Sleeping Beauty: Current applications and future strategies CAR-TCR Summit 2017 Partow Kebriaei, MD
Outline Chimeric antigen receptor (CAR) technology Viral versus nonviral vectors Results of current clinical trials utilizing the Sleeping Beauty (SB) platform Future strategies utilizing the SB platform to target hematologic malignancies with CARs and solid tumors with T- cell receptor (TCR)s Support provided by ZIOPHARM Oncology
a b Current CAR, TCR T-cell technology Uses a genetically-engineered CAR or TCR that is: Transduced into T cells using viral and non-vectors and Expressed in T cells which are expanded ex vivo and then administered to patients to target tumor cells in the body The introduced CAR and TCR redirect T-cell specificity to target cancer cells Recipient Endogenous immune response fails to halt cancer Intracellular targets T cell TCR CAR T cell T cell genetically modified ex vivo to redirect specificity Cell surface targets
Allogeneic, autologous CAR T-cells One allogeneic donor into multiple recipients Made in advance of need and infused on demand Allogeneic Donor Viral gene transfer Off-the-shelf (OTS) Engineered T cells Recipients One autologous donor/recipient Made as needed Readily available Barriers: Requirement for lymphodepletion product. Rejection Potential of for infused T cells rejection curtails of anti-tumor modified effect Only T cells. applicable to CARs (not solid tumors) Very little clinical data. Current practice Autologous Donor Viral gene transfer Engineered T cells Recipient Most commonly Barriers: Requirement used. for lymphodepletion Potential for Cost Time manufacture to manufacture failure. (during Long which time to patients make. condition deteriorates) Autologous
Non-viral versus viral transduction Release DNA plasmid Packaging cells Lentivirus Ex vivo gene transfer Sleeping Beauty Release DNA plasmid Non-viral gene transfer: Sleeping Beauty Cost effective Slower transduction rate More nimble production: Customizable Viral gene transfer: Retrovirus & Lentivirus High cost Faster transduction rate Labor intensive, challenging to customize
Limitations of current approach to CAR + (& TCR + ) T Cells Heterogeneity in end-product after ex vivo culture Failure to manufacture. Risk of insertional mutagenesis Time for manufacture Lack of control of T cells after infusion Need for lymphodepletion Specialized treatment centers for administration
SLEEPING BEAUTY: CLINICAL TRIALS AT MD ANDERSON CANCER CENTER
SB transposon/transposase Transposon DNA plasmid IR/DR hef1a Transposase DNA plasmid (or in vitro transcribed mrna) CMVIE IR/DR CAR SB11 Transposase Transposase CAR Co-delivery into cells by nucleofection Transposon Nucleus Cytoplasm scfv Extracellular scaffold Non-viral gene transfer using Sleeping Beauty system to express CAR, TCR and membranebound IL-15 (mbil15) Sleeping Beauty DNA plasmid Release Intracellular domains Showing signaling motifs Chimeric antigen receptor (CAR) a b T-cell receptor (TCR) Membrane bound IL-15 (mbil15)
First-in-human application of SB system CAR + T cells infused after hematopoietic stem-cell transplantation Methods available at: J Vis Exp. 2013 Feb 1;(72):e50070. Irradiated AaPC (feeder cells) derived from K-562 cells and modified to co-express CD19, CD86, CD137L, membranebound IL-15 (mbil15, and CD64) 9
Trial schema Study populations CD19 + lymphoid malignancies beyond first remission, induction failure, or relapse at time of HSCT 1-65 yrs-old for allo-hsct; up to 75 yrs-old for auto-hsct Preparative treatment Auto-HSCT BEAM prep PBSC day 0, CAR T cells day +2 Allo-HSCT HSCT prep per MD choice Donor-derived T cells 6-12 weeks post HSCT GVHD prophylaxis maintained J Clin Invest. 2016 Sep 1;126(9):3363-76. Dose Level Single T-cell dose* A >5 x 10 7 /m 2 but < 5 x 10 8 /m 2 B >5 x 10 8 /m 2 but < 5 x 10 9 /m 2 Dose Level Single T-cell dose* A Not to exceed 10 6 /m 2 B >10 6 /m 2 but < 10 7 /m 2 C >10 7 /m 2 but < 5x 10 7 /m 2 D >5x10 7 /m 2 but < 10 8 /m 2 *Per body surface area
Trial Summary I Successful manufacture of T-cell products 200 ml of peripheral blood (avoiding costs & inconvenience of apheresis) Safely infuse patients No immediate or late toxicity Decreased GVHD rate at 11% Administered up to 10 8 /m 2 genetically modified haplo-identical T cells Decreased CMV reaction, 24% vs. 41% 1 Outpatient infusions Cytokines Low levels of cytokine at time of T-cell infusion Mild elevation, peak at ~2 weeks No cytokine storm 1. J Oncol Parm Practice, 2014, 20:257 1 st generation complete
First-in-human use of SB system: Summary II Persistence of CAR + T cells Average, days Max, days Autologous 201 360 Allogeneic 51 180 Survival of recipients after CAR + T cells PFS OS Autologous 83%, 3-yr 100% 3-yr Allogeneic (all) 53%, 1-yr 63%, 1-yr Allo, haploidentical 75%, 1-yr 100%, 1-yr CAR + T cells exhibit longer persistence in the auto-hsct group. No apparent positive correlation with T-cell dose or disease burden. J Clin Invest. 2016 Sep 1;126(9):3363-76
1 st generation trial: Evaluate SB system (first-in-human studies) 1 st generation SB platform in two trials infusing CAR + T cells after HSCT showed favorable PFS and OS in both autologous and allogeneic cohorts 1 compared with historical controls. 2,3 1. J Clin Invest. 2016 Sep 1;126(9):3363-76 2. Blood 125, 2579-2581 (2015) 3. Curr Hematol Malig Rep 7, 144-152 (2012)
Shortening manufacturing time for CD19-specific CAR + T cells 1 st generation complete 2 nd generation ongoing J Clin Invest. 2016 Sep 1;126(9):3363 Shortening manufacturing process time from 4 weeks to ~ 2 weeks as T cells co-cultured on feeder cells
2 nd generation trial with SB-modified T cells for active CD19 + malignancies clinicaltrials.gov: NCT02529813 CD19-specific CAR + T cells; SB-based gene transfer T cells modified to express CD19-specific CARs with a CD8 stalk region that signals through CD28 and CD3z Shortened manufacturing time CAR + T cells infused following lymphodepletion in active disease Phase 1, dose escalation Actively recruiting Dose level -1 10 5 CAR + T-cell dose/kg +1 > 10 5, but 10 6 +2 > 10 6, but 10 7 +3 > 10 7, but 10 8 +4 > 10 8, but 10 9
SLEEPING BEAUTY: COMBINING CAR WITH CYTOKINE SIGNALING
High serum levels of IL-15 associated with tumor regression J Clin Oncol. 2017 Jun 1;35(16):1803-1813
Improving CAR + T cells by cosignaling through IL-15 receptor Co-expression of CAR and mbil-15 Combination CAR IL-15 Type I cytokine Signal 3 Signals 1 & 2 T cell Membrane bound IL-15 (mbil15) scfv Extracellular scaffold Intracellular domains Showing signaling motifs JAK STAT Proc Natl Acad Sci U S A. 2016 Nov 29;113(48):E7788-E7797
Membrane bound IL-15 augments antitumor activity of CAR + T cells
3 rd generation SB technology based on point-of-care (POC) Rapid manufacture: Manufactured as needed in <2 days at POC without need to ex vivo expand cells Target tumors with CARs or TCRs Sleeping Beauty non-viral system mbil15 to keep T-cells in naive, powerful state Distributed manufacture and infusion similar to blood-banking practices Safety switch will allow T-cells to be tuned to patient s need 3 rd generation progressing
Pre-clinical data supporting POC Transposon DNA plasmid Transposase DNA plasmid (or in vitro transcribed mrna) IR/DR IR/DR hef1a 1 2 CAR CMVIE SB11 Transposase Transposase CAR Transposon Co-delivery into cells by electroporation Cytoplasm Nucleus Days: 0 1 7 14 21 28 Standard Manufacture (4- Stim) Mononuclear Cells 2-Stim P-O-C: < 2 days T N T SCM T CM T EM T Eff Nucleofection (DNA transposon & DNA transposase) Stimulation with K562 AaPC 21
Disease-free survival (%) Tumor flux (p/s/cm 2 /sr) Tumor flux (p/s/cm 2 /sr) Tumor flux (p/s/cm 2 /sr) POC-generated T cells co-expressing CAR and mb IL-15 Harnessing mbil15 so that infused T cells propagate inside, removing the need to pre-expand before administration 10 10 11 11 Tumor Tumor only only 10 10 10 10 10 10 9 9 10 10 8 8 POC-CAR neg T T cells cells 10 11 10 10 10 9 10 8 POC-mbIL15-CAR T cells POC-mbIL15-CAR T cells POC-CAR T cells POC CAR neg T cells 10 10 7 7 10 10 6 6 10 7 10 6 10 10 5 10 5 5 10 15 20 25 30 35 40 45 50 5 10 15 20 25 30 35 40 45 50 100 75 50 25 Days after tumor injection 0 0 10 20 30 40 50 Days after tumor injection Days after tumor injection (3/5) (3/5) Days after tumor injection Tumor only * POC-CAR T cells POC-mbIL15-CAR T cells POC-CAR neg T cells Electroporated (Day 0) <2 days infusion 10 6 CAR + T cells/mouse (Day 1 expression*) *Integrated & episomal expression * * POC CAR T cells POC mbil15-car T cells ASH: December 4 Publication Number: 2807
Competitive advantage achieved by POC Viral-based POC Solutions Reduce concerns regarding heterogeneity of product Major reduction in costs Improve scalability Very rapidly deliver T-cell therapy T cells targeting solid tumors, as CAR is replaced with TCR 23
SLEEPING BEAUTY: TARGETING SOLID TUMORS WITH TCR-MODIFIED T CELLS
Personalized TCR-modified T cells Tumor antigen is not known before the patient arrives Tumor and normal cells are interrogated to determine the neoantigen TCRs against known tumor antigen are prepared in real time TCRs rapidly expressed in autologous T cells using SB platform via POC Donation a T cell b Targeting neoantigen(s) Endogenous TCR Tumor a Genetically modified a Generated using P-O-C b b Introduced TCR mbil15 Control persistence Recipient
TCR + T cells specific for neoantigens using SB system Mol Ther. 2016 Jun;24(6):1078-89
Competitive advantage of POC Reduce concerns regarding heterogeneity of product Major reduction in costs Improve scalability Rapidly deliver T-cell therapy T cells targeting solid tumors, as CAR is replaced with TCR 27
Harnessing genetically modified T cells using SB system Potential of point-of-care (P-O-C) 1 st era Viral-based gene transfer 2 nd era Sleeping Beauty non-viral gene transfer CAR + Hematologic cancers 3 rd era Shorten manufacture with IL-15 signaling Final era Point-ofcare TCR + Solid tumors 28
Summary First generation trial demonstrated safety and feasibility of the SB platform Second generation trial is testing a modified CAR with shorter manufacturing time using SB platform Third generation trial to rapidly infuse CAR + mbil15 + T cells in < 2 days using SB platform
THANK YOU pkebriae@mdanderson.org