Engineered Anti-CD70 Antibody-Drug Conjugate with Increased Therapeutic Index

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1 Engineered Anti-CD70 Antibody-Drug Conjugate with Increased Therapeutic Index C. F. McDonagh et al. Supplementary Material Methods Construction and Expression of Anti-CD70 Antibody Variants and Fcγ Receptors Complementary DNA clones for hufcγri (IMAGE clone ), hufcγriiia V158 (IMAGE clone ) and hufcεri γ chain (IMAGE clone ) were obtained from Invitrogen (Carlsbad, CA) and cloned into pdef38. A hufcγriib cdna clone (TC110889) was obtained from Origene (Rockville, MD) and cloned into pdef38. The hufcγri and hufcγriib expression constructs were linearized (100 μg) and then electroporated into CHO-DG44 (1) cells. Linearized hufcγriiia V158 and hufcεri γ chain expression constructs (50 μg each) were coelectroporated into CHO-DG44 cells. Stable cell lines expressing the hufcγr were selected as described for IgG expression. Highly expressing clones were identified by flow cytometry and recovered by limiting dilution cloning. Complementary DNA clones for mufcγriib (porf-mfcgr2b), mufcγriiia (porf-mfcgr3) and mufcγriv (porf-mfcgr4) were obtained from InvivoGen (San Diego, CA) and cloned into pdef38. Complementary DNA clones for mufcγri and mufcεrγ chain were synthesized (Biobasic, Markham, Ontario) and cloned into pdef38. The mufcγriib expression construct was linearized (100 μg) and then electroporated into CHO-DG44 cells. Linearized mufcγri, mufcγriiia and mufcγriv were cotransfected with mufcεrγ chain (50 μg each) by electroporation into CHO- DG44 cells. Stable cell lines expressing the various mufcγr were selected as described for IgG expression. Highly expressing clones were identified by flow cytometry and recovered by limitingdilution cloning. McDonagh et al., 5/17/

2 Antibody Purification The anti-cd70 IgG variants were purified by protein A chromatography using an ÄKTAexplorer FPLC (GE Healthcare, Piscataway, NJ). Endotoxin removal as well as purification of anti-cd70 IgG1 and IgG1v1 from cell culture conditioned media were performed as previously described for cac10 anti-cd30 IgG1 variants (2) with the following modifications. Antibody was eluted from the protein A columns with 45 mmol/l sodium acetate, ph 3.4, 20 mmol/l NaCl. The pooled fractions were dialyzed against 45 mmol/l sodium acetate buffer containing 75 mmol/l NaCl, 5% v/v glycerol, ph and precipitated contaminants removed by centrifugation. The antibody remained in solution and was used directly or alternatively dialyzed into PBS, ph 7.4. Purification of anti-cd70 IgG4 and IgG4v3 was performed as for the IgG1, except that 16 mmol/l Na 2 HPO 4, 3 mmol/l KH 2 PO 4, 5 mmol/l KCl, mol/l NaCl, ph buffer was used for diafiltration, column equilibration, and column washes. For IgG2 purification 24 mmol/l Na 2 HPO 4, 4.4 mmol/l KH 2 PO 4, 8 mmol/l KCl, 0.41 mol/l NaCl, ph 7.2 buffer was used for diafiltration, column equilibration, and column washes, and endotoxin was removed by treating the sample with 0.5% v/v Triton X-100 with gentle stirring for1 h at 4 C followed by washing the column with 5 column volumes 0.5% v/v Triton X-100 after sample was loaded onto the column. Purified antibodies were analyzed by SDS-PAGE and by TSK-Gel G3000SW HPLC size exclusion chromatography (Tosoh Bioscience, Montgomeryville, PA). Endotoxin Measurement Endotoxin levels in all antibody and ADC preparations were 0.3 EU/mg as determined by kinetic quantitative chromogenic Limulus amebocyte assay (Cambrex, Walkersville, MD) performed as previously described (2). ELISA Analysis of Pharmacokinetic Samples Serum concentrations of ADCs were measured by sandwich ELISA for total antibody. 96-well plates were coated with 1.0 μg/ml anti-1f6 (anti-idiotype) antibody in 100 mmol/l sodium carbonate-bicarbonate buffer, ph 9.6 at 4 o C overnight. After the plates were blocked with PBS, ph 7.4, 1% BSA, 0.05% Tween 20), 100 μl standard or diluted serum samples in triplicate were added McDonagh et al., 5/17/

3 and incubated for 1 h. Biotinylated anti-1f6 antibody was added to each well (0.1 μg/ml) and plates were incubated for 1 h at room temperature followed by addition of horseradish peroxidaseconjugated streptavidin (Pierce) at a 1:50,000 dilution and incubation for an additional 1 h. 100 μl of horseradish peroxidase substrate, 3,3,5,5 -tetramethylbenzadine (Sigma, St. Louis, MO) was added to each well and the reaction was quenched with 100 μl 1.0 mol/l sulfuric acid. Absorbance was measured using a VMax Kinetic Microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm and a blank at 630 nm. Serum concentration data for each animal were subjected to noncompartmental and compartmental analysis using WinNonlin version (Pharsight, Mountain View, CA). SDS-PAGE Humanized anti-cd70 IgG variant samples were prepared in 4x NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA) supplemented with NuPAGE reducing agent, 0.5 mol/l DTT, or alternatively with 0.1 mol/l N-ethylmaleimide (NEM) to suppress disulfide bond scrambling as described by Taylor et al. (3). The samples (~4.5 μg) were incubated for 20 min at 87 o C, loaded on the NuPAGE 4-12% Bis-Tris gels, and electrophoresed using NuPAGE MES SDS running buffer under reducing or non-reducing conditions as instructed by the manufacturer (Invitrogen). The gels were fixed in 10% acetic acid for 10 min at room temperature and stained with SimplyBlue TM staining solution (Invitrogen) by microwaving for 25 s, followed by 45 min incubation at room temperature. The gel staining was repeated with fresh new staining solution as indicated above. Gels were destained in 50% MeOH for 30 min, then in 10% MeOH/10% acetic acid for 3.5 h at room temperature. The gels were scanned using EPSON Perfection 2400 Photo (Epson) at 16-bit grayscale, 2400 dpi interfaced with Adobe Photoshop Elements software. McDonagh et al., 5/17/

4 Supplementary Figure 1. SDS-PAGE analysis of humanized anti-cd70 IgG variants under nonreducing and reducing conditions. Samples were prepared as described in the Supplementary Material Methods. Supplementary Figure 2. Single and multiple dose efficacy of anti-cd70 IgG variant ADCs against established tumor xenografts. Median tumor volume plots for the same experiments for which mean tumor volume plots are shown in Fig. 4. Data were plotted for each group until one or more animals died or was euthanized (see Materials and Methods). A, vcf4 ADCs in 786-O renal cell cancer model. B, vcf4 ADCs in DBTRG05-MG glioblastoma model. C, vcf4 ADCs in UMRC3 renal cell cancer model. D, mcf4 ADCs in 786-O model. Groups of nude mice with ~90 mm 3 subcutaneous tumors were dosed (arrows) by intravenous injection once for 786-O and DBTRG05-MG models or every 4 days times 4 for the UMRC3 model. There were 8 or 9 mice per group, except for the untreated group in A (n = 6). Data shown are representative of independent experiments. McDonagh et al., 5/17/

5 References 1. Urlaub G, Mitchell PJ, Kas E, et al. Effect of gamma rays at the dihydrofolate reductase locus: deletions and inversions. Somat Cell Mol Genet 1986;12: McDonagh CF, Turcott E, Westendorf L, et al. Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment. Protein Eng Des Sel 2006;19: Taylor FR, Prentice HL, Garber EA, Fajardo HA, Vasilyeva E, Blake Pepinsky R. Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody. Anal Biochem 2006;353: McDonagh et al., 5/17/