Isolation and characterization of human antibodies targeting human aspartyl (asparaginyl) beta-hydroxylase.

Over-expression of the enzyme human aspartyl (asparaginyl) beta-hydroxylase (HAAH) has been detected in a variety of cancers. It is proposed that upon cellular transformation, HAAH is overexpressed and translocated to the tumor cell surface, rendering it a specific surface antigen for tumor cells. In this work, twelve human single-chain Fv fragments (scFv) against HAAH were isolated from a human non-immune scFv library displayed on the surface of yeast. Five of the twelve were reformatted as human IgG1. Two of the five IgGs, 6-22 and 6-23, showed significant binding to recombinant HAAH in ELISA, tumor cell lines, and tumor tissues. The apparent dissociation constants of 6-22 and 6-23 IgG were 1.0 +/- 0.2 nM and 20 +/- 10 nM respectively. These two antibodies were shown to target different domains of HAAH, with 6-22 targeting the catalytic domain of HAAH and 6-23 targeting the N-terminal non-catalytic domain of HAAH. 6-22 IgG was further characterized, as it had high affinity and targeted the catalytic domain. 6-22 IgG alone does not exhibit significant cytotoxicity toward the tumor cells. However, 6-22 internalizes into tumor cells and can therefore be employed to deliver cytotoxic moieties. A goat anti-human IgG-saporin conjugate was delivered into tumor cells by 6-22 IgG and hence elicited cytotoxicity toward the tumor cells in vitro. These tumor-binding human antibodies can potentially be used in both diagnosis and immunotherapy targeting HAAH-expressing tumor cells.

Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody.

Here, we compare an antibody with the highest known engineered affinity (K(d)=270 fM) to its high affinity wild-type (K(d)=700 pM) through thermodynamic, kinetic, structural, and theoretical analyses. The 4M5.3 anti-fluorescein single chain antibody fragment (scFv) contains 14 mutations from the wild-type 4-4-20 scFv and has a 1800-fold increase in fluorescein-binding affinity. The dissociation rate is approximately 16,000 times slower in the mutant; however, this substantial improvement is offset somewhat by the association rate, which is ninefold slower in the mutant. Enthalpic contributions to binding were found by calorimetry to predominate in the differential binding free energy. The crystal structure of the 4M5.3 mutant complexed with antigen was solved to 1.5A resolution and compared with a previously solved structure of an antigen-bound 4-4-20 Fab fragment. Strikingly, the structural comparison shows little difference between the two scFv molecules (backbone RMSD of 0.6A), despite the large difference in affinity. Shape complementarity exhibits a small improvement between the variable light chain and variable heavy chain domains within the antibody, but no significant improvement in shape complementarity of the antibody with the antigen is observed in the mutant over the wild-type. Theoretical modeling calculations show electrostatic contributions to binding account for -1.2 kcal/mol to -3.5 kcal/mol of the binding free energy change, of which -1.1 kcal/mol is directly associated with the mutated residue side-chains. The electrostatic analysis reveals several mechanistic explanations for a portion of the improvement. Collectively, these data provide an example where very high binding affinity is achieved through the cumulative effect of many small structural alterations.

Protein engineering by cell-surface display.

Protein libraries displayed on cell surfaces can be labeled with soluble ligands exhibiting well-characterized binding equilibria and dissociation kinetics, and then quantitatively screened by flow cytometry at a rate of >10(4) clones/second. The promise of cell-surface display for directed evolution is being realized, with significant improvements recently reported in protein ligand binding affinity, stability, expression and enzymatic activity.

High affinity T cell receptors from yeast display libraries block T cell activation by superantigens.

The alphabeta T cell receptor (TCR) can be triggered by a class of ligands called superantigens. Enterotoxins secreted by bacteria act as superantigens by simultaneously binding to an MHC class II molecule on an antigen- presenting cell and to a TCR beta-chain, thereby causing activation of the T cell. The cross-reactivity of enterotoxins with different Vbeta regions can lead to stimulation of a large fraction of T cells. To understand the molecular details of TCR-enterotoxin interactions and to generate potential antagonists of these serious hyperimmune reactions, we engineered soluble TCR mutants with improved affinity for staphylococcal enterotoxin C3 (SEC3). A library of randomly mutated, single-chain TCRs (Vbeta-linker-Valpha) were expressed as fusions to the Aga2p protein on the surface of yeast cells. Mutants were selected by flow cytometric cell sorting with a fluorescent-labeled SEC3. Various mutations were identified, primarily in Vbeta residues that are located at the TCR:SEC3 interface. The combined mutations created a remodeled SEC3-binding surface and yielded a Vbeta domain with an affinity that was increased by 1000-fold (K(D)=7 nM). A soluble form of this Vbeta mutant was a potent inhibitor of SEC3-mediated T cell activity, suggesting that these engineered proteins may be useful as antagonists.

Yeast surface display for directed evolution of protein expression, affinity, and stability.

The described protocols enable thorough screening of polypeptide libraries with high confidence in the isolation of improved clones. It should be emphasized that the protocols have been fashioned for thoroughness, rather than speed. With library plasmid DNA in hand, the time to plated candidate yeast display mutants is typically 2-3 weeks. Each of the experimental approaches required for this method is fairly standard: yeast culture, immunofluorescent labeling, flow cytometry. Protocols that are more rapid could conceivably be developed by using solid substrate separations with magnetic beads, for instance. However, loss of the two-color normalization possible with flow cytometry would remove the quantitative advantage of the method. Yeast display complements existing polypeptide library methods and opens the possibility of examining extracellular eukaryotic proteins, an important class of proteins not generally amenable to yeast two-hybrid or phage display methodologies.

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 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 surface-displayed 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 wild-type 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.

Directed evolution of a stable scaffold for T-cell receptor engineering.

Here we have constructed a single-chain T-cell receptor (scTCR) scaffold with high stability and soluble expression efficiency by directed evolution and yeast surface display. We evolved scTCRs in parallel for either enhanced resistance to thermal denaturation at 46 degrees C, or improved intracellular processing at 37 degrees C, with essentially equivalent results. This indicates that the efficiency of the consecutive kinetic processes of membrane translocation, protein folding, quality control, and vesicular transport can be well predicted by the single thermodynamic parameter of thermal stability. Selected mutations were recombined to create an scTCR scaffold that was stable for over an hour at 65 degrees C, had solubility of over 4 mg ml(-1), and shake-flask expression levels of 7.5 mg l(-1), while retaining specific ligand binding to peptide-major histocompatibility complexes (pMHCs) and bacterial superantigen. These properties are comparable to those for stable single-chain antibodies, but are markedly improved over existing scTCR constructs. Availability of this scaffold allows engineering of high-affinity soluble scTCRs as antigen-specific antagonists of cell-mediated immunity. Moreover, yeast displaying the scTCR formed specific conjugates with antigen-presenting cells (APCs), which could allow development of novel cell-to-cell selection strategies for evolving scTCRs with improved binding to various pMHC ligands in situ.

In vitro evolution of a T cell receptor with high affinity for peptide/MHC.

T cell receptors (TCRs) exhibit genetic and structural diversity similar to antibodies, but they have binding affinities that are several orders of magnitude lower. It has been suggested that TCRs undergo selection in vivo to maintain lower affinities. Here, we show that there is not an inherent genetic or structural limitation on higher affinity. Higher-affinity TCR variants were generated in the absence of in vivo selective pressures by using yeast display and selection from a library of Valpha CDR3 mutants. Selected mutants had greater than 100-fold higher affinity (K(D) approximately 9 nM) for the peptide/MHC ligand while retaining a high degree of peptide specificity. Among the high-affinity TCR mutants, a strong preference was found for CDR3alpha that contained Pro or Gly residues. Finally, unlike the wild-type TCR, a soluble monomeric form of a high-affinity TCR was capable of directly detecting peptide/MHC complexes on antigen-presenting cells. These findings prove that affinity maturation of TCRs is possible and suggest a strategy for engineering TCRs that can be used in targeting specific peptide/MHC complexes for diagnostic and therapeutic purposes.

Glutathione excretion in response to heterologous protein secretion in Saccharomyces cerevisiae.

Glutathione is excreted in a dose-dependent, non-stoichiometric fashion from Saccharomyces cerevisiae cells expressing and secreting Bovine Pancreatic Trypsin Inhibitor (BPTI), a small, disulfide-bonded protein. Glutathione excretion commences 40 hours following induction of BPTI synthesis. Expression of several secretory proteins with varying disulfide and cysteine contents results in glutathione excretion with no apparent requirement for protein disulfide content. Glutathione excretion is also triggered by overexpression of Kar2p/BiP, a native ER-resident protein-folding chaperone, indicating that the response is a general one not restricted to overexpression of thiol-containing heterologous proteins. Functional vesicular transport is not required at the time of glutathione excretion, and glutathione excretion requires the presence of molecular oxygen. These data are consistent with a delayed oxidative stress response potentiated by earlier heterologous secretion, but are inconsistent with secretory transport of glutathione spent as oxidizing equivalents for disulfide-bond formation in the endoplasmic reticulum.

Fine affinity discrimination by yeast surface display and flow cytometry.

Yeast surface display is a eucaryotic system for the directed evolution of protein binding and stability. For antibody affinity maturation, achievable single-pass enrichment factors are a critical variable. Both reliable recovery of rare clones (yield) and effective differentiation between clones of only slightly improved affinity (purity) are paramount. To validate yeast display's purification potential, trial sorting experiments were performed. The D1.3 (anti-hen egg lysozyme) single chain variable fragment antibody and a 2-fold higher affinity mutant (M3) were each displayed on the surface of Saccharomyces cerevisiae. M3-displaying cells were mixed into the D1.3-displaying cells at a ratio of 1:1000. Cells were fluorescently labeled according to antigen equilibrium binding and then sorted using a flow cytometer. Single-pass enrichment of M3-displaying cells was 125-fold (+/- 65-fold). This level of performance is achievable because of the precision and reproducibility of optimal labeling conditions. This work further demonstrates the capability of yeast display for very fine discrimination between mutant clones of similar affinity. Because large improvements in affinity typically result from combinations of small changes, this capability to identify subtle improvements is essential for rapid affinity maturation.

Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency.

Efficiency of yeast cell surface display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins. Several single-chain T cell receptor (scTCR) single-site mutants that enable yeast surface display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the yeast Saccharomyces cerevisiae. While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in yeast supernatants, with increased expression resulting from the double and triple mutants. Soluble secretion levels were strongly correlated with the quantity of active scTCR displayed as a fusion to Aga2p on the surface of yeast. Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates display efficiency. Thus, yeast display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties.

Expression of the 180-kD ribosome receptor induces membrane proliferation and increased secretory activity in yeast.

Expression of the canine 180-kD ribosome receptor (p180) in yeast cells resulted in a marked proliferation of intracellular membranes. The type of membranes observed varied with the expression of specific portions of p180. Rough membranes predominated when the ribosome binding domain of p180 was present, whereas expression constructs lacking this region resulted in smooth membranes. Northern analysis indicated that expression of the NH(2)-terminal 767 amino acids (DeltaCT), which include the ribosome binding domain, upregulated the transcription and translation of genes involved in exocytosis. The membranes that were proliferated were functional as these cells overcame a temperature-sensitive translocation defect. Most significantly, cells that overexpressed DeltaCT and proliferated rough endoplasmic reticulum exhibited severalfold higher levels of secretion of an ectopically expressed secretory protein. We conclude that p180 expression triggers a cascade of events leading to an increase in secretory potential akin to the terminal differentiation of mammalian secretory cells and tissues.

Selection of functional T cell receptor mutants from a yeast surface-display library.

The heterodimeric alphabeta T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the alpha- and beta-chains and in the beta-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.

Biosynthetic polypeptide libraries.

Methodological advances and new applications have fueled significant growth in the practice of polypeptide library screening.

A small-molecule catalyst of protein folding in vitro and in vivo.

BACKGROUND: The formation of native disulfide bonds between cysteine residues often limits the rate and yield of protein folding. The enzyme protein disulfide isomerase (PDI) catalyzes the interchange of disulfide bonds in substrate proteins. The two -Cys-Gly-His-Cys- active sites of PDI provide a thiol that has a low pKa value and a disulfide bond of high reduction potential (Eo'). RESULTS: A synthetic small-molecule dithiol, (+/-)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), has a pKa value of 8.3 and an Eo' value of -0.24 V. These values are similar to those of the PDI active sites. BMC catalyzes the activation of scrambled ribonuclease A, an inactive enzyme with non-native disulfide bonds, and doubles the yield of active enzyme. A monothiol analog of BMC, N-methylmercaptoacetamide, is a less efficient catalyst than BMC. BMC in the growth medium of Saccharomyces cerevisiae cells increases by > threefold the heterologous secretion of Schizosaccharomyces pombe acid phosphatase, which has eight disulfide bonds. This effect is similar to that from the overproduction of PDI in the S. cerevisiae cells, indicating that BMC, like PDI, can catalyze protein folding in vivo. CONCLUSIONS: A small-molecule dithiol with a low thiol pKa value and high disulfide Eo' value can mimic PDI by catalyzing the formation of native disulfide bonds in proteins, both in vitro and in vivo.

A yeast surface display system for the discovery of ligands that trigger cell activation.

Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell surface. For example, T cells are typically activated when the T cell receptor (TCR) and its associated costimulatory molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell. In this report, yeast expressing high cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vbeta domain) were shown to act as 'pseudo' antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by downregulation of cell surface TCR. Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors and thereby trigger specific cellular responses.

Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments.

We have produced single-chain antibody fragments (scFv) in Saccharomyces cerevisiae at levels up to 20 mg/L in shake flask culture by a combination of expression level tuning and overexpression of folding assistants. Overexpression of the chaperone BiP or protein disulfide isomerase (PDI) increases secretion titers 2-8 fold for five scFvs. The increases occur for scFv expression levels ranging from low copy to ER-saturating overexpression. The disulfide isomerase activity of PDI, rather than its chaperone activity, is responsible for the secretion increases. A synergistic increase in scFv production occurs upon cooverexpression of BiP and PDI.

Purification and functional characterization of a chaperone from Methanococcus jannaschii.

A chaperone from Methanococcus jannaschii has been purified to homogeneity with a single chromatographic step. The chaperone was identified and characterized using activity assays for characteristic chaperone abilities. The M. jannaschii chaperone binds unfolded proteins, protects proteins against heat-induced aggregation, and has a strongly temperature dependent ATPase activity. The chaperone has also been shown to inhibit the spontaneous refolding of a mesophilic protein at low temperatures. The purified chaperone complex has a M(r) of about 1,000,000 and consists of a single type of subunit with an approximate M(r) of 60,000. Analysis of partial sequence data reveals that this chaperone is the predicted protein product of the previously identified chaperonin gene in M. jannaschii (BULT et al., 1996). To our knowledge, this is the first functional characterization of a chaperone from a methanogen.