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...

Attempts to target antibody-ricin conjugates (immunotoxins) to designated cell types in vivo may be thwarted by their rapid clearance by hepatic reticuloendothelial cells which have receptors that recognise oligosaccharide side chains on the toxin. The B-chain of ricin contains high mannose type oligosaccharides and the A-chain contains a complex unit (GlcNAc)2-Fuc-Xyl-(Man)4-6, all of which potentially could be recognised by the reticuloendothelial system.Treatment of ricin with a mixture of sodium metaperiodate and sodium cyanoborohydride at pH 3.5 resulted in oxidative cleavage of the carbohydrates and reduction of the aldehyde groups thus formed to primary alcohols. By conducting the modification procedure at acidic pH, both the possibility of Schiff s base formation between the aldehyde groups and amino groups in the protein and the possibility of non-specific oxidation of amino acids were minimised. The extent of the carbohydrate modification depended on the duration of treatment, resulting maximally in the destruction of 13 of the 18 mannose residues and of all xylose and fucose.The toxicity of the modified toxin to cells in culture declined by up to 90% as the carbohydrate was destroyed. This was not due to a reduced ability of the B-chain to bind to cells or of the A-chain to inactivate ribosomes. In contrast to the in vitro results, the toxicity of the modified toxin to mice and rats was elevated by up to fourfold. The modification greatly reduced the clearance of the toxin by non-parenchymal cells in the liver and prevented the damage to hepatic Kupffer and sinusoidal cells and to the red pulp of the spleen that is inflicted by the native toxin. The elevated toxicity to animals appears to be because the modified toxin evades the reticuloendothelial system and persists in the bloodstream for longer periods, thus resulting in lethal damage to vital tissues in the animal at lower dosage.The results suggest that immunotoxins prepared from modified ricin would not be readily cleared by the reticuloendothelial system and so be more effective at killing their target cells.Attempts have been made in several laboratories to target ricin to designated cell types in animals by linking it to specific antibodies (reviewed in [l -31).Ricin, the toxin from the castor bean, is a glycoprotein comprising two polypeptide chains, A and B, joined by a disulphide bond. The B-chain binds to galactose-containing glycoproteins and glycolipids on cell surfaces and the A-chain then penetrates the membrane (probably from an endocytic vesicle) and kills the cell by inactivating ribosomes (reviewed in 141).When injected intravenously into rats or mice, rich rapidly accumulates in the liver and, to a lesser extent, in the spleen [5] wise be cleared from the bloodstream in animals and prompted us to devise ways of modifying the carbohydrate side chains in the toxin to prevent hepatic recognition. A previous attempt by Simeral et al. [8] to destroy the carbohydrate in ricin using sodium metaperiodate resulted in virtually complete ...

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococoal protein A (PA) have been produced in E. coii. Construcb consisting of N-terminal RTA and C-terminal PA (RTA-PA) or N-terminal PA and C-terminal (PA-RTA) were capable of binding to immunoglobulin G (via PA) and of specitically depurinating 28 S ribosomal RNA (via RTA). Howevtx, neither fusion protein was cytotoxic to antigen-bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short ammo acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a dis~~de-links loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA-DT-PA to the two chain form consisting of RTA Iinked by a d&hide bond to PA. The cleaved fusion protein was highly toxic to Saudi cells coated with ~~-~~~oglob~in antibody suiting that the RTA could be released from the PA by reduction witbin the oytosol.Ricin A chain; Fusion protein; Protein A, ~oteol~ti~ily-cl~~ble sequexe

Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigen, CD22, were prepared from 4 monoclonal antibodies (MAbs) or their Fab' fragments: RFB4, HD6, UV22-I and UV22-2. The ITs were tested for their ability to kill cells from the Burkitt lymphoma line, Daudi, the pre-B-cell leukemia line, NALM-6, and the myeloma cell line, ARH-77. Daudi expresses high levels of CD22, whereas NALM-6 and ARH-77 express low levels of CD22. The IgG-RFB4-A was highly toxic to all 3 cell lines; it killed 50% of the Daudi cells at a concentration of 1.2 x 10(-12) M and 50% of NALM-6 and ARH-77 cells at concentrations of 1.5 to 2.1 x 10(-11) M. IgG-RFB4-A was 10-30 times more toxic to Daudi cells than were the IgG-As constructed from the other 3 CD22 MAbs and 10 times more toxic than ricin itself. IT-As constructed from the Fab' fragments of the 4 CD22 antibodies were 2 to 5 times less toxic to Daudi cells than their IgG-A counterparts. Fab'-RFB4-A was twice as toxic to Daudi cells as ricin, whereas the other Fab'-As were about 7 times less toxic than ricin. Scatchard analyses of the binding of the radio-iodinated antibodies to Daudi cells showed that the intact RFB4 antibody bound 3-10 times more strongly than the other antibodies, whereas the Fab'-RFB4 bound 1.2 to 3.5 times more strongly than the Fab' fragments prepared from the other antibodies. Thus, the potent cytotoxic activity of the RFB4-As appears to derive, in part, from their superior binding affinity. Prior studies have shown that UV22-I and HD6 cross-react with certain normal human tissues lacking cells of B-cell lineage, whereas UV22-2 and RFB4 are B-cell-specific. This fact, together with its superior potency as an IT-A, suggests that RFB4 is the antibody of choice for preparing Fab'-As or IgG-As for in vivo therapy of human B-cell leukemias and lymphomas.