Selección de componentes eléctricos y electrónicos

Anti-tumour activity of abrin and ricin w as n o t e d as early as 1951. Since then several laboratories have studied the effects of the toxic lectins on animal tumours a n d neoplastic cell lines (Olsnes and Pihl, 1982 and Shionoya et a l ., 1982). The toxic lectins exert their effect through inhibition of protein synthesis which is different to the mech a n i s m of action of other chemotherapeutic agents such as vinblastin, methotrexate and cyclophosphamide. In several instances abrin and ricin were found to be more effective as cancerostatic agents than the drug most commonly used to treat t h e tumour. In addition, in combined therapy where ricin or ab r i n were administered in conjunction with a chemotherapeutic agent, a synergeneic effect was seen (Walker et a l. 1984). Phase I studies of ricin as a cancerostatic agent have been c arried out

(Fodstad et a l., 1984).

The mechanism by which these toxic lectins c a n suppress tumour growth may be in part due to the direct cytotoxic effects on tumour cells and stimulation of host immunity with non-specific suppression of tumour cells. The bin d i n g of a few

molecules of abrin or ricin to a tumour cell m a y be insufficient for inactivation but may cause incr e a s e d destruction of toxin-coated cells by the reticuloendothelial system. APA enhances the natural cytotoxicity of p e r i p h e r a l blood lymphocytes in vitro and in humans with n asopharangeal carcinoma (Won et a l ., 1988). Certain tumour cells have bee n shown to require fewer molecules of toxin to be bound for cytotoxicity (Chan et a l ., 1985).

Abrin has been used as the positive control in h u m a n tumour clonogenic assays for comparison of cytotoxic e f f e c t s of therapeutic agents (Salmon et a l ., 1983)

M O IHMPyOTOXXMS

1.10.1 Overview

An immunotoxin is a cytotoxic agent which consists of a cell-binding moeity, linked via a chemical cross-link, a peptide linker or disulphide bond, to a toxic moeity. The c e l l ­ binding moeity may be an antibody, or antibody fragment, o r a hormone which has selectivity for a particular cell type. The toxic moeity may be a holotoxin, an A-chain or a sing l e - c h a i n ribosome-inactivating protein. Immunotoxins therefore c o m b i n e ligand specificity with the exquisite toxicity of the toxin.

Despite the conceptual simplicity of immunotoxins t h e y are complex molecules and each component continues to be s t u d i e d and improved. Immunotoxins have been extensively reviewed (see Frankel, 1990, Lord et a l ., 1989, Blakey et a l ., 1988, P a s t a n et a l ., 1986 and Vitetta and Thorpe, 1989) and only the s a l i e n t points will be mentioned here.

1.1 0 . 2 Th« ligand portion of ianunotoxjns Antibodies

Antibodies or fragments thereof have been most f r e q uently utilised as the ligand portion of immunotoxins. The speci f i c i t y

of monoclonal antibodies can be extremely high although non- neoplastic cells of the same lineage may also be a target. Whole antibodies are large immunogenic molecules and therefore may penetrate solid tumours poorly. Fab' and F(ab')2 fragments are less immunogenic, lack Fc receptors and therefore do not bind to reticuloendothelial cells, and are smaller. However, the antibody fragments are more rapidly cleared from the body and the monovalent Fab' fragments bind with reduced avidity compared to the bivalent or polyvalent counterparts.

Non-antifrrtY carriers

Non-antibody carriers, such as the interleukins and epidermal growth factor, have been used as the ligand in immunotoxins. Generally these ligands will also bind to normal cells but since many neoplastic cells express large amounts of the receptor compared to the normal cell and the normal cells that are killed can be replaced from progenitor cells which lack the receptor, applications continue to be sought.

1.10.3 Tbs toxin portion of antibodies

The extreme toxicity of the toxic lectins has made them the focus of interest in immunotoxin research. The toxin is modified by removal of the B-chain, or the blocking of the galactose binding sites, and specificity is conferred by the addition of a specific ligand. An attractive feature of the toxic-lectins is that resting cells are susceptible to the action of ribosome-inactivating proteins. The single-chain ribosome-inactivating proteins naturally have low toxicity but can be rendered specifically cytotoxic by linkage to a ligand moeity. Bacterial toxins such as diphtheria toxin and Pseudomonas exotoxin A have also been extensively utilised to generate immunotoxins.

*.10.4 F f paratjop of

A variety of linkers have been used to joi n ligands, with a free thiol group, to the A-chains of the to x i c lectins, the holotoxins or single-chain ribosome-inactivating proteins. In the case of the holotoxins the galactose b i n d i n g sites are stearically hindered by the ligand and are s e l ected by failure to bind to galactose, or have a chemically reactive group covalently attached in the vicinity of the bin d i n g sites. For review see Cumber et a l. (1985).

1.10.5 Cytotoxic properties of iBUBunotoxlna in vitro

Immunotoxins containing the A-chain of r i c i n or abrin or the single-chain ribosome-inactivating proteins can be highly specific because the toxin portion does not c o n t ribute to cell binding. However, these immunotoxins show a wide degree of cytotoxic potency. It is now generally a c c e p t e d that the binding affinity of the ligand plays a major role in determining efficacy. A ligand with high a f f i n i t y generally gives high potency to the immunotoxin. The receptor for the ligand is also important. A receptor t h a t is rapidly internalised is preferential. The routing of the conjugate after internalisation may be disadvantageous e.g. to lysosomal compartments. Immunotoxins constructed with the A-chain of diphtheria toxin are consistently less effective than those constructed with the A-chain of ricin or abrin. T his has led to the idea that the toxic lectin A-chains contain a translocation domain, lacking in diphtheria toxin A-chain, that is necessary for transport to the cytosol.

Virtually without exception, the potency of an immunotoxin containing a holotoxin is exceptional but the non-specific binding associated with the B-chain makes their use in vivo problematical. The blocked forms of these inununotoxins have been shown to have potent and specific cytotoxicity in vitro.

Of importance is that these inununotoxins have faster kinetics of cell killing which will be of particular relevence in vivo.

1.10.6 cytotoxic properties of Immunotoxins i n v j v c

Many of the ribosome-inactivating proteins are glycosylated and the B-chains of abrin and ricin are highly glycosylated (Kimura et a l ., 1988). The mannose- and fucose- containing oligosaccharides are recognised by cells of the reticuloendothelial system. Intact ricin has been u s e d to selectively deplete Kuppfer cells in rodents (Zenilman et a l ., 1988). Therefore immunotoxins containing native glycosylated ribosome-inactivating proteins are rapidly cleared from the blood and tissues, and liver damage can occur. Deglycosylation of the ribosome-inactivating protein, or the A-chain, has been shown not to alter enzymatic ability (Blakey et a l., 1987 and Foxwell et a l., 1987). Blocking of the galactose binding sites, commonly performed using asialofetuin affinity labels w h i c h are rich in galactose, would therefore only increase liver toxicity. In immunotoxins where the B-chain was deglycosylated, the potentiating effect of the B-chain was reduced by 5 to 50 fold.

From studies in tumour models and in rodents the features desirable in an immunotoxin have become apparent. Ideally the immunotoxin should be highly specific and highly cytotoxic with fast kinetics of killing. The immunogenicity should be low, the half life in sera and tissues should be high and the linkage between the toxic and lectin moeity should be stable. The immunotoxin should be unglycosylated and 100% homogeneous.