Antibodies are widely used for diagnostic and therapeutic applications because of their sensitive and specific recognition of a wide range of targets; however, their application is limited by their structural complexity. More demanding applications require greater stability than can be achieved by immunoglobulin-based reagents. Highly stable, protein-based affinity reagents are being investigated for this role with the goal of identifying a suitable scaffold that can attain specificity and sensitivity similar to that of antibodies while performing under conditions where antibodies fail. We have engineered Top7--a highly stable, computationally designed protein--to specifically bind human CD4 by inserting a peptide sequence derived from a CD4-specific antibody. Molecular dynamics simulations were used to evaluate the structural effect of the peptide insertion at a specific site within Top7 and suggest that this Top7 variant retains conformational stability over 100 degrees C. This engineered protein specifically binds CD4 and, consistent with simulations, is extremely resistant to thermal and chemical denaturation--retaining its secondary structure up to at least 95 degrees C and requiring 6 M guanidine to completely unfold. This CD4-specific protein demonstrates the functionality of Top7 as a viable scaffold for use as a general affinity reagent which could serve as a robust and inexpensive alternative to antibodies.

The primary toxicity associated with repeated oral administration of the PDE4 inhibitor IC542 to the rat is an inflammatory response leading to tissue damage primarily in the gastrointestinal tract and mesentery. Although necrotizing vasculitis is frequently seen with other PDE4 inhibitors, blood vessel injury was rare following IC542 administration and was always associated with inflammation in the surrounding tissue. The incidence and severity of the histologic changes in these studies correlated with elevated peripheral blood leukocytes, serum IL-6, haptoglobin, and fibrinogen, and with decreased serum albumin. By monitoring haptoglobin, fibrinogen and serum albumin changes in IC542-treated rats, it was possible to identify rats with early histologic changes that were reversible. Since PDE4 inhibition is generally associated with anti-inflammatory activity, extensive inflammation in multiple tissues was unexpected with IC542. Co-administration of dexamethasone completely blocked IC542-induced clinical and histologic changes in the rat, confirming the toxicity resulted from inflammatory response. In addition, IC542 augmented release of the proinflammatory cytokine IL-6 in LPS-activated whole blood from rats but not monkeys or humans. The effect of IC542 on IL-6 release from rat leukocytes in vitro is consistent with the proinflammatory response observed in vivo and demonstrates species differences to PDE4 inhibition.

Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.

These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.

Bispecific T-cell engagers (TcEs) are antibody-based immunotherapeutic drugs that specifically direct lymphocyte effector functions against tumors. TcEs have one arm with affinity for an activating immunoreceptor connected through a flexible hinge to a distinct arm with affinity for a tumor antigen to achieve tumor killing by cytotoxic immune cells. Understanding the structure-function relationships between TcE architecture, immunological synapse formation and function could accelerate design of new TcE-based cancer therapies. Here, we engineer and systematically characterize TcE formats with antigen binding antibody fragments or single chain variable fragments linked together in cis through immunoglobulin G1 hinge or in trans across the antibody constant fragment. The TcEs were tested in CD8+ T-cell killing of Her2+ breast cancer cells and evaluated by high-content imaging of immunological synapse formation on a supported lipid bilayer (SLB) platform. We find that cis TcEs perform better than a trans TcE for T-cell mediated killing. Quantification of synapse formation dynamics revealed that all three cis TcEs tested, created close contacts of < 16 nm leading to rapid synapse formation and integrin activation. In contrast, the trans TcE formed close contacts averaging ≥ 16 nm and formed synapses more slowly with weaker integrin activation. We conclude that segmental flexibility is important for TcE function, but adding additional degrees of freedom through trans formats has a cost for killing efficiency that may be explained by failure of close contact formation and integrin activation.