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. 1988 Jun;26(3):231–236. doi: 10.1007/BF00199934

An immunotoxin containing a rat IgM monoclonal antibody (Campath 1) and saporin 6: effect on T lymphocytes and hemopoietic cells

Pier Luigi Tazzari 1,✉, Luigi Barbieri 2, Marco Gobbi 1, Angelo Dinota 1, Simonetta Rizzi 1, Andrea Bontadini 1, Annalisa Pession 2, Sante Tura 1, Fiorenzo Stirpe 2
PMCID: PMC11038324  PMID: 3260131

Abstract

The elimination of the cells responsible for graft-versus-host disease in allogeneic bone marrow transplantation has been attempted with a variety of methods, including the use of the ribosome-inactivating toxin ricin bound to monoclonal antibodies acting as carriers. However the high nonspecific toxicity of these immunotoxins containing the whole toxin greatly limited clinical application. Toxicity can be reduced using the A-chain of ricin or other ribosome-inactivating proteins (RIPs) which are devoid of a B-chain with lectin properties. We used saporin 6 purified from Saponaria officinalis seeds, which was conjugated with the rat IgM monoclonal antibody Campath 1 specific for mature T and B lymphocytes as well as for monocytes. The immunotoxin retained both RIP and antibody activity, inhibiting protein synthesis both in a cellfree system and in cells bearing the Campath 1 antigen; it also abolished methyl 3H-thymidine uptake in phytohemagglutinin-stimulated T lymphocytes. Myeloid progenitors were largely spared as shown by myeloid stem cell (CFUGM) growth which was scarcely affected. Toxicity of the immunotoxin to cell lines not expressing the antigen recognized by Campath 1 monoclonal antibody was not greater than the toxicity due to free saporin 6, while the immunotoxin was more toxic to mice than free saporin.

Keywords: Stem Cell, Monoclonal Antibody, Bone Marrow Transplantation, Marrow Transplantation, Allogeneic Bone

Footnotes

Work supported by grants of Regione Emilia Romagna, delibera n. 1970, 13/5/86, by the Italian National Research Council, Roma, Finalized Project “Oncologia”, contracts n. 86.00589.44 and n. 86.00603.44 and by the Pallotti's Legacy for Cancer Research

Angelo Dinota is supported by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy.

References

  • 1.Allen EH, Schweet RS. Synthesis of hemoglobin in a cell-free system. Properties of the complete system. J Biol Chem. 1962;237:760. [PubMed] [Google Scholar]
  • 2.Bandini G, Ricci P, Gobbi M, Tazzari PL, Motta MR, Guardigli C, Rizzi S, Baccarani M, Tura S. T-cell depletion with the monoclonal antibody Campath 1 to prevent graft versus host disease in ten high risk patients. Haematologica. 1986;71:307. [PubMed] [Google Scholar]
  • 3.Barbieri L, Stirpe F. Ribosome-inactivating protein from plants: properties and possible uses. Cancer Surveys. 1982;1:489. [Google Scholar]
  • 4.Berenson RJ, Bensinger WI, Kalamasz D, Martin P. Elimination of Daudi lymphoblasts from human bone marrow using avidin-biotin immunoadsorbtion. Blood. 1986;67:509. [PubMed] [Google Scholar]
  • 5.Bjorn MJ, Larrik J, Platak M, Wilson KJ. Characterization of translational inhibitors from Phytolacca americana. Amino terminal sequence determination and antibody-inhibitor conjugates. Biochem Biophys Acta. 1984;790:154. doi: 10.1016/0167-4838(84)90219-x. [DOI] [PubMed] [Google Scholar]
  • 6.Bregni M, De Fabritiis P, Raso V, Greenberger J, Lipton J, Nadler L, Rothstein L, Ritz J, Bast RC., Jr Elimination of clonogenic tumor cells from human bone marrow using a combination of monoclonal antibody: ricin A chain conjugates. Cancer Res. 1986;46:1208. [PubMed] [Google Scholar]
  • 7.Carlsson J, Previn D, Axen R. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio) propionate, a new hetero-bifunctional reagent. Biochemistry. 1978;173:723. doi: 10.1042/bj1730723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Colombatti M, Nabholz M, Gros O, Bron C. Selective killing of target cells by antibody-ricin A chain or antibody-gelonin hybrid molecules: comparison of cytotoxic potency and use in immunoselection procedures. J Immunol. 1983;131:3091. [PubMed] [Google Scholar]
  • 9.Dicke KA, Spitzer G. Evaluation of the use of high-dose cytoreduction with autologous marrow rescue in various malignancies. Transplantation. 1986;41:4. doi: 10.1097/00007890-198601000-00002. [DOI] [PubMed] [Google Scholar]
  • 10.Eiklid K, Olsnes S, Pihl A. Entry of lethal dose of abrin, ricin and modeccin into the cytosol of HeLa cells. Exp Cell Res. 1980;126:321. doi: 10.1016/0014-4827(80)90270-0. [DOI] [PubMed] [Google Scholar]
  • 11.Douay L, Gorin NC, Lopez M, Casellas P, Liance MC, Jansen FK, Voisin GA, Baillou C, Laporte JP, Najman A, Duhamel G. Evidence for absence of toxicity of T 101 immunotoxin on human hematopoietic progenitor cells prior to bone marrow transplantation. Cancer Res. 1985;45:438. [PubMed] [Google Scholar]
  • 12.Filipovich AH, Vallera D, Youle R, Quinones RA, Neville D, Kersey JH. Ex vivo treatment of donor bone marrow with anti-T cell immunotoxins for the prevention of graft versus host disease. Lancet. 1984;I:469. doi: 10.1016/s0140-6736(84)92847-2. [DOI] [PubMed] [Google Scholar]
  • 13.Fisher A, Durandy A, Villartay JP, Vilmer EE, Le Deist F, Gerota I, Griscelli C. HLA-haploidentical bone marrow transplantation for severe combined immunodeficiency using E-rosette fractionation and Cyclosporin. Blood. 1986;67:444. [PubMed] [Google Scholar]
  • 14.Goldmacher VS, Anderson J, Blatter WA, Lambert JM, Senter PD. Antibody-complement-mediated cytotoxicity is enhanced by ribosome-inactivating proteins. J Immunol. 1985;135:3648. [PubMed] [Google Scholar]
  • 15.Hale G, Waldmann H. Depletion of T-cells with Campath 1 and human complement: analysis of GVHD and graft failure in a multi-centre study. Bone Marrow Transplantation. 1986;1(1):93. [Google Scholar]
  • 16.Hale G, Broight S, Chumbley G, Hoang T, Metcalf D, Munro AJ, Waldmann H. Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement. Blood. 1983;62:873. [PubMed] [Google Scholar]
  • 17.Hale G, Swirski D, Waldmann H, Chan LC. Reactivity of rat monoclonal antibody Campath 1 with human leukaemia cells and its possible application for autologous bone marrow transplantation. Br J Haematol. 1985;60:41. doi: 10.1111/j.1365-2141.1985.tb07383.x. [DOI] [PubMed] [Google Scholar]
  • 18.Hudson L, Hay FC. Practical Immunology. 2 ed. Oxford: Blackwell; 1980. [Google Scholar]
  • 19.Iscove NN, Senn JS, Till JE, McCulloch EA. Colony formation by normal and leukemic human bone marrow cells in culture: effect of conditioned medium from human leukocytes. Blood. 1971;37:1. [PubMed] [Google Scholar]
  • 20.Jones L, Apperley J, Rombos G, Tsatalas C, Hale G, Waldmann H, James D, Hows J, Gordon-Smith E, Goolden A, Goldman J. Use of Campath 1 for T-depletion in matched and mismatched transplants for CGL. Bone Marrow Transplantation. 1986;1(1):96. [Google Scholar]
  • 21.Kernan NA, Knowles RW, Burns MJ, Broxmeyer HE, Lu L, Lee HM, Kawahata RT, Scannon PJ, Du Pont B. Specific inhibition of in vitro lymphocyte transformation an antipan T cell (gp 67) ricin A-chain immunotoxin. J Immunol. 1984;133:137. [PubMed] [Google Scholar]
  • 22.Lambert JM, Senter PD, Yau-Young A, Blatter WA, Goldmacher VS. Purified immunotoxins that are reactive with human lymphoid cells. Monoclonal antibodies conjugated to the ribosome-inactivating proteins gelonin and the pokeweed antiviral proteins. J Biol Chem. 1985;260:12035. [PubMed] [Google Scholar]
  • 23.Lappi DA, Esch FS, Barbieri L, Stirpe F, Soria M. Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies. Biochem Biophys Res Commun. 1985;129:934. doi: 10.1016/0006-291x(85)91981-3. [DOI] [PubMed] [Google Scholar]
  • 24.Masuho Y, Kishida K, Hara T. Targeting of antiviral protein from Phytolacca americana with an antibody. Biochem Biophys Res Commun. 1982;105:462. doi: 10.1016/0006-291x(82)91457-7. [DOI] [PubMed] [Google Scholar]
  • 25.Molday RS, Yen SP, Rembaum A. Application of magnetic microspheres in labelling and separation of cells. Nature. 1977;268:437. doi: 10.1038/268437a0. [DOI] [PubMed] [Google Scholar]
  • 26.Muirhead M, Martin PJ, Torok-Storb B, Uhr JW, Vitetta ES. Use of an antibody-ricin A-chain conjugate to delete neoplastic cells from human bone marrow. Blood. 1983;62:327. [PubMed] [Google Scholar]
  • 27.Olsnes S, Pihl A. Toxic lectins and related proteins. In: Cohen P, Van Heyningen S, editors. Molecular actions of toxins and viruses. Amsterdam: Elsevier North Holland; 1981. p. 51. [Google Scholar]
  • 28.Prentice HG, Blacklock HA, Janossy G. Use of the anti-T monoclonal antibody OKT3 for the prevention of acute graft-versus-host disease (GvHD) in allogeneic bone marrow transplantation for acute leukaemia. Lancet. 1982;I:700. doi: 10.1016/s0140-6736(82)92619-8. [DOI] [PubMed] [Google Scholar]
  • 29.Prentice HG, Blacklock HA, Janossy G, Gilmore MJML, Price-Jones L, Tidman N, Treidosiewicz LK, Skegos DB, Panjvani D, Ball S, Graphakos S, Patterson J, Ivory K, Hoffbrand AV. Depletion of T-lymphocytes in donor marrow prevents significant graft-versus-host disease in matched allogeneic leukaemic marrow recipients. Lancet. 1984;I:472. doi: 10.1016/s0140-6736(84)92848-4. [DOI] [PubMed] [Google Scholar]
  • 30.Ramakrishnan S, Houston LL. Comparison of selective cytotoxic effects of immunotoxins containing ricin A chain or pokeweed antiviral protein and anti-Thy 1.1 monoclonal antibody. Cancer Res. 1984;44:201. [PubMed] [Google Scholar]
  • 31.Ramakrishnan S, Houston LL. Prevention of growth of leukemia cells in mice by monoclonal antibodies directed against Thy 1.1 antigen disulfide linked to ribosomal inhibitors: pokeweed antiviral protein or ricin A chain. Cancer Res. 1984;44:1398. [PubMed] [Google Scholar]
  • 32.Ramakrishnan S, Houston LL. Inhibition of human acute lymphoblastic leukaemia cells by immunotoxins: potentiation by chloroquine. Science. 1984;223:58. doi: 10.1126/science.6318313. [DOI] [PubMed] [Google Scholar]
  • 33.Ramakrishnan S, Houston LL. Immunological and biological stability of immunotoxins in vivo as studied by the clearance of disulfide-linked pokeweed antiviral protein-antibody conjugates from blood. Cancer Res. 1985;45:2031. [PubMed] [Google Scholar]
  • 34.Ramakrishnan S, Uckun FM, Houston LL. Anti-T cell immunotoxins containing pokeweed antiviral protein: potential purging agents for human autologous bone marrow transplantation. J Immunol. 1985;135:3616. [PubMed] [Google Scholar]
  • 35.Roberts WK, Selitrennikoff CP (1986) Plant protein that inactivate foreign ribosomes. Biosci Rep 6:19 [DOI] [PubMed]
  • 36.Sandvig K, Olsnes S. Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. II. Effect of pH, metabolic inhibitors and ionophors and evidence for toxin penetration from endocytic vesicles. J Biol Chem. 1982;257:7504. [PubMed] [Google Scholar]
  • 37.Stirpe F, Barbieri L. Ribosome-inactivating protein up to date. FEBS Lett. 1986;195:1. doi: 10.1016/0014-5793(86)80118-1. [DOI] [PubMed] [Google Scholar]
  • 38.Stirpe F, Gasperi-Campani A, Barbieri L, Falasca A, Abbondanza A, Stevens WA. Ribosome-inactivating protein from seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree) Biochemistry. 1983;216:617. doi: 10.1042/bj2160617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Stirpe F, Derenzini M, Barbieri L, Farabegoli F, Brown ANF, Knowles PP, Thorpe PE. Hepatotoxicity of immunotoxins made with saporin, a ribosome-inactivating protein from Saponaria officinalis . Virchows Arch [b] 1987;53:259. doi: 10.1007/BF02890252. [DOI] [PubMed] [Google Scholar]
  • 40.Stong RC, Uckun F, Youle RJ, Kersey JH, Vallera DA. Use of multiple T cell-directed intact ricin immunotoxins for autologous bone marrow transplantation. Blood. 1985;66:627. [PubMed] [Google Scholar]
  • 41.Stuart AE, Habeshaw JA, Davidson AE. Phagocytes in vitro. In: Weir DM, editor. Handbook of experimental immunology. Oxford: Blackwell Scientific Publications; 1973. p. 241. [Google Scholar]
  • 42.Tazzari PL, Gobbi M, Dinota A, Bontadini A, Grassi G, Cerato C, Cavo M, Pileri S, Caligaris-Cappio F, Tura S. Normal and neoplastic plasma cell membrane phenotype. Studies with new monoclonal antibodies. Clin Exp Immunol. 1987;70:192. [PMC free article] [PubMed] [Google Scholar]
  • 43.Thorpe PE, Brown ANF, Ross WCJ, Cumber AJ, Detre SJ, Edwards DC, Davies AJS, Stirpe F. Cytotoxicity acquired by conjugation of an anti-Thy 1.1 monoclonal antibody and the ribosome-inactivating protein, gelonin. Eur J Biochem 1981. 1981;116:447. doi: 10.1111/j.1432-1033.1981.tb05356.x. [DOI] [PubMed] [Google Scholar]
  • 44.Thorpe PE, Brown ANF, Bremner JAG, Foxwell BMJ, Stirpe F. An immunotoxin composed of monoclonal anti-Thy 1.1 antibody and a ribosome-inactivating protein from Saponaria officinalis: potent antitumor effects in vitro and in vivo. J Natl Cancer Inst. 1985;75:151. [PubMed] [Google Scholar]
  • 45.Uckun FM, Ramakrishnan S, Houston LL. Increased efficiency in selective elimination of leukaemic cells by a combination of cyclophosphamide and a human B-cell specific immunotoxin containing pokeweed antiviral protein. Cancer Res. 1985;45:69. [PubMed] [Google Scholar]
  • 46.Uckun FM, Ramakrishnan S, Houston LL. Immunotoxin-mediated elimination of clonogenic tumor cells in the presence of human bone marrow. J Immunol. 1985;134:2010. [PubMed] [Google Scholar]
  • 47.Uckun FM, Gajl-Peczalska KJ, Kersey JH, Houston LL, Vallera DA. Use of a novel colony assay to evaluate the cytotoxicity of an immunotoxin containing pokeweed antiviral protein against blast progenitor cells freshly obtained from patients with common B-lineage acute lymphoblastic leukaemia. J Exp Med. 1986;163:347. doi: 10.1084/jem.163.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Vallera DA, Ash RC, Zanjanie D, Kersey JH, Lebien TW, Beverley PCL, Neville DM, Jr, Youle RJ. Anti-T cell reagents for human bone marrow transplantation: ricin linked to three monoclonal antibodies. Science. 1983;222:8512. doi: 10.1126/science.6353579. [DOI] [PubMed] [Google Scholar]
  • 49.Vitetta ES, Uhr JW. Immunotoxins. Ann Rev Immunol. 1985;3:197. doi: 10.1146/annurev.iy.03.040185.001213. [DOI] [PubMed] [Google Scholar]
  • 50.Wiels J, Junqua S, Dujardin P, Lepecq JB, Tursz T. Properties of immunotoxins against a glycolipidic antigen associated with Burkitt's lymphoma. Cancer Res. 1984;44:129. [PubMed] [Google Scholar]

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