FAQS – PHAGE DISPLAY

Antibody libraries represent a collection of the antibody repertoire from a specific host species. Libraries are sourced from B lymphocytes (B cells) or recovered from peripheral blood mononuclear cells (PBMCs) obtained by density gradient centrifugation. These cells are subsequently used for mRNA isolation and cDNA synthesis to produce a DNA template in sufficient quality and quantity for PCR amplification of antibody encoding genes (VL and VH chain).

Libraries can be produced in different formats, namely VHH (from camelid species), Fab (antigen-binding fragment), and scFv (single-chain variable fragment). The two latter formats require a random pairing step of VH and VL chains since this information is lost during library construction. Antibody libraries can also be classified as:

• Naïve – sourced from naïve hosts never challenged by a specific antigen
• Immune – sourced from hosts after immunization with a specific antigen
• Synthetic – generated by computational (in silico) and/or experimental methods (random or site-directed mutagenesis) from available antibody sequences
• Semi-synthetic – a mixture of synthetic and natural (naïve/immune) antibody sequences

To learn more about the different types of antibody libraries, read the complete article: What is an antibody library?

Antibody libraries are very versatile tools, since they are used in display techniques, they ultimately allow us to study antibody-ligand interactions. For this reason, libraries can serve for antibody discovery for therapeutic, diagnostics, and research applications. Additionally, if mutagenesis is applied, antibody libraries can serve as an efficient approach to engineer specific properties of a given antibody, namely affinity and developability.

In general terms, naïve libraries are more advantageous than immune or synthetic libraries for antibody discovery. High-quality naïve libraries typically gather the diversity of dozens of hosts or individuals with up to 10¹¹ different clones. The high diversity guarantees board functionality and the ability to target many different antigens.

For these reasons, naïve libraries are the preferred format when generating antibodies with a “novel” function. Another advantage is that despite being a labor-intensive process, naïve libraries are only built a single time and can be recurrently used for a multitude of different projects and antigens. Immune libraries, in contrast, are reserved for more specific projects where antibody affinity may play a more preponderant role. Furthermore, these libraries serve only for a single project making them less cost-effective than naïve libraries.

Antibody libraries are stored depending on the display method they are designed to serve. For instance, libraries for antibody phage display are typically cloned in phagemid/phage vectors and amplified in the phage’s natural host organism – typically a strain of Escherichia coli.

The phage/bacteria system used in this technology presents important advantages in comparison to other display methods. For instance, E. coli is fast-growing with particularly simple requirements in terms of nutrient availability. For this reason, the amplification of phagemid/phage libraries is very straightforward, and it can be done rather quickly before its use in display campaigns.

Early attempts at antibody discovery using complex libraries were made using time and labor-intensive colony screening assays with modified (radio-labeled) antigens. Today, antibodies are more easily selected from high-diversity libraries by applying an initial enrichment step whereas the binders with the lowest affinity are eliminated before the screening step.

Over the decades, many antibody display techniques have been developed to allow efficient enrichment (biopanning) and screening of antibodies. But the most robust and cost-effective technique continues to be the phage display technology. Based on phage/Escherichia coli systems, phage display is versatile and flexible allowing its quick adaptation to multiple different antigens and assay conditions.

The phage display technology is a binary system encompassing the use of phage particles coated with proteins, antibodies, or peptides, that can be easily propagated in its corresponding bacterial host. As a general principle, phage display is an in vitro approach to study protein-ligand interactions. The protein or peptide are generally fused to one of the capsid proteins and expressed as a hybrid construct. This link between the genotype and the phenotype allows the straightforward screening of vast protein/peptide libraries by a simple process of biopanning.

In general terms, biopanning involves the incubation of several phage particles carrying different proteins or peptides with a specific target immobilized on a bead (in suspension) or plate (semi-solid phase assays). Phage libraries can be negative or positively targeted. In the first case, negative targeting can be used to remove phage-protein particles that bind to an undesired ligand, while positive targeting (the most common approach) can be used to enrich binders with high affinity towards the desired target.

The desired phages are recovered after each panning step and amplified in E. coli. The process is repeated 4-6 times allowing a stepwise enrichment of the desired binders. Afterward, individual clones are screened in the desired format (ELISA, WB, or Flow Cytometry) and characterized by DNA sequencing.

To learn more about the phage display technology, read the complete article: What is the phage display technology?

The filamentous E. coli phage M13 is the most widely used system for antibody phage display alongside the closely related E. coli phages fd and f1. A clear advantage of these phages over others is their non-lytic nature, in this way, they propagate in E. coli without lysing the cells, allowing the fast purification of phages using a simple PEG precipitation process to separate them from cellular proteins in-between biopanning rounds.

Other phages have been used for display like T7, T4, and lambda E. coli phages, however, they have yet to find efficient applicability due to their lytic nature. Since they lyse E. coli during amplification, this complicates the multiple purification steps in-between rounds of biopanning. For this reason, these systems are only sparsely used for phage display.

In M13, the proteins/peptides are linked to one of the capsid proteins – the minor coat protein pIII (or gp3) or the major coat protein pVIII (or gp8). The pIII is present at 5 copies per phage particle, while the pVIII is present 2700 copies per virion. As a result, pIII fusions are present at a lower valency and pVIII at a higher valency, for this reason, pIII is favored for the selection of binders with strong affinity and pVIII constructs can be used to recover binders with lower affinity.

However, one of the challenges of pIII system resides in the key role of this capsid protein in infectivity. M13 adheres to E. coli via pIII/F factor interactions, for this reason, it is essential to preserve its original function to allow the replication of positively selected phages. A solution to this challenge arose with the creation of phagemid systems and using them in conjugation with helper phages. Phagemids contains only essential elements:

• A gene encoding for the pIII-protein/peptide/antibody fusion protein
• An origin of replication
• A selection marker (typically an antibiotic resistance gene)

In contrast, helper phages contain all elements essential for infectivity, replication, and virion assembly but lack the protein/peptide fragment fused to the pIII and contain a weakened origin of replication. As a result, the phagemid/helper phage system produces virions with a mixed phenotype of native pIII and pIII-protein fusion constructs (1-5 copies).

Since phage display allows the study of protein-ligand interactions, its applications are quite diverse. One of the most important applications of phage display can be found in the field of antibody discovery. Allied to naïve, immune, or synthetic libraries, phage display can help researchers finding antibodies with novel functions for therapeutic, diagnostics, and research applications.

Another use of this technology lies in antibody engineering and lead optimization. Phage display has been extensively used as a tool to optimize antigen selectivity (cross-reactivity), affinity, and developability. This is recurrently achieved by building synthetic libraries obtained from computational or experimental mutagenesis (random or site-directed), enriching promising variants, and screening their activity in the desired conditions and application.

Finally, phage display has been recently gaining interest as a tool for epitope mapping. By displaying peptides or proteins against a single purified antibody, it is possible to determine its epitope-specificity – invaluable information when developing particularly complex therapies like oligoclonal antibodies (antibody cocktails) or antibody pairs for Sandwich ELISA tests.

To learn more about the applications of phage display, read the complete article: What are the applications of phage display?

In comparison to other techniques, phage display for monoclonal antibody production can be an extremely advantageous approach. Unlike hybridomas, phage display allows researchers to skip animal immunization to generate novel antibodies (naïve libraries) or forgo the need for time-consuming antibody humanization when fully human antibody libraries are used.

Moreover, the technique is Compatible with toxic and non-immunogenic molecules, making it invaluable for the development of envenoming treatments (e.g., snakebites) or for the creation of environmental/food monitoring tools that typically detect small non-immunogenic molecules.

In contrast to hybridoma, phage display technologies allow the development of fully human antibodies. That way, no further humanization is needed, allowing researchers to save efforts, time, and costs, greatly speeding up preclinical and clinical development. Nowadays, in the fast-paced global competition environment, this is a key advantage for accelerating the development of invaluable and novel biotherapies.

Antibody libraries are very flexible tools for the discovery of molecules with “novel” functions. All naïve and immune antibody libraries produced by ProteoGenix are available for purchase in the form of a Library Kit where the following components are supplied:

• Phagemid library (scFv, Fab, or VHH) with M13 pIII fusion system
• Phagemid information and guidelines for use
• Helper phage
• Host bacteria stocks
• Forward and reverse sequencing primers

Forward and reverse sequencing primers
We use PBMCs from a wide number of donors of different breeds/ethnic origins to maximize the diversity of the naïve antibody repertoire. This allowed us to achieve a diversity of over 10 billion different variants. For example, our human library LiAb-SFMAXTM was made using samples from 368 healthy donors belonging to 5 different ethnic groups (Caucasian, Arabic, Black-African, Latino & Asian) and has a diversity of 5.37×10¹⁰ variants. Our rabbit and camelid libraries were created from different breeds/species, respectively, and contain over 1×1010 different variants.

Yes, we can build custom libraries using biological samples like PBMCs or RNA either sourced by us or provided by clients. In any case, clients can choose between a wide variety of formats like scFv, Fab, or VHH, and a wide variety of species including rabbit, mouse, camel, and alpaca.

Due to the high diversity of our naïve libraries and an optimized protocol for efficient antibody phage display, we can guarantee to identification and delivery of at least 3 unique binders against your antigen in each phage campaign. Otherwise, clients pay only part of the service.

Given our extended capabilities, we offer all clients the possibility to synthesize their antigens in-house. We can manufacture peptides, proteins, small molecules, and cells to be used in phage display campaigns, greatly reducing lead times in phage display projects.

The type of antigen used in phage display depends, first and foremost, on its intended application and nature and structure of the antigen.

For instance, clinical targets are often complex membrane-bound proteins, and, for this reason, a successful phage display campaign will depend on the antibody’s ability to recognize the native protein conformation. In this case, whole cells overexpressing the desired antigen often make more suitable targets than the recombinant proteins themselves. In contrast, if the antibody is intended for analytical, diagnostic, or research applications, the sample may be subjected to a pre-treatment where the antigen is denatured prior to the analysis (e.g., WB applications). In this case, using peptides as targets in phage display campaigns may be a more suitable approach.

Deliverables are 100% customizable but typically include:

• Detailed report
• Antibody DNA/amino acid sequence
• Full ownership over the sequences

Additional deliverables are available on request and may typically include:

• Additional screening of single phage clones by ELISA, WB, or flow cytometry on whole cells (suitable to assess affinity towards membrane-bound proteins)
• Antibody cDNA cloned into vectors
• Expression of recombinant antibodies in a variety of formats
• Antibody engineering like the design of bispecific or ADC, and optimization of expression or effector functions
• Antibody characterization like KD determination, stability/aggregation analysis, ADCC/CDC evaluation