Display on the yeast cell wall is well suited for engineering mammalian cell-surface and secreted proteins (e.g., antibodies, receptors, cytokines) that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity. C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library. A eukaryotic host should alleviate expression biases present in bacterially propagated combinatorial libraries. Quantitative flow cytometric analysis enables fine discrimination of kinetic parameters for protein binding to soluble ligands.

This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.

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...

Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue.

Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant Kd ‫؍‬ 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10 5 -10 7 yeast surfacedisplayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wildtype complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.