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•
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
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
T cell genetically modified ex vivo to redirect specificity
ab
TCR
CAR
Intracellulartargets
Cell surfacetargets
T cell
T cell
Allogeneic, autologous CAR T-cells
Allogeneic
Autologous
Donor RecipientsEngineered
T cells
Off-the-shelf (OTS)
Barriers:· Requirement for
lymphodepletion· Rejection of infused T
cells curtails anti-tumor effect
· Only applicable to CARs (not solid tumors)
Donor RecipientEngineered
T cells
Autologous
Barriers:· Requirement for
lymphodepletion· Cost· Time to manufacture
(during which patients condition deteriorates)
One allogeneic donor into multiple recipientsMade in advance of need and infused on demand
One autologous donor/recipientMade as needed
Viral gene transfer
Viral gene transfer
Curr
ent
prac
tice
• Readily available product.
• Potential for rejection of modified T cells.
• Very little clinical data.
• Most commonly used.
• Potential for manufacture failure.
• Long time to make.
Non-viral versus viral transduction
Non-viral gene transfer: Sleeping Beauty Viral gene transfer: Retrovirus & Lentivirus
Cost effective High cost
Slower transduction rate Faster transduction rate
More nimble production: Customizable Labor intensive, challenging to customize
Packaging cells Lentivirus
Release
Release
DNA plasmid
Sleeping
Beauty
DNA plasmidEx vivo gene transfer
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
Nucleus
Transposase
Transposon
CAR
Cytoplasm
IR/DR
IR/DR
hEF1a
CAR
CMVIE
SB11
Transposase
Co-delivery into cells by nucleofection
Transposon DNA plasmid Transposase DNA plasmid
(or in vitro transcribed mRNA)
Release
DNA plasmid
Sleeping
Beauty
a b
T-cell receptor(TCR)
scFv
Extracellular scaffold
Intracellular domainsShowing signaling motifs
Chimeric antigen receptor(CAR)
Membrane bound IL-15
(mbIL15)
Non-viral gene transfer using Sleeping Beauty
system to express CAR, TCR and 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, membrane-bound 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
Dose Level Single T-cell dose*
A >5 x 107/m2 but < 5 x 108/m2
B >5 x 108/m2 but < 5 x 109/m2
Dose Level Single T-cell dose*
A Not to exceed 106/m2
B >106/m2 but < 107/m2
C >107/m2 but < 5x 107/m2
D >5x107/m2 but < 108/m2
*Per body surface area
J Clin Invest. 2016 Sep 1;126(9):3363-76.
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 108/m2 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
1st generation complete
First-in-human use of SB system: Summary II • Persistence of CAR+ T cells
• Survival of recipients after CAR+ T cells
Average, days Max, days
Autologous 201 360
Allogeneic 51 180
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
1st generation trial: Evaluate SB system (first-in-human studies)
1st generation SB platform in two trials infusing CAR+ T cells after HSCT showed favorable PFS and OS in both autologous and allogeneic cohorts1 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
1st generation complete 2nd generation ongoing
Shortening manufacturing process time from 4 weeks to ~ 2 weeks as T cells co-cultured on feeder cells J Clin Invest. 2016 Sep 1;126(9):3363
2nd 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 CAR+ T-cell dose/kg
-1 105
+1 > 105, but 106
+2 > 106, but 107
+3 > 107, but 108
+4 > 108, but 109
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 co-signaling through IL-15 receptor
Co-expression of CAR and mbIL-15
T cell
Signal 3
Signals 1 & 2
Combination
IL-15
Type I cytokine
CAR
Membrane bound IL-15
(mbIL15)
scFv
Extracellular scaffold
Intracellular domainsShowing 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
3rd 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
3rd generation progressing
Pre-clinical data supporting POC
21
Standard
Manufacture (4-
Stim)
P-O-C: < 2 days
Mononuclear Cells 2-Stim
Days: 0 1 7 14 21 28
Nucleofection
(DNA transposon & DNA transposase) Stimulation with K562 AaPC
TN TSCM TCM TEM TEff
Nucleus
Transposase
Cytoplasm
CAR
Transposon
IR/DR
IR/DR
hEF1a
CAR
CMVIE
SB11
Transposase
Co-delivery into cells by electroporation
Transposon DNA plasmid Transposase DNA plasmid
(or in vitro transcribed mRNA)
1 2
POC-generated T cells co-expressing CAR and mb IL-15
Tu
mo
r fl
ux
(p
/s/c
m2/s
r)
5 10 15 20 25 30 35 40 45 50
105
106
107
108
109
1010
1011 Tumor only
Days after tumor injection
POC-CARneg
T cells
Tu
mo
r fl
ux
(p
/s/c
m2/s
r)
5 10 15 20 25 30 35 40 45 50
105
106
107
108
109
1010
1011
Days after tumor injection
POC-mbIL15-CAR T cells
POC-CAR T cells
Days after tumor injection
Tum
or
flux (
p/s
/cm
2/s
r)
POC-CARneg T cells
Tumor only POC-mbIL15-CAR T cells
POC-CAR T cells
5 10 15 20 25 30 35 40 45 50 5 10 15 20 25 30 35 40 45 50
1011
1010
109
108
107
106
105
Harnessing mbIL15 so that infused T cells propagate inside, removing the need to pre-expand before administration
0 10 20 30 40 500
25
50
75
100
Days after tumor injection
Dis
ease
-fre
e s
urv
ival (%
) Tumor only
POC-CAR T cells
POC-mbIL15-CAR T cells
POC-CARneg
T cells
**
(3/5)
*
(3/5)
ASH: December 4 Publication Number: 2807
• Electroporated (Day 0) • <2 days infusion • 106 CAR+ T cells/mouse (Day 1
expression*) *Integrated & episomal expression
POC
CAR T cells
POC
mbIL15-CAR T cells
POC
CARneg
T cells
Competitive advantage achieved by POC
23
• 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
Viral-based POC Solutions
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 RecipientGenerated using P-O-C
IntroducedTCR
Genetically modified
a b
a b
EndogenousTCR
T cell
a b
TumorTargeting
neoantigen(s)
mbIL15
Control persistence
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)
1st era • Viral-based gene
transfer
2nd era • Sleeping Beauty non-viral gene
transfer
3rd era • Shorten manufacture
with IL-15 signaling
Final era • Point-of-
care
CAR+
Hematologic cancers
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