Supplementary Figure S1 A NormalLuminal A Luminal B Basal- like HER2- enriched Normal-like Ly6E Log2 (Expression Value) 8 9 10 11 12 13 14 B ER+ Triple.

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Transcrição da apresentação:

Supplementary Figure S1 A NormalLuminal A Luminal B Basal- like HER2- enriched Normal-like Ly6E Log2 (Expression Value) B ER+ Triple Negative Subtype:  Ct ** NormalLuminal ALuminal BHer2+Normal-like Fold Change p-value Basal-like vs. <3x <2x <3x <2x10 -5 <4x10 -15

Supplementary Figure S2

Supplementary Figure S3 None MBC CPZ LAMP1 Merge Transferrin None MBC CPZ LAMP1 Merge LY6E A B

Supplementary Figure S4 Vehicle Ctrl(2) LY6E(12) LY6E(4) LY6E(2) Ctrl(12) LY6E(8) Ctrl(8) Tumor volume (100 X mm 3 ) Day 2 Tumor volume (100 X mm 3 ) Day Vehicle T-DM1(3) T-DM1(10) B C A MAXF /2+ (heterogeneous)

Supplementary Table S1 Primer and probe sets used for qRT-PCR; LY6E specific siRNA Taqman (RT-PCR) Primer and Probe sets Human LY6E Taqman Forward Primer5' GCT CCG ACC AGG ACA ACT ACT G Taqman Reverse Primer5' TGT CAC GAG ATT CCC AAT GC Taqman Cy 5 -BHQ-1 or MGB-FAM Probe5' TGA CTG TGT CTG CTA GTG Human VPS33B Taqman Forward Primer5' GGC TCG AGA CCA GCT CAT CTA Taqman Reverse Primer5' GAG ATC TGC CTC AAT GAA TAA ATC C Taqman MGB-FAM Probe 5' TGG AGC AGC TTC CT Human GAPDH Taqman Forward Primer 5' GAG TCC CTG CCA CAC TCA Taqman Reverse Primer 5' GGG GTC TAC ATG GCA ACT G Taqman FAM-BHQ-1 Probe 5' CCC CAC CAC ACT GAA TCT CCC C Human TMEM55B Assay ID : Hs _m1 (Life Techonologies) LY6E siRNA LY6E_siRNA_1F 5' AGA GCA ATC TGT ACT GCC TTT LY6E_siRNA_1R 5' AGG CAG TAC AGA TTG CTC TTC LY6E_siRNA_2F 5' GGA CAA CTA CTG CGT GAC TTT LY6E_siRNA_2R 5' AGT CAC GCA GTA GTT GTC CTG LY6E_siRNA_3F 5' GTG CCG GCA TTG GGA ATC TTT LY6E_siRNA_3R 5' AGA TTC CCA ATG CCG GCA CTA LY6E_siRNA_4F 5' CCA GAG CTT TCT GTG CAA TTT LY6E_siRNA_4R 5' ATT GCA CAG AAA GCT CTG GCA

Normal TissueLy6ENormal TissueLy6E Adrenal glandWEAK OvaryNO Bone MarrowNOPancreasNO BrainMODPituitaryNO BreastWEAKProstateNO CervixWEAKSalivary GlandWEAK ColonNOSkeletal MuscleNO EsophagusNOSkinNO EyeNOSpleenWEAK HeartNOStomachNO Intestine SmallNOTestisNO KidneyNOThymusNO LarynxNOThyroidNO LiverNOTonsilNO LungNOUterus (Endometrium)WEAK Supplementary Table S2 Expression of LY6E protein in normal tissues

Supplementary Table S3 9B12 affinity to human and cynomolgus monkey LY6E Ave.  average; K d  equilibrium binding constant. Test MaterialAssay KDKD aveK D ± SD nM 9B12 with13 4±1 Human LY6E B12 with16 7±1 Cynomolgus monkey LY6E28 37

Supplementary Table S4 Correlation of MMAE killing to LY6E amplification Cell LineEC 50 Copy number Anti-LY6E vcEFree MMAE ug/mL MMAE (nM)nM RMG DOV EFO A COV TOV-112D COV OVCAR OVISE KURAMOCHI OAW42N/A 16645

Supplementary Table S5: Tumor growth data for xenograft studies in Fig. 6 and Supplementary Fig. S4B PAXF-1657 Grp.Name AUC/Day %TGI (lower,upper) N Day 0TTP 2XTTP 5XPRCR 01 - Vehicle 0 (0, 0) Hu anti-LY6E 9B12.v12 naked, 12 mg/kg 14 (-78, 59) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 2 mg/kg 8 (-88, 54) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 8 mg/kg 27 (-60, 67) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 12 mg/kg 73 (36, 90) Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 2 mg/kg 95 (82, 100)1032.5NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 4 mg/kg 104 (101, 111)10NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 8 mg/kg 105 (102, 113)10NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 12 mg/kg 105 (102, 111)10NA 46 MAXF-1162 vs anti-LY6E ADC (Supplementary Fig. 4B) Grp.Name AUC/Day %TGI (lower,upper) N Day 0TTP 2XTTP 5XPRCR 01 - Vehicle 0 (0, 0) Hu anti-LY6E 9B12.v12 naked, 12 mg/kg -20 (-131, 33) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 2 mg/kg -5 (-89, 45) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 8 mg/kg 36 (-19, 64)107NA Hu anti-gD 5B6-MC-vc-PAB-MMAE, 12 mg/kg 46 (0, 72)106.5NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 2 mg/kg 47 (3, 72)106.5NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 4 mg/kg 94 (83, 102)10NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 8 mg/kg 107 (100, 115)10NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 12 mg/kg 108 (102, 117)10NA 100 SU Grp.Name AUC/Day %TGI (lower,upper) N Day 0TTP 2XTTP 5XPRCR 01 - Vehicle 0 (0, 0) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 4 mg/kg 5 (-108, 59) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 8 mg/kg 55 (-2, 82) Hu anti-LY6E 9B12.v12 naked, 8 mg/kg 14 (-108, 60) Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 0.1 mg/kg -19 (-160, 47) Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 0.5 mg/kg 66 (17, 89) Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 1 mg/kg 94 (76, 104)1031.5NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 2 mg/kg 110 (104, 122)10NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 4 mg/kg 111 (105, 121)10NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 8 mg/kg 112 (106, 123)10NA 82 SW900 Grp.Name AUC/Day %TGI (lower,upper) N Day 0TTP 2XTTP 5XPRCR 01 - Vehicle0 (0, 0) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 6 mg/kg 72 (51, 84)920.5NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 1 mg/kg 122 (115, 133)9NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 3 mg/kg 128 (120, 140)9NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 6 mg/kg 128 (120, 142)9NA 81 HBCx-9 Grp.Name AUC/Day %TGI (lower,upper) N Day 0TTP 2XTTP 5XPRCR 01 - Vehicle0 (0, 0) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 2 mg/kg24 (-63, 65) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 8 mg/kg39 (-37, 72) Hu anti-gD 5B6-MC-vc-PAB-MMAE, 12 mg/kg24 (-73, 68) Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 2 mg/kg33 (-49, 72) Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 4 mg/kg71 (29, 90) Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 8 mg/kg119 (109, 143)9NA Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 12 mg/kg129 (115, 157)9NA 09

Supplementary Table S6: Statistical analysis between test groups in xenograft efficacy studies (corresponding to Fig. 6 and Supplementary Fig. 4B) Table Analyzed SU Fig. 6B Columns Analyzed (Vehicle vs Test) Vehicle vs. Ctrl ADC 8mpkLy6E ADC 0.5mpkLy6E ADC 1mpkLy6E ADC 2mpkLy6E ADC 4mpkLy6E ADC 8mpk P value * Significantly different? (P < 0.05)No Yes Welch-corrected t, dft= df=18.94t=1.116 df=19.09t=2.722 df=11.93t=3.820 df=10.09t=3.788 df=10.23t=3.948 df=10.20 Table Analyzed SW900 Fig. 6C Columns Analyzed (Vehicle vs Test) Vehicle vs. Ctrl ADC 6 mpkLy6E ADC 1mpkLy6E ADC 3 mpkLy6E ADC 6 mpk P value * Significantly different? (P < 0.05)NoYes Welch-corrected t, dft=1.423 df=14.85t=4.389 df=8.138t=4.689 df=8.250t=4.712 df=8.319 Table Analyzed PAXF-1657 Fig. 6D Columns Analyzed (Vehicle vs Test) Vehicle vs. Ctrl ADC 12mpkLy6E ADC 2mpkLy6E ADC 4mpkLy6E ADC 8mpkLy6E ADC 12mpk P value * < Significantly different? (P < 0.05)Yes Welch-corrected t, dft=3.084 df=20.02t=5.374 df=12.82t=6.501 df=11.48t=6.633 df=11.52t=6.653 df=11.55 Table Analyzed HBCx-9 Fig. 6E Columns Analyzed (Vehicle vs Test) Vehicle vs. Ctrl ADC 12mpkLy6E ADC 2mpkLy6E ADC 4mpkLy6E ADC 8mpkLy6E ADC 12mpk P value * Significantly different? (P < 0.05)No Yes Welch-corrected t, dft= df=18.95t= df=17.27t=2.055 df=13.49t=3.964 df=10.38t=4.231 df=10.37 Table Analyzed MAXF-1162 Fig. S4B Columns Analyzed (Vehicle vs Test) Vehicle vs. Ctrl ADC 8mpkCtrl ADC 12mpkLy6E ADC 2mpkLy6E ADC 4mpkLy6E ADC 8mpkLy6E ADC 12mpk P value * < Significantly different? (P < 0.05)NoYes Welch-corrected t, dft=1.654 df=18.34t=2.131 df=16.28t=1.997 df=16.38t=4.612 df=11.99t=5.270 df=12.85t=5.345 df=13.41 * One-tailed P value by unpaired t-test with Welch’s correction