Journal of Clinical Immunology, Vol. 10, No. 6 (November Supplement 1990)
Immunotoxins: New Therapeutic Reagents for Autoimmunity, Cancer, and AIDS E L L E N S. V I T E T T A 1
cases, this ligand is a cell-reactive antibody, hormone, or growth factor (1-4). There are very few monoclonal antibodies (mAbs) that are lineage specific and even fewer that are tumor specific. This problem has been circumvented to some degree by (i) using mAbs that either eliminate an entire lineage of cells that can be readily replaced by precursor cells [e.g., hematopoietic tissue (5)], (ii) using ITs directed against target cells (e.g., lymphoid tumors) expressing higher than normal densities of the antigen in question (6), or (iii) utilizing targeting vehicles which bind only to an infected cell, e.g., targeting to viral proteins on the surface of such cells (7, 8). In selecting a ligand it is crucial to screen a large panel of normal tissues since unexpected cross-reactions are frequently encountered (9). In selecting a ligand with which to prepare an IT, it is crucial that it bind to a determinant on the cell which is endocytosed into a compartment in the cell that is favorable for A-chain translocation into the cytosol [where it can exert its cytotoxic effect (1-4)]. In general, those ITs that are routed preferentially to Golgi-endosomes are more potent than those routed to lysosomes. The latter pathway results in rapid degradation and decreases the probability that an intact A chain will translocate into the cytosol (10-12). When an mAb is used as the ligand, it plays a major role in both the potency and the specificity of the IT. Hence, it was important to develop an assay which can predict the ability of an mAb to make an effective IT. This was accomplished by the development of an "'indirect I T " screening assay to evaluate panels of mAbs directed against specific markers on different types of cells (13, 14). The effectiveness of the antibody in this indirect assay accurately predicts its effectiveness as a direct IT, i.e., w h e n it is purified and coupled
Immunotoxins consist of cell-reactive ligands coupled to toxins or their toxic subunits. The ligands are usually antibodies, hormones, or growth factors and the toxins are of bacterial or plant origin. In vitro studies using A chain-containing immunotoxins specifically to kill tumor cells were successful and led to further experiments in vivo. Such studies, carried out over the past 5 years in both animals and humans, have demonstrated that the efficacy of immunotoxins in vivo is often poor, due to problems involving instability of the conjugate, inferior potency, inaccessibility of tumor cells, nonspecific binding to ceils other than the target cells, and survival of antigen-negative mutants. In addition, immune responses against both the ligand and the A chain are usually elicited, precluding repeated therapy. During the past several years, there have been attempts to solve these problems and develop more effective immunotoxins. KEY WORDS: Immunotoxin (IT); m o n o c l o n a l antibodies (mAbs); ricin A chain (A chain).
INTRODUCTION An immunotoxin (IT) consists of cell-binding ligands coupled to toxins or their toxic subunits. Such reagents are designed to specifically kill neoplastic cells, viralty infected cells or subsets of normal cells. In this brief review, I focus on the development of ITs containing ricin A chain, emphasizing problems in their in vivo use and modifications of these reagents that have solved some of these problems and led to the availability of secondgeneration reagents. LIGANDS The specificity of an IT is determined primarily by the ligand used as the targeting vehicle. In most 1Department of Microbiology and Cancer Immunobiology Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
15S 0271-9142/90/1100-015S$06.00/0 © 1990 PlenumPublishingCorporation
t6S
VITETTA
directly to the A chain. This assay has made it possible to screen many mAbs and to select those that will make the most potent ITs. Similar assays could be readily developed for other types of ligands. Another approach to selecting a ligand is to use one directed against an antigen expressed at abnormally high levels on target cells and lower levels on normal cells (6), e.g., growth factor receptors or products of viruses or oncogenes. Such ligands coupled to A chain are less likely to kill normal cells expressing lower levels of these target molecules. One problem remaining with nonautologous ligands, such as murine mAbs, is the generation of an immune response that precludes repeated use. Strategies to avoid this problem include the use of immunosuppressive agents during IT therapy (15, 16) and the "humanizing" of murine mAbs (17, 18). RICIN A CHAIN
Ricin is a plant toxin made up of two subunits. The A chain is a ribosome-inactivating protein of 32 kD linked by a disulfide bond to a galactose-speclfic lectin (B chain) of approximately 30 kD (19). The A and B chains of ricin can be separated following reduction and the A chain can then be purified (20) and chemically linked to different ligands to generate a cell-reactive conjugate (1-4). Since native ricin A chain contains a large amount of mannose (21) and both parenchymat and nonparenchymal cells of the liver have high-aflSnity mannose receptors (reviewed in Ref. 1), ITs containing native ricin A chain home to the liver and are frequently hepatotoxic. In addition, the diversion to the liver decreases the proportion of ITs that reach the target cells. By using recombinant A chain lacking carbohydrate (22, 23) or by chemically "deglycosylating" native A chain (24), it was possible to eliminate this problem and to demonstrate that the IT containing the degtycosylated or recombinant A chain could localize more effectively (25) and destroy the target tissue (26, 27). CONSTRUCTION OF ITs For an IT containing a ricin A chain to be effective, there must be a disulfide bond between the ligand and the A chain (1, 4). This introduces the potential problem of disulfide bond instability in vivo. Disulfide bonds can be split by either enzymes or thiols in the tissues or in the circulation (1, 27).
Hence, there has been a strong impetus to develop more stable cross-linkers that allow ITs to remain intact in the circulation. This problem has been surmounted by developing cross-linkers containing hindered disulfide bonds (27-29) or by the direct use of the cysteine on the antibody (Fab' portion) and the cysteine on an A chain to form a disulfide bond (25). Both IT constructs are more stable in vivo and facilitate effective localization (25) and killing of target cells (25, 28). ITs containing more stable disulfide bonds have proven more potent in animal models (25, 28). CLINICAL EXPERIENCE WITH ITs
Several clinical trials with "first-generation" ITs have been reported (reviewed in Refs. 1 and 2) and other clinical trials involving their improved "second-generation" versions are in progress. The major dose-limiting side effects in humans include allergic reactions, hypoalbuminemia resulting in edema (capillary leak), fatigue, and myalgia (2). The mechanisms underlying capillary leak and myalgia have not been defined. Based on initial clinical trials, it appears that cells that are accessible to the circulation and that internalize the IT effectively, e.g., normal lymphocytes, macrophages, lymphoma cells, etc., are better cell targets for IT therapy than are solid tumors (1, 2). Thus, ITs containing an anti-T-cell antibody coupled to ricin A chain have proven effective in treating steroid-resistant graft-vs-host disease in humans (2) and are under study for the treatment of autoimmune disorders (30). Similar approaches could be used to eliminate B cells in other autoimmune disorders. The problem of killing virally infected cells has been addressed using cells infected with the human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS) (31, 32). In in vitro studies, it has been demonstrated that ricin A chain-containing ITs directed against the gp41 protein of the HIV can effectively kill infected cells (8). In addition, the soluble receptor for the gp120 envelope protein of HIV, i.e., CD4, has been cloned (33--37). Soluble recombinant CD4 (rCD4) has been coupled to A chain and such conjugates kill cells expressing the HIV-encoded envelope protein, gpl20 (7). Both approaches are attractive because the anti-gp41 antibody and the rCD4 molecule should not bind to other cells in the host, and hence, conjugates containing rCD4 or anti-gp41 should be relatively spe-
Journal of Clinical Immunology, Vol. 10, No. 6 (November Supplement 1990)
IMMUNOTOXINS AND AUTOIMMUNITY
cific in their cytotoxicity. In addition, it is possible that the target cells in the very early stages of the disease are confined largely to sites accessible to the circulation and therefore would not present problems. A major limitation of this approach is that HIV can cause a latent infection in both T cells and monocytes. Such cells probably lack HIV envelope proteins on their surfaces and therefore would not be susceptible to ITs.
17S
7.
8.
9.
FUTURE DEVELOPMENTS OF ITs The cloning of the genes for the relevant toxins, mAbs, growth factors, soluble receptors, and other ligands is now making possible the generation of ITs by recombinant DNA technology (38-42). Such genetically engineered ITs can be produced in large quantity. More importantly, structure-function relationships can be elucidated and such ITs can be quickly redesigned to generate more effective reagents. Results of current in vivo experiments using these reagents in animals are encouraging.
10.
11.
12.
13.
ACKNOWLEDGMENTS This work is supported by N I H Grants CA-28149 and CA-41081, Texas Technologies Grant 18512, Welch Foundation Grant 1-947, and Cancer Immunology Training Grant CA-09082. I thank Dr. J. Uhr for helpful comments concerning this review and Ms. G. A. Cheek and N. Stephens for secretarial assistance.
14.
15.
16.
REFERENCES 1. Blakey DC, Wawrzynczak EJ, Wallace PM, Thorpe PE: Antibody toxin conjugates: A perspective. In Progress in Allergy (Monoclonal Antibody Therapy), H. Waldmann (ed). Basel, Switzerland, Karger, 1988, pp 50-90 2. Byers VS, Baldwin RW. Therapeutic strategies with monoclonal antibodies and immunoconjugates. Immunology 65:329-335, 1988 3. Pastan I, Willingham MC, FitzGerald DJ: Immunotoxins. Cell 47:641-648, 1986 4. Vitetta ES, Fulton ILl, May RD, Till M, Uhr JW: Redesigning nature's poisons to create anti-tumor reagents. Science 238:1098-1104, 1987 5. Krolick KA, Uhr JW, Slavin S, Vitetta ES: In vivo therapy of a murine B cell tumor (BCL1) using antibody-ricin A chain immunotoxins. J Exp Med 155:1797-1809, 1982 6. Kr6nke M, Schlick E, Waldmann TA, Vitetta ES, Greene WC: Selective killing of human T-lymphotropic virus-I infected leukemic T-cells by monoclonal anti-interleukin 2 receptor antibody-ricin A chain conjugates: Potentiation by
17.
18.
19.
20.
21.
ammonium chloride and monensin. Cancer Res 46:32953298, 1986 Till M, Ghetie V, Gregory T, Patzer E J, Porter JP, Uhr JW, Capon DJ, Vitetta ES: HIV-infected cells are killed by rCD4-ricin A chain. Science 242:1166-1168, 1988 Till MA, Zolla-Pazner S, Gorny MK, Patton JS, Uhr JW, Vitetta ES: Human immunodeficiency virus-infected T ceils and monocytes are killed by monoclonal human anti-gp41 antibodies coupled to ricin A chain. Proc Natl Acad Sci USA 86:1987-1991, 1989 Li J-L, Shen G-L, Ghetie M-A, May RD, Till M, Ghetie V, Uhr JW, Janossy G, Thorpe PE, Amlot P, Vitetta ES: The epitope specificity and tissue reactivity of four murine monoclonal anti-CD22 antibodies. Cell Immunol 118:85-99, 1989 Lambert JM, Senter PD, Yau-Young A, Bl~ittler 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 260:12035-12041, 1985 Press OW, Vitetta ES, Farr AG, Hansen JA, Martin PJ: Evaluation of ricin A-chain immunotoxins directed against human T cells. Cell Immunol 102:10-20, 1986 Raso V, Basala M: Study of the transferrin receptor using a cytotoxic human transferrin-ricin A conjugate. In ReceptorMediated Targeting of Drugs, G Gregoriadis, G Poste, J Senior, A Trouet (eds). New York, Plenum Press, 1984, pp 73-86 Till M, May RD, Uhr JW, Thorpe PE, Vitetta ES: An assay that predicts the ability of monoclonal antibodies to form potent ricin A chain-containing immunotoxins. Cancer Res 48:1119-1123, 1988 Vitetta ES, Krolick KA, Miyama-Inaba M, Cushley W, Uhr JW: Immunotoxins: A new approach to cancer therapy. Science 219:644-650, 1983 Garnett MC, Baldwin RW: Endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen visualised using fluorescent conjugates. Eur J Cell Biol 41:214-221, 1986 Ledermann JA, Begent RHJ, Riggs SJ, Scarle F, Read D, Bagshawe HD: Suppression of the human anti-mouse antibody response by cyclosporin A (CsA) during therapy with a monoclonal antibody. Proc Am Assoc Cancer Res 29:423, 1988 Jones PT, Dear PH, Foote J, Neuberger MS, Winter G: Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321:522525, 1986 Morrison SL, Johnson MJ, Herzenberg LA, Oi VT: Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci USA 81:6851-6855, 1984 Olsnes S, Pihl A: Toxic lectins and related proteins. In Molecular Action of Toxins and Viruses, P Cohen, S yon Heyningen (eds). New York, Elsevier, 1982, pp 51-105 Fulton RJ, Blakey DC, Knowles PP, Uhr JW, Thorpe PE, Vitetta ES: Purification of ricin A1, A2 and B chains and characterization of their toxicity. J Biol Chem 261:53145319, 1986 Funatsu M: Structure of toxic function of ricin. Subunit structure of ricin D. Proc Jap Acad 47:718, 1971
Journal of Clinical Immunology, Vol. 10, No. 6 (November Supplement 1990)
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22. Lamb FI, Roberts LM, Lord JM: Nucleotide sequence of cloned cDNA coding for preproricin. Eur J Biochem t48:265-270, 1985 23. OHare M, Roberts LM, Thorpe PE, Watson GJ, Prior B, Lord JM: Expression of ricin A chain in Escherichia coti. FEBS Lett 216:73-78, 1987 24. Thorpe PE, Detre SI, FoxweI1 BM, Brown AN, Skilleter DN, Wilson G, Forrester JA, Stirpe F: Modification of the carbohydrate in ricin with metaperiodate-cyanoborohydride mixtures: Effects on toxicity and in vivo distribution. Eur J Biochem 147:197-206, 1985 25. Fulton RJ, Tucker TF, Vitetta ES, Uhr JW: Pharmacokinetics of tumor-reactive immunotoxins in tumor-bearing mice: Effect of antibody valency and deglycosylation of the ricin A chain on clearance and tumor localization. Cancer Res 48:2618-2625, 1988 26. Fulton RJ, Uhr JW, Vitetta ES: In vivo therapy of the BCL1 tumor: Effect of immunotoxin valency and deglycosylation of the ricin A chain. Cancer Res 48:2626-2631, 1988 27. Thorpe PE, Wallace PM, Knowles PP, Retf MG, Brown AN, Watson GJ, Blakey DC, Newell DR: Improved antitumor effects of immunotoxins prepared with deglycosylated ricin A-chain and hindered disulfide linkages. Cancer Res 48:6396-6403, 1988 28. Thorpe PE, Wallace PM, Knowles PP, Relf MG, Brown AN, Watson GJ, Knyba RE, Wawrzynczak EJ, Blakey DC: New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo. Cancer Res 47:5924-5931, 1987 29. Worreli NR, Cumber AJ, Parnell GD, Mirza A, Forrester JA, Ross WC: Effect of linkage variation on pharmacokinetics of ricin A chain-antibody conjugates in normal rats. Anticancer Drug Des 1:179, 1986 30. Byers VS, Caperton E, Ackerman S, Shepard J, Scannon P: Modification of the immune system in patients with rheumatoid arthritis treated with anti-CD5 ricin A chain immunotoxin. FASEB J 3:Al122, 1989 31. Curran JW, Jaffe HW, Hardy AM, Morgan WM, Selik RM, Dondero TJ: Epidemiology of HIV infection and AIDS in the United States. Science 239:610-616, 1988
32. Fauci AS: The human immunodeficiency virus: Infectivity and mechanisms of pathogenesis. Science 239:617-622, 1987 33. Deen KC, McDougal JS, Inacker R, Folena-Wasserman G, Arthos J, Rosenberg J, Maddon PJ, Axel R, Sweet RW: A soluble form of CD4 (T4) protein inhibits AIDS virus infection. Nature 331:82-84, 1988 34. Fisher RA, Bertonis JM, Meier W, Johnson VA, Costopoulos DS, Liu T, Tizard R, Walker BD, Hirsch MS, Schooley RT, et aL: HIV infection is blocked in vitro by recombinant soluble CD4. Nature 331:76-78, 1988 35. Hussey RE, Richardson NE, Kowalski M, Brown NR, Chang HC, Siliciano RF, Dorfman T, Walker B, Sodroski J, Reinherz EL: A soluble CD4 protein selectively inhibits HIV replication and syncytium formation. Nature 331:78-81, 1988 36, Smith DH, Byrn RA, Marsters SA, Gregory T, Groopman JE, Capon DJ: Blocking of HIV-1 infectivity by a soluble, secreted form of the CD4 antigen. Science 238:1704-1707, 1987 37. Traunecker A, Luke W, Karjalainen K: Soluble CD4 molecules neutralize human immunodeficiency virus type 1. Nature 331:84-86, 1988 38. Chang MS, Russell DW, Uhr JW, Vitetta ES: Cloning and expression of recombinant, functional ricin B chain. Proc Natl Acad Sci USA 84:5640-5644, 1987 39. Chaudhary VK, FitzGerald DJ, Adyha S, Pastan I: Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudornonas toxin. Proc Natl Acad Sci USA 84:4538-4542, 1987 40. Colombatti M, Greenfield L, Youle RJ: Cloned fragment of diphtheria toxin linked to T cell-specific antibody identifies regions of B chain active in cell entry. J Biol Chem 261:30303035, 1986 41. Murphy JR, Bishai W, Borowski M, Miyanohara A, Boyd J, Nagle S: Genetic construction, expression and melanomaselective cytotoxicity of a diphtheria toxin-related alphamelanocyte-stimulating hormone fusion protein. Proc Natl Acad Sci USA 83:8258-8262, 1986 42. Williams D, Bacha P, Parker K, Strom TB, Murphy JR: Diphtheria toxin receptor-binding domain substitution with interteukin-2. Protein Eng 1:239, 1987
Journal of Clinical Immunology, VoL 10, No. 6 (November Supplement 1990)
Immunotoxins: New Therapeutic Reagents for Autoimmunity, Cancer, and AIDS E L L E N S. V I T E T T A 1
cases, this ligand is a cell-reactive antibody, hormone, or growth factor (1-4). There are very few monoclonal antibodies (mAbs) that are lineage specific and even fewer that are tumor specific. This problem has been circumvented to some degree by (i) using mAbs that either eliminate an entire lineage of cells that can be readily replaced by precursor cells [e.g., hematopoietic tissue (5)], (ii) using ITs directed against target cells (e.g., lymphoid tumors) expressing higher than normal densities of the antigen in question (6), or (iii) utilizing targeting vehicles which bind only to an infected cell, e.g., targeting to viral proteins on the surface of such cells (7, 8). In selecting a ligand it is crucial to screen a large panel of normal tissues since unexpected cross-reactions are frequently encountered (9). In selecting a ligand with which to prepare an IT, it is crucial that it bind to a determinant on the cell which is endocytosed into a compartment in the cell that is favorable for A-chain translocation into the cytosol [where it can exert its cytotoxic effect (1-4)]. In general, those ITs that are routed preferentially to Golgi-endosomes are more potent than those routed to lysosomes. The latter pathway results in rapid degradation and decreases the probability that an intact A chain will translocate into the cytosol (10-12). When an mAb is used as the ligand, it plays a major role in both the potency and the specificity of the IT. Hence, it was important to develop an assay which can predict the ability of an mAb to make an effective IT. This was accomplished by the development of an "'indirect I T " screening assay to evaluate panels of mAbs directed against specific markers on different types of cells (13, 14). The effectiveness of the antibody in this indirect assay accurately predicts its effectiveness as a direct IT, i.e., w h e n it is purified and coupled
Immunotoxins consist of cell-reactive ligands coupled to toxins or their toxic subunits. The ligands are usually antibodies, hormones, or growth factors and the toxins are of bacterial or plant origin. In vitro studies using A chain-containing immunotoxins specifically to kill tumor cells were successful and led to further experiments in vivo. Such studies, carried out over the past 5 years in both animals and humans, have demonstrated that the efficacy of immunotoxins in vivo is often poor, due to problems involving instability of the conjugate, inferior potency, inaccessibility of tumor cells, nonspecific binding to ceils other than the target cells, and survival of antigen-negative mutants. In addition, immune responses against both the ligand and the A chain are usually elicited, precluding repeated therapy. During the past several years, there have been attempts to solve these problems and develop more effective immunotoxins. KEY WORDS: Immunotoxin (IT); m o n o c l o n a l antibodies (mAbs); ricin A chain (A chain).
INTRODUCTION An immunotoxin (IT) consists of cell-binding ligands coupled to toxins or their toxic subunits. Such reagents are designed to specifically kill neoplastic cells, viralty infected cells or subsets of normal cells. In this brief review, I focus on the development of ITs containing ricin A chain, emphasizing problems in their in vivo use and modifications of these reagents that have solved some of these problems and led to the availability of secondgeneration reagents. LIGANDS The specificity of an IT is determined primarily by the ligand used as the targeting vehicle. In most 1Department of Microbiology and Cancer Immunobiology Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
15S 0271-9142/90/1100-015S$06.00/0 © 1990 PlenumPublishingCorporation
t6S
VITETTA
directly to the A chain. This assay has made it possible to screen many mAbs and to select those that will make the most potent ITs. Similar assays could be readily developed for other types of ligands. Another approach to selecting a ligand is to use one directed against an antigen expressed at abnormally high levels on target cells and lower levels on normal cells (6), e.g., growth factor receptors or products of viruses or oncogenes. Such ligands coupled to A chain are less likely to kill normal cells expressing lower levels of these target molecules. One problem remaining with nonautologous ligands, such as murine mAbs, is the generation of an immune response that precludes repeated use. Strategies to avoid this problem include the use of immunosuppressive agents during IT therapy (15, 16) and the "humanizing" of murine mAbs (17, 18). RICIN A CHAIN
Ricin is a plant toxin made up of two subunits. The A chain is a ribosome-inactivating protein of 32 kD linked by a disulfide bond to a galactose-speclfic lectin (B chain) of approximately 30 kD (19). The A and B chains of ricin can be separated following reduction and the A chain can then be purified (20) and chemically linked to different ligands to generate a cell-reactive conjugate (1-4). Since native ricin A chain contains a large amount of mannose (21) and both parenchymat and nonparenchymal cells of the liver have high-aflSnity mannose receptors (reviewed in Ref. 1), ITs containing native ricin A chain home to the liver and are frequently hepatotoxic. In addition, the diversion to the liver decreases the proportion of ITs that reach the target cells. By using recombinant A chain lacking carbohydrate (22, 23) or by chemically "deglycosylating" native A chain (24), it was possible to eliminate this problem and to demonstrate that the IT containing the degtycosylated or recombinant A chain could localize more effectively (25) and destroy the target tissue (26, 27). CONSTRUCTION OF ITs For an IT containing a ricin A chain to be effective, there must be a disulfide bond between the ligand and the A chain (1, 4). This introduces the potential problem of disulfide bond instability in vivo. Disulfide bonds can be split by either enzymes or thiols in the tissues or in the circulation (1, 27).
Hence, there has been a strong impetus to develop more stable cross-linkers that allow ITs to remain intact in the circulation. This problem has been surmounted by developing cross-linkers containing hindered disulfide bonds (27-29) or by the direct use of the cysteine on the antibody (Fab' portion) and the cysteine on an A chain to form a disulfide bond (25). Both IT constructs are more stable in vivo and facilitate effective localization (25) and killing of target cells (25, 28). ITs containing more stable disulfide bonds have proven more potent in animal models (25, 28). CLINICAL EXPERIENCE WITH ITs
Several clinical trials with "first-generation" ITs have been reported (reviewed in Refs. 1 and 2) and other clinical trials involving their improved "second-generation" versions are in progress. The major dose-limiting side effects in humans include allergic reactions, hypoalbuminemia resulting in edema (capillary leak), fatigue, and myalgia (2). The mechanisms underlying capillary leak and myalgia have not been defined. Based on initial clinical trials, it appears that cells that are accessible to the circulation and that internalize the IT effectively, e.g., normal lymphocytes, macrophages, lymphoma cells, etc., are better cell targets for IT therapy than are solid tumors (1, 2). Thus, ITs containing an anti-T-cell antibody coupled to ricin A chain have proven effective in treating steroid-resistant graft-vs-host disease in humans (2) and are under study for the treatment of autoimmune disorders (30). Similar approaches could be used to eliminate B cells in other autoimmune disorders. The problem of killing virally infected cells has been addressed using cells infected with the human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS) (31, 32). In in vitro studies, it has been demonstrated that ricin A chain-containing ITs directed against the gp41 protein of the HIV can effectively kill infected cells (8). In addition, the soluble receptor for the gp120 envelope protein of HIV, i.e., CD4, has been cloned (33--37). Soluble recombinant CD4 (rCD4) has been coupled to A chain and such conjugates kill cells expressing the HIV-encoded envelope protein, gpl20 (7). Both approaches are attractive because the anti-gp41 antibody and the rCD4 molecule should not bind to other cells in the host, and hence, conjugates containing rCD4 or anti-gp41 should be relatively spe-
Journal of Clinical Immunology, Vol. 10, No. 6 (November Supplement 1990)
IMMUNOTOXINS AND AUTOIMMUNITY
cific in their cytotoxicity. In addition, it is possible that the target cells in the very early stages of the disease are confined largely to sites accessible to the circulation and therefore would not present problems. A major limitation of this approach is that HIV can cause a latent infection in both T cells and monocytes. Such cells probably lack HIV envelope proteins on their surfaces and therefore would not be susceptible to ITs.
17S
7.
8.
9.
FUTURE DEVELOPMENTS OF ITs The cloning of the genes for the relevant toxins, mAbs, growth factors, soluble receptors, and other ligands is now making possible the generation of ITs by recombinant DNA technology (38-42). Such genetically engineered ITs can be produced in large quantity. More importantly, structure-function relationships can be elucidated and such ITs can be quickly redesigned to generate more effective reagents. Results of current in vivo experiments using these reagents in animals are encouraging.
10.
11.
12.
13.
ACKNOWLEDGMENTS This work is supported by N I H Grants CA-28149 and CA-41081, Texas Technologies Grant 18512, Welch Foundation Grant 1-947, and Cancer Immunology Training Grant CA-09082. I thank Dr. J. Uhr for helpful comments concerning this review and Ms. G. A. Cheek and N. Stephens for secretarial assistance.
14.
15.
16.
REFERENCES 1. Blakey DC, Wawrzynczak EJ, Wallace PM, Thorpe PE: Antibody toxin conjugates: A perspective. In Progress in Allergy (Monoclonal Antibody Therapy), H. Waldmann (ed). Basel, Switzerland, Karger, 1988, pp 50-90 2. Byers VS, Baldwin RW. Therapeutic strategies with monoclonal antibodies and immunoconjugates. Immunology 65:329-335, 1988 3. Pastan I, Willingham MC, FitzGerald DJ: Immunotoxins. Cell 47:641-648, 1986 4. Vitetta ES, Fulton ILl, May RD, Till M, Uhr JW: Redesigning nature's poisons to create anti-tumor reagents. Science 238:1098-1104, 1987 5. Krolick KA, Uhr JW, Slavin S, Vitetta ES: In vivo therapy of a murine B cell tumor (BCL1) using antibody-ricin A chain immunotoxins. J Exp Med 155:1797-1809, 1982 6. Kr6nke M, Schlick E, Waldmann TA, Vitetta ES, Greene WC: Selective killing of human T-lymphotropic virus-I infected leukemic T-cells by monoclonal anti-interleukin 2 receptor antibody-ricin A chain conjugates: Potentiation by
17.
18.
19.
20.
21.
ammonium chloride and monensin. Cancer Res 46:32953298, 1986 Till M, Ghetie V, Gregory T, Patzer E J, Porter JP, Uhr JW, Capon DJ, Vitetta ES: HIV-infected cells are killed by rCD4-ricin A chain. Science 242:1166-1168, 1988 Till MA, Zolla-Pazner S, Gorny MK, Patton JS, Uhr JW, Vitetta ES: Human immunodeficiency virus-infected T ceils and monocytes are killed by monoclonal human anti-gp41 antibodies coupled to ricin A chain. Proc Natl Acad Sci USA 86:1987-1991, 1989 Li J-L, Shen G-L, Ghetie M-A, May RD, Till M, Ghetie V, Uhr JW, Janossy G, Thorpe PE, Amlot P, Vitetta ES: The epitope specificity and tissue reactivity of four murine monoclonal anti-CD22 antibodies. Cell Immunol 118:85-99, 1989 Lambert JM, Senter PD, Yau-Young A, Bl~ittler 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 260:12035-12041, 1985 Press OW, Vitetta ES, Farr AG, Hansen JA, Martin PJ: Evaluation of ricin A-chain immunotoxins directed against human T cells. Cell Immunol 102:10-20, 1986 Raso V, Basala M: Study of the transferrin receptor using a cytotoxic human transferrin-ricin A conjugate. In ReceptorMediated Targeting of Drugs, G Gregoriadis, G Poste, J Senior, A Trouet (eds). New York, Plenum Press, 1984, pp 73-86 Till M, May RD, Uhr JW, Thorpe PE, Vitetta ES: An assay that predicts the ability of monoclonal antibodies to form potent ricin A chain-containing immunotoxins. Cancer Res 48:1119-1123, 1988 Vitetta ES, Krolick KA, Miyama-Inaba M, Cushley W, Uhr JW: Immunotoxins: A new approach to cancer therapy. Science 219:644-650, 1983 Garnett MC, Baldwin RW: Endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen visualised using fluorescent conjugates. Eur J Cell Biol 41:214-221, 1986 Ledermann JA, Begent RHJ, Riggs SJ, Scarle F, Read D, Bagshawe HD: Suppression of the human anti-mouse antibody response by cyclosporin A (CsA) during therapy with a monoclonal antibody. Proc Am Assoc Cancer Res 29:423, 1988 Jones PT, Dear PH, Foote J, Neuberger MS, Winter G: Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321:522525, 1986 Morrison SL, Johnson MJ, Herzenberg LA, Oi VT: Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci USA 81:6851-6855, 1984 Olsnes S, Pihl A: Toxic lectins and related proteins. In Molecular Action of Toxins and Viruses, P Cohen, S yon Heyningen (eds). New York, Elsevier, 1982, pp 51-105 Fulton RJ, Blakey DC, Knowles PP, Uhr JW, Thorpe PE, Vitetta ES: Purification of ricin A1, A2 and B chains and characterization of their toxicity. J Biol Chem 261:53145319, 1986 Funatsu M: Structure of toxic function of ricin. Subunit structure of ricin D. Proc Jap Acad 47:718, 1971
Journal of Clinical Immunology, Vol. 10, No. 6 (November Supplement 1990)
18S
VITETTA
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Journal of Clinical Immunology, VoL 10, No. 6 (November Supplement 1990)
