Phage Display Custom Services

Phage display is a biotechnological technique that uses bacteriophages to display proteins on their surface while linking them to their genetic information. By inserting a gene for a desired protein into a phage coat protein gene, the phage can present the protein externally and carry the gene internally. This method is crucial for studying interactions such as protein-protein, protein-peptide, and protein-DNA, using in vitro selection similar to natural selection. Crucially, phage display is widely used to select antibodies against specific antigens, a key process in protein-protein interaction studies. This is especially important for developing therapeutic antibodies, as it allows for the discovery of antibodies that are highly specific and have a strong affinity for their target antigens, streamlining the path to innovative treatments.

Phage display offers several key advantages:

• High Throughput: Ability to screen billions of variants simultaneously.
• Versatility: Applicable to a wide range of targets, including challenging ones like toxic antigens or even membrane proteins.
• Specificity and Affinity: Facilitates the identification of molecules with high specificity and affinity for targets.
• Functional Linkage: Directly links phenotype (protein function) with genotype (DNA sequence), enabling efficient identification and optimization of binders.

Phage display is widely utilized in:

• Antibody Discovery and Development: For identifying antibodies with high specificity and affinity, including therapeutic antibodies.
• Protein Interaction Studies: To explore protein-protein, protein-peptide, and protein-DNA interactions.
• Drug Discovery: In finding new ligands, enzyme inhibitors, receptor agonists, and antagonists.
• Vaccine Development: For identifying antigens and developing vaccines.
• Cancer Research: In determining tumor antigens for diagnostic and therapeutic targeting.

The benefits of using Phage Display for therapeutic antibody development are significant and multifaceted. Unlike traditional hybridoma techniques, phage display technology facilitates the creation of fully human antibodies directly. This direct approach eliminates the need for subsequent humanization processes, offering a streamlined path from discovery to development. Here are the key advantages:

• Fully Human Antibodies: Phage display generates antibodies that are fully human from the outset, reducing the risk of immunogenicity when administered to patients. This is a crucial factor in therapeutic antibody development, ensuring better safety and efficacy profiles.
• Efficiency and Speed: By bypassing the need for humanization, phage display significantly accelerates the timeline from antibody discovery to clinical application. In today’s competitive landscape, this speed can be a decisive factor in the success of developing new biotherapeutics.
• Cost Reduction: The streamlined process not only saves time but also reduces the costs associated with complex humanization procedures and the subsequent validation of humanized antibodies. This cost-effectiveness makes phage display an attractive option for therapeutic antibody development.
• Versatility and Diversity: Phage display libraries can be constructed to represent a vast diversity of antibody sequences, increasing the likelihood of identifying high-affinity and highly specific antibodies against a wide range of targets, including challenging ones like membrane proteins.
• Facilitates Complex Target Engagement: The technology allows for the selection of antibodies that can engage with targets in their native conformation, crucial for the development of therapeutics aimed at complex or conformationally sensitive targets.

A phage library is a comprehensive collection of the antibody repertoire from a specific host species, crucial for antibody discovery and research. These libraries are typically derived from B lymphocytes (B cells) or peripheral blood mononuclear cells (PBMCs), which are isolated using density gradient centrifugation. The extracted cells undergo mRNA isolation and cDNA synthesis, providing a high-quality DNA template for PCR amplification of antibody-encoding genes, including the variable light (VL) and variable heavy (VH) chains.

Antibody libraries can be formatted in various ways, such as VHH (from camelid species), Fab (antigen-binding fragment), and scFv (single-chain variable fragment). The Fab and scFv formats necessitate a random pairing of VH and VL chains, a step required due to the loss of this information during the library’s construction.

Antibody libraries are categorized into several types based on their source and method of generation:

• Naïve Libraries: Created from hosts that have not been exposed to the antigen of interest, providing a broad and unbiased antibody repertoire.
• Immune Libraries: Derived from hosts immunized with a specific antigen, containing antibodies with higher specificity and affinity towards that antigen.
• Synthetic Libraries: Constructed using computational and experimental techniques, such as random or site-directed mutagenesis, based on known antibody sequences.
• Semi-synthetic Libraries: A hybrid approach that combines synthetic sequences with sequences from naïve or immune sources, enhancing the diversity and potential of the library.

Antibody libraries are exceptionally versatile tools that, through display techniques, enable the detailed study of antibody-ligand interactions. Consequently, these libraries are instrumental in discovering antibodies for therapeutic, diagnostic, and research purposes. Furthermore, when mutagenesis is employed, antibody libraries become a powerful method for engineering specific antibody characteristics, such as enhanced affinity and improved developability.

Naïve antibody libraries are a valuable resource for antibody discovery, offering a balance alongside immune or synthetic libraries. Their key advantage is the diversity they offer, derived from the antibody repertoires of multiple donors or animals, potentially including up to 10^11 different clones. This diversity facilitates the identification of antibodies with a wide range of functionalities, capable of recognizing a broad spectrum of antigens. However, it’s important to note that immune libraries also play a crucial role, especially when targeting specific antigens that require a more focused approach.

The preference for naïve libraries in generating antibodies with novel functions stems from their ability to be used across multiple projects and against various antigens, thanks to their comprehensive coverage of potential antibody sequences. While the construction of naïve libraries is a labor-intensive process, their one-time creation offers long-term utility, making them a cost-effective choice for diverse antibody discovery endeavors.

In contrast, immune libraries are tailored for specific projects where high antibody affinity is crucial. Although they provide antibodies with enhanced specificity and affinity due to the immunization process, their use is generally limited to the project they were created for, which can make them less cost-efficient compared to the reusable nature of naïve libraries.

Antibody libraries are cloned using a meticulous process that begins with the collection of genetic material from a source, such as B lymphocytes from immunized or non-immunized hosts for immune and naïve libraries, respectively. The process involves several key steps:

• Isolation of mRNA: The first step involves isolating mRNA from the cells of interest. This mRNA contains the genetic information for the antibodies produced by those cells.
• cDNA Synthesis: The isolated mRNA is then reverse-transcribed into complementary DNA (cDNA). This cDNA serves as a template for amplifying the antibody genes.
• PCR Amplification: Using polymerase chain reaction (PCR), the variable regions of the antibody genes (both heavy and light chains) are amplified. This step is crucial for generating a diverse library.
• Library Construction: The amplified genes are then inserted into a vector – a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. For phage display libraries, these vectors are phagemids or phages that can display the antibody fragments on their surface.
• Transformation into Host Cells: The vectors containing the antibody genes are introduced into host cells, typically E. coli, which are then infected with helper phages if phagemids are used. This results in the display of the antibody fragments on the surface of phage particles.
• Screening and Selection (Biopanning): The library of phage-displayed antibodies is then screened against the target antigen to identify and select clones that bind with high affinity. This process, known as biopanning, involves several rounds of selection to enrich the library for high-affinity binders.

In the M13 phage, proteins or peptides are attached to one of its capsid proteins, either the minor coat protein pIII (gp3) or the major coat protein pVIII (gp8). With only 5 copies of pIII per phage but 2700 copies of pVIII, pIII-linked fusions are displayed at a lower density, making them ideal for isolating high-affinity binders. In contrast, pVIII-linked constructs, due to their higher display density, are more suitable for identifying binders of lower affinity.
The pIII system, however, faces a challenge due to its crucial role in the phage’s infectivity. M13 binds to E. coli through interactions involving pIII and the F factor, necessitating the preservation of pIII’s function to ensure the replication of selected phages. This issue is addressed by employing phagemid systems in combination with helper phages. Phagemids are simplified vectors that carry:

• A gene for the fusion protein linked to pIII
• A replication origin
• A selection marker, usually for antibiotic resistance

Meanwhile, helper phages provide all necessary components for phage infectivity and assembly but exclude the fusion protein linked to pIII and have a modified, less efficient replication origin. This arrangement allows for the production of virions that display both native pIII and the pIII-linked fusion proteins, typically in 1 to 5 copies, offering a balanced approach to phage display.

Our antibody libraries, instrumental in identifying molecules with unique functionalities, are indeed available for acquisition. ProteoGenix offers Naïve and Immune Antibody Library Kits, which include:

• A Phagemid library (scFv, Fab, or VHH) utilizing the M13 pIII fusion system
• Comprehensive phagemid documentation and usage instructions
• Helper phage
• Stocks of host bacteria
• Sequencing primers (forward and reverse)

Absolutely, we specialize in creating tailor-made libraries from biological samples such as PBMCs or RNA, which can either be procured by us or supplied by the client. Clients have the flexibility to select from an array of formats including scFv, Fab, or VHH, and from a diverse range of species like rabbit, mouse, camel, and alpaca.

Leveraging our comprehensive capabilities, we provide every client with the option to internally synthesize antigens. Our facility is equipped to produce a wide range of antigens including peptides, proteins, small molecules, and even cells, specifically for use in phage display endeavors. This approach significantly shortens the timeline for phage display projects.

The choice of antigen for phage display is crucial and varies based on the specific application and the antigen’s characteristics.
For example, targets for clinical applications are frequently complex, membrane-bound proteins. Achieving success in phage display in these scenarios often hinges on the antibody’s capacity to recognize the antigen in its native conformation. Therefore, whole cells that overexpress the target antigen are typically more effective than recombinant proteins for these purposes. Conversely, for antibodies aimed at analytical, diagnostic, or research uses, where antigens might be denatured for analysis (such as in Western Blotting), employing peptides as targets in phage display might be the preferable strategy.

• A comprehensive report
• The antibody’s DNA and amino acid sequences
• Complete rights to the sequences

Upon request, we can provide additional services, including:

• Further screening of individual phage clones through ELISA, Western Blot, or flow cytometry on whole cells (ideal for evaluating affinity to membrane-bound proteins)
• Cloning of antibody cDNA into vectors
• Production of recombinant antibodies in various formats
• Advanced antibody engineering, such as creating bispecific antibodies or antibody-drug conjugates (ADCs), and enhancing expression or effector functions
• In-depth antibody characterization, including affinity (KD) measurement, stability/aggregation testing, and ADCC/CDC assessment

With our naïve libraries’ vast diversity and a refined protocol for effective antibody phage display, we assure the identification and delivery of a minimum of three distinct binders targeting your antigen in every phage display campaign. Should this not be achieved, clients are only responsible for a portion of the service fee.