The Preparation and Cytotoxic Properties of Antibody‐Toxin Conjugates

L INTRODUCTION Interest has burgeoned recently in the possibility of attacking tumor ceils with cytotoxic agents covalently linked to specific antibodies. This follows the innovation of cell hybridization techniques for producing monoclonal antibodies which has stimulated a world-wide search for antibodies with specificity for cancer cells. In anticipation that these endeavors will meet with success, a number of groups have attempted to devise ways of arming the antibody molecule to endow it with potent cytotoxic activity against the cells to which it can bind. Our decision to use toxins as the cytotoxic component ofthe conjugate was provoked by their supreme potency. One molecule of the bacterial exotoxin, diphtheria toxin, or of the plant toxins, abrin, from Abrus precatorius or ricin, from Ricinus communis, may be sufficient to kill a cell which it penetrates (Yamaizumiet al. 1978, Eiklid et al. 1980). This maximizes the chance of killing cancer cells which do not express specific antigens at high density or which reside in solid tumors not freely permeable to the conjugate. The modes of cytotoxic action of diphtheria toxin, abrin and ricin have been reviewed elsewhere (Collier 1976, Olsnes & Pihl 1976, Pappenheimer 1977, Gill 1978). Briefly, the toxins ali comprise two polypeptide chains, denoted A and B, which are linked by a disulfide bond and which, when separated, are virtually devoid of cytotoxic action. The toxins bind via a recognition site on the B-chain to receptors on the cell surface and the A-chain then penetrates (or is translocated across) the cell membrane into the cytosol where it terminates protein synthesis. Diphtheria toxin A-chain inactivates elongation factor 2 by

[1]  P. Casellas,et al.  High specific cytotoxicity of antibody-toxin hybrid molecules (immunotoxins) for target cells , 1980 .

[2]  The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. III. The chymotryptic peptides, the peptides derived by cleavage at tryptophan residues, and the complete sequence of the protein. , 1979, The Journal of biological chemistry.

[3]  W. Ross.  547. Aryl-2-halogenoalkylamines. Part III. The reactions of NN-di-2-chloroethyl-p-anisidine and β-naphthyldi-2-chloroethylamine in aqueous-acetone salt solutions , 1949 .

[4]  G. Nicolson.  Dynamic changes on the surfaces of normal and transformed cells and their relationship to the entry of pharmacology active proteins. , 1975, American journal of clinical pathology.

[5]  F. Stirpe,et al.  Gelonin, a new inhibitor of protein synthesis, nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A. , 1980, The Journal of biological chemistry.

[6]  Phil Gold,et al.  SPECIFIC CARCINOEMBRYONIC ANTIGENS OF THE HUMAN DIGESTIVE SYSTEM , 1965, The Journal of experimental medicine.

[7]  K. Sandvig,et al.  Rates of different steps involved in the inhibition of protein synthesis by the toxic lectins abrin and ricin. , 1976, The Journal of biological chemistry.

[8]  D. C. Edwards,et al.  Toxicity of diphtheria toxin for lymphoblastoid cells is increased by conjugation to antilymphocytic globulin , 1978, Nature.

[9]  E. Mekada,et al.  One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell , 1978, Cell.

[10]  J Carlsson,et al.  Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. , 1978, The Biochemical journal.

[11]  R. Rubens.  Antibodies as carriers of anticancer agents. , 1974, Lancet.

[12]  J. Mach,et al.  Isolation and characterization of carcinoembryonic antigen (CEA) extracted from normal human colon mucosa. , 1977, Immunochemistry.

[13]  I. Pastan,et al.  Collection of insulin, EGF and α 2-Macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization , 1978, Cell.

[14]  P. Casellas,et al.  Immunotoxins: hybrid molecules of monoclonal antibodies and a toxin subunit specifically kill tumour cells , 1981, Nature.

[15]  E. Mekada,et al.  Hybrid toxin of the A chain of ricin toxin and a subunit of Wistaria floribunda lectin. Possible importance of the hydrophobic region for entry of toxin into the cell. , 1980, The Journal of biological chemistry.

[16]  P. Isakson,et al.  Selective killing of normal or neoplastic B cells by antibodies coupled to the A chain of ricin. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[17]  D. C. Edwards,et al.  Abrogation of the non-specific toxicity of abrin conjugated to anti-lymphocyte globulin. , 1981, Clinical and experimental immunology.

[18]  W. Ross.  The interaction of chlorambucil with human γ-globulin , 1974 .

[19]  A. Barrett.  Proteinases in Mammalian Cells and Tissues , 1977 .

[20]  K. Sandvig,et al.  Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells. , 1976, The Journal of biological chemistry.

[21]  N. Capparell,et al.  Antitumor effects of antibody-diphtheria toxin conjugates. III. Cyclophosphamide-induced immune unresponsiveness to conjugates. , 1975, Journal of the National Cancer Institute.

[22]  Y. Masuho,et al.  Target-cell cytotoxicity of a hybrid of Fab' on immunoglobulin and A-chain of ricin. , 1980, Gan.

[23]  S. Cooperband,et al.  Antitumor effects of antibody-diphtheria toxin conjugates: use of hapten-coated tumor cells as an antigenic target. , 1972, Journal of the National Cancer Institute.

[24]  T. Oeltmann,et al.  A hybrid protein containing the toxic subunit of ricin and the cell-specific subunit of human chorionic gonadotropin. II. Biologic properties. , 1979, The Journal of biological chemistry.

[25]  L. Barbieri,et al.  Seed extracts inhibiting protein synthesis in vitro. , 1980, The Biochemical journal.

[26]  M. Beppu,et al.  Preparation of antibody (IgG)-ricin A-chain conjugate and its biologic activity. , 1980, Gan.

[27]  N. Groman,et al.  In Vitro Inhibition of Diphtheria Toxin Action by Ammonium Salts and Amines , 1965, Journal of bacteriology.

[28]  G. Nicolson.  Ultrastructural analysis of toxin binding and entry into mammalian cells , 1974, Nature.

[29]  H. Herschman,et al.  Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[30]  D. C. Edwards,et al.  Cytotoxicity acquired by conjugation of an anti-Thy1.1 monoclonal antibody and the ribosome-inactivating protein, gelonin. , 1981, European journal of biochemistry.

[31]  A. Sehon,et al.  A method for the preparation of protein-protein conjugates of predetermined composition. , 1978, Journal of immunological methods.

[32]  A. Pihl,et al.  Entry of lethal doses of abrin, ricin and modeccin into the cytosol of HeLa cells. , 1980, Experimental cell research.

[33]  D. Fiete,et al.  Structural determinants of Ricinus communis agglutinin and toxin specificity for oligosaccharides. , 1979, The Journal of biological chemistry.

[34]  Y. Aoki.  [Lysosomal enzymes]. , 1983, Ryumachi. [Rheumatism].

[35]  D. Neville,et al.  Ricin linked to monophosphopentamannose binds to fibroblast lysosomal hydrolase receptors, resulting in a cell-type-specific toxin. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[36]  S. Cooperband,et al.  Antitumor effects of antibody-diphtheria toxin conjugates. II. Immunotherapy with conjugates directed against tumor antigens induced by simian virus 40. , 1975, Journal of the National Cancer Institute.

[37]  D. Cameron,et al.  Immunochemotherapy of Cancer with Chlorambucil-carrying Antibody , 1972, British medical journal.

[38]  T. Chang,et al.  Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen. , 1977, The Journal of biological chemistry.

[39]  Y. Ikawa,et al.  Preparation of concanavalin A-ricin A-chain conjugate and its biologic activity against various cultured cells. , 1979, Journal of the National Cancer Institute.

[40]  H. Stukenbrok,et al.  An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. II. Intracellular fates of the 125I- ligands , 1979, The Journal of cell biology.

[41]  R. Collier,et al.  Amino-acid sequence of fragment A, an enzymically active fragment from diphtheria toxin. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[42]  D. Mason,et al.  The kinetics of antibody binding to membrane antigens in solution and at the cell surface. , 1980, The Biochemical journal.

[43]  S. Cooperband,et al.  Selective Destruction of Target Cells by Diphtheria Toxin Conjugated to Antibody Directed against Antigens on the Cells , 1970, Science.

[44]  A. M. Pappenheimer.  Diphtheria toxin. , 1952, Annual review of biochemistry.

[45]  E. Mekada,et al.  Reconstitution of hybrid toxin from Fragment A of diphtheria toxin and a subunit of Wistaria floribunda lectin. , 1978, The Journal of biological chemistry.

[46]  K. Sandvig,et al.  Effect of temperature on the uptake, excretion and degradation of abrin and ricin by HeLa cells. , 1979, Experimental cell research.

[47]  M. Simon,et al.  The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement , 1980, The Journal of cell biology.

[48]  W. Miskimins,et al.  Synthesis of a cytotoxic insulin cross-linked to diphtheria toxin fragment A capable of recognizing insulin receptors. , 1979, Biochemical and biophysical research communications.

[49]  Robert A. H. White,et al.  Monoclonal Antibodies That Define T-Lymphocyte Subsets in the Rat , 1980 .

[50]  K. Sandvig,et al.  Chemical modifications of the toxic lectins abrin and ricin. , 1978, European journal of biochemistry.

[51]  D. Gilliland,et al.  Epidermal growth factor-toxin A chain conjugates: EGF-Ricin A is a potent toxin while EGF-diphtheria fragment A is nontoxic , 1980, Cell.

[52]  V. Raso,et al.  Specific cytotoxicity of a human immunoglobulin-directed Fab'-ricin A chain conjugate. , 1980, Journal of immunology.

[53]  D. C. Edwards,et al.  Increased toxicity of diphtheria toxin for human lymphoblastoid cells following covalent linkage to anti-(human lymphocyte) globulin or its F(ab')2 fragment. , 1980, European journal of biochemistry.

[54]  Y. Masuho,et al.  Preparation of a hybrid of fragment Fab' of antibody and fragment A of diphtheria toxin and its cytotoxicity. , 1979, Biochemical and biophysical research communications.

[55]  A. Habeeb,et al.  Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. , 1966, Analytical biochemistry.

[56]  L. Orci,et al.  Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[57]  A. Pihl,et al.  Isolation and comparison of galactose-binding lectins from Abrus precatorius and Ricinus communis. , 1974, The Journal of biological chemistry.

[58]  D. Neville,et al.  Anti-Thy 1.2 monoclonal antibody linked to ricin is a potent cell-type-specific toxin. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[59]  D. Gilliland,et al.  A model system involving anti-concanavalin A for antibody targeting of diphtheria toxin fragment A1. , 1980, Cancer research.

[60]  T. Uchida,et al.  Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. , 1973, The Journal of biological chemistry.

[61]  D. Wall,et al.  The galactose-specific recognition system of mammalian liver: The route of ligand internalization in rat hepatocytes , 1980, Cell.

[62]  D. Neville,et al.  Studies on the galactose-binding site of ricin and the hybrid toxin man6P-ricin , 1981, Cell.

[63]  A. Pihl,et al.  Abrin, Ricin, and their Associated Agglutinins , 1977 .

[64]  D. Gilliland,et al.  Chimeric toxins: toxic, disulfide-linked conjugate of concanavalin A with fragment A from diphtheria toxin. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[65]  D. Gilliland,et al.  Antibody-directed cytotoxic agents: use of monoclonal antibody to direct the action of toxin A chains to colorectal carcinoma cells. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[66]  M. Bretscher,et al.  Coated pits act as molecular filters. , 1980, Proceedings of the National Academy of Sciences of the United States of America.