Date of Award

2008

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Natalie Sutkowski

Second Advisor

Michael Nishimura

Third Advisor

David T. Kurtz

Fourth Advisor

Azizul Haque

Fifth Advisor

Stephen Tomlinson

Abstract

We have developed a novel approach for the production of potentially therapeutic human monoclonal antibodies in vitro. This method holds great promise for generating therapeutic antibodies against infectious disease, cancer, and inflammation. The complex nature of current methods for producing human monoclonal antibodies is a major impediment to large scale passive transfer immunotherapy. Our novel technique utilizes Epstein-Barr virus immortalized tonsil or peripheral blood B cells from healthy human volunteers. These B cells are forced to undergo non-specific differentiation to become mature IgG antibody producing plasma "like" cells that continuously proliferate in vitro. Using this method, millions of transformed B cells can be generated and screened for production of antibodies of desired specificity. Optimization of EBV infection, reaching nearly 100%, was achieved by combining viral concentration, using centrifugal ultrafiltration with "spinfection". This allowed for transformation of the largest possible B cell repertoire from human tonsil samples, a rich source of B cells. EBY immortalized tonsil B cells were forced to undergo differentiation into IgG secreting plasma "like" cells through addition of anti-Igm (Fab')2, Interleukin (IL)-4 and IL-6 or anti-IgM (Fab')2, soluble CD40L and BAFF once a week for three weeks. We hypothesized that anti-IgM (Fab')2 would mimic antigen activation of the BCR and CD40L would mimic T cell costimulation. ELISA analysis of supematants from the immortalized cells indicated that we efficiently induced immunoglobulin isotype class switching from IgM to IgG after several weeks, and that the transformed B cells continued to secrete IgG for months in vitro. Flow cytometry analysis revealed that IgG secreting immortalized B cells have a differentiated surface expression phenotype that resembles plasma cells. We are currently applying this differentiation method to produce therapeutic and diagnostic antibodies against avian influenza virus hemagglutinin (H5 HA). EBY immortalized tonsil and peripheral blood B cells produced IgG antibodies specific for H5 HA. Overall, the results indicate that differentiation of immortalized human B cells can be influenced in vitro, with the ultimate purpose of exploiting their therapeutic potential.

Rights

All rights reserved. Copyright is held by the author.

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