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 (T_m1 > 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.

Purpose: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy. Experimental Design: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ϵ transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti–PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. Results: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ϵ bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell–mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti–PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. Conclusions: These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

LOX‐1 is a scavenger receptor that functions as the primary receptor for oxidized low‐density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX‐1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX‐1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand‐binding function of cell surface LOX‐1, we generated a series of recombinant human LOX‐1–crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand‐binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX‐1 is required for high‐avidity binding of ligands, we found that LOX‐1–Fc fusion protein containing four ligand‐binding domains per Fc dimer, but not the one containing two ligand‐binding domains, exhibited ligand‐binding activity. Optimal ligand‐binding activity could be achieved via crosslinking of LOX‐1–Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX‐1–Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX‐1. These findings demonstrate that functional oligomerization is required for recombinant LOX‐1–Fc to function as an effective antagonist.Structured digital abstract MINT‐7213199: LOX1 (uniprotkb:P78380) and LOX1 (uniprotkb:P78380) physically interact (MI:0914) by molecular sieving (MI:0071)

Mouse body weights of CT26-mGUCY2C tumor bearing hCD3ε transgenic mice treated with PF-07062119

PF-07062119 binds to Cynomolgus Monkey T cells