Cloning and expression of single chain antibody fragments in Escherichia coli and Pichia pastoris

The potential of recombinant antibody fragments is likely to be fulfilled only if they can be produced routinely at high concentrations. We have compared the ability of Escherichia coli and Pichia pastoris to produce functional recombinant single chain antibody (scAb) fragments. Two scAb fragments were expressed, an antihuman type V acid phosphatase (TRAP) and an anti-Pseudomonas aeruginosa lipoprotein I. We report here that, while expression from P. pastoris resulted in a significantly increased level of expression of the anti-TRAP scAb compared to E. coli, neither fragment was able to bind its target antigen as well as the bacterial product.     

Reduced toxicity of expression, in Escherichia coli, of antipollutant antibody fragments and their use as sensitive diagnostic molecules

Single-chain antibody fragments (scAb), specific for the chlorophenoxy acid herbicide mecoprop, have been expressed and purified from the bacterium Escherichia coli. Co-expression with the colE1-compatible, arabinose-inducible, skp expression vector pHELP1 prevented bacterial lysis and significantly increased both total and functional expression yield. The periplasmic protein, SKP, may have a role as a generic detoxification protein. Surface plasmon resonance (BIAcore 2000) analysis confirmed that the purified scAb retained similar binding kinetics to the monoclonal antibody (Mab) from which it was cloned. In competition ELISA, the bacterial scAb showed the same specificity for mecoprop and a related herbicide, MCPA, as the Mab but an increase in sensitivity for free antigen in all ELISA formats. Bacterially expressed antibody fragments provide a simple, sensitive and cost-effective alternative to the traditional production of diagnostic Mabs via tissue culture.     

Single-chain antibody displayed on a recombinant measles virus confers entry through the tumor-associated carcinoembryonic antigen

To redirect the tropism of the vaccine strain of measles virus (MV), Edmonston B, to a targeted cell population, we displayed on the viral hemagglutinin (H) a single-chain antibody (scAb) specific for the tumor-associated carcinoembryonic antigen (CEA). We generated H fusion proteins with three forms of the scAb appended, differing in the lengths of the linkers separating the VH and VL domains and thus in the oligomerization states of the scAbs. All proteins were stable, appeared properly folded, and were transported to the cell surface, but only H displaying the long-linker form of scAb was functional in supporting cell-cell fusion. This protein induced extensive syncytia in cells expressing the normal virus receptor CD46 and also in CD46-negative cells expressing the targeted receptor, human CEA. Replication-competent MV with H replaced by H displaying the long-linker form of scAb was recovered and replicated efficiently in both CD46-positive and CD46-negative, CEA-positive cells. Thus, MV not only tolerates the addition of a scAb on its H protein but also infects cells via a novel interaction between the scAb and its targeted receptor.     

Production of soluble and functional engineered antibodies in Escherichia coli improved by FkpA

Overproduction of genetically engineered antibodies, such as single-chain antibodies (scAbs) in Escherichia coli often results in insoluble and inactive products known as inclusion bodies. We now report that fusion or co-expression of FkpA, the E. coli periplasmic peptidyl-prolyl-isomerase with chaperone activity, substantially improves soluble and functional expression of scAbs. Anti-human bladder carcinoma scAb (PG) and anti-human CD3 x anti-human ovarian carcinoma-bispecific scAb (BH1) were fused with FkpA on the pTMF-based plasmid and expressed in E. coli. More than half of the amount of each expressed fusion protein FkpA-PG or FkpA-BH1 was soluble. In addition, the fusion protein cellulose-binding domain from Cellulomonas fimi (CBD)-PG and anti-human CD3 x anti-human CD28 x anti-human ovarian carcinoma-trispecific scAb (TRI) fused to the pelB (a signal peptide from pectate lysase B of a Bacillus sp.) signal sequence were co-expressed with FkpA under the control of the T7 promoter. A substantial portion of the co-expressed CBD-PG or TRI was soluble. Furthermore, PG, BH1, and TRI were biologically active as judged by ELISA and in vitro cytotoxicity assay. These results suggest that overexpression of FkpA should be useful in expressing heterologous proteins in E. coli.     

Improved expression characteristics of single-chain Fv fragments when fused downstream of the Escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain

The expression of single-chain Fv fragments (scFv) targeted to the periplasm of Escherichia coli often results in very low yields of soluble protein frequently accompanied by host cell growth arrest and sometimes lysis. Single-chain antibody fragments (scAb) are scFv with a human kappa light chain constant (HuCkappa) domain attached C-terminally and share similar problems of expression. By fusing the E. coli maltose-binding protein (mbp) gene either 3' or 5' to a scAb specific for the herbicide atrazine, a reduction in growth arrest was observed that was dependent on the order of gene fusion. The scAb-mbp fusion delayed the onset of growth arrest following induction while the mbp-scAb fusion appeared to ablate growth arrest completely. Cell fractionation revealed barely detectable levels of scAb-mbp in the periplasm while mbp-scAb was detected at equivalent levels as scAb in the periplasmic compartment, indicating that periplasmic scAb solubility is unrelated to propensity to cause growth arrest. IMAC purification of scAb and mbp-scAb proteins followed by liquid competition ELISA revealed the IC(50) for atrazine to be approximately 1 nM for both proteins demonstrating that 5'-mbp fusion does not alter antigen binding. The equivalent scFv and mbp-scFv vectors expressed far less material in both periplasmic and insoluble fractions indicating that the HuCkappa domain can have a positive effect on scFv expression when expressed either alone or as a mbp fusion. The ablation of growth arrest by a 5'-mbp fusion and enhancement of expression by a 3'-HuCkappa domain fusion were extended to a second scFv specific for the herbicide diuron. Therefore, by expressing scFv as tripartite fusions (mbp-scFv-HuCkappa) enhanced levels of soluble periplasmic expression can be achieved without causing growth arrest of the host cell, realizing the potential for constitutive expression of hapten-binding scFv in the E. coli periplasm.     

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.     

High-level production of a single chain antibody against anthrax toxin in Escherichia coli by high cell density cultivation

Previously, we isolated the M18 scFv, which is an affinity matured antibody against the anthrax toxin PA, and observed that its single chain antibody (scAb) form (M18 scAb) exhibited superior stability compared to the scFv. Here, we report high cell density cultivations for preparative scale production of M18 scAb in a 3.5 L fermenter. Briefly, a pH–stat feeding strategy was employed in fed-batch cultivation, and four different cell densities (OD₆₀₀ of 40, 80, 120, and 150) were examined for the induction of scAb gene expression. Among the four cell densities investigated, lower cell densities (OD₆₀₀ of 40) showed higher post-induction cell growth and production yields (665 mg/L of scAb). Even though lower solubility (51%) of scAb was achieved at lower cell density (OD₆₀₀ of 40), monomeric scAb could be purified with high purity (>95%) using simple purification procedures. The purified scAb from high cell density cultures showed biological activity equivalent to that of scAb purified from shake flask cultivation.     

Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water

A na?ve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 microg liter(-1)) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 micro g of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.     

Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter⁻¹) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.     

Biomedicals from a soil bug: expanding scFv production host range

Recombinant antibody fragments have a wide range of applications from research to diagnostics and therapy. Of special interest are small fragments like fragment antigen binding (Fab) or single chain fragment variables (scFv) fragments as they can be produced inexpensively in bacterial expression systems. However, recombinant production efficiencies from established production hosts vary significantly leading to inadequate yields. Gene sequences that have been synthetically adapted to match the codon preferences and respective genomic tRNA pool of the host have been used to improve yields but cannot resolve the principal problem. The development of inducible broad host range scFv expression plasmid constructs leads the way to an easy and efficient screening method for the identification of the optimal bacterial expression host.     

Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and sc-dsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv.     

Surface plasmon resonance (SPR) based binding studies of refolded single chain antibody fragments

Recent advances in Recombinant antibody technology / Antibody Engineering has given impetus to the genetic manipulation of antibody fragments that has paved the way for better understanding of the structure and functions of immunoglobulins and also has escalated their use in immunotherapy. Bacterial expression system such as Escherichia coli has complemented this technique through the expression of recombinant antibodies. Present communication has attempted to optimize the expression and refolding protocol of single chain fragment variable (ScFv) and single chain antigen binding fragment (ScFab) using E.coli expression system. Efficiency of refolding protocol was validated by structural analysis by CD, native folding by fluorescence and functional analysis by its binding with full length HIV-1 gp120 via SPR. Results show the predominant β–sheet (CD) as secondary structural content and native folding via red shift (tryptophan fluorescence). The single chain fragments have shown good binding with HIV-1 gp120 thus validating the expression and refolding strategy and also reinstating E.coli as model expression system for recombinant antibody engineering. SPR based binding analysis coupled with E.coli based expression and purification will have implication for HIV therapeutics and will set a benchmark for future studies of similar kind.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.

In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments

Expression of single-chain antibody fragments (scAb)in the periplasm of Escherichia coli often results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing the E. coli Skp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl beta-d-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

Escherichia coliSkp Chaperone Coexpression Improves Solubility and Phage Display of Single-Chain Antibody Fragments

Expression of single-chain antibody fragments (scAb)in the periplasm ofEscherichia colioften results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing theE. coliSkp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl β- -thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.     

Generation of functional scFv intrabodies for triggering anti-tumor immunity

Intracellular expression of recombinant antibodies (intrabodies) allows to interfere with the functions of oncogenic or viral molecules expressed in different cell compartments and has therefore a vast clinical potential in therapy. Although the use of phage-display libraries has made it possible to select Fab or single chain Fv (scFv) antibody fragments usable for intracellular targeting, a major source of recombinant antibodies for therapeutic use still remains hybridoma B cells producing well-characterized monoclonal antibodies (mAbs). However, the cloning and the intracellular expression of antibody fragments derived from mAbs can be markedly hampered by a number of technical difficulties that include failure of cloning functional variable regions as well as lack of binding of the antibody fragments to the targeted molecule in an intracellular environment. We discuss herein various molecular methods that have been developed to generate functional recombinant antibody fragments usable as anti-tumor triggering agents when expressed in tumor cells. Such antibodies can neutralize or modify the activity of oncogenic molecules when addressed in specific subcellular compartments and/or they can be used to trigger anti-tumor immunity when expressed on tumor cell surface.     

Microarray of recombinant antibodies using a streptavidin sensor surface self-assembled onto a gold layer

We have developed a sensitive method for the detection of recombinant antibody-antigen interactions in a microarray format. The biochip sensor platform used in this study is based on an oriented streptavidin monolayer that provides a biological interface with well-defined surface architecture that dramatically reduces nonspecific binding interactions. All the antibody or antigen probes were biotinylated and coupled onto streptavidin-coated biochip surfaces (1 microL total volume). The detection limits for the immobilized probes on the microarray surface were 0.5 microgram/mL (200 fmol/spot) for the peptide antigen and 0.1 microgram/mL (3 fmol/spot) for the recombinant antibodies. Optimal concentrations for the detection of the Cy5-labeled protein target were in the range of 20 micrograms/mL. Protein microchips were used to measure antibody-antigen kinetics, to find optimal temperature conditions, and to establish the shelf life of recombinant antibodies immobilized on the streptavidin surface. For recombinant antibody fragments with a kDa of 10-100 nM, we have established an easy and direct immunoassay. In addition, we developed an indirect method for antibody detection with no need for expensive and time-consuming antibody purifications and modifications. Such a method was shown to be useful for large-scale screening of recombinant antibody fragments directly after their functional expression in bacteria. Our data demonstrate that recombinant antibody fragments are suitable components in the construction of antibody chips.     

Expression of the catalytic antibodies in eukaryotic systems

Expression of recombinant antibodies in mammalian cells is one of key problems in immunobiotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively done in yeast cells. We obtained expression strains of the methylotrophic beast Pichia pastoris producing single chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab) and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (Phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and single chain antibody A.17 expressed in CHO and E. coli cells respectively.     

VL position 34 is a key determinant for the engineering of stable antibodies with fast dissociation rates

Predictive engineering of antibodies exhibiting fast kinetic properties could provide reagents for biotechnological applications such as continuous monitoring of compounds or affinity chromatography. Based on covariance analysis of murine germline antibody variable domains, we selected position L34 (Kabat numbering) for mutational studies. This position is located at the VL/VH interface, at the base of the paratope but with limited antigen contacts, thus making it an attractive position for mild alterations of antigen binding properties. We introduced a serine at position L34 in two different antibodies: Fab (fragment antigen binding) 57P (Asn34Ser) and scFv (single chain fragment variable) 1F4 (Gln34Ser), that recognize peptides derived from the coat protein of tobacco mosaic virus and the oncoprotein E6, respectively. Both mutated antibodies exhibited similar properties: (i) expression levels of active fragments in Escherichia coli were markedly improved; (ii) thermostability was enhanced; and (iii) dissociation rate parameters (k(off)) were increased by 2- and at least 57-fold for scFv1F4 and Fab57P, respectively, while their association rate parameters (k(on)) remained unchanged. The L34 Ala and Thr mutants of both antibody fragments did not possess these properties. This first demontration of similar effects observed in two antibodies with different specificities may open the way to the predictive design of molecules with enhanced stability and fast dissociation rates.