Genetic Delivery of Fusion Proteins for Immune Gene Therapy of Cancer

Abstract

AbstractThe University of ManchesterMajid Mostafaie-MehrDegree of Doctor of PhilosophyGenetic Delivery of Fusion Proteins for Immune Gene Therapy of Cancer2013Background: Monoclonal antibodies have widely been used in cancer therapy andtheir number is growing. There were 165 candidate mAbs as anti-cancer agents inclinical trials in 2012 (Reichert and Dhimolea 2012) and of this number there were11(7%) in phase III. The administration of monoclonal antibodies to patients, inprotein form, have several draw backs including: cost, pharmacokinetic issues,difficulty in production and purification all which require to be addressed. However,the delivery of the same monoclonal antibody in nucleotide form and in vivoproduction of the same protein has the potential to bypass many of these currentdrawbacks.Hypothesis: The TAA binding scFv linked to a Fc region gene with the CH1 genedeleted to make its size smaller for better penetration into tumour mass, might bebiologically active when expressed, first systemically by adenoviral vectors or byretroviral vectors in TILs to be expressed at the vicinity of tumour cells to mediateADCC or CDC leading to tumour elimination.Aims and objectives: Identify whether the constructed twelve fusion proteins werebiologically active and if these fusion genes can successfully be introduced into anadenoviral or retroviral vector. Subsequently, investigate if peripheral blood T cellshave the required machinery to express bioactive soluble recombinant mAb andquantify serum concentration. Finally, investigate whether the expressed fusionproteins are able to trigger ADCC or CDC.Methods: MFE23 which recognises CEA and B1.8 which recognises hapten NIPwere used as target and control scFvs respectively. Both of these scFvs were fused tomouse Fc regions of mIgG1, IgG2a and mIgG3. These fusion genes were transferredto adenoviral vector for systemic expression and to retroviral vectors for expressionby T cells. The fusion protein expressed by these vectors if biologically active may beable to mediate cytotoxicity in tumour cells.Results: All constructs showed biological activity in ELISA. The expressedMFE23.mIgG1 (M1) and MFE23.mIgG2a (M2) proteins from HeLa cells weredetectable by Western Blot and were shown to be able to target CEA expressing cells.The recombinant retrovirus transduced T cells were shown to be able to secreteapproximately 25-45ng/ml of recombinant protein from 1x106 T cells/ml/24 hours.None of the constructs showed any significant anti-tumour effect in an ADCC assaybut a CDC assay showed a weak trend towards specific killing with MFE23.mIgG2acompared to the control.Conclusion: These results showed M1, M2 and M3 and their controls werebiologically active when transiently transfected and when expressed in HeLa cells byadenoviral and retroviral vectors. The results also demonstrate T cells have thenecessary machinery to produce soluble recombinant antibodies. The ADCC andCDC assays require additional optimisation with these constructs before be able toconclude whether it is better to switch to other scFvs.Fc fusion constructs.

Date of Award	3 Jan 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Robert Hawkins (Supervisor) & Waquas Waheed (Supervisor)