Overcoming Challenges in
Manufacturing of Transmembrane
Proteins to Accelerate Development
of Immunotherapeutics

What are transmembrane proteins and why are they important to research?

Transmembrane proteins are these large macromolecules embedded into the cell surface. Think of the cell surface as like Earth’s surface, but on a microscopic scale. The random assortment of carbohydrates, lipids, and other proteins stain the surface as mountains, trenches, and trees do. In this metaphor, transmembrane proteins are like volcanoes, connecting the deep molten core with our atmosphere. However, on the cellular level, transmembrane proteins will act as an important channel connecting the intracellular and extracellular environments. These proteins enable the transportation of various ions and molecules in and out of the cell, as well as relaying activation or responding to extracellular stimuli. Many of these external stimuli will subsequently propagate intracellularly through internal signaling pathways and result in the regulation of cell metabolism, cellular activity, and cellular fate.

How does the role of transmembrane proteins in the signaling pathway translate to its potential as an immunotherapeutic target?

In general, transmembrane proteins are the cell’ s gatekeepers, preferentially letting in signals while keeping out the rest. Diseases are generally associated with abnormally functioning transmembrane proteins, resulting in discordant cellular signaling, and subsequently, cell function. For example, the Claudin transmembrane protein family is a critical part as a signaling platform to coordinate cellular behaviors as well as forming tight junctions between cells.

In cancers, Claudins are frequently upregulated and overexpressed leading to abnormal activation of downstream signaling. In some other contexts, abnormal function of Claudins will disrupt the tight-junction function, leading to cancer stem cell breaching, metastasis, migration, and tumor invasion. As such, drug therapies inhibiting transmembrane proteins such as the Claudin family have an immense potential in contributing to the fight against cancer.

With transmembrane proteins such as Claudin showing promise as an immunotherapeutic target, what is limiting transmembrane protein research?

A major limiting factor in transmembrane protein research is the development of a viable antigen for research use. Unlike the manufacturing of conventional intra-or extra-cellular proteins, the manufacturing of transmembrane proteins for research use is an incredibly complex and difficult process. This is a significant bottleneck to the transition of an identified immunotherapeutic target to the development of its corresponding drug therapy. Three main difficulties exist: low expression levels, low abundances, and aggregation. Low expression levels are a direct result of the size of transmembrane proteins. As large, surface-bound macromolecules, transmembrane proteins are extremely limited by the available surface area on a cell. Secondly, as a signaling gatekeeper, the overexpression of these proteins is likely to cause cytotoxicity.

Finally, transmembrane proteins are generally hydrophobic, therefore are prone to aggregate and have conformational changes in less-than-optimal environments. Altogether, scaling-up and producing high purity, high quality, and viable transmembrane proteins for use is a significant challenge.

What is FLAG and how does it overcome the challenges in current transmembrane protein manufacturing?

FLAG, which stands for Full-length Active Gallery, is our technology platform developed to directly overcome the challenges in manufacturing. We re-designed our entire development system and optimized the expression interval, expression system, and culture conditions to maximize the amount of full-length, multi-pass transmembrane proteins. We also established a stringent quality control system including monitoring of structural homogeneity through dynamic light scattering evaluations. The manufacturing of transmembrane proteins is also only half of the solution. We also developed three platforms to stabilize the transmembrane proteins for delivery to our customers, including virus-like particles, detergent micelles, and Nanodiscs. Each of them has their own advantages for different applications such as for quantitation, drug screening, and affinity verification.

The term ‘Full-length’ seems to be a big emphasis point for FLAG. Could you please explain why this is an important factor in drug development?

Due to the limitations in the development and expression of complete transmembrane proteins, drug research is generally performed using shortened or a portion of the transmembrane protein, mostly the target area for interaction.

Only a portion of the transmembrane protein is reproduced and used with the extracellular domain (ECD) being the primary component. However, this provides an incomplete picture of the drug to transmembrane protein interaction. One notable example is rituximab (RTX) and CD20. Since CD20 has two ECDs, CD20-targeting therapeutics such as RTX can potentially bind to both extracellular loops. Without expression of both ECDs, comprehensive evaluation of complement-dependent cytotoxicity and mechanism of action analysis is impossible, therefore emphasizing the use of full-length transmembrane proteins as antigens. Our FLAG technology enables us to provide our customers with native, ‘full-length’ transmembrane proteins for use in research and drug therapy evaluations.

Beyond research and development, how does ACROBiosystems contribute to immunotherapy manufacturing?

Drug development is a long and exploratory process, where there are many uncertain factors. Since we produce key reagents for drugs and therapies that are revolutionary towards healthcare, quality is one of our priorities when it comes to all of our products.In accordance to this mission, we have established strict quality management systems that are certified and audited under both ISO 9001:2015 and ISO13485:2016 guidelines. Through this approach, we are able to systematically standardize our entire production process for our products, including recombinant proteins, antibodies, enzymes, cell culture cytokines, assay kits, and many other products.