Developing Antibodies Against Membrane Protein Targets

Membrane proteins, such as G protein-coupled receptors (GPCRs), ion channels, transporter, and membrane-bound enzymes, are involved in many biologic functions such as ion transport and cell signaling. Alterations to these proteins have been associated with diseases such as cystic fibrosis, cancer, and neurological disorders. “The primary cause of these diseases are alterations in the signaling pathways that control cell function or metabolism,” says Sreethu Sankar, Product Manager at Proteintech. “Due to this reason, membrane proteins are preferred targets for drug development.” Ross Chambers, VP of Antibody Discovery at Integral Molecular, agrees. “They have naturally become very important targets for clinical medicine,” he adds.

Anne Sloan is a Product Scientist at Cell Sciences, which produces biosimilar antibodies targeting membrane proteins for research use. She says that there’s a “huge potential market for membrane protein targets” as they comprise about 30% of the sequenced human proteome. Around 60% of all approved drugs target membrane proteins, but the majority of these are small molecule drugs.

While there are numerous small molecule drugs, antibody drugs are a new modality that has gained more interest in the last decades. Therapeutic antibodies have benefits including low toxicity and long residence times in the body, explains Sloan. However, generating antibodies against membrane proteins can come with many challenges that exist both on the antigen side and the antibody side. This is particularly true for multipass membrane proteins.

Single-pass vs. multipass membrane proteins

Of the 6,000 membrane proteins in the human proteome, approximately half of them are single-pass membrane proteins. These proteins only pass through the membrane once and include a large extracellular domain. “It’s very easy to make antibodies against single-pass membrane proteins,” says Chambers. “The challenging area is what we call the multipass membrane proteins, the ones that thread through the membrane many times.” These proteins require the lipid bilayer to fold properly and maintain the proper conformation. “Immunization using soluble protein domains, proteoliposomes, or intact cells, does not always result in antibodies that can effectively bind to the proteins in their native state,” explains Sankar.

Challenges in purifying membrane proteins

In order to generate antibodies, you need purified antigens to stimulate antibody production. “A big problem is expression,” says Chambers. “Cells generally don’t need many copies of a membrane protein and they’re not evolved to support high levels.” Sloan cites other problems: “The small amounts of these proteins are hydrophobic, with low solubility that make these proteins difficult to purify.”

Moreover, it’s best to purify proteins in their native states. “Because membrane proteins undergo conformational changes during binding with ligands, effective antibodies should target the correct native conformational state,” says Sloan. To capture membrane proteins in their native state, you’d need to purify them with the membrane even though a small portion of the protein is extracellular. This is particularly true for multipass membrane proteins that require the lipid membrane to stabilize their conformation.

“It is becoming common to co-express lipid binding partners that can stabilize membrane protein structure,” says Sankar. This strategy would involve identifying the lipids that interact with the membrane protein and expressing them alongside the protein.

Integral Molecular uses lipoparticles to overcome the above challenges. “Lipoparticles were actually the technology that launched our company,” says Chambers. These particles are virus-like particles that concentrate proteins onto a lipid membrane to both increase their abundance and maintain the proper structure.

Overcoming immune tolerance

Once you have adequate protein, the challenge becomes generating an immune response against them. Mouse models are often used to study human processes because of their similarities to humans in genetics and biological processes. However, this can be a problem for antibody generation. “Highly conserved structure and sequences equates to low immunogenicity,” explains Sloan. Thus, it can be difficult to generate antibodies against the membrane protein of interest.

One way to overcome low immunogenicity is to use “knock-out mice deficient in the target protein,” says Sloan. At Integral Molecular, Chambers says that they immunize chickens to overcome the low immunogenicity in mice. “The reason we use chickens is because they’re so evolutionarily distinct from humans,” he explains. “This enables a strong immune response that allows us to isolate high-affinity monoclonal antibodies.” But there’s still a long way for the industry to go to turn away from immunizing mice.

Another way to generate antibodies is to use synthetic phage display libraries. “However, it really hasn’t been successful for multipass membranes, so the industry is reliant on animals for making antibodies to these proteins,” says Chambers.

At Proteintech, Sankar says they use peptide immunogens for modified proteins. “These peptide immunogens are typically conjugated to larger carrier proteins to enhance immunogenicity,” he adds.

Advances in antibody development against membrane proteins

In recent years, the industry has developed new ways to prepare antigens for antibody generation. Aside from some of the approaches mentioned above, it’s also possible to use DNA or RNA immunization where the host animal produces the membrane protein in their native conformation. “A major innovation for being able to make antibodies to multipass membrane proteins is nucleic acid immunization,” says Chambers. “We use a combination of RNA and lipoparticles and this approach has proven successful for nearly every target that we’ve tried.” Integral Molecular recently launched an antibody reagent company, Cell Surface Bio, based on this approach.

Sankar also sees the potential of AI and machine learning. “These approaches are becoming mainstream to predict epitopes on membrane proteins,” he says. “This approach speeds up development by directing antibody generation toward more promising candidates, minimizing the trial-and-error usually required for targeting membrane proteins.”

With the development of new tools to both purify membrane proteins and generate antibodies against them, it’s likely there will be an increase in membrane protein-targeted drugs in the form of antibodies.