Transfer of EBV-specific CTL to prevent EBV lymphoma post bone marrow transplant

Journal of Clinical Apheresis 14:154–156 (1999)

Concise Review Transfer of EBV-Specific CTL to Prevent EBV Lymphoma Post Bone Marrow Transplant Helen E. Heslop,1* Margot Perez,1 Ely Benaim,2 Richard Rochester,2 Malcolm K. Brenner,1 and Cliona M. Rooney1 1

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas Division of Bone Marrow Transplantation, St. Jude Children’s Research Hospital, Memphis, Tennessee

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EBV-associated lymphoproliferative disorders (EBV-LPD) are a significant problem after hemopoietic stem cell transplantation from unrelated donors or mismatched family members. Risk factors include T-cell depletion, MHC mismatch, and intensity of immunosuppression. New therapeutic strategies involve cellular immunotherapy approaches and both donor T-cells and EBV-specific cytotoxic T lymphocytes (CTLs) have proven to be effective therapies. EBV-specific CTL has also proved to have a major impact on the incidence of this complication when used prophylactically. J. Clin. Apheresis 14:154–156, 1999. © 1999 Wiley-Liss, Inc.

INTRODUCTION

Epstein-Barr virus (EBV) is a latent herpesvirus that infects over 90% of the world’s population. Primary infection usually begins in the oropharynx, and virus produced in these cells then infects neighboring epithelial cells and circulating B-cells. In normal seropositive individuals, B-cells expressing EBV-associated proteins are tightly controlled by the host immune system and in particular EBV-specific T-cells [1]. By contrast, reactivation of EBV in severely immunocompromised individuals can lead to the development of aggressive B-cell lymphomas. EBV-associated lymphoproliferation (EBVLPD) is a highly immunogenic tumor expressing all nine EBV latency-associated proteins, and occurs only in severely immunosuppressed individuals [1]. Recipients of T-cell-depleted stem cell transplants, obtained from matched unrelated or mismatched family member donors, are at particular risk of developing EBV-LPD [2]. If the infused donor marrow is treated with antibodies that selectively deplete T-cells to decrease the risk of GVHD, the incidence of EBV-LPD ranges from 12–25% [3–6]. However, the risk is lowered if methods that deplete donor B-cells as well as donor T-cells are used. In a large review of patients whose transplants were treated with the Campath series of antibodies, the incidence of EBV-LPD was less than 2% [7] and low incidence was also seen after elutriation, which removes over 90% of B-cells from the donor graft [8]. In another report, addition of a monoclonal antibody depleting B-cells to the T-cell depletion regimen in a group of high-risk patients transplanted for immunodeficiency, reduced the incidence from 7 in 19 historical controls to 0 in the next 19 patients [9]. © 1999 Wiley-Liss, Inc.

Patients usually present with fever and localized or disseminated lymphadenopathy and organ infiltration. However, diffuse disease may present as multiorgan system failure and a high index of suspicion is needed to make this diagnosis. The onset of EBV-LPD seems to be preceded by rises in EBV DNA levels [5,10] and monitoring EBV DNA can assist with diagnosis. The pathology ranges from polymorphic B-cell lymphomas to immunoblastic lymphomas [11] that are usually oligoclonal or monoclonal. THERAPY OF EBV-LPD WITH UNMANIPULATED DONOR T CELLS

Until recently, EBV-LPD arising after stem cell transplantation was invariably fatal. Over the past few years, an improved understanding of the biology of the disease has stimulated interest in cellular immunotherapies. EBV is a latent virus and most seropositive individuals have high precursor frequencies of cytotoxic T-lymphocytes (CTL) reactive with EBV so that transfer of donor PBMC includes small numbers of EBV-specific T-lymphocytes, which may expand in vivo. Investigators at Memorial Sloan Kettering Cancer Center originally reported on five patients with EBV-LPD, all of who responded to

Contract grant sponsor: National Institute of Health; Contract grant numbers: CA 74126, CA 61384. *Correspondence to: Dr. Helen Heslop, Center for Cell and Gene Therapy, Baylor College of Medicine, 1102 Bates St., Suite 1140, Houston, TX 77030. Received 8 May 1999; accepted 23 July 1999

Concise Review: EBV-Specific CTL

small doses of unmanipulated donor lymphocytes [12]. In an update of their experience, 17/19 patients responded to donor T-cells [4]. However, such unmanipulated products may also contain a high frequency of alloreactive T-lymphocytes and induce GVHD [5,13]. The Indiana group also noted a less impressive response rate with five of the nine patients dying of disease progression within 10 days of receiving donor leukocytes. This different response rate may reflect better outcome with early diagnosis or tumors with varying biological behavior. Two approaches have been utilized to overcome the problem of alloreactivity with administration of unmanipulated donor T-cells. Bonini and colleagues [14] transduced T-cells with a “suicide gene,” that renders transduced cells sensitive to Ganciclovir, so that this agent may be administered to induce cell death if the patient develops GVHD. An alternative approach that we have explored is to administer EBV-antigen-specific CTLs rather than unmanipulated polyspecific donor Tcells. EBV is an excellent model to evaluate the efficacy of adoptively transferred antigen-specific CTL therapy. The tumor cells have the same phenotype as EBVtransformed B-lymphoblastoid cell lines (LCLs) that can readily be prepared from any donor and provide a source of antigen-presenting cell expressing the appropriate viral antigens. The tumor cells express all latent-cycle virus-encoded antigens (EBNA 1, 2, 3a, 3b, 3c, and LMP1, 2a, 2b), which can act as targets for CTL killing. Finally, most donors are immune to EBV, and because the virus persists in latent form, the EBV-specific CTL precursors persist long term at a high frequency.

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of EBV-specific CTL, there is reconstitution of immunity to EBV with a rise in virus-specific CTL activity in peripheral blood and a rise in CTLp frequencies to the high end of the normal range [17]. In addition, there is evidence of anti-viral activity. Seven of the patients in the prophylaxis study developed high levels of EBV DNA prior to CTL infusion. In all cases, EBV DNA levels dropped two to four logs within 3 weeks of infusion [18]. The approach also had a major impact on our incidence of EBV-LPD as none of the patients in the prophylaxis study have developed EBV-LPD, compared with 11.5% of controls [18]. EBV SPECIFIC CTLS AS THERAPY

Three patients who had not received CTL (two were ineligible and one declined prophylaxis) developed EBV-LPD and received CTL as treatment. All presented with aggressive disseminated disease. Two patients recovered fully, although one with bulky disease developed a severe inflammatory response, requiring intubation illustrating the advantages of prevention over treatment [18]. The third patient died with extensive disease, and preliminary data suggest that a mutation in the EBV EBNA-3 gene in the tumor resulted in loss of the two immunodominant epitopes and resistance to killing by donor derived EBV specific CTLs [19]. The occurrence of such mutants argues for treating patients when they have a low viral/tumor burden and hence a lower probability of mutation and engenders caution about using T-cell clones instead of polyclonal lines as target recognition will be more restricted.

PROPHYLAXIS STUDY

We have evaluated whether adoptive transfer of donor-derived EBV-specific cytotoxic T-lymphocytes is an effective prophylaxis for EBV lymphomas arising after marrow transplantation in high-risk recipients [15]. Over 55 recipients of T-cell-depleted marrow received from one to four doses of donor-derived EBV-specific CTL at St. Jude Children’s Research Hospital between September 1993 and December 1997 and at Baylor College of Medicine since 1998. Gene marking of infused CTL in the first 26 patients allowed us to demonstrate that these adoptively transferred cells persist in blood mononuclear cells for up to 18 weeks and for up to 55 months in regenerated EBV-specific CTL [16,17]. The marker gene is detected in both CD4 and CD8 components and the level of detection is 0.1–1%, which is similar to levels in input lines [17]. The ability of the CTL to respond to antigenic challenge in vivo was seen in two patients in whom the marker gene reappeared in peripheral blood over one year post-infusion co-incident with sub-clinical EBV reactivation [17]. EBV-specific CTL responses rarely recover before 8 months after a T-cell-depleted BMT. Following infusion

CONCLUSIONS OF EBV CTL STUDY

In summary, prophylactic EBV-specific CTL appear to be safe and effective, both in restoring EBV-specific immune responses and in preventing EBV-LPD [17,18]. Prophylaxis has advantages over therapy, since treatment of bulky disease can cause severe morbidity from tissue inflammation and may be ineffective in cases where virus strain differences affect important CTL epitopes. Recently, we have been evaluating the use of CD20 monoclonal antibody in conjunction with EBV-specific CTL in patients with bulky or infiltrative disease. FUTURE DIRECTIONS

This approach is now being extended to adenoviruses [20] and other viruses that produce post-transplant morbidity and additional EBV-associated malignancies. Hodgkin disease and nasopharyngeal cancer are two EBV-associated primary malignancies in which tumor cells may be less susceptible to immunotherapeutic approaches because they express a more restricted array of subdominant EBV-encoded antigens [21]. For example,

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Reed Sternberg cells, in patients with EBV genome– positive Hodgkin’s disease, express only LMP-1, LMP2, and EBNA-1. In polyclonal CTL lines, the majority of clones recognize the more immunodominant EBNA-3 family of antigens and only a few clones, if any, will recognize subdominant antigens. Furthermore, CTL must be generated from an immunosuppressed patient rather than a normal donor. We have been evaluating the use of autologous polyclonal CTLs in a clinical study for patients with relapsed EBV genome positive Hodgkin disease [22]. Five patients with multiply relapsed Hodgkin disease have received gene-marked EBV-specific CTL on the lowest dose of a phase-1 dose escalation study. Adoptively transferred cells have persisted in peripheral blood for up to 12 weeks and in one patient were detected in a malignant pleural effusion [22]. This study with polyclonal EBV-specific CTL is continuing and in preclinical studies we are exploring the use of gene modified dendritic cells to generate LMP-2 specific CTLs. REFERENCES 1. Rickinson AB, Moss DJ. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu Rev Immunol 1997; 15:405–431. 2. Aguilar LK, Rooney DM, Heslop HE. Lymphoproliferative disorders involving Epstein-Barr virus after hemopoietic stem cell transplantation. Curr Opin Oncol 1999;11:96–101. 3. Heslop HE, Rooney CM. Adoptive immunotherapy of EBV lymphoproliferative diseases. Immunol Rev 1997;157:217–222. 4. O’Reilly RJ, Small TN, Papadopoulos E, Lucas K, Lacerda J, Koulova L. Biology and adoptive cell therapy of Epstein-Barr virus-associated lymphoproliferative disorders in recipients of marrow allografts. Immunol Rev 1997;157:195–216. 5. Lucas KG, Burton RL, Zimmerman SE, Wang J, Cornetta KG, Robertson KA, et al. Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation. Blood 1998;91:3654–3661. 6. Gerritsen EJ, Stam ED, Hermans J, van den Berg H, Haraldsson A, van Tol MJ, et al. Risk factors for developing EBV-related B cell lymphoproliferative disorders (BLPD) after non-HLAidentical BMT in children. Bone Marrow Transplant 1996;18: 377–382. 7. Hale G, Waldmann H, for CAMPATH users. Risks of developing Epstein-Barr virus-related lymphoproliferative disorders after Tcell-depleted marrow transplants. Blood 1998;91:3079–3083. 8. Gross TG, Steinbuch M, DeFor T, Shapiro RS, McGlave P, Ramsay NKC, et al. B cell lymphoproliferative disorders following hematopoietic stem cell transplantation: Risk factors, treatment and outcome. Bone Marrow Transplant 1999;23:251–258. 9. Cavazzana-Calvo M, Bensoussan D, Jabado N, Haddad E, Yvon

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