Professor John Gribben Barts Cancer Institute New Frontiers in CAR T Cell Therapy: What Are TheirFuture Roles in CLL? Professor John Gribben Barts Cancer Institute

The Power of the Chimeric Antigen Receptor (CAR)! The basic parts of the typical CAR are shown here.

Redirecting the Specificity of T Cells Targeting Leukemia with Chimeric Antigen Receptor Modified T cells T cell CD19 Native TCR CLL cell CARs combine an Ag recognition domain of antibody with intracellular signaling domain into single chimeric protein. Gene transfer (lentivirus vector) to stably express CAR on T cells confers novel Ag specificity. CTL019 cell Dead CLL cell Lentiviral vector Anti-CD19 CAR construct

Designing a Chimeric Antigen Receptor y 5’LTR 3’LTR scFv Hinge Costim z ζ SIGNAL 2 Costimulatory molecule The basic parts of the typical CAR are shown here. Antibody TCR zeta chain SIGNAL 1

Increasing Potency Across Generations: Not All CARs Created Equal Classic CARs Sports CARs Muscle CARs

Clinical Trials Using CAR-T Lymphocytes Major limitation to overcome is a lack of persistence of CAR-transduced T cells in vivo CAR-CD19 studies are attempting to improve T-cell persistence using different strategies 6

Incomplete Activation of First-Generation CAR-Directed T Cells Improved T-cell activation and proliferation Tumor B7 1st gen CAR CD28 2nd gen CAR T cell ζ T cell ζ CD28 Killing of tumor cells 7

“Second-Generation” CARs Potential costim domains: CD28 OR 41BBL OX40L Spacer Linker Anti-IgG1. CH2-CH3 scFv zeta CD28 zeta Add 2 costims = third-generation CAR Increased IL-2 production Increased T-cell persistence?? Maher et al. Nat Biotechnol. 2002.

CD19 CARs: Original CAR and CARs in Clinical Trials Juno Kite Bluebird bio Novartis Juno

Are Second-Generation Superior to First-Generation CAR T Cells Targeting CD19? Infuse 2 autologous activated T-cell lines expressing CAR.CD19z CAR.CD19-28z into each patient Track each CAR T-cell population in vivo First generation Second generation BCM

✔ Only T Cells Transduced With Second-Generation CAR Expanded In Vivo CAR-CD19z+ T cells CD20+ cells CAR-CD19.28z+ T cells 350 10 First infusion ✔ 300 8 250 6 200 CD20+ Cells (%) Copy Numbers × 103 ng of DNA 150 4 100 2 50 Week 1 Week 2 Week 4 Week 6 Pre-infusion 3hr post-infusion Savoldo et al. J Clin Invest. 2011.

Improving Persistence 2 Improving costimulation (second-gen CAR.CD28) PLUS Make space for CAT T cells to expand CD28 Lymphodepletion MSKCC and NCI approach

Using T Cells Transduced With Second-Generation CD19. CAR Using T Cells Transduced With Second-Generation CD19.CAR.28z After Lymphodepleting Chemotherapy (± IL-2 ) Days –5 to –1 fludarabine administration Day 0 to 5 administration of IL-2 Day –7 and –6 cyclophosphamide administration Day 0 Infusion of RETROVIRUS CD19-CAR.28z transduced T cells DEFINED LYMPHODEPLETING REGIMEN ± IL-2 Cyclophosphamide dose is 60 mg/kg for 2 doses Fludarabine dose is 25 mg/m2 for 5 doses IL-2 dose is 720,000 IU every 8 hours to tolerance (maximum 15 doses)

Persistence of CAR-T Cells Correlates With Response PR, Steroids CR PR NR 0 12 Months After Infusion Porter. Sci Transl Med. 2015

Bone Marrow Disease Cleared Within 14 Weeks After CD19.CAR.28z T Cells Before treatment: extensive involvement H&E CD19 CD79a CD20 14 weeks after treatment: absence of all B cells H&E CD19 CD79a CD20 Kochenderfer et al. Blood. 2010.

CD19-CAR.28z T Cells Using Low-Dose Conditioning: Disease Responses With Reduced Toxicities: NCI CD19-CAR.28z T Cells Using Low-Dose Conditioning: Disease Responses With Reduced Toxicities: NCI Patient 2 had a PR of chemotherapy-refractory triple-hit DLBCL that lasted 9 months after infusion of anti-CD19 CAR T cells Before Treatment 6 Months After Treatment <50% patients receiving anti-CD19 CAR T cells after low-dose chemo required ICU admission. Overall response rate: 73%, CR in 55%, PR in 18%

CD19 CAR Therapy Toxicities No Pain No Gain?? The Cytokine Release Syndrome High spiking fevers Neurologic Toxicities 50% ICU admission Deaths

Issues to Resolve CLL T cells “damaged” by previous therapy and by disease COST Manufacturing issues Patient selection Other targets than CD19 – this loses all normal B cells ROR1 CD38 Will it ever by NICE approved????