Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of "developability." Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotypematched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.monoclonal antibody | developability | biophysical properties | manufacturability | nonspecificity T arget binding is the predominant first concern in development of any drug. However, once a lead molecule attains the desired potency of biological modification, a suite of characteristics termed "developability" assumes critical importance. For monoclonal antibodies, these properties include high-level expression, high solubility, covalent integrity, conformational and colloidal stability, low polyspecificity, and low immunogenicity. The high cost of failing any of these criteria at a late stage in drug development has led to considerable efforts at predicting developability on the basis of sequence motifs and experimentally determined biophysical properties (1-15).In a landmark study of small-molecule drugs over 2,000 molecules with United States Adopted Names (USAN) designations and known to have oral availability were collected and computationally analyzed (16). A simple set of thresholds, encapsulated as the "Lipinski rule of fives," was formulated and has been used by many to prioritize small molecules for entry into clinical development. To date, analogous guiding principles for antibody drugs have not emerged-we therefore endeavor here to do so. By analogy to the Lipinski effort, we first collected the sequences of antibodies that had reached at least phase-2 trials and had USAN or WHO International Nonproprietary Names (INN) designations (137 in total as of the start of this project). As a common basis for comparison of intrinsic variable domain phenotypes we expressed each antibody as the human IgG1 isotype and formulated them in simple Hepes-buffered saline. Each antibody was then subjected to a battery of 12 different biophysical assays in common use for developability assessment.Unexpectedly, for many of the measures the distribution of values was not symmetrically Gaussian, but instead was lon...

Antibody therapy is coming of age, with 15 monoclonal antibodies approved for therapeutic use in the United States and many others currently undergoing clinical trials (1). The advent of antibody engineering over the past two decades has contributed to the recent clinical success of therapeutic antibodies. The development of chimeric (2) and humanized (3) antibodies not only reduced the potent immunogenicity of rodent antibodies in humans but also improved the serum halflives and efficacy of such therapeutics compared with rodent antibodies. Phage display (4) and other display technologies have led to the ability to increase the affinity of antibodies for their target antigens. More recently, antibody engineering has been used to modify the effector functions of antibodies by altering their binding to C1q (5) and various Fc␥ receptors (6).The neonatal Fc receptor (FcRn) 1 is a heterodimer that comprises a transmembrane ␣ chain with structural homology to the extracellular domains of the ␣ chain of major histocompatibility complex class I molecules, and a soluble light chain consisting of ␤2-microglubulin (␤2m) (7). FcRn mediates both transcytosis of maternal IgG to the fetus or neonate and IgG homeostasis in adults (8). Evidence for the latter role initially came from studies indicating an unusually short serum halflife for IgG antibodies in ␤2m-deficient mice (9 -11). This observation led to the generation of mutant mouse hinge-Fc fragments with enhanced binding to FcRn and increased serum persistence in mice (12). Recently, several studies have identified human IgG 1 mutants with enhanced FcRn binding (6, 13), although no improvement in the serum half-lives of these mutants was observed in mice (13) or reported in primates.The binding of IgG to FcRn is sharply pH-dependent; IgG binds to FcRn under mildly acidic conditions and is released under slightly basic conditions (14). It has been hypothesized that pinocytosed IgG antibodies are captured by FcRn in acidified endosomes, rescued from degradation in lysosomes, recycled back to the cell surface, and returned to the circulation (8). Mutagenesis studies have identified both the mouse (15, 16) and human (17) Fc residues believed to be important in mediating pH-dependent binding. The results of the mutagenesis studies are consistent with the interpretation of a crystallographic study of the Fc⅐FcRn interaction (18). In the current study, molecular modeling was used to identify residues in the human IgG Fc near the FcRn binding site that, when mutated, might alter binding to FcRn without affecting the pH dependence of this interaction. Following exhaustive mutagenesis at these positions, several IgG 2 mutants were identified with improved binding to FcRn at pH 6.0 that retained the property of pH-dependent release. A pharmacokinetics study in rhesus monkeys showed that two mutant IgG 2 antibodies with increased FcRn binding affinity had considerably longer serum half-lives than the wild-type antibody. EXPERIMENTAL PROCEDURESMolecular Modeling-Molecular models o...

Low expression, poor solubility, and polyspecificity are significant obstacles that have impeded the development of antibodies discovered from in vitro display libraries. Current biophysical characterization tools that identify these 'developability' problems are typically only applied after the discovery process, and thus limited to perhaps a few hundred candidates. We report a flow cytometric assay using a polyspecificity reagent (PSR) that allows for the identification and counter selection of polyspecific antibodies both during and after the selection process. The reported assay correlates well with cross-interaction chromatography, a surrogate for antibody solubility, as well as a baculovirus particle enzyme-linked immunosorbent assay, a surrogate for in vivo clearance. However, unlike these assays, PSR labeling is compatible both with screening of individual antibodies as well as selections of large antibody libraries. To this end, we demonstrate the ability to counter-select against polyspecificity while enriching for antigen affinity from a diverse antibody library, which enables simultaneous evolution of both antigen binding and superior non-target-related properties during the discovery process.

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by one- and two-dimensional NMR techniques in aqueous solution: acetyl-Phe(8)-Leu(9)-Ala(10)-Glu-(11)-Gly(12)-Gly(13)-Gly(14)-Val(15)-Ar g(16)- Gly(17)-Pro(18)-NHMe (F6), acetyl-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16) (tF6), acetyl-Asp(7)-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16)-Gly(17)-Pro- Arg(19)-Val(20)-NHMe (F8), and acetyl-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16) (tF8). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds in both F6 and F8 were cleaved instantaneously in the presence of 0.5 mM thrombin, producing truncated peptides tF6 and tF8 and other peptide fragments. On the basis of observations of line broadening, thrombin was found to bind to the cleavage products, tF6 and tF8, of peptides F6 and F8. Peptide tF8 may have a higher affinity for thrombin than peptide tF6, as suggested by the more pronounced thrombin-induced line broadening on the proton resonances in peptide tF8. Transferred NOE (TRNOE) measurements were made of the complexes between thrombin and peptides tF6 and tF8. Medium- and long-range NOE interactions were found between the NH proton of Asp(7) and the C beta H protons of Ala(10), between the C alpha H proton of Glu(11) and the NH proton of Gly(13), and between the ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). Sets of structures of the decapeptide tF8 were deduced by use of distance geometry calculations based on sequential and medium- and long-range TRNOEs from the thrombin-bound peptide. A predominant feature of these structures is the nonpolar cluster formed by the side chains of residues Phe(8), Leu(9), and Val(15) that are directly involved in binding to thrombin. This structural feature is brought about by an alpha-helical segment involving residues Phe(8)-Ala(10), followed by a multiple-turn structure involving residues Glu(11)-Val(15). These results provide an explanation for the observations that Asp(7), Phe(8), and Gly(12) are strongly conserved in mammalian fibrinogens and that the mutations of Asp(7) to Asn(7) and of Gly(12) to Val(12), result in delayed release of fibrinopeptide A, producing human bleeding disorders.

Several hydration models for peptides and proteins based on solvent accessible surface area have been proposed previously. We have evaluated some of these models as well as four new ones in the context of near-native conformations of a protein. In addition, we propose an empirical site-site distance-dependent correction that can be used in conjunction with any of these models. The set of near-native structures consisted of 39 conformations of bovine pancreatic trypsin inhibitor (BPTI) each of which was a local minimum of an empirical energy function (ECEPP) in the absence of solvent. Root-mean-square (rms) deviations from the crystallographically determined structure were in the following ranges: 1.06-1.94 A for all heavy atoms, 0.77-1.36 A for all backbone heavy atoms, 0.68-1.33 A for all alpha-carbon atoms, and 1.41-2.72 A for all side-chain heavy atoms. We have found that there is considerable variation among the solvent models when evaluated in terms of concordance between the solvation free energy and the rms deviations from the crystallographically determined conformation. The solvation model for which the best concordance (0.939) with the rms deviations of the C alpha atoms was found was derived from NMR coupling constants of peptides in water combined with an exponential site-site distance dependence of the potential of mean force. Our results indicate that solvation free energy parameters derived from nonpeptide free energies of hydration may not be transferrable to peptides. Parameters derived from peptide and protein data may be more applicable to conformational analysis of proteins. A general approach to derive parameters for free energy of hydration from ensemble-averaged properties of peptides in solution is described.

(2014) High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy, mAbs, 6:2, 483-492,

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Summary We describe the proceedings and conclusions from a “Workshop on Applications of Protein Models in Biomedical Research” that was held at University of California at San Francisco on 11 and 12 July, 2008. At the workshop, international scientists involved with structure modeling explored (i) how models are currently used in biomedical research, (ii) what the requirements and challenges for different applications are, and (iii) how the interaction between the computational and experimental research communities could be strengthened to advance the field.