HEK293 Expressed Full Length CD20 Protein with Nanodisc / DDM / VLP

The 61st annual meeting of the American society of hematology (ASH) in 2019 presented bi-specific antibodies targeting CD20 on the surface of B cells and CD3 on the surface of T cells, such as CD20- TCB, with preliminary resultsof clinical studies showing acceptable safety and promising efficacy of this pair of specific antibodies.

CD20 is a hot target for the treatment of lymphoma, leukemia and some autoimmune diseases. However, due to low expression titer and the unstable bioactivity, it is so difficult to find an active CD20 protein on the market for antibody drug development and the CD20 CAR expression.

To address these issues, ACROBiosystems has developed the highly active full length multi-transmembrane CD20 proteins, expressed by HEK293 cells. The active full length CD20 proteins carry both big and small ECD loops, and bind anti-CD20 antibodies with high binding affinity in ELISA and SPR assay. We have different types of CD20 proteins include unconjugated protein, biotinylated proteins with DDM, Nanodisc and VLP, which are suitable for immunization, ELISA, SPR, BLI and FACS.

DDM can disintegrate the lipid membrane to release the membrane protein and provide a hydrophobic environment, maintain and protect the hydrophobic transmembrane structure of membrane proteins. But it is not recommended for cell experiments because it can destroy the structure of the cell membrane.

Nanodisc is a non-covalent structure of phospholipid bilayer and membrane scaffold protein. Membrane proteins can be integrated into nanodiscs to maintain their biological activity, CD20 protein with nanodisc was recommended for cell experiments.

The VLP (Virus like particles) technology platform based on the HEK293 expression system is specially set up by ACROBiosystems to express transmembrane proteins on the host cell surface. The viral envelop/capsid protein then turns these cell surfaces into soluble lipid bilayer particles with highly-concentrated proteins amenable for antibody immunization and screening. The membrane protein-VLP complex displays correctly folded in its cell membrane induce and screen functional antibodies that recognize the natural conformation of the target.

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

Full length CD20 protein has better bioactivity

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

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).

Protocol

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).

Protocol

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

Protocol

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

Protocol

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).

Protocol

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).

Protocol

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).

Protocol

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).

Protocol

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).

Protocol

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).

Protocol