Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531243
Title: A functional comparison of Campath-IH antibodies expressed in, and isolated from, different cellular sources
Author: Hale, Christine Betty
Awarding Body: University of London
Current Institution: King's College London (University of London)
Date of Award: 2001
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Abstract:
Campath-IH is a humanised antibody which has entered clinical trials for both rheumatoid arthritis and lymphoma. The clinical trial material was expressed in, and isolated from, Chinese Hamster Ovary (CHO) cells. It is proposed that variations observed in preliminary antibody-dependent cellular-cytotoxicity (ADCC) assays, in which human mononuclear cells were used as effectors and CHO Campath-1H was compared with Campath-IH prepared from rat YO cells, are correlated to host and/or culture condition related changes in antibody N-linked glycosylation. To investigate the hypothesis, CHO Campath-1H mediated assays including ADCC, monocyte mediated cytostasis, antigen engagement and subsequent crosslinking were analysed as was the antibody N-linked carbohydrate composition. The total removal of the carbohydrate ablated ADCC activity, decreased the cytostatic effect seen with intact antibody but did not alter antigen binding. Campath-IH cDNA was recloned into Celltech Glutamine Synthetase expression vectors and transfected into mouse NSO cells for antibody isolation. Comparisons of antibody made during NSO Campath-1H development from clone to fermentor and between CHO and YO Campath-1H with the various stages of NSO antibody revealed variations in both assay response and N-linked carbohydrate structure. The glycosylphophatidylinositol (GPI)-anchored antigen CDw52 recognised by Campath-1H was isolated from Wien 133 B cell cDNA by polymerase chain reaction and sequenced in preparation for expression cloning. Two sequence variants of the antigen were present in the cDNA, differing by two amino acids outside of the antigen coding region but, at a site controlling GPI-anchor attachment. Both cDNAs were expressed in CHO cell lines for comparison but only one could be detected by Campath-IH. In vivo and in vitro studies on the two forms are described. Chimeric forms of the T cell antigen CD4, linked to either of the CDw52 antigen GPI attachment sequences, were shown to be both expressed in CHO cells and detected by anti-CD4 antibodies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.531243  DOI: Not available
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