Cellular Therapy in Acute Myeloid Leukemia. Prof. Richard Champlin

1. 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.

2. Cell Therapy for AML
Richard Champlin, MD
University of Texas- MD Anderson
Cancer Center

3. 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

5. 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.

6. 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 Specific
Idiotype, Fusion peptide of
translocation (bcr-abl)

7. Chimeric antigen receptors (CARs)
vL
vH
CH1
CL
Antibody
Fab
vH vL
Chimeric antigen receptor
a b
TCR-complex
ge e d
z z
(Eshhar et al; PNAS 1993)

8. 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

9. 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)

10. 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

11. 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 on
CD123+ aAPC with exogenous IL-2/21
Antigen-specific activation &
proliferation of CAR+ T cells
CD28 or CD137

12. Inducible Caspase 9 Self Destruct
Switch
12

13. CD123-CD28CAR CD123-41BBCAR
Conditional ablation of CAR+ T cells by chemical-
induced dimerization of iCaspase9

14. Redirected killing of CD123+ AML cells by
CAR+ T cells
CD123-CD28 CAR CD123-41BB CAR

15. CD123-41BB CARUntreated CD123-CD28 CAR
In vivo killing of AML tumor cells by
CAR+ T cells

16. 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

17. (1) 8F4: A T cell receptor-like antibody
PR1
P3
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

18. (2) 8F4-CAR gene modified T cells
CD28
CD3z
sch8F4
8F4-CAR+
T cell
PR1
P3
NE
PR1-
CTL
TCR
AML, CML, & MDS
PR1
P3 - Proteinase 3
NE - Neutrophil elastase
Lysis
8F4 (anti-PR1/HLA-A2) CAR
PR1/HLA-A2tetramer
CD3
96.6%
18

19. 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. 20
Alloreactive NK Cells and Haploidentical
Transplantation
Reduce GVHD
Reduce rejection
Reduce relapse

21. Manufacturing mbIL21-expanded NK cells
= 5 x 109
NK cells/kg
Cryopreserve in
aliquots
Antigen-specific proliferation of
CAR+Tcells
Cryopreservation
Infusion
Cell Culture Bags
Volume Reduction
Biosafe Sepax
ells
n
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
FoldExpansion
Lee et al Plos One 2012 21

22. 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
Supernatant[pg/ml]
Lysis of 721.221 targets
by fresh vs. expanded NK cells
10:1
5:1
2.5:1
1.25:1
0.625:1
0.3125:1
0
20
40
60
80
100
Fresh NK cells
mbIL15-expanded
mbIL21-expanded
E:T Ratio
PercentSpecificLysis
C
lone
4
(m
bIL15)
C
lone9.m
bIL21
-20
-10
0
10
20 ***

23. 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

24. 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

25. 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