[CANCER RESEARCH 32, 280-284, February 1972] The Use of the Proteolytic Enzyme Brinase to Produce Autocytotoxicity in Patients with Acute Leukemia and Its Possible Role in Immunotherapy1 R. Douglas Thornes, Patrick F. Deasy, Robert Carroll, Denis J. Reen,2 and J. Desmond MacDonell Department of Experimental Medicine, Royal College of Surgeons in Ireland [R. D. T.J, Our Lady's Hospital for Sick Children [P. F. D., R. C.J, and St. Laurence's Hospital, Dublin, Ireland ¡D.J.R., J. D. M.¡ SUMMARY investigated further in 3 patients and is described below. It was necessary to know whether brinase could be combined with Daily infusion of brinase (Protease 1 of Aspergillus oryzae) conventional antileukemic therapy or whether it could be of for 10 to 30 days in six patients (three children, three adults) value if given after the patient became resistant to with acute leukemia resulted in the production of conventional therapy. complement-dependent autocytotoxicity against leukemic The ability to reproduce autocytotoxicity was the main cells and lymphocytes and, in some instances, against platelets. purpose of this study, and this was accomplished on each of This appeared to be due to the production of autoantibody, the 5 occasions on which it was attempted in 3 patients. but its nature remains to be elucidated. The autocytotoxicity can be demonstrated in vivo by blood transfusion from a healthy donor or in vitro at 37° by Terasaki's MATERIALS AND METHODS microcytotoxicity test. The autocytotoxicity is transient, Hrinasc. Protease 1 of Aspergillus oryzae is produced by lasting from 3 to 15 days, but repeat courses of brinase, Astra AB, Sweden, as a fibrinolytic agent. Astra 1652 (Lot No. whether they are given alone or in combination with 17042). Brinase was given daily by i.v. infusion for 1 hr, as antileukemic drugs, produce further autocytotoxic previously described (20). The dosage was 2.5 mg/kg of body "antibodies." Remission was obtained in three of five patients weight in children and 100 to 350 mg daily in adults. who were given combination therapy and in the one patient Serum antiplasmin levels were used to estimate dosage, with acute myeloblastic leukemia who was treated by brinase which was varied to keep antiplasmin levels between 50 and alone. 10% of normal (100%). Full hematological analysis was performed before and after each infusion, as described (8). INTRODUCTION Bone marrow examinations were carried out weekly during therapy. A proteolytic enzyme similar to plasmin has been isolated Assay of Autocytotoxicity. This was performed by D.J.R. from Aspergillus oryzae (2) and is now called brinase (4). Like at the Transplantation-Immunology Unit of Jervis Street and plasmin, this enzyme can kill cancer cells in culture and, when St. Laurence's Hospitals, Dublin, under the direction of Dr. J. given i.V., it lowers antiplasmin levels. In patients with acute G. Devlin. leukemia, serum antiplasmin levels are abnormally high; the We used the microdroplet method of Terasaki and reduction of these antiplasmin levels to below 50% of normal McClelland (16) for the detection of lymphocytotoxic values rapidly decreases the number of circulating leukemic antibodies. Serum samples from patients were tested against cells (18). the patient's own lymphocytes. This test consists essentially of the incubation of the patient's serum for 2 hrs at 37°,in While investigating the therapeutic effects of brinase in 25 cases of leukemia complement-dependent autocytotoxicity 0.001-ml quantities with 1000 lymphocytes and 0.005 ml of against leukemic cells, lymphocytes and platelets were complement. The killed cells were identified by eosin dye observed in 4 patients (19). This autocytotoxicity was first inclusion or by complete cell lysis. A test was considered demonstrated in vivo by a dramatic fall in white cell and negative unless more than 20% of the cells were killed. In the platelet counts when whole-blood transfusion was given at the patients described below, all positive tests showed more than end of an 18-day course of brinase. The 22-year-old patient 30% killed and the negative tests showed less than 10%. had acute lymphoblastic leukemia (20). Warfarin sodium was The cells from the leukemic patients were prepared by the given as maintenance therapy to inhibit malignant cell method of Terasaki and McClelland (16). In the patients with locomotion and growth (21). lymphatic leukemia, all the separated cells tested were The production of autocytotoxicity by brinase was lymphocytes, but confusion arose in the case of acute 1Supported by the Irish Cancer Society. myeloblastic leukemia because leukemic blast cells separated with the lymphocytes. Neither the lymphocytes nor the blast J Holder of the Lady Tata Memorial Foundation Leukaemia cells were killed when the patients' sera were not toxic. When Fellowship. Received May 26, 1971;accepted October 21, 1971. the sera became cytotoxic, both lymphocytes and blast cells 280 CANCER RESEARCH VOL. 32 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1972 American Association for Cancer Research. Brinoseand Autocytotoxicity in Leukemic Patients were killed. The percentage of each cell type was not 7 or more days. Therefore, 3 more patients were specifically calculated, but from 40 to 80% of the mixed cells were killed treated with brinase to induce autocytotoxicity under on each occasion of testing during the period of cytotoxicity; different conditions. All the cases are summarized in Table 1. approximately 50% of these cells were lysed completely after When the 3 patients in this study were treated with daily 2 hr of incubation. The term "autocytotoxicity" was therefore doses of brinase, it produced autocytotoxicity on each of 5 used instead of "lymphocytotoxicity" because both occasions. These are described below. lymphocytes and leukemic cells appear to be affected by positive sera. Case 4 Controls. All patients had negative tests before and after the K. M. (treated by Dr. H. E. Counihan), a 45-year-old woman period when autocytotoxicity was present. with acute myeloblastic leukemia, was treated with brinase We used 3 controls for each test in which 0.9% NaCl and anticoagulated with warfarin sodium. The 1st course of solution replaced autologous serum or complement and brinase lasted 30 days and was interrupted 1 day in 7. The heat-inactivated complement replaced active complement. In daily dosage was 200 mg (total 5200 mg). Autocytotoxicity addition, the patient acted as his own control because was detectable for 7 days for 30 to 40% of her leukocytes. autologous serum taken when autocytotoxicity was not present did not kill with the patient's own cells or with other There was a partial remission which relapsed 5 weeks later. patients' cells. The 2nd course of brinase was 300 mg for 4 days. After 7 weeks, a 3rd course of 300 mg was given for 5 days. Complete The complement source used was nontoxic rabbit serum. remission for 2 months followed, but there was no evidence of Modifications of the hemolysin test (10) were used to estimate autocytotoxicity. After 10 weeks, 3 courses of 200 mg were complement levels (7) and complement fixation (11). given daily for 5 days without producing autocytotoxicity, but Cross-reaction Tests. We used the autocytotoxic serum from remission ensued for 13 months before bone marrow relapse. 1 patient (Case 5) to study cross-reaction with lymphocytes This condition was then treated by continuous daily infusions from patients with other forms of cancer. In positive of 250 mg of brinase. After 10 days, autocytotoxicity cross-reaction tests 30 to 50% of the lymphocytes were killed. developed against 75% of the patient's lymphocyte When 10% or fewer lymphocytes were killed, the preparations (50% of these cells were completely lysed after 2 cross-reaction was considered negative. The control for these hr of incubation). Brinase was continued for another 15 days tests was serum from the same patient (Case 5) when and autocytotoxicity remained throughout this time, varying autocytotoxicity was absent. between 40 and 80%. The patient was discharged in hematological remission from the hospital, and 1 week later RESULTS no autocytotoxicity was detectable. Comment. The necessity of continued daily infusions of an The reexamination of previous cases showed that adequate dosage of brinase to produce autocytotoxicity was autocytotoxicity occurred when brinase therapy was given for revealed. Table 1 Occurrence of autocytotoxicity in relation to brinase therapy Cases 1 and 2 were tested after an unexplained fall in white cell and platelet counts following whole blood transfusion. doseDaysgiven to (days) of Case1. mg18 remissions20000011120004Dayscytotoxicity181328301013131116Durationcytotoxicity127NoneNoneNoneNoneNone15126310ConcomitanttherapyBloodBlood, T.W.2. 1007 remission6Complete B.F.3. 5016 VCBloodWarfarinWarfarinWarfarinWarfarinWarfarinWarfarinWarfarin6MP,maintainedonremission mo.ShortVC and 6MP for 12 O.M.4. 2026 remissionClinicalcomplete K.M.5. 2004 improvementClinical 3005 improvementComplete 3005 mo.NilNilCompleteremission for 2 2005 2005 20025 mo.Hematologicalremission for 13 25018 remissionNilNilComplete P.M.6. 17514 VCSteroidsL-AsparaginaseNoneResultPeripheral 15028 15022 maintainedonremission steroidsSepsis,VC, 6MP, and P. B.SexFFMFMMAge227345406DiagnosisALL"ALLALLAMLALLALLAv.30Previous hemorrhage, death. " The abbreviations used in the table are: ALL, acute lymphoblastic leukemia; VC, vincristine; 6MP, 6-mercaptopurine; AML, acute myeloblastic