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.     

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.     

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 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.     

A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration

GM-CSF (granulocyte-macrophage colony stimulating factor) plays a central role in inflammatory processes. Treatment with antibodies neutralizing murine GM-CSF showed significant therapeutic effects in mouse models of inflammatory diseases. We constructed by phage display technology a human scFv, which could potently neutralize human GM-CSF. At first, a human V(L) repertoire was combined with the V(H) domain of a parental GM-CSF-neutralizing rat antibody. One dominant rat/human scFv clone was selected, neutralizing human GM-CSF with an IC50 of 7.3 nM. The human V(L) of this clone was then combined with a human V(H) repertoire. The latter preserved the CDR 3 of the parental rat V(H) domain to retain binding specificity. Several human scFvs were selected, which neutralized human GM-CSF at low nanomolar concentrations (IC50 > or = 2.6 nM). To increase serum half-life, a branched 40 kDa PEG-polymer was coupled to the most potent GM-CSF-neutralizing scFv (3077) via an additional C-terminal cysteine. PEG conjugation had a negligible effect on the in vitro neutralizing potential of the scFv, although it caused a significant drop in binding affinity owing to a reduced on-rate. It also significantly increased the stability of the scFv at elevated temperatures. In mouse experiments, the PEGylated scFv 3077 showed a significantly prolonged elimination half-life of 59 h as compared with 2 h for the unconjugated scFv version. PEGylated scFv 3077 is a potential candidate for development of a novel antibody therapy to treat pro-inflammatory human diseases.     

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.     

Expression in COS cells of a mouse--human chimaeric B72.3 antibody

B72.3 is a mouse hybndoma cell-line secreting an IgG1 antibodywhich recognises an epitope on a tumour-associated antigen,TAG-72. This high molecular weight mucin-like molecule is foundon a variety of human neoplasms, including colon, breast andovarian carcinomas. Chimaeric Immunoglobulin genes with theB72.3 specificity have been constructed by joining the mousevariable regions from cDNA clones to human genomic constantregions using recombinant DNA techniques. The chimaeric heavyand light chain Immunoglobulin genes were placed under the controlof a strong viral promoter, and co-transfected into COS-1 cells.SDS–PAGE analysis of the ³⁵S-labelled products demonstratedthat the transiently expressed antibodies were correctly synthesisedand assembled. The specific binding characteristics of the parentB72.3 antibody were retained by the chimaeric antibody in anantigen-based ELISA. This system gave sufficiently high transientexpression of the chlmaerlc antibody molecules to allow rapidphysical and Immunological characterisation of the engineeredgene products.     

Antigen selection from an HIV-1 immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage

Phage display of antibody libraries has been widely used for over a decade to generate monoclonal antibodies. Yeast display has been developed more recently. Here the two approaches were directly compared using the same HIV-1 immune scFv cDNA library expressed in phage and yeast display vectors and using the same selecting antigen (HIV-1 gp120). Yeast display was shown to sample the immune antibody repertoire considerably more fully than phage display, selecting all the scFv identified by phage display and twice as many novel antibodies. Positive phage display selection appeared to largely reflect those antibodies that as phage-scFv gave the highest signal in phage ELISAs assessing antigen binding. This signal is thought to reflect the efficiency of expression of folded scFv at the phage surface. Increased access to immune repertoires may increase the rescue of novel antibodies of therapeutic or analytical value that often form a minor part of a typical antibody response.     

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.     

Recombinant anti-polyamine antibodies: identification of a conserved binding site motif

Polyamines are small linear polycations found ubiquitously ineukaryotic cells. They are involved in nucleic acid and proteinsynthesis and rises in cellular polyamine levels have been correlatedwith cell proliferation. Antibodies to these molecules havepotential as prognostic indicators of disease conditions andindicators of treatment efficacy. Antipolyamine monoclonal antibodiesof differing but defined specificities have been generated inour laboratory using polyamine ovalbumin conjugates as immunogens.These antibodies show small but significant cross reactivitieswith other polyamine species; IAG-1 cross reacts with spermidine(8%), JAC-1 with spermine (6%) and JSJ-1 with both putrescine(11%) and spermine (6%). We have rescued and sequenced the heavyand light chain variable regions of all three of these antibodies.While the light chains of two antibodies, IAG-1 and JSJ-1, were93% homologous at the amino acid level, none of the heavy chainsdisplayed any significant sequence homology. However, computer-generatedmodels of all three antibody binding sites revealed a three-dimensionallyconserved polyamine binding site motif. The polyamine appearsto bind into a negatively charged cleft lined with acidic andpolar residues. The cleft is partially or completely closedat one end and the specificity of the interaction is determinedby placement of acidic residues in the cleft. Aromatic residuescontribute to polyamine binding interacting with the carbonbackbone. The polyamine-binding motif we have identified isvery similar to that observed in the crystal structure of PotD,the primary receptor of the polyamine transport system in Escherichiacoli.     

Study of antibody-antigen interaction through site-directed mutagenesis of the VH region of a hybrid phage-antibody fragment

An important aspect of the study of antibody structure–functionrelationships involves analysis of natural or synthetic mutationsof antigen-combining sites. The anti-hen egg lysozyme monoclonalantibody HyHEL-10 has been a focus for antibody structure–functionstudies. We have displayed on bacteriophage of a hybrid singlechain Fv, containing the light chain variable region of HyHEL-10and the heavy chain variable region of a structurally relatedbut functionally distinct antibody, AS32. By using a combinationof site-directed mutagenesis, complementary determining regiongrafting and molecular modeling, we have identified a numberof contact and non-contact residues that are important in theaffinity of HyHEL-10 for lysozyme. In particular, the heavychain variable region framework residue at position 94 was shownto be an important determinant of high-affinity binding. Thephage display approach eliminates the need for purificationof antibodies and, when used in combination with polymerasechain reaction for variable region sequence mutagenesis, facilitatesthe rapid generation and characterization of mutant antibodies.     

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.     

A functional antibody mutant with an insertion in the framework region 3 loop of the VH domain: implications for antibody engineering

We have studied the effects of a four residue insertion intothe FR3 loop of the heavy chain variable region from the anti-NPantibody Bl-8. The insertion mutant is obtained as secretedantibody without major defects in biosynthesis, indicating thatantibody variable domains can accommodate length variation notonly in complementarity determining regions (CDRs), but alsoin framework region (FR) loops. The Bl-8 antigen binding siteis not affected by the change in a neighbouring loop. FR3 insertionsrepresent a new method of antibody engineering with a potentialto obtain strong antigen binding by designing additional antigencontacting residues.     

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.     

Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting

An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody 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.     

A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G

The therapeutic application of small recombinant antibody molecules is often limited by a short serum half-life. In order to improve the pharmacokinetic properties, we have investigated a strategy utilizing fusion with an albumin-binding domain (ABD) from streptococcal protein G. This strategy was applied to a bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. This novel tri-functional fusion protein (scDb-ABD) was expressed in mammalian cells and recognized both antigens as well as human and mouse serum albumin. scDb-ABD was capable to retarget T cells to CEA-expressing target cells in vitro and to activate the effector cells as measured by stimulation of IL-2 release. Although activity was reduced 3-fold compared with scDb and further reduced 4-fold in the presences of human serum albumin, this assay demonstrated that scDb-ABD is active when exposed to all three antigens. Compared with scDb, the circulation time of scDb-ABD in mice was prolonged 5- to 6-fold similar to a previously described scDb-HSA fusion protein. This strategy, which adds only a small protein domain (46 amino acids) and which utilizes high-affinity, non-covalent albumin interaction, should be broadly applicable to improve serum half-lives of small recombinant antibody molecules.     

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.     

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli

Single-chain antibodies consist of the variable, antigen-bindingdomains of antibodies joined to a continuous polypeptide bygenetically engineered peptide linkers. We have used the flexibleinterdomain linker region of a fungal cellulase to link togetherthe variable domains of an anti-2-phenyloxazolone IgGl and showhere that the resulting single-chain antibody is efficientlysecreted and released to the culture medium of Escherichia coli.The yield of affinity-purified single-chain antibody is 1 -2mg/1 of culture medium and its affinity and stability are comparableto those of the corresponding native IgG.     

A predicted three-dimensional structure for the human immunodeficiency virus binding domains of CD4 antigen

A predicted three-dimensional structure of the two N-terminalextracellular domains of human CD4 antigen, a cell surface glycoprotein,is reported. This region of CD4, particularly the first domain,has been identified as containing the binding region for theenvelope gp120 protein of the human immuno-deficiency virus.The model was predicted based on the sequence homology of eachdomain with the variable light chain of immunoglobulins. Theframework ß-sheet regions were taken from the crystalcoordinates of REI. For one region in the first domain of CD4there was an ambiguity in the alignment with REI and two alternatemodels are presented. Loops connecting the framework were modeledfrom fragments selected from a database of main chain coordinatesfrom all known protein structures. Residues identified as involvedin binding gp120 have been located in several other studieswithin the first domain of CD4. Epitopes from eight monoclonalantibodies have been mapped onto residues in both domains. Competitionof these antibodies with each other and with gp120 can be interpretedfrom the structural model.