Vahid Dastjerdi, F; (2016) Novel Recombinant Antibodies to EGFRvIII. Doctoral thesis , UCL (University College London).

Abstract

Epidermal growth factor receptor variant III (EGFRvIII) is the most common mutant form of EGFR which is constitutively active independent of ligand binding. EGFRvIII i s a tumour specific antigen and only expressed in cancer cells, which make it an ideal target for therapeutic application. It has been shown that EGFRvIII expression drive tumour progression and is associated with poor prognosis. It is expressed in different types of cancer, particularly glioblastoma but is rarely observed in normal tissue. Antibodies to EGFRvIII have potential to deliver potent new cancer therapies such as example chimeric antigen receptor (CAR) T-cells, but available antibodies are few and tend to be limited by low affinity or cross reactivity with EGFR. EGFRvIII protein is characterised by deletion of 267 amino acids in the extracellular domain, fusing exons 1 and 8 to create a novel junctional peptide with a unique glycine. Mice do not naturally make EGFRvIII but the region around the murine EGFRvIII junction is identical to that in humans, indicating that vaccinating mice with artificially generated murine EGFRvIII (mEGFRvIII) could give rise to anti-EGFRvIII antibodies that have minimum cross-reactivity with human EGFR. A vaccination approach was developed using mEGFRvIII protein and the junctional peptide peptide (PEP3). EGFRvIII sero-conversion was confirmed by flow cytometry and splenic lymphocytes of the immunised mice were subsequently used as a source of V-regions to generate single chain Fv (scFv) phage-display libraries of 8 a pp roxi m ate ly 10independent clones. Five different anti-EGFRvIII-specific scFvs were isolated from the libraries through a combination of panning strategies using peptide, recombinant antigen and cells. The lead clone (C18), was generated with a h exa h i sti d i n e tag (his tag) and purified by immobilised metal affinity chromatography. C18 bound to EGFRvIII with a KD of 72 nM and showed no binding to EGFR. It also bound specifically to EGFRvIII expressing cells. The C18 scFv was therefore taken forward to make a second generation human CAR T-cell to assess killing of E G F RvIII-expressing tumour cells in vitro. The cytotoxicity results showed that C18-CAR-T was selectively able to kill EGFRvIII-expressing tumour cells without showing any toxicity to cells expressing EGFR. These results (i) exemplify a means to obtain anti-EGFRvIII scFvs by a combination of immunisation and antibody phage display technology and (ii) and support the underlying hypothesis that EGFRvIII-specific antibodies have potential to deliver potent T-cell mediated anti-cancer therapy.

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