Cell lines. EL4 thymoma cell line (ATCC), modified to express human CD19 (EL4hCD19+), was ... Murine ID8 ovarian tumor cells, modified to expresses.
Cell Reports, Volume 23
Supplemental Information
Engineered Tumor-Targeted T Cells Mediate Enhanced Anti-Tumor Efficacy Both Directly and through Activation of the Endogenous Immune System Mauro P. Avanzi, Oladapo Yeku, Xinghuo Li, Dinali P. Wijewarnasuriya, Dayenne G. van Leeuwen, Kenneth Cheung, Hyebin Park, Terence J. Purdon, Anthony F. Daniyan, Matthew H. Spitzer, and Renier J. Brentjens
Experimental Procedures Cell lines EL4 thymoma cell line (ATCC), modified to express human CD19 (EL4hCD19+), was maintained in RPMI 1640 medium (Gibco) supplemented with HEPES (N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid), and L-glutamine. Nalm6 (ATCC) tumor cells were maintained in RPMI 1640 (Invitrogen, Grand Island, NY, USA) supplemented with 0.1mM nonessential amino acids, 1mM sodium pyruvate, 5x10-2 mM 2ME (Invitrogen). NALM6 tumor cells were modified to express a GFP-firefly luciferase fusion protein (NALM6-GFP+/Luc+). Murine ID8 ovarian tumor cells, modified to expresses the retained domain of MUC16, were maintained in DMEM. Retroviral producer cell lines (293 Glv9) producing 1928z, 1928z-P2A-IL-18, 4H1128z or 4H1128z-P2A-IL-18 encoding virus were maintained in DMEM. Phoenix ecotropic packaging cells (ATCC) producing 19m28mz, 19m28mz-mIL18, 4H11m28mz, or 4H11m29mz-mIL18 CARs were maintained in DMEM. Murine T cells were maintained in RPMI 1640 medium with the addition of 60 IU/mL rhIL-2 (Novartis Pharmaceuticals). All media were supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin and streptomycin, and 2mM L-glutamine. All cells were routinely tested for mycoplasma contamination. Flow cytometry Flow cytometric analyses were performed using a Gallios Flow Cytometer with Kaluza software (Beckman Coulter, Brea, CA, USA). Flow sorting of CAR-positive T cells was done using a BD Biosciences (San Jose, CA, USA) FACSARIA IIu Special Order System with BD FACSDIVA software. NALM6 and EL4hCD19+ tumor cells were stained with mouse anti-human CD19 (Life Technologies). CD19-targeted CAR T cell expression was detected using Alexa Fluor 647-labeled Armenian hamster 12D11 antibody and Muc16ecto-targeted CAR T cell was detected with an Alexa Fluor 647–labeled anti4H11 idiotype antibody. B cell aplasia was detected by staining mouse cells with mouse anti-human CD19 (Life Technologies) and rat anti-mouse CD3 (Life Technologies). Mouse B cells were considered hCD19+ and mCD3-. Flow cytometry of blood cells was performed after red cell lysis with ACK lysis buffer (Lonza) and Ficoll mononuclear cell separation (GE Healthcare). In vitro T-cell dysfunction studies utilized mouse anti-human CD279 (PD1), mouse anti-human CD223 (LAG-3), and mouse antihuman TIM-3. Macrophages were analyzed with F4/80 and CD11b antibodies. Cells were stained with antibodies at 4°C for 20 minutes in all flow cytometry experiments. The antibodies identifications, catalog numbers and antibody concentration/test were the following: mouse anti-human CD19 (Life Technologies, MHCD1905, 0.1mg/ml); hamster anti-mouse CD3 (Life Technologies, HM3401, 0.1µg/test); mouse anti-human CD279 (BD Biosciences, 557860, 1.5µg/test); mouse anti-human CD223 (Ebioscience, 48-2239-42, 5µl/test); mouse anti-human TIM-3 (Ebioscience, 48-3109-42, 5µl/test); rat anti-mouse F4/80 (Ebioscience, 17-4801-82, 0.2µg/test); rat anti-mouse CD11b (Ebioscience, 12-011282, 0.2µg/test); rat anti-mouse NKp46 (Ebioscience, 46-3351-82, 0.2µg/test) Analysis of cytokine production For in vitro cytokine secretion experiments, murine CAR T cells were cocultured with either EL4hCD19+ or ID8 tumor cells and human CAR T cells were cocultured with NALM6 tumor cells at a 1:1 E:T ratio. Supernatant was collected after 24 hours of coculture and frozen at -80°C until analysis. Cytokine detection was performed using the MILLIPLEX MAP Human or Mouse Cytokine/Chemokine, Premixed 13 Plex kit, MILLIPLEX MAP Non-Human Primate Cytokine Interleukin 18, MILLIPLEX MAP Rat Cytokine Interleukin 18, and the Luminex IS100 system.
A 1928z-hIL18
80
100
50
0
60
1928z 1928z-hIL18
40 20
D 8
19
28
z-
C
hI
D 4
L1
28
8
z
0 19
CAR
ns ns
C
CAR (MFI)
B
ns
150
Percent Cell (CAR+)
1928z
1928z
1928z-hIL18
Percent Cell (CAR+)
CD62L CD45RA
1928z 1928z-hIL18
ns
100
D
19m28mz
19m28mz-mIL18
ns
80
ns
60
ns
40 20
) -
(T
em
) C D 45 R A
-
(T
cm
em (T
-
C D 45 R A
CAR
C
C
R
7
-
+
R C C
C
C
D
D
62
62
L
L
7
-
+
C D 45 R A
-
(T
1928z-hIL18
C D 45 R A
1928z
cm
)
0 )
C
19m28mz
CCR7
F
19m28mz-mIL18
80
ns
60
CD62L
19m28mz 19m28mz-mIL18 Percent Cell (CAR+)
100
ns
40 20
8 D
19m28mz
80 60
19m28mz-IL18 ns
40 20
C
C
D
4
0
CD4
ns
0
C
D
62
G
L+ C D 44 + C (T D cm 62 L) C D 44 + (T em )
19m28mz-mIL18 Percent Cell (CAR+)
19m28mz
CD8
E
CD44
CD5RA
Figure S1. Characterization of 1928z-hIL18 CAR T cells in vitro. Related to Figure 1. (A) Transduction efficiency of human 1928z and 1928z-hIL18 CAR T cells. (B) Percentages of CD4+ and CD8+ 1928z and 1928z-hIL18 CAR T cells. (C) Central (Tcm) and effector (Tem) memory characterization of 1928z and 1928z-hIL18 CAR T cells. (D) Transduction efficiency of mouse 19m28mz and 19m28mz-mIL18 CAR T cells. (E) Percentages of CD4+ and CD8+ murine 19m28mz and 19m28mz-mIL18 CAR T cells. (F) Central (Tcm) and effector (Tem) memory characterization of 19m28mz and 19m28mz-mIL18 CAR T cells. (G) T cell dysfunction markers analyzed by flow cytometry and compared between 1928z and 1928z-hIL18 CAR T cells on day 7 and day 14. Data presented as means ± SEM *P < 0.05. ns=non significant. Analyses were determined with Student’s t test.
A
B
Figure S2. 19m28mz-mIL18 CAR T cells display similar expansion and anti-tumor cytotoxicity in vitro. Related to Figure 2. (A) In vitro proliferation assay with 19m28mz and 19m28mz-mIL18 CAR T cells after stimulation with EL4hCD19 cells (E:T 1:1) on day 0 and day 7. (B) 4h 51Cr release cytotoxicity assay comparing 19m28mz and 19m28mz-mIL18 CAR T cells, when cocultured with EL4hCD19+ tumor cells.
B Cell Aplasia B Cell Aplasia **
40
ns
3040
60
**
20
**** ****
7
010 0
7
D 8 C
****
****
1020
0
**
ns
ns
2030
40
**
ns
14
80
C
CD4
B
14
100
D 4
CD8
% of 19m28mz-mIL18 CAR
CAR Phenotype
mouse B Cells (hCD19+ mCD3-) (%) mouse B Cells (hCD19+ mCD3-) (%)
A
Figure S3. 19m28mz-mIL18 CAR T cells maintain increased CD8 to CD4 ratio in vivo. Related to Figure 3 (A) CD4+ and CD8+ ratios of circulating 19m28mz-mIL18 CAR T cells, 7 days after infusion into tumor-bearing mice. No second generation 19m28mz CAR T cells are detectable at this time point. (B) B cell counts in tumorbearing untreated, 19m28mz or 19m28mz-mIL18 CAR T cell-treated mice. Experiments performed without preconditioning. Data presented as means ± SEM. Analyses determined with Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.
Untreated 19m28mz 19m28mz-mIL18 Untreated 19m28mz 19m28mz-mIL18
A
B
IFN-γ
TNF-α 800
50000
WT +
30000
600
MFI
MFI
40000
IL18RKO +
400
20000 10000
200
TNF-α 800
19m28mz
150
19m28mz-IL18 100
0 19m28mz-IL18 IL18R-KO 19m28mz-mIL18
400 200 0
50
2
4
8
1:
1:
1:
1:
0
1
Specific Cytotoxicity (%)
C
MFI
600
0
Effector:Target ratio
Figure S4. 19m28mz-mIL18 CAR T cells lacking intracellular signaling domains fail to enhance syngeneic mice survival and functional evaluation of IL18R-KO T cells. Related to Figure 3. (A) Survival of mice inoculated with EL4hCD19+ tumor cells on day 0 and treated with 19mDel or 19mDel-mIL18 CAR T cells on day 1. (B) WT or IL18R-KO-derived CAR T cells cocultured with CD19+ EL4 cells for 24 hr. Cytokine secretion determined by luminex. (C) WT or IL18R-KO-derived CAR T cells cocultured with CD19+ EL4 cells at different E:T ratio for 24 hr. Specific cytotoxicity was determined using a bioluminescence-based assay. Survival curve analysis performed using Mantel-Cox test. Data from B and C presented as means ± SEM. ns=non significant.
A
B
Figure S5. Anti-Muc16ecto 4H11m28mz-mIL18 CAR T cells enhance survival in syngeneic metastatic ovarian tumor-bearing mice. Related to Figure 3. C57BL/6 mice were inoculated with ID8 tumor cells I.P. on day 0 and treated with 2x106 anti-Muc16ecto 4H11m28mz or 4H11m28mz-mIL18 CAR T cells either on day 24 (A) or day 42 (B). Survival curve analysis performed using Mantel-Cox test **p=0.002, ***p=0.0008
Experiment: sort 1.2 million Sample ID: (Sample 4) #19Cells Sorted: Total Events: Sort Logic: Sort Directions: 1-way Fluorochromes: What was Sorted: + Fl12 DAPI
A
Operator(s): Experiment: sort 1.2 million #18-2 Cells Sorted: Sample ID: (Sample 3) Total Events: Sort Logic: Sort Directions: 1-way Fluorochromes:What was Sorted: + Fl12 DAPI
Untreated
Left
1,200,000
19m28mz
Half Left # Cells Sorted: Sort Logic: What was Sorted:
Left 1,200,000
19m28mz-mIL18
Half Left
Half Right
Right
1,200,000
B
CD3 CAR
Figure S6. Flow cytometry (FACS) sorting results of splenocytes derived from mice treated with various CAR T cells. Related to Figure 5. (A) 19m28mz and 19m28mz-mIL18 or (B) Thy1.1 mice-derived 19m28mz and 19m28mz-mIL18 CAR T cells.
A
B Untreated (n=4)
Percent survival
100
Clodronate Liposomes (n=6) PBS liposomes (n=6) 50
ns 0
FSC
0
10
20
30
40
50
Days since tumor injection
C
FSC
Figure S7. Related to Figure 5. Flow cytometry (FACS) analysis of bone marrow macrophages stained with F4/80 antibody from mice treated with (A) PBS Liposome or Clodronate Liposome. (B) Untreated tumor-bearing mice or mice treated with either Clodronate liposomes or PBS liposomes evaluated for survival. (C) FACS analysis of bone marrow NK cells from mice treated with IgG2a antibody or NK1.1 antibody.
Supplementary Table 1. Related to experimental procedures: Antibody panel used for mass cytometry experiment
Channel
Metal
Protein
113 115 139 140 141 142 143 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 209
In In La Ce Pr Nd Nd Nd Nd Sm Nd Sm Nd Eu Sm Eu Sm Gd Gd Gd Gd Tb Gd Dy Dy Dy Dy Ho Er Er Er Tm Er Yb Yb Yb Yb Lu Yb Bi
Ter119 CD45 Ly6G IgD CD16/32 CD49b CD11c CD27 CD138 PD-L1 CD103 SiglecF PDCA-1 Ly6C Ki67 CD11b cKit CD8 CD4 CD3 PD-1 B220 NK1.1 T-bet TCRgd CD62L-FITC CD86 CD69 FcER1a Foxp3 RORgt F4/80 CD115 CAR GATA3 CD19 IgM CD44 CD90 MHC II
Concentration (ug/ml) 3 3 1.5 2.5 6 0.1875 0.75 2.8 1.5 3 2.4 1.5 1.5 0.1875 1.5 0.75 1.5 0.1875 0.1875 1.5 3 1.5 6 2 1.5 3.5 0.75 6 0.1875 3 1.5 1.5 3 0.1875 3 0.75 2.25 0.1875 0.5 0.75
Clone
Vendor
TER119 30-F11 1A8 11-26c.2a 2.4G2 HMα2 HL3 LG.3A10 281-2 10F.9G2 2E7 E50-2440 120g8 HK1.4 SolA15 M1/70 2B8 53-6.7 RM4-5 17A2 29F.1A12 RA3-6B2 PK136 04-46 GL3 MEL-14 GL-1 H1.2F3 MAR-1 NRRF-30 B2D BM8 AFS98
Biolegend Biolegend Biolegend BD BD Biolegend BD Biolegend Biolegend Biolegend Biolegend BD Imgenex Biolegend eBioscience Biolegend Biolegend Biolegend Biolegend BD Biolegend BD Biolegend BD Biolegend Biolegend Biolegend Biolegend Biolegend eBioscience eBioscience Biolegend Biolegend
L50-823 6D5 RMM-1 IM7 G7 M5/114.15.2
BD Biolegend Biolegend BD Biolegend Biolegend
