Technology platforms for multi-pass transmembrane protein development

Transmembrane proteins (TPs) are proteins that traverse both the intracellular and extracellular environments and are embedded in the cell membrane. The transmembrane area, which interacts directly with the phospholipid bilayer, is a critical link between two environments, allowing for the transit of ions and molecules as well as conveying activation and response reactions to external stimuli.

These processes govern cell metabolism, cellular activity and cellular destiny by propagating intracellularly through downstream signal pathways. TPs could be characterized structurally as beta-barrels or alpha-helical, and topologically as single-pass or multi-pass molecules, depending on how the N and C termini are oriented.

The aberrant actions of these TPs have been linked to a range of human illnesses, and their position in a variety of signaling pathways makes them excellent therapeutic targets.

A full-length and physiologically relevant version of these proteins must be identified to create efficient therapeutics targeting these TPs.

Multi-pass TPs are notably hard to isolate due to their complicated architectures with numerous hydrophobic transmembrane sections and low expression levels in host cells, yet they have emerged as a popular disease target.

Rituximab detects CD20 not only at its primary epitope in extracellular loop 2 (ECL2) but also at its secondary epitope in extracellular loop 1 (ECL1). Similarly, Rituximab’s CDC activity is affected by this secondary epitope. To properly evaluate antibody activity and mode of action, full-length TPs with native folding are required.

Multiple technological platforms have been specifically developed by ACROBiosystems to solve the structural complexity of multi-pass TPs and to fulfill the demands of various applications.

To enhance drug development and mechanism research, ACRO has created a large variety of full-length multi-pass TPs with stable structure and high activity, such as CD20, Claudin18.2, GPRC5D, CD133, CCR5 and CCR8.

Figure 1. Multi-pass transmembrane target proteins. Image Credit: ACROBiosystems

Technology platforms

VLP platform

Principle

ACROBiosystems' VLP (Virus Like Particles) technological platform depends on the HEK293 expression system to express TPs on the host cell surface. The viral envelop or capsid protein subsequently converts these cell surfaces into soluble lipid bilayer particles containing highly concentrated proteins, which may be used for antibody immunization and screening.

The membrane protein-VLP complex induces and screens functional antibodies that identify the target’s normal configuration by displaying appropriately folded multi-pass TPs in its cell membrane. In addition to the range of VLP-based multi-pass TPs, ACRO also offers customized services.

Advantages

• Immunogenicity is higher
• Higher abundance than that of overexpressing cells
• Immunization, ELISA, SPR, BLI, cell experiment and CAR detection are all compatible
• Full ECD epitopes in full-length TPs
• Due to their 100–200 nm size, they may be employed as the ideal targets for dendritic cells for phage display in vivo.

Image Credit: ACROBiosystems

Detergent micelle platform

Principle

As the transmembrane portion of multi-pass TPs is known to be highly hydrophobic, it is challenging to keep it in the right conformation once it has been removed from the cell membrane in standard buffers. Detergents can be added to resolve this problem.

For challenging therapeutic target TPs, ACROBiosystems has developed a full platform for insect cell and mammalian cell expression, purification and stabilization. To enhance solubility and assure the natural fold of this protein in vitro, ACRO uses detergent screening, including DDM/CHS (Cat. No. DC-11).

Advantages

• Can be precisely measured
• Immunization, ELISA, SPR and BL are all compatible
• TPs with complete conformation

Image Credit: ACROBiosystems

Nanodisc platform

Principle

Membrane scaffold proteins (MSPs) and phospholipid molecules make up the so-called “Nanodisc,” a synthetic phospholipid bilayer membrane structure. After the detergent is removed, TP may be incorporated into the structure of Nanodisc to maintain its natural folding, biological activity and increased hydrophilicity for numerous applications.

In CAR expression assays, for example, the detergent-free formulation of Nanodisc-based TP is acceptable. The Nanodisc technology was licensed from the University of Illinois, and ACROBiosystems has been continuously optimizing and improving the assembly process to make it appropriate for commercial scale-up manufacturing.

ACRO’s efforts have provided the biopharma industry with a long-term steady supply of Nanodisc-based TP products.

Advantages

• Immunization/ELISA/SPR/BLI/cell experiment/CAR detection compatible
• Detergent-free high hydrophilicity
• Patent holder with a valid license
• Full-length TPs reside in a natural membrane environment, which allows them to retain high biological activity.

Image Credit: ACROBiosystems

Product list

VLP

Table 1. Source: ACROBiosystems

Click here to learn about many more VLP.

Detergent micelle

Table 2. Source: ACROBiosystems

Click here to learn about many more detergent micelles.

Nanodisc

Table 3. Source: ACROBiosystems

Click here to learn about many more nanodiscs.

Case display

Claudin18.2-VLP

Claudin18.2-VLP. Image Credit: ACROBiosystems

Correct assembly validated by SEM

Under an electron microscope, full-length Claudin18.2-VLP (Cat. No.CL2-H5547) was examined to confirm that it was assembled appropriately.

Image Credit: ACROBiosystems

Good bioactivity validated by ELISA

Immobilized Human Claudin-18.2 Full Length Protein-VLP (Cat. No. CL2-H5547) at 5 μg/mL (100 μL/well) can bind Monoclonal Anti-Chimeric Claudin-18.2 Antibody, Human IgG1 with a linear range of 0.8–3 ng/mL (QC tested). Image Credit: ACROBiosystems

High affinity validated by SPR

Human Claudin-18.2 Full Length Protein-VLP (Cat. No. CL2-H5547) captured on CM5 Chip via Anti-Claudin-18.2 antibody can bind Anti-Claudin-18.2 antibody with an affinity constant of 1.24 nM as determined in an SPR assay (Biacore T200) (Routinely tested). Image Credit: ACROBiosystems

Suitable for CAR detection

2e5 of Anti-Claudin-18.2 CAR-293 cells were stained with 100 μL of 3 μg/mL of Fluorescent Human Claudin-18.2 Full Length Protein-VLP (Cat. No.CL2-HF218) and negative control protein respectively, FITC signals was used to evaluate the binding activity (Routinely tested). Image Credit: ACROBiosystems

CD20-DDM/CHS

CD20-DDM/CHS. Image Credit: ACROBiosystems

Good bioactivity validation of full-length CD20-DDM/CHS (Cat. No.CD0-H52H3) by ELISA

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4–3 ng/mL (in presence of DDM and CHS). Image Credit: ACROBiosystems

Good bioactivity validation of CD20-DDM/CHS (Cat. No.CD0-H82E5) by ELISA

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His, Avitag (HEK293) (Cat. No. CD0-H82E5) with a linear range of 4–63 ng/mL (in presence of DDM and CHS). Image Credit: ACROBiosystems

High affinity validation of CD20-DDM/CHS (Cat. No.CD0-H82E5) by SPR

Biotinylated Human CD20, His, Avitag (HEK293) (Cat. No. CD0-H82E5) captured on Biotin CAP-Series S Sensor Chip can bind Rituximab with an affinity constant of 1.73 nM as determined in an SPR assay (in presence of DDM and CHS) (Biacore T200). Image Credit: ACROBiosystems

CD133-nanodisc

CD133-Nanodisc. Image Credit: ACROBiosystems

Purity greater than 90% by SDS-PAGE

Human CD133 Full Length, His Tag (Nanodisc) (Cat. No. CD3-H52H1) on SDS-PAGE under reducing (R) condition. The gel was stained overnight with Coomassie Blue. The purity of the protein is greater than 90%. Image Credit: ACROBiosystems

Good bioactivity validated by ELISA

Immobilized Human CD133 Full Length, His Tag (Nanodisc) (Cat. No. CD3-H52H1) at 1 μg/mL (100 μL/well) can bind Monoclonal Anti-Human CD133 (133-3) Antibody, Human IgG1 with a linear range of 0.2–4 ng/mL (QC tested). Image Credit: ACROBiosystems