Immunotoxins

Cell, Vol. 47, 641-646,

December

5, 1986, Copyright

0 1986 by Cell Press

mmunotoxins

Ira Pastan, Mark C. Willingham, and David J. R FitzGerald National Institutes of Health National Cancer Institute Laboratory of Molecular Biology 9000 Rockville Pike Bethesda, Maryland 20892

The use of chemotherapeutic agents to treat cancer has resulted in the cure or amelioration of some types of human cancer. However, many forms of cancer are either initially unresponsive to chemotherapy or eventually acquire resistance to it. Clearly, alternative types of treatment are necessary. Paul Ehrlich originated the idea of treating cancer with a “magic bullet,” a molecule consisting of a toxic compound attached to an antibody that specifically recognizes cancer cells. Over the last few years, such molecules-now known as immunotoxins (ITS)-have been constructed by conjugating toxins from bacteria or plants to monoclonal antibodies. Here, we discuss the factors that appear to contribute to the biological activity of immunotoxins and, in light of this information, reexamine the potential use of these molecules in treating disease. Toxins-Structure

and Function

Many different toxins are produced by bacteria and plants, but only a few have so far been used to make ITS. Some properties of the most commonly used toxins are shown in Table 1. Like many proteins, toxins are composed of several different domains with different functions. Usually, one domain contains information for cell recognition, a second domain is used to translocate the toxin across the cell membrane to the cytosol where the targets of most toxins reside, and a third domain catalyzes the reaction responsible for cell killing. To construct a cell-specific IT, it is necessary both to prevent the binding of the toxin to its normal cellular receptor and to attach the toxin to an appropriate antibody that will direct it to the target cell. Cellspecific ITS have been constructed in several different ways depending on the properties of the toxin. Diphtheria toxin has been most extensively studied. Although it is synthesized as a single chain, it is considered a two-chain toxin since it must be nicked to be fully active on human cells (Sandvig and Olsnes, 1981). Diphtheria toxin is extremely potent-a single molecule in the cytoplasm is sufficient to kill a human cell (Yamaizumi et al., 1979). Mutant forms of the toxin have been identified which retain full enzymatic activity but are much less toxic to human cells, presumably because the cell-binding domain is altered or removed. Colombatti et al. (1986) have found that immunotoxins constructed from one such mutant form (i.e., containing the CRM 45 mutation) are very active and specific. Pseudomonas exotoxin A (PE) is made by the bacte-

eview

rium Pseudomonas aeruginosa, and has the same enzymatic activity as diphtheria toxin, although the two proteins are not related in sequence. Crystallographic analysis shows that PE has three structurally distinct regions (Allured et al., 1986) which probably correspond to a cell-binding domain, a domain that catalyzes the ADPribosylation of elongation-factor-2, and a domain participating in the transport of the toxin across the cell membrane. Treatment of intact PE with 2iminothiciane produces a molecule with greatly reduced cellular toxicity but full enzymatic activity (Pirker et al., 1985a), apparently because this reagent reacts with lysine residues involved in cellular binding. The sulfhydryl groups generated by this treatment have been used to couple PE to antibodies (Pirker et al., 1985b) and growth factors (FitzGerald et al., 1983a). Ricin and abrin are ribosome-inactivating proteins. Ritin is synthesized as a single precursor molecule and is subsequently processed into two chains, A and B, which are linked by disulfide bonds. The A chain catalyzes the inactivation of ribosomes, whereas the B chain is responsible for both cell binding and translocation of the toxin across the cell membrane (Olsnes and Pihl, 1982; Olsnes and Sandvig, 1985; Youle and Neville, 1982). ITS made with intact ricin are very active but not specific, because the B chain binds to galactosyl residues of glycoproteins and glycolipids present on the surface of many cell types. The addition of excess galactose or lactose to cultured cells exposed to a ricin IT can block the toxin’s nonspecific cell killing without affecting its antibody-directed toxicity (Youle and Neville, 1980). ITS made with ricin A chain are much more specific than those made with intact ricin but, unfortunately, are as much as lOO-fold less active (Weil-Hillman et al., 1985). While the B chain of ricin appears to be responsible for the translocation of native ricin into the cell cytoplasm, the A chain may possess a similar but much less efficient activity; otherwise, immunotoxins made with the A chain alone would be devoid of cytotoxic activity. Another difficulty with ricin A chain is that it contains carbohydrate residues which affect the distribution and metabolism of fTs in animals (Skilleter et al., 1985). Recently, cDNA clones encoding ricin have been identified and sequenced (Lamb et al., 1985) and carbohydrate-free recombinant ricin A chain has been synthesized in E. coli (Piatak et al., unpublished data). Recombinant ricin A chain has been used to prepare ITS; these are as active as ITS containing native ricin A chain when tested in cell culture and in animal experiments (FitzGerald et al., 1986b). Other toxins that have been used to make ITS include pokeweed antiviral protein, saporin, and gelonin (see Table 1). These toxins exist naturally as A chains and, when introduced into cells, inhibit protein synthesis by inactivating 60s subunits of ribosomes. Internalization

of Toxins and Imm

Like other molecules that bind to receptors on the cell surface, iTs are internalized by the pathway of receptor-

Ceil 642

Table 1. Properties

of Toxins Used to Construct

lmmunotoxins Comments

References

Chains

Target

A+B

ADP-ribosylates

elongation-factor-2

Humans

One

ADP-ribosylates

elongation-factor-2

A+B

Inactivates

ribosomes

A+B A

Inactivates Inactivates

ribosomes ribosomes

Saporin

A

Inactivates

ribosomes

Gelonin

A

Inactivates

ribosomes

Makes very potent immunotoxin since “B” region is maintained A-chain immunotoxins less active than those with whole toxin Similar to ricin Makes active immunotoxin Makes active immunotoxin Makes active immunotoxin

Toxin Diphtheria

toxin

Pseudomonas

exotoxin

A

Ricin

Abrin Pokeweed

antiviral protein

mediated endocytosis (Olsnes and Sandvig, 1985; Middlebrook and Dorland, 1984). This process is depicted in Figure 1 (for detailed reviews of receptor-mediated endocytosis, see Pastan and Willingham, 1983; 1985). In the first step, a ligand binds to its receptor. The receptor-ligand complex then diffuses laterally within the cell membrane until it encounters a clathrin-coated pit. The coated pit gives rise to an endocytic vesicle termed a receptosome (Willingham and Pastan, 1980) or endosome (Helenius et al., 1980), which moves away from the cell surface by saltatory motion. These vesicles fuse with interconnected tubular elements, called the TR Golgi (Willingham and Pastan, 1982) or CURL (Geuze et al., 1983). As a result of this fusion, receptors and ligands enter the TR Golgi, where their fate is determined: the receptor and ligand may both be recycled to the cell surface (e.g., the transferrin system; Dickson et al., 1983b), the receptor and ligand may both be directed to the lysosomes (e.g., the epidermal growth factor system; Willingham and Pastan, 1982) or the receptor may be recycled to the cell surface and the ligand directed to the lysosomes (e.g., the low density lipoprotein and as-macroglobulin systems). Many toxins are internalized by receptor-mediated endocytosis. Because the target molecules for many toxins are located in the cytosol, it is clear that the toxins must cross a membrane. Most information is available about the entry of diphtheria toxin, which is translocated across a membrane when it encounters an acidic environment (Sandvig and Olsnes, 1980; Draper and Simon, 1980). The interior of endocytic vesicles (receptosomes, endosomes) and lysosomes is at pH 5.5, about 2 pH units lower than that of the extracellular fluid (Tycko and Maxfield, 1982). Thus, soon after toxins enter a cell by endocytosis, they reach an acidic environment. In the case of diphtheria toxin, and probably also PE, this low pH environment promotes translocation of the toxin across the membrane of the endocytic vesicle into the cytosol. The toxicity of diphtheria toxin and PE is blocked by treating cells with ammonium chloride, chloroquine and monensin, agents that raise the pH in intracellular vesicles, and thereby prevent translocation (Olsnes and Sandvig, 1985). These

immunized

Gilliland et al., 1980 Columbatti at al., 1986 Fitzgerald et al., 1984 Pirker et al., 1985a; 19&b Sjorn et al., 1986 Youle and Neville, 1982

Hwang et al., 1984 Ramakrishnan and Houston, 1984a; 1984b Thorpe et al., 1985 Lamberf et al., 1985

agents do not block the toxicity of ricin or abrin, indicating that these toxins use a different mechanism or site of membrane translocation. When antibodies are coupled to ricin A chain or other A chain toxins, the antibody replaces the B chain (or binding domain) and forms a complex with antigen, thereby linking the A domain to the antigen on the cell surface. If the antigen is internalized, the IT is internalized with it (Figure 1). After internalization, most of the IT is often directed to lysosomes where it is degraded. However, a small portion of the IT escapes from some intracellular location into the cytosol where its targets are located. There, the toxin inhibits protein synthesis by inactivating elongation-factor-2 (diphtheria toxin, PE) or ribosomes (ricin, abrin, pokeweed antiviral protein). If the antigen is a molecule that is not internalized, and its internalization is not induced by antibody, the IT is likely to have low activity. It has been speculated that toxins coupled to antibodies against the transferrin receptor would make effective immunotoxins, since this receptor is present in large numbers on cancer cells and is efficiently internalized (Trowbridge and Domingo, 1981). Antibodies to the transferrin receptor have been conjugated to various toxins and the action of these ITS studied in some detail (FitzGerald et al., 1983b; Ramakrishnan and Houston, 1984c; Domingo and Trowbridge, 1985; Bjorn et al., 1985; Pirker et al., 1985a). These studies illustrate some of the interesting complexities encountered in studying IT action. The physiological role of the transferrin receptor is to internalize the iron-carrier protein, transferrin, bring it to an acidic compartment (receptosome, endosome) where iron is released, and then recycle apotransferrin to the extracellular environment. During this process, transferrin may spend as little as 10 min within the cell and very little transferrin or its receptor is delivered to lysosomes (Dickson et al., 1983b). However, when a monoclonal antibody to the transferrin receptor (conjugated to a toxin or not) is allowed to bind to the receptor, the result is somewhat different. The antibody-transferrin receptor complex is internalized like the transferrin-receptor complex but, rather than all of the ligand reappearing in the medium as

Review: immunotoxins 643

Construction

Figure 1. Pathway of lmmunotoxin

Entry

The figure illustrates receptor-mediated endocytosis, the pathway by which immunotoxins (ITS) are internalized into cells. First, the antibody component of the IT binds to an antigen on the cell surface. The entire complex (IT + antigen) then diffuses laterally in the cell membrane to a coated pit. The coated pit gives rise to an endocytic vesicle (receptosome, endosome), which moves away from the cell surface by saltatory motion and fuses with another organelle, the TR Golgi (also called CURL). As a result of a sorting process that occurs in the TR Golgi, much of the IT is sent on to lysosomes to be degraded, some is released into the extracellular fluid, and a smali amount escapes into the cytosol (either as intact IT or a toxic fragment). Within the cytosol, the IT acts on its substrates-inactivating either elongation-factor-2 (EFa) or ribosomes. The fate of the antigen is not shown. Conceivably, it is also degraded in the lysosomes, resulting in “down-regulation: or else is returned to the cell surface to associate with other IT molecules.

One convenient way to make an 1T is to coupie toxin and antibody by a disulfide bond. Reduction of the native forms of ricin and abrin, for example, yields A and 6 chains with free sulfhydryl groups that can be used for the coupling reaction. Alternatively, ~u~fhydry~ groups can be introduced by reacting both the toxin and the antibody with iminothiolane, which reacts preferentially with amine residues. Then, either the antibody or the toxin is treated with 5,5’-dithio-bis(2nitrobenzoic acid) and mixed with the other component to form the disulfide-linked conjugate. Using this procedure, it is possible to introduce at least two sulfhydryl groups into PE, both of which are available for conjugation with antibody (see FitzGerald, 1966c). ITS have also been made using N-succinimidyl3(2-pyridyldithio) proprionate; this and other methods have been reviewed by Thorpe and colleagues (Cumber et al., 1985). It is also possible to conjugate toxins to antibodies by a thioether (S-C) bond. In principle, such conjugates should be much more stable in blood and tissue fluids than disulfide conjugates; whereas a disulfide bond is susceptible to both enzymatic and chemical cleavage in vivo, there is no known enzyme in animals that can cleave a thioether bond. Nevertheless, thioether conjugates of ritin A chain and diphtheria toxin A chains have been reported to be much less active in killing cells than comparable disulfide conjugates (Youle and Neville, 1982; Weil-Hillman et al., 1985; Masuho et al., 1982). Thioether conjugates of PE have also been prepared. These conjugates have not lost any ADP-ribosylati when tested in a cell killing assay were fou as active as disulfide conjugates (Djorn et al., 1986; FitzGerald et al., unpublished data). PE has also been coupled to epidermal growth factor (EGF) by a thioether bond (FitzGerald et al., 1983a); the cytotoxic activity of this conjugate is proportional to the number of EGF receptors on the cells (Clark et al., 1985). Activity

does transferrin (Hopkins and Trowbridge, 1963) much of the antibody is delivered to lysosomes. Some of the internalized receptor (with antibody attached to it) is also delivered to lysosomes. Thus, for example, a 12 hr exposure of KB cells to an antibody raised against the transferrin receptor resulted in a 25% decrease in the number of transferrin receptors. Similarly, exposure of erythroleukemia cells to this antibody induced a marked downregulation of the transferrin receptor (Weissman et al., 1966). In a study designed to investigate whether one could predict the effectiveness of an antibody as an IT, Pirker et al. (196513) exposed ovarian cancer cells to a group of different antibodies and measured their internalization. They also made ITS from these antibodies and assessed their cell killing activity. Their results showed that antibodies which were effectively internalized made better ITS than those that were not.

of lmmunotoxins

of lmmunotoxins

Generally, an IT has a lower activity than the native toxin with which it is coupled, although exceptions exist. A number of variables contribute to their diminished activity. The sensitivity of a cell to a toxin is dependent on the presence of an appropriate receptor and a suitable intracellular environment. Diphtheria toxin, for example, is very active against human cells but not against mouse cells, since the latter do not appear to contain significant amounts of the receptor for this toxin (Pappenheimer, 1977). Additional factors associated with mouse cells also may contribute to their extreme resistance (Heagy and Neville, 1981; Kaneda et al., 1984). Conversely, human cells are generally somewhat resistant to PE, whereas mouse cells are very sensitive to this toxin (~id~lebr~ok and Dorland, 1977). This is also probably due to the presence of more receptors for PE on mouse cells than o cell-free extracts of either mouse or human cells, diphtheria toxin and PE toxin have approximately equal activities

Cell 644

when assayed for their ability to ADP-ribosylate elongation-factor-2, and thereby inhibit protein synthesis (Lory and Collier, 1980). Pokeweed antiviral protein behaves like a naturally occurring A chain toxin and, by itself, is not very toxic to living cells (Ramakrishnan and Houston, 1984a, 1984b). No cellular receptor is known for this toxin. The efficiency of an IT in crossing a cell membrane is much lower than that of the original toxin alone. Presumably, the size and other characteristics of the antibody molecule hinder the translocation step, but the inability of the toxin to bind to its own receptor may also contribute. It seems likely that somewhere within the cell the thiol bond of disulfide-linked conjugates is broken, probably by reduction, generating free toxin which then escapes from a vesicular compartment into the cytosol. How efficient this cleavage process is and where it occurs have not been established. Other factors contributing to the activity of ITS are the affinity of the antibody for the antigen, the efficiency of antigen (and therefore IT) internalization, the presence or absence of protein sequences in the toxin promoting translocation across the membrane, and the presence or absence of the IT in the appropriate compartment for translocation (i.e., acidic for diphtheria toxin and PE, neutral for ricin and abrin). The endocytic vesicle into which the IT initially enters, and the lysosomes to which they are ultimately delivered, are acidic. Taking advantage of this, Shen and Ryser (1981) and Blattler et. al. (1985) have used an acid-labile cross-linking agent to generate an active IT The activity of these types of conjugates merits further study. An important limiting step in IT action is the rate of transfer of the toxin moiety of the IT across a membrane. When the membrane of a vesicle containing an IT is disrupted, IT activity is greatly enhanced. One way vesicle disruption can be achieved is to expose cells simultaneously to adenovirus and an IT The virus and the IT enter cells in the same endocytic vesicle. Adenovirus escapes into the cytosol by disrupting the membrane of the endocytic vesicle (Seth et al., 1986) and, as a consequence, the contents of the vesicle, including the IT, are released into the cytosol. IT action is greatly enhanced by this procedure (FitzGerald et al., 19834 1984). It would be useful to develop agents that could selectively destabilize the membrane of endocytic vesicles and to direct these agents to cells with the same antibodies used to construct ITS. Chloroquine, ammonium chloride, and monensin (agents that raise pH in endocyticvesicles) have been found to enhance the action of ITS containing intact ricin and ricin A chains, indicating that the same environment is required for both types of molecules to reach the cytosol (Casellas et al., 1982, 1984). As mentioned above, these agents block the action of (unconjugated) diphtheria toxin and PE. Colombatti et al. (1986) have shown that ammonium chloride inhibits the activity of immunotoxins made with intact diphtheria toxin or a fragmented toxin that includes part of the B chain, but does not inhibit immunotoxins that consist of only the A chain conjugated to the antibody. The effect of these agents on the activity of immunotoxins containing PE is not known. The addition of ricin B chain to ITS made with A chain

has also been shown to be a useful strategy to enhance activity (Youle and Neville, 1982). An increase in target cell cytotoxicity can be demonstrated by individually coupling the same antibody to ricin A chain and B chain, and then exposing cells to both conjugates simultaneously (Vitetta et al., 1983, 1984). Thus, the A and B chains are brought together within one target cell. Animal Studies In the past five years various reports have demonstrated antitumor effects mediated by ITS. With one exception, these studies have been carried out in mice. Either normal mice have been injected with syngeneic tumor cells, or immunodeficient nude mice have been injected with nonsyngeneic murine cancer cells or human cancer cells. In all cases, monoclonal antibodies of rodent origin were used to construct ITS. Data summarized in Table 2 show a spectrum of antitumor effects, ranging from a 25%-300/o increase in the survival of tumor-bearing animals to a 2000/o-300% extension of life. In some instances, the injection of antibody alone also gave a significant antitumor effect, bringing into question the efficacy of the IT. However, in one set of experiments (human ovarian cancer cells growing in the peritoneal cavity of nude mice), it was shown that excess unconjugated antibody blocked IT action; this confirms that the toxic moiety of the IT is solely responsible for tumor cell killing (FitzGerald et al., 1986a, 1986b). In cell culture systems, ITS are subjected to metabolism by only one cell type. In vivo, however, they must interact with a wide variety of cells, including cells that bear Fc receptors and cells that may react with other sites on the antibody or toxin. The stability in rabbits of a disulfidelinked IT made with pokeweed antiviral protein has been studied by Ramakrishnan and Houston (1985). They found that the IT disappeared from the blood much more rapidly than unconjugated antibody, with a half-life of about 20 hr. Free toxin was not detected in the blood at any time. The stability of ricin A chain ITS has also been evaluated (Bourrie et al., 1986; Worrell et al., 1986). Both studies showed that disappearance of the IT is much more rapid than that of unmodified antibody, and is not accompanied by rapid cleavage of disulfide bonds to generate free ricin A chain. Interestingly, ricin A chain was found to contribute to rapid clearance of the IT by the liver. Diseases Currently Being Considerad lmmunotoxin Therapy

for

The in vivo administration of ITS for the treatment of cancer is only one possible application of these agents. One other use is in bone marrow transplantation for the destruction of activated T lymphocytes that contribute to the development of graft-versus-host disease. After its removal from a donor, bone marrow is treated in vitro with ITs such as anti-Thy-1.2-ricin, which are directed at T cells. Since this treatment is carried out in vitro, an IT containing whole ricin can be used if lactose is added to block the binding of ricin B chain to the cellular ricin receptor (see

WEHI-

Guinea piga hepatorna

into tumor

IV

IV

IP

IP

Tl 01 -ricin

D3-RTA

anti-thy 1.1~RTA

anti-TFR-PE

anti-TFR-RTA

10 w Human ovarian

LlO

AKR-A

OVCAR-3

OVCAA-3

6 x lo7

IP

IP

1 x 10s 6 x lo7

IP

Prolongs

1 x 10s

1 x 105

Inhibits tumor growth

1 x 108

Prolongs

Prolongs

Prolongs

survival

survival

survival

inhibits tumor growth

Inhibits tumor growth

survival

Inhibits tumor growth

2.4 x IO6

from

2 x 107

intraderma?

-

SC

SC

SC

Response

Reference

inactive

--

A few animals tumor-free at end of experiment; excess mAb blocks IT action

TlOl-RTA

Intact mAb alone also effective except for W.%

et al., 1983

et al.,

FitzGerald 1986b

FitzGerald 1986a

et al.,

et al.,

Thorpe et al., 1985

Bernard

Weil-Hiilman 1985

Ramakrishnan and Houston, 1984b

Bumol et al., 1983

mAb alone also effective

et al., 1981

Trowbridge and Domingo, 1981

Blythman

mAb alone also effective

Comments

-~

-.

tumor transplantation. The routes of injection of the ITS are intraperitoneal (IP), intravenous (IV), or subcutaneous (SC). Dose time after tu;Oor implantation that the IT was administered. Other abbreviations used are: anti-thy 1.2 (antibody to a murine of AKR mice), TFR (transferrin receptor), TlOl (antibody to a 67,000 M, glycoprotein found on ali human T cells), RTA (&in (pseudomonas exotoxin), mAb (monoclonal antibody), ___--~-“l__(----..---~-_

Human ovarian

Days 5, 6, 7, 8

Days 5, 8, 11

Mouse leukemia

Day 1

Day 7

Mouse leukemia AKR SL3, AKR SLl

Day 0, 2, 4, 8, 16

GEM,

Human melanoma

Day 3, then every 3 days

a Experiments performed in guinea pigs. lmmunotoxins were assayed by injecting them into animals at various times after refers to the amount of IT administered in each injection. Schedule refers to the T-lymphocyte differention antigen), anti-thy 1.1 (antibody to a thymocyte antigen A chain), DTA (diphtheria toxin A chain), PAP (pokeweed antiviral protein), PE -_____-

-------?-

Human leukemia Daudi

SC

anti-thy 1 .l-F(ab’)z-PAP

19 lag

40 w

IF

M21

9.2.27-DTA

melanoma

50 Irg

Days 0, 7, 74

Human

IV

of

__I__-

Prolongs survival 35 days to 45 None

Mouse leukemia

anti-TFR-RTA

1 hr and day 7

38 WI

6 x 10s

IP

IP

Number cells

Site

____-

Tumor

anti-thy l.P-RTA

Schedule

Route

Dose

on Tumor Growth in Mice

lmmunotoxin

Table 2. Effects of tmmunotoxins -

Ceil 646

above). Such a reagent has been successfully used in mice (Vallera et al., 1983), and clinical studies are in progress (Filipovich et al., 1984). Another use of ITS is in autologous bone marrow transplantation (Thorpe et al., 1982; Filipovich et al., 1984). In the treatment of certain leukemias and lymphomas, bone marrow is harvested from a patient, treated with an IT to kill contaminating cancer cells, and placed in storage. Subsequently, the patient is given extremely large doses of drugs and radiation to kill all remaining cancer cells, a treatment that also results in total destruction of the bone marrow. To rescue the patient, the purged bone marrowfree of cancer cells-is reinfused. Other possible uses for ITS include destruction of activated T cells in vivo in order to allow organ graft survival, or to arrest or prevent diseases such as diabetes, which may be caused by such cells. Autoimmune disorders, where anti-“self” antibodies are responsible for disease, might be treated with immunotoxins directed against the idiotypes of the offending antibody-producing B cells. Finally, certain parasitic diseases may also be amenable to IT therapy.

Prospects For IT therapy to be effective, suitable antigens must be identified which are selectively expressed on cancer cells. An ideal target antigen would be expressed only on cancer cells. However, since this level of specificity may not be attainable, a more workable approach is to seek antigens expressed at high levels on target (cancer) cells and at low levels on normal cells. If the normal cells are nonessential, easily renewed, or “end-stage” cells, higher antigen expression could be tolerated on nontarget cells. A number of researchers have described monoclonal antibodies that are relatively tumor-specific (reviewed in Schlom and Weeks, 1985). For the most part, these antibodies have been detected by screening hybridomas produced by injecting mice with human cancer cells. In some cases, however, the antibody has been produced against an antigen known to be expressed on a limited number of cells. One of these is an antibody to the interleukin-2 receptor, a molecule found mainly on activated T cells but also found in large amounts in cells from patients with adult T cell leukemia. A number of antiidiotypic antibodies specific for B cell lymphomas have been prepared and have been used by themselves (i.e., without a conjugated toxin) with some success to treat this disease (Meeker et al., 1986). Antibodies to antigens present on a variety of cancers have also been tested by themselves. With some exceptions, they have been found to be ineffective or to give a partial response. Nevertheless, such antibodies have been shown to accumulate in the tumor and some antibodies of this type are being used as diagnostic agents for tumor imaging. All these studies suggest that if sufficient amounts of an IT can reach a tumor, the IT should be able to kill the cells making up the tumor. Because ITS (M, ~200,000) are larger than IgG antibodies (M, -150,000) and have altered chemical structures, they may not be distributed to tumors as effectively as an IgG,

but may still accumulate in tumors in sufficient quantities to act. It is also possible to take advantage of the fact the 1% slowly leave the compartment into which they are injected in order to treat cancers confined to these compartments. Cancers in this category include leukemias, which are mainly confined to the circulatory system, ovarian cancer, and possibly tumors in the central nervous system. Death from ovarian cancer usually occurs at a time when cancer cells are still mainly confined to the peritoneal cavity and surrounding tissues. Death is due to obstruction of essential organs such as the urinary system and gastrointestinal tract, and to the invasion of nearby lymphatics, producing lymphatic obstruction and massive ascites. Injection of an IT into the peritoneal cavity temporarily concentrates the active agent near the cancer cells, before it is diluted within the circulation. In addition, the IT leaves the peritoneal cavity by the same lymphatics as many of the cancer cells, giving the IT access to these cells. In a xenograft model in which human ovarian cancer cells grow in the peritoneal cavity and surrounding tissues of immunodeficient mice, ITS composed of antibodies to the human transferrin receptor and either ricin A chain (FitzGerald at al., 19866) or PE (FitzGerald et al., 1986a) are vary effective. The action of these immunotoxins is readily demonstrable even when they are administered after the tumor is well established and growing. Nevertheless, ITS containing antibodies to the human transferrin receptor may not be very useful clinically because of the widespread distribution of that receptor (Gatter et al., 1983). ITS made with other monoclonal antibodies that are more “specific” for human cancers are now being evaluated. What other information or experiments are there to indicate that ITS will be effective therapeutic agents? First is our knowledge that the immune system and the endocrine system produce molecules that are in some cases quite large, and yet act effectively at sites remote from their site of synthesis. Second is a set of experiments with diphtheria toxin. As mentioned above, human cells are lOOOfold more sensitive than mouse cells to the action of this toxin. In 1979, DiPersio et al. reported that human tumors growing in nude mice regressed by 70% upon treatment with a single dose of diphtheria toxin. In many laboratories including our own, diphtheria toxin is often used as a positive control in immunotoxin experiments to show that regression of human tumors in mice can occur. For example, injection of 10” human KB cells will produce a lethal subcutaneous tumor in a nude mouse. lntravenous injection of 1 pg diphtheria toxin when the tumor dimensions are (1 cm)2 causes complete regression of the tumor within four days. If ITS prove successful in treating cancers or other diseases, still other problems need to be considered. One is the potential emergence of unresponsive cells caused by loss of the target antigen or by mutations in the internalization pathway of the IT Another is the development of antibodies to the toxin or to the monoclonal antibody to which it is attached. One obvious solution to these problems is to have a variety of monoclonal antibodies and toxins available. A large number of tumor-specific antibodies have been, and are being, generated, yet only a few have

Review: lmmunotoxins 547

been evaluated for use as ITS. Similarly, of the hundreds, and probably thousands, of toxins found in nature, only a handful have been studied and even fewer evaluated as ITS. With so many unexplored avenues of research available, the lT field is rapidly expanding. At least three clinical phase I trials are now underway. Whether these first generation ITS will be useful therapeutic agents is a question that may soon be answered.

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