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Establishment and Application of Anti-CD20 Monoclonal Antibodies using the Cell-based Immunization and Screening Method for the Detection of B Cells

古澤, 慶一 筑波大学 DOI:10.15068/0002005699

2022.11.25

概要

In the field of Biochemistry and Medicine, there is immense importance of specific antibodies against a target protein, and thus, a number of such antibodies have been generated, with varied properties. In general, the hybridoma method developed by Kohler and Milstein is used for antibody production. However, it is difficult to produce a few types of antibodies, such as those against proteins with complex structures and multiple transmembrane proteins, using this method. CD20, the target of this study, is one such protein.

CD20 is a small protein with four membrane-spanning domains and two regions consisting of 50 amino acid exposed to the outside of the cell. Since CD20 is specifically expressed on B cells, it is used as a marker for the detection of B cells and is a target molecule for antibody drugs used in B-cell lymphoma. Owing to its importance, several anti-CD20 antibodies have been developed. However, there is still a bottleneck: no single antibody developed till date can be used for many varied applications. For example, antibodies such as rituximab used in lymphoma treatment recognize the extracellular region of CD20, while antibodies used for immunohistochemical analyses, such as L26, recognize the intracellular region. Due to the difference in the epitopes of these antibodies, there may be a difference in diagnostic results and therapeutic effects when CD20 is mutated. Thus, using several antibodies to analyze a target is an obstacle to obtaining accurate data, because of differences in the epitope and reactivity of the antibodies. However, there are many antibodies, other than anti-CD20 antibodies, that also have limited applications. Therefore, development of antibodies that can be used in various applications, as well as, the methods for producing such antibodies are very important.

In this study, I aimed to develop anti-CD20 antibodies that can be used in different applications, such as flow cytometry (FCM), western blot (WB), and immunohistochemistry (IHC). In addition, this study also used the Cell-Based Immunization and Screening (CBIS) method, a novel method for antibody production, and tested whether it is useful for producing antibodies for difficult targets and multiple applications.

Upon antibody production, 180 of the 7680 samples tested were found to be positive in the first screening, from which 56 clones of monoclonal antibodies were established. These 56 clones were then used to perform WB and IHC against CD20, of which 13 clones showed reactivity in FCM, WB, and IHC. Two of these clones, C20Mab- 11 (Immunoglobulin [Ig]M, kappa) and C20Mab-60 (IgG2a, kappa), were then used for further analysis. Upon doing so, it was found that C20Mab-11 and C20Mab-60 are useful for FCM and WB in endogenous CD20-expressing cell lines, such as BALL-1 (acute lymphoblastoid leukemia cell line) and Raji (Burkitt lymphoma cell line). Upon carrying out IHC of lymph nodes, both the antibodies reacted strongly with B cells in the lymphoid follicles. However, on carrying out analysis of 38 sections of B-cell lymphoma, C20Mab- 11 showed reactivity in only 2 of the 38 sections, while C20Mab-60 showed reactivity in 35 sections. These results suggested that C20Mab-11 and C20Mab-60 can be used in FCM, WB, and IHC, and are useful for CD20 research. In addition, C20Mab-60 detected CD20 with high sensitivity when applied for IHC. Epitope analysis revealed that C20Mab-60 recognizes the same region as the anti-CD20 antibody used in the treatment of lymphoma. This suggests that C20Mab-60 may be particularly useful for IHC-based lymphoma analysis. Thus, the antibody production study resulted in the generation of several antibodies against CD20, and many of these antibodies had multi-use properties.

Therefore, the CBIS method can produce antibodies against difficult proteins and is considered to be a useful technique for boosting research in Biochemistry and Medicine.

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