Supplementary Tables 1-2 from Biochemical Characterization of AMG 102: A Neutralizing, Fully Human Monoclonal Antibody to Human and Nonhuman Primate Hepatocyte Growth Factor

Abstract AMG 102 is a fully human monoclonal antibody that selectively targets and neutralizes hepatocyte growth factor/scatter factor (HGF/SF). A detailed biochemical and functional characterization of AMG 102 was done to support its clinical development for the treatment of cancers dependent on signaling through the HGF/SF:c-Met pathway. In competitive equilibrium binding experiments, AMG 102 bound to human and cynomolgus monkey HGF with affinities of approximately 19 pmol/L and 41 pmol/L, respectively. However, AMG 102 did not detect mouse or rabbit HGF on immunoblots. Immunoprecipitation experiments showed that AMG 102 preferentially bound to the mature, active form of HGF, and incubation of AMG 102/HGF complexes with kallikrein protease indicated that AMG 102 had no apparent effect on proteolytic processing of the inactive HGF precursor. AMG 102 inhibited human and cynomolgus monkey HGF-induced c-Met autophosphorylation in PC3 cells with IC50 values of 0.12 nmol/L and 0.24 nmol/L, respectively. AMG 102 also inhibited cynomolgus monkey HGF-induced migration of human MDA-MB-435 cells but not rat HGF-induced migration of mouse 4T1 cells. Epitope-mapping studies of recombinant HGF molecules comprising human/mouse chimeras and human-to-mouse amino acid substitutions showed that amino acid residues near the NH2-terminus of the β-chain are critical for AMG 102 binding. Bound AMG 102 protected one trypsin protease cleavage site near the NH2-terminus of the β-chain of human HGF, further substantiating the importance of this region for AMG 102 binding. Currently, AMG 102 is in phase II clinical trials in a variety of solid tumor indications. Mol Cancer Ther; 9(2); 400–9

Flow cytometry (FCM) is an important technology with a broad spectrum of applications ranging from basic research to clinical diagnostics. In a typical FCM experiment, thousands of cells are queried with respect to size, shape, and abundance of multiple cell surface antigens. Recent advances in FCM techniques and instrumentation have enabled researchers to raise the throughput of experimentation dramatically. However, data analysis has remained a time-consuming activity requiring significant manual intervention for gating as well as for overall data reduction and interpretation. Presented in this article is a novel, algorithmically flexible, internally developed, software framework for the analysis of plate-based FCM data for high-throughput screening (HTS). Utilizing a post-treatment pooling strategy, >87,000 individual wells representing over 240,000 compounds were automatically gated, percent of control (POC) calculated, results assembled, deconvolved, and sorted, allowing researchers to visually assess wells of interest in minutes.

As technologies for high throughput and high content screening continue to evolve, new platforms for quantitative cellular imaging will play an increasingly important role in identifying and profiling lead compounds. To gain insight into the effects of a compound on cell morphology or intracellular events, it is necessary to have quality images and the ability to enumerate thousands of data points for statistical relevance. Imaging flow cytometry combines many of the features of flow cytometry, microscopy and imaging as well as a number of unique characteristics. The result is an instrument capable of highly quantitative analysis of cellular behaviors such as receptor internalization, phagocytosis, cell-cell communication, apoptosis and nuclear translocation. This promising new technology and unique type of flow cytometry provides enhanced capabilities for highly multiplexed assays. Here, we review the capabilities of the ImageStream imaging cytometer and discuss several applications relevant to compound screening and profiling.

Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for reducing excess hepatic glucose production in patients with type 2 diabetes. Despite extensive efforts, there is currently no human therapeutic that directly inhibits the glucagon/glucagon receptor pathway. We undertook a novel approach by generating high-affinity human monoclonal antibodies (mAbs) to the human glucagon receptor (GCGR) that display potent antagonistic activity in vitro and in vivo. A single injection of a lead antibody, mAb B, at 3 mg/kg, normalized blood glucose levels in ob/ob mice for 8 days. In addition, a single injection of mAb B dose-dependently lowered fasting blood glucose levels without inducing hypoglycemia and improved glucose tolerance in normal C57BL/6 mice. In normal cynomolgus monkeys, a single injection improved glucose tolerance while increasing glucagon and active glucagon-like peptide-1 levels. Thus, the anti-GCGR mAb could represent an effective new therapeutic for the treatment of type 2 diabetes.

Purpose: Bendamustine has shown clinical activity in patients with disease refractory to conventional alkylator chemotherapy. The purpose of this study was to characterize the mechanisms of action of bendamustine and to compare it with structurally related compounds. Experimental Design: Bendamustine was profiled in the National Cancer Institute in vitro antitumor screen. Microarray-based gene expression profiling, real-time PCR, immunoblot, cell cycle, and functional DNA damage repair analyses were used to characterize response to bendamustine and compare it with chlorambucil and phosphoramide mustard. Results: Bendamustine displays a distinct pattern of activity unrelated to other DNA-alkylating agents. Its mechanisms of action include activation of DNA-damage stress response and apoptosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. In addition, unlike other alkylators, bendamustine activates a base excision DNA repair pathway rather than an alkyltransferase DNA repair mechanism. Conclusion: These results suggest that bendamustine possesses mechanistic features that differentiate it from other alkylating agents and may contribute to its distinct clinical efficacy profile.

14536 Background: Panitumumab, a fully human monoclonal antibody against epidermal growth factor receptor (EGFR), has anti- tumor activity as monotherapy in both preclinical models and clinical trials. The objective of this study was to identify the epitope on EGFR for panitumumab and compare it to that of cetuximab, a chimeric anti-EGFR Ab. Methods: The extracellular domain of EGFR (amino acids 1–618) and domains I, II, III, IV individually were expressed to determine the domain on EGFR that was necessary for panitumumab binding. Chromatography was used to compare the binding affinity of panitumumab vs cetuximab. By flow cytometry and amino acid replacement scanning, the critical residues involved in panitumumab and cetuximab binding for EGFR were determined. Using diffraction data from the panitumumab Fab'2 and published data for cetuximab, crystal structure models of EGFR and the residues determined to be critical for panitumumab or cetuximab binding were built. To evaluate in vitro activity, A549 NSCLC cells were treated with 20μg/ml of panitumumab or cetuximab for 1 hour prior to a 15-minute stimulation with known EGFR ligands (TGF-α, amphiregulin, epiregulin, HB-EGF, betacellulin, and EGF). Inhibition of ligand-induced phosphorylation of EGFR was determined using a specific anti-pEGFR (pY1068) antibody. Results: Truncation analysis by flow cytometry narrowed the binding epitopes of panitumumab and cetuximab to domain III of EGFR. Point mutations revealed that amino acids 349, 355, 412, and 438 were critical for panitumumab and cetuximab binding. A 2.9 Angstrom crystal structure of the panitumumab Fab'2 was solved. Models of the panitumumab-EGFR and cetuximab-EGFR complexes were generated and predicted that the CDRs for both light and heavy chains would be proximal to domain III of EGFR. From in vitro studies, panitumumab and cetuximab can inhibit receptor activation of all known EGFR ligands in A549 NSCLC tumor cells. Conclusion: Panitumumab and cetuximab bind to comparable surface exposed amino acids in domain III of EGFR and inhibit all known EGFR ligands, resulting in inhibition of receptor activation. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Amgen Inc. Amgen Inc.

B121 Background: Understanding an antibody’s binding epitope may shed light on its mechanism of action (MOA). Panitumumab, a fully human monoclonal antibody directed against epidermal growth factor receptor (EGFR), has demonstrated anti-tumor efficacy as a monotherapy in both preclinical models and in clinical trials. The objective of this study was to identify the residues on EGFR that are critical for panitumumab binding and to gain further information on the mechanism of action of panitumumab. Methods: The extracellular region of EGFR (amino acids 1-618) was expressed as an avidin fusion protein in 293T cells and purified using biotin coated polystytene beads. Fusion proteins were used to determine the binding affinity of panitumumab using BIAcore technology and were used as a control for further epitope mapping studies using a flow cytometry based assay. EGFR truncation mutations, including individual domains I, II, III and IV, were also expressed to determine the domain on EGFR that was necessary for panitumumab binding. Replacement scanning was performed on solvent-accessibleamino acids in domain III (ligand binding domain) to further define the critical residues involved in panitumumab binding. Using diffraction data from the panitumumab Fab’2, the crystal structure of the EGFR and the residues determined to be critical for panitumumab binding, a model for panitumumab binding that minimize steric clashes was built. To evaluate the potential MOA, A549 NSCLC cells were treated with 20μg/ml of panitumumab 1 hour prior to a 15 minute stimulation with known EGFR ligands (TGF-α, amphiregulin, epiregulin, HB-EGF, betacellulin and EGF). Inhibition of ligand-induced phosphorylation of EGFR was determined using a specific anti-pEGFR (pY1068) antibody. Results: The binding affinity of panitumumab for the extracellular domain of EGFR was 50 pM by BIAcore. Further truncation analysis by flow cytometry narrowed the binding epitope of panitumumab to domain III of the EGFR. Point mutations revealed that amino acids 349, 355, 412 and 438 were critical for panitumumab binding. A 2.9 Angstrom crystal structure of the panitumumab Fab’2 was solved. A model of the panitumumab-EGFR complex was generated and predicts that the CDRs for both light and heavy chains of panitumumab would be proximal to domain III of EGFR. In vitro studies determined that panitumumab can inhibit receptor activation of all known EGFR ligands in A549 NSCLC tumor cells. Conclusion: From our model using the point mutation and crystal structure data, we determined that panitumumab binds to surface exposed amino acids in the ligand binding domain of EGFR (domain III). Panitumumab inhibits all known EGFR ligands, resulting in inhibition of receptor activation.

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Non-steroidal anti-inflammatory drugs have been shown to inhibit carcinogenesis in colon cancer, and to induce apoptosis in a variety of tumor cell lines. Some anti-tumor effects are thought to be related to their cyclooxygenase-2-inhibitory activity, but recent studies have shown that non-steroidal anti-inflammatory drugs exert their anti-tumor effect via cyclooxygenase-2-independent mechanism. SDX-308 (CEP-18082) is a non-cyclooxygenase-2-inhibiting indole-pyran analog and is structurally related to SDX-101, an R-enantiomer of etodolac. SDX-308 has a potent anti-myeloma effect and shows synergism in combination with other drugs for the treatment of chronic lymphocytic leukemia. In addition SDX-308 inhibits osteoclast formation and activity and thereby might be an attractive drug for the treatment of diseases with increased osteoclast activity such as osteolytic lesions in multiple myeloma and metastatic carcinomas, as well as osteoporosis. This review covers future application of SDX-308 as an anti-myeloma drug regulating increased osteoclast activity.

SDX-308 and SDX-309 are potent indole-pyran analogues of SDX-101 (R-etodolac) which has anti-tumour activity unrelated to cyclooxygenase-2 inhibition. Their cytotoxic activity was further studied herein using a well-characterized human tumour cell-line panel containing ten cell lines, as well as in 58 primary tumour cell samples from a variety of diagnoses. The indole-pyran analogues of SDX-101 were in general considerably more active in both cancer cell lines and primary tumour samples. Low cross-reactivity with standard agents was observed, indicating a unique mechanism of action. No apparent influence on efficacy was observed via classical mechanisms of multidrug-resistance. SDX-101 and SDX-309 showed higher relative activity in haematological compared to solid tumour samples, while SDX-308 had pronounced solid-tumour activity. High SDX-308 cytotoxic efficacy was observed in non-small cell lung cancer, renal cancer and ovarian cancer samples, and also in chronic lymphocytic leukaemia. In conclusion, the indole-pyran analogues showed a favourable pharmacological profile and represent a potentially important new class of drugs for cancer treatment.

In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.

Objective SDX-101 is the non-cyclooxygenase 2-inhibiting R-enantiomer of the non-steroid anti-inflammatory drug etodolac, and has anti-tumour activity in chronic lymphocytic leukaemia (CLL). SDX-308 and SDX-309 are more potent, structurally related indole-pyran analogues of SDX-101. The current study was performed to investigate and quantify the cytotoxic potentiating effects resulting from a combination of either SDX-101, SDX-308 or SDX-309 with standard cytotoxic agents used in the CLL treatment today.Methods The lymphoma cell line U937-gtb was used, together with primary tumour cells isolated from seven CLL patients. Combinations between chlorambucil and each one of the agents etodolac, SDX-101, SDX-308 and SDX-309 were studied. In addition, SDX-309 was combined with fludarabine, doxorubicin or vincristine. Both simultaneous and sequential exposures were explored using the median-effect method.Results Most combinations were additive, which could be of clinical benefit since SDX-101 has been shown to be well tolerated. At the 70% effect level, synergy was observed between SDX-308 and chlorambucil in U937-gtb cells and in two-third of the CLL samples. Since chlorambucil is the most important drug in CLL therapy today and SDX-308 is presently targeted as the lead clinical candidate, this combination would be interesting for further studies. Vincristine and SDX-309 were synergistic in two-fourth of CLL samples.Conclusions To conclude, the non-COX-inhibiting etodolac-derivatives SDX-101, SDX-308 and SDX-309 are potential candidates for combination treatment of CLL. Especially, SDX-308 in combination with chlorambucil warrants further evaluation.

L-Alanosine (SDX-102) exerts its cytotoxicity through inhibition of de novo purine biosynthesis, an effect potentiated by methylthioadenosine phosphorylase (MTAP) deficiency. The relevance of circadian dosing time was investigated for chronotherapeutic optimization of SDX-102. Toxicity was assessed in healthy mice following single (1, 150, 1,650, or 1,850 mg/kg/d) or multiple doses (250 or 270 mg/kg/d). Efficacy was tested in mice with P388 leukemia receiving multiple doses (225 or 250 mg/kg/d). SDX-102 was administered at six circadian times 4 hours apart in mice synchronized with 12 hours of light alternating with 12 hours of darkness. MTAP expression was determined in liver, bone marrow, small intestinal mucosa, and P388 cells. Dosing at 19 hours after light onset reduced lethality 5-fold after single administration and 3-fold after multiple doses as compared with worst time [P < 0.001 and P < 0.01, respectively (chi(2) test)]. Neutropenia, lymphopenia, and bone marrow hemorrhagic lesions were significantly less in mice dosed at 19 hours after light onset as compared with 7 hours after light onset. SDX-102 at 7 hours after light onset transiently ablated the 24-hour patterns in body temperature and activity. A circadian rhythm characterized small intestinal MTAP expression with a maximum at 6:30 hours after light onset (P = 0.04). A minor survival improvement was found in MTAP-deficient P388 mice receiving SDX-102 at 7 or 23 hours after light onset as compared with other times (P = 0.03, log-rank test). In conclusion, the therapeutic index of SDX-102 was improved by the delivery of SDX-102 in the mid to late activity span. These results support the concept of chronomodulated infusion of SDX-102 in cancer patients.