Development of PF-06671008, a Highly Potent Anti-P-cadherin/Anti-CD3 Bispecific DART Molecule with Extended Half-Life for the Treatment of Cancer

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART®) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (~4.4 days in human FcRn knock-in mice), high stability (Tm1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

commercially-available anti-human P-cadherin monoclonal and polyclonal antibodies (R & D Systems, Minneapolis, MN, USA). Purified recombinant human CD3 epsilon-delta protein was acquired from MacroGenics.

S.2. Development of Engineered Cell Lines Expressing P-Cadherin
P-cadherin ortholog expressing cell lines were generated by performing stable transfections in Chinese Hamster Ovary (CHO-Dukx) or SW480 primary colon (ATCC CCL-228) cell lines. cDNA was obtained for human (catalog number SC303069; accession number NM_001793.3) and mouse P-cadherin ECD (catalog number MC221848; accession number NM_007665.3) from a commercial source (Origene, Rockville, MD, USA). Using conventional molecular biological techniques, the full gene (including pro-peptide region) was cloned into a Pfizer-proprietary mammalian expression vector sequence-confirmed. Pilot expression analysis indicated that the pro-peptide processing of P-cadherin ECD was about 50% or less. For enhanced pro-peptide processing, CHO cells harboring soluble PACE stably integrated in its genome were used [42]. Following transfection of the expression vector encoding the P-cadherin ortholog with the Lipofectamine LTX and Plus™ reagent (Life Technologies, Grand Island, NY, USA), CHO cells were selected in 20, 50, 100 nM Methotrexate (MTX) + 1 mg/mL G418 (Geneticin, Life Technologies, Grand Island, NY, USA) with 20 nM MTX chosen for lead human clonal line and 100 nM for lead murine clonal line.

S.3. Preparation of Phage Expressing scFv for Use in ELISAs
To prepare phage expressing scFv on their surface, 96-deep well plates containing 1 mL 2× YT media with 2% glucose/100 μg/mL ampicillin were inoculated with 0.5-1 μL from thawed glycerol stocks (one clone per well) using the QPix™ 2 Colony picker (Molecular Devices, Sunnyvale, CA, USA) and grown at 37 °C (900 rpm) for ~4 h. Next, 5 μL of a 1:29 dilution of helper phage (8.3 × 10 13 pfu) was added and the plates and incubated for a further 30 min at 37 °C with no shaking then 1 h at 300 rpm. Plates were centrifuged and the media was replaced with a kanamycin/non-glucose containing media (2× YT with 50 μg/mL kanamycin and 100 μg/mL ampicillin). Plates were grown overnight at 25 °C (900 rpm), and phage were harvested in the supernatant following centrifugation.

S.3.1. Transient DART Protein Expression
For transient transfections, expression vectors encoding the DART protein variants were co-transfected into HEK FreeStyle™ 293 cells as previously described (Johnson et al., 2010). Supernatants were collected on Day 6 and screened by ELISA for binding to P-cadherin recombinant protein and expressed on the cell surface. Expression yields were determined by Octet concentration assay (Pall, Port Washington, NY, USA). Protein A-labeled biosensors dipped in conditioned medium supernatants or controls using the Octet QK, according to the manufacturer's instructions, and DART protein concentrations were calculated using a pre-determined standard curve.

S.3.2. Purification of E-K DART Protein
DART proteins retaining P-cadherin affinity were selected for purification. DART proteins were affinity purified using an anti-coiled-coil mAb 15F1 coupled to CNBr-activiated sepharose 4B (GE Healthcare, Piscataway, NJ, USA). The affinity Sepharose resin was equilibrated in PBS, pH 7.2 before loading. Following loading, bound protein was washed with 5 column volumes (CV) equilibration buffer and eluted with 2 CV 50 mM glycine, pH 2.5 then immediately neutralized with 1 M Tris-HCl pH 8.5. The DART proteins were further purified by size exclusion chromatography (SEC) using a Superdex200HR 10/30 according to the manufacturer's protocol (GE Healthcare, Piscataway, NJ, USA). Purified DART proteins were analyzed by SDS-PAGE and analytical SEC as previously described [16].