Efficient generation of a reshaped human mAb specific for the {alpha} toxin of Clostridium perfringens

We have used the technique of antibody reshaping to producea humanized antibody specific for the a toxin of Clostridiumperfringens. The starting antibody was from a mouse hybridomafrom which variable (V) region nucleo-tide sequences were determined.The complementarity-determining regions (CDRs) from these Vregions were then inserted into human heavy and light chainV region genes with human constant region gene fragments subsequentlyadded. The insertion of CDRs alone into human frameworks didnot produce a functional reshaped antibody and modificationsto the V region framework were required. With minor frameworkmodifications, the affinity of the original murine mAb was restoredand even exceeded. Where affinity was increased, an alteredbinding profile to overlapping peptides was observed. Computermodelling of the reshaped heavy chain V regions suggested thatamino acids adjacent to CDRs can either contribute to, or distort,CDR loop conformation and must be adjusted to achieve high bindingaffinity.     

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.     

Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life

Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly(4)Ser)(n) to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His(6) tag fused to the heavy chain and the Strep-tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.     

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment

Europium chelates provide a non-radioactive alternative forsensitive labelling of antibodies for diagnostic immunoassays.Lysine residues at antibody surfaces are ready targets for labellingby an isothiocyanate derivative of the europium chelate (Eu³⁺).Here the labelling efficiency of a recombinant anti-human -fetoprotein(hAFP) Fab fragment has been improved by increasing its lysinecontent by protein engineering. Molecular modelling was usedto identify three light chain constant domain surface arginineresidues, R154, R187 and R210, which were mutated to lysineresidues. The mutations did not influence the affinity of thelysine-enriched Fab fragment and its labelling efficiency wasfound to be 40% higher than that of the wildtype Fab fragmentWith low degree of labelling, the affinities of the two Fabfragments were identical and comparable with that of the originalmonoclonal anti-hAFP IgG. With a higher degree of labellingthe affinities of both Fab fragments decreased more than thatof the intact IgG since more lysine residues are available forlabelling in the additional heavy chain constant domains ofthe larger molecule. Electrostatic adsorption and covalent immobilizationof the Fab fragments were characterized by BIAcore^TM and thelysine-enriched Fab fragment was found to be more efficientlyimmobilized to an activated carboxymethyl surface.     

Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation

A mouse monoclonal antibody (mAb 425) with therapeutic potentialwas ‘humanized’ in two ways. Firstly the mouse variableregions from mAb 425 were spliced onto human constant regionsto create a chimeric 425 antibody. Secondly, the mouse complementarity–determiningregions (CDRs) from mAb 425 were grafted into human variableregions, which were then joined to human constant regions, tocreate a reshaped human 425 antibody. Using a molecular modelof the mouse mAb 425 variable regions, framework residues (FRs)that might be critical for antigen-binding were identified.To test the importance of these residues, nine versions of thereshaped human 425 heavy chain variable (VH) regions and twoversions of the reshaped human 425 light chain variable (VJregions were designed and constructed. The recombinant DNAscoding for the chimeric and reshaped human light and heavy chainswere coexpressed transiently in COS cells. In antigen-bindingassays and competition-binding assays, the reshaped human antibodieswere compared with mouse 425 antibody and to chimeric 425 antibody.The different versions of 425–reshaped human antibodyshowed a wide range of avidities for antigen, indicating thatsubstitutions at certain positions in the human FRs significantlyinfluenced binding to antigen. Why certain individual FR residuesinfluence antigen-binding is discussed. One version of reshapedhuman 425 antibody bound to antigen with an avidity approachingthat of the mouse 425 antibody.     

Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment

By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. In an analysis of 30 non-redundant Fab structures it was found that at the former variable/constant domain interface of the Fv fragment the frequency of exposed hydrophobic residues is much higher than in the rest of the Fv fragment surface. We investigated the importance of these residues for different properties such as folding in vivo and in vitro, thermodynamic stability, solubility of the native protein and antigen affinity. The experimental model system was the scFv fragment of the anti-fluorescein antibody 4-4-20, of which only 2% is native when expressed in the periplasm of Escherichia coli. To improve its in vivo folding, a mutagenesis study of three newly exposed interfacial residues in various combinations was carried out. The replacement of one of the residues (V84D in VH) led to a 25-fold increase of the functional periplasmic expression yield of the scFv fragment of the antibody 4-4-20. With the purified scFv fragment it was shown that the thermodynamic stability and the antigen binding constant are not influenced by these mutations, but the rate of the thermally induced aggregation reaction is decreased. Only a minor effect on the solubility of the native protein was observed, demonstrating that the mutations prevent aggregation during folding and not of the native protein. Since the construction of all scFv fragments leads to the exposure of these residues at the former variable/constant domain interface, this strategy should be generally applicable for improving the in vivo folding of scFv fragments and, by analogy, also the in vivo folding of other engineered protein domains.      

Bacterial secretion of the Fab fragment of a mouse monoclonal IgM that reacts with IgG variable regions

In this report, we describe the expression system that enabledus to produce in Escherichia coli the Fab fragment of a mouseIgM that has previously been shown to inhibit the binding ofIgG to autoantigens by interacting with their variable regions.In our system, both light chain and heavy chain fragments wereput under the control of the malE promoter. The light chainwas fused to the MalE signal sequence, while the heavy chainvariable and first constant region were fused to the alkalinephosphatase signal sequence. In this system, after inductionof the promoter with maltose, the Fab fragment could be detectedin a periplasmic extract of the bacteria by Western blottingand also by ELISA. This Fab fragment was purified on a goatanti-mouse immunoglobulin immunoadsorbent and biotinylated.The Fab fragment produced by E.coli reacted with the trinitrophenyl(TNP) hapten and F(ab')₂ fragments of mouse IgG and these reactivitiescould be specifically inhibited by the corresponding solubleantigens. The dissociation constants of this Fab were 1.65 x10^–6 M for TNP and 5 x 10^–6 M for IgG F(ab')₂ fragments,indicating that the affinity of the Fab fragment compared withthat of the whole IgM molecule was similar for TNP but was lowerfor IgG F(ab')₂ fragments     

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.     

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity

Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30- fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.      

Humanization of 60.3, an anti-CD18 antibody; importance of the L2 loop

Monoclonal antibody 60.3 binds to the CD18 component of theß2 integrin family of adhesion molecules. 60.3 haspotential clinical application in blocking the neutrophilmediatedorgan damage which occurs following a myocardial infarct orhemorrhagic shock. Analysis of the nucleotide and deduced aminoacid sequences of murine 60.3 shows that the light chain containsno amino acid substitutions relative to the closest germlinesequence, while the heavy chain is heavily substituted. We reporthere the humanization of 60.3. The humanized antibody bindsto CD18-bearing cells with {small tilde}4-fold less affinitythan the murine or chimeric antibody. We have shown that modificationof amino acid L50 in the L2 loop of the humanized antibody resultsin loss of binding, while modification of a structural determinant(H71) for the H2 loop has no effect.     

Structure activity relationships of monocyte chemoattractant proteins in complex with a blocking antibody

Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 A structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody-chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.     

A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin

T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.     

The pharmacokinetics of an albumin-binding Fab (AB.Fab) can be modulated as a function of affinity for albumin

An AB.Fab (albumin-binding Fab) consists of a Fab and a phage-derived albumin-binding peptide. This molecule is capable of binding both antigen and albumin simultaneously. Using a Fab derived from Herceptin we generated a panel of AB.Fab variants with wide-ranging affinities for albumin. An assay that measured AB.Fab binding to albumin in solution was developed to most accurately reflect the binding affinity for albumin in vivo. Affinity varied depending upon the species of albumin tested. For rat and rabbit albumin, affinities ranged from 0.04 to 2.5 microM. Reduced affinity for albumin correlated with a reduced half-life and higher clearance rates in both species; the beta half-life ranged 6-fold while clearance ranged over 50-fold in rats and 20-fold in rabbits. To estimate the pharmacokinetic properties of an AB.Fab in humans, AB.Fab variants with similar affinities for rat and rabbit albumin were selected. Using their pharmacokinetic parameters and the principles of allometric scaling for albumin, we estimate an approximate beta half-life for an AB.Fab with 0.5 microM affinity for albumin of up to 4 days in humans with a clearance of 76 ml/h. These variants demonstrate the ability to modulate the clearance of a Fab fragment in vivo and help to establish guidelines for pharmacokinetic engineering of molecules through albumin binding.     

Humanization of mouse anti-human IL-2 receptor antibody B-B10

Mouse monoclonal anti–human IL–2 receptor antibody(BB10) inhibits EL–2–dependent human T–cellproliferation. It has been used in clinical trials in the transplantationfield and promising results are being accumulated. Mouse B–B10antibody was humanized by grafting all CDRs and some frameworkamino add residues onto human antibodies, KAS for V_H and PAYfor V_x. Nine humanized B–BlOs with differently graftedframework residues were constructed and assessed for their biologicalactivities. One of these humanized B–B10, M5, showed nearlythe same activity as the mouse B–B10. The 49th residueof V_x was demonstrated to play a crucial role in the antigen–antibodyinteraction by 3–D structure analysis with a computermodeling system.     

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.     

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.     

Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity

We developed a novel bivalent antibody fragment, the linear(L-) F(ab')₂, comprising tandem repeats of a heavy chain fragmentV_H–C_H1–V_H–C_H1 cosecreted with a light chain.Functional humanized L-F(ab')₂ directed against p185^HER2 wassecreted from Escherichia coli at high titer (100 mg/l) andpurified to homogeneity. The L-F(ab')₂ binds two equivalentsof antigen with an apparent affinity (K_d = 0.46 nM) that iswithin 3-fold of the corresponding thioether-linked F(ab')₂fragment The N-terminal site binds antigen with an affinity(K_d = 1.2 nM) that is 4-fold greater than that for the C-terminalsite, as shown by the comparison of L-F(ab')₂ variants containinga single functional binding site. L-F(ab')₂ has greater antiproliferativeactivity than the thioether-linked F(ab')₂ against the p185^HER2-overexpressingtumor cell line BT474. Linear and thioether-linked F(ab')₂ havevery similar pharmacokinetic properties in normal mice, andtheir serum permanence times are respectively 7- and 8-foldlonger than the corresponding Fab fragment L-F(ab')₂ offersa facile route to bivalent antibody fragments that are potentiallysuitable for clinical applications, and that may have improvedbiological activity compared with thioether-linked F(ab')₂ fragments.     

Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding

The crystallographic study of chimeric B72.3 antibody illustratedthat there are three FR side-chain interactions with eitherCDR residue's side chain or main chain. For example, hydrogenbonds are formed between the hydroxyl group of threonine atL5 in FR1 and the guanidinal nitrogen group of arginine at L24in CDR1, between the hydroxyl group of tyrosine at L36 in FR2and the amide nitrogen group of glutamine at L89 in CDR3 andbetween the hydroxyl group of tyrosine at L71 in FR3 and thecarbonyl group of isoleucine at L29 as well as the amide nitrogengroup of serine at L31 in CDR1. Elimination of these hydrogenbonds at these FR positions may affect CDR loop conformations.To confirm these assumptions, we altered these FR residues bysite-directed mutagenesis and determined binding affinitiesof these mutant chimeric antibodies for the TAG72 antigen. Wefound that the substitution of tyrosine by phenylalanine atL71, altering main-chain hydrogen bonds, significantly reducedthe binding affinity for the TAG72 antigen by 23-fold, whereasthe substitution of threonine and tyrosine by alanine and phenylalanineat L5 and L36, eliminating hydrogen bonds to side-chain atoms,did not affect the binding affinity for the TAG72 antigen. Ourresults indicate that the light-chain FR residue tyrosine atL71 of chimeric B72.3 antibody plays an important role in influencingthe TAG72 antigen binding. Our results will thus be of importancewhen the humanized B72.3 antibody is constructed, since thisimportant mouse FR residue tyrosine at L71 must be maintained.     

鼠源性抗EHEC O157:H7 Stx2噬菌体Fab抗体库的构建及筛选

目的构建鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,并从中筛选特异性的抗体。方法用EHEC O157∶H7 Stx2类毒素免疫BALB/c小鼠,取脾分离淋巴细胞,提取总RNA,RT-PCR分别扩增抗体轻、重链(к和Fd)基因,经双酶切依次克隆入噬粒载体pComb3X中,电转化大肠杆菌XL1-Blue,以辅助噬菌体M13K07进行超感染,构建抗EHEC O157∶H7 Stx2的Fab噬菌体抗体库。以纯化的Stx2为抗原进行筛选,获得抗EHECO157∶H7Stx2的特异性Fab抗体,Western blot法检测噬菌体抗体与毒素抗原的结合活性,并对所得阳性克隆进行基因序列分析。结果构建了一个库容为1.56×107的Fab抗体库,筛选出3株特异性较强的阳性克隆,其中2个可与Stx2A1亚单位抗原反应,1个可与Stx2B亚单位抗原反应。基因序列分析显示,轻、重链可变区氨基酸序列与GenBank中已注册的鼠免疫球蛋白可变区氨基酸序列同源性分别为98.5%和99.6%。结论已成功构建了鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,为进一步制备抗EHECO157∶H7Stx2的治疗性人源化抗体奠定了基础。