Targeting potential of antibody conjugates

Pharmac. Ther. Vol. 23, pp. 147 to 177, 1983

0163-7258/83 $0.00+0.50 Copyright © 1983 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

Specialist Subject Editor: G. M. IHLER

TARGETING

POTENTIAL

OF ANTIBODY

CONJUGATES

D. C. EDWARDS Institute of Cancer Research, Royal Cancer Hospital, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, U.K.

1. INTRODUCTION The assumption underlying the concepts discussed in this article is that cells display characteristic molecules on their surface membranes that can be defined by complementary ligands. These ligands (e.g. antibodies) may in turn be used as carriers for the delivery of other moieties (e.g. toxic molecules) to the cell surface. The therapeutic potential of complexes of such 'haptophores' and 'toxophores' was foreseen by Ehrlich in 1913 but was not pursued until 1958 when the use ofmethotrexate linked to an antibody directed against L1210 leukemia cells was reported (Math~ et al., 1958). Since that time two main types of adduct with antibody have been constructed using la) conventional drugs or (b) toxins of bacterial or plant origin. In favor of the use of drugs is that, being already part of the pharmacopoeia and having passed stringent statutory safety tests, conjugates containing them may be more readily acceptable to licencing agencies. Further, a vast body of information on mode of action, side effects and acute and chronic toxicity has been established and is available to the designers of drug conjugates. The argument for the use of toxins such as diphtheria toxin, abrin and ricin is that, acting catalytically, they are many times more effective than chemicals with stoichiometric mechanisms. Thus dosages are minimized while the chances of a successful outcome are maximized, particularly in those instances where a target antigen is not strongly expressed. Justification for the general principle of designing agents based on the separate functioning of relatively independent molecular domains is given by examples from nature such as antibodies, hormones, and the toxins themselves. In these, primary interaction of one part of the molecule with a cell surface component leads to a secondary event which may involve activation of another part of the molecule, interaction with another membrane component, or full insertion into the cytosol. But despite such respectable antecedents and even with Ehrlich's imprimatur, exploitation has been long delayed. The reasons for this include the success enjoyed by conventional medicinal chemistry, the problems of producing antibodies of sufficient purity and selectivity, and the lack of good conjugation procedures. However, growing dissatisfaction with currently available drugs and particuJarly with the cytotoxic agents used in cancer chemotherapy has caused many workers to consider the possibility of using antibody-directed therapy. Improved methods of immune purification of antibodies and better chemical linkage techniques have combined to make conjugates more attractive. But, it is probably the development of hybridoma technology for the production of monoclonal antibodies, discovered in 1975 by Kohler and Milstein, that explains the current vogue for investigations into the use of antibody conjugates. These methods have revolutionized antibody production. Large quantities of pure, highly specific immunoglobulins can be produced with relative ease and are being ~ncreasingly exploited in conjugate technology. 2. ANTIBODIES AS CARRIER MOLECULES Although various authors have reported on the use of lectins (Uchida et al., 1978; Gilliiand et al., 1978) and hormones (Change et al., 1977; Oeltman and Heath, 1979; 147

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Cawley et al., 1980), it is antibodies that seem to present the most promising vehicles for the transport of cytotoxic agents to specific targets, on the grounds both of availability and localization potential. Other advantages are that antibodies tend to be fairly robust molecules which stand up well to labeling methods and are little affected by the chemical manipulations required for conjugate synthesis (Ross et al., 1980). They can be broken down to given Fab fragments (Nisonoff et al., 1960), when the consequent decrease in molecular weight might aid diffusion and the removal of Fc binding improve specificity of action. 2.1. AVAILABILITY OF TARGET ANTIGENS 2.1.1. Tumor Associated Antigens

The effectiveness of a given antibody as a carrier will depend in the first instance on its ability to bind to highly specific and preferably unique elements expressed by target cells. In the case of tumors, early optimism for the existence of tumor specific antigens was formalized in the immune surveillance hypothesis proposed by Thomas (1959) and espoused by Burnet (1970). There is, however, little direct evidence that autochthonous tumors are antigenically unique on account of their malignancy and Hewitt et al. (1976) have argued that, in fact, tumors are not antigenic. However, the emphasis has shifted towards the possibility that there exist tumor associated antigens, due to exaggerated expression or chronological abnormality, that might be used as targets. Examples are carcino-embryonic antigen (Gold and Freedman, 1965), stage specific embryonic antigen (Solter and Knowles, 1978), inappropriate expression of histocompatibility antigens (Bonavida and Roman, 1979), TL antigen (Old, 1981), and the Thomson-Friedenreich antigen borne by some human malignant breast cells (Springer et al., 1976). Various other antigens may be used opportunistically. Ectopic hormone production, e.g. human chronionic gonadotropin (Begent et al., 1980) might provide targets, the receptor for transferrin (Trowbridge and Domingo, 1981), viral antigens such as those coded for by hepatitis B virus (Shouval et al., 1982) and, in the case of certain lymphomas, idiotypic determinants (Stevenson et al., 1977) have been suggested. In the case of tumors arising from cells of non-vital organs, tissue specific antigens provide potential targets. The existence of such antigens can be inferred from the development of autoimmunity. Direct evidence is provided by examples such as antilymphocytic globulin (Medawar, 1969) from which Shigeno et al. (1968) prepared a mouse specific lymphocyte antibody, from the existence of antibodies to prostatic tissue (Flocks et al., 1960) and breast epithelium (Arklie et al., 1981). Tumors might thus be attacked not via abnormally but via normally expressed antigens, destruction of the healthy tissue being acceptable. Another possibility is the selective destruction of proliferating endothelial cells to cause metabolic starvation of tumors (Denekamp, 1982). More direct evidence for the existence of potentially exploitable antigens comes from the studies of Old and his colleagues, who have concluded that malignant melanomas (Carey et al., 1976; Shiku et al., 1976, 1977), astrocytomas (Pfreundschuh et al., 1978), renal tumours (Ueda et al., 1979) and leukemias (Garrett et al., 1977) all display antigens which vary in their uniqueness of expression. These antigens have been divided into three classes: (1) antigens apparently unique to an individual tumor, (2) shared tumor antigens present on autologous tumors and some allogeneic tumors as well, and (3) antigens present on normal cells including components adsorbed from sera onto the cell surface (Pfreundschuh et al., 1980). Related to the question of antigenic identity of tumors is the expression of viral gene products on cell surfaces. Thus monoclonal antibodies have been described which bind to all Epstein-Barr virus carrying lymphoblastoid cell lines and most lines derived from EB virus-positive lymphomas (Kintner and Sugden, 1981). An increasing number of antigens is being discovered by the application of hybridoma technology although not all have received the same depth of appraisal given to the monoclonal antibody Ca 1 (McGee et al., 1982). This antibody has recently been described

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149

by Ashall et al. (1982) and apparently defines an antigen, Ca, common to many malignant epithelial tumors. Mesenchymal but not neuroectodermal tumors also possess Ca, though in any given population of cells not all express the antigen equally (McGee et al., 1982). The status of Ca antigen vis-a-vis stage specific antigens is not yet clear. It should be noted that Greaves (1981) has pointed out that many monoclonal antibodies may discriminate quantitatively rather than qualitatively and a number of unsuspected crossreactions of monoclonal antibodies have been reported (Lane and Koprowski, 1982). These may limit the usefulness of some preparations in the construction of therapeutic agents. 2.1.2. Lymphocytes

Apart from their projected role as carriers of drugs and toxins to target tissues in vivo I:here is growing interest in antibodies as vectors in ridding bone marrow preparations of unwanted cells prior to injection into a patient. A major barrier to the extended use of allogeneic bone marrow grafting is the development of potentially lethal graft versus host disease. This syndrome, caused by the presence of mature T lymphocytes of donor origin in the graft might be prevented by pretreatment of the graft with, for example, a toxin attached to an antibody of suitably restricted reactivity. A study by Mason (1981) has shown that in the rat two populations of lymphocytes ('helper' and 'suppressor') can be defined by two monoclonal antibodies W3/25 and MRC 0X8. In man both these T-cell subsets appear to be recognized by the OKT3 monoclonal antibody (Reinherz and Schlossman, 1980), which migh therefore be a useful carrier in this context. The use of unconjugated OKT3 for bone marrow cleansing has already been reported (Filipovich et al., 1982). The advantage of conjugated form would be to abolish the requirement for complement and thus the risk of complement mediated stem cell death (Vallera et al., 1982). A more direct means to avoid the risk of graft versus host disease is to use autologous bone marrow replacement. This stratagem is however flawed by the risk of micrometastatic deposits of malignant cells being present and thus reintroducing the tumor to the patient. The need is thus for antibodies recognizing tumor cells but not crossreacting with the pluripotential stem cells of the marrow. The monoclonal antibody J-5 seems suitable for conjugation (Ritz et al., 1980). It is said to bind to cells of non-T-cell acute lymphoblastic leukemia and to cells from some cases of chronic lymphocytic leukemia but not to normal adult or fetal bone marrow cells. Another possible carrier in the same field is the monoclonal antibody A2B5 described by Eisenbarth et al. (1979) and shown by Kemshead et al. (1981) to discriminate between neuroblastoma and hemopoietic stem cells in human bone marrow. In addition to being possible targets in bone marrow, lymphocyte subpopulations provide special examples where destruction in vivo would be advantageous. This would be in the field of immunosuppression and in clonal deletion in autoimmune disease. In the former case the monoclonal antibodies of the OKT series (Reinherz and Schlossman, 1980) can be seen as useful. The latter might rely on the use not of antibody conjugates but of antigen conjugates which would react in primary fashion with lymphocytes bearing the complementary antibody. 2.1.3. Other Target Antigens Although monoclonal antibodies have now been described for a number of bacteria, Mycobaeterium tuberculosis (Coates et al., 1981), Lancefield Group A and Group B

strepococci (Herbst and Braun, 1981; Polin and Kennett, 1980), there seems so far to have been no reported attempt to couple such antibodies to antibacterial agents and use the products therapeutically. In principle at least, there seems to be no reason for this omission, which may owe much to the availability of a large selection of antibiotics and other successful antibacterial agents. The rapid evolution of resistant strains of some pathogenic bacteria may make the antibody option more attractive at a later time.

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Other interesting possibilities are opening up with the development of monoclonal antibodies against parasitic antigens. These have been recently reviewed (Cohen, 1982). The complexities of the life cycles of many parasites and the ability of some to modify their surface antigens create formidable obstancles to chemotherapy. It seems likely that similar problems will complicate the use of antibody conjugate therapy. The ability to use the most potent toxic agents regardless of their usual side effects may, however, prove advantageous. 2.2. LOCALIZATION OF ANTIBODIES IN VIVO If antibodies are to be successful carriers of toxic moieties to selected tissues in vivo, they should preferentially bind in those tissues. Until fairly recently this proved none to easy to demonstrate. During the course of long and painstaking studies, Pressman and his colleagues were able to show only moderate specific localization of antibodies to a number of tissues or tumors (Pressman, 1949; Pressman et al., 1951, 1952; Korngold and Pressman, 1954). Later they showed the interpretation of their results on tumor localization to be complicated by the presence of antifibrinogen activity in the antibody preparation (Day et al., 1959a,b). In 1971, Reif, using the paired label method developed by Pressman et al. (1957) studied localization of antibody to a y-G-type myeloma protein secreted by a transplantable plasma cell tumor. Specific uptake by the tumor was found but only if antibody was injected four days after tumor cell implantation and the animals were dissected eight days following antibody injection. Goldenberg et al. (1974) used a photoscanning method to follow the localization of an anti-CEA antibody to a tumor growing in the cheek pouch of the hamster. They concluded that an increase in labeling of 7.5-20 fold had occurred in the tumor as compared to other tissues. Later, using the more discriminating paired label method, the same group (Primus et al., 1977) found 3-4~ of the injected dose per gram of tumor tissue and localization ratios Anti CEA IgG/normal IgG in tissue Anti CEA IgG/normal IgG injected of between 7 and 8, eight days after injection. Moshakis et al. (1981) used an affinity purified anti-CEA and looked for uptake onto human breast carcinomas growing as xenografts in immune-deprived mice. Antibody localized in the tumors but not in normal tissue, the degree of localization being correlated with the amount of tumor CEA but being independent of the level of circulating CEA or of CEA/anti-CEA complexes. Along similar lines Koji et al. (1980) have shown localization of anti-alpha fetoprotein to a transplanted hepato-cellular carcinoma growing in rats. The tumor/blood ratio of antibody at one week was four times greater in antibody treated than in control animals. Somewhat better results have been obtained with monoclonal antibodies. Houston et al. (1980), using monoclonal anti-Thyl.1 and the paired-label method, demonstrated localization factors of between 250-475 for uptake into lymph nodes of mice of Thyl. 1 phenotype. Interestingly the antibody failed to accumulate in the brain, although the ceils were Thyl. 1 positive. Uptake in the thymus was also low, both observations being taken as indicating a failure of antibody to extravasate into the tissue. Another set of results showing good levels of localization for a monoclonal antibody have been reported by Levine et al. (1980). In this an anti-stage-specific embryonic antigen (SSEA-1) was found to localize 150 times more in tumor than in muscle and the tumor to blood ratio was 15:1. Thus there seems little doubt that satisfactory levels of specific localization have been and can be achieved. The question now remains whether such localized materials will assure effective delivery of drugs or toxins to all or at least to a large majority of the cells forming the tissue mass. Evidence from two sources suggest that some optimism may be justified. It has been shown that diphtheria toxin (MW 62,000) can destroy human tumors growing as xenografts in nude (Reid et al., 1978) or immune-deprived mice (Thorpe et al., 1982). This is despite the fact that the highest level of localization shown for [~25I]diphtheria

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151

toxin was 3:1 for human tumors compared to other tissues in the grafted mouse (Edwards, unpublished). Secondly, working with a monoclonal antitransferrin receptor antibody, Trowbridge and Domingo (1981) showed that growth of a subcutaneous inoculum of 2 × 107 melanoma cells could be prevented by an intravenous injection of antibody given seven days after the tumor cells. 2.3. SUMMARY On the grounds of availability of targets, selectivity, ease of preparation and handling, and of ability to localize and probably to penetrate tissues, antibodies appear to provide the best vectors currently available for the delivery of other molecules to cell surfaces.

3. THE CHEMISTRY OF CONJUGATION While it is beyond the scope of this review to cover all the developments that have occurred in the art of producing protein conjugates during the last few years, it is necessary to discuss some of the issues raised by the production and use of antibody conjugates. 3.1. NON-COVALENT LINKAGE In studies designed to explain the in vivo activity of alkylating agents, Israels and Linford (1963) showed chlorambucil to be bound avidly to serum proteins and the rate of hydrolysis of the drug to be reduced in the presence of serum proteins particularly serum albumin (Linford, 1961, 1963). The protective effect was shown by Hopwood and Stock (1971) as likely to be due to interaction with hydrophobic regions in the protein, again serum albumin was implicated, ~-globulin was relatively ineffective. However, Blakeslee and Kennedy (1974) claimed that chlorambucil could be linked non-covalently to rabbit IgC, provided that suitable reaction conditions, e.g. high pH, pertained and that as many as 64.5 moles drug per mole IgG could be incorporated. Protein-protein conjugates have also been prepared using non-covalent forces for adhesion. These rely on antigen-antibody interaction. Refsnes and Munthe-Kaas (1976), for example, used an anti-ricin B chain antibody-ricin complex. A recent review by Raso (1982) also deals with this subject. Using the method of Nisonoff and Rivers (1961), hybrid antibodies were produced with the ability to bind human IgG through one V-region while the other held neocarzinostatin. Similar molecules were constructed where the hybrid contained an anti-ricin A chain V-region and could thus be used to bind and deliver ricin A chain (Raso and Griffin, 1981). There are some evident questions regarding the use of non-covalent bonds in conjugate construction. The most obvious problem regarding the use of non-covalent conjugates concerns their stability in the body fluids. The issue has been raised by Ross (1974) but information on this score is generally scanty. The problem is less evident when antigen-antibody reactions are considered but their use gives rise to a curious paradox. Antibodies are classically the means of protecting cells and animals from the lethal effects of proteinaceous cytotoxins, and antibody-toxin complexes (such as diphtheria toxin-anti-toxin) have been demonstrated to be safe (for review of early work see Pope, 1954). Also it has been shown that in the case of ricin A chain the appropriate (intact) antibody will block A chain activity in vitro; however it is not clear that this would apply to the more easily dissociable monovalent association of hybrid antibody. 3.2. COVALENT LINKAGE

3.3.1. Antibody-Drug Conjugates Proteins present many options for chemical substitution with small molecular weight compounds and, subject to checks for retention of activity, it is now usually fairly straightforward to effect addition of any required constituent. Much of the early work on

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production of substituted proteins was carried out by Landsteiner (see Landesteiner, 1945), whose concern was with the construction of immunogenic molecules. More recently, accounts of the various methods of protein conjugation have been given in a series of articles in Methods in Enzymology, starting with one by Erlanger (1980). For the production of antibody conjugates a number of methods have been used. Math6 et al. (1958) used the diazonium reaction to couple methotrexate to their antibody. De Carvalho et al. (1964) described the coupling of methotrexate, uracil mustard, 5-fluorouracil, tetracycline, 6-mercaptopurine and chlorambucil, but their method was found by Ghose and Blair (1978) to lead to extensive protein precipitation. Harding (1971) and Robinson et al. (1973) preferred to use a carbodiimide, finding that diazotization was accompanied by many side reactions. Ross (1975) coupled chlorambucil to human 7-globulin using a water-soluble carbodiimide but later devised a more elegant technique in which the acid anhydride derivative of chlorambucil was used (Thorpe et al., 1978). In a later modification of this method, the N-hydroxysuccinimide ester was preferred (Thorpe and Ross, 1982). Hurwitz et al. (1975) compared three methods of conjugating daunomycin or adriamycin to antibody, (1) reaction of the periodate product of the drug with antibody oxidation followed by reduction with borohydride, (2) glutaraldehyde, (3) a carbodiimide. Of the three, glutaraldehyde linkage preserved most drug activity but caused variable amounts of antibody aggregation and the preferred method was that using periodate oxidation. Rowland et al. (1975) used a carbodiimide in a novel manner to link N - N - b i s ( 2 chloroethyl)-p-phenylenediamine (PDM) to immunoglobulin. First, the drug was reacted with poly-L-c~-glutamic acid (PGA) using a carbodiimide. The P D M - P G A complex so produced was then isolated and coupled to Ig in a second carbodiimide coupling reaction. With the alkylating agent, trenimon (2,3,5-trisethyleneimino-l:4 benzoquinone), Linford et al. (1974) used a different approach. By adding dithiothreitol to their immunoglobulin to produce partial reduction they were able, after dialysis of the reduced Ig, to bind the drug to it in covalent fashion. The binding possibly involved a reaction between a sulfydryl group on the globulin molecule and the 6 carbon atom of the drug (Linford, 1973). Recently attempts have been made to introduce bonds susceptible to cleavage in the conditions prevailing in cellular lysosomes. Shen and Ryser (1981) described a conjugate of daunomycin and polylysine prepared with N-cisaconitic anhydride that was believed to be subject to pH sensitive cleavage. Trouet et al. (1982) argued along similar lines and coupled daunomycin through a spacer arm comprising a tri- or tetrapeptide and finally reacted with succinylated protein. The resulting lysosomotropic conjugates were sensitive to hydrolysis by lysosomal enzymes with consequent release of drug.

3.3.2. Antibody-Protein Conjugates A new set of problems is presented by the use of proteins as the active constituents of conjugates, since the requirement now is to couple chemically similar molecules. In their first experiments, Moolten and Cooperband (1970) used toluene diisocyanate as a heterobifunctional agent, to join antibody to diphtheria toxin, in a two-stage process. In later work, however, this group reverted to the use of the homobifunctional agent glutaraldehyde (Moolten et al., 1972; Moolten et al., 1975). Difluorodinitrophenylsulfone was used by Samagh and Gregory (1972), while Philpott et al. (1973a) favored diethyl malonimidate. Although such methods may often be appropriate for coupling small molecules to proteins, they are distinctly disadvantageous for coupling one protein to another. The general problem is that mixtures of aggregate molecules of unknown composition are formed involving both species of parent molecule. These mixtures are almost impossible to resolve either analytically or preparatively. With toxins, a specific problem arises due to the fact that, as will be discussed more fully later, they comprise two polypeptide chains

Targeting potential of antibody conjugates

153

joined by a disulfide bridge, and homobifunctional agents covalently link the chains and inactivate the toxin. To overcome this problem, it is usual now to devise two stage linkage processes. One such, already mentioned, is based on the work of Ross and uses derivatives of chlorambucil. In the first version, the acid anhydride of chlorambucil was used (Thorpe et al., 1978), in a later modification the N-hydroxysuccinimidyl ester was preferred (Thorpe and Ross, 1982) on the grounds of greater water solublity. The principle underlying this procedure is that the chloroethylamino groups of the drug are inactive at low temperatures, and the first reaction, usually with the antibody, is carried out at 4°C and pH 9. ~O Antibody.,, NH2 +

/CH2CH2CI N

- - O CO(CH2)3~

\CH2CH2Ct (I)

_

~ Antib°dy~NH'OC(CH2)3~

_

/CHECHECt N~CH2CH2CI.

Intermediate (1) can be freed from unreacted linking agent by simple gel filtration. The second protein is then added, usually in considerable molar excess and the temperature raised to 37°C. /CH2CH2Ct Antibody ~ NHOC(CH2)3 ~ N

+ NH2 ~ Toxin

~CH2CH2Ct

(2) Antibody ,-~ NHOC(CH2)3

jCH2CH2Ct~ N N,~ Toxin ~CH2CH2Ct /

The product of the reaction is isolated by gel filtration and conditions can usually be arranged to give a reasonable yield of a conjugate with antibody-toxin in 1:1 molecular combination. Antibody binding and toxin A and B chain activities are well preserved. Rector et al. (1978) described another method for the preparation of well-defined protein-protein conjugates, in which they iodoacetylated immunoglobulin and allowed the derivatized product to react with ovalbumin into which a sulfydryl group had been introduced. The number of ovalbumin molecules coupled per immunoglobulin molecule was controlled by the degree of iodoacetylation. Several modifications of this method are described by Thorpe and Ross (1982), the use of acetylhomocysteine thiolactone or of N-succinimidyl-3(2-pyridyldithio)propionate for the thiolation stage and the use of bromoacetyl-p-aminobenzoic acid either in the acid anhydride or N-hydroxy-succinimidyl form for the introduction of a reactive halogen into the protein. The conjugation of the enzyme glucose oxidase to rabbit immunoglobulin has been studied by Yoshitake et al. (1979). They used the N-hydroxy-succinimide ester of N(4-carboxycyclohexylmethyl)-maleimide to introduce meleimide groups onto the enzyme which was then allowed to react with thiol groups of reduced immunoglobulin or Fab~ fragments. There was only slight loss of enzyme activity and antibody binding was well preserved. J F I 23/I- J'

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D.C. EDWARDS

The methods considered so far have dealt with the introduction of non-reducible covalent bonds. Since the subunits of toxins are joined by disulfide bridges and since separation of the chains may be an essential feature of the expression of cytotoxicity (see later), many workers have preferred to utilize disulfide bonds in the generation of conjugates. For this purpose, various methods have been described. Swan's (1957) method for the synthesis of asymetric disulfides was developed by Chang and Neville (1977), who coupled the A chain of diphtheria toxin to human placental lactogen. Following amidination of the hormone amino group with methyl-5-bromovalerimidate, the 5-bromovalerimidated protein was converted into the S-sulfonated derivative by reaction with sodium thiosulphate. The toxin A chain sulfydryl was then used in the substitution reaction. DipA-S + Hormone-SSO~- ~ DipA-SS-Hormone + $2O2Masuho et al. (1979) used a similar technique to couple diphtheria toxin A chain to an Fab~ fragment ofimmunoglobulin. In this case they sulfonated the A chain and mixed with Fabl-SH to complete the formation of the hybrid. A thiol disulphide exchange reaction has been used by Raso and Griffin (1980). Using Ellman's reagent they formed an adduct with Fabl-SH which reacted with the free sulphydryl of ricin A chain to give the conjugates. Gilliland et al. (1978) also made use of an exchange reaction. In this case they coupled the first protein to cystamine using a carbodiimide, followed by addition of toxin A chain under conditions favouring disulfide exchange. A further variation is described by Yamaguchi et al. (1979), who derivatized the first protein by reaction with 3-Y-dimethyldithiobispropionimidate. The product was then reduced to yield a sulfydryl substituted derivative which was allowed to react with Ellman's reagent. The resulting disulfide was allowed to interact with toxin A chain to form the required conjugate. Currently, the most commonly favoured method for producing covalent binding through disulfide groups involves the use of N-succinimidyl-3-(2 pyridyldithio) propionate (SPDP) developed by Carlsson et al. (1978) and now commercially available. The reactions are as follows:-

O Ab~NH2 + ~~o.OCO. (CH2)2 -- Sk S N - ~ (3)

AbNNHCO(CH2)2--S--SN - ~ (3) is reduced with dithiothreitol to give Ab ~ NHCO (CH2)2-SH

(4)

The second protein may also be reacted with the reagent to give

ToxN NH--CO -- (CH2)2--S --S

(5)

Disulfide exchange between (4) and (5) gives the desired product. In cases where the second protein contains a free sulfydryl group, direct exchange with (3) is utilized. 3.4. SUMMARY Methods are available for the construction of covalently and non-covalently linked conjugates, and products of reasonably well-defined chemical composition can be isolated.

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However, for any particular combination or application it is necessary to consider the requirements on an individual basis. The retention of the fundamental biological activities of each component part of the conjugate is mandatory and is normally achieved. In cases where penetration of the cytosoi is required for the expression of cytotoxicity some capacity for dissociation of the conjugate might be desirable. This in turn could be incompatible with the need for stability in the body fluids when in vivo application is in mind. Enzyme labile linkages have been proposed to overcome this dilema and further developments seem likely to follow.

4. ANTIBODIES CONJUGATED TO LOW MOLECULAR WEIGHT COMPOUNDS 4.1. CONJUGATES WITH METHOTREXATE

In 1958, Math6 et al., resuscitating Ehrlich's earlier idea, joined methotrexate to an antibody directed agonist mouse L1210 leukemia cells. The antibody had been raised in hamsters and absorbed with normal mouse tissue. Mice were injected with 10~ L1210 cells and the next day were treated with the conjugate. Control animals received either methotrexate or antibody alone. Median survival times of the mice were measured and the conjugate was found to have doubled the life-time expectancy of treated animals compared with untreated animals. There was also an improvement in survival compared to the other control groups. Later, Robinson et al. (1973) also linked methotrexate to anti-L1210 antibodies and used the conjugate to attack L1210 leukemia cells growing in mice. The conjugate was shown to be more effective therapeutically than unconjugated antibody and drug given together, i.e. there was no synergism. More recently, Burstein and Knapp (1977) coupled methotrexate to an antibody against a partially purified antigen from an ovarian carcinoma from C3HebFej mice. They employed both a carbodiimide and a mixed anhydride reaction to affect conjugation. Used in mice which had been previously challenged with tumour cells, both conjugates gave increases in mean survival time compared with those obtained with antiserum alone or with methotrexate coupled to a normal rabbit immunoglobulin. Latif et al. (1980) on the other hand found a conjugate produced by the mixed anhydride reaction to be inactive.

4.2. CONJUGATESWITH CHLORAMBUCIL A considerable stimulus to the investigation of the potential of antibody-drug conjugates was provided by the work of Chose and his colleagues. In 1972, Chose et al. described their first experiments with non-covalent conjugates of antibody and chlorambucil. In these, antibody to EL4 lymphoma cells was complexed with the drug and the product used to treat animals which had received an intraperitoneal inoculation of EL4 cells. If the cells were followed by an injection of conjugate two hours later and then by daily injections for five consecutive days, the mice all survived and no tumors developed. Similar injections of chlorambucil either free or complexed with a normal rabbit immunoglobulin gave no long term survivors and were essentially without effect. As the time between cell inoculation and the commencement of treatment was increased, the number of survivors decreased but, even after a delay of five days, the median survival time was noticably increased by injection of the complex. This paper also contains what is presumably the first description of the application of antibody-drug targetting to man. A male patient with disseminated malignant melanoma was injected locally and intravenously with an antibody-chlorambucil complex and regression of all metastatic nodules was reported. The same year, Chose and Nigam (1972) used chlorambucil in non-covalent association with an anti-Ehrlich ascites antibody. Treatment was begun two hours after tumor injection and continued daily for five days. Under these conditions the use of the complex resulted in the survival of all the animals, whereas a comparable complex of drug with a

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normal immunoglobulin gave no long term survivors and only a moderate increase in mean survival time. Pretreatment of cells prior to injection with antibody complex was completely effective in preventing tumor development but the control complex was inactive. This work received confirmation in the experiments reported by Flechner (1973). He used chlorambucil complexed with an anti-Ehrlich ascites cell antibody to attack EAC cells growing in the mouse peritoneal cavity. Like Ghose and Nigman, he was able to prevent growth of the tumor by the specific treatment. He also found surviving mice to be (immunologically) resistant to further challenge with EAC cells. Ghose et al. (1975) raised an antiserum against L2 lymphoma cells from AKR mice. The anti-serum was absorbed with normal AKR tissue and the globulin was precipitated with ammonium sulfate, prior to being complexed with chlorambucil. In a test system in which even one tumor cell was sufficient for tumor development and consequent death of the animal, intraperitoneal injection of complex two hours after tumor cell inoculation and five consecutive daily doses saved the mice (16) in the test group. Smith et al. (1975) used an unadsorbed rabbit anti-Novikoff heptoma cell antibody complexed with chlorambucil to treat animals previously injected with an ascites form of the tumor. In two groups of four animals injected intraperitoneally with the complex 3 or 24 hr after the tumor, no growth of tumor cells was observed. The results seem to be an improvement over those seen in the control animals, though in all control groups save one, at least one animal produced no tumor. Complexes of antibodies with chlorambucil have been used by De Weger et al. (1982) in two experimental systems, SL2 lymphoma in DBA/2 mice and the Harding Passey melanoma in BALB/C mice. Only in the latter was a significant therapeutic effect seen, the complex being more effective than a mixture of drug and antibody, successively injected drug and antibody or either antibody or drug alone. Papachristou et al. (1977) used a non-tumor model viz. immunosuppression with antilymphocyte globulin and chlorambucil. The survival of skin allografts in rats was increased for a mean of 7.4 days to 54.9 days when antibody-drug therapy was given one day prior to grafting and on each of the two days following grafting. When the globulin and drug were given simultaneously but separately into different sites, the mean graft survival time was 35.3 days. The figures for antibody or drug alone were 16.5 days and 8.0 days respectively. Somewhat similar results were found for the survival of heart grafts. Hirschberg et al. (1978) also used anti-lymphocytic antibody linked non-covalently to chlorambucil and tested the complex for its ability to inhibit lymphocytes in tissue culture. The complex was more effective than either antibody or drug alone in preventing the proliferative response of human mixed lymphocyte culture and the ability of activated T-cells to lyse S~Cr-labeled lymphoblasts. In addition to the work already mentioned, there have been several reports on the use of antibody-chlorambucil complexes in man. In 1974, Oon et al. described their work using autologous human antibodies complexed with chlorambucil both to attack cells in tissue culture and to treat patients. 18 hr cultures of melanoma cells treated with antibody-drug complex gave 35% 'survival', survival being undefined. The corresponding figures for antibody or for chlorambucil alone were 56% and 86% respectively. Patient treatment was to give the complex intravenously twice weekly. No serious side effects were seen and no anti-haptene antibody developed. The evidence pointed to a therapeutic effect of the treatment although the patient died after ten months. Somewhat similar effects were seen in two children with disseminated neuroblastoma. Parental antibody coupled to chlorambucil was an effective cytotoxic agent which also gave good initial results when used in the patients. Grant et al. (1974) also attempted to treat melanoma. Of ten patients receiving BCG therapy, four were given antitumor antibody-chlorambucil complex in addition. One of the four survived for 16 months after first being given BCG and the authors found the part played by the conjugate 'difficult to assess'. An anti-IgE antibody complexed with chlorambucil was used by Clancy et al. (1976)

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to treat a patient suffering from a mast cell leukemia. The complex was given intravenously and on two occasions gave a fall in mast cell count. However, the third time it was used it was without effect and the authors speculate that this might have been due to immune elimination of the complex. Ghose et al. (1977) used a xenogenic anti-human melanoma antibody in complex with chlorambucil to treat thirteen patients with recurrent malignant melanoma. Within this group, two patients responded to therapy, in five their condition stabilized and six showed no response. The median survival times of responders and aon-responders were 20 months and 3-5 months respectively. None of a group of eleven patients treated with DTK showed tumor regression and all died in eleven months with a median survival time of three months. The interpretation of the results from experiments, particularly those in vivo, involving the use of non-covalent complexes has been questioned. Ross (1974) produced evidence that the two molecules would be likely to dissociate rapidly after injection and thus the ability of the antibody to act as a homing device for the drug seemed to him doubtful. In accord with this view is that many of the effects seen were due to synergism of action between the antibody and the drug. Davies and O'Neill (1973), using the EL4 and SB1 lymphomas of the mouse, showed that antibody-drug complexes were better able to protect mice against death in these model systems than either drug or antibody alone, but were not better than the same amounts of antibody or drug injected one hour apart. Likewise, Rubens and Dulbecco (1974), using polyoma transformed BHK21/CI3 cells showed that, whereas antibody-chlorambucil complex was better than antibody or drug alone, it was not superior to antibody or drug added separately to the culture. Further to this point, in an elegant experiment O'Neill et al. (1975) showed that anti-Thyl. 1 antibody complexed with chlorambucil was also toxic to cells bearing the Thyl. 2 antigen and that this toxicity was increased in the presence of anti-Thyl. 2 antibody. Additionally, chlorambucil followed by antibody was shown to be somewhat more toxic than either alone or antibody followed by drug. Synergism is not confined to the chlorambucil-antibody system. Davies (1974), using the EL4 mouse lymphoma and a rabbit anti-EL4 antibody, found melphalan also acted synergistically. Further, he extended his observation outside the alkylating agents to cytosine arabinoside and showed that this too showed improved efficacy if followed by specific antibody. O'Neill (1980) has also shown the synergistic action of cytosine arabinoside with an antilymphoma antibody (SB1 lymphoma of mice). Although synergism undoubtedly occurs and may explain most of the improvements seen when non-covalent conjugation is practised, it does not appear to cover all cases. Ghose et al. (1975), using the L2 lymphoma, showed a complex to be superior to antibody and drug given separately and one hour apart . Hirschberg et al. (1978) also ruled out synergism as an explanation for their findings. Attempts have been made to exploit the synergistic effect in man, with some limited success. Newman et al. (1977) treated patients suffering from carcinoma of the bronchus with a variety of drugs (adriamycin, bleomycin, methotrexate, vincristin, cyclophosphamide and 5-fluorouracil) followed by antibody, but no statistically significant effects were observed among those treated compared with groups not receiving antibody. Everall et al. (1977) also tried to exploit the synergistic effect and claimed that four patients with metastatic melanoma all lived beyond the expected median survival time. There have been fewer reports on the use of covalent conjugates of antibody and chlorambucil. Tai et al. (1976) used a carbodiimide method to link chlorambucil to an anti-EL4 antibody. The product was used with some degree of success to attack EL4 lymphoma growing in C57 B1 mice. Latif et al. (1980) coupled chlorambucil covalently to a goat anti-human leukemia cell line antibody, using the method described by Rowland (1977). The leukaemia cell was grown as a xenograft in nude mice. While the conjugate retained in vitro cytotoxicity, it appeared less effective than unconjugated antibody. This was also true in the in vivo model and the possibility that the drug was in fact playing no part in the activity expressed by the conjugate must be considered.

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Apart from chlorambucil, various other alkylating agents have been used to form adducts with antibody. Linford et al. (1974) bound trenimon to a partially reduced antibody raised against a 3-methylcholanthrene induced sarcoma (MCA-D of strain 13 guinea-pigs). The resulting conjugate was between two and three times more cytotoxic for MCA-D cells than for BHK cells. The same group (Froese et al. 1976) studied the effect of a similarly prepared conjugate of trenimon and an antibody to L51787 lymphoma cells. The antibody (from rabbits) was absorbed with normal DRA/2 mouse spleen, thymus and blood cells. Their experiment was to inject 10 6 tumor cells subcutaneously into mice and treat the animals intraperitoneally with conjugate on the following three days. The mean survival time for animals given conjugate was extended to 35.6 days compared to 19.1 for control animals receiving saline. Drug alone produced a mean survival time of 20.7 days and, when given with antibody, the m.s.t, was 2.8 days. In addition to the improvement in therapeutic response, the authors also commented that the drug was less acutely toxic when given in the conjugated form. This effect had also been noted by Wade et al. (1967), who coupled a range of nitrogen mustards to various carrier proteins and showed large increases in LDs0. Kadin and Otterness (1980) have discussed this particular issue, and concluded that drug toxicity reversal may be a viable proposition using antibody conjugation provided that Fab fragments are used. Trenimon has also been used for the preparation of conjugates by Ghose et al. (1982). In their experiments the drug was linked to rabbit anti-mouse H6 heptoma antibody by the method used by Linford et al. (1974). Groups of at least six mice were injected subcutaneously with 105 H6 tumor cells, and 24 hr later the first of five daily intravenous injections was given. 6 #g trenimon conjugated to antibody was an effective and non-toxic dose and the potentiation of trenimon activity by antibody was present whether or not the drug was in the conjugated form. An interesting observation concerned the in vitro cytotoxicity of free and conjugated drug which suggested that, in contrast to the free drug, conjugated trenimon acted not on intracellular DNA but on some cell surface component. The drug PDM has been used by Rowland et al. (1975), who attached it to polyglutamic acid and then coupled the product to a rabbit anti-mouse lymphoma (EL4) antibody by a carbodiimide reaction. In this way the amount of drug substituted per molecule of antibody could be greatly increased without damaging the antigen combining site. C57B1/6 mice were injected intraperitoneally with 5 x 104 EL4 cells and 24 hr later were given the first of four daily intraperitoneal injections of conjugate. The median survival time of mice given saline was 13 days. When antibody and drug-polyglutamic acid were given together but unconjugated the m.s.t, increased to 38 days, two out of five mice being long term survivors. However, when the complex was used, all the mice in the group survived and the median survival time was in excess of 100 days. Later, Rowland (1977) substituted dextran for the polyglutamic acid, on the grounds that the conjugate might be preferentially removed by the reticulo-endothelial system. Again using an antibody against EL4 lymphoma, a conjugate was prepared. As before, mice were injected intraperitoneally with 5 x 104 EL4 cells and next day the first of four daily injections by the same route was given. The anti-tumor effect seemed very clear with mean survival times of over 150 days and 4/4 survivors in one experiment and 3/5 in another. However, the author was not convinced that he was demonstrating a true 'homing' effect mediated by antibody as there was free IgG present in the conjugate preparation and the possibility of synergy between this and the conjugate could not be excluded. Everall et al. (1977) treated a melanoma patient with a conjugate based on immunoglobulin, melphalan and polyglutamic acid. Treatment was given intravenously. Over a period of a year in which intermittant dosage was used, the patient received over 6 g of immunoglobulin, showed no side effects and survived for several months longer than expected.

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Hirschberg et al. (1978), in addition to the anti-lymphocyte globulin and chlorambucil complex already mentioned, also used a conjugate of the antibody with melphalanpolyglutamic acid. Mixed lymphocyte cultures and T effector cells were strongly inhibited, but the potency was no greater than that of the simpler chlorambucil complex. 4.4. CONJUGATES WITH ANTIBIOTICS The use of conjugates of antibody with adriamycin and daunomycin has been studied particularly by the group from the Weizmann Institute (for review see Arnon and Sela, 1982). A conjugate prepared by reaction of the product of periodate oxidation of drug with antibody followed by borohydride reduction was found to be toxic in vitro against cells bearing the appropriate antigens and not against a cell that did not crossreact with the antibody (Hurwitz et al., 1975, Levy et al., 1975). Hurwitz et al. (1978) extended the study to the use of in vivo model systems. Two tumors were used, a plasmacytoma (PL5) and a lymphoma (YAC). With the former, both tumor and treatment were given by the intraperitoneal route. Antibody-drug conjugate was no better than free drug but was superior to a normal immunoglobulin-drug conjugate. The best results were seen when the drug was simply mixed with the antibody. In the YAC model, the tumor was again given intraperitoneally but treatment was administered intravenously. Also the authors used a dextran bridge carrier to increase the amount of drug carried on the antibody. The results showed the antibody conjugate to be superior to a normal immunoglobulin conjugate at low but not at high doses. Other results were reported by Hurwitz et al. (1979). Antisera to the Lewis lung carcinoma (3LL) were prepared in rabbits or in mice. The sera were adsorbed, radiolabeled and shown to localize preferentially in lungs bearing metastases. When antibody-daunamycin conjugate was used against tumor cells in vitro, it was more active than either free drug or a conjugate made with a normal immunoglobulin. It appears, however, that conjugates ofmonoclonal antibodies with daunomycin were ineffective when tested against 3LL in vivo (Arnon and Sela, 1982). The same authors (Arnon and Sela, 1982) describe an attempt to extend the observations on the YAC system described by Hurwitz et al. (1978). To do this they employed a monoclonal anti-YAC antibody (KH 3 4). However, the use of the conjugate in vivo is described as 'not highly beneficial'. Latif et al. (1980) also used daunomycin conjugates. In this case an antibody directed against a human myelogenous leukemia cell line (K-562) was used. While the antibody itself was effective in vitro and in vivo against cells growing as a xenograft in nude mice, the conjugate was without effect. The reason for the disparity between the results and those of the Weizman group is not clear. However, they did find that, when daunomycin was attached to antibody via a dextran bridge, some drug activity remained. Another conjugate which apparently failed was prepared with an antimouse nerve growth factor antibody and adriamycin, which was used unsuccessfully against the S-180 mouse sarcoma (Vinores and Perez-Polo, 1979). The use of antibodies to oncofetal antigens has been considered. Belles-Isles and Pag6 (1980) and Pag6 and Belles-Isles (1980) coupled daunomycin to anti-alphafetoprotein antibody using glutaraldehyde. The conjugate appeared to be more cytotoxic for antigenbearing BW-7756 mouse hepatoma cells than free drug or antibody-drug mixture. When the tumor was treated in vivo, a higher survival rate, smaller tumors and increased latency were reported. Tsukada et al. (1982) have also utilized alphafetoprotein antibody. A rat ascites cell line AH66 was treated with conjugate and its ability to grow was examined in vitro and in vivo. Although synergism was seen in tissue culture, the antibody-drug conjugate was 100 times more effective than the admixture. When treated cells were injected intraperitoneally and survival of the injected animals was considered, there was once more a slight synergy of action but the highest survival rate was seen amongst the rats receiving specific conjugate.

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In this case five out of ten rats survived and were resistant to further challenge by tumor cells. In a further experiment, conjugate was used therapeutically. Rats were injected intraperitoneally with I x 104 hepatoma cells and were treated with conjugate on the third, fifth and seventh days by the same route. The mean survival times for rats given saline or normal IgG were 16.2 and 20.4 days respectively. For these given antibody, drug, normal IgG drug conjugate or antibody-drug mixtures, this improved to between 33.3 and 45.2 days. The best result (64 days) was seen in the group receiving antibody-daunomycin conjugate. A monoclonal antibody to rat mammary carcinoma Sp4 was shown to localize preferentially onto Sp4 tumors growing subcutaneously (Pimm et al., 1982). A conjugate was prepared with adriamycin, using a dextran bridge, with a substitution ratio of around 26:1. The product was used to treat tumor cells in vitro and more interestingly tumors growing in vivo. In a series of experiments the conjugate produced significant reductions in mean tumor diameter. No synergism was seen in a single trial and a control adriamycin-normal Ig conjugate was ineffective in all cases. In a further experiment the antibody conjugate was found to prolong by a significant period the survival of tumor bearing rats. In the light of the known propensity of fibrinogen to accumulate in tumors (Day et al., 1959a,b), Lee et al. (1978) coupled daunomycin to an anti-fibrin antibody with glutaraldehyde. While the conjugate was not significantly more directly cytotoxic than free drug, it was shown by indirect immunofluorescence to localize in the fibrin matrix of tumor (methyl cholanthrene-induced sarcoma, MC-D). For in vivo testing, guinea-pigs were inoculated subcutaneously in the flank with 106 MC-D sarcoma cells. Nine days later, a series of seven intraperitoneal injections on alternate days commenced, giving conjugate, free drug or antibody. An untreated control group was also included. None of the animals in the control groups showed tumor regression. However, of those treated with conjugate, three regressed completely and recovered for the tumor, while in the other three, tumor growth was slowed though ultimately the cancer grew and the animals died. Raso (1982) used the hybrid antibody method to produce a conjugate containing the polypeptide antibiotic, neocarzinostatin. An antibody to the drug was raised in a rabbit and was purified by affinity chromatography. This was hybridized with similarly obtained anti-human IgG F(ab)2. Ig positive cells exposed to the hybrid antibody were shown to bind it and be capable subsequently of uptake of drug. Although by this device an increased cell surface concentration of drug could be achieved when compared to control treatments, increased cytotoxicity did not appear to result. The suggestion is that this arises from the high cytotoxicity displayed by neocarzinostatin on its own. 4.5. CONJUGATE WITH VINDESINE In 1981 Johnson et al. coupled the anti-mitotic agent, vindesine, to an antibody against carcinoembryonic antigen (CEA), to give a conjugate with a molar ratio of drug to antibody of 4.6:1. A control conjugate with normal sheep Ig was also prepared. The target cell was a human lung tumor cell line Calu-6 and inhibition of incorporation of radiolabelled uridine was used to assess cytotoxicity. The results showed the specific conjugate to be much more effective than drug or antibody alone or in admixture. It was also more active than the control conjugate. Embleton et al. (1983) also preferred to use vindesine and coupled it to a monoclonal antibody directed against an antigen borne by a human osteogenic sarcoma line (791 T). Effectiveness was measured as the inhibition of uptake of [75Se]-methionine and the results were somewhat mixed. When the 791 T cells were exposed to the agents for 24 hr, free drug was 2000 times more cytotoxic than the conjugate. With shorter exposure (15min) vindesine was 1000 times less active compared with the 24 hr culture but antibody (and presumably conjugate) binding was almost complete in this period. Four osteosarcoma lines were tested for sensitivity to the agents using the short exposure and it was found that antibody alone was without effect while the conjugate was inhibitory

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for all four cell lines, being more active than free drug in three cases and less active in the fourth. In a series of controls with cells not bearing the antigen, the conjugate was ineffective although the cells were all affected by free vindesine. The authors conclude that these experiments demonstrate the imposition of selectivity on videsine by conjugation to an antibody. 4.6. CONJUGATESWITH RADIONUCLIDES Implicit in the experimental use of radiolabeled antibodies to demonstrate localization on to target tissue is their potential as chemotherapeutic agents. Bale et al. (1960) tried to exploit the ability of anti-fibrin antibodies to localize in tumors to attack transplantable Murphy-Sturm lymphosarcomas growing in rats. Antibody to rat fibrin was raised in rabbits and radiolabeled with 131I. Tumors were grown as subcutaneous implants and were allowed grow to between 2-3 g. A single intravenous injection of iodinated antibody sufficed to produce consistent, rapid and permanent tumor regression. In one experiment, four out of twenty-five rats were apparently cured of tumors. Nairn et al. (1963) also recognized the potential of radiolabeled antibodies and the idea was tested experimentally by Ghose et al. (1967). An antibody to Ehrlich ascites tumor cells was raised in rabbits and radiolabeled with 131I. Ehrlich cells were incubated with iodinated antibody and complement or with iodinated non-immune globulin. Later it was shown (Ghose et al., 1975) that mice injected intraperitoneally with ['31I]antibody, two hours after receiving tumor cells, survived three times longer than controls. The properties of radioactive antilymphocytic globulin were investigated by Dresser (1971). Using 1251as the radionuclide, he showed labeled antibody to give more prolonged protection of mouse skin grafts against immunological rejection than unlabeled antibody. Similarly, Anderson and Dresser (1971), with an immunopurified antilymphocyte globulin labeled with 1251,showed skin allograft survival to be improved by the use of radiolabeled antibody and that even better results were obtained if the animals were treated prior to being grafted. An alternative idea was developed by Hawthorne et al. (1972). Their suggestion was to use the ability of the l°B nucleus to liberate high energy fragments following neutron capture. Boron was incorporated into antibodies against human and mouse histocompatibility antigens. These conjugates were shown to produce destruction of specific cell populations when the treated cultures were subjected to neutron bombardment. 4.7. SUMMARY

In general the results using antibody-drug conjugates have shown some improvement over those produced with antibody or drug alone. In addition a reduction in whole toxicity of the drug was sometimes noted. Exploitation in man has not been widespread. The exact role of the antibody as a carrier in many experiments can be questioned since a lot of work involved the use of non-covalent complexes and synergism of action between antibody and drug given separately has been recognized. Most of the work with conjugates involving low molecular weight compounds was carried out before monoclonal antibodies were generally available and some appraisal of the possibilities of commercial drugs in association with monoclonal antibodies seems both appropriate and likely.

5. ANTIBODIES C O N J U G A T E D TO H I G H M O L E C U L A R W E I G H T COMPOUNDS 5.1. CONJUGATES WITH ENZYMES The amplification effect offered by the use of enzymes has attracted interest. Flickinger and Trost (1976) coupled phospholipase C to an antibody raised against cells carrying Friend leukemia virus. Normal spleen cells from DBA/2 mice were unaffected when treated in tissue culture with 0.60 or 1.2~o of conjugate. However, leukemic cells

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expressing the appropriate antigen were reduced to 2.1~o viability by the 0.6~ concentration of conjugate and completely killed by 1.2~o. The substrate for phospholipase is widely distributed and might be expected to prove troublesome were in vivo application considered. Philpott et al. (1973b,c) sought to overcome this difficulty by the use of glucose oxidase, conjugated to antibody. At the target cell surface the oxidase became bound via the antibody and oxidized glucose to gluconic acids with the release of hydrogen peroxide. In the presence of iodide and lactoperoxidase, the cells would be lethally iodinated. Experimentally, hapten (2.4.6-trinitrophenyl) labeled cells were shown to be selectively killed by an antihapten-glucose oxidase conjugate in the presence of lactoperoxidase and iodide. The method worked for TNP-HeLa and TNPHep-2 cells. To test the method in vivo, TNP-cells were treated with antibody-enzyme conjugate prior to injection into the left thighs of recipient mice. TNP-cells without conjugate were put into the right thighs. Lactoperoxidase was given intravenously (or intraperitoneally) immediately after the cells and was followed by Na125I. Under such conditions a significant increase in iodination was evident in the cells in the left thigh, i.e. those cells pretreated with antibody-glucose oxidase conjugate. The same group (Shearer et al., 1974) coupled glucose oxidase to antibodies against a human colonic cancer cell line (HT-29) and to anti-CEA antibody. In both cases the conjugates were more effective cytotoxic agents than the same antibodies in unconjugated form and in the presence of complement. Another modification involved a different cytotoxic agent, arsphenamine replacing iodide (Philpott et al., 1974). This compound was activated by horseradish peroxidase in the presence of glucose and glucose oxidase. As with the iodination procedure, it was the hydrogen peroxide that released the active species, in this case an arsenical compound. Yet a further variation on this theme was developed by Parker et al. (1975). Here the authors made use of the reactivity of 5-amino-2-3-hydrol,4-phthalazinedione (luminol). This compound when oxidised with horseradish peroxidase gave an unstable free radical capable of rapid reaction with cell membranes. As this reaction would link to the membrane any molecule to which luminol might be conjugated, this was seen as a potential method for catalytic entrapment of enzymes, toxins and haptens at the cell surface. 5.2. CONJUGATES WITH TOXINS

The current trend has been away from the use of drugs in favor of toxins, their A chain or other ribosome damaging proteins. The argument in their favor is that, being enzymic in action, they are many times more active than drugs acting stoichrometrically. In fact it has been estimated that entry into the cytosol of only one molecule of A chain from diphtheria toxin or ricin is sufficient to kill the cell (Yamaizumi et al., 1978, Eiklid et al., 1980). The toxins under consideration are diphteria toxin, the exotoxin from Corynebacterium diphtheriae and the two toxins of plant origin, abrin and ricin isolted from the seeds of Abrus precatorius and Rieinus communis respectively. Their modes of action have been described in reviews (Collier, 1977; Olsnes and Pihl, 1977) but will be briefly recapitualated here as being germane to the rest of the discussion. A short description of ribosome inhibiting proteins is also given. 5.2.1. Toxins and Ribosome Inhibiting Proteins 5.2.1.1. Diphtheria toxin. The structural information for diphtheria toxin synthesis is carried by a phage gene in the bacterium (Uchida et al., 1971). The toxin is produced as a single polypeptide chain containing two disulfide bridges, one of which subtends a loop containing three arginine residues (Michel et al., 1972). This site is readily cleaved by enzymic hydrolysis giving rise to the so-called 'nicked' toxin, the fully active form comprising two polypeptide chains joined by a single disulfide bridge (Collier and Kandel, 1971; Gill and Dinius, 1971).

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Nicked toxin can be further split to give an A chain and a B chain with different functions within the intact molecule. Briefly, the B chain serves to bind the toxin onto a receptor (as yet only partially characterized) on the cell surface (Proia et al., 1979). It is also presumed to facilitate the entry of the A chain into the cytosol of the cell. The mechanism has not been fully elucidated. A chain activity is better understood. Acting enzymically it catalyses the transfer of ADP-ribose from nicotinamide adenine dinucleotide (Kandel et al., 1974) to elongation factor 2 (EF2), an enzyme involved in the translocation of polypeptidyl-transfer RNA from the acceptor to the donor site on the eukaryotic ribosome (Honjo et al., 1968). ][nactivation of EF2 leads to cessation of protein synthesis and death of the cell ensues. :5.2.1.2. Abrin and ricin. The seeds of Abrus precatorius and Ricinus communis contain the toxin glycoproteins, abrin and ricin, respectively. Both are similar in structure to nicked diphtheria toxin, comprising as they do an A chain and a B chain linked by a single disulphide bridge. The B chains of abrin and ricin bind to the cell surface via glycoproteins containing galactose, in the terminal or subterminal position. The number of such receptors has been put at 10 6 for human erythrocytes and 3 × 10 7 for HeLa cells (Sandvig et al., 1976). Binding of the toxins and hence their cytotoxicity, is subject to competitive inhibition by galactose (Olsnes et al., 1974), although high concentrations are required (Sandvig et al., 1976). As with diphtheria toxin, the mechanism by which the A chain reaches the cytosol is obscure, but once inside, or at least in contact, with the cytosol, it inactivates the 60S ribosome subunit and protein synthesis ceases. Since one molecule of a chain can inactivate 1,500 ribosomes per minute in a reticulocyte extract, the action is presumed to be enzymatic in nature (Olsnes et al., 1975). ADP-ribosylation of EF2 does not appear to be involved in the mechanism of action of abrin or ricin. 5.2.1.3 Ribosome inhibiting proteins. There also exist in the plant kingdom a number of proteins which appear to be analogous to the A chains of abrin and ricin. They are relatively non-toxic, probably due to the absence of an equivalent B chain. These ribosome inhibiting proteins (RIP) have been described inter alia from Phytolacca americana (Obrig et al., 1973), Dianthus caryophyllus (Stirpe et al., 1981),Triticum aestivum (Stewart et al., ]977), Gelonium multiflorum (Stirpe et al., 1980), Momordica charantia (Barbieri et al., il980) and Croton tiglium and Jatrophe curcas (Stirpe et al., 1976). They are found in different parts of different plants and are present in variable quantities. The function of these RIPs (or of the holotoxins for that matter) in the plant is unknown, but their wide distribution and sometimes great abundance suggest some important role. They tend not to inhibit homologous ribosomes. Thus the product from Phycolacca americana, PAP, did not inactivate pokeweed ribosomes (Owens et al., 1973), neither did the wheatgerm product, tritin, affect wheatgerm ribosomes (Coleman and Roberts, 1981). An anti-viral function has been suggested (Grasso et al., 1980, Stevens et al., 1981, Stirpe et al., 1981). 5.3. CONJUGATES WITH DIPHTHERIA TOXIN The first attempts to harness the specificity of antibodies to the extreme potency of the toxins was reported by Moolten and Cooperband in 1970. In this pioneering work, diphtheria toxin was coupled to an anti-mumps virus antibody, using toluene diisocyanate in a two step procedure. The product was used to attack mumps virus infected monkey kidney cells growing in tissue culture. The antibody-toxin conjugate was more toxic for mumps infected than for uninfected cells. There was also a considerable loss of cytotoxicity relative to free toxin. In the next few years, the same group (Moolten et al., 1972, 1975, 1976) reported results from a variety of experimental systems, but they changed to the use of glutaraldehyde as a linking agent. This method might induce intra chain cross links and inactivation of the

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toxin, a reservation supported by the low whole body toxicity of their conjugates (MLD was 20-140/~ g). One way used to increase the number of antigenic sites and to provide suitable target specificity is to use hapten coated cells. This method was used by Philpott et al. (1973a) who coated HeLa cells with trinitrophenyl (TNP) groups and attacked the cells in tissue culture with a conjugate of anti-TNP antibodies and diphtheria toxin. TNP coated HeLa cells were selectively killed relative to uncoated cells and the cytotoxicity was inhibited by free hapten. In the Moolten experiments an antibody to the dinitrophenyl moiety (DNP) was coupled to diphtheria toxin and used against TT101 hamster sarcoma cells which had been labeled with DNP. Hamsters were implanted subcutaneously with tumor and treated concurrently with a subcutaneous injection in the opposite flank. Treatment with conjugate delayed the appearance of palpable tumor and prolonged the lifespan of the animals. If the inoculum size was reduced to 3 × 102 cells, there was also a reduction in the incidence of tumor occurrence. Established TT101 sarcomas were refractory to treatment with even repeated doses of conjugate but with established TRD14 lymphomas, three doses at weekly intervals sufficed to induce complete regression in 12 out of 28 animals. In another model, antibodies against Simian virus 40 transformed hamster sarcoma or lymphoma cells were immunopurified and coupled to diphtheria toxin. The resulting conjugate was specifically cytotoxic for SV40 transformed cells in tissue culture. When hamsters were implanted subcutaneously in the left flank with 10 3 tumor cells and injected in the right side with 0.2 MLD of conjugate or control, the groups receiving conjugate showed a modest but significant protection. Following this early work, the problems alluded to earlier concerning the use of glutaraldehyde were overcome by Thorpe et al. (1978), who employed a derivative of chlorambucil in a two-stage process as already discussed. Thorpe et al. (1978) and Ross et al. (1980) linked diphtheria toxin to a horse anti-human lymphocyte globulin (AHLG) (and to F(abl)2 fragments from it). The conjugate was over 100 times more effective as an inhibitor of protein synthesis by the human lymphoblastoid cell lines CLA4 and DAUD1 than the free toxin. A conjugate made with a normal horse immunoglobulin (NIgG) was fifty to one hundred times less active than the free toxin. The activity required the expression of both binding through the antibody and the cytotoxicity of the toxin as shown by competitive inhibition by excess free antibody and blockade by diphtheria antitoxin. Further when non-lymphoid cells, human fibroblasts, were used as targets, the AHLG and NIgG conjugates were equipotent. There was no synergism between free antibody and toxin. This was the first occasion on which such potent agents had been reported and as it still provides one of the few examples in which the cytotoxicity of the conjugate exceeds that of the free toxin, it is worthwhile to examine the result in more detail. A surprising finding was that lymphoblastoid cells were relatively resistant to the toxin. Previously it had been thought that cells from species of animals sensitive to the toxin would likewise to sensitive. The only exceptions were reticulocytes (Collier, 1977) and deliberately selected variants of KB human epidermoid carcinomas and chinese hamster ovary cell lines (Moehring and Moehring, 1972, 1977) and of HeLa cells (Venter and Kaplan, 1976). The resistance of the lymphoid lines seemed to indicate poor expression of the receptor. By attaching the toxin to the antibody, an increase in toxin concentration at the cell membrane could be achieved with increased likelihood of cell kill. While such a mechanism could account for the effects seen with the cells of human origin, it did not explain the observations made with mouse cells. In this case, a conjugate of anti-mouse lymphocyte globulin and diphtheria toxin was not cytotoxic to mouse lymphocytes, although it did bind to them (Ross et al., 1980). Thus murine cells seem to be non-permissive for the translocation of diphtheria toxin A chain into their cytosol. The AHLG used in these experiments was polyclonal and would have contained

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relatively little specific antibody. The use of a monoclonal antibody might have been expected to give an improvement in potency provided that there was no paucity of antigen on the surface and that no other restriction to toxin expression was operating. When diphtheria toxin was conjugated to MAI/34, a monoclonal antibody recognizing the HTA1 on human lymphocytes (McMichael et al., 1979), the resulting product was highly effective :and specific for the thymic leukemia cell line MOLT4 and was about ten times more potent than the conjugate made with the polyclonal AHLG (Thorpe et al., 1982). 5.4. CONJUGATES WITH ABRIN

Using the derivatized chlorambucil conjugation procedure, Edwards et al. (1981) joined a horse anti-mouse lymphocyte globulin (AMLG) to abrin. The conjugate was tested in vivo using suppression of the ability of the mouse to respond immunologically to an injection of sheep red blood cells as the criterion for activity. The AMLG-abrin proved about twice as effective as NIgG-abrin and this differential was the same as that found for the two conjugates in vitro. By comparison with antibody alone, AMLG-abrin was 50,000 times more active. In a repeat of this work using a conjugate that had been purified by a second gel filtration stage, a differential of four fold was found (Edwards, unpublished). The immunosuppression model has also been used to compare the effect of the nature of the linkage on the biological activities of conjugates (Edwards et al., 1982). AMLG-abrin conjugated by the chlorambucil method was compared with AMLG-S-Sabrin made by the Carlsson et al. (1978) technique. The activities of both conjugates on a reticulocyte ribosome preparation, i.e. A chain activity, were identical but the AMLG-S-S-abrin was the more effective cytotoxin when used against mouse spleen cells in tissue culture. It was also more acutely toxic for mice when injectedintraperitoneally. However, it was markedly less effective as an immunosuppressive agent and premature dissociation by reduction or sulfydryl exchange in the tissues was postulated. Although in these studies the carrier effect of antibody could clearly be demonstrated, it was obvious that the abrin component of the conjugate retained much of its ability to bind and kill independently of the antibody. To overcome this, Thorpe et al. (1981) used the known property of galactose as a competitive inhibitor. With a conjugate of AHLG and abrin they showed that, in the absence of the sugar, AMLG-abrin was ten times more active than NIgG-abrin when used against Daudi cells in tissue culture. When 100 mM lactose was present, the toxicity of both abrin and NIgG-abrin was abolished but that of AHLG-abrin was only slightly affected. This device, which could be useful in the removal of one population of undesirable cells from a mixture with another desired population, has not so far proved useful in vivo. The highest maintained plasma concentration of lactose achieved was 5-10 mM and was not sufficient to affect the immunosuppressive potency of either NIgG-abrin or of NIgG-ricin (Edwards, unpublished). 5.5. CONJUGATES WITH RICIN

Refsnes and Munthe-Kaas (1976) used ricin-antiricin B chain antibody complexes and found that they could inhibit protein synthesis by Kupffer cells and by peritoneal macrophages. The cells were sensitive to free ricin, and toxicity was subject to lactose competition. The ricin-anti-B chain complex was not affected by lactose, although it was blocked by soluble complexes of rabbit IgG-anti-rabbit IgG. HeLa cells were unaffected by the ricin-anti-B chain and it seems clear that Fc receptors were responsible for the effects reported. By contrast, ricin-anti-A chain complexes were non-cytotoxic. A somewhat similar approach was adopted by Raso and Griffin 0979, 1981). They constucted hybrid antibodies from anti-ricin A chain and anti-human IgG antibodies by the method of Nisonoff and Rivers (1961). The hybrid was reacted with IgG bearing cells to which it bound through the anti-IgG variable region. Ricin was then added in the presence of an inhibitory concentration of lactose so that the only binding to occur was via the second (anti-A chain) are of the hybrid. Under these conditions cytotoxicity resulted. These results contrast with those of Refsnes and Munthe-Kass (1976), in that JPT23/I K

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when Fc receptor mediated internalization was used, the A chain (anti-A chain) complex was inactive. When binding to surface Ig was involved, A chain anti-A chain appears to be cytotoxic, probably indicating A chain entry to the cytosol to be mediated by a different pathway. Other workers have preferred to use covalent conjugates of ricin and antibody. Moolten et al. (1976) reported that an antibody-ricin conjugate failed to show selective toxicity. Youle and Neville (1980) also coupled ricin to antibody, in this case a monoclonal anti-Thyl. 1 antibody. This conjugate, they found to be as toxic for EL4 cells as ricin itself but, whereas the free toxin was inhibited by 100 nM lactose, the conjugate was not. The specificity of the conjugate was shown by the fact that in the presence of lactose, neither AKR T-cells (Thyl. 1) nor HeLa cells (Thyl negative) were affected. Vallera et al. (1982) have used these observations as a basis for the elimination of T-cells from bone marrow grafts in order to obviate the risk of graft-versus-host disease when cells of one histocompatibility phenotype are transferred into an animal of another. In a similar way, Thorpe et al. (1982) have demonstrated the removal of malignant cells from bone marrow of a leukemic rat, using monoclonal antibody W3/25 conjugated to ricin and by abrogating non-selective toxicity by working in the presence of lactose. 5.6. CONJUGATES WITH A CHAINS OR RIBOSOME INHIBITING PROTEINS

Theoretically there are several ways of decreasing the influence of the toxin B chain in the expression of conjugate activity. Competitive inhibition by lactose has already been mentioned. Non-reversible substitution of the sugar into the binding site is feasible but has not been reported. The most usual strategy is to use isolated A chain and thus remove B chain influence entirely. The first experiments along this line were carried out by Chang et al. (1977) using not antibody but human placental lactogen bound to diphtheria toxin A chain. Later, Oeltmann and Heath (1979) followed the same general line but used the cell specific subunit of human chorionic gonadotropin attached to diphtheria toxin A chain. Among the first to publish a substantive paper on the use of antibody-A chain conjugates were Masuho et al. (1979), who coupled Fab~ fragments of rabbit anti-mouse L1210 leukemia antibody to the A chain of diphtheria toxin. The conjugate was cytotoxic for L1210 cells at a concentration of 10 -9 to 10 -8 M whereas diphtheria toxin, A chain or a conjugate of A chain with an anti-DNP antibody were ineffective. Similar results were later reported by Masuho and Hara (1980) using A chain from ricin in place of that from diphtheria toxin. These results, although encouraging, were well short of the potency levels recorded for free toxin and probably indicate a deficiency in the ability of the conjugate to induce delivery of A chain into the cytosol. The propensity of the lectin concanavalin A to bind to cell surfaces has been used by Gilliland and Collier (1980) to provide target molecules on cell membranes. By coupling anti-concanavalin A antibodies to diphtheria toxin A chain, they produced an agent cytotoxic for con A coated murine 3T3 cells and Chinese hamster ovary cells but not to uncoated cells. These results and those of Masuho et al. (1979) indicate that mouse cells normally resistant to diphtheria toxin may become sensitive if an anchorage point for the A chain is provided. This is in contrast to the results of Ross et al. (1980) whose anti-mouse lymphocyte globulin-diphtheria toxin conjugate was inactive against mouse cells. The reasons for the discrepancy are unclear but may reflect qualitative differences in the nature of the membrane antigen being recognized by the various antibodies. In 1980, Raso and Griffin described the first of several of their attempts to produce effective agents from antibody and ricin A chain. In this they split an anti-tumor IgG antibody to give the Fabl fragment which was treated with Ellman's reagent prior to being used in thiol-disulfide exchange reaction with isolted ricin A chain. The resulting conjugate was selective for human cells bearing Ig on their surface. In co-cultures of Daudi (Ig +) and CEM (Ig-) cells, only the Daudi appeared to be affected and there was no concurrent killing of the second cell type.

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A similar approach was tried by Miyazaki et al. (1980). They produced a conjugate of a rabbit anti-mouse IgG with ricin A chain and showed it to be selectively cytotoxic for mouse B lymphocytes. Both the Raso and Griffin and the Japanese conjugates were considerably (50 x and 1000 x respectively) less cytotoxic than free ricin. Around this time (1980), work from other groups began to appear. Jansen et al. (1980) coupled an immunopuriffed rabbit anti-dinitrophenol (DNP) antibody to ricin A chain. Their product was used against TNP bearing HeLa cells in tissue culture and, while it was between 10-100 times less cytotoxic than free ricin, it was 50-500 times more active than free A chain. When given intraperitoneally to nude mice which had received an intraperitoneal injection of hapten coated HeLa cells one hour previously, the conjugate reduced tumour incidence from 14 out of 15 (93~o) in untreated mice to 1 in 15 (7~o). Somewhat similar experiments were conducted by Blythman et al. (1981) using an alternative antigen, in this case the alloantigen Thyl. 2. The antibody was a monoclonally derived IgM which, when coupled to ricin A chain, inhibited protein synthesis in a Thyl. 2 bearing mouse leukemia cell (WEHI-7) by 50~o at a concentration (IDs0) of 10 -~° M, and was 5000 fold more effective than ricin A chain alone. Free ricin was 25 times more active lhan the conjugate. When used to treat WEHI-7 cells in the peritoneum of the mouse, the conjugate gave significant prolongation of survival when 6 × 105 cells were used. At higher cell inoculum (2.4 × 106) no beneficial effect was seen. Gilliland et al. (1980) also reported on the use of a monoclonal antibody, in this case one directed against colorectal carcinoma cells of human origin. The antibody (IgG) was coupled to a chain from either diphtheria toxin or ricin. Although the conjugates appeared not to be particularly potent, killing cells in the region of 10 7 to 10 -8 M, they were specific in that among seven cell types tested only the two of colorectal origin were affected. The adoption of surface immunoglobulin as a potential target antigen by Raso and Griffin (1980) and by Miyazaki et al. (1980) has been further developed by Vitetta et al., 1982). They prepared a number of antibodies directed against several antigens expressed on the surface of normal mouse B lymphocytes and murine B cell tumors (BCL0. The ,antibodies were conjugated to ricin A chains. Protein synthesis in normal B lymphocytes from mice was shown to be inhibited by a conjugate of anti-~ chain and ricin A chain. Also when monoclonal antibodies against either the a or b allotypes of IgD were used, conjugates were inhibitory and displayed specificity for cells of appropriate allotype. Also, ,an antiidiotype antibody was prepared against the surface immunoglobulin expressed by the BCL~ tumor cell, which when linked to ricin A chain proved cytotoxic for the appropriate target cells (Krolick et al., 1980). In a later study (Krolick et al., 1982), the application of the same technology to the therapy of BCL~ growing as a tumour in vivo was made. Antiidiotype and anti-6-chain antibodies were prepared and joined to ricin A chain. BALB/C mice were injected intravenously with 106 BCL~ cells and treatment begun 6-8 weeks later. Tumor burden was reduced by X-irradiation and splenectomy, prior to the use of conjugate. Intravenous administration of the anti-chain-ricin A chain conjugate was shown to eliminate the residue of BCL~ cells not removed by the non-specific cytoreductive therapy in three out of four experiments. By contrast, all animals treated with a control conjugate or with antibody alone relapsed between three and four weeks after splenectomy. Specific killing of a human breast carcinoma cell line by a conjugate of a monoclonal ,antibody and ricin A chain has been shown (Krolick et al., 1981). In this instance the .antibody was a monoclonal IgM and the conjugate was found to kill only cells bearing the appropriate antigen. A control conjugate was ineffective. An anti-CEA antibody has been studied as a carrier of ricin A chain by Griffin et al. (1982). The conjugate was 40 times as potent as A chain alone, or in admixture with antibody, when used against a CEA-bearing human adenocarcinomas cell line, but its activity was similar to that of free A chain when both were tested against a CEA-negative human melanoma cell line. Most work involving the use of conugates has concentrated on producing effects in tissue culture, and by and large the results have been more encouraging in this system than in

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the more demanding/n vivo work. This had led to a recent interest in the application of conjugate technology to the removal of unwanted cells from bone marrow transplants. These unwanted cells are of two types. If autologous grafts are being considered for cancer patients, the requirement is to remove malignant cells present as micro-metastatic deposits in the marrow. In the case of allografts, the need is to eliminate T lymphocytes responsible for the development of graft-versus-host disease in the recipients. Several studies have been addressed to these problems. Krolick et al. (1980) used a conjugate of affinity purified anti-mouse immunoglobulin and ricin A chain to treat spleen cells (8~o tumor cells) from BCL1 bearing mice. After treatment in vitro, 104 cells were injected into normal BALB/C mice. In animals receiving specific conjugate treated cells there was no increase in leukemic cells in spleen or blood over a period of up to twelve months. Since test transfers showed that 95~ of animals injected with 10 BCLI cells and 43~o of those given a single cell developed leukemia, it appeared that in the 104 conjugate treated cells injected, less than 10 viable tumor cells were present. Similarly, it was estimated that of mice rescued from lethal irradiation by 10 6 conjugate treated bone marrow cell from BCLI bearing mice, only 1-10 tumor cells remained. In both instances cell kills of 99.9~o were estimated. An antibody of potential use in human bone marrow cleansing has been described. This is the monoclonal J-5 said to be specific for the common acute lymphoblastic leukaemia antigen (CALLA) (Ritz et al., 1980). This has been coupled to ricin A chain by Raso et al. (1982) and the conjugate shown to be a highly active cytotoxic agent for Nalm-I cells which express the antigen. The difference in senstivities of CALLA + and C A L L A - cells was around 5000 fold. The same workers also showed F(ab~)2 fragments of the antibody to be more efficient carriers of ricin A chain than F a b , possibly due to the extra valency of the dimer. Selective killing of malignant cells in bone marrow has also been studied by Thorpe et al. (1982) using a monoclonal antibody W3/25 which has a high affinity for rat thymocytes, T helper cells or macrophages. A conjugate with ricin A chain was ineffective as a cytotoxic agent against rat PVG leukemia cells. The suggestion that this was due to a failure of endocytosis was disproved by the use of a rabbit anti-mouse immunoglobulin antibody which induced patching and capping but with no effect on protein synthesis in the cell (Thorpe and Ross, 1982). A W3/25 conjugate with ricin was, however, very effective as a cytotoxic agent for PVG cells and was used to remove leukemic cells in admixture with bone marrow cells prior to repopulating sublethally irradiated recipient rats. Using a chimera, the authors were able to show, as had Krolick et al. (1982) that their conjugate was capable of killing 99.9~o of leukemic cells. Around 50~ of the hemopoietic stem cells were spared. The presence of lactose in their medium not only reduced non-specific cytotoxicity but also prevented the transfer of lethal quantities of ricin via the cells of the graft. The prevention of graft-versus-host disease following bone marrow transplantation across a major histocompatibility barrier has been studied in the mouse by Vallera et al. (1982). For this purpose they used anti-Thyl. 2 antibody coupled to ricin. BALB/C spleen and bone marrow cells were given to irradiated C57 B1/6 recipient mice. Provided the cells were pretreated with conjugate in the presence of lactose, the incidence of fatal graftversus-host disease was reduced from around 90~ to around 9~o (1 out of 11 mice died). The possibility of using ribosome inhibiting proteins (RIP) of plant origin not derived from toxins has been discussed earlier and so far two such attempts have been reported. One involved the use of gelonin by Thorpe et al. (1981). The experiment was to couple gelonin to a monoclonal anti-Thyl. 1 antibody and to use the product(s) against Thyl. l bearing lymphocytes from A K R strain mice. When cells were treated with a high molecular weight complex and subsequently stimulated to divide with phytohemagglutinin or concanavalin A in tissue culture, the complex was shown to be as effective a cytotoxin as was free abrin. There was on the other hand little or not toxicity exerted on B-lymphocytes or on Thyl. 2 expressing T-cells from CBA mice. In further work reported by Thorpe and Ross (1982), it was shown that gelonin linked by the chlorambucil method, i.e. through

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a non-reducible bond, to the antibody was less active by 20 fold than the comparable disulfide linked conjugate. As it is often assumed that dissociation of the A chain and its release in free form into the cytosol is an integral part of the expression of toxicity, the potency of the chlorambucil-linked conjugate was unexpectedly high. It is not clear whether the whole conjugate was translocated or whether some degradation occurs within the lysosomes leading to release of active A chain. Recently Masuho et al. (1982) coupled the anti-viral protein (PAP) from Phytolacca americans to the Fabl from an anti-L1210 antibody raised in the rabbit. The conjugate was prepared using SPDP and was effectively cytotoxic against L1210 cells, the activity was subject to competitive inhibition by F(ab02, and was comparable in level with that of a similarly prepared conjugate of the antibody and the A chain from ricin. 5.7. SUMMARY Although the use of conjugates of enzymes has been considered, their use has not been very actively pursued. Much more interest has focused on the possibilities offered by the ase of toxins, their subunits or of ribosome inhibitors. Conjugates of holotoxin which were very active and specific in vitro have been prepared. High in vivo activity and some specificity together with reduced whole body toxicity has been described. When A chain subunits or ribosome inhibiting proteins are used, the results are more mixed. Some very active and specific conjugates are known but others have relatively low activity. The reasons for this may be associated with the particular antigen to which the antibody component is directed, the nature of the A chain or inhibitor and the type and physiological state of the target cell. Application to man seems likely in the first instance to involve the removal of undesirable cells from bone marrow transplants. 6. CONCLUSION Although the idea of using antibodies as vectors to carry destructive chemicals to specified target tissues has been around for some 70 years, it is only now being actively pursued. The interval has seen our understanding of the chemistry of proteins in general and of antibodies in particular expand to the stage at which confident manipulation and even modification are commonplace. The development of the hybridoma technique has given additional impetus by providing antibodies that are, potentially at least, chemically pure. For these reasons and by virtue of their ability to circulate and localize, antibodies are currently the favored vehicles to act as carriers of non-specific cell poisons. The choice of poison, lying somewhere between conventional drugs and enzymaticallyacting toxins, will probably never resolve to a single preferred compound. Constraints of premature clearance from the blood stream, immunogenicity, natural or acquired immunity and licence requirements will apply, and the issue will finally depend on a favorable therapeutic index being demonstrated. From results in tissue culture it is quite clear that antibody will act as a carrier and that very effective and specific cytotoxic agents can be constructed. In the more demanding field of in vivo experimentation, there has been less success, nevertheless the indications encourage optimism that with a proper choice of carrier, effector, chemical linkage and target, a highly active, specific and safe agent will be manufactured. Although much of the effort is at present devoted to the search for a specific for tumour chemotherapy, it may well be that application will be most readily made in some other field. Immunosuppression in transplantation, clonal deletion in autoimmune states, destruction of parasites, bacteria or virus infected cells, all provide potential candidates. The retiarii, gladiators in ancient Rome, who used nets to trap their opponents before attacking them with tridents, provide a neat anthromorphic model of conjugate action. Antibodies provide the net, poisons are the trident and retiarian therapy is the name of the game.

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