Supplementary Information
Development of a High Affinity, Non-covalent Biologic to Add Functionality to Fabs
Kendra N. Avery, Cindy Zer, Krzysztof P. Bzymek
and John C. Williams*
Department of Molecular Medicine,
Beckman Research Institute at City of Hope
Materials and Methods:
Protein Expression and Purification
Protein L and meditope-protein L fusion protein DNA coding sequences were inserted between BamH1 and Xho1 sites of a modified pET28b vector encoding an N-terminal 6-His-tagged SMT3. Protein expression was induced with 200 M final concentration IPTG in BL21 (DE3) E. coli in LB media under Kanamycin selection for 4 hrs at 37 C. Cells were lysed by french pressure cell, clarified by centrifugation, filtered through 0.22 m filter and loaded onto a Ni-NTA column (Thermo Scientific HisPur). After washing with 10 mM imidazole in PBS, the protein was eluted with 250-500 mM imidazole. The His6-SMT3 fusion was cleaved by ULP1-His6 protease with 10-20 mM DTT while being dialyzed into PBS at 4C. The cleaved protein was further purified from ULP1 and SMT3 by reverse Ni-NTA and concentrated to 2.5-3 mL. The concentrated product was applied to a Superdex G75 preparative column (GE Healthcare) for final purification. The final product was concentrated,aliquot into small volumes and flash frozen. Protein aliquots were stored at -80 C. Each mutant construct of the meditope-proL fusion, protein L and MPL6-GFP were purified in a similar manner. Meditope peptide was synthesized at the City of Hope Synthetic Biopolymer Chemistry Core by solid phase synthesis on a semi-automated peptide synthesizer, purified by HPLC and confirmed by mass spectrum analysis. Prescription grade parental trastuzumab and cetuximab were obtained from the City of Hope Pharmacy and used without additional purification. The extracellular portion of sHER2 (sErbB2) was expressed in High 5™ insect cells and purified by Ni-NTA affinity and size exclusion chromatography as previously described1. Meditope-enabled trastuzumab was expressed in NS0 cells and purified by protein A affinity chromatography and size exclusion gel chromatography as previously described1.
Sequences
(S) is not counted in the numbering scheme as it is a residual amino acid from the cleavage of the His6-SMT3 tag by ULP1 protease.
MPL6
(S)CQFDLSTRRLKCGGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
meditope
CQFDLSTRRLKC
protein L
(S)EVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL6 variant F3A R8A
(S)CQADLSTARLKCGGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL6 variant Y51W L55H
(S)CQFDLSTRRLKCGGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRWAALHAKVNGEYTADLEDGGNHMNIKFAG
MPL no linker
(S)CQFDLSTRRLKCVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL3 (GSE)
(S)CQFDLSTRRLKCGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL9 (GSGSGGGSE)
(S)CQFDLSTRRLKCGSGSGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL3 (PSE)
(S)CQFDLSTRRLKCPSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL3 (PPE)
(S)CQFDLSTRRLKCPPEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL3 (PPP)
(S)CQFDLSTRRLKCPPPVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL6 Pro (PPPPPP)
(S)CQFDLSTRRLKCPPPPPPVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL6 (PPPGPP)
(S)CQFDLSTRRLKCPPPGPPVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL6 (GGPGSE)
(S)CQFDLSTRRLKCGGPGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAG
MPL6-GFP
(S)CQFDLSTRRLKCGGGGSEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYAALLAKVNGEYTADLEDGGNHMNIKFAGGGSGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
SPR Measurements
Protein concentrations were calculated by measuring the absorbance at 280 nm using extinction coefficients estimated by Expasy Protparam tool. All SPR measurements were performed at 25 °C on the GE Biacore T100 instrument using Series S CM5 sensor chips. All ligands (trastuzumab, cetuximab, memAb trastuzumab or sHER2) were amine coupledusing EDC/NHS chemistry in GE acetate buffer pH 5.5 at densities suitable for kinetics experimentsusing the equation:
RL=Rmax X (ligand MW/analyte MW) X 1/Sm
(Rmax values between 50-150 RU were used and typically 10% more ligand was immobilized to account for non-productive coupling orientations). Typical experiments with MPL variants as analyte had memAb trastuzumab immobilized at ~1200 RU and meditope as analyte ~5000 RU. Surface densities of 300-700 RU were necessary to achieve Rmax signals from 50-150 for the immobilization of HER2 binding to MPL(GFP)/Fab experiments.The running buffer for all experiments was GE buffer HBS-EP+ (10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% v/v surfactant P20). Kinetics experiments were carried out at a flow rate of 30 L/min using HBS-EP+ as both running and regeneration buffer in the case of the meditope binding experiments. For the MPL to memAb and Fab binding to HER2 experiments, 10 mM glycine pH 2.0, 1 M NaCl was used as regeneration buffer, followed by a wash with HBS-EP+. Due to the stringent regeneration condition, experiments could not all be carried out on the same chip due to ligand damage over time, therefore replicate experiments were frequently performed on different chips with similar coupling densities. This ligand damage over time due to the pH 2.0 regeneration is likely the reason for the relatively high standard deviation values on some of our measurements in addition to the lack of curvature in the off rate sensograms. In the experiments with memAb trastuzumab Fab and memAb trastuzumab Fab + 500 nM MPL6, the same concentrations of Fab were used in each experiment, but 500 nM MPL6 remained constant in each of the memAb Fab with MPL samples. The 0.625 concentration was removed from the data fit in figure 3B due to an air bubble. Figure 3 data were performed on the same chip for direct comparison and figure S4 shows data collected from different chips. For the memAb Fab with MPL6-GFP experiments, the memAb Fab concentration was varied while MPL6-GFP was held constant at 100 nM in each sample. Kinetic parameters were calculated using Biacore T100 Evaluation software version 2.0.1 using the 1:1 binding model.Kinetic parameters were calculated for each individual experiment and theaverages and standard deviations of replicate measurements are reported.
Alexa Fluor labeling of MPL6 and mAbs
Parental and memAb trastuzumab were labeled with Alexa Fluor 488 according to manufacturer's protocol (Invitrogen). MPL6 was labeled at 7 mg/ml with ten fold more Alexa Fluor 647 than recommended in the protocol and three hours of labeling reaction at room temperature to account for the inefficiencies in labeling a small protein.
FACS Analysis
SKBR3 cells were maintained in 10% FBS-supplemented DMEM at 37 C and 5% CO2 humidifed atmosphere. Each sample contained 1 million SKBR3 cells. Cells were trypsinized and washed once with 1 mL wash buffer (0.3% BSA in PBS), and then incubated with 100 L of 10 nM either mAb and 50 nM of MPL6 for 30 min on ice. Alternatively, cells were first incubated with 100 L of 10 nM either mAb for 15 min, washed once with 1 ml wash buffer, then another 15 min with 100 L of 50 nM MPL6 on ice. At the end of the incubation, the cells were washed twice with 1 mL wash buffer, and resuspended in 1 mL of wash buffer supplemented with SYTOX® Blue dead cell stain (Invitrogen). FACS were performed on a CyAn™ ADP Analyzer (Beckman Coulter), and the data were analyzed with Flowjo software.
Microscopy
50,000 SKBR3 cells were seeded onto cover slips in 12 well plates over night. The culture medium was replaced with 250 L of medium containing 50 nM Alexa Fluor 555 labeled-memAb or parental trastuzumab and 250 nM MPL6-GFP at 37°C for 30 min. The cells were washed twice with pre-warmed PBS, fixed with room temperature 3.7% formaldehyde diluted in PBS, washed twice again with room temperature PBS, and then mounted with ProLong® Gold Antifade Reagent with DAPI (Invitrogen) onto glass slides. Images were taken with Olympus IX81 automated inverted microscope at 64 x magnification. Images were analyzed with Photoshop.
Supplemental Figures and Tables:
Supplemental Figure 1RepresentativeSPR sensograms with residuals showing binding avidity of A) MPL6 and the loss of avidity for B) monomeric meditope, C) protein L, D) MPL F3A R8A and E) MPL Y51W L55H with immobilized memAb trasstuzumab IgG. Analyte concentrations include A) 10 nM - 78 pM, B) 16 µM - 62.5 nM, C) 1 µM - 15.6 nM, and D-E) 2 µM -20 nM.
Supplemental Figure 2Representative SPR sensograms with residuals showing the binding of A) MPL no linker, B) MPL3 (GSE), and C) MPL9 (GSGSGGGSE) to immobilized memAb trastuzumab. Analyte concentrations include 78 pM - 10 nM.
Supplemental Figure 3 Representative SPR sensograms with residuals showing the binding interaction of A) MPL3 (PSE), B) MPL3 (PPE), C) MPL3 (PPP), D) MPL6 (PPPPPP)E) MPL6 (PPPGPP) and F) MPL6 (GGPGSE) with immobilized meditope-enabled trastuzumab. Analyte concentrations include 78 pM to 10 nM for A-C, E-F and 2.5 nM - 320 nM for D).
Supplemental Figure4Representative SPR sensograms with residuals showing the binding of A) memAb Fab with immobilized HER2, B) memAb Fab pre-incubated with excess MPL6 to immobilized HER2, C) MPL6-GFP to immobilized memAb and D) memAb Fab pre-incubated with excess MPL6-GFP with immobilized HER2. Analyte concentrations of memAb Fab in A-B and D include 78 pM - 10 nM, MPL6 remained constant at 500 nM in all dilutions for B) and MPL6-GFP remained constant at 100 nM in all dilutions for D). MPL6-GFP concentrations in C) were 10 nM - 78 pM.
Supplemental Figure 5Representative SPR sensogram with residuals showing the binding interaction of MPL6 with immobilized parental trastuzumab. Analyte concentrations include 7.8 nM - 2 µM.
Supplemental Table 1. Summary of dissociation and kinetic constants calculated from SPR analysis for engineered IgG binding proteins with memAb and memAb Fab complexed to IgG binding proteins with antigen.
Analyte / Immobilized Ligand / kon(X 105) / koff
(X 10-4) / KD
(X 10-10)
MPL6 / memAb trastuzumab / 7.9 +/- 3.2 / 1.8 +/-1.6 / 1.8 +/- 0.4
MPL6-GFP / memAb trastuzumab / 3.1 +/- 3.7 / 1.9 +/- 1.1 / 13 +/- 8
memAb Fab / HER2 / 14 +/- 6 / 2.4 +/- 1.1 / 1.9 +/- 1.0
memAb Fab + MPL6 / HER2 / 14 +/- 1 / 1.9 +/- 0.5 / 1.3 +/- 0.4
memAb Fab + MPL6-GFP / HER2 / 6.5 +/- 2.7 / 3.1 +/- 1.8 / 4.5 +/- 0.7
References
1) J. M. Donaldson, C. Zer, K. N. Avery, K. P. Bzymek, D. A. Horne, J. C. Williams, Proc Natl Acad Sci U S A 2013, 110, 17456-17461.