Phage-derived Antibody Research Articles

Monoclonal Antibody Development for Cancer Treatment Using the Phage Display Library Platform

Thirty-four years ago, the groundbreaking work of John McCafferty and Sir Gregory Winter in developing phage display technology revolutionized the discovery of human antibodies, paving the way for diverse applications. Since then, numerous phage-derived antibodies have been successfully developed and advanced into clinical studies, resulting in the approval of more than a dozen therapeutic antibodies. These antibodies have demonstrated efficacy across a spectrum of medical conditions, ranging from autoimmune diseases to various cancers. In this article, we provide an in-depth review of the development of phage display libraries as powerful platforms for therapeutic antibody discovery, elucidating the intricate procedures involved in antibody development. Additionally, we conduct a review of the current ntibody drugs for cancer treatment that have been developed using the phage display platform. Furthermore, we discuss the challenges inherent in this technology, offering insights into potential solutions to enhance crucial steps and facilitate more efficient drug discovery in the field of phage display technology.

A rapid colorimetric lateral flow test strip for detection of live Salmonella Enteritidis using whole phage as a specific binder.

Specific antibodies are essential components of immunoassay, which can be applied for the detection of pathogens. However, producing an antibody specific to live bacterial pathogens by the classical method of immunizing animals with live pathogens can be impractical. Phage display technology is an effective alternative method to obtain antibodies with the desired specificity against selected antigenic molecules. In this study, we demonstrated the power of a microarray-based technique for obtaining specific phage-derived antibody fragments against Salmonella, an important foodborne pathogen. The selected phage-displayed antibody fragments were subsequently employed to develop a lateral flow test strip assay for the detection of live Salmonella. The test strips showed specificity to Salmonella Enteritidis without cross-reactivity to eight serovars of Salmonella or other bacteria strains. The test strip assay requires 15 min, whereas the conventional biochemical and serological confirmation test requires at least 24 h. The microarray screening technique for specific phage-based binders and the test strip method can be further applied to other foodborne pathogens.

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel's method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity.

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel's method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification

We describe a novel cloning method, referred to as insert-tagged (InTag) positive selection, for the rapid one-step reformatting of phage-displayed antibody fragments to full-length immunoglobulin Gs (IgGs). InTag positive selection enables recombinant clones of interest to be directly selected without cloning background, bypassing the laborious process of plating out cultures and colony screening and enabling the cloning procedure to be automated and performed in a high-throughput format. This removes a significant bottleneck in the functional screening of phage-derived antibody candidates and enables a large number of clones to be directly reformatted into IgG without the intermediate step of Escherichia coli expression and testing of soluble antibody fragments. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated, and optimized methods for the small-scale transient expression of IgGs at high levels are described. InTag positive selection cloning has the potential for wide application in high-throughput DNA cloning involving multiple inserts, markedly improving the speed and quality of selections from protein libraries.

Modulation of flavonoid metabolism in Arabidopsis using a phage-derived antibody

The utility of recombinant antibodies for immunomodulation of plant metabolism was tested using the Arabidopsis flavonoid biosynthetic pathway as a target. Two genes encoding antibodies against chalcone isomerase (CHI) were isolated from a human synthetic single chain variable fragment (scFv) library and expressed in the cytoplasm of transgenic plants. In one line, low-level expression of the scFv resulted in reduced visible pigmentation in seedlings as well as lower accumulation of phenolics in plants throughout development. Surprisingly, other transgenic lines with much higher expression of the same antibody showed no phenotype. Protein mobility shift assays indicated that the scFv is bound to the target enzyme, but only in the affected plants. This work shows that recombinant scFv antibody technology offers a viable approach to disrupting protein activity in plants, but that further refinement is required before it will be of general utility for metabolic engineering and other antibody-based applications in plants.

Protein-protein interactions in hematology and phage display.

Phage display, which exploits fundamental tools and principles of immune repertoire diversity, antigen-antibody interactions, and clonal and immunologic selection, is used increasingly to advance experimental and clinical hematology. Phage display is based on the ability of bacteriophage to present engineered proteins on their surface coat. Diverse libraries of proteins such as peptides, antibody fragments, and protein domains corresponding to gene fragments or cDNAs may be displayed. Interactions between phage-displayed proteins and target antigens can be identified rapidly and characterized using high throughput methodologies. Peptide and gene fragment libraries are particularly useful to characterize binding interactions between proteins, such as ligand-receptor interactions. This approach allows rapid generation of human antibodies, often against nonimmunogenic, conserved proteins. Phage antibodies against surface and intracellular antigens are used as reagents for flow cytometry, in vivo imaging, and therapeutic targeting. Phage-derived antibodies also facilitate analyses of the humoral antibody response. Finally, cellular delivery of phage-displayed peptides and gene fragments can be used to modulate functional pathways and molecules in vitro and in vivo. The combinatorial power of phage display enables identification of candidate epitopes without knowledge of the protein interaction, a priori. Overall, these capabilities provide a versatile, high-throughput approach to develop tools and reagents useful for a plethora of experimental hematology applications. This paper focuses on current and future applications of antibody and epitope phage display technology in hematology.

Infrared photodetection for the in vivo localisation of phage-derived antibodies directed against angiogenic markers

Angiogenesis, the formation of new blood vessels from pre-existing ones, is a characteristic process which underlies many diseases, including cancer, rheumatoid arthritis and blinding ocular disorders. Antibodies capable of selective targeting and occlusion of neovasculature would open diagnostic and therapeutic opportunities. We have recently demonstrated that phage-derived human antibody fragments with high affinity for the extra-domain B (ED-B) of fibronectin, a marker of angiogenesis, selectively localise in new-forming blood vessels upon intravenous injection. Here, we show that infrared fluorescence methodologies nicely complement radioactive techniques for the study of the antibody-mediated targeting of angiogenesis in a variety of animal models. Methods are presented for the construction and use of infrared fluorescence imagers, as well as for the production and characterisation of recombinant antibodies labeled with infrared fluorophores.

Display of antibody chains on filamentous bacteriophage.

The construction of very large combinatorial libraries whereby antigen-binding functional domains of antibodies (Abs) are cloned and displayed on the surface of bacteriophage is proving to be a powerful technology in providing reagents for immunotherapy. Combinations of randomly assembled pairs of immunoglobulin heavy (H) and light (L) chain genes are cloned and expressed as fusions of bacteriophage coat proteins, allowing the bacteriophage expressing them to be selected by methods analogous to affinity chromatography. Purified phage can then be used for the production of large amounts of the pure antibody in a soluble form. Although phage-derived antibodies lack the effector functions of the parent antibodies since they have no Fc regions, these can subsequently be engineered into place or substituted with others, such as a toxin, with relative ease if required. Various antigen-recognition regions of the Ab can be cloned and selected, including: 1. Fv fragments that are noncovalently associated heterodimers of the heavy variable (VH) and light variable (VL) domains 2. Single-chain Fv fragments (scFv) consisting of VH and VL regions linked by a flexible peptide 3. Fabs that are noncovalently associated VH-CH and VL-CL pairings of Ab chains.

Inhibition of murine leukaemia virus retrotranscription by the intracellular expression of a phage-derived anti-reverse transcriptase antibody fragment.

The intracellular targeting of recombinant antibodies is an experimental strategy to interfere with the function of selected molecules that is being utilized in a variety of different systems for research and medical applications. Since recombinant antibodies are increasingly being derived from phage display libraries, we have exploited phage technology to isolate, from a large combinatorial library, human antibody fragments directed against human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). We describe in this paper the in vitro and in vivo properties of a neutralizing anti-RT antibody fragment. We demonstrate that the heavy chain domain (VH-CH1) of the phage-derived antibody is able to inhibit the retroviral enzyme, in that it neutralizes the RNA-dependent DNA polymerase activity of HIV-1 RT. The VH-CH1 antibody fragment also neutralizes the activity of RT of drug-resistant HIV-1 mutants as well as that of murine retrovirus RT. To confirm the broad reactivity of the synthetic antibody fragment, we have assessed the ability of the intracellularly expressed VH-CH1 protein to interfere with murine retroviral infection. To this end, we developed an in vivo selection procedure based on the antibody-mediated resistance to a cytotoxic retrovirus and used this selection procedure to rescue, from a heterogeneous population, cells expressing the VH-CH1 antibody fragment. We finally demonstrate that the intracellular expression of the recombinant heavy chain antibody fragment leads to an efficient inhibition of viral retrotranscription by murine-based retrovirus.

Dimerization of Fab fragments enables ready screening of phage antibodies that affect hepatocyte growth factor/scatter factor activity on target cells.

A number of applications of antibodies in diagnosis and therapy require multivalent reagents either because of the polymeric nature of the antigens or because biological activity depends on an effect on the formation of homodimeric species. Here, we report a procedure for mass screening of phage-derived monomeric antibody fragments that depend on valency for activity. As a model system, a set of 13 phage-derived human Fab fragments were first selected against mouse and human recombinant hepatocyte growth factor/scatter factor (HGF/SF), a high molecular weight polypeptide growth factor related to the blood protease plasminogen and involved in development and cancer. These Fab fragments were subsequently screened for an effect on HGF/SF activity either as monomeric fragments or after dimerization with a monoclonal antibody (9E10) directed against a peptide tag on the fragments. Fab were identified that either inhibited or enhanced HGF/SF activity on target cell lines, but dimerization was required for this effect. The approach proposed should facilitate mass screening of phage-derived antibody fragments that depend on multiple valency for activity.

Sequence and structure specific antibodies from phage display libraries

A large combinatorial phage display library was panned against five nucleic acid antigens, calf thymus DNA, poly[d(GC)], poly[d(AT)], poly(dA) · poly(dT) and poly(rA) · poly(dT). After the third and fourth rounds of panning, many positive clones were selected against poly[d(GC)], poly(dA) · poly(dT) and poly(rA) · poly(dT). The specificity of these antibodies was tested by both direct and competitive solid phase radioimmune assays. All the clones derived from panning with poly[d(GC)] were non-specific and bound to all nucleic acids. The poly(rA) · poly(dT) derived clones were specific for single-stranded nucleic acids, with some sequence preferences, and the poly(dA) · poly(dT) derived clones showed considerable specificity for this antigen. The sequences of these phage-derived antibodies showed no similarities with DNA-binding antibodies from other sources. Even after six rounds of panning no positive clones were detected which bound to poly[d(AT)] and after seven rounds only two were derived from panning with calf thymus DNA. Therefore, sequence-and structure specific antibodies can be recovered from phage display libraries but not all sequences may be represented in the repertoire.