Sharma, V;
(2006)
Generation of single chain Fv protein a fusion molecules for melanoma therapy.
Doctoral thesis , UCL (University College London).
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Abstract
The high incidence rate and absence of a suitable treatment pose a great threat for melanoma patients. Surgery, the main form of treatment for clinically detectable melanoma is unsuitable for micrometastatic disease. Therefore there is a clear need for the development of new adjuvant therapies. We have cloned RAFT3 a single chain Fv specific for melanoma associated chondroitin sulphate proteoglycan (mCSP) fused with 2 artificial S. aureus protein A domains (ZZ). The fusion molecule (R3ZZ) has the potential to redirect both a humoral and a cellular immune response to melanoma. R3ZZ was expressed, secreted in E.coli and the purified protein tested in vitro and in vivo. The binding ability of the fusion protein to bind to human IgG sepharose, mCSP and the Clq component of the complement system were determined. In vitro antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays showed that RAFT3ZZ caused 80% specific ADCC after 4 hours and 65% specific CDC after 48 hours. The ability of the fusion molecule to prevent tumour formation was tested in vivo in Balb/c nude mice bearing human melanoma xenografts. The dosing regimes for the therapy were also investigated but remain inconclusive. Melanoma growth retardation was observed in some experiments but not in others. Immunocompetent CBA mice were used to evaluate the potential side effects of repeated administration of RAFT3ZZ. Tail bleeds analysed showed only a weak immune response against scFv part of the therapeutic molecule after 5 boosters. The blood samples from immunised CBA mice showed RAFT3 scFv to be much more immunogenic than the fusion molecule. This phenomenon was contrary to all published literature and thus investigated further. The therapeutic potential of RAFT3 scFv was tested in vivo in Balb/c nude mice with results similar to RAFT3ZZ Extensive work was carried out to locate the epitope inducing the immune response against single chain Fv fragments. We have tenetively identified two potential epitopes responsible for the immunogenic properties of scFvs. The human c-myc detection tail and the former interphase of the V domains with the Cl and ChI domains.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | Generation of single chain Fv protein a fusion molecules for melanoma therapy |
| Identifier: | PQ ETD:593238 |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1445914 |
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