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

Abstract

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 m 1 > 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.

A Modeling Framework to Characterize Cytokine Release upon T‐Cell–Engaging Bispecific Antibody Treatment: Methodology and Opportunities

“…P‐cadherin LP DART (PF‐06671008) is an anti‐P‐cadherin × anti‐CD3 bispecific dual affinity retargeting (DART, MacroGenics, Rockville, MD) molecule with a human Fc domain fusion developed for the treatment of solid tumors. In nonclinical in vitro studies, it mediated redirected T‐lymphocyte cytotoxicity toward P‐cadherin‐expressing tumor cell lines and induced release of cytokines . In this manuscript, it was used as a case study for solid tumor indications.…”

Development of a Target cell-Biologics-Effector cell (TBE) complex-based cell killing model to characterize target cell depletion by T cell redirecting bispecific agents

“…In doing so, our TBE model was able to capture the in vitro cytotoxicity data at various effector cell concentrations using one single intrinsic engagement potency value EC 50 (Table 1, S1, and S2, Figure 2, S2, 3e and 3f), suggesting that the intrinsic engagement potency of a bsAb, i.e., the number of TBE complexes between one effector cell and one target cell sufficient to activate the engaged T cells, is a critical parameter for assessing T-cell redirecting bsAb-mediated target cell killing. The EC 50 for a T-cell redirecting bsAb is expected to be determined by CD3 and target receptor binding epitopes, 53,54 the bsAb architecture, 31 and the sensitivity of individual target cells to T cell killing (Tables 1 and 2, Figure 2 and S2) . 2,33,53 The exact mechanism for how T-cell redirecting bsAbs trigger T cell activation and subsequent target cell killing has yet to be elucidated, but some bsAb architectures and epitopes appear to be more potent than others.…”

“…The EC 50 for a T-cell redirecting bsAb is expected to be determined by CD3 and target receptor binding epitopes, 53,54 the bsAb architecture, 31 and the sensitivity of individual target cells to T cell killing (Tables 1 and 2, Figure 2 and S2) . 2,33,53 The exact mechanism for how T-cell redirecting bsAbs trigger T cell activation and subsequent target cell killing has yet to be elucidated, but some bsAb architectures and epitopes appear to be more potent than others. 31,53,54 Taken together, these findings support the mechanistic nature and biological relevance of our TBE model.…”

“…2,33,53 The exact mechanism for how T-cell redirecting bsAbs trigger T cell activation and subsequent target cell killing has yet to be elucidated, but some bsAb architectures and epitopes appear to be more potent than others. 31,53,54 Taken together, these findings support the mechanistic nature and biological relevance of our TBE model.…”

Envelope-specific antibodies and antibody-derived molecules for treating and curing HIV infection

“…The crystal structure of a disulfide-constrained DART molecule showed that it assembles into a novel compact structure, with the two antigen-binding sites separated from each other by approximately 30 Å and facing approximately 90° apart 162 . The orientation and short distance between the antigen-binding domains of the DART protein may facilitate more efficient synapse formation between the T cell and the target cell and contribute to an increase in potency of T cell-directed lysis compared with other bsAb formats.…”