Donna Marie Stone

The Application of Target Information and Preclinical Pharmacokinetic/Pharmacodynamic Modeling in Predicting Clinical Doses of a Dickkopf-1 Antibody for Osteoporosis

PF-04840082 is a humanized prototype anti-Dickkopf-1 (Dkk-1) immunoglobulin isotype G2 (IgG2) antibody for the treatment of osteoporosis. In vitro, PF-04840082 binds to human, monkey, rat, and mouse Dkk-1 with high affinity. After administration of PF-04840082 to rat and monkey, free Dkk-1 concentrations decreased rapidly and returned to baseline in a dose-dependent manner. In rat and monkey, PF-04840082 exhibited nonlinear pharmacokinetics (PK) and a target-mediated drug disposition (TMDD) model was used to characterize PF-04840082 versus Dkk-1 concentration response relationship. PK/pharmacodynamic (PK/PD) modeling enabled estimation of antibody non-target-mediated elimination, Dkk-1 turnover, complex formation, and complex elimination. The TMDD model was translated to human to predict efficacious dose and minimum anticipated biological effect level (MABEL) by incorporating information on typical IgG2 human PK, antibody-target association/dissociation rates, Dkk-1 expression, and turnover rates. The PK/PD approach to MABEL was compared with the standard “no adverse effect level” (NOAEL) approach to calculating clinical starting doses and a pharmacological equilibrium method. The NOAEL method gave estimates of dose that were too high to ensure safety of clinical trials. The pharmacological equilibrium approach calculated receptor occupancy (RO) based on equilibrium dissociation constant alone and did not take into account rate of turnover of the target or antibody–target complex kinetics and, as a result, it likely produced a substantial overprediction of RO at a given dose. It was concluded that the calculation of MABEL according to the TMDD model was the most appropriate means for ensuring safety and efficacy in clinical studies.

Specific inhibition of Notch1 signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells

Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly, Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs, with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival.

Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (KD values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent KD and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.

Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another.

Here we describe how real-time label-free biosensors can be used to identify antibodies that compete for closely adjacent or minimally overlapping epitopes on their specific antigen via a mechanism of antibody displacement. By kinetically perturbing one another’s binding towards their antigen via the formation of a transient trimolecular complex, antibodies can displace one another in a fully reversible and dose-dependent manner. Displacements can be readily identified when epitope binning assays are performed in a classical sandwich assay format whereby a solution antibody (analyte) is tested for binding to its antigen that is first captured via an immobilized antibody (ligand) because an inverted sandwiching response is observed when an analyte displaces a ligand, signifying the antigen’s unusually rapid dissociation from its ligand. In addition to classifying antibodies within a panel in terms of their ability to block or sandwich pair with one another, displacement provides a hybrid mechanism of competition. Using high-throughput epitope binning studies we demonstrate that displacements can be observed on any target, if the antibody panel contains appropriate epitope diversity. Unidirectional displacements occurring between disparate-affinity antibodies can generate apparent asymmetries in a cross-blocking experiment, confounding their interpretation. However, examining competition across a wide enough concentration range will often reveal that these displacements are reversible. Displacement provides a gentle and efficient way of eluting antigen from an otherwise high affinity binding partner which can be leveraged in designing reagents or therapeutic antibodies with unique properties.

Abstract 3485: Inhibition of Notch signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Triple negative breast cancer (TNBC) is characterized by lack of estrogen receptor (ER), progesterone receptor (PR) or Her2/neu. It is known to be particularly aggressive and refractory to current chemotherapies. Tumor-initiating cells (TICs) have been postulated as responsible for treatment failure for this type of cancer. Recent evidence suggests that Notch signaling may play a role in regulation of TIC self-renewal and differentiation hence present a promising target for development of novel therapies for TNBC. We previously discovered a mouse monoclonal Ab (mu-hN1) that produces robust efficacy in both T-cell acute lymphoblastic leukemia and TNBC by specifically blocking human Notch1 signaling in tumor cells. We subsequently produced a humanized Notch1 mAb (hu-hN1) that retained similar characteristics and activities compared to the mouse prototype mu-hN1. To gain insights into the mechanisms of Notch1 blockade on TNBC tumor grow inhibition (TGI), we evaluated the effect of hu-hN1 on apoptosis, proliferation and TIC self-renewal in Notch-driven Sum149 and a patient derived xenograft (PDX) TNBC models. We showed that hu-hN1 inhibited Notch signaling by reduction of NICD (Notch intracellular domain) and target genes Hes-1 and cMyc in Sum149 tumors. Inhibition of Notch signaling led to significant TGI either as a single agent or in combination with Docetaxel. TGI of hu-hN1 appeared to be in part due to induction of apoptosis in the tumor cells. More interestingly, treatment with this Ab caused reduction in mammosphere formation and CD44+/CD133+/ESA+ cell population. It also resulted in decrease in tumor incidence and delay in tumor recurrence, suggesting an anti-TIC activity of the agent. Further investigation in a PDX TNBC model confirmed the robust tumor efficacy and anti-TIC effect of hu-hN1. Taken together, our findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional treatments by directly targeting the TIC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3485. doi:1538-7445.AM2012-3485

Abstract 1765: Inhibition of Notch signaling by a Notch1 monoclonal antibody induces robust anti-tumor efficacy in T-cell acute lymphoblastic leukemia and breast cancer

Notch signaling is deregulated in T-cell acute lymphoblastic leukemia (T-ALL) and advanced solid tumors, making it an attractive target for oncology drug development. In this present report, we describe a novel mouse monoclonal antibody, Notch1 mAb, that specifically binds to the negative regulatory region (NRR) of human Notch1 receptor. Using a T-ALL cell line, HPB-ALL, that harbors mutations in Notch1 heterodimerization and PEST domains, we demonstrated that Notch1 mAb blocked Notch signaling by reduction of Notch1 intracellular domain (NICD) and down-regulation of Notch target genes, Hes-1 and cMyc. Notch1 mAb caused cell growth inhibition of HPB-ALL and several other T-ALL cell lines, via induction of cell cycle arrest and apoptosis. Notch1 mAb treatment resulted in robust NICD reduction and marked antitumor efficacy in HBP-ALL xenograft model. Additional mechanism-of-action studies revealed inhibition of tumor cell proliferation and induction of apoptosis in HPB-ALL tumors, suggesting that the anti-tumor activity of Notch1 mAb may be mediated by its direct effects on tumor cell growth or survival. Furthermore, this antibody led to a significant reduction in leukemia progenitor cells (LPCs) baring NOTCH1 mutation in bioluminescent humanized T-ALL LPC mouse models. In addition to its inhibitory effect on mutant Notch1, Notch1 mAb also displayed robust neutralization activity on wild type Notch1 and caused tumor growth inhibition in breast cancer models MDA-MB-231 and MX-1 by targeting the wild type receptor in these tumor types. Interestingly, using a gamma secretase inhibitor PF-03084014, we showed that Notch1 mAb and PF-03084014 elicited similar degree of biology responses in HPB-ALL cells. Our results indicate that antibodies that bind to NRR can act as potent inhibitors of Notch1 signaling and provide opportunities for development of novel cancer therapeutics for T-ALL and solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1765. doi:10.1158/1538-7445.AM2011-1765