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 and humanization of a rat monoclonal Fab to human interleukin-5

The X-ray crystal structure of a rat monoclonal Fab JES1-39D10,raised against recombinant human interleukin-5, has been determinedwith the use of molecular replacement techniques and refinedat 2.7 Å resolution by simulated annealing. The overallstructure is similar to a murine Fab HyHEL-10 that is specificfor hen egg white lysozyme. An interesting feature of the structureis the presence of leucine residues to support the H1 complementarity-determiningregion (CDR) loop. To our knowledge this is the first Fab crystalstructure containing this unusual HI loop support pattern. Theactivity of three humanized versions of 39D10 is explained byanalysis of Fv interface residues and H1 support patterns of39D10 and the human template HLL.     

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.     

Humanization of a mouse anti-human IgE antibody: a potential therapeutic for IgE-mediated allergies

Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and,therefore, has potential as a therapeutic agent in patientswith IgE-mediated allergies such as hay fever, food and drugallergies and extrinsic asthma. The clinical usefulness of mouseantibodies is limited, however, due to their immunogenidty inhumans. Mouse C21 antibody was humanized by complementaritydetermining region (CDR) grafting with the aim of developingan effective and safe therapeutic for the treatment of IgE-mediatedallergies. The CDR-grafted, or reshaped human, C21 variableregions were carefully designed using a specially constructedmolecular model of the mouse C21 variable regions. A key stepin the design of reshaped human variable regions is the selectionof the human framework regions (FRs) to serve as the backbonesof the reshaped human variable regions. Two approaches to theselection of human FRs were tested: (i) selection from humanconsensus sequences and (ii) selection from individual humanantibodies. The reshaped human and mouse C21 antibodies weretested and compared using a biosensor to measure the kineticsof binding to human IgE. Surprisingly, a few of the reshapedhuman C21 antibodies exhibited patterns of binding and affinitiesthat were essentially identical to those of mouse C21 antibody.     

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.     

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.     

Design and synthesis of germline-based hemi-humanized single-chain Fv against the CD18 surface antigen

The 6.7 murine monoclonal antibody (mAb) recognizes the humanCD18 antigen and is therefore of interest as an anti-inflammatoryagent. The 6.7 heavy variable chain (VH) was humanized usingthe closest human germline sequence as the template on to whichto graft the murine complementary determining regions (CDRs).Two versions were proposed, one in which the residue proline45 of the murine form was maintained and another in which thisframework residue was changed to the leucine found in the humansequence. These VH humanized versions were expressed in theyeast Pichia pastoris as hemi-humanized single-chain Fv (scFvs),with the VL from the murine antibody. The scFv from the murineantibody was also expressed. The binding activities of the murineand both hemi-humanized scFvs were determined by flow cytometryanalysis. All the constructions were able to recognize humanlymphocytes harboring CD18, indicating successful humanizationwith transfer of the original binding capability. Some differencesbetween the two hemi-humanized versions were observed. The methodused was simple and straightforward, with no need for refinedstructural analyses and could be used for the humanization ofother antibodies.     

Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach

CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the murine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an alphaCD30 x alphaCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.     

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.     

Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations

To determine whether mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis, the ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis. The vivarium of a research laboratory Balb/c mice were challenged with an LD₈₀ dose of either lipopolysaccharide (LPS/endotoxin), TNFα, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis. Each group of mice was checked daily for survivors, and Kaplan–Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG_1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time- and dose-dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animals if administered early and at a high dose. Our conclusions are that MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAb’s efficacy is both time- and dose-dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens of thousands of lives annually and could result in improved antibiotic stewardship.     

The effects of induction conditions on production of a soluble antitumor sFv in Escherichia coli

CC49 is a second generation monoclonal antibody(mAb) with highaffinity to a pancarcinoma antigen, TAG-72. A single-chain Fv(sFv)ofCC49 may have a role in managing human carcinomas. Most reportedsFvs have been expressed as insoluble products that must besolubilized and renatured. Soluble sFv expression is advantageousas activity can be assayed directly from the periplasmic fraction.Also, gene-level immunoconjugates may not be amenable to refoldingprotocols. Using a vector that carries the tac promoter andomp A signal, we have examined the effects of four variableson the expression and accumulation of soluble CC49 sFv: (i)linker sequence joining VL and VH, (ii) isopropylthio-ß-D-galactosideconcentration for induction, (iii) temprature, and (iv) theaddition of nonmetabolizable sugars to the medium. We have beenable to demonstrate, using rapidly prepared periplasmic extracts,that the yield of soluble sFv improves by the addition of 0.4Msucrose to the medium and by inducing expression with a verylow concentration of IPTG (0.02–0.03 mM). Under theseinduction conditions periplasmic extracts demonstrate increasedexpression of the sFv, as shown by the larger amount of a 27kDa band on SDS-polyacrylamide gel, and an increased abilityto inhibit binding of the mAb CC49 to immobilized tumor extracts.     

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

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

Humanization strategies for an anti-idiotypic antibody mimicking HIV-1 gp41

Anti-idiotypic antibodies could represent an alternative vaccination approach in human therapy. The anti-idiotypic antibody Ab2/3H6 was generated in mouse and is directed against the human monoclonal antibody 2F5, which broadly and potently neutralizes primary HIV-1 isolates. Ab2/3H6 is able to mimic the antigen recognition site of 2F5 making it a putative candidate for HIV-1 vaccine purposes. In order to reduce immunogenicity of therapeutic proteins, humanization methods have been developed. The mouse variable regions of Ab2/3H6 were subjected to three different humanization approaches, namely resurfacing, complementarity determining region (CDR)-grafting and superhumanization. Four different humanized Ab2/3H6 variants were characterized for their binding affinity to 2F5 in comparison to the chimeric Ab2/3H6. The resurfaced and the 'conservative' CDR-grafted variants showed similar binding properties to 2F5 when compared to the chimeric version, while the 'aggressive' CDR-grafted antibody showed reduced affinity and the superhumanized type lost its binding ability. In this study, we developed humanized Ab2/3H6 variants that retained the same affinity as the parental antibody, and are therefore of potential interest for future clinical trails.     

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.     

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].     

The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment

The facile detection and purification of a recombinant proteinwithout detailed knowledge about its individual biochemicalproperties constitutes a problem of general interest in proteinengineering. The use of a novel kind of random peptide libraryfor the stepwise engineering of a C-terminal fusion peptidewhich confers binding activity towards streptavidin is describedin this study. Because of its widespread use as part of a varietyof conjugates and other affinity reagents, streptavidin constitutesthe binding partner of choice both for detection and purificationpurposes. The streptavidin-affinity tag was engineered at theC-terminus of the V_H domain as part of the D1.3 Fv fragmentwhich was functionally expressed in Escherichia coli. Irrespectiveof whether it was displayed by the V_H or the V_L domain, theoptimized version of the affinity peptide termed ‘Strep-tag’allowed the detection of the Fv fragment both on Western blotsand in ELISAs by a streptavidin–alkaline phosphatase conjugate.In addition, the one-step purification of the intact Fv fragmentcarrying a single Strep-tag at the C-terminus of only one ofits domains was achieved by affinity chromatography with streptavidin-agaroseusing very mild elution conditions.     

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

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

Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv

Using molecular modeling technology, we have recently identifiedtwo positions in conserved framework regions of antibody Fvfragments (Fvs) that are distant from CDRs, and potentiallycan be used to make recombinant Fv fragments in which the unstableV_H and V_L heterodimer is stabilized by an interchain disulfidebond inserted between structurally conserved framework positions.A disulfide bond has been introduced at one of these positions,V_H44-V_L105, and shown to stabilize various Fvs that retain fullbinding and specificity. Recombinant immunotoxias, e.g. B3(dsFv)-PE38KDELin which this disulfide-stabilized Fv moiety is connected toa truncated form of Pseudomonas exotoxin (PE; PE38KDEL) whichcontains the translocation and ADP ribosylation domains, areindistinguishable in binding and specificity from its single-chainimmunotoxin counterparts. We have now analyzed the alternativeposition, (V_H111-V_L48), predicted by the modeling methodology,for disulfide stabilization of mAb B3(Fv) by producing a recombinantimmunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxinwas also very active and retained full specificity to B3 antigen-positivecells. However, it was 2- to 3-fold less active than the V_H44-V_L105dsFv-molecule. We also tested various biochemical features ofV_H44-V_L105 and V_H111-V_L48 dsFv immunotoxins and compared themwith the corresponding single-chain immunotoxin. We found thedsFv immunotoxins were more stable in human serum and more resistantto thermal and chemical denaturation than the single chain (sc)Fv immunotoxin. Because dsFv immunotoxins and dsFvs have fullactivity and specificity and improved stability, they may bemore useful than scFv immunotoxins as therapeutic and diagnosticagents.     

Genetically engineered brain drug delivery vectors: cloning, expression and in vivo application of an anti-transferrin receptor single chain antibodystreptavidin fusion gene and protein

A single chain Fv antibody–streptavidin fusion proteinwas expressed and purified from bacterial inclusion bodies followingcloning of the genes encoding the variable region of the heavychain and light chain of the murine OX26 monoclonal antibodyto the rat transferrin receptor. The latter undergoes receptormediated transcytosis through the brain capillary endothelialwall in vivo, which makes up the blood–brain barrier (BBB);therefore, the OX26 monoclonal antibody and its single chainFv analog may act as brain drug delivery vectors in vivo. Attachmentof biotinylated drugs to the antibody vector is facilitatedby production of the streptavidin fusion protein. The bi-functionalityof the OX26 single chain Fv antibody–streptavidin fusionprotein was retained, as the product both bound biotin and therat transferrin receptor in vitro and in vivo, based on pharmacokineticand brain uptake analyses in anesthetized rats. The attachmentof biotin–polyethyleneglycol–fluorescein to theOX26 single chain Fv antibody–streptavidin fusion proteinresulted in illumination of isolated rat brain capillaries inconfocal fluorescent microscopy. In conclusion, these studiesdemonstrate that genetically engineered single chain Fv antibody–streptavidinfusion proteins may be used for non-invasive neurotherapeuticdelivery to the brain using endogenous BBB transport systemssuch as the transferrin receptor.