scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptidelinker to tether the V_H and V_L domains together. An scFv moleculewith a linker 5–12 residues long cannot fold into a functionalFv domain and instead associates with a second scFv moleculeto form a bivalent dimer (diabody). Direct ligation of V_H andV_L domains further restricts association and forces three scFvmolecules to associate into a trivalent trimer (triabody). Wehave defined the effect of linker length on scFv associationby constructing a series of scFvs from anti-neuraminidase antibodyNC10 in which the linker varied from one to four glycine residues.NC10 scFv molecules containing linkers of three and four residuesshowed a strong preference for dimer formation (diabodies),whereas a linker length of one or two glycine residues preventedthe formation of diabodies and directed scFv association intotrimers (triabodies). The data suggest a relatively strict transitionfrom dimer (diabody) to trimer (triabody) upon reduction ofthe linker length from three to two glycine residues. Modellingstudies are consistent with three residues as the minimum linkerlength compatible with diabody formation. Electron microscopeimages of complexes formed between the NC10 scFv multimers andan anti-idiotype Fab' showed that the dimer was bivalent forantigen binding and the trimer was trivalent.     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus

HNK20 is a mouse monoclonal IgA that binds to the F glycoproteinof respiratory syncytial virus (RSV) and neutralizes the virus,both in vitro and in vivo. The single-chain antibody fragment(scFv) derived from HNK20 is equally active and has allowedus to assess rapidly the effect of mutations on affinity andantiviral activity. Humanization by variable domain resurfacingrequires that surface residues not normally found in a humanFv be mutated to the expected human amino acid, thereby eliminatingpotentially immunogenic sites. We describe the constructionand characterization of two humanized scFvs, hu7 and hu10, bearing7 and 10 mutations, respectively. Both molecules show unalteredbinding affinities to the RSV antigen (purified F protein) asdetermined by ELISA and surface plasmon resonance measurementsof binding kinetics (K_a 1x10⁹ M^–1). A competition ELISAusing captured whole virus confirmed that the binding affinitiesof the parental scFv and also of hu7 and hu10 scFvs were identical.However, when compared with the original scFv, hu10 scFv wasshown to have significantly decreased antiviral activity bothin vitro and in a mouse model. Our observations suggest thatbinding of the scFv to the viral antigen is not sufficient forneutralization. We speculate that neutralization may involvethe inhibition or induction of conformational changes in thebound antigen, thereby interfering with the F protein-mediatedfusion of virus and cell membranes in the initial steps of infection.     

Analysis of IgG heavy chain to light chain ratio with mutant Encephalomyocarditis virus internal ribosome entry site

Immunoglobulin G (IgG) is a heterotetrameric protein assembled from two identical heavy chain (HC) and two identical light chain (LC) polypeptides. The HC and LC folding and assembly are a crucial step for IgG production. It is affected by the ratio of HC to LC expression (HC:LC). To date, the HC:LC ratio was analysed mainly by cotransfection of different amounts of two monocistronic HC and LC expression plasmids, an approach biased by different transfection efficiencies. To circumvent this problem, a series of Encephalomyocarditis virus internal ribosome entry site (EMCV IRES) variants with different translation efficiencies were created and used to mediate HC translation in bicistronic constructs. HC and LC were translated from the same mRNA, which provides a more accurate method for the evaluation of the optimal ratio of HC:LC. The results show that the IgG optimal expression levels were obtained when the IRES mediated translation efficiency of the HC was about 50% compared to the cap-dependent translation of the LC. A surprisingly sharp transition to low production was shown when the ratios were below 40%. This study provides a new method to investigate the production of heterodimeric proteins in mammalian cells and adds understanding to the mechanisms of IgG folding and assembly.     

Extracellular self-assembly of virus-like particles from secreted recombinant polyoma virus major coat protein

Mouse polyoma virus major coat protein (VP1) expressed from a recombinant baculovirus is efficiently transported to infected cell nuclei and assembles into protein nanospheres morphologically similar to natural capsids. The nanospheres readily combine with plasmid DNA to form a hybrid gene therapy agent known as virus-like particles (VLPs). To facilitate large-scale production of VLPs free from cellular contaminants, the use of stable Drosophila cell lines expressing either wild-type protein, or VP1 tagged with a secretion signal for targeting to the extracellular medium, was investigated. Both wild-type and tagged VP1 expressed at 2-4 mg VP1/litre of culture. As expected, the wild-type protein self-assembled into VLPs. The tagged VP1 was efficiently secreted to the extracellular medium but was also glycosylated, unlike wild-type VP1. Despite this fact, a small fraction of the recombinant secreted protein assembled into VLP-like structures that had altered disulphide bonding, but were still biologically active. These results demonstrate the considerable tolerance in the nanosphere assembly to structural (i.e. aberrant glycosylation) and environmental (i.e. extracellular medium vs. nuclear milieu) changes. Thus, with modifications to improve nanosphere assembly, the secretion method could be adapted to large-scale preparation of VLPs, providing significant advantages over current methods of production of the vector.     

Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease

To explore the role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of human foamy virus (HFV) protease (PR) in comparison with human immunodeficiency virus type 1 (HIV-1) protease, single (Q8R, H22L, S25T, T28D) and double (Q8R-T28D, H22L-T28D) mutants were created based on sequence alignments and on the molecular model of HFV PR. The wild-type and mutant enzymes were expressed in fusion with maltose binding protein in Escherichia coli and the fusion proteins were purified by affinity chromatography. Specificity constant of most mutants was lower, but the value of Q8R-T28D double mutant enzyme was higher than that of the wild-type HFV PR. Furthermore, urea denaturation at two pH values and pH optimum values showed an increased stability and pH optimum for most mutants. These results suggest that the mutated residues may not be responsible for the higher pH optimum of HFV PR, but they may contribute to the lower dimer stability as compared with that of HIV-1 PR.     

Interaction between a Fab fragment against gp41 of human immunodeficiency virus 1 and its peptide epitope: characterization using a peptide epitope library and molecular modeling

The molecular interaction of the Fab fragment of the human monoclonalantibody 3D6, directed against the transmembrane protein gp41of human immunodeficiency virus (HTV) 1, with its peptide epitopeis characterized by a panel of overlapping peptides, a peptideepitope library and molecular modeling techniques. The sequenceCSGKLICTTAVPW, corresponding to amino acids 605–617 ofgp41, was identified as the best binding peptide (K_D = 1x10^-8mol/1). This peptide served as a starting point to prepare acellulose-bound peptide epitope library in which each residueof the epitope is substituted by all L- and D-amino acids, resultingin 494 epitope peptide variants which were subsequently analyzedfor binding 3D6. The library was synthesized to identify residuescritical for binding and to obtain information about the molecularenvironment of the epitope peptide bound to 3D6. Both cysteineresidues, as well as isoleucine 6, threonine 8 and proline 12,of the epitope were highly sensitive to substitution. Usingthe data obtained from the epitope characterization, as wellas a low-resolution electron density map of a 3D6 Fab-peptidecomplex, a 3-D model of the Fab-peptide complex was generatedby molecular modeling. The modeling experiments predict bindingof the peptide, which is cyclized via the two cysteine residues,to a pocket formed dominantly by the hypervariable loops complementaritydetermining regions CDR3L, CDR2H and CDR3H.     

Dissecting carbohydrate-Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition

The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-N(mutDA) and CV-N(mutDB), compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the trimannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.     

Fimbriae of Bacteroides nodosus: protein engineering of the structural subunit for the production of an exogenous peptide

The pattern of sequence variation between Bacteroides nodosusfimbrial subuits of different serotypes suggests a degree offlexibility, which might be exploited for protein engineeringapproaches for the expression of other peptides. We have testedthis using the well-characterized peptide epitope from VP1 offoot-and-mouth disease virus (FMDV), residues 144–159:LRGDLQVLAQKVARTL (strain 01-BFS). Using bacterial codon usage,several oligonucleotides were designed for the substitutionof this sequence internally at hypervariable regions of thefimbrial subunit (aligned for maximum homology), and for itsaddition at the carboxy-terminus with a diglycine spacer asa flexible hinge. Following site-directed mutagenesis in phageM13, the modified genes were placed under P_L promoter controland placed in a broad host range vector. Analysis of the variantproteins expressed in E.coli showed that these substitutionsaffected, to varying extents, recognition by both anti-fimbrialand anti-FMDV antibodies, indicating that hypervariable region2 is a major antigenic determinant of the fimbrial subunit andthat local stereochemical effects can influence antibody bindingto the FMDV peptide antigenic determinant. In Pseudomonas aeruginosa,viable transformants could only be obtained with the mutantgene encoding the carboxy-terminal graft. These cells providedfimbrial preparations comprised of the modified subunit. Thisthen constitutes the prototype for the development of a generalexpression system for the production of vaccine epitopes andother bioactive peptides. Furthermore, there is considerablescope for further modification of the system, for example byengineering specific chemical or protease cleavage sites forrelease of the grafted peptide. Alternatively, the fimbriaethemselves may serve as a useful supramoleuclar carrier or adjuvantfor immune provocation.     

Expression in insect cells and purification of a catalytically active recombinant human gastric lipase

Human gastric lipase (HGL) cDNA was synthesized by RT-PCR amplificationand cloned into the PVL 1392 baculovirus transfer vector. Therecombinant transfer vector was cotransfected with a modifiedbaculovirus DNA (Baculogold^TM) which contains a lethal deletion.Cotransfection of baculovirus DNA with the recombinant transfervector rescues the lethal deletion of this virus DNA and reconstitutesviable virus particles inside the transfected insect cells.BTI-TN-5B1-4 insect cells (also called High Five^TM cells) wereused to express recombinant HGL. The level of HGL secretionwas {small tilde}32 mg/1 of culture medium. The insect cellsalso accumulated HGL intracellularly, which indicated the existenceof rate-limiting steps in the secretion of HGL. Therefore weinvestigated the effect of replacing the HGL signal peptide(SP) by other SP of secreted proteins. The honeybee melittinSP and the human pancreatic lipase (HPL) SP were tested. Thefusion of HGL with HPL SP resulted in a 2-fold increase in theamount of lipase secreted from the insect cells. The recombinantactive HGL was not processed at the expected cleavage site ofthe natural enzyme, however, but at residue +3. On the otherhand, High Five^TM cells transfected with the vector encodingHGL fused to the melittin SP did not secrete any detectableactive HGL. Recombinant HGL was identified using the Westernblot procedure with rabbit polyclonal antibodies. The proteinmigrated with an apparent molecular mass of 45 kDa under SDS–PAGEanalysis (compared with 50 kDa in the case of natural HGL),indicating that the insect cells have only a limited capacityto glycosylate HGL. The maximum specific activities of the recombinantlipase were 434, 730 and 562 units/mg using long-chain (Intralipid^TM),medium-chain (trioctanoylglycerol) and short-chain (tributyroylglycerol)triacylglycerols, respectively.     

Normal-mode analysis suggests important flexibility between the two N-terminal domains of CD4 and supports the hypothesis of a conformational change in CD4 upon HIV binding

Human CD4 is the receptor for human immunodeficiency virus (HTV).It is well established that the first domain of CD4 binds withhigh affinity to gp120, an envelope protein of HIV, but it hasalso been demonstrated that amino acids located in its seconddomain, within or close to residues 120–127 or 163–166(lying 15 Å away from the binding site), play a role invirus infectivity. We show here that these two stretches ofamino acids happen to be important for the largest amplitudemotion obtained with the normal-mode theory for the two N-terminaldomains of human CD4: an overall rigid-body displacement ofone domain with respect to the other. Such a ‘hinge-bending’motion is unexpected since these two domains were found by crystallographersto be tightly abutting. On the other hand, since for severalproteins the hinge-bending motion experimentally observed uponligand binding was found to be similar to the largest amplitudemotion obtained with the normal-mode theory for these proteins,our results suggest that CD4 may undergo such a kind of conformationalchange upon HTV binding.     

A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII

We have constructed new B domain deletion derivatives of humanfactor Vm (FVm) by manipulating the cDNA using recombinant DNAtechniques. One of these new derivatives, FVIII II, in whichamino acids 771(pro)–1666(asp) have been deleted, no longercontains the protease cleavage site at amino acid position 1648(arg)–1649(glu) known to be involved in the initial step ofFVin processing. We have expressed this molecule in both babyhamster kidney (BHK) 21 cells using the vaccinia virus (VV)expression system and have established Chinese hamster ovary(CHO) derived permanent cell lines expressing either recombinant(r)FVIII or FVIII II AD. The characteristics of FVIII II ADhave been compared to those of rFVIII and/or plasma derived(pd) FVIII. FVIII II All has the following properties: (i) itexhibits FVDI procoagulant activity; (ii) it is expressed at5-fold higher levels than is the complete molecule in comparablesystems; (iii) it migrates for the most part as a single majorband on SDS-PAGE, hi contrast to the complete molecule; (iv)it is activated to a greater extent by thrombin than is eitherrFVm or pdFVIII; and (v) it retains the ability to bind vonWillebrand factor (vWf).     

Revisiting the correlation between proteins' thermoresistance and organisms' thermophilicity

The possibility to rationally design protein mutants that remain structured and active at high temperatures strongly depends on a better understanding of the mechanisms of protein thermostability. Studies devoted to this issue often rely on the living temperature (T(env)) of the host organism rather than on the melting temperature (T(m)) of the analyzed protein. To investigate the scale of this approximation, we probed the relationship between T(m) and T(env) on a dataset of 127 proteins, and found a much weaker correlation than previously expected: the correlation coefficient is equal to 0.59 and the regression line is T(m) approximately 42.9 degrees C + 0.62T(env). To illustrate the effect of using T(env) rather than T(m) to analyze protein thermoresistance, we derive statistical distance potentials, describing Glu-Arg and Asp-Arg salt bridges, from protein structure sets with high or low T(m) or T(env). The results show that the more favorable nature of salt bridges, relative to other interactions, at high temperatures is more clear-cut when defining thermoresistance in terms of T(m). The T(env)-based sets nevertheless remain informative.     

Design of four-helix bundle protein as a candidate for HIV vaccine

To be efficient, a synthetic vaccine should contain differentT and B cell epitopes of human immunodeficiency virus (HIV)antigens, and the B epitope regions in the vaccine and in theHIV should be conformationally similar. We have suggested previouslythe construction of vaccines in the form of a protein with apredetermined tertiary structure, namely a four--helix bundle.Antigenic determinants of cellular and humoral immunity areblocks for the vaccine design. From experimentally studied HIV-1T and B cell epitopes, we constructed a sequence of a four-helixprotein TBI (T and B cell epitopes containing immunogen). Thegene of the protein was synthesized and the protein was producedin C600 Escherichia coli cells under recA promoter from Proteusmirabelis. CD spectroscopy of the protein demonstrated that30% of amino acid residues adopt an -helical conformation. Miceimmunized with TBI have shown both humoral and cellular immuneresponses to HIV-1. The obtained data show that the design ofTBI was successful. The synthesized gene structure makes possiblefurther reconstruction and improvement of the protein vaccinestructure.     

Prediction of the antigenic sites of the cystic fibrosis transmembrane conductance regulator protein by molecular modelling

Antibodies are powerful tools for studying the in situ localizationand physiology of proteins. The prediction of epitopes by molecularmodelling has been used successfully for the papilloma virus,and valuable antibodies have been raised [Muller et aL (1990)J. Gen. Virol, 71, 2709–2717]. We have improved the modellingapproach to allow us to predict epitopes from the primary sequencesof the cystic fibrosis transmembrane conductance regulator.The procedure involves searching for fragments of primary sequenceslikely to make amphipathic secondary structures, which are hydrophilicenough to be at the surface of the folded protein and thus accessibleto antibodies. Amphipathic helices were predicted using themethods of Berzofsky, Eisenberg and Jahnig. Their hydrophobichydrophilicinterface was calculated and drawn, and used to predict theorientation of the helices at the surface of the native protein.Amino acids involved in turns were selected using the algorithmof Eisenberg. Tertiary structures were calculated using ‘FOLDING’,a software developed by R.Brasseur for the prediction of smallprotein structures [Brasseur (1995) J. MoL Graphics, in press].We selected sequences that folded as turns with at least fiveprotruding polar residues. One important property of antibodiesis selectivity. To optimize the selectivity of the raised antibodies,each sequence was screened for similarity (FASTA) to the proteinsequences from several databanks. Ubiquitous sequences werediscarded. This approach led to the identification of 13 potentialepitopes in the cystic fibrosis transmembrane conductance regulator:seven helices and six loops.     

Modular mutagenesis of a TEM-barrel enzyme: the crystal structure of a chimeric E.coli TIM having the eighth {beta}{alpha}-unit replaced by the equivalent unit of chicken TIM

Abstract The crystal structure of a hybrid Escherichia coli triosephosphateisomerase (TIM) has been determined at 2.8 Å resolution.The hybrid TIM (ETIM8CHI) was constructed by replacing the eighthß-unit of E.coli TIM with the equivalent unit of chickenTIM. This replacement involves 10 sequence changes. One of thechanges concerns the mutation of a buried alanine (Ala232 instrand 8) into a phenylalanine. The ETIM8CHI structure showsthat the A232F sequence change can be incorporated by a side-chainrotation of Phe224 (in helix 7). No cavities or strained dihedralsare observed in ETIM8CHI in the region near position 232, whichis in agreement with the observation that ETIM8CHI and E.coliTIM have similar stabilities. The largest CA (C-alpha atom)movements, 3 Å, are seen for the C-terminal end of helix8 (associated with the outward rotation of Phe224) and for theresidues in the loop after helix 1 (associated with sequencechanges in helix 8). From the structure it is not clear whythe k_cat of ETIM8CHI is 10 times lower than in wild type E.coliTIM     

Search for the stable state of a short chain in a molecular field

A general approach is developed to search for stable structuresof short chain fragments (e.g. of loops or bound oligopeptides)in a given molecular field. This molecular field is producedby the remaining part of a globule or by any other surface witha defined spatial structure. The fragment must be short enoughto have no pronounced long-range interactions within itself.The method is illustrated by calculation of the 3-D structuresof two loops of bovine pancreatic trypsin inhibitor (BPTI).Computations are based on a lattice model of conformationalspace and on strict and fast algorithms of 1-D statistical mechanicsand dynamic programming (which are very similar in essence).This makes a search of oligopeptide structures only severaltimes (and not several orders of magnitude) longer than thatof a dipeptide.     

Lipid modification of proteins and their membrane transport

An effective method for artificial attachment of lipid anchorsto water-soluble proteins has been developed. To this end, aprotein molecule is modified in a system of reversed micellesby a water-insoluble reagent, e.g. fatty acid chloride. Fattyacylated proteins acquire an ability to translocate across lipidmembranes and penetrate intact cells. This principle of impartingtransmembrane properties to water-soluble proteins makes itpossible to realize in vivo a direct transport of antibodiesacross the hemato-encephalic barrier into the brain and to developa method for virus suppression by fatty acylated anti-viralantibodies capable of penetrating infected cells. The effectof a drastic increase in the biological activity of exogenousprotein factors, e.g. Staphylococcus aureus enterotoxin A, asa result of their artificial fatty acylation has been discovered.The above-mentioned phenomena are discussed in relation to thein vivo data, indicating that post-translational modificationof proteins by fatty acids and phospholipids is very widespreadin nature and evidently plays an important role in protein transportand sorting. In this connection, lipid modification of proteinsis regarded as a possible general step of protein transportin vivo.     

Humanization of the anti-CEA T84.66 antibody based on crystal structure data

Chimeric T84.66 (cT84.66) is a monoclonal antibody (mAb) of high specificity and affinity for the tumor-associated carcinoembryonic antigen (CEA). Radiolabeled cT84.66 has demonstrated utility in the clinic as a reagent for the radioimmunoscintigraphy and radioimmunotherapy of CEA-positive colorectal and breast malignancies. To extend the therapeutic efficacy of T84.66, humanization by complementary determining region (CDR) grafting was employed. CDR grafting is a well-established technique, though often a series of framework back-mutations is required to restore high affinity. Recently, the crystal structure of the T84.66 diabody (scFv dimer) derived from the murine T84.66 mAb was determined, facilitating the humanization process by the availability of crystal structure data for both the graft donor and graft acceptor. A search of the Protein Data Bank revealed close structural similarity (r.m.s.d. of 1.07 A) between the Fv of T84.66 and the Fv of 4D5v8, a humanized anti-p185HER2 antibody marketed as Herceptin (Trastuzumab). This resulted in two humanized versions of the T84.66 M5A and M5B mAbs that differed only in the number of murine residues present in the C-terminal half of CDR-H2. Biochemical analysis and animal biodistribution studies were conducted to evaluate the humanized mAbs. The M5A, M5B and cT84.66 mAbs showed sub-nanomolar affinity for CEA and as radiolabeled mAbs exhibited specific tumor localization in tumor bearing mice. The T84.66 M5A mAb was selected for clinical development due to a slightly higher tumor uptake and a larger content of human residues, and was renamed hT84.66. A limited-scale production and animal imaging study have demonstrated hT84.66's ability to support clinical trials. Planned clinical trials will determine the effective utilization of this structure-based approach in the development of a promising new therapeutic.     

SARFing the PDB

Fast growth of the number of the solved protein structures isincreasing the role of their comparative analysis. In this paperI describe a new program, SARF2, for protein structure comparisonand discuss new examples of the non-topological structural resemblance.SARF2 is designed to detect ensembles of secondary structureelements, which form similar spatial arrangements with possibledifferent topological connections. The program is availableto everyone through the World Wide Web (URL http://wwwhnmb.ncifcrf.gov/{small tilde}nicka/sarf2.html). The performance of the programis demonstrated by previously unnoticed cases of the significantsimilarities. One similarity discussed in this paper, betweenheme-binding proteins (cytochrome P450 and globin), consistsof six -helices, arranged into a globin fold. Another pair ofstructures (pectate lyase and snowdrop lectin) achieve similarß-prism architecture through different topologies.The significance of these similarities is validated by (i) thedistribution of a similarity score, (ii) the comparison of thealigned contact maps and/or (iii) the location of the activesite. The observation of recurrent non-topological structuralmotifs implies their energetic stability and opens new possibilitiesfor sequence-structure alignment (threading) methods.