Robert Anthony Spooner

Applications of monoclonal antibodies in clinical oncology.

Application of monoclonal antibodies (mAbs) to clinical settings has proved to be slower than originally hoped. However, a recent conference provided evidence that the field is coming of age, and that antibodies, and their recombinant derivatives, will continue to find a role in the clinic.

Construction, characterisation and kinetics of a single chain antibody recognising the tumour associated antigen placental alkaline phosphatase

The murine monoclonal antibody H17E2 recognises placental alkaline phosphatase (PLAP), an antigen present in the human term placenta and also expressed by many tumours. The antibody is of value in both immunoscintigraphy and radioimmunotherapy in testicular and ovarian cancer. The small size of genetically engineered single chain antibodies (SCAs) should give diagnostic and therapeutic advantages of improved tumour penetration and increased blood clearance compared to IgG. Employing recombinant DNA techniques a SCA based on H17E2 has been expressed in Escherichia coli and has been shown to bind placental alkaline phosphatase specifically. When administered to nude mice bearing human tumour xenografts, the H17E2 SCA effectively localised to tumour whilst a co-administered non-specific SCA did not. H17E2 SCA achieves tumour: blood ratios that are superior to those achieved with whole IgG, probably owing to its rapid blood clearance. We conclude that the H17E2 SCA is suitable for further investigation as an agent for clinical imaging and therapy. Additionally, the SCA can also be used for the construction of antibody based fusion proteins to target other effector functions to tumour cells.

Genetically engineered antibodies for diagnostic pathology

Antibody genes can be cloned, genetically manipulated, and expressed in both homologous and heterologous expression systems to produce viable antigen-binding proteins complete with natural effector functions. Manipulation of antibody genes permits the expression of fusion proteins or truncated proteins that retain antigen-binding activity. The new antibody technologies are becoming increasingly sophisticated, permitting the alteration of antigen-binding responses, the transfer of antigen specificity between antibodies, and the expression of minimal-size antigen-binding protein domains. These new molecules have been made mostly for studies on function or to provide molecules suited for in vivo diagnosis and therapy; very few have been specifically designed for, or used for, diagnostic histopathology. We describe here the adaptation of small antibody derivatives for use in immunohistochemistry. Molecules suitable for this purpose need only to possess specific antigen-binding ability and some means of detection of antigen-bound material. Detection could be by recognition of a genetically fused flag or tag epitope, by the fusion of an enzyme whose activity can be assayed, or by fusion with a protein that can interact with pre-existing histopathological reagents.

Deoxyribonuclease I (DNase I) A Novel Approach for Targeted Cancer Therapy

A number of phosphodiesterases, some of which possess additional biological activities (e.g., antitumor, immunosupressive, and so on), have been considered for use in targeted tumor therapy. We propose Deoxyribonuclease I (DNase I), a compact, monomeric enzyme, as a very attractive candidate for targeting to tumor cells. Only a small amount of enzyme targeted to a cell needs to enter the nucleus in order to degrade the chromosomal DNA, making a cell incapable of further replication. We describe preliminary data on the construction of a potent single-chain antibody (scFv) immunotoxin based on bovine pancreatic DNase I. The use of a mammalian enzyme should be much less toxic and less immunogenic than current immunotoxins and may expand the current limits of immunotoxin therapy.

Expression of immunoglobulin heavy chain-ricin a chain fusions in mammalian cells

Mammalian cell lines were transfected with antibody heavy (H) chain-ricin A chain gene fusions in attempts to assemble a recombinant immunotoxin. We found that a light chain-secreting mouse plasmacytoma cell line can be transfected stably with such a chimaeric gene, but only if the ricin A chain portion is disarmed by genetic means prior to transfection; if not, stable transfection appears to select for genetic inactivation of the transfected gene. Co-expression of an antibody heavy chain-ricin A chain fusion with light chain in non-lymphoid cells results in cell death. We conclude that the ricin A chain moiety retains biological activity precluding the expression of biologically active antibody-ricin A chain fusion proteins in mammalian cells.

A recombinant single-chain antibody interleukin-2 fusion protein.

Recombinant interleukin-2 (rIL-2) therapy has been shown to be of value in the treatment of some cases of melanoma and renal cell carcinoma. However its use can be limited by severe systemic toxicity. Targeting rIL-2 to the tumour should improve the anti-tumour immune response and decrease the systemic toxicity. With this aim we have employed recombinant DNA techniques to construct a single chain antibody interleukin-2 fusion protein (SCA-IL-2). The protein used in this model system comprises the variable domains of the anti-lysozyme antibody D1.3 fused to human IL-2. It has been expressed by secretion from Escherichia coli and the purified product possesses antigen binding specificity and retains the immunostimulatory activities of rIL-2. This approach can be taken to generate SCA-IL-2 proteins that bind to appropriate cellular antigens. In vivo administration of a tumour binding SCA-IL-2 should result in a localised high concentration of IL-2 in tumour tissues, maximising the anti-tumour immune response, whilst keeping systemic side effects to a minimum.

A universal antibody-derived targeting agent

We report bacterial expression of a single-chain antibody (ScFv) reactive against the haptens 4-hydroxy-3 nitrophenylacetic acid (NP) and 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) that is suitable for targeting to mammalian cells in vitro in a novel two-step targeting strategy. Hapten-derivatized primary antibodies of known specificity, bound to target cells, can capture the ScFv. Specificity resides in the interaction of the primary targeting antibody with the target and the interaction of the ScFv for NP/NIP, since the ScFv does not bind cells and nonderivatized antibodies bound at cells cannot capture the ScFv. The ScFv described here can therefore be considered as a universal agent for delivery of drugs, toxins, or radionuclides to any cell type for which a previously characterized antibody exists.

Patent Update Oncologic, Endocrine & Metabolic: Anticancer antibodies: patent activity in 1993

1993 saw a number of patent applications in the field of anticancer antibodies. This review is limited to three areas that have been improved by work in this year.The first of these is the extension of the range of antigenic targets available in the antitumour repertoire, partly by the development of new monoclonal antibodies (mAbs) to previously characterised targets, and partly by the characterisation of new targets and mAbs that recognise them. Of necessity, only a handful of these have been mentioned here, but they should give a broad view of the years activities. Such inventions are, of course, useful not only for therapy, but also for diagnosis. Indeed, many of the applications are supported by in vitro staining data.The second area under consideration is that of radio-labelling mAbs. The emphasis here is on in vivo use. Two examples given here show the development of novel macrocyclic compounds that can chelate a range of radiometals: the third describes improvements to a completely different appr...