Targeting tumor-overexpressed EGFR with an antibody-drug conjugate (ADC) is an attractive therapeutic strategy; however, normal tissue expression represents a significant toxicity risk. The anti-EGFR antibody ABT-806 targets a unique tumor-specific epitope and exhibits minimal reactivity to EGFR in normal tissue, suggesting its suitability for the development of an ADC. We describe the binding properties and preclinical activity of ABT-414, an ABT-806 monomethyl auristatin F conjugate. In vitro, ABT-414 selectively kills tumor cells overexpressing wild-type or mutant forms of EGFR. ABT-414 inhibits the growth of xenograft tumors with high EGFR expression and causes complete regressions and cures in the most sensitive models. Tumor growth inhibition is also observed in tumor models with EGFR mutations, including activating mutations and those with the exon 2-7 deletion [EGFR variant III (EGFRvIII)], commonly found in glioblastoma multiforme. ABT-414 exhibits potent cytotoxicity against glioblastoma multiforme patient-derived xenograft models expressing either wild-type EGFR or EGFRvIII, with sustained regressions and cures observed at clinically relevant doses. ABT-414 also combines with standard-of-care treatment of radiation and temozolomide, providing significant therapeutic benefit in a glioblastoma multiforme xenograft model. On the basis of these results, ABT-414 has advanced to phase I/II clinical trials, and objective responses have been observed in patients with both amplified wild-type and EGFRvIII-expressing tumors. Mol Cancer Ther; 15(4);

The interaction between leukocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1) has been implicated in inflammatory and immune diseases. Recently, a novel series of p-arylthio cinnamides has been described as potent antagonists of the LFA-1/ICAM-1 interaction. These compounds were found to bind to the I domain of LFA-1 using two-dimensional NMR spectroscopy of 15N-labeled LFA-1 I domain. On the basis of NOE studies between compound 1 and the I domain of LFA-1, a model of the complex was constructed. This model revealed that compound 1 does not directly inhibit ICAM-1 binding by interacting with the metal ion dependent adhesion site (MIDAS). Instead, it binds to the previously proposed I domain allosteric site (IDAS) of LFA-1 and likely modulates the activation of LFA-1 through its interaction with this regulatory site. A fragment-based NMR screening strategy was applied to identify small, more water-soluble ligands that bind to a specific region of the IDAS. When incorporated into the parent cinnamide template, the resulting analogues exhibited increased aqueous solubility and improved pharmacokinetic profiles in rats, demonstrating the power of this NMR-based screening approach for rapidly modifying high-affinity ligands.

Despite clinical efficacy, current approved agents targeting EGFR are associated with on-target toxicities as a consequence of disrupting normal EGFR function. MAb 806 is a novel EGFR antibody that selectively targets a tumor-selective epitope suggesting that a mAb 806-based therapeutic would retain antitumor activity without the on-target toxicities associated with EGFR inhibition. To enable clinical development, a humanized variant of mAb 806 designated ABT-806 was generated and is currently in phase 1 trials. We describe the characterization of binding and functional properties of ABT-806 compared with the clinically validated anti-EGFR antibody cetuximab. ABT-806 binds the mutant EGFRvIII with high affinity and, relative to cetuximab, exhibits increased potency against glioblastoma multiforme cell line and patient-derived xenografts expressing this form of the receptor. ABT-806 also inhibits the growth of squamous cell carcinoma xenograft models expressing high levels of wild-type EGFR, associated with inhibition of EGFR signaling, although higher doses of ABT-806 than cetuximab are required for similar activity. ABT-806 enhances in vivo potency of standard-of-care therapies used to treat glioblastoma multiforme and head and neck squamous cell carcinoma. An indiumlabeled version of ABT-806, [ 111 In]-ABT-806, used to investigate the relationship between dose and receptor occupancy, revealed greater receptor occupancy at lowers doses in an EGFRvIII-expressing model and significant uptake in an orthotopic model. Collectively, these results suggest that ABT-806 may have antitumor activity superior to cetuximab in EGFRvIIIexpressing tumors, and similar activity to cetuximab in tumors highly overexpressing wild-type EGFR with reduced toxicity.

The study of many biological processes requires the analysis of three-dimensional (3D) structures that change overtime. Optical sectioning techniques can provide 3D data from living specimens; however, when 3D data are collected over a period of time, the quantity of image information produced leads to difficulties in interpretation. A computer-based system is described that permits the analysis and archiving of 3D image data taken over time. The system allows a user to roam through the full range of time points and focal planes in the data set. The user can animate images as an aid to visualization and can append multicolored labels and text notes to identified structures during data analysis. The system provides a valuable tool for the study of embryogenesis and cytoplasmic movements within cells and has considerable potential as an educational tool.Living things, by their very nature, are dynamic. Individual cells undergo complex rearrangements of their cytoarchitecture as they progress through the cell cycle or become terminally differentiated. Groups of cells undergo coordinated shape changes and migrations during the development of shape and form in an embryo. Understanding these processes is a fundamental goal of the developmental or cell biologist. Traditionally, biologists have studied these phenomena by fixing the specimen at a defined time point, embedding it in a plastic or wax matrix, and cutting sections for observation under the microscope. By repeating this process at differing time points, it is possible to infer dynamic behavior, provided the behavior being studied is sufficiently stereotyped to enable reconstruction from discrete time points taken from different specimens. However, even in these cases, the inference of continuous behavior from sparsely sampled discrete time points can lead to misinterpretation, particularly if the processes being studied have intrinsic variability.Optical sectioning microscopy has become a very powerful tool for the biologist because it allows a single focal plane to be observed within an intact specimen with minimal image degradation caused by outof-focus interference. Optical sectioning is noninvasive and is therefore well suited to the study of living specimens. It allows the 3D dynamic behavior of a given organism to be directly and unambiguously inferred.

Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the erythropoietin receptor (EPOR) in erythroid progenitor cells, leading to proliferation and differentiation into mature red blood cells. To allow less frequent dosing, a hyperglycosylated version of EPO has been developed with a longer halflife. In principle, an agonistic antibody targeting EPOR would offer an even longer half-life, support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell aplasia. IntroductionEPO, a naturally occurring hematopoietic growth factor produced by the kidney, is the primary regulator of erythropoiesis. 1 Recombinant human EPO (rHu-EPO) has important clinical uses in patients with anemia associated with renal disease and cancer. Analogs rHu-EPO with extended serum half-lives have been developed and of shown to provide a clinical advantage by allowing maintenance of stable hemoglobin levels with less frequent dosing. 2,3 A fulllength human agonistic antibody targeting the EPO receptor (EPOR) would offer a longer serum half-life and may support even less frequent dosing regimens that could better match with many chemotherapy regimens and may provide better convenience for both predialysis and peritoneal dialysis patients who need to attend the clinic only infrequently. In addition, an antibody EPO mimic is unlikely to induce pure red cell aplasia, a condition associated with some forms of rHu-EPO due to the formation of rHu-EPO-induced neutralizing antibodies. 4 Mouse monoclonal antibodies (mAbs) that are raised to the soluble extracellular domain (ECD) of the human EPOR and that mimic EPO activation by inducing ligand-dependent cell proliferation and differentiation have been described. 5,6 These mAbs, however, activate the EPOR less efficiently than the natural hormone does and consequently are less potent agonists and unsuitable for clinical use. Crystal structure of the EPO-(EPOR)2 complex reveals that EPO binds 2 distinct sites of the 2 cell-surface EPORs and that asymmetric molecules may therefore be required for optimal signaling. 7 This report describes a fully human agonistic antibody, ABT-007, that effectively stimulates both proliferation and erythroid differentiation. Since ABT007 exhibits a high degree of selectivity and does not recognize rodent EPOR, mice expressing the human EPOR transgene were used to establish its in vivo efficacy. Surprisingly, the activation signal achieved with the symmetric molecule ABT007 is sufficient to support potent and more sustained erythropoiesis in animal models compared with standard doses of rHu-EPO. We examined the crystal structure of human monomeric EPOR ECD complexed with a single antibody fragment of ABT007 (Fab-EPOR) to better understand the molecular basis for the erythropoietic potency of ABT007. Resolution of the resulting crystal structure identified a unique EPOR nonlinear epitope distinct from the EPO binding site, resulting in a receptor conformation that supports activation. In vivo properties of ABT007 may b...