Engineering Bifunctional Antibodies with Constant Region Fusion Architectures

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1 Engineering Bifunctional Antibodies with Constant Region Fusion Architectures Juanjuan Du, Yu Cao, Yan Liu, Ying Wang, Yong Zhang, Guangsen Fu, Yuhan Zhang, Lucy Lu, Xiaozhou Luo, Chan Hyuk Kim, Peter G. Schultz, Feng Wang California Institute for Biomedical Research, N. Torrey Pines Road, La Jolla, CA 92037, United States Department of Chemistry, The Scripps Research Institute, N. Torrey Pines Road, La Jolla, CA 92037, United States Supporting Information Materials All chemicals were purchased from common commercial sources and used as received unless otherwise specified. MDA-MB-231, MDA-MB-468, MDA-MB-453, SK-BR-3, Jurkat, HT-29 cells were purchased from ATCC (Rockville, MD) and cultured under conditions and in media recommended by ATCC. The cell line MDA- MB-435 and its respective Her2-transfected cell line, MDA-MB-435/Her2, were provided by Dr. Brunhilde H. Felding (The Scripps Research Institute, La Jolla, CA). PBMCs were purified from healthy human donor blood (provided by normal blood donor service from The Scripps Research Institute) by conventional Ficoll-Hypaque gradient centrifugation (GE Healthcare, San Diego, CA) and freshly used. Methods Gel filtration chromatography. 500 µl EPO-Herceptin-CH1 (10.5 mg/ml) or EPO- Herceptin-CL (8.2 mg/ml) in PBS was injected into a Superdex 200 column (90 ml column volume), followed by elution using the same buffer at a flow rate of 1 ml/min. Her2ScFv-SP34-CL (2 mg/ml) in PBS (ph7.4) was injected into Superdex /300 GL column, followed by elution using the same buffer at a flow rate of 0.4 ml/min. UV absorbance at 280 nm was plotted versus the elution time or elution volume. The apparent molecular weight of EPO-Herceptin-CH1, EPO-Herceptin-CL or Her2ScFv-SP34-CL was estimated on the basis of their corresponding elution volume by referring to the elution profiles of a series of standard proteins. Her2 binding affinity of EPO-Herceptin-CL and EPO-Herceptin-CH1 determined by flow cytometry. Cell-based antibody binding assays were performed using flow cytometry. Her2+ SK-BR-3 cells were maintained in DMEM supplemented with 10%FBS, 1% Pen/Strep. When SK-BR-3 cells reach 70% confluency, they were harvested by trypsinization, washed with PBS and blocked with PBS/1% BSA for 1 hour at 4 ºC. Cells were then re-suspended in PBS/1% BSA at a density of /100 µl with or without 100 nm EPO-Herceptin-CL, EPO- Herceptin-CH1 and Herceptin (positive control). The cells were then gently shaken at 4ºC for 2 hours, and were then washed with PBS for 3 times and re-suspended in PBS/1% BSA with APC-anti-human Fc (Clone HP6017, Biolegend, CA). After an additional incubation for 2 hours at 4 ºC, the cellular fluorescence was analyzed with a BD LSR II Flow cytometry. Coiled coil structure prediction. The coiled coil stalk contains ascending peptide S 1

2 (NH 2 -AKLAALKAKLAALK-COOH) and descending peptide (H2N- ELAALEAELAALEA-COOH). The structure of the stalk is predicted with PepFold 3 by simulating the structure of NH 2 -AKLAALKAKLAALKGGGGSGGGGSGGGGS ELAALEAELAALEA-COOH. 1 Thermal stability of Her2ScFv-SP34-CL. The thermal stability of Her2ScFv-SP34- CL was determined with Thermal Shift Dye Kit (ThermoFisher Scientific) according to manufacturer s instruction. Briefly, 12.5 µl 0.2 mg/ml Her2ScFv-SP34-CL in PBS was added to 5 µl Protein Thermal shift buffer, followed by 2.5 µl Diluted Protein Thermal Shift TM Dye (8x). The mixture was loaded onto a 384 well plate and the protein denaturation curve was determined with ViiA TM 7 Real-Time PCR System according to the manufacturer s manual. Dynamic light scattering studies. Dynamic light scattering (DLS) experiments were performed with a Zetasizer Nano instrument (Malvern Instruments Ltd., UK) equipped with a 10-mW helium-neon laser (λ = nm) and thermoelectric temperature controller. 0.5 ml 10 mg/ml Her2ScFv-SP34-CL in PBS is added into the cuvette. Measurements were taken at 90 scattering angle. The volume Particle Size Distribution (PSD) is plotted to characterize the aggregation of Her2ScFv-SP34- CL. Fluorescent staining. SK-BR-3 and Jurkat cells were stained with CellTracker Orange CMRA Dye (Life Technolgoy) and Calcein AM (R&D Systems), respectively, following the manufacturer s protocol. In 24-well plate, Jurkat cells ( ) and SK-BR-3 cells ( ) were incubated with Her2ScFv-SP34-CL (100 nm) or PBS in 200 μl for 12 hours at 37 ºC. Wells were gently washed with PBS (500 μl) 3 times and imaged on a fluorescent microscope (Eclipse Ti, Nikon) under FITC (for Calcein AM) and rhodamine (for CellTracker Orange CMRA) filters. The images from each channel were combined to produce an overlay images. T cell activation analysis. PBMCs were purified from fresh healthy human donor blood (from The Scripps Research Institute normal blood donor service) by conventional Ficoll-Hypaque gradient centrifugation (GE Healthcare). PBMCs were incubated with target cells in the presence of Her2ScFv-SP34-CL antibodies. After 20 h, cells were labeled with PE anti-human CD69 (BD Biosciences) and APC antihuman CD3 cells (Biolegend) and analyzed by flow cytometry. PBMCs activated with plate-bound anti-cd3 antibody (clone OKT3, ebioscience) and 2 µg/ml of soluble anti-cd28 antibody (clone CD28.2, ebioscience) were used as the positive control. The percentage of CD3+/CD69+ cells in all CD3+ cells was used to quantify the autoactivation of T cells. Serum Stability of Her2ScFv-SP34-CL. 20 µm Her2ScFv-SP34-CL was added to mouse serum to a final concentration of 1 µm. Aliquots of the mixtures were then incubated for 0 hr, 6 hr, 24 hr, 48 hr, 72 hr and 96 hr. The residual Her2ScFv-SP34- CL concentration was determined by ELISA against immobilized herbb2-fc Fc (R&D Systems) with HRP anti-human Kappa (Abcam) as secondary antibody. S 2

3 Supporting Figures. Fig. S1. A comparison of the distance between the N- and C-termini of human erythropoietin (EPO) and the axial distance of coiled-coil stalk. (a) NMR minimized average structure of human erythropoietin (EPO), PDB# 1BUY. (b) Predicted structure of coiled-coil stalk by simulating the folding of AKLAALKAKLAALKGGGGSGGGGSGGGGSELAALEAELAALEA with PepFold 3 (GGGGSGGGGSGGGGS is hidden in the figure). 1 Fig. S2. SDS-PAGE gel of purified Herceptin, EPO-Herceptin-CL (hepo.cl) and EPO-Herceptin-CH1 (hepo.ch1) with and without the treatment of 50 um DTT. S 3

4 Fig. S3. Mass spectrometry of the Herceptin full-length IgG treated with PNGase and DTT. Heavy Chain (blue): theoretical molecular weight 49252, observed (due to protease cleavage of the C-terminal lysine); light chain (green): theoretical molecular weight 23457, observed and (due to mis-processed IL2 signal peptide, calculated mass: 23972). Fig. S4. Mass spectrometry of the EPO-Herceptin-CH1 full-length IgG treated with PNGase and DTT. Heavy Chain (red): theoretical molecular weight 71268, observed (due to protease cleavage of the C-terminal lysine) and (multiple peaks, due to o-glycosylation on EPO insert); light chain (blue): theoretical molecular weight 23457, observed and (due to mis-processed IL2 signaling peptide, calculated mass: 23972). S 4

5 A280 Fig. S5. Mass spectrometry of the EPO-Herceptin-CL full-length IgG treated with PNGase and DTT. Heavy Chain (green): theoretical molecular weight Da, observed Da (due to protease cleavage of the C-terminal lysine); light chain (red): theoretical molecular weight Da, observed and Da (multiple peaks, due to o-glycosylation on EPO insert). EPO-Herceptin-CH1 EPO-Herceptin-CL time (min) Fig. S6. Gel filtration analysis of EPO-Herceptin-CH1 and EPO-Herceptin-CL in PBS (ph7.4). EPO-Herceptin-CH1 (10.5 mg/ml) or EPO-Herceptin-CL (8.2 mg/ml) was injected into Superdex 200 column (100 ml column volume), followed by elution using the same buffer. UV absorbance at 280 nm was plotted versus the elution volume. The fusions elute out at 61 min, which is slightly earlier than an average IgG molecule (64 min under the same conditions), corresponding to their slightly larger molecular weights than IgG (200 vs 160 kda). The humps eluted before the main peak might be dimers and oligomers. S 5

6 Mean Fluorescence Herceptin EPO-Herceptin-CL EPO-Herceptin-CH conc. (nm) Fig. S7. Cellular mean fluorescence (mean±sd from duplicated experiments) plotted as a function of protein concentration. For Herceptin, EC 50 = 15.3±4.0 nm; EPO- Herceptin-CL, EC 50 = 7.5±4.3 nm; EPO-Herceptin-CH1, EC 50 = 11.0±2.3 nm. Fig. S8. The distance between the N-terminus and C-terminus of Herceptin scfv is approximately 3.4 nm (PDB# 4x4x). S 6

7 mau ml Fig. S9. Size exclusion chromatography (SEC) of Her2ScFv-SP34-CL in PBS buffer. 2 mg/ml Her2ScFv-SP34-CL (after protein G chromatography purification) is loaded on the Superdex /300 GL column and eluted with PBS. The absorbance at 280 nm is recorded and plotted as a function of elution time (blue curve). The major peak is collected, concentrated and re-loaded on the Superdex /300 GL column. The absorbance at 280 nm is recorded and plotted as a function of elution time (magenta curve). Figure S10. SDS-PAGE gel of purified SP34 Fab (Lane 1) and Her2ScFv-SP34-CL (Lane 2) with and without the treatment by 50 um DTT. S 7

8 volumn % Fig. S11. Mass spectrometry of Her2ScFv-SP34-CL after reducing with 50 mm DTT. Heavy Chain (black): theoretical molecular weight Da, observed Da. Light chain (green): theoretical molecular weight Da, observed Da size (nm) Fig. S12. The hydrodynamic radius distribution of 10 mg/ml Her2ScFv-SP34-CL in PBS buffer at room temperature determined by dynamic light scattering. 0.5 ml 10 mg/ml Her2ScFv-SP34-CL in PBS is added into the cuvette. Measurements were taken at 90 scattering angle. The result shows the hydrodynamic diameter of Her2ScFv-SP34-CL is 11±4 nm without observable aggregation. S 8

9 d (F lu o r e s c e n c e )/d T H e r2 S c F v -S P 3 4 -C L -1 H e r2 S c F v -S P 3 4 -C L T ( C ) Fig. S13. The thermal stability of Her2ScFv-SP34-CL. 0.2 mg/ml Her2ScFv-SP34- CL in PBS mixed with the Thermal shift buffer and Thermal shift dye in the Thermal Shift Dye Kit (ThermoFisher Scientific). The protein denaturation curve was determined with ViiA TM 7 Real-Time PCR System. Her2ScFv-SP34-CL-1 and Her2ScFv-SP34-CL-2 are duplicate experimental curves. Fig. S14. The body weight change of NSG mice inoculated with MDA-MB-453 (Her2 2+) or MDA-MB-435 (Her2 1+) cells, treated with Her2ScFv-SP34-CL or saline (n=5). S 9

10 Supporting Tables Table S1. Plasmids used for fusion protein expression Protein Heavy Chain Light Chain EPO-Herceptin-CH1 EPO-Herceptin-CH1 HC 20 µg Herceptin LC 10 µg EPO-Herceptin-CL Herceptin HC 15 µg EPO-Herceptin-CL LC 15 µg Her2ScFv-SP34-CL SP34 Fab HC 15 µg Her2ScFv-SP34-CL LC 15 µg Table S2. Cytotoxic activity of PBMCs induced by different Her2ScFv-SP34-CL against various human breast cancer cells after 24 h treatment. MDA-MB-435/Her2 SK-BR-3 MDA-MB-231 EC50 (nm) Reference: (1) Lamiable, A.; Thévenet, P.; Rey, J.; Vavrusa, M.; Derreumaux, P.; Tufféry, P. Nucleic Acids Research 2016, 44 (W1), W449. S 10