Opportunities of Full-length CD20 in Evaluation of CAR-T Expression

CD20 is present in B cell development except for early and last steps and has been targeted by mAbs like Rituxima* and ofatumumab as part of treatment against B-cell lymphomas, leukemias and B cell mediated autoimmune diseases. CD20 exists as a dimer as shown in the crystal structure (Fig. 1A) and multiple reports indicate two extracellular loops form epitopes that are strong antibody/CAR-T targets (Fig. 1B), and full-length CD20 with native folding is needed to fully characterize antibody activity and mechanism of action. However, CD20 is difficult to express and purify, which seriously restricts its applications.

A

B

Figure 1. Structural complexity of MP CD20. A) Schematic showing antigen binding site located on extracellular loops (ECLs) B) Crystal structure of CD20 revealing multiple hydrophobic including p-p stacking and hydrophilic interaction stabilizing the dimerization of transmembrane region.^[1,2]

ACROBiosystems has been able to successfully purify full-length CD20 by means of several rounds of optimization during each step with expression, extraction, affinity and size exclusion chromatography (Fig. 2).

Figure 2. CD20 production process and indicated optimization for high purity product.

Once purified, two methods were utilized to stabilize full-length CD20: detergent stabilization and Nanodisc stabilization. Detergent can separate the lipid membrane from the protein and result in protein-detergent complexes (Fig. 3A). Nanodisc is a type of detergent free non-valent synthetic model membrane consisting of phospholipid bilayer and membrane scaffold protein (MSPs) that stabilize membrane proteins and help retain their biological activity (Fig. 3B). A big advantage of Nanodisc assembly is powder formulation and use in CAR-T expression.

A •   TM regions coated by detergent molecules •   Liquid formulation •   CAR-T incompatible

B •   Detergent free •   TM regions coated by lipids  mimicking cell membrane •   Tied by scaffold protein •   Powder formulation •   Can detect CAR-T expression

Figure 3. Two methods employed by ACRO to stabilize full-length CD20. A) Detergent stabilization. B) Nanodisc assembly.

With fully verification, Nanodisc assembled full-length CD20 performed very well against two known commercial therapeutic mAbs Rituxima* and Ofatumumab (Fig. 4).

Immobilized Rituxima* 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.

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Immobilized Human CD20 Full Length, His Tag (Cat. No. CD0-H52H1) at 5 μg/mL (100 μL/well) can bind Biosimilar of Obinutuzumab with a linear range of 0.3-5 ng/mL (Routinely tested).  

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Figure 4. ELISA binding analysis of Nanodisc assembled full-length CD20 by Rituxima* and Ofatumuma respectively.

Furthermore, Nanodisc assembly platform makes full-length CD20 suitable for evaluation of CAR-T expression. Good bioactivity of full-length CD20-Nanodisc (Cat. No.  CD0-H52H1 & CD0-H82E3) was verified by FACS (Fig. 5).

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).

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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).

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Figure 5. FACS analysis of Nanodisc assembled full-length CD20.

In summary, with optimized HEK293 membrane protein expression system, recombinant membrane protein purification strategy, self-optimized and scalable Nanodisc assembly with its technical platform, ACRO overcomes the problem of full-length CD20 expression and purification. Full-length CD20 produced by Nanodisc assembly platform greatly broadens its application in evaluation of CAR-T expression and CD133, GPRC5D benefit from it as well.

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References

1.    L. Rougé et al., Structure of CD20 in complex with the therapeutic monoclonal antibody Rituxima*. Science (New York, N.Y.) 367,  (2020).

2.    A. Kumar, C. Planchais, R. Fronzes, H. Mouquet, N. Reyes, Binding mechanisms of therapeutic antibodies to human CD20. Science 369,  (2020).