Minor histocompatibility antigens as targets of cellular immunotherapy in leukaemia

Best Practice & Research Clinical Haematology Vol. 17, No. 3, pp. 415–425, 2004 doi:10.1016/j.beha.2004.05.008 available online at http://www.sciencedirect.com

4 Minor histocompatibility antigens as targets of cellular immunotherapy in leukaemia J.H. Frederik Falkenburg* Roel Willemze MD, PhD

MD, PhD

Department of Hematology, Leiden University Medical Centre, P.O. Box 9600, C2-R, 2300 RC Leiden, The Netherlands

Allogeneic human-leukocyte-antigen-matched stem cell transplantation is associated with a lower risk of relapse of leukaemia than autologous transplantation due to a T-cell-mediated graft-vs.leukaemia effect. Replacement of patient haematopoiesis by donor haematopoiesis allows the application of donor-derived specifically targeted cellular immunotherapy for the treatment of leukaemia. Following allogeneic transplantation, donor-derived T cells recognizing minor histocompatibility antigens expressed on haematopoietic cells from the patient may result in eradication of all haematopoietic cells of recipient origin. Since after transplantation, normal haematopoiesis is of donor origin, these T-cell responses may result in establishment of full donor chimerism associated with elimination of the haematological malignancy. By targeting the immune response to minor histocompatibility antigens that are not expressed on non-haematopoietic tissues, graft-vs.-host reactions may be limited. Several methods can be used for in vitro selection of T-cell responses with high specificity for malignant cells, and in vitro manipulation of donor T cells including transfer of antigen-specific T-cell receptors may greatly enhance specificity and efficacy of donor-derived cellular immunotherapy of haematological malignancies. Key words: graft-vs.-leukaemia; minor histocompatibility antigens; allogeneic stem cell transplantation.

Allogeneic human-leukocyte-antigen (HLA)-matched haematopoietic stem cell transplantation (SCT) has been applied for decades as a method of stem cell reconstitution in patients who have received myeloablative chemotherapy for the treatment of leukaemia. Initially, the only purpose of allogeneic SCT was to replace the irreversibly damaged haematopoietic stem cell compartment after chemotherapy and irradiation at the maximum doses that could be tolerated by non-haematopoietic tissues.1 Since allogeneic SCT is accompanied by immunologically mediated complications including graft-vs.-host disease (GVHD) or graft rejection, the use of autologous SCT or stem cells derived from a syngeneic twin appeared to be a better source of haematopoietic * Corresponding author. Tel.: þ31-71-526-2271; Fax: þ31-71-526-6755. E-mail address: [email protected] (J.H.F. Falkenburg). 1521-6926/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

416 J. H. F. Falkenburg and R. Willemze

stem cells for transplantation. Clinical studies, however, have illustrated that the counterbalance of the occurrence of GVHD was a decreased likelihood of relapse after allogeneic SCT.2 Allogeneic SCT is associated with a lower risk of relapse after transplantation and better disease-free survival.3 Following HLA-matched transplantation, it became evident that donor-derived T cells were responsible for the reduced incidence of recurrence of the disease. Depletion of T cells from the graft to prevent GVHD resulted in an increased incidence of relapse after transplantation, illustrating the capacity of donor T cells to mediate a graft-vs.-leukaemia (GVL) effect.4 – 6 From the observation that transplantation between homozygous twins did not result in a clinically significant GVL effect, it was concluded that the mere presence of T cells in the graft was not sufficient to induce GVL reactivity, but that the administration of allogeneic T cells into the patient was essential to the GVL effect.7,8 Further proof of the ability of donor T cells to exert a GVL effect came from the administration of donor lymphocytes to patients with recurrence of their malignancy after allogeneic SCT. Donor lymphocyte infusion (DLI) as a treatment for relapses of leukaemia after transplantation has resulted in 20– 80% complete remissions depending on the nature of the leukaemia.9 – 13 Chronic myeloid leukaemia (CML) in the chronic phase was found to be most susceptible to DLI, whereas patients treated for relapsed acute leukaemia after transplantation have shown limited responses. From these observations, it can be concluded that the potential advantage of allogeneic SCT over autologous SCT appears to be the possibility of exploitation of the immune system to eradicate the malignancy after transplantation. The presence of donor haematopoiesis in the patient after allogeneic SCT provides the unique opportunity to introduce donor T cells into the patient without the risk of rejection, and without the risk that these T cells will attack the recipient’s haematopoietic system since these cells are of donor origin after transplantation. Insight into the nature of alloreactive immune responses after HLA-identical transplantation may allow the detrimental GVHD effect to be separated from the beneficial GVL effect, leading to new exploitation of the immune system in the treatment of leukaemia. Minor histocompatibility antigens (mHAGs) are the most likely targets for these immune reactivities after allogeneic HLA-matched SCT.14

MINOR HISTOCOMPATIBILITY ANTIGENS mHAGs can be defined as alloantigens that are capable of eliciting an allogeneic T-cellmediated immune response between HLA-identical individuals.15 Characterization of the human genome has illustrated that many single nucleotide polymorphisms (SNPs) exist within the human population. If an SNP is present within the coding region of a gene, amino acid substitutions in the protein may occur. In most of these cases, these small changes are not likely to influence the biological activity of the protein. These differences in amino acids may result in the processing of potentially immunogenic peptides that can be presented by HLA molecules. The immunogenic peptides derived from the polymorphic proteins can be presented in the context of HLA class I or HLA class II molecules, which can be recognized by CD8- or CD4-positive cells, respectively. If an HLA-identical allogeneic individual does not contain the same SNP, T cells from this individual may recognize these immunogenic peptides, leading to destruction of the cells expressing these polymorphic genes. In conclusion, mHAGs are peptides derived from intracellular polymorphic proteins that can be presented by HLA class I or class II

Minor histocompatibility antigens as targets of cellular immunotherapy in leukaemia 417

molecules, and can be recognized as non-self antigens by T cells from HLA-identical individuals. A prerequisite for mHAGs to be recognized by alloreactive T cells is adequate intracellular processing of the polymorphic proteins, leading to presentation of the peptide in HLA molecules expressed on the cell membrane. First, the protein must be degraded by intracellular proteases (the proteasome) into smaller peptide fragments. Peptides consisting of eight to 11 amino acids can then be transported by the ‘transporter in antigen processing’ (TAP) into the endoplasmic reticulum and bound to HLA class I molecules. The likelihood that a specific peptide is actually presented by HLA molecules on the cell membrane is therefore dependent on several intracellular processes, including the activity of intracellular proteases, trimming of the protein by amino peptidases, binding to TAP and binding to HLA molecules.16,17 Based on our current understanding of antigen processing, it can be hypothesized that immunogenic mHAGs can arise from polymorphic proteins by several intracellular mechanisms. First, amino acid substitutions present in the peptide that is actually bound into the groove of the HLA molecules may lead to specific recognition of the peptide –HLA complex by allogeneic T cells. Examples of such mHAGs include a number of male-specific HY antigens and the mHAGs HA-1 and BCL2A1.18 – 23 In addition, differential expression of the peptide – MHC complex may be the result of differential intracellular processing due to amino acid substitutions that may take place within the peptide-binding region or adjacent to the epitope that can be recognized by T cells.24 – 27 This may result in the presence or absence of the complex on the cell membrane. If the peptide is not processed in donor cells, donor T cells may not consider this specific peptide-MHC complex to be ‘self’, and an immune response against the protein may occur after allogeneic transplantation. The mHAGs HA-2 and HA-8 are likely to be members of this family of mHAGs. In addition, (partial) deletion of the gene coding for the protein involved has been described as a mechanism by which mHAGs can arise.28 Polymorphic peptides of various lengths can also be presented in the context of HLA class II molecules. The intracellular mechanism by which these endogenous proteins are processed and presented in HLA class II molecules are less clearly understood, but it has been found that similar amino acid substitutions in the peptide can act as mHAGs.29 A number of mHAGs that have been characterized and their restriction molecules are summarized in Table 1.

TISSUE DISTRIBUTION OF MHAGS The tissue distribution of the mHAG – HLA complex plays a crucial role in the clinical effect of T-cell responses against these antigens. It determines the likelihood that T-cell responses against these antigens are responsible for graft rejection, GVHD or GVL activity. The presentation of mHAGs in specific tissues is dependent on the presence of genes encoding mHAGs in the specific tissues, as well as HLA expression. Since most nucleated cells express HLA class I molecules, the presentation of class-I-associated mHAGs appears to be mainly dependent on the presence of the polymorphic gene encoding the peptide in the specific tissues, providing that the intracellular processing mechanism is operational. The expression of HLA class II molecules is more restricted to cells of haematopoietic origin and other cells during inflammatory reactions. Therefore, not only the genes encoding class-II-associated mHAGs but also the relative expression of HLA class II molecules on the target cells may determine the clinical

418 J. H. F. Falkenburg and R. Willemze

Table 1. Minor histocompatibility antigens involved in graft-vs.-leukaemia reactivity. Common name HLA restriction HA-1 HA-2 HA-3 HA-8 HB-1 HY-A1 HY-A2 HY-B7 HY-B8 HY-B60 ACC-1 BCL HY-DQ5

HLA-A2 HLA-A2 HLA-A1 HLA-A2 HLA-B44 HLA-A1 HLA-A2 HLA-B7 HLA-B8 HLA-B60 HLA-A24 HLA-B44 HLA-DQ5

Gene

Distribution

Reference

KIAA0223 Myosin-related gene LBC KIAA0020 HB1 DFFRY SMCY SMCY UTY UTY BCL2A1 BCL2A1 DBY

Haematopoiesis restricted Haematopoiesis restricted Ubiquitous Ubiquitous Pre-B cells Ubiquitous Ubiquitous Ubiquitous Ubiquitous Ubiquitous Relatively haematopoiesis specific Relatively haematopoiesis specific Ubiquitous

22 24 27 26 30 19 31 18 21 20 23 23 29

outcome of T-cell responses against these antigens. During inflammation, upregulation of HLA class II molecules may lead to amplification of the immune response. The polymorphic proteins encoding the mHAGs may be differentially expressed in normal tissues, leading to restricted tissue expression of these antigens. Certain mHAGs including many male-specific mHAGs, HA-3 and HA-8 can be expressed in all or most tissues. Other mHAGs such as CD31 appear to be primarily expressed in nonhaematopoietic cells.33 A number of mHAGs have been found to be expressed selectively in cells of haematopoietic origin. These mHAGs include HA-1 and HA-2, and possibly the recently described mHAG derived from the BCL2A1 proteins.23,31,32,34 Other mHAGs such as HB-1 have been reported to be expressed by a subpopulation of B cells within the haematopoietic compartment.35 The absence of expression of mHAGs on specific tissues under steady-state circumstances does, however, not preclude induced expression during inflammatory responses. Some mHAGs are only expressed in normal non-haematopoietic cells when these cells are treated with interferon gamma or tumour necrosis factor (TNF).36

MHAGS AND GVHD Since it had been demonstrated that donor-derived allogeneic T cells were essential for the development of clinically significant GVHD after HLA-matched SCT, it was thought that the recognition of mHAGs by donor T cells plays an important role in the development of this complication. Initially, it was predicted that only disparities between donor and recipient for mHAGs that are broadly expressed on all tissues from the recipient may be associated with a T-cell response against these antigens, leading to severe GVHD. In favour of this hypothesis was the observation that transplantation of male patients with female donors that are capable of eliciting an antiHY response was associated with an increase in GVHD after transplantation.37,38 However, if this type of immune response was solely responsible for the occurrence of GVHD, it could be expected that the alloreactivity would lead to complete destruction of all tissues from the recipient. However, GVHD preferentially involves specific tissues that contain

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significant numbers of professional antigen-presenting cells (APCs).39 Recently, elegant mouse studies have illustrated that T-cell responses against mHAGs that are highly and specifically expressed on recipient-derived dendritic cells (DCs) may be an essential step in the development of GVHD, possibly partly due to the induction of ‘collateral damage’ even in the absence of T-cell responses against broadly expressed antigens.40 – 42 In the absence of a T-cell response against DCs, no acute GVHD occurred. Recent experiments performed by Dickinson et al indicated that T-cell responses specific for haematopoietic mHAGs such as HA-1 do not destroy skin tissues directly, as illustrated using a skin explant model.43 This would indicate that T-cell responses against antigens expressed solely on haematopoietic cells do not cause major tissue damage directly. However, as reported in a study by Goulmy et al, differential expression of haematopoiesis-specific mHAGs between donor and recipient was associated with the development of acute GVHD.44 This has led to the hypothesis that the development of GVHD is based on multistep pathogenesis. First, T-cell responses against mHAGs that are broadly expressed on multiple tissues including DCs or against mHAGs expressed on haematopoietic cells that are present in high frequency in the GVHD-associated organs may lead to local inflammation, resulting in immune activation and cytokine-mediated destruction of both haematopoietic and collatoral nonhaematopoietic tissues. This ‘cytokine storm’ was shown to be responsible for many of the clinical features associated with acute GVHD. This immune response may lead to the amplification of T-cell-mediated cytolytic reactivity against antigens that are more broadly expressed on GVHD target organs. This observation was supported by the finding that during acute GVHD in male patients transplanted with female grafts, a high frequency of T cells against broadly expressed male-specific antigens was measured using mHAG peptide-HLA-specific tetrameric complexes.45 The contribution of tissuespecific mHAGs in the development of chronic GVHD has been less well defined. The clinical features of chronic GVHD make it likely that T cells directed against more broadly expressed mHAGs play a more significant role in this complication.

MHAGS AS TARGET STRUCTURES FOR ANTILEUKAEMIA REACTIVITY Clinical studies have revealed that T-cell depletion of the haematopoietic stem cell graft to abolish GVHD results in a relapse rate that may not differ significantly from the likelihood of relapse after autologous transplantation. This observation indicates that donor T cells which are derived from donor stem cells in the patient after transplantation and are thus educated in the patient are tolerant to potential immunogenic antigens expressed on the malignant cells. Apparently the immunological education of T cells in the donor in the absence of the immunological environment of the patient is essential for the antileukaemic potential of the donor-derived T cells. Thus, antigens expressed on malignant cells that are not normally present on tissues from the donor may serve as primary targets for T-cell-mediated GVL reactivity. These target antigens may include antigens derived from proteins that are highly overexpressed in the malignant cells and not normally expressed in their normal counterpart. The non-polymorphic proteinase-3 derived peptide PR-1 may be an example of an overexpressed target structure that can elicit a donor-derived T-cell response capable of recognizing malignant cells that express these proteins.46,47 However, since transplantation of non-T-cell-depleted grafts from homozygous twins does not exert a GVL effect, it is likely that T cells recognizing alloantigens may

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contribute to the antileukaemic effect more frequently.48 Since the treatment of relapsed leukaemia after allogeneic SCT with DLI is often associated with the conversion of mixed chimerism to full donor chimerism, it seems likely that T cells may frequently recognize mHAGs that are expressed on all haematopoietic cells from the recipient including, but not limited to, the malignant cell population.49 – 51 Both mHAGs that are expressed on multiple tissues from the recipient and haematopoiesis-associated mHAGs may elicit a T-cell response leading to antileukaemic reactivity with high or relatively low risk of concurrent development of GVHD, respectively.38,52 The major prerequisite for eradication of leukaemic cells after transplantation is T-cell recognition of antigens that are expressed on the clonogenic leukaemic precursor cell essential for sustaining the malignancy. Most of the mHAGs described, including all male-specific antigens as well as the mHAGs HA-1 to HA-5, HA8, HB-1 and BCL2A1, are present on clonogenic leukaemic precursor cells.23,26,30,53 The observation that T-cell responses against these mHAGs are capable of eliminating leukaemic precursor cells capable of engrafting into immunodeficient NOD/SCID mice confirmed the ability of these T cells to prevent outgrowth of leukaemia.54 The observation that successful elimination of leukaemic cells following DLI associated with conversion of mixed chimerism to full donor chimerism may occur both in the presence and in the absence of GVHD, has led to the hypothesis that mHAGs expressed on both normal and malignant haematopoietic cells may serve as tumour-specific targets for cellular immunotherapy in the absence of GVHD. The haematopoiesis-associated mHAGs HA-1 and HA-2 have been studied for their ability to act as tumour-specific antigens after allogeneic SCT. The functions of the proteins encoding the HA-1 or HA-2 peptides are unknown, but the observation that HA-1- and HA-2-specific T cells are capable of eliminating all normal and leukaemic cells expressing these antigens indicates that these proteins are expressed in normal and leukaemic haematopoietic (stem) cells. It has been demonstrated that patients with CML who were positive for mHAGs HA-1 or HA-2, receiving DLI from their mHAGnegative stem cell donors, showed specific T-cell responses against these antigens during the clinical antileukaemic response.52 Using HLA-A2/HA-1- or HA-2-specific tetramers, disappearance of the malignancy and conversion to full donor chimerism was associated with the emergence and persistence of antigen-specific T cells. After isolation of these HA-1- or HA-2-specific T cells using fluorescent tetramers, the ability of these T cells to eliminate clonogenic leukaemic precursor cells was confirmed. In several patients, full clinical responses were associated with the absence of severe GVHD, but in most patients, grade 1 skin GVHD that did not require systemic treatment was observed. These observations may confirm the previous hypothesis that T-cell responses against haematopoiesis-associated mHAGs in the patient may lead to local inflammatory reactions resulting in limited tissue damage. It can be hypothesized that the relative expression of normal as well as malignant APCs from recipient origin in GVHD-associated tissues may determine whether or not T-cell responses against these antigens will result in clinically relevant GVHD. Similarly, T-cell responses against mHAGs derived from BCL2A1 have been reported to be associated with an antileukaemic effect in patients with acute myeloid leukaemia (AML) in the absence of clinically significant GVHD.23 Although it has been reported that BCL2A1 is expressed in non-haematopoietic tissues, the relatively low expression in non-haematopoietic tissues compared with haematopoietic tissues may result in specific elimination of the leukaemia in the absence of GVHD. However, we recently illustrated that under the influence of interferon-gamma and TNF, non-haematopoietic tissues may upregulate the expression of BCL2A1, leading to specific recognition by antigen-specific T cells

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(unpublished observations). These results may indicate that the activation state of nonhaematopoietic tissues from the patient may determine whether or not a clinically relevant antileukaemic T-cell response may be associated with (chronic) GVHD.

MHAG-SPECIFIC INDUCTION OF GVL REACTIVITY IN VIVO Based on the polymorphic nature of the human genome, and the likelihood of disparity of mHAGs between donors and recipients in HLA-matched SCT, it seems likely that an alloresponse against the leukaemic cells may occur after transplantation or DLI in most cases. However, although high frequencies of complete responses have been observed in patients with relapsed CML after transplantation, treatment with DLI of patients with relapsed AML or acute lymphoblastic leukaemia (ALL) is less successful.9 – 13 It has been hypothesized that the ability of CML cells to induce a potent GVL response may be attributed to the capacity of precursor cells from CML to develop into DCs of leukaemic origin.55,56 In contrast, cells from patients with acute leukaemia may be poor APCs, not expressing the relevant co-stimulatory and/or adhesion molecules necessary for the induction of an antileukaemic response in vivo.57,58 The poor APC function of these malignant cells in combination with inappropriate cytokine production may even downregulate the alloreactive T-cell response capable of eliminating the malignant cells. In particular, in patients who develop a relapse of AML or ALL relatively late after transplantation when all or most APCs from the patient are replaced by donor cells, no appropriate initiation of the T-cell response may occur in vivo. It is possible that vaccination of the patient with mHAG-specific synthetic peptides or proteins in the context of DLI may augment the alloreactive GVL response. Alternatively, vaccination of the donor with peptides expressed on the malignant cells from the patient prior to harvesting T cells may potentiate GVL reactivity by donor T cells. This strategy may favour GVL reactivity over GVHD by selectively increasing the T-cell response against haematopoiesis-associated mHAGs, although limited GVHD may also occur due to ‘collateral damage’ in these cases.

IN VITRO GENERATION OF MHAG-SPECIFIC T CELLS FOR TREATMENT OF LEUKAEMIA In vivo vaccination studies using mHAG-specific peptides or proteins may be successful in cases of minimal residual disease after transplantation. However, in patients with overt leukaemia, the induction of a T-cell response in vivo is likely to be insufficient or may not be evoked at the appropriate time. Alternatively, it may be possible to isolate mHAG-specific T cells from the donor, activate and expand them in vitro, and infuse them into the patient in case of relapse. Treatment of patients with viral infections after allogeneic SCT with cytomegalovirus (CMV)- or Epsteir-Barr-virus-specific T cells has demonstrated the feasibility of this approach.59,60 The ability of in vitro cultured T cells to induce complete remission of resistant leukaemia after allogeneic transplantation was demonstrated by the administration of leukaemia-reactive T cells.61,62 By generating donor-derived DCs from CD34-positive cells or CD14-positive cells in the presence of multiple cytokines and loading these DCs with synthetic mHAGspecific peptides, a primary T-cell response against these antigens may be generated in vitro.63 In vitro initiation of the immune response may bypass the silencing effects of

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the malignant cells in vivo. Although this approach may be attractive, the logistics of generating large numbers of mHAG-specific T cells for clinical application have been complex. At present, little is known about the requirements for large-scale ex vivo expansion of T cells while preserving their ability to proliferate and execute their function in vivo. Direct isolation methods using tetrameric complexes or by exploring the cytokine release assay following activation by mHAG-specific peptides may, in future, lead to more rapid enrichment of large numbers of antigen-specific T cells for adoptive immunotherapy.

RE-ENGINEERING OF MHAG-SPECIFIC T-CELL REACTIVITY Since a number of mHAG-specific T cells have been isolated clonally and the T-cell receptors (TCRs) have been characterized in detail, gene transfer of these TCRs into immunocompetent cells from the donor or from the patient after transplantation may be an attractive alternative to circumvent the logistical problems of ex vivo isolation of large numbers of antigen-specific T cells. The feasibility of redirection of mHAG-specific T cells by gene transfer was demonstrated recently.64 This procedure can lead to the generation of large numbers of mHAG-specific T cells with appropriate recognition of haematopoietic precursor cells and leukaemic cells. The transfer of TCR-alpha and TCR-beta genes into primary unselected T cells may, however, also result in unpredictable specificities due to the undesired pairing of the exogenous TCR chains with endogenous TCR chains. Furthermore, introduction of mHAG-specific TCRs into randomly selected T cells may also potentially activate circulating self-reactive T cells. To circumvent a number of these potential problems, TCR gene transfer may be performed selectively in virus-specific T cells destined to be activated and to persist after the transplantation. The feasibility of this approach was demonstrated recently by successfully transferring mHAG-specific TCRs into CMV-specific T cells with preservation of both antiCMV- and antimHAG-specific reactivity.65 An alternative strategy may be the transfer of mHAG-specific TCR into gamma-delta T cells since the alpha and beta chains do not have the capacity to pair with the endogenous gamma and delta chains. This may lead to the generation of T cells with a single specificity. The in vivo potential of redirected TCRs has been illustrated in murine models.66 In humans, this approach has not yet been applied. Although concern has been raised about the possibility of retroviral gene insertions causing malignancies in cases of gene transfer into CD34-positive stem cells, the large number of (pre)clinical experiments of retroviral gene transfer into mature T cells without transformation of these cells into antigen-independent proliferating cells may allow the clinical application of this approach.67

SUMMARY mHAGs are likely to play a major role in induction of beneficial GVL reactivity after allogeneic SCT. The tissue distribution of mHAGs determines the clinical outcome of Tcell responses against these targets, and manipulation of the specificity of mHAGreactive T cells may improve GVL reactivity while decreasing the likelihood of developing GVHD. T-cell responses against mHAGs preferentially expressed on haematopoietic cells may increase the specificity and efficacy of T-cell-mediated

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immunotherapy in the context of allogeneic transplantation. Vaccination of patients or donors with mHAG-specific peptides or proteins may boost the antileukaemic T-cell response in cases of minimal residual disease. In vitro isolation and expansion of mHAG-specific T cells may result in the production of cellular immunotherapeutic products that may be explored for the treatment of haematological malignancies in which an in vivo immune response cannot be evoked. Furthermore, by redirection of T-cell specificity by gene transfer of TCRs, large numbers of specifically targeted T cells may be generated for a future treatment of resistant haematological malignancies after allogeneic transplantation.

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