Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).     

Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.     

A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli

Although Escherichia coli is in wide use for preparative protein expression, problems with the folding of the recombinant gene product and protein aggregation are frequently encountered. Apart from cytoplasmic expression, this is also true for secretion into the bacterial periplasm, the method of choice for the production of proteins that carry structural disulfide bonds. Here we report the construction of the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC that catalyze the formation and isomerization of disulfide bridges and the peptidyl-prolyl cis/trans-isomerases with chaperone activity, FkpA and SurA. pTUM4 carries a p15a origin of replication and a chloramphenicol resistance gene and, thus, it is compatible with many conventional expression vectors that use the ColEI origin and an ampicillin resistance. Its positive effects on the yield of soluble recombinant protein and the homogeneity of disulfide pattern are illustrated here using the human plasma retinol-binding protein as well as the extracellular carbohydrate recognition domain of the dendritic cell membrane receptor DC-SIGN. Hence, pTUM4 represents a novel helper vector which complements existing cytosolic chaperone coexpression plasmids and should be useful for the functional secretion of various recombinant proteins with hampered folding efficiency.     

A33scFv-green fluorescent protein, a recombinant single-chain fusion protein for tumor targeting

Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv-green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by approximately 95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.     

A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli

Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFNalpha2b as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli.     

Rapid isolation of high-affinity protein binding peptides using bacterial display

A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.     

In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants

The use of random mutagenesis in concert with protein display technologies to rapidly select high affinity antibody variants is an established methodology. In some cases, DNA recombination has been included in the strategy to enable selection of mutations which act cooperatively to improve antibody function. In this study, the impact of L-Shuffling DNA recombination on the eventual outcome of an in vitro affinity maturation has been experimentally determined. Parallel evolution strategies, with and without a recombination step, were carried out and both methods improved the affinity of an anti-Fas single chain variable fragment (scFv). The recombination step resulted in an increased population of affinity-improved variants. Moreover, the most improved variant, with a 22-fold affinity gain, emerged only from the recombination-based approach. An analysis of mutations preferentially selected in the recombined population demonstrated strong cooperative effects when tested in combination with other mutations but small, or even negative, effects on affinity when tested in isolation. These results underline the ability of combinatorial library approaches to explore very large regions of sequence space to find optimal solutions in antibody evolution studies.     

Engineering of the Escherichia coli Im7 immunity protein as a loop display scaffold

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.     

Can three-dimensional contacts in protein structures be predicted by analysis of correlated mutations?

A method has been developed to detect pairs of positions withcorrelated mutations in protein multiple sequence alignments.The method is based on reconstruction of the phylogenetic treefor a set of sequences and statistical analysis of the distributionof mutations in the branches of the tree. The database of homology-derivedprotein structures (HSSP) is used as the source of multiplesequence alignments for proteins of known three-dimensionalstructure. We analyse pairs of positions with correlated mutationsin 67 protein families and show quantitatively that the presenceof such positions is a typical feature of protein families.A significant but weak tendency is observed for correlated residuepairs to be close in the three-dimensional structure. With furtherimprovements, methods of this type may be useful for the predictionof residue-residue contacts and subsequent prediction of proteinstructure using distance geometry algorithms. In conclusion,we suggest a new experimental approach to protein structuredetermination in which selection of functional mutants afterrandom mutagenesis and analysis of correlated mutations providesufficient proximity constraints for calculation of the proteinfold     

Monovalent antibody scFv fragments selected to modulate T-cell activation by inhibition of CD86-CD28 interaction

Beside the interaction of the antigen-presenting major histocompatibility complex with the T-cell receptor, a co-stimulatory signal is required for T-cell activation in an immune response. To reduce immune-mediated graft rejection in corneal transplantation, where topical application of drugs in ointments or eye-drops may be possible, we selected single-chain antibody fragments (scFv) with binding affinity to rat CD86 (B7.2) that inhibit the co-stimulatory signal. We produced the IgV-like domain of rat CD86 as a fusion protein in Escherichia coli by refolding from inclusion bodies. This protein was used as a target for phage display selection of scFv from HuCAL-1, a fully artificial human antibody library. Selected binding molecules were shown to specifically bind to rat CD86 and inhibit the interaction of CD86 with CD28 and CTLA4 (CD152) in flow cytometry experiments. In an assay for CD86-dependent co-stimulation, the selected scFv fragment successfully inhibited the proliferation of T-cells induced by CD86-expressing P815 cells.     

A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry

Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 10(9) variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host ( approximately 10(6) variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.     

Isolating recombinant antibodies against specific protein morphologies using atomic force microscopy and phage display technologies

Isolation of antibodies to antigens that are either unstable, exist in multiple morphologies or have very limited availability can be prohibitively difficult. Here we describe a novel technique combining the capabilities of phage display antibody technology and atomic force microscopy (AFM) that is used to isolate antibody fragments that bind to a specific morphology of the target antigen, alpha-synuclein. AFM imaging allows us to both visualize the presence and morphology of the target antigen as well as to monitor the efficiency of each step in the bio-panning process. We demonstrate that phage displayed antibodies specific to the target antigen morphology can be isolated after only two rounds of selection. The target antigen, alpha-synuclein, has been correlated with the Parkinson's disease (PD). Accumulation of alpha-synuclein fibrillar aggregates into Lewy body inclusions is a hallmark feature of PD. While alpha-synuclein can form several different aggregate morphologies including oligomers, protofibrils and fibrils, the role of these morphologies in the progression of PD is not known. The successful selection of the recombinant antibody described here can have potential therapeutic value since the single-chain fragment variable (scFv) can be expressed intracellularly to control folding and toxicity of the specific protein aggregates.     

Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion

In antigen–antibody interactions, the high avidity ofantibodies depends on the affinity and number of the individualbinding sites. To develop artificial antibodies with multiplevalency, we have fused the single-chain antibody Fv fragmentsto core streptavidin. The resulting fusion protein, termed scFv::strep,was found after expression in Escherichia coli in periplasmicinclusion bodies. After purification of the recombinant productby immobilized metal affinity chromatography, refolding andsize-exclusion FPLC, tetrameric complexes resembling those ofmature streptavidin were formed. The purified tetrameric scFv::strepcomplexes demonstrated both antigen- and biotin-binding activity,were stable over a wide range of pH and did not dissociate athigh temperatures (up to 70°C). Surface plasmon resonancemeasurements in a BIAlite system showed that the pure scFv::streptetramers bound immobilized antigen very tightly and no dissociationwas measurable. The association rate constant for scFv::streptetramers was higher than those for scFv monomers and dimers.This was also reflected in the apparent constants, which wasfound to be 35 times higher for pure scFv::strep tetramers thanmonomeric singlechain antibodies. We could also show that mostof biotin binding sites were accessible and not blocked by biotinylatedE.coli proteins or free biotin from the medium. These sitesshould therefore facilitate the construction of bispecific multivalentantibodies by the addition of biotinylated ligands.     

Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37 degrees C

An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37 degrees C. Two mutations responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry. Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for improved yeast surface display level. This scFv will be useful as a limiting case for evaluating the significance of affinity in tumor targeting to non-internalizing antigens.     

Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability

The glutamate dehydrogenase gene from the hyperthermophilicarchaeon Pyrococcus furiosus has been functionally expressedin Escherichia coli under the control of the X, PL promoter.The P.furiosus glutamate dehydrogenase amounted to 20% of thetotal E.coli cell protein, and the vast majority consisted ofhexamers. Following activation by heat treatment, an enzymecould be purified from E.coli that was indistinguishable fromthe glutamate dehydrogenase purified from P.furiosus. Hybridgenes, that consisted of the coding regions for the homologousglutamate dehydrogenases from P.furiosus and the mesophilicbacterium Clostridium difficile, were constructed and successfullyexpressed in E.coli. One of the resulting hybrid proteins, containingthe glutamate binding domain of the C.difficile enzyme and thecofactor binding domain of the P.furiosus enzyme, did not showa detectable activity. In contrast, the complementary hybridcontaining the P.furiosus glutamate and the C.difficile cofactorbinding domain was a catalytically active hexamer that showeda reduced substrate affinity but maintained efficient cofactorbinding with the specificity found in the Clostridium symbiosumenzyme. Compared with the C.difficile glutamate dehydrogenase,the archaeal-bacterial hybrid is slightly more thermoactive,less thermostable but much more stable towards guanidinium chloride-inducedinactivation and denaturation     

Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state

Phosphotriesterase from Pseudomonas diminuta (PTE) is an extremely efficient metalloenzyme that hydrolyses a variety of compounds including organophosphorus nerve agents. Study of PTE has been hampered by difficulties with efficient expression of the recombinant form of this highly interesting and potentially useful enzyme. We identified a low-level esterolytic activity of PTE and then screened PTE gene libraries for improvements in 2-naphthyl acetate hydrolysis. However, the attempt to evolve this promiscuous esterase activity led to a variant (S5) containing three point mutations that resulted in a 20-fold increase in functional expression. Interestingly, the zinc holoenzyme form of S5 appears to be more sensitive than wild-type PTE to both thermal denaturation and addition of metal chelators. Higher functional expression of the S5 variant seems to lie in a higher stability of the metal-free apoenzyme. The results obtained in this work point out another-and often overlooked-possible determinant of protein expression and purification yields, i.e. the stability of intermediates during protein folding and processing.     

A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities

Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. We have noted that two anti-CD3epsilon scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH(2) terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3epsilongamma was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH(2) terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3epsilongamma. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3epsilongamma increased approximately 3-fold when displayed on pYD5 compared with pYD1. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand-scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements.     

The correlation between protein stability and dipole moment: a critical test

Improving the stability of proteins is a major aim in basic and applied protein science. Querol and coworkers calculated changes in the quasi-electric dipole moment of a protein and used it as a simple criterion to predict stabilizing charge mutations. They employed this method to propose for the bacterial cold shock protein Bc-Csp a number of charge mutations that should have a strong influence on stability. We produced eight variants of Bc-Csp with such mutations and measured their stabilities experimentally. However, we could not find a correlation between the stability and the quasi dipole moment of these variants. Possibly, the quasi dipole moment reflects only a secondary aspect of the changes that are caused by charge mutations in a protein.     

Bacterial lipid modification of proteins for novel protein engineering applications

Functioning of proteins efficiently at the solid-liquid interface is critical to not only biological but also modern man-made systems such as ELISA, liposomes and biosensors. Anchoring hydrophilic proteins poses a major challenge in this regard. Lipid modification, N-acyl-S-diacylglyceryl-Cys, providing an N-terminal hydrophobic membrane anchor is a viable solution that bacteria have successfully evolved but remains unexploited. Based on the current understanding of this ubiquitous and unique bacterial lipid modification it is possible to use Escherichia coli, the popular recombinant protein expression host, for converting a non-lipoprotein to a lipoprotein with a hydrophobic anchor at the N-terminal end. We report two strategies applicable to non-lipoproteins (with or without signal sequences) employing minimal sequence change. Taking periplasmic Shigella apyrase as an example, its signal sequence was engineered to include a lipobox, an essential determinant for lipid modification, or its mature sequence was fused to the signal sequence of abundant outer membrane lipoprotein, Lpp. Lipid modification was proved by membrane localization, electrophoretic mobility shift and mass spectrometric analysis. Substrate specificity and specific activity measurements indicated functional integrity after modification. In conclusion, a convenient protein engineering strategy for converting non-lipoprotein to lipoprotein for commercial application has been devised and tested successfully.     

The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli

In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from V_H manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation.