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

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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

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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

08 NOV 2018

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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

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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

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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)

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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

IKK P

TRAF2

LTR LTR CAR

NF-kB

CELL DEATH

Fas Chimeric Antigen Receptor (CAR)

+

cepAR)

Self- amplification

CAR(

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 22222 22222222222222222222222222222222222AAFAFAFFFFFAAFAFAAAAAAAA 2222AFAAFF2RAAAAAAAAAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTRRRRRRTRRRRRRTRTRRRTRTRRRRRRRRRRRRRRTTTRTTTTTRRTRTTRTRRRRRRTRRTRTRRRRRRRRRRRRRRRRRRRRRRRRRRTRRRTTRRRRRRRRRRTRRRATRAF2

Gomes-Silva et al. Cell Rep 2017

•  When expressed from a gammaRV vector,

4-1BB.zeta CARs may enhance their own expression

and amplify tonic signaling

•  High tonic CAR signaling can reduce viability and

anti-tumor activity of CAR T cells

4-1BB

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Interaction between the viral vector and its CARgo

IKK P

TRAF2

LTR LTR CAR

NF-kB

CELL DEATH

Fas Chimeric Antigen Receptor (CAR)

+

cepAR)

Self- amplification

CAR(

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 2222 22222 22222222222222222222222222222222AFAFFFAFFFFFAAAAAFAAAAA 2222AFAAFFF2RAAAAAAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTRRRRRTRRRTRRTRRTRRTRRTRRRRTRRRRRRRRTRRTRRTRTTRTRTRTTRTRRRRTTRRRTTRRRRRRRTRRRRRRRRRRRRRTRTRRRTRRRRRRRTRRRRATRAF2

Gomes-Silva et al. Cell Rep 2017

•  When expressed from a gammaRV vector,

4-1BB.zeta CARs may enhance their own expression

and amplify tonic signaling

•  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

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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)

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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

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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

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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

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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

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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

• Biological (expansion, persistence…..)• Clinical

GMP seminar series

• Investigators present • Upcoming clinical protocols• Results of ongoing protocols• New manufacturing strategies

• Duties of the PI• Other

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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

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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

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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

• Where there may be risk to cells

• To validate final manufacturing process

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Release Testing Assays• Microbiology (Sterility) • Mycoplasma• Identity • Purity• Viability• Potency• Dose

Release Testing - Microbiology• 21 CFR Part 610.12 -

approved method• Rapid methods e.g.

Bactec/BacT ALERT –more readily approved +/-validation

• 14 day incubation• Often extended for fungal

cultures• Gram stain additional for

fresh products

Release Testing - Mycoplasma• FDA approved Culture assay

– USP >63 need not be validated if followed directly

• PCR – Roche MycoToolMycoplasma Detection Kit, Approved 2012

• Rapid Test – MycoAlert (Validation)

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Release Testing – Identity/Purity

• 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

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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

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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;

CAR T Cell -Potency Assays

Kassim S. H., BioInsights 2017

4 Cytotoxicity: 75% Responders6 IFN- : 53% Responders

Future Testing MethodsFrom meeting of Cell Manufacturing Technologies Consortium

at Georgia Institute of Technology, November 2018

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Release Testing – Potency

Release Testing – Additional• Replication-competent virus

• Send for Testing or Obtain results before release

https://www.cellandgene.com/doc/fda-s-new-guidance-on-replication-competent-retrovirus-testing-during-gene-therapy-manufacture-0001?vm_tId=2079484&user=4d90e9af-4a74-417a-ba1f-44813e67eb43

Release Testing – Additional• Replication-competent virus – expensive to

establish, validate & perform

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CCertificate of Analysis

• Used for product release• Details

• Tests performed & assay used• Assay sensitivity• Lab. performing assay• Assay criteria for release• Results obtained

• Reviewed and signed by Quality Unit representative

Conclusions• Release assays for CAR-T cells

follow the usual criteria for other cellular therapy products

• Microbiology (Sterility) • Mycoplasma• Identity • Purity• Viability• Potency – most problematic• Dose

• Addition of replication-competence assay

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Issues Involved in ssues nvo ved nStarting CAR T Cell Sttaaartt ng CAR T Ce

Manufacturing

Speaker Contact Email

Adrian Geeapgee@txch.org

Cliona Rooneycmrooney@txch.org

Maksim Mamonkinmamonkin@bcm.edu

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