Antibodies in the Treatment of Aplastic Anemia

Antibodies have been the cornerstone of treatment of acquired aplastic anemia for more than 25 years. Treatment with antithymocyte globulin (ATG) is considered pivotal and the addition of cyclosporine improves the overall response rate. This antibody is heterogeneous and horse ATG is apparently more effective than rabbit ATG. Several issues remain unsolved in relation to the combination of ATG and cyclosporine: cost, toxicity and late clonal disorders. In recent years, alternative immunosuppressive therapy has been proposed and new antibodies have emerged: porcine ATG, alemtuzumab, daclizumab, and rituximab. Experience with these antibodies is limited to a few studies with alemtuzumab being the most promising, but the results are interesting and provocative. More studies are needed to find the perfect antibody.     

Therapy of severe aplastic anemia with anti-human thymocyte globulin (ATGAM) with and without HLA-haploidentical bone-marrow infusion

Six patients with severe aplastic anemia treated with horse anti-human thymocyte globulin (ATG) and androgen. Four of these patients were only given ATG (ATGAMR), 16 mg/Kg/dose x 10 doses. The remaining two cases received an infusion of maternal HLA-haploidentical marrow cells following ATG therapy. One patient had a complete response, three had a partial response, one showed minimal improvement and two were non-responders. The two patients who received the additional haploidentical marrow cells showed a hematologic recovery sooner than the ATG alone cases. The toxicity of the ATG therapy was tolerable. Long term follow up of there patients and further studies of this treatment in aplastic anemia with pediatric age group are under way.     

Heterogeneous clearance of antithymocyte globulin after CD34+-selected allogeneic hematopoietic progenitor cell transplantation.

Antithymocyte globulins (ATG) are purified, concentrated preparations of polyclonal immunoglobulin G from hyperimmune serum of horses or rabbits immunized with human thymus lymphocytes. Both the horse and the rabbit products induce immunosuppression as a result of lymphocyte depletion and immune modulation. The exact mechanism of action is unknown but may include T-cell clearance from the circulation and modulation of T-cell activation, homing, and cytotoxic activities. Both horse and rabbit ATG include multiple antibodies against T-cell surface antigens and have been used extensively in allogeneic hematopoietic progenitor cell transplantation (HPCT) for the treatment and prevention of graft-versus-host disease or graft rejection. To quantify the active ATG after HPCT, we developed a flow-based assay to measure residual ATG capable of binding to lymphocytes. In contrast to prior assays that measure total rabbit or horse immunoglobulin, this assay quantitates only the antibody capable of binding to lymphocytes, which presumably reflects the functionally active fraction of the xenoantiserum. Thirty patients with hematologic malignancies underwent T cell-depleted HPCT and had ATG levels assayed during the peritransplantation period. The time required for ATG levels to decay to background was quite variable (mean, 46 days; range, 14-91 days), although most patients demonstrated a rapid early clearance followed by a slower decline. The actual mean half-life was 6.8 days (range, 2.4-14.0 days). The persistence of ATG for months after administration has significant implications for the pace of immune reconstitution after transplantation and is a potentially confounding variable in any study that involves early administration of donor lymphocyte infusions or other cellular transfer. These findings indicate that ATG levels should be explicitly measured in studies that involve early donor lymphocyte administration so that proper conclusions regarding dose, safety, and efficacy can be reached.     

Immune monitoring of anti-thymocyte globulin (ATG) treatment in transplant patients

Over the past few years there has been an increasing array of monoclonal antibodies directed against immunocompetent lymphocytes available for use in the prevention and treatment of rejection in solid organ transplantation. Despite this there has been a continued and expanding use of polyclonal anti-T cell globulins in the management of cardiac, lung and renal transplants in many centres. As with all immunosuppressive drugs over and under-immunosuppression remains a major problem. A number of different methods have been described for monitoring anti-thymocyte globulin (ATG) therapy in transplant patients. These include: monitoring serum IgG levels, the E- rosette assay; flow cytometry and monoclonal antibodies. The measurement of serum IgG levels of the host animal (rabbit or horse) has been shown not to correlate with the presence of acute rejection episodes. The E-rosette assay has been found to be extremely time consuming and unreliable. With flow cytometric techniques, monoclonal antibodies directed against the T lymphocytes have been used to monitor ATG therapy and this has allowed the adjustment of the ATG administered to each individual patient. In patients receiving, renal, cardiac, lung or heart lung transplants we have developed flow cytometric assays to monitor ATG therapy on a daily basis with adjustment of the dosage of ATG administered according to this result. We have progressed from a dual platform assay where the T-cell percent from flow cytometry is combined with the lymphocyte count determined from a Haematology Cell Analyser to a single platform flow cytometric assay using commercially available polyfluorescent beads of two different types.An increasing problem with transplant patients is that ATG once given may induce antibodies against the heterologous rabbit or horse IgG. With time, a number of renal, cardiac and lung transplant patients have lost their grafts and are retransplanted. A similar situation is seen in patients who are given regular ATG therapy for the treatment of aplastic anemia. We have developed a flow cytometric duplex assay for the detection of such antibodies. This has allowed us to screen the sera of transplant patients for the presence of such antibodies before therapy is given and to suggest for that patient the most appropriate ATG to be used. The cellular and humoral assays we have developed are rapid and reproducible and are now used as routine in the clinical management of patients who are receiving or about to receive ATG therapy in transplantation.     

Studies on antilymphocyte antibody in the chicken. I. Effect of rabbit antithymus and antibursa globulin on chicken embryos

Antithymus globulin (ATG) and antibursa globulin (ABG) prepared in rabbits with thymus and bursa cells were used in experiments on New Hampshire embryos. The in vitro assay of potency of ATG and ABG by means of absorption, leucoagglutination, cytotoxicity test and passive haemagglutination showed that ATG and ABG react equally well with both thymus and bursa antigens.

Seven-day-old embryos were treated with eleven intrachorioallantoic injections of ATG, ABG and NRG (normal rabbit globulin) from the seventh to seventeenth day of incubation. The incidence of death in embryos receiving ATG and ABG was very high when compared with controls injected with NRG and saline. The ATG affected the cellular make-up of the spleen and bursa, while ABG influenced only the bursa. The cytotoxic-like and development-retarding activity of ATG and ABG were possible only in the presence of guinea-pig complement. The embryonic thymus proved to be resistant to the action of heterologous antilymphocyte antibody.

     

Studies on antilymphocyte antibody in the chicken. II. Effect of antithymus and rabbit antibursa globulin on immune reactions in young chickens

Four-week-old chickens repeatedly injected intraperitoneally with heterologous antithymus globulin (ATG) and antibursa globulin (ABG) were immunized, in the course of treatment, with bovine γ-globulin (BGG). The ATG and ABG depressed the production of anti-BGG antibody. On the other hand, only ATG was effective in suppressing experimental allergic encephalomyelitis. Immunoelectrophoretic analysis of sera from chickens treated with ATG, ABG and NRG (normal rabbit globulin) revealed that those globulins are themselves immunogenic. ATG and ABG induced in the spleen a moderate depletion of lymphocytes and plasma cells respectively. These reagents produced lymphocytopenia and granulocytopenia, but a similar, although less expressed, effect on leucocytes in the peripheral blood could be induced by NRG. Thymus, bursa, caecal tonsil, Peyer's patches and lymphoid masses of the intestine were not influenced by ATG, ABG and NRG. The results are discussed and interpreted as further evidence for the delineation between immune functions of the thymus and the bursa of Fabricius in the chicken.     

Activation of human platelets by antibodies to thymocytes and beta 2-microglobulin. I. Qualitative and quantitative aspects of the platelet aggregation induced by HATG and SA beta 2mG.

The platelet effect of antibody preparations known to have immunosuppressive action was investigated by a turbidimetric method in vitro. Both horse anti-human thymocyte globulin (HATG) and sheep anti-human beta 2-microglobulin IgG (SA beta 2mG) caused the platelets to aggregate in all human platelet-rich plasmas (PRP) tested. The aggregation was usually irreversible and characterized by a sigmoid curve. The action is specific for the antibodies in HATG and SA beta 2mG because control preparations (horse normal IgG and IgG fractions of sheep normal, anti-dog IgG and anti-human IgA sera) were ineffective; further, heating (30 min at 56 degrees C) and BaSO4 or Al(OH)3 adsorption of HATG and SA beta 2mG did not alter their aggregating capability. When HATG and SA beta 2mG were added together to PRP, they induced aggregation in a simple additive manner. High antibody doses tended to decrease the extent of aggregation. The effect of platelet count on aggregation varied with both the dose level ('low' or 'high') and type (HATG or SA beta 2mG) of the inducer antibody. Using fixed submaximal doses, four main aggregation patterns could be recognized among 60 PRP: (i) high responses to both HATG and SA beta 2mG; (ii) high to HATG, low to SA beta 2mG; (iii) low to HATG, high to SA beta 2mG; and (iv) low to both. The results provide guidelines for quantitative aggregation studies with platelet antibodies and suggest that HATG and SA beta 2mG act through distinct platelet membrane components, the receptor for the latter being the best characterized protein of the mammalian cell membrane.     

High Serum Level of Antithymocyte Globulin Immediately before Graft Infusion Is Associated with a Low Likelihood of Chronic,But Not Acute,Graft-versus-Host Disease

Rabbit antithymocyte globulin (ATG) is administered during transplant conditioning to decrease the risk of both acute graft-versus-host disease (aGVHD) and chronic graft-versus-host disease (cGVHD). Here we evaluated the relationship between the serum concentration of ATG (capable of binding to lymphocytes) immediately before graft infusion (day 0) or on day +7 or +28 post-transplantation and the development of aGVHD or cGVHD. We studied 180 patients whose conditioning included 4.5 mg/kg antithymocyte globulin (ATG; Thymoglobulin). For aGVHD, we found no association with ATG levels on day 0. Nevertheless, high day +7 and +28 ATG levels were associated with a low likelihood of aGVHD. For cGVHD, high ATG levels at all 3 time points (days 0, +7, and +28) were associated with a low likelihood of cGVHD. In conclusion, high-dose ATG administration at the time of graft infusion appears to inhibit the development of cGVHD, but not aGVHD; however, higher ATG levels on days +7 and +28 are associated with lower rates of both aGVHD and cGVHD.     

Reverse Passive Cutaneous Anaphylaxis in the Guinea Pig with Horse, Sheep or Hen Antibodies

Reverse passive cutaneous anaphylaxis experiments were performed in guinea pigs with rabbit gamma globulin and human gamma globulin as antigens and horse, sheep or hen sera containing antibodies against these antigens. These antibodies were unable to directly sensitize the guinea-pig skin but when the reverse technique was used, namely, the antigen (gamma globulin) was injected before the antibody, characteristic anaphylactic reactions were obtained. It is concluded that, if a suitable antigen is used, which can be fixed to the host by the reverse technique, sera from species which cannot directly sensitize the guinea pig can nevertheless give typical anaphylactic reactions.     

Antithymocyte globulin is associated with complement deposition in cardiac transplant biopsies

Polyclonal antithymocyte globulin preparations contain antibodies with reactivity to endothelial cells. Therefore, we investigated whether treatment with this reagent caused complement deposition in human cardiac transplants. Frozen tissue was available from endomyocardial biopsies of 75 patients, who were transplanted between April 1995 and April 2000. Nine of these patients were converted from cyclosporin A (CsA) to horse antithymocyte globulin (ATGAM) in the first month after transplantation. All of the biopsies were stained by immunofluorescence for C4d as evidence of activation of the classical pathway of complement. In addition, biopsies from patients treated with ATGAM and control patients were stained for deposition of horse immunoglobulin (Ig)G. All nine patients who received ATGAM had deposition of horse IgG and C4d. Two color stains demonstrated that the horse IgG colocalized with the C4d staining. No staining for horse IgG or C4d was evident in biopsies obtained before ATGAM treatment. Likewise, no staining for horse IgG was detected in seven control patients who had C4d staining. Most patients treated with ATGAM had no histologic evidence of rejection, but did have myocyte damage and macrophage infiltration. Thus prophylactic treatment with ATGAM is associated with the deposition of horse IgG and activation of complement in the transplant.     

Case reports of evaluation and desensitization for anti-thymocyte globulin hypersensitivity.

BACKGROUND: Biologic products of heterologous sera have been used to treat a variety of conditions. One example is anti-thymocyte globulin (ATG), which is approved for use in the management of renal transplantation and for aplastic anemia. As ATG is a product of heterologous sera it has the potential for adverse reactions, including anaphylaxis. Patients can be skin tested prior to ATG administration to aid in determining hypersensitivity status to ATG. OBJECTIVE: To provide case reports to illustrate evaluation for ATG hypersensitivity. Also, to discuss desensitization procedures for patients who are found to have ATG hypersensitivity, and yet are to receive the medication as it is judged to be essential. CASE REPORTS: We report four patients who were to receive ATG. The results of skin testing and each patient's response to ATG are reviewed to illustrate problems that can occur in evaluating the hypersensitivity status of these patients. Further, some patients also underwent ATG desensitization, but none completed the entire protocol successfully. Their outcomes are reviewed to illustrate problems that can occur with the desensitization procedure. CONCLUSION: Anti-thymocyte globulin is a product of heterologous sera and has the potential to produce anaphylaxis. It is recommended that patients be skin tested prior to administration to aid in determining hypersensitivity status. Those patients who demonstrate hypersensitivity to ATG should not receive ATG unless it is deemed essential and benefits are judged to outweigh risks. In these circumstances, patients are candidates for ATG desensitization. Complications with desensitization occurred in the cases attempted, and highlights that desensitization to ATG, a xenogeneic protein, carries risk and can be difficult. Physicians involved in such cases should be familiar with interpretation of skin tests and problems that can occur with desensitization.     

Uptake of antilymphocyte globulin by human peripheral lymphocytes

The uptake by human peripheral lymphocytes of horse anti-human lymphocyte globulin was studied using a fluorescein-labelled sheep anti-horse globulin serum. The aim was to see whether a subpopulation of B cells, which can be identified by their abundant surface immunoglobulin, would take it up less readily than T cells. No difference in uptake could be detected between B and T cells.     

Interaction of gelatin with stereospecific binding proteins and its enhancement of competitive binding assays

The effect of gelatin (0.5 g/l) on binding curves at high dilution of three classes of stereo-specific binding proteins was studied. These included two antibodies (to oestradiol and aldosterone), six transins (horse and dog transcortins, human thyroxine-binding globulin, human sex steroid-binding globulin, and guinea pig transprogestin), and one receptor (bovine adrenal protein kinase). Gelatin increased the apparent binding of all these proteins, particularly at the highest dilutions and sometimes in a striking manner. While much of this action can be attributed to its decreasing the adhesion of the dilute binding protein to glass, gelatin also increased the apparent uptake of some tracers by certain adsorbents. Similar findings were obtained using human gamma globulin (2 g/l). These effects resulted in increased sensitivity and improved reproducibility in the assays employing them.     

Reduction in false antigenemia in the second generation Histoplasma antigen assay.

We have previously reported false Histoplasma antigenemia in solid organ transplant patients who received rabbit antithymocyte globulin (ATG). False antigenemia was caused by human anti-rabbit antibody, produced in response to ATG administration. Those specimens were obtained through a research study to assess the development of anti-rabbit antibodies following ATG administration. Here we compare the likelihood of false antigenemia between the original and second generation assays in clinical specimens obtained specifically for the diagnosis of histoplasmosis. Of 12 serum specimens that were falsely positive in the original assay, four (33%) remained positive in the second generation assay. The sensitivity was 98% in urine was and 80% in serum in patients with AIDS complicated by disseminated histoplasmosis. False-positive results were not observed in serum or urine from healthy subjects. Thus, the second generation assay is less susceptible to false antigenemia caused by anti-rabbit antibodies.     

Antithymocyte globulin treatment of retrovirus-induced feline erythroid aplasia: in vivo and in vitro studies

The Kawakami-Theilen strain of feline leukemia virus (FeLV-KT) was used experimentally to produce erythroid aplasia in cats. The in vivo effects of goat anti-feline-thymocyte globulin (ATG) on hematopoiesis were investigated in FeLV-negative normal and FeLV-positive anemic cats. Treatment was initiated in anemic cats between 4 and 6 weeks postinoculation (PI) when erythroid progenitors were reduced to 10% of normal levels. During the first 2 weeks of treatment, ATG significantly increased the numbers of erythroid precursors in bone marrow from 15 to 35% in anemic cats and from 28 to 43% in normal cats. ATG stimulated a twofold increase of CFU-E and a threefold increase of CFU-GM in normal cats between 2 and 4 weeks after initiation of treatment but had no effect on CFU-E or CFU-GM in anemic cats. The in vivo effects of ATG were transient despite weekly treatment. Cats treated with normal globulin were not significantly different from untreated anemic control cats. In vitro treatment of low density bone marrow mononuclear cells with ATG plus complement increased CFU-E and BFU-E of bone marrow from cats prior to inoculation but not from viremic cats. These results indicate that, although ATG stimulates erythropoiesis and granulopoiesis in normal cats, it does not reverse retrovirus-induced erythroid aplasia.     

Anti-horse globulin antibodies in human sera

Organ transplant patients who had received ALG, patients with rheumatoid arthritis and normal persons were studied for serum antibodies to horse globulins. Although normal individuals rarely show the presence of anti-horse antibody, rheumatoid patients with high titres of rheumatoid factor usually show anti-horse globulin agglutinins in their IgM globulins and these agglutinins are considered to be a manifestation of their `rheumatoid factors'. These agglutinins are readily absorbable with aggregated human γ-globulin. On the other hand, although most transplant patients administered ALG produce anti-horse antibody, it is usually produced as an IgG globulin and it is not absorbable with aggregated human γ-globulin.     

The identification of allo-and auto-lymphocytotoxic antibodies in serum, in the presence of rabbit antithymocyte globulin

An enzyme-linked immunosorbent assay (ELISA) for rabbit immunoglobulin (Ig) was developed in order to investigate serum levels achieved by therapeutic doses of rabbit antithymocyte globulin (ATG) and their relationship to in vitro serum lymphocytotoxic activity. Twenty renal allograft recipients treated for acute steroid resistant rejection with ATG were studied, where possible, before, during and following their treatment with ATG. During therapy, peak serum levels of rabbit Ig were in the range 20-101 micrograms/ml with one exception of 404 micrograms/ml. After treatment, levels gradually declined to zero within 12 weeks. All sera with lymphocytotoxic activity were absorbed with platelets and treated with dithiothreitol so that reactivity due to anti-HLA antibodies and autoantibodies produced by the patient could be differentiated from each other and from serum ATG. Nine patients had detectable serum ATG associated with in vitro lymphocytotoxic activity. In two cases the lymphocytotoxicity was attributed solely to ATG; two had only anti-HLA antibodies; in one patient the lymphocytotoxicity was due to a combination of ATG and anti-HLA antibodies; two had autoantibodies; and for two, cytotoxicity was initially due to ATG and subsequently to the development of autoantibodies. The serum levels of ATG achieved during treatment could thus be quantified, and the important distinction between anti-HLA antibodies, autoantibodies and ATG clearly made.     

Granulocyte-macrophage colony-stimulating factor in the sera of patients with aplastic anemia

Summary To clarify the role of growth factors in the pathophysiology of aplastic anemia we measured serum granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in 33 aplastic anemia patients by a specific and sensitive enzyme-linked immunosorbent assay. GM-CSF serum levels of patients with aplastic anemia were significantly higher than in healthy volunteers. GM-CSF levels were correlated with the severity of aplastic anemia but not with the absolute neutrophil count. Since T lymphocytes are one of the main sources of GM-CSF, our data provide further evidence for in vivo T lymphocyte activation in aplastic anemia. GM-CSF serum levels are higher in patients responding to immunosuppressive treatment than in nonresponders. Elevated serum GM-CSF might be predictive of a good response to immuno-suppressive therapy. GM-CSF serum levels are lower immediately after treatment with antilymphocyte globulin/antithymocyte globulin (ALG/ATG) than corresponding pretreatment values. Thus we cannot confirm the hypothesis that ALG/ATG effects in vivo are mediated by stimulating the release of growth factors. We conclude that in aplastic anemia the primary defect is a failure in GM-CSF response rather than in GM-CSF supply.Abbreviations AA aplastic anemia - ALG antilymphocyte globulin - ATG antithymocyte globulin - CyA cyclosporine A - ELISA enzyme-linked immunosorbent assay - Epo erythropoietin - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor - IFN- interferon- - IL interleukin - MP methylprednisolone - nSAA nonsevere aplastic anemia - rh recombinant human - SAA severe aplastic anemia - TNF- tumor necrosis factor- Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

T-cell depleting antibodies: new hope for induction of allograft tolerance in bone marrow transplantation?

Graft versus host disease (GVHD) remains the main barrier to successful allogeneic bone marrow transplant outcomes. Depletion of graft T cells is an effective way of reducing the incidence of acute and chronic GVHD, and a variety of methods have been used to achieve this depletion. Donor CD8+ T cells seem to be the critical effector cells; GVHD is reduced when the depletion process eliminates these cells, but not when CD4 cells are targeted alone. However, despite the successful reduction in GVHD, transplant outcomes are usually inferior with T-cell depleted transplants, because of increased graft failure, infections and relapse. Alternative approaches are needed. In vivo T-cell depletion, using antithymocyte globulin (ATG) as part of the conditioning regimen, seems an attractive option. Pre-transplant ATG lingers in the bone marrow to deplete engrafting donor T cells, but also depletes host T cells to prevent graft rejection and allow de-escalation of the conditioning regimen. It also avoids the need for graft manipulation with its associated costs, need for expertise and CD34+ cell loss. The efficacy of pre-transplant horse ATG remains anecdotal but it has been reported to modestly lower GVHD in single arm studies. Rabbit ATG has been studied in prospective randomised trials. There is evidence of a dose-response effect in reducing GVHD; however, there was no improvement in outcome, because of increased mortality associated with infection. In contrast, pre-transplant alemtuzumab (campath-1H) or an earlier version of this molecule (campath-1G), which target CD52+ cells, do appear to be effective in reducing both acute and chronic GVHD. There is speculation that this is not solely due to the effect of campath on T cells but that it may also be due to the elimination of host antigen-presenting cells (APC), which seem to be important in GVHD pathogenesis. Host APC are more efficient at expressing endogenous and exogenous host antigens on class I MHC to donor CD8+ cells than donor APC, which need to cross-prime exogenous antigen. Campath-1G eliminates host dendritic cells by the time of graft infusion, supporting this as a possible mechanism of action. Pre-transplant alemtuzumab has not yet been studied in a prospective randomised study, and this is required to quantify any benefit on outcome; despite this, published studies do show cause for optimism.     

Crossreaction of antilymphocyte globulin with human granulocyte colony-forming cells.

Clinical preparations of horse antilymphocyteglobulin (ALG) were found to inhibit human bone marrow granulocyte colony growth. This effect was enhanced by complement and was dose dependent, being almost complete at ALG concentrations of 100 microgram/ml. Inhibition was a property of ALG but not of normal horse globulin. However, short incubation of ALG with bone marrow cells occasionally stimulated colony growth and normal horse globulin regularly stimulated it. Three hours' incubation of bone marrow cells with ALG was needed to produce consistent colony inhibition, which was measurable as a reduction in the expected number of colonies and as a fall in the colony: cluster ratio of surviving cell aggregates. Absorption of ALG on acute myeloid leukaemia blast cells removed the inhibiting property of the ALG while preserving its lymphocytotoxic action. Serum from two patients receiving ALG treatment inhibited colony growth for up to 48 hours after ALG administration. The results suggest the presence in ALG of antibodies specifically cytotoxic to myeloid stem cells which may relate to its myleosuppressive properties in vivo, and also indicate that it should be possible to remove antimyeloid antibodies from ALG by absorption. The use of such purified ALG would have advantages in clinical bone marrow transplantation.