(D The Macmillan Press Ltd., 1987
`Br. J. Cancer (1987), 55, 227-231
`Br.~~~~~~~~~~~~~~~~~ J. Cacr(97,5,2721-TeMcilnPesLd,18
`
`EDITORIAL
`Drug-targeting by monoclonal antibodies
`
`The advent of monoclonal antibodies (MoAbs) has already made an impact on cancer diagnosis,
`particularly in the areas of immunoscintigraphy and immunohistology, and the great hope of many
`oncologists is that MoAbs will also come to have a major role in therapy. There are already several
`reports of trials in which anti-tumour monoclonal antibodies have been administered to cancer patients,
`often with transient or quite long-lasting clinical benefits (Levy & Miller, 1983; Ritz et al., 1981;
`Koprowski et al.,
`1984). However, in objective terms the effects observed cannot be described as
`dramatic. No MoAb treatment has yet resulted unequivocally in permanent tumour regression, and
`most studies reporting clinical improvements still require independent confirmation. It is thought that in
`most cases the reported responses have been due to interaction of MoAbs with host effector
`mechanisms. In the short term the most important mechanism appears to be antibody-dependent cellular
`cytotoxicity (Herlyn et al., 1980; Levy & Miller, 1983), and there is evidence suggesting that in the
`longer term there may, in some cases, be immunomodulation via
`the immunological network
`(Koprowski et al., 1984). Many patients mount an immune response to the injected MoAb, some of
`these reactions being directed against the antigen-binding portion of the antibody molecule, referred to
`as the idiotype. It has been suggested that anti-idiotype antibodies produced by these patients can
`behave as 'mirror images' of the MoAb defined tumour-associated antigen, and may ultimately stimulate
`systemic host immunity against the tumour. Certainly, prolonged stabilisation or regression tends to be
`associated with an anti-idiotype response on the part of the patient (Koprowski et al., 1984).
`The number of MoAbs capable of inducing such host-mediated effects is likely to remain small,
`however, and it is by no means certain that such interactions can be amplified sufficiently to achieve a
`major therapeutic response. It is considered by many researchers that the greatest therapeutic potential
`of MoAbs lies in the targeting of anti-cancer agents (drugs or toxins) rather than their use in
`unmodified form. Monoclonal antibodies are much more suitable than polyclonal antibodies for this
`purpose because of their defined specificity and their constant physico-chemical properties. Although
`some clinical trials of MoAb-targeted drugs or toxins have begun and others are imminent, this
`approach is generally still very much at the experimental stage, and the present discussion aims to
`highlight some of the problems which have to be overcome rather than describe the positive
`achievements to date. The areas discussed will refer particularly to the systemic administration of
`MoAb-linked therapautic agents. An alternative use for them is the elimination of neoplastic cells (or T
`cells in the case of allografts) from bone marrow, ex vivo, with a view to marrow replacement in
`patients treated with high-dose chemotherapy or whole-body irradiation (Filipovich et al., 1984; Casellas
`1985). However, the attendant problems in this application are less severe than in systemic
`et al.,
`administration because the patient is not directly exposed to the antibody-targeted drug or toxin.
`
`Construction of conjugates
`(a) Antibody vector The overall aim of drug targeting is two-fold: to increase the uptake of the anti-
`tumour agent by the tumour and to decrease uptake by other tissues and thereby avoid, or substantially
`reduce, toxicity. Either of these objectives should by itself increase the therapeutic index of the targeted
`drug, and the two together should in theory result in significant advantages over conventional
`chemotherapy. It is self-evident that the first requirement of any potential targeting MoAb is that it
`should recognise antigens expressed upon tumour cells but not on normal cells, and thus be capable of
`localising to tumour deposits in vivo. Many MoAbs have now been raised against human tumours, and
`some are indeed claimed to be specific for particular tumour types (Wright, 1984; Sell & Reisfeld, 1985).
`However, rigorous testing (e.g. by immunohistology) usually reveals cross-reactions with some normal
`tissues albeit often at a low level. It is more realistic to think of currently available anti-tumour MoAbs
`as detecting antigens expressed on tumour cells at a quantitatively higher level than in normal tissues,
`rather than being qualitatively tumour-specific. This raises the possibility of drug delivery to innocent
`cells, but so long as binding is at a low level or the cells involved are not important to the survival of
`the host, this may be considered an acceptable risk.
`Other obvious requirements are that the antigen detected should be expressed at the cell surface and,
`according to current thinking, that the antibody and any agent linked to it should be internalised
`following binding to the antigen. Another important property is the ability of the MoAb to withstand
`the chemical treatments involved in attaching sufficient molecules of the anti-cancer agent without
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`severely impairing antigen-binding activity. In practice different MoAbs vary widely in their ability to
`withstand substitution, as exemplified by retention of activity ranging from 2% to 98% by different
`antibodies substituted with vindesine (Rowland et al., 1983). Resistance to inactivation shows little
`correlation with antibody isotype other than the fact that some IgM antibodies are particularly sensitive,
`and the picture is further complicated by the finding that a single antibody may be affected to different
`degrees by coupling to different drugs (Embleton, 1986). At our present level of understanding, the
`suitability of any candidate IgG antibody from this viewpoint can only be determined empirically.
`Fortunately it is possible to minimise damage to the antibody by coupling the drug to an 'inert' carrier
`(such as a protein, polypeptide or dextran) which is then attached to the MoAb on an equimolar basis,
`rather than overloading the antibody with multiple drug residues (Garnett & Baldwin, 1986; Rowland et
`al., 1975; Hurwitz et al., 1978).
`
`Cytotoxic agents exploited to date in experimental or preliminary
`(b) Targeted anti-tumour agent
`clinical studies include cytotoxic drugs, toxins of plant or bacterial origin, and radionuclides (see reviews
`in Moller, 1982; Davies & Crumpton, 1982; Baldwin & Byers, 1985). Cytotoxic drugs have a potential
`advantage in that there is already considerable experience in their use clinically, and their side-effects (in
`un-conjugated form) are well known. However, they have several disadvantages with regard to targeting.
`In the first instance it is necessary for the drug to have a chemical group, separate from its active site,
`which will allow it to be coupled by a covalent linkage to the antibody or carrier molecule without
`is possible to introduce suitable groups if necessary by
`losing its cytotoxic activity. In some cases it
`chemical substitution, but certain drugs are clearly not amenable to conjugation. Another difficulty is
`posed by the relatively high intracellular concentration of drug required to kill target cells. Even
`combinations of the most active drugs and sensitive tumour cells probably depend upon intracellular
`106 or more drug molecules. While this can be achieved by tissue fluid concentrations
`accumulation of
`is necessary in the case of MoAb targeting for the drug to be
`obtained at clinically used doses, it
`internalised following binding of the conjugate to cell surface antigen sites. This demands a high antigen
`concentration and preferably a high antigen turnover and recycling rate at the cell surface, as well as
`reasonably high levels of drug substitution. The problem is further exacerbated by the finding that
`usually the drug loses much of its activity following binding to protein (Embleton, 1986). Reasons for
`this are not clear, but possibly stearic hindrance or altered mechanism of uptake are contributory
`factors. The most effective solution is to couple the drug to a carrier molecule, which can be heavily
`substituted in order to deliver increased amounts of drug to each antibody-binding site (Garnett &
`Baldwin, 1986; Rowland et al., 1975; Hurwitz et al., 1978). To some extent the need to deliver large
`quantitities of drug may enhance the specificity of such conjugates, since only cells bearing a high
`antigen density are likely to receive enough drug to result in death. Weakly antigenic cells which bind
`only small amounts of conjugate may not accumulate enough to bring about irreversible damage.
`Plant toxins are attractive agents for targeting because they are extremely cytotoxic. It has been
`suggested that entry of perhaps a single molecule of toxin can kill a target cell (Eiklid et al., 1980). This
`is because, unlike drugs which mostly behave in a stochiometric fashion, the toxins act enzymatically.
`Being proteins, they are easily conjugated to antibodies by means of heterobifunctional reagents (Thorpe
`& Ross, 1982). Toxins with lectin properties (e.g. ricin) can be coupled to antibody in such a way that
`the lectin site is inactivated, or in the form of a toxic A chain sub-unit from which the sugar-binding B
`chain has been enzymatically cleaved (Thorpe & Ross, 1982). The conjugate is then able to bind to cells
`bearing the antigen recognised by the antibody moiety, but does not bind indiscriminately to other cells
`as it would if the lectin activity remained intact. In a sense, the A-chain (and likewise non-B chain
`toxins such as gelonin) is a prodrug which only becomes active when targeted to and internalised by the
`appropriate cell. Because such conjugates, termed 'immunotoxins', are often much more cytotoxic than
`drug-antibody conjugates the requirement for antibody specificity is more stringent. Discrimination
`between strongly and weakly antigenic cells is less clear-cut, which could increase the risk of damage to
`innocent cells expressing low levels of antigen (Embleton et al., 1986). For systemic use of the dosage of
`immunotoxins will thus need to be carefully regulated, although in situations where cytotoxicity against
`weakly antigenic cells is desired immunotoxins have a distinct advantage.
`Some A-chain immunotoxins may be poorly internalised by target cells, an important role for the
`B-chain being implicated in the process of trans-membrane transport. To overcome this, one approach
`has been to supplement the A-chain immunotoxin with a MoAb-targeted B-chain, directed either towards
`the same tumour antigen as the A-chain immunotoxin or to the bound A-chain immunotoxin itself
`(Vitetta et al., 1983, 1984). The two immunotoxins can be demonstrated to have synergistic cytotoxic
`activity in vitro. There is, however, a theoretical objection in that it may be possible for free A and B
`chains to be released from killed cells in a form in which they could recombine. This could conceivably
`result in the accumulation of intact toxin, with undesirable consequences for the host when used in vivo.
`Another method of enhancing the cytotoxicity of immunotoxins in vitro is treatment of target cells with
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`lysosomotropic amines such as ammonium chloride or methylamine, or ionophores like monensin, which
`can greatly facilitate the cellular uptake of the immunotoxin (Casellas et al., 1984; Carriere et al., 1985).
`This may well be applicable to ex vivo treatment of bone marrow aspirates (Casellas et al., 1985) but it
`is difficult to see how it could safely be applied to patients systemically, unless through the development
`of novel, non-toxic potentiators active in small doses.
`Antibody-targeted radionuclides are, strictly speaking, outside the scope of this article but they
`deserve a mention because they constitute an active area of MoAb targeting. There are a variety of
`radioactive isotopes with ox- and fl-emitting properties which make them attractive as potential targeted
`therapeutic agents, but most pose technical problems of labelling or toxicity. Therapeutic trials have
`been undertaken with 1311-labelled antibodies (Order et al., 1984; Pectasides et al., 1986) with little
`reported acute toxicity. However, further development of this field creates a series of radiobiological
`problems owing to the high doses of radiation involved in therapeutic applications, and demands special
`facilities for labelling and patient isolation.
`
`Evaluation of conjugates and potential problems
`The initial testing of antibody-binding activity and drug activity is normally carried out in
`vitro.
`Antibody binding activity of conjugates can be compared with that of unmodified MoAb by a variety of
`methods, the most reliable being radio-immunoassay or flow cytofluorimetry. Drug or toxin activity can
`often be determined by appropriate cell-free assays, but the definitive test must be the cytotoxic effect of
`conjugate, compared to free drug or antibody, on cultured cells as measured by radiometric or
`it necessary to demonstrate cytotoxicity, but also specificity. The
`clonogenic assays. Not only is
`conjugate must eventually be able to distinguish between tumour and normal cells, so it follows that it
`should be highly cytotoxic in vitro for cells with high antigen expression, but non-toxic (or relatively so)
`for cells with weak or negative antigenicity. Conjugates with poor selectivity in vitro are poor candidates
`for in vivo targeting. However, satisfactory in vitro performance is not necessarily indicative of success in
`vivo, which means that animal models must be available. Unfortunately these only partially simulate
`clinical situations in that although they can provide much valuable pharmacokinetic and toxicological
`data as well as information on therapeutic efficacy, they present an artificially 'clean' situation with
`regard to antigenic specificity. Rodent tumour models are normally tuned so that the antibody under
`study is reactive only with the tumour and not with normal host tissues. Human tumour xenografts
`grown in immune-deprived rodents as targets for anti-human tumour MoAbs present themselves as the
`only human tissue in the animal. However, it is likely that most anti-human tumour MoAbs will react at
`a low level with at least some normal cells when administered to patients. Also, most transplanted or
`cultured model tumour lines are relatively homogeneous with regard to antigen expression and drug
`sensitivity. The tumour cell populations in primary and secondary human tumours are highly
`heterogeneous with regard to both these properties, and moreover the behaviour of any given clone may
`be influenced by other cells (e.g. host cells) within the tumour milieu. For drug-antibody conjugates and
`immunotoxins it is necessary to develop strategies to overcome any consequent inability to bind to a
`proportion of the cells. It is just possible that antibodies will be found which bind preferentially to all
`clonogenic or potentially clonogenic cells, but this does not seem likely. One possible solution may be to
`devise linkages by which a drug is released following binding to tumour cells recognised by the
`conjugated MoAb, in a form in which it is able to be taken up also by other cells within the immediate
`vicinity. Alternatively, conjugates could be applied as 'cocktails' constructed from different MoAbs and
`different toxic agents. (It is sometimes erroneously argued that if cocktails of MoAbs are considered
`necessary, one may as well use polyclonal antibodies; however, mixtures of MoAbs with individually
`defined properties are very different from the ill-defined immunoglobulin mixtures in polyclonal sera).
`Perhaps the main potential advantage of targeted radionuclides is that, in theory at least, therapeutic
`effects may be expected against non-antibody binding bystander cells.
`Animal models have already revealed several problems associated with the trafficking of conjugates.
`The stability of drug-antibody conjugates and immunotoxins in
`vivo is variable. In some cases
`circulating antibody and drug may remain associated for periods of several days, and in others they may
`become separated within hours or even minutes after injection into the host. This can be modified by
`alternative conjugation methods and it has been shown, for example, that immunotoxins prepared using
`a thio-ether linkage have a longer half-life in the circulation than those constructed using a disulphide
`linkage (Cumber et al., 1985). At the present time the optimum half-life for the various types of
`conjugate in terms of therapeutic effect is unknown, and it
`is by using animal models that this
`information will be gained and fed back to the design and construction of conjugates. One fairly safe
`prediction is that conjugates which are large in size are likely to show poor tumour localisation owing to
`poor penetration through capillary walls and/or rapid uptake by the reticulo-endothelial system. The
`larger conjugates include drug-carrier-antibody conjugates and immunotoxins, which typically have
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`molecular weights in excess of 200,000 daltons. There is a need for development of smaller conjugates,
`for example by using antibody fragments (Fab or F(ab')2) and smaller carrier molecules, although the
`optimum size for targeting has yet to be defined.
`that of host immune
`Another complication, indicated also by clinical immunoscinitigraphy, is
`responses to the components of conjugates (Levy & Miller, 1983). This will lead to increased clearance
`rates, and could also give rise to adverse reactions in patients, although experience suggests that the
`latter may not prove to be a serious problem (Levy & Miller, 1983). It is well established that patients
`make antibodies against murine MoAbs, and for this reason many researchers feel that human MoAbs
`would be preferable for targeting purposes, particularly since multiple-dose schedules will almost
`certainly be required. However, at the present time human anti-tumour MoAbs have poor specificity
`and activity, and in any case a significant element of the anti-immunoglobulin response is directed
`towards the antibody idiotype. This will equally be the case with whole human antibodies and with
`fragments of either human or rodent antibodies. Moreover, immune responses can be expected against
`carriers (unless small non-immunogenic molecules can be used), against toxins and against drugs as
`is not clear at present how such responses would affect therapeutic efficacy, but this
`haptens. It
`information will undoubtedly come from clinical trials. Whatever the effect on conjugate survival, anti-
`idiotype antibodies in themselves may not be a bad thing, as noted above.
`One important snag often overlooked by enthusiasts is that of cost. MoAbs are expensive to produce
`at the present time and they will be required in large quantities for therapeutic trials. Fortunately there
`is rapid development of large scale production methods and it is to be expected that this will lead to
`comparatively low-cost MoAbs within the next few years, so that by the time Phase I and II trials have
`been completed it will hopefully become economically feasible to produce conjugates on a large scale if
`the trials indicate the desirability of proceeding. A number of groups have experienced chemical
`difficulties in preliminary scale-up of conjugate synthesis, but this is a problem which presumably can be
`dealt with by appropriate modifications to procedure.
`
`Future developments
`It is clear that more basic research and development is required to refine MoAb-targeted conjugates for
`clinical use. I am, of course, referring to systemic administration; for extra-corporeal manipulations such
`as removal of neoplastic cells from bone marrow, the currently available types of conjugate may well be
`adequate. Probably the most fruitful area for improvement will be in conjugate construction, but this
`will depend equally upon the pursuance of extensive in vivo pharmacological and biodistribution studies.
`It will be necessary to determine the optimum size and chemical stability for therapy, and to develop
`conjugation methods and carriers to fit these requirements.
`More difficult will be the development of improved basic reagents, i.e. MoAb and drug. It remains to
`be seen whether better human MoAbs can be made than currently available, for example by in vitro
`immunisation techniques. Epstein-Barr virus infection or fusion of patients' lymphocytes with myeloma
`or lymphoblastoid cells has to date not produced sufficiently effective MoAbs for targeting, and indeed
`there is good evidence that lymphocytes from normal individuals can produce antibodies with similar
`binding specificities (Campbell et al., 1986; Winger et al., 1983). Human (and also hybrid) MoAbs will,
`in any case, still induce immune responses in patients and I suspect that Fab or F(ab')2 fragments of
`rodent antibodies will ultimately be the reagents of choice. Whether better rodent MoAbs can be made
`is also debatable; at present we are still working with 'first generation' anti-tumour MoAbs, and the
`hoped-for 'second generation' antibodies of greater specificity and affinity remain elusive.
`Many of the drugs and toxins currently in experimental use for conjugates may be of limited clinical
`application or efficacy. There is a need for a new generation of prodrugs of small molecular size and
`with potent cytotoxic activity (once introduced into the cell), developed especially with antibody
`targeting in mind. This would presumably be a job for the pharmaceutical industry rather than cancer
`research institutes. Indeed, it is quite likely that within the next decade the whole area of antibody-
`targeted chemotherapy will be taken over by industry.
`Finally, and most important, antibody-targeted therapy is now entering the field of clinical oncology.
`The consequences of immune responses to the components of conjugates remain to be fully elucidated,
`and ways of modulating these responses may need to be developed. Other aspects of toxicology will also
`is already known that, on the basis of percentage of injected dose, the
`need to be resolved. It
`localisation of radio-labelled antibodies to tumours in patients is less than that to xenografts of similar
`tumours in immune-deprived mice, so there is good reason to believe that the patient will be required to
`handle substantial amounts of conjugate which fails to reach its target. Added to this, of course, it is
`only by clinical trials that we will learn whether or not MoAb-targeted chemotherapy will be a
`successful form of therapy.
`The next five or ten years will be a critical phase for MoAb therapy of any description, and if some
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`
`of the foregoing comments appear to be less than optimistic this is because the lessons learned from past
`attempts to exploit immunology for cancer therapy have led many of us to be cautious and to anticipate
`as far as possible the obstacles likely to occur. However, there are numerous encouraging reports of
`selective cytotoxicity in
`vitro by drug-antibody conjugates and immunotoxins, and of therapeutic
`responses with low toxicity against tumour xenografts (see review articles in Moller, 1982; Davies &
`Crumpton, 1982; Baldwin & Byers, 1985). Thus, even allowing for caution, it is generally felt that if
`basic and clinical research continue along the right lines the promise of MoAb-targeted therapy is
`substantial.
`
`M.J. Embleton
`Cancer Research Campaign Laboratories,
`University of Nottingham,
`University Park,
`Nottingham, NG7 2RD.
`
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