NK Cells for Therapy - PACT GROUPpactgroup.net/system/files/cagt_wkshp_lapteva_12.pdfNK Expansion in Bags and G-Rexes in 40 197-mL bags Day 10 Bags Day 0 harvest 2x106 NK no processing

PACT Meeting, Houston Lapteva

1

NK Cells for Therapy

Natasha Lapteva, Ph.D.

Center for Cell and Gene Therapy Baylor College of Mediciney g

Houston, TX

Natural Killer (NK) cells

o Cytotoxic lymphocytes of innate immune systemo Mediate innate responses to viruses and intracellular

NK

o Mediate innate responses to viruses and intracellular pathogens

o Recognize alterations in MHC-I and ligands expressed in stressed/malignant cells

o Tumor rejection: destroy cancer cells resistant to chemotherapy with little or no toxicity to the hostchemotherapy with little or no toxicity to the host

o Used for treatment of malignancies, e.g. AML, melanoma, multiple myeloma


2

Inhibitory KIR + self HLA = Tolerance

Inhibitory and Activation Signaling Determine NK Function

Activating receptors: activating KIR, NCR, NKG2D

Lack of self HLA-I

Protocols for Manufacture NK Products

o NK clinical trials require high doses of cells

o Starting material are apheresis cells, depletion of CD3+/CD19+ cells

o Expansion protocols with cytokines (without feeder cells) require prolonged cultures

o Expansion protocols with feeder cells generate high yields and high potency cells in short time


3

Expansion of NK Cells Ex-vivo

4-1 BBL

mb-IL-15K562 vr

NKK562 vr

NK

NK

NK

NK

NKNK

NK

NK

NK

10 K562vr: 1NK

MCB906K562

10 U/mL IL-2SCGM medium

MNC

MNC

MNCMNC

Described by Dario Campana et al

NKNK

Questions for Manufacturing Transfer

o Can we grow NK cells in G-Rexes ?o Can we grow NK cells in G-Rexes ?

o Shipment of cryopreserved or fresh NK cells ?

o Can we generate more potent NKs?


4

Growth in Bags

o Low ratio of medium to surface area for optimal gas exchange

o Requires large numbers of bags

o Frequent feeding and culture manipulations

o Large harvest volume

Gas Permeable Rapid Expansion Device (Wilson Wolf - G-Rex)

o Gas permeable membrane pallows optimal exchange

o Supports cell growth with large volumes of media on a small surface area

o Waste dilutiono Waste dilutiono Minimizes manipulationo Low harvest volume


5

Optimization of NK Expansion in G-Rex

ansi

on

109 )

>200-fold expansion in CCD100 up to 1.2x109 cells/G-Rex100

1 300 2x106 NK4x106 NK

2x106 NK4x106 NK

seeded

Fold

NK

expa

Abs

# c

ells

(x1

0.5 150

6 7 86 7 8days days

L) 500

4x10 NK8x106 NK

4x10 NK8x106 NK

T cells

NKs

0 5 6 7 8 days

Glu

cose

(mg/

dL

2502x106 NK4x106 NK8x106 NK

Expression of NK Activation Markers

10% 39% 85% 91%Day 0 Day 4 Day 6 Day 10

CD

56

CD3

96%

Days7-10

coun

t

IgG NKp44 NKp30 NKG2D

2% 86% 91% 96%


6

Cytotoxicity of Expanded NK cells

K562 CEL100s K562 CEL

Raji BL

U266 MM

OPM-2 MM

00

60

20

% S

peci

fic L

ysis

5:1 10:1 20:1 40:1 E:T

Day 0 Day 2 Day 4 Day 6 Day 8 Day 10

18x109 cellsh t

Feeding and splitting2x106 NK

NK Expansion in Bags and G-Rexes

in 40 197-mL

bags

Day 10

Bags

Day 0

harvest

2x106 NK

harvestno processing for up to 10 days 18x109 cellsin 20

G-Rex100sG-Rex


7

ssel

1000

NK Expansion in Bags and G-Rexes

G-RexVueLife Culture Bags

4 6 8 10 Days

*

6 8 10 Days

# N

K c

ells

(x10

6 ) p

er v

es

200

600

1000

100

Fold

NK

Exp

ansi

on

200

300

400

500

**

Days Days

NK Expansion in WAVE Bioreactor

200

400

Ks (x

106 )

56+ C

D3-

40

80G-RexWAVE

# N

K

% C

D 40

pans

ion

8080

s

3 4 5 6 7 8 9 3 4 5 6 7Days Days

G-RexWAVE

8 9

Fold

of N

K e

xp

40

20

60

% K

562

Lysi

s

3 4 5 6 7

Days 40:1 10:1 2.5:1 1:1

E:T8 9


8

Release specification G-Rex WAVE

WAVE Evaluation: Final Product Release Specifications

%CD56+CD3- >50% 61.3% 73.5%Viability (7-AAD) >70% 86.5% 84.2%

%GFP+ 20% 84.8% 85%

Clinical Protocol Logistics

Apheresis

Texas Arkansas

CAGT: Expansion of NK cells, QC, CoA and

shipment of fresh TC-NK product to UAMSproduct to UAMS


9

Treatment Schema for Allo-NK In Myeloma Patients

Bzb

CyMesna

Treatment Dose Purpose eBortezomib 1.0 mg/m2 Reduce HLA, increase TRAIL on MM

0 2Days

Daily IL2Flu

-6 -2 4 6

Exp-NK

y

-4 12-7-9

Dex

Mesna 30 mg/kg i.v. Prevention of hemorrhagic cystitisCyclophos 60 mg/kg i.v. Tumor debulking, immunosuppressionDex 40 mg PO Tumor debulking, immunosuppressionFludarabine 25 mg/m2 i.v. ImmunosuppressionExp-NK cells 2-10x107/kg Lysis of MMInterleukin-2 3x106 U s.c. Support NK activity, persistence

Expansion K562-MCB ~12 days

Receive apheresis product

NK Cell Production Outline

AllogeneicCo-culture apheresis product K562-MCB

Ficoll apheresis product

Click-iT Assay

Irradiation K562-MCB

Formulation and Shipment

CliniMACS depletion of CD3+ T cells

For ~8 daysAutologous


10

CERTIFICATE OF ANALYSISCenter for Cell & Gene Therapy, GMP Cell Processing Facility

Baylor College of Medicine, Houston, Texas 77030TC-NK CELLS

Overnight release criteria for shipped fresh NK cells

Test SpecificationTest SpecificationViability >70%

%CD56+CD3- >50% for auto->70% for allo-# CD3+CD56-


11

Subject NK % T % Viability % Potency

Val1 52 34 72 63.4V l2 69 19 86 61 9

NK Purity, Potency and Viability

Non CD3 Depleted products

Subject NK % T % Viability % Potency

Val 1 93 0.1 91 92V l 2 93 0 04 9 9

Val2 69 19 86 61.91 88 4 83 654 84 6 92 39

autologous donors

CD3 Depleted products

Val 2 93 0.04 97 792A 86 0.21 96 742B 89 1.02 89 743 85 0.09 91 65

Potency = % lysis of K562 at 20:1 E:T ratio

allogeneic donors

Subject Cryopreserved NK cell dose

Recovery(%)

Actualinfused NK

ViabilityBy flow

Potency

Recovery After NK Cryopreservation Was Suboptimal

NK cell dose for infusion

(%) infused NK cell Dose

By flow

Val 1 4.7x107/kg 61 2.9x107/kg 78% 77%Val 2 4.9x107/kg 100 4.9x107/kg 99% 90%1 4.9x107/kg 65 3.2x107/kg 94% 94%2A 2.1x107/kg 100 75%2B 1.3x107/kg 100 83%3.4x10

7/kg 79%

3 5.0x107/kg 100 5.0x107/kg 77% 70%4 3.4x107/kg 65 2.2x107/kg 94% ND


12

Frozen NK Cells Are Suboptimal

Within 1 hour post-thaw16 24h post thaw 500

600

100

60

20% V

iabl

e N

K ce

lls

16-24h post-thaw

NK

/µL

100

200

300

400

500

0%

1 2 3 Donors

0‐9 0 1 3 5 7 10 14

Day of Protocol

Can we ship fresh NK products?


13

Frozen or Fresh NK Products

Frozen Fresh

One product for multiple infusions One product for one infusion

Post- thawo Good viability right after thaw

One product for multiple infusions One product for one infusion

o Good viability ?

40% HBSS10% DMSO12.5% HSA

5% HSA

o Good viability right after thawo Recovery low after 24ho Not cytotoxic right after thawo Do not expand well in vivo

yo Cytotoxic ?o Expand well in vivo ?o Continue to expand during

shipment ?

Fresh NK Potency is Retained After Shipping

48h after formulation24h after formulation

100

20

60

100

% S

peci

fic ly

sis

Donor 1Donor 2Donor 3 20

60

100

2.5:1 10:1 40:1E:T

2.5:1 10:1 40:1

E:T


14

Fresh NK Continue to Expand During Shipping

Time post formulation (h) 0 24 48 0 24 48

Sample Manual Count Manual Count Manual Count Viability Viability ViabilitySample Manual Count (M/ml)

Manual Count (M/ml)/

Recovery (%)

Manual Count (M/ml)/

Recovery (%)

Viability Trypan/7AAD (%)



Donor 1 10 12.1/121% 13.7/137% NA/85.8 81/97.8 87.8/98.3Donor 2 10 15.0/150% 9.7/97% NA/80.8 85/98.9 75.8/97.9Donor 3 10 18.8/188% 17.1/171% NA/86.9 87/99.3 89.5/98.6

Time post

Fresh NK Purity and T cell ContentTime post

formulation (h) 0 24 48 0 24 48Sample CD3‐CD56+

(%)CD3‐CD56+

(%)CD3‐CD56+

(%)CD3+CD56‐

(%)CD3+CD56‐

(%)CD3+CD56‐

(%)Donor 1 71.1 68.9 70.7 19.7 16.7 15.9Donor 2 62.8 65 67.1 24.7 21.8 18.6Donor 3 86.2 85.5 87.3 8 7.8 6.1

500

600

Fresh NKFrozen NK

500

600

Fresh Auto-NKs Expand in vivo

100

200

300

400

500

NK

/µL

100

200

300

400

500

0‐9 0 1 3 5 7 9 14 28

Day of Protocol

0‐9 0 1 3 5 7 10 14

Day of Protocol


15

Fresh Allo-NKs Expand in vivoFresh NKFrozen NK

500

600

500

600

100

200

300

400

500

100

200

300

400

500

NK

/µL

0‐11 0 1 3 9 14 29

Day of Protocol

0‐34 0 1 3 5 7 9

Day of Protocol

Can we grow more potent NK cells?


16

Improved Potency of NKs Generated with High IL-2 Doses

0101002003004005001000

20

60

100

% O

PM

-2 ly

sis

20

60

100

% K

562

lysi

s

IL-2 (U/mL)

10:1 5:1 2.5:1 1.25:1 10:1 5:1 2.5:1 1.25:1

E:T E:T

400

er G

-Rex

)

CD56+CD3neg NKsCD3+CD56neg Ts

Comparable Expansion for 10 U/mL and 500 U/mL IL-2 Protocols

200

xpan

sion

100

s

10 500

200

Abs

# c

ells

pe

(x10

6

100

ble

10 500

100

Fold

of N

K E

x

10 500

20

60

% C

ell

IL-2 (U/mL)10 500 IL-2 (U/mL)

20

60

% V

iab


17

600

Fresh NK low IL-2 Fresh NK high IL-2

Expansion in vivo Auto-NKs

7000

200

300

400

500

NK

/l

2000

3000

4000

5000

6000

0

100

-9 0 1 3 5 7 9 14 28

Days of protocol

0

1000

0 1 3 5 7 9 14

Days of protocol

Conclusionso NK cells efficiently expand in G-Rexes within 10 days

w/o manipulations Fold expansion is donor dependent (25 to 160-fold)

o Shipment fresh NK cells: Retain viability and potency after 48h in 5% HSA

at RT and frozen ice-packs Allow for higher infused NK dose Expand further after infusion in vivo Expand further after infusion in vivo

o Potency in vitro is improved with higher IL-2 during manufacturing


18

Acknowledgement

Cliona Rooney, Ph.D.Jian Fang, M.S.

Cindy Gu& M b f D R ’ L b

CAGT CAGT/CTL groupOumar Diouf

Huimin ZhangWeili Liu& Members of Dr Rooney’s Lab

Mei Zhuyong, M.D. and VPF group

Juan Vera, M.D.Antonio DiStasi, M.D.

April Durett & Flow Cytometry group members

Frits van Rhee, M.D.Susann SzmaniaBijay Nair, M.D.

J h E t i D S

UAMS

Weili LiuJoyce Ku

Pallavi Mohapatra

& Flow Cytometry group members

Debbie Lyon& QC group members

Adrian Gee, Ph.D.Helen Heslop, M.D.

Malcolm Brenner, M.D., Ph.D.

Joshua Epstein, D.Sc.

Funding: PACT, NHLBI

Documents

NK Cells for Therapy - PACT GROUPpactgroup.net/system/files/cagt_wkshp_lapteva_12.pdfNK Expansion in Bags and G-Rexes in 40 197-mL bags Day 10 Bags Day 0 harvest 2x106 NK no processing