Summary Effective radioimmunotherapy is limited by slow antibody clearance from the circulation, which results in low tumour to blood ratios and restricts the dose of radiolabelled anti-tumour antibody that can be safely administered. Avidin and streptavidin clearing agents have been shown to effectively complex and clear radioactive biotinylated antibodies from the circulation, but their immunogenicity may limit their repeated use. We have investigated whether polyethylene glycol (PEG) modification can reduce the immunogenicity of our galactosylated streptavidin (gal-streptavidin) clearing agent without altering its effectiveness as a clearing agent. The immune response evoked in mice after intraperitoneal injection of 30 ,g of gal-streptavidin was decreased after PEG modification, as shown by lower antibody titres and a reduction in the number of mice that elicited an anti-gal-streptavidin response. The effect of PEG-modified gal-streptavidin on the blood clearance and tumour localisation of a "5I-labelled biotinylated anti-CEA was investigated in the LS174T human colon carcinoma xenograft in nude mice. Although PEG modified gal-streptavidin bound the ['25I]biotinylated antibody in vivo, effective clearance from the circulation was inhibited, resulting in very little reduction in the levels of circulating radioactivity, together with a decrease in the antibody localised to the tumour.
Summary The therapeautic efficacy of intact and F(ab')2 fragments of a "'I anti-CEA antibody were compared in an established LS174T colonic xenograft model in nude mice. A single IV dose of either 0.5 mCi (18.5 MBq) intact or 1.0 mCi (37 MBq) F(ab')2 fragments significantly delayed tumour growth, and increased survival time to the same extent. Biodistribution studies showed that the more rapid clearance of the fragments from the circulation improved the tumour:normal tissue ratios found for the intact antibody, but reduced the duration and therefore absolute amount of radioantibody localisation (% injected dose/gram) at the tumour site. The tumours received a similar accumulated beta radiation dose, with 4,065 cGy from 0.5 mCi intact antibody and 4,500 cGy from 1.0 mCi F(ab')2 fragments. The dose rate to the tumour was initially higher for the fragments, but fell off more rapidly as clearance occurred. However, the rapid circulatory clearance resulted in a radiation dose of only 995 cGy to the blood, compared with 2,300 cGy for the intact antibody. This suggests that twice the radiation dose could be delivered to the tumour in the form of fragments for the same blood dose from the intact antibody. Fractionating the 1.0 mCi dose of F(ab')2 into three doses of 0.33 mCi (12.2 MBq), given on days 1, 3 and 5, significantly reduced the therapeutic effect of the treatment. The clinical relevance of these findings is discussed.The successful tumour localisation of radiolabelled antibodies raised against carcinoembryonic antigen (CEA), a tumour associated marker of epithelial carcinomas, has led to the investigation of radioimmunotherapy as a form of cancer treatment in both animal xenograft models (Buchegger et al., 1988;Sharkey et al., 1987;Pedley et al., 1991) and in man (Begent et al., 1989;DeNardo et al., 1988). Antibodies labelled with isotopes emitting medium-to high-energy beta particules such as 13'I and 90Y are promising for solid tumour therapy, because they can deposit their energy over a range of more than 40 cells without requiring either binding to each individual cell or internalisation.However, a major drawback to the use of radioimmunotherapy is the potential damage to normal tissues from the high doses due to circulating radioantibody. The more rapid circulatory clearance and increased tumour penetration normally produced by antibody fragments make them an attractive alternative to intact antibody for tumour localisation and therapy, although they do have the disadvantage of also clearing more rapidly from the tumour itself.We have previously reported on the comparative tumour localisation and clearance patterns of intact IgG and antibody fragments in the nude mouse model (Harwood et al., 1985). The present study compares the therapeutic efficacy of a radiolabelled intact antibody and its F(ab')2 fragments. We have compared the effect of a single dose of "3'I-Fab(ab')2 A5B7, an anti-CEA antibody, with that of the intact antibody on the colonic tumour xenograft LS174T grown in nude (nu/nu) mice, and ha...
Antibody directed enzyme prodrug therapy (ADEPT) has been studied as a two‐ and three‐phase system in which an antibody to a tumor‐associated antigen has been used to deliver an enzyme to tumor sites where it can convert a relatively nontoxic prodrug to a cytotoxic agent. In such a system, it is necessary to allow the enzyme activity to clear from the blood before prodrug injection to avoid toxicity caused by prodrug activation in plasma. To accelerate plasma clearance of enzyme activity, two approaches have been studied. The studies have been performed with a monoclonal anticarcinoembryonic‐antigen antibody fragment A5B7‐F(ab′)2 conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), in LS174T xenografted mice. In the first approach, a monoclonal antibody (SB43), directed at CPG2, was used, which inactivates CPG2 in vitro and in vivo. SB43 was galactosylated so that it had sufficient time to form a complex with plasma CPG2, resulting in the inactivation and clearance of the complex from plasma via the carbohydrate‐specific receptors in the liver. Injection of SB43gal 19 hours after administration of the radiolabeled conjugate reduced the percentage of injected dose per gram in blood without affecting levels in the tumor. The second approach involved galactosylation of the conjugate so that it cleared rapidly from blood via the asialoglycoprotein receptors in the liver. Localization of the radiolabeled conjugate was achieved by blocking this receptor for about 8 hours with a single injection (8 mg/mouse) of an inhibitor that binds competitively to the receptor. This allowed tumor localization of the conjugate followed by a rapid clearance of the galactosylated conjugate from blood as the inhibitor was consumed. A tumor‐to‐blood ratio of 45:1 was obtained at 24 hours, which increased to 100:1 at 72 hours after the conjugate injection. These accelerated clearance mechanisms have been applied in antitumor studies in ADEPT. Cancer 1994; 73:1114–20.
SummaryThe improved tumour to non-tumour ratios needed for effective tumour targeting with antibodies requires that blood background radioactivity is reduced. We investigated the effect of streptavidin as a clearing agent for '25l-labelled biotinylated anti-CEA antibodies in a human colon carcinoma xenograft model. By comparing the biodistribution of the monoclonal antibody A5B7 with four, nine or 22 biotins per antibody molecule, we investigated how the degree of biotinylation of the primary radiolabelled antibody affects its clearance with streptavidin. Limiting the degree of biotinylation limited blood clearance, whereas nine or 22 biotins per antibody molecule resulted in a 13-to 14-fold reduction in blood radioactivity, the streptavidinbiotinylated antibody complexes clearing rapidly via the liver and spleen. Although a reduction in tumour activity was also seen, a 6.6-fold improvement in the tumour to blood ratio was achieved. A comparative study of streptavidin versus second antibody clearance was carried out using the polyclonal antibody PK4S biotinylated with 12 biotins per antibody molecule. This study indicated that second antibody was superior for clearance of the polyclonal antibody, resulting in a larger and faster reduction in blood radioactivity and improved tumour to blood ratios. In this case the primary antibody was polyclonal, and therefore nonuniformity of biotinylation may affect complexation with streptavidin. Therefore, the degree of biotinylation and type of antibody must be carefully considered before the use of streptavidin clearance.
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