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.     

Improved design of an antigen with enhanced specificity for the broadly HIV-neutralizing antibody b12

In an attempt to design immunogens that elicit broadly HIV-neutralizing antibodies, we recently engineered monomeric HIV-1 gp120 to bind preferentially b12, a broadly neutralizing antibody to the CD4-binding site (CD4bs) on gp120, by mutating four central residues in the CD4bs to alanine and introducing extra N-glycosylation sites potentially to mask unwanted B-cell epitopes. Despite the favorable antigenicity of this mutant, it harbors two potential caveats that may limit its effectiveness to elicit b12-like antibodies: (i) b12-binding affinity is reduced relative to wild-type gp120 and (ii) binding of some non-neutralizing antibodies to the N-terminal C1 region of gp120 is still observed. Here, we sought to correct these potential limitations. By reverting one of the added N-glycosylation sites on the gp120 core, b12 binding was improved without affecting the epitope-masking properties of the original mutant. Furthermore, truncation of the gp120 N-terminus eliminated binding of the anti-C1 antibodies. Finally, based on the binding profiles of additional non-neutralizing antibodies tested here, further N-glycosylation sites were incorporated to mask their corresponding epitopes. The resulting hyperglycosylated gp120 variants bind b12 and another broadly neutralizing antibody, 2G12, with apparent affinities approaching that of wild-type gp120, but do not bind 21 non- or weakly neutralizing antibodies to seven different epitopes on gp120. These hyperglycosylated variants expand our panel of glycoengineered gp120s that are currently being evaluated for their ability to elicit broadly neutralizing antibodies.     

Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis

One approach to the analysis of leucocyte cell surface proteinsis to express their domains with part of another protein asa carrier. We report the use of two immunoglobulin superfamily(IgSF) domains from rat CD4 (CD4d3+4) in producing domains fromvarious superfamilies as chimeric proteins in Chinese hamsterovary cell lines. Four types of construct were successfullyexpressed containing: (i) the two IgSF domains of CD48; (ii)the IgSF domain of mb-1 which is part of the B cell antigenrecognition complex; (iii) a T cell receptor V domain; and (iv)the N-terminal domain of CD5 which belongs to the scavengerreceptor superfamily. This CD5 chimeric protein was antigenkfor a panel of CD5 mAbs showing that mAbs with functional effectsreacted with the N-terminal domain of CD5. The CD48 chimericprotein has been used both as multivalent complexes producedby crosslinking with mAbs recognizing CD4 and in a monomericform to analyse the kinetics of the interaction between CD48and CD2 [van der Merwe et al. (1993) EMBO J., 12, 4945–4954].     

Domain organization of the extracellular region of CD45

CD45 is a large, heavily glycosylated, transmembrane proteinphosphotyrosine phosphatase found on all nucleated cells ofhaematopoietic origin. In lymphocytes, the cytoplasmic phosphataseis necessary for efficient signalling through the antigen receptorbut in contrast little is known about the interactions of theextracellular region of the molecule. This consists of a mucin-likeregion, a novel cysteine-containing region and a region containingthree putative fibronectin type III domains. To confirm thisorganization and to identify parts potentially important forfunction, we have expressed fragments of the extracellular domainof rat CD45 as recombinant soluble proteins. Proteins correspondingto two, three and four domains of CD45 were expressed in secretedforms. Single domains and constructs for proteins with truncationsof the predicted domains were not expressed. This is consistentwith the proposed structural organization. Determination ofthe positions of the disulphide bonds in the N-terminal cysteine-containingregion and the first fibronectin type III domain identifiednovel disulphide bonds within the fibronectin type III domainand an unusual inter-domain disulphide linkage. Circular dichroismspectroscopy indicated that this region of rat CD45 has mainlyß-strand secondary structure and no -helical content.These studies support the proposed domain organization of CD45.     

Structural analysis of the CD2 T lymphocyte antigen by site-directed mutagenesis to introduce a disulphide bond into domain 1

Many proteins have been predicted to contain domains with immunoglobulin-Iikefolds and hence to be members of the immunoglobulin superfamily(IgSF). However, several members lack the Cys residues capableof forming the disulphide bond that forms a characteristic bridgebetween the ß sheets in the Ig fold, e.g. domain 1of the lymphocyte antigen CD2. The assignment of ßstrands in CD2 by sequence analysis was tested by attemptingto introduce a disulphide bridge between the ß sheetsby mutating two residues in the relevant positions to Cys. Mutant,soluble forms of CD2 were expressed in Chinese hamster ovarycell lines and amino add sequencing showed that a disulphidebond was formed as predicted, but not in the control where oneCys residue was misplaced by four residues. Evidence that bothmutated molecules folded correctly is given by the indistinguishablebinding of three monoclonal antibodies recognising differentepftopes on CD2. The 3-D structure of rat CD2 domain 1 has beendetermined by NMR spectroscopy and X-ray crystallography, confirmingthe predictions from the sequence. Applications of this methodof insertion of disulphide residues for probing protein structuresare discussed, together with other structures of IgSF domainslacking the typical inter-sheet disulphide bond.     

'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization

‘Knobs-into-holes’ was originally proposed by Crickin 1952 as a model for the packing of amino acid side chainsbetween adjacent -helices. ‘Knobs-into-holes’ isdemonstrated here as a novel and effective design strategy forengineering antibody heavy chain homodimers for heterodimerization.In this approach a ‘knob’ variant was first obtainedby replacement of a small amino acid with a larger one in theC_H3 domain of a CD4-IgG immuno-adhesin: T366Y. The knob wasdesigned to insert into a ‘hole’ in the C_H3 domainof a humanized anti-CD3 antibody created by judicious replacementof a large residue with a smaller one: Y407T. The anti-CD3/CD4-IgGhybrid represents up to 92% of the protein A purified proteinpool following co-expression of these two different heavy chainstogether with the anti-CD3 light chain. In contrast, only upto 57% of the anti-CD3/CD4-IgG hybrid is recovered followingco-expression in which heavy chains contained wild-type C_H3domains. Thus knobs-into-holes engineering facilitates the constructionof an antibody/ immunoadhesin hybrid and likely other Fc-containingbifunctional therapeutics including bispecific immuno-adhesinsand bispecific antibodies.     

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

A model is presented for the 3-D structure of the catalyticdomain of the putative leader peptidase NisP of Lactococcuslactis, and the interaction with its specific substrate, theprecursor of the lantibiotic nisin. This homology model is basedon the crystal structures of subtilisin BPN' and thermitasein complex with the inhibitor eglin. Predictions are made ofthe general protein fold, inserted loops, Ca²⁺ binding sites,aromatic interactions and electrostatic interactions of NisP.Cleavage of the leader peptide from precursor nisin by NisPis the last step in maturation of nisin. A detailed predictionof the substrate binding site attempts to explain the basisof specificity of NisP for precursor nisin. Specific acidicresidues in the SI subsite of the substrate binding region ofNisP appear to be of particular importance for electrostaticinteraction with the PI Arg residue of precursor nisin afterwhich cleavage occurs. The hydrophobic S4 subsite of NisP mayalso contribute to substrate binding as it does in subtilisins.Predictions of enzyme-substrate interaction were tested by proteinengineering of precursor nisin and determining susceptibilityof mutant precursors to cleavage by NisP. An unusual propertyof NisP predicted from this catalytic domain model is a surfacepatch near the substrate binding region which is extremely richin aromatic residues. It may be involved in binding to the cellmembrane or to hydrophobic membrane proteins, or it may serveas the recognition and binding region for the modified, hydrophobicC-terminal segment of precursor nisin. Similar predictions forthe tertiary structure and substrate binding are made for thehighly homologous protein EpiP, the putative leader peptidasefor the lantibiotic epidermin from Staphylococcus epidermidis,but EpiP lacks the aromatic patch. Based on these models, proteinengineering can be employed not only to test the predicted enzyme-substrateinteractions, but also to design lantibiotic leader peptidaseswith a desired specificity.     

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.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

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.     

Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold

In order to probe the structural constraints on binding of RGDsequences to the platelet receptor _IIbß₃ we have usedrecombinant DNA techniques to install the RGD sequence into‘presentation scaffolds’, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinV_L domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to _IIbß₃ up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 42–57 or 44–55from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC₅₀s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto _IIbß₃, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents.     

Chimeric protein for selective cell attachment onto cellulosic substrates

We have developed a fusion protein (CBD-LG) incorporating a cellulose-binding domain and an antibody binding domain, protein LG, to provide an adaptor molecule for cell separation with regenerated cellulose hollow fiber arrays. A single hollow fiber cell adhesion assay utilizing a CD34+ cell line, KG1a, was used to investigate whether ligand affinity interactions were strong enough for cell attachment and separation. CBD-LG efficiently captured CD34+ cells labeled with the mouse IgG2a monoclonal antibody MHCD3400. However, it was not possible to bind CD34+ cells labeled with an IgG1 antibody (HPCA-2). The low affinity of HPCA-2 for LG was overcome by secondary antibodies: KG1a cells that were dual labeled with HPCA-2 followed by rat anti-mouse IgG1 adhered inside hollow fibers coated with CBD-LG. Alternatively, immobilized rabbit polyclonal anti-mouse IgG1 captured cells labeled with HPCA-2. The development of an adaptor molecule to display recombinant domains at the surface of hollow fibers will be an effective tool to investigate cellular ligand-receptor interactions, a necessary step in the development of hollow fiber bioreactors for manufacture of human cellular products.     

Molecular modeling of antibody-antigen complexes between the Brucella abortus O-chain polysaccharide and a specific monoclonal antibody

A molecular model of the binding site of an anti-carbohydrateantibody (YsT9.1) has been developed using computer-assistedmodeling techniques and molecular dynamics calculations. Sequencehomologies among YsT9.1 and the Fv regions of McPC603, J539and human Bence-Jones protein REI, all of which have solvedcrystal structures, provided the basis for the modeling. Thegroove-type combining site model had a topography which wascomplementary to low energy confonners of the polysaccharide,a Brucella O-antigen, and the site could be almost completelyfilled by a pentasaccharide epitope in either of two dockingmodes. Putative interactions between this epitope and the antibodyare consistent with the known structural requirements for bindingand lead to the design of oligosaccharide inhibitors that probethe veracity of the modeled docked complex. Ultimately boththe Fv model and the docked complex will be compared with independentcrystal structures of YsT9.1 Fab with and without pentasaccharideinhibitor, currently at the stage of refinement.     

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).     

Synthesis and mutagenesis of an IgG-binding protein based upon protein A of Staphylococcus aureus

A novel protein able to bind with high affinity to the Fc fragmentof IgG from a variety of animals has been produced by a genesynthesis approach. The IgG binding is accomplished by the presenceof a single or two consecutive domains based upon domain B fromprotein A of Staphylo-coccus aureus. The IgG-binding moietyis fused to a peptide containing 21, 53 or 81 amino acids derivedfrom the N-terminus of bovine DNase I. The latter is presentto guide the expression of the protein in Escherichia coli intoan inclusion body. This facilitates the high expression andrecovery of the IgG-binding domains. The binding activity ofthis fusion protein is very close to that of the native proteinA. Site-directed mutagenesis of the fusion protein and subsequentidentification of changed binding interactions is reported.     

Enhancement and Induction of HIV‐1 Infection through an Assembled Peptide Derived from the CD4 Binding Site of gp120

Contact between the human immunodeficiency virus (HIV‐1) and its target cell is initiated by the interaction of viral gp120 with cellular CD4. An assembled peptide (CD4bs‐M) that presents the CD4 binding site of gp120 was previously shown to inhibit the gp120–CD4 interaction. Here, we demonstrate that CD4bs‐M selectively enhances infection of cells with HIV‐1, whereas infection with herpes simplex virus remains largely unaffected. The effects of CD4bs‐M variants containing D ‐amino acids, or prolines at selected positions, point to the importance of side chain orientation and spatial orientation of this fragment. Furthermore, CD4bs‐M was shown to assemble into amyloid‐like fibrils that capture HIV‐1 particles, which likely contributes to the infection‐enhancing effect. Beyond infection enhancement, CD4bs‐M enabled HIV‐1 infection of CD4‐negative cells, suggesting that binding of the peptide to gp120 facilitates interaction of gp120 with coreceptors, which might in turn enhance HIV‐1 entry.     

A comparison of two murine monoclonal antibodies humanized by CDR-grafting and variable domain resurfacing

The variable domain resurfacing and CDR-grafting approachesto antibody humanization were compared directly on the two murinemonoclonal antibodies N901 (anti-CD56) and anti-B4 (anti-CD19).Resurfacing replaces the set of surface residues of a rodentvariable region with a human set of surface residues. The methodof CDR-grafting conceptually consists of transferring the CDRsfrom a rodent antibody onto the Fv framework of a human antibody.Computer-aided molecular modeling was used to design the initialCDR-grafted and resurfaced versions of these two antibodies.The initial versions of resurfaced N901 and resurfaced anti-B4maintained the full binding affinity of the original murineparent antibodies and further refinements to these versionsdescribed herein generated five new resurfaced antibodies thatcontain fewer murine residues at surface positions, four ofwhich also have the full parental binding affinity. A mutationalstudy of three surface positions within 5 Å of the CDRsof resurfaced anti-B4 revealed a remarkable ability of the resurfacedantibodies to maintain binding affinity despite dramatic changesof charges near their antigen recognition surfaces, suggestingthat the resurfacing approach can be used with a high degreeof confidence to design humanized antibodies that maintain thefull parental binding affinity. By comparison CDR-grafted anti-B4antibodies with parental affinity were produced only after seventeenversions were attempted using two different strategies for selectingthe human acceptor frameworks. For both the CDR-grafted anti-B4and N901 antibodies, full restoration of antigen binding affinitywas achieved when the most identical human acceptor frameworkswere selected. The CDR-grafted anti-B4 antibodies that maintainedhigh affinity binding for CD19 had more murine residues at surfacepositions than any of the three versions of the resurfaced anti-B4antibody. This observation suggests that the resurfacing approachcan be used to produce humanized antibodies with reduced antigenicpotential relative to their corresponding CDR-grafted versions.     

Binary discontinuous compact protein domains

Few methods exist that identify discontinuous protein domainscontaining more than one polypeptide chain. This paper describesa new method for locating such discontinuous domains based ontheir compactness, and applies the methodology to locate themost compact domains in bovine pancreatic trypsin inhibitor,ribonuclease, cytochrome c and myoglobin. The compactness ofall binary discontinuous peptide combinations is first exhaustivelyevaluated. Several screening steps are then used to locate thosecompact units that represent global minima of compactness. Sincedomains are generally taken to be large, mutually exclusivestructures that span most of the protein's sequence, compactdomains were found by examining all compact units (both continuousand discontinuous) to locate two or three units that span mostof the protein's sequence, have little mutual overlap and goodoverall compactness. Compact domains compare well with domainsfound by other methods and with experimental evidence that maydifferentiate domain structure. The strongest experimental evidencefor the existence of compact discontinuous domains comes fromthe work of Oas and Kim [(1988) Nature, 336, 42–48] wherea peptide that corresponds almost exactly to a compact domainhas been synthesized and shown to have native-like structurein solution.     

Homology modeling study of the human interleukin-7 receptor complex

Following a recent model of human interleukin-7 (IL-7), we presenthere a modeling study of the extracellular part of the humanIL-7 receptor complex, including the IL-7 specific (IL-7R) andthe common gamma (_c) chains. The investigation is based on structuralhomology to the complex of human growth hormone (hGH) boundto its receptor (hGHR). For domain 1 of IL-7R two differentmodels are presented which differ in the alignment to hGHR inthree regions. However, these differences affect binding toIL-7 in only one region, at the interface between loop EF ofdomain 1 of IL-7R and helix C of IL-7. The disulfide patternin domain 1 of IL-7R is predicted to deviate from that observedin hGHR in that the C'–E disulfide (hGHR) is replacedby a C-C' cross-link. The prediction for the _c chain is comparedwith two previous studies. The models of the complex provideinsight into the binding of IL-7 to its receptor and have implicationsfor the suggestion of mutagenesis experiments and the designof (ant)agonists.     

Improving solubility and refolding efficiency of human V(H)s by a novel mutational approach

The antibody V(H) domains of camelids tend to be soluble and to resist aggregation, in contrast to human V(H) domains. For immunotherapy, attempts have therefore been made to improve the properties of human V(H)s by camelization of a small set of framework residues. Here, we have identified through sequence comparison of well-folded llama V(H) domains an alternative set of residues (not typically camelid) for mutation. Thus, the solubility and thermal refolding efficiency of a typical human V(H), derived from the human antibody BT32/A6, were improved by introduction of two mutations in framework region (FR) 1 and 4 to generate BT32/A6.L1. Three more mutations in FR3 of BT32/A6.L1 further improved the thermal refolding efficiency while retaining solubility and cooperative melting profiles. To demonstrate practical utility, BT32/A6.L1 was used to construct a phage display library from which were isolated human V(H)s with good antigen binding activity and solubility. The engineered human V(H) domains described here may be useful for immunotherapy, due to their expected low immunogenicity, and in applications involving transient high temperatures, due to their efficient refolding after thermal denaturation.