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Patients with multiple myeloma (MM) undergoing high dose therapy and autologous stem cell transplantation (SCT) remain at risk for disease progression. Induction of the expression of highly immunogenic cancer testis antigens (CTA) in malignant plasma cells in MM patients may trigger a protective immune response following SCT. We initiated a phase II clinical trial of the DNA hypomethylating agent, azacitidine (Aza) administered sequentially with lenalidomide (Rev) in patients with MM. Three cycles of Aza and Rev were administered and autologous lymphocytes were collected following the 2nd and 3rd cycles of Aza-Rev and cryopreserved. Subsequent stem cell mobilization was followed by high-dose melphalan and SCT. Autologous lymphocyte infusion (ALI) was performed in the second month following transplantation. Fourteen patients have completed the investigational therapy; autologous lymphocytes were collected from all of the patients. Thirteen patients have successfully completed SCT and 11 have undergone ALI. Six patients tested have demonstrated CTA up-regulation in either unfractionated bone marrow (n = 4) or CD138+ cells (n = 2). CTA (CTAG1B)-specific T cell response has been observed in all three patients tested and persists following SCT. Epigenetic induction of an adaptive immune response to cancer testis antigens is safe and feasible in MM patients undergoing SCT.
Allogeneic stem cell transplantation (allo-SCT) is associated with a reduction in relapse rate in patients with multiple myeloma (MM) on the basis of an allo-immune graft vs. myeloma effect, mediated by donor immune cells targeting tumour (myeloma)-specific antigens, resulting in prolonged remission. Allografting is however complicated by graft-versus-host disease and unacceptable treatment-related mortality, obviating the survival benefit particularly if newly diagnosed myeloma patients are considered. On the other hand, patients undergoing high dose therapy with autologous stem cell transplantation (SCT) remain at risk for relapse, despite maintenance and consolidation regimens (Barlogie et al, 2006). There are additional toxicities, such as thromboembolic disease and disabling neuropathy, to be considered with such maintenance regimens. An alternative strategy is needed to relieve the burden of treatment toxicity observed in patients with myeloma whilst maintaining and prolonging current treatment efficacy. Immunotherapeutic interventions mimicking graft-versus-myeloma effect in the SCT setting may provide such an option. However efficacious, safe, and widely applicable strategies for immunotherapy remain elusive, limiting this option only to a select number of participants in clinical trials at tertiary cancer centers (Rapoport et al, 2009).
Cancer testis antigens (CTA) represent potential targets for immunotherapy in myeloma. These proteins are highly immunogenic with no natural self-tolerance because, under normal circumstances, they are only expressed in ‘immunologically privileged’ germ cells, and in the placenta (Simpson et al, 2005). Aberrant CTA expression has been observed in both solid tumours and in haematological malignancies, particularly in MM (Meklat et al, 2007). This often elicits a broad range of cellular and humoral immune responses. In myeloma, several reports have described sporadic over-expression of CTA and accompanying CTA-specific T cell and B cell responses (Lim et al, 2001; Wang et al, 2003; Jungbluth et al, 2005; Van Rhee et al, 2005; Condomines et al, 2007). Induced CTA alloreactivity has also been reported in MM patients undergoing allografting, possibly associated with freedom from relapse (Atanackovic et al, 2007). It is noteworthy that CTA expression is regulated by methylation of CpG islands in the promoters of these genes, which are mostly located on the X chromosome. There is evidence suggesting that therapy with azacitidine (Aza), a potent DNA methyl-transferase inhibitor, increases the expression of various CTA in a variety of in vitro and in vivo tumour models (Coral et al, 2002, 2006; Guo et al, 2006).
We hypothesized that in vivo induction of CTA by Aza may induce a CTA-specific T cell response if it is sequentially administered with an immuno-modulatory agent, lenalidomide (Rev), which is widely used in the therapy of MM. Rev works in part by increasing T cell and NK cell tumour cytotoxicity in vitro (LeBlanc et al, 2004; Hayashi et al, 2005). Additionally, Rev stimulates T cell proliferation and secretion of interleukin 2 (IL2) and γ-interferon (IFNG) in T cell co-stimulation assays (Corral et al, 1999; Davies et al, 2001). Further, CTA-specific T cells generated by this combination of Aza and Rev, when adoptively transferred to autologous SCT recipients, could expand in vivo and provide robust protection from disease progression. In this setting, SCT produces both a minimal residual disease state, as well as lympho-depletion, promoting the preferential proliferation of adoptively transferred CTA-specific T cells, setting the scene for effective adaptive immunotherapy (Rapoport et al, 2005).
In 2009, our programme initiated a multi-step phase II study to determine the feasibility of generating CTA-specific T cells in MM patients and their application in post-transplant maintenance (NCT01050790). MM patients received sequential Aza and Rev (Aza-Rev) to induce the expression of immunogenic CTA on malignant plasma cells and elicit a CTA-specific cellular immune response. The patients had autologous lymphocytes collected and cryopreserved following the second and third cycle of this regimen. After completion of the investigational regimen patients underwent stem cell mobilization and eventually SCT. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered post-transplant to facilitate haematopoietic recovery and augment dendritic cell (DC) function. The autologous lymphocytes were adoptively transferred to the patients in the second month after transplant. We report the promising early results of this clinical trial demonstrating the feasibility of administering Aza-Rev to MM patients prior to SCT, as well as the feasibility of collecting and reinfusing autologous lymphocytes following SCT. Further, we demonstrate induction of the expression of CTA in bone marrow of MM patients and an increase in CTA reactive T cell responses monitored by using human recombinant CTAG1B (NY-ESO-1) in vitro.
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This clinical trial demonstrated the in vivo epigenetic induction of highly immunogenic CTAs in patients with MM. This induction was associated with a subsequent cell-mediated immune response, which was transferable at the time of SCT and persisted following transplant. This was accomplished with the administration of a well-tolerated regimen of chemo-immunotherapy, which is easy to deliver and widely applicable making such adaptive immunotherapy available wherever SCT is performed for MM.
At the present time the only known curative therapy for patients with MM is allo-SCT. The graft-versus-myeloma effect observed following allografting has been linked to the emergence of tumour antigen-specific cellular and humoral immune response (such as against B cell maturation antigen), particularly following donor lymphocyte infusion (Bellucci et al, 2004, 2005). Similarly, antibodies against HY antigens in male recipients of female donor stem cells are correlated with freedom from relapse following allo-SCT (Miklos et al, 2005). A guiding principle in understanding such graft-versus-tumour and graft-versus-host responses is the allo-reactivity of donor T cells to ‘non-self’ minor histocompatibility antigens, oligopeptides that differ between human leucocyte antigen (HLA)-matched donors and recipients (Shlomchik, 2007), such as the HY antigens in the above example. Unfortunately, the recognition of such non-self antigens also triggers graft-versus-host disease, which erodes the benefit observed in terms of relapse protection following allografting. Therefore, if the paradigm of immune recognition of ‘non-self’ can be extended to the autologous setting, one may then observe the graft-versus-tumour benefit without the risk of graft-versus-host disease.
CTA offer such a target, and have been subject of intense study because of their aberrant expression in a variety of tumours. In patients with malignant melanoma, CTAG1B reactive T cells have been isolated and expanded ex vivo and re-infused into autologous recipients with dramatic responses recorded (Hunder et al, 2008). Recently, in vitro evidence of CTA overexpression by epigenetic modification and an adaptive T cell response has been demonstrated against MAGEA4 in patients with Hodgkin lymphoma treated with decitabine (Cruz et al, 2011). Similar findings have been reported with acute myeloid leukaemia and myelodysplasia, where therapy with Aza and valproic acid (a histone deacytelase inhibitor), has led to the emergence of cytotoxic T cells reactive to MAGEA1, MAGEA2, MAGEC2 and RAGEA1 peptides over the course of treatment (Goodyear et al, 2010). This synergy was also demonstrated in myeloma cells lines with Aza and MGCD-0103, up-regulating the expression of MAGEA3 in myeloma cell lines, which in turn stimulated cytokine responses from MAGEA3-specific cytotoxic T cells (Moreno-Bost et al, 2011). Our finding of CTA up-regulation in bone marrow and plasma cells from myeloma patients treated with Aza-Rev in vivo, corroborates these in vitro findings. Evaluation of the expression of a limited panel of CTA showed over-expression of multiple CTA in each patient tested following hypomethylating therapy with Aza, suggesting a broad-spectrum effect of such epigenetic modification. Such poly-antigen overexpression should in theory overcome the limitation of differential immunogenicity of different CTA. Further, it is likely that, depending on the patients HLA repertoire, one may see a polyclonal antigen-driven T cell response against a number of antigens, facilitating more effective tumour control compared with situations where single antigens are targeted using ex vivo vaccine generation.
Certain CTA transcripts have rarely been identified in normal tissues other than testis, albeit at a low level. These are termed testis selective (MAGEA3 to A6, AKAP4, SPA17 in our panel) as opposed to testis restricted (MAGEC1, CTAG1B, SPACA3, SPANXB1, SPANXB2) CTA (Hofmann et al, 2008). This implies that the CTA overexpression seen in unfractionated marrow may derive from normal haematopoietic elements. However, when tested in fractionated marrow cell populations, CTA induction following Aza-Rev in our patients appears to be limited to CD138+ plasma cells, which could be primed to overexpress these antigens, as opposed to normal haematopoietic cells. This may be related to the abnormal regulation of, or activity of epigenetic modifiers, such as DNA methyltransferase, in cancer cells. Recently, altered histone methylation with a more open chromatin structure has been demonstrated in patients with chromosomal translocation (4:14) in myeloma, related to aberrant MMSET activity (Martinez-Garcia et al, 2011). A similar mechanism may be invoked in explaining the differential response to Aza between normal and malignant marrow elements. Particularly intriguing is the observation of disease responses in two patients with abnormal cytogenetics following investigational therapy, despite a very low dose of lenalidomide being used, suggesting a drug sensitization effect of epigenetic modification on the malignant clone in selected patients. Aza is known to promote susceptibility to apoptosis in myeloma cell lines by restoring the expression of proteins such as RASD1, (Nojima et al, 2009) DAPK1 (Chim et al, 2007) and SPI1 (PU1) (Tatetsu et al, 2007). In such instances Aza has shown restoration of dexamethasone or doxorubicin sensitivity in resistant cell lines (Kiziltepe et al, 2007). A similar mechanism may be invoked in the responses we have observed.
Aside from the augmenting the direct cytotoxicity of lenalidomide against myeloma cells, synergy may be observed in terms of immuno-modulation. Hypomethylating therapy may facilitate autologous anti-tumour immune response by augmenting HLA class I expression on antigen-presenting cells as well as tumour cells in a variety of tumour models (Coral et al, 2006; Fonsatti et al, 2007; Natsume et al, 2008). Immuno-modulation by hypo-methylating therapy was demonstrated when decitabine + interferon-γ treated target neuroblastoma cell lysis was accomplished in an HLA class I-restricted context by CTA-specific cytotoxic T cells derived from normal donors (Bao et al, 2011). Such synergy may be partly derived from demethylation of HLA class II transcriptional activator (CII TA) gene promoters augmenting HLA class II expression in response to extraneous interferon- γ (De Lerma Barbaro et al, 2008). These data support the validity of our immunotherapeutic approach combining Aza with Rev.
CTA reactivity has been invoked as a possible mechanism for graft-versus-leukaemia responses in allogeneic SCT recipients (McLarnon et al, 2010). We tested CTAG1B reactivity by incubating peripheral blood lymphocytes and stem cell products with recombinant CTAG1B pulsed autologous DCs. CTAG1B-reactive T cells were observed in 3 patients tested, with reactivity correlating with level of CTAG1B expression observed post Aza-Rev therapy, and being maintained for 2–11 months following SCT. The post-transplant maintenance of CTAG1B-reactivity was protracted in one patient (Patient 1) with high levels of CTAG1B expression and MRD. Additionally two other patients (Patients 2 and 4) improved their response from VGPR to CR in the months following ALI. This suggests ongoing disease suppressive activity in the patient with MRD and possible maintenance of CTAG1B (and other CTA) expression in clonal plasma cells. Further, the CTA-specific T cell response elicited may be polyclonal and synergistic, targeting several different CTA simultaneously providing redundancy in terms of protective capability.
CTA-reactive T cells were identified in both the stem cell product as well as the ALI, which were given relatively early after transplantation to utilize the cytokine-rich milieu present with lymphocyte depletion following high dose therapy (Klebanoff et al, 2005; Rapoport et al, 2005, 2009). The absence of competing lymphocyte populations could, in this setting, lead to preferential proliferation of oligoclonal T cells in the ALI. Additionally we used GM-CSF for haematopoietic reconstitution rather than G-CSF to avoid the T-helper cell type 2 skewing and T cell hypo-responsiveness reported with the use of G-CSF (Sloand et al, 2000). GM-CSF also promotes DC differentiation, potentially triggering T cell reactivity against clonal plasma cells over-expressing CTA in the MRD state. Whether this results in a higher rate of CR achievement and improved survival will not be known definitively until randomized trials are conducted, comparing standard high dose and maintenance therapy with the current regimen. Further survival advantage may be observed if immunomodulatory drugs, particularly Rev, are used following SCT in conjunction with ALI to augment and prolong CTA-specific T cell reactivity. This will be explored in future patients enrolled on a follow up clinical trial.
At the current time we do not have adequate follow-up, nor the number of patients necessary to define the value of this immunotherapeutic approach in maintaining remission in patients with MM. When compared with the current standard of consolidation and maintenance therapy reported by many of the larger groups performing myeloma trials, ALI, if proven comparable in a larger cohort of patients, would represent a paradigm shift in the management of myeloma. This will be particularly true for patients with standard risk disease and may allow the management of these patients without protracted maintenance therapy with its own inherent toxicities.
Two issues that arose during this trial have been the problem of inadequate disease control by this regimen, and the potential unmasking of auto-immune toxicities. The protocol as it is currently executed safeguards against the former by incorporating this immunotherapy in a stem cell transplant scheme, using the ALI to target MRD following SCT. On the other hand, the potential unmasking of auto-immune toxicities is a matter that will need protracted follow up and early detection through careful follow up of patients undergoing this immunotherapy. Additionally, antigen loss as a mechanism of tumour escape will also need to be monitored for, by evaluating post-transplant persistence of CTA expression on plasma cells in bone marrow to determine the durability of this epigenetic modification.
In conclusion, we demonstrate the safety and feasibility of epigenetic modification resulting in over-expression of antigenic targets in MM. This may then be exploited in formulating adaptive immunotherapy protocols in these patients. Adoptively transferred cells may maintain long-term surveillance against malignant plasma cells in patients with MM and translate into prolonged freedom from progression in this otherwise incurable disease.