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HEK293 Expressed Full Length CD20 Proteins

Interaction Between CD20 and the Critical Molecular Determinants of Rituximab (RTX)

DOI: 10.1126/science.aaz9356

Interaction Between CD20 and the Critical Molecular Determinants of Rituximab (RTX)

CD20 is a tetraspan membrane protein selectively expressed on the surface of B cells. It is composed of 297 amino acids with an apparent molecular weight of roughly 33–37 kDa. As a key regulator of B-cell development and function, CD20 is highly expressed on more than 95% of B-cell lymphomas, making it a “gold-standard” therapeutic target in B-cell malignancies and autoimmune disorders.

Structurally, CD20 contains four transmembrane domains that form two extracellular loops (ECL1 and ECL2). Studies have shown that rituximab (RTX) exerts its immune-mediated killing primarily by engaging a dominant epitope within ECL2, while ECL1 contributes indirectly by stabilizing the protein’s conformation and supporting secondary interactions. The integrity of these epitope structures directly influences antibody affinity and complement-dependent cytotoxicity. As a result, using full-length CD20 with a native conformation is essential throughout drug discovery and development.

Leveraging multi-pass transmembrane protein development platform—Full Length Active Gallery—we have successfully developed full-length CD20 proteins,with high bioactivity across ELISA, BLI, SPR, and cell-based assays, enabling robust candidate screening and mechanism exploration. We also provide CD20 stable cell lines and comprehensive SPR/BLI interaction analysis services to further accelerate therapeutic development and regulatory submission.

Different Types of CD20 Proteins

VLP Platform
VLP Platform

The VLP (virus-like particle) platform clusters full-length, correctly folded membrane proteins on the surface of producer cells, converting these otherwise challenging multispan targets into soluble, high-concentration immunogens for antibody generation and screening. The resulting enveloped VLPs present native, properly folded multipass membrane proteins within their original lipid membrane, enabling the induction and selection of functional antibodies that recognize the target in its natural physiological conformation.

Detergent Platform
Detergent Platform

Multi-pass transmembrane proteins are highly hydrophobic within their transmembrane domains and therefore struggle to maintain their native conformation in standard aqueous buffers. To address this, we established a dedicated detergent-based platform built on both insect and mammalian expression systems. Through systematic screening, we identified a set of high-performance detergents—including our optimized Detergent formulations—that substantially improve target protein solubility and preserve correct folding in solution.

Nanodisc Platform
Nanodisc Platform

Nanodiscs are phospholipid bilayer structures assembled from membrane scaffold proteins (MSPs) and defined phospholipids. This format recreates a near-native membrane environment while incorporating membrane proteins in a way that preserves their biological activity and maintains excellent solubility, substantially broadening their utility across downstream applications. We hold official patent authorization for this technology, ensuring your R&D programs advance on a secure and fully compliant technical path.

Nanodisc-pro Platform
Nanodisc-pro Platform

The "Nanodisc-pro" technology platform employs an innovative scaffold in-situ assembly strategy that directly encircles target proteins along with their closely associated native phospholipids in a natural membrane environment, forming a stable phospholipid bilayer structure. By eliminating the disruptive detergent stripping and reconstitution steps required by traditional methods, this approach effectively preserves the native conformation and biological activity of transmembrane proteins.

Product List

VLP Platform
Detergent Platform
Nanodisc Platform
Nanodisc-pro Platform
Stable Cell Lines
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Product Features

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

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)

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).

Cat. No. CD0-H52H1 (HEK293 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).

ELISA
Cat. No. CD0-H82E3 (HEK293 expression)
ELISA

Immobilized Rituximab biosimilar 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 2-62.5 ng/mL (QC tested).

Cat. No. CD0-H52H1 (HEK293 expression)
ELISA

Immobilized Rituximab 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 (QC tested).

Cat. No. CD0-H82E5 (HEK293 expression)
ELISA

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

Cat. No. CD0-H52H3 (HEK293 expression)
ELISA

Immobilized Human CD20 Full Length Protein, His Tag (Cat. No. CD0-H52H3) at 5 μg/mL (100 μL/well) can bind Biosimilar of Obinutuzumab with a linear range of 0.3-5 ng/mL (Routinely tested).

Cat. No. CDP-HF2P8 (HEK293 expression)
ELISA

Immobilized Fluorescent Human CD20 Full Length Protein-VLP (Cat. No. CDP-HF2P8) at 5 μg/mL (100 μL/well) can bind Rituximab biosimilar (Cat. No. CD0-M36) with a linear range of 0.2-3 μg/mL (QC tested).

Cat. No. CDP-H52P6 (HEK293 expression)
ELISA

Immobilized Human CD20 Full Length Protein-VLP (Cat. No. CDP-H52P6) at 5 μg/mL (100 μL/well) can bind Rituximab biosimilar (Cat. No. CD0-M36) with a linear range of 0.2-3 μg/mL (QC tested).

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).

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).


FAQ

Q

Why does CD20 remain a gold-standard lineage marker and therapeutic target for B-cell malignancies and autoimmune disorders?

CD20 is a non-glycosylated transmembrane phosphoprotein expressed strictly during specific stages of B-cell ontogeny, from the pre-B-cell phase to mature B-lymphocytes. Crucially, it is absent on pluripotent hematopoietic stem cells and mature plasma cells, minimizing systemic toxicity during targeted ablation. CD20 acts as a store-operated calcium channel, fundamentally modulating B-cell activation, proliferation, and differentiation. Its high expression across various B-cell malignancies, such as non-Hodgkin lymphoma and chronic lymphocytic leukemia, makes it an ideal oncology target. Consequently, therapeutic strategies leveraging CD20 enable precise depletion of pathogenic B-cell populations while preserving the regenerative capacity of the bone marrow.
Q

What strategies are recommended to eliminate nonspecific antibody background arising from VLP, detergent, and nanodisc preparations used for CD20 immunization?

To mitigate non-specific antibody responses, several critical steps are required. First, rigorous purification protocols should be implemented: VLPs require multi-step purification including ion exchange chromatography to remove carrier protein contaminants . Second, for detergent-solubilized CD20, careful selection of mild detergents minimizes epitope distortion while complete detergent removal prevents anti-detergent antibodies. Third, nanodisc-based immunization offers advantages as high-purity full-length transmembrane proteins with controlled composition reduce off-target reactivity . Finally, pre-absorption of immune sera against empty carriers helps eliminate cross-reactive antibodies.
Q

What advanced biochemical platforms are required to preserve the native tetrameric conformation of CD20 during antibody screening?

Isolating CD20 is notoriously difficult because its hydrophobic multi-pass segments make it highly unstable and prone to denaturation when extracted from cellular membranes. Furthermore, functional CD20 naturally homooligomerizes into tetrameric complexes within native lipid bilayers. To preserve this physiological conformation, researchers must utilize sophisticated mammalian expression systems paired with optimized detergent solubilization protocols. Reconstituting the protein into advanced membrane-mimetic environments, such as virus-like particles, liposomes, or synthetic nanodiscs, nanodisc-pro, is highly recommended. These specialized platforms shield the hydrophobic domains while successfully presenting the delicate extracellular loops, ensuring that screening assays capture biologically relevant binding kinetics.
Q

How do therapeutic anti-CD20 antibodies achieve B-cell depletion through distinct mechanisms of action?

Anti-CD20 antibodies are classified into Type I (rituximab, ocrelizumab) and Type II (obinutuzumab) based on epitope binding and effector functions. Type I antibodies efficiently induce complement-dependent cytotoxicity (CDC) and bind to the large extracellular loop. Type II antibodies demonstrate enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) with superior direct cell-killing activity. All therapeutic antibodies target the extracellular regions while the membrane-anchored structure prevents internalization or dissociation. Ofatumumab exhibits notably slower dissociation rates, facilitating prolonged complement activation. The relative contribution of these mechanisms varies across clinical settings but collectively enable profound B-cell depletion in B-cell malignancies.
Q

What therapeutic modalities targeting CD20 have demonstrated clinical efficacy in treating B-cell malignancies and autoimmune disorders?

Multiple therapeutic modalities targeting CD20 are clinically established. Rituximab, the first chimeric anti-CD20 antibody approved two decades ago, remains the gold standard for non-Hodgkin lymphoma and chronic lymphocytic leukemia treatment. Newer agents include second-generation antibodies and CD20×CD3 bispecific T-cell engagers showing significant efficacy in CAR-T relapsed large B-cell lymphoma. Dual-target CAR-T therapies (CD19/CD20) are advancing in clinical trials for hematologic malignancies and autoimmune diseases like systemic lupus erythematosus. These approaches exploit CD20's surface localization, high cancer-specific expression, and absence from plasma cells, enabling selective B-cell depletion while preserving stem cell reserves
  • Background
  • Different Types of CD20 Proteins
  • Product List
  • Product Features
  • FAQ
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