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Investigation of the effect of physical parameters on the design of tumour targeting agents

Casey, Joanne Lois; (1997) Investigation of the effect of physical parameters on the design of tumour targeting agents. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Tumour targeting using radiolabelled antibodies for radioimmunodetection (RAID) and radioimmunotherapy (RIT) has been studied for many years. The main factors that have limited clinical success are low tumour uptake, immunogenicity and poor therapeutic ratios. This thesis has applied current technology to make advances in this area of research. The effect of physical parameters (antibody size, valency, affinity and charge) on the design of tumour targeting agents was studied by constructing divalent (DFM) and trivalent (TFM) forms of the murine anti-CEA antibody A5B7 Fab' by chemical cross-linking. This involves partial reduction of the hinge disulphides to expose thiol (-SH) groups and subsequent reaction with a maleimide cross-linker to form a thioether bond at the hinge region. Previous studies have suggested that the stability of thioether bonds is superior to naturally occurring disulphide bonds present at the hinge region of IgG and F(ab')2. The aim was to compare the functional affinities and in vivo tumour targeting in nude mice bearing human tumour xenografts of DFM and TFM to similar sized parent IgG and F(ab')2. Radiolabelling with 131I and 90Y was also compared with a view to determine which combination would be optimal for RIT. Results clearly demonstrated a significantly faster on-rate of DFM compared to all other antibody forms and estimated dosimetry analysis suggested that DFM would be the most suitable antibody form radiolabelled with 131I for RIT. Both F(ab')2 and DFM showed high kidney uptake levels on labelling with which is unacceptable for RIT. Despite the improved tumour: blood ratios for TFM, the increased estimated dose to normal tissues and lower therapeutic effect in RIT studies suggests that the most promising combination with the radionuclide appears to be IgG. A humanised version of A5B7 hFab' has been constructed previously in order to reduce its immunogenicity in man. The in vivo stability of hDFM proved to be superior to hF(ab')2 in the nude mouse xenograft model. To study the safety, stability and tumour targeting of hDFM a clinical trial using 131I was described here including details of production, characterisation, pharmacokinetics and dosimetry. ScFv's are known to have favourable tumour targeting characteristics compared to whole antibodies for RAID. To evaluate the clinical potential of a scFv, the methodology to prepare a phage derived scFv with the aid of a subcloned hexahistidine tail was described here. To enhance the clinical potential of scFv's a construct consisting of a hinge region containing a single cysteine residue was constructed. This enabled site-specific 99mTc-labelling and could facilitate multimerisation. One of the major limitations revealed by this and other studies is the problem associated with renal accretion of antibody fragments. Various modification techniques and blocking effects were used here in attempt to reduce the kidney uptake levels in mouse models. Reduction of the pi of A5B7 Fab by attachment of NHS-ester groups was effective in lowering kidney uptake levels, but losses in immunoreactivity could limit this approach. Attachment of PEG (5kD) to DFM did not adversely affect immunoreactivity and increased the circulation time of DFM in vivo. This has implications for reducing kidney uptake levels at early time points, in addition PEG is known to reduce immunogenicity of proteins.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Investigation of the effect of physical parameters on the design of tumour targeting agents
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Health and environmental sciences; Radiotherapy
URI: https://discovery.ucl.ac.uk/id/eprint/10105812
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