Full-length CD20 membrane proteins for research and development

The newly launched article Structure of CD20 in complex with the therapeutic monoclonal antibody RTX reported on Science by the Genentech team stresses the significance of featuring intact CD20 for antibody drug development.

The team has found numerous RTX mutants that, despite holding cell-binding activity similar to that of OBZ, and highly unaffected binding to the main ECL2 epitope, are not capable of eliciting CDC. This verifies that the secondary ECL1/2 epitope and Fab:Fab interface contribute to the special binding properties and high CDC activity of RTX.

Structure of CD20 in complex with the therapeutic monoclonal antibody RTX.

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

To support CD20 antibody drug development, the HEK293-expressed complete-length CD20 from ACROBiosystems has been highly appealing to customers.

Background

The 61st annual meeting of the American Society of Hematology (ASH) conducted in 2019 presented bi-specific antibodies that target CD20 on the surface of B cells and CD3 on the surface of T cells, like CD20-TCB. The preliminary clinical study results display acceptable safety and potential efficacy of this pair of particular antibodies.

CD20 is crucial for the treatment of leukemia, lymphoma and certain autoimmune diseases. But as a result of the low expression titer and the unstable bioactivity, it is so hard to determine an active CD20 protein that is commercially available for the CD20 CAR expression and antibody drug development.

To overcome these problems, ACROBiosystems has come up with the highly active full-length multi-transmembrane CD20 proteins that have been expressed by HEK293 cells. The active full-length CD20 proteins feature both big and small ECD loops and bind anti-CD20 antibodies with high binding affinity in SPR and ELISA assay.

The company offers various kinds of CD20 proteins, such as unconjugated protein, biotinylated proteins with DDM and Nanodisc, which are appropriate for immunization, FACS, BLI, SPR and ELISA.

Different types of CD20 proteins

Protein with DDM

ACROBiosystems has created a VLP (Virus Like Particles) technology platform depending on the HEK293 expression system to produce transmembrane proteins on the surface of the host cell.

The viral envelop or capsid protein subsequently converts these cell surfaces into soluble lipid bilayer particles containing highly concentrated proteins.  This may be used for antibody immunization and screening.

The membrane protein-VLP complex is appropriately folded in its cell membrane, thereby allowing researchers to generate and evaluate functional antibodies that identify the target’s native shape.

Protein with nanodisc

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

Product list

Detergent Micelle

Table 1. Source: ACROBiosystems

Molecule Cat .No. Product Description Application
CD20 CD0-H52H3 Human CD20 / MS4A1 Full Length Protein, His Tag (HEK293) (SPR verified) Immunization
ELISA
SPR
BLI
CD20 CD0-H82E5 Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag™ (HEK293)
CD20 CD0-C52H8 Cynomolgus CD20 / MS4A1 Full Length Protein, His Tag
Buffer DC-11 200x DDM CHS buffer Maintain solubilization and activity of membrane proteins
Dilute membrane proteins or antibodies Prepare running buffer in SPR or BLI assay, etc.

 

Nanodisc

Table 2. Source: ACROBiosystems

Molecule Cat .No. Product Description Application
CD20 CD0-H52H1 Human CD20 / MS4A1 Full Length Protein, His Tag (Nanodisc) (HEK293) Immunizationa
ELISA
SPR
BLI
Cell-based assay
CAR detection
CD20 CD0-H82E3 Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag™ (Nanodisc) (HEK293)
MSP1D1 APO-H51H3 Human MSP1D1 Protein, His Tag (Nanodisc) isotype control
MSP1D1 APO-H81Q5 Biotinylated Human MSP1D1 Protein, His,Avitag™ (Nanodisc) isotype control

 

VLP

Table 3. Source: ACROBiosystems

Molecule Cat .No. Product Description Application
CD20 CDP-H52P6 Human CD20 Full Length Protein-VLP (HEK293) Immunization
ELISA
SPR
BLI
Cell-based assay
CAR detection
VLP VLP-H52C5 Virus-Like Particle (VLP) isotype control VLP isotype control

 

Product features

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

Full-length CD20 membrane proteins for research and development

Image Credit: ACROBiosystems

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)

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

(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.

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

The bioactivity of full-length CD20 protein was validated by FACS/ELISA/SPR and the protocols are provided.

FACS

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).

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

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).

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

ELISA

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.

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

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.

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

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 the presence of DDM and CHS).

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 the presence of DDM and CHS). Image Credit: ACROBiosystems

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 the presence of DDM and CHS).

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 the presence of DDM and CHS). Image Credit: ACROBiosystems

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 the presence of DDM and CHS).

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 the presence of DDM and CHS). Image Credit: ACROBiosystems

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 the presence of DDM and CHS).

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 the presence of DDM and CHS). Image Credit: ACROBiosystems

SPR

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 an SPR assay (in the presence of DDM and CHS) (Biacore T200).

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 an SPR assay (in the presence of DDM and CHS) (Biacore T200). Image Credit: ACROBiosystems

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 an SPR assay (in presence of DDM and CHS) (Biacore 8K).

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 an SPR assay (in presence of DDM and CHS) (Biacore 8K). Image Credit: ACROBiosystems