Educational Web Seminar Issues Involved in Starting CAR T Cell Manufacturing Thursday, 08 November 2018 12:00 noo n n – – 1:00 PM ET Adrian P. Gee, MI Biol Director Clinical Applications Laboratory, Center for Cell and Gene Therapy Baylor College of Medicine Cliona M. Rooney, PhD Director Translational Research Laboratories, Center for Cell and Gene Therapy Baylor College of Medicine Maksim Mamonkin, PhD Assistant Professor Center for Cell and Gene Therapy Baylor College of Medicine It is the policy of the University of Minnesota Office of Continuing Professional Development to ensure balance, independence, objectivity and scientific rigor in all of its educational activities. All individuals (including spouse/partner) who have influence over activity content are required to disclose to the learners any financial with a commercial interest related to the subject matter of this activity. A commercial interest is any entity producing, marketing, re-selling, or distributing health care goods or services consumed by or used on, patients. Disclosure information is reviewed in advance in order to manage and resolve any possible conflicts of interests. Specific disclosure information for each presenter, course director, and planning committee member will be shared with the learner prior to presenter's presentation. Persons who fail to complete and sign this form in advance of the activity are not eligible to be involved in this activity. Unless otherwise noted, individuals did not indicate any relevant affiliations or financial interests Faculty Disclosure Role Name of Company Adrian P. Gee None Director, Clinical Applications Laboratory, Baylor College of Medicine None Cliona M. Rooney Yes Director, Translational Research Laboratory, Baylor College of Medicine SAB/Consulting: Cell Medica, CellGenix, Tessa Therapeutics; Research Support: Tessa Therapeutics and CoFounder: Viracyte, Marker Maksim Mamonkin None Assistant Professor, Baylor College of Medicine None Debbie Wood None Project Director, The Emmes Corporation None Laarni Ibenana None Project Manager, The Emmes Corporation None Aisha Khan None Executive Director of Laboratory Operations, Interdisciplinary Stem Cell Institute, University of Miami None David McKenna None Medical Director, Molecular and Cellular Therapeutics, UMN None Joseph Gold None Manufacturing Director, Center for Biomedicine and Genetics, City of Hope None Linda Kelley None Director, cGMP Cell Therapy Facility, Moffitt Cancer Center None Jodi Brenden Amir None Education Consultant, Office of Continuing Professional Development, UMN None Dasha Dobrinina None Education Coordinator, Office of Continuing Professional Development, UMN None 08 NOV 2018 1
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Educational Web Seminar
Issues Involved in Starting CAR T Cell Manufacturing
Thursday, 08 November 2018
12:00 noon n –– 1:00 PM ET
Adrian P. Gee, MI BiolDirector
Clinical Applications Laboratory, Center for Cell and Gene TherapyBaylor College of Medicine
Cliona M. Rooney, PhDDirector
Translational Research Laboratories, Center for Cell and Gene TherapyBaylor College of Medicine
Maksim Mamonkin, PhDAssistant Professor
Center for Cell and Gene TherapyBaylor College of Medicine
It is the policy of the University of Minnesota Office of Continuing Professional Development to ensure balance,independence, objectivity and scientific rigor in all of its educational activities. All individuals (including spouse/partner)who have influence over activity content are required to disclose to the learners any financial with a commercial interestrelated to the subject matter of this activity. A commercial interest is any entity producing, marketing, re-selling, ordistributing health care goods or services consumed by or used on, patients. Disclosure information is reviewed in advancein order to manage and resolve any possible conflicts of interests. Specific disclosure information for each presenter,course director, and planning committee member will be shared with the learner prior to presenter's presentation. Personswho fail to complete and sign this form in advance of the activity are not eligible to be involved in this activity.
Unless otherwise noted, individuals did not indicate any relevant affiliations or financial interests
Faculty Disclosure Role Name of Company
Adrian P. Gee None Director, Clinical Applications Laboratory, Baylor College of Medicine None
Cliona M. Rooney YesDirector, Translational Research Laboratory, Baylor College of Medicine
SAB/Consulting: Cell Medica, CellGenix, Tessa Therapeutics; Research Support: Tessa Therapeutics and CoFounder: Viracyte, Marker
Maksim Mamonkin None Assistant Professor, Baylor College of Medicine None
Debbie Wood None Project Director, The Emmes Corporation None
Laarni Ibenana None Project Manager, The Emmes Corporation None
Aisha Khan None Executive Director of Laboratory Operations, Interdisciplinary Stem Cell Institute, University of Miami None
David McKenna None Medical Director, Molecular and Cellular Therapeutics, UMN None
Joseph Gold None Manufacturing Director, Center for Biomedicine and Genetics, City of Hope None
Linda Kelley None Director, cGMP Cell Therapy Facility, Moffitt Cancer Center None
Jodi Brenden Amir None Education Consultant, Office of Continuing Professional Development, UMN None
Dasha Dobrinina None Education Coordinator, Office of Continuing Professional Development, UMN None
08 NOV 2018
1
Accreditation StatementIn support of improving patient care, this activity has been planned and implemented by University of Minnesota,Interprofessional Continuing Education and The Emmes Corporation. The University of Minnesota,Interprofessional Continuing Education is jointly accredited by the Accreditation Council for Continuing MedicalEducation (ACCME), the Accreditation Council for Pharmacy Education (ACPE), and the American NursesCredentialing Center (ANCC), to provide continuing education for the healthcare team.
Credit Designation StatementsAmerican Medical Association (AMA) The University of Minnesota, Interprofessional Continuing Education designates this live activity for a maximum of1.0 AMA PRA Category 1 Credits™. Physicians should claim only the credit commensurate with the extent of theirparticipation in the activity.
Laboratory Professionals1.0 hour of P.A.C.E. credit (CEU) through the University of Minnesota Medical Laboratory Sciences Program will be offered for this session.
Florida Clinical Laboratory PersonnelThe University of Minnesota Medical School, Office of Continuing Professional Development has been approved bythe Florida Board of Clinical Laboratory Personnel, CE Provider #50-21144. This activity has been approved by theFlorida Board of Clinical Laboratory Personnel, CE Broker Tracking # 20-678872 and will offer 1.0 hour ofcontinuing education.Other Healthcare Professionals Other healthcare professionals who participate in this CE activity may submit their statement of participation to their appropriate accrediting organizations or state boards for consideration of credit. The participant is responsible for determining whether this activity meets the requirements for acceptable continuing education.
Complete the online attendee roster w/in 72 hrs. of the webseminarhttps://z.umn.edu/PACTWebSeminarAttendanceRoster
Complete the online survey w/in 72 hrs of the web seminar:1. Survey will display when you exit the web seminar2. Survey link provided in your email reminder sent 08 November 2018
3. PACT website: Education>PACT web seminars>November 08 Web Seminar
CE credit is only offered to participants who have attended this live web seminar
Each attendee must:
Note: After the web seminar, on-line rosters and surveys have been processed, a Statement of Participation will be issued via email to each participant listed on the attendee rosters requesting CE.
Educational Web Seminar
Issues Involved in Starting CAR T Cell Manufacturing
Thursday, 08 November 2018
12:00 noon n –– 1:00 PM ET
08 NOV 2018
2
Identify key requirements when selecting a vector forCAR T cell transduction.
Identify significant and unique challenges regardingthe manufacture of CAR T cells
Acquire knowledge of the in-process and releasetests required for CAR T cells.
Choosing Optimal Viral Vector for T-cell Transduction
PACT Webinar Nov 08, 2018
Max Mamonkin, PhD
Center for Cell and Gene Therapy Baylor College of Medicine
Viral vectors for blood cells Short/long term gene expression Stable gene expression
Adenoviral vectors (AdV)
Gamma- retroviral vectors
Lentiviral vectors
Adenoviralvectors (AdV)
Gamma-retroviral vectors
Lentiviralvectors
Adeno-associated viral vectors (AAV)
Non-integrating Integrating
T cell
08 NOV 2018
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Adeno-associated viral vectors (AAV)
Short/long term gene expression
Viral vectors for blood cells Stable gene expression
Adenoviral vectors (AdV)
Gamma- retroviral vectors
Lentiviral vectors
Gamma-retroviral vectors
Lentiviralvectors
Adenoviralvectors (AdV)
Adeno-associated viral vectors (AAV)
Short/long term n gene expressioo T cell
Retroviruses Retrovirus
MoMLV, MSCV etc HIV, FIV, SIV etc
• ssRNA viruses capable ofintegrating into hostgenome
• Can be modified to efficientlytransduce T cells
Intechopen.org
Gammaretroviral vector Gammaretroviral genome
Adapted from Intechopen.org
poly-A
• In vectors most viral genes aredeleted to make room for transgene
• Gag, pol, and env are provided inseparate plasmids in packaging cells
• Host specificity is dictated by env genespoly-A +
Packaging plasmids (not included in the virion)
Ψ
Ψ ΨΨΨΨΨΨΨΨdeleted Ψ
Ψ
Ψ
08 NOV 2018
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Gammaretro- vs SIN lentiviral vectors
CAR
LTR
Ψ
LTR
CAR
LTR
Ψ
LTR
ext. promoter
Prom plasmid
Gammaretroviral Lentiviral (SIN)
Gammaretro- vs SIN lentiviral vectors
CAR
LTR
Ψ
LTR
CAR AAAA
+gag-pol +env packaging
infection, RT
CAR
LTR
Ψ
Ψ
CAR
LTR
Ψ
LTR
CAR AAAA
ext. promoter
Prom
viral particle
plasmid
integrated provirus
Gammaretroviral Lentiviral (SIN)
Gammaretro- vs SIN lentiviral vectors
CAR
LTR
Ψ
LTR
CAR AAAA
+gag-pol +env packaging
infection, RT
CAR
LTR
Ψ
Ψ
CAR
LTR
Ψ
LTR
+gag-pol +env +rev
packaging
infection, RT
Ψ
CAR AAAA
ext. promoter
Prom
CAR Prom AAAA
CAR Prom AAAA viral
particle
plasmid
integrated provirus
Ψ
CAR Prom
Gammaretroviral Lentiviral (SIN)
08 NOV 2018
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Gammaretro- vs SIN lentiviral vectors Gammaretroviral Lentiviral (SIN)
• High efficiency of T cell transduction
• Pseudotypes: RD114, GALV
• Cheap(er) to manufacture in cGMP
• Stable producer cell lines available
• Integrates in dividing cells only
• Smaller genome -> limited cargo (~4-5Kb)
• Transgene expression is driven by LTR
• Tends to integrate near promoters, potential for
insertional mutagenesis (HSC)
Gammaretro- vs SIN lentiviral vectors Gammaretroviral Lentiviral (SIN)
• High efficiency of T cell transduction
• Pseudotypes: RD114, GALV
• Cheap(er) to manufacture in cGMP
• Stable producer cell lines available
• Integrates in dividing cells only
• Smaller genome -> limited cargo (~4-5Kb)
• Transgene expression is driven by LTR
• Tends to integrate near promoters, potential for
insertional mutagenesis (HSC)
• Lower rates of transduction
• Mainly VSV-G pseudotype
• More complex production
• No cGMP-grade producer lines yet
• Dividing and non-dividing cells
• Higher cargo capacity
• Transgene expression is driven by an
internal promoter
• More random integration in gDNA, self-
inactivating LTR promoter
Interaction between the viral vector and its CARgo
• High tonic CAR signaling can reduce viability and
anti-tumor activity of CAR T cells
SOLUTIONS
• Reduce CAR expression
• Modify CAR structure to reduce tonic signaling
• Use non-LTR (SIN lentiviral) vectors
4-1BB
Gammaretroviral vector production cGMP production of gamma RV and LV vectors
Gammaretroviral vector production cGMP production of gammaRV vectors – stable producer line
gag pol
env (GALV)
PG-13
gag pol
env (GALV)
CAR
Ψ CAR AAAA
viral transduction
Ψ
RD114 pseudotyped RV (GLP)
Transduced PG-13
08 NOV 2018
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Gammaretroviral vector production cGMP production of gammaRV vectors – stable producer line
gag pol
env (GALV)
PG-13
gag pol
env (GALV)
CAR
Transduced PG-13
Ψ CAR AAAA
viral transduction
Ψ
cloning selection
expansion MCB
gag pol
env (GALV)
CAR Ψ
Ψ CAR AAAA
Ψ CAR AAAA
Ψ CAR AAAA
RD114 pseudotyped RV (GLP)
GALV pseudotyped RV (cGMP)
continuous production
Stable producer (PG-13)
aliquot, freeze, test
Gammaretroviral vector production cGMP production of gammaRV vectors – transient transfection
gag pol
env (GALV)
293Vec-GALV
DNA transfection CAR
Ψ
plasmid
Gammaretroviral vector production cGMP production of gammaRV vectors – transient transfection
gag pol
env (GALV)
293Vec-GALV
gag pol
env (GALV)
CAR
Transiently transfected 293Vec-GALV
DNA transfection
Ψ gag pol
env (GALV)
CAR Ψ
Ψ CAR AAAA
Ψ CAR AAAA
Ψ CAR AAAA
GALV pseudotyped RV (cGMP)
transient production
CAR Ψ
plasmid
48h, 72h, 96h
Transiently transfected 293Vec-GALV
aliquot, freeze, test
Phase 1 CD5 CAR T cells (MAGENTA)
08 NOV 2018
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Stable producer lines vs transient transfection
Gammaretroviral vector production cGMP production of lentiviral vectors – transient transfection HEK293
Transiently transfected HEK293
DNA transfection
VSV-G pseudotyped LV
transient production
LV plasmid
CAR
LTR
Ψ
Prom
LTR
gag pol env (VSV-G) rev
+ +
gag pol
env (VSV-G)
rev
CAR
Ψ
Prom
CAR
Ψ
Prom CAR
Ψ
PromCAR
Ψ
Prom CAR
ΨΨ
Prom
CAR
Ψ
Prom CAR
Ψ
Prom
Gammaretroviral vector production cGMP production of lentiviral vectors – transient transfection HEK293
Transiently transfected HEK293
DNA transfection
VSV-G pseudotyped LV
transient production
LV plasmid
CAR
LTR
Ψ
Prom
LTR
gag pol env (VSV-G) rev
+ +
gag pol
env (VSV-G)
rev
CAR
Ψ
Prom
gag pol
env (VSV-G)
rev
CAR
Ψ
Prom
Toxicity to producer cell
CAR
Ψ
Prom CAR
Ψ
PromCAR
Ψ
Prom CAR
ΨΨ
Prom
CAR
Ψ
Prom CAR
Ψ
Prom
• Concentration • Purification • Formulation
08 NOV 2018
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Gammaretroviral vs lentiviral vectors
% %
Taking CAR T-Cells From The Research Laboratory To The GMP Facility
Cliona RooneyDirector of the Translational Research Laboratories (TRL) of
The Center for Cell & Gene TherapyBaylor College of Medicine
Houston, Texas
Taking a Manufacturing Strategy from Research Lab to GMP facility
• The Translational Research Laboratories (TRL) of the CAGT• Role of the Principal Investigator
• scientist who developed the strategy• CAR T-cell manufacturing at the CAGT• Closed systems
CAR T-cell
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Is the Methodology GMP Compliant?• Are reagents and supplies GMP
compliant?• Media• Other reagents• Culture vessels
• Manufacturing strategy• Cell selection• Transduction• Expansion• Scale up?• Can it be simplified?
Translational Research Laboratories• One floor above GMP facility (16th floor)
• GMP staff, QA, QC and a CLIA-certified flow cytometry group
• 17th floor• 15 Principal Investigators (PI’s) and their labs• Research Coordinators and Regulatory Affairs group• Facilitates regular exchange of information
• TRL Investigators aware of technologies used in GMP facility• Discussion of translation early in project development• Reagents and supplies• T-Cell culture methodology• Transduction
• Facilitates translation of laboratory methods to GMP compliant SOPs
Cell Culture Medium • TRL Investigators use the T-cell culture medium used in the
GMP• Selected and reselected for T-cell culture over 30 years
08 NOV 2018
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Translational Research Staff• Trained to work in GMP and in research laboratories
• Understand both sides• Highly versatile
• Work with TRL Investigators to facilitate translation• Help with
• Bringing the manufacturing method into GMP compliance• Writing SOPs and worksheets/batch records• Validating new procedures• Equipment qualifications• Stability programs
Role of the TRL Investigator
1. Work with translational staff to develop SOPs• Start with existing similar SOP• Investigator modifies for his/her purpose
CAR T-cell SOPs at CAGT• 9 current CAR trials
• 7 different CARs• ATCs (CD3/28-activated), VSTs and NKTs
• For ATCs we attempt to use same SOP for all• Investigators (or their CARs) can be picky
• Not all CARs are equal• CARs affect T-cell growth and differentiation• Important factors for clinical outcome
08 NOV 2018
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Sometimes we must compromise
• “Do you really need to seed only 1 x 105 cells per well for transduction?
• For 10 x 106 transduced T-cells we will need 5 plates!!!”
“Do you want to compromise clinical results because of feasibility”?
1 x 105
NT2.5 x 105
5 x 105
Cotransduction efficiency Fold CAR T-cell expansion
1 x 105 2.5 x 105 5 x 105
Cells per well
68.9% 56.2% 35.8%
10% 20% 40%
Role of the TRL Investigator
1. Work with translational staff to develop SOPs• Write the SOP
2. TRL Investigator will train the translational staff on the SOP in TRL laboratory
3. Translational staff train GMP staff in GMP facility
08 NOV 2018
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Role of the TRL Investigator
1. Work with translational staff to develop SOPs• Write the SOP
2. Train translational staff on SOP in TRL laboratory3. Translational staff train GMP staff in GMP facility4. TRL Investigator can maintain involvement with production
• Scrutinize batch records (not sign off)• Help with trouble shooting
• Perform functional assays on final product5. Work closely with clinical PI and research co-ordinator
Weekly clinical protocol meetings• Attended by
• TRL Investigators, Clinical PI’s, Research co-ordinators, GMP staff, QC, QA, GLP staff (for follow up samples)
• Discuss • Patient referrals to protocols
• Consents and procurement • Manufacturing progress and release• Infusions• Follow up
• Investigators present • Upcoming clinical protocols• Results of ongoing protocols• New manufacturing strategies
• Duties of the PI• Other
08 NOV 2018
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Do we need a closed manufacturing system?
• GRex is good for small-to-large scale T-cell expansion • Seed 5 million CART cells in a GRex 10, • Harvest 1 to 2 billion from 1 Grex 100• Compatible with closed seeding and harvest
Not suited to retroviral transductionRequires adhesion to retronectin-coated surface
CCAR T-ccell manufacture using retroviral vectors according to CAGT
T-cell activationon
CD3/28-coated NTC plates
24 x 106 PBMCs
1 x 106 per well
1 x 24 well plate
Transductionon
Retronectin-coated wells
20 x 106 cells
0.375 x 106 per well
3 x 24 well plates
CryopreservationExpansion
in GRex
Day 1 Day 2/3 Day 5 to 13
What is “in vogue” for a CAR T-cell?• Minimally differentiated
• Retains naïve or central memory phenotype
• Short culture period• High numbers
• Transduce more T-cells• More 24 well plates
• Validation of transduction in T75 flasks
08 NOV 2018
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CCan we use closed Systems?
•Desirable•Expensive
• Hardware, tubing•Not suited to small scale manufacturing•Not suited to retroviral transduction•Not required for phase I/II clinical trials
If Phase I/II is successful
• Worth expense of closing manufacturing and scale up• Likely supported by industry
Summary • Close communication is crucial for effective SOP transfer• The PI can help with troubleshooting in early days
• Listen to hem/her• You cannot make T-cells fit your needs and wants
• They have highly specific growth and media requirements• CAR-T-cells grow exponentially, only if those requirements are met
• Can be manufactured from small blood volumes• The field requires new closed systems for small blood
volumes• Closed systems not required for phase I/II trials
08 NOV 2018
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Testing CAR-T Cell Products
Adrian GeeCenter for Cell & Gene Therapy
Baylor College of MedicineHouston, Texas
Texas Medical Center
CAR-T Vector Production
Manufacturing & Testing RecordsCertificate of Analysis
CAR T cells are genetically modified, often using viral vectors, these require extensive testing in their own right
In-Process Testing
• To evaluate changes to manufacturing procedure
• Test at critical control points• Where there are Go/No Go criteria
• Flow Cytometry• T cell markers• CAR Expression• Contaminating cells
Release Testing – Purity
• Removal of manufacturing reagents
• Animal sera• Antibiotics• DMSO etc.
• Calculate residual reagent by dilution factor
• Perform assay for residual reagent
Release Testing – Viability
• Manual: e.g. Trypan Blue exclusion
• Automated: Flow cytometry – e.g. 7AAD staining +/-apoptosis markers
• Pre-freeze• Post-thaw?
• Used also for stability testing
08 NOV 2018
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Potency - Choice of AssaysNo single test adequately predicts clinical efficacy• Provide “substantial evidence” that
the product will have the effect it purports to have under the conditions of use prescribed
• Obtained from “ adequate and well-controlled investigations conducted with a consistently manufactured product
Release Testing – Potency
• Not “formally” required before Phase 3 trial
• Should correlate with in vivo activity
• Use Phase 1 &2 to refine and validate the potency assay
Why Assay Potency in Phase 1?• To show activity, quality &
consistency during development• Generate data to define
specifications for lot release• To assess effects of
manufacturing changes• To evaluate product stability• To detect problems• Evaluate a variety of assays and
their correlation
08 NOV 2018
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Release Testing - Potency
Potency for CAR-T• Cytotoxicity against cells line
bearing the specific antigen e.g. 51Cr release
• ELISpot assay• Proliferation assay
An Innovative in Vitro Potency Assay Designed to Predict the Fate of Chimeric Antigen Receptors Modified T Cell Therapy Post Infusion in ALLJunxia Wang, Mark Dudley, Therese Choquette, MargitJeschke, Erik Rutjens and Sadik KassimBlood 2016 128:5830;