CD20, Why is It Such Popular in Antibody Drug Development?

1. What is CD20?

CD20, also known as cluster of Differentiation 20, is a transmembrane protein encoded by the MS4A1 gene in human. The MS4A1 gene is 16 kb long located on chromosome 11q12.2, comprises eight exons, and several different CD20 mRNA transcripts have been annotated. The dominant CD20 mRNA variant is 2.8 kb long and uses all eight exons. The second most common form is 263 bases shorter, as it skips exon II. A minor 3.5 kb mRNA results from splicing exons in the upstream region into an internal 3' splice site located in exon I. However, all three transcripts are translated into identical full-length CD20 protein as the translation start codon is localized within exon III ^[1].

CD20 protein is a 33-37 kDa non-glycosylated protein expressed on the surface of normal and malignant B lymphocytes ^[2]. As the figure 1 shows, it consists of four hydrophobic transmembrane domains, one intracellular and two extracellular domains (large and small loops) with both N- and C- termini residing within the cytosol ^[3]. Currently, three CD20 isoforms (33, 35 and 37 kDa) resulting from different phosphorylation have been identified, and CD20 phosphorylation was reported to be higher in proliferating malignant B cells than in resting B cells ^[4].

Figure 1. The structure of CD20 molecule.

*This diagram is derived from reference 5.

2. What is the Function of CD20?

CD20 is a cell surface tetraspan receptor expressed exclusively on B-lymphocytes, which are differentiated from pluripotent stem cells in the bone marrow and develop through the pro-B, pre-B, immature B and mature B stages. CD20 is present in the Pro-B to mature B cell stages, but is not expressed on haematopoietic stem cells, progenitor B cells or mature plasma cells. In addition to being expressed in normal B cells, CD20 is also expressed in tumor cells of B-cell origin, such as lymphomas and leukaemias. Despite it is, the function of CD20 has not been determined ^[6].

CD20 exists as an oligomer on the surface of B lymphocytes and available studies have demonstrated that CD20 forms tetramers on the surface of B cells. The interaction of CD20 with the membrane IgM (sIgM) of the B cell antigen receptor (BCR) has been demonstrated and upon activation of B cells, the BCR-CD20 complex dissociates and Phosphoproteins, calmodulin-binding proteins, are temporarily recruited to CD20 and are thus involved in intracellular signaling. Calcium flow has an important impact on the biological function of cells. Calcium pool-regulated calcium entry (SOCE), which increases intracellular calcium concentration through calcium-release-activated calcium (CRAC) channels, is the main way in which lymphocytes increase their intracellular calcium concentration. There is increasing evidence that CD20 is involved in SOCE and that CD20 has an important role in regulating intracellular calcium concentration ^{[7] [8] [9]}.

3. What are the Ways of Treatment Targeting CD20?

As mentioned before, In addition to being expressed in normal B cells, CD20 is also expressed in tumor cells of B cell origin such as lymphoma and leukaemia, as well as in B cells involved in immune and inflammatory diseases. This makes CD20 become a target for the treatment of diseases such as lymphoma, leukaemia and certain autoimmune diseases. Currently, there are three main ways of treatment targeting CD20 in these disease, including anti-CD20 monoclonal antibodies, CAR-T and bispecific antibodies. In this section, we focus on anti-CD20 monoclonal antibodies.

Anti-CD20 monoclonal antibodies refer to monoclonal antibodies targeting CD20. Accumulating evidence suggests that anti-CD20 monoclonal antibodies kill tumors of B-cell origin and are associated with three mechanisms of action, namely antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and direct effects caused by the binding of antibodies to CD20 molecules, including inhibition of cell growth, alteration of the cell cycle and apoptosis. ADCC refers that B cells coated with anti-CD20 are killed by a cell-based mechanism. CDC refers that the complement membrane attack complex is assembled on the cell surface (Figure 2).

Figure 2. Mechanism of action of anti-CD20 antibodies

*This diagram is derived from reference 10.

Since the approval of the world's first anti-CD20 monoclonal antibody (Roche's rituximab) in 1997, several anti-CD20 monoclonal antibodies have been approved worldwide. Depending on the degree of humanization and Fc fragment modification, anti-CD20 monoclonal antibodies can be divided into three generations. The first generation is represented by rituximab as a chimeric or mouse-derived monoclonal antibody, the second generation is represented by Arzerra from Genmab Inc. as a humanized monoclonal antibody and the third generation of anti-CD20 monoclonal antibodies is represented by Gazyva from Roche, whose Fc fragment of the antibody has been modified by glycosylation.

4. The Collection of Biologics targeting CD20 in Clinic

As mentioned before, there are 93 biologics targeting CD20 in the clinical stage of development. Here, we list projects in the clinical stage II and III.

5. What Reagents You May Need for CD20-Targeted Research?

Recombinant Human B-lymphocyte antigen CD20(MS4A1)-VLPs (Active) (CSB-MP015007HU)

Human B-lymphocyte antigen (CD20) ELISA Kit (CSB-E14007h)

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