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Integration of work flows for the generation of gene-modified cell productsBoro Dropulić, PhD, MBAChief Science Officer and General ManagerLentigen Technology Inc., A Miltenyi Biotec Company

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Miltenyi Biotec: Enabling Clinical Centers and Industry

• Miltenyi Biotec is a 1800 person company with global operations• Employs over 600 people in R&D and engineering• Headquarters near Cologne, Germany• Committed to supporting investigators with solutions that enable the

clinical translation and practice of novel cell and gene therapies• Lentigen was purchased in August 2014 by Miltenyi Biotec GmbH• Integration of LV manufacturing competency with MB work flow

solutions for the manufacture of gene-modified cell therapy products

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+ + ++ ++

Donor / patient

BloodLeukapheresis

Cell selection

Cell transduction

Cell product

Administration to patient

Cell activation

Cell expansion

Generation of CAR-T cell products using manual processes are complex and difficult to integrate

CD4 and CD8or CD62L selection

Cytokine IL-2and Media

CD3 + CD28stimulation

CAR inRV or LV

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Donor / patient CliniMACS

Reagents

MACS GMPIL-2

TexMACS

MACS GMPCD3 + CD28

CART/TCRRV or LV

The CliniMACS Prodigy

Integration of unit operations into a single device: Automation of cell processing

BloodLeukapheresis

Cell selection

Cell transduction

Cell product

Administration to patient

Cell activation

Cell expansion

• Integrated cell processing from starting material to final cellular product:- Sample preparation- Cell washing & density gradient

separation- MACS cell separation- Cell activation - Genetic modification (LV)- Cell culture & expansion- Final product formulation

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Automated cell processing provides options for the manufacture of patient specific cell therapies

Centralized Point-of-careRegional

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Clinical success of CD19 CAR T cells in Phase I Trials

0 200 400 6000

20

40

60

80

100

Days after CAR Infusion

Leuk

emia

Fre

e S

urvi

val

Patients Achieving MRD Negative Remission (n=12)Patients Achieving MRD Negative Remission

Group CR RateNCI, Ped. Onc. Branch 85%

UPenn, CHOP 90%MSKCC 88%

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CD19 Negative Relapse in 10-50% of CAR-T 19 treated patients

Pre-CD19 CAR Therapy

CD

45

CD10

CD

22

CD19

Relapse

CD

22

CD19

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Improving CAR-T function: geometry of binding

IntracellularTM

1 2 3 4 5 6 7

CD19

Extracellular

CD22

HA22 m971

FMC63

Rimas OrentasWaleed HasoTerry FryCrystal MacKall

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Site of binding matters for CAR potency

Intracellular TM Extracellular

CD8 HA22CD28ζCD8 m971CD28ζ

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4-1BB costimulation is important for T cell persistence

Intracellular TM Extracellular

TMD

TMD m971

m971

CD28

4-1BB

ζ

ζ

M971- 28zM971-BBz

M971- 28z M971-BBz

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PC USERS:

Reset the slide back to its original form via the menu bar:Home | Slides | Reset

Folie in Ursprungsform bringen über Menu:Start | Folien | Zurücksetzen

MAC USERS:

Reset the slide back to its original form via the menu bar:Home | Slides | Layouts | Reset

Folie in Ursprungsform bringen über MenuStart |

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Picture Tools | Format |

Bildtools | Format |

Format Picture | Compress

Development of Tandem bispecific CD19-CD20 targeting CAR T cells for Adult Leukemia

CAR T cells targeting two B cell tumor antigens at once are postulated tobe more efficient, and to prevent tumor antigen escape

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Specific detection of surface expression of tandem and single CD19/20 CAR in primary human T cells

N.T.

GFP

19B

19A

20A

1920

2019Protein L-PE CD19-AF647(APC) CD20-PE

% % %

0.3

3.6

62.0

93.4

93.6

85.1

89.3

0.5

0.3

0.7

0.3

92.9

68.3

85.4

0.5

1.0

87.4

95.7

0.9

80.4

80.7

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6050

40

30

20

CAR T protein expression in primary human T cells as detected by Western Blotting

Tandem CARs81kDa

Single CARs54kDa

Native CD3 zeta~16kDa

N.T. – non-transduced T cell control

Construct

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Reduction of CD19 downregulation by Tandem CARs

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Planned Phase I clinical trial at Medical College of Wisconsin, Milwaukee, WI, USA

Shah, Taylor, MCW

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Point-of-care cell processing facility blueprint

• Hospitals could be leveraged to decrease overall cost of manufacture• Patients would benefit by local manufacture their patients’ cell products

- better control, no transportation, no cryopreservation à safer product• If given a choice, hospitals would prefer a point-of-care model

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Donor / patient

Robust work-flows requires seamless integration of work-flow components and analytics

TEXTDeviceandProtocols

VectorDesign

IsolationBeadsandStimuationReagents

MediaandConditions

Real-timeAnalytics

Single-source supply avoids costly qualification and validation of outsourced materials

BloodLeukapheresis

Cell selection

Cell transduction

Cell product

Administration to patient

Cell activation

Cell expansion

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Summary• Miltenyi Biotec is an enabler for gene and cell therapy

• Enable companies with custom LVs, automated work-flows, reagents, regulatory support• Enable clinical centers with automated work-flows, reagents, “generic” products – CAR19 LV

• Miltenyi’s vision for commercializing gene-modified cell therapy • Automation improves economics and robustness of cell processing work-flows• Automation provides options for manufacturing: Point-of-Care models now possible

• Development of “generic” & novel LV GMP CARs for clinical testing• Manufacture of patient CAR-T cells in Prodigy device is robust

• Programs for Hematopoietic Stem Cells and other cell types: available or in development

• New products – Prodigy electroporation unit and Tyto flow-though microchip sorting of specific cell types

• Committed to helping investigators to turn their innovations into clinical and commercial realities

boro.dropulic@lentigen.com