Purpose: Angiopoietin-1 (Ang1) plays a key role in maintaining stable vasculature, whereas in a tumor Ang2 antagonizes Ang1's function and promotes the initiation of the angiogenic switch. Specifically targeting Ang2 is a promising anticancer strategy. Here we describe the development and characterization of a new class of biotherapeutics referred to as CovX-Bodies, which are created by chemical fusion of a peptide and a carrier antibody scaffold.Experimental Design: Various linker tethering sites on peptides were examined for their effect on CovXBody in vitro potency and pharmacokinetics. Ang2 CovX-Bodies with low nmol/L IC 50 s and significantly improved pharmacokinetics were tested in tumor xenograft studies alone or in combination with standard of care agents. Tumor samples were analyzed for target engagement, via Ang2 protein level, CD31-positive tumor vasculature, and Tie2 expressing monocyte penetration.Results: Bivalent Ang2 CovX-Bodies selectively block the Ang2-Tie2 interaction (IC 50 < 1 nmol/L) with dramatically improved pharmacokinetics (T ½ > 100 hours). Using a staged Colo-205 xenograft model, significant tumor growth inhibition (TGI) was observed (40%-63%, P < 0.01). Ang2 protein levels were reduced by approximately 50% inside tumors (P < 0.01), whereas tumor microvessel density (P < 0.01) and intratumor proangiogenic Tie2 CD11bþ cells (P < 0.05) were significantly reduced. When combined with sunitinib, sorafenib, bevacizumab, irinotecan, or docetaxel, Ang2 CovX-Bodies produced even greater efficacy ($80% TGI, P < 0.01). Conclusion: CovX-Bodies provide an elegant solution to overcome the pharmacokinetic-pharmacodynamic problems of peptides. Long-acting Ang2 specific CovX-Bodies will be useful as single agents and in combination with standard-of-care agents.

Bispecific antibodies (BsAbs) are regarded as promising therapeutic agents due to their ability to simultaneously bind two different antigens. Several bispecific modalities have been developed, but their utility is limited due to problems with stability and manufacturing complexity. Here we report a versatile technology, based on a scaffold antibody and pharmacophore peptide heterodimers, that enables rapid generation and chemical optimization of bispecific antibodies, which are termed bispecific CovX-Bodies. Two different peptides are joined together using a branched azetidinone linker and fused to the scaffold antibody under mild conditions in a site-specific manner. Whereas the pharmacophores are responsible for functional activities, the antibody scaffold imparts long half-life and Ig-like distribution. The pharmacophores can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. As a prototype, we developed a bispecific antibody that binds both vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2) simultaneously, inhibits their function, shows efficacy in tumor xenograft studies, and greatly augments the antitumor effects of standard chemotherapy. This unique antiangiogenic bispecific antibody is in phase-1 clinical trials.

The central roles played by protein–tyrosine kinase (PTK)‐dependent signal transduction in normal cellular regulation and homeostasis have made inappropriate or aberrant functions of certain of these pathways contributing factors to a variety of diseases, including several cancers. For this reason, development of PTK signaling inhibitors has evolved into an important approach toward new therapeutics. Since in these pathways phosphotyrosyl (pTyr) residues provide unique and defining functions either by their creation under the catalysis of PTKs, their recognition and binding by protein modules such as SH2 and phosphotyrosyl binding (PTB) domains, or their destruction by protein–tyrosine phosphatases, pTyr mimetics provide useful general starting points for inhibitor design. Important considerations in the development of such pTyr mimetics include enzymatic stability (particularly toward PTPs), high affinity recognition by target pTyr binding proteins, and good cellular bioavailability. Although small molecule, nonpeptide inhibitors may be ultimate objectives of inhibitor development, peptides frequently serve as display platforms for pTyr mimetics, which afford useful and conceptually straightforward starting points in the development process. Reported herein is a limited overview of pTyr mimetic development as it relates to peptide‐based agents. Of particular interest are recent findings that highlight potential limitations of peptides as display platforms for the identification of small molecule leads. One conclusion that results from this work is that while peptide‐based approaches toward small molecule inhibitor design are often intellectually satisfying from a structure‐based perspective, extrapolation of negative findings to small molecule, nonpeptide contexts should be undertaken with extreme caution. © 2001 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 60: 32–44, 2001

Previously we reported synthesis and characterization of water-soluble chiral conducting polymer nanocomposites via template-guided synthesis. Experimental procedures and parameters need to be carefully controlled to achieve synthesis of water-soluble nanocomposites. Here, we describe a modified synthetic procedure with higher molecular weight poly(acrylic acid) (PAA) (MW ∼ 250 000) as a template. This new system allows synthesis of chiral water-soluble nanocomposites over a more broad range of conditions making the synthesis more reproducible and easier to carry out at large scale. Another objective of this work is to further understand the underlying formation mechanism of chiral polyaniline nanocomposites. We carried out a detailed study of how temperature, template, and solvent affect the final morphology and properties of these nanocomposites. We found that the extent of interaction and stability of the template/monomer/acid adduct (nanocomposite precursor) and population density of monomer surrounding the template are crucial in determining the degree of nanocomposite chirality. Detailed characterization of the nanocomposites and their precursors was carried out by circular dichroism (CD), UV-vis, FTIR, NMR, and TEM spectroscopy. On the basis of experimental results and synthetic procedures, a simplified model for the formation mechanism of chiral polyaniline nanocomposite is proposed.