Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery

One of the most commonly used recombinant antibody formats is the single-chain variable fragment (scFv) that consists of the antibody variable heavy chain connected to the variable light chain by a flexible linker. Since disulfide bonds are often necessary for scFv folding, it can be challenging to express scFvs in the reducing environment of the cytosol. Thus, we sought to develop a method for antigen-independent selection of scFvs that are stable in the reducing cytosol of bacteria. To this end, we applied a recently developed genetic selection for protein folding and solubility based on the quality control feature of the Escherichia coli twin-arginine translocation (Tat) pathway. This selection employs a tripartite sandwich fusion of a protein-of-interest with an N-terminal Tat-specific signal peptide and C-terminal TEM1 β-lactamase, thereby coupling antibiotic resistance with Tat pathway export. Here, we adapted this assay to develop intrabody selection after Tat export (ISELATE), a high-throughput selection strategy for the identification of solubility-enhanced scFv sequences. Using ISELATE for three rounds of laboratory evolution, it was possible to evolve a soluble scFv from an insoluble parental sequence. We show also that ISELATE enables focusing of an scFv library in soluble sequence space before functional screening and thus can be used to increase the likelihood of finding functional intrabodies. Finally, the technique was used to screen a large repertoire of naïve scFvs for clones that conferred significant levels of soluble accumulation. Our results reveal that the Tat quality control mechanism can be harnessed for molecular evolution of scFvs that are soluble in the reducing cytoplasm of E. coli.     

Cloning and characterization of heavy and light chain genes encoding the FimA-specific monoclonal antibodies that inhibit Porphyromonas gingivalis adhesion

FimA of Porphyromonas gingivalis, a major pathogen in periodontitis, is known to be closely related to the virulence of these bacteria and has been suggested as a candidate for development of a vaccine against periodontal disease. In order to develop a passive immunization method for inhibiting the establishment of periodontal disease, B hybridoma clones 123-123-10 and 256-265-9, which produce monoclonal antibodies (Mabs) specific to purified fimbriae, were established. Both mAbs reacted with the conformational epitopes displayed by partially dissociated oligomers of FimA, but not with the 43 kDa FimA monomer. Gene sequence analyses of full-length cDNAs encoding heavy and light chain immunoglobulins enabled classification of the genes of mAb 123-123-10 as members of the mVh II (A) and mVκ I subgroups, and those of mAb 256-265-9 as members of the mVh III (D) and mVκ I subgroups. More importantly, 50 ng/mL of antibodies purified from the culture supernatant of antibody gene-transfected CHO cells inhibited, by approximately 50%, binding of P. gingivalis to saliva-coated hydroxyapatite bead surfaces. It is expected that these mAbs could be used as a basis for passive immunization against P. gingivalis-mediated periodontitis.     

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction(PCR) technique.We found that 2 and 3 mAbs utilized genes of the VHIV and VHⅢ families,respectively.The former 2 VH segments were in germline configuration.A common VH segment,with the best similarity of 90.1% to the published VHⅢ germline genes,was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs.This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHⅢ gene.All these polyreactive mAbs displayed a large NDN region(VH-D-JH junction).The entire H chain V regions of these polyreactive mAbs are unusually basic.The analysis of the charge properties of these mAbs as well as those of other poly-and mono-reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites(CDRs),may be an important structural feature involved in antibody polyreactivity.     

Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain

Currently, almost all U.S. Food and Drug Administration-approved therapeutic antibodies and the vast majority of those in clinical trials are full-size antibodies mostly in an immunoglobulin G1 format of about 150 kDa in size. Two fundamental problems for such large molecules are their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules [e.g., on the human immunodeficiency virus envelope glycoprotein (Env)] that are accessible by molecules of smaller size. We have identified a phage-displayed heavy chain-only antibody by panning of a large (size, ∼ 1.5 × 10¹⁰) human naive Fab (antigen-binding fragment) library against an Env and found that the heavy chain variable domain (V_H) of this antibody, designated as m0, was independently folded, stable, highly soluble, monomeric, and expressed at high levels in bacteria. m0 was used as a scaffold to construct a large (size, ∼ 2.5 × 10¹⁰), highly diversified phage-displayed human V_H library by grafting naturally occurring complementarity-determining regions (CDRs) 2 and 3 of heavy chains from five human antibody Fab libraries and by randomly mutating four putative solvent-accessible residues in CDR1 to A, D, S, or Y. The sequence diversity of all CDRs was determined from 143 randomly selected clones. Most of these V_Hs were with different CDR2 origins (six of seven groups of V_H germlines) or CDR3 lengths (ranging from 7 to 24 residues) and could be purified directly from the soluble fraction of the Escherichia coli periplasm. The quality of the library was also validated by successful selection of high-affinity V_Hs against viral and cancer-related antigens; all selected V_Hs were monomeric, easily expressed, and purified with high solubility and yield. This library could be a valuable source of antibodies targeting size-restricted epitopes and antigens in obstructed locations where efficient penetration could be critical for successful treatment.     

Analysis of a conjugate between anti-carcinoembryonic antigen monoclonal antibody and alkaline phosphatase for specific activation of the prodrug etoposide phosphate

Summary The selective targeting of tumours by enzymes conjugated to monoclonal antibodies (mAb) may be an ideal approach to convert relatively nontoxic prodrugs into active agents at the tumour site. We used the anti-carcinoembryonic antigen mAb BW431/26 conjugated to alkaline phosphatase (AP) and phosphorylated etoposide (etoposide-P) as a prodrug to study the feasibility of this concept. Etoposide was phosphorylated with POCl₃. Quantitative hydrolysis of etoposide-P to etoposide occurred within 10 min in the presence of AP. BW431/26 and AP were conjugated using a thioether bond. The AP conjugate retained 93% of its calculated activity.¹²⁵I-labelled AP conjugate did not show a reduction of immunoreactivity as determined by a cell-binding assay. SW1398 colon cancer cells were used to analyse the cytotoxicity of etoposide and etoposide-P. Etoposide (IC₅₀ 22 µM) was 100 times more toxic than etoposide-P (20% growth inhibition at 200 µM). Pretreatment of the cells with BW431/26-AP prior to etoposide-P exposure resulted in a dramatic increase in cytotoxicity (IC₅₀ 70 µM). The pharmacokinetics and tumour-localizing properties of BW431/27 and the AP conjugate were assessed in nude mice bearing SW1398 tumours. BW431/26 showed excellent tumour localization (10% of the injected dose/g tissue retained from 8 h to 120 h), whereas the AP conjugate showed a reduced tumour uptake (3%-0.3% of the injected dose/g tissue at 8–120 h), a faster clearance from the circulation and a high liver uptake. Radiolabelled AP showed a similar pharmacokinetic profile to the AP conjugate. Gel filtration analysis of blood, liver, and tumour samples indicated good stability of the conjugate.     

Convenient preparation and characterization of a monoclonal antibody for the N-linked sugar chain of a glycoprotein using a microbial endoglycosidase

We attempted to obtain the monoclonal antibody specific for the N-linked complex-type sialo-oligosaccharide in glycoproteins. We first synthesized a chimeric immunoantigen having an N-linked complex-type of oligosaccharide of glycopeptide, which was bound to a p-formylphenyl compound and conjugated with phosphatidylethanolamine dimylistoyl using the transglycosylation activity of a microbial endoglycosidase (Endo-M) and a reductive amination reaction. This preparative method was convenient and provided a good yield. By immunizing mice with this chimeric neoglycolipid, the monoclonal antibody for the complex-type of sialo-oligosaccharide was obtained in the culture fluid of the cell line even though it was relatively unstable. The monoclonal antibody reacted with various glycoproteins having complex-type sialo-oligosaccharides, but not with those having complex-type asialo-oligosaccharides and high mannose types of oligosaccharides, or with any glycosphingolipids. One of epitopes of this monoclonal antibody seemed to be an α-2,6-linked sialic acid at the non-reducing end of the sialo-oligosaccharide of the glycoprotein.     

Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain

This report describes the preparation and partial characterization of monoclonal antibodies that are reactive specifically with the nascently produced non-triple helical form of the type IV collagen α1 chain, designated as NTH α1(IV). These antibodies were nonreactive with the α1 chain of the type IV collagen in the triple-helical conformation. Three antibodies, #141, #179 and #370, with different epitopes in NTH α1(IV) were found to be reactive with the nascent polypeptide secreted from human normal cells and a human carcinoma cell line. The antibodies with different epitopes may provide a key method for elucidating the physiological function and tissue distribution of NTH α1(IV), which is distinct from the chain derived from triple-helical type IV collagen.     

Reactivity of elementary and reticulate bodies of Chlamydia trachomatis LGV2 with monoclonal antibodies specific for the major outer membrane protein

The reactivity of the major outer membrane protein (MOMP) of Chlamydia trachomatis (LGV2 serotype) with 15 monoclonal antibodies was studied during the course of developmental cycle by immunoblotting and immunofluorescence. The monoclonal antibodies reacted in immunoblots with the MOMP of both elementary bodies (EBs) and reticulate bodies (RBs). Using an immunofluorescence test with LGV2-infected cell cultures, the 15 monoclonal antibodies could be divided into 5 groups, according to the time of appearance of their reactivity with the cell culture.     

Production and characterization of a novel monoclonal antibody against Vibrio parahaemolyticus F0F1 ATP synthase's delta subunit and its application for rapid identification of the pathogen

We raised monoclonal antibodies (MAbs) against Vibrio parahaemolyticus cell extracts. One of the MAbs, designated MAb-VP34, reacted in enzyme-linked immunosorbent assays (ELISAs) with 140 V. parahaemolyticus strains, regardless of serotype or origin. MAb-VP34 did not detectably react with 96 strains belonging to 27 other Vibrio species (except for Vibrio natriegens) or with 29 non-Vibrio species. These results show that MAb-VP34 is highly specific for V. parahaemolyticus. Western blotting and mass spectrometry analyses revealed that MAb-VP34 recognized V. parahaemolyticus F₀F₁ ATP synthase's delta subunit.Using MAb-VP34, a rapid and simple immunodot blotting assay (VP-Dot) was developed to determine whether bacterial colonies growing on selective agar, represented V. parahaemolyticus. To evaluate VP-Dot, 20 V. parahaemolyticus strains and 19 non-related strains were tested. The results indicated that VP-Dot is a reliable tool for identification of V. parahaemolyticus colonies. The simple VP-Dot procedure took 40 min, indicating that the MAb-VP34 based immunological method will greatly reduce labor, time, and costs required to verify V. parahaemolyticus colonies as compared with the conventional biochemical test.     

Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies

The somatic mutations accumulated in variable and framework regions of antibodies produce structural changes that increase the affinity towards the antigen. This implies conformational and non covalent bonding changes at the paratope, as well as possible quaternary structure changes and rearrangements at the V_H-V_L interface. The consequences of the affinity maturation on the stability of the Fv domain were studied in a system composed of two closely related antibodies, F10.6.6 and D44.1, which recognize the same hen egg-white lysozyme (HEL) epitope. The mAb F10.6.6 has an affinity constant 700 times higher than D44.1, due to a higher surface complementarity to HEL. The structure of the free form of the Fab F10.6.6 presented here allows a comparative study of the conformational changes produced upon binding to antigen. By means of structural comparison, kinetics and thermodynamics of binding and stability studies on Fab and Fv fragments of both antibodies, we have determined that the affinity maturation process of anti-protein antibodies affects the shape of the combining site and the secondary structure content of the variable domain, stabilizes the V_H-V_L interaction, and consequently produces an increase of the Fv domain stability, improving the binding to antigen.     

Structural and functional characterization of the single-chain Fv fragment from a unique HCV E1E2-specific monoclonal antibody

The nucleotide sequence of the unique neutralizing monoclonal antibody D32.10 raised against a conserved conformational epitope shared between E1 and E2 on the serum-derived hepatitis C virus (HCV) envelope was determined. Subsequently, the recombinant single-chain Fv fragment (scFv) was cloned and expressed in Escherichia coli, and its molecular characterization was assessed using multi-angle laser light scattering. The scFv mimicked the antibody in binding to the native serum-derived HCV particles from patients, as well as to envelope E1E2 complexes and E1, E2 glycoproteins carrying the viral epitope. The scFv D32.10 competed with the parental IgG for binding to antigen, and therefore could be a promising candidate for therapeutics and diagnostics.     

Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus

In contrast to canonical proteases, myelin basic protein (MBP)-Sepharose-purified IgG from multiple sclerosis (MS) and systemic lupus erythematosus (SLE) patients efficiently hydrolyze only MBP, but not many other tested proteins. It was shown that anti-MBP SLE IgGs cleave nonspecific tri- and tetrapeptides with an extremely low efficiency and cannot efficiently hydrolyse longer oligopeptides corresponding to antigenic determinants (AGDs) of HIV-1 integrase. To identify all sites of IgG-mediated proteolysis corresponding to two AGDs of MBP, we have used a combination of reverse-phase chromatography (RPhC), MALDI spectrometry, and TLC to analyze the cleavage products of two (17- and 19-mer) encephalytogenic oligopeptides corresponding to these AGDs. Both oligopeptides contained several clustered major and minor sites of cleavage. The active sites of anti-MBP abzymes are localized on their light chains, while the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high specificity of MBP hydrolysis. The affinity of anti-MBP abzymes for intact MBP was ～10(3)-fold higher than for the oligopeptides. The data suggest that both oligopeptides interact mainly with the light chain of different monoclonal abzymes of total pool of IgGs, which possesses lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific.