A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities

Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. We have noted that two anti-CD3epsilon scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH(2) terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3epsilongamma was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH(2) terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3epsilongamma. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3epsilongamma increased approximately 3-fold when displayed on pYD5 compared with pYD1. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand-scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements.     

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.     

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.     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs

The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptide–heavy chain–linker–lightchain–(His)₅ tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 10–30% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with K_d values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay.     

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].     

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.     

Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).     

A33scFv-green fluorescent protein, a recombinant single-chain fusion protein for tumor targeting

Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv-green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by approximately 95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.     

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.     

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.     

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.     

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.     

Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion

In antigen–antibody interactions, the high avidity ofantibodies depends on the affinity and number of the individualbinding sites. To develop artificial antibodies with multiplevalency, we have fused the single-chain antibody Fv fragmentsto core streptavidin. The resulting fusion protein, termed scFv::strep,was found after expression in Escherichia coli in periplasmicinclusion bodies. After purification of the recombinant productby immobilized metal affinity chromatography, refolding andsize-exclusion FPLC, tetrameric complexes resembling those ofmature streptavidin were formed. The purified tetrameric scFv::strepcomplexes demonstrated both antigen- and biotin-binding activity,were stable over a wide range of pH and did not dissociate athigh temperatures (up to 70°C). Surface plasmon resonancemeasurements in a BIAlite system showed that the pure scFv::streptetramers bound immobilized antigen very tightly and no dissociationwas measurable. The association rate constant for scFv::streptetramers was higher than those for scFv monomers and dimers.This was also reflected in the apparent constants, which wasfound to be 35 times higher for pure scFv::strep tetramers thanmonomeric singlechain antibodies. We could also show that mostof biotin binding sites were accessible and not blocked by biotinylatedE.coli proteins or free biotin from the medium. These sitesshould therefore facilitate the construction of bispecific multivalentantibodies by the addition of biotinylated ligands.     

Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding

MUC1 mucin expressed in epithelial cancer, such as prostate and breast, is aberrantly glycosylated providing unique targets for imaging and therapy. In order to create a broadly applicable construct to target these unique epitopes on metastatic cancer, we selected an antibody fragment (scFv) that binds both synthetic MUC1 core peptide and epithelial cancer cell-expressed MUC1, and developed a recombinant bivalent molecule (di-scFv). Genetically engineered modifications of the di-scFv were constructed to create five molecular versions, each having a free cysteine (di-scFv-c) at different locations for site-specific conjugation. The effects of the engineered cysteine in the varied sites were studied relative to tumor binding and polyethylene glycol-maleimide (PEG-Mal) conjugation (PEGylation). Escherichia coli production as well as binding to MUC1 core peptide, human tumor cell lines and human tumor biopsies, were comparable. However, the location of the engineered cysteine in these di-scFv-c did influence PEGylation efficiency of this free thiol; higher PEGylation efficiency occurred with this cysteine in the inter-scFv linkage. Di-scFv-c PEG, with the cysteine engineered after the fifth amino acid in the linker, was used as an example to demonstrate comparable antigen-binding to non-PEGylated di-scFv-c. In summary, novel anti-MUC1 di-scFv-c molecules can be efficiently produced, purified and conjugated by site-specific PEGylation without loss of immunoreactivity, thus providing flexible multidentate constructs for cancer-targeted imaging and therapy.     

scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptidelinker to tether the V_H and V_L domains together. An scFv moleculewith a linker 5–12 residues long cannot fold into a functionalFv domain and instead associates with a second scFv moleculeto form a bivalent dimer (diabody). Direct ligation of V_H andV_L domains further restricts association and forces three scFvmolecules to associate into a trivalent trimer (triabody). Wehave defined the effect of linker length on scFv associationby constructing a series of scFvs from anti-neuraminidase antibodyNC10 in which the linker varied from one to four glycine residues.NC10 scFv molecules containing linkers of three and four residuesshowed a strong preference for dimer formation (diabodies),whereas a linker length of one or two glycine residues preventedthe formation of diabodies and directed scFv association intotrimers (triabodies). The data suggest a relatively strict transitionfrom dimer (diabody) to trimer (triabody) upon reduction ofthe linker length from three to two glycine residues. Modellingstudies are consistent with three residues as the minimum linkerlength compatible with diabody formation. Electron microscopeimages of complexes formed between the NC10 scFv multimers andan anti-idiotype Fab' showed that the dimer was bivalent forantigen binding and the trimer was trivalent.     

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.     

Normal-mode analysis suggests important flexibility between the two N-terminal domains of CD4 and supports the hypothesis of a conformational change in CD4 upon HIV binding

Human CD4 is the receptor for human immunodeficiency virus (HTV).It is well established that the first domain of CD4 binds withhigh affinity to gp120, an envelope protein of HIV, but it hasalso been demonstrated that amino acids located in its seconddomain, within or close to residues 120–127 or 163–166(lying 15 Å away from the binding site), play a role invirus infectivity. We show here that these two stretches ofamino acids happen to be important for the largest amplitudemotion obtained with the normal-mode theory for the two N-terminaldomains of human CD4: an overall rigid-body displacement ofone domain with respect to the other. Such a ‘hinge-bending’motion is unexpected since these two domains were found by crystallographersto be tightly abutting. On the other hand, since for severalproteins the hinge-bending motion experimentally observed uponligand binding was found to be similar to the largest amplitudemotion obtained with the normal-mode theory for these proteins,our results suggest that CD4 may undergo such a kind of conformationalchange upon HTV binding.     

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.     

In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants

The use of random mutagenesis in concert with protein display technologies to rapidly select high affinity antibody variants is an established methodology. In some cases, DNA recombination has been included in the strategy to enable selection of mutations which act cooperatively to improve antibody function. In this study, the impact of L-Shuffling DNA recombination on the eventual outcome of an in vitro affinity maturation has been experimentally determined. Parallel evolution strategies, with and without a recombination step, were carried out and both methods improved the affinity of an anti-Fas single chain variable fragment (scFv). The recombination step resulted in an increased population of affinity-improved variants. Moreover, the most improved variant, with a 22-fold affinity gain, emerged only from the recombination-based approach. An analysis of mutations preferentially selected in the recombined population demonstrated strong cooperative effects when tested in combination with other mutations but small, or even negative, effects on affinity when tested in isolation. These results underline the ability of combinatorial library approaches to explore very large regions of sequence space to find optimal solutions in antibody evolution studies.     

Human monoclonal antibodies to domain C of tenascin-C selectively target solid tumors in vivo

We had previously reported that splice isoforms of tenascin-C containing the extra-domain C are virtually absent in normal adult tissues but are highly abundant in high-grade astrocytomas, with a prominent peri-vascular pattern of expression. We now report that the extra-domain C of tenascin-C is strongly expressed in the majority of lung cancers, with a vascular and stromal pattern of expression. Using antibody phage technology, we have generated a human monoclonal antibody (G11), with a dissociation constant K(D) = 4.2 nM for the human domain C. The G11 antibody, expressed in scFv and in mini-antibody (SIP) format, as well as a scFv-interleukin-2 fusion protein, was then characterized in quantitative biodistribution studies using mice grafted subcutaneously with U87 gliomas, revealing a selective tumor uptake, with tumor/blood ratios up to 11.8:1 at 24 h. A radioiodinated preparation of SIP(G11) was also investigated in a double tracer study using an orthotopic rat glioma model, confirming the antibody's ability to preferentially localize at the tumor site, with tumor/brain ratios superior to the ones observed with (18)F-fluorodeoxyglucose. These tumor-targeting properties, together with the strong immunohistochemical staining of human tumor sections, indicate that the G11 antibody may be used as a portable targeting moiety for the selective delivery of imaging and therapeutic agents to gliomas and lung tumors.