Eugenia Kraynov

Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (Kd = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC50 = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC50 = 4.7 ± 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC50 value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.

Breaching the DNA damage checkpoint via PF-00477736, a novel small-molecule inhibitor of checkpoint kinase 1

Checkpoints are present in all phases of the cell cycle and are regarded as the gatekeepers maintaining the integrity of the genome. Many conventional agents used to treat cancer impart damage to the genome and activate cell cycle checkpoints. Many tumors are defective in the tumor suppressor p53 and therefore lack a functional G1 checkpoint. In these tumors, however, the S-G2 checkpoints remain intact and, in response to DNA damage, arrest cell cycle progression allowing time for DNA repair. Checkpoint kinase 1 (Chk1) is a key element in the DNA damage response pathway and plays a crucial role in the S-G2-phase checkpoints. Inhibiting Chk1 represents a therapeutic strategy for creating a “synthetic lethal” response by overriding the last checkpoint defense of tumor cells against the lethal damage induced by DNA-directed chemotherapeutic agents. Chk1 inhibition is consistent with emerging targeted therapies aiming to exploit molecular differences between normal and cancer cells. Adding a Chk1 inhibitor to DNA-damaging cytotoxic therapy selectively targets tumors with intrinsic checkpoint defects while minimizing toxicity in checkpoint-competent normal cells. PF-00477736 was identified as a potent, selective ATP-competitive small-molecule inhibitor that inhibits Chk1 with a Ki of 0.49 nM. PF-00477736 abrogates cell cycle arrest induced by DNA damage and enhances cytotoxicity of clinically important chemotherapeutic agents, including gemcitabine and carboplatin. In xenografts, PF-00477736 enhanced the antitumor activity of gemcitabine in a dose-dependent manner. PF-00477736 combinations were well tolerated with no exacerbation of side effects commonly associated with cytotoxic agents. [Mol Cancer Ther 2008;7(8):2394–404]

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.

PF-03732010: a fully human monoclonal antibody against P-cadherin with antitumor and antimetastatic activity.

Purpose: P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion, proliferation, and invasiveness. We characterized the biological properties of PF-03732010, a human monoclonal antibody against P-cadherin, in cell-based assays and tumor models. Experimental Design: The affinity, selectivity, and cellular inhibitory activity of PF-03732010 were tested in vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumor and antimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were also investigated. Results: PF-03732010 selectively inhibits P-cadherin–mediated cell adhesion and aggregation in vitro. In the P-cadherin–overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-435HAL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth of primary tumors and metastatic progression, as determined by bioluminescence imaging. Computed tomography imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of PF-03732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the counterpart tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence, immunohistochemical analyses, and 3′-[18F]fluoro-3′-deoxythymidine–positron emission tomography imaging revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membrane β-catenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capacity. Changes in the levels of Ki67, caspase-3, and 3′-[18F]fluoro-3′-deoxythymidine tracer uptake also indicated antiproliferative activity and increased apoptosis in the tested xenografts. Conclusions: These findings suggest that interrupting the P-cadherin signaling pathway may be a novel therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clinical trials. Clin Cancer Res; 16(21); 5177–88. ©2010 AACR.

A Model-Based Approach to Predicting the Human Pharmacokinetics of a Monoclonal Antibody Exhibiting Target-Mediated Drug Disposition

In the drug discovery and development setting, the ability to accurately predict the human pharmacokinetics (PK) of a candidate compound from preclinical data is critical for informing the effective design of the first-in-human trial. PK prediction is especially challenging for monoclonal antibodies exhibiting nonlinear PK attributed to target-mediated drug disposition (TMDD). Here, we present a model-based method for predicting the PK of PF-03446962, an IgG2 antibody directed against human ALK1 (activin receptor-like kinase 1) receptor. Systems parameters as determined experimentally or obtained from the literature, such as binding affinity (kon and koff), internalization of the drug-target complex (kint), target degradation rate (kdeg), and target abundance (R0), were directly integrated into the modeling and prediction. NONMEM 7 was used to model monkey PK data and simulate human PK profiles based on the construct of a TMDD model using a population-based approach. As validated by actual patient data from a phase I study, the human PK of PF-03446962 were predicted within 1- to 2-fold of observations. Whereas traditional approaches fail, this approach successfully predicted the human PK of a monoclonal antibody exhibiting nonlinearity because of TMDD.

Critical Review of Preclinical Approaches to Investigate Cytochrome P450–Mediated Therapeutic Protein Drug-Drug Interactions and Recommendations for Best Practices: A White Paper

Drug-drug interactions (DDIs) between therapeutic proteins (TPs) and small-molecule drugs have recently drawn the attention of regulatory agencies, the pharmaceutical industry, and academia. TP-DDIs are mainly caused by proinflammatory cytokine or cytokine modulator–mediated effects on the expression of cytochrome P450 enzymes. To build consensus among industry and regulatory agencies on expectations and challenges in this area, a working group was initiated to review the preclinical state of the art. This white paper represents the observations and recommendations of the working group on the value of in vitro human hepatocyte studies for the prediction of clinical TP-DDI. The white paper was developed following a “Workshop on Recent Advances in the Investigation of Therapeutic Protein Drug-Drug Interactions: Preclinical and Clinical Approaches” held at the Food and Drug Administration White Oak Conference Center on June 4 and 5, 2012. Results of a workshop poll, cross-laboratory data comparisons, and the overall recommendations of the in vitro working group are presented herein. The working group observed that evaluation of TP-DDI for anticytokine monoclonal antibodies is currently best accomplished with a clinical study in patients with inflammatory disease. Treatment-induced changes in appropriate biomarkers in phase 2 and 3 studies may indicate the potential for a clinically measurable treatment effect on cytochrome P450 enzymes. Cytokine-mediated DDIs observed with anti-inflammatory TPs cannot currently be predicted using in vitro data. Future success in predicting clinical TP-DDIs will require an understanding of disease biology, physiologically relevant in vitro systems, and more examples of well conducted clinical TP-DDI trials.

Identification and quantification of osteopontin splice variants in the plasma of lung cancer patients using immunoaffinity capture and targeted mass spectrometry

The expression patterns and functional roles of three osteopontin splice variants (OPNa, b, and c) in cancer metastasis and progression are not well understood due to the lack of reliable assays to differentiate the isoforms. We have developed a mass spectrometric method to quantify OPN isoforms in human plasma. The method is based on the immunocapture of all OPN isoforms, followed by MRM–MS analysis of isoform-specific tryptic peptides. We were able to simultaneously identify and quantify all three isoforms in the plasma of 10 healthy individuals and 10 non-small cell lung cancer (NSCLC) patients. Our results show that none of the OPN splice variants is cancer specific. However, OPNa, the major isoform in healthy and NSCLC plasma, is substantially elevated in NSCLC patients, whereas OPNb and OPNc are at equivalent levels in two populations.

Discovery of Pyrroloaminopyrazoles as Novel Pak Inhibitors.

The P21-activated kinases (PAK) are emerging antitumor therapeutic targets. In this paper, we describe the discovery of potent PAK inhibitors guided by structure-based drug design. In addition, the efflux of the pyrrolopyrazole series was effectively reduced by applying multiple medicinal chemistry strategies, leading to a series of PAK inhibitors that are orally active in inhibiting tumor growth in vivo.

Osteopontin induces growth of metastatic tumors in a preclinical model of non-small lung cancer

Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity a nti-O PN m onoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvβ3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvβ3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvβ3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a KrasG12D-LSLp53fl/fl subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.

A Mathematical Model of the Effect of Immunogenicity on Therapeutic Protein Pharmacokinetics

A mathematical pharmacokinetic/anti-drug-antibody (PK/ADA) model was constructed for quantitatively assessing immunogenicity for therapeutic proteins. The model is inspired by traditional pharmacokinetic/pharmacodynamic (PK/PD) models, and is based on the observed impact of ADA on protein drug clearance. The hypothesis for this work is that altered drug PK contains information about the extent and timing of ADA generation. By fitting drug PK profiles while accounting for ADA-mediated drug clearance, the model provides an approach to characterize ADA generation during the study, including the maximum ADA response, sensitivity of ADA response to drug dose level, affinity maturation rate, time lag to observe an ADA response, and the elimination rate for ADA–drug complex. The model also provides a mean to estimate putative concentration–time profiles for ADA, ADA–drug complex, and ADA binding affinity-time profile. When simulating ADA responses to various drug dose levels, bell-shaped dose–response curves were generated. The model contains simultaneous quantitative modeling and provides estimation of the characteristics of therapeutic protein drug PK and ADA responses in vivo. With further experimental validation, the model may be applied to the simulation of ADA response to therapeutic protein drugs in silico, or be applied in subsequent PK/PD models.