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Background: In a pilot trial we evaluated the toxicity and efficacy of sirolimus (rapamycin) as second-line therapy for the treatment of acute graft-versus-host disease (GVHD) in 21 patients (1-46 years of age) after allogeneic hematopoietic stem cell transplantation (HSCT)., Methods: All patients were treated with methylprednisolone at 2 mg/kg/day, but failed to respond satisfactorily. Sirolimus was started 19-78 (median 37) days after HSCT when 10 patients had grade III and 11 had grade IV GVHD. The first four patients received a loading dose (15 mg/m2) of oral sirolimus on day 1 followed by 5 mg/m2/day for 13 days. The next 17 patients received either 5 (n=7) or 4 (n=10) mg/m2/day for 14 days without a loading dose. Eleven patients completed the 14-day sirolimus course. Five patients were treated for 9-13 days, two for 6 days, and three for 1-3 days., Results: Sirolimus was discontinued early in 10 patients because of lack of improvement in GVHD (n=5), myelosuppression (n=2), seizure (n=2), and attending physician preference (n=1). The most common and significant adverse events were thrombocytopenia (n=7) and neutropenia (n=4). Other side effects included increased blood triglycerides (n=8) and cholesterol (n=3). Five patients had evidence of a hemolytic uremic syndrome concurrently with or after sirolimus treatment. Eighteen of the 21 patients received 6 or more doses of sirolimus and 12 responded, 5 with complete and 7 with partial responses. Six of the 12 responders (28% of all patients enrolled) and 1 nonresponder are currently alive at 400-907 days after HSCT, 3 with chronic GVHD. Fourteen of the 21 patients (66%) died 40-263 days after transplant., Conclusion: These data suggest that sirolimus has activity in the treatment of steroid-refractory acute GVHD. However, there was considerable toxicity and further dose optimization studies seem warranted.

Pretransplant blood transfusions from a dog leukocyte antigen (DLA)-identical canine littermate marrow donor will sensitize the recipient to non-DLA-linked polymorphic minor histocompatibility antigens, which uniformly results in graft rejection. We observed previously that 2000 cGy gamma-irradiation of marrow donor blood transfusions prevented this sensitization and subsequent marrow graft rejection. The purpose of the present study was to determine whether treatment of unrelated blood transfusions with gamma-irradiation would also prevent sensitization. Conceivably, sensitization to minor histocompatibility antigens might be more efficient or potent and thus more difficult to prevent when those antigens are seen in the context of disparity for DLA antigens. Furthermore, this model, in which sensitization to DLA-identical littermate marrow is caused by unrelated blood transfusions, is directly relevant to the clinical circumstances of human marrow transplantation. We assessed sensitization caused by unrelated blood transfusions by monitoring graft outcome in recipients transplanted with DLA-identical littermate marrow after conditioning with 920 cGy total body irradiation. Two thousand cGy gamma-irradiation of unrelated blood transfusions significantly reduced the incidence of transfusion-induced sensitization of recipients. There was successful marrow engraftment in 15 of 16 (94%, P < 0.003) of these animals in contrast to the previous study in which only 7 of 16 (44%) animals engrafted after they were transfused with unmodified blood on the same schedule. These results suggest that blood transfusions for use in humans, especially for patients with aplastic anemia, should be gamma-irradiated in order to reduce the incidence of marrow graft rejection caused by sensitization to minor histocompatibility antigens.

FK-506 was evaluated either alone or combined with methotrexate (MTX) for prevention of graft-versus-host disease (GVHD) in dogs given 9.2 Gy total body irradiation and dog leukocyte antigen-nonidentical unrelated marrow grafts. Studies with marrow autografts showed gut toxicity and weight loss to be major side effects of FK-506. There was no hematopoietic toxicity with FK-506. In an initial allograft study, 5 dogs were given FK-506 intramuscularly at 0.3 mg/kg/day from days 0 to 8 and then orally at 0.5 mg/kg/day. All 5 died, 3 with intussusception most likely due to FK-506 toxicity, 1 with graft failure, and 1 with GVHD. Subsequently, the FK-506 dose was reduced and these drug schedules were used: FK-506 days 0-8 at 0.15 mg/kg/day i.m. and then orally at 0.5 mg/kg/day until day 90, with or without MTX intravenously at 0.4 mg/kg days 1, 3, 6, and 11. Twenty allografts were done, 10 with FK-506 alone, and 10 with MTX/FK-506. Results were compared with those in concurrent and historical controls given either no immunosuppression (n = 64), MTX (n = 114), CsA (n = 15), or MTX/CsA (n = 17). Five of 20 current dogs died with intussusception, too early to be evaluated for GVHD. The 10 dogs given FK-506 alone survived significantly better than those not given immunosuppression but not differently from those given short-term MTX or CsA alone. Three died from toxicity, 2 with graft failure, and 4 with GVHD. Only 1 dog became a long-term survivor, and this dog inadvertently received a single dose of MTX on day 7. Two of 10 dogs given MTX/FK-506 died from toxicity, 1 died with graft failure, 2 died with GVHD, and 5 became long-term survivors, a result that is significantly better than seen with either drug alone and similar to that seen with MTX/CsA. Four of the 5 survivors had no clinical GVHD. FK-506 blood levels were 15-35 ng/ml between days 8 and 15, when gut toxicity was most severe. Thereafter, levels were approximately 5 ng/ml. In conclusion, FK-506 prolonged survival of recipients of dog leukocyte antigen-nonidentical unrelated marrow grafts. When FK-506 was combined with MTX, graft-host tolerance was induced in 50% of dogs, even though FK-506 was stopped on day 90.

L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is a lysosomotropic agent that selectively kills cytotoxic T cells and their precursors, natural killer cells, and monocytes but not helper T cells or other cells of hematopoietic origin. In this study, the effects of treatment of bone marrow and peripheral blood buffy coat with Leu-Leu-OMe on the outcome of allogeneic marrow transplantation were studied in several canine models. Whereas incubation of autologous marrow with Leu-Leu-OMe had no adverse effects on subsequent engraftment, incubation of marrow from dog leukocyte antigen (DLA)-identical littermates resulted in a high rate of graft failure. Previous studies have demonstrated that the addition of peripheral blood buffy coat allows engraftment of unrelated DLA-nonidentical marrow, and in this study we found that incubation of buffy coat with Leu-Leu-OMe did not alter this graft promoting effect. In a final experiment it was demonstrated that incubation of both marrow and peripheral blood buffy coat did not prevent the development of graft-versus-host disease in recipients of marrow from DLA-haploidentical littermates. In considering the eventual application of Leu-Leu-OMe in the clinic, these results are less encouraging than those previously reported using murine models.

Disease recurrence remains a major limitation to the use of marrow transplantation to treat leukemia. Previous transplant studies have demonstrated that higher doses of total-body irradiation result in less disease recurrence, but more toxicity. In this study, the possibility of delivering radiotherapy specifically to marrow using a radiolabeled anti-CD33 antibody (p67) was explored. Biodistribution studies were performed in nine patients using .05-.5 mg/kg p67 trace-labeled with 131I. In most patients initial specific uptake of 131I-p67 in the marrow was seen, but the half-life of the radiolabel in the marrow space was relatively brief, ranging from 9-41 hr, presumably due to modulation of the 131I-p67-CD33 complex with subsequent digestion and release of 131I from the marrow space. In four of nine patients these biodistribution studies demonstrated that with 131I-p67 marrow and spleen would receive more radiation than any normal nonhematopoietic organ, and therefore these four patients were treated with 110-330 mCi 131I conjugated to p67 followed by a standard transplant regimen of cyclophosphamide plus 12 Gy TBI. All four patients tolerated the procedure well and three of the four are alive in remission 195-477 days posttransplant. This study demonstrates the feasibility of using a radiolabeled antimyeloid antibody as part of a marrow transplant preparative regimen and also highlights a major limitation of using conventionally labeled anti-CD33--namely, the short residence time in marrow. Strategies to overcome this limitation include the use of alternative labeling techniques or the selection of cell surface stable antigens as targets.

Treatment with the monoclonal antibody OKT3 specific for the CD3 complex associated with the T cell antigen receptor can reverse acute rejection of human renal allografts. However, efficacy of anti-CD3 antibodies for treatment of patients with acute graft-versus-host disease after marrow transplantation has not been established. The dose-limiting side effects resulting from T cell activation induced by some anti-CD3 antibodies in vivo have discouraged their use for this application. We now report a phase I-II study of GVHD treatment with the anti-CD3 antibody BC3, a monoclonal murine IgG2b that, unlike OKT3, does not activate T cells. Fourteen patients were treated with BC3 after progression of acute GVHD despite treatment with cyclosporine and corticosteroids, and three patients received BC3 as primary treatment for GVHD. BC3 was administered at a dose of 0.1 or 0.2 mg/kg/day for seven or eight days. Five patients achieved complete resolution of GVHD, eight patients had partial improvement, two patients had no change, and two patients had progression of GVHD on therapy. Responses were sustained in 8 of 13 patients. Mild chills, fever, hypertension, and chest discomfort occurred in various combinations following 6 of 17 (35%) initial infusions of BC3 and following 4 of 99 (4%) subsequent infusions. In each instance it was possible to continue BC3 therapy without adjusting the dose or treatment schedule. In each patient treated, the absolute count of peripheral blood lymphocytes decreased transiently but returned to baseline within 22 hr after the first infusion. Circulating T cells had surface CD3 molecules saturated by the infused antibody in all but one patient. Four patients survived longer than one year after treatment with antibody BC3, and 13 patients died of infection or organ failure. Administration of the nonmitogenic anti-CD3 antibody BC3 was associated with improvement in the clinical manifestations of GVHD with minimal acute toxicity. Efficacy of antibody treatment did not depend on depletion of circulating T cells. Therefore, antibody BC3 may be achieving therapeutic immunosuppression by modulating T cell function. Controlled studies in patients treated earlier in the course of GVHD should determine whether antibody BC3 can improve survival.

Dogs given total-body irradiation and marrow transplants from DLA-identical littermates exhibit prompt and sustained hematopoietic engraftment. However, animals given three preceding blood transfusions from the marrow donor before transplant become sensitized and reject the marrow graft. Rejection is due to exposure to polymorphic minor non-DLA histocompatibility antigens expressed on blood mononuclear cells. We sought to determine whether heat treatment would prevent blood from sensitizing recipients in this model since heating blood to 45 degrees C for 45 min abrogates the ability of blood mononuclear cells to stimulate in mixed lymphocyte culture. Three of 4 evaluable dogs given heat-treated blood before transplant rejected their marrow grafts. To prevent possible reexpression/reacquisition of mononuclear cell functional activity in vivo after transfusion, subsequent dogs were given heated blood that was additionally exposed to 2000 cGy gamma irradiation. Eight of 10 evaluable dogs given blood treated in this fashion engrafted. Unexpectedly, 9 out of 10 evaluable dogs transfused with blood treated only with gamma irradiation also engrafted. These results demonstrate that treatment of blood with gamma irradiation alone or in combination with heat prevents transfusion-induced sensitization to minor histocompatibility antigens. Results from this canine model suggest that blood products be gamma irradiated before transfusion in patients who are transplant candidates in order to prevent sensitization to minor histocompatibility antigens and reduce the risk of marrow graft rejection.

A murine IgG1 antibody specific for the IL-2-binding site on the human lymphocyte IL-2 receptor beta chain (CD25) was evaluated in 11 patients who developed acute graft-versus-host disease following allogeneic marrow transplantation. All patients had received cyclosporine and methotrexate for prophylaxis of GVHD, either alone (4 cases), or in combination with antithymocyte globulin (4 cases) or with prednisone (3 cases). Patients had developed GVHD at 7-53 days (median 12) after transplantation and had failed treatment with corticosteroids for 3-44 days (median 19). Residual GVHD was of grade II severity in 4 patients, grade III in 5 patients, and grade IV in 2 patients. Sequential patients received monoclonal antibody in escalating doses from 0.1 mg/kg/day to 1.0 mg/kg/day for 7 days. Side effects were fever, respiratory distress, hypertension, hypotension, and chills occurring in 11 of 72 (14%) antibody infusions. Trough antibody levels greater than 6 micrograms/ml were achieved in patients treated with 0.5 or 1.0 mg/kg/day. Four of eight evaluable patients had an IgM antibody response, and one had an IgG response to the murine immunoglobulin. Clinical response of GVHD was evaluated in 10 patients who received the entire course of the antibody treatment. Among 7 patients treated within 40 days from transplantation, one patient had a complete response in the skin as the only involved organ, and 3 patients had a partial response, 2 in the skin and one in the gastrointestinal tract. No responses were achieved with liver disease at anytime or in any organ in patients treated beyond 40 days after transplantation. Since administration of this antibody was well tolerated and some efficacy was observed in patients with acute GVHD treated early after transplantation, there is a rationale for testing this antibody as an agent for prophylaxis of GVHD.

Past studies in dogs have suggested that marrow graft rejection was mediated by major histocompatibility complex (MHC) class II antigen-positive non-T cells that survived standard doses of total-body irradiation (TBI). We have now raised 4 monoclonal antibodies (mAbs) against marrow cells harvested 6 days after TBI. The mAbs are highly reactive (greater than 70%) with marrow cells surviving radiation and also bind strongly (greater than 50%) to normal marrow cells, lymphocytes, monocytes, and granulocytes. One of the mAbs (34-S3) reacted strongly with NK-like cells. In vitro treatment of marrow with mAb and rabbit complement (C') did not affect erythroid colony-forming unit (CFU-E) growth, whereas 2 of the 4 mAbs inhibited granulocyte-macrophage colony-forming unit (CFU-GM) growth, and all 3 mAbs tested suppressed autologous marrow engraftment. One of the mAbs, 69-S5 (IgG1), bound to a 95,000 dalton antigen. It crossreacted with human cells, but not with cells from Rhesus monkeys, baboons, and cats. We administered this mAb intravenously at 0.2 mg/kg/day on days -5 to 0 to dogs given 9.2 Gy TBI on day 0 followed by marrow grafts (less than or equal to 4 x 10(8) cells/kg) from DLA-nonidentical unrelated donors. Three of five dogs had sustained grafts. Increasing the dose of mAb ten-fold (2 mg/kg/day) resulted in graft failure (2 of 2 dogs). Treatment with a dose of 0.2 mg/kg/day from day -7 to -2 showed sustained engraftment in 7 of 10 dogs. This result is in contrast to sustained grafts in 3 of 36 dogs not given mAb, and in 1 of 7 dogs treated with an irrelevant mAb (P = 0.0002 and 0.04, respectively). We conclude that treatment of recipients with a mAb raised against marrow cells surviving radiation and not directed at major histocompatibility complex (MHC) class II antigens and NK-like cells can also facilitate engraftment of DLA-nonidentical canine marrow. These results may be relevant for the transplantation of HLA-incompatible marrow in man, particularly after in vivo T cell depletion, where graft failure is frequent.

We examined the effect of methotrexate (MTX) combined with cyclosporine (CSP) on the prevention of graft-versus-host disease (GVHD) in dogs. Ten recipients were prepared for grafting with 9 Gy of total-body irradiation and given marrow and buffy coat cells from littermate donors differing for one DLA haplotype (AA leads to AB or AB leads to AC). MTX (0.4 mg/kg) was given i.v. on days 1, 3, 6, and 11. CSP was given i.m. on days 0 to 7 and orally on days 8 to 100. The dose was 15 mg/kg/day on days 0 to 25, 10 mg/kg/day on days 26 to 50, 5 mg/kg/day on days 51 to 75, and 5 mg/kg every other day on days 75 to 100. All ten dogs had sustained engraftment. Three dogs died, one with pneumonia (day 57), one with pneumonia and GVHD (day 107), and one with convulsions (day 204). Seven dogs are surviving at 210 to 493 days, and all are complete chimeras; five are well and two have chronic GVHD that developed after immunosuppressive treatment had been stopped. These results with a combination of MTX and CSP as GVHD prophylaxis are superior to those obtained previously with MTX alone in dogs given marrow grafts from DLA-haploidentical littermates.

Marrow transplants were carried out between unrelated DLA-nonidentical dogs. Recipients were prepared for transplantation by 9.2 Gy single-dose total-body irradiation (TBI) administered at 7 cGy/min. All recipient dogs received marrow, 4 X 10(8) cells/kg, and no buffy coat cells. In addition to TBI, 19 dogs were given anti-class II monoclonal antibody (mAb) 7.2, either alone or combined with a second anti-class II mAb, HB10a, at doses of 0.2 or 0.4 mg/kg/day on days -5 to 0 or -1 to +3. Seven had hemopoietic engraftment. Ten dogs were given, in addition to TBI, methotrexate (MTX), 0.4 mg/kg/day on days 1, 3, 6, and 11 after grafting, and six had engraftment. Ten dogs were given a combination of mAb 7.2 pregrafting and MTX after grafting, and 9 had evidence of engraftment. Among 7 dogs treated with the irrelevant mAb 31E6.4, only one had engraftment. Thus the administration of anti-class II mAb pregrafting and MTX postgrafting had a beneficial effect in overcoming resistance to histoincompatible marrow grafts. Possibly this effect is more pronounced when both approaches are combined.

Incubation of canine marrow and peripheral blood mononuclear cells with L-leucyl-L-leucine methyl ester resulted in the inhibition of mitogen- and alloantigen-induced blastogenesis, the elimination of allosensitized CTL and NK activity, and prevented the development of CTL from pCTL. The effects of these incubations were similar to those described in mice and humans. Additionally, in vitro CFU-GM growth from treated canine marrow was reduced, but could be regained when the Leu-Leu-OMe-treated marrow was cocultured with either untreated autologous peripheral blood mononuclear cells or monocyte-enriched PBMC but not with untreated monocyte-depleted PBMC. Six of seven dogs conditioned with 920 cGy total-body irradiation engrafted successfully after receiving autologous marrow that was incubated with Leu-Leu-OMe prior to infusion. These cumulative results indicate that incubation with Leu-Leu-OMe is a feasible method to deplete canine marrows of alloreactive and cytotoxic T cells prior to transplantation.

Ninety-five dogs with spontaneous malignant lymphoma in chemotherapy-induced remission were treated with total-body irradiation (TBI) and bone marrow transplantation. Among 38 dogs treated with 8.4 Gy delivered at 4 cGy/min, 9 (24%) became long-term disease-free survivors. Ten of the 38 (26%) died of transplant-related complications and the actuarial relapse rate was approximately 65%. Forty animals were treated with higher-dose TBI (13.5 Gy). The higher-dose TBI led to an increased incidence of transplant-related deaths (55% vs. 26%) and did not reduce the actuarial relapse rate. Eight animals were treated with 8.4 Gy at 4 cGy/min, allogeneic marrow from unrelated donors, and posttransplant immunosuppression with methotrexate and cyclosporine. Of 8 animals, 6 died within 2 weeks of transplant of infection and 2 died later of graft-versus-host disease. Finally, 9 dogs were treated with 8.4 Gy at 4 cGy/min, autologous marrow, and posttransplant methotrexate and cyclosporine. Six of these animals died within 2 weeks of transplant. These studies thus demonstrated that dogs with malignant lymphoma in remission can be cured with high-dose TBI and autologous marrow transplantation, that increasing the total dose of TBI led to increased toxicity without a decrease in the relapse rate, and that post-transplant therapy with methotrexate and cyclosporine was poorly tolerated in these animals.

The incidence of interstitial pneumonitis (IP) was reviewed in 70 consecutive patients who received autologous marrow transplants for hematologic malignancies. All patients were treated with total-body irradiation (TBI), with or without other chemotherapeutic agents. Seven patients (10%) developed IP, 3 were due to cytomegalovirus, 1 due to Pneumomcystis carinii, and 3 of unknown cause (idiopathic). Risk factors for developing IP were increasing age and a prior history of irradiation to the chest. The use of methotrexate posttransplant did not increase the incidence of IP.

In an effort to produce stable, long-term, complete hematopoietic chimerism in unrelated RLA-matched adult rabbits, the maximum dose of total-body irradiation (TBI) tolerated both with and without autologous marrow support was defined, and it was then determined whether the maximum tolerated dose was sufficiently immunosuppressive and myeloablative to allow engraftment of allogeneic marrow. Forty rabbits received TBI at 4.8 cGy/min at doses from 525-925 cGy. All rabbits receiving 525, 625, 725 and 825 cGy survived without marrow transplantation. At 875 cGy 50% of animals died and at 925 cGy all animals died within 96 hr of TBI. Survival was not changed with autologous marrow support, with all animals receiving 825 cGy surviving, while 50% survived at 875 cGy, and none at 925 cGy. Two RLA-matched, sex and immunoglobulin allotype-mismatched pairs were transplanted following 825 cGy and were given posttransplant cyclosporine. Both recipients survived beyond 200 days posttransplant, with cytogenetic and immunoglobulin allotype evidence of complete, or nearly complete, hematopoietic engraftment. Thus, stable, long-term complete chimerism can be achieved in adult RLA-matched unrelated rabbits prepared by TBI, and this animal model can be used for bone marrow transplantation studies.

Twelve dogs with spontaneous malignant lymphoma in chemotherapy-induced remission were treated with total-body irradiation (TBI) and transplantation of autologous peripheral blood mononuclear cells collected following chemotherapy-induced expansion of the stem cell pool. Four animals (33%) died of transplant-related complications, five (41%) relapsed, and three (25%) are long-term disease-free survivors. Recovery to 1000 white cells/mm3 occurred by day 16 and dogs no longer required platelet transfusions by day 37. With the exception of delayed platelet recovery these results are virtually identical to those previously reported using autologous bone marrow transplantation for canine lymphoma. This study therefore suggests that autologous peripheral blood mononuclear cells collected following chemotherapy-induced expansion of the stem cell pool may offer a realistic alternative to autologous marrow transplantation in patients with malignant lymphoma for whom autologous marrow is not available.

Forty-eight patients with hematologic malignancies treated by allogeneic marrow transplantation developed acute graft-versus-host disease (GVHD), grades II-IV, despite prophylaxis with methotrexate. They were treated with a combination of antithymocyte globulin (ATG) and cyclosporine (CsA), with or without the addition of methylprednisolone (MP). Thirty patients had received HLA-identical and 18 HLA-nonidentical transplants. Median onset of GVHD was day 13 (range 8-60) for patients with HLA-nonidentical grafts and day 18 (range 7-48) for patients given HLA-identical grafts (P = 0.01). Forty-five patients could be evaluated for response on day 7 of therapy. Among these, 13 of 27 given ATG/CSP and 6 of 18 given ATG/CSP/MP improved. Among 33 patients evaluable on day 14 of therapy 13 of 19 given ATG/CSP and 5 of 14 given ATG/CSP/MP showed improvement of GVHD. Patients given HLA-nonidentical grafts responded somewhat (although not significantly) less frequently than patients given HLA-identical grafts. Chronic GVHD developed in 16 of 18 evaluable patients given ATG/CSP and in 5 of 6 given ATG/CSP/MP. Viral, bacterial, and fungal infections were the major cause of death in both groups. Interstitial pneumonitis was more frequent among patients given ATG/CSP/MP. Survival beyond 6 months was 67% among patients treated with ATG/CSP and 25% with ATG/CSP/MP. These data indicate that a regimen of ATG/CSP is of value in the treatment of acute GVHD. The addition of MP was not beneficial and resulted in decreased survival--presumably because of excessive immunosuppression and associated complications.

We determined the incidence of venocclusive disease of the liver (VOD) in 96 recipients of autologous bone marrow transplants (BMT) to be 9.4%, a figure less than that reported for allogeneic transplantation. The development of VOD was compared in a cohort of 21 autologous BMT recipients and in 56 randomly chosen, comparably conditioned, concurrent allogeneic BMT recipients. One of these 21 (4.8%) autologous recipients developed VOD, versus 14 of 56 (25%) allogeneic recipients (P less than 0.05). Logistic regression analysis confirmed pretransplant hepatocellular dysfunction as a risk factor for VOD, and suggested that the use of methotrexate and/or cyclosporine contributes to the development of VOD after chemoradiation therapy. However, a graft-versus-host reaction cannot be excluded as a cause of the higher incidence of VOD in allogeneic recipients.