HEK293 expressed full length CD20 proteins

The recently published article in Science by the Genentech team presents the structure of CD20 in complex with the therapeutic monoclonal antibody RTX. This study underscores the significance of preserving intact CD20 for effective antibody drug development.

The researchers identified several RTX mutants that, although they retain cell-binding activity similar to OBZ and maintain substantial binding to the primary ECL2 epitope, fail to trigger complement-dependent cytotoxicity (CDC). This finding highlights that the secondary ECL1/2 epitope and the Fabinterface are crucial for RTX's distinct binding characteristics and its high CDC activity.

Image Credit: Lionel Rougé, et al. Structure of CD20 in complex with the therapeutic monoclonal antibody RTX. Science. 2020

Image Credit: ACROBiosystems Inc

To support the development of CD20 antibody drugs, the full-length CD20 expressed in HEK293 cells by ACROBiosystems has become highly appealing to researchers and developers.

Background

At the 61st Annual Meeting of the American Society of Hematology (ASH) in 2019, bi-specific antibodies targeting CD20 on B cells and CD3 on T cells, such as CD20-TCB, were presented. Preliminary results from clinical studies showed that these antibodies have acceptable safety profiles and promising efficacy.

CD20 remains a key target for treating lymphoma, leukemia, and some autoimmune diseases. However, finding a stable, active CD20 protein for antibody drug development and CD20 CAR expression is challenging due to issues with low expression levels and unstable bioactivity.

To address these challenges, ACROBiosystems has developed highly active full-length CD20 proteins expressed in HEK293 cells. These proteins feature both large and small extracellular domain (ECD) loops and exhibit high binding affinity for anti-CD20 antibodies in ELISA and SPR assays. ACROBiosystems offers various forms of CD20 proteins, including unconjugated, biotinylated proteins with DDM, Nanodisc, and VLP formats, suitable for applications such as immunization, ELISA, SPR, BLI, and FACS.

Image Credit: ACROBiosystems Inc

Different types of CD20 proteins

Protein with DDM

DDM (n-dodecyl-β-D-maltoside) is a detergent that disrupts lipid membranes to release membrane proteins while providing a hydrophobic environment to preserve their transmembrane structures. However, DDM is not suitable for cell experiments as it can damage the structure of the cell membranes.

Protein with Nanodisc

Nanodiscs are non-covalent structures comprising a phospholipid bilayer and membrane scaffold proteins. Membrane proteins can be integrated into nanodiscs to maintain their biological activity. CD20 protein with nanodisc is recommended for cell experiments.

Proteins with VLP

Virus-like particles (VLPs) are a technology platform developed by ACROBiosystems using the HEK293 expression system. VLPs involve expressing transmembrane proteins on the surface of host cells, which are then converted into soluble lipid bilayer particles. This approach concentrates the proteins and allows for high-quality antibody immunization and screening. The membrane protein-VLP complexes ensure that proteins are properly folded in their native conformation, facilitating the identification of functional antibodies that recognize the target's natural structure.

Product list

Detergent micelle

Source: ACROBiosystems Inc

Nanodisc

Source: ACROBiosystems Inc

VLP

Source: ACROBiosystems Inc

Product features

Full length CD20 protein has a complete conformation with both small and big loops

Image Credit: ACROBiosystems Inc

Full length CD20 protein has better bioactivity

A) HEK293 cell (Full length)

Immobilized RTX 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 Inc

B) E. coli (Extracellular)

Immobilized RTX at 5 μg/mL (100 μL/well) can bind Human CD20, TrxA Tag, His Tag (E. coli) with a linear range of 78-2500 ng/mL. Image Credit: ACROBiosystems Inc

The bioactivity of full length CD20 protein was verified by FACS/ELISA/ SPR and the protocols are offered

FACS

Cat. No. CD0-H82E3 (HEK293 expression)

FACS analysis of anti-CD20 CAR expression

2e5 of CD20-CAR-293 cells transfected with anti-CD20-scFv were stained with 100 μL of 3 μg/mL of Biotinylated Human CD20 Full Length, His,Avitag (Cat. No. CD0-H82E3) and negative control protein respectively, washed and then followed by PE-SA and analyzed with FACS (QC tested). Image Credit: ACROBiosystems Inc

Cat. No. CD0-H52H1 (HEK293 expression)

FACS analysis of anti-CD20 CAR expression

2e5 of CD20-CAR-293 cells transfected with anti-CD20-scFv were stained with 100 μL of 3 μg/mL of Human CD20 / MS4A1 Full Length Protein, His Tag (Nanodisc) (HEK293) (Cat. No. CD0-H52H1) and negative control protein respectively, washed and then followed by PE anti-His antibody and analyzed with FACS (QC tested). Image Credit: ACROBiosystems Inc

ELISA

Cat. No. CD0-H82E3 (HEK293 expression)

Immobilized RTX at 5 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (Cat. No. CD0-H82E3) with a linear range of 0.008-0.125 μg/mL. Image Credit: ACROBiosystems Inc

Cat. No. CD0-H52H1 (HEK293 expression)

Immobilized RTX at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag (Cat. No. CD0-H52H1) with a linear range of 1-63 ng/mL. Image Credit: ACROBiosystems Inc

Cat. No.CD0-H82E5 (HEK293 expression)

Immobilized RTX 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 Inc

Cat. No.CD0-H82E5 (HEK293 expression)

Immobilized anti-CD20 antibody 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 Inc

Cat. No. CD0-H52H3 (HEK293 expression)

Immobilized RTX 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 Inc

Cat. No. CD0-H52H3 (HEK293 expression)

Immobilized anti-CD20 antibody 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 0.4-6 ng/mL (in presence of DDM and CHS). Image Credit: ACROBiosystems Inc

SPR

Cat. No. CD0-H82E5 (HEK293 expression)

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

Cat. No. CD0-H52H3 (HEK293 expression)

Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) captured on CM5 chip via Anti-human IgG Fc antibodies surface can bind RTX with an affinity constant of 6.21 nM as determined in a SPR assay (in presence of DDM and CHS) (Biacore 8K). Image Credit: ACROBiosystems Inc