- Email: [email protected]
Interleukin 2 Toxin: A Step Toward Selective Immunomodulation
Interleukin 2 Toxin: A Step Toward Selective Immunomodulation John R. Murphy, PhD, Vicki E. Kelley, PhD, and Terry B. Strom, MD • We have used protein engineering and recombinant DNA methodologies to genetically replace the eukaryotic cell receptor binding domain of diphtheria toxin with interleukin 2 (IL-2). The toxin-related T cell growth factor fusion gene has been cloned in Escherichia coli K12. Recombinant strains of E coli produce a 68,086 K hybrid toxin, IL-2 toxin that retains immunologic properties intrinsic to both its diphtheria toxin and IL-2 components. IL-2 toxin has been found to selectively inhibit protein synthesis in both human and murine T cell lines that bear high affinity IL-2 receptors, whereas the hybrid toxin is not active against cells that do not bear this receptor. The cytotoxic action of IL-2 toxin is specifically blocked by free IL-2 and monoclonal antibodies that bind to the p55 (Tac antigen) subunit of the high affinity IL-2 receptor. In addition, IL-2 toxin, like diphtheria toxin itself, must pass through an acidic compartment in order to deliver its adenosine diphosphate ribosyl transferase activity to the cytosol of target T cells. In a murine delayed type hypersensitivity (DTH) model system, we have shown that IL-2 toxin treament induces a marked immunosuppression. © 1988 by the Nat/onal Kidney Foundation, Inc. INDEX WORDS: Diphtheria toxin; immunomodulation; chimeric toxin; Interleukin-2; interleukin-2 receptor; protein engineering; recombinant DNA.
T
HERE IS LITTLE doubt that the therapeutic use of some, but not all, monoclonal pan-T cell antibodies in transplantation have prolonged engraftment. Nonetheless , the broad spectrum of reactivity of these monoclonal antibodies with T cells may not be ideal. Since a low percentage (eg, < 5 %) of the total T cell population is involved in an acute rejection episode, several investigators have examined the therapeutic effect of monoclonal antibodies directed against the interleukin 2 (lL-2) receptor.! This receptor is found primarily on the surface of activated proliferating T cells . Such an approach would sharpen the focus of therapy and direct it toward those lymphocytes that are committed to the unwanted immune reaction. The expression ofIL-2 and the appearance of the IL-2 receptor marks a critical and pivotal event in the development of the immune response. 2,3 The interaction of IL-2 with IL-2 receptor is a prerequisite for clonal expansion and continued viability of most, if not all, activated T cells. 4 -7 Because of the critical role that the IL-2 receptor plays in mediating signals that are required for proliferation, it has been of considerable interest to examine the therapeutic effectiveness of monoclonal antibodies directed against this receptor. In the case of both monoclonal anti-IL-2 receptor antibodies that recognize the IL-2 receptor on the surface of murine T cells (M7/20) and rat T cells (ART 18), a single course of therapy was found to greatly extend allograft survival. 8\0 In addition, tolerance was often noted . These studies
clearly demonstrated that the ability to target only activated proliferating T cell populations may have clinical relevance in the human instance. Grantstein et aP! and Kelley et aP2 have further shown that anti-IL-2 receptor monoclonal antibodies are immunosuppressive. While it has generally been assumed that anti-T cell antibodies mediate immunosuppression by cell killing , it is possible that other mechanisms are involved. It has been shown that low doses of M7/20, which competitively blocks IL-2 binding to its receptor, inhibit a delayed type hypersensitivity (OTH) response in BALB/c mice; whereas, another monoclonal antibody that does not block IL-2 binding has little immunosuppressive effect. 13 Furthermore, Kelley et aP3 have shown that a functional complement sysFrom the Evans Department of Clinical Research and Department of Medicine, The University Hospital, Boston University Medical Center; the Department of Medicine, Brigham and Women's Hospital , and Charles A. Dana Research Institute, Harvard Thorndike Laboratory of the Beth Israel Hospital, Harvard Medical School , BoslOn. Supported in part by Public Health Service Grants Nos. AI21628 (JRM), and AI-22882 (TBS) from the National Institute of Allergy and Infectious Diseases, and No . CA-41746 (JRM) from the National Cancer Institute, and a grant from Seragen, Inc, Hopkinton, MA. Consensus Conference on Monoclonal Antibodies in Transplantation sponsored by the National Kidney Foundation, Scollsdale, AZ, June 26-27, 1987. Address reprint requests 10 John R. Murphy, PhD, Evans Department of Clinical Research. The University Hospital , Boston University Medical Center, BoslOn, MA 02118. © 1988 by the National Kidney Foundation, Inc. 0272-6386/88/1102-0020$3.00/0
American Journal of Kidney Diseases, Vol XI, No 2 (February), 1988: pp 159-162
159
160
tem is required for M7/20 treatment to be effective in the DTH model. In this instance, therefore, both blockade of the IL-2 receptor and the presence of C5 were required for immunosuppression. These results clearly suggest that the development of potent cytotoxic agents that are directed against the IL-2 receptor may have therapeutic utility. A large number of mouse antihuman lymphocyte monoclonal antibodies have been tested for their ability to lyse human lymphocytes via activation of human complement. Without exception these antibodies fix human complement poorly (unpublished observations). It is not surprising therefore, that mouse anti-human IL-2 receptor monoclonal antibodies prolong engraftment of monkeyl4 and human kidneyl5 grafts, but never induce tolerance. In order to develop more potent IL-2 receptor directed biologicals, we have recently turned to protein engineering and recombinant DNA methodologies in order to genetically construct a fusion gene that is composed of portions of diphtheria toxin linked through peptide bond to the T cell specific growth factor IL-2.16 This approach to the development cell receptor specific target chimeric toxins was based on the observation that amelanocyte stimulating hormone (a-MSH) could be used to replace the diphtheria toxin receptor binding domain, and thus form a new toxin that was directed toward the a-MSH receptor on human malignant melanoma cells. I? The potency of these fusion proteins is based on the well known action of diphtheria toxin; whereas, the target cell specificity is dependent on the ligand portion of the chimera. The intoxication of sensitive eukaryotic cells by diphtheria toxin has been shown to be dependent on an order series of interactions between structural/functional domains of the toxin molecule and the cell. This interaction leads to the delivery of diphtheria toxin fragment A to cytosol, and the subsequent inhibition of protein synthesis. The steps involved in the intoxication process are at least: (a) the binding of toxin to its receptor on the cell surface l8 ; (b) internalization of toxin by receptor mediated endocytosisl 9 ; (c) and on acidification of the endosome,20 a partial unfolding of fragment B that allows for the lipid associating domains to interact with the endocytic vesicle membrane,21.22 thereby facilitating the translocation of fragment A into the cytosol. Once delivered to the cytosol, fragment A catalyzes the adenosine
MURPHY, KELLEY, AND STROM
diphosphoribosylation of elongation factor 2. This reaction results in the inhibition of cellular protein synthesis and death of the cell. It is remarkable that a single molecule of diphtheria toxin fragment A delivered to the cytosol can be lethal for the cell. 23 Since the target cell sensitivity for diphtheria toxin is dependent on the presence of the diphtheria toxin receptor, we reasoned that the substitution of the toxin's receptor binding domain with polypeptide hormones, or cell specific growth factors should result in the formation of new toxins with defined target cell specificity., We have now attempted to target the IL-2 receptor with a chimeric toxin. It is important to note that monoclonal antibodies may not provide the best vehicle for targeting IL-2 receptor positive cells. For example, it has been shown that anti-Tac antibody, as well as all other tested monoclonal antibodies that bind to the p55 subunit of the IL-2 receptor are not endocytosed. Since internalization is a required step in the intoxication process, immunotoxins assembled with these monoclonal antibodies are unlikely to efficiently deliver a fragment of microbial or plant toxin to the cytosol of target cells. In marked contrast, however, IL-2 undergoes obligatory endocytosis following binding to the high affinity IL-2 receptor. As a result one would anticipate that a chimeric toxin in which IL-2 serves as the ligand should be internalized and deliver the toxophore to the target cell. Moreover, since only high affinity IL-2 receptors mediate internalization, an IL-2 directed toxin is likely to be a more selectively targeted therapeutic probe than an antiIL-2 receptor antibody. The affinity of IL-2 receptor antibodies are inferior to that of IL-2 itself; therefore, we have chosen to pursue the genetic construction of an IL-2toxin. 16 We have used protein engineering and recombinant DNA methodologies for the following reasons: chemical linkage between a toxophore and a ligand requires activation of E-amino groups with heterobifunctional reagents. As a result, the subsequent crosslinking step to form the conjugate toxin yields a family of isomers rather than a single defined molecular species. Furthermore, such a mixture is likely to contain a mixture of biologically active, as well as inactive and inhibitory molecules. In addition, the stability of the disulfide bond in an in vivo setting is uncertain. The amino acid sequence of IL-2-toxin is the
161
IL-2-TOXIN
product of a gene fusion between a portion of the structural gene for diphtheria toxin in which the receptor binding domain has been replaced with IL-2 sequences. In mature form IL-2-toxin is a 617 amino acid protein with a deduced molecular weight of 68,086 k. The chimeric IL-2-toxin has been found to possess antigenic determinants associated with both its diphtheria toxin-related and IL2 components. 16 IL-2-toxin dramatically inhibits protein synthesis in HTLV-I infected transformed human T cell lines that express high affinity IL-2 receptors. 24 In this instance the IC so for IL-2-toxin is in the range of 10 to 50 pM. The chimeric toxin has also been found to inhibit protein synthesis in lecthin activated murine and human T cell blasts (J. Williams, personal communcation). In contrast, IL-2-toxin fails to inhibit protein synthesis in a large variety of malignant and normal cell types, including cells that bear either the p55 or p75 subunits of the multimeric high affinity IL-2 receptor. The mechanism of IL-2-toxin action involves the classic diphtheria toxin A fragment mediated ADP-ribosylation of target cell elongation factor 2. The IL-2-toxin mediated inhibition of protein synthesis can be selectively blocked by either a molar excess of free IL-2, or an anti-IL-2 receptor monoclonal antibody that has been shown to block IL-2 from binding to its receptor. Cytotoxicity, however, is not affected by agents that bind to other cell surface receptors (eg, transferrin), or bind to other activation antigens (eg, 4F2). Since low concentrations of chloroquine also blocks the
cytotoxicity of IL-2-toxin, we conclude that this chimeric toxin must pass through an acidic compartment in order to deliver its fragment A to the cytosol of target high affinity IL-2 receptor positive T cells. 24 We have recently tested IL-2-toxin in the murine DTH model. In all instances we have found that treatment with this chimeric toxin results in a dramatic antigen specific immunosuppression. Moreover, characterization of lymphocyte populations in treated and control groups strongly suggests that the only cell type that was affected is that of the activated and proliferating high affinity IL-2 receptor positive T cell (unpublished obervations). While the sensitivity ofIL-2 receptor positive cells to IL-2-toxin is within the range of an IC so of 10 to 50 pM,24 it is remarkable to note that the LDso for the whole animal is approximately 3 mg/k (P. Bacha, personal communication). This broad range of concentration between targeted cytotoxicity and nonspecific toxicity offers a potentially large therapeutic window for the further development of IL-2 toxin for human therapeutic trial. Monoclonal antibodies, immunotoxins, or exotic IL-2-toxins afford several potential means to selectively target the small proportion of recipient lymphocytes that are activated by the donor graft. While the potency of IL-2-toxin in clinical practice is unproven, therapeutic targeting of high affinity IL-2 receptor positive activated lymphocytes affords the most selective means of immunotherapy available for testing.
REFERENCES 1. Shapiro ME, Kirkman RL, Murphy JR, et al: The IL-2 receptor as a target for immunosuppressive therapy. Transplant Proc (in press) 2. Leonard WI , Depper 1M, Uchiyama T, et al: A monoclonal antibody that appears to recognize the receptor for human T-cell growth factor; partial characterization of the receptor. Nature (London) 300:267-268, 1982 3. Cantrell PA, Smith KA : The interleukin 2 T-cell system: A new cell growth model. Science 224:1312-1315, 1984 4. Schreier MH, Iscover NN, Tess R, et al: Clones of killer and helper T-cells; growth requirements, specificity, and retention of function in long-term culture. Immunol Rev 51:315336, 1980 5. Farrar 11, Benjamin WR, Hilfiker ML, et al: The biochemistry, biology, and role of interleukin 2 in the induction of cytotoxic T cell and antibody forming B cell responses . Immunol Rev 63:129-166,1982 6. Leonard WI , Depper 1M, Robb RJ , et al: Characterization of the human receptor for T-cell growth factor. Proc Natl Acad Sci USA 80:6957-6962, 1983
7 . Malek TB, Robb RJ, Shevach EM: Identification and initial characterization of a rat monoclonal antibody reactive with the murine interleukin 2 receptor-ligand complex. Proc Natl Acad Sci USA 80:5694-5698, 1983 8. Kirkman RL, Barrett LV, Gaulton GN, et al : Administration of an anti-interleukin 2 receptor monoclonal antibody prolongs allograft survival in mice. 1 Exp Med 162:358-365 , 1985 9. Kirkman RL, Barrett LV, Gaulton GN, et al: The effect of anti-interleukin 2 receptor monoclonal antibody on allograft rejection. Transplantation 40:719-728 , 1985 10. Kupiec-Weglinski lW, Diamantstein T, Tilney N, et al: Anti-interleukin 2 receptor monoclonal antibody spares T suppressor cells and prevents and reverses acute allograft rejection . Proc Natl Acad Sci USA 83 :2624-2627, 1986 II. Granstein RD, Goulston C, Gaulton GN: Prolongation of murine skin allograft survival by immunologic manipulation with anti-interleukin 2 receptor antibody. 1 Immunol 136:898906, 1986 12 . Kelley VE, Naor D, Tarcic N, et al: Anti-interleukin 2 receptor antibody suppresses delayed type hypersensitivity to
162 foreign and syngenic antigens. J Immunol 137:2122-2127, 1986 13. Kelley VE, Gaulton GN, Strom TB: Inhibition effects of anti-interleukin 2 receptor and anti-L3T4 antibodies on delayed type hypersensitivity: The role of complement and epitope. J Immunol 138:2771-2775 14. Shapiro ME, Kirkman RL , Read MH, et al: Monoclonal anti-interleukin 2 receptor antibody in primate renal transplantation. Transplant Proc 19:594-598, 1987 15. Soulillou JP, Peyronet P, Lamauff B, et al: Prevention of rejection of kidney transplants by monoclonal antibodies directed against interleukin 2 receptors. Lancet I: 1339-1342 , 1987 16. Williams DP, Parker K, Bacha P, et al: Diphtheria toxin receptor binding domain substitution with interleukin 2: Genetic construction and properties of a diphtheria toxin-related interleukin 2 fusion protein. Protein Eng (in press) 17. Murphy JR, Bishai W, Borowski M, et al: Genetic construction , expression, and melanoma-selective cytotoxicity of a diphtheria toxin-related a-melanocyte stimulating hormone fusion protein. Proc Nat! Acad Sci USA 83 :8258-8262, 1986 18. Middlebrook JL , Dorland RB , Leppla SH: Association
MURPHY, KELLEY, AND STROM of diphtheria toxin with Vero cells . J Bioi Chern 253:73257330, 1978 19. Moya MA, Dautry-Versat A, Goud B, et al: Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin. JCell Bioi 191 :548553 , 1985 20. Sandvig K, Tonnessen TI, Sand 0, et al : Requirement of a transmembrane pH gradient for the entry of diphtheria toxin into cells at low pH. J Bioi Chern 261 : 11639-11645, 1986 21. Eisenberg D, Schwartz E, Komaromy M, et al: Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Bioi 179: 125-142, 1984 22. Lambotte P, Falmagne P, Capiau C, et al: Primary structure of diphtheria toxin fragment B: Structural similarities with lipid-binding domains. J Cell Bioi 87:837-840, 1980 23. Yamaizumi M, Mekada M, Uchida T, et al: One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15:245-250, 1978 24. Bacha P, Williams J, Waters C, et al: Interleukin-2 receptor targeted cytotoxicity: Interleukin-2 receptor mediated action of a diphtheria toxin-related interleukin-2 fusion protein . J Exp Med (in press)
T
HERE IS LITTLE doubt that the therapeutic use of some, but not all, monoclonal pan-T cell antibodies in transplantation have prolonged engraftment. Nonetheless , the broad spectrum of reactivity of these monoclonal antibodies with T cells may not be ideal. Since a low percentage (eg, < 5 %) of the total T cell population is involved in an acute rejection episode, several investigators have examined the therapeutic effect of monoclonal antibodies directed against the interleukin 2 (lL-2) receptor.! This receptor is found primarily on the surface of activated proliferating T cells . Such an approach would sharpen the focus of therapy and direct it toward those lymphocytes that are committed to the unwanted immune reaction. The expression ofIL-2 and the appearance of the IL-2 receptor marks a critical and pivotal event in the development of the immune response. 2,3 The interaction of IL-2 with IL-2 receptor is a prerequisite for clonal expansion and continued viability of most, if not all, activated T cells. 4 -7 Because of the critical role that the IL-2 receptor plays in mediating signals that are required for proliferation, it has been of considerable interest to examine the therapeutic effectiveness of monoclonal antibodies directed against this receptor. In the case of both monoclonal anti-IL-2 receptor antibodies that recognize the IL-2 receptor on the surface of murine T cells (M7/20) and rat T cells (ART 18), a single course of therapy was found to greatly extend allograft survival. 8\0 In addition, tolerance was often noted . These studies
clearly demonstrated that the ability to target only activated proliferating T cell populations may have clinical relevance in the human instance. Grantstein et aP! and Kelley et aP2 have further shown that anti-IL-2 receptor monoclonal antibodies are immunosuppressive. While it has generally been assumed that anti-T cell antibodies mediate immunosuppression by cell killing , it is possible that other mechanisms are involved. It has been shown that low doses of M7/20, which competitively blocks IL-2 binding to its receptor, inhibit a delayed type hypersensitivity (OTH) response in BALB/c mice; whereas, another monoclonal antibody that does not block IL-2 binding has little immunosuppressive effect. 13 Furthermore, Kelley et aP3 have shown that a functional complement sysFrom the Evans Department of Clinical Research and Department of Medicine, The University Hospital, Boston University Medical Center; the Department of Medicine, Brigham and Women's Hospital , and Charles A. Dana Research Institute, Harvard Thorndike Laboratory of the Beth Israel Hospital, Harvard Medical School , BoslOn. Supported in part by Public Health Service Grants Nos. AI21628 (JRM), and AI-22882 (TBS) from the National Institute of Allergy and Infectious Diseases, and No . CA-41746 (JRM) from the National Cancer Institute, and a grant from Seragen, Inc, Hopkinton, MA. Consensus Conference on Monoclonal Antibodies in Transplantation sponsored by the National Kidney Foundation, Scollsdale, AZ, June 26-27, 1987. Address reprint requests 10 John R. Murphy, PhD, Evans Department of Clinical Research. The University Hospital , Boston University Medical Center, BoslOn, MA 02118. © 1988 by the National Kidney Foundation, Inc. 0272-6386/88/1102-0020$3.00/0
American Journal of Kidney Diseases, Vol XI, No 2 (February), 1988: pp 159-162
159
160
tem is required for M7/20 treatment to be effective in the DTH model. In this instance, therefore, both blockade of the IL-2 receptor and the presence of C5 were required for immunosuppression. These results clearly suggest that the development of potent cytotoxic agents that are directed against the IL-2 receptor may have therapeutic utility. A large number of mouse antihuman lymphocyte monoclonal antibodies have been tested for their ability to lyse human lymphocytes via activation of human complement. Without exception these antibodies fix human complement poorly (unpublished observations). It is not surprising therefore, that mouse anti-human IL-2 receptor monoclonal antibodies prolong engraftment of monkeyl4 and human kidneyl5 grafts, but never induce tolerance. In order to develop more potent IL-2 receptor directed biologicals, we have recently turned to protein engineering and recombinant DNA methodologies in order to genetically construct a fusion gene that is composed of portions of diphtheria toxin linked through peptide bond to the T cell specific growth factor IL-2.16 This approach to the development cell receptor specific target chimeric toxins was based on the observation that amelanocyte stimulating hormone (a-MSH) could be used to replace the diphtheria toxin receptor binding domain, and thus form a new toxin that was directed toward the a-MSH receptor on human malignant melanoma cells. I? The potency of these fusion proteins is based on the well known action of diphtheria toxin; whereas, the target cell specificity is dependent on the ligand portion of the chimera. The intoxication of sensitive eukaryotic cells by diphtheria toxin has been shown to be dependent on an order series of interactions between structural/functional domains of the toxin molecule and the cell. This interaction leads to the delivery of diphtheria toxin fragment A to cytosol, and the subsequent inhibition of protein synthesis. The steps involved in the intoxication process are at least: (a) the binding of toxin to its receptor on the cell surface l8 ; (b) internalization of toxin by receptor mediated endocytosisl 9 ; (c) and on acidification of the endosome,20 a partial unfolding of fragment B that allows for the lipid associating domains to interact with the endocytic vesicle membrane,21.22 thereby facilitating the translocation of fragment A into the cytosol. Once delivered to the cytosol, fragment A catalyzes the adenosine
MURPHY, KELLEY, AND STROM
diphosphoribosylation of elongation factor 2. This reaction results in the inhibition of cellular protein synthesis and death of the cell. It is remarkable that a single molecule of diphtheria toxin fragment A delivered to the cytosol can be lethal for the cell. 23 Since the target cell sensitivity for diphtheria toxin is dependent on the presence of the diphtheria toxin receptor, we reasoned that the substitution of the toxin's receptor binding domain with polypeptide hormones, or cell specific growth factors should result in the formation of new toxins with defined target cell specificity., We have now attempted to target the IL-2 receptor with a chimeric toxin. It is important to note that monoclonal antibodies may not provide the best vehicle for targeting IL-2 receptor positive cells. For example, it has been shown that anti-Tac antibody, as well as all other tested monoclonal antibodies that bind to the p55 subunit of the IL-2 receptor are not endocytosed. Since internalization is a required step in the intoxication process, immunotoxins assembled with these monoclonal antibodies are unlikely to efficiently deliver a fragment of microbial or plant toxin to the cytosol of target cells. In marked contrast, however, IL-2 undergoes obligatory endocytosis following binding to the high affinity IL-2 receptor. As a result one would anticipate that a chimeric toxin in which IL-2 serves as the ligand should be internalized and deliver the toxophore to the target cell. Moreover, since only high affinity IL-2 receptors mediate internalization, an IL-2 directed toxin is likely to be a more selectively targeted therapeutic probe than an antiIL-2 receptor antibody. The affinity of IL-2 receptor antibodies are inferior to that of IL-2 itself; therefore, we have chosen to pursue the genetic construction of an IL-2toxin. 16 We have used protein engineering and recombinant DNA methodologies for the following reasons: chemical linkage between a toxophore and a ligand requires activation of E-amino groups with heterobifunctional reagents. As a result, the subsequent crosslinking step to form the conjugate toxin yields a family of isomers rather than a single defined molecular species. Furthermore, such a mixture is likely to contain a mixture of biologically active, as well as inactive and inhibitory molecules. In addition, the stability of the disulfide bond in an in vivo setting is uncertain. The amino acid sequence of IL-2-toxin is the
161
IL-2-TOXIN
product of a gene fusion between a portion of the structural gene for diphtheria toxin in which the receptor binding domain has been replaced with IL-2 sequences. In mature form IL-2-toxin is a 617 amino acid protein with a deduced molecular weight of 68,086 k. The chimeric IL-2-toxin has been found to possess antigenic determinants associated with both its diphtheria toxin-related and IL2 components. 16 IL-2-toxin dramatically inhibits protein synthesis in HTLV-I infected transformed human T cell lines that express high affinity IL-2 receptors. 24 In this instance the IC so for IL-2-toxin is in the range of 10 to 50 pM. The chimeric toxin has also been found to inhibit protein synthesis in lecthin activated murine and human T cell blasts (J. Williams, personal communcation). In contrast, IL-2-toxin fails to inhibit protein synthesis in a large variety of malignant and normal cell types, including cells that bear either the p55 or p75 subunits of the multimeric high affinity IL-2 receptor. The mechanism of IL-2-toxin action involves the classic diphtheria toxin A fragment mediated ADP-ribosylation of target cell elongation factor 2. The IL-2-toxin mediated inhibition of protein synthesis can be selectively blocked by either a molar excess of free IL-2, or an anti-IL-2 receptor monoclonal antibody that has been shown to block IL-2 from binding to its receptor. Cytotoxicity, however, is not affected by agents that bind to other cell surface receptors (eg, transferrin), or bind to other activation antigens (eg, 4F2). Since low concentrations of chloroquine also blocks the
cytotoxicity of IL-2-toxin, we conclude that this chimeric toxin must pass through an acidic compartment in order to deliver its fragment A to the cytosol of target high affinity IL-2 receptor positive T cells. 24 We have recently tested IL-2-toxin in the murine DTH model. In all instances we have found that treatment with this chimeric toxin results in a dramatic antigen specific immunosuppression. Moreover, characterization of lymphocyte populations in treated and control groups strongly suggests that the only cell type that was affected is that of the activated and proliferating high affinity IL-2 receptor positive T cell (unpublished obervations). While the sensitivity ofIL-2 receptor positive cells to IL-2-toxin is within the range of an IC so of 10 to 50 pM,24 it is remarkable to note that the LDso for the whole animal is approximately 3 mg/k (P. Bacha, personal communication). This broad range of concentration between targeted cytotoxicity and nonspecific toxicity offers a potentially large therapeutic window for the further development of IL-2 toxin for human therapeutic trial. Monoclonal antibodies, immunotoxins, or exotic IL-2-toxins afford several potential means to selectively target the small proportion of recipient lymphocytes that are activated by the donor graft. While the potency of IL-2-toxin in clinical practice is unproven, therapeutic targeting of high affinity IL-2 receptor positive activated lymphocytes affords the most selective means of immunotherapy available for testing.
REFERENCES 1. Shapiro ME, Kirkman RL, Murphy JR, et al: The IL-2 receptor as a target for immunosuppressive therapy. Transplant Proc (in press) 2. Leonard WI , Depper 1M, Uchiyama T, et al: A monoclonal antibody that appears to recognize the receptor for human T-cell growth factor; partial characterization of the receptor. Nature (London) 300:267-268, 1982 3. Cantrell PA, Smith KA : The interleukin 2 T-cell system: A new cell growth model. Science 224:1312-1315, 1984 4. Schreier MH, Iscover NN, Tess R, et al: Clones of killer and helper T-cells; growth requirements, specificity, and retention of function in long-term culture. Immunol Rev 51:315336, 1980 5. Farrar 11, Benjamin WR, Hilfiker ML, et al: The biochemistry, biology, and role of interleukin 2 in the induction of cytotoxic T cell and antibody forming B cell responses . Immunol Rev 63:129-166,1982 6. Leonard WI , Depper 1M, Robb RJ , et al: Characterization of the human receptor for T-cell growth factor. Proc Natl Acad Sci USA 80:6957-6962, 1983
7 . Malek TB, Robb RJ, Shevach EM: Identification and initial characterization of a rat monoclonal antibody reactive with the murine interleukin 2 receptor-ligand complex. Proc Natl Acad Sci USA 80:5694-5698, 1983 8. Kirkman RL, Barrett LV, Gaulton GN, et al : Administration of an anti-interleukin 2 receptor monoclonal antibody prolongs allograft survival in mice. 1 Exp Med 162:358-365 , 1985 9. Kirkman RL, Barrett LV, Gaulton GN, et al: The effect of anti-interleukin 2 receptor monoclonal antibody on allograft rejection. Transplantation 40:719-728 , 1985 10. Kupiec-Weglinski lW, Diamantstein T, Tilney N, et al: Anti-interleukin 2 receptor monoclonal antibody spares T suppressor cells and prevents and reverses acute allograft rejection . Proc Natl Acad Sci USA 83 :2624-2627, 1986 II. Granstein RD, Goulston C, Gaulton GN: Prolongation of murine skin allograft survival by immunologic manipulation with anti-interleukin 2 receptor antibody. 1 Immunol 136:898906, 1986 12 . Kelley VE, Naor D, Tarcic N, et al: Anti-interleukin 2 receptor antibody suppresses delayed type hypersensitivity to
162 foreign and syngenic antigens. J Immunol 137:2122-2127, 1986 13. Kelley VE, Gaulton GN, Strom TB: Inhibition effects of anti-interleukin 2 receptor and anti-L3T4 antibodies on delayed type hypersensitivity: The role of complement and epitope. J Immunol 138:2771-2775 14. Shapiro ME, Kirkman RL , Read MH, et al: Monoclonal anti-interleukin 2 receptor antibody in primate renal transplantation. Transplant Proc 19:594-598, 1987 15. Soulillou JP, Peyronet P, Lamauff B, et al: Prevention of rejection of kidney transplants by monoclonal antibodies directed against interleukin 2 receptors. Lancet I: 1339-1342 , 1987 16. Williams DP, Parker K, Bacha P, et al: Diphtheria toxin receptor binding domain substitution with interleukin 2: Genetic construction and properties of a diphtheria toxin-related interleukin 2 fusion protein. Protein Eng (in press) 17. Murphy JR, Bishai W, Borowski M, et al: Genetic construction , expression, and melanoma-selective cytotoxicity of a diphtheria toxin-related a-melanocyte stimulating hormone fusion protein. Proc Nat! Acad Sci USA 83 :8258-8262, 1986 18. Middlebrook JL , Dorland RB , Leppla SH: Association
MURPHY, KELLEY, AND STROM of diphtheria toxin with Vero cells . J Bioi Chern 253:73257330, 1978 19. Moya MA, Dautry-Versat A, Goud B, et al: Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin. JCell Bioi 191 :548553 , 1985 20. Sandvig K, Tonnessen TI, Sand 0, et al : Requirement of a transmembrane pH gradient for the entry of diphtheria toxin into cells at low pH. J Bioi Chern 261 : 11639-11645, 1986 21. Eisenberg D, Schwartz E, Komaromy M, et al: Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Bioi 179: 125-142, 1984 22. Lambotte P, Falmagne P, Capiau C, et al: Primary structure of diphtheria toxin fragment B: Structural similarities with lipid-binding domains. J Cell Bioi 87:837-840, 1980 23. Yamaizumi M, Mekada M, Uchida T, et al: One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15:245-250, 1978 24. Bacha P, Williams J, Waters C, et al: Interleukin-2 receptor targeted cytotoxicity: Interleukin-2 receptor mediated action of a diphtheria toxin-related interleukin-2 fusion protein . J Exp Med (in press)