CAR treated. 46. CD19- CD22+ CD127+ B220+ BP1- CD93-. CD43+ KIT- Gr1- CD11b-. +. 44-6. CAR treated. 47. CD19- CD22+ CD127dim B220+ BP1+ CD93+.
Supplementary figure 1 a 262144
10
10
10
4
3
10
10
5
CD38% PerCP% Cy55
CD19% PC7
SSC)A
131072
5
CD58% APC
10
196608
4
3
65536
10
2
10
2
10
10
10
10
5
4
3
2
0
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2
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3
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4
CD10% PE
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5
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2
10
3
10
4
CD45% AH7
10
5
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2
10
3
10
4
CD19% PC7
10
5
10
2
10
3
10
4
CD10% PE
10
5
b 262144
5
5
5
10
CD38% PerCP% Cy55
10
131072
CD58% APC
SSC)A
CD19% PC7
196608
4
10
3
10
4
10
3
10
65536
2
2
10
10 2
10 3
10 4
CD10% PE
10 5
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2
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3
10
4
CD45% AH7
10
4
10
3
10
2
10
10
0
10
5
10
2
10
3
10
4
CD19% PC7
10
5
10
2
10
3
10
4
CD10% PE
10
5
Supplementary Figure 1: Flow cytometry plots of bone marrow from patient ALL_H0082 pre-CAR (a) and post CAR (b). panels are gated on CD10+ SSC leukemic blasts.
1
Day 90 10
5
0.607
: CD45-1
12
10
4
9
10 3
CD45.1
10
6
2
8.92
91.1
3
0 64.2 0
10 2
CD45.2
10 3 10 4 : CD45-2
10 5
0 0
CAR
10 2
10 3 10 4 : CAR
10 5
Supplementary Figure 2: C57Bl/6 mice injected with E2a:PBX leukemia and treated with mCD19 CAR were sacrifriced on dy +90. Splenocytes were harvested and stained for CD45 isoforms, T cell markers and protein L for CAR T cell detection, and analyzed by flow cytometry. The histogram on the right represents adoptively transferred T cells gated on CD45.1 isoform.
2
mCD19
E2a:PBX Em-RET 289 68-32 68-12 30-4 44-6 59-61 25-2 24-6
Sample / Mouse ID
Primary
DNA exon 1-3 mRNA exon 1-3 500bp
Early
mRNA Dex2 233bp
Late
DNA exon 3-4 mRNA exon 3-4 428bp
CD19
mRNA exon 4-7 317bp
Supplementary Figure 3: RNA was extracted from parent E2a:PBX and Eu-RET cell lines, untreated splenocytes from E2a:PBX leukemic mice, and post-CD19-CAR CD19relapses. Left panel: CD19 exon specific primers were used in a PCR reaction and run on a gel. Locations of specific DNA and RNA sites are marked, and were confirmed by band sequencing. Right panel: screen-shot from RNA-sequencing directed at the CD19 transcript.
3
Supplementary figure 4
Log Fold Change ∆∆CT
6 5
Nalm6 Pre-CAR sample Post-CAR sample Molm13
4 3 2 1 0 -1
CD19
PAX5
EBF1
Supplementary Figure 4: mRNA expression of CD19, PAX5 and EBF1 by real-time RTPCR expression of NALM6 (pre-B ALL cell line, black bars), Molm13 (AML cell line, grey bars), and leukemic cells from patient ALL_H0140 (figure 1g-h) pre CD19CAR (blue bars) and post CD19 CAR (red bars).
4
Supplementary Figure 5: Unsupervised hierarchical clustering on primary and postCD19 CAR relapses of E2a:PBX murine leukemia, clustering 2 post-CAR sapmles with CD19- B-ALL (marked in red) with the primary/ untreated samples , and separate from the lineage switch samples (marked in blue).
5
CD19 CAR
50
Mock T-cells
100
200
0
300
Days
f
c
g
SSC
CD19
b
CD45.2
CD22
250K 10
5
10
4
80.3
200
CD22
250K
Mock
10 3 100K
10
5
10
4
10
3
10
10
50K
9.38 0
10
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250K 10
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49.4
100K
0
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50.4
150K
2
0
300
200K
150K
50K
100
CD45.2
9.63
200K
Mock
0
SSC
0
CD19 CAR Mock T-cells
50
0 Days
Eµ-RET 289
100
CD19
Percent survival
e
Eµ-RET 309
100
Percent survival
a
10
10
3
10
4
0.0137
10
2
0
0.681 2
0.166
0
5
0
0.0137
10
2
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3
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10
5
250K
0
10 5
10
5.2e-3 2
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3
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4
0.499
10
5
0.18
200K
CAR
CAR
200K
150K
100K
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150K
100K 2
10
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0
0
3.74 10
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10
97.1
0
5
0
50-31
289 Em-RET
Marrow
E2a:PBX
Spleen
2
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3
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4
10
5
0
2.24 10
2
10
3
10
4
10
5
h
mCD19 ex2-in2
309 Em-RET
E2a:PBX
289 Em-RET
Marrow
Spleen
Em-RET Trangene
309 Em-RET
d
10
CAR T cells
96.2 0
10 2
50K
0 0
50-21
10
50K
Supplementary Figure 6: Eμ-RET leukemia does not relapse following CD19 CAR and does not develop a lineage switch. (A-D) experiments performed on 309-EμRET cell line. (a) survival curve of Balb/c mice treated as in figure 1a, with syngeneic 3x105 CD19 CAR T cells (n=5 mice per group). (b) Phenotype of a spleen of a mouse that died following CAR treatment, demonstrating sclerosis. Size bar is 1cm. (c) flow cytometry plots of mock treated (top) or CAR treated (bottom) Balb/C mice bearing 309-Eμ-RET leukemia. (d) PCR on genomic DNA for the Eμ-RET transgene was performed in tissues of the mouse found to die following CD19 CAR treatment. Controls include parent 309 and 289 Eμ-RET leukemia as positive controls, as well as E2a:PBX as a negative control. (e-g) similar graphs as in (a-c) on 289-Eu-RET ALL, 6
demonstrating late mortality in 1 of 5 mice following CD19 CAR, same phenotype of post-CAR mice with sclerotic spleen and absence of CD19+ cells or of a leukemic population. (h) PCR for the CD19 CAR was done in genomic DNA from mice dying late post CAR following 289 (50-21) or 309 (50-31) Eμ-RET leukemia. DNA extracted from CAR T cells used a control.
7
100
% of Max
80
60
40
20
0
100
100
80
80
% of Max
% of Max
0
60
40
20
20
0
10 3
10 4
10 5
60
40
CD19
10 2
0 0
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2
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3
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4
10
5
CD11b 0
10
2
10
3
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4
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5
100
% of Max
80
Single Clones_2_G4_003.fcs e2a clones 6-22_Single_cell_G4.fcs Singe cell E2a:PBX, G4, Day 7 Sample Singe cell E2a:PBX, G4, Day 1
60
40
20
0 0
10
2
10
3
10
4
10
5
KIT100
% of Max
80
60
Supplementary Figure 7: Flow cytometry plots showing single cell cloning of E2a:PBX which resulted in a clone with high cKIT expression (red), with absence of cKIT positive cells following 1 week in culture (grey line). 40
20
0
0
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8
70.7 93 %
2x107 cells
Day 5 Post-sort 0
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0 10
CD19
2x107 cells High sensitivity CD19 depletion
Culture
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1.26
98.7
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0.0105 0
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CD11b
10 3
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CD19
1 colony growth
2
B220
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B220
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GR1
0.119
0.0296
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KIT
0.0455
6.5e-3
10 3
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99.5 0
0.361 10
2
CD11b
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CD9
Bulk Culture
Pre-sort Post-sort
GR1
CD19 depletion
2
0
99.5 0
0.481 10
2
10
3
10
4
10
5
KIT
Supplementary Figure 8: Lack of myeloid cells upon antibody-based CD19 depletion of E2a:PBX cell lines. Upper panel: 20x107 E2a:PBX cells were sorted using CD19 labeled magnetic beads in Miltenyi AutoMACS system, with the negative fraction kept in culture. Flow cytometry on pre-sorted cells (grey), immediate post-sort negative fraction (blue) and day 5 in culture following sort (green). Bottom panel: We conducted this experiment using a high sensitivity LD column for sorting, and single cell cloned immediately. Flow cytometry plots of the single colony that grew shown.
9
Murine CD19- B-ALL post CD19 CAR relapse
5
10
4
10
3
10
2
2.41e-3
0.138
10
5
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3
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2
0
Off-pressure
4.44e-3
0.204 : CD19
10
: CD19
CD19
: CD19
Off-pressure
10
5
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4
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3
10
2
0
3.81
CD22
103 104 : CD22
0.0484
0
96.1
0 102
7.63e-3
0.733
105
99.1
0 102
103 104 : CD22
2.58
105
97.4
0 102
103 104 : CD22
105
Human CD19- B-ALL post CD19 CAR relapse 2nd passage, NSG mice
4
10
3
10
2
0
0
10
96.1 100K
50K
0 0
10 2
10 3
CD19
SSC-A
150K
10 4
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2
0.0134
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3
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0
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5
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0.0124
66.2
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10 30
10 2 10 4
3
10 0
10 5
92.5 10 2
2
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10 10
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10 310 5
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6.63e-3
0.159
0.524 10
4
6.63e-3
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99.3 2
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: CD22
0.159
0
0.524 0
10 2
: CD22 : CD45
CD22
4
hCD45
0
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92.5 10
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0
: CD22 : CD45
66.2
7.52 10 5
: CD45
10
0.0124
10 5
: mCD45
: CD19
10 5
2
: CD45
hCD45 200K
5
7.52
0
250K
10
0
: CD19
SSC
50K
10
0.0134
: CD19
96.1 100K
5
: mCD45
150K
10
CD19
SSC-A
200K
mCD45
: CD19
250K
99.3 10 3
10 4
10 5
: CD22
CD22
Supplementary Figure 9: Stability of CD19- B-ALL in murine and patient samples upon re-passaging off immune-pressure. Upper panel: Murine post-CAR sample 30-4 was passaged twice into mice off immunopressure, causing stable CD19- B-ALL in recipients. Lower panel: Patient sample with CD19- B –ALL following CD19 CAR was passaged twice in NSG mice. Flow cytometry analysis shown.
10
Supplementary figure 10
Percent survival
100
CD19+ CD19+ CAR
75
CD19ko CD19ko CAR
50 25 0
0
10
20
30
40
50
60
Days
Supplementary Figure 10: Survival curve of E2a:PBX ALL injected mice untreated (cyan) or following CD19-CAR (blue); and of CD19ko-E2a:PBX (generated using the CRISPR/CAS9 system) untreated (orange) or CD19-CAR treated (red).
11
Supplementary Table 1: Primary E2a:PBX cell lines and mouse relapse IDs Sample
Description
E2a:PBX
Primary bulk cell line
Days to relapse NA
E2a:PBX B3
Single cell clone
NA
E2a:PBX D3
Single cell clone
NA
E2a:PBX G4
Single cell clone
NA
38-1
Mock treated in vivo
18
68-32
24
78-51
Single cell clone in vivo, untreated Single cell clone in vivo, untreated Cy/ARA-C treated
78-53
Cy/ARA-C treated
28
78-54
Cy/ARA-C treated
31
78-55
Cy/ARA-C treated
41
30-4
CAR treated
46
44-6
CAR treated
47
59-61
CAR treated
58
25-2
CAR treated
76
43-1
CAR treated
76
A001
CAR treated
90
24-6
CAR treated
110
30-42
CAR treated
121
32-2
CAR treated
160
25-8
CAR treated
210
48-21
CAR treated
225
48-22
CAR treated
232
21-1
CAR treated
268
47-24
21-1, in vivo passage
NA
53-41
44-6, in vivo passage
NA
53-42
44-6, in vivo passage
NA
68-12
25 20
Immunophenotype CD19+ CD22+ CD127+ B220+ BP1dim CD93+ CD43+ KIT- CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220+ BP1dim CD93+ CD43+ KIT- CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220dim BP1+ CD93+ CD43+ KIT- CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220+ BP1dim CD93+ CD43+ KIT+ CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220+ BP1dim CD93+ CD43+ KITdim CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220+ BP1dim CD93+ CD43+ KIT- CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220+ BP1dim CD93+ CD43+ KIT- CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220+ BP1+ CD93+ CD43+ KITdim CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220+ BP1dim CD93+ CD43+ KIT- CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220+ BP1+ CD93+ CD43+ KITdim CD9- Gr1- CD11bCD19+ CD22+ CD127+ B220+ BP1+ CD93+ CD43+ KITdim CD9- Gr1- CD11bCD19- CD22+ CD127+ B220+ BP1- CD93CD43+ KIT- Gr1- CD11bCD19- CD22+ CD127dim B220+ BP1+ CD93+ CD43+ KIT- CD9- Gr1- CD11bCD19- CD22+ CD127- B220+ BP1- CD93dim CD43+ KIT- CD9+ Gr1+ CD11b+ CD19- CD22+ CD127dim B220dim BP1+ CD93+ CD43+ KIT- CD9dim Gr1- CD11b+ CD19- CD22+ CD127+ B220- BP1dim CD93+ CD43+ KITdim CD9+ Gr1+ CD11b+ CD19- CD22- CD127- B220- BP1dim CD93dim CD43+ KITdim CD9+ Gr1- CD11bCD19- CD22- CD127- B220- BP1- CD93+ CD43+ KITdim CD9dim Gr1dim CD11b+ CD19- CD22- CD127- B220- BP1- CD93CD43+ KIT- CD9+ Gr1- CD11bCD19- CD22- CD127- B220- BP1- CD93dim CD43+ KITdim CD9+ Gr1+ CD11b+ CD19- CD22- CD127dim B220- BP1- CD93+ CD43+ KIT+ Gr1- CD11bCD19- CD22- CD127- B220- BP1- CD93dim CD43+ KIT- CD9- Gr1- CD11bCD19- CD22- CD127- B220- BP1- CD93dim CD43+ KIT+ CD9- Gr1+ CD11bCD19- CD22- CD127- B220- BP1- CD93CD43+ KIT- Gr1+ CD11b+ CD19- CD22- CD127- B220- BP1- CD93CD43+ KIT- Gr1+ CD11b+ CD19- CD22+ CD127dim B220+ BP1+ CD93+ CD43+ KIT- CD9- Gr1- CD11bCD19- CD22+ CD127dim B220+ BP1+ CD93+ CD43+ KIT- CD9- Gr1- CD11b-
12
E2a:PBX1 PCR + + + + + + + + + + + + + + + + + + + + + + + +
Supplementary Table 2: Primers Name
Forward (5’3’)
Reverse (5’3’)
E2a:PBX transgene Eu-RET transgene mCD19 ex2-in2 mCD19 ex1-3 mCD19 ex3-4 mCD19 ex4-7 mCD19 ex4-6 mPAX5 mEBF1 hCD19 ex1-3 hCD19 ex3-4 hCD19 hPAX5 hEBF1 mCD19_CRISPR mEBF1_CRISPR mPAX5_CRISPR
GCACAACCACGCGGCCC CATTGGGGACACATTGAGCAG GGAGAGCACCCGGTCAGAGAG CCCAGCATCCTCTGCGCAG CAGGGACCTGGACTGTGACC GGGTCTCTTCTGCTTCTGCCC GGCACCTATTATTGTCTCCG GCATAGTGTCTACAGGCTCCG CGAGATCATGTGCAGCCGC CACCCCCATGGAAGTCAGGC GGAGGGAGAGCCTCCGTG GCACTGGCTGCTGAGGACT GGGAGATCAGGGACCGGC TGCCGAGTCTTGCTCACAC CACCGTGACGTCTGAAGCATTCCAC CACCGGATTGAGAGGACGGCCTTTG CACCGGGGCAAGTTCCACTATCCTT
CCACGCCTTCCGCTAACAGC TAGCAGTGGATGCAGAAGGCAGAC CCTTGTCTGTCTGTCTGTCTGTCTGTC GAAGTGGACCTGTGGCTCCC CCTTCACGTGCCTCTCGATGG CCGTACTGGTTCTGGGTCCCG GGGTCAGTCATTCGCTTC GATGGGTTCCGTGGTGGT CCTGGAATCTCCGCATGTCACG GGTGAGGTCCTGGCTGAGG GGGCAACAACAGACCCGTCTC CTTTTCCTCCTCAGGACCAGGG GCTGTGACTGGAAGCTGGGAC CATTGACTGTCGTAGACACCAC AAACGTGGAATGCTTCAGACGTCAC AAACCAAAGGCCGTCCTCTCAATCC AAACAAGGATAGTGGAACTTGCCCC
13
