Ka Chester

Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library

We present a system for cancer targeting based on single–chain Fv (scFv) antibodies selected from combinatorial libraries, produced in bacteria and purified by using an engineered tag. Combinatorial libraries of scFv genes contain great diversity, and scFv antibodies with characteristics optimized for a particular task can be selected from them using filamentous bacteriophage. We illustrate the benefits of this system by imaging patients with carcinoembryonic antigen (CEA)–producing cancers using an iodine–123 labeled scFv anti–CEA selected for high affinity. All known tumor deposits were located, and advantages over current imaging technology are illustrated. ScFvs are produced in a cloned form and can be readily engineered to have localizing and therapeutic functions that will be applicable in cancer and other diseases.

PHAGE LIBRARIES FOR GENERATION OF CLINICALLY USEFUL ANTIBODIES

Insertion of antibody genes into filamentous bacteriophage makes it possible to generate and screen libraries of 10(7) or more antibodies. Each phage expresses an antibody on its surface and contains the corresponding antibody gene. Genes that encode antibodies with desired characteristics are readily selected and their antibodies expressed as soluble proteins in Escherichia coli. We used this system to produce an antibody to carcinoembryonic antigen with higher affinity and better tumour specificity than antibodies currently in use.

Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography

A new procedure is described for the purification of an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), referred to as MFE-23, from bacterial supernatant. A simple insertion of a hexa-histidine tail fused at the C-terminus (MFE-23 His) provides an affinity tag which selectively binds to transition metal ions immobilised on an iminodiacetic acid (IDA) derivitised solid phase matrix. This method proved to be superior to standard CEA antigen affinity chromatography in the following ways. (1) A higher yield was produced (10 mg/l as opposed to 2.2 mg/l of bacterial supernatant). The latter figure was largely affected by the limited availability (size of the column) of immobilised CEA antigen. (2) Scale-up was relatively simple and less costly. (3) The risk of tumour derived antigen leaching from the column is eliminated. Results showed that immobilised Cu2+ ions were more effective than Ni2+ and Zn2+ ions in retaining the His tagged product giving a 90% pure product on elution. Clinical grade material was generated using size exclusion chromatography to remove aggregated material, and Detoxi gel to remove bacterial endotoxins. Validation assays to measure DNA, copper and endotoxins were performed to assess the levels of contaminants. MFE-23 His retained 84% antigen binding after 6 months storage at 4 degrees C and > 75% after radiolabelling. Further experiments confirmed that the His tail did not affect biodistribution and tumour localisation in nude mice bearing human colorectal tumour xenografts.

Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT)

Carboxypeptidase G2 (CP) is a bacterial enzyme, which is targeted to tumours by an antitumour antibody for local prodrug activation in antibody-directed enzyme prodrug therapy (ADEPT). Repeated cycles of ADEPT are desirable but are hampered by human antibody response to CP (HACA). To address this, we aimed to identify and modify clinically important immunogenic sites on MFECP, a recombinant fusion protein of CP with MFE-23, a single chain Fv (scFv) antibody. A discontinuous conformational epitope at the C-terminus of the CP previously identified by the CM79 scFv antibody (CM79-identified epitope) was chosen for study. Modification of MFECP was achieved by mutations of the CM79-identified epitope or by addition of a hexahistidine tag (His-tag) to the C-terminus of MFECP, which forms part of the epitope. Murine immunisation experiments with modified MFECP showed no significant antibody response to the CM79-identified epitope compared to A5CP, an unmodified version of CP chemically conjugated to an F(ab)2 antibody. Success of modification was also demonstrated in humans because patients treated with His-tagged MFECP had a significantly reduced antibody response to the CM79-identified epitope, compared to patients given A5CP. Moreover, the polyclonal antibody response to CP was delayed in both mice and patients given modified MFECP. This increases the prospect of repeated treatment with ADEPT for effective cancer treatment.

Recombinant anti-carcinoembryonic antigen antibodies for targeting cancer

Abstract Antibodies can be used to target cancer therapies to malignant tissue; the approach is attractive because conventional treatments such as chemo- and radiotherapy are dose limited due to toxicity in normal tissues. Effective targeting relies on appropriate pharmacokinetics of antibody-based therapeutics, ideally showing maximum uptake and retention in tumor and rapid clearance from normal tissue. We have studied the factors influencing these dynamics for antibodies against carcinoembryonic antigen (CEA). Protein engineering of anti-CEA antibodies, in vivo biodistribution models, and mathematical models have been employed to improve understanding of targeting parameters, define optimal characteristics for the antibody-based molecules employed, and develop new therapies for the clinic. Engineering antibodies to obtain the desired therapeutic characteristics is most readily achieved using recombinant antibody technology, and we have taken the approach of immunizing mice to provide high-affinity anti-CEA single-chain Fv antibodies (sFvs) from filamentous bacteriophage libraries. MFE-23, the most characterized of these sFvs, has been expressed in bacteria and purified in our laboratory for two clinical trials: a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumor deposits are detected by a hand-held probe during surgery. Both these trials showed that MFE-23 is safe and effective in localizing tumor deposits in patients with cancer. We are now developing fusion proteins that use the MFE-23 antibody to deliver a therapeutic moiety; MFE-23:: carboxypeptidase G2 (CPG2) targets the enzyme CPG2 for use in the antibody-directed enzyme prodrug therapy system and MFE::tumor necrosis factor alpha (TNFα) aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

Taking engineered anti-CEA antibodies to the clinic.

There is a need to improve on existing targeting technologies in order to develop effective cancer therapy. We have investigated this for colorectal cancer using antibodies directed against carcinoembryonic antigen (CEA). Chemical and molecular protein engineering has been used to produce antibody molecules which differ in molecular weight, affinity, valency and specificity. These have been characterised and tested in animal tumour models and clinical trials to test the parameters important for optimising tumour penetration, increasing residence time in viable areas of the tumour, accelerating clearance from normal tissues and improving therapeutic efficacy.

Clinical Applications of Phage-Derived sFvs and sFv Fusion Proteins

Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNFα aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

Exemplifying guidelines for preparation of recombinant DNA products in phase I trials in cancer: preparation of a genetically engineered anti-CEA single chain Fv antibody

Products of recombinant DNA technology have potential for the diagnosis or treatment of cancer. There is a need to investigate whether they function by the intended mechanism in small phase I clinical trials before their suitability for more extensive studies can be assessed. Quality and safety of these products should be assured prior to their use in humans in a way which is appropriate to the preliminary nature of the trials but not inhibitory to progress. The Cancer Research Campaign control recommendations for products derived from recombinant DNA technology (Begent RHJ and associates. Eur J Cancer 1993, 29A, 13, 1907-1910) provide guidelines for the production of new biotechnology products in academic research units within a relatively short time, while ensuring appropriate quality and safety. The practical application of the guidelines requires that solutions are found for the quality and safety issues during the production of recombinant products. We describe an approach to the relevant quality and safety issues during and after the production and purification of a genetically engineered anti-carcinoembryonic antigen (CEA) single chain Fv (scFv) antibody for a phase I trial of radioimmunoguided surgery with the intention of providing a model for other products.