anderson Cancer Center

3 dose levels. Patient receive one infusion at: 1x10e5/kg, 1x10e6/kg, 1x10e7/kg, NK cells NK NK derived Transduction NK NK NKNK from CB NK NK NK Expansion of CAR NK of NK cells NK NK NK NK NK NK NK NKNK NK cells NK NK with NK NK NK NK NK retroviral NK NK NK NK NK NK NK NK NK vectors NK NK Cyclophosphamide, 300 mg/m2 Antigen Specific CAR+ NK cells Fludarabine 30 mg/m2 Cell Infusion

Day -14 Day -12 Day -10 Day -4 Day -3 Day -2 Day 0

Banked umbilical cord blood

Courtesy K. Rezvani Approved by IRB and FDA 2016-0641 (IND 17321) Patient 5- CLL With Richter’s Transformation Achieved Complete Response in Lymph Nodes Pre-admission Day 30 post CAR NK

Courtesy K. Rezvani Future Approaches to CAR-T Therapy: Conclusions • CAR-T cell design will allow endless possibilities • Controllable CAR expression • Logic driven CAR expression and killing • Mitigation of toxicity • Enhanced proliferation, killing and resistance to inhibition • Combinations will likely improve outcome • Checkpoint mAbs • Ibrutinib? • Universal CAR-T (or NK-T) approaches • Improve delivery of an “off the shelf” product • Reduce cost (??) May be challenging to get new approaches approved in current era Immunotherapy is changing the way cancer is treated!

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