Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris

We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF). The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains. Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen. We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment. More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P. pastoris culture supernatant by one-step affinity chromatography.     

A single-chain Fv reactive with the Goodpasture antigen

Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.     

Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.     

The middle school experience: effects on the math and science achievement of adolescents with LD

A recombinant Fv construct of the B1 monoclonal antibody that recognizes the LewisY-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the VH and VL domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues VL100 and VH44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023-1029, 1995). The B1 disulfide-stabilized Fv (BldsFv) crystallizes in space group P6(1)22 with the unit cell parameters a = b = 80.1 A, and c = 138.1 A. The crystal structure of the BldsFv has been determined at 2.1-A resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 A and in bond angle of 2.74 degrees. Comparisons of the BldsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of BldsFv is a deep depression 10-A wide and 17-A long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the LewisY tetrasaccharide, Fuc-Gal-Nag-Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781-3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab.     

Cytotoxic and antitumor activity of a recombinant tumor necrosis factor-B1(Fv) fusion protein on LeY antigen-expressing human cancer cells

We have constructed a fusion protein composed of tumor necrosis factor alpha (TNF-alpha) fused at its COOH terminus to the scFv region of monoclonal antibody (mAb) B1, an antibody that recognizes LeY antigen present on many human cancer cells. Our rationale for fusing the scFv to the COOH terminus of TNF was to diminish the binding of the fusion protein to TNF receptors because the COOH terminus of TNF is involved in binding, and thus to partially inactivate (detoxify) the molecule. The Fv region should then target and accumulate the fusion protein on cancer cells, which should compensate for the reduced binding affinity of the TNF moiety and lead to selective killing of TNF-sensitive antigen-expressing cancer cells. The fusion protein was expressed in Escherichia coli and found in insoluble inclusion bodies. After refolding and purification by anion exchange, Ni-NTA affinity, and size-exclusion chromatography, we obtained monomeric TNF-B1(Fv). This molecule binds to LeY antigen on cancer cells with the same affinity as B1(scFv) and B1(scFv) immunotoxins but with significantly lower affinity to the TNF receptor compared to the TNF trimer. TNF-B1(Fv) is very toxic to LeY antigen-expressing cancer cells that are sensitive to TNF (e.g., MCF-7 breast or CRL-1739 gastric cancer cells). This cytotoxicity is antibody targeted and TNF mediated because it can be prevented (as shown on MCF-7 cells) by an antibody competing for LeY antigen binding and by an antibody that neutralizes TNF-alpha. TNF-B1(Fv) kills TNF-alpha-sensitive cells that do not express the target antigen only at much higher doses than TNF trimer, and it does not kill LeY-bearing but TNF-alpha-resistant cells. TNF-B1(Fv) can cause significant tumor regression of MCF-7 tumor xenografts in mice at doses that are not toxic to the mice. Thus, the reduced binding of the TNF moiety to TNF receptors, combined with binding of the B1(Fv) portion to LeY antigen, makes TNF-B1(Fv) an agent for selective killing of LeY-expressing TNF-sensitive cancer cells.     

Folding and assembly of an antibody Fv fragment, a heterodimer stabilized by antigen

The folding and assembly of the Fv fragment of the phosphorylcholine binding antibody McPC603, a non-covalent heterodimer of the variable domains VH and VL, was investigated. Since both domains, each engineered for stability and folding efficiency, could now be obtained in native and soluble form by themselves, fluorescence spectra of VH and VL in unfolded, folded and associated states can be reported. VH and VL only associate when they are native, and the stability of the heterodimer is strongly increased in the presence of antigen. VH rapidly folds into an hyperfluorescent intermediate, and the native state is reached in two parallel, proline-independent reactions. VL displays two fast refolding reactions, which are followed by two slower phases, limited by proline cis/trans-isomerization. The rate-limiting step for both the Fv and the scFv (single-chain Fv) fragment is the formation of the native VH-VL interface, which depends on ProL95 being in cis. The folding of the Fv fragment is fast after short-term denaturation or in the presence of proline cis/trans-isomerase catalysis, but the scFv fragment falls into a kinetic trap, observed by the persistence of the slow phases under all conditions. Furthermore, the scFv fragment, but not the Fv fragment, gives rise to premature interface formation, indicated by the fluorescence spectra and a much higher transient binding of 8-anilino-1-naphthalene sulfonate. The analysis of the folding pathway of the domains VH and VL in isolation and in non-covalent and covalent assemblies should provide helpful insights into the folding of multimeric proteins in general, and for the further engineering of stable and well-folding antibody fragments in particular.     

Diffusing capacity of the lung for carbon monoxide

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (VH) and light (VL) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.     

Production and characterization of a recombinant anti-MUC1 scFv reactive with human carcinomas

Recombinant single-chain fragments (scFv) of the murine anti-MUC1 monoclonal antibody C595 have been produced using the original hybridoma cells as a source of variable heavy (V(H))- and variable light (V(L))-chain-encoding antibody genes. The use of the polymerase chain reaction (PCR), bacteriophage (phage) display technology and gene expression systems in E. coli has led to the production of soluble C595 scFv. The scFv has been purified from the bacterial supernatant by peptide epitope affinity chromatography, leading to the recovery of immunoreactive C595 scFv, which was similar in activity to the C595 parent antibody. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the scFv, while ELISA, FACScan analysis, fluorescence quenching, quantitative immunoreactivity experiments and immunohistochemistry confirm that the activity of the scFv compares favourably with that of the parent antibody. The retention of binding activity to MUC1 antigen on human bladder and breast carcinoma tissue specimens illustrates the potential application of this novel product as an immunodiagnostic and immunotherapeutic reagent.     

Localized primary non-Hodgkin lymphoma of the breast

The efficiency of both phage display in Escherichia coli and periplasmic expression of recombinant proteins may be limited by the same periplasmic folding steps. To search for E. coli factors that improve the efficiency of both procedures, a library of E. coli proteins was coexpressed in a phagemid vector that contained a poorly folding single-chain Fv antibody (scFv) fragment fused to g3p. We enriched, by panning for antigen binding, those phagemids in which the amount of displayed scFv is highest. We thus identified the periplasmic protein Skp/OmpH/HlpA as improving phage display of a wide range of scFv fragments. This occurs as a result of an increase in the amount of hybrid protein displayed on the phage. Coexpression of skp also increases the functional yield of scFv fragments when expressed by secretion to the periplasm.     

Recombinant anti-human melanoma antibodies are versatile molecules

The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.     

Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression

Mutated ras genes are found in a large number of human tumors and, therefore, constitute one of the primary targets for cancer treatment. Microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 was previously reported to induce transient phenotypic reversion of ras-transformed rodent fibroblasts in vitro. We have prepared a single-chain Fv fragment (scFv) derived from Y13-259, and here, we show that intracellular expression of the scFv led to the specific inhibition of the Ras signaling pathway in Xenopus laevis oocytes and NIH3T3 fibroblasts. Moreover, neutralizing Ras with the scFv specifically promoted apoptosis in vitro in human cancer cells but not in untransformed cells. As a step toward cancer gene therapy, we finally demonstrated that intratumor transduction of HCT116 colon carcinoma cells with the anti-Ras scFv using an adenoviral vector elicited sustained tumor regression in nude mice.     

Construction of a semisynthetic antibody library using trinucleotide oligos

A semisynthetic antibody library composed of single chain Fv fragments (scFv) was constructed by replacing the heavy chain CDR3 region of a human scFv by a random sequence of eight amino acids using trinucleotide codons. After cloning into a phage display vector, an antibody library was generated with a complexity of 8 x 10(8) independent clones. The library was screened for binders to dinitrophenol, fluorescein isothiocyanate and 3-nitro-4-hydroxy-5-iodophenylacetic acid. scFv antibodies that specifically bound the antigen were obtained in each case.     

Expression of monovalent fragments derived from a human IgM autoantibody in E. coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity

A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFc1.1. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni(2+)-agarose or a c-myc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 micrograms from 1 litre bacterial culture), (ii) scFv fragments were obtained in suitable amounts from bacterial periplasm (800-1000 micrograms/l), (iii) the monovalent recombinant fragments as well as the Fab obtained by tryptic digestion reflected the polyspecific antigen binding features of the complete IgM antibody, but did bind to the antigens with much lower affinity, and (iv) the CDR3 was found to be of critical importance for the antigen binding pattern of this particular IgM. We discuss the expression of recombinant scFv fragments in E. coli as a suitable method in studying the role of the somatic mutation in autoantibody generation.     

Inhibition of vascular endothelial growth factor-induced receptor activation with anti-kinase insert domain-containing receptor single-chain antibodies from a phage display library

A single-chain antibody phage display library was constructed from spleen cells of mice immunized with a soluble form of a human vascular endothelial growth factor (VEGF) receptor, kinase insert domain-containing receptor (KDR). After two rounds of biopanning, >90% of the clones recovered were specifically reactive to KDR. Subsequent selection identified two clones that blocked VEGF binding to KDR. The clones were expressed in Escherichia coli and purified as soluble single-chain Fv (scFv) antibodies. The affinities of the scFv for binding to KDR were determined by BIAcore analysis (2.1 x 10(-9)-5.9 x 10(-9) M). One scFv, p1C11, was shown to inhibit VEGF-induced KDR phosphorylation and VEGF-stimulated DNA synthesis in human umbilical vein endothelial cells. There is much experimental evidence to suggest that the VEGF/KDR/Flk-1 pathway plays an important role in tumor angiogenesis, a process that is essential for tumor growth and metastasis. The antibodies discussed here, which block VEGF binding to KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved.     

A quantitative immunoassay utilizing Escherichia coli cells possessing surface-expressed single chain Fv molecules

A facile, quantitative immunoassay is described that utilizes Escherichia coli (E. coli) bacteria expressing single chain Fv (scFv) antibody fragments attached to the cell surface. A Scatchard analysis demonstrated that the antibodies on the surface of the cells retained full binding activity (Kd = 2.2 x 10(-9) M) and that there are 60,000 scFv molecules per cell. The cells are used as the antibody reagent in the assay, and, following incubation with analyte, simple centrifugation is used to separate the antibody-bound from unbound analyte. The immunoassay is rapid and accurate down to the nanomolar level. In addition, a variety of detection strategies can be used, and the immunoassay is not adversely affected by the presence of animal serum. A key advantage of the new immunoassay is that the antibody reagent can be inexpensively produced in a "ready to use" form by simply growing cultures of the bacteria.     

Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone

The heavy-chain and kappa light-chain variable region genes of an antizearalenone hybridoma cell line (2G3-6E3-2E2) were isolated by PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a single-chain Fv (scFv) DNA fragment. The scFv DNA fragment was cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 in Escherichia coli TG1. In the presence of helper phage M13K07, the scFv fusion protein was displayed on the surfaces of recombinant phages. High-affinity scFv phages were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjugate. The selected recombinant phages were used to infect E. coli HB2151 for the production of soluble scFv antibodies. One selected clone (pQY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5) with a high zearalenone-binding affinity (concentration required for 50% inhibition of binding, 14 ng/ml), similar to that of parent monoclonal antibody in a competitive indirect enzyme-linked immunosorbent assay. However, scFv QY1.5 exhibited higher cross-reactivity with zearalenone analogs and had greater sensitivity to methanol destabilization than the parent monoclonal antibody did. Nucleotide sequence analyses revealed that the light-chain portion of scFv QY1.5 had a nucleotide sequence identity of 97% to a mouse germ line gene VK23.32 in mouse kappa light-chain variable region subgroup V, whereas the heavy-chain nucleotide sequence was classified as mouse heavy-chain subgroup III (D) but without any closely related members having highly homologous complementarity-determining region sequences. The potential of soluble scFv QY1.5 for routine screening of zearalenone and its analogs was demonstrated with zearalenone-spiked corn extracts.     

High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.