Custom anti-GPCR antibodies

Find the Perfect Method for Making your custom GPCR Antibody

ProteoGenix uses one of three different antibody engineering strategies to make custom monoclonal antibodies that bind GPCR antibodies. These include creating hybridoma cell lines, isolating anti-GPCR-specific B-cells, and screening for specific anti-GPCR antibodies using phage display. ProteoGenix can assist you in determining which method is best suited to your project and budget.

Antibody Phage Display

Antibody phage display is a method that involves obtaining peripheral blood mononuclear cells (PBMCs) from either immunized animal hosts (immune libraries) or unimmunized hosts (naïve libraries). After PBMC collection, the antibody gene segments are converted into cDNA and cloned into a phagemid to create an antibody-phage fusion protein, which displays the antibody on the outer surface of the bacteriophage.
Next, the target antigen is cloned and immobilized to a surface so the antibody on the phage surface can bind the immobilized antigen, a process called biopanning. Once the antibody binds to the antigen, the DNA inside the bacteriophage, containing the antibody-related genes, is extracted, sequenced, and expressed to confirm its efficacy. Learn more about our antibody phage display service.

Why Antibody Phage Display is Ideal for Making Human Therapeutic Antibodies

Antibody phage display is a valuable technique for producing monoclonal antibodies that target cytotoxic antigens, rare antigens (or epitopes), or antigens with low immunogenicity. In addition, antibody phage display is particularly useful for making custom antibodies used clinically because of ProteoGenix’s flexible cancer and autoimmune human phage display libraries. These new libraries offer our clients the flexibility to quickly, identify unique human antibodies that bind their GPCR of interest cost-effectively.
If your biomedical research lab or pharmaceutical company aims to expedite human immunotherapy projects aimed at treating cancer, autoimmunity, or other diseases, then antibody phage display technology is the ideal choice.

Phage Display Has No Antibody Species Limitations

Antibody phage display is a powerful technique for custom antibody generation because there is no species limitation. Whether you’re interested in making unique anti-GPCR antibodies to perform in a specific biomedical research experiment or you need to build complex immunotherapy, ProteoGenix can meet your needs. Our team can immunize rabbits, mice, and even camelids to generate custom anti-GPCR antibodies that target your favorite GPCR with precision. We can also screen your GPCR of interest using our pre-built naive human autoimmune and cancer libraries to quickly identify antibodies that bind rare GPCR epitopes. Our comprehensive approach ensures that you receive high-quality, fully customized antibodies for your specific research needs.

Phage Display Delivers Antibody DNA Sequences, That You Own

Once we discover at least three different antibodies that bind your GPCR with precision, we will deliver the antibody DNA (cDNA) sequences to you. These sequences become your intellectual property (IP) giving your institution or company the following benefits:

• The right to patent the antibody.
• Access to potential revenue streams from licensing agreements, royalties, or other financial arrangements.
• Greater control over the antibody’s development, production, and distribution.
• Protecting the antibody from being copied or exploited by competitors and provide legal recourse in case of infringement.
• Enhancing your scientific reputation and credibility as a leader in the field. This can help to attract funding, partnerships, and help strengthen patent applications related to the custom antibody.

ProteoGenix Can Therapeutically Modify Your Phage Display Antibodies

We can adapt your monoclonal phage display antibody into a bispecific antibody or conjugate it to cytotoxic drugs (ADC antibody) to target diseased tissue. We can also adapt your custom GPCR antibody for diagnostic applications such as ELISA, flow cytometry, or clinical imaging.

Hybridoma Cell Line Generation

Generating your custom anti-GPCR antibody by making a hybridoma cell line is a great way to make anti-GPCR antibodies that bind with high affinity. The first step in producing a hybridoma cell line involves immunizing mice or rats with the GPCR antigen. Next, we collect splenocytes from the immunized mice or rats and fuse the appropriate B-cells with a myeloma cell line.
We select the hybrid cells by screening the supernatant for antibodies that bind the target antigen by ELISA. Lastly, we subject the positive binders to limited dilutions to isolate individual cells and expanded them into colonies. We then screen each colony and further verify their potential to bind antigens by ELISA. The entire process takes 10 weeks from start to finish. Read more about ProteoGenix’s hybridoma technology.

B-cell Screening

Our B-cell screening process follows a rigorous three-step procedure that ensures the production of monoclonal antibodies with exceptional affinity. To begin, we immunize rodents with purified antigens, which we carefully assess using SDS-PAGE. This process lasts for 6 to 8 weeks and involves our optimized immunization protocol, which consists of 4 to 6 rounds of injections.
The next step involves a 2–3-week process of sorting and screening B-cells, where lymphocytes are isolated from the PBMC compartment and spleen of immunized rodents. These B-cells are then cultured in vitro, and the supernatants are analyzed via ELISA to identify the presence of antibodies that bind to the target antigen. The top-performing antibodies are then sequenced, cloned, and expressed in XtenCHO cells, followed by further ELISA screening to confirm the production of high-quality monoclonal antibodies. Learn more about ProteoGenix’s B-cell screening and isolation methods.

G Protein Coupled Receptors

G protein-coupled receptors (GPCRs) are a large family of cell surface receptors representing roughly four percent of the protein-coding genome. They are known as 7-(pass)-transmembrane domain receptors, as they span the cell membrane seven times, with their N-terminus extending into the extracellular space and the C-terminus extending into the cytoplasm. GPCRs are involved in a wide range of physiological processes, including vision, smell, taste, hormone regulation, and neurotransmission. Drugs affecting these receptors constitute the single largest drug class currently on the market. In fact, over 30% of all FDA-approved drugs target GPCRs.

G Protein Coupled Receptors in Health and Disease

GPCRs play a vital role in mediating the response of cells and tissues to extracellular signals, such as hormones, neurotransmitters, and sensory stimuli. Upon activation by a ligand, GPCRs undergo a conformational change that activates a downstream signaling pathway allowing the membrane-associated palmitoylated G alpha subunit to exchange GDP for GTP. The GTP-bound G alpha subunit dissociates from the receptor promoting the activation of a variety of downstream effectors, including adenylyl cyclase, phosphodiesterases, phospholipase C, and ion channels. These effectors, influence the intracellular concentrations of secondary messengers such as cAMP, diacylglycerol, sodium, or calcium cations, leading to a physiological response.
Abnormalities in GPCR signaling have been implicated in a wide range of diseases, including cancer, neurological disorders, cardiovascular disease, and metabolic disorders. For example, mutations in GPCRs can lead to constitutive receptor activation and downstream signaling, which can promote tumor growth and metastasis. Additionally, dysregulation of GPCR-mediated signaling has been linked to the development of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease.
As a result of their critical role in human health and disease, GPCRs are an important area of research for drug development. Scientists are investigating new ways to modulate GPCR signaling, including the development of custom GPCR antibodies that block receptor activity. While this novel approach could lead to more targeted and effective therapies for a wide range of diseases there are many challenges to making custom GPCR antibodies.

The Challenges of Making Custom GPCR Antibodies

Engineering custom G protein-coupled receptor (GPCR) antibodies are challenging for several reasons. First, the majority of a GPCR’s amino acid sequence is embedded in a lipid membrane, resulting in the accessibility of a small number of extracellular epitopes to antibodies. Furthermore, GPCRs share similar sequences and structural domains, making them difficult for antibodies to distinguish specific GPCRs from closely related GPCRs.
Secondly, GPCRs are difficult to express and purify because they are lipophilic. Lipophilic antigens are difficult to use in the immunization process because they tend to form aggregates and become improperly folded preventing the formation of an adaptive immune response against conformational epitopes. Therefore, recombinant expression and isolation of GPCRs to produce natively folded antigens must occur using membrane preparations such as micelles or nanodiscs. The alternative antigen preparation method is to overexpress GPCRs using a cell line and immunize the animal hosts using the entire cell instead of purified GPCRs. These approaches ensure the host’s immune system is stimulated with a properly folded GPCR.
Lastly, making custom antibodies against GPCRs is difficult due to the low immunogenicity profile of GPCRs. To overcome this challenge, ProteoGenix uses various techniques, such as increasing their immunogenicity by coupling GPCR peptides to an immunogenic carrier protein like ovalbumin. This strategy allows us to overcome the limitations of generating a high-quality anti-GPCR antibody using single B cell screening and hybridoma generation resulting in the production of anti-GPCR antibodies that perform in a range of experimental applications.

How GPCRs are Expressed to Make Custom anti-GPCR Antibodies

To overcome the challenges of GPCR solubility, several methods have been developed to express and stabilize GPCR antigens, including expressing GPCRs in micelles, nanodiscs, or cell lines that express a GPCR of interest. These approaches can enhance the solubility and stability of the GPCR antigen, making it more immunogenic and increasing the likelihood of generating high-quality antibodies.
In this context, immunizing animals using the GPCR-loaded nanodiscs, micelles, or GPCR overexpressing cell lines, can stimulate the immune response and improve the specificity of the generated antibodies. In this way, these methods offer promising strategies for producing custom anti-GPCR monoclonal antibodies with high specificity and affinity.

Micelles

Micelles are spherical structures composed of lipid molecules, such as lipids or surfactants, that self-assemble in an aqueous solution due to their amphiphilic properties. Micelles have a hydrophobic core and a hydrophilic outer layer, allowing them to solubilize and stabilize hydrophobic molecules, such as integral membrane proteins.
Micelles have been utilized as a tool for producing antibodies that target integral membrane proteins, such as G protein-coupled receptors (GPCRs). In this approach, the GPCR is solubilized in a micelle composed of a detergent, which can stabilize the protein. The solubilized GPCR-micelle complex is then used as an immunogen to generate antibodies that recognize the GPCR.
The use of micelles as immunogens has several advantages. First, micelles can help stabilize the GPCR in its native conformation, increasing the likelihood of generating antibodies that recognize functional epitopes. Micelles can also increase the solubility of the GPCR to prevent protein aggregation and degradation.

Nanodiscs

Nanodiscs are self-assembling lipid bilayer nanodiscs that have become a popular tool for studying membrane proteins, including integral membrane proteins such as G protein-coupled receptors (GPCRs).
Nanodiscs consist of a lipid bilayer surrounded by two copies of a membrane scaffold protein (MSP) that stabilizes the lipid bilayer and allows for the incorporation of the target membrane protein into the nanodisc. Specifically, the MSP forms a belt-like structure around the lipid bilayer, holding it together and allowing for the formation of a stable, water-soluble complex that contains the embedded GPCR. The MSP itself is a soluble protein that is engineered to bind to the lipids in a specific way, which allows for the formation of the nanodisc.
Nanodiscs have several advantages over other methods of stabilizing and solubilizing membrane proteins. First, nanodiscs can incorporate the target protein in an environment that mimics a cell membrane, allowing for the presentation of functional epitopes that are more likely to elicit specific antibodies. Second, the use of MSPs can improve the stability of the GPCR, reducing the likelihood of protein aggregation and degradation.
The final step in the process involves the purification of the nanodisc-bound protein complex, typically using chromatography techniques such as size-exclusion chromatography or affinity chromatography. Once purified, the nanodisc-bound protein complex can be used for a variety of downstream applications, including antibody production.

Whole Cell Immunization

Immunization using GPCR-overexpressing cells is a technique used to generate antibodies against integral membrane proteins, such as G protein-coupled receptors (GPCRs). In this approach, animal models are immunized with whole cells that overexpress the GPCR of interest on their surface. This approach provides a more native-like presentation of the GPCR in its natural membrane environment, and as a result, it can generate antibodies that recognize conformational epitopes that are only present in the properly folded and assembled receptor.
To prepare the GPCR-overexpressing cells for immunization, the target GPCR gene is cloned into a vector, which is then transfected into a suitable host cell line, such as HEK293 or CHO cells. The overexpression of the GPCR on the cell surface is confirmed by various techniques such as immunofluorescence, flow cytometry, or Western blotting. After overexpression has been confirmed the cells are used as immunogens to generate antibodies.
Animal models are immunized with the overexpressing cells subcutaneously or intraperitoneally, usually combined with an adjuvant to enhance the immune response. After several rounds of immunization, the animal’s immune response is tested for the presence of specific antibodies against the target GPCR. The most promising animals are then selected for antibody production.
This approach has been used successfully to generate antibodies against a wide range of GPCRs, including those that are difficult to produce using other methods. In addition, antibodies generated using GPCR-overexpressing cells can recognize a wider range of epitopes, including those that are post-translationally modified or present on the extracellular domain of the receptor, which are important targets for drug development. Overall, immunizing using GPCR overexpressing cells is a powerful technique for generating antibodies against integral membrane proteins.