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Most proteins are flexible molecules that coexist in an ensemble of several conformations. Point mutations in the amino acid sequence of a protein can trigger structural changes that drive the protein population to a conformation distinct from the native state. Here, we report a protein engineering approach to better understand protein dynamics and ligand binding of the FK506-binding protein 51 (FKBP51), a prospective target for stress-related diseases, metabolic disorders, some types of cancers and chronic pain. By randomizing selected regions of its ligand-binding domain and sorting yeast display libraries expressing these variants, mutants with high affinity to conformation-specific FKBP51 selective ligands were identified. These improved mutants are valuable tools for the discovery of novel selective ligands that preferentially and specifically bind the FKBP51 active site in its open conformation state. Moreover, they will help us understand the conformational dynamics and ligand binding mechanics of the FKBP51 binding pocket. [ABSTRACT FROM AUTHOR]

The programmed death-ligand 1 (PD-L1) is a major checkpoint protein that helps cancer cells evade the immune system. A non-invasive imaging agent with rapid clearance rate would be an ideal tool to predict and monitor the efficacy of anti-PD-L1 therapy. The aim of this research was to engineer a subnanomolar, high-affinity fibronectin type 3 domain (FN3)-based small binder targeted against human PD-L1 (hPD-L1) present on tumor cells. A naive yeast G4 library containing the FN3 gene with three binding loop sequences was used to isolate high-affinity binders targeted to purified full-length hPD-L1. The selected binder clones displayed several mutations in the loop regions of the FN3 domain. One unique clone (FN3hPD-L1-01) with a 6x His-tag at the C-terminus had a protein yield of >5 mg/L and a protein mass of 12 kDa. In vitro binding assays on six different human cancer cell lines (MDA-MB-231, DLD1, U87, 293 T, Raji and Jurkat) and murine CT26 colon carcinoma cells stably expressing hPD-L1 showed that CT26/hPD-L1 cells had the highest expression of hPD-L1 in both basal and IFN-γ-induced states, with a binding affinity of 2.38 ± 0.26 nM for FN3hPD-L1-01. The binding ability of FN3hPD-L1-01 was further confirmed by immunofluorescence staining on ex vivo CT26/hPD-L1 tumors sections. The FN3hPD-L1-01 binder represents a novel, small, high-affinity binder for imaging hPD-L1 expression on tumor cells and would aid in earlier imaging of tumors. Future clinical validation studies of the labeled FN3hPD-L1 binder(s) have the potential to monitor immune checkpoint inhibitors therapy and predict responders. [ABSTRACT FROM AUTHOR]

It has been observed that converting scFv formatted antibodies to full-length IgG often associates with loss of affinity. We aim to address this issue in this paper by establishing an integrated affinity maturation method applying yeast display technology platform. To demonstrate that, we employed a human thrombopoietin receptor targeting antibody named 3D9 which was identified previously from a combinational antibody library in scFv-Fc fusion protein form. We have observed that significant potency loss happened when 3D9 was transformed to full-length IgG form. In this study, we tested whether the potency of the full-length IgG can be improved by affinity maturation of 3D9 using a modified Fab yeast display platform. An efficient CDR3 targeted mutagenesis strategy was designed for Fab library with pre-designed CDR diversity. Next generation sequencing was also used for evaluation of the enrichment process and investigation of sequence-function relationship of the antibody. A variant with improved affinity and higher potency was identified. The study demonstrates that the strategy we used here are efficient for optimizing affinity and activity of full-length IgGs. [ABSTRACT FROM AUTHOR]

Fc fragment with antigen-binding (Fcab) is a novel construct which can be selected to recognize specifically a wide variety of target proteins. We describe the selection and affinity maturation of Fcab clones targeting VEGF, an important pro-angiogenesis factor. To investigate the extent of engineering permissible to Fcabs we applied targeted mutagenesis to all three C-terminal loop structures and the C-terminus of the CH3 domain to isolate high-affinity binders by directed evolution and yeast display. The matured clone, CT6, binds to VEGF with low nanomolar affinity and inhibits VEGF-stimulated proliferation of human umbilical vein endothelial cells in vitro. Molecular dynamics simulations were performed to address flexibility of the molecular structure of CT6 and to approximate a structural ensemble in aqueous solution. Significantly higher RMSF levels of CT6 in comparison to wild-type Fc were limited to the elongated CD-loop in the CH3 domain, while the overall structural integrity was retained. This allowed the Fcab to replace the Fc portion of a mAb, in which both the CH3 and Fab are capable of antigen engagement: a construct called mAb2 was assembled with CT6 and the Fab of bevacizumab. This bispecific molecule showed more potent antagonistic activity than bevacizumab in vitro. Further evaluation for the potential of the CT6 Fcab in targeted therapy is warranted due to the possibility of being combined with other therapeutically meaningful targets. [ABSTRACT FROM AUTHOR]

Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human high-affinity CSPG4 antibodies or antibody fragments. To overcome this limitation, we performed affinity maturation on a novel human CSPG4 single-chain Fv fragment (scFv) using the random mutagenesis approach and screened for improved variants from a yeast display library using a modified whole-cell panning method followed by fluorescence-activated cell sorting. After six rounds of panning and sorting, the top seven mutant scFvs were isolated and their binding affinities were characterized by flow cytometry and surface plasmon resonance. These highly specific, affinity-matured variants displayed nanomolar to picomolar binding affinities to the CSPG4 antigen. While each of the mutants harbored only two to six amino acid substitutions, they represented ~270-3000-fold improvement in affinity compared to the parental clone. Our study has generated affinity-matured scFvs for the development of antibody-based clinical therapeutics targeting CSPG4-expressing tumors. [ABSTRACT FROM AUTHOR]

The combination of protein display technologies and noncanonical amino acids (ncAAs) offers unprecedented opportunities for the high throughput discovery and characterization of molecules suitable for addressing fundamental and applied problems in biological systems. Here we demonstrate that ncAA-compatible yeast display facilitates evaluations of conjugation chemistry and stability that would be challenging or impossible to perform with existing mRNA, phage, or E. coli platforms. Our approach enables site-specific introduction of ncAAs into displayed proteins, robust modification at azide-containing residues, and quantitative evaluation of conjugates directly on the yeast surface. Moreover, screening allows for the selective enrichment of chemically modified constructs while maintaining a genotype-phenotype linkage with encoded azide functionalities. Thus, this platform is suitable for the high throughput characterization and screening of libraries of chemically modified polypeptides for therapeutic lead discovery and other biological applications. [ABSTRACT FROM AUTHOR]

Display technologies such as yeast and phage display offer powerful alternatives to traditional immunization-based antibody discovery, but require conversion of displayed proteins into soluble form prior to downstream characterization. Here we utilize amber suppression to implement a yeast-based switchable display/secretion system that enables the immediate production of soluble, antibody-like reagents at the end of screening efforts. Model selections in the switchable format remain efficient, and library screening in the switchable format yields renewable sources of affinity reagents exhibiting nanomolar binding affinities. These results confirm that this system provides a seamless link between display-based screening and the production and evaluation of soluble forms of candidate binding proteins. Switchable display/secretion libraries provide a cloning-free, accessible approach to affinity reagent generation. [ABSTRACT FROM AUTHOR]

The application of deep sequencing to in vitro display technologies has been invaluable for the straightforward analysis of enriched clones. After sequencing in vitro selected populations, clones are binned into identical or similar groups and ordered by abundance, allowing identification of those that are most enriched. However, the greatest strength of deep sequencing is also its greatest weakness: clones are easily identified by their DNA sequences, but are not physically available for testing without a laborious multistep process involving several rounds of polymerization chain reaction (PCR), assembly and cloning. Here, using the isolation of antibody genes from a phage and yeast display selection as an example, we show the power of a rapid and simple inverse PCR-based method to easily isolate clones identified by deep sequencing. Once primers have been received, clone isolation can be carried out in a single day, rather than two days. Furthermore the reduced number of PCRs required will reduce PCR mutations correspondingly. We have observed a 100% success rate in amplifying clones with an abundance as low as 0.5% in a polyclonal population. This approach allows us to obtain full-length clones even when an incomplete sequence is available, and greatly simplifies the subcloning process. Moreover, rarer, but functional clones missed by traditional screening can be easily isolated using this method, and the approach can be extended to any selected library (scFv, cDNA, libraries based on scaffold proteins) where a unique sequence signature for the desired clones of interest is available. [ABSTRACT FROM AUTHOR]

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. [ABSTRACT FROM AUTHOR]

The inserted (I) domain of αLβ2 integrin (LFA-1) contains the entire binding site of the molecule. It mediates both rolling and firm adhesion of leukocytes at sites of inflammation depending on the activation state of the integrin. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High-affinity mutants of the I domain have been discovered previously using both rational design and directed evolution. We have found that binding affinity fails to dictate the behavior of I domain adhesion under shear flow. In order to better understand I domain adhesion, we have developed a novel panning method to separate yeast expressing a library of I domain variants on the surface by adhesion under flow. Using conditions analogous to those experienced by cells interacting with the post-capillary vascular endothelium, we have identified mutations supporting firm adhesion that are not found using typical directed evolution techniques that select for tight binding to soluble ligands. Mutants isolated using this method do not cluster with those found by sorting with soluble ligand. Furthermore, these mutants mediate shear-driven cell rolling dynamics decorrelated from binding affinity, as previously observed for I domains bearing engineered disulfide bridges to stabilize activated conformational states. Characterization of these mutants supports a greater understanding of the structure–function relationship of the αL I domain, and of the relationship between applied force and bioadhesion in a broader context. [ABSTRACT FROM AUTHOR]

Superantigens (SAgs) are a class of immunostimulatory exotoxins that activate large numbers of T cells, leading to overproduction of cytokines and subsequent inflammatory reactions and systemic toxicity. Staphylococcal enterotoxin C (SEC), a SAg secreted by Staphylococcus aureus, has been implicated in various illnesses including non-menstrual toxic shock syndrome (TSS) and necrotizing pneumonia. SEC has been shown to cause TSS illness in rabbits and the toxin contributes to lethality associated with methicillin-resistant S.aureus (MRSA) in a rabbit model of pneumonia. With the goal of reducing morbidity and mortality associated with SEC, a high-affinity variant of the extracellular variable domain of the T-cell receptor beta-chain for SEC (∼14 kDa) was generated by directed evolution using yeast display. This protein was characterized biochemically and shown to cross-react with the homologous (65% identical) SAg staphylococcal enterotoxin B (SEB). The soluble, high-affinity T-cell receptor protein neutralized SEC and SEB in vitro and also significantly reduced the bacterial burden of an SEC-positive strain of MRSA (USA400 MW2) in an infective endocarditis model. The neutralizing agent also prevented lethality due to MW2 in a necrotizing pneumonia rabbit model. These studies characterize a soluble high-affinity neutralizing agent against SEC, which is cross-reactive with SEB, and that has potential to be used intravenously with antibiotics to manage staphylococcal diseases that involve these SAgs. [ABSTRACT FROM AUTHOR]

No single engineered protein has been shown previously to robustly downregulate epidermal growth factor receptor (EGFR), a validated cancer target. A panel of fibronectin-based domains was engineered to bind with picomolar to nanomolar affinity to multiple epitopes of EGFR. Monovalent and homo- and hetero-bivalent dimers of these domains were tested for EGFR downregulation. Selected orientations of non-competitive heterodimers decrease EGFR levels by up to 80% in multiple cell types, without activating receptor signaling. These heterodimers inhibit autophosphorylation, proliferation and migration, and are synergistic with the monoclonal antibody cetuximab in these activities. These small (25 kDa) heterodimers represent a novel modality for modulating surface receptor levels. [ABSTRACT FROM PUBLISHER]

Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2–MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs. [ABSTRACT FROM AUTHOR]

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput. [ABSTRACT FROM AUTHOR]

This report describes a new cell-surface display system, the Secretion and Capture Technology (SECANT™) platform, which relies on in vivo biotinylation of the protein of interest followed by its capture on the avidinated surface of the parent cell. Cell sorting techniques are then used to isolate clones that display target-binding protein. A distinguishing feature of this method is its ability to display complex proteins, such as full-length immunoglobulin G (IgG) antibodies, on living cells. In this proof-of-concept study, Saccharomyces cerevisiae cells that displayed Herceptin IgG were isolated from a 10 000-fold excess of cells that displayed a lysozyme-binding antibody. [ABSTRACT FROM AUTHOR]

Single-chain antibody fragments (scFv), consisting of two linked variable regions (VH and VL), are a versatile format for engineering and as potential antigen-specific therapeutics. Although the analogous format for T cell receptors (TCRs), consisting of two linked V regions (Vα and Vβ; referred to here as scTv), could provide similar opportunities, all wild-type scTv proteins examined to date are unstable. This obstacle has prevented scTv fragments from being widely used for engineering or therapeutics. To further explore whether some stable human scTv fragments could be expressed, we used a yeast system in which display of properly folded domains correlates with ability to express the folded scTv in soluble form. We discovered that, unexpectedly, scTv fragments that contained the human Vα2 region (IMGT: TRAV12 family) were displayed and properly associated with different Vβ regions. Furthermore, a single polymorphic residue (Serα49) in the framework region conferred additional thermal stability. These stabilized Vα2-containing scTv fragments could be expressed at high levels in Escherichia coli, and used to stain target cells that expressed the specific pep-HLA-A2 complexes. Thus, the scTv fragments can serve as a platform for engineering TCRs with diverse specificities, and possibly for therapeutic or diagnostic applications. [ABSTRACT FROM AUTHOR]

Botulism, a disease of humans characterized by prolonged paralysis, is caused by botulinum neurotoxins (BoNTs), the most poisonous substances known. There are seven serotypes of BoNT (A–G) which differ from each other by 34–64% at the amino acid level. Each serotype is uniquely recognized by polyclonal antibodies, which originally were used to classify serotypes. To determine if there existed monoclonal antibodies (mAbs) capable of binding two or more serotypes, we evaluated the ability of 35 yeast-displayed single-chain variable fragment antibodies generated from vaccinated humans or mice for their ability to bind multiple BoNT serotypes. Two such clonally related human mAbs (1B18 and 4E17) were identified that bound BoNT serotype A (BoNT/A) and B or BoNT/A, B, E and F, respectively, with high affinity. Using molecular evolution techniques, it proved possible to both increase affinity and maintain cross-serotype reactivity for the 4E17 mAb. Both 1B18 and 4E17 bound to a relatively conserved epitope at the tip of the BoNT translocation domain. Immunoglobulin G constructed from affinity matured variants of 1B18 and 4E17 were evaluated for their ability to neutralize BoNT/B and E, respectively, in vivo. Both antibodies potently neutralized BoNT in vivo demonstrating that this epitope is functionally important in the intoxication pathway. Such cross-serotype binding and neutralizing mAbs should simplify the development of antibody-based BoNT diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

High-throughput generation of antibodies against cellular components is currently a challenge in proteomics, therapeutic development and other biological applications. It is particularly challenging to raise antibodies that target membrane proteins due to their insolubility in aqueous solutions. To address these issues, a yeast display library of human single-chain antibody fragments (scFvs) was efficiently screened directly against detergent-solubilized and biotinylated lysates of a target cell line, thereby avoiding issues with membrane protein insolubility and eliminating the need for heterologous expression or purification of antigens. Antibody clones that specifically bind plasma membrane proteins or intracellular proteins were identified, depending on the biotinylation method applied. Antibodies against a predetermined target could also be identified using cell lysate as an antigen source as demonstrated by selecting an scFv against the transferrin receptor (TfR). When secreted from yeast and purified, the selected scFvs are active under physiological conditions in the absence of detergents. In addition, this method allows facile characterization of target antigens because it is compatible with yeast display immunoprecipitation. We expect that this method will prove useful for multiplex affinity reagent generation and in targeted antibody screens. [ABSTRACT FROM AUTHOR]