P-MUC1C-101: A Stem Cell Memory CAR-T Therapy for Epithelial-Derived Solid Tumors
Devon J. Shedlock, PhDVP, Preclinical Development
September 5th, 2018
piggyBac™ Enables Multiple Differentiated CAR-T Product Attributes
• Unprecedented cargo capacity (>30X lentivirus) – three-in-one transgene and possibility of multiple CAR or TCR molecules
• Creates highly desirable T Stem Cell Memory (Tscm) Phenotype
• Non-viral delivery system – non-oncogenic and non-mutagenic
• High insertion efficiency and stable transgene expression
• Faster to clinic with lower cost than viral methods
• Substantial IP portfolio with no dominant or competing IP
1
piggyBac™ is a superior DNA delivery system for developing CAR-T and other gene therapy products
GENOMIC DNAITR ITR
“Paste”
CARGO
ITR ITR
“Cut”
CARGO
piggyBacTM
Transposase
ITR ITRCARGO
piggyBacTM
Transposon
+
piggyBac™ Unmatched Cargo Capacity Increases Optionality
2
piggyBac™ effectively delivers multiple full-length CARs in single transposon system
1
2
4
# Full-length CARs* Function (Killing)
* Plus selection gene and marker gene
BCMA
3
BCMA PSMA
BCMA PSMA CD19
BCMA PSMA CD19 GD2
Massive piggyBac™ Cargo Capacity Allows for Delivery of Three-In-One Transgene for CAR-T products
3
CAR-T MOLECULE
Superior binding molecule
• Molecule (Centyrin, VH, scFv, etc…) with high-specificity binding
• Fully human and not susceptible to tonic signaling
1
POSITIVE SELECTION
Drug resistance gene permits positive selection
• ~100% of T-cells in final product express the CAR molecule
• Predicted to result in better therapeutic index
2
SAFETY SWITCH
Incorporates proprietary safety switch
• Rapid, dose-dependent elimination of engineered T-cells if needed
• Management of Cytokine Release Syndrome (CRS) or other AEs
3
TTAA
ITR
Insulator
Promoter Safety Switch CAR Molecule
Selection Gene
Poly(A)
TTAA
Insulator
ITR
Poseida CAR-T Products Comprised of Highly Favorable Stem Cell Memory T Cells
• Ability to develop product with high percentage of Tscmcells is a distinct competitive advantage
• piggyBac™ preferentially transposes in Tscm cells
⎼ Tscm cells persist and live longer than effector cells
⎼ Tscm cells can produce potentially unlimited effectors cells
• Tscm-rich product should lead to better engraftment and better duration of response with the potential for re-response
• Lentivirus-produced products have not achieved high Tscmpublished percentages ranging from less than 1% to ~14%
4
Tscm phenotype should increase duration of response and allow for relapse control without re-administration
TEFF
TEM TCM
TSCM
CD62L
CD
45
RA
Teff Tscm
TcmTem
Tscm May Be Key to Potent and Durable Responses
5
Gattinoni et al. (2009) Nat Med; Hinrichs et al. (2009) PNAS; Hinrichs et al (2011) Blood; Gattinoni et al. (2011) Nat Med; Lugli et al. (2013) JCI; Klebanoff et al
(2016) JCI; Sukumar et al (2016) Cell Met; Sabatino et al. (2016) Blood;
• Correlates with clinical response
⎼ Melenhorst J. et al., UPenn (2017) 20th ASGCT
⎼ Basu et al., Adaptimmune (2017) CAR-TCR Summit
⎼ TCM: Larson, Juno (2018) AACR
• Without prior fractionation of TN/TCM, Akt inhibitors
and shortened process are mostly successful in
increasing TCM during viral manufacture
• “The extreme longevity, the robust proliferative
potential and the capacity to reconstitute a wide-
ranging diversity of the T cell compartment make
the Tscm cell type an ideal cell population to employ
in adoptive immunotherapy”
Poseida CAR-T Products Comprised of Highly Favorable Stem Cell Memory T Cells
6
Adapted from Gattinoni et al. (2017) Nat. Med.
CD62L
CD
45
RA
Teff Tscm
TcmTem
P-BCMA-101
+-++++++++++-
Naïve/TscmTcmTemTeff
P-BCMA-1010 10
310
410
5
Perforin
CD
45R
A85.8%0.5%
13.1%0.6%
CD62L
Poseida CAR-T Comprised of Highly Favorable Tscm
piggyBac™ and Lentivirus Modify Different T Cell Subsets
0
2 0
4 0
6 0
% T
ra
ns
du
ce
d C D 4 + T c e lls
C D 8 + T c e lls
L e n tiv iru s
Cell graphics adapted from Henning et al., Nat. Rev. Immunol., 2018
0
2 0
4 0
6 0
%
Tr
an
sp
os
ed
p i g g y B a c
T M
7
We purified donor cells to these T-cell subsets and then performed optimized piggyBac™ or optimized lentivirus manufacturing on each subset
piggyBac™ preferentially transposes early Tscm cells, while lentivirus prefers differentiated T cells
Poseida CAR-Tscm Unprecedented Preclinical Efficacy
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P-BCMA-101: Liquid tumor (MM.1S) disseminated IV implantation in NSG mice
P-PSMA-101: Solid tumor (LNCaP) SC implantation in NSG mice
0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0
0
5 0 0
1 , 0 0 0
1 , 5 0 0
2 , 0 0 0
2 , 5 0 0
3 , 0 0 0
P r o s t a t e C a n c e r ( L N C a P ) S . C .
D a y s p o s t C A R - T A d m i n i s t r a t i o n
Tu
mo
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T c e l l s ( N o C A R ; 4 e 6 )
P - B C M A - 1 0 1 ( 4 e 6 )
P - P S M A - 1 0 1 ( 4 e 6 )
C l i n c i a l P S M A s c F v C A R ( 4 e 6 )
L O D
- 1 4 - 7 0 7 1 4 2 1 2 8 3 5 4 2 4 9 5 6 6 3 7 0 7 7 8 4 9 1
1 05
1 06
1 07
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D a y s P o s t C A R - T A d m i n i s t r a t i o n
Tu
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To
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T u m o r O n l y ( n = 1 0 )
P - B C M A - 1 0 1 ( 1 2 e 6 ( n = 2 0 ) )
P - B C M A - 1 0 1 ( 4 e 6 ( n = 1 9 ^ ) )
M u l t i p l e M y e l o m a ( M M . 1 S ) I . V .
9
• Design⎼ Single dose, 3+3 dose escalation + selected expansion⎼ Adult subjects with relapsed or refractory MM
• Key Objectives⎼ Safety, maximum tolerated dose and/or optimal dose⎼ Anti-myeloma efficacy⎼ Expansion and functional persistence of the CARTyrin-T cells
Poseida P-BCMA-101: Phase I Study Ongoing
Mucin-1 (MUC1) is highly expressed in most
epithelial-derived cancers
• MUC1 normally expressed on apical surface of
epithelium
• Aberrant form of MUC1 is expressed on
cancer cells, which is specifically recognized by
our binder
• Target for multiple immunotherapies
(e.g. cancer vaccines, antibody therapies)
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Tumor TypeMUC1
ExpressionNumber
of TissuesReference
Breast 91% 1,447 Rakha et al (2005)
NSCLC 99% 231 Merck Serono. Data on file
RCCa 84% 133 Langner et al (2004)
Colorectal 81% 243 Baldus et al (2002)
Ovarian 83% 63 Chauhan et al (2006)
H&N SCCa 82% 29 Croce et al (2001)
Nasopharyngeal 100% 38 Zhong et al (1993)
Gastric 77% 136 Utsunomiya et al (1998)
Prostate 79% 89 DeNardo et al (2005)
Pancreatic 81% 53 Qu et al (2004)
Mesothelioma 75% 20 Saad et al (2005)
Multiple myeloma 59% 125 Cloosen et al (2006)
Esophageal 32% 53 Kijima et al (2001)
MUC1 Target Has Broad Pan-Tumor Potential
Lambrechts et al
MUC1 is a Highly Complex Protein
Sousa et al 2016
• Single pass Type I transmembrane protein
⎼ N-terminal subunit (MUC1-N) and C-terminal subunit (MUC1-C) form stable heterodimeric complex
• Canonical isoform 1 is a 1,255aa protein
⎼ 42 20aa repeats
• MUC1 is highly polymorphic; various alleles change number of tandem repeats in N’
⎼ >90 isoforms; many with 20-200 VNTR repeats
12
MUC1 is a Highly Complex Protein
Sousa et al 2016
Roulois et al 2013
• MUC1 confined to apical surface of normal epithelial cells
⎼ Hyperglycosylated on normal cells
• Polarity is lost in tumor cells
⎼ Hypoglycosylated on tumor cells; branches seem highly
immunogenic
13
MUC1 can be cleaved by proteases
• MUC1 remains heterodimeric under normal growth conditions
• ECD is cleaved (ADAM 17/TACE and MMP-MT1) to generate shorter membrane-associated peptide fragments (MUC1-C)
⎼ MUC1* and MUC1-CTF15
• Cleaved MUC1-N is shed from cell and triggers inflammation
⎼ Soluble MUC1-N is target for MUC1-N-specific
immunotherapies and may limit their efficacy
14
MUC1 is a Highly Complex Protein
Nath et al 2014
MUC1 Comprises Various Epitopes for CAR Targeting
• Hypoglycosylated branches on tumor cells are highly immunogenic
⎼ Tn-specific binders recognize cancer-associated Tn glycoforms occurring in VNTR (Posey et al, 2016)
• MUC1-C specific binders
⎼ Target epitopes in region proximal to cell membrane
• May be retained post-cleavage
⎼ Are difficult to produce and currently more rare
Tn CARs
MUC1-C CARs
Adapted from Nath et al 201415
16
Many MUC1 Isoforms, But Not All Comprise Tn Epitopes
Exon1 Exon2 Exon3
TnEpitopes
Exon3 Exon4 Exon5 Exon6 Exon7 Exon8
TMMUC1-C Epitopes
Screening CARs Recognizing MUC1
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Several CAR candidates exhibited antitumor activity against a MUC1+ cancer cell line
Co
ntr
ol
P C
AR
T C
AR
F C
AR
Y C
AR
S C
AR
A C
AR
G C
AR
E C
AR
B C
AR
K C
AR
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
% C
D1
07
a+
R a ji (M U C 1 -)
R P M I8 2 2 6 (M U C 1 + )
We constructed multiple MUC1-C
CARs and tested:
• mRNA CAR Expression in primary
human pan T cells
⎼ Confirmed surface expression of all
• Evaluated antitumor activity against
a MUC1+ cancer cell line
• A few CARs (A,G, and E) expressed
MUC1-specific antitumor activity
Screening CARs Recognizing MUC1
18
G MUC1 CAR exhibited strong antitumor activity against multiple MUC1+ cancer cell lines
We assessed mRNA CAR activity against MUC1 (full-length Isoform 10):
A C
AR
G C
AR
E C
AR
0
2 0
4 0
6 0
8 0
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CD
10
7a
+%
e R a ji (F L M U C 1 -Is o 1 0 )
R a ji (M U C 1 -)
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0
1 0
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6 0
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8 0
9 0
CD
10
7a
+%
M D A - M B - 4 6 8
M C F - 7
R a j i ( M U C 1 - )
N o t a r g e t
We compared mRNA MUC1 (MUC1-C vs TN) CAR activity against several breast cell lines:
MUC1 is Expressed on Numerous Cancers with High Unmet Need
19
Poseida MUC1-C CAR mediates robust anti-tumor activity against multiple tumor lines
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Raw
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CD
10
7a
+%
N o ta rg e t
B re a s t M e s o th e lio m a
& N S C L C
P a n c re a tic C M L & M M
M U C 1 -C C A R
N o rm a l M u rin e
M
A G 1 2 2 1 1 : F ib ro b la s t - L u n g (M U C 1l o
)
A G 0 2 1 0 2 : F ib ro b la s t - U te ru s e n d o m e tr ia l (M U C 1-)
A G 1 1 1 3 2 : E p ith e lia l - B re a s t o rg a n o id (M U C 1-)
N o rm a l c e ll lin e s
C R &
C e rv ic a l
MUC1 is Expressed on Numerous Cancers with High Unmet Need
20
Poseida MUC1-C CAR mediates robust anti-tumor activity against multiple tumor lines including ovarian
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to
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M C F 7 ( B r e a s t )
2 4 h r C y t o t o x i c i t yWe assessed P-MUC1C-101 activity against
ovarian tumor line (OVCAR3):
• Strong cytotoxicity
• CAR-Ts also proliferated and secreted IFNg
• Studies evaluating additional ovarian lines
underway
Poseida piggyBac™ manufacture of P-MUC1C-101
21
piggyBac™ manufacturing process yields high levels of CAR-Tscm
Q1
2
Q2
59
Q3
33
Q4
6
0 103
104
105
CD62L
0
103
104
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CD
45R
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Q2
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Q4
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Q3
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Q2
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Q3
58
Q4
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0 103
104
105
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0
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104
105
CD
45R
A
Mock P-BCMA-101 P-MUC1C-101 Tn CAR
CD62L
CD
45
RA
Teff Tscm
TcmTem
Evaluation of P-MUC1C-101 in vitro
22
Robust in vitro Tumor Killing by MUC1-C CAR-Tscm
1 : 2 1 : 5 1 : 1 0
- 5 0
- 2 5
0
2 5
5 0
7 5
1 0 0B r e a s t c a n c e r M C F - 7
%
Ki
ll
in
g
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- 2 5
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1 0 0B r e a s t c a n c e r M D A - M B - 4 6 8
P B C M A 1 0 1
P - M U C 1 C - 1 0 1
T n C A R
M o c k
1 : 2 1 : 5 1 : 1 0
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%
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1 0 0N o r m a l c e l l l i n e A G 0 2 1 0 2 ( M U C 1
-
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Evaluation of P-MUC1C-101 in vivo
23
MUC1 CAR-Tscm were evaluated in MCF-7 breast cancer orthotopic tumor model
Day 0: CAR-T Treatment1) Tumor only control (n=8)2) Tumor + P-BCMA-101 control (12e6; n=4)3) Tumor + P-MUC1C-101 (12e6; n=4)4) Tumor + Tn CAR (12e6; n=4)
Days -21
Day -21:
MCF-7.luc 3.5x106
Left FP (n=20)
Caliper
7
BLI ImagingBlood Draws
14 21 28 35 42 490-2
Efficacy of MUC1 CAR-Tscm(12e6) in breast cancer model
• MCF-7.luc tumor implanted into left fat pad 21 days prior to CAR-T treatment
• Also serves as tox model since P-MUC1-101 cross-reacts with murine MUC1
Potent in vivo Efficacy of P-MUC1C-101
24
Tumor elimination in 100% of animals at standard dose in human breast cancer xenograft model
0 7 1 4 2 1 2 8 3 5 4 2 4 9
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(
mm
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D a y s p o s t T c e l l i n j e c t i o n
*
**
* a n i m a l s u c c u m e d
t o t u m o r
0 7 1 4 2 1 2 8 3 5 4 2 4 9
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D a y s p o s t T c e l l i n j e c t i o n
* ** a n i m a l s u c c u m e d
t o t u m o r
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D a y s p o s t T c e l l i n j e c t i o n
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D a y s p o s t T c e l l i n j e c t i o n
*
* a n i m a l s u c c u m e d
t o t u m o r
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c a l i p e r m e a s u r e m e n t s
Tu
mo
r
Vo
lu
me
(
mm
3)
D a y s p o s t T c e l l i n j e c t i o n
T u m o r o n l y c o n t r o l ( n = 8 )
P - B C M A 1 0 1 c o n t r o l ( n = 4 )
P - M U C 1 C - 1 0 1 ( n = 4 )
T n C A R ( n = 4 )
*
*
*
*
*
*
* a n i m a l s u c c u m e d t o t u m o r
Summary: Developing MUC1 CAR-T to Address Multiple Indications
25
Discovery program with compelling preclinical data and multiple development options
• P-MUC1C-101 demonstrated robust antitumor activity against multiple tumor types
• P-MUC1C-101 Tscm mediated rapid, sustained tumor regression and completely eliminated established tumors in an MCF-7 based orthotopic mouse model
• Compelling data suggesting molecule is binding tumor-specific MUC1
• Preclinical data portends broad product potential
AcknowledgmentsEric Ostertag, M.D., Ph.D, CEOMark J. Gergen, J.D., CBO & CFOMatthew Spear, M.D., CMO
Devon J Shedlock, Ph.D., VP of Preclinical Development
Immuno-OncologyJenessa Smith, Ph.D.Xinxin Wang, Ph.D.*Burton Barnett, Ph.D.Stacey Cranert, Ph.D.Christopher Martin, Ph.D.Elvira Khialeeva, Ph.D.Srinivas Rengarajan, M.S.Yenning Tan, M.S.Rebecca Codde