We obtain the cancer-reactive scFvs from filamentous phage libraries that contain many millions of diverse antibody specificities and we engineer the scFvs in a format designed or a particular therapy. For example, antibody-directed enzyme-prodrug therapy (ADEPT) of cancer, a treatment that uses a systemically administered anti-tumour antibody-enzyme complex to localize enzyme in tumours. In a second stage, a prodrug is administered and is selectively converted into an active cytotoxic drug by enzyme at the tumour site. [Fig 2].

ADEPT has the potential to generate high concentrations of cytotoxic agent selectively within tumours but it is key for success that non-tumour associated enzyme is effectively cleared from blood and other normal tissues before prodrug administration. A recombinant fusion protein, comprising MFE-23 fused to the enzyme carboxypeptidase G2 (CPG2), has been designed and produced by the group to meet this challenge [Sharma et al 2005, Kogelberg et al 2006]. The therapeutic protein is expressed and purified from yeast Pichia pastoris, and the post-translational glycosylation added by this organism is being exploited to control blood clearance via the mannose receptor [Fig 3] and modify bio-distribution. The therapeutic system is effective in pre-clinical tests [Sharma et al., 2006] and is currently in Phase I/II Clinical Trials [Mayer et al., 2006].

The strong translational theme of the group is central to our research thinking. The therapeutics designed by the team can be brought to clinical trial because the group have a dedicated facility which can produce clinical grade microbially-expressed recombinant proteins in compliance with Good Manufacturing Practice (GMP) [Fig 4] [Tolner et al, 2006 a,b;].

The Group are also developing new molecular formats for radioimmunotherapy (RIT) and drug delivery and we are exploring a series of scFv fusion proteins with human serum albumin (HSAbodies). These molecules are designed to be multivalent for antigen, have controllable pharmacokinetics and avoid the human immune system. We have shown that HSAbodies have specific tumour uptake and retention [Fig 5] and that there is little kidney accumulation of radiolabelled material during elimination, which is particularly important for RIT.

We have recently been using scFv molecules to target iron oxide magnetic nanoparticles [Fig 6] which could be used for specific cancer imaging using MRI. A therapeutic application, by creating localized hyperthermia of cancerous tissue upon application of an alternating magnetic field to targeted nanoparticles is also being explored.

Related Groups

Effect of Tumour Biology on Therapeutic Response
- Barbara Pedley

Fig 1. IgG (150 kD) and scFv (27kD). For scFv, antibody VH and VL chains are genetically tethered with a flexible linker to produce a single polypeptide chain.

Fig 2. ScFv targets enzyme (blue) to antigen on cancer cell (red). Targeted enzyme subsequently catalyzes prodrug into active cytotoxic (green). The toxic drug is thereby restricted to cancerous areas and does not harm healthy organs.

Fig 3. Indirect fluorescence confocal microscopy of single cell (cytoplasm green, nucleus blue) showing MFECP fusion protein (pink) internalized by human mannose receptor.

Fig 4. Making recombinant therapeutics in GMP facility.

Fig 5. In vivo localisation of 131I HSAbody to viable regions of human tumour xenografts, 24hr post injection. Phosphor image (LHS) and H&E of tumour sections.

Fig 6. Specific localisation of scFv-functionalized magnetic particles to human cancer cells in vitro.

Kogelberg H, Tolner B, Sharma SK, Lowdell MW, Qureshi U, Robson M, Hillyer T, Pedley RB, Vervecken W, Contreras R, Begent RHJ, Chester KA (2006) Clearance Mechanism of Mannosylated Antibody-Enzyme Fusion Protein used in Experimental Cancer Therapy. Glycobiology 17: 36-45. (Pubmed)

Mayer A, Francis RJ, Sharma SK, Tolner B, Springer CJS, Martin J, Boxer GM, Bell J, Green AJ, Hartley JA, Cruickshank C, Wren J, Chester KA, Begent RHJ (2006). A Phase I Study of Single Administration of Antibody-Directed Enzyme Prodrug Therapy with the Recombinant Anti-Carcinoembryonic Antigen Antibody-Enzyme Fusion Protein MFECP1 and a Bis-Iodo Phenol Mustard Prodrug. Clin Cancer Res.12: 6509-16. (Pubmed)

Tolner B, Smith L, Begent RHJ & Chester KA. (2006a) Production of Recombinant Protein in Picha pastoris by Fermentation. Nature Protocols 1:1006-1021.

Tolner B, Smith L, Begent RHJ & Chester KA. (2006b) Expanded Bed Adsorption Immobilized Metal Affinity Chromatography. Nature Protocols 1:1213–1222.

Sainz-Pastor N, Tolner B, Huhalov A , Kogelberg H, Lee YC, Zhu D, Begent RHJ, Chester KA (2005) Deglycosylation to obtain stable and homogeneous Pichia pastoris–expressed N-A1 Domains of Carcinoembryonic Antigen. International Journal of Biological Macromolecules 39: 141-150. (Pubmed)

Sharma SK, Pedley RB, Bhatia J, Boxer GM, El Emir E, Qureshi U, Tolner B, Lowe H, Michael NP, Minton N, Begent RH, Chester KA (2005) Sustained tumor regression of human colorectal cancer xenografts using a multifunctional mannosylated fusion protein in antibody-directed enzyme prodrug therapy. Clin Cancer Res 11: 814-825. (Pubmed)

Chester K, Pedley B, Tolner B, Violet J, Mayer A, Sharma S, Boxer G, Green A, Nagl S, Begent R (2004) Engineering antibodies for clinical applications in cancer Tumour Biol 25: 91-98. (Pubmed)