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|Title:||Model systems for the retargeting of T cell-mediated immunity to tumor antigens|
|Author(s):||Wilson, Erik Robert|
|Doctoral Committee Chair(s):||Kranz, David M.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
Health Sciences, Immunology
|Abstract:||B cells and T cells are involved in the response against foreign antigens. Specificity of B cells is determined by antibodies while the specificity of T cells is determined by similar proteins called T-cell receptors. One function of T cells is surveillance against tumor cells. Progressing tumors have escaped T cell surveillance, but the immune system could still be effective if the T cell immune response could be retargeted against the tumor. A strategy for accomplishing this is the construction of a bispecific antibody which binds a tumor cell antigen and a T-cell receptor, bringing the T cell into contact with the tumor cell and activating the T cell to destroy the tumor cell.
A bispecific antibody model system was constructed to examine two questions: First, what is the affinity of the anti-tumor antibody needed to transmit an activation signal to the T cell? Second how many antigens are required on the target cell to activate T cells? The model system utilized fluorescein as a "surrogate" tumor antigen and an anti-fluorescein antibody (4-4-20) as one half of the bispecific antibody (the other half being an anti-T cell receptor antibody). Conjugated forms of fluorescein with nearly a 100-fold range of affinities for 4-4-20 were synthesized and characterized. These fluorescein conjugates were attached to tumor cells as targets at a range of antigen densities. T cell activation was measured by the killing of these target cells. A clear density threshold was established of between 2,000 and 24,000 antigen molecules per cell for the highest-affinity fluorescein conjugate. The density threshold increased to between 19,000 and 54,000 antigen per cell for the next-highest-affinity fluorescein conjugate. The density threshold increased to over 270,000 antigen per cell for the lowest affinity fluorescein conjugate. These antigen densities are within the range seen for many tumor antigens and the affinities are within the range seen for anti-tumor antibodies. The results demonstrate the antibody affinities that should be used when targeting particular tumor antigens in therapy with bispecific antibodies.
Because very little is known about T-cell receptor affinity in normal T cell function, another system was developed to address this issue. A chimeric antibody/T-cell receptor was constructed from the variable regions of anti-fluorescein antibody (9-40) and the constant regions of a T-cell receptor with the intention of using it to study affinity and ligand density requirements for the activation of native T cells. DNA for the chimeric receptor was transfected into a T lymphoma. To detect and select for the chimeric receptor, an antibody (4B5) was produced that was specific for the variable regions of antibody 9-40. Antibody 4B5 was attached to magnetic beads and used to select transfectants that expressed the chimeric receptor. The transfectants were shown to bind fluorescein but did not show significant levels of activation upon binding. Nevertheless, the magnetic selection method should be useful in obtaining cells that are transfected with genes that encode multimeric surface proteins such as other chimeric receptors.
|Rights Information:||Copyright 1994 Wilson, Erik Robert|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9512597|