Gail Rowlinson-Busza

Targeted delivery of biologic and other antineoplastic agents.

This review summarizes several strategies under investigation for targeted delivery of antineoplastic agents to tumor cells, which avoids normal tissue damage. Monoclonal antibodies remain the molecules of choice for targeted therapy, and several improvements to immunotargeting are discussed. These improvements include the use of novel radioisotopes, cytokines, new linkers for chemotherapeutic drugs, more potent drugs, and improved immunotoxins. Bifunctional antibodies, in which one antigen-binding site recognizes a tumor-associated antigen and the other an antineoplastic agent, have been investigated for radioimmunotherapy and for the activation of cytotoxic cells for targeted immunotherapy. The activation of relatively nontoxic prodrugs by antibody-enzyme conjugates is increasingly being investigated in an effort to reduce the systemic toxicity associated with conventional chemotherapy. Finally, the use of liposomes as carriers of drugs or as activators of macrophages is described.

Effect of tumour necrosis factor on the uptake of specific and control monoclonal antibodies in a human tumour xenograft model

The investigations reported in this paper aim to exploit tumour necrosis factor (TNF)-induced vascular changes in an attempt to increase the tumour uptake of specific monoclonal antibody. The vascular permeability to monoclonal antibody of a human tumour xenograft increased 2.6-fold by 1 h post injection of 2.5 x 10(3) U of TNF, although this effect was lost by 3 h. The normal tissues also demonstrated increased vascular permeability to IgG, but to a lesser extent. Liver permeability increased 1.5-fold at 1 h but returned to the control value by 6 h. Lung permeability increased 1.4-fold at 1 h post injection and returned to normal by 3 h. Muscle values were not significantly increased compared with controls. The blood activity was cleared more quickly in the TNF-treated mice (t1/2 beta = 101 h, compared with 121 h in control mice). This was probably due to the increased vascular permeability in normal organs of treated mice. At 1 day and 3 days post injection, the tumour uptake of the specific, but not the control, antibody was significantly increased by 25% and 29% respectively. This resulted in an increase in the area under the tumour activity curve, and therefore tumour radiation dose, of 25% in treated compared with control mice. In addition, a consequence of the faster blood clearance of the isotope in the TNF-treated mice was a reduction in the area under the blood activity curve of 12%, thereby reducing systemic toxicity. The increase in vascular permeability to IgG following TNF injection resulted in both specific and control antibodies having improved access to the tumour antigens, and a transient increase in uptake was observed. Only in the case of the specific antibody was the increase maintained, since this antibody binds to the available antigenic sites, whereas the control antibody was cleared from the tumour without binding. No evidence of tumour necrosis was observed at the TNF doses given, nor was there any toxicity to the mice.

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.

90Y-Labeled antibody uptake by human tumor xenografts and the effect of systemic administration of EDTA

PURPOSE A human tumor xenograft model was used to compare the tumor and normal tissue uptake of a tumor-associated monoclonal antibody radiolabeled with 125I or 90Y. METHODS AND MATERIALS Nude mice bearing SC xenografts of the human colon adenocarcinoma, HT29, were injected with a mixture of 125I- and 90Y-DTPA-labeled AUA1 monoclonal antibody, which recognizes an antigen expressed on the surface of the tumor cells. In addition, the effect of systemic ethylenediaminetetraacetic acid (EDTA) administration on 90Y-labeled antibody clearance, tumor uptake of antibody and bone accumulation of 90Y was studied in a nude mouse model of intraperitoneal cancer. RESULTS Both the absolute amount (%id.g-1) and the tumor:normal tissue ratios were superior for the 90Y-labeled antibody, compared with the iodinated antibody, with the notable exception of bone. These results suggest that 90Y is a preferable isotope to iodine for radioimmunotherapy of solid masses, but that myelotoxicity, due to bone uptake of released 90Y, will limit the radiation dose which can be given when DTPA is used to chelate the 90Y. The 90Y-labeled antibody showed similar serum stability in vitro in the presence or absence of EDTA after incubation for up to 48 h. In vivo, urine excretion of 90Y was significantly enhanced in mice receiving daily injections of 20 mg EDTA for 3 days, commencing 2 h after intraperitoneal antibody administration, compared with control mice. There was no significant difference in the tumor uptake of 90Y-labeled antibody in EDTA-treated and control mice at any time-point up to 9 days postinjection. However, the bone levels of 90Y were significantly reduced in EDTA-treated mice at all times from 1 to 9 days. CONCLUSION Based on these results, it should be possible to increase the amount of 90Y-labeled antibody administered, by chelating the released 90Y with systemic EDTA to facilitate its excretion, without compromising tumor uptake of radiolabeled antibody.

Improving tumour targeting and decreasing normal tissue uptake by optimizing the stoichiometry of a two‐step biotinylated‐antibody/streptavidin‐based targeting strategy: Studies in a nude mouse xenograft model

We aimed to study in detail the in vivo stoichiometry of the individual elements of the 2‐step streptavidin based approach to tumour targeting, in a nude mouse xenograft model, by the administration of a first step consisting of biotinylated anti‐tumour specific antibody and a second step consisting of streptavidin. This process was undertaken to identify the optimum conditions for radiotherapeutic tumour targeting using this approach. Antibody was biotinylated to various degrees (1–25 biotins per antibody). Protein stoichiometry of the 2 steps was studied over a range of 2 logs. Both steps, i.e., the biotinylated‐antibody (1st step) and streptavidin (2nd step) were radiolabelled (125I and 131I, respectively). A 24‐hr interval between 1st and 2nd step was studied, animals being killed 24 hr after the 2nd step. Streptavidin excess led to a decrease in levels of monobiotinylated‐antibody in the circulation and in the tumour. Biotinylated‐antibody excess led to an increase in circulating levels of streptavidin, a decrease in renal uptake of streptavidin and increased targeting of streptavidin to tumour. At a constant protein molar ratio of biotinylated antibody to streptavidin of 10:1, increasing biotinylation density resulted in an increase in circulating levels, increase in tumour uptake, decrease in renal uptake and increase in liver uptake of streptavidin. As early as 24 hr, the tumour‐to‐blood ratios of streptavidin already exceeded 1 (max 1.27). Compared with antibody tumour‐to‐blood ratios, they were better by a factor of between 2 and 3. Tumour‐to‐normal tissue ratios of radiolabelled streptavidin (with the exception of liver) were also significantly improved when polybiotinylated‐antibody was administered first. We have thus shown that the 2‐step biotinylated antibody/ streptavidin approach can lead to a significant increase in absolute amounts of activity in the tumour under appropriate stoichiometric conditions. This was accompanied by high levels of circulating streptavidin and relatively favourable tumour‐to‐blood and normal tissue ratios of streptavidin. Int. J. Cancer 78:610–617, 1998.

Xenograft Mouse Models for Tumour Targeting

Human tumour xenografts in nude or SCID mice are a useful model for studying the tumour targeting ability of a new antibody, scFv or fusion protein. Nude mice are athymic and therefore immunologically incompetent, allowing the growth of implanted human tumours. These xenografts have been shown to retain the histology of the original tumour and the relevant human tumour markers. In order to compare different targeting molecules in vivo, the nude mouse provides a convenient model, since genetically identical tumours can be induced in several animals, allowing direct comparisons to be made.