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Koide S.,University of Chicago | Koide A.,University of Chicago | Lipovsek D.,Adnexus
Methods in Enzymology | Year: 2012

We describe concepts and methods for generating a family of engineered target-binding proteins designed on the scaffold of the 10th human fibronectin type III domain ( 10Fn3), an extremely stable, single-domain protein with an immunoglobulin-like fold but lacking disulfide bonds. Large libraries of possible target-binding proteins can be constructed on the 10Fn3 scaffold by diversifying the sequence and length of its surface loops, which are structurally analogous to antibody complementarity-determining regions. Target-binding proteins with high affinity and specificity are selected from 10Fn3-based libraries using in vitro evolution technologies such as phage display, mRNA display, or yeast-surface display. 10Fn3-based target-binding proteins have binding properties comparable to those of antibodies, but they are smaller, simpler in architecture, and more user-friendly; as a consequence, these proteins are excellent building blocks for the construction of multidomain, multifunctional chains. The ease of engineering and robust properties of 10Fn3-based target-binding proteins have been validated by multiple independent academic and industrial groups. In addition to performing well as specific in vitro detection reagents and research tools, 10Fn3-based binding proteins are being developed as therapeutics, with the most advanced candidate currently in Phase II clinical trials. © 2012 Elsevier Inc. All rights reserved. Source

Waters J.D.,University of California at San Diego | Sanchez C.,Center for Nervous System Repair | Sahin A.,Center for Nervous System Repair | Futalan D.,University of California at San Diego | And 13 more authors.
Journal of Neuro-Oncology | Year: 2012

Glioblastomas are among the most aggressive human cancers, and prognosis remains poor despite presently available therapies. Angiogenesis is a hallmark of glioblastoma, and the resultant vascularity is associated with poor prognosis. The proteins that mediate angiogenesis, including vascular endothelial growth factor (VEGF) signaling proteins, have emerged as attractive targets for therapeutic development. Since VEGF receptor-2 (VEGFR-2) is thought to be the primary receptor mediating angiogenesis, direct inhibition of this receptor may produce an ideal therapeutic effect. In this context, we tested the therapeutic effect of CT322, a selective inhibitor of VEGFR-2. Using an intracranial murine xenograft model (U87-EGFRvIII-luciferase), we demonstrate that CT322 inhibited glioblastoma growth in vivo and prolonged survival. Of note, the anti-neoplastic effect of CT322 is augmented by the incorporation of temozolomide or temozolomide with radiation therapy. Immunohistochemical analysis of CT322 treated tumors revealed decreased CD31 staining, suggesting that the tumoricidal effect is mediated by inhibition of angiogenesis. These pre-clinical results provide the foundation to further understand long term response and tumor escape mechanisms to anti-angiogenic treatments on EGFR over-expressing glioblastomas. © 2012 Springer Science+Business Media, LLC. Source

Khan J.A.,Bristol Myers Squibb | Camac D.M.,Bristol Myers Squibb | Low S.,Adnexus | Tebben A.J.,Bristol Myers Squibb | And 17 more authors.
Journal of Molecular Biology | Year: 2015

The human pregnane X receptor (PXR) is a promiscuous nuclear receptor that functions as a sensor to a wide variety of xenobiotics and regulates expression of several drug metabolizing enzymes and transporters. We have generated "Adnectins", derived from 10th fibronectin type III domain (10Fn3), that target the PXR ligand binding domain (LBD) interactions with the steroid receptor co-activator-1 (SRC-1) peptide, displacing SRC-1 binding. Adnectins are structurally homologous to the immunoglobulin superfamily. Three different co-crystal structures of PXR LBD with Adnectin-1 and CCR1 (CC chemokine receptor-1) antagonist Compound-1 were determined. This structural information was used to modulate PXR affinity for a related CCR1 antagonist compound that entered into clinical trials for rheumatoid arthritis. The structures of PXR with Adnectin-1 reveal specificity of Adnectin-1 in not only targeting the interface of the SRC-1 interactions but also engaging the same set of residues that are involved in binding of SRC-1 to PXR. Substituting SRC-1 with Adnectin-1 does not alter the binding conformation of Compound-1 in the ligand binding pocket. The structure also reveals the possibility of using Adnectins as crystallization chaperones to generate structures of PXR with compounds of interest. © 2015 Elsevier Ltd. Source

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