METHOD FOR TREATING MALIGNANCY AND AUTOIMMUNE DISORDERS IN HUMANS

Abstract: The present invention relates to a method for treating malignancy and autoimmune disorders and for preventing allograft rejection. Conjugated or unconjugated monoclonal anti-Tac antibodies are employed to treat the above conditions.

Description: 1 METHOD FOR TREATING MALIGNANCY AND 2 AUTOIMMUNE DISORDERS IN HUMANS 3 BACKGROUND OF THE INVENTION 4 Technical Field: 5 The present invention is related to a method for 6- treating malignancy and autoimmune disorders and for 7 preventing allograft rejection. More particularly, the 8. present invention is directed to treating any human 9- condition or disorder related to the expression of Tac 10 antigen or involving abnormal IL-2- receptor expression, 1.1 by reacting Tac antigen or IL-2 receptor expressing cells 12 with anti-Tac monoclonal antibody or a preparation 13 thereof. 1.4 State of the Art: 15 The normal resting cells of the body, including T 16 cells, do not express IL-2 receptors and thus do not 17 react with .a monoclonal antibody anti-Tac that recognizes IB. the human IL-2 receptor. However, in certain conditions, Sf. such as in leukemic T cells of • patients infected with 2G human T-cell lymphotrophiσ virus I (HTLV-I-associated 21 Adult T Cell Leukemia), large numbers of IL-2 receptors 22 are constitutively expressed. The Tac antigen is also 3 expressed in other malignant conditions including the 4 malignant B lymphocytes of hairy cell leukemia, 1 follicular lymphoma and the Reed-Sternberg cells of 2 Hσdgkin's disease. Furthermore, activated T cells 3 expressing the Tac antigen also appear to play a 4 pathogenic role in certain forms of autoimmune disorders, 5 such as type I diabetes and a subset of patients with 6 aplastic anemia. In addition, when cells responding to 7 foreign histocompatibility antigens become activated, S they express the Tac antigen and participate in allograft 9 rejection such as in patients receiving vascularized Id organ allografts and in graft-versus-host disease in 12L patients receiving marrow allografts. Thus, there are a 12 number of clinical circumstances where the expression of 13 Tac-antigen is involved. Clearly, therefore, the 1.4 elimination of Tac-positive cells using the anti-Tac S monoclonal antibodies would be of value in treating or 1.6 controlling such pathological states. 1.7 SUMMARY OF THE INVENTION IBs It is, therefore, an object of the present IS. ! invention to provide a r method of eliminating 2SC disease-associated Tac-positive cells. 23L It is a further object of the present invention to 22 provide a method of treating adult T-cell leukemia or 23 T-cell-mediated autoimmune disorders. 24 It is another object of the present invention to 25> provide a method of treating B-cell malignancy. 26 It is yet another object of the present invention 27 tα provide a method of controlling allograft rejection 28: reactions. 3} Other objects and advantages of the present 3XE invention will become apparent from the Detailed 3EL Description of the Invention. BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: Fig. 1 shows the results of anti-Tac therapy of patient with Tac-positive ATL. The patient was treated with four infusions (20, 40, 50, and 50 mg) of anti-Tac monoclonal antibody over a 12 day period (indicated by solid bars). After the anti-Tac therapy, the number of circulating T cells bearing the Tac antigen declined from 8000 to less than lOO/mm^. There was a parallel decline of cells expressing another tumor-associated marker of the transferrin receptor from over 9000 before therapy to less than 100/mm 3 ; Fig. 2 shows the effect of anti-Tac therapy on CT 3 chain gene arrangement in a patient with ATL. The remission of the T-cell leukemia in this patient after anti-Tac therapy was confirmed using molecular genetic analysis of the arrangement of the genes encoding the chain of the antigen-specific T-cell receptor. Southern analysis of the arrangement of the T-cell receptor 3 chain was performed on BamHI digests of DNA from the peripheral blood mononuclear cells of the patient by using a radiolabeled probe to the constant region of the T 3 chain. The constant T 3 genes are universally present on a 24-kb BamHI fragment in germline tissues of normal individuals and in a B-cell line from the patient. However, before therapy there was an additional 22-kb BamHI band hybridizing with the constant T 3 probe when digests of the patient's circulating T cells were examined, a hallmak of a clonal expansion of T lymphocytes. This band reflecting the clonally 1 rearranged T-cell receptor gene was not demonstrable on 2 specimens obtained after anti-Tac therapy when the 3 patient was in remission. Six months after the initial 4 remission the leukemia recurred with reappearance of 5 leukemic cells identified by a molecular genetic 6 analysis. A second course of infusions of anti-Tac was 7 followed by a virtual .disappearance of the skin lesions 8 and the circulating leukemic cells (data not shown); and S Fig. 3 shows the effect of anti-Tac therapy on 10 leukemic mononuclear cells with integrated HTLV-I. 1 HTLV-I is clonally integrated into the cells of patients 12 with HTLV-I-associated ATL. Such integrated HTLV-I can 13 be identified by Southern analysis using a radiolabeled 14 HTLV-I probe. In the case shown, there are two lines on 15 the Southern gel indicating the integration of two HTLV-I 16 viruses per cell. After anti-Tac therapy, the 17 circulating cells of this patient did not contain IS integrated HTLV-I as shown by the clear Southern gel 19 radioautograph. After relaps, integrated HTLV-I could 2Q again be demonstrated in the circulation T cells. 21 DETAILED DESCRIPTION OF THE INVENTION 2, The above and various other objects and advantages 3 of the present invention are achieved by a method of 24 treating T-cell mediated disorders in humans comprising 25 administering to a human afflicted with T-cell mediated 26 discorder, therapeutic amounts of conjugated or 27 unconjugated anti-Tac monoclonal antibodies to eliminate 28 disease-associated Tac-positive cells without affecting 29" normal cell populations. 3Q Unless defined otherwise, all technical and 31 scientific terms used herein have the same meaning as 32 commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Prior to the present studies, little was known about the inducible IL-2 receptor, and no antibodies to IL-2 receptor had been made. Using hybridoma technology, an IgG2a mouse monoclonal antibody called anti-Tac was prepared. This anti-Tac antibody reacted with activated but not resting T cell (Uchiyama et al, J. Immunol. 126:1393-1397, 1981; Uchiyama et al, J. Immunol. 126:1398-1403, 1981). Furthermore, this antibody identified the IL-2 receptor and blocked IL-2 binding to its receptor (Leonard et al, Nature 300:267-269, 1981). The structure, function and expression of the IL-2 receptors on normal and malignant lymphocytes has been reviewed by Waldmann (Science, 232:727-732, 1986). Based on the known unique properties of anti-Tac antibodies, a novel approach to immunotherapy was developed for the first time to eliminate leukemic cells and activated T cells in autoimmune disorders and in organ allograft protocols. These therapeutic studies were extended by coupling toxins to anti-Tac and showing that they killed tumor cells at doses that did not affect normal cells. Furthermore, anti-Tac was coupled to the alpha-emitting radionuclide such as bismuth 212 ( • ^ ■ '■ -^B± ) or a -emitting radionuclide such as yttrium-90 by the use of a bifunctional chelate. This agent was also shown to be an effective and specific immunocytotoxic agent for the elimination of IL-2 receptor-positive cells. The details of the procedure for the use of anti-Tac in the therapy of patients with adult T-cell leukemia and in organ allograft protocols are described below. 1 A. Treatment of ATL with Unmodified Anti-Tac 2 Patients with adult T-cell leukemia (ATL) are 3 treated by intravenous infusions of unmodified anti-Tac. 4- ATL is an aggressive leukemia of polymorphic mature T 5 cells with a propensity to infiltrate the skin. This 6 leukemia is frequently associated with hyperσalcemia and 7 pulmonary involvement. The leukemic cells always contain 8 the C-type retrovirus Human T-Cell Lymphotrophic Virus I 9 (HTLV-I). There is no curative threapy for patients with 1Q ATL, and such patients have a mean survival time of only 11 about 20 weeks. In contrast to normal cells, the 12 malignant cells of patients with ATL display the cell 13 surface receptor for interleukin-2 identified by the 14 anti-Tac monoclonal antibody. 15 The anti-Tac murine-derived monoclonal antibody 16 used for these therapeutic studies has been produced by 17 fusing NS-1 cells with spleen cells of mice immunized 18 with a cell line derived from an ATL patient. Large 19 quantities of the monoclonal antibody are produced by 2D, . inoculating hybrid cells into the peritoneum of BALB/c 21. " mice and purifying this IgG2a K antibody from the 221 resulting ascites fluid by DEAE chromatography with 23 elution by 0.1 Tris buffer as the eluting agent. The 24 material is dialyzed against saline, centrifuged, 25 filtered, precipitated with 20% sodium sulfate, and then 26 diluted in saline at pH 7.4 to a concentration of about 27 2 mg/ml. Each lot of the product is shown to be pure by 23 assays that include immunoelectrophoresis, diffusion in 29 agar plates using antisera to IgG2a, IgGl, IgM, and 30. transferrin, as well as polyvalent antibodies to major 31. mouse proteins. Furthermore, the lots are shown to be 32 homogenous by HPLC. The monoclonal preparations are 3S sterilized by passge through a 0.22 millipore filter and 34 are shown to be nonpyrogenic and sterile. Patients with 3 Tac-expressing ATL receive anti-Tac antibody by 1 intravenous administration of a dose in 100 cc of normal 2 saline with 5% human albumin over a 2-hour period. 3 Patients receive anti-Tac at a higher dosage each week 4 for 2 weeks. During the first week, they receive two 5 doses of 20 g per patient and, in the second week, two 6 doses of 50 mg per patient. Patients undergoing partial 7 or complete remission or those with leukemic cells with 8 persistent Tac receptors unblocked by the anti-Tac 9 monoclonal antibodies may receive additional biweekly 10 doses of 50 mg of anti-Tac. Unblocked IL-2 receptors can 11 be identified with flow cytometry using 12 fluorochrome-labeled anti-Tac monoclonal antibodies using 13 conventional procedures. 14 Ten patients with ATL have been treated with 15 intravenously administered anti-Tac monoclonal antibody 16 according to the above protocol. None of the patients 17 suffered any untoward reactions or produced antibodies to 18 mouse immunoglobulin or to the idiotype of the anti-Tac 19 monoclonal. There was no reduction in the number of any 20 of the normally formed elements of the blood. Three of 21 the patients had a reduction in the number of circulating 22 leukemic cells or a complete remission after anti-Tac 23 therapy. In one of these patients, therapy was followed 24 by a 5-month remission as assessed by routine 25 hematological tests, immunofluorescence analysis of 26 circulating T cells (Figure 1), molecular genetic 27 analysis of the arangement of the genes encoding the 28 chain of the T-cell receptor (Figure 2), as well as the 29 genes of the retrovirus HTLV-1 (Figure 3). After the 30 5-month remission, the patient's disease relapsed, but a 31 new course of anti-Tac therapy was followed by a virtual 32. disappearance of the skin lesions and an over 80% 33 reduction in the number of circulating leukemic cells. 2 B. Treatment of ATL with Anti-Tac Conjugated with 2. Cytotoxic Agents 3 . Anti-Tac Antibody Coupled to Ricin A Chain 4 Using conventional procedures, purified anti-Tac 5. monoclonal antibody is conjugated to purified or 6 recombinant ricin A chain using a thiol-containing 7 crosslinker, N-succinimidyl-3-(2-pyridyldithio)propionate 3 (Kronke et al Blood 65:1416-1421, 1985). The resulting <£ conjugates are separated from the majority of- free ricin 10: A chains by Sephacryl S-200 gel filtration. Conjugates UL are, adjusted to 1 mg/ml with reduced and alkyla ed human 121 IgG and stored at -20°C. The addition of carrier protein 13 assures stability of the conjugates, and the alkylation 14. prevents disulfide toxin exchange between specific 15 " antibody and carrier"protein. The addition of anti-Tac 16. antibody coupled to the A chain of the toxin (ricin) 17. " effectively inhibited protein synthesis and led to cell 13 death of an HTLV-I-associated, Tac-positive ATL cell 19 ~ - line, HUT102-B2. In contrast, conjugates of ricin A with 20) a control monoclonal of the same isotype did not inhibit 21. protein synthesis when used in the same concentration. 22: The inhibitory action of anti-Tac conjugated with ricin A 23 could be abolished by the addition of excess unlabeled 24 anti-Tac or IL-2. 25E 2- Anti-Tac Coupled to Pseudomonas Toxin 26r The immunotoxin Pseudomonas exotoxin anti-tac is 27 made from purified pyrogen-free anti-Tac and purified 28:- Pseudomonas exotoxin (PE) according to published methods 22- (Fitsgerald et al Proc. Natl. Acad. Sci. USA 3£T 80:4134-4138, 1983). Two mg (30 nM) of PE in KP0 4 0.1 M, 3H EGTA 1 iπM, pH 8.0, is incubated with 500 nM of NAD and 2. 50Q0 nM of 2-iminothiolane-HCl for 1 hour at 37°C. NAD is 33 added to protect the enzyme-active site of the 1 toxin. This derivatized PE preparation is separated on 2 HPLC from a small amount of aggregated toxin by the other 3 reactants. Dithio-bis(2-nitrobenzoiσ acid) (DTNB) is 4 added to the derivatized PE to a final concentration of 5 about ImM. The addition of DTNB and its reaction with 6 free sulfhydryl groups serves to activate the toxin for 7 future disulfide exchange with antibody. 8 The antibody (5-8 mg) in KPO4 0.1 M, EGTA 1 mM, 9 pH 8.0, is incubated with 120 nMol of 2-iminothiolane- 10 HCL for 1 hour at 37 β C. At the end . of the incubation 11 period, the antibody is separated from iminothiolane by 12 gel filtration on a G-25 column. An aliquot of the 13 derivatized antibody is reacted with DTNB to determine 14 the number of new sulfhydryl groups introduced. The 15 remainder is mixed with the activated PE. Activated PE " 4 16 is reacted with derivatized anti-Tac antibody. The 17 reaction is followed by measuring the release of TNB 18 (thionitro-benzoiσ acid - nitrophenol ) at OD412• τhe 19 antibody-SH releases routinely half of the TNB from the 20 activated PE molecules. The balance is released by 21 adding excess cysteine. The reaction mixture is 22 separated by HPLC. The PE-antibody-(cys)2 has the most 23 activity and is used for patient therapy. The 24 PE-anti-Tac is stored at -20°C in 0.15 M NaCl, 10 mM 25 ■ KPO4, 1 mM EGTA, pH 7.2. 26. Patients with ATL receive PE-anti-Tac antibody by 27 intravenous administration in 100 cc of normal saline 28 with 1% albumin over 2 hours. Each patient received 29 about 200 g of PE-anti-Tac twice during the first week 30 and 2 mg twice a week during the second week. Therapy is 31 stopped if the patient manifests grade III hepatic 32 toxicity, that is, a bilirubin over 3.0 mg/ml or an SGOT 3 or alkaline phosphatase 3-5 times the base line. 4 Four patients have been treated with PE-anti-Tac 5 according to this protocol. One of the four patients 1. manifested hepatic dysfunction, including abdominal pain 2. and a transient disorder of the liver function tests. One 3 of the patients had a response to the PE-anti-Tac therapy 4 manifested by an over 50% decline in the number of 5 circulating leukemic cells. 6 It is noted that other cytotoxic conjugates of 7 anti-Tac can be similarly prepared and used. The S examples provided herein being only exemplary. S C. Therapy of ATL with Anti-Tac Conjugated with IO Radionuclides 11. • Anti-Tac has been successfully conjucated to the 12: α-particle-emitting radionuclide bismuth-212 and to the 13 3-emitting yttrium-90 by use of bifunctional ligands, 14 such as isobutylcarboxycarbonic anhydride of 15 diethylenetriamine-pentaacetic acid (DTPA). The physical 16 properties of 212gi are appropriate for 17 radioimmunotherapy in that it has a short half-life, IS deposits its high energy over a short distance, and can 19 be obtained in large quantities from a radium generator. 2Ω The labeling protocols have been described by Gansow et 21. al (Am. Chem. Soc. Symp. Ser. 241:215-227). DTPA is 22 linked to anti-Tac with 14 C-labeied DTPA used to identify 23 chelate-antibody ratio. DPA (0.2 mM)was dissolved in 2 24 ml of H2O by addition of triethylamine (1.38 mM) and 25 lyophilized. The solid formed is taken up in 1 ml of 26 acetonitrile at 4°C and treated with 27 isobutylchlorσformate (0.27 mM) for about 30 minutes, 28 centrifuged, and a 20-μl aliquot of 29" isobutylcarboxycarbonic anhydride solution is reacted 30; with anti-Tac at 4°C for about 1.5 hours. Sequential 31. dialyses in metal-free buffer are used to purify the 33 protein. A comparable procedure is used to couple 33 anti-Tac to the 3-emitting radionuclide yttrium-90. 34. Conjugates with other α or 3 emitting ' nuclides are similarly prepared and used. The examples provided herein being only exemplary. Activity levels of 0.5 μCi or the equivalent of 12 rad/ml of or irradiation targeted by eliminated more than 98% of the proliferative capacity of the HUT102-B2 cells with only minimal effect on IL-2 receptor-negative lines. This specific cytotoxicity was blocked by excess unlabeled anti-Tac but not by human IgG. Thus, 212Bi_ an -ti_ ι r ac i s an effective and specific immunocytotoxic agent for the elimination of IL-2 receptor-positive ATL cells. D. Protocol for Treatment of Autoimmune Disorders Patients with certain forms of autoimmune disease, including subsets of patients with the disease aplastic anemia, have increased number of circulating and marrow Tac-positive T cells. In this group of patients, the Tac-positive but not the Tac-negative T cells inhibit hematopoiesis when cocultured with normal bone marrow cells. Patients with elevated number of Tac-positive T cells and associated aplastic ,anemia receive unmodified anti-Tac monoclonal antibody in 100 ml normal saline with 5% alumin by intravenous administration over a 2 hour period. Patients are treated with 20 mg of anti-Tac three times over a 7 to 10 day period. This course may be modified and repeated if Tac positive cells remain elevated. An alternative therapeutic approach with anti-Tac is the use of Pseudomonas exotoxin anti-Tac according to protocols described above. Patients receive about 200 ug of PE-anti-Tac twice a week during the first week of treatment and at doses of about 2 mg twice a week during the second week. I. E. Protocol for Treatment to Prevent Allograft Rejection 2 After renal or cardiac allografts and during 3 graft-versus-host diesase, certain host T lymphocytes 4 recognize the foreign histocompatibility antigens 5 expressed on the donor organs and thus become activated 6 and express the Tac antigen. Such Tac-expressing 7 activated T cells participate in the rejection of the 3 allografts and in the graft-versus-host disease. The survival of renal allografts was prolonged in cynomolgus IOJ monkey recipients treated with the anti-Tac monoclonal II. antibody. 12 " . In patient studies, intravenously administered 13-' anti-Tac is added to conventional immunosuppression to 14- prevent allograft rejection. The patients receive 15 anti-Tac monoclonal antibody by intravenous adminstration 16 in 100 ml of glucose or saline with 5% albumin carrier 17 over about 2 hours. The patients are treated with about 18 20 mg of anti-Tac daily for about 10 days between the 19 first and tenth day after their receipt of the organ 20: allograft. 21 Eight patients receiving renal allografts have 22 " been treated with the above protocol of anti-Tac in 23 addition to conventional immunsupression. None of these 2M patients manifested toxicity due to the anti-Tac 25: monoclonal antibody. Furthermore, none of them have 26- rejected the transplanted kidney. 7' It is understood that the examples and embodiments 23 described herein are for illustrative purposes only and 2SF that various modifications or changes in light thereof 3CB will be suggested to persons skilled in the art and are 31. to be included within the spirit and purview of this 32. application and scope of the appended claims.

Claims: WHAT IS CLAIMED IS 1. A method of treating T-cell mediated disorders in humans, comprising administering to a human afflicted with T-cell mediated disorder, therapeutic amount of conjugated or unconjugated anti-Tac monoclonal antibody to eliminate disease-associated Tac-positive cells without affecting normal cells. - 2. The method of claim 1 wherein T-cell mediated disorder is Adult-T-Cell Leukemia, autoimmune disfunction, or allograft incompatibility. ' 3. The method of claim 2 wherein said disorder is Adult T-Cell Leukemia. 4. The method of claim 1 wherein said disorder is autoimmune disfunction. 5. The method of claim 2 wherein said disorder is allograft incompatability. 6. The method of claim 1 wherein said anti-Tac ? monoclonal antibody is conjugated with a cytotoxic agent. 7. The method of claim 6 wherein said cytotoxic , agent is selected from the group consisting of toxin and radionuclide. 8. The method of claim 7, wherein said toxin is selected from the group consiting of ricin-A and pseudomonas toxin. 9. The method of claim 8 wherein said toxin is ricin-A. 10. The method of claim 8 wherein said toxin is pseudomonas toxin. 11. The method of claim 7 wherein said radionuclide is alpha-emitting or beta-emitting radionuclide. 12. The method of claim 11, wherein said alpha-emitting radionuclid is 212 Bi. 13. The method of claim 11, wherein said beta-emitting radionuclide is yttrium-90.

Inventors: WALDMANN THOMAS A, WALDMANN, THOMAS, A.
Applicants: US ARMY, THE UNITED STATES OF AMERICA, AS REPRESENTED BY TH
Application year: 1988
Main classification: A61K43
IPC codes: A61K38, A61K39, A61K45, A61K51, A61P35, A61P37, C07K16