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.     

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.     

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

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

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.     

Study of B72.3 combining sites by molecular modeling and site-directed mutagenesis

A B72.3 Fab/sTn² complex was modeled from the known structureof B72.3 Fab and the dimeric Tn-serine cluster (sTn²). In thecomplex model, the side chains of 15 heavy- and light-chaincomplementarity-determining region (CDR) residues and the mainchains of two light-chain CDR residues contact the sTn² epitope.Among 15 CDR residues which contact sTn² in the model, two heavy-chainresidues (Ser95 and Tyr97) and light-chain CDR residue (Tyr96)have been confirmed in a previous study. To test the accuracyof the computational model, further site-directed mutagenesiswas performed by alanine scanning on the remaining 12 residuesthat are predicted in the model to have side-chain interactionswith sTn². Of these 12 mutants, eight that are all from theheavy-chain (His32Ala, Ala33Leu, Tyr50Ala, Ser52Ala, Asn52Ala,Asp56Ala, Lys58Ala and Tyr96Ala) had significantly reduced sTn²affinities, and four consisting of three light-chain mutations(Asn32Ala, Trp92Ala and Thr94Ala) and one heavy-chain mutation(His35Ala) retained wild-type sTn² affinity. On the whole, thisevidence suggests that the complex model, although not perfect,is correct in many of its features. In a more general vein,these results lend credibility to the computational modelingapproach for the study of the molecular basis of antigen–antibodycomplexes.     

Molecular recognition of the Lewis Y antigen by monoclonal antibodies

The murine monoclonal antibody BR55-2 is directed against thetumor-associated antigen Lewis Y oligosaccharide. The LewisY core antigen is a difucosylated structure consisting of fourhexose units. Analysis of binding profiles of lactoseries isomericstructures by BR55–2 suggest that the binding epitopeincludes the OH-4 and OH-3 groups of the ß-D-galactoseunit, the 6-CH₃ groups of the two fucose units and the N-acetylgroup of the subterminal ß-D-N-acetylglucosamine (ßDGlcNAc).To elucidate the molecular recognition properties of BR55–2for the Y antigen, BR55–2 was cloned, sequenced and itsthree-dimensional structure was examined by molecular modeling.The crystal structure of BR96, another anti-Lewis Y antibody,solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide,and the lectin IV protein in complex with a Lewis b tetrasaccharidecore were used as a guide to probe the molecular basis for BR55–2antigen recognition and specificity. Our modeling study showsthat BR55–2 shares similar recognition features for thedifucosylated type 2 lactoseries Lewis Y structure observedin the BR96-sugar complex. We observe that a major source ofspecificity for the Lewis Y structure by anti-Y antibodies emanatesfrom interaction with the ß-D-N-acetylglucosamineresidue and the nature of the structures extended at the reducingsite of the fucosylated lactosoamine.     

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.     

Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (V(H) and V(L)) domains of two different specificities connected by three linkers. When assembled in the order V(H)(A)-linker(1)-V(L)(B)-linker(2)-V(H)(B)-linker(3)-V(L)(A), the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary V(H) and V(L) domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains. We generated a number of single-chain constructs using four V(H) and V(L) domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6-27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites.     

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].     

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.     

Human aldolase B: liver-specific properties of the isozyme depend on type B isozyme group-specific sequences

A series of chimeric enzymes between two human aldolases A,B or C were constructed to identify the molecular regions responsiblefor isozyme-specific functions. Chimeras constructed betweenaldolases A and B were AB34 (an AB chimera connected at position34), ABA34–306 and ABA212–306 (the ABA chimeras).Those between aldolases B and C are BC243, BC263 and BC306 (theBC chimeras connected at positions as indicated), as well asCB55, CB243, CB263 and CB306 (the CB chimeras connected at positionsas indicated), CBC55–263 (a CBC chimera), and BCB55–193,BCB55–306, BCB79–193 and BCB79–306 (the BCBchimeric enzymes). Through the analysis of the properties ofthese chimeras, it was found that for aldolase B, isozyme Bgroup-specific sequences (IGSs)-l and-4 were required for exertingtype B-specific functions, while the IGSs-2 and -3 enhanced,in collaboration with the IGS-1, the catalytic activity of aldolaseB. In addition, the /ß-barrel and the restricted stretches,which were not specified but occupied two distinct regions spanningthe amino acid positions 108–137 (designated connector1) and 243–306 (designated connector 2), were found tobe indispensable for showing full catalytic activity of aldolaseB.     

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.     

Analysis of stability and catalytic properties of two tryptophanases from a thermophile

Two tryptophanases, Tna1 and Tna2, both of which were clonedfrom the thermophile Symbiobacterium thermophilum, differ intheir enzymatic properties, such as thermal stability, catalyticefficiency and activation energy of catalysis, despite the greatsimilarity (92%) in their amino acid sequences. Chimeric tryptophanaseswere constructed by recombination of the two genes to try toelucidate the molecular basis for the difference. The stabilityof each chimeric enzyme was roughly proportional to the contentof amino acid residues from Tna1. Three regions, tentativelynamed regions 2, 4 and 5, which contained the amino acid residues70–129, 192–298 and 299–453, respectively,were especially important for the increase in thermal stability.Site-directed mutagenesis revealed that V104 in region 2 andY198 in region 4 of Tna1 were involved in the increase in thermalstability of Tna1. Amino acid residues contributing to the highercatalytic efficiency of Tna1 were similarly analyzed, usingthe chimeric tryptophanases, and found to be located in region5. Site-directed mutagenesis revealed that I383 and G395 inTna1, which were presumably located close to the putative activecenter, played an active role in the increase of catalytic efficiencyof Tna1. The activation energy of catalysis was proportionalto the content of amino acid residues from Tna2, suggestingthe amino acid residues responsible for the difference weredispersed over the whole molecule.     

Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains

We cloned 17 different PCR fragments encoding V_H genes of camel(Camelus dromedarius). These clones were derived from the camelheavy chain immunoglobulins lacking the light chain counterpartof normal immunoglobulins. Insight into the camel V_H sequencesand structure may help the development of single domain antibodies.The most remarkable difference in the camel V_H, consistent withthe absence of the V_L interaction, is the substitution of theconserved Leu45 by an Arg or Cys. Another noteworthy substitutionis the Leu11 to Ser. This amino acid normally interacts withthe C_H1 domain, a domain missing in the camel heavy chain immunoglobulins.The nature of these substitutions agrees with the increasedsolubility behavior of an isolated camel V_H domain. The V_H domainsof the camels are also characterized by a long CDR3, possiblycompensating for the absence of the V_L contacts with the antigen.The CDR3 lacks the salt bridge between Arg94 and Asp101. However,the frequent occurrence of additional Cys residues in both theCDR1 and CDR3 might lead to the formation of a second internaldisulfide bridge, thereby stabilizing the CDR structure as inthe DAW antibody. Within CDRs of the camel V_H domains we observea broad size distribution and a different amino acid patterncompared with the mouse or human V_H. Therefore the camel hypervariableregions might adopt structures which differ substantially fromthe known canonical structures, thereby increasing the repertoireof the camel antigen binding sites within a V_H.     

Site-specific attachment to recombinant antibodies via introduced surface cysteine residues

Many diagnostic and therapeutic applications of monoclonal antibodiesrequire the covalent linking of effector or reporter moleculesto the immunoglobulin polypeptides. Existing methods generallyinvolve the non-selective modification of amino acid side chains,producing one or more randomly distributed attachment sites.This results in heterogeneous labelling of the antibody moleculesand often to a decrease in antigen-binding due to the modificationof residues close to the antigen-binding site. We report a novelstrategy for site-specifically labelling antibodies throughsurface cysteine residues. Examination of molecular structureswas used to identify amino acids of the C_H1 domain of the IgGheavy chain that were accessible to solvent but not to largermolecules. Site-directed mutagenesis was used to substitutecysteine residues at these positions in the heavy chain of amouse/human chimaeric version of the tumour-binding monoclonalantibody, B72.3. Expression of the modified antibody genes inmammalian cells yielded correctly assembled proteins that hadthiol groups in pre-determined positions and showed no lossof antigen-binding activity. One of the mutants was used todemonstrate the site-specific attachment of a radio-iodinatedligand to the chimaeric B72.3 antibody.     

Structure and functional complementation of engineered fragments from yeast phosphoglycerate kinase

Previous studies have shown that, although the isolated structuraldomains of yeast phosphoglycerate kinase recover a quasi-nativestructure in vitro as well as in vivo, they do not reassociatenor generate a functional enzyme. The aim of this work was firstto study the folding of complementary fragments different fromstructural domains and second to determine the requirementsfor their reassociation and functional complementation. Themethod used for producing rigorously defined fragments consistsof the introduction of a unique cysteinyl residue in the proteinfollowed by a specific cleavage by 5'5'-dithiobis(2-nitrobenzoate)/potassiumcyanide at this residue. Two pairs of complementary fragmentswere thus obtained, 1–96/97–415 and 1–248/249–415.The structure and stabilities of the different fragments werestudied. The short fragments, i.e. 1–96 and 249–415were found to contain some secondary structure, but to havea low stability. Each large fragment has a high structural contentand a stability close to that of the corresponding domain. Incontrast to that observed with the isolated domains, a weakbut significant complementation was observed for the two pairsof fragments; the pair of fragments 1–248/249–415recovered 8% of the activity of the native enzyme upon complementation.An independent refolding of the complementary fragments beforereassociation decreased the yield of complementation for thepair of fragments 1–96/97–415, but did not affectthe complementation for the other pair (1–248/249–415).From the present data and previous work on the isolated domains,it appears that the correct folding of the isolated fragmentsis not a prerequisite for their complementation.     

Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide

The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H(2)O(2)-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in K(m,HOOH) for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening >3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35-55 degrees C) and maintained approximately 1.4-fold higher activity than QM at 45 degrees C for 6 h. In addition, QM/L295H showed a significant increase in T(m,app) compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5-15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a >2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.     

The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

The DNA binding mechanism of box B in HMG1, a member of thesequence non-specific DNA binding HMG1/2-box family of proteins,has been examined by both mutation analyses and molecular modelingtechniques. Substitution of the residue 102F, which is characteristicallyexposed to solvent, with a small hydrophobic amino acid affectedits DNA binding activity. However, no additional effect wasobserved by the further mutation of flanking 101F. Moleculardynamics simulation and modeling studies revealed that 102Fintercalates into DNA base-pairs, being supported by the flanking101F. The mutants with a small hydrophobic residue at position102 tolerated the substitution for 101F because the side chainat position 102 is too short to intercalate. Thus the intercalationof 102F and the positive effect of the flanking 101F residueare important for the sequence non-specific DNA binding of theHMG1/2-box. The conserved basic residues of 95K, 96R and 109Rwere also examined for their roles in DNA binding. These residuesinteracted with DNA mainly by electrostatic interaction andmaintained the location of the box on the DNA, which prescribedthe intercalation of 102F. The DNA intercalation by HMG1 consistsof an ingenious mechanism which brings DNA conformational changesnecessary for biological functions.     

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.