Molecular design and assembly
The anti-CEA M5A mAb served as a human IgG1 scaffold for fusion of the anti-DOTA VH-VL C8.2.5 scFv Ab. The various BsAb cDNA constructs were formed by splice overlap PCR (Horton et al., 1989
) with PCR primers that incorporate flexible amino acid linkers. The C-terminal (Coloma and Morrison, 1997
) and N-terminal BsAbs fuse the C8.2.5 scFv by a glycine serine (G4
amino acid linker to the 5′ or 3′ end of the M5A IgG1 heavy chain cDNA, respectively. The LC fusion has the C8.2.5 scFv attached to the 3′ end of the human kappa LC cDNA (Orcutt et al., 2010
). For the DVD, the cDNA encoding the C8.2.5 scFv cDNA was reformatted into a full-length IgG1. The M5A variable domains were then fused to the 5′ end of the C8.2.5 variable domains by a VKappa
linker (Wu et al., 2009
). The M5A-C825 BsAb heavy and LC genes were cloned into pEE12/6 dual-gene vector as part of the glutamine synthetase mammalian expression/selection system (Lonza Biologics, Slough, UK).
Expression and purification
Transient transfection of the plasmids encoding the BsAbs was conducted using linear 25 kDa PEI (Polysciences, Warrington, PA, USA) into Freestyle™ 293-F cell line following the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Five- to seven–day-old culture supernatants were screened for anti-CEA activity by enzyme-linked immunosorbent assay and protein quantified by Protein A as previously described (Yazaki and Wu, 2004
Purification of the BsAbs used a two-step procedure consisting of Protein A capture and ceramic hydroxyapatite chromatography. Briefly, the culture supernatant was clarified in batch with 5% v/v AG1-X8 resin (Bio-Rad Laboratories, Hercules, CA, USA), sterile filtered and loaded on a Prosep rA high-capacity column (4.6 mm × 100 mm, 1 ml/min; Millipore, Billerica, MA, USA). The column was washed in 0.01 M sodium phosphate, 1 M NaCl, 0.05% Tween-80, pH 7 and eluted with 0.05 M NaCl, 0.01 M sodium phosphate, 0.05% Tween-80, pH 3. Eluted fractions were collected in tubes containing 10% v/v of 50 mM sodium phosphate, pH 8 for neutralization. Pooled fractions were pH adjusted with 25% v/v of 0.1 M MES pH 6.5 buffer and loaded on a ceramic hydroxyapatite CHT® type I column (4.6 mm × 100 mm, 1 ml/min; Bio-Rad Laboratories) pre-equilibrated in 0.05 M MES, 0.01 M potassium phosphate, pH 6.5. A linear gradient from 0.01 to 0.5 M potassium phosphate pH 6.5 eluted the fusion protein in a single peak and the purified material was dialyzed vs. phosphate-buffered saline prior to concentration (Ultracel—30 k, Millipore).
Characterization of the BsAbs
Aliquots of the purified proteins were analyzed for protein quantification by UV absorbance 280 nm. Purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions and high-performance liquid chromatography (HPLC) size-exclusion chromatography (SEC) on a Superdex 200 10/300 column (GE Healthcare, Piscataway, NJ, USA) as previously described (Yazaki and Wu, 2004
Anti-CEA and anti-DOTA binding affinities were measured by surface plasmon resonance (SPR) using a Biacore X100 instrument (GE Healthcare). For the anti-CEA measurements, biotinylated CEA (~500 RU) was immobilized on a Sensor Chip SA, following the manufacturer's instructions. HBS-EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA and 0.005% surfactant P20) was used as running and diluent buffer (GE Healthcare). Using the kinetic affinity program, serial concentrations (15.6–500 nM) of the BsAbs and parental M5A mAb were injected at a flow rate of 30 µl/min at 25°C. The data were analyzed using the bivalent analyte model on the Biacore X100 BIAevaluation 2.0 software (GE Healthcare). The kd1/ka1 data were used to calculate KD1, to compare the relative binding affinity of the Ab variants. To measure anti-DOTA affinity, p-SCN-Bn-DOTA (Macrocyclics, Dallas, TX, USA) was conjugated to human serum albumin (HSA) at a ratio of 50 : 1, purified by SEC Superdex 75 (GE Healthcare) and loaded with non-radioactive 89YCl metal. The 89Y-DOTA-Bn-HSA (~1000 RU) was conjugated to a CM5 sensor chip following the manufacturer's instructions.
Binding to FcRn was also measured by SPR using a Biacore 3000 as previously described (Andersen et al., 2008
). Flow cells of CM5 sensor chips were coupled with recombinant forms of soluble human or mouse FcRn (shFcRn or smFcRn; ~500 RU) using amine-coupling chemistry. The coupling was performed by injecting 1 μg/ml of the protein in 10 mM sodium acetate pH 5.0. Phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20, at pH 6.0), or HBS-EP buffer was used as running and dilution buffer. Titrated amounts of the BsAbs and parental M5A mAb (3–100 nM) were injected over immobilized receptor at pH 6.0 with a flow rate of 50 μl/min at 25°C. Regeneration of the surfaces was done using injections of HBS-EP buffer at pH 7.4 In all experiments, data were zero adjusted and the reference cell subtracted. Data were evaluated using the heterogeneous ligand binding model supplied with the BIAevaluation 4.1 software and reported as KD1 and KD2.
The BsAbs were radiolabeled with Iodine-125 (125
I), using the Iodogen methodology as previously described (Yazaki et al., 2008
). All radiolabeled Abs were purified by HPLC SEC on Superdex 200, 10/300 (GE Healthcare).
For dual radiolabeling, the LC BsAb and M5A mAb were conjugated with NHS-DOTA (Macrocyclics) at a ratio of 50 : 1. Each DOTA-mAb was radiolabeled separately with 111InCl (specific activity = 5.4 µCi/µg) or Na125I (specific activity = 10.1 µCi/µg) and purified by HPLC SEC. The two radiolabeled versions were mixed, 4 µCi 111In-LC-BsAb to 10 µCi 125I-LC-BsAb, and co-injected into athymic mice bearing tumor xenografts (see below). Tissues were counted in a calibrated dual-isotope window.
All radiolabeled antibodies were analyzed for immunoreactivity to soluble CEA by a liquid phase assay incubating the radiolabeled protein with 20 equivalents by mass of purified CEA at 37°C for 15 min. The resultant solution was analyzed by HPLC-SEC using a Superose 6 10/300 GL column (GE Healthcare). Anti-CEA immunoreactivity was determined by integrating the area on the HPLC radiochromatogram and calculating the percentage of radioactivity shifting to higher molecular weights, consistent with binding to CEA (180 kDa).
Protein stability studies were performed on the 125I-LC BsAb incubated in either saline or fresh mouse serum at 37°C. Aliquots were analyzed on an HPLC SEC Superose 6 column at the time points of 0, 4, 24 and 48 h.
Animal biodistribution and imaging studies in tumor-bearing mice
All animal handling was done in accordance with protocols approved by the City of Hope Institutional Animal Care and Use Committee. Seven- to eight-week-old female athymic mice (Charles River Laboratories, Wilmington, MA, USA) were injected subcutaneously in the flank region with 106 LS-174T human colon carcinoma cells obtained from ATCC. After 10 days, when tumor masses were in the range of 0.1–0.5 g, 1–2 µg (specific activity ~10 µCi/µg) of the purified 125I-M5A-C8.2.5 BsAbs were injected via the tail vein per animal. At selected time points (0, 4, 24, 48 and 72 h), groups of four to five mice were euthanized, necropsy performed, organs weighed and counted for radioactivity. All data presented are mean values with the standard error (±SEM) and have been corrected for background and radioactive decay from the time of injection, allowing organ uptake to be reported as percentage of the injected dose per gram (% ID/g).