Total and active rabbit antithymocyte globulin (rATG;Thymoglobulin) pharmacokinetics in pediatric patients undergoing unrelated donor bone marrow transplantation.

Rabbit antithymocyte globulin (rATG; Thymoglobulin) is currently used to prevent or treat graft-versus-host disease (GVHD) during hematopoietic stem cell transplantation (HSCT). The dose and schedule of rATG as part of the preparative regimen for unrelated donor (URD) bone marrow transplantation (BMT) have not been optimized in pediatric patients. We conducted a prospective study of 13 pediatric patients with hematologic malignancies undergoing URD BMT at St. Jude Children's Research Hospital from October 2003 to March 2005, to determine the pharmacokinetics and toxicities of active and total rATG. The conditioning regimen comprised total body irradiation (TBI), thiotepa, and cyclophosphamide (Cy); cyclosporine (CsA) and methotrexate (MTX) were administered as GVHD prophylaxis. Patients received a total dose of 10 mg/kg rATG, and serial blood samples were assayed for total rATG by enzyme linked immunosorbent assay (ELISA) and active rATG by florescein activated cell sorting (FACS). We found that our weight-based dosing regimen for rATG was effective and well tolerated by patients. The half-lives of total and active rATG were comparable to those from previous studies, and despite high doses our patients had low maximum concentrations of active and total rATG. There were no occurrences of grade iii-iv GVHD even in patients having low peak rATG levels, and the overall incidence of grade II GVHD was only 15%. None of the patients had serious infections following transplantation. These data support the use of a 10 mg/kg dose of rATG in children with hematologic malignancies because it can be administered without increasing the risk of graft rejection, or serious infection in pediatric patients with a low rate of GVHD. These conclusions may not apply to patients with nonmalignant disorders.

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. IV. Cytotoxic effect of an anti-T11-gelonin immunotoxin.

The cytotoxic effect of a lymphocyte-specific immunotoxin formed by disulfide conjugation of an anti-T11 monoclonal antibody with the ribosome-inactivating protein gelonin was assessed in vitro on peripheral blood T cells and in vivo on splenic and lymph node T cells of macaque monkeys. This immunotoxin was cytotoxic to proliferating peripheral blood T cells in vitro as measured by both direct and indirect assays. Two sequential intravenous infusions into macaque monkeys achieved plasma concentrations of immunotoxin far in excess of those shown to be cytotoxic for cultured T cells and coated all T cells in lymph nodes and spleen with intact immunotoxin for four days. However, the cytotoxic effect of the immunotoxin on T cells in vivo was considerably less than that predicted by the in vitro studies. Further experiments suggested that the state of activation of the targeted T cell population in vivo, or the appearance of anti-immunotoxin antibodies, which occurred in all infused monkeys, might attenuate immunotoxin-mediated cell killing in vivo. These studies illustrate the significant differences between the action of immunotoxin conjugates in vitro, and those seen when these conjugates are utilized as therapeutic agents in vivo.

Subcutaneous alemtuzumab vs ATG in adjusted conditioning for allogeneic transplantation: influence of Campath dose on lymphoid recovery, mixed chimerism and survival.

Sixty-nine consecutive patients (median age 54 years) were prospectively enrolled in a single-institution protocol for allogeneic transplantation with adjusted non-myeloablative fludarabine-melfalan-based conditioning including cyclosporin A and MMF, and one of three modes of serotherapy. Thirty-one donors (45%) were unrelated. The first cohort of 29 had ATG (Thymoglobulin 2 mg/kg x 3 days), the subsequent 26 had Campath 30 mg x 3 days subcutaneously, and the final cohort of 14 had 30 mg Campath once. The groups were similar as regards age, diagnosis and risk factors. Campath-patients had no acute toxicity, fewer days with fever and antibiotics, and required fewer transfusions than ATG-treated patients. 3-d-Campath patients showed lower lymphocyte counts from day +4, and CD4+, CD8+, CD19+ and NK cells recovered slower than in ATG-treated patients. More Campath patients developed mixed chimerism that required DLI. 3-d-Campath induced more serious and opportunistic infections than ATG, which resulted in a greater non-relapse mortality and an impaired overall survival despite a low tumor-related mortality. The change of the Campath dosing schedule to one dose abrogated the deleterious effect of 3-d-Campath on immune recovery, severe infections and survival. Subcutaneous Campath is simple and provides strong immune suppression with no early toxicity, but dose limitation to 30 mg once is recommended.

Treatment of aplastic anemia with antithymocyte globulin, high-dose corticosteroids, and androgens.

A total of 46 patients with aplastic anemia (34 severe; 12 moderate) were treated with antihuman thymocyte globulin (ATG), high-dose methylprednisolone, and oxymetholone. Early symptoms of ATG toxicity included fever, rash, and bronchospasm. Signs of serum sickness also developed in 23 patients. Complications associated with high doses of steroids were hyperglycemia, hypertension, fluid retention, gastrointestinal hemorrhage, and aseptic necrosis of the hip. Other morbidity possible associated with steroid administration included seizures, arrhythmias, and headache with papilledema. Studies of elevated liver function necessitated discontinuation of androgen therapy in eight patients. A complete or partial hematological response was noted in 19 patients (41%). Of these, three have had recurrent cytopenias, of whom one has developed a myelodysplastic syndrome. There are currently 34 patients surviving, and 12 who have died. Actuarial survival at three years is 65%. These response and survival data are comparable to those of previous trials using ATG and androgens without high-dose steroids. A prospective, randomized trial is needed to determine whether the addition of high-dose corticosteroids to ATG does significantly increase the rate and frequency of response in order to justify the toxicity of this additional immunosuppressive therapy in the treatment of aplastic anemia.

Treatment of severe aplastic anemia using antithymocyte globulin with or without an infusion of HLA haploidentical marrow.

Nineteen patients with severe aplastic anemia were treated with a 4-day course of horse-anti-human thymocyte globulin (ATG). Thirteen of these patients also received an infusion of HLA one haplotype-identical bone marrow. Toxicity of ATG included fever, chills, rash, arthralgias and elevated liver function tests. Platelet transfusion requirements increased during therapy. Eleven patients died 0.2-9.4 months after beginning ATG therapy. None of the 11 patients had any improvement in hematologic status prior to death. The eight surviving patients have been followed for at least 24 months. Six had evidence of hematologic improvement within 6-8 weeks after ATG therapy and are transfusion-independent. The other two patients improved more than one year after treatment. Survival after ATG therapy did not correlate with the presumed etiology of aplasia, duration of aplasia, patient age or sex, prior therapy, or admission granulocyte count. Addition of bone marrow infusion to ATG treatment also did not affect survival. This study demonstrated the necessity for a randomized trial of ATG versus supportive care alone for the treatment of severe aplastic anemia.

In vitro assessment of dose-dependent platelet activation by polyclonal antithymocyte globulins: a flow-cytometric analysis.

Polyclonal antithymocyte globulins (ATGs) are immunosuppressive drugs widely used in transplantation and hematologic disorders. Treatment with ATGs can induce side effects such as neutropenia and thrombocytopenia because of unspecific antibodies directed against nonmyeloid cells present in these preparations. Depletion, activation, and expression of adhesion molecules on platelets in vitro were studied in the whole blood of healthy volunteers by means of flow cytometry after incubation with different doses of three polyclonal ATGs. Our data show no ATG-mediated cytotoxic activity against platelets. ATGs are able to induce activation of platelets through increased expression of P-selectin and hLAMP-1 and higher percentages of gated thrombocytes expressing these molecules. Furthermore, increased expression of hLAMP-1 presented a dose-dependent pattern. ATGs induced activation and enhanced expression of adhesion molecules in unstimulated platelets. Increased adhesion may be responsible for undesirable side effects such as thrombocytopenia and reticulopenia.

Manufacture of antithymocyte globulin (ATGAM) for clinical trials.

Methods are described for preparing large amounts of horse anti-human thymocyte globulin (ATG, ATGAM; The Upjohn Company) for clinical use. These methods have been used since 1968 to provide material for clinical trials. Characteristics of 40 lots of ATG are summarized.

Beneficial and detrimental effects of RBC-adsorbed antilymphocyte globulin and prednisone on purified canine islet autograft and allograft function.

We have examined the effects of prednisone, cyclosporine, azathioprine, and RBC-adsorbed goat antidog antilymphocyte globulin on islet graft function in totally pancreatectomized canines with purified, quantitatively defined, autologous, or allogeneic islets transplanted to the liver. The objectives were twofold: (1) to determine the potential detrimental effects to islet autograft function of the aforementioned agents, and (2) to determine the relative efficacy of the "nontoxic" agents in prolonging purified islet allograft function administered in doses that would be considered tolerable in human. The islet autograft studies demonstrated that prednisone given in doses of 1-2 mg/kg/day had a detrimental effect on islet autograft function, and that the combinations of immunosuppression involving CsA, azathioprine, and ALG were not detrimental to islet autograft function to the extent that hyperglycemia would ensue. In the subsequent allograft studies, three groups of canines received islet transplants: (1) controls (n = 5; 7860 +/- 750 islets/kg/weight), (2) canines given CsA and azathioprine (n = 6; 6810 +/- 890 islets/kg/body weight), and (3) canines given CsA, azathioprine, and RBC-adsorbed goat antidog ALG (n = 8; 6540 +/- 710 islets/kg/body weight). The mean (+/- SE) day of rejection (serum glucose greater than or equal to 200 mg/dl) in the group of canine islet allograft recipients receiving CsA, azathioprine, and ALG was 11.8 +/- 1.4 days--significantly prolonged versus islet allograft recipients receiving no immunosuppression (mean survival 4.8 +/- 1.1 days, P less than 0.03), and versus allograft recipients receiving CsA/azathioprine without ALG (mean survival 4.4 +/- 1.4 days, P less than 0.05). Prednisone appears to be detrimental to islet graft function, even at low doses. ALG was not toxic, and significantly extended the survival of canine islet allografts. The inclusion of steroids as part of maintenance immunosuppression, or as treatment for acute rejection of islets, in human islet transplants should be reconsidered, whereas ALG or other antilymphocyte agents should continue to be used.

Side effects and immunogenicity of murine lymphocyte-specific monoclonal antibodies in subhuman primates.

The immediate side effects of lymphocyte-specific monoclonal antibody treatment of nearly 150 monkeys is documented in this study. Immediate side effects were only seen with antibodies specific for CD3 and CD8. These side effects are most likely related to stimulation of T cells to produce lymphokines (CD3) and/or to the rapid cell clearance (CD3 and CD8). No immediate effects were observed when CD4 or major histocompatibility complex class II-specific antibodies were injected. These antibodies may therefore be considered for the treatment of graft rejection or autoimmune diseases. Of the 43 animals that received a monoclonal antibody (MoAb) at least 2 years and up to 5 years prior to this study, none has shown any late effects of MoAb treatment. Most animals tested had a vigorous immune response to the injected MoAbs, both antiidiotypic as well as anti-isotypic antibodies were formed. This response was reduced by using Fab2 fragments or by additional immunosuppression, but it was still high enough to prevent further effectiveness of the MoAb treatment.

The impact of antithymocyte globulin on short-term toxicity after allogeneic stem cell transplantation.

Antithymocyte globulin (ATG) is commonly used in allogeneic haematopoietic stem cell transplantation (HSCT). Little information is available, however, as to the optimal protocol for use and the side-effects occurring if ATG is administered in high daily doses (10-30 mg/kg). We report our experience with ATG Fresenius (ATG-F) in conditioning for allogeneic HSCT. During a period of 3 days, 47 patients received doses between 10 and 30 mg/kg either over 4 h preceded by 1-1.5 mg/kg prednisolone 30 min before the start of ATG-F (protocol A) or alternatively, over 12 h with 3-4 mg/kg prednisolone being administered before and 6 h after start of ATG (protocol B). During treatment with ATG-F, the side-effects observed included inflammation, disseminated intravascular coagulation, hyperdynamic circulation and renal dysfunction. Although these complications caused substantial morbidity, they were reversible within a few days. Side-effects were significantly more severe in patients treated according to protocol A than in those treated according to protocol B. As prolonged infusion of ATG-F does not reduce T cell clearance due to the long half-life of ATG-F, and since less cytokine release during conditioning might have beneficial long-term effects, we recommend administering ATG-F over 12 h preceded by high-dose steroid treatment.

Evaluation of a monoclonal IgM antibody for purging of bone marrow for autologous transplantation.

A monoclonal antibody of the IgM class, reacting with the CD9 (p24) antigen is described. The antibody (FMC27) is cytotoxic against cells of the common type of acute lymphoblastic leukaemia (c-ALL), giving killing at higher dilutions than an IgG antibody (FMC8) against the same antigen. FMC27 and FMC8 recognise different epitopes, and FMC27 may thus be used in a cocktail together with FMC8 and an antibody against the c-ALL antigen, WM21. Furthermore, the IgM antibody can be coated directly onto magnetic microparticles for magnetic purging, unlike the IgG antibody which must be used in a two-layer procedure.

Immune reconstitution following allogeneic stem cell transplantation in recipients conditioned by low intensity vs myeloablative regimen.

We have investigated the immune status of patients with hematologic malignancies treated with a low intensity conditioning in preparation for allogeneic stem cell transplantation. Conditioning consisted of fludarabine, anti-T lymphocyte globulin and low-dose busulfan, followed by infusion of allogeneic blood stem cells. This protocol resulted in rapid engraftment and complete replacement of host with donor hematopoietic cells. Immunological parameters of these patients were compared to those patients who were conditioned by an aggressive myeloablative regimen. Distribution of cell surface markers of lymphocyte subsets from both groups of patients was similar, but different from that of normal control cells. Reduced intensity or non-myeloablative conditioning prior to allogeneic stem cell transplantation (NST), hardly lowered the normal T cell-dependent mitogenic response even during the early period following transplant, while the myeloablative treatments resulted in a suppressed mitogenic reaction and in slow immune recovery. Reactivity of non-MHC restricted cytotoxic T cells was also at a normal level in patients who were treated with NST. We conclude that stem cell engraftment following reduced conditioning may result in early reconstitution of immune responses assessed in vitro. We hypothesize that clinical application of NST may lead to faster development of effective immune responses against residual host-type malignant and abnormal non-malignant hematopoietic cells, although the role of fludarabine on post-transplant infections remains to be investigated in a larger cohort of patients.

Effect of anti-thymocyte serum on acquisition of resistance to infestation by Rhipicephalus appendiculatus larvae in rabbits.

Administration of specific goat anti-thymocyte serum (ATS) to rabbits, prior to a primary infestation by Rhipicephalus appendiculatus larvae, blocked the acquisition of resistance significantly only in the third infestation. The larvae which fed on these rabbits had higher engorgement masses than did those feeding on untreated control rabbits. Also, a higher percentage (92%) of larval ticks fed on these animals than on the controls (88%). ATS also induced a leucopenia due to a lymphopenia in the treated rabbits. It was concluded that a T-cell-dependent component might be involved in acquired resistance to infestation by R. appendiculatus.