RICHARD CHAMPLIN, MD

Houston, USA

• Professor and Chair of the Department of Stem Cell Transplantation and Cellular Therapy at the University of Texas MD Anderson Cancer Center

• Dr. Richard Champlin has over 30 years of experience in clinical and translational research involving hematopoietic stem cell transplantation. He has been the President of the American Society of Blood and Marrow Transplantation as well as the Center for International Center for Blood and Marrow Transplantation Research. He was also the board Member of the National Marrow Donor Program and is a current member of the HRSA Advisory Committee for Cord and Stem Cell Transplantation at the MD Anderson Cancer Center. Professor Champlin chairs the Working Committee on Alternative Donors and Cell Sources of the International Bone Marrow Transplant Registry.

Cell Therapy for AML

Richard Champlin, MD

University of Texas- MD Anderson Cancer Center

AML- Sensitivity to immunotherapy

• Allogeneic hematopoietic transplantation is curative through the immune graft-vs-leukemia effect

– T-cells

– NK cells

• Major cause of treatment failure is relapse

• Can we develop more effective cellular immune therapy with T- and NK- cells

Problems

• Cancer cells are poorly immunogenic

• Cancer cells induce hyporesponsiveness/tolerance

• Allogeneic transplantation can provide nontolerized effector cells reactive against alloantigens as well as leukemia related targets.

Targets for Graft-vs.-Malignancy

Broadly expressed minor

histocompatibility antigen (GVHD)

Lineage restricted

minor histocompatibility

antigen (G-vs-hematopoietic),

or Redirected CAR T-cells vs CD19

Overexpressed normal

cellular constituent

(Proteinase 3, WT1,

telomerase)

Allo-Specific Malignancy SpecificIdiotype, Fusion peptide of

translocation (bcr-abl)

Chimeric antigen receptors (CARs)

vL

vH

CH1

CL

Antibody

Fab

vH vL

Chimeric antigen receptor

a b

TCR-complex

g e e dz z

(Eshhar et al; PNAS 1993)

Lessons from Anti CD19 CAR T-cells

• Effective against B-cell malignancies, ALL, lymphoma, CLL

• Responders also eradicate normal B-cells

• Patients can survive without B cells - IVIG

• Cross reactivity and destruction of normal hematopoiesis is a major problem with CARs against AML– May ablate cells with suicide gene

– Can give prior to hematopoietic transplantation

Targeting AML with CAR- T cells

• Myeloid cell surface antigens

– CD123 (IL-3 receptor)

– CD33 (may be absent on leukemia stem cells)

• Leukemia related antigens

– WT1

– PR1 (derived from Proteinase 3)

Targeting CD123 (IL-3Ra)

a subunit of IL-3 receptor required for cell

proliferation, differentiation, and survival

Expression

Over expressed on leukemic stem cells (AML, B-

ALL, CML) and leukemic blasts

Weak expression on monocytes, neutrophils,

basophils, and megakaryocytes

Absent on peripheral T cells, natural killer cells,

platelets, red blood cells 10

Chimeric Antigen Receptors

Cooper et al.

Major issue is separation of cytotoxicity AML , sparing normal myeloid cells.Possible Targets CD123, CD33

Numeric expansion of CAR+ T cells onCD123+ aAPC with exogenous IL-2/21

Antigen-specific activation & proliferation of CAR+ T cells

CD28 or CD137

Inducible Caspase 9 Self Destruct Switch

12

CD123-CD28CAR CD123-41BBCAR

Conditional ablation of CAR+ T cells by chemical-induced dimerization of iCaspase9

Redirected killing of CD123+ AML cells by CAR+ T cells

CD123-CD28 CAR CD123-41BB CAR

CD123-41BB CARUntreated CD123-CD28 CAR

In vivo killing of AML tumor cells by CAR+ T cells

Conclusions

CD123-specific CAR+ T cells co-expressing iCaspase9 can be generated using approach suitable for human application

T cells showed anti-tumor efficacy in vitro and in vivo

iCAR+ T cells conditionally ablated upon addition of dimerizer

(1) 8F4: A T cell receptor-like antibody

PR1P3

NE

PR1-CTL

TCR

PR1

P3 - Proteinase 3

NE - Neutrophil elastase

Lysis

AML, CML, & MDS

Anti-PR1/HLA-A2 monoclonal antibody (8F4)

Molldrem et al Blood 2011

17

(2) 8F4-CAR gene modified T cells

CD28

CD3z

sch8F4

8F4-CAR+T cell

PR1P3

NE

PR1-CTL

TCR

AML, CML, & MDS

PR1

P3 - Proteinase 3

NE - Neutrophil elastase

Lysis

8F4 (anti-PR1/HLA-A2) CAR

PR

1/H

LA-A

2 t

etra

mer

CD3

96.6%

18

Anti PR1-HLA A2 CAR T-cell

• Targets PR1 HLA A2 complex

• Should spare normal hematopoiesis

• If myelosuppressive, could use in haploidentical transplants from HLA A2 negative donor

• Limitation- only targets disease in HLA A2 positive recipients, but these cells can be produced against a panel of HLA antigens

20

Alloreactive NK Cells and Haploidentical

Transplantation

Reduce GVHD

Reduce rejection

Reduce relapse

Manufacturing mbIL21-expanded NK cells

= 5 x 109

NK cells/kg

Cryopreserve in aliquots

Amaxa Nucleofector

scFv

Hinge

Stalk

TM

T-cell

signaling

Transposase Transposon

CARSB11

K562 aAPC

K562 aAPC

Master Cell Bank/Working Cell Bank

Antigen-specific proliferation ofCAR +Tcells

Cryopreservation

Infusion

T-75cm2 flasks

Cell Culture Bags

Cell Culture Bags

Apheresis

Product

PBMC separation

Biosafe Sepax PBMC

Volume Reduction

Biosafe Sepax

Numeric expansion of CAR+ Tcells

with integrated transposon on

g-irradiated K562-aAPC

IL-2 (50U/mL)

IL-21 (50U/mL)

Wave Bioreactor

Wave Bioreactor

0 7 14 21 28 35

100

101

102

103

104

105

106

107

108

WAVE GMP Validation

Lab data, n=19

Days

Fo

ld E

xp

an

sio

n

Lee et al Plos One 2012 21

mbIL21-expanded NK cells have increased

cytotoxicity, telomeres, and cytokine secretion

PLoSOne, Denman 2012 7:e30264

1

10

100

1000

10000

IFNg TNF IL-2 IL-6IFNg TNF IL-2 IL-6

IFN Fresh

mbIL15

mbIL21

Su

pe

rna

tan

t [p

g/m

l]

Lysis of 721.221 targetsby fresh vs. expanded NK cells

10:1 5:

12.

5:1

1.25

:1

0.62

5:1

0.31

25:1

0

20

40

60

80

100

Fresh NK cells

mbIL15-expanded

mbIL21-expanded

E:T Ratio

Pe

rce

nt S

pe

cific

Lysis

Clone

4 (m

bIL1

5)

Clone

9.m

bIL2

1-20

-10

0

10

20 ***

NK Cell Clinical Trials

• We have initiated first in man phase I/II studies of ex vivo expanded NK cells to augment GVL effects of allogeneic HSCT

– Developed GMP procedure, completed validation, FDA IND

– 2012-0819 tests addition of NK cells to HLA matched transplants 8 patients enrolled (Champlin, Lee)

– 2012-0708 tests addition of NK cells to haploidentical transplants 3 patients enrolled (Ciurea, Champlin)

– 2011-0943 NK cells with cord blood transplants- 4 patients enrolled (Hosing, Shpall)

• All studies open in dose escalation phase. No NK cell related toxicities. Low rate GVHD. Too early to assess antileukemia effects.

23

NK-cell Expansion and CAR

transduction

Antigen-specific

CAR+ NK cells

Expand NK cells on

clone 9

CB-NK culture with

cytokine (IL-2 at 200

units/ml or IL-15 at

10ng/ml)

and clone 9

Transduction of NK

cells with retroviral

vector

Conclusions

• Cellular immunotherapy is a highly promising approach to treatment of AML

• Leukemia antigens have been identified• Challenge is to avoid damage to normal hematopoiesis;

may be overcome with suicide switch or use prior to HSCT

• Targeting PR1, aberantly expressed on AML, may spare normal hematopoiesis

• NK cells naturally target AML; NK CARs further increase cytotoxicity

• Clinical trials using CAR T-cells, other antigen specific T-cells and NK cells are in progress