Date of Award

8-2007

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Microbiology

Advisor

Yu, Xianzhong

Committee Member

Wei , Yanzhang

Committee Member

Wagner , Thomas

Abstract

CD20 is a non-glycosylated transmembrane protein expressed on normal B cells, malignant B cells, and plasma cells, but not their stem cell precursors. It is an ideal target for antibody therapy because it is not significantly shed or internalized, and CD20 expression is generally not lost after antibody binding. Depleted B cells can also be regenerated after antibody therapy. Rituximab, a chimeric monoclonal antibody against CD20, was the first monoclonal antibody to be approved by the FDA for lymphoma. Results from clinical trials have shown that anti-CD20 monoclonal antibodies, which can be used unchanged or as carriers for radionuclides or toxins, have significant therapeutic effects and can be administered with minimal side effects. Several studies have also shown anti-CD20 monoclonal antibodies to be useful against autoimmune diseases such as rheumatoid arthritis.
Phage display is an effective tool in genomics and drug development. The ability to obtain high affinity binders for any antigen with decreased cost and time expenditure has been useful in the development of therapeutics. Single chain variable fragments (scfvs) are the smallest antibody fragments which maintain the specificity and the affinity of the parent antibody. ScFvs can be conjugated to radioisotopes, toxins, drugs, highly effective Fc region isotypes, and gene delivery vectors, which can be employed in diagnosis and therapy.
Studies have shown that chimeric and humanized forms of anti-CD20 demonstrate improved clinical efficacy, as compared with murine anti-CD20 monoclonal antibody, as well as a decrease in HAMA (human anti-mouse antibody) response, which is frequently observed with murine antibodies. Although chimerization of murine antibodies through protein engineering can retain the affinity and specificity of the parental molecule, this strategy is time-consuming and does not yield the benefit of fully human antibodies.
To further improve the efficacy of anti-CD20 antibodies, a na•ve human scFv phage library (Tomlinson I+J) was used in the present study to screen for human antibodies against CD20. A cell based screening strategy was employed in this study by establishing a tetracycline inducible CD20 CHO cell line, to better mimic the microenvironment where CD20 and anti-CD20 antibody interaction would occur. Panning of the phage library against the CD20 transfected cell lines yielded clones with affinity for CD20 antigen. PCR analysis showed the expected 935bp scFv band, and monoclonal ELISA showed an affinity for CD20 antigen. Periplasmic protein was extracted from the clone and subjected to dot blot analysis, showing that in its native form, Clone 3 had an affinity for CD20. A fusion gene with the CD20 extracellular domain and an in vivo biotinylation domain was constructed for the isolation and characterization of antibody fragments. A functional biotinylated CD20 fusion protein was obtained from bacterial production and western blot analysis under denaturing conditions yielded a 20kDa band as expected.

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Microbiology Commons

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