Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes.

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated beta2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33-41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33-41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8(+) T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.

Assembly of a functional immunoglobulin Fv fragment in Escherichia coli.

An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli. The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the Fv fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The Fv fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant Fv fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the Fv fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.

Protein-fold evolution in the test tube.

Currently, the combination of library selection and directed evolution is the most powerful approach for finding proteins with novel folds or functions. In the past, most studies concentrated either on protein scaffolds with a given fold or on short peptides. With the recent development of potent in vitro selection and evolution techniques, the screening of much larger sequence space is possible, allowing for the de novo generation of proteins.

Unravelling Receptor and RGD Motif Dependence of Retargeted Adenoviral Vectors using Advanced Tumor Model Systems.

Recent advances in engineering adenoviruses are paving the way for new therapeutic gene delivery approaches in cancer. However, there is limited knowledge regarding the impact of adenoviral retargeting on transduction efficiency in more complex tumor architectures, and the role of the RGD loop at the penton base in retargeting is unclear. To address this gap, we used tumor models of increasing complexity to study the role of the receptor and the RGD motif. Employing tumor-fibroblast co-culture models, we demonstrate the importance of the RGD motif for efficient transduction in 2D through the epithelial cell adhesion molecule (EpCAM), but not the epidermal growth factor receptor (EGFR). Via optical clearing of co-culture spheroids, we show that the RGD motif is required for transduction via both receptors in 3D tumor architectures. We subsequently employed a custom-designed microfluidic model containing collagen-embedded tumor spheroids, mimicking the interplay between interstitial flow, extracellular matrix and adenoviral transduction. Image analysis of on-chip cleared spheroids indicated the importance of the RGD motif for on-chip adenoviral transduction. Together, our results show the interrelationship between receptor characteristics, the RGD motif, the 3D tumor architecture and retargeted adenoviral transduction efficiency. The findings are important for the rational design of next-generation therapeutic adenoviruses.

Beyond binding: using phage display to select for structure, folding and enzymatic activity in proteins.

Phage display of a wide range of polypeptides has been increasingly used to identify novel molecules with useful binding properties for research, medical and industrial applications. Recent developments include methods for the selection of stabilized variants of a protein, the selection of regulatable enzymes and promising strategies for the selection and evolution of protein catalysts.

Selection for a periplasmic factor improving phage display and functional periplasmic expression.

The efficiency of both phage display in Escherichia coli and periplasmic expression of recombinant proteins may be limited by the same periplasmic folding steps. To search for E. coli factors that improve the efficiency of both procedures, a library of E. coli proteins was coexpressed in a phagemid vector that contained a poorly folding single-chain Fv antibody (scFv) fragment fused to g3p. We enriched, by panning for antigen binding, those phagemids in which the amount of displayed scFv is highest. We thus identified the periplasmic protein Skp/OmpH/HlpA as improving phage display of a wide range of scFv fragments. This occurs as a result of an increase in the amount of hybrid protein displayed on the phage. Coexpression of skp also increases the functional yield of scFv fragments when expressed by secretion to the periplasm.

Selecting proteins with improved stability by a phage-based method.

We describe a method for the stabilization of proteins that links the protease resistance of stabilized variants of a protein with the infectivity of a filamentous phage. A repertoire of variants of the protein to be stabilized is inserted between two domains (N2 and CT) of the gene-3-protein of the fd phage. The infectivity of fd phage is lost when the three domains are disconnected by the proteolytic cleavage of unstable protein inserts. Rounds of in vitro proteolysis, infection, and propagation can thus be performed to enrich those phage containing the most stable variants of the protein insert. This strategy discriminates between variants of a model protein (ribonuclease T1) differing in conformational stability and selects from a large repertoire variants that are only marginally more stable than others. Because fd phage are exceptionally stable and the proteolysis in the selection step takes place in vitro a wide range of solvent conditions can be used, tailored for the protein to be stabilized.

Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display.

Here we applied ribosome display to in vitro selection and evolution of single-chain antibody fragments (scFvs) from a large synthetic library (Human Combinatorial Antibody Library; HuCAL) against bovine insulin. In three independent ribosome display experiments different clusters of closely related scFvs were selected, all of which bound the antigen with high affinity and specificity. All selected scFvs had affinity-matured up to 40-fold compared to their HuCAL progenitors, by accumulating point mutations during the ribosome display cycles. The dissociation constants of the isolated scFvs were as low as 82 pM, which validates the design of the naïve library and the power of this evolutionary method. We have thus mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.

Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones.

To create a rapid system to test the effect of sequence changes on recombinant antibody binding, we have developed a procedure for producing functional scFv fragments in an Escherichia coli cell-free translation system. Functional antibodies with antigen-binding activity are obtained only if disulfide formation and rearrangement is allowed to take place during the translation reaction. The inclusion of protein disulfide isomerase (PDI) leads to a threefold increase in yield over that obtained in the presence of glutathione redox systems. DsbA had no such effect, indicating that disulfide shuffing, and not net formation, is the crucial yield-limiting step. The addition of the molecular chaperones DnaK and DnaJ increased the amount of soluble protein but not the amount of functional scFv, which appears to be limited entirely by correct disulfide formation. None of these factors significantly influenced total protein synthesis. In the presence of PDI, chaperones, reduced glutathione and oxidized glutathione, 50% of the scFv produced (about 8 micrograms/ml in only 15 min) could be recovered from immobilized antigen.

An in vivo library-versus-library selection of optimized protein-protein interactions.

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

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.

Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.

BACKGROUND: Infection of male Escherichia coli cells by filamentous Ff bacteriophages (M13, fd, and f1) involves interaction of the phage minor coat gene 3 protein (g3p) with the bacterial F pilus (primary receptor), and subsequently with the integral membrane protein TolA (coreceptor). G3p consists of three domains (N1, N2, and CT). The N2 domain interacts with the F pilus, whereas the N1 domain--connected to N2 by a flexible glycine-rich linker and tightly interacting with it on the phage--forms a complex with the C-terminal domain of TolA at later stages of the infection process. RESULTS: The crystal structure of the complex between g3p N1 and TolA D3 was obtained by fusing these domains with a long flexible linker, which was not visible in the structure, indicating its very high disorder and presumably a lack of interference with the formation of the complex. The interface between both domains, corresponding to approximately 1768 A2 of buried molecular surface, is clearly defined. Despite the lack of topological similarity between TolA D3 and g3p N2, both domains interact with the same region of the g3p N1 domain. The fold of TolA D3 is not similar to any previously known protein motifs. CONCLUSIONS: The structure of the fusion protein presented here clearly shows that, during the infection process, the g3p N2 domain is displaced by the TolA D3 domain. The folds of g3p N2 and TolA D3 are entirely different, leading to distinctive interdomain contacts observed in their complexes with g3p N1. We can now also explain how the interactions between the g3p N2 domain and the F pilus enable the g3p N1 domain to form a complex with TolA.

High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment.

The epithelial glycoprotein-2 is abundantly expressed on many solid tumors and is a suitable target for antibody-based therapy. In the present study, an antiepithelial glycoprotein-2 single-chain Fv (scFv) was derived from the hybridoma MOC31 by phage display. Despite its high affinity (KD = 3.9 x 10(-9) M), however, this antibody fragment failed to significantly enrich at lung tumor xenografts in mice, mostly because of its insufficient thermal stability. To overcome this limitation, the antigen-binding residues of the MOC31 scFv fragment were grafted onto the framework of the highly stable and well-folding anti-c-erbB2 scFv 4D5. Further modification of the resulting 4D5 MOC-A, which was performed by transferring eight additional residues of the heavy chain variable domain core of the parent MOC31 antibody, produced 4D5 MOC-B, resulting in increased serum stability at 37 degrees C and also significantly improved expression behavior while retaining the antigen specificity and affinity of the parent MOC31 scFv. In mice, the scFv 4D5 MOC-B, which was radiolabeled with 99mtechnetium using a new histidine-tag specific labeling method (Waibel et al., Nature Biotechnol., 17: 897-901, 1999), showed favorable blood clearance and efficient enriches at lung tumor xenografts, with a tumor:blood ratio of 5.25 and a total dose of 1.47% injected dose per gram after 24 h. Biophysical properties such as high thermal stability are thus decisive for whether these molecules are useful in vivo, and our approach may provide a general strategy to solve this problem. This is also the first report of using a humanized anti-EGP-2 scFv in vivo for targeting solid tumors, which is a promising targeting moiety for the diagnostics and therapy of EGP-2-positive tumors in patients.

Conformation of fatty acyl chains in alpha- and beta-phosphatidylcholine and phosphatidylethanolamine derivatives in sonicated vesicles.

Mono- and dimethylated derivatives constitute important intermediates in the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in eucaryote membranes. 1H-NMR techniques were utilized to examine the conformation of the region of the fatty acyl chains that is close to the polar group in the series of alpha-phospholipids: PE, N-methyl-PE, N,N-dimethyl-PE, and PC. The same series of polar groups, but on phospholipid containing sn-1 and/or sn-3 fatty acyl chains (beta-phospholipids) were also examined. All of the phospholipids were in the form of small sonicated vesicles which are widely utilized as membrane models. The alpha-methylene group of the sn-1 and sn-2 fatty acyl chains of the alpha-phospholipids give rise to separate signals due to the non-equivalency of these chains with respect to the glycerol phosphate backbone on all alpha-phospholipids tested. Additionally, differences in the environment of the PC molecules as well as N-methyl-PE, and N,N-dimethyl-PE, but not PE itself on the inside and outside of the vesicles are reflected in the chemical shift of the alpha-methylene protons. On the other hand, all of the beta-phospholipids (including beta-PE) were found to reflect the inside/outside packing differences in their alpha-methylene groups. The bilayer packing does not induce any nonequivalence in the chemically equivalent acyl chains. In mixed micelles with detergents, beta-phospholipids showed one alpha-CH2 signal for all phospholipids. These results are consistent with a common conformational arrangement for the fatty acyl chains in all alpha-phospholipids that have been investigated no matter what aggregated form. The conformational arrangement in the beta-phospholipids is different, but again is similar for all of the compounds tested in various aggregated forms.

Folding in vitro and transport in vivo of pre-ß-lactamase are SecB independent.

The rate of folding of the precursor of ß-lactamase is not influenced by the presence of SecB under conditions in which GroEL/ES retards the folding. Wild-type ß-lactamase and several mutants in the signal or the mature protein, affecting either transport or enzyme kinetics and probably folding, were examined for total expression, total enzymatic activity, and transported ß-lactamase (in vivo resistance) in secB- and secB+ strains. We conclude that there is no indication of any relevant interaction between SecB and pre-ß-lactamase in vitro, nor did the secB- mutation affect the transport of wild-type ß-lactamase or any of the mutants in vivo. Thus, putative Escherichia coli'folding modulators'must be of limited specificity.

Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL. II. GroEL recognizes thermally unfolded mature beta-lactamase.

By thermal equilibrium measurements we found a three-state folding behavior of mature Escherichia coli beta-lactamase TEM2. The thermodynamically stable intermediate H had no enzymatic activity, but a native-like secondary structure. State H was 9 kcal mol-1 less stable than the native state N and 4 kcal mol-1 more stable than the totally unfolded state U, which is consistent with urea equilibrium measurements of mature beta-lactamase measured under similar conditions. Between 38 degrees C and 50 degrees C there was a decrease in the apparent equilibrium constant for dissociation K'D of the complex between GroEL and mature beta-lactamase, at least partially caused by a decrease in the thermodynamic stability of the native form of mature beta-lactamase. GroEL-bound beta-lactamase was released either after addition of ATP, or in the presence of a competing substrate (i.e. a single-chain antibody), or after lowering the temperature. Whereas at 10 degrees C the folding reaction of mature beta-lactamase was rate limiting, at 37 degrees C the release reaction was the rate-determining step for the regain of beta-lactamase activity, consistent with a decrease of the equilibrium constant for dissociation KD of the complex with temperature. A temperature dependent behavior of GroEL was also observed, when measuring the anilinonaphthalene sulfonic acid (ANS) fluorescence of the chaperone. Similar to all other substrate proteins studied so far, the maximal tryptohan fluorescence of GroEL-bound beta-lactamase was observed at 342 nm. Our results are compatible with a hydrophobic binding pocket of GroEL and confirm the suggested thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL.

Correctly folded T-cell receptor fragments in the periplasm of Escherichia coli. Influence of folding catalysts.

The T-cell receptor is the central recognition molecule in cellular immunity. Its extracellular domains are homologous with and thought to be structurally similar to an antibody Fab fragment. Despite the biological importance of the TCR and the ease of bacterial expression of antibody fragments, there are only few reports of TCR-fragment expression in E. coli. In order to understand the difficulties of expressing correctly folded TCR fragments in E. coli, we have characterized the expression behavior of single-chain Fv analogs of three different TCRs (scTCR). All of them can be folded into the correct conformation in the periplasm of E. coli, yet the extent of correct folding varies greatly. In order to overcome the folding problems of some of the scTCRs, we have developed a system with enhanced in vivo folding capability based on the simultaneous induction of the heat-shock response and over-expression of the E. coli disulfide isomerase DsbA at low temperature. We present a model describing the folding of the scTCRs in the periplasm of E. coli and possible points of folding assistance. The role of the periplasm as an independent folding compartment is emphasized and the existence of a general periplasmic chaperone is postulated. We have also shown that a bivalent scTCR, dimerized in vivo with helix-turn-helix modules, can be expressed in a correctly folded form.

Folding and assembly of an antibody Fv fragment, a heterodimer stabilized by antigen.

The folding and assembly of the Fv fragment of the phosphorylcholine binding antibody McPC603, a non-covalent heterodimer of the variable domains VH and VL, was investigated. Since both domains, each engineered for stability and folding efficiency, could now be obtained in native and soluble form by themselves, fluorescence spectra of VH and VL in unfolded, folded and associated states can be reported. VH and VL only associate when they are native, and the stability of the heterodimer is strongly increased in the presence of antigen. VH rapidly folds into an hyperfluorescent intermediate, and the native state is reached in two parallel, proline-independent reactions. VL displays two fast refolding reactions, which are followed by two slower phases, limited by proline cis/trans-isomerization. The rate-limiting step for both the Fv and the scFv (single-chain Fv) fragment is the formation of the native VH-VL interface, which depends on ProL95 being in cis. The folding of the Fv fragment is fast after short-term denaturation or in the presence of proline cis/trans-isomerase catalysis, but the scFv fragment falls into a kinetic trap, observed by the persistence of the slow phases under all conditions. Furthermore, the scFv fragment, but not the Fv fragment, gives rise to premature interface formation, indicated by the fluorescence spectra and a much higher transient binding of 8-anilino-1-naphthalene sulfonate. The analysis of the folding pathway of the domains VH and VL in isolation and in non-covalent and covalent assemblies should provide helpful insights into the folding of multimeric proteins in general, and for the further engineering of stable and well-folding antibody fragments in particular.