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Radiolabelled synthetic branched chain polypeptides (BCP) represent a new and novel range of materials with potential as radiopharmaceuticals. Preliminary imaging studies have been undertaken with 111In-labelled BCP in mice with subcutaneously transplanted mammary carcinoma. Four polypeptides each with a poly(L-lysine) backbone and side chains of DL-alanine residues were studied. These were AK, which is polycationic, EAK which is amphoteric, having additional glutamic acid residues at the end of the side chains, and AcEAK (anionic) and SucEAK (highly polyanionic) where the terminal glutamic acid amino groups were acetylated or succinylated respectively. Radiolabelling was achieved by previous conjugation with DTPA. Serial images up to 48 hours showed marked retention of 111In-labelled polycationic AK and polyanionic SucEAK in the liver and spleen, with renal uptake also being visible in the case of AK. 111In-labelled EAK and AcEAK showed longer blood survival with some liver uptake, but tumour uptake was also visualized by 24 hours with both of these polypeptides. These studies demonstrate the feasibility of using 111In-labelled synthetic branched chain polypeptides as radiopharmaceuticals for gamma scintigraphy and the visualization of tumours by modification of the side chain structure. These materials warrant further study.

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The feasibility of using immobilised anti-combining site (anti-idiotypic) antibodies as targets for assessing the immunoreactivity of radiolabelled anti-tumour monoclonal antibodies has been assessed. With two anti-tumour monoclonal antibodies (anti-CEA and anti-gp72) it was possible to quantify binding of their 125I labelled Fab fragment preparations to their anti-idiotypic monoclonal antibodies immobilised on cyanogen bromide activated Sepharose. Binding was specific for immobilised anti-idiotypic antibodies reactive with the anti-tumour antibody fragments. Moreover binding was inhibited by unlabelled Fab or intact monoclonal antibody, but not by an irrelevant antibody or its Fab fragment. The use of anti-idiotypic antibodies for quantifying immunoreactions of radiolabelled antibodies has advantages over the use of initial target antigen, which may be available only in inconvenient forms, such as cultured tumour cells.

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A branched polypeptide drug carrier, with a poly(L-lysine) backbone, has been labelled with 111In and its biodistribution imaged in mice with transplanted B16 melanoma. Levels of tracer in tumours were not great enough for tumours to be discerned on gamma-camera images, and this was confirmed by subsequent dissection analysis. Tumour levels of 111In from labelled polymer were about 3% of the dose g-1 two days after injection. Similar levels of tracer were found in tumour tissue of mice injected with mouse immunoglobulin or serum albumin labelled with 111In by DTPA chelation, or injected with free 111In-chloride to label serum transferrin. There was rapid excretion of a sub-component of the 111In-labelled polymer visible in the images. Gel permeation chromatography suggested that the polymer was heterogeneous, some components having Stoke's radii below that allowing renal clearance. Gel permeation chromatography was used to separate labelled polymer into fractions having high, intermediate and low renal clearance. The low-excretion fraction showed a two-fold increase in tumour levels, compared with native polymer, although as this fraction showed greater survival in the blood and body as a whole, discrimination between tumour and normal tissue was not increased.

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Systemic administration of lysine to mice injected with indium-111-labelled Fab fragment of a monoclonal antibody has been shown to reduce renal uptake/retention of the radiometal. This treatment should be applicable to clinical immunoscintigraphy and radioimmunotherapy with radiometal-labelled antibody fragments.

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It has previously been shown that a syngeneic anti-idiotypic (anti-id) antibody response generated in Balb/c mice to the monoclonal antibody (Mab) NCRC-2 can cause its clearance from the circulation. However, there was a positive prolongation of the survival of the Fab fragment, which is monovalent but also lacks Fc. The present study was carried out to examine the effect against the Fab/c fragment of this Mab, since this is also monovalent but has intact Fc. There was accelerated clearance, rather than retention. This suggests that anti-id responses prolong survival of fragments because they lack Fc, rather than only because of their monovalency. Such monovalent, Fc deficient fragments, are the type of human antibody fragments most easily obtained by genetic engineering techniques for clinical use. The present findings have implication for the effects of patients' immune responses on the biodistribution of such fragments.

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The effect of syngeneic anti-idiotypic (anti-id) antibodies on the biodistribution of three murine monoclonal antibodies (mAb) against human tumour-associated antigens, and also on that of their fragments, has been examined in mice using, as a model system, purified anti-id mAb against three different target mAb. With the IgG2b mAb NCRC-2, pretreatment of mice 24 h previously with its IgG1 anti-id mAb NCRC-60 caused clearance of subsequently administered NCRC-2. With the univalent Fab/c fragment of NCRC-2 there was little effect, even with anti-id to Fab/c pretreatment ratios of 20:1, although immune complexes were present in the circulation. With Fab of NCRC-2, anti-id mAb prolonged blood survival by reducing renal clearance, immune complexes surviving in the circulation. With the IgG1 mAb NCRC-23, immune complexes formed in vivo with the IgG2b anti-id mAb NCRC-59, but with only little hepatic clearance. With the Fab and F(ab')2 fragments of NCRC-23, blood survival was increased in mice pretreated with anti-id mAb, and with Fab this was clearly due to reduced renal clearance. The third mAb, the IgG3 NCRC-48, formed complexes with its IgG2a anti-id mAb NCRC-62, but these survived in the circulation with no accelerated clearance of the target mAb. These results are different from those previously seen with endogenous anti-id responses. They indicate the diversity of effects that anti-id mAb can have on the biodistribution of their target mAb, and emphasise the difficulty of using such anti-id mAb to modulate the pharmacokinetics of target mAb.

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Balb/c mice were immunised with three different syngeneic monoclonal antibodies (Mabs). Following injection of radiolabelled Mabs into mice with anti-antibody responses they were cleared from the circulation, predominantly to the liver, due to the formation of immune complexes. Anti-antibody titres as low as 1/100 were virtually as effective as 1/3,000 in perturbing biodistribution of Mabs. There were no effects against isotype matched control Mabs. There was a much lesser effect against Fab or F (ab')2 of one Mab, although these did form immune complexes in the circulation of immune mice. With Fab of another Mab its blood survival was actually prolonged, due to immune complex formation. With this Fab, although blood survival was prolonged, localisation into human tumour xenografts expressing its target antigen in Nude mice was reduced by pre-treatment with immune serum. These studies show that syngeneic (anti-idiotypic) responses have a detrimental effect on the pharmacokinetics of Mabs, but with more diverse effects on their fragments. With fragments the indication is that even when biodistribution is not grossly perturbed, tumour recognition may still be restricted.

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Three tumour-specific monoclonal antibodies (MoAbs) showed localisation in human tumour xenografts in nude mice, although the tumour discrimination was limited by the survival of a greater proportion of the MoAb in the blood and body as a whole. An attempt was made to increase tumour discrimination by the subsequent administration of syngeneic idiotypic-specific MoAbs (anti-id) directed against the first antibodies, in the expectation of clearing excess of the first MoAb from the circulation. With one MoAb (NCRC-2), its anti-id (NCRC-60) did effectively clear it from the blood, and, at least within a few hours, the tumour-to-blood ratios were increased. After longer periods, however, the tumour levels of NCRC-2 were also reduced, and the tumour discrimination was no longer increased. With another MoAb (NCRC-23) the tumour levels were reduced to a greater extent than were the blood levels in mice treated with its anti-id (NCRC-59), so that rather than being increased the tumour discrimination was actually reduced to about a third of that in control mice. With a third MoAb (NCRC-48), there was no effect on the tumour or blood levels within a few hours of injection of its anti-id (NCRC-62), and so there was no short-term effect on tumour discrimination. Subsequently, however, the tumour levels were slightly reduced, while the blood levels increased in mice treated with anti-id compared with control mice, so that the tumour-to-blood ratios decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

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We have previously shown that Balb/c mice immunised against syngeneic monoclonal antibodies (mAbs), so that they have anti-idiotypic responses against those mAbs, will clear the mAbs from the circulation as a result of immune complex formation. We report here that with three separate mAbs, this clearance of complexes can result in toxicity to the animals. This was particularly severe with one mAb (NCRC-2), in some cases leading to death. It was not seen with Fab/c or Fab fragments of NCRC-2. This toxic response could be passively transferred with serum, indicating that it was due to formation of immune complexes or to their subsequent clearance. Treatment of mice with cobra venom factor, to reduce complement levels, reduced clearance of complexes but had no effect on toxicity. The anti-histamine pyrilamine did not reduce toxicity. This is one of only a few situations in which an anti-(mouse antibody) response has been reported to be potentially dangerous, and is particularly remarkable since it occurs as a result of such a limited anti-antibody response.

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A reduction-mediated technetium-99m labelling method has been evaluated with a range of tumour-specific monoclonal antibodies. Antibodies reduced with 2-mercaptoethanol (2-ME) had free sulphydryl groups, but their number was much higher than could be accounted for by only limited intra-chain reduction of disulphide bonds. Reduced antibody could be labelled efficiently with 99mTc using an methylene diphosphonate (MDP) bone-scanning kit, although this seemed to depend on the presence of residual 2-ME in the preparations. With a carcinoembryonic antigen (CEA)-specific antibody, immunoreactivity of labelled antibody was confirmed, and after injection into nude with CEA-producing xenografts there was localisation into the tumours. Sephacryl S300 gel filtration showed the radiolabel eluting at a single discrete peak at the expected 150 kDa. However, examination of all of the labelled antibodies by polyacrylamide gel electrophoresis (PAGE) and autoradiography showed the presence of a large number of radiolabelled low molecular weight degradation products of the antibodies. These degradation products seemed to be formed in previously reduced antibodies during processing for PAGE, indicating some fragility of the reduced antibody.

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Mice with s.c. grafts of gastric carcinoma MKN45 or osteosarcoma 788T were injected i.v. with recombinant tumour necrosis factor alpha (rTNF alpha) and tumour blood flow rates were determined 4 h later as a fraction of the cardiac output g tissue-1. With MKN45, the tumour blood flow rate was significantly reduced from a mean of 1.86% cardiac output g-1 to 0.84% and 0.65% with 50 and 200 micrograms kg-1 rTNF alpha respectively. With 788T, the tumour blood flow rate was reduced at 50 micrograms kg-1 rTNF alpha from 1.13% cardiac output g-1 to 0.56%. There were essentially no changes in blood flow rates in other organs. The effect of rTNF alpha on localisation of monoclonal antibodies into these xenografts were examined. When a single dose of rTNF alpha (50 micrograms kg-1) was given at the same time as labelled NCRC-2 antibody there was a significant reduction in localisation into 788T osteosarcoma xenografts. In other tests, mice were injected daily for 3 days with 50 micrograms kg-1 rTNF alpha. They were injected i.v. with monoclonal antibody 4 h after the first injection and dissected on the 4th day. With 788T there was a small but not statistically significant reduction in the absolute amount of NCRC-2 antibody localising in tumour, although this reduction was greater when results were expressed as tumour-to-blood ratios. With MKN45 xenografts, treatment with rTNF alpha had little effect on tumour localisation of an anti-(carcinoembryonic antigen) monoclonal antibody (NCRC-24). These studies show that TNF can be administered so as to reduce tumour blood flow and with little effect on tumour localisation of antibody, suggesting that combination therapy with TNF and antibodies or their immunoconjugates is feasible. Other studies have suggested that TNF can increase antibody localisation into tumours, but this was not seen here, and in some cases administration could reduce tumour localisation. It appears that this method of enhancing antibody localisation may be critically dependent on scheduling, and therefore it may not be extensively applicable.

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As a model for human mouse-specific antibody responses in patients undergoing immunoscintigraphy, we have investigated in rats the production of mouse-specific antibodies (MA) to the mouse monoclonal antibody 791T/36. At intervals of between 5 and 16 weeks the rats were given repeated cycles of intravenous (IV) injections of antibody with or without a simultaneous intradermal (ID) injection. The IV dose was 60 micrograms/kg, a dose similar to that used in many clinical immunoscintigraphy studies. The ID injection was 2 micrograms, which mimicks the skin test dose often given in clinical imaging protocols. The study was carried out with both 131I-labelled antibody and with antibody labelled with 111In by diethylenetriamine-penta-acetic acid (DTPA) chelation. MA was measured with a passive haemagglutination assay using sheep red blood cells coated with the monoclonal antibody. Of rats given ID injections of unlabelled antibody at the same time as the IV imaging doses, 9/20 produced MA during 4 cycles of injections. In contrast, only 2/16 rats given only the IV dose produced MA. Both 131I- and 111In-labelled antibody appeared equally immunogenic with 5/18 and 6/18 overall responders, respectively. The production of MA was associated with a significant perturbation in the biodistribution of the IV dose of labelled antibody as seen by gamma-camera imaging of the rats given 111In-labelled antibody. There was clearance of immune complexes to the liver, this organ accumulating up to 90% of the whole body count rate of radiolabel. MA titres of between 1/100 and 1/78,000 caused equal perturbation of biodistribution, although below 1/100 the effect was more variable.

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