Effects of inter-alpha-inhibitor and several of its derivatives on calcium oxalate crystallization in vitro.

The bikunin peptide chain of the protease inhibitor inter-alpha-inhibitor (IalphaI) has been reported to be an inhibitor of calcium oxalate (CaOx) crystallization, and hence has been proposed as having a role in CaOx kidney stone formation. However, further experimental evidence is required to assess if fragments of IalphaI other than bikunin may play a role in the regulation of crystallization events in stone formation. The aim of the present study was to assess the effects of IalphaI and several of its derivatives on CaOx crystallization in a seeded inorganic system and to compare these effects with those of a known inhibitor of crystallization, prothrombin. IalphaI was purified from a preparation of human plasma and fragmented by alkaline hydrolysis, and two of its peptide chains, bikunin and heavy chain 1 (H1), were purified further by HPLC. Their purity was confirmed by SDS/PAGE. Using Coulter counter and [(14)C]oxalate analysis and scanning electron microscopy, IalphaI, its H1 chain and bikunin from urine and from plasma were shown to be relatively weak inhibitors of CaOx crystallization in vitro at expected physiological concentrations. It was concluded that members of the IalphaI family may not be as important in kidney stone formation as has been generally proposed, although further studies are required before a possible role for IalphaI and its fragments in stone formation can be unambiguously discounted.

Inter-alpha-inhibitor in calcium stones.

1. A broad spectrum of proteins has been detected within calcium stones. A newcomer to the field of urolithiasis is the blood protein inter-alpha-inhibitor. Inter-alpha-inhibitor comprises three protein chains linked by chondroitin sulphate: two heavy chains, H1 (65 kDa) and H2 (70 kDa) and a light chain (approx. 30 kDa) most commonly known as bikunin. The physiological function of the two heavy chains is unknown; nor has their presence been reported in urine. However, bikunin has been implicated in various renal diseases, including urolithiasis. 2. This study was undertaken to determine which chains of inter-alpha-inhibitor are actually present in calcium kidney stones. Organic extracts were obtained from 10 calcium stones and analysed by SDS/PAGE and Western blotting. The H1 and H2 chains of inter-alpha-inhibitor were detected in 9 of the 10 stones, but only one stone contained a protein with a molecular mass close to that of bikunin (30-35 kDa). 3. These results demonstrate for the first time that H1 and H2 are present in stones and show that the bikunin chain of inter-alpha-inhibitor may not be the only part of the molecule implicated in stone formation.

Monoclonal antibodies to human apolipoprotein AI: probing the putative receptor binding domain of apolipoprotein AI.

We have used four monoclonal antibodies (MAbs) specific for human apolipoprotein (apo) AI, designated AI-1, AI-3, AI-4.1 and AI-4.2, to study the interaction between high-density lipoprotein HDL3 and rat liver plasma membranes. MAbs AI-1 and AI-3 recognize epitopes within residues 28-47 and 140-147 respectively of apoA-I [Allan, Tetaz and Fidge (1991) J. Lipid Res. 32, 595-601]. Two previously unreported MAbs, AI-4.1 and AI-4.2, were raised against purified CNBr fragment 4 (CF4) of apoAI, the C-terminal region. Using e.l.i.s.a. and immunoblotting techniques, we have demonstrated that all four MAbs recognize distinct epitopes within apoAI. Epitope mapping studies using endoproteinase cleavage peptides of CF4 showed that AI-4.1 binds to an epitope within residues 223-233, which is poorly exposed on apoAI molecules associated with lipid. Fab fragments derived from MAb AI-4.2 inhibited the binding of 125I-labelled HDL3 to rat liver plasma membranes, whereas Fab fragments from AI-4.1, AI-3 and AI-1 had little or no effect. In ligand blotting studies with purified CNBr fragments of apoAI and using apoAI-specific antibodies for detection, CF4 showed the highest capacity to recognize two HDL-binding proteins previously identified in rat liver plasma membranes. We propose that the specific interaction between HDL and liver plasma membranes is largely mediated through a binding domain in the C-terminus of apoAI, which is consistent with the involvement of specific receptors for the apolipoprotein moiety of HDL.

Antibodies to the carboxyl terminus of human apolipoprotein A-I. The putative cellular binding domain of high density lipoprotein 3 and carboxyl-terminal structural homology between apolipoproteins A-I and A-II.

We have studied the binding of 125I-labeled high density lipoproteins (HDL3) to liver plasma membranes, which are thought to contain specific HDL receptor sites, using anti-peptide antibodies directed against two sites in the carboxyl-terminal region of human apoA-I. Two distinct antibody populations raised to peptides corresponding to amino acid residues 205-220 and 230-243, respectively, recognized regions of apoA-I that are exposed in the lipid environment of HDL3. However, anti-AI[230-243] IgG, but not anti-AI[205-220] IgG, recognized HDL2, suggesting that residues 205-220 of apoA-I are expressed differently in the two HDL populations. In addition, anti-AI[230-243] IgG showed strong cross-reactivity toward apoA-II. Epitope mapping studies showed that anti-AI[230-243] binds to an epitope located in the carboxyl-terminus of apoA-II, demonstrating significant structural homology between the carboxyl-terminal of apoA-II, demonstrating significant structural homology between the carboxyl-terminal regions of apoA-I and A-II, two candidate proteins for mediating the specific cellular interaction of HDL3. Fab fragments from anti-AI[205-220] and anti-AI[230-243] inhibited the binding of 125I-HDL3 to liver plasma membranes by approximately 80% and 60%, respectively. These findings are in agreement with our recent work using isolated CNBr fragments of apoA-I (Morrison, J., Fidge, N. H., and Tozuka, M. (1991) J. Biol. Chem. 266, 18780-18785), which suggest that the carboxyl-terminal region of apoA-I contains a binding domain which mediates the specific interaction of HDL3 with liver plasma membranes, possibly through the involvement of specific HDL receptors.

Intratumour therapy of solid tumours with ricin-antibody conjugates.

Immunotoxin conjugates of whole ricin with monoclonal antibody were prepared with the galactose binding site on the ricin B chain blocked. These whole ricin-antibody conjugates were then injected directly into tumours (IT) in mice with established solid tumours. The conjugates were found to be effective, in vivo, in (C57BL/6XBALB/c)F1 mice carrying thymoma grafts and in nude mice bearing human tumour xenografts. Thymomas (Ly-1.1-, 2.1+) completely regressed following IT injection of either ricin-anti-Ly-2.1 or 'modified' (periodate treated to remove carbohydrate) ricin-anti-Ly-2.1 but did not regress when treated with the non-reactive ricin-anti-Ly-1.1. Similarly, established CEM (transferrin receptor+) or HT-29 (17.1/2+) tumours in nude mice completely regressed following IT injection of ricin-anti-transferrin receptor antibody or ricin-17.1/2 antibody conjugates. The tumours disappeared within 48 h, and in 80-100% of these there was no recurrence. Intact ricin-antibody conjugates did not require the presence of lactose to block the binding of native ricin and selective activity was entirely dependent on the reactivity of the monoclonal antibodies (MoAb). Further, the killing of target cells was specific because non-reactive ricin-antibody conjugates did not cause regression of tumours and MoAb alone did not inhibit tumour growth. In addition, there was no systemic toxicity evident in mice treated with reactive conjugates. By contrast, in mice treated with only ricin or with non-reactive ricin-antibody conjugates there was toxicity to liver and spleen due to diffusion from the tumour; thus the MoAb moiety of the immunotoxin serves to target the ricin to the tumour, to hold ricin in the tumour, and little escapes. It was found that ricin-antibody conjugate treatment (IT) with 1-2 micrograms of ricin-antibody conjugate was not harmful to mice, in contrast to ricin alone, which killed all treated tumour bearing mice at a dose of 0.5 micrograms. Thus whole ricin-antibody conjugates can be used successfully in vivo for local therapy, leading to the eradication of solid tumours by direct injection of the tumour.

In vivo studies of whole ricin monoclonal antibody immunoconjugates for the treatment of murine tumours.

Ricin is a highly potent toxin which binds to cells via galactose binding sites on the B chain; the toxicity is manifest by the A chain and most studies with immunotoxins have used ricin A chain-antibody conjugates. We have previously described a method for the coupling of whole ricin to monoclonal antibody (MoAb) so that the galactose binding sites on the B chain are blocked; these conjugates are not non-specifically toxic. Whole ricin-MoAb conjugates were examined in vivo for their stability, clearance rates and toxicity, and these were compared with modified ricin-MoAb conjugates produced by periodate treatment of ricin. First, the biological half-life of whole ricin or modified ricin was determined and indicated that while ricin was rapidly cleared from the circulation (t 1/2 = 4.5 +/- 0.5 min) compared with modified ricin (t 1/2 = 17 +/- 1 min), covalent linkage of native ricin to MoAb increased the blood circulation time of the toxin (t 1/2 = 22 +/- 1 min). Immunotoxins synthesized with the Ly-2.1 MoAb using native or modified (deglycosylated) ricin were compared in vivo. In two different models anti-Ly-2.1 immunotoxins could be shown to be effective in vivo: (i) intraperitoneal tumours, thymomas grown in the peritoneal cavity could be completely eradicated by ricin-MoAb; (ii) subcutaneous tumours, mice with tumours approximately 0.75 cm in diameter received intravenous doses of the whole ricin-MoAb and a substantial reduction in tumour size was achieved. Thus whole ricin-antibody conjugates made with the galactose binding site blocked combine the advantage of high potency with high specificity (which was previously lacking in intact ricin conjugates) and can be successfully used in vivo to treat tumours.

Novel synthesis and in vitro characterization of disulfide-linked ricin-monoclonal antibody conjugates devoid of galactose binding activity.

The use of tumor immunotherapy using whole ricin-antibody conjugates is complicated by the nonspecific lectin activity of the ricin B-chain which leads to toxic side effects. A novel method of coupling whole intact ricin to monoclonal antibody (MoAb) is described herein, where the nonspecific binding of the ricin B-chain is blocked. The coupling was done using the bifunctional reagents S-acetylmercaptosuccinic anhydride for antibody and succinimidyl 3-(2-pyridyldithio)propionate for ricin, and this resulted in the loss of B-chain binding activity, while impairing neither the toxic potential of the A-chain nor the activity of the MoAb. The purified immunotoxins could not bind to lactose-Sepharose and were equally cytotoxic in vitro to MoAb-reactive cell lines in the presence or absence of lactose. The coupling method was suitable for six different ricin-antibody conjugates and also using ricin deglycosylated by treatment with periodate. However, the blocking of the ricin B-chain was only effective with whole IgG molecules as F(ab')2-ricin immunotoxins could, like ricin, bind to lactose-Sepharose. Ricin-antibody conjugates reduced the [3H]leucine incorporation of appropriate target cells by 50% at a concentration of 6 to 45 ng/ml, whereas nonreactive antibody immunotoxins were not toxic to the target cells at concentrations as high as 10(4) ng/ml. The specific cytotoxicity of these immunotoxins could be inhibited by the addition of unconjugated reactive MoAb; the presence of lactose or a nonreactive MoAb did not significantly affect the observed cytotoxicity. Thus, whole ricin-antibody conjugates produced in this way do not bind nonspecifically to target cells, the most important implication being that such immunotoxins should be more potent that ricin A-chain conjugates and capable of being used in vivo.

Anti-tumour activity of aminopterin-monoclonal antibody conjugates; in vitro and in vivo comparison with methotrexate-monoclonal antibody conjugates.

Two related folate antagonists aminopterin (AMN) and methotrexate (MTX) were used to produce drug-antibody conjugates and their tumouricidal effects compared in vitro and in vivo. Active ester derivatives were produced with the use of N-hydroxysuccinimide (NHS) and covalently coupled to monoclonal antibodies (MoAb) reactive with murine cell surface antigens; approximately 11 molecules of AMN or 13 molecules of MTX were specifically bound per molecule of anti-Ly-2.1, with good retention of antibody activity and protein recovery. AMN was a more effective inhibitor of tumour cell growth in vitro than MTX, and AMN-anti-Ly-2.1 conjugates were also more potent in vitro than MTX-anti-Ly-2.1 conjugates. Although there was some loss of drug activity on binding to antibody, AMN-MoAb conjugates were as toxic as free MTX. However, in contrast to free drugs (which can act on any target), the toxicity of drug-MoAb conjugates was entirely specific for the target cells. In addition, AMN-MoAb conjugates were effective anti-tumour agents in vivo, and in mice bearing established thymoma grafts AMN-MoAb conjugates inhibited tumour proliferation better than MTX-MoAb, free AMN or MTX or antibody alone. AMN coupled to specific MoAb is a potentially useful agent for immunotherapy and is of particular relevance in man as free AMN has been discarded because of its systemic toxicity. Now, coupled with antibody, there will be specific tumouricidal effects in the absence of toxicity.

Coupling of the 99mtechnetium-nitrido group to monoclonal antibody and use of the complexes for the detection of tumors in mice.

The in vivo detection of tumors by immunoscintigraphy with the use of radiolabeled monoclonal antibodies (MoAb) is a new diagnostic procedure currently undergoing clinical evaluation. In the present study the use of 99mtechnetium (99mTc) for this purpose was explored. A simple method for the labeling of microgram quantities of MoAb with 99mTc based on the substitution reaction of MoAb and tetrachloronitridotechnetate ion (99mTcNCl4-) is described. The selective activity of the 99mtechnetium-nitrido-MoAb (99mTcN-MoAb) complexes was proved in vitro by a binding assay with different target cells. The 99mTcN-MoAb complexes were shown to bind reactive cells up to 20 times more avidly than nonreactive cells. The specificity of the 99mTcN-MoAb complexes was shown in vivo. (C57BL/6 X BALB/c)F1 mice bearing palpable tumors (0.3-1.5 cm in diameter) were given an iv injection of 1 of 2 MoAb (one reactive and the other nonreactive) identically labeled with 99mTcNCl4- and then scanned with a gamma camera, and/or the tissues were removed and the localization of 99mTc-nitrido group-labeled MoAb was measured. Tumor localization of the reactive MoAb (1.8-2.2% of the injected dose) was four times greater than that of the nonreactive 99mTcN-MoAb (0.3-0.4% of the injected dose). The localization of specific 99mTcN-MoAb to a murine thymoma was observed in the gamma camera image at just 2 hours after injection. At 27 hours, tumors could readily be detected by 99mTcN-MoAb without the need for background subtraction. Nonreactive 99mTcN-MoAb did not image the tumors. The use of 99mTcN-MoAb offers substantial improvement over radioiodinated (125I or 131I) MoAb for the detection of tumors. The use of 99mTcNCl4- as a labeling agent results in 99mTc-labeled MoAb with high specific activity and specificity when compared with the specific activity and specificity of the 99mTc-MoAb prepared by using the conventional SnCl2 reduction of pertechnetate.

Studies of methotrexate-monoclonal antibody conjugates for immunotherapy.

Methotrexate (MTX) was covalently bound to two different murine monoclonal antibodies, one reactive with human colon carcinoma and the other reactive with the transferrin receptor. The drug-antibody complexes were examined for their in vitro and in vivo potency against tumors. The conditions of coupling were closely monitored, with particular attention being paid to the preservation of both drug and antibody activity. After activation of MTX with N-hydroxysuccinimide, MTX was bound to the antibodies under conditions leading to maximum protein and antibody recovery. Although the coupling conditions for both antibodies were different, up to 13 molecules of MTX-antibody molecule could be attached with retention of antibody activity. Such conjugates were active in vitro and could inhibit the growth [( 3H]deoxyuridine uptake) of cells in culture. The conjugates were highly specific, having no effect on tumors lacking the antigen; however, MTX complexed to antibody was less potent than the free drug. In vivo, the MTX-antibody impaired the growth of established tumors. Thus MTX-antibody complexes can be successfully produced and can be used for the immunotherapy of tumors.