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Background: Antibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV., Results: In the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni-NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests., Conclusions: For the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed immunoassays eliminate the use of commercial secondary antibodies and shorten detection time. Meanwhile, both assays display great developmental prospect for further commercial production and application.

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

A mammalian expression vector comprised of the PreS2-TLM (translocation motif), a single-chain variable fragment (ScFv) that binds to hepatitis B surface antigen (HBsAg) and the EGFP gene was constructed. A stably transformed cell line that could express and secrete the fusion protein (PreS2-TLM-ScFv-EGFP) was established. HBsAg-positive HepG2.2.15 cells and HepG2 and HeLa cells were incubated with the supernatant of the transformed cell line cultures for evaluating the cellular permeability of PreS2-TLM-ScFv-EGFP. The location of the fusion protein PreS2-TLM-ScFv-EGFP in HepG2.2.15 cells was observed with immunofluorescence staining. EGFP was next replaced by a dominant negative mutant of the hepatitis B virus core gene (HBcDN) for producing fusion protein PreS2-TLM-ScFv-HBcDN, which was detected by western blot. The supernatant containing fusion protein PreS2-TLM-ScFv-HBcDN was added to the cultures of HepG2.2.15 cells, and the packaged hepatitis B virus (HBV) pregenomic RNA expression levels in the cells were measured using qRT-PCR. The results of the in vitro study indicated that the packaged HBV pregenomic RNA expression levels in HepG2.2.15 cells significantly decreased when these cells were exposed to the supernatant at the dose of 25% for 24, 48 and 72 h, or at the dose of 12.5% for 72 h., (© 2011 Blackwell Publishing Ltd.)

Single-chain antibody variable fragment (scFv) proteins consist of an antibody heavy chain variable sequence joined via a flexible linker to a light chain variable sequence. Prior work has shown that ScFv 18-2 binds the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and sensitizes cancer cells to radiation following nuclear microinjection. A potential clinical delivery strategy is based on modification of the scFv so that it can be taken up into cells and imported to the nucleus. This will require development of an expression system for a nuclear localization signal (NLS)-tagged scFv derivative. We found, however, that addition of the highly basic NLS severely compromised expression in the host-vector system used for the parental scFv. After testing a variety of host strains, fusion partners, and NLS sequences and placements, successful expression was obtained with a construct containing a stabilizing N-terminal maltose binding protein tag and a single, optimized, C-terminal NLS moiety. Amylose affinity-purified ScFv 18-2 NLS protein was stable to storage at 4 degrees C in the presence of glycerol or trehalose, bound selectively to an epitope peptide, and was cleavable at an engineered Factor Xa protease site. Following lipid-mediated uptake into cultured cells, NLS-tagged ScFv 18-2, unlike the parental ScFv 18-2, localized predominantly in the cell nucleus.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with NADH oxidase activity that has a vital function in healthy cells but is also an important mediator of caspase-independent programmed cell death in stressed and damaged cells. Here, we generated a truncated AIF derivative (AIF(Delta100)) that lacks the mitochondrial import signal of the protein. Bacterially expressed AIF(Delta100) was functionally active and induced cell death on microinjection into Vero cells accompanied by clear signs of apoptosis. For specific targeting to tumor cells, AIF(Delta100) was genetically fused to the scFv(FRP5) antibody fragment that recognizes the ErbB2 (HER2) receptor tyrosine kinase frequently overexpressed in many human cancers. Recombinant scFv(FRP5)-AIF(Delta100) (5-AIF(Delta100)) protein and a similar scFv(FRP5)-ETA(252-366)-AIF(Delta100) (5-E-AIF(Delta100)) molecule harboring in addition the nontoxic translocation domain of Pseudomonas exotoxin A as an endosome escape function displayed binding to ErbB2-expressing cells followed by protein internalization and accumulation in intracellular vesicles. In the presence of the endosomolytic reagent chloroquine 5-E-AIF(Delta100) but not the similar 5-AIF(Delta100) protein displayed potent cell killing activity, which was strictly dependent on the expression of ErbB2 on the target cell surface. Our results show that recombinant AIF specifically targeted to human cancer cells and delivered into the cytosol has potent cell killing activity, suggesting this molecule as an effector function suitable for the development of humanized immunotoxin-like molecules.

A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut. In the second variant, the murine CDRs were grafted to the human acceptor framework huVkappa3-huV(H)3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic applications.

Antibody variable domains differ considerably in stability. Single-chain Fv (scFv) fragments derived from natural repertoires frequently lack the high stability needed for therapeutic application, necessitating reengineering not only to humanize their sequence, but also to improve their biophysical properties. The human V(H)3 domain has been identified as having the best biophysical properties among human subtypes. However, complementarity determining region (CDR) grafts from highly divergent V(H) domains to huV(H)3 frequently fail to reach its superior stability. In previous experiments involving a CDR graft from a murine V(H)9 domain of very poor stability to huV(H)3, a hybrid V(H) framework was obtained which combines the lower core residues of muV(H)9 with the surface residues of huV(H)3. It resulted in a scFv with far better biophysical properties than the corresponding grafts to the consensus huV(H)3 framework. To better understand the origin of the superior properties of the hybrid framework, we constructed further hybrids, but now in the context of the consensus CDR-H1 and -H2 of the original human V(H)3 domain. The new hybrids included elements from either murine V(H)9, human V(H)1 or human V(H)5 domains. From guanidinium chloride-induced equilibrium denaturation measurements, kinetic denaturation experiments, measurements of heat-induced aggregation and comparison of soluble expression yield in Escherichia coli, we conclude that the optimal V(H) framework is CDR-dependent. The present work pinpoints structural features responsible for this dependency and helps to explain why the immune system uses more than one framework with different structural subtypes in framework 1 to optimally support widely different CDRs.

The isolation of mammalian cell lines capable of high-yield expression of recombinant antibodies is typically performed by screening multiple individual clones by limiting dilution techniques. A number of experimental strategies have recently been devised to identify high-expressing clones, but protocols are often difficult to implement, time consuming, costly and limited in terms of number of clones which can be screened. In this article, we describe new vectors for the expression of recombinant antibodies in IgG format and in other formats, based on the single-chain Fv module, as well as a high-throughput screening procedure, based on the direct staining of antibodies transiting the membrane of a stably transfected cell, followed by preparative sorting using a high-speed cell sorter. This procedure allows, in one step, to deposit single cells into individual wells of a 96-well microtiter plate (thus facilitating cloning) and to preferentially recover those rare cell populations which express dramatically higher levels of recombinant antibody. Using cell cultures followed by affinity purification techniques, we could confirm that the new vectors and the new screening procedure reliably yield high-expression clones and homogenous protein preparations. We expect that these techniques should find broad applicability for both academic and industrial antibody engineering research.

Most neurodegenerative disorders, such as Alzheimer's (AD), Parkinson's, Huntington's and Creutzfeldt-Jakob disease, are characterised by the accumulation of insoluble filamentous aggregates known as amyloid. These pathologies share common pathways involving protein aggregation which can lead to fibril formation and amyloid plaques. The 4 kDa Abeta peptide (39-43 amino acids) derived from the proteolysis of the amyloid precursor protein is currently a validated target for therapy in AD. Both active and passive immunisation studies against Abeta are being trialled as potential AD therapeutic approaches. In this study, we have characterised engineered antibody fragments derived from the monoclonal antibody, WO-2 which recognises an epitope in the N-terminal region of Abeta (amino acids 2-8 of Abeta). A chimeric recombinant Fab (rFab) and single chain fragments (scFvs) of WO-2 were constructed and expressed in Escherichia coli. Rationally designed mutants to improve the stability of antibody fragments were also constructed. All antibody formats retained high affinity (K(D) approximately 8 x 10(-9) M) for the Abeta peptide, comparable with the intact parental IgG as measured by surface plasmon resonance. Likewise, all engineered fragments were able to: (i) prevent amyloid fibrillisation, (ii) disaggregate preformed Abeta(1-42) fibrils and (iii) inhibit Abeta(1-42) oligomer-mediated neurotoxicity in vitro as efficiently as the whole IgG molecule. These data indicate that the WO-2 antibody and its fragments have immunotherapeutic potential. The perceived advantages of using small Fab and scFv engineered antibody formats which lack the effector function include more efficient passage across the blood-brain barrier and minimising the risk of triggering inflammatory side reactions. Hence, these recombinant antibody fragments represent attractive candidates and safer formulations of passive immunotherapy for AD.

Nanodisks (ND) are nanometer scale complexes of phospholipid and apolipoprotein that have been shown to function as drug delivery vehicles. ND harboring significant quantities of the antifungal agent, amphotericin B, or the bioactive isoprenoid, all trans retinoic acid, have been generated and characterized. As currently formulated, ND possess limited targeting capability. In this study, we constructed a single chain variable antibody (scFv).apolipoprotein chimera and assessed the ability of this fusion protein to form ND and recognize the antigen to which the scFv is directed. Data obtained revealed that alpha-vimentin scFv.apolipoprotein A-I is functional in ND formation and antigen recognition, opening the door to the use of such chimeras in targeting drug-enriched ND to specific tissues.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7, a primary enteric pathogen, has been implicated in a wide spectrum of food/water-borne infectious diseases such as hemorrhagic colitis (HC) and hemolyticuremic syndrome (HUS). Effective treatments for EHEC O157:H7 induced disease are not available yet. Shiga-toxin 2 (Stx2) has been related to clinical manifestations of HUS, suggesting its critical role in pathology following infection with EHEC O157:H7. Here we report the development of four anti-Stx2-Monoclonal antibodies (McAbs) (5F3and 5C11 for Stx2A, and 1A4 and 1A5 for Stx2B), all of which have strong immunogenicity and neutralization activities in vitro and in vivo. The full-length cDNA coding for anti-Stx2A McAb, 5F3, was cloned and an engineered antibody was developed whose therapeutic effects were evaluated. Our data indicate that the engineered scFv together with two new McAbs may be applicable for the prevention and therapy of EHEC induced pathology.

Since many cell functions are regulated by members of the cytokine receptor superfamily, the artificial mimicry of the cytokine receptor system would be attractive for cellular engineering. We previously showed that an antibody/cytokine receptor chimera can transduce a growth signal in response to non-natural ligands, such as fluorescein-conjugated BSA. However, considerable background of cell proliferation was observed without antigen. Therefore, we redesigned chimeric receptor constructs with different combinations of domains containing anti-fluorescein single chain Fv (ScFv), extracellular D1/D2 as well as transmembrane domains of erythropoietin receptor (EpoR), and the intracellular domain of glycoprotein 130 (gp130), to obtain strictly fluorescein-dependent chimeric receptors. When interleukin-3-dependent Ba/F3 cells were transduced with retroviral vectors encoding individual chimeric receptors, the chimeras either with both D1 and D2 domains or without any EpoR extracellular domain attained a strict ligand-dependent ON/OFF regulation.

Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.

Aim: To investigate the antitumor activities of an anti-ErbB2 scFv-Fc-interleukin 2 (IL-2) fusion protein (HFI) in vitro and in vivo., Methods: Fusion protein HFI was constructed. The efficacy of HFI in mediating tumor cell lysis was determined by colorimetric lactate dehydrogenase release assays. The antitumor activity of HFI was evaluated in tumor xenograft models., Results: The fusion protein was folded as a homodimer formed by covalently linking Fc portions and it retained ErbB2 specificity and IL-2 biological activity. HFI mediated antibody-dependent cell-mediated cytotoxicity (ADCC) at low effector-to-target ratios in vitro and improved the therapeutic efficacy of IL-2 in experiments in vivo., Conclusion: The genetically-engineered anti-ErbB2 scFv-Fc-IL-2 fusion protein exhibited high efficiency both in mediating ADCC in vitro and significant antitumor activity in tumor xenograft models.

We have developed a universal ovarian cancer cell targeting vehicle that can deliver biotinylated therapeutic drugs. A single-chain antibody variable domain (scFv) that recognizes the CA125 antigen of ovarian cancer cells was fused with a core-streptavidin domain (core-streptavidin-VL-VH and VL-VH-core-streptavidin orientations) using recombinant DNA technology and then expressed in Escherichia coli using the T7 expression system. The bifunctional fusion protein (bfFp) was expressed in a shaker flask culture, extracted from the periplasmic soluble protein, and affinity purified using an IMAC column. The two distinct activities (biotin binding and anti-CA125) of the bfFp were demonstrated using ELISA, Western blot and confocal laser-scanning microscopy (CLSM). The ELISA method utilized human NIH OVCAR-3 cells along with biotinylated bovine serum albumin (B-BSA) or biotinylated liposomes, whereas, the Western blot involved probing with B-BSA. The CLSM study has shown specificity in binding to the OVCAR-3 cell-line. ELISA and Western blot studies have confirmed the bifunctional activity and specificity. In the presence of bfFp, there was enhanced binding of biotinylated antigen and liposome to OVCAR-3 cells. In contrast, the control EMT6 cells, which do not express the CA125 antigen, showed minimal binding of the bfFp. Consequently, bfFp based targeting of biotinylated therapeutic drugs, proteins, liposomes, or nanoparticles could be an alternative, convenient method to deliver effective therapy to ovarian cancer patients. Peritoneal infusion of the bfFp-therapeutic complex could also be effective in locally targeting the most common site of metastatic spread.

Two novel engineered antibody fragments binding to antigen CD20 were generated by fusing a murine IgM-type anti-CD20 single-chain Fv fragment (scFv) to the human IgG1 CH2 (i.e., Cgamma2) and CH3 (i.e., Cgamma3) domains with the human IgG1 hinge (i.e. Hgamma). Given the relationship between structure and function of protein, the 3-D structures of the two engineered antibody fragments were modeled using computer-aided homology modeling method. Furthermore, the relationship between 3-D conformation and their binding activity was evaluated theoretically. Due to the change of active pocket formed by CDRs, the HL23 (VH-Linker-VL-Hgamma-Cgamma2-Cgamma3) remained its activity because of its preserved conformation, while the binding activity of the LH23 (VL-Linker-VH-Hgamma-Cgamma2-Cgamma3) was impaired severely. Experimental studies by flow cytometry and fluorescence microscopy showed that HL23 possessed significantly superior binding activity to CD20-expressing target cells than LH23. That is to say, the order of variable regions could influence the binding activity of the fusion protein to CD20+ cell lines, which was in accordance with the theoretical results. The study highlights the potential relationship between the antibody binding activity and their 3-D conformation, which appears to be worthwhile in providing direction for future antibody design of recombinant antibody.

The use of transgenic avian species as a transgenic bioreactor for the production of recombinant proteins has been proposed. In recent years, although various procedures for generating transgenic chickens have been reported, the expression of a useful protein at a commercially feasible level has rarely been attained. In this study, we injected a concentrated retroviral vector into quail embryos to generate genetically manipulated quails that produce recombinant proteins. We found that transgene expression in the whole body at a high level was observed for viral injection into the heart of the developing embryos after a 48-h incubation. For the practical production of a useful protein, a retroviral vector encoding an anti-prion scFv-Fc gene under the control of the beta-actin promoter was injected into quail embryos. The quails that hatched stably produced scFv-Fc at a high level in their serum and egg white. The production of scFv-Fc was maintained throughout the breeding period. scFv-Fc purified from the egg white retained the antigen-binding activity. This system exhibited the potential of transgenic quails for the commercial production of recombinant proteins.

The immunogenicity of therapeutic Abs is a concern as anti-drug Abs may impact negatively on the pharmacodynamics and safety profile of Ab drugs. The factors governing induction of anti-drug Abs are not fully understood. In this study, we describe a model based on mouse-human chimeric Abs for the study of Ab immunogenicity in vivo. Six chimeric Abs containing human V regions and mouse C regions were generated from six human anti-Rhesus D Abs and the Ag-binding characteristics of the parental human Abs were retained. Analysis of the immune response toward the individual chimeric Abs revealed the induction of anti-variable domain Abs including anti-idiotypic Abs against some of these, thereby demonstrating the applicability of the model for studying anti-drug Ab responses in vivo. Immunization of BALB/c, C57, and outbred NMRI mice with a polyclonal composition consisting of all six chimeric Abs demonstrated that the immunogenicity of the individual Abs was haplotype dependent. Chimeric Abs, which were nonimmunogenic when administered individually, did not become immunogenic as part of the polyclonal composition, implying the absence of epitope spreading. Ex vivo Ab-binding studies established a clear correlation between the level of immunogenicity of the Abs comprised in the composition and the impact on the pharmacology of the Abs. These analyses demonstrate that under these conditions this polyclonal Ab composition was generally less susceptible to blocking Abs than the respective mAbs.

TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and of both fusion proteins with TNFR type 1 (TNF-R1) using surface plasmon resonance correlated well with in vitro and in vivo toxicity data. There is strong evidence that TNF subunits in a trimeric state display similar toxicity profiles despite genetic fusion to single-chain variable fragment domains. However, LD(50) of either immunodeficient BALB/c nu/nu or immunocompetent BALB/c mice was significantly decreased following administration of TNF in the formation of IgG1-derived dimeric fusion protein. Reduction of unspecific peripheral complexation of TNF-R1 resulted in higher anticancer potency by immunotargeting of fibroblast-activating protein-expressing xenografts. The broader therapeutic window of the IgG1-derived TNF fusion protein favors the dimeric TNF-immunokine format for systemic TNF-based tumor immunotherapy.

Aim: To construct and produce a recombinant bispecific humanized single-chain Fv (sFv) /Interleukin-2 (IL-2) fusion protein by using mammalian cells., Methods: The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency., Results: The fusion protein was constructed and high efficiently expressed with yields up to 102 +/- 4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy (V(H)) and light (V(L)) domains of monoclonal antibody (mAb) 520C9 directed against the human HER-2/neu (c-erbB2) proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL-2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells., Conclusion: The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.

In this study we investigate the effect of thioredoxin (Trx1) protein fusions in the production, oxidation, and folding of single chain Fv (scFv) antibodies in the cytoplasm of Escherichia coli. We analyze the expression levels, solubility, disulfide-bond formation, and antigen-binding properties of Trx1-scFv fusions in E. coli wild-type cells and isogenic strains carrying mutations in the glutathione oxidoreductase (gor) and/or thioredoxin reductase (trxB) genes. We compare the Trx1-scFv fusions with other reported systems for production of scFv in the cytoplasm of E. coli, including protein fusions to the maltose-binding protein. In addition, we analyze the effect of co-expressing a signal-sequence-less derivative of the periplasmic chaperone and disulfide-bond isomerase DsbC (DeltassDsbC), which has been shown to act as a chaperone for scFvs in the cytoplasm. The results reported here demonstrate that Trx1 fusions produce the highest expression level and induce the correct folding of scFvs even in the absence of DeltassDsbC in the cytoplasm of E. coli trxB gor cells. The disulfide bridges of Trx1-scFv fusions were formed correctly in E. coli trxB gor cells, but not in trxB single mutants. Antigen-binding assays showed that Trx1 has only a minor influence in the affinity of the scFv, indicating that Trx1-scFv fusions can be used without removal of the Trx1 moiety. In addition, we proved that a Trx1"AGPA" variant, having its catalytic cysteine residues mutated to alanine, was fully capable of assisting the folding of the fused scFvs. Taken together, our data indicate that the Trx1 moiety acts largely as an intramolecular protein chaperone, not as a disulfide bond catalyst, inducing the correct folding of scFvs in the cytoplasm of E. coli trxB gor cells.

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is overexpressed and exposed on the cell surface in a large fraction of solid malignancies. We constructed a single-chain fragment (scFv) based on CC49 and fused it to beta-lactamase (BLA). Following optimization of the scFv domain by combinatorial consensus mutagenesis (CCM) for increased expression and stability, we characterized the protein variant for binding, in vivo pharmacokinetics (PK), and antitumor efficacy. The fusion protein TAB2.5 possessed a similar binding specificity relative to the parent antibody CC49. TAB2.5 also showed prolonged retention (T(1/2) = 36.9 h) in tumor-bearing mice with tumor/plasma ratios of up to 1000. Preliminary evaluation of TAB2.5, in combination with a novel prodrug, GC-Mel, resulted in significant efficacy in a colorectal xenograft tumor model and supports the utility of the protein as an agent for tumor-selective prodrug activation.

The insecticidal protoxin from Bacillus thuringiensis has been shown to be a major component of the spore coat. We have developed a novel surface display system using B. thuringiensis spores in which the N-terminal portion of the protoxin is replaced with a heterologous protein. The expression vector with a sporulation-specific promoter was successfully used to display green fluorescent protein and a single-chain antibody (scFv) gene that encodes anti-4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (anti-phOx) antibody. The spores that carry the anti-phOx antibody can bind to phOx specifically.

Persistent activation of T-lymphocytes requires two signals: one is initiated by T-cell receptor binding to antigenic peptide presented by MHC molecules. In addition, binding of the B7 family members CD80 or CD86 on professional antigen presenting cells to CD28 on T cells is considered to provide an important costimulatory signal. Activation without costimulation induces T-cell unresponsiveness or anergy. To selectively localize costimulatory activity to the surface of tumor cells and enhance activation of tumor-specific T cells, we have developed a novel molecular design for bispecific costimulatory proteins with antibody-like structure. Within a single polypeptide chain we have assembled the IgV-like, CD28-binding domain of human CD86 (CD86(111)) together with hinge, CH2 and CH3 domains of human IgG1, and the scFv(FRP5) antibody fragment which recognizes the ErbB2 (HER2) protooncogene present at high levels on the surface of many human tumor cells. Upon expression in the yeast Pichia pastoris, the resulting CD86(111)-IgG-scFv(FRP5) protein could be purified as a homodimeric, tetravalent molecule from culture supernatants using single-step affinity chromatography. Bispecific binding of the molecule to ErbB2 on the surface of tumor cells and to the B7 counter receptor CTLA-4 was demonstrated by FACS analysis. Potent costimulatory activity of chimeric CD86(111)-IgG-scFv(FRP5) was confirmed by its ability to stimulate the proliferation of primary human lymphocytes pre-activated by low concentrations of anti-CD3 antibody. Our results suggest that such multivalent soluble proteins which combine specific targeting to tumor cells with costimulatory activity may become useful tools to elicit and/or improve T-cell mediated, tumor-specific immune responses.

The ability of intracellular antibodies (intrabodies) to block the function of a target protein can be dependent on the stability of the single-chain antibody (sFv) when expressed in the intracellular environment. Low-affinity sFvs capable of reaching high steady-state levels can be more effective modulators of target proteins than high-affinity, unstable sFvs. In an effort to enhance the intracellular stability of sFvs when expressed as intrabodies, we have generated novel sFv-Fc fusion intrabodies. Fusion of the native sFv sequence with the entire heavy chain constant region fragment of IgG results in decreased turnover of the intrabody and enhanced steady-state accumulation of sFv-Fc protein, while maintaining the ability to target intrabody expression to sub-cellular compartments. Here, we describe the rationale and design for this strategy using two anti-cyclin E sFvs constructed for use as intrabodies.

The apparent tumor selective apoptosis-inducing activity of recombinant soluble TNF-related apoptosis-inducing ligand (TRAIL) has aroused much interest for use in clinical application. However, to exploit fully its therapeutic potential, the characteristics of both the TRAIL receptor system and soluble TRAIL (sTRAIL) should be taken into account: first, the widespread expression of the various TRAIL receptors throughout the human body; second, the differential binding affinities and crosslinking requirements of the agonistic receptors TRAIL-R1 and TRAIL-R2; and third, the solution behavior of particular sTRAIL preparations. Therefore, we constructed a novel TRAIL fusion protein, designated scFvC54:sTRAIL, comprising the human scFv antibody fragment C54 genetically linked to the N-terminus of human sTRAIL. The scFvC54:sTRAIL fusion protein was designed to induce apoptosis by crosslinking of agonistic TRAIL receptors only after specific binding of scFvC54:sTRAIL to the abundantly expressed carcinoma-associated cell surface antigen EGP2 (alias EpCAM). Target antigen-restricted apoptosis induction was demonstrated for various EGP2-positive tumor cells and could be inhibited by an EGP2 competing antibody. Target antigen binding converted soluble scFvC54:sTRAIL into a membrane-bound form of TRAIL that was capable of signaling apoptosis not only through TRAIL-R1 but also through TRAIL-R2. Size-exclusion fast protein liquid chromatography (FPLC) indicated that scFvC54:sTRAIL was produced as stable and homogeneous trimers in the absence of detectable TRAIL aggregates. The favorable characteristics of the scFvC54:sTRAIL fusion protein potentially reduce the amount of sTRAIL required for antitumor activity and may be of value for the treatment of various human carcinomas., (Copyright 2003 Wiley-Liss, Inc.)

Two sets of expression vectors were constructed that permitted the efficient expression of single-chain Fv fragments (scFvs) fused N-terminally to an enhanced mutant of the green fluorescent protein GFP+ or the red fluorescent protein DsRed in insect and mammalian cells. The vectors allowed rapid cloning of scFv fragments and secretion of the fusion proteins in a native conformation. Fluorescent scFv fusion proteins directed against a series of cluster of differentiation (CD) antigens were efficiently secreted by transiently transfected mammalian cells and insect cells infected with baculoviral expression constructs. Yields of the secreted proteins varied from 100 microg/l to 3 mg/l. The purified proteins were functionally active in flow cytometry, immunofluorescent microscopy, and competition binding experiments performed to delineate the epitopes recognized by different monoclonal antibodies against the same polypeptide. The use of two different scFv fragments fused with red and green fluorescent proteins and reacting with T- and B-cell lineage markers (CD7 and CD19), respectively, allowed a simplified quantitation of both subsets in two-color flow cytometry experiments with mixed populations of T- and B-lymphoid cells. Due to the lack of Fc domains in the scFv proteins, the fluorescent fusion proteins showed more than 20-fold reduced background fluorescence compared with whole antibodies of the same specificity in experiments with effector cells expressing the high affinity FcgammaRI receptor CD64. Thus, for a number of analytical applications, fluorescent scFv fusion proteins offer advantages over the use of complete primary antibodies and chemically labeled fluorescent secondary antibodies.

Generation of specific antibodies against enriched subcellular fractions is a powerful strategy to identify and characterize cellular components. We show that recombinant antibodies can be selected in vitro by phage display against complex subcellular fractions, namely microtubule-binding proteins and Golgi stacks. This technique has allowed us to overcome many limitations of the classical animal-based approach and generate cell biology-compliant antibodies. In addition, we show that intracellular expression of GFP-tagged recombinant antibodies can reveal the dynamics of endogenous proteins in vivo. Endogenous Giantin is very static and outlines the Golgi in living cells. It accumulates neither onto Golgi-derived tubules upon Brefeldin A treatment before Golgi disappearance, nor onto de novo formed Golgi mini-stacks upon microtubule depolymerization, and remains instead on the 'old' pericentriolar Golgi. This suggests that, in contrast to other Golgi matrix proteins, endogenous Giantin is very stably associated with the Golgi and does not efficiently recycle to the ER. Altogether, we show that the antibody phage display technique represents an efficient alternative to rapidly generate versatile antibodies that represent new tools to study protein function.

The atrazine-specific single-chain variable antibody fragments (scFv) K411B was produced by expression in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the pelB leader was N-terminally fused to scFv, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly. Expression of scFv with leader was 2.3 times higher than that of the protein without leader. This was further investigated by generating the respective translation profiles using coupled in vitro transcription/translation assays, the results of which were in agreement. This comparative approach was also applied to functionality: Periplasmic expression and in vitro expression resulted in only 10% correctly folded scFv, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of scFv with enhanced green fluorescent protein (EGFP) fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile as for scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.

The intracellular expression of single-chain Fv antibody fragments (scFv) in eukaryotic cells has an enormous potential in functional genomics and therapeutics [Marasco (1997) Gene Ther. 4, 11-15; Richardson and Marasco (1995) Trends Biotechnol. 13, 306-310]. However, the application of these so-called intrabodies is currently limited by their unpredictable behavior under the reducing conditions encountered inside eukaryotic cells, which can affect their stability and solubility properties [Wörn et al. (2000) J. Biol. Chem. 275, 2795-2803; Biocca et al. (1995) Bio/Technology 13, 1110-1115]. We present a novel system that enables selection of stable and soluble intrabody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single-chain antibodies fused to a selectable marker that can control gene expression and cell growth. Our results show that the activity of a selectable marker fused to well characterized scFvs [Wörn et al. (2000) J. Biol. Chem. 275, 2795-2803] correlates with the solubility and stability of the scFv moieties. This method provides a unique tool to identify stable and soluble scFv frameworks, which subsequently serve as acceptor backbones to construct intrabody complementarity determining region libraries by randomization of hypervariable loops.

A novel recombinant single-chain fragment variable (scFv) antibody against western equine encephalitis (WEE) virus has been previously constructed and partially characterized. The RS10B5huFc antibody was made by fusing an anti-WEE scFv to a human heavy-chain IgG1 constant region. The RS10B5huFc antibody was functional in binding to WEE virus in enzyme-linked immunosorbent assays (ELISAs), and the Fc domain of the antibody was capable of effector functions, such as binding to protein G and human complement. In this study, the RS10B5huFc antibody was further characterized by BIAcore analyses and was found to possess a binding affinity to a WEE virus epitope (K[D] = 9.14 x 10(-6) M), 4.5-fold lower than its parental mouse monoclonal antibody (MAb) 10B5 E7E2 (K[D] = 2 x 10(-6) M). No cross-reactivity was found between the RS10B5huFc antibody and three other alphaviruses (Sindbis virus [SIN], Venezuelan equine encephalitis [VEE] virus, and eastern equine encephalitis [EEE] virus). Pharmacokinetics studies showed that the RS10B5huFc antibody (free and encapsulated) was found to be retained in the lungs of mice for greater than 48 h when administered intranasally. In contrast, when administered intramuscularly to mice, the RS10B5huFc antibody was not detected in the lungs and only found in the liver and kidneys.

The provision of the T-cell costimulatory molecule B7 to tumor cells can be an effective way to trigger a tumor-specific cytolytic T-cell response. One way to provide B7 to tumor cells would be to couple an antitumor antibody directly to B7. Such a molecule should target tumors displaying antigen and provide the costimulatory signal to T cells, resulting in the initiation of an antitumor T-cell response. To this end, a fusion protein was designed that incorporates a single-chain antibody fragment (scFv) to erbB-2 (Her2/neu), an oncogene product overexpressed by 30% to 50% of breast carcinomas, and the ECD of B7-2 (CD86). This fusion protein, expressed and purified from Pichia pastoris, was shown to retain binding activity to both counter receptors, erbB-2 and CD28. The fusion protein was also shown to target erbB-2-positive tumor cells and to deliver a CD28-specific T-cell costimulatory signal. These results suggest that a fusion protein engineered to target tumor cells and signal T cells for activation may be an effective means of cancer immunotherapy. Further studies should be performed to characterize the fusion protein in erbB-2 tumor-bearing mice for in vivo tumor targeting, biodistribution, and efficacy.

Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow's medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Fd chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Fd chains were used, secretion of L:Fd heterodimers was highly successful. Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75-100 kDa). These disulfide-stabilized bispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)(2) ("tribody") heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties. Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.

We describe a method for the isolation of recombinant single-chain antibodies in a biologically active form. The single-chain antibodies are fused to a cellulose binding domain as a single-chain protein that accumulates as insoluble inclusion bodies upon expression in Escherichia coli. The inclusion bodies are then solubilized and denatured by an appropriate chaotropic solvent, then reversibly immobilized onto a cellulose matrix via specific interaction of the matrix with the cellulose binding domain (CBD) moiety. The efficient immobilization that minimizes the contact between folding protein molecules, thus preventing their aggregation, is facilitated by the robustness of the Clostridium thermocellum CBD we use. This CBD is unique in retaining its specific cellulose binding capability when solubilized in up to 6 M urea, while the proteins fused to it are fully denatured. Refolding of the fusion proteins is induced by reducing with time the concentration of the denaturing solvent while in contact with the cellulose matrix. The refolded single-chain antibodies in their native state are then recovered by releasing them from the cellulose matrix in high yield of 60% or better, which is threefold or higher than the yield obtained by using published refolding protocols to recover the same scFvs. The described method should have general applicability for the production of many protein-CBD fusions in which the fusion partner is insoluble upon expression., (Copyright 1999 Academic Press.)

An anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody, designated as 3B10, inhibits the biological activity of human TNF-alpha. In the present study, we constructed humanized version of the antibody by grafting its complementarity-determining regions (CDRs) onto a human antibody, HBS-1. Using a molecular model of mouse 3B10, framework residues affecting the CDR conformation were identified. Thus, these residues were also introduced into the framework together with the CDRs in a stepwise manner, depending on the degree of the possible importance of the residues. As a result, one humanized version (h3B10-9) which possesses nine mouse framework residues showed the same binding activity as that of the chimeric version. This humanized anti-TNF-alpha antibody is expected to be less immunogenic and thus more suitable for possible clinical use.

Background: To perform an ELISA or for panning phage particles which display recombinant proteins, one of the reactants is immobilized on solid phase. Immobilization in ELISA is generally performed by passive adsorption of ligands to plastic. However, protein is denatured during the adsorption process. This may result in low efficiency interaction between ligands and receptors which depend on native structures. In contrast, indirect immobilization has been shown to prevent protein denaturation., Objectives: The aim was to develop a system that allows efficient and stable indirect immobilization of a variety of recombinant multimeric proteins to solid phase., Results: A new vector was constructed which allows the expression of up to three proteins linked by the Jun/Fos leucine zipper. Purification of the resulting protein was achieved by Ni+ affinity chromatography utilizing the 6xHis-ABP (albumin binding protein) protein fused to the N-terminus of the Jun polypeptide. The high binding affinity of ABP to rat serum albumin (RSA) was exploited for indirect immobilization of recombinant proteins to solid phase. In an enzyme linked assay, the binding of ABP to immobilized RSA was shown to be 10-1000 times more efficient than other immobilization systems. Using the ZZ IgG binding domain of staphylococcal protein A as bait, the RSA-ABP immobilization system was successfully used to screen and enrich IgG Fc encoding DNA fragments from a cDNA phage library., Conclusion: The newly designed vector termed pJuFoexpress allows production and purification of multimeric protein complexes linked by the Jun/Fos leucine zipper. Without chemical modifications, the recombinant proteins can be immobilized indirectly to solid phase. The immobilization results in the stable display of native, biologically active proteins which can be used in ELISA and phage display systems.

Monomeric single chain antibody (scFv) fragments lack both the avidity of the bivalent IgG, or (Fab')2 fragment, and the effector functions conferred by the Fc domain. For certain diagnostic or therapeutic applications it may be desirable to link these molecules to other proteins, antibodies, enzymes or peptide ligands, and chemical or recombinant methods have been developed to produce many of these crosslinked reagents. One approach has been to link an antibody fragment to streptavidin which can bind a second biotinylated molecule to create a higher affinity, bifunctional or bispecific molecule. To demonstrate the applicability of this technology, an anti-neuraminidase NC10 scFv-streptavidin fusion was expressed in E. coli and the product was refolded and purified to homogeneity from 6 M guanidine hydrochloride. Analysis in a BIAcore biosensor showed that the NC10 scFv moiety reacted with immobilised neuraminidase and that the core streptavidin moiety was able to bind biotinylated anti-ferritin Fab' to produce a new model bispecific reagent which bound ferritin. Conceptually, this design principle can be applied to the creation of useful diagnostic and possibly therapeutic molecules.

The surrogate light chain, composed of VpreB and lambda 5/14.1 proteins, is selectively expressed on B cell precursors, and is important for B cell development. The surrogate light chain associates with cell surface mu-chains on preB cells, but little is known about the ligand specificity and affinity of VpreB binding. To analyze its interactions with Igs, we made recombinant human VpreB protein and measured its affinity for H and L chain V domains using surface plasmon resonance. The recombinant VpreB protein existed as a homodimer in solution. VpreB chains associated with each other with an apparent Kd = 5 x 10(-7) M, and bound to a human VH domain, a mouse VH domain, and a human VL domain with a similar affinity. VpreB protein also bound to human Fab fragments of IgG with an apparent Kd = 6 x 10(-7) M, but showed a very low affinity for human Fc fragments of IgG. VpreB-Fab complex formation was reproduced by the formation of a trimolecular VpreB-VH-VL complex. Thus, VpreB proteins can associate with each other and also form complexes with Ig at sites different from those involved in VH-VL interaction. By flow cytometry, biotinylated VpreB protein bound to surface Ig-positive B cells but not T cells. Receptors that contain VpreB could be cross-linked by either Ig or by self-association.

A recombinant chimeric Fab (rcFab), with Fv derived from the monoclonal A5B7 antibody to carcinoembryonic antigen (CEA), and with human CHI and C kappa was cloned into pUC 19 and expressed in Escherichia coli. rcFab (10 to 12 mg per litre) was produced in bacterial culture fluid, and functional purified rcFab was isolated by affinity chromatography (using antibody to human C kappa) and size-exclusion gel filtration. The rcFab did not show reduced affinity for CEA, and reacted with human colorectal tumours showing a typical anti-CEA pattern by immunocytochemistry; it was also stable after iodination. Biodistribution studies in nude mice bearing human tumour xenografts showed no toxicity and good tumour localization. Therapeutic ratios at early time points were better than those obtained with whole murine antibody. The results demonstrate that bacterially produced anti-CEA Fab is of use for tumour targeting.

One amino acid, tyrosine at position 96 and 97 in the VH CDR3 region of a mouse/human chimeric anti-TAG72 antibody cB72.3m4 was substituted by the phenylalanine residue and by a number of amino acids from different amino acid groups by the site-directed mutagenesis technique. The expression vector mpSV2neo-EP1-Vm11-16C1 containing mutant VH region fragments (Vm11-16) as well as the immunoglobulin enhances (E), promoter (P1) and the human genomic C1 region fragments, were transfected into a heavy-chain-loss mutant cell line B72.3Mut(K), respectively. Mutant chimeric cB72.3m11-16 antibodies were purified from the transfectant supernates and compared based upon their binding affinity for the TAG72 antigen relative to that of the original cB72.3m4 antibody. The data showed that a single amino acid substitution of tyrosine by phenylalanine and a number of amino acids including serine, asparagine, histidine and arginine at position 97 in the VH CDR3 region all resulted in approximate 18-fold lower binding affinity, whereas the substitution of tyrosine by phenylalanine at position 96 in the VH CDR3 region did not affect the binding affinity of the cB72.3m4 antibody. This suggests that the tyrosine residue at position 97 in the VH CDR3 region is in a contact position in the B72.3/TAG72 antibody/antigen interaction, and that the terminal hydroxyl group of the position 97 tyrosine side-chain contributes hydrogen bonding to the TAG72 antigen, whereas the position 96 tyrosine side-chain does not.

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)

We have generated a chimeric protein molecule composed of the alpha- and beta-chains of the MHC class II I-E molecule fused to antibody V regions derived from anti-human CD4 mAb MT310. Expression vectors were constructed containing the functional, rearranged gene segments coding for the V region domains of the antibody H and L chains in place of the first domains of the complete structural genes of the I-E alpha- and beta-chains, respectively. Cells transfected with both hybrid genes expressed a stable protein product on the cell surface. The chimeric molecule exhibited the idiotype of the antibody MT310 as shown by binding to the anti-idiotypic mAb 20-46. A protein of the anticipated molecular mass was immunoprecipitated with anti-mouse IgG antiserum. Furthermore, human soluble CD4 did bind to the transfected cell line, demonstrating that the chimeric protein possessed the binding capacity of the original mAb. Thus, the hybrid molecule retained: 1) the properties of a MHC class II protein with regard to correct chain assembly and transport to the cell surface; as well as 2) the Ag binding capacity of the antibody genes used. The generation of hybrid MHC class II molecules with highly specific, non-MHC-restricted binding capacities will be useful for studying MHC class II-mediated effector functions such as selection of the T cell repertoire in thymus of transgenic mice.