A B

BT956_CC

JSRP1JSRP1 TSIX FAM166A ZACN RPL21 AMN Patient MMP3CXCR5 EDN2 APOA1 GRIN3B XIST %CD70+ 5 PP2D1 HIST1H4I TMEM184A APCDD1LRIPK4 HCRTR1 LHFPL5 TFPI2EPHA1 DKK1 CD70 MRAP derived BTIC TWIST2 WDR38 TH ALDH3A1 MAP3K15 cells lines MYCN MET BT241 94

TERT EGFR TP53 MBT190 69 CCND1 CDK4 CCNE1 ATRX SOX2 CDKN1A CDK6 PTEN MDM2 NF1

50 PDGFRA − MBT225 68 PROM1 CDKN2A BT618 46 10

− MBT173 41 log2( Our Sample / Median TCGA Expr ) CSF3RELAVL4ADGRL4NOTCH2NLNOTCH2NLBCANRGS18NRXN1LRRTM4RGPD4RGPD6RGPD6WNT7AFAM107A SERF1AGAPTC7PCDHA1 PIK3CGKCND2GIMAP6VIPR2ST18FOCADITIH5PTERPRF1LY V E 1 VWFACVRL1ARL11SMOC1RASGRF1SV2B GALR1 PTPRT ANKRD44LRRC3B HPGDSADCY2IRX1TXNDC5UBDADGRF5LHFPL3LRRC61 APBB1IPMS4A6A RNASE2GCOM1ACSM5CNTNAP4SPN AQP4 SLC52A3PWP2PWP2CLDN5CSF2RBSPRY3SPN BT972 11.3 FCGR3APTPN7 MMRN1IBSP MYCT1 TSPYL5KLHL9TEK FOLH1FOLH1ZBTB16 CELF6 ARHGEF15 CELF5SIGLEC14FPR1MYT1OLIG1GPR34ITM2A SELL AMIGO3 ETNPPLEMCNSPOCK3 TREM1 GIMAP7HACD4WDFY4MGMT SLCO1A2 FAM20A IFI30 SRXN1 SLC6A1ATRIPLTFP2RY13 HAPLN1 TUSC1 AS3MT TEN1ZNF177NCANGPR4 GUSBP11TLR7VSIG4UTYZFY CDH10 PI16NXPH1 CAAP1AKAP2 PIRT NLGN4YEIF1AYUSP9Y

15 MOG PIK3R2 CMC4 BRINP3 SCN3A APLNR SLX1B SMIM11ASMIM11A DCXSLX1B − HBB NDN LRRC55 OMGOMG DDX3Y SB2b 4 MFRPLMO3 BOLA2 BOLA2CD99 ALOX5AP HBA2 CD93 SCHIP1 OLIG2 BT566 3

12345 6 78 9 10 11 12 13 15 16 17 18 19 20 22 X GBM4 72 Chromosome GBM8 70

BT698_CC BT698 65

JSRP1JSRP1 RN1 35 ZACN TSIX LCN2 FAM166A POU2F3 RPL21 AMN PLA2G3 PP2D1 CXCR5 CD70 XIST MBT103 35 5 OPRD1 MYCNOS LHFPL5 TMEM213 HCRTR1 HIST1H4IBMP5 EPHA1MAPK15 BEST1COL2A1 WNT10A AKR1B10 SLC18A2 GRIN3B MRAPRIPK4 CSAG3 MBT121 12 MYCN BT458 9 TERT MET

PROM1 CDK4 BT428 5 CDK6 CCND1 SOX2 TP53NF1 ATRX CDKN1A PTEN MDM2 50 PDGFRA CCNE1 − EGFR BT954 4

CDKN2A WK1 3 MGMT 10

− BT799 2.2

FCGR1B RASGRF1 GPR34 PLA2G5S1PR1FCGR1ARGS18PTPRCTNR LRRTM1RGPD4DPP10CX3CR1CCRL2ITIH4 CDS1SERF1AGAPTSERPINB9TXNDC5LY86MOG CDKN2BGDAPALM2 LYSYT13MS4A7 V E 1 RPH3AARL11RNASE4 ATP6V0CCYBACCL4CD300LFGALR1BCKDHASIGLEC8LILRB2PTPRTCTSZ SASH3 ADGRL4SLC6A17BCANCD48NTSR2GPR17CYTIPLRRC3BHHATLCCR5CADM2HCLS1KLHL6HPGDSPCDH10LIX1 UBDSOGA3MYO1G FAM225BMRC1ZNF488EMX2 GCOM1ACSM5CNTNAP4SPNDPEP1 COX7A1MAGSIGLEC14SIGLEC7ADAMTS5NCAM2CSF2RBSPRY3SPN log2( Our Sample / Median TCGA Expr ) CD84PTPN7 MSTN RASGEF1BMMRN1CD14 GPR37GIMAP7HACD4SYKH2AFY2FOLH1FOLH1ZBTB16HSPB2VWF CELF6 PLVAPAPOC1LILRA2LILRB4SRXN1 ANOS1 GBM123 1.5 TNFAIP8L2CD53TSTD1RGS1 GALNT15SLC6A1AMIGO3ALDH1L1ETNPPLEMCNSPOCK3ESM1EMB NRSN1FGD2ENPP5 GRM3GIMAP8EPHB6MSR1FAM135BSLAMTAP WDFY4LUZP2MFRPCD163 SHISA9BOLA2 HAMPSIGLEC9CYYR1ERG BOLA2 GRIK3 MNDA ST6GAL2 P2RY13SNCAHAPLN1 GGTA1PLRRTM3MS4A6ACLEC7ADCNALOX5APRNASE1 HBA1 TEN1VAV1HAPLN4FPR3THBDGUSBP11NCF4TLR7 UTYZFY IGSF21C1QA TREM2 LHFPL3CLEC5AGIMAP5 AKAP2 FOLR2SLCO1C1 CLEC14A PRKCBIRF8PIRT CHST9PIK3R2TYROBP SAMSN1CLDN5CYBBVSIG4NLGN4YEIF1AYUSP9YCMC4 15 CCR1 C1QCC1QBHEYLFCGR3A GIMAP6DOCK8 MS4A4A RNASE6 SLX1B EVI2B SLC24A3SMIM11ASMIM11A SLX1B − FYB GIMAP4 OLR1 SMOC1 AQP4MRO KLHL9APBB1IPHBB FPR1 DDX3Y MBT63 1.3 ADORA3 AIF1 LMO3 TSPAN7 HBA2 CD93 SCHIP1 MBT27 1 CD99 20 − BT935 <1 12345 6 78 9 10 11 12 13 15 16 17 18 19 20 22 X BT594 0.5 Chromosome BT459 <1 BT618_CC BT778 <1 ZACN JSRP1JSRP1 TSIX AMN NSC195 1.8 PP2D1 HIST1H4I FAM166A RPL21 GRIN3B DKK1 BEST1CXCR5 5 PRDM13FEZF1 SLC39A5 HOXB8 XIST HCRTR1DCDC2B GPR39 AKR1B10 ATP12A MRAP PITX2 TFPI2EPHA1FOXH1 KRT16KLHL14CD70 NSC201FT 8.5 Astrocytes 1.2

MET MGMT TERT CDKN1A CDK6 CCND1 SOX2 MDM2 TP53 CCNE1 CDK4 NF1 ATRX 50 − PDGFRA EGFR

MYCN CDKN2A PROM1 10 −

GALNT15CCK SLC16A10KBTBD11 PRF1 SHANK2CACNA2D4 RASGRF1SULT1A3EIF3CLCDH1 CD300LFSYT4FXYD7 CLDN5MAGED4BSULT1A3 GABRDPADI2ELAVL4NOTCH2NLNOTCH2NLSLC6A17RGS18 RGPD6RGPD6DPP10WNT7ACCRL2ALDH1L1 GAPT WBSCR17PIK3CGGFRA2MSR1ST18CDKN2BRASGEF1ACSNK2A3SLC1A2PTPN5IL10RANCKAP1LDCNRFX4ARL11SMOC1 SV2BACSM5CNTNAP4DPEP1HP CD300ACA10 LILRB2PTPRTHCKNKAIN4OSMZNF674RAI2BEX5GLUD2SPRY3 RUNX3LAPTM5 CD48 ARHGAP15TFCP2L1CYTIPCCR5KLHL6HPGDSBASP1ECSCRPACSIN1UBDCARD11CNTNAP2GIMAP1CSMD1SLAKLHL9SUSD3PTGS1STAMBPL1MS4A6AMS4A7SLCO2B1VWF FLT1RNASE2GCOM1HBA2FA2HCYBAEIF4A1CCL3ST8SIA3ATCAYPLVAPCHST8IFI30SIGLEC14SIGLEC7SLC52A3SIRPB2BMP7MYT1CSF2RBKIAA2022PGAM4RP2 log2( Our Sample / Median TCGA Expr ) GSTM5CD84PTPN7FMOD KIF1ACX3CR1TM4SF18 TREM1AIF1MYCT1GTF2IP1TFECCTHRC1TSPYL5LY6HHACD4ANKRD22ADIRFPTENFOLH1FOLH1ZBTB16 RNASE1 CELF6 LYL1 LILRA2CD33KLK6 ADORA3S100A8ATP1A2 NHEJ1AMIGO3 SPOCK3ENPP6ESM1TXNDC5FGD2SMOC2GIMAP8MTAP SORCS3AS3MT PDE1BGALNT4 GPR65 TMC8SLC14A1FXYD1HAMPSIGLEC9HIF3ASIGLEC1CYYR1 VSIG4 CD53FCGR3AMNDA ST6GAL2SLC6A11P2RY13 HAPLN1 PI16 GIMAP5MLLT3 GPR183PRKCH ATP6V0CHBA1 HAPLN4NCANIGLON5LILRB1PPP1R16BTHBDGUSBP11TLR7 UTYZFY C1QCC1QAGRIK3FCGR1B ZNF518B SERPINB9CPLX2MOG TMEM176BCLEC5A AKAP2 FOLR2IL18LPAR5CD163 CLEC14A RAP1GAP2IRF8 PIK3R2SIGLEC8 SIK1KRBOX4NLGN4YEIF1AYUSP9YCMC4 15 C1QB TMEFF1 FAM181A NAPSB S100A9RCSD1 CD86 LY86 GIMAP6 ALOX5 TMEM132EKCNJ16ABI3 MAGSLC24A3SMIM11ASMIM11AMFNGFUNDC1 − PTPRC SERPINB1ADGRF5 GIMAP4 SYKAPBB1IPATAD1 OLR1 LILRB4 SASH3DDX3Y SERF1A SOGA3TMEM176A MFRP BOLA2 BCKDHAFPR1LAIR1SRXN1 GPR34NDPBOLA2 TREM2 ALOX5AP TYROBPCD93 ITM2A RGS1 SCHIP1 HBB MAOB CD99 20 −

12345 6 78 9 10 11 12 13 15 16 17 18 19 20 22 X

Chromosome A B CD70neg 15000 100 shGFP * CD70pos shCD70 80

10000 60 * 40 * 25 5000 20 15

% CD70 population CD70 % 10 5 Flourescence intensity (A.U.) 0 0

BT698 BT428 BT458 BT241 GBM8 GBM4

C GBM4 D 150 GBM4 100 shGFP **

) shCD70 2 100 * 50 50 Percent Survival Percent Tumor area (mm area Tumor

0 0 0 50 100 150 Days Elapsed shGFP shCD70 A MOSERLE_IFNA_RESPONSE PID_IL1_PATHWAY REACTOME_ANTIVIRAL_MECHANISM_BY_IFN_STIMULATED_GENES PID_IL1_PATHWAY HALLMARK_INTERFERON_ALPHA_RESPONSE IL1-MEDIATED SIGNALING EVENTS CD70-dependent activation ISG15 ANTIVIRAL MECHANISM ANTIVIRAL MECHANISM BY IFN-STIMULATED GENES HALLMARK_HYPOXIA HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION BMI1_DN_MEL18_DN.V1_UP CD70-dependent repression MEL18_DN.V1_UP ESC_V6.5_UP_EARLY.V1_DN MENSE_HYPOXIA_UP ELVIDGE_HYPOXIA_UP ELVIDGE_HIF1A_TARGETS_DN -3 -2 -1 0 1 2 3 NES score

B Geranylgeranylation ATM Pathway

Resolution of D-loop Structures Folate Metabolism

Hypoxia Cell cycle

Epigenetic Modifications

HIF1A Pathway TLR Cascade

UV Response Protein degradation and antigen processing TRAF6-mediated NF KB Signaling Protein sumoylation and uiquitinylation

IFN Signaling UV Response

Complement Cascade mRNA Translation EGF Signaling HIF1A/HIP2A Signaling

H3K27ME3 Activity PKA Signaling

Collagen Processing

CK1 Pathway EMT Process

mRNA decay

Cancer Cell Metastasis Apoptosis Cell Junction STAT3 Target Response TCA Cycle A B C ConCAR ConCAR BT241 CD70CAR CD70CAR 150 25000 200 **** 20000 **** **** 150 100 15000 (pg/mL)

(pg/mL) 100 α γ 10000 50 ** ns

IFN- **** TNF-

einMI(A.U) MFI Median 50 5000

0 0 0 BT935 GBM4 GBM8 BT935 GBM4 GBM8

4°C, 2h 37°C, 2h D GBM8 100 ConCAR

80 CD70CAR * * 60 * 40

% Cell lysis 20

0 0:1 1:1 2:1 3:1 4:1 -20 E:T Ratio A B 1010 ConCAR 100 109 CD70CAR ConCAR 8 10 CD70CAR

107 50 106 CAR Injection Total flux (p/s) Percent Percent survival *** 105

104 0 0 20 40 60 80 100 120 140 0 50 100 150 Days post-tumor injection Days post-tumor injection C

60 ConCAR CD70CAR ) 2 40

20 Tumor area (mm area Tumor

5 mm 0

ConCAR CD70CAR A

CD27 in CD45+CD3+ pop. 100 CD8+ in CD45+CD3+ pop. 80

60

40 %CD27+ Cells 20 Respective of parental gating) ( 0

MBT155MBT162MBT163MBT168MBT189MBT190MBT191 MBT155MBT162MBT163MBT168MBT189MBT190MBT191 B Experimental setup %CD3+ %CD3+/CD27+ T cells alone 84 78 T cells + BT241 crAAVS1 (supernatant) 76 78 T cells + BT241 crCD70-A (supernatant) 76 78 T cells + BT241 crCD70-B (supernatant) 78 78 T cells + BT241 crAAVS1 (cells) 77 43 T cells + BT241 crCD70-A (cells) 77 79 T cells + BT241 crCD70-B (cells) 80 76

T cells T cells + BT241 crAAVS1 T cells + BT241 crCD70-A