Achieving T-cell Immunotherapy's Promise
3
Current challenges facing autologous T-cell immuno-therapies:
• "One Patient, One Product" difficult to scale to meet needs of large populations
• Chemo washout and delayed treatment
• Immuno-modulatory tumor microenvironments in solid tumors
• Limited speed of development for novel, exploratory CARs/TCRs due to the need for viral vector development
Sangamo Universal T-cell Program
4
• Builds on T-cell engineering process from previous HIV trials; T-cell engineering / expansion process maintains TSCM subpopulation that led to long-term (3+ year) engraftment in all patients
• Highly efficiency (>90%) simultaneous KO and TI; possible due to extreme design densities of new ZFN architecture
• TI for physiological regulation of CAR/TCR expression and increased potency
• Eliminates TCR repertoire, HLA Class I on T-cells; exploring additional novel functionalities
• Efficient editing and expansion process enables QC steps in manufacturing that improve product quality
PD-1 Knockout for More Potent TIL Adoptive Cell TransferBeane et al – Molecular Therapy, June 2015
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TCR Knockout – Previous Results
7
• Gene editing of TCR alpha constant
or TCR beta constant alone is
sufficient to eliminate formation of
TCR complex
• KO of endogenous TCR eliminated
GvHD in mouse models
• ZFNs used in this study was built
~2010
New ZFN Platform Enables Optimal Targeting Capability
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C C A A C G C G A A T T A T G G C G G C G T G C G C T T A A C G C A T G G G T
G G T T G C G C T T A A T A C C G C C G C A C G C G A A T T G C G T A C C C A
T A C
A T G3’
5’
module module module
ZFP-Fok
linker module module module
intermodule linkers
ZFP-Fok
linkerintermodule
linkers
Modules: 1- and 2-finger units that >8000 hexamer / module combinations
recognize base sequence
Intermodule connect adjacent modules 6 alternatives for skipping 0, 1, or 2 bp
linkers:
ZFP-Fok linker: links the Fok and ZFP 5 alternatives for skipping 5-9 bp
domains 4 alternatives for reversing Fok-ZFP polarity
Yields Highly Potent, Specific Nucleases from Unparalleled Design Density
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1 2 3
Target base
Design
quality
score
exon 1 exon 2
20
30
40
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200
20
30
40
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200
20
30
40
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200
exon 3
Example: TCRα constant region
• 3 exons / 374 bp
• 374 potential locations for cleavage
218 TRAC locations (1 per 1.7 base pairs, or 58%)
immediately targetable with current ZFN library
Able to construct new ZFNs to target virtually
any base pair
• HiFi CRISPR has a theoretical design density of 2-3%, or ~10 targetable
locations in TRAC
• High design density (20X relative to HiFi CRISPR) enables selection of most
potent and specific nucleases to target TRAC and B2M
Concordant TRAC Genomic Modification Observed
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0
20
40
60
80
100
Untransfected Transfected
% N
HEJ
(MIS
EQ)
Gene Modification
% Gene Modified
% C
D3
Ne
g.
Targeting b2-microglobulin (B2M) to Knockout HLA Class I
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• Secretion of MHC Class I proteins requires intracellular B2M (Ploegh et. al. PNAS 1979)
• B2M is a relatively small protein (119 aa), located on Chr 15
• Bi-allelic KO likely required to eliminate MHC Class I expression
http://www.slideshare.net/drshahinhameed/tests-in-organ-transplantation
MHC Class I Structure
Codelivery Enables >80% Double Knockout
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Control
CD3 expression
HL
A e
xp
ressio
nTCRα
ZFNs
B2M
ZFNs
TCRα + B2M
ZFNs
96% CD3(-)
82% double
knockout
92% HLA(-)
Specificity Assessed Via End Capture Assay
Cleave genome
Integrate donor
Doublestrand break
donor
donor
Sequence genome
adjacent to donor
K562 cells
delivery via nucleofection
400 ng RNA / ZFN, 1 uM oligo duplex
Assess indels at candidate
off-target lociCD4 T cells
delivery via RNA electroporation
Sequence reveals candidate cleavage site.
off-target ?
adaptor
Linear amplification,
adaptor ligation, PCR
genome
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Locus # of sequences % indels in
recovered followup study
chr14 22550606 8784 82.6%
chr2 102757228 57 0.2%
chr10 69524194 40 0.3%
chr17 26885875 39 0.1%
chrX 47506726 36 0.1%
chr8 45001673 35 0.2%
chr8 44052523 31 0.2%
chr8 44586530 27 0.1%
chr7 61023155 25 0.2%
chr8 44428079 25 0.2%
chr8 44632025 25
chr8 45163917 25 0.1%
chr8 45285156 25 0.2%
chr8 44855417 24 0.1%
chr4 139988255 22
chr8 44693889 22 0.1%
chr8 44921744 22 0.1%
chr8 45092809 21
chr8 44192064 20 0.3%
chr8 44464487 20 0.1%
chr8 44621162 20 0.1%
chr2 55427491 19 0.1%
chr8 44753420 19 0.1%
chr8 44871959 19 0.2%
assay background
not significant
TCRα
nd
nd
nd
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TCRa ZFN Exhibit No Evidence of Cleavage at Non-Target Loci in Human Primary T-cells
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Locus sequences % indels in
recovered followup study
chr15 44711566 6387 84.93% < 0.00001chr3 43763206 23 0.22% nschr7 137998072 22 0.02% nschr12 133265000 21 ndchr22 50808098 -8454 21 ndchr2 28690516 21 0.07% nschr5 119308938 16 0.06% nschr4 76952328 15 0.08% nschr10 10310 14 ndchr11 1850324 12 0.02% nschr2 72868420 12 ndchr21 8398712 11 0.11% nschr15 44695230 10 0.29% nschr1 168688026 10 0.35% nschr1 143239626 9 0.24% nschr21 8460366 -1230 9 ndchr17 47080328 9 ndchr4 138367446 9 ndchr6 163866136 8 0.20% 0.01chr17 41493978 8 0.06% nschr5 135481282 8 ndchr1 121767656 8 0.65% ns
p-value
B2M ZFN Exhibit No Evidence of Cleavage at Non-Target Loci in Human Primary T-cells
B2M
assay background
not significant
ZFN-Mediated Targeted Integration With AAV Donors
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5’
3’
Cap Poly-A tailZFNs provided as mRNA
Donor provided as rAAV2/6
Integrated Donor
+
ZFN-Mediated Targeted Integration of an GFP Expression Cassette
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5’
3’
Cap Poly-A tailZFNs provided as mRNA
CD19 CAR Donor provided as rAAV2/6
Integrated Donor
+
pGK GFP pA
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Gene KO Targeted Integration
TCR
locus
B2M
locus
88%
CD3-
93%
HLA-
CD3
HLA
71% TI
(CD3- / GFP+)
72% TI
(HLA- / GFP+)
Concerted Knockout + Integration in T-cells
90% Double KO of TCR and HLA Class I, with 90% Targeted Gene Insertion
Sham ZFNs only ZFNs + B2M Donor ZFNs + TRAC Donor
HLA-/CD3-
HLA-/CD3-/GFP+
0%
CD3
HLA
Cla
ss I
GFP
FSC
-HCD3
HLA
Cla
ss I
CD3
HLA
Cla
ss I
CD3
HLA
Cla
ss I
GFP
FSC
-H
GFP
FSC
-H
83.4%92.7% 89.1%
90.4% 86.6%
0%
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ZFN-Mediated Targeted Integration of CD19 CAR
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5’
3’
Cap Poly-A tailZFNs provided as mRNA
CD19 CAR Donor provided as rAAV2/6
Integrated Donor
+
pGK CD19 CAR pA
ZFN-Mediated Editing Enable Efficient Targeted Insertion of CD19 CAR into the TRAC Locus
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83.1%
61.2%
96.5%
TCR
HLA
CD19 CAR
T-cell Killing Assay: CD19 Antigen Specific Cytotoxicity
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Target cells only Target cells + CAR neg T-cells
Sham Treated T-cells TCR KO T-cellsNo T-cells
ZFN Engineering CD19 CAR-T Efficiently Kills Target Cells In Vitro
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0.4%99.6% 9.9%90.1% 27.4%72.6% 40.6%59.4% 43.1%56.9%
T-cell: Target cell Ratio
2:1 1:1 0.5:1 0.25:1 0.125:1
0.1%99.9% 0.1%99.9% 1.7%98.3% 25.7%74.3% 35.1%64.9%
TI into
B2M
TI into
TRAC
Highly Efficient T-cell Engineering Enables One-Step Universal T-cell Manufacturing Process
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TCR and B2M
Knockouts Expansion
& Selection
Testing &
Release
CAR Targeted
Insertion
AAV6
Transduction~150 x 109
T-cells Post
Expansion and
Selection
Apheresis: Cell
Procurement
Product
Infusion
mRNA
Electroporation
~500 Treatment Doses
Per T-cell run
3 x 108 Dose
per Treatment
5 x 109 T-cells
Post
Enrichment
Cell
Purification
Summary
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• Highly active and specific ZFN reagents to KO TCR and HLA Class I
developed
• 90+% double KO achieved
• T-cell engineering process optimized to mediate >90% TI using a
AAV2/6 GFP donor
• TI of CAR or antigen-specific TCR • T-cell effector function against antigen positive target cells demonstrated
• ZFN mediated T-cell engineering may be deployed to engineer and
develop next generation ACT products
Acknowledgements
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Research
Lynn Truong
Nimisha Gandhi, M.S.
Anthony Conway, PhD
Matthew Mendel
Ken Kim
Michael Holmes, PhD
ZFN Technology
Lei Zhang, PhD
David Paschon, PhD
Danny Xia
Sarah Hinkley
Nicholas Scarlott
Anna Vincent
Stephen Lam
Jeff Miller, PhD
Patrick Li, PhD
Ed Rebar, PhD
AAV
Alicia Goodwin
Tim Gabriele
Hung Tran
Andrea Kang
Jennifer Huang
Richard Surosky, PhD