Kerry A. Chester

Clinical issues in antibody design

Antibody genes can now be cloned and expressed in various ways to give new versions of antibodies that possess reduced immunogenicity, improved affinity, altered size, increased avidity and novel effector functions. The task for any clinical application is, first, to define a relevant target, and then to design the optimal antibody-based therapeutic molecule to react with that target. This article reviews these improved antibody-based molecules and examines their role in cancer therapy.

In vitro characterization of a recombinant 32P-phosphorylated anti-(carcinoembryonic antigen) single-chain antibody

Abstract The major limitations of monoclonal antibody conjugates as therapeutic agents have been their poor tumour targeting, inadequate tumour penetration and immunogenicity. More even and deeper tissue penetration has been demonstrated with smaller antibody fragments. The smaller size and absence of an Fc segment may contribute to a lowered immunogenicity with single-chain antibodies (scFv) and also permit their recombinant engineering and bacterial expression. We describe the successful engineering, expression and pre-clinical characterisation of a phosphorylatable “kemptide” (Leu-Arg-Arg-Ala-Ser-Gly) anti-carcinoembryonic antigen (anti-CEA) scFv (PKS-scFv), for use as a radioimmunotherapeutic agent. Specifically, a yield of 6 mg/l induced culture was obtained. Site-specific phosphorylation was demonstrated without loss of specificity. In vitro assays revealed a selective cytotoxicity of 32P-PKS-scFv for high-CEA-expressing LS-174T cells compared to the low-CEA-expressing HT-29 cells, with a rapid internalisation rate.

Opportunities with phage technology and antibody engineering of fusion proteins

Abstract Directed enzyme therapy has enormous potential for cancer treatment but is a complex and technically demanding approach requiring optimisation of several components. The choice of enzyme/prodrug combinations are reviewed elsewhere in this book and in this chapter we concentrate on the antibody/enzyme component. We consider the recent advances in protein engineering that have resulted in new methods of producing antibodies which may have major advantages when incorporated into ADEPT. In addition we review the progress with the new fusion protein approach and the prospects for the use of non-immunogenic antibody-enzymes.

The use of a surface active agent in the protection of a fusion protein during bioprocessing: BLAS et al.

The bioprocessing of a fusion protein is characterised by low yields and at a series of recovery and purification stages that leads to an overall 90% loss. Much of this apparent loss is due to the denaturation of a protein, missing a vital affinity ligand. However, there is evidence of the protection of degradation products which occurs in the presence of shear plus air/liquid interfaces. This study seeks out to characterise the loss and use ultra‐scale‐down studies to predict its occurrence and hence shows these may be diminished by the use of protective reagents such as Pluronic F68.

A Strategy for Characterizing Antibody/Antigen Interactions Using ProteinChip® Arrays

If antibody-based therapeutics are to be rationally designed to give optimal interactions with their target antigens, it is essential to understand these antibody/antigen interactions as fully as possible. This has been greatly advanced by the recent developments in mass spectrometry (MS), structural identification, and bioinformatics, which allow the relatively simple and rapid characterization of protein—protein interactions in a high-throughput manner. Previous methods of mapping an antibody-binding site (epitope) on an antigen have relied on peptide libraries (1). Such methods are suitable for the identification of continuous amino acid sequences (2) but not for those that are conformationally dependent. This latter group forms a major category of antibody/antigen interactions.

Phage Technology for Producing Antibody-Enzyme Fusion Proteins

For ADEPT approaches to be successful, site-specific delivery of the enzyme to the tumor is clearly essential. This is most readily achieved with antibodies, of which an extensive range exists covering a wide variety of antigens. Most of the early trials of ADEPT systems, from preclinical studies through to clinical trials, used conjugates whose attachment of antibody to enzyme was performed by chemical conjugation. Now these molecules can be joined at a molecular level by linking the genes. The resulting expressed product, known as a fusion protein, has both antibody and enzymic activities. For effective therapy with ADEPT, the antibody—enzyme conjugate must localize in the tumor and remain there after it clears from normal tissues. Then a nontoxic prodrug can be administered, and it is activated by the localized enzyme to produce a cytotoxic drug at the tumor site. A perceived advantage of ADEPT systems is that they potentially include an amplification step (each enzyme molecule produces many molecules of the active drug within the tumor) and a bystander effect (destruction of neighboring cells and those binding the antibody fragment) because the final cytotoxic molecule is produced at the cell surface and is small, allowing diffusion throughout the tumor. The potential disadvantages of the approach are that the antibody—enzyme conjugate must be stable for long periods in the tumoral environment and it must be nonimmunogenic to allow repeated therapy. The use of fusion proteins should result in very stable antibody enzyme conjugates, and there is the potential to construct them from either human proteins or proteins engineered to minimize immunogenicity.