Secretion of a functional Fab fragment in Escherichia coli and the influence of culture conditions

Summary Genes encoding a light chain and an Fd region (a variable region and a CH1 domain of a heavy chain) of a mouse-human chimeric antibody with specificity for human carcinoembryonic antigen (CEA) were fused to a DNA segment coding for the signal peptide of Escherichia coli ompF. E. coli cells harbouring an expression vector containing these genes downstream of a tac promoter were able to secrete a Fab fragment of the antibody efficiently. When the cells were cultured at 37° C and the inducer (isopropyl-\-d-thiogalactopyranoside, IPTG) concentration was 1 mm (standard conditions), production of functional Fab was very low (medium; 200 ng/l culture and periplasm; <90 ng/l culture). In order to optimize functional Fab production, we examined the influence of culture conditions (i.e. temperature and the inducer concentration) on secretion of the product. It was found that a 12.7-fold higher amount of Fab fragment could be produced at 30° C using 0.1 mm IPTG, as compared with standard conditions. Under these optimal conditions, functional Fab accumulated in the periplasm and culture medium for 10 h after induction and the total production level was found to reach approximately 4.5 mg/l culture. Correspondence to: T. Shibui     

Single chain Fab (scFab) fragment

Background  

The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.     

抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

我们采用RT-PCR,从小鼠杂交瘤细胞中扩增并克隆了抗破伤风类毒素(TT)抗体轻、重链可变区,重链Fd区基因,测定了其VH、Vk序列。并在大肠杆菌中表达了Fd片段,ELISA分析的结果表明Fd片段具有抗原结合的能力,但特异性很差。进一步采用SOE,和PCR技术,将VH、VK基因与ScFv连接片段组装成单链抗体(ScFv)基因片段,以及将人重链CH1和Fab基因连接片段组装成Fab基因片段。将它们分别插入含噬菌体fd外壳蛋白3基因的phagem-id pHEN 1中,在辅助噬菌体M 13-VCS作用下,噬菌体表面表达了抗TT的噬菌体单链抗体(phage-ScFv)与噬菌体Fab(phage-Fab),经ELISA检测,表明它们都能与TT特异结合。     

抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

The heavy and light chain variable region genes of anti-tetanus toxoid (TT) antibody and the heavy chain Fd genes were amplified and cloned through RT-PCR from mouse hybridoma cells. The sequences of VH and VK were determined. Fd gene fragments were expressed in E. coli. The ELISA results indicated that the expressed Fd showed antigen binding activity but was nonspecific. Furthermore, through SOE and PCR techniques, the VH and VK gene fragments together with ScFv linker were assembled into single chain antibody (ScFv) gene fragment. While together with human heavy chain CH 1 gene fragment and Fab linker, they were assembled into chimeric Fab gene fragment. The two assembled gene fragments were separately inserted into phagemid pHEN 1, which was a fd-based vector containing gene 3 encoding the minor coat protein. In presence of helper phage M 13-VCS the anti-TT phage-ScFv or phage-Fab were displayed on the surface of phage particles respectively. Results from phage-ELISA indicated that both phage antibodies were TT-specific.     

Comparison of two forms of catalytic antibody displayed on yeast-cell surface

Two forms of the Fab fragment of the catalytic antibody 6D9 were individually displayed on yeast-cell surface in fusion to the C-terminal half of -agglutinin: one was 6D9 Fab1, in which the light chain of the Fab (Lc) fragment is displayed on cell surface and the heavy chain of the Fab (Fd) fragment is secreted and linked to the Lc fragment with a disulfide bond; the other was 6D9 Fab2, in which the Fd fragment is displayed on cell surface and the Lc fragment is secreted and linked to the Fd fragment with a disulfide bond. Analysis by flow cytometry indicated that some 6D9 Fab2 fragments were unable to construct an appropriate conformation, and that most of the 6D9 Fab1 fragments displayed on yeast-cell surface exhibited higher binding affinity, stability, and catalytic activity. Conformation of the surface-displayed hetero-dimeric Fab fragment mainly depended on the intermolecular disulfide bond between the Lc and Fd fragments. The conformation of 6D9 Fab1 was more stable than that of Fab2. In the reducing environment of solution containing 25 nM DTT, the function of 6D9 Fab2 was almost completely lost. The successful display of 6D9 Fab1 on yeast-cell surface provides a novel approach to the engineering of catalytic antibodies.     

Neutralization of Burkholderia pseudomallei Protease by Fabs Generated through Phage Display

The isolation of therapeutic and functional protease inhibitors in vitro via combinatorial chemistry and phage display technology has been described previously. Here we report the construction of a combinatorial mouse-human chimeric antibody fragment (Fab) antibody library targeted against the protease of the tropical pathogen, Burkholderia pseudomallei. The resulting library was biopanned against the protease, and selected clones were analyzed for their ability to function as protease inhibitors. Three families of Fabs were identified by restriction fingerprinting, all of which demonstrated high specificity towards the protease of B. pseudomallei. Purified Fabs also demonstrated the capacity to inhibit B. pseudomallei protease activity in vitro, and this inhibitory property was exclusive to the pathogenic protease. Thus these recombinant antibodies are candidates for immunotherapy and tools to aid in further elucidation of the mechanism of action of the B. pseudomallei protease.     

Efficient production of a functional mouse/human chimeric Fab′ against human urokinase-type plasminogen activator by Bacillus brevis

Expression/secretion vectors for the production of Fab′ and single-chain (sc) Fab′ by Bacillus brevis have been constructed. For the production of Fab′, the cDNAs encoding the L chain and Fd′ fragment (Fd with the hinge region) of a mouse-human chimeric Fab′ against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab′, the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab′ was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab′ remained at a level of a few milligrams per liter in the culture medium. The Fab′ produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd′ fragment, constituting the Fab′, had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments. Received: 25 April 1997 / Received revision: 3 June 1997 / Accepted: 29 June 1997     

Guided selection of an anti-gamma-seminoprotein human Fab for antibody directed enzyme prodrug therapy of prostate cancer

Background The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug therapy in vivo. In this study we have achieved humanization of the anti-γ-seminoprotein E₄B₇ murine mAb by guided selection. Methods Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients. The human Lc gene repertoire was first paired with the murine Fd gene of E₄B₇ mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified γ-seminoprotein antigen. The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four more rounds of panning, completely human Fab antibodies specific for γ-seminoprotein were selected and further identified. Results First, using the E₄B₇ Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a template, a human Fab antibody against γ-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA, and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E₄B₇ mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry analysis further confirmed that this newly constructed human anti-γ-seminoprotein Fab antibody indeed specifically bound prostate cancer cells and tissue. Conclusions Through guided-selection, we successfully produced a human anti-γ-seminoprotein Fab antibody. This work lays the foundation for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody. Zhang Qing and Zhang Si-He are co-first authors on the publication.     

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface

A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of alpha-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.     

Disulfide bond bridged divalent antibody-toxin, (Fab-PE38fl)2, with the toxin PE38 fused to the light chain

B3 antibody specifically binds the LewisY-related carbohydrate antigen of many carcinomas, and it is used as a model antibody in this study. In a previous study, the Fab fragment of the antibody was fused to a 38 kDa truncated form of Pseudomonas exotoxin A, PE38, to make Fab- PE38, where PE38 is fused to the Fd fragment of the Fab domain. This parent monomer molecule, Fab-PE38, had no cysteine in the hinge region, and it could not make a disulfide bond to form a disulfide bond bridged homodimer [7]. In this study, we constructed three different kinds of divalent Fab-toxin fusion homodimers where the toxin is fused to the light chain of Fab, (Fab-PE38fl)2. In addition to the PE38 toxin fused to the light chain, these three molecules have different hinge sequences h1, h2, and h3 making Fabh1-, Fabh2-, and Fabh3-PE38fl monomers, respectively. These hinges contain only one cysteine on different positions of the hinge sequence. The disulfide bond between the hinge region of two monomers forms homodimers (Fabh1-PE38fl)2, (Fabh2-PE38fl)2, and (Fabh3- PE38fl)2. The refolding yields of these dimers were 5- 16-fold higher than a previously constructed dimer where the PE38 was fused to the Fd fragment (Fabh1-PE38)2 [8]. Our data suggest that the steric repulsion between the two PE38s in (Fabh1-PE38)2 during disulfide bridge formation is relieved by fusing it at the end of the light chain. The best cytotoxicity value of these dimers showed about 2.5-fold higher on an MCF7 cell line than that of the monovalent reference molecule in ng/ml scale, which is 15-fold higher in pM scale.     

Production of single chain Fab (scFab) fragments in Bacillus megaterium

Background

The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli.

Results

The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli.

Conclusion

B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.     

Expression of a functional mouse-human chimeric anti-CD19 antibody in the milk of transgenic mice

Human B cell lymphomas are suitable targets for immunotherapy. Clinical trials with mouse-human chimeric B cell-specific monoclonal antibodies (mAbs) have already shown promising results. However, limitations for their use in clinical trials can be the lack of sufficient amounts and high production costs. Expression of mAbs in the mammary gland of transgenic animals provides an economically advantageous possibility for production of sufficient quantities of a promising antibody for clinical trials and beyond. In this paper, we show the feasibility of this approach, by generating transgenic mice expressing mouse-human chimeric anti-CD19 mAbs in their milk. Mouse anti-CD19 variable (V) region genes were combined with human IgG1 heavy (H) and kappa light (L) chain constant (C) region genes and fused to the bovine -lactoglobulin (BLG) promoter in two separate expression cassettes. Co-injection resulted in five transgenic lines. In one of these lines completely assembled chimeric mAbs were secreted into the milk, at an approximate level of 0.5mg/ml. These mAbs were able to bind specifically to the CD19 surface antigen on human B cells.     

The mechanism of reassembly of immunoglobulin G.

The noncovalent interaction of light (L) chain with heavy (H) chain or Fd isolated from a human myeloma protein Jo (IgG1, kappa) was studied by following circular dichroic (CD) change at 235 nm. The dimerization constants of Jo-L chain determined by measuring the CD change at 293 nm with protein concentration showed that the Jo-L chain exists as the monomeric form under the experimental conditions used for recombination with H chain. The second-order rate constants for the interaction between H and L chains were in good agreement with those for the interaction between Fd and L chain at various pH values. The binding behavior of L chain to Fd could be described by a single association constant. In the interpretation of the binding of L chain to H chain, however, it was necessary to assume that the binding of L chain to one of the two sites on H chain dimer (H2) decreases the affinity of the other site for L chain. The binding constant of the first L chain to H2 was the same as that of L chain to Fd. Renaturation processes of L chain, Fd, Fab(SS) fragment (with intact interchain disulfide bond), and Fab(RA) fragment (in which the interchain disulfide bond had been reduced and alkylated) from the denatured states in 0.5 or 1 M acetic acid on neutralization were studied. The renaturation of Fd occurred very rapidly, while that of L chain consisted of a very rapid process and a slow process which followed first-order kinetics. The renaturation process of Fab(SS) consisted of rapid and slow phases, of which the latter followed first-order kinetics. The renaturation process of Fab(RA) also consisted of rapid and slow phases, but the latter process followed second-order kinetics. The overall rate constant of renaturation of Fab(RA) was the same as that of the reformation of Fab(RA) from isolated Fd and L chain. On the basis of these facts, the kinetic mechanism by which Fd and L chain recombine to yield Fab(RA) can be described in terms of the scheme Fd + L in equilibrium Fd ... L leads to Fab(RA), where Fd ... L is an intermediate, and CD change is only observed in the second unimolecular process and not in the first bimolecular process.     

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (C_H1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between V_H-C_H1 and V_L-C_L was improved by the C_H1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91–62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.     

Fc and Fab Fragments from IgG<Subscript>2</Subscript> Human Immunoglobulins Characterized

Papain digestion of 7S immunoglobulin G (IgG) produces two 3.5S Fab fragments and one 3.5S Fc fragment^1–8. The Fab fragment contains one light chain and one Fd fragment and is still able to combine specifically univalently with antigen. The Fc fragment is a dimer of the carboxyl terminal half of the heavy chain. Pepsin splits 7S IgG into some small peptides derived from Fc and one 5S F(ab′)₂ fragment, which contains both antigen-binding sites. Based on this information, some investigators^6,7 have postulated that pepsin splits the γ chains at the C-terminal side of the inter-heavy chain disulphide bridges, whereas papain splits at the N-terminal side of the inter-heavy chain disulphide bridges. We report here evidence that this model does not apply to all IgG subclasses. In the case of human IgG₂ subclass myeloma proteins, papain splits initially at the C-terminal side of inter-heavy chain disulphide bridges. We also show that the amino-acid sequence of the Fc fragment of human IgG₂ subclass so far determined has approximately 95% homology with that of human IgG₁ and IgG₄ subclasses reported by others^9–15.     

Directed evolution of an anti-human red blood cell antibody

We have earlier described a haemagglutination-based assay for on-site detection of antibodies to HIV using whole blood. The reagent in this assay comprises of monovalent Fab fragment of an anti-human RBC antibody fused to immunodominant antigens of HIV-1 and HIV-2. In the present work, we describe a rational and systematic method for directed evolution of scFv and Fab antihuman RBC antibody fragments. Based on homology modeling and germline sequence alignments of antibodies, target residues in the anti-RBC MAb B6 sequence were identified for mutagenesis. A combinatorial library of 10⁷ clones was constructed and subjected to selection on whole RBC under competitive binding conditions to identify several phage-displayed B6 scFv clones with improved binding as determined in an agglutination assay. Selected V_L and V_H sequences were shuffled to generate a second generation phage-displayed Fab library which on panning yielded Fab clones with several fold better binding than wild type. The mutants with better binding also displayed more Fab molecules per phage particle indicating improved in vivo folding which was also confirmed by their increased periplasmic secretion compared to the wild type. The mutant Fab molecules also showed superior characteristics in large scale production by in vitro folding of LC and Fd. The biophysical measurements involving thermal and chemical denaturation and renaturation kinetics clearly showed that two of the mutant Fab molecules possessed significantly improved characteristics as compared to the wild type B6 Fab. Structural modelling revealed that B6 Fab mutants had increased hydrogen bonding resulting in increased stability. Our approach provides a novel and useful strategy to obtain recombinant antibodies with improved characteristics.Key words: phage display, antibody maturation, Fab, antibody folding, scFv, agglutination     

Fermenter production of an artificial fab fragment,rationally designed for the antigen cystatin,and its optimized crystallization through constant domain shuffling

The synthetic antibody model “M41” was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine C_K and C_H 1_γ1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P2₁2₁2₁ with unit cell dimensions a = 104.7 Å, b = 113.9 Å, c = 98.8 Å and diffracted X-rays to a nominal resolution of 2.5 Å. © 1995 Wiley-Liss, Inc.