Soluble Antibody Fragments Research Articles

A Novel Approach to Human Anti-HLA mABs Production: Use of Phage Display Libraries

ABSTRACT: The production of human monoclonal antibodies was previously limited to very laborious and time-consuming processes involving EBV-transformation and/or hybridoma generation. Due to the development of molecular cloning techniques, it is now possible to produce human monoclonal antibody fragments quickly by panning phage display libraries against predefined antigenic specificities. Therefore, we tested this technology for producing human single chain Fv fragments (scFvs) against HLA-DR1 purified molecules immobilized on solid phase. Enrichment of DR1-specific phages was measured through five selection rounds of a synthetic library and revealed a 100-fold amplification. Soluble antibody fragments were then expressed and 7 out of 48 clones were found to secrete scFvs which specifically bind to DR1 molecules in ELISA. Further analysis revealed binding of the scFvs also to DR3 but not to DR5 or DR7 molecules correlating with the presence of particular polymorphic aminoacid residues in the DRβ chain. Western blot analysis indicated that the 7 scFvs react with the DR1 α/β-dimer but not with free α- or β- chains. This study shows that the innovative approach of phage display libraries can efficiently provide scFv fragments as useful reagents for the identification and dissection of HLA polymorphic epitopes.

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity

Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30-fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15–25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80–150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20°C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.

Selection of binders from phage displayed antibody libraries using the BIAcore™ biosensor

In this report we show that phage displayed antibodies can be selected based on dissociation rate constants, using a BIAcore™ biosensor. To demonstrate the principle, two Fab phage stocks displaying antibodies specific for hen egg lysozyme or phenyloxazolone were mixed in a ratio of 1:10 and injected over the biosensor chip containing immobilized lysozyme. Antigen-specific bound phages were eluted and analysed for specificity and phage titer. This procedure enriched for phages carrying specific antibodies. Selection of high affinity binders from phage libraries was then demonstrated with the BIAcore™ when phages were eluted and collected at different time points. Soluble antibody fragments were subsequently expressed and their kinetic parameters were determined. The time of elution was directly proportional to the affinity, due to decreased dissociation rate constants. This procedure offers a rapid and simple approach for selecting binders from phage libraries differing in antibody dissociation rate constants.

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.

Functional Immunoliposomes Harboring a Biosynthetically Lipid-Tagged Single-Chain Antibody

An anti-2-phenyloxazolone single-chain antibody was expressed in Escherichia coli as a lipoprotein fusion in order to generate a biosynthetically lipid-tagged molecule [Laukkanen et al. (1993) Protein Eng. 6, 449-454]. For purification, a hexahistidinyl tag was introduced to the C-terminus of the protein. The resulting antibody, termed Ox lpp-scFv-H6, was membrane-bound, displayed hapten-binding activity, and contained the lipoprotein-specific lipid modification as indicated by metabolic [3H]palmitic acid labeling. The Ox lpp-scFv-H6 was purified by immobilized metal affinity chromatography followed by hapten-based affinity chromatography to essential homogeneity with a yield of 0.4-1.6 mg/L of culture. In detergent dialysis, the purified antibody partitioned quantitatively into phospholipid liposomes. The immunoliposome preparation consisting of a homogeneous population of unilamellar 100-200 nm vesicles displayed specific hapten-binding activity as measured by using ELISA and surface plasmon resonance (SPR)-based real-time biospecific interaction analysis. In SPR experiments, the immunoliposomes exhibited virtually irreversible binding to immobilized hapten compared to soluble antibody fragments, consistent with the predicted multivalent binding. Biosynthetic lipid-tagging of antibodies may prove useful for immunoliposome-based diagnostic and therapeutic applications.

Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display.

Functional antibody fragments may be displayed on the surface of filamentous bacteriophage by introducing variable region genes into the viral genome at a gene encoding a viral coat protein. "Phage display" enables the isolation of antibody genes from large libraries according to the binding specificities they encode. We have constructed a new phage-display vector encoding a polyhistidine tag that has been used for rapid purification of soluble antibody fragments. An antibody library derived from immunized mice was cloned into this vector. This library was panned against the transition state analog RT3, and a high proportion of binders isolated after two rounds of panning. PCR analysis revealed that there were 24 different pattern groups. Sequencing of 15 clones within the major pattern group revealed 10 related clones with a range of point mutations. Thus, phage display can provide a large diverse repertoire of candidate catalytic antibodies based on TSA selection and screening.

Making antibodies by phage display technology.

Antibody fragments of predetermined binding specificity have recently been constructed from repertoires of antibody V genes, bypassing hybridoma technology and even immunization. The V gene repertoires are harvested from populations of lymphocytes, or assembled in vitro, and cloned for display of associated heavy and light chain variable domains on the surface of filamentous bacteriophage. Rare phage are selected from the repertoire by binding to antigen; soluble antibody fragments are expressed from infected bacteria; and the affinity of binding of selected antibodies is improved by mutation. The process mimics immune selection, and antibodies with many different binding specificities have been isolated from the same phage repertoire. Thus human antibody fragments have been isolated with specificities against both foreign and self antigens, including haptens, carbohydrates, secreted and cell surface proteins, viral coat proteins, and intracellular antigens from the lumen of the endoplasmic reticulum and the nucleus. Such antibodies have potential as reagents for research and in therapy.

Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector

Phagemid vectors have been developed which promise to supersede hybridoma technology for the selection and production of human antibodies. We have modified an existing phagemid vector to improve the stability of synthesized soluble antibody fragments. The vector allows the antibody fragment to be produced: i) as a soluble protein incorporating a stable carboxyl terminal octapeptide (FLAG) or, ii) on the surface of a bacteriophage fused to a minor coat protein (the gene III protein). The antibody gene encoding the well characterized monoclonal antibody NC10 (an antibody that recognizes the neuraminidase of the influenza strain N9) was inserted as a single chain Fv construct into the phagemid vectors pHFA and pHFA/SacII. Western blotting, ELISA and electron microscopy studies showed that recombinant clones could be manipulated to either synthesize soluble protein into the periplasm or present the protein on the surface of bacteriophage. Cosynthesis of GroEL and GroES chaperonins resulted in complete proteolysis of the scFvNC10-FLAG-gIIIp fusion product and did not improve total phage production. Coexpression of chaperonins should be used with caution for library construction due to the expected selection pressure for protease resistant gene III fusions.

By-passing immunization: Human antibodies from V-gene libraries displayed on phage

We have mimicked features of immune selection to make human antibodies in bacteria. Diverse libraries of immunoglobulin heavy (V H) and light (V κ and V λ chain variable (V) genes were prepared from peripheral blood lymphocytes (PBLs) of unimmunized donors by polymerase chain reaction (PCR) amplification. Genes encoding single chain Fv fragments were made by randomly combining heavy and light chain V-genes using PCR, and the combinatorial library (>10 7 members) cloned for display on the surface of a phage. Rare phage with “antigenbinding” activities were selected by four rounds of growth and panning with “antigen” (turkey egg-white lysozyme (TEL) or bovine serum albumin) or “hapten” (2-phenyloxazol-5-one (phOx)), and the encoding heavy and light chain genes were sequenced. The V-genes were human with some nearly identical to known germ-line V-genes, while others were more heavily mutated. Soluble antibody fragments were prepared and shown to bind specifically to antigen or hapten and with good affinities, K a (TEL) = 10 7 m −1; K a (phOx) = 2 × 10 6 m −1. Isolation of higher-affinity fragments may require the use of larger primary libraries or the construction of secondary libraries from the binders. Nevertheless, our results suggest that a single large phage display library can be used to isolate human antibodies against any antigen, by-passing both hybridoma technology and immunization.

Phage antibodies: filamentous phage displaying antibody variable domains

New ways of making antibodies have recently been demonstrated using gene technology. Immunoglobulin variable (V) genes are amplified from hybridomas or B cells using the polymerase chain reaction, and cloned into expression vectors. Soluble antibody fragments secreted from bacteria are then screened for binding activities. Screening of V genes would, however, be revolutionized if they could be expressed on the surface of bacteriophage. Phage carrying V genes that encode binding activities could then be selected directly with antigen. Here we show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage (one in a million) can be isolated after affinity chromatography.

The Interaction of Muscle Phosphorylase with Soluble Antibody Fragments

The Interaction of Muscle Phosphorylase with Soluble Antibody Fragments