Gelonin, a plant protein which can powerfully reduce the protein-synthetic capacity of ribosome preparations, was covalently coupled to anti-Thy1 .I antibody. The conjugate was prepared using N-succinimidyl-3-(2-pyridyldithi0)propionate which generates a disulphide linkage between the component molecules. Two conjugate fractions were obtained with M, of 180000 and > 200000.After its linkage to the antibody, gelonin suppressed those Thy1.1-bearing T lymphocytes from AKR mice which will respond to phytohaemagglutinin and concanavalin A in tissue culture. The [3H]leucine incorporation with the T-cell mitogens was inhibited by 50% with the 180000-M, fraction at a concentration of 0.4nM and with the > 200000-Mr fraction of pM. Unconjugated gelonin induced comparable reductions in T-cell responsiveness but at concentrations of 30 nM. The conjugates exerted little or n o effect upon B lymphocytes or T lyniphocytes from CBA mice (Thyl.z+ve). Two Thyl.l-expressing AKR lymphoma cell lines, AKR-A and BW5147, were found to be sensitive to the conjugates, albeit much less so than the norinal T lymphocytes, The conjugates injected in vivo significantly prolonged the life of CBA mice bearing an AKR-A lymphoma allograft.It is concluded that gelonin can, by its linkage to an antibody, be rendered cytotoxic with a potency to match or exceed those of the toxins abrin and ricin.Gelonin, a glycoprotein from the seeds of Gelonium nzulti-,florum, inhibits protein synthesis in rabbit reticulocyte lysates by damaging the 60-S subunit of the ribosomes. The inactivation of the ribosomes is irreversible, seems not to involve cofactors, and occurs with an efficiency that suggests lhat gelonin acts enzymically [l]. Several other plants have been found to contain proteins which can inactivate isolated eukaryotic ribosomes apparently by the same mechanism as does gelonin. The proteins can be divided into those with and those without the capacity to recognise and bind to carbohydrates on cell surfaces.The inhibitors which d o not bind to cells are single polypeptide chains and are more or less devoid of toxic effects upon mammalian cells. This group, to which gelonin belongs, also includes the pokeweed antiviral protein [2,3] and inhibitors from wheat germ [4,5], Momordica charuntia [6] and probably also from Croton tiglium and Jatropha curcus [7].The proteins which bind to cells d o so because the ribosomc-inhibitory subunit (the A chain) is linked by a disulphide bond to a second subunit (the B chain) which has the cell-binding property. These proteins are either monovalent or, as with the lectins, divalent with two A-B units associated noncovalently. They differ in toxicity both to intact animals and to cells in tissue culture. Some, such as ricin and abrin (reviewed in [S]), modeccin [9,10] and a toxic lectin from Viscum album [Ill, are extremely potent toxins whereas others are less toxic or non-toxic, such as Ricinus communis agglutinin [12], Ahrus precatorius agglutinin [8,13] and thc lectins from iz4. charantia, Vicia craccu and Crotulu...

A method is described for preparing specific cytotoxic agents by linking intact rich to antibodies in a manner that produces obstruction of the galactose-binding sites on the B chain of the toxin and so diminishes the capacity of the conjugate to bind non-specifically to cells. The conjugates were synthesised by reacting iodoacetylated ricin with thiolated immunoglobulin and the components of conjugate with reduced galactose-binding capacity were separated by affinity chromatography on Sepharose (a P-galactosyl matrix) and asialofetuin-Sepharose.Fluorescence-activated cell sorter (FACS) analyses revealed that the fraction of a monoclonal anti-Thy 1.1 -rich conjugate that passed through a Sepharose column had markedly diminished capacity to bind non-specifically to Thyl.2-expressing CBA thymocytes and EL4 lymphoma cells. The fraction of conjugate that passed through an asialofetuin-Sepharose column displayed no detectable non-specific binding. Both fractions of conjugate were potent cytotoxic agents for Thyl. 1-expressing AKR-A lymphoma cells in tissue culture. They reduced the [3H]leucine incorporation of the cells by 50 % at a concentration of 2 -5 pM. Comparable inhibition of EL4 cells was only achieved with 3000 -7500-fold greater concentrations of conjugate. By contrast, the fraction of anti-Thyl.1-ricin that retained Sepharose-binding capacity showed marked non-specific binding and toxicity to EL4 cells.A conjugate with diminished galactose-binding capacity was also prepared from the W3/25 monoclonal antibody which recognises an antigen upon helper T-lymphocytes in the rat. It elicited powerful and specific toxic effects upon W3/25 antigen-expressing rat T-leukaemia cells. This finding is of particular importance because isolated ricin A-chain disulphide-linked to W3/25 antibody is not cytotoxic. The property of the B-chain in intact rich conjugates that facilitates delivery of the A-chain to the cytosol thus appears to be independent of galactose recognition.It is concluded that the 'blocked' ricin conjugates combine the advantages of high potency, which is often lacking in antibody-A-chain conjugates, with high specificity, which previously was lacking in intact rich conjugates.Cell-type-specific cytotoxic agents have been synthesised in several laboratories by covalently linking antibody molecules to highly potent plant and bacterial toxins (reviewed in [l, 21). Ricin, from Ricinus comrnunis, and abrin, from Abrus precatorius, have been the two plant toxins most widely used. Abrin and ricin bind to virtually all nucleated eukaryotic cells with which they come in contact and kill them by essentially the same mechanism (reviewed in [3,4]). They both comprise two polypeptide chains, A and B, joined by a disulphide bond. The B-chain binds to galactose-containing glycoproteins and glycolipids on the cell surface and the A-chain (or the whole toxin) appears to traverse either the plasma membrane itself or the membrane of an endocytic vesicle and stops protein synthesis by catalysing a modification to the 60s s...

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We report the conversion of a non-cytotoxic atibody-ricin A chain conjugate to one displaying specific cytotoxic effects comparable with that of native ricin, by the addition of ricin B chain as a second stage reagent. The results suggest that this conversion is achieved by the association of the added B chain with the A chain of the conjugate, and not through a primary binding of B chain at the cell surface. RickA chain Targeting

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