Engineering Artificial Antigen Presenting Cells for

Efficient Expansion of T Cell Subsets

Bruce Levine, Ph.D.

Division of Transfusion Medicine

Department of Pathology and Laboratory Medicine

Abramson Cancer Center Translational Research Program

University of Pennsylvania

J Immunol 1997; 159: 5921

Science 1997; 276: 273

Immunol. Rev. 1997; 160: 43

Mol. Ther. 2004; 9; 902

Exp. Opin. Biol. Ther. 2008; 8: 475

Anti-CD3 Anti-CD28

Artificial APC: Bead

Signal 1

Growth

CD28 CTLA4TcR/CD4

+

Magnetic Bead-Based Expansion of

Effector T Cells wo/Feeder Cells

Polyclonal Replicative Potential of

Adult CD4 T Cells In VitroFigure 3

1E+06

1E+07

1E+08

1E+09

1E+10

1E+11

1E+12

1E+13

1E+14

1E+15

1E+16

1E+17

1E+18

1E+19

1E+20

0 10 20 30 40 50 60 70 80 90

Day

To

tal

Cell

s

IL-2 Added Day 49

No IL-2 Day 50

Exponential growth (days) 60-100

Mean log10 growth (fold) 9-12

Mean Pop Doubling 30-40

Interaction of LV-aAPC with CD8+ T Cell

2 microns

CD8 T LV-aAPC

Suhoski, M., Molecular Therapy 2007 May;15(5):981-8

1st and 2nd Generation aAPC for

T Cell Expansion

CD3/28 coated beads

•GMP reagents available

•Safety record in humans, ~800 infusions

Cell Based aAPC

•Ability to use natural ligands

•Multiple costimulatory signals

•Secretion of cytokines

•Antigen specific expansion

CD3/28 coated beads Cell Based aAPC

Preparation

-purchase Dynal/Invitrogen ClinExVivo

or

-coat “naked” beads w/mAbs, QC

Preparation

-expand in culture

-clear non-specific Ab from CD64

-irradiate

-load anti-CD3 (CD28) mAb

1st and 2nd Generation aAPC for

T Cell Expansion

Master Cell Bank Qualification- Quality control

Sterility Testing- to test for absence of bacteria, fungi, mycoplasmas, viruses other than lentiviral vector (CMV, HIV-1 & 2, HTLV1 &2, EBV, HBV, HCV, etc).

Identity Testing- to establish that all significant properties of the cells are stable and maintained throughout the entire manufacturing process. Includes lentiviral transgene expression and RCL testing.

Purity Testing- to detemine identity and quantity of other cells- determine % of target aAPCs in MCB

Functional Testing- to determine potency of aAPCs in stimulating Tregs (as measured by CD4+, CD25+, and FoxP3 expression, functional assay)

Two Tiered Cell Banking System

Closed System Production of MCB and WCB

Source Cells

Expand in Wave

Harvest and

Cryopreserve MCB

Thaw and

Expand in Wave

Antibody Clearance

Irradiation

Anti-CD3 mAb Loading

Cryopreserve WCB

Tregs are uncommon: rigorous in vitro selection

process is required.

Tregs are hypoproliferative: ex vivo culture can

select for effector T cells

Establishing the best aAPC to expand human PB

and UCB Tregs: Cell based aAPCs are superior

to beads in expanding functional T reg cells

Challenges of Treg Isolation,

Expansion, and Testing

Adult Peripheral Blood Cord Blood

Adult CD25+ Selected Cord CD25 Selected

CD4

CD

25

Godfrey, W. R. et al. Blood 105:750-758, 2005

Treg Expansion with aAPC

CD4+25+127-

(Miltenyi)

Golovina et al. J Immunol. 2008 81(4):2855-68.

Foxp3 Expression on In Vitro

Expanded Tregs

Golovina et al. J Immunol. 2008 81(4):2855-68.

Day 14

Expanded Treg In Vitro Potency Assay

1:1

1:2

1:4

1:8

Tregs: PBMC

PBMC control

Cell-Based aAPCs are Superior to CD3/28 Bead Expanded

Cord Blood TREGS in Suppression of CD8+ T Cell Proliferation

No beads control

+ CD3 beads control

+ Tregs 1/2

+ Tregs 1/8

+ Tregs 1/4

CD3/CD28 Beads KT64 86 aAPC

K562 aAPC Effector T Cell Expansion

Expansion of CD8+ T cells by KT.A2.41BBL.64

0

2

4

6

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10

12

14

16

18

0 5 10 15 20 25

Po

pu

lati

on

Do

ub

lin

g Le

vel

Day of Culture

KTA2.41BBL.64

KTA2.41BBL.64 (fresh/cryo)

CD3/CD28 beads

restimulation

K562 aAPC’s Cell Bank StatusResearch grade aAPC’s

cGMP WCB

BMF 4/09

KT64:86

“Treg” aAPC

cGMP MCB

KTA2.41BBL.64

“CTL” aAPC

cGMP MCB

IND #1 IND #2

Clinical

Trials

Clinical

Trials

GLP WCB’s

-validations

-to collaborators

cGMP WCB

BMF

(future)

IND #1 IND #2

Clinical

Trials

Clinical

Trials

-validations

-to collaborators

-BMF’s

-IND’s

-Clinical Trials

aAPC Line #3

GMP MCB

GLP/GMP

WCB’s prn

PENN - TRP• Tatiana Golovina

• Samik Basu

• Xiaochuan Shan

• Rong Liu

• Tatiana Mikheeva

• Carl June

• Jim Riley

PENN - CVPF• Andrea Brennan

• Anne Chew

• Julio Cotte

• Dawn Maier

• Dan Powell

• Tamara Tripic

• Ashley Vogel

• Zoe Zheng

Univ. Minnesota• Stephen Porter

• Keli Hippen

• Paul Harker-Murray

• Aryel Londer

• Megan Bina

• Bruce Blazar

• Claudio Brunstein

• Margaret Macmillan

• David McKenna

• Diane Kadidlo

UCSF, BSRI• Jeff Bluestone

• Greg Szot

• Amy Putnam

• EJ Read

Univ. Florida• Mark Atkinson

NCI, DTM• Crystal Mackall

• David Stroncek

• Naomi Taylor

• Marianna Sabatino

• Mark Dudley

NCI R01-CA-105216

NCI Intramural B2B

JDRF

Becton Dickinson