supplementary figure s1 a normalluminal a luminal b basal- like her2- enriched normal-like ly6e log2...

Supplementary Figure S1

A

Normal Luminal A

Luminal B

Basal-like

HER2-enriched

Normal-like

Ly6E

Log

2 (E

xpre

ssio

n Va

lue)

8

9

10

11

12

13

14

B

ER+ Triple NegativeSubtype:

4

6

8

10

- D

Ct

**

Normal Luminal A Luminal B Her2+ Normal-likeFold Change 2.14 1.76 1.51 1.29 1.51

p-value

Basal-like vs.

<3x10-39 <2x10-33 <3x10-15 <2x10-5 <4x10-15


None MBC CPZ

LAM

P1

M

erge

Tr

ansf

errin

None MBC CPZ

LAM

P1

Mer

ge

LY

6E

A

B


VehicleCtrl(2)

LY6E(12)

LY6E(4)

LY6E(2)Ctrl(12)

LY6E(8)

Ctrl(8)

Tum

or v

olum

e (1

00 X

mm

3 )

10 20 30 400

2

4

6

8

10

Day

2Tum

or v

olum

e (1

00 X

mm

3 )

10 30 400Day

2

4

6

8

20

Vehicle

T-DM1(3)T-DM1(10)

B C

AMAXF-1162

MAXF-1162MAXF-1162

1+/2+ (heterogeneous)

Supplementary Table S1Primer and probe sets used for qRT-PCR; LY6E specific siRNA

Taqman (RT-PCR) Primer and Probe sets Human LY6E Taqman Forward Primer 5' GCT CCG ACC AGG ACA ACT ACT GTaqman Reverse Primer 5' TGT CAC GAG ATT CCC AAT GCTaqman Cy5-BHQ-1 or MGB-FAM Probe 5' TGA CTG TGT CTG CTA GTGHuman VPS33B Taqman Forward Primer 5' GGC TCG AGA CCA GCT CAT CTATaqman Reverse Primer 5' GAG ATC TGC CTC AAT GAA TAA ATC CTaqman MGB-FAM Probe 5' TGG AGC AGC TTC CTHuman GAPDH Taqman Forward Primer 5' GAG TCC CTG CCA CAC TCATaqman Reverse Primer 5' GGG GTC TAC ATG GCA ACT GTaqman FAM-BHQ-1 Probe 5' CCC CAC CAC ACT GAA TCT CCC CHuman TMEM55B Assay ID : Hs00292741_m1 (Life Techonologies) LY6E siRNA LY6E_siRNA_1F 5' AGA GCA ATC TGT ACT GCC TTTLY6E_siRNA_1R 5' AGG CAG TAC AGA TTG CTC TTC LY6E_siRNA_2F 5' GGA CAA CTA CTG CGT GAC TTTLY6E_siRNA_2R 5' AGT CAC GCA GTA GTT GTC CTG LY6E_siRNA_3F 5' GTG CCG GCA TTG GGA ATC TTTLY6E_siRNA_3R 5' AGA TTC CCA ATG CCG GCA CTA LY6E_siRNA_4F 5' CCA GAG CTT TCT GTG CAA TTTLY6E_siRNA_4R 5' ATT GCA CAG AAA GCT CTG GCA

Normal Tissue Ly6E Normal Tissue Ly6E

Adrenal gland WEAK Ovary NOBone Marrow NO Pancreas NO

Brain MOD Pituitary NO

Breast WEAK Prostate NO

Cervix WEAK Salivary Gland WEAK

Colon NO Skeletal Muscle NO

Esophagus NO Skin NO

Eye NO Spleen WEAK

Heart NO Stomach NO

Intestine Small NO Testis NO

Kidney NO Thymus NO

Larynx NO Thyroid NO

Liver NO Tonsil NO

Lung NO Uterus (Endometrium) WEAK

Supplementary Table S2Expression of LY6E protein in normal tissues

Supplementary Table S3 9B12 affinity to human and cynomolgus monkey LY6E

Ave. average; Kd equilibrium binding constant.

Test Material AssayKD aveKD ± SD

nM nM9B12 with 1 3

4±1Human LY6E 2 5

3 3

9B12 with 1 6

7±1Cynomolgus monkey LY6E 2 8

3 7

Supplementary Table S4 Correlation of MMAE killing to LY6E amplification

Cell Line EC 50 Copy

number Anti-LY6E vcE Free MMAE

ug/mLMMAE

(nM) nM RMG-1 2.14 51.8 0.2334 2DOV 13 661 16313.3 0.3761 2EFO-27 726 17915.4 0.5406 2A2780 4304 106219.6 0.6619 2

COV318 500 12339.5 1.521 2

TOV-112D 5.14 125.8 0.2257 3COV362 6.053 148 0.3333 3

OVCAR433 3.46 85.1 0.5132 3

OVISE 0.8774 22.2 0.1747 4KURAMOCHI 0.4404 11.1 0.3048 4

OAW42 N/A N/A 1664 5

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 0 TTP 2X TTP 5X PR CR

01 - Vehicle 0 (0, 0) 10 3 8.5 0 002 - Hu anti-LY6E 9B12.v12 naked, 12 mg/kg 14 (-78, 59) 10 3 7.5 0 003 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 2 mg/kg 8 (-88, 54) 10 3.5 9 0 004 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 8 mg/kg 27 (-60, 67) 10 4 15.5 0 005 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 12 mg/kg 73 (36, 90) 10 5.5 31.5 1 006 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 2 mg/kg 95 (82, 100) 10 32.5 NA 2 007 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 4 mg/kg 104 (101, 111) 10 NA NA 8 208 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 8 mg/kg 105 (102, 113) 10 NA NA 3 709 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 12 mg/kg 105 (102, 111) 10 NA NA 4 6

MAXF-1162 vs anti-LY6E ADC (Supplementary Fig. 4B)


01 - Vehicle 0 (0, 0) 10 7.5 28.5 0 002 - Hu anti-LY6E 9B12.v12 naked, 12 mg/kg -20 (-131, 33) 10 6.5 24 0 003 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 2 mg/kg -5 (-89, 45) 10 6 26.5 0 004 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 8 mg/kg 36 (-19, 64) 10 7 NA 0 005 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 12 mg/kg 46 (0, 72) 10 6.5 NA 0 006 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 2 mg/kg 47 (3, 72) 10 6.5 NA 0 007 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 4 mg/kg 94 (83, 102) 10 NA NA 4 008 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 8 mg/kg 107 (100, 115) 10 NA NA 9 009 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 12 mg/kg 108 (102, 117) 10 NA NA 10 0

SU.86.86


01 - Vehicle 0 (0, 0) 10 9 21 0 002 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 4 mg/kg 5 (-108, 59) 10 11 22 0 003 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 8 mg/kg 55 (-2, 82) 10 18 30 1 004 - Hu anti-LY6E 9B12.v12 naked, 8 mg/kg 14 (-108, 60) 10 10.5 23 0 005 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 0.1 mg/kg -19 (-160, 47) 10 8 18.5 0 006 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 0.5 mg/kg 66 (17, 89) 10 20 32 0 007 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 1 mg/kg 94 (76, 104) 10 31.5 NA 1 008 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 2 mg/kg 110 (104, 122) 10 NA NA 8 109 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 4 mg/kg 111 (105, 121) 10 NA NA 8 010 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 8 mg/kg 112 (106, 123) 10 NA NA 8 2

SW900


01 - Vehicle 0 (0, 0) 9 8 24.5 0 002 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 6 mg/kg 72 (51, 84) 9 20.5 NA 0 003 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 1 mg/kg 122 (115, 133) 9 NA NA 8 004 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 3 mg/kg 128 (120, 140) 9 NA NA 9 005 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 6 mg/kg 128 (120, 142) 9 NA NA 8 1

HBCx-9


01 - Vehicle 0 (0, 0) 9 7.5 20 0 002 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 2 mg/kg 24 (-63, 65) 9 8 23.5 0 003 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 8 mg/kg 39 (-37, 72) 9 15 25.5 0 004 - Hu anti-gD 5B6-MC-vc-PAB-MMAE, 12 mg/kg 24 (-73, 68) 9 6.5 23 0 005 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 2 mg/kg 33 (-49, 72) 9 10 25 0 006 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 4 mg/kg 71 (29, 90) 9 20 33.5 0 007 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 8 mg/kg 119 (109, 143) 9 NA NA 4 508 - Hu anti-LY6E 9B12.v12-MC-vc-PAB-MMAE, 12 mg/kg 129 (115, 157) 9 NA NA 0 9

Supplementary Table S6: Statistical analysis between test groups in xenograft efficacy studies (corresponding to Fig. 6 and Supplementary Fig. 4B)

Table Analyzed SU.86.86 Fig. 6B

Columns Analyzed (Vehicle vs Test)

Vehicle Vehicle Vehicle Vehicle Vehicle Vehiclevs. vs. vs. vs. vs. vs.Ctrl ADC 8mpk Ly6E ADC 0.5mpk Ly6E ADC 1mpk Ly6E ADC 2mpk Ly6E ADC 4mpk Ly6E ADC 8mpk

P value* 0.2512 0.1391 0.0093 0.0017 0.0017 0.0013Significantly different? (P < 0.05) No No Yes Yes Yes YesWelch-corrected t, df t=0.6837 df=18.94 t=1.116 df=19.09 t=2.722 df=11.93 t=3.820 df=10.09 t=3.788 df=10.23 t=3.948 df=10.20

Table Analyzed SW900 Fig. 6C


Vehicle Vehicle Vehicle Vehiclevs. vs. vs. vs.Ctrl ADC 6 mpk Ly6E ADC 1mpk Ly6E ADC 3 mpk Ly6E ADC 6 mpk

P value* 0.0877 0.0011 0.0007 0.0007Significantly different? (P < 0.05) No Yes Yes YesWelch-corrected t, df t=1.423 df=14.85 t=4.389 df=8.138 t=4.689 df=8.250 t=4.712 df=8.319

Table Analyzed PAXF-1657 Fig. 6D


Vehicle Vehicle Vehicle Vehicle Vehiclevs. vs. vs. vs. vs.Ctrl ADC 12mpk Ly6E ADC 2mpk Ly6E ADC 4mpk Ly6E ADC 8mpk Ly6E ADC 12mpk

P value* 0.0029 < 0.0001 < 0.0001 < 0.0001 < 0.0001Significantly different? (P < 0.05) Yes Yes Yes Yes YesWelch-corrected t, df t=3.084 df=20.02 t=5.374 df=12.82 t=6.501 df=11.48 t=6.633 df=11.52 t=6.653 df=11.55

Table Analyzed HBCx-9 Fig. 6E


Vehicle Vehicle Vehicle Vehicle Vehiclevs. vs. vs. vs. vs.Ctrl ADC 12mpk Ly6E ADC 2mpk Ly6E ADC 4mpk Ly6E ADC 8mpk Ly6E ADC 12mpk

P value* 0.2764 0.1809 0.0299 0.0012 0.0008Significantly different? (P < 0.05) No No Yes Yes YesWelch-corrected t, df t=0.6042 df=18.95 t=0.9369 df=17.27 t=2.055 df=13.49 t=3.964 df=10.38 t=4.231 df=10.37

Table Analyzed MAXF-1162 Fig. S4B


Vehicle Vehicle Vehicle Vehicle Vehicle Vehiclevs. vs. vs. vs. vs. vs.Ctrl ADC 8mpk Ctrl ADC 12mpk Ly6E ADC 2mpk Ly6E ADC 4mpk Ly6E ADC 8mpk Ly6E ADC 12mpk

P value* 0.0576 0.0243 0.0314 0.0003 < 0.0001 < 0.0001Significantly different? (P < 0.05) No Yes Yes Yes Yes YesWelch-corrected t, df t=1.654 df=18.34 t=2.131 df=16.28 t=1.997 df=16.38 t=4.612 df=11.99 t=5.270 df=12.85 t=5.345 df=13.41

* One-tailed P value by unpaired t-test with Welch’s correction

supplementary figure s1 a normalluminal a luminal b basal- like her2- enriched normal-like ly6e log2...

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