Monoclonal antibodies to drugs: Novel diagnostic and therapeutic reagents

Pharmac. Ther. Vol. 28, pp. 273 to 285, 1985 Printed in Great Britain. All rights reserved

Copyright 3

0163-7258/85 $0.00+0.50 1985 Pergamon Press Ltd

Specialist Sub/ect Editors: C. M. FRASER a n d J. C. VENTER

MONOCLONAL DIAGNOSTIC

ANTIBODIES TO DRUGS: NOVEL AND THERAPEUTIC REAGENTS J. R. ZALCBERG

Department of Medicine, Princess Margaret Hospital, 500 Sherbourne Street, Toronto, Ontario, Canada M4X IK9

1. INTRODUCTION Within the last decade the use of monoclonal antibodies has revolutionized the study of biology in such diverse fields as organ transplantation, cancer, pharmacology, endocrinology, genetics and cell biology (Lennox, 1984). In this review, I will discuss the application of the monoclonal antibody technology to pharmacology. Within this discipline these reagents have been used widely--from the development of new and sensitive assay systems to the treatment of drug toxicity. The following discussion will consider the diagnostic and therapeutic uses of antibodies which recognize individual drugs, or drug classes. Diagnostic uses of antibodies to drugs have included: (a) drug assays; (b) pharmacokinetic studies; (c) the analysis of cell surface receptors; and, (d) the study of conformational changes within drug molecules. The therapeutic application of antibodies to drugs have been principally to reverse the toxic complications of digoxin overdose. However, this approach may be useful for other drug classes. As the monoclonal antibody technology has been developed only recently, this review will consider conventional polyclonal as well as monoclonal reagents. Data based on the use of conventional polyclonal antisera will be used to illustrate the potential and theoretical advantages of monoclonal antisera, in instances where the latter have not been tested. Two areas which will not be considered further are: (a) drug-antibody conjugates in which the antibody serves as a drug delivery system (Krolick et al., 1982); and, (b) antibodies that react with cell surface antigens (Thompson et al., 1983) or endogenous hormones such as renin (Dzau et al., 1983) and thus may be considered as being drugs themselves. 2. MONOCLONAL ANTIBODIES 2.1. DEFINITION The scientific basis of monoclonal antibody production has recently been reviewed (Zalcberg and McKenzie, 1982). In brief, when the host animal is immunized with a foreign antigen, there is an immediate humoral response involving the transient proliferation of B-lymphocytes. These cells produce a complex mixture of antibodies, which are directed against the numerous antigenic determinants (epitopes) on each molecule. This polyclonal response to an antigenic stimulus is made up of a large number of monoclonai antibodies, each produced by a single clone of B-lymphocytes. Thus, the total response is polyclonal with many B-cells contributing to theoverall result. By fusing tile B-lymphocyte with a myeloma cell--a malignant cell intrinsically capable of antibody production, usually of undefined specificity--a hybridoma is produced and the B-cell is immortalized (Kohler and Milstein. 1975). Hybridomas can be selectively cloned and will continue producing large quantities of a monoclonal antibody whose specificity is determined by the fused B-cell. JPT

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2.2° PRODUCTION The principles of producing monoclonal antibodies to drugs are similar to those described for cellular antigens and will not be reviewed in detail. The techniques of somatic cell fusion, screening for immunoglobulins of required specificity, cloning particular hybridomas and antibody characterization are all well established (Zalcberg and McKenzie, 1982). However, unlike cellular antigens, drugs are immunologically inert and must be chemically modified, usually by linking to large molecular weight carrier proteins, before they can act as immunogens. Thus, to produce monoclonal anti-digoxin antibodies, digoxin has been conjugated covalently to lysosyme (Zalcberg et al., 1983) or bovine serum albumin (Mudgett-Hunter et al., 1982). 2.2.1. The Drug-carrier C o m p l e x The efficiency of monoclonal antibody production may be increased by manipulation of the drug-carrier complex. In a recent report, immunization with highly substituted protein carriers was shown to increase the yield of drug-specific monoclonal antibodies (Kato et al., 1984). This study involved the coupling of methotrexate (MTX) to keyhole limpet hemocyanin (KLH) in varying molar ratios. Immunization with conjugates containing MTX and KLH in a ratio of 430:1, resulted in antibodies that specifically recognized the drug moiety in 58% of the hybrids tested. In comparison, a ratio of M T X : K L H of 14:343 resulted in only 4% of the hybrids producing drug-specific antibodies. Antibody specificity, defined as the ability of immunoglobulins to distinguish biologically related molecules, will vary depending on the site chosen to chemically link the drug to its carrier. To develop polyclonal antibodies that distinguished testosterone from 5-~-dihydrotestosterone, testosterone was linked to a macromolecular protein at several independent sites on the drug molecule (Ismail et al., 1972). However, these attempts to produce testosterone-specific antibodies were only partially successful as the level of antibody cross-reactivity was never less than 10%. Tateishi et al. (1980) described a novel approach to immunologically resolving the two testosterone derivatives. Animals were immunized with testosterone coupled to a co-polymer of D-glutamic acid and D-lysine. The polymer rendered B-cells tolerant to the relevant drug so that subsequent immunization of the same animals with 5-~-testosterone linked to KLH resulted in the production of relatively specific antisera. More recently, monoclonal antibodies to testosterone that showed minimal (2%) cross-reactivity with 5-~-testosterone have been described (Kohen et al., 1982). Two studies reporting the production of antibodies to morphine, illustrate the importance of structural variations in the drug-carrier conjugate. When morphine was coupled to bovine serum albumin (BSA) via the 3 hydroxyl position, the resultant antisera could not distinguish morphine from heroin or codeine (Spector and Parker, 1970). However, when azo-morphine was synthesized and then coupled to KLH, the resultant antibodies were readily able to differentiate these reagents (Gross et al., 1974). The cross-reactivities of codeine and heroin with morphine in the latter report were 11% and 7%, respectively. Recently monoclonal antibodies to MTX have been produced, and these reagents are capable of distinguishing this drug from its metabolite 7-hydroxy-methotrexate (Kato et al., 1984). Two derivatives which differ from MTX at the glutamic acid carboxyl group (methotrexate-~-n-butylester and methotrexate-,/-n-butylester) showed the strongest cross-reactivity with the parent molecule. However~ in these cases, loss of antibody specificity was almost certainly due to the linkage of MTX to KLH via this carboxyl group. 2.3. SCREENING In producing monoclonal antibodies to drugs, it is important to develop appropriate screening assays that define the required antibody specificity early in the course of antibody characterization. Thus, in order to distinguish certain drug metabolites or functionally active intermediates, appropriate antibodies must be selected at the outset. The yield and diversity of antibodies recognizing a particular antigen may be increased by the use of

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TABLE 1. Monoclonal Antibodies to Drugs Drug

Reference

Morphine Methotrexate Digoxin L-Asparaginase Gentamicin

(Glasel et al., 1983) (Kato et al., 1984) (Zalcberg et al., 1983) (Kitao and Hattori, 1983) (Dobbins-Place et al., 1983)

multiple screening assays. Mierendorf and Dimond (1983), identified three fundamentally distinct classes of monoclonal antibody to lysosomal-ct-mannosidase by employing a combination of enzyme immunoassay and immunoprecipitation screening techniques. Some of the monoclonal antibodies to drugs which have been reported are listed in Table 1. At present, this list is rather limited, but in view of the potential advantages of monoclonal antibodies (see below), it seems certain that monoclonai antibodies to drugs will ultimately replace polyclonal antibodies in both the laboratory and the clinic. 2.4. ADVANTAGESAND DISADVANTAGESOF MONOCLONALANTIBODIES 2.4.1. Advantages Monoclonal antibodies have a number of advantages over conventional antisera (Table 2). The latter are contaminated with immunoglobulins of multiple specificity only some of which recognize the molecule of interest. However, this problem can be overcome by using chromatographic techniques to produce affinity-purified polyclonal antibodies. The specificity of these reagents is significantly increased, but affinity-purified antiserum still constitutes a heterogeneous mixture of immunoglobulins with the variability in affinity, immunoglobulin subclass and proportion of individual immunoglobulin molecules all contributing to the efficacy of these reagents. In addition, affinity-purified polyclonal antibodies are subject to the same batch to batch variation that characterizes conventional polyclonal antisera. The capacity of the humoral immune system to recognize any one antigenic determinant seems almost limitless (Haber, 1982). This response can now be dissected using hybridoma techniques so that any one immunoglobulin in an antibody repertoire can be selectively expanded. At present, this technology represents the only reliable means of obtaining a standardized laboratory or pharmacological reagent that conforms to the rigid specifications demanded by scientists and physicians from manufacturers and distributors of biological products. 2.4.2. Disadvantages Monoclonal antibodies may have some disadvantages (Davie, 1982) and these will be briefly discussed. 2.4.2.1. Specificity. Hybridomas have demonstrated chromosomal instability resulting in a change in immunoglobulin specificity (Lennox, 1984). However, this potential difficulty can be avoided by adequate storage of the original cell line, ensuring essentially an unlimited supply of any one antibody. The intrinsic tissue cross-reactivity of monoclonal antibodies may limit their specificity. Monoclonal antibodies to complex glycolipid and glycoprotein cell surface determinants may show major unexpected cross-reactions or variations in activity because of minor changes in antigen carbohydrate or amino acid TABLE2. Comparison o f Monoclonal and Conventional Antisera

Monoclonal

Polyclonal

Homogeneous High specificity Low variability High sensitivity One Ig class

Heterogeneous Low specificities Batch to batch variability Variable sensitivity Mixture of classes

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structure (Krolick et al., 1982). This phenomenon is less of a problem when dealing with less complex drug molecules available as relatively pure reagents. However, antibody specificity must be determined experimentally in each case. 2.4.2.2. Sensitivity. Monoclonal antibodies were thought initially to have a decreased affinity for their respective antigens. However, this has no~ been substantiated by all workers. Monoclonal antibodies with affinities of the same or greater magnitude than that observed for conventional antisera have now been defined. Two separate laboratories have described high affinity anti-digoxin monoclonal antibodies with association constants (K,,) of 3 × 109 M - l (Zalcberg et al., 1983; Mudgett-Hunter et al., 1982). These reagents compare favorably with an affinity purified polyclonal sheep antibody to digoxin which had a K, of 1.4 × 10~°M-~ (Smith et al., 1974). In the case of MTX, monoclonal antibodies were shown to have a higher affinity to the drug than polyclonal antibodies (Kato et al., 1984). Mixing monoclonal antibodies together may further enhance antibody affinity (Moyle et al., 1983). The affinity of a mixture of two monoclonal antibodies to human chorionic gonadotropin, each of which recognized a different epitope on the same molecule, was 10 times greater than the affinity of either antibody alone. As the humoral immune response includes the production of both high and low affinity antibodies, the isolation of high affinity reagents should be incorporated into the immunization and initial screening methods. Fantyl and Wang (1984) have suggested that high affinity monoclonal antibodies are more likely to be detected following prolonged immunization protocols. Using this approach, these investigators isolated monoclonal antibodies to progesterone with a-K,, of 3 × 10l° M-I.

3. DIAGNOSTIC USES OF ANTIBODIES TO DRUGS In the following section, I will consider the diagnostic use of antibodies to various drugs, emphasizing the theoretical or proven advantage of monoclonal reagents. 3.1. IMMUNOASSAY

Currently available monoclonal antibodies to drugs have been listed in Table 1. However, numerous drugs have been assayed using polyclonal antisera (Table 3; Butler, 1978). Insulin levels were first determined by radioimmunoassay in 1959 and since then TABLE 3. Conventional (Polyclonal) Antibodies to Drugs *t Drug Diphenylhydantoin Digoxin Gentamicin Morphine Propranolol Tetra-hydro-cannibol Theophylline Adriamycin Acetylsalicylic acid Fluphenazine Glibenclamide Clonidine Prednisone Valium Desmethylimipramine Barbiturates Warfarin *This is a representative rather than exhaustive list of the numerous drugs, each representing a particular class of drug, used to produce conventional antisera. ~'Adapted from Butler, 1978.

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immunoassays have been used to detect a wide variety of reagents. The advantages of immunoassay methods are their high sensitivity, rapidity and simplicity. 3.1.1. A s s a y Sensitivity

The sensitivity of an immunoassay is primarily related to the affinity of the antibody for the drug. It would be theoretically possible to detect digoxin in the concentration range of femptomole per milliliter (ml), by using a monoclonal antidigoxin antibody with a K, of approximately 109 M- ~ (Zalcberg' and McKenzie, 1982). This estimate assumes that the sensitivity of an antibody is equal to one-tenth the dissociation constant (Ka), where Kd = 1/Ka (Walker, 1977). Monoclonal antibodies to MTX were able to detect this reagent at concentrations of less than 1 ng/ml (Kato et al., 1984). However, this degree of sensitivity was similar to that achieved by conventional polyclonal antisera to MTX. Several drug assays in which monoclonal and polyclonal reagents were directly compared, have now been reported. Gentamicin levels in serum can be readily measured by a substrate-labelled fluorescent immunoassay (Dobbins-Place et al., 1983). When conventional antisera employed by the commercial gentamicin assay kit were replaced by monoclonal antibodies, comparable results were obtained. The sensitivity of a conventional enzyme immunoassay for ~-fetoprotein, was slightly increased by the use of monoclonal reagents (Portsmann et al., 1984). Monocional antibodies were shown to be as effective as commercially available radioimmunoassay kits in which sheep antibodies were used to measure serum digoxin levels (Mudgett-Hunter et al., 1982). Thus, it appears that compared to polyclonal reagents, monoclonal antibodies have not produced a major advance in immunoassay sensitivity. However, their homogeneity, stability and batch to batch reproducibility justifies the replacement of conventional antisera by monoclonal reagents. 3.1.2. A n t i b o d y Specificity

In immunoassays, the other major potential advantage of monoclonal antibodies to drugs relates to antibody specificity. The ability of an antibody to distinguish pharmacologically related compounds (antibody specificity), determines the specificity of an immunoassay (Walker, 1977). But the importance of this parameter partly depends on the circumstances in which the assay is to be used. Thus, a monoclonal antibody to gentamicin which also reacts with netilmicin can be used to assay either drug, as these two antibiotics are rarely administered concurrently. Similarly, antiserum which binds to functionally reactive metabolites, as well as the parent compound may be preferred when used to treat drug toxicity (see below). For many diagnostic purposes, antibody specificity is an important criterion that can be defined by comparative immunoassay techniques. A specific radioimmunoassay for 5-fluorodeoxyuridine (Schreiber and Raso, 1978) could distinguish this drug from endogenous, biologically related compounds, such as thymidine and uridine (the cross-reactivity was less than 0.04%). Conventional antisera to puromycin (Fujiwara et al., t982) and macromomycin (Wiskelhake and Buckmire, 1977) did not cross-react with any other cytotoxic drugs. Antibodies that can differentiate between morphine and structurally very similar molecules, such as heroin or codeine have also been produced (Glasel et al., 1983). However, pharmacological concentrations of cortisone and corticosterone have been shown to cross-react with polyclonal antisera to digoxin (Soldin et al., 1984). In addition, several glycolipids in high concentration can also cross-react with digoxin in an immunoassay (Soldin et al., 1984). Monoclonal antibodies that can distinguish methotrexate from folic acid (Kato et al., 1984), digoxin from spironolactone (Zalcberg et al., 1983) or ouabain (Mudgett-Hunter et al., 1982), testosterone from related steroids (Kohen et al., 1982) and morphine from nalorphine (Glasel et al., 1983) have all been reported. However, monoclonal antibodies were unable to separate digoxin from digitoxin (the two glycosides differ by a single hydroxyl group), despite the fact that both molecules were easily resolved by polyclonal antisera (Mudgett-Hunter et al., 1982). In instances where the specificity of monoclonal and polyclonal reagents have been compared, similar results have been observed. The

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specificity of conventional and monoclonal antisera to testosterone were compared by measuring the cross-reactivity of these reagents to 14 pharmacologically related steroids (Kohen et al., 1982). The results were virtually identical, although the cross-reactivity of 5-or-testosterone was reduced from 10-2~ by using one particular monoclonal antibody. However, two other hy,bridoma antibodies produced in the same fusion were unable to distinguish testosterone from its derivative. These variable results may be explained by the diversity of the immune response which makes it highly unlikely that any two monoclonal antibodies to a particular drug will be identical, or that monoclonal and polyclonal antisera to the same antigen will have similar characteristics. Nevertheless, manipulation of the immune system is a powerful pharmacological tool, perhaps best exemplified by the use of antibodies that recognize drug metabolites in pharmacokinetic studies. 3.1.3. Pharmacokinetics Studies

Drugs and their metabolites can be monitored by a variety of chromatographic or other non-immunochemical techniques. However, these procedures are often technically more difficult, time consuming, more expensive and less sensitive than immunological methods. It has been suggested that the sensitivity of high pressure liquid chromatography approximates 20 ~g/l (Soldin, 1982). Thus this technique is inadequate for assaying drugs with therapeutic activity at concentrations below this level. As a result, immunoassays have been developed for a wide variety of drugs (Butler, 1978). Conventional antisera have been used to distinguish theophylline (1,3 dimethylxanthine) and diazepam from their major metabolites, 3-methylxanthine and N-desmethyldiazepam, respectively (Singh et al., 1980; Peskar and Spector, 1973). Antisera to desmethylimipramine (Spector et al., 1975) and clonidine (Jarrott and Spector, 1978) were developed as a means of following the distribution of these drugs in vivo. In both instances, the antibodies recognized both the parent drug and its active metabolite. Thus, prior to performing the immunoassay in the last example, the metabolite 4-hydroxyclonidine was removed from plasma by using a solvent extraction step. In this manner, clonidine was shown to conform to a two compartment open model. Nelson et aL (1979) have used a combination of chromatographic and immunoassay techniques to distinguish digoxin from several metabolites that cross-react significantly with digoxin in a radioimmunoassay. These inactive metabolites only accumulate when renal function is impaired, but under these circumstances, an immunoassay may incorrectly suggest that digoxin plasma concentrations are elevated. However, in patients with normal renal function, the chromatographic step did not improve assay sensitivity over a simpler direct radioimmunoassay. 3.1.4. Which Assay?

The previous sections have discussed the use of monoclonal or polyclonal antibodies to assay numerous drugs. Recently, attempts have been made to compare various assay systems directly. For example, an immunoassay for methotrexate was found to have similar specificity and precision to a high pressure liquid chromatographic (HPLC) method, although the latter was 500 times less sensitive (Ferrua et al., 1983). A radioimmunoassay technique was found to be as accurate and precise as HPLC methods for measuring the digoxin content of commercially available tablets (Beasley et al., 1983). Similarly, there were no significant differences between an immunoassay and HPLC method in measuring serum tobramicin levels (Stobberingh et al., 1982). However, the specificity and cost-effectiveness of HPLC are the major advantages of this method. Thus, both radioimmunoassay and chromatographic technii:lues are important laboratory aids which may be used individually or conjointly depending on the assay requirements. More recently, other types of immunoassays representing minor modifications of the original radioimmunoassay technique have been introduced. Thus, enzyme immunoassays and fluorescent immunoassays have been used to measure various drugs, although the former are usually preferred. Five assays of gentamicin were directly compared (Ratcliff et al., 1981) and the enzyme immunoassay was found to be the most accurate and specific in addition to being the simplest and most rapid to perform. However, a second report

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claimed that a substrate-labelled gentamicin immunoassay based on the use of monoclonal antibodies, was superior to fluorescent, enzyme or radioimmunoassay methods (DobbinsPlace et al., 1983). Clearly immunoassays are an establishment pharmacological tool, but the development of specific, high affinity monoclonal antibodies which can be produced in large amounts should ultimately lead to the replacement of polyclonal antisera in diagnostic drug assays. 3.2'. R£CEVTOR ANALYSIS 3.2.1. Receptor Purification Antibodies to drugs have recently been used to isolate various receptors expressed on the cell surface. Polyclonal antibodies to an alprenolol-albumin conjugate were produced by conventional means and this antisera was purified by affinity chromatography techniques (Schreiber et al., 1980). The resultant antibodies were then used to immunize syngeneic rabbits. As a result, idiotypic antibodies, i.e. immunoglobulins specific for the antigen combining site (hypervariable region) of anti-alprenolol immunoglobulin molecules were isolated (Homcy et al., 1982). These anti-idiotypic antibodies competitively inhibited the binding of both fl-receptor agonists and antagonists to the fl-receptor as well as the isoproterenol-mediated adenyl cyclase activity of this cell surface protein. Thus, the agonist drug alprenolol had effectively served as a template for the production of antibodies to the functional fl-receptor. Similarly, antibodies to the propranolol binding site of cardiac muscle have been defined (Wrenn and Haber, 1979). This antiserum was able to distinguish structural differences between the fl-receptors present in cardiac muscle and hepatic membranes. Using an analogous experimental protocol, attempts have also been made to structurally differentiate between ~1 and ~2 adrenergic receptors using the respective agonists prazosin and yohimbine (Graham et al., 1982). Such antibodies could be used to screen potential receptor-blocking drugs, to analyze the basis of structural variations between 0~1 and ~2 adrenergic receptors and ultimately to purify the receptor and its gene. Although monoclonal antibodies to alprenolol have been reported, the anti-idiotypic studies discussed above have involved the use of conventional polyclonal antisera. However, the hybridoma technology allow the selection and expansion of those antiidiotypic antibodies that best conform to the three-dimensional structure of the fl-receptor. Alternatively, by immunizing mice with affinity purified, fl-receptors obtained from appropriate tissues, it may be possible to produce monoclonal antibodies to the receptor without using drug agonists as immunological templates. Whichever approach is followed, the application of immunological techniques to an understanding of drug receptors promises to be an exciting area of pharmacology and one in which monoclonal antibodies will undoubtedly play a major role. 3.2.2. Drug Conformation and Antibody Binding Sites Antibodies have also been used to study the structural and stereochemical requirements of antigen-antibody binding as a model for drug-receptor interaction. The capacity of antibodies to distinguish conformational, or other structural changes in the hapten may mimic characteristics of the drug-receptor complex. For example, antibodies to D-methamphetamine have been shown to prefer the trans conformation of the phenethylamine skeleton, as well as the D isomer of this drug (Faraj et al.o 1976). This data predicts that 1-amphetamine would have a reduced pharmacological activity--an observation that has been confirmed experimentally. These principles have also been used for the study of varioushallucinogenic drugs that exhibit considerable structural differences. On the basis of biological cross-tolerance, it was suggested that drugs such as N,N-diethyl-o-lysergamide (LSD) and mescaline may act via a common neuronal receptor (Vunaki et al., 1971). Antibodies to LSD were produced and as predicted, cross-reactivity with mescaline and other related compounds was observed. The data seemed to suggest that in vivo, structurally dissimilar but biologically related

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hallucinogenic drugs, assumed a conformation resembling the prototype hallucinogen, LSD. Interactions between other drugs, such as phenylhydantoin (Rowell and Paxton, 1976) and fluphenazine (Rowell et al., 1980) and their respective antibodies have also been used to study characteristics of the drug-receptor complex. With the use of monoclonal antibodies to drugs, further studies of drug structure-function relationships and drugreceptor interactions can be expected and should lead to improved drug design and an increased understanding of cell biology. 4. THERAPEUTIC USES OF ANTIBODIES TO DRUGS Antibodies have been shown to reverse the adverse effects of a wide variety of biologically active molecules. These include bacterial, plant, animal and environmental toxins, endogenous peptides and hormones, as well as a number of drugs (Butler, 1982). In this section, I will concentrate on studies which involve the use of antibodies to alter the biological activity of several drugs. Initially, the general immunological and pharmacological principles governing the therapeutic use of antibodies to drugs will be discussed, following which specific examples of antibody-mediated drug detoxification will be reviewed in more detail. 4.1. GENERAL PRINCIPLES For simplification, the general principles governing the therapeutic use of antibodies to drugs have been arbitrarily divided into immunological and pharmacological subgroups. 4.1.1. Immunological Principles In previous sections, the importance of antibody specificity has been emphasized. For drug kinetic studies, it is important to use antibodies capable of differentiating structurally related molecules. However, for therapeutic rather than diagnostic purposes, antibodies that recognize pharmacologically active metabolites, as well as the native drug would be preferred. This is an important consideration when producing antibodies to individual drugs, illustrating the importance of determining the intended application of the required antibodies prior to screening. Drugs that are reversibly bound to cell surface receptors are ideally suited for antibody-mediated drug detoxification, as in the presence of irreversible receptor binding or intracellular targets, high affinity antibodies could possibly prevent further toxicity but would not be able to reverse established side-effects. Several potential problems may arise following the therapeutic use of antibodies to drugs. Antibody molecules contain a species-specific constant region (Fc) portion which renders such reagents immunogenic unless a human antibody source is available. However, as drug detoxification is likely to be an isolated event used to treat accidental or suicidal overdose, allergic reactions to foreign proteins should not be a major problem, provided that multiple injections of antibody are administered over a short period of time. In addition, antibody chimeras consisting of a murine variable region linked to a human constant region have now been described (Boulianne et al., 1984). When generally available, this methodology should further reduce the risk of immune complex disease. Intact immunoglobulin molecules are cleared following metabolic degradation by the reticuloendotheliai system, rather than by glomerular filtration in the kidney. By contrast, antibody fragments such as Fab molecules which lack the Fc portion of immunoglobulin but retain immunological specificity, are rapidly excreted via the kidney with an elimination half-life of approximately 4 hr (Colburn, 1980). The relatively prolonged serum half-life of intact antibodies in comparison to antibody fragments, may not only delay the plasma clearance of the drug but the metabolic breakdown of immunoglobulin may theoretically cause a secondary release of the previously bound toxic reagent, a phenomenon which has not been seen experimentally to date. Finally, drug withdrawal phenomena may complicate the clinical use of antibodies to drugs. Thus, cardiac output may be decreased in the case of anti-digoxin antibodies, or narcotic withdrawal may be precipitated by the use of anti-morphine antisera. However,

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as the clinical use of antibodies to drugs primarily involves the treatment of life-threatening drug toxicity, the potential risk of drug withdrawal must be a secondary consideration. Individual antibodies must conform to rigid criteria for antibody-mediated drug detoxification to t:e widely available and acceptable as a reasonable form of therapy. Thus, specificity, sensitivity and availability must be clearly defined and not subject to random variation. As stated previously this can best be achieved by using monoclonal antibodies, rather than conventional antisera. The restricted specificity of monoclonal immunoglobulins may be a potential limilation in circumstances where pharmacologically active metabolites are present, although this problem can be overcome by using panels of different monoclonal antibodies. This limitation is a theoretical one at present, as monoclonal antibodies have not been used clinically to trea ~ drug toxicity.

4.1.2. Pharmacological Principles The important pharmacological principles for antibody-mediated drug detoxification are best illustrated by considering several different detoxification methods. In general, methods of detoxification can involve supportive care, close observation of the patient while serum drug levels decline due to expected clearance mechanisms, the establishment of alternative routes of elimination, such as hemodialysis or hemoperfusion, or the stimulation of endogenous clearance pathways of excretion. In life-threatening situations requiring active intervention, hemodialysis and hemoperfusion are the most efficient methods of increasing drug clearance (Colburn, 1980). Hemodialysis is best suited for drugs that are excreted mainly via the kidney, have a limited binding to plasma proteins and a small volume of distribution. Hemoperfusion does not depend on renal excretion or plasma protein binding of the particular drug but like hemodialysis is inefficient when the drug in question has a large volume of distribution. However, Fab-mediated drug clearance is less dependent on the distribution volume of a drug as circulating Fab antibody fragments can also reach the interstitial space. Colburn (1980), compared the theoretical efficacy of various methods of detoxification for two hypothetical compounds, both of which were cleared entirely by glomerular filtration, but differed significantly in their volume of distribution. In the case of a molecule with a small volume of distribution, a conventional detoxification approach such as hemodialysis, was more effective than the use of drug-specific antibodies in reducing the elimination half-life of the drug. However, it was predicted that for a reagent with a much larger distribution volume, the use of Fab fragments would reduce the elimination half-life of this drug 10-fold when compared to hemodialysis. Furthermore, these calculations do not take into account the fact that drugs bound to immunoglobulin are unable to exert any pharmacological activity. As a result of these studies, it was recommended that antibody-mediated drug detoxification should be tested for various compounds characterized by a large distribution volume and a low therapeutic index. 4.2. DRUG DETOXIFICATION This section will discuss specific examples of antibody-mediated drug detoxification. 4.2.1. Digoxin Antibodies to the digitalis glycosides have been used to reverse the pharmacological effects of these drugs in vitro, in animal models and more recently in patients (Butler et al., 1977a). Toxic electrophysiological effects of digoxin on isolated Purkinje fibers have been reversed by digoxin-specific antibodies. The addition of sheep anti-digoxin antibodies to human erythrocyte preparations previously exposed to digoxin, produced a rapid fall in intracellular digoxin concentratio.ns and a slower decline in Inembrane-bound digoxin levels (Gardner et al., 1973). In these experiments, potassium uptake was measured simultaneously and a slow, but consistent fall in the inhibition of potassium transport that characterizes the effects of digitalis on the cell membrane, was also observed. This increase in potassium flux was temporally related to the observed decrease in membrane digoxin levels, suggesting that digoxin exerted its pharmacological effect at the level of the cell

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membrane, rather than intracellularly. In addition, it was postulated that the antibodymediated reduction ofintracellular and membrane bound digoxin was almost certainly due to the rapid fall in the extracellular concentration of free drug. The effects of digoxin on monovalent cation transport in cardiac tissue obtained from dogs that were hemodynamically monitored, has also been investigated. (Houghen et al., 1979). Lethal doses of digoxin were associated with a 59~ to 80~o fall in the uptake of Rubidium--an ion which mimics potassium transport. Anti-digoxin antibodies were able to reverse digoxininduced arrhythmias in these animals. At the time of reversion to sinus rhythm, Rubidium transport in cardiac tissue obtained from the antibody treated dogs, had increased to 51% of controls. Using a feline model, Hess et al. (1983) demonstrated that anti-digoxin antibodies were able to inhibit digoxin-induced mesenteric vasoconstriction in isolated strips of feline mesenteric artery. In these experiments, antibodies administered after digitalis toxicity was fully established, did not reverse pharmacological toxicity completely. Nevertheless, when lethal doses of digoxin were injected into the whole animal, only those that had subsequently received anti-digoxin antibodies survived. The influence of anti-digoxin antibodies on digoxin pharmacokinetics have been determined experimentally. Both intact antibodies and their Fab fragments produced a significant increase in total serum digoxin levels (Butler et al., 1977b), As this increase was associated with a decrease in drug side-effects, it was suggested that digoxin was being mobilized from an extravascular compartment. Attempts to measure myocardial digoxin levels have resulted in conflicting data. In a murine system, we did not observe any change in myocardial digoxin levels following the administration of monoclonal antidigoxin antibodies (Zalcberg et al., 1983). In contrast, Anderson et al. (1981), observed a moderate decrease in myocardial digoxin concentrations in a dog treated with sheep anti-digoxin antibodies. However, total myocardial drug levels will reflect the presence of both bound and free drug moieties, and thus represent at best only indirect evidence for drug redistribution. The theoretical benefits of antibody fragments over intact immunoglobulin (see above), have been confirmed experimentally. Digitalis toxicity was reversed more rapidly with Fab fragments than intact molecules, correlating with the fact that the former have a larger volume of distribution (nine-fold) than whole immunoglobulin (Smith et al., 1979). When intact molecules were used, antibody treatment of digitalized animals resulted in elevated digoxin levels for 5-7 days. By contrast, in the baboon, digoxin levels fell within 4-10 hr following the injection of Fab fragments, corresponding to the detection of both Fab fragments and digoxin in the urine. Finally, Fab fragments were shown to be less immunogenic than intact molecules, although no clinical immune complex disease was documented with use of the latter (Smith et al., 1982). Numerous case reports have now demonstrated the clinical usefulness of digoxin-specific antibodies for the reversal of accidental or suicidal digoxin overdose. For example, a child who accidently ingested 10mg of digoxin developed refractory ventricular fibrillation (Zucker et al., 1982). The serum free digoxin level prior to antibody administration was greater than 100 ng/ml. Following treatment with digoxin-specific Fab fragments, a normal rhythm and circulation were rapidly restored and the free digoxin level was undetectable in less than 12 hr. Recently, the results of a multicenter clinical trial using digoxin-specific Fab antibody fragments to treat life-threatening digitalis intoxication were reported (Smith et al., 1982). All patients had advanced cardiac arrhythmias and/or hyperkalemia which had not responded to conventional therapy. Of the 26 patients treated in this study, five died. Four of these deaths were in patients treated afte'r prolonged hypotension and in the fifth case, inadequate supplies of antibody were available. The remaining 21 patients recovered completely with no treatment related side-effects. Three of these survivors had ingested digitoxin with serum concentrations greater than 100 ng/ml prior to treatment, but despite the reduced affinity (10-fold), of digoxin-specific antibodies for this cardiac glycoside, antibody-mediated detoxification was still successful (Smith et al., 1982). The conventional treatment of significant digitalis toxicity includes careful observation, correction of electrolyte imbalance and the use of appropriate antiarrhythmics or other

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interventions for serious cardiac arrhythmias. Digoxin-specific antibodies do not replace any of these therapeutic modalities, but rather add to the clinician's armamentarium when faced with a life threatening situation. For wider applicability, the antibodies must exist as standardized reagents, a requirement that can now be satisfied by the use of monoclonal antibodies. 4.2.2. Other Drugs Many of these studies are preliminary brief reports and are mentioned here for the sake of completeness. Adriamycin is a potent cytotoxic agent used to treat a wide variety of human malignancies. However its administration is limited by the occurrence of doserelated myocardial dysfunction. Conventional antibodies to Adriamycin have been used experimentally in an attempt to reverse Adriamycin-induced cardiac toxicity (Savaraj et al., 1980). However, further data supporting these preliminary observations are necessary, before this approach can be adopted clinically. From the results presented, it is not certain whether Adriamycin-specific antibodies can reverse established Adriamycin effects. Mice that had been injected with both Adriamycin and the specific antibody had higher serum drug levels than controls, although it is not clear whether the drug was free or bound nor the extent to which antibody treatment compromised anti-tumor responses. Experimentally, antibodies to morphine have been used to reverse the depressant effect of this drug on electrically stimulated contractions of guinea-pig ileum (De Cato and Adler, 1973). In addition, the biological activity and pharmacokinetics of morphine were altered in animals that had previously been actively immunized against this narcotic (Hill et al., 1975). These experiments illustrate the potential therapeutic use of anti-morphine antibodies. But in view of the ready availability of rapidly acting narcotic antagonists such as nalorphine, further research using monoclonal antibodies to this drug, has concentrated on the diagnostic uses of these reagents (Glasel et al., 1983). In experimental models, conventional antibodies to pentabarbitol have been shown to alter drug pharmacokinetics, as well as reduce pentabarbitol-induced ataxia (Cerreta et al., 1979) and passively administered antibodies to Dg-tetrahydrocannibol (THC) have been shown to alter the effect of THC on the barbiturate sleeping time (Chiarroti et al., 1980). 5. CONCLUSIONS Antibodies to drugs have indisputably made a major contribution to pharmacological research. This review has discussed the use of these reagents for diagnostic and therapeutic purposes. The therapeutic potential of antisera to drugs has been clearly demonstrated by the use of such antibodies to treat serious digitalis toxicity. However, antibodies to drugs have not been used as extensively for therapy as they have as diagnostic aids. This trend probably reflects the various problems associated with the use of conventional antisera. But, the development of the hybridoma technology should result in an increase in the pharmacological application of monoclonal antibodies directed against various drug classes. Acknowledgements--I would like to thank Dr C. Erlichman and Dr S. Soldin for assistance in the preparation

of this manuscript and Miss Marcella D'Orazio for secretarial assistance.

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