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CAR-T Cell Therapies:
From Origin to Acceleration
Bruce L. Levine, Ph.D.Center for Cellular Immunotherapies, University of Pennsylvania
Co-Founder, Tmunity Therapeutics
President, International Society for Cell and Gene Therapy
@BLLPHD
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Conflict of Interest Statement
• Declaration of financial interest due to intellectual property and patents
in the field of cell and gene therapy.
• University of Pennsylvania Alliance with Novartis
• Consultant for Terumo
• Scientific Advisory Board for Akron, Avectas, Immuneel, Immusoft,
In8bio, Ori Biotech, Vycellix
• Co-Founder and equity holder Tmunity Therapeutics
• Conflict of interest is managed in accordance with University of
Pennsylvania policy and oversight
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Redirecting Orion to Hunt Cancer
Ignace-Gaston Pardies, Star Atlas 2nd edition 1693, Paris
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Engineered Immunity:
Chimeric Antigen Receptor (CAR) T Cells
To Kill Cancer
CAR T Cell
Tumor Cell
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Redirection of Specificity and
Effector Function:
The Beginning
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B Cell Receptors + T Cell Receptors: 1987-1989MATERIAL OWNED BY BRUCE LEVINE UNIVERSITY OF PENNSYLVANIA
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First CAR Publications in Academia
First CAR Clinical Trials in Industry
RECOMBINANT DNA ADVISORY COMMITTEE MEETING
March 3-4, 1994
CD4-zeta in HIV+ Subjects
First CAR T Trial in Cancer
Conducted 1997
Published 2017!
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Women of Early CAR T Cell Development
• CellTech (UCB)
• Chimeric receptors providing
both primary and costimulatory
signaling in T cells from a
single gene product. Journal of
Immunology. 1998;161:2791-7
Helene Finney
• Cell Genesys
• Celgene
• Led first ever clinical trial
of CAR T Cells in cancer
1997
Kristen Hege
• Cell Genesys
• Kite
• Chimeric receptor molecules
for delivery of co-stimulatory
signals US Patent 5,686,281
filed May 31, 1995, granted
November 11, 1997
Margo Roberts
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Agenda
• CAR T cells in Liquid Tumors
• CAR T cells in Solid Tumors
• Multiplex Engineering
• Where are We Going?
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August, 2010CAR T Cells Targeting CD19 in CLL
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August, 2010 Pt #12.9 (1.3)
Pt#25.5 (2.5)
Pt #37.7 (3.5)
Before CAR T Cells
After CAR T Cells
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April, 2012CAR T Cells Targeting CD19 in ALL
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Global Regulatory Approvals
US, CAN, EU, UK, CH, IS, JP, AU
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J Clin Invest. 2020 Feb 3;130(2):673-685.
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Outstanding Fate Mapping Questions
▪Do you “get what you get?”
▪Should you select (patients or cells) and how do we know we are not missing important cells or denying patients?
▪Passengers or Drivers?
▪Experimentation and Iteration
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Kymriah for CLL: summary
▪ A subset of patients with refractory CLL have durable remissions after Kymriah
▪ For refractory CLL, the CR rate with Kymriah is about ~30%
• Frey NV et al. J Clin Oncol. 2020;38(25):2862-71
▪ CLL cells induce CAR T dysfunction
• Singh N et al. Cancer Discov. 2020;10(4):552-67
▪ BTK/ITK inhibitors appear to synergize with CD19 CAR T
• Turtle CJ. J Clin Oncol. 2017;35(26):3010-20, Gill, S. et al, unpublished
▪ CLL is emblematic of the tug-of-war between effective but non-curative targeted reagents (ibrutinib, venetoclax, etc) and a potentially one-time curative T cell-based therapy!
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Solid Tumor Challenges
• Antigen Coverage
• Antigen Escape
• Antigen Specificity
• Immunosuppressive Microenvironment
• Trafficking
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Solid Tumor Challenges
• Antigen Coverage
• Antigen Escape
• Antigen Specificity
• Immunosuppressive Microenvironment
• Trafficking
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EGFRviii as a CAR Target for GBM
Pros and Cons as a CAR Target
▪ Pro: tumor specific mutation so that on-target, off-tumor toxicity is unlikely
▪ Con: EGFRviii is a subclonal mutation: tumor heterogeneity
▪ Francis JM. EGFR variant heterogeneity in glioblastoma resolved through single-nucleus sequencing. Cancer discovery. 2014;4(8):956-71.
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Decadron 4mgAnti-epileptics
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CART engraftment and persistence in bloodFlow Cytometry
A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma, O’Rourke, D.M., et al. Sci Transl Med. 2017 Jul 19;9(399):eaaa0984
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CART Trafficking to Brain and Downregulation of EGFRviii
A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma, O’Rourke, D.M., et al. Sci Transl Med. 2017 Jul 19;9(399):eaaa0984
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CART EGFRviii in Glioblastoma Conclusions
Screening for EGFRvIII and CART-EGFRvIII manufacturing is feasible for GBM patients
Infusion of CART-EGFRvIII is safe, but may induce seizures.
There is no EGFR-directed toxicity in 8 of 8 patients (not cetuximab!)
Mechanism of Action established: ➢CART-EGFRvIII expand in blood and traffic to brain.➢CART-EGFRvIII detectable (Q-PCR analysis) in brain tumor in 3 of 5 patients➢EGFRvIII antigen loss (NGS analysis) in brain tumor in 4 of 5 patients
Induction of new T cell infiltrates by IHC in tumor resection specimens may suggest antigenic spreading or bystander T cells
Additional strategies: target other antigens to prevent tumor escape, combine with checkpoint Ab
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Solid Tumor Challenges
• Antigen Coverage
• Antigen Escape
• Antigen Specificity
• Immunosuppressive Microenvironment
• Trafficking
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Mesothelin as a Target for CAR T cells
Expression by malignant tissues• Pancreatic adenocarcinoma (100%)
• Epithelial mesothelioma (100%)
• Ovarian carcinoma (85-100%)
• Lung adenocarcinoma (50%)
Description and characterization• GPI anchored membrane protein (40 kDa) that can be shed from cells. Elevated levels
of soluble mesothelin in ~50% of patients with mesothelioma and ovarian cancer but
have not been observed in pancreatic adenocarcinoma.
• Normal expression on mesothelial cells lining pleura, peritoneum, and pericardium.
• Biological function remains unclear but has been shown to bind to CA-125 (MUC16)
and may have a role in cellular adhesion, tumor invasion and metastasis.
H&E MesothelinLiver met
Patient
with
metastatic
pancreatic
adenocarcinoma
Pastan and Hassan Cancer Res 2014; Chang and Pastan PNAS 1996; Carpenito et al, PNAS 2009
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Penn Clinical Trials using SS1 and M5 Mesothelin-targeting
CAR T cells for the treatment of solid tumors
RNA ss1 CART
• Safety and feasibility
• Transient persistence and limited anti-tumor activity
• CAR Immunogenicity
• Mesothelioma, Ovarian and Pancreatic Cancer
LV M5 CART
• Safety and feasibility
• Cohort 1 and 2 completed
• 1 DLT in Cohort 3
• Mesothelioma, Ovarian and Lung Cancer
NCT03054298
2017
LV ss1 CART
• Safety and feasibility
• Expand, transient persistence and limited anti-tumor activity
• CAR Immunogenicity
• Mesothelioma, Ovarian and Pancreatic Cancer
NCT02159716
2014
NCT01897415
2013
LV M5 CART
• Trial in Pancreatic Cancer
• IV and regional infusionNCT03323944
2017
-Zhao, Y et al., Cancer
Res, 2010
-Maus, MV et al., Cancer
Immunol. Res., 2013
-Beatty, GL et al., Cancer
Immunol. Res., 2014
-Beatty, GL et al.,
Gastroenterology, 2018
-Haas, AR et al., Mol.
Therapy, 2019
-Ko, AH et al., Mol.
Therapy, 2020
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Activity of SS1 Mesothelin RNA CAR in Metastatic PDA:
Clinical Responses
Beatty et al. Gastroenterology, 2018
G. Beatty M. O’Hara
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Summary: Mesothelin CAR T Cells
▪ CAR T targeting mesothelin have antitumor activity in mesothelioma and pancreatic cancer
▪ The optimal schedule and route of administration of CAR T cells remains to be defined
▪ CAR T cell trafficking to tumor and evidence of spreading immunity
▪ Checkpoint blockade with PD-1 and CTLA-4 antagonists augments the activity of CAR T cells in preclinical models and early stage clinical trials
1. Beatty GL, et al . Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity
in solid malignancies. Cancer Immunol Res. 2014;2:112.
2. Beatty GL, et al. Activity of Mesothelin-specific Chimeric Antigen Receptor T cells Against Pancreatic Carcinoma
Metastases in a Phase 1 Trial. Gastroenterology. 2018; pii: S0016-5085(18)30323.
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Multiplex Engineered T Cells in Cancer
32
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• Relapse associated with loss of gene modified cells
• Evidence suggests that NY-ESO-1 T cells become exhausted
• How to enhance survival and function?
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Hypothesis and Pre-clinical Work
• Hypothesis:‒ Removal of genes encoding the endogenous TCR, TCRα (TRAC) and TCRβ (TRBC), would
enhance NY-ESO-1 TCR activity and reduce autoimmunity
‒ Removal of PD-1 (PDCD1) would enhance activity and persistence
Ren et al, Clin. Cancer Res. 2016
Overall Survival
0
50
100
0 10 20 30 40 50 60 70 80
Days Post-A549 Injection%
Su
rviv
al
NY-ESO-1 TCR
NY-ESO-1 TCR
CRISPR
T cells alone
CRISPR/Cas9 Editing of T cells
Cas9 + TRAC gRNA
Cas9 + TRBC gRNACD
3
FSC
Cas9 Mock
Cas9 + PDCD1 gRNA
PD
-1
FSC
• We previously demonstrated CRISPR/Cas9 and TRAC, TRBC and PDCD1 targeting gRNAs could
be successfully introduced via electroporation in preclinical models to disrupt gene expression
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• Logic Gated Boolean CAR’s- “And”, “Or”, Not
• Safety Switches
• Conditional/Stealth CAR’s
• Armored CAR’s
The Era of Synthetic Biology
Advanced Cellular Re-Programming
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NY-ESO-1 TCR CRISPR Triple Edited (TCRα TCRβ PD1) T Cell Study Objectives
Adult patients HLA-A2*0201 positive who have relapsed/refractory tumors expressing NY-
ESO-1 antigen. Patients with myeloma, synovial sarcoma, melanoma
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NY-ESO-1 TCR CRISPR Triple Edited (TCRα TCRβ PD1) T Cell
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Feasibility of multiplex human genome editing with CRISPR-Cas9: Cell Product
Stadtmauer et al., Science. 2020 Feb 28;367(6481):eaba7365
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23
53
83
11
3
14
3- 7
17
0
- 10
0 02
04
06
08
0
10
0
- 20
- 10
60
13
0
20
0
27
0
e - 1
e 0
e 1
e 2
e 3
e 4
e 5
34
0
P e r i p h e r a l B l o o d
T u m o r B i o p s y
B o n e M a r r o w
NY
-ES
O-1
TC
R
(co
pie
s/
g D
NA
)
Time (Days)
• There is rapid expansion and stable persistence of T cells expressing the NY-ESO-1
transgenic TCR as measured by qPCR in all 3 patients
- The stable PK of NY-ESO-1 expressing T cells is very different from the PK of CAR
T cells which tends to decrease more quickly
• Clear trafficking of T cells to the tumor
- The levels of T cells expressing the NY-ESO-1 TCR in bone marrow and tumor is
similar to blood
Expansion, Persistence and Trafficking of NYCE T cells: NY-ESO-1 TCR T cells (PCR)
25416-3925416-35 25416-07
Stadtmauer et al., Science. 2020 Feb 28;367(6481):eaba7365
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NYCE CRISPR Genome-Edited Cells: Persistence in Bloodof TRAC, TRBC, and PDCD1 edits by dPCR
• TRAC and PD1 have highest frequency• TRBC was lowest efficiency in vitro, and also in vivo• Pt 07 shortest follow up
Stadtmauer et al., Science. 2020 Feb 28;367(6481):eaba7365
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Di-genic and Tri-genic Editing at the level of a single human genome
Stadtmauer et al., Science. 2020 Feb 28;367(6481):eaba7365
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NY-ESO-1 CRISPR (TCR-PD1) Triple Edited T Cell Study Summary
IND 17297 and Clinicaltrials.gov NCT03399448
Sponsor: Tmunity and Parker Institute for Cancer Immunotherapy
1. A single infusion of CRISPR/Cas9 triple edited T cells is safe in 3 patients- No cytokine release syndrome, no severe adverse events- Best response is stable disease
2. Persistent engraftment of the edited T cells for at least 6 months- Suggests immunogenicity of Cas9 is not occurring under these conditions- Studies to determine impact of T cells on NY-ESO1 and LAGE1 expression are pending
3. Up to 10% of the TCR-modified T cells have PD1 disruption at 1 to 6 months- No evidence of autoimmunity
4. This trial: 2016-17 gene editing technology. New trial: latest improved gene editing technology
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Stadtmauer EA, et al. Science. 2020 Feb 28;367(6481):eaba7365. doi: 10.1126/science.aba7365.
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What a Gene Editing Investigator May Be Asked
• Worst case scenario: induced off-target effects that induce
transformation, and a form of hematologic malignancy
• FDA: Off target sites identification needs to be based on not only
in silico analysis but also on unbiased assays.
• # gRNAs evaluated, detailed description of manufacture, # non-
complementary bases tolerated, sequence of Cas9, purity, ratio of
free vs complexed protein, stability, residual
• Laboratory test, validated laboratory test, CLIA test
• Long term culture assay
• Cell product potency
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Synthetic Biology:
The Advanced CAR Toolbox
• Logic Gated Boolean CAR’s- “And”, “Or”, Not
• Checkpoint Resistant CAR’s
• Safety Switches
• Conditional/Stealth CAR’s
SOLID TUMORS
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Evolution in Tools
Evolution in Manufacturing
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Neolithic Cell Therapy
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Ex Vivo Immune Cell Engineering
Processing Flow
Courtesy of Krish Roy
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Anthropocene Cell Therapy
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Ex Vivo Immune Cell Engineering
Processing Flow
• Activators
• Cytokines
• Metabolism /
transcriptional
regulation
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Manufacturing Improvements:
Faster CAR’s
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Ghassemi et al, 2018
Making Better and Cheaper CAR TMATERIAL OWNED BY BRUCE LEVINE UNIVERSITY OF PENNSYLVANIA
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Successful 24-Hours Manufacture of Anti-CD19/CD22 Dual Chimeric
Antigen Receptor (CAR) T Cell Therapy for B-Cell Acute
Lymphoblastic Leukemia (B-ALL) – Gracell, Shanghai, China
ASH 2020
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Activated T Cell Ex Vivo
Processing Flow
X
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Genomic disruption (and insertion) as
therapeutic cell engineering tools
Trends in Neurosciences 2014 37, 106-123DOI: (10.1016/j.tins.2013.11.001)
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Cellular Door Dash: Gene/Cargo Delivery
NextGenElectroporation Mechanoporation Soluporation
Squeeze or Shake or Stir Martini
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(Some) Critical Path Issues for
Wider Patient Access
• Enhancing potency, especially for solid tumors
–Armor, switches, combinations (CAR’s)
• Manufacturing complexity: cell-based living drugs, viral vectors
–Automation/shortened ex vivo culture
• Education & Training at all levels
– ISCT (isctglobal.org), et al.
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58
WWW.ISCT2021.COM
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59WWW.ISCT2021.COM
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60
www.isctglobal.org/news-events/upcoming-events
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(Some) Critical Path Issues for
Wider Patient Access
• Enhancing potency, especially for solid tumors
–Armor, switches, combinations (CAR’s)
• Manufacturing complexity: cell-based living drugs, viral vectors
–Automation/shortened ex vivo culture
• Education & Training at all levels
– ISCT (isctglobal.org), et al.
• Financial complexity/affordability
–Predictive biomarkers
–Value based payment links price to outcome
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CAR Cells Move Beyond T Cells in Oncology
• CAR T Cells for HIV/AIDS
• CAR T Cells for Autoimmunity and Organ Transplantation
• CAR Macrophages for Cancer
• CAR T Cells for Heart Failure and Fibrosis
• CAR T Cell for Aging?
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ACKNOWLEDGEMENTS
Study
Participants
@BLLPHDMATERIAL OWNED BY BRUCE LEVINE UNIVERSITY OF PENNSYLVANIA
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It Takes A VillageMATERIAL OWNED BY BRUCE LEVINE UNIVERSITY OF PENNSYLVANIA