www.sciencetranslationalmedicine.org/cgi/content/full/5/207/207ra144/DC1

Supplementary Materials for

Tumor-Specific Activation of an EGFR-Targeting Probody Enhances Therapeutic Index

Luc R. Desnoyers, Olga Vasiljeva, Jennifer H. Richardson, Annie Yang,

Elizabeth E.M. Menendez, Tony W. Liang, Chihunt Wong, Paul H. Bessette, Kathy Kamath, Stephen J. Moore, Jason G. Sagert, Daniel R. Hostetter, Fei Han,

Jason Gee, Jeanne Flandez, Kate Markham, Margaret Nguyen, Michael Krimm, Kenneth R. Wong, Shouchun Liu, Patrick S. Daugherty, James W. West, Henry B. Lowman*

*Corresponding author. E-mail: henry@cytomx.com

Published 16 October 2013, Sci. Transl. Med. 5, 207ra144 (2013)

DOI: 10.1126/scitranslmed.3006682

The PDF file includes:

Fig. S1. Selectivity of PB1 Probody linker cleavage. Fig. S2. FACS binding of Probody PB1 and cetuximab to H292 cells. Fig. S3. Time course of ex vivo Probody activation. Fig. S4. Quantification of tumor/normal Probody and cetuximab accumulation in mice. Fig. S5. EGFR pathway inhibition in tumors from mice treated with cetuximab or Probody PB1. Fig. S6. EGFR expression and lack of Probody activation in skin from normal cynomolgus monkey. Table S1. EGFR ELISA binding data from individual experiments. Table S2. H292 cell–based activity inhibition data from individual experiments. Table S3. Tumor volumes from H292 efficacy study. Table S4. Tumor volumes from LXFA677 efficacy study. Table S5. Anti-drug antibody observations in nonhuman primates. Table S6. Probody IHZ screening of NSCLC patient tumor samples. Table S7. Probody IHZ screening of CRC patient tumor samples.

Figure S1. Selectivity of PB1 Probody linker cleavage. PB1 Probody (500 nm) was incubated in the presence of 40 nM of uPA, MTSP1, or tPA (prepared in 50 mM Tris-HC1, 150 mM NaCl, 0.05% Tween-20, pH=7.4), or in the presence of plasmin or KLK5 (prepared in 50 mM Tri-HCl, 100 mM NaCl, 1 mM EDTA, 0.01% Tween-20, pH=7.4), or in the presence of ADAM10 or ADAM17 (prepared in 25 mM Tris-HCl, 0.005% Brij-35, pH=9.0). In all cases, proteases were shown to be active against synthetic peptide substrates. Samples were incubated at 37°C for 2.5 hours or 20h. To stop the reaction, 5 μl samples were incubated with 7 μl of HT Protein Express Sample Buffer (Caliper LifeSciences) containing 20 mM 2-mercaptoethanol for 10 minutes at 95°C and run on the LabChip GXII as described in the text. The LabChip GXII software was used to quantify light chain (LC) peak area (A) and to generate a gel image (B). Product conversion was calculated according to the following equation: Product conversion= (cleaved LC)/(cleaved LC + uncleaved LC).

Figure S2. FACS binding of Probody PB1 and cetuximab to H292 cells. H292 cells (0.5-1 x 106) were plated in 96-well U-bottom plates. Cells were incubated with 0.25 ug/ml of AF488-conjugated cetuximab and indicated amounts of competing unlabeled PB1, uPA activated PB1, or cetuximab diluted in MACS buffer in 0.5% BSA (Miltenyi) for 30 min on ice. Cells were washed 2 times. Mean fluorescence intensity (MFI) was measured on an Accuri flow cytometer (BD Biosciences). Percent fluorescence was calculated: (MFI of the sample)/(MFI of sample without competing antibodies) x 100. Experiments were repeated three times. Data are presented as mean of relative fluorescence ± SEM. Data were fitted on a 4-parameter curve and the EC50 was calculated (Prism Graphpad). Fold change was calculated as 76-fold by dividing EC50 of PB1 (21.5 nM) by EC50 of cetuximab (0.28 nM).

Figure S3. Time course of ex vivo Probody activation. Incubation of PB1 with H292 xenograft tumor tissue for 30, 45 or 60 min resulted in a gradual increase in Probody activation by tissue-derived proteases leading to subsequent binding of the activated Probody to EGFR cell-surface receptor.

Figure S4. Quantification of tumor/normal Probody and cetuximab accumulation in mice. The tumor to normal tissue ratios for Alexa750 fluorescence intensity are displayed. Higher ratios of the fluorescent signal were detected in the tumors of mice dosed with PB1 or cetuximab, suggesting that PB1 was activated and accumulated in the tumor through EGFR binding. Data are represented as mean ± s.d. (n = 3 mice).

Figure S5. EGFR pathway inhibition in tumors from mice treated with cetuximab or Probody PB1. Immunohistochemical staining for pERK1/2 in H292 xenograft tumors resected 72 h after treatment with vehicle, 25 mg/kg of PB-NSUB, 25 mg/kg cetuximab or 25 mg/kg of PB1. Scale bars, 100 μm.

Figure S6. EGFR expression and lack of Probody activation in skin from normal cynomolgus monkey. Cetuximab staining indicates the presence of EGFR receptor in normal skin tissue. No signal of Alexa-680 labeled PB1 could be detected on normal skin tissue by IHZ analysis.

Table S1. EGFR ELISA binding data from individual experiments.

Apparent Kd (nM)* Experiments No.

Cetuximab Parental Ab Activated PB1

PB1

1 0.007 0.003 0.004 0.216 2 0.016 0.014 0.016 0.361 3 0.01 0.014 0.017 0.990 Mean Kd (nM) 0.011 0.01 0.012 0.523 SD 0.004 0.006 0.007 0.411 SEM 0.002 0.004 0.004 0.238 Relative Apparent Kd (fold) Experiment No.

Fold over Cetuximab

Fold over Parental Ab

Fold over Activated

PB1

Fold over PB1

1 30 63 52 -1- 2 23 25 23 -1- 3 100 73 60 -1- Mean Relative Kd (fold)

52 54 45 -1-

SD 44 25 19 - SEM 25 15 11 -

*Calculated from 9 point curve (0.0004 to 31.25 nM). Curve fitting was performed with GraphPad Prism, one site specific binding, least squares fit.

Table S2. H292 cell-based activity inhibition data from individual experiments.

*Calculated from 9 point curve (0.0006 to 40 μg/ml). Curve fitting was performed with GraphPad Prism, log (inhibitor) versus response – variable slope (four-parameter fit).

IC50 (μg/ml)* Experiment No.

Cetuximab Activated PB1

PB1

1 0.008 0.006 2.27 2 0.019 0.034 7.26 3 0.013 0.032 5.36 4 0.007 0.01 4.45 Mean IC50 (nM) 0.012 0.020 4.84 SD 0.006 0.014 2.07 SEM 0.003 0.007 1.04 Relative IC50 (fold) Experiment No.

Fold over Cetuximab

Fold over Activated

PB1

Fold over PB1

1 300 400 -1- 2 380 220 -1- 3 410 170 -1- 4 640 460 Mean Relative IC50 (fold) 430 310

-1-

SD 150 140 - SEM 74 69 -

Table S3. Tumor volumes from H292 efficacy study.

Complete Response (CR) was defined as the disappearance of the tumor. Partial Response (PR) was defined as at least a 70% decrease in the tumor growth compared to the average tumor volume in the vehicle group (824 mm3).

PR CR Cetuximab 8/8 0/8 PB-NSUB 1/8 0/8 PB1 7/8 0/8

Mouse #

Study Day

1 3 6 8 10 13 15 17 20

Vehicle 1 179.4 185.9 164.2 140.0 144.2 299.1 242.7 457.4 531.2

Vehicle 2 247.4 490.9 550.6 983.5 1077.1 1012.8 1101.2 1622.5 1732.0

Vehicle 3 116.8 128.3 116.2 288.5 195.5 275.4 296.2 289.2 483.3

Vehicle 4 182.4 277.6 350.2 493.0 482.2 651.8 714.0 811.3 1024.8

Vehicle 5 94.2 63.9 93.1 157.0 178.4 218.8 242.0 302.9 489.1

Vehicle 6 117.7 91.5 205.8 279.1 339.5 456.2 323.9 433.5 659.8

Vehicle 7 94.1 105.5 125.0 183.6 167.7 228.5 224.2 234.7 343.9

Vehicle 8 258.3 339.2 629.7 466.9 873.1 1110.0 1222.6 1252.1 1323.5

cetuximab 9 116.1 60.4 39.6 33.6 22.3 25.6 29.2 19.0 67.4

cetuximab 10 117.9 171.4 69.3 54.8 43.4 37.9 28.5 27.8 85.3

cetuximab 11 93.5 109.4 44.1 29.4 16.0 13.6 16.7 14.3 80.8

cetuximab 12 238.1 179.7 178.5 111.7 120.2 90.6 93.7 79.3 118.3

cetuximab 13 173.4 340.7 139.8 82.7 136.4 64.5 67.1 55.4 152.8

cetuximab 14 189.8 187.0 155.8 135.8 81.8 124.2 124.8 69.7 169.5

cetuximab 15 99.5 111.5 81.8 51.5 53.0 51.7 44.3 21.1 96.7

cetuximab 16 260.6 290.5 139.1 73.1 85.3 66.9 59.9 59.9 96.9

PB-NSUB 17 273.0 192.6 280.1 373.7 379.7 349.5 289.4 429.1 404.2

PB-NSUB 18 228.7 598.9 985.2 1288.6 1267.4 1490.4 1953.7 1990.6 2143.8

PB-NSUB 19 192.6 207.8 284.8 446.3 415.8 594.0 665.9 713.5 798.0

PB-NSUB 20 91.0 46.2 69.4 105.1 98.1 100.6 115.5 154.3 211.9

PB-NSUB 21 121.0 122.1 137.4 166.5 79.0 138.1 130.9 210.5 255.2

PB-NSUB 22 171.4 197.1 279.8 285.4 286.5 311.5 376.8 442.0 448.7

PB-NSUB 23 107.4 119.0 114.9 166.2 142.1 231.1 242.5 265.3 368.8

PB-NSUB 24 113.4 128.0 157.9 225.8 156.5 208.5 212.5 293.6 335.9

PB1 25 282.7 342.6 280.9 375.4 378.2 498.4 526.7 616.5 686.9

PB1 26 124.0 67.3 106.0 89.3 80.0 77.9 69.1 61.6 102.8

PB1 27 111.4 91.8 97.4 192.8 141.4 153.9 84.2 84.7 159.3

PB1 28 162.4 116.9 120.4 95.4 76.8 68.8 47.9 64.7 81.1

PB1 29 210.9 198.1 137.1 156.5 127.4 98.8 117.9 88.2 153.9

PB1 30 215.4 406.3 252.9 176.4 192.2 84.0 42.1 34.3 87.5

PB1 31 110.9 127.0 99.2 120.7 79.8 61.2 40.5 81.9 134.0

PB1 32 86.4 119.2 77.1 82.5 68.8 57.1 52.0 59.8 96.9

Table S4. Tumor volumes from LXFA677 efficacy study.

Mouse #

Study Day 0 3 7 11 14 18 21 25

Vehicle 1 159.7 268.3 351.3 518.9 550.5 941.7 1208.4 1439.8 Vehicle 2 159.5 123.5 145.1 184.3 190.1 227.9 307.2 518.9 Vehicle 3 96.9 121.1 142.9 154.2 163.4 194.1 288 316.9 Vehicle 4 95.5 134 174.2 244.7 255.9 254.1 496.4 598.2 Vehicle 5 191.8 330.8 425.9 747.2 747.2 1540 1912.5 2641.9 Vehicle 6 249.3 484 518.4 695 825.7 1350.6 1921.3 2870.9 Vehicle 7 194.4 271.5 282.9 531.6 555.1 1149.8 1332.6 1859.9 Vehicle 8 242.6 509 567.6 738.1 905.1 1048.4 1520.9 1530.8 Vehicle 9 129.6 183.8 196 320.3 327.2 539.1 842.8 893.8 Vehicle 10 130.5 177.5 273.8 543.7 569.2 1233.7 1485.3 2078.2 Cetuximab 11 158.8 50.8 19.7 16.3 16.3 0.1 0.1 0.1 Cetuximab 12 94.8 42.3 16.4 11.7 10 0.1 0.1 Cetuximab 13 161.2 34.7 14 12.2 10.9 0.1 0.1 0.1 Cetuximab 14 240.9 101.3 45.6 23.4 21.2 16.3 16.7 0.1 Cetuximab 15 249.9 113.4 30.3 29.6 29.6 0.1 0.1 0.1 Cetuximab 16 191.6 54 22.7 15.1 13.3 0.1 0.1 0.1 Cetuximab 17 131.6 58.7 26 20.7 18.3 6.6 0.1 0.1 Cetuximab 18 194.5 36.8 56.4 64.5 63.5 217.8 352 1104.8 Cetuximab 19 98.3 51.8 25.1 24.5 21.8 35.3 50.3 73.7 Cetuximab 20 126.5 58.8 28.2 13.5 13.5 0.1 0.1 PB1 21 78.4 24.5 24.5 11.4 7.8 0.1 0.1 0.1 PB1 22 111.3 38.8 21.2 17.8 9.5 0.1 0.1 0.1 PB1 23 117 66.3 47.6 38.8 29.4 0.1 0.1 0.1 PB1 24 221.2 106.6 45.3 25.1 23.9 0.1 0.1 0.1 PB1 25 142.2 82.5 38.8 37 28.2 0.1 0.1 0.1 PB1 26 203.4 104.4 33.2 28.5 27 0.1 0.1 0.1 PB1 27 289.1 102.3 46.7 21.8 18.3 0.1 0.1 0.1 PB1 28 173.8 89.2 32 18.4 16.2 0.1 0.1 0.1 PB1 29 148.8 68 27.4 20.2 16.8 0.1 0.1 0.1 PB1 30 142.2 43.5 17.3 0.1 0.1 0.1 0.1 0.1 Complete Response (CR) was defined as the disappearance of tumor; partial Response (PR) was defined as >= 70% decrease compared to the average tumor volume in the vehicle group (1475 mm3).

PR CR Cetuximab 1/10 8/10 PB1 0/10 10/10

Table S5. Anti-drug antibody observations in nonhuman primates.

Test Articlea Dose; mg/kg (Loading; weekly)

Frequency of ADA b (animals positive/animals treated)

Cetuximab 40 mg/kg; 25 mg/kg 1/3 PB1 40 mg/kg; 25 mg/kg 0/3 PB1 120 mg/kg; 75 mg/kg 1/3 a Cynomolgus monkeys (n = 3 females/group) were given cetuximab or PB1 weekly for

5 weeks followed by a 4-week recovery period as described in the text. b ADA assays were carried out as follows: serum samples were incubated with

biotinylated cetuximab or PB1 in microtiter plates coated with either cetuximab or PB1. After incubation, wells were washed and bridged biotinylated drug was detected using poly-horseradish peroxidase (HRP)-conjugated streptavidin and visualized by 3,3′,5,5′-tetramethylbenzidine (TMB). Color development was stopped and the absorbance at 450 nm was measured. All samples and standards were analyzed in triplicate wells. Samples yielding a signal above that of the non-specific binding control (assay buffer used in place of test sample) at the lowest dilution tested were considered positive.

Table S6. Probody IHZ screening of NSCLC patient tumor samples.

*Cetuximab staining scored from “-” to 3+ indicates the amount of antibody binding: “-”, no staining; 1+, weak staining; 2+, moderate staining; and 3+, strong staining. **PB1 staining scoring is based on comparison with parental antibody (cetuximab) staining: “-”, no staining; 1+, weak staining as compared to parental antibody; 2+, moderate staining as compared to parental antibody; and 3+, similar staining to parental antibody.

Table S7. Probody IHZ screening of CRC patient tumor samples.

*Cetuximab staining scored from “-” to 3+ indicates the amount of antibody binding: “-”, no staining; 1+, weak staining; 2+, moderate staining; and 3+, strong staining. **PB1 staining scoring is based on comparison with parental antibody (cetuximab) staining: “-”, no staining; 1+, weak staining as compared to parental antibody; 2+, moderate staining as compared to parental antibody; and 3+, similar staining to parental antibody.