CD123 Sex
UPN
11 F
Age
SampleType
Status
Diagnosis
Subtype
Pheresis
Blast Crisis AML
48 Pheresis
Relapsed AML
M4 [myelomonocytic]
55 M
90
39 Pheresis
Pheresis
100 M
91
1
2
3
Relapsed
Unknown
WBC Blast %
Cytogenetics_Abnormality_t
N/A
65.9 N/A
54-­‐55, xx, +6, +8, +8, +13, +14, +15, +19, +20, +22 [6]//
AML
AML
M2 without t(8;21) [with maturation]
94.5 N/A
M2 without t(8;21) [with maturation]
382.2 N/A
74 BM-­‐MNC
AML
M1 [without maturation]
80 F
90 F
29 BM-­‐MNC
37 Pheresis
Refractory AML
De Novo
AML
52-­‐53, xx, +4, +6, +8, +8, +11, +13, +15, +19 [15]
46, xy
46,XX[51]
SEE NOTE
46,XY[25]
4
5
6
36.9 95
Cytogenetics_SourceMaterial Positive_LTxResults
None Performed
t(4;11) [MLL-­‐AF4]+
BM
BM
BM
FLT3
NPM
ITD
ITD
CD13+ CD14 5% + CD15+ CD33+ CD64+
CD13+ CD15+ CD33+ CD34-­‐ HLA-­‐DR+
BM
5.3
47
None Performed
AML with inv(16)(p13;q22) or t(16;16)(p13;q22)
129.3 N/A
46,XX,inv(16)(p13.3q22)[4]/47,XX,idem+8[17]/FISH inv(16) POSITIVE 189/200 BM
(94.5%)
All_FlowResults_List
CD13+ CD15+ CD33+ CD34-­‐ CD64+ HLA-­‐DR+
CD13+ CD14-­‐ CD15+ CD33+ CD34-­‐ CD64-­‐ HLA-­‐DR+ TdT-­‐
CD13+ CD15-­‐ CD33+ CD34+ HLA-­‐DR+
CD13+ CD15-­‐ CD19-­‐ CD20-­‐ CD33+ CD34+ CD64+ CD79-­‐ HLA-­‐DR+ TdT-­‐
inv16 [CBFb-­‐MYH11]+
7
8
9
78 M
66 BM-­‐MNC
AML
M2 without t(8;21) [with maturation]
32.9
100 M
94 M
58 Pheresis
22 Pheresis
Relapsed
De Novo
AML
AML
AML-­‐MLD no prior MDS or MPN 58.8 N/A
M5a
137.1 N/A
66 47,XY,+21[8]/46,XY[16]
97 M
66 Pheresis
De Novo
AML
AML with inv(16)(p13;q22) or t(16;16)(p13;q22)
137.8 N/A
NO GROWTH
PB
96 F
38 Pheresis
De Novo
AML
M1 [without maturation]
71 M
57 Pheresis
De Novo
AML
M4 [myelomonocytic]
99 M
79 Pheresis
De Novo
AML
M1 [without maturation]
5.9 M
58 Pheresis
t-­‐AML
AML
AML with 11q23 abnormalities
58 M
70 Pheresis
t-­‐AML
AML
74
76
95
98
70
73
83
53
Pheresis
Pheresis
Pheresis
Pheresis
De Novo
De Novo
De Novo
De Novo
94 M
97 M
57 PB-­‐MNC
52 Pheresis
De Novo
99 F
56 M
59 Pheresis
53 Pheresis
Relapsed
Relapsed
85 F
43 Pheresis
46,XY[25]
46,XY[4]
BM
CD10-­‐ CD13 dim + CD14-­‐ CD15-­‐ CD19-­‐ CD20-­‐ CD33+ CD34 dim + CD64-­‐ CD79-­‐ HLA-­‐DR+ TdT-­‐
BM
PB
ITD
835
CD13 variable + CD14 subset + CD15+ CD33+ CD34 subset + CD64+ HLA-­‐DR+
10
inv16 [CBFb-­‐MYH11]+ Wildtype
CD10-­‐ CD13+ CD14-­‐ CD15-­‐ CD19-­‐ CD20-­‐ CD33+ CD34+ CD64-­‐ CD79-­‐ HLA-­‐DR+ TdT-­‐
11
63.4 N/A
46,XX[25]
BM
ITD
CD13+ CD15+ CD33+ CD34+ CD64+ HLA-­‐DR+ TdT-­‐
136.4 N/A
46,XY[2]
BM
ITD
CD33+ CD34-­‐ HLA-­‐DR+
71.3 N/A
46,XY[25]
BM
ITD
CD10-­‐ CD13 dim + CD14-­‐ CD15-­‐ CD19-­‐ CD33+ CD34-­‐ CD64-­‐ CD79-­‐ HLA-­‐DR-­‐ TdT-­‐
137.5 N/A
46,XY,t(11;22)(q23;q13)[25]
BM
Wildtype
CD10-­‐ CD13-­‐ CD14+ CD15+ CD19-­‐ CD20-­‐ CD33+ CD34-­‐ CD64+ CD79-­‐ HLA-­‐DR-­‐ TdT-­‐
AML with t(8;21)(q22;q22)
170.9 N/A
46,XY,t(8;21)(q22;q22),del(13)(q12q22)[16] FISH MLL negativein 200
PB
Wildtype
CD10-­‐ CD13+ CD14-­‐ CD15 var + CD19 dim + CD20-­‐ CD33+ CD34 dim + CD64 dim + CD79-­‐ HLA-­‐DR+ TdT-­‐
AML
AML
AML
AML
M1 [without maturation]
M5a
AML with 11q23 abnormalities
M4 [myelomonocytic]
96.2
129.0
115.0
139.1
46,XX[25]
46,XY,t(3;9)(p14;p21)[20]
45,XY,t(9;11)(p22;q23),der(13;14)(q10;q10)[20]
47,XX,+8[8]/46,XX[14]
BM
BM
PB
BM
ITD
Wildtype
ITD
CD10-­‐ CD13-­‐ CD14-­‐ CD15-­‐ CD19-­‐ CD20-­‐ CD33-­‐ CD34+ CD64-­‐ CD79-­‐ HLA-­‐DR+ TdT-­‐
CD13 variable + CD14 variable + CD15+ CD33+ CD34-­‐ CD64+ HLA-­‐DR+ TdT-­‐
CD10-­‐ CD13 variable + CD14 variable + CD15 variable + CD19-­‐ CD20-­‐ CD33+ CD34-­‐ CD64+ CD79-­‐ HLA-­‐DR-­‐ TdT-­‐
CD10-­‐ CD13+ CD14-­‐ CD15-­‐ CD19-­‐ CD20-­‐ CD33+ CD34+ CD64-­‐ CD79-­‐ HLA-­‐DR+ TdT-­‐
AML
AML
M5a
M4 [myelomonocytic]
42.9
77
113.0 N/A
46,XY,add(6)(p21),del(8)(p21),add(12)(q24.1)[13]/46,XY,del(1)(q32),del(7)(q BM
22q32),der(6;12)(q10;p10),add(22)(q?11.2),+mar[3]/45,XY,t(1;2)(p?22;q11.2),
-­‐21[1]/46,XY[3]
ITD
CD10-­‐ CD13-­‐ CD14-­‐ CD15 variable + CD19-­‐ CD20-­‐ CD33+ CD34-­‐ CD64+ CD79-­‐ HLA-­‐DR+ TdT-­‐
CD15 VAR + CD33+ CD64 VAR + HLA-­‐DR+
AML
AML
M4 [myelomonocytic]
NOS
287.8 N/A
131.7 N/A
AML
12
13
14
15
16
17
18
19
20
21
22
23
F
M
M
F
24
25
26
27
28
29
30
31
97
85
72
90
40
M
M
M
F
M
71
83
74
76
54
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
97
91
36
ND
77
79
80
96
61
23
81
75
97
98
96
84
F
M
M
19
69 PB
53 PB
PB
66 PB
67 PB
54 PB
52 PB
66 PB
59 PB
PB
62 PB
PB
49 PB
45 PB
27 PB
M
M
F
M
F
F
?
M
F
F
M
M
?
?
PB
PB
PB
PB
PB
PB
N/A
N/A
N/A
N/A
t(8;21) [AML1-­‐ETO]+
None Performed
46,XY,del(2)(p13p?23),t(4;13)(q31;q34),add(4)(q?25),del(6)(q13q25),t(9;22)( PB
q34;q11.2),del(10)(q24),add(16)(q24)[20]
ITD
CD33+ CD34+ CD64 dim subset + HLA-­‐DR+
CD13 variable + CD34 bright +
NOS
86.4 N/A
47,XX,+8[13]
BM
ITD
Wildtype
CD10-­‐ CD13+ CD14-­‐ CD15-­‐ CD19-­‐ CD20-­‐ CD33+ CD34 var + CD56-­‐ CD64-­‐ CD79-­‐ HLA-­‐DR+ TdT-­‐
AML
RELAPSE
AML
de novo
AML
de novo
transformedAML
AML
de novo
AML
NOS
AML-­‐MLD
MDS/MPN
AML from MDS
M4
10.1
69.7
28.2
10.2
51.9
46,XY[20]
47,XY,+8,del(20)(q11.2)[13]/47,idem,?del(12)(p12)[7]
47,XY,+8,del(20)(q11.2)[19]/45,XY[1]
NA
46,XY
BM
BM
BM
NA
BM
negative
negative
negative
NA
negative
WT
ITD
WT
WT
WT
NA
WT
de novo
AML
RELAPSE (post allo)
AML
de novo
AML
AML
de novo
AML
de novo
AML
de novo
AML
RELAPSE (post allo)
AML
de novo
AML
de novo
AML
?
AML
de novo
AML
de novo
AML
de novo
AML
RELAPSE
AML
de novo
M4 (inv 16)
AML-­‐MLD
MDS/MPN (myeloid sarcoma)
BM
BM
BM
CBFB-­‐MYH11
NA
negative
ITD
ITD
WT
WT
NA
WT
CD4+ CD13(Var) CD15(var) CD33+ CD64(small) CD117(var) HLADR+ MPO+
CD4+ CD11b(subset) CD15 (hetero) CD33 (hetero) CD64+ HLADR+ MPO (hetero)
CD34+ CD13(dim) CD33 (var) HLADR+
CD13/CD33/CD34/HLADR/MPO (subset var)
CD4+ CD11b+ CD13+ CD15+ CD33+ CD64+ HLA-­‐DR+ MPO+with subset expression of CD14, CD16, CD34 and CD117.
CD2(var)+ CD4(var)+ CD7(var)+ CD13+ CD15(var)+CD33(var)+ CD34+ CD117+ HLA-­‐DR+ MPO+ MYELOBLASTS and ATYPICAL CD2(var)+ CD4+ CD11b(var)+ CD13+ CD14(var)+CD15(var)+ CD33+ CD34(var)+ CD64+ HLA-­‐DR+ MPO+ MONOCYTES
PB
BM
BM
BM
BM
BM
?
BM
NA
BM
BM
BM
negative
negative
WT
ITD
WT
WT
WT
WT
?
ITD, D835
NA
ITD
WT
wt
NA
POS
CD34+, CD117+, HLA-­‐DR+, MPO (variable dim)+, TDT-­‐ CD11b-­‐, CD13-­‐, CD33-­‐, CD56-­‐, CD64-­‐
CD11b+ CD33+ CD34-­‐ CD64+ CD117-­‐ HLADR+ TdT-­‐ CD4/CD13/CD14/CD25/MPO(var)
WT
WT
WT
?
POS
NA
POS
BLASTS WHICH ARE CD34(bright)+ MPO+ CD25(major subset)+ CD15(small subset)+HLA-­‐DR(small subset)+ AND VARIABLY POSITIVE FOR CD4, CD13, CD33, AND CD117
CD2(dim) CD4(dim het) CD13+ CD15 (hetero) CD33+ CD34+ CD117+ HLADR+ MPO+
CD7+ CD34+ HLADR+ CD13(dim/var) CD33 (dim/var) CD117 (dim/var)
?
CD4(heterogeneous)+ CD33(heterogeneous)+ CD56(heterogeneous)+ CD64(heterogeneous)+ MPO(heterogeneous)+
NA
see comment
CD13, CD33, CD34, CD117, HLA-­‐DR, cMPO, CD71, CD19(dim/partial), cTdT (dim/partial) and cCD79a(dim/partial)
CD33+ HLADR+ MPO/CD4/CD11b/CD13/CD14/CD15/CD34/CD64/CD117 (var)
CD123 defined by flow cytometry using FMO controls
NOS
M5
M0/M1
AML M1 M$ (inv 16)
CML-­‐BC
?
M5
?M4
M5
NA
M4 (inv 16)
60
83%
25
56
65%
28.5
17.5
131.2
55 46,XX,inv(16)(p13.2q22)[3]/46,idem,del(7)(q32)[15]/47,idem,+22[2]
55 46,XY
10 46,XY,del(7)(p11.2)[2]/46,XY[18]
79.3
31.3
8.5
61.8
20.7
70.2
?
80
91%
84
88
47
20
?
204.4
60.0 ?
79.5
8.3
16.6
95
67
84
42
46,XY
46,XY
45,XX,-­‐7[3]/46,XX[4]
46 XY
46,XX,inv(16)(p13.1q22)[20]
46,XX,t(9;22)(q34;q11.2)[20]
?
46,XY
NA
46,XX[20]
20q (normal CG at presentation)
46,XY,del(7)(q32),inv(16)(p13.3q22)[2]/47,idem,+22[14]/46,XY[7]
negative
CBFB-­‐MYH11
BCRABL b3a2
?
negative
NA
CBFB-­‐MYH11
NA
WT
wt
Supplementary Figure 1. CD123 expression among CD34/CD38 subsets
within leukemia blasts.
Supplementary Figure 2. CAR123 constructs.
32716 scFv
72
VL
VH
CD8αH
4-1BB
TCR-ζ
73
VH
VL
CD8αH
4-1BB
TCR-ζ
74
VL
VH
IgG4H
4-1BB
TCR-ζ
75
VH
VL
IgG4H
4-1BB
TCR-ζ
26292 scFv
76
VL
VH
CD8αH
4-1BB
TCR-ζ
77
VH
VL
CD8αH
4-1BB
TCR-ζ
78
VL
VH
IgG4H
4-1BB
TCR-ζ
79
VH
VL
IgG4H
4-1BB
TCR-ζ
Supplementary Figure 3. Preclinical testing of the optimal CAR123 construct.
A. Gating
CD3
Unstimulated T cells
CAR
CAR (goat anti-mouse Fab)
T cell gate
CD3
none
T cells + MOLM14
CAR
SSC
FSC
CD107a / CAR FMO
none
T cell gate
none
Morphology
CD107a
CD107a
Supplementary Figure 3. Preclinical testing of the optimal CAR123 construct.
B. Degranulation
All comparisons are made with
construct 72 as control,
ANOVA, Dunnett’s multiple
comparisons post-test.
C. Cytokine production
Supplementary Figure 4. T cell proliferation upon non-specific or specific
stimulation. CFSE-labeled UTD, CART19 or CART123 were left unstimulated (red),
or stimulated with the indicated conditions (blue) for 96 hours. Gating strategy is
shown for a representative sample of CART123 cells.
Supplementary Figure 5. Flow cytometric analysis of CART123-treated
MOLM14-engrafted xenograft mice.
Supplementary Figure 6A. CD123 expression among the primary human
AML specimens xenografted in mice.
UPN024
UPN034
CD123
UPN022
CD45
Blood from a representative mouse injected with AML UPN022, followed by T
cells, which demonstrates that T cells are CD45bright pos CD3+ cells and that
blasts are CD45pos CD123+ cells.
Supplementary Figure 6B. Dim CD123 expression on primary human AML
specimen UPN034.
UPN034
Supplementary Figure 7A. Reduction in marrow AML burden at time of
death in mice treated with CART123.
Marrow
Supplementary Figure 7B. Long-term survivors after primary AML have
persistent T cells and no residual AML in marrow.
Mouse #1 marrow
Mouse #2 marrow
Mouse #3 marrow
Supplementary Figure 8.
Gating strategy
Supplementary Figure 9A.
Gating strategy for normal
bone marrow.
Supplementary Figure 9B. CD123 gating on normal bone marrow (NBM)
cells.
Supplementary Figure 9C. CD123 gating on bone marrow subsets.
CD19
CD19
Supplementary Figure 10. Expression of CD123 on normal peripheral blood
cells.
CD3
CD33
CD14
CD123
CD123
CD123
Supplementary Figure 11A. Gating strategy for analyses of mouse
peripheral blood.
Supplementary Figure 11B. Gating strategy for analyses of mouse bone
marrow.
Supplementary Figure 11C. Number of human cells per bone from humanized
mice treated as indicated and harvest at one month post-treatment.