Gene Modified T Cells
Oncology Immunotherapeutics-I
Laurence J.N. Cooper
ljncooper@mdanderson.org
10-23-2014
10:30 am to 12:00 pm
Thanks to Dr. George McNamara
Disclosure. Dr. Laurence J. N. Cooper has been a consultant for American Stem Cells, Inc., GE Healthcare, Ferring Pharmaceuticals, Inc., and Bristol-Myers
Squibb. Dr. Cooper has received multiple grants from foundations in the state of Texas, including CPRIT and UT STAR, and Federal to support research. Dr.
Cooper has received honoraria and payment for the development of education presentations including service on speakers’ bureaus from Miltenyi Biotec.
Dr. Cooper has received travel/accommodations expenses covered/reimbursed by Lonza.
Broadening the clinical appeal of
genetically modified T cells
Tumor-associated antigens targeted by
CARs
Torikai H, Moyes JS, Cooper LJN 2014 engineering T cells to target tumor cells. In W. Cai (ed) Engineering
in Translational Medicine, Springer-Verlag London 2014.
Identify tumor-associated antigens
Collaboration with Immatics and XPRESIDENT® Discovery Platform
Targeting tumor-associated antigens
Requires targeting self antigens?
Targeting neoantigens possible?
LB Alexandrov et al. Nature, 1-7 (2013) doi:10.1038/nature12477
• Boundaries on this slide are arbitrary
• Immunogenic neoantigens may be identified by exome sequencing with sufficient probability only on
mutation-rich tumors
• Identifying immunogenic self antigens will require a different technology, e.g. mass spectrometry
applicable to all HLA expressing tumors
Broadening the clinical appeal of
genetically modified T cells
Approaches to gene transfer
Viral
• Retrovirus
• Lentivirus
•
GOF Gain of function
LOF Loss of function
DGF Dominant gain of function
DNF Dominant negative form
Enhancer deleted, self inactivating (SIN) long terminal repeat lentivirus vectors.
• Adenovirus
• AAV
• other
Non-viral
• DNA (electroporation or other)
• Sleeping Beauty (SB11, SB100X)
• piggyBac
Cytoplasm
Transposase
mRNA
mRNA
•
•
Transient production
Artificial nucleases, transposases, recombinases
CAR
Transposon
Nucleus
Genetic modification of T cells to
redirect specificity
Kershaw, Westwood, Darcy Nature Reviews Cancer 13, 525–541 (2013)
Personalizing the therapy:
Manipulating the T cell by harvesting and re-expressing
melanoma-specific TCR
6 OCTOBER 2006 VOL 314 SCIENCE
TCR gene therapy in patients with metastatic melanoma
Target Antigen
TCR
Patient
Response
MART-1/A2
human
31
MART-1/A2
human
high avidity
gp100/A2
NYESO1/A2
CR
PR
Reference
4
4
Science 2006; 314:126-129
Blood. 2009;114:535-546
20
6
6 (3, 4, 9, 16+,
17+, 17+)
Blood. 2009;114:535-546
mouse
16
3
1 (14+)
2 (4, 3)
Blood. 2009;114:535-546
human
11
5
2 (22+, 20+)
3 (3, 8, 9+)
J Clin Oncol 2011;29:917-924
Avoiding TCR miss-pairing
Torikai, Moyes, Cooper 2014 Engineering T cells to target
tumor cells. In W. Cai (ed) Engineering in Translational
Medicine, Springer-Verlag London 2014.
Zhang, Morgan Adv Drug Delivery Rev 2012 64: 756–762.
Provasi et al., Nat Med. 2012 May;18(5):807-15.
Torikai et al., Blood. 2012 Jun 14;119(24):5697-705.
Govers et al 2014 J Immunol
ε
not CD3ζ
CD3
(epsilon)
(zeta)
Identification of target antigens and
TCRs for CD8+ T cells
Siewert et al., Unbiased identification of target
antigens of CD8+ T cells with combinatorial
libraries coding for short peptides. Nat Med. 2012.
Outcomes and toxicities infusing CD19specific CAR+ T cells
Approaches to gene editing, genome
engineering
Nucleases
CRISPR/Cas9
Meganucleases
TALENs
ZFNs
 site specific integration by HR, mutations by NHEJ.
clustered regularly interspaced short palindromic repeat (RNA)/ Cas9
I-CreI meganucleases derived from self-splicing introns/inteins
TAL Effector Nucleases
Zinc Finger Nucleases
Recombinases /
Transposases
Adenovirus
Cre
SB11, SB100X
piggyBac
Adenovirus delivery of TALENs+DNA with protein capped ends (Holkers 2014)
Cre recombinase
Sleeping Beauty transposase (2 of various versions), random AT sites
piggyBac transposases (various versions), random AATT sites
Activation
sgRNA:dCas9-TF
TALE
ZFP-TF
Synthetic guide RNA:disabled Cas9 nuclease-Transcription Factor
Transcription Activator like-Effector
Zinc Finger Protein-Transcription Factor
Alternative functional domains include: Paired Nickases (increase selectivity for double stranded breaks), recombinases, repressors, epigenomic
modifiers (DNA methyltransferases, histone acetylases or deacetylases, etc), fluorescent proteins (SUNtags, TALEcolor, TALE-Lights).
NHEJ: Non-homologous end joining (& micro-homologous recombination).
HR: Homologous recombination.
Select companies in this field are highlighted later.
Artificial nucleases
Hard
Clinical
Easy
Research
http://www.toolgen.com/html/eng/technology/engineered_nucleases.php
Cellular substrates
Delete
TILs, CAR or
Hypoxia
High adenosine, H2O2,
lactate, PGE2, TGFβ
Arg, Trp depletion
Tumor
Adenosine
A2AR (drug or delete gene)
BCM: “Rewire”: TGFβRex-TLR4in co-stimulation
Arginase
and iNOS
deplete Arg,
iNOS generates
H2O2
Adapted from: Gattinoni, Klebanoff, Restifo 2012 Nat Rev Cancer 12: 671-684
CAR designs
Jena et al. Curr Hematol Malig Rep. 2014 Mar;9(1):50-6.
CAR design
scFv
• affinity
• Other binders … ex. D-CAR based on Dectin-1
(Kumarasen et al. Proc Natl Acad Sci U S A.
2014 Jul 22;111(29):10660-5. )
Stalk
• Which stalk … Ig vs. CD8 vs. short
• Length of hinge+ stalk for proximal vs. distal
epitopes
• Reduce FcR binding
Signaling domains
• Signal 1, 2, 3
Combinatorial CARs
2nd: inhibitory
costimulation
Abate-Daga, et al., Oncoimmunology. 2014 Jun 18;3:e29194.
modulatory
ligands-> signal(s)
K = kinase docking sites
P = phosphorylation sites
Other sites possible
(ITAMs, ITIMS …)
Conditional ablation of T cells
Casucci, Bondanza 2011 J of Cancer
HSV-tk: phosphorylates pro-drug ganciclovir, that is then incorporated into DNA.
HSV-tk can be immunogenic
Approaches to propagation of
genetically modified T cells
• CD3 with CD28 beads (Novartis)
• CD3 and CD28 beads (TransACT, Miltenyi Biotec)
• Activating and propagating cells (AaPC),
• many variations … different outputs
Huls et al., J Vis Exp. 2013 Feb 1;(72):e50070. doi: 10.3791/50070.
Forget et al. 2014 J Immunother 37: 448-460.
T-cell subpopulations for genetic
engineering
Gattinoni, Klebanoff, Restifo 2012 Nat Rev Cancer 12: 671-684.
Lymphocyte populations for
engineering
CD16 T cells Deniger, … Cooper 2014 Clin Cancer Res
 T cells
NK cells
NK cells*
NKT cells
*
Kudo, … Campana 2014 Cancer Res
Chu, ... Yu, Hofmeister 2014 Leukemia
Denman, ... Cooper, Lee 2012 PLoS One
Heczey, ... Metelitsa 2014 Blood
mbIL-21 AaPC
Broadening the clinical appeal of
genetically modified T cells
Approaches to manufacture and
distribution of engineered T cells
T cells are rendered as a drug
Insert CAR or TCR
Eliminating TCR on CAR+ T cells
Patient
Gene insertion:
Retrovirus/lentivirus
Sleeping Beauty
mRNA
CD19
Intended response
CAR
HLAs
B-cell leukemia/lymphoma
TCRαβ
HLAs
Artificial nuclease
Blood. 2013 Aug 22;122(8):1341-9
Blood. 2012 Jun 14;119(24):5697-705
Normal cells
Cellectis/Pfizer
Summary
Inter- and intra-tumor heterogeneity
T cells are precision tools
CAR
TCR++ T
cells
CAR
TCR+ T
cells
CAR
TCR+ T
cells
CAR
TCR+ T
cells
CAR
TCR+ T
cells
Infuse T cells with one or more specificity
Personalized for the patient and the disease
“N=1” trial paradigm
27
Power-law curve
The teaching of Beyoncé
Number of purchases
Brick &
Mortar
Cost of distribution
iTunes
Number of artists
Power-law curve
The new industrialization of T cells
Number of patients
Traditional
Med Centers
Cost of distribution
Immuno-oncology at Med Centers
1
Number of trials
The N=1 approach has been shown for
non-genetically modified T cells
Evidence of tumor regression
after treatment with a highly
pure population of V22+
ERBB2IP mutation–reactive
CD4+ T cells.
COMMERCIALIZATION OF T-CELL
THERAPIES
Cell Immunotherapy of Cancer – Deals 2012-2014
Institution
Amount
Year
Emphasis
Adaptimmune
$104M
2014
Series A financing, enhanced TCR(s) to cancer specific antigens (NY-ESO-1, …)
Amgen-Micromet
$1.16B
2012
Acquisition. BiTES, including Blinatumomab (CD3&CD19 bispecific antibody)
$42M
2013
Dendritic cell personalized kidney cancer vaccine AGS-003
$225M*
2013
PD-1 inhibitor AMP-514; preclinical B7 pathway molecules. * up to $275M milestones.
2014
Research – Moon Shot Program’s Immunotherapy platform
$69M*
2014
CAR T-cells, iCasp-9 inducible suicide gene switch (2014 series C $55M; 2014 series B $14M)
$50M
2014
Bellicum worldwide exclusive license to Dimerizer (ex. AP1903) for use in human cell therapies for all diseases.
$225M*
2013
Chimeric antigen receptor (CAR) T-cells; *up to $225M per product
$50M*
2014
Two immune checkpoint pathways. * up to $300M milestones
2014
Nivolumab (anti-PD-1) FDA approval (Ipilimumab, anti-CTLA, was approved in 2011)
$15
2012
CPRIT Commercialization award. EBV+ tumor cells antigen specific T-cells in collaboration with BCM.
$335M*
2012
OX40L mimicking mAbs (co-stimulation)
2014
Research – Moon Shot Program’s Immunotherapy platform
$47M
2013
Series A funding. Founders: Allison, Gajewski, Pardoll, Sharma, Weiner
$300M
2013
Chimeric antigen receptor (CAR) T-cells (MSKCC, FHCRC CAR T-cells)
Lion Biotechnologies
$23M
2013
Tumor infiltrating lymphocytes (TILs, Rosenberg-NCI). SAB: Hwu, Radvanyi, Yee.
Kite Pharma-NCI/NIH
$128M
2014
Rosenberg CAR T-cell therapies (CART’s)
Merck-Ablynx
$20M*
2014
Immune checkpoint targeting nanobodies. * plus up to $2.3B royalties
Merck-Pfizer
2014
Collaboration: PD-1 inhibitor pembrolizumab (MK-3475); TKI Inlyta; 4-1BB agonist
Merck-Incyte
2014
Collaboration: PD-1 inhibitor pembrolizumab (MK-3475) plus INCB24360 IDO inhibitor
Novartis-CoStim
2014
Acquisition. Co-stimulatory pathways pipeline
$43M
2012
Novartis bought cell manufacturing plant (i.e. for UPenn CAR T-cells)
Novartis-UPenn
$20M*
2012
Chimeric antigen receptor (CAR) T-cells. * plus milestones and royalties.
Pfizer-Cellectis
$110M
2014
CAR T-cells, TALEN genome engineering. * plus up to $2.9B milestones
Pfizer-MDA
2014
Research – Moon Shot Program’s Immunotherapy platform
Pierre Fabre-Aurigene
2014
AUNP-12, 29 amino acid peptide based PD-1 inhibitor (license)
Argos Therapeutics
AstroZeneca/MedImmune-Amplimmune
AstroZeneca/MedImmune-MDA
Bellicum Pharmaceuticals
Bellicum Pharmaceuticals-Ariad
Bluebird Bio-Celgene
Bristol-Myers Squibb-Five Prime Ther.
Bristol-Myers Squibb
Cell Medica
GlaxoSmithKline-MDA
Johnson&Johnson/Janssen Biotech-MDA
Jounce Therapeutics
Juno Therapeutics
Novartis-Dendreon
Roche-Immatics
$17M*
2013
Access to Immatics proprietary TUMAP peptide vaccines. * plus up to $1B research funding and milestones.
Roche-Inovia
$10M*
2013
Two of Inovia’s preclinical therapies. * Plus up to $412M in funding and potential milestones.
$12M
2014
Series A funding. Founder: Dario Campana. T-cells with CD16 FcReceptors to bind mAbs.
UNUM Therapeutics
LC 10/22/2014. Data from Sheridan 2013, 2014 Nat Biotechnol; Herper 2014 Forbes; MDA Strategic Alliances web page; Companies Internet web sites; current news.