Abstract
M195 antibodies recognize CD33, an antigen present on acute myeloid leukemia blasts as well as some myeloid progenitor cells, but not on the ultimate hematopoietic progenitor stem cell. Immunotoxins (IT) reactive with human myeloid leukemias were constructed by conjugating gelonin, a single-chain ribosome-inactivating protein, to murine and genetically engineered, humanized M195 antibodies via anN-succinimidyl-3-(2-pyridyldithio)-propionate linkage. No losses of gelonin cytotoxic activity or M195 binding activity were observed after conjugation of up to two toxin molecules per antibody. Toxin conjugates displayed specific, potent toxicity for CD33+ cells. The murine and humanized IT were not toxic to CD33− cells and were 600 and 4500 times more potent, respectively, than free gelonin in inhibiting CD33+ HL60 cells. Treatment of HL60 cells with 1 μg/ml HuM195-gelonin resulted in more than 1000 times lower colony formation; normal bone marrow mononuclear cell colonyforming units treated with HuM195-IT were reduced by a factor of 10. HL60 leukemia cells could be effectively purged from an excess of normal bone marrow cells. Exposure of target cells to IT for as little as 30 min was as effective as continuous exposure of IT for up to 6 days. However, measures of the efficacy of the immunotoxin were directly related to the length of time of observation after IT exposure and were inversely related to cell concentration. M195-gelonin immunoconjugates are potential candidates for therapeutic use in in vivo or ex vivo bone marrow purging of myeloid leukemias.
Similar content being viewed by others
References
Andrews RG, Torok-Storb B, Bernstein ID (1983) Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies. Blood 62: 124
Barbieri L, Stirpe F (1982) Ribosome-inactivating proteins from plants: properties and possible uses. Cancer Surv 1: 490
Bernstein ID, Singer JW, Andrews RG, Keeting A, Powell JS, Bjornson BH, Cuttner J, Najfeld V, Reaman G, Raskind W, Sutton DMC, Fialkow PJ (1987) Treatment of acute myeloid leukemia cells in vitro with monoclonal antibody recognizing a myeloid differentiation antigen allows normal progenitor cells to be expressed. J Clin Invest 79: 1153
Blakey DC, Thorpe PE (1988) An overview of therapy with immunotoxins containing ricin or its A-chain. Antibody Immunoconjugates Radiopharm 1: 1
Blattler WA, Lambert JM, Goldmacher VD (1989) Realizing the full potential of immunotoxins. Cancer Cells 1: 50
Brigotti M, Rambelli F, Zamboni M, Montanaro L, Sperti S (1989) Effects of alpha-sarcin and ribosome inactivating proteins on the interaction of elongation factors with ribosomes. Biochem J 257: 723
Brown PS, Parenteau GL, Dirbas FM, Garsia RJ, Goldman CK, Bukowski MA, Junghans RP, Queen C, Hakimi J, Benjamin WR, Clark RE, Waldmann TA (1991) Anti-Tac-H, a humanized antibody to the interleukin 2 receptor, prolongs primate cardiac allograft survival. Proc Natl Acad Sci USA 88: 2663
Caron PC, Co MS, Bull MK, Avdalovic NM, Queen C, Scheinberg DA (1992) Biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies. Cancer Res 52: 6761
Caron PC, Jurcic JG, Scott AM, Finn RD, Divgi CR, Graham MC, Jureidini IM, Sgouros G, Tyson D, Old LJ, Larson SM, Scheinberg DA (1993) A phase 1 B trial of humanized monoclonal antibody M195 (anti-CD33) in myeloid leukemia: specific targeting without immunogenicity. Blood 83 (in press)
Chapman PB, Scheinberg DA, DiMaggio JJ, Houghton AN (1991) Unconjugated monoclonal antibodies as anticancer agents. In: Oettgen HF (ed) Immunology and allergy clinics of north america. Saunders, Philadelphia, p 257
Co MS, Deschamps M, Whitley RJ, Queen C (1991) Humanized antibodies for antiviral therapy. Proc Natl Acad Sci USA 88: 2869
Co MS, Avdalovic NM, Caron PC, Avdalovic MV, Scheinberg DA, Queen C (1992) Chimeric and humanized antibodies with specificity for the CD33 antigen. J Hematol 148: 1149
Co MS, Scheinberg DA, Avdalovic NM, McGraw KJ, Vasquez MH, Caron PC, Queen C (1993) Genetically engineered deglycosylation of the variable domain increases the affinity of an anti-CD33 monoclonal antibody. Mol Immunol 30: 1361
Divgi CR, Minniti JG, Old LJ, et al. (1989) Internalization of radiolabeled monoclonal antibody (mAb) into specific cells: effect of the radionuclide and antibody form. Cancer Res [Suppl] 30: 494a
Engert A, Brown A, Thorpe P (1991) Resistance of myeloid leukemia cell lines to ricin A-chain immunotoxins. Leukemia Res 15: 1079
Gabrilove JL, Wong G, Bollenbacher E, White K, Kojima S, Wilson EL (1993) Basic fibroblast growth factor counteracts the suppressive effect of transforming growth factor-beta 1 on human progenitor cells. Blood 81: 909
Goldmacher VS, Lambert JM, Blattler WA (1992) Evidence that the membrane binding and A-chain translocation activities of ricin cannot be separated. Biochem Biophys Res Commun 183: 758
Griffin D, Linch D, Sabbath K, Larcom P, Schlossman SF (1984) A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells. Leuk Res 8: 521
Grossbard ML, Lambert JM, Goldmacher VS, Blattler WA, Nadler LM (1992) Correlation between in vivo toxicity and preclinical in vitro parameters for the immunotoxin anti B4-blocked ricin. Cancer Res 52: 4200
Grossbard ML, Press OW, Appelbaum FR, Bernstein ID, Nadler LM (1992) Monoclonal antibody-based therapies of leukemia and lymphoma. Blood 80: 863
Grossbard ML, Freedman AS, Ritz J, Coral F, Goldmacher VS, Eliseo L, Spector N, Dear K, Lambert JM, Blattler WA, Taylor JA, Nadler LM (1992) Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase I trial of daily bolus infusion. Blood 79: 576
Hertler AA, Frankel AE (1989) Immunotoxins: a clinical review of their use in the treatment of malignancies. J Clin Oncol 7: 1932
La Russa VF, Griffin JD, Kessler SW, Cutting MA, Knight RD, Blattler WA, Lambert JM, Wright DG (1992) Effects of anti-CD33 blocked ricin immunotoxin on the capacity of CD34+ human marrow cells to establish in vitro hematopoiesis in long term marrow culture. Exp Hematol 20: 442
Peiper SC, Ashmun RA, Look AT (1988) Molecular cloning expression and chromosomal localization of a human gene encoding the CD33 myeloid differentiation antigen. Blood 72: 314
Preijers FWMB, De Witte T, Wessels JMC, Gast GC de, Van Leeuwen E, Capel PJA, Haanen C (1989) Autologous transplantation of bome marrow purged in vitro with anti-CD7-(WT1)ricin A immunotoxin in T-cell lymphoblastic leukemia and lymphoma. Blood 74: 1152
Raso V, Watkins SC, Slayter H, Fehrmann C (1987) Intracellular pathways of ricin A chain cytotoxins. Ann N Y Acad Sci 507: 172
Reichman L, Clark M, Waldman H, Winter G (1988) Reshaping human antibodies for therapy. Nature 332: 323
Robertson MJ, Soiffer RJ, Freedman AS, Rabinowe SL, Anderson KC, Ervin TJ, Murray C, Dear K, Griffin JD, Nadler LM, Ritz J (1992) Human bone marrow depleted of CD33-positive cells mediated delayed but durable reconstitution of hematopoiesis: clinical trial of MY9 monoclonal antibody-purged autografts for the treatment of acute myeloid leukemia. Blood 79: 2229
Rosenblum MG, Murray JL, Cheung L, Rifkin R, Salmon S, Bartholomew R (1991) A specific and potent immunotoxin composed of antibody ZME-018 and the plant toxin gelonin. Mol Biother 3: 6
Rosenblum MG, Kohr W, Beatty K, Beatty WF, Friedman P (1993) Sequencing, cloning and expression of recombinant gelonin toxin. Proc Am Assoc Cancer Res 34: 478
Roy DC, Griffin JD, Belvin M, Blattler WA, Lambert JM, Ritz J (1991) Anti-MY9-blocked ricin: an immunotoxin for selective targeting of acute myeloid leukemia cells. Blood 77: 2404
Scheinberg DA, Tanimoto M, Mc Kenzie S, Strife A, Old LJ, Clarkson BD (1989) Monoclonal antibody M195: a diagnostic marker for acute myelogenous leukemia. Leukemia 3: 440
Scheinberg DA, Lovett D, Divgi CR, Graham MC, Berman E, Pentlow K, Feirt N, Finn RD, Clarkson BD, Gee TS, Larson SM, Oettgen HF, Old LJ (1991) A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide. J Clin Oncol 9: 478
Schwartz MA, Scheinberg DA, Houghton AN (1992) Monoclonal antibody therapy. In: Longo D (ed) Cancer chemotherapy and biological response modifiers, vol 13. Elsevier Amsterdam p 156
Schwartz MA, Lovett DR, Redner A, Finn RD, Graham MC, Divgi CR, Dantis L, Gee TS, Andreeff M, Old LJ, Larson SM, Scheinberg DA (1993) Dose-escalation trial of M195 labeled with iodine 131 for cytoreduction and marrow ablation in relapsed or refractory myeloid leukemias. J Clin Oncol 11: 294
Sivam G, Pearson JW, Bohn W, Oldham RK, Sadoff JC, Morgan AC (1987) Immunotoxins to a human melanoma-associated antigen: comparison of gelonin with ricin and other A chain conjugates. Cancer Res 47: 3169
Stirpe F, Olsnes SE, Pihl A (1980) Gelonin, a new inhibitor of protein synthesis, nontoxic to intact cells. J Biol Chem 255: 6947
Tanimoto M, Scheinberg DA, Cordon-Cardo C, Huie D, Clarkson BD, Old LJ (1989) Restricted expression of an early myeloid and monocytic cell surface antigen defined by monoclonal antibody M195. Leukemia 3: 339
Trowbridge IS, Domingo DL (1982) Prospects for the clinical use of cytotoxic monoclonal antibody. Conjugates in the treatment of cancer. Cancer Surv 1: 544
Uckun FM, Gail-Peczalska KJ, Kersey JH, Houston LL, Vallera DA (1986) 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 leukemia. J Exp Med 163: 347
Vitetta E (1990) Immunotoxins: new therapeutic reagents for autoimmunity, cancer, and AIDS. J Clin Immunol [Suppl] 10: 15S
Author information
Authors and Affiliations
Additional information
These studies were supported in part by the Lucille P. Markey Charitable Trust, ACS Grant No. IM551, NIH PO1CA33049, NIH RO1CA55349. Research conducted, in part, by the Clayton Foundation for Research. David A Scheinberg is a Lucille P. Markey Scholar
Rights and permissions
About this article
Cite this article
McGraw, K.J., Rosenblum, M.G., Cheung, L. et al. Characterization of murine and humanized anti-CD33, gelonin immunotoxins reactive against myeloid leukemias. Cancer Immunol Immunother 39, 367–374 (1994). https://doi.org/10.1007/BF01534423
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01534423