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Lipoparticles Enable Discovery and Characterization of Antibodies Against Challenging Membrane Protein Targets

Scientist on October 26, 2022

Tech Snapshot® captures today’s cutting-edge tools and technologies that will help drive drug discovery tomorrow. This installment was written by Integral Molecular, a research-driven biotechnology company creating innovative technologies and therapeutic antibodies for under-exploited membrane protein targets, including GPCRs, ion channels, transporters, and viral envelopes.

Integral membrane proteins such as G protein-coupled receptors (GPCRs) and ion channels represent key molecules involved in cell signaling, cell homeostasis and human disease, with one-third of all approved drugs targeting such proteins. Despite the clinical relevance of these targets, monoclonal antibodies (MAbs) against these complex membrane proteins have been challenging to generate and characterize due to the structural dependence on an intact lipid bilayer.

Lipoparticles represent a key technology for immunization, antibody discovery and antibody characterization for complex membrane proteins (Banik et al., 2011).1 They are virus-like particles (VLPs) produced from mammalian cells by co-expressing the retroviral structural core protein Gag, along with a user-specified membrane protein. The target membrane proteins retain their native structure and orientation because they are derived directly from the cell surface without mechanical disruption or detergents and are expressed at high concentrations of 50-200 pmol/mg.

Characteristics of Lipoparticles

The high concentration of membrane proteins on Lipoparticles allows for a robust immune response, and native-conformation antigen enables the generation of antibodies that recognize biologically relevant structures. Native-conformation antigen also allows for screening and isolation of antibodies using techniques such as phage panning and B-cell screening. Lipoparticles can be biotinylated or fluorescently-labeled, allowing their use in diverse applications.

A wide range of membrane proteins can be displayed on Lipoparticles including GPCRs, ion channels, transporters, single-pass membrane proteins and viral glycoproteins. Lipoparticles have been used in over 2,000 research projects to date and have enabled R&D at numerous biotech and pharmaceutical companies.

Ad Alta Discovers Therapeutic Candidate for Phase 1 Study Using CXCR4-expressing Lipoparticles

Phage Panning with CXCR4 Lipoparticles

Like all GPCRs, CXCR4 has a complex membrane-spanning structure that makes in vitro panning and binding assays challenging for antibody discovery. CXCR4 plays critical roles in many therapeutic areas, including cancer, fibrosis and HIV, making it an important target of potential therapeutics. Ad Alta used Lipoparticles for high-throughput phage panning and isolation of CXCR4-targeting i-bodies that are based on shark antibodies. This strategy enabled isolation of antibodies that showed functional activity including successful inhibition of inflammatory cell recruitment in an in vivo mouse model and inhibition of cell infectivity with HIV in an in vitro assay. Epitope maps of several i-body candidates were generated, revealing critical residues bound by the i-bodies, showing that three of these molecules bound in the major sub-pocket of CXCR4 (Griffiths et al., 2016).2 CXCR4 Lipoparticles were also used in surface plasmon resonance (SPR) binding assays that assessed i-body binding kinetics and inhibitory activity in comparison with CXCL12, its native ligand.

Ad Alta’s lead candidate was re-engineered to develop a therapeutic (AD-214) that entered a Phase 1 study (NCT04415671) for Idiopathic Pulmonary Fibrosis (IPF) and demonstrated excellent tolerability. This MAb drug candidate is planned to progress to phase 2 trials in IPF patients with an inhaled delivery formulation.

Lipoparticles Enable First-ever Clinical Trial of a MAb Targeting CX3CR1

The chemokine receptor CX3CR1 is a GPCR that has been associated with atherosclerosis. It mediates cell survival and migration in areas where plaque has been developed. Fractalkine (CX3CL1), the unique ligand of this membrane protein, is a chemotactic agent that is released into circulation and can be used as a systemic biomarker for CX3CR1 activity.

Boehringer Ingelheim (BI) and Ablynx proposed using a VHH antibody to modulate CX3CR1 because the unique small size and long CDR regions of these molecules could promote engagement with the binding pockets of GPCRs. The researchers immunized llamas and then tried both conventional panning on cells and panning using Lipoparticles. Typical cell panning did not produce any antibodies with the desired properties. With Lipoparticle panning, the researchers were able to identify a diverse set of VHH antibodies. One family showed all the desired functionalities with strong inhibition and binding to both human and cynomolgus CX3CR1 (Lowe et al., 2020).3 This work led to their clinical candidate BI 655088, which entered Phase 1 clinical development for the treatment of chronic kidney disease (NCT02696616), representing the first clinical trial of a MAb targeting CX3CR1.

Panning Using CX3CR1 Lipoparticles Led to Discovery of MAbs with Desired Characteristics

argenx Uses Lipoparticles to Isolate Functional Antibodies Against the Glucagon Receptor

The glucagon receptor is a therapeutic target for diabetes and is involved in the regulation of blood glucose levels in the body. In addition to being a challenging target as a 7TM GPCR, the glucagon receptor also has a large N-terminal extracellular domain (ECD) that covers the transmembrane extracellular loops (ECLs) and many potential binding sites.

Antibodies Targeting the ECD and ECL Were Isolated Using Lipoparticle Panning

To isolate antibodies against the glucagon receptor, argenx used both Lipoparticles and soluble ECD for phage panning against the glucagon receptor. Using soluble ECD, five VH antibody families were found that targeted the extracellular domain. When panning on Lipoparticles, the researchers isolated a diverse group of 25 VH antibody families, 23 of which targeted ECDs and two of which targeted ECLs.

This was the first report of antibodies isolated against the ECLs of a class B GPCR. Panning on Lipoparticles yielded a greater number and greater diversity of MAbs, several of which also had antagonist activity (van der Woning, 2014, Discovery on Target; van der Woning et al., 2016).4

GPCRs are extremely challenging targets for antibody discovery. Lipoparticles provide high-quality, native-conformation antigen at a high concentration that can be used to discover MAbs against these complex membrane proteins. The combination of immunization and panning with Lipoparticles has led to success for many researchers in finding lead MAb candidates for therapeutics.