Graft versus host disease

Abstract: Reconstitution of an individual's haemopoietic stem cells by bone marrow transplantation (BMT) is the recommended treatment for a number of haematological conditions, both malignant and non-malignant. Despite evolution in BMT technology over the past forty years, graft-versus-host disease (GvHD) remains a major, potentially lethal complication. GvHD normally affects the skin, liver and gastrointestinal tract, resulting in a high rate of morbidity. The standard prophylaxis for GvHD is a combination of methotrexate and cyclosporin A, but this is only partially effective. Acute GvHD is difficult to treat and many patients are resistant to steroid therapy. Alternative methods of prevention and treatment are now being sought, and include monoclonal antibodies (MAbs) which target T cells and cytokines. T-cell depletion of donor marrow using rat MAbs reduces the incidence of GvHD but can increase the chances of leukaemic relapse. Mouse MAbs also have been used but some produce severe side-effects. The most successful MAbs are those linked to toxins, and these immunotoxins (IT) have proved very effective in reversing steroidresistant acute GvHD. MAb therapies are becoming increasingly important in the treatment and prevention of GvHD, and could replace steroids as the main treatment option in some situations. It is predicted that, ultimately, peripheral blood stem-cell transplantation will replace the use of BMT; however, this alternative stem-cell source will not remove the GvHD risks associated with allogeneic stem-cell transplantation. Therefore, reducing the risks will remain a major challenge in the successful allogeneic transplantation of haemopoeitic stem cells for the foreseeable future.

Key words: Antibodies, monoclonal. Bone marrow transplantation. Graft vs host disease.

Introduction

Bone marrow (BM) was first used as a clinical treatment for the victims of an irradiation accident' and is now an established treatment for numerous haematological conditions, including aplastic anaemia and acute and chronic leukaemia.'

Bone marrow can be obtained from a variety of sources. In some haematological conditions, such as leukaemia, the patient's own BM is used; recently, however, this has been overtaken by the use of autologous stem cells (CD34+) harvested from peripheral blood (PBSC). Material sourced from other individuals can take four forms. One form carries no immunological risk and involves BM donation from an identical twin (syngeneic transplantation). The other three forms of bone marrow transplantation (BMT) are referred to as allogeneic and involve donations from human leucocyte antigen (HLA)-matched siblings, other HLA-matched close relatives, or from unrelated donors-ideally matched, but often unmatched.

Early BMT experiments-reviewed by Billingham in 19663-described a wasting disease that occurred when mice received a transplant from unrelated mice. This complication is now accepted as a description of murine graft-versus-host disease (GvHD). Despite advances in tissue typing, GvHD remains a major cause of disease and morbidity in BMT recipients, and occurs in up to 70% of patients receiving HLAmatched transplants, with a mortality rate of approximately 20%.4 Some 60% of patients requiring BMT do not have a suitable HLA-matched siblings and, consequently, increasing numbers of partially matched, related or unrelated, transplants are performed. Recently, trials using unrelated BMT in acute myeloid leukaemia (AML)6 have commenced, and it seems likely that BMT and/or allogeneic PBSC transplantation will continue to increase. The occurrence of GvHD is greater when donor and recipient are only partially matched, compared with that for matched BMT.'

Effective GvHD therapy should have a number of qualities. Mortality rates due to acute GvHD and morbidity associated with chronic GvHD both should be reduced. When used as part of a therapeutic protocol in leukaemia, treatment should not affect the graftversus-leukaemia effect adversely, otherwise graft rejection will increase and the chance of leukaemic relapse will rise.8 Prophylactic therapy against GvHD typically has used combinations of immunosuppressive agents-for example, the cytotoxic agent methotrexate (MTX) in conjunction with cyclosporin A (CsA)administered after transplantation. However, these drugs are non-specific in their immunosuppressive action and their use can lead to opportunistic infection by pathogens. Steroids are also used to treat GvHD but cases of steroid-resistant acute GvHD are common.9

Monoclonal antibodies (MAbs) are produced against a wide variety of cell-surface receptors and antigens expressed by different cell types, and this technology permits the targeting of effector cells in numerous diseases, including GvHD. Rat MAbs have been employed widely for T cell depletion of donor marrow. Good results have been reported for the prevention of GvHD,'>' but there have been problems with increased graft rejection." Rat MAbs also have been used to treat acute GvHD.12

Despite success in reversing rejection in renal transplants,'3 some antibodies (both mouse and rat) failed to achieve significant results, and in some instances were associated with severe adverse reactions.'4 Human MAbs have been produced","16 and one such antibody (Campath-1H) has been used to induce remission in non-Hodgkin's lymphoma.'s

As an alternative to relying on complement- or effector-cell-mediated, targeted cell death, MAbs have been conjugated to various toxins to produce immunotoxins (ITs), and their efficacy has been demonstrated both in vitro and in vivo.1-19

Pathophysiology of graft-versus-host disease

Development of GvHD in BMT patients is initiated by donor T cells which recognise minute differences between donor and recipient major histocompatibility complex (MHC). Once T cells identify foreign peptides in the MHC, a graft-versus-host (GvH) reaction occurs." A second co-stimulatory signal is then needed and this triggers an immunological reaction (GvHD), a partial reaction, or a shut down of response (anergy).11 If activation occurs, interleukin-2 (IL-2) is secreted and the IL-2 receptor on the donor T cells is stimulated, resulting in clonal expansion and proliferation.

Cytokines, such as IL-I and tumour necrosis factor a (TNFa), are released by T cells and monocytes, and these recruit effector cells (e.g. natural killer cells). These cytokines produce tissue damage and further activation, resulting in the release of more cytokines. Therefore, although donor T cells initiate the GvH response, the disease is mediated primarily by the host's cells. In BMT, the risk of GvHD is increased because the host is either inherently immunologically incompetent or deliberately immunosuppressed, and cannot, therefore, eliminate the donor T cells effectively. Furthermore, BMT contains a significant number of T lymphocytes, but the figure is several-fold higher in a typical PBSC harvest.

Clinical manifestations

GvHD can be subdivided into chronic and acute disease. Acute GvHD normally occurs during the first 100 days following BMT, and chronic GvHD after this period. Acute GvHD often evolves into the chronic form of the disease. The most common manifestations of acute and chronic GvHD occur in the skin, liver and gastrointestinal tract.22

The first indication of GvHD is a maculopapular rash, which often develops at the time of leucocyte engraftment. Initially, the rash affects the palms of the hands, the soles of the feet and the ears, but may spread to the entire surface. In severe cases, the rash develops into bullous lesions and epidermal necrolysis.

Liver disease is the next most common manifestation, and increases in bilirubin and alkaline phosphatase levels are detected by hepatic function tests." However, these are non-specific and histological diagnosis of disease is often required to confirm GvHD. Evidence for GvHD includes bile duct damage, which can lead to severe cholestasis."

The most obvious indication of gastrointestinal GvHD is diarrhoea,10 and this is often accompanied by nausea, vomiting and severe abdominal pain. Excess fluid loss can cause problems in maintaining fluid balance in patients.20In addition, bacterial infections often occur as a result of gastrointestinal tract GvHD.22

Staging

Severity of GvHD is indicated by the degree of involvement in the three organs. The surface area covered by the rash and the appearance of bullous lesions is considered when grading GvHD in the skin. Bilirubin level is used to grade hepatic involvement and gastrointestinal disease is staged according to the volume of diarrhoea produced.24 Overall clinical severity is determined by establishing a grade, based on scoring (i.e. + to . . . . ) the degree of organ involvement (Table 1).24

The scoring system, developed by Glucksberg et al.23 and subsequently modified the following year,24 has formed the basis for predicting leukaemia-free survival and overall survival for the past 20 years. Some researchers have simplified acute GvHD staging to either severe, moderate or absent. In a recent study of chronic myeloid leukaemia, the Glucksberg23 method was found to be more valuable clinically, as different survival outcomes were identified for the different gradings assigned using detailed GvHD staging.zs

Pharmacological strategies for treatment Since GvHD was first described, treatment and prevention regimes have concentrated on the use of drugs. MTX proved effective in reducing the incidence of GvHD but the rate of development remained extremely high,23 and cyclophosphamide was shown to be an effective prophylactic drug. 26

Evidence that prophylaxis could significantly improve survival rates in patients undergoing BMT was seen with CsA,22 which permits development of antigen-specific T cells but inhibits both helper T-cell and cytotoxic T-cell development.2' CsA became the most widely used prophylactic agent for GvHD in the early 1980s, and was cited recently as the most important factor in the improved survival rates for aplastic anaemia patients, post-BMT.zB

Numerous immunosuppressive agents have been used in isolation to treat acute GvHD, including high doses of prednisolone,29 antithymocyte globulin (ATG),30 CsA31 and thalidomide;32 however, these have proved largely unsuccessful. Conversely, chronic GvHD can be treated with CsA, with or without prednisolone.33

Research by Storb et al.8 was pivotal in developing the current most effective pharmacological prophylaxis against acute GvHD. This study compared the effect of combining MTX and CsA against monotherapies with either drug. The incidence of acute GvHD was significantly lower when the drug combination was used, and engraftment was not inhibited; however, there was an increase in the incidence of chronic GvHD. Presently, a combined regime of MTX and CsA is the most common form of prophylaxis against GvHD.s

Monoclonal antibodies as an alternative approach to therapy

Cell and cytokine targets

Prevention and treatment methods which utilise MAbs have concentrated on either removing donor T cells prior to BMT or destroying the cells after GvHD has developed. Rat MAbs of the Campath series recognise the CDw52 cluster, expressed on the majority of human lymphocyteS,14 and have been used in prophylaxis extensively. Mouse MAbs, including anti-CD3 which targets the majority of the Tcell population, have been used in the treatment of steroid-resistant GvHD. 13

The complex nature of GvHD is being uncovered gradually, aided by murine experiments. These indicate that CD4' T cells are activated initially and these stimulate the GvH reaction and activate CD8' cells, which later become involved in GvHD. The application of anti-CD4 MAbs prevented the development of GvHD.II Li et al.36 also demonstrated that the effector cells in murine hepatic GvHD are CD4' T cells. Hepatic GvHD is mediated by CD4' cells via two pathways; one pathway is independent of CD8' cells, the other involves CD8' cells, activated by CD4' cells. This data confirms the important role of CD4' T cells in murine GvHD.

The role of cytokines in the pathogenesis of GvHD has yet to be fully explained; however, it is known that IL-2 and TNFa are involved in mediating acute GvHD" and that activated T cells express IL receptors not found on resting cells. Tong et al.38 established that patients who developed grades III and IV acute GvHD had higher pre-BMT TNFa levels. As GvHD developed in all patients, TNFa levels subsequently increased, but at different stages depending on the severity of the disease. Levels of serum IL-2 were not significantly high in those with GvHD, neither before nor after development of disease symptoms. In addition, murine experiments have confirmed the apparent up-regulation of GvHD by TNFa,39 and MAbs specific for this cytokine target have been used to treat human GvHD.40

Mouse monoclonal antibodies

Successful application of the mouse anti-CD3 MAb OKT3 in reversing kidney graft rejection 13 prompted its use in GvM therapy. Initial trials produced promising results, particularly in the treatment of steroid-resistant GvHD." However, recovery was mainly temporary and serious side-effects were noted. In some studies, treatment with OKT3 41 and another anti-CD3 MAb,42 64.1, led to an increase in lymphoproliferative disorders. In vitro studies have shown that these particular antibodies mediate cross-linking of the CD3 complex on T cells, resulting in cytokine production, Tcell activation and proliferation. Conversely, in prophylactic regimes using OKT3 combined with other immunosuppressive therapies,41 none of the patients who received OKT3 developed a lymphoproliferative disorder in the year following treatment.

The potentially serious side-effects witnessed with cross-linking MAbs has led to the search for alternative anti-CD3 MAbs. One such alternative, BC3, reversed acute GvHD but did not promote either cross-linking of the CD3 complex or T cell activation. 14 In addition, the incidence of lymphoproliferative disorders was reduced. In this context, it is significant that immunosuppression by BC3 is achieved through inhibition of T-cell function rather than destruction of circulating T cells, as is the case with OKT3 and 64. 1.

Murine MAbs have been developed against IL-2 receptors and used to treat steroid-resistant GvHD.',44 Patients treated in the early stages of disease responded better than those with more established disease.' AntiIL-2 receptor antibodies also have been employed as a first-line treatment for acute GvHD but the results have been disappointing.41

Anti-TNF(x antibodies have demonstrated both prophylactic and therapeutic properties against GvHD in mice, 39 and have been developed for human GvHD therapy.40 Initially, patient symptoms improved, but when treatment was stopped, the symptoms returned. This suggests that cytokine modulation in isolation is insufficient to inhibit the immunological processes involved in established GvHD.

Recently, combinations of murine MAbs targeting both T cells and cytokines were used to treat corticosteroid-resistant acute GvHD;46 however, the results were not significantly better than those obtained with the individual MAbs.

In vitro and in vivo use of rat monoclonal antibodies Depletion of bone marrow T cells prior to transplantation has centred on the use of rat MAbs, owing to their ability to fix human complement in vivo and because, historically, rat MAbs could be generated in large amounts in ascites. The MAbs used normally for Tcell depletion are those directed against the CDw52 antigen, Campath-1. This is expressed on the majority of human lymphocytes but not on erythrocytes, granulocytes, platelets and myeloid precursor cells. Initial clinical trials were carried out with an immunoglobulin M (IgM) MAb of the Campath-1 series.41 T cells were removed by complement-mediated lysis, without affecting engraftment adversely. The frequency of GvHD was reduced but graft-rejection problems were encountered.

In 1985, it was demonstrated that rat MAbs of the IgG2b isotype were most effective in promoting antibody-dependent, cell-mediated cytotoxicity (ADCC) with human effector cells,41 and subsequent research has centred on the use of this isotype, particularly Campath-IG. When this MAb was used for Tcell depletion 41,50 the incidence of GvHD was reduced. In one study," patients were treated with MAb both prior to and after BMT.

A combination of in vitro and in vivo MAb therapy has been used in BMT from unrelated donors to treat acute lymphoblastic leukaemia (ALL).' Here, Campath- I G was given to patients prior to BMT, and donor bone marrow was treated with Campath-IG and Campath-IM in vitro, prior to transplantation. The results of this trial were encouraging and showed lower leukaemic relapse levels. Furthermore, it suggested that unrelated BMT for ALL is a viable option, as BMT from related donors did not give significantly better results.

The efficient removal of donor T cells can lead to an increase in leukaemic relapse. In an attempt to rectify this problem, donor lymphocytes have been given to BMT recipients after transplantation;" however, refinement of this approach is needed to ensure a graftversus-leukaemia effect, and successful engraftment, without the attendant risk of GvHD.

Immunotoxins

Immunotoxins are MAbs conjugated to either plant or bacterial toxins, and offer an alternative approach to other MAb therapies because their action is not complement- or effector-cell-dependent. In treating GvHD, the toxin used is normally ricin-A-chain (RTA).

Steroid-resistant acute GvHD has been treated successfully with RTA linked to an anti-CD5 MAb,I' and the success of this approach has been endorsed by subsequent studies.'Z Treatment with this IT is more effective than other regimes and has led to the use of ITs as a second-line treatment when steroids fail to improve acute GvHD.

Recently, ITs were used in combination with steroids as the initial treatment for acute GvHD.53 This combined approach proved more effective than steroids alone in reversing the symptoms of acute GvHD, and response to the CD5-based IT was quicker than that with steroids used in isolation. Side-effects resulting from IT treatment (Figure 1) tend to be related to the toxin component of the conjugate?

Disadvantages of monoclonal antibody therapies

The most significant problem encountered with mouse MAbs is the increased incidence of Epstein-Barr virus (EBV)-mediated lymphoproliferative disorders following the use of T-cell-activating anti-CD3 MAbs.41 It appears that the T-cell-activating qualities of these antibodies, and subsequent release of cytokines, facilitate EBV infection and B-cell transformation, and the immunosuppressed nature of BMT patients predisposes them to EBV infection. The other major disadvantage to the use of mouse MAbs is that the positive effects of therapy often are only temporary -a particular characteristic of anti-cytokine MAbs.9

Pretransplant T cell depletion of bone marrow using rat MAbs has two major disadvantages. Use of Campath-IM is associated with an increased incidence of graft rejection,55 and that of Campath-IM or Campath-1G increases the level of leukaemic relapse.49 These complications are due to the two antibodies' very efficient action in clearing T cells from the donor marrow. It has been shown that few donor T cells are needed to mediate the graft-versus-leukaemia effect (i.e. donor T cells react with residual leukaemic cells in the transplant patient by recognising their foreign MHC antigens2z). A recent study56 showed that grade I acute GvHD favours increased leukaemia-free survival in allogeneic BMT recipients, and this explains why T cell depletion is effective in reducing the rate of acute GvHD development, whilst increasing the chances of leukaemic relapse.

Production of antibodies by the transplant recipient against therapeutic MAbs is not generally a problem because the patient is severely immunocompromised. However, significant antibody responses which have inhibited the action of the MAb have been reported, and have led to cessation of treatment. Such antibody responses have been overcome successfully either by administration of CsA (alone, or in conjunction with the MAbS*) prior to treatment, the `humanisation' of the antibodies, or the use of human MAbs.15

Advantages of monoclonal antibody therapy

Specificity is the major advantage of MAbs in GvHD therapy. This permits targeting of distinct cell populations, rather than the general immunosuppressive effects achieved with pharmacological treatments. Despite the problems of graft rejection and leukaemic relapse associated with T cell depletion techniques, this use of MAbs considerably reduces the incidence of acute GvHD. The treatment of steroid-resistant GvHD remains a major problem and the use of IT offers one of the best chances of recovery.52

An additional advantage of the success of T cell depletion in reducing acute GvHD has been the increased opportunties for BMT using mismatched donors, 7 as patients given T-cell-depleted marrow can tolerate less-well-matched grafts. Therefore, BMT can be carried out earlier as the need to find a perfectly match donor is not imperative. T cell depletion also reduces the risk of mucositis, kidney and liver toxicity,56 and infection by opportunistic pathogens.

Novel approaches to GvHD therapy using monoclonal antibodies

Murine models have been used to test novel approaches to both the treatment and prophylaxis of GvHD. For example, an anti-T-cell-receptor mouse MAb given to the donor mouse prior to BMT proved to be an effective prophylactic measure against acute GvHD.58 Immunosuppression of the donor provided protection against GvHD in both MHC-matched and -mismatched transplants.

Studies in mice have shown that neutralisation of the immunoregulatory cytokine IL-12 by a monoclonal antibody can transform the helper T cell response from Thl to Th2.59 The Thl response mediates lethal acute GvHD, with the production of IL-2 and interferon-y, whereas the Th2 response promotes chronic GvHD, with high levels of IL-4, IL-5 and IL-10. The immune response was polarised into a Th2-type response in this animal model; however, it is of interest to note that there was no evidence of chronic GvHD-associated symptoms in the mice.

Another approach to prevention of GvHD using MAbs is to modulate the co-stimulatory pathway B7: CD28, which could promote tolerance in vivo and prevent GvHD developing. Initial results suggest that blocking of this pathway with an MAb may have prophylactic efficacy in human GvHD.z*

Conclusions

BMT has been the standard form of treatment for a variety of haematological disorders. Unfortunately, most patients requiring BMT do not receive marrow from an HLA-matched, related donor.' Consequently, unrelated, mismatched BMT is now more common and minor differences between donor and recipient HLA can lead to the development of GvHD.

With a clearer understanding of the immunobiology of GvH, it is envisaged that additional MAb targets will be revealed. The targeting of specific T cell populations and/or cytokines may help to overcome the problems of graft rejection and graft-versus-leukaemia effect that can complicate the treatment of GvHD.

With the advent of PBSC transplants (and increased availability of cord blood stem-cell sources), the future role of BMT is now questioned." However, GvHD will remain a risk whenever allogeneic transplantation is contemplated. For the foreseeable future, there remains a role for allogeneic BMT, and, regardless of the allogeneic stem-cell source, MAbs have an important contribution to make in the prevention and treatment of GvHD.

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A. HISCOTT and D. S. McLELLAN*

Centre for Applied Microbiology and Research, Porton Down, Salisbury, alts SP4 03C, and *School of Pharmacy E5 Biomedical Sciences, University of Portsmouth, Portsmouth, UK

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