Supplemental Information for MS# MCT-13-0933

Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using a IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex

Figure S1. Comparison of tumor time-activity data for three successive treatment cycles (12.3 MBq/cycle) compared with a single cycle of 37 MBq, derived from biodistribution experiments conducted with groups of GD2(+)-NB tumor-bearing mice given 1.75 mg of hu3F8-C825, 250 µg of CA (14% (w/w) of 61 (Y)-DOTA-Bn/dextran), and ~5.6 MBq of 177Lu-DOTA-Bn (~30 pmol). The cumulative activities (kBq-hr/MBq/g) for a total of 37 MBq injected using the fractionated approach are as follows: for 3 cycles, first injection: 2,418; second injection: 3,415; and third injection: 6,140. The cumulative activity for a total of 37 MBq injected as a single dose is 10,043 kBq-hr/MBq/g. The 3-injection tumor cumulated activity was 11,973 kBq-h/MBq/g of Lu-177, ~20% more than the single-injection cumulated activity. The 3-injection tumor absorbed dose was 107.6 cGy/MBq Lu-177, also ~20% more than the single-injection cumulated activity of 84.9 cGy/MBq Lu-177.

Figure S2. Blood clearance (red symbols) and tumor uptake curves (black symbols; for (▲) n = 1, and for (■) n = 4; average ± SEM) derived from serial PET images up to 64 h (8.5-10.2 MBq of 124I- hu3F8-C825 (280-500 pmol). Inset: representative PET images of 124I-hu3F8-C825 (8.55 MBq, 56 ug, 280 pmol) in a mouse bearing a s.c. GD2(+)-NB xenograft in the lower flank (Th. = thyroid, T = tumor) after 22 h p.i.. The transverse (trans.) and coronal planar images intersect the center of the tumor. Figure S3. Biodistribution analysis of 124I-hu3F8-C825 in GD2(+)-NB tumor and various tissues determined ex vivo immediately following PET scanning at 48 h p.i. n = 4; as %ID/g, mean ± SD). Table S1. PRIT of 177Lu-DOTA-Bn with hu3F8-C825 BsAb in athymic nude mice bearing s.c. GD2(-) human breast cancer cell line BT474. Animals were given 1.75 mg of hu3F8-C825 followed 28 h later with 5.6 MBq (~30 pmol) 177Lu-DOTA-Bn. Biodistribution was conducted 24 h p.i. of 177Lu-DOTA-Bn. Results are given as mean %ID/g ± SEM. PRIT with Tissues hu3F8-C825 GD2(-) tumors (n = 3) Blood 1.40 ± 0.59 Heart 0.66 ± 0.22 Lungs 1.53 ± 0.37 Liver 1.33 ± 0.30 Spleen 5.69 ± 1.33 Stomach 0.33 ± 0.09 Small Int. 0.33 ± 0.16 Large Int. 0.36 ± 0.06 Kidneys 1.87 ± 0.39 Muscle 0.46 ± 0.08 Bone 0.32 ± 0.10

Tumor 1.31 ± 1.01

Tumor-to-tissue ratios Blood 0.9 ± 0.8 Heart 2.0 ± 1.7 Lungs 0.9 ± 0.7 Liver 1.0 ± 0.8 Spleen 0.2 ± 0.2 Stomach 4.0 ± 3.6 Small Int. 3.6 ± 2.8 Large Int. 0.7 ± 0.6 Kidneys 0.1 ± 0.0 Muscle 2.8 ± 2.2 Bone 4.2 ± 3.4

Table S2. Comparison of PRIT of 177Lu-DOTA-Bn with either control IgG-scFv BsAb (A33- C825) or hu3F8-C825 in athymic nude mice bearing s.c. GD2(+)-NB tumors. Groups of animals were given 0.25 mg of BsAb followed 28 h later with 5.6 MBq (~30 pmol) 177Lu-DOTA-Bn. Biodistribution was conducted 24 h p.i. of 177Lu-DOTA-Bn. Results are given as mean %ID/g ± SEM.

PRIT with PRIT with Tissues control IgG-scFv hu3F8-C825 (n = 3) (n = 3) Blood 7.91 ± 0.43 2.50 ± 0.19 Heart 2.33 ± 0.15 0.72 ± 0.10 Lungs 3.91 ± 0.68 1.12 ± 0.08 Liver 3.55 ± 0.34 1.57 ± 0.07 Spleen 1.82 ± 0.26 1.81 ± 0.67 Stomach 0.26 ± 0.07 0.06 ± 0.00 Small Int. 0.95 ± 0.13 0.31 ± 0.01 Large Int. 0.59 ± 0.20 0.30 ± 0.02 Kidneys 3.08 ± 0.39 1.83 ± 0.16 Muscle 0.60 ± 0.15 0.27 ± 0.00 Bone 0.82 ± 0.05 0.23 ± 0.03

Tumor 2.25 ± 0.21 8.21 ± 1.50

Tumor-to-tissue ratios Blood 0.3 ± 0.0 3.3 ± 0.6 Heart 1.0 ± 0.1 11.5 ± 2.6 Lungs 0.6 ± 0.1 7.3 ± 1.4 Liver 0.6 ± 0.1 5.2 ± 1.0 Spleen 1.2 ± 0.2 4.5 ± 1.9 Stomach 8.7 ± 2.5 133.0 ± 24.3 Small Int. 2.4 ± 0.4 26.9 ± 4.9 Large Int. 3.8 ± 1.4 27.7 ± 5.4 Kidneys 0.7 ± 0.1 4.5 ± 0.9 Muscle 3.8 ± 1.0 30.4 ± 5.6 Bone 2.7 ± 0.3 35.3 ± 7.6

Table S3. Hematological and clinical chemistry analyses of athymic female nude mice bearing s.c. GD2(+)-NB tumors following PRIT therapy with hu3F8-C825, CA, and 33.3 MBq total of 177Lu-DOTA-Bn at days 10 and 28 post-treatment. Data is provided as mean ± SD. N/A = not available, WBC = white blood cells, BUN = blood urea nitrogen, CREA = creatinine, ALT = alanine aminotransferase, and AST = aspartate aminotransferase.

Normala Day 10 Day 28 post-treatment post-treatment (n = 10) (n = 4-5) (n = 5) Hematology WBC (K/uL) 2.6 ± 1.0 3.87 ± 0.36b 5.86 ± 1.28 Hemoglobin (g/dL) 15.0 ± 0.7 15.6 ± 0.83b 14.0 ± 0.15 Platelets (K/uL) 1099.6 ± 142.8 617.25 ± 212.34b 735.2 ± 45.93 Lymphocytes (K/uL) N/A 2.47 ± 0.21 b 3.27 ± 0.82 Neutrophils (K/uL) N/A 1.12 ± 0.27 b 1.9 ± 0.55 Monocytes (K/uL) N/A 0.12 ± 0.06 b 0.554 ± 0.19 Clinical Chemistry BUN (mg/dL) 30.4 ± 2.6 31.8 ± 7.92c 28.2 ± 3.83 CREA (mg/dL) 0.3 ± 0.0 0.210 ± 0.142c 0.232 ± 0.0228 ALT (U/L) 33.4 ± 5.2 N/A 37.6 ± 7.13 AST (U/L) 110.5 ± 19.3 N/A 88.0 ± 14.11 a Data from Harlan Sprague Dawley, Athymic Nude, Hsd:Athymic Nude-Foxn1nu, 16-17 week old females b (n = 4) c (n = 5)