Epstein-Barr Virus Lymphoproliferative Disease Research Articles

Virus-specific T-cells from third party or transplant donors for treatment of EBV lymphoproliferative diseases arising post hematopoietic cell or solid organ transplantation.

EBV+ lymphomas constitute a significant cause of morbidity and mortality in recipients of allogeneic hematopoietic cell (HCT) and solid organ transplants (SOT). Phase I and II trials have shown that in HCT recipients, adoptive transfer of EBV-specific T-cells from the HCT donor can safely induce durable remissions of EBV+ lymphomas including 70->90% of patients who have failed to respond to treatment with Rituximab. More recently, EBV-specific T-cells generated from allogeneic 3rd party donors have also been shown to induce durable remission of EBV+ lymphomas in Rituximab refractory HCT and SOT recipients. In this review, we compare results of phase I and II trials of 3rd party and donor derived EBV-specific T-cells. We focus on the attributes and limitations of each product in terms of access, safety, responses achieved and durability. The limited data available regarding donor and host factors contributing to T cell persistence is also described. We examine factors contributing to treatment failures and approaches to prevent or salvage relapse. Lastly, we summarize strategies to further improve results for virus-specific immunotherapies for post-transplant EBV lymphomas.

Primary EBV Lymphoproliferative Disease Responsive to Therapy in a Patient with CTLA4-Haploinsufficiency

Epstein-Barr virus lymphoproliferative disease (EBV-LPD) occurs most often in patients with acquired immunosuppression. However, several inborn errors of immunity have been associated with primary EBV-LPD, including pathogenic variants in SH2D1A, ITK, MAGT1, CORO1A, CD27, CTPS1, RASGRP1, and ZAP70 (PMID29942301,34239742). Here, we present a patient with primary EBV-LPD in the setting of CTLA-4 haploinsufficiency. JC is a 25yo male with Evans syndrome, hypogammaglobulinemia, and central nervous system vasculitis who presented to the NIH for evaluation of EBV-LPD. After a complicated course of legionella pneumonia at age 23, he developed recurrent fevers, respiratory distress, hypoxia, and night sweats responsive to dexamethasone. Chest CT revealed innumerable bilateral pulmonary nodules and masses with mediastinal hilar lymphadenopathy and scattered ground glass opacities. Transbronchial biopsy demonstrated EBV-positive B-cell LPD with extensive necrosis and clonal gene rearrangements. He responded well to 1 cycle of rituximab. Two years later, he again had fever, dyspnea on exertion, and hypoxia requiring glucocorticoids. Chest CT revealed ground glass opacities of the lungs, lung nodules/masses, and consolidation. Lung biopsy showed T and B-cell infiltrates, EBV+ B-cells by immunohistochemistry, and a clonal B-cell population (Figure 1). His course was also complicated by concurrent Pneumocystis Jirovecii (PJP) infection. He received 1 cycle of rituximab, trimethoprim-sulfamethoxazole, and sirolimus for treatment of his EBV-LPD, PJP, and underlying CTLA-4 haploinsufficiency respectively with symptomatic and radiologic response (Figure 2). [Display omitted] [Display omitted] NIAID whole exome sequencing (WES) revealed 2 compound heterozygous variants in CTLA-4 in trans: a novel pathogenic variant c.373G > A, p.Gly125Arg, and a non-pathogenic c.326G > A, p.Gly109Glu, shared with his mother(allele frequency of 0.000367 popmaxv.3.1.1 and Cadd score 17). Other variants that may contribute to EBV-LPD are under investigation.EBV-LPD in CTLA-4 haploinsufficiency is rare, previously reported in only 2 pediatric cases (PMID34329649). Additionally, WES revealed compound heterozygous variants in CTLA-4; however, only 1 variant was proven pathogenic. While this patient initially demonstrated excellent response to first line therapy, long-term prognosis is guarded. Abatacept was not included in this patient’s treatment because of an association with progressive LPD in patients with and without CTLA-4 haploinsufficiency. Immune reconstitution with bone marrow transplantation will likely be required for permanent disease control.

Familial CD45RA- T cells to treat severe refractory infections in immunocompromised patients.

Immunocompromised patients are susceptible to high-risk opportunistic infections and malignant diseases. Most antiviral and antifungal drugs are quite toxic, relatively ineffective, and induce resistance in the long term. The transfer of pathogen-specific Cytotoxic T-Lymphocytes has shown a minimal toxicity profile and effectiveness in treating Cytomegalovirus, Adenovirus, Epstein - Barr virus, BK Virus and Aspergillus infections, but this therapy have the main limitations of regulatory issues, high cost, and absence of public cell banks. However, CD45RA- cells containing pathogen-specific memory T-cells involve a less complex manufacturing and regulatory process and are cheaper, feasible, safe, and potentially effective. We present preliminary data from six immunocompromised patients: four who had severe infectious diseases and two who had EBV lymphoproliferative disease. All of them underwent multiple safe familial CD45RA- T-cell infusions as adoptive passive cell therapy, containing Cytomegalovirus, Epstein - Barr virus, BK virus, and Aspergillus-specific memory T-cells. We also present the method for selecting the best donors for CD45RA- cells in each case and the procedure to isolate and store these cells. The infusions were safe, there was no case of graft-versus host disease, and they showed a clear clinical benefit. The patients treated for BK virus nephritis, Cytomegalovirus encephalitis, Cytomegalovirus reactivation, and disseminated invasive aspergillosis experienced pathogen clearance, complete resolution of symptoms in 4-6 weeks and a lymphocyte increase in 3 of 4 cases after 3-4 months. Donor T cell transient microchimerism was detected in one patient. The two patients treated for EBV lymphoproliferative disease underwent chemotherapy and several infusions of CD45RA- memory T-cells containing EBV cytotoxic lymphocytes. Donor T-cell microchimerism was observed in both patients. The viremia cleared in one of the patients, and in the other, despite the viremia not clearing, hepatic lymphoproliferative disease remained stable and was ultimately cured with EBV-specific Cytotoxic T-Lymphocytes. The use of familial CD45RA- T-cells containing specific Cytotoxic T-lymphocytes is a feasible, safe and potential effective approach for treating severe pathogen infections in immunocompromised patients through a third party donor. Furthermore, this approach might be of universal use with fewer institutional and regulatory barriers.

Highly Efficient & Specific Repair of MAGT1 Mutation in Xmen Patient T Cells and Hematopoietic Stem Cells

Introduction: X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN) is a congenital glycosylation disorder with combined immune deficiency due to Magnesium Transporter 1 (MAGT1) mutations. Defective glycosylation of Natural Killer Group 2D (NKG2D), an activating receptor expressed on CD8 and NK cells critical for cytotoxicity, results in EBV lymphoproliferative disease and an inverted CD4:CD8 ratio in many XMEN patients. Oral magnesium supplementation for XMEN patients was not helpful, and hematopoietic stem cell transplants (HSCT) are complicated by high mortality. CRISPR-Cas9-AAV inserts MAGT1 specifically at endogenous locus but relies on double strand DNA breaks and adeno-associated virus donor that triggers DNA damage response. To address this, we evaluated base editing approach to repair a MAGT1 mutation that affects multiple patients with XMEN disease. We observed highly efficient mutation repair of XMEN T cells (75.5%) and hematopoietic stem cells (71.9%) to restore MAGT1 function and persistence of gene correction following transplant into immunodeficient mice. Materials and methods: Peripheral blood hematopoietic stem cells (HSPCs, CD34+) and mononuclear cells (PBMC) were collected from XMEN patients and healthy donors (HD) after G-CSF and plerixafor mobilization. PBMCs were stimulated (anti-CD3/anti-CD28, IL-2 (100 IU/mL), in ImmunoCult™-XF T cell expansion medium for 5-7 days prior to editing. HSPCs were cultured in StemSpan™ SFEM II Stem Cell Factor, Thrombopoietin, Fms-like tyrosine kinase 3 ligand (all 100ng/mL) and UM171 for two days prior to editing. Base editing is performed by electroporation (MaxCyte Inc.) of cells with ABE8e SpRY (CELLSCRIPT LLC.) and synthetic guide (Synthego) RNAs. For in vitro killing activity, T cells were co-cultured with EBV-B cells at a ratio of 1:1 for 48 hours and analyzed for apoptosis (Apoptracker Green+ 7AAD-), necrosis (Apoptracker Green+ 7AAD+) and degranulation (CD8+ CD107a+). HSPCs were transplanted into mice two days after EP and harvested 17-20 weeks after transplant. Results:Base editing of XMEN T cells: Flow cytometric analysis 2 days post BE show restored expression of biomarker NKG2D in XMEN CD8+ T cells (75.5%) compared to HD (93.2%) and naïve XMEN (2.39%). CD8+ cytotoxic activity against EBV-B cells was restored in BE XMEN patient cells; as indicated by increased apoptosis (BE: 6.78%, naïve: 4.82%, HD: 8.17%), necrosis (BE: 9.35%, naïve: 4.54%, HD: 7.19%), and degranulation (BE: 22.80%, naïve: 15.50%, HD: 21.40%), compared to naïve MAGT1 CD8+ T cells.In an in vivo tumor model, BE XMEN T cells injected into mice with induced luminescent EBV-B cell tumors lysed tumors and reduced average bioluminescence flux (photons/sec) (7 weeks) (8.23 × 107 ± 1.06 × 108) compared to naïve T cells (3.39 × 109 ± 2.22 × 109 and HD T cells (1.45 × 109 ± 2.22 × 109). Base editing of HSPCs: Immunodeficient NSGS pups transplanted with BE XMEN HSPCs engrafted and differentiated normally into lymphocytes with a normalized CD4:CD8 ratio in spleen (BE: 0.79 ± 0.14, naïve: 3.45 ± 1.1, HD: 1.3 ± 0.40) and peripheral blood (BE: 2.16 ± 0.84, naïve: 5.56 ± 2.32, HD: 1.79 ± 0.70) (Fig 1a). Importantly, restoration of biomarker NKG2D expression selectively on CD8+ T cells in spleen (BE: 80.5% ± 8.61%, naïve: 3.63% ± 1.45%, HD: 68.9% ± 10.32%) and peripheral blood (BE: 86.1% ± 2.88%, naïve: 2.25% ± 1.45%, HD: 62.6% ± 12.44%) (Fig 1b) confirmed correction of MAGT1 function in the glycosylation of critical NKG2D protein. High throughput targeted sequencing of on-target locus shows minimal bystander edits and additional off-target analysis is ongoing. Conclusion: Our data show highly efficient and specific base editing repair of MAGT1 mutation in XMEN T lymphocytes and CD34+ HSPCs. BE XMEN T cells increased cytotoxic activity in vitro and killing of tumor cells in mice. In vivo studies with mice transplanted with BE CD34+ HSPCs also confirm long term engraftment and differentiation into CD8+ T cells with restored NKG2D necessary for activation and cytolytic activity of CD8+ T and NK cells. The highly efficient and specific correction with base editing holds great promise for gene therapy for XMEN disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeted Delivery of BZLF1 to DEC205 Drives EBV-Protective Immunity in a Spontaneous Model of EBV-Driven Lymphoproliferative Disease.

Epstein-Barr virus (EBV) is a human herpes virus that infects over 90% of the world’s population and is linked to development of cancer. In immune-competent individuals, EBV infection is mitigated by a highly efficient virus-specific memory T-cell response. Risk of EBV-driven cancers increases with immune suppression (IS). EBV-seronegative recipients of solid organ transplants are at high risk of developing post-transplant lymphoproliferative disease (PTLD) due to iatrogenic IS. While reducing the level of IS may improve EBV-specific immunity and regression of PTLD, patients are at high risk for allograft rejection and need for immune-chemotherapy. Strategies to prevent PTLD in this vulnerable patient population represents an unmet need. We have previously shown that BZLF1-specific cytotoxic T-cell (CTL) expansion following reduced IS correlated with immune-mediated PTLD regression and improved patient survival. We have developed a vaccine to bolster EBV-specific immunity to the BZLF1 protein and show that co-culture of dendritic cells (DCs) loaded with a αDEC205-BZLF1 fusion protein with peripheral blood mononuclear cells (PMBCs) leads to expansion and increased cytotoxic activity of central-effector memory CTLs against EBV-transformed B-cells. Human–murine chimeric Hu-PBL-SCID mice were vaccinated with DCs loaded with αDEC205-BZLF1 or control to assess prevention of fatal human EBV lymphoproliferative disease. Despite a profoundly immunosuppressive environment, vaccination with αDEC205-BZLF1 stimulated clonal expansion of antigen-specific T-cells that produced abundant IFNγ and significantly prolonged survival. These results support preclinical and clinical development of vaccine approaches using BZLF1 as an immunogen to harness adaptive cellular responses and prevent PTLD in vulnerable patient populations.

CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease

Epstein-Barr virus (EBV) is a latent and oncogenic human herpesvirus. Lytic viral protein expression plays an important role in EBV-associated malignancies. The EBV envelope glycoprotein 350 (gp350) is expressed abundantly during EBV lytic reactivation and sporadically on the surface of latently infected cells. Here we tested T cells expressing gp350-specific chimeric antigen receptors (CARs) containing scFvs derived from two novel gp350-binding, highly neutralizing monoclonal antibodies. The scFvs were fused to CD28/CD3ζ signaling domains in a retroviral vector. The produced gp350CAR-T cells specifically recognized and killed gp350+ 293T cells in vitro. The best-performing 7A1-gp350CAR-T cells were cytotoxic against the EBV+ B95-8 cell line, showing selectivity against gp350+ cells. Fully humanized Nod.Rag.Gamma mice transplanted with cord blood CD34+ cells and infected with the EBV/M81/fLuc lytic strain were monitored dynamically for viral spread. Infected mice recapitulated EBV-induced lymphoproliferation, tumor development, and systemic inflammation. We tested adoptive transfer of autologous CD8+gp350CAR-T cells administered protectively or therapeutically. After gp350CAR-T cell therapy, 75% of mice controlled or reduced EBV spread and showed lower frequencies of EBER+ B cell malignant lymphoproliferation, lack of tumor development, and reduced inflammation. In summary, CD8+gp350CAR-T cells showed proof-of-concept preclinical efficacy against impending EBV+ lymphoproliferation and lymphomagenesis.

The Morphologic Features of Primary Epstein-Barr Virus Infection in the Gastrointestinal Tract: An Approach to Correct Diagnosis.

Epstein-Barr virus (EBV) is a herpes virus that has been shown to contribute to the development of multiple tumor types. There are isolated reports on EBV infection in the gastrointestinal tract, but, notably, there are none with detailed descriptions of the morphologic features. It is believed to be difficult to generalize the histologic features, as EBV does not cause viral inclusions in infected cells. This study was aimed at describing the pathologic changes of nonlymphomatous gastrointestinal EBV lymphoproliferative diseases in both immunocompetent and immunocompromised settings; the former was focused on chronic active EBV infection, and the latter was about the infection in posttransplantation patients. A retrospective search identified a total of 26 cases of EBV lymphoproliferative disease in the gastrointestinal tract-8 in immunocompetent patients and 18 in immunocompromised patients. We found that there was no difference in pathologic findings in immunocompetent and immunocompromised patients, which ranged from essentially subtle to severe mucosal architectural distortion with prominent lymphoid aggregates. Most cases showed either focal or diffuse lymphocytic infiltration comprised of small to intermediate-sized lymphocytes with a round shape and increased cytoplasm when compared with mature small lymphocytes. Focal lymphoepithelial lesions and karyorrhexis can be seen. Although gastrointestinal primary EBV lymphoproliferative diseases are not common, the mortality is high. Awareness of the potential histologic features combined with suspicion of EBV infection from clinical presentation, radiographic findings, and/or EBV serologies can aid in the diagnosis of primary EBV infection in the gastrointestinal tract.

EBV-lymphoproliferative Disease During Treatment of Childhood Acute Lymphoblastic Leukemia/Lymphoma

EBV-lymphoproliferative Disease During Treatment of Childhood Acute Lymphoblastic Leukemia/Lymphoma

Long-Term Follow-up of Myeloablative Haploidentical (HID) Peripheral Blood Stem Cell Transplantation and Post-Transplant Cyclophosphamide (PTCy) Following Fludarabine and Total Body Irradiation (TBI)

▪Haploidentical donor (HID) transplant with post-transplant cyclophosphamide (PTCy) is being increasingly utilized worldwide. Although the original “Baltimore” protocol employed nonmyeloablative conditioning, there has been increasing interest in the use of myeloablative conditioning (MAC) in an attempt to decrease the risk of disease recurrence and graft failure. We present the long-term follow-up results of 82 consecutive patients receiving a MAC HID transplant at a single institution between 2011-2017, following a uniform protocol of Fludarabine/total body irradiation (TBI) 1200cGy conditioning, PTCy, tacrolimus and mycophenolate mofetil. PBSC was utilized as the graft source for all transplants and graft CD34 dose was capped at 5 x 106/kg. Median patient age was 42 (range 19-61) years. The most common diagnoses were AML (51%) and ALL (33%), and 39% were high/very high disease risk index (DRI). Donor was a child, sibling or parent in 35%, 43% and 22% respectively. Median follow-up time for surviving patients was 37 months. Primary engraftment occurred in all patients, with all patients achieving complete donor T cell and myeloid chimerism by day +30. There were no cases of late graft failure. The cumulative incidence of grade 3-4 acute graft-versus-host disease (GVHD) and moderate-to-severe chronic GVHD was 17% and 22%, respectively. Non-relapse mortality (NRM) was 7% at one year and 12% at three years post-transplant. Overall survival at 1, 2, and 3 years post-transplant was 85%, 72% and 66% respectively (figure 1). Estimated 3-year DFS, current GVHD-, relapse-free survival (CGRFS), conventional GRFS, and relapse was 67%, 62%, 43%, and 21% respectively. DFS at 3-yr was significantly improved in patients with low/intermediate vs high/very high DRI (79% vs. 48%, figure 2) due to a lower risk of relapse (12% vs. 36%). Infectious complications included CMV reactivation in 55% of patients (CMV disease - 2%) and BK virus infection in 38% (severe - 2%). There were no reported cases of EBV lymphoproliferative disease. This large single institution analysis with long-term follow-up confirms the feasibility of Fludarabine/TBI-based MAC HID transplant with PBSC/PTCy in younger adult patients. This is supported by both the high rate of engraftment, low NRM and a 3-yr CGRFS of 62% (suggesting freedom from relapse and need for long-term systemic immunosuppression for GVHD). The achievement of long term DFS in approximately 80% of low/intermediate DRI and half of high/very high DRI patients is promising. [Display omitted] DisclosuresSolh:ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Amgen: Speakers Bureau.

Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300.

Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

Herpesviruses in the Activated Phosphatidylinositol-3-Kinase-δ Syndrome.

The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is important for multiple stages of herpesvirus replication including virus entry, replication, latency, and reactivation. Recently, patients with gain-of-function mutations in the p110δ-catalytic subunit of PI3K or in the p85-regulatory subunit of PI3K have been reported. These patients have constitutively active PI3K with hyperactivation of Akt. They present with lymphoproliferation and often have infections, particularly recurrent respiratory infections and/or severe virus infections. The most frequent virus infections are due to Epstein–Barr virus (EBV) and cytomegalovirus (CMV); patients often present with persistent EBV and/or CMV viremia, EBV lymphoproliferative disease, or CMV lymphadenitis. No patients have been reported with CMV pneumonia, colitis, or retinitis. Other herpesvirus infections have included herpes simplex pneumonia, recurrent zoster, and varicella after vaccination with the varicella vaccine. Additional viral infections have included adenovirus viremia, severe warts, and extensive Molluscum contagiosum virus infection. The increased susceptibility to virus infections in these patients is likely due to a reduced number of long-lived memory CD8 T cells and an increased number of terminally differentiated effector CD8 T cells.

Anti-TNFα therapy for inflammatory bowel diseases is associated with Epstein-Barr virus lytic activation.

Anti-TNFα therapy, known to suppress T-cell immunity, is increasingly gaining popularity for treatment of autoimmune diseases including inflammatory bowel diseases (IBD). T-cell suppression increases the risk of B-cell EBV-lymphoproliferative diseases and lymphomas. Since EBV-lytic activation is essential for development of EBV-lymphomas and there have been reports of EBV-lymphomas in patients treated with anti-TNFα therapy, we investigated if patients treated with anti-TNFα antibodies demonstrate greater EBV-lytic activity in blood. Peripheral blood mononuclear cells from 10 IBD patients solely on anti-TNFα therapy compared to 3 control groups (10 IBD patients not on immunosuppressive therapy, 10 patients with abdominal pain but without IBD, and 10 healthy subjects) were examined for the percentage of T-cells, EBV load and EBV-lytic transcripts. Patients on anti-TNFα therapy had significantly fewer T-cells, greater EBV load, and increased levels of transcripts from EBV-lytic genes of all kinetic classes compared to controls. Furthermore, exposure of EBV-infected B-cell lines to anti-TNFα antibodies resulted in increased levels of BZLF1 mRNA; BZLF1 encodes for ZEBRA, the viral latency-to-lytic cycle switch. Thus, IBD patients treated with anti-TNFα antibodies have greater EBV loads likely due to enhanced EBV-lytic gene expression and anti-TNFα antibodies may be sufficient to activate the EBV lytic cycle. Findings from this pilot study lay the groundwork for additional scientific and clinical investigation into the effects of anti-TNFα therapy on the life cycle of EBV, a ubiquitous oncovirus that causes lymphomas in the setting of immunocompromise.

FDA Actions for Drugs for EBV Lymphoproliferative Disease and Fallopian Tube Cancer

FDA Actions for Drugs for EBV Lymphoproliferative Disease and Fallopian Tube Cancer

Epstein-Barr virus lymphoproliferative disease after hematopoietic stem cell transplant.

Epstein-Barr virus (EBV) reactivation can cause significant morbidity and mortality after allogeneic hematopoietic stem cell transplant. Delays in reconstitution of EBV-specific T lymphocyte activity can lead to life-threatening EBV lymphoproliferative disease (EBV-PTLD). This review highlights recent advances in the understanding of pathophysiology, risk factors, diagnosis, and management of EBV viremia and PTLD. During the past decade, early detection strategies, such as serial measurement of EBV-DNA load, have helped identify high-risk patients and diagnose early lymphoproliferation. The most significant advances have come in the form of innovative treatment options, including manipulation of the balance between outgrowing EBV-infected B cells and the EBV cytotoxic T lymphocyte response, and targeting infected B cells with monoclonal antibodies, chemotherapy, unmanipulated donor lymphocytes, and donor or more recently third-party EBV cytotoxic T lymphocytes. Defining criteria for preemptive therapy remains a challenge. EBV reactivation is a significant complication after stem cell transplant. Continued improvements in risk stratification and treatment options are required to improve the morbidity and mortality caused by EBV-associated diseases. Current approaches use rituximab to deplete B cells or adoptive transfer of EBV cytotoxic T lymphocyte to reconstitute immunity. The availability of rapid EBV-specific T cell products offers the possibility of improved outcomes.

Adoptive Treatment Of EBV-Associated Leiomyosarcoma In Immunodeficient Patients With EBV Specific Cytotoxic T Cells

▪Epstein-Barr virus (EBV)-associated smooth muscle tumors or leiomyosarcomas (EBV LMS) are extremely rare. These tumors develop in patients with acquired or underlying immune deficiency including those after solid organ transplant (SOT) and exhibit a latency type II phenotype. EBV LMSs are typically aggressive and respond poorly to conventional therapy. As part of an on-going trial of EBV specific cytotoxic T lymphocytes for the treatment of EBV related malignancies we have treated 5 patients with EBV LMS with cytotoxic T cell lines generated from normal donors. Five patients, three after SOT (kidney N=1, small bowel N=2), one after solid organ (kidney) and hematopoetic stem cell transplant, and one with a poorly defined underlying immune deficiency syndrome developed EBV LMS. Two patients were successfully treated for EBV lymphoproliferative disease prior to developing EBV LMS. EBV LMS was diagnosed 18 – 46 months after SOT in 4 patients, at 9 years old in the fifth and were multifocal at diagnosis in all 5 patients. All four patients who had undergone SOT failed to respond to decreased immune suppression, one had failed treatment with autologous EBV CTLs and two had failed cytotoxic chemotherapy. The EBV LMS was of host origin in 4/5 patients and of mixed donor and host origin in one. Lines of EBV CTLs were generated as previously described by co-culture of donor peripheral blood T lymphocytes with autologous EBV transformed BLCLs. Lines are tested for EBV specific cytotoxicity, sterility and absence of alloreactivity. Patients with EBV LMS were treated with cell lines generated from unrelated as well as related donors. Three patients received EBV CTLs generated from related haplo-identical parent donors including one whose parental donor was also the donor of her transplanted kidney. One patient received EBV CTLs from an HLA identical related donor. All other lines were generated from 3rd parties. Lines from unrelated third party donors were matched at 2 – 4/8 HLA alleles. In all instances EBV directed cytotoxicity of infused lines was restricted by HLA alleles shared by the patient and the EBV LMS. Three patients achieved partial remissions (PR) lasting 18 – 122 months and one patient has had stable disease for >80 months. One patient had stable disease for 14 months before progression. Expansion of EBV CTLp was demonstrated in all patients after infusion of EBV CTLs lasting from 0.5 to 4 months. Based on the prediction that 3rd party partially matched EBV CTLs are unlikely to persist long term in vivo four of the patients have received multiple cycles of EBV CTLs in an effort to maintain these responses. EBV directed cytotoxic T cell therapy is a novel treatment strategy for EBV LMS, a malignancy that is typically associated with a very poor prognosis. EBV CTLs from immunologically normal, partially HLA-matched third party donors, appropriately specific and HLA restricted can induce significant and durable responses to EBV LMS. Approaches designed to selectively expand T-cells specific for EBV antigens expressed by these malignancies may further improve responses. Disclosures:No relevant conflicts of interest to declare.

Third Party Donor Derived EBV Specific T Cells for the Treatment of Refractory EBV-Related Malignancies in Immunodeficient Recipients

AbstractAbstract ▪577▪This icon denotes a clinically relevant abstractAdoptive immunotherapy is an effective strategy for the treatment of EBV+ lymphoproliferative diseases (EBV-LPD) arising after an allogeneic hematopoietic stem cell (HSCT) or solid organ transplant (SOT). This approach is, however, often limited by an inability to generate donor derived in vitro expanded EBV-specific cytotoxic T-lymphocyte (EBV-CTL) lines in a timely manner and/or the fact that EBV CTL lines derived from HLA non-identical donors may be restricted by non-shared HLA alleles.To date, we have treated 25 consecutive patients with an EBV LPD (N=20) or EBV Leiomyosarcoma (LMS) (N=5) with in vitro expanded EBV-CTLs derived from a donor other than the patient or their transplant (HSCT or SOT) donor. EBV CTLS were selected from a bank of 345 lines generated under GMP conditions from normal HSCT donors. Each donor was specifically consented for use of their T cells in patients other than their designated transplant recipient. Patients were recipients of unmodified (n=4), T cell depleted (n=5) or unrelated cord blood (n=5) HSCT, a solid organ transplant (n=6), a combined SOT and HSCT (n=1), or were non-transplanted patients with a primary immunodeficiency disease (n=4).EBV disease in transplanted patients was of host origin in 5 of 10 evaluable HSCT recipients, in 3 of 4 evaluable solid organ recipients, and in the one patient who underwent a combined HSCT/solid organ transplant. Third party EBV-CTLs were selected on the basis of HLA matching at a minimum of 2/8 recipient alleles. Where possible EBV-CTLs were selected that were restricted through HLA alleles present on the EBV+ tumor. HLA restriction was evaluated in vitro in 20 EBV-CTL donor lines. The restriction was at a single HLA allele (n=12), at two alleles (n=6) and at >than two alleles (n=2).Patients received infusions of 3rd party EBV-CTLs after failing a median of 2 prior therapies including rituximab in all but one case of EBV LPD. Four patients failed prior infusions with EBV-CTLs which were autologous (n=1), derived from their original HSCT (n=2) or from their solid organ donor (n=1). In two patients who progressed after treatment with EBV CTLs generated from their HSCT or organ donor, it was demonstrated that the donor derived EBV CTLs were restricted by a non-shared HLA allele.Patients received a median of 5 infusions most at 1×106 EBV-CTL/kg/infusion. Four patients received EBV-CTLs from >1 3rd party donor. Nine patients achieved a completed response. Nine patients died of progressive disease, 6 shortly after the first infusion (17–29 days). Two patients with LMS achieved long term stable disease (46 and 8 months); 5 achieved partial remissions which have been sustained in 4 (11- 68 months), and 1 patient progressed after 10 months in a partial remission. Response to EBV CTL therapy did not correlate with the degree of HLA matching between donor and recipient or donor and tumor. Radiographic and clinical responses correlated with detectable increases in the frequency of CTL precursors in the blood. However durable EBV CTL engraftment was not seen.One patient developed mild skin GvHD after infusion with 3rd party EBV-CTLs, but tolerated subsequent infusion of EBV-CTLs from an alternate 3rd party donor. Although no SOT recipient developed anti-HLA antibodies, one developed and episode of steroid responsive renal transplant rejection more than 6 months after infusion of EBV CTLs without evidence of donor (by STR analysis) in biopsied tissue.This study demonstrates a high response rate among patients with otherwise refractory EBV malignancy treated with EBV specific 3rd party CTLs restricted by HLA alleles shared by the tumor. Treatment failures correlated with the use of EBV CTLs restricted by HLA alleles not shared by the tumor. In addition two patients with a primary immunodeficiency disease who were unable to mount an endogenous EBV T cell response had transient but not durable responses to 3rd party cells. EBV CTLs can be effective when selected based on restriction to shared alleles despite significant HLA disparity. The bank of EBV specific T cells can provide an immediate source of HLA partially matched appropriately restricted T cells for adoptive immunotherapy to treat EBV associated malignancy. This enables treatment early in the course of disease and the use of EBV-CTL lines previously prepared and characterized in terms of HLA restriction. This is anticipated to maximize the response rate. Disclosures:No relevant conflicts of interest to declare.

A Novel Reduced Intensity Conditioning Regimen Induces a High Incidence of Sustained Donor Neutrophil and Platelet Engraftment After Double-Unit Cord Blood Transplantation

AbstractAbstract 4155 Introduction:Cord blood (CB) transplant (CBT) can be curative for patients with high-risk hematologic malignancies. However, patients of older age and/or with significant co-morbidities do not tolerate CBT with high-dose myeloablative conditioning. Non-myeloablative (NMA) conditioning can reduce transplant-related mortality (TRM) and extend transplant access to older or infirm patients, but it is limited by the risks of graft rejection in patients without extensive prior chemotherapy and relapse. While the addition of anti-thymocyte globulin (ATG) may reduce rejection, it increases the risk of viral infections, including Epstein-Barr virus lymphoproliferative disease, and may also increase relapse risk. Methods:We investigated the safety and efficacy of an ATG-free regimen of intermediate intensity prior to double-unit CBT in 30 patients with acute leukemias and myelodysplasia. Units were 4–6/6 HLA-A, B antigen, DRB1 allele matched to the patient. The conditioning regimen included cyclophosphamide 50 mg/kg (day -6), fludarabine 30 mg/m2/day × 5 (days -6 to -2), thiotepa 5 mg/kg/day × 2 (days -5 and -4), total body irradiation 200 cGy × 2 (days -2 and -1), and cyclosporine-A/mycophenolate mofetil immunosuppression. The indication for this regimen was one or more risk factors for TRM including age > 50 years, extensive prior therapy, and/or significant co-morbidities. The hematopoietic cell transplant co-morbidity index (HCT-CI) score of Sorror was retrospectively assigned. Results:The median age was 56 years (range 18–69). All but one patient had high-risk disease. Twenty-one had AML (16 CR1, 5 CR2) with all CR1 patients having high-risk features, including high-risk cytogenetics (n = 3), FLT-3 ITD mutation (n = 5), therapy-related disease or prior MDS (n = 6), and/or > 3 consecutive induction chemotherapies (n = 2). Five had ALL (4 CR1, 1 CR3); the 4 in CR1 had BCR/ABL mutations (n = 3) or prior refractory CNS disease (n = 1). Four patients had MDS with 3 having an IPSS score > 2. The median HCT-CI score was 2.5 (range 1–5). Median infused TNC doses were 2.6 (larger unit) and 1.9 (smaller unit) × 107/kg, respectively. Ninety-seven percent of patients engrafted (95%CI: 87–100) at a median of 26 days (range 13–43). The median day 21 total donor bone marrow chimerism was 100% (range 71–100). All surviving patients were 100% donor by day 100, and sustained hematopoiesis has been mediated by a single unit in all but one patient. The cumulative incidence of platelet recovery > 20 × 109/L by day 180 was 93% (95%CI: 83–100), and occurred at a median of 46 days (range 30–79). Day 180 TRM and 2-year relapse incidences were 20% and 11%, respectively. With a median 26.5 months (range 9–53) follow-up of survivors, the 2-year overall survival and disease-free survival (DFS) are both 60% (95%CI: 44–82). There was a hierarchy in 2-year DFS according to the Sorror HCT-CI score (Figure): the 11 patients (median age 55 years) with a score of 1 had a DFS of 82%. This compared with a 2-year DFS of 62% in the 9 patients (median age 51 years) with a score of 2–3, and 40% in the 11 patients (median age 58 years) with a score of 4–5 (p = 0.13). Discussion:This reduced intensity regimen combined with double-unit CBT reliably facilitates sustained donor engraftment without ATG. This regimen is associated with less toxicity than high-dose myeloablative conditioning. While other approaches are needed in patients with high comorbidity scores, this regimen is highly effective in older patients who are otherwise reasonably fit, as evidenced by the 82% 2-year DFS in patients with a median age of 55 years. Given the relatively low risk of relapse, it also represents a promising alternative to high-dose conditioning in younger patients. [Display omitted] Disclosures:Giralt:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Membership on an entity's Board of Directors or advisory committees.

Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation

AbstractWe evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.

Moving Successful Virus-specific T-cell Therapy for Hematopoietic Stem Cell Recipients to Late Phase Clinical Trials

There are now multiple effective strategies for the activation and expansion, or direct selection of virus-specific T cells that may be able to eliminate a range of virus infections in the immunocompromised host. In this mini-review, we explain how the stage is set for their rigorous evaluation in large-scale clinical trials. Virus infections are the cause of 30% or more of transplant-related deaths in recipients of T-cells depleted, allogeneic hematopoietic stem cell transplants (HSCT).1 Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenoviruses (AdV) are the most common culprits, but other common viruses like parainfluenza virus, respiratory syncytial virus, influenza viruses, polyomaviruses, and human herpesvirus 6 together contribute significant morbidity and mortality (Figure 1).2,3,4 Small molecule therapies are often ineffectual, always costly and frequently produce significant adverse effects. Figure 1 Relative frequency of viral infections after HSCT. Adv, adenovirus; BK, JC, KI, and WU (polyomaviruses); CMV, cytomegalovirus; H1N1, influenza strain Hemagglutinnin 1 Neuraminidase 1); HHV, human herpes virus; HSCT, hematopoietic stem cell transplant; ... Virus-specific T-cells derived from stem cell donors can prevent and treat post-transplant viral infections, in the recipients for whom they were intended, and also in partially human leukocyte antigen (HLA)-matched, third party recipients.5,6,7 The low toxicity and long-term protection provided by virus-specific T-cells compares favorably with the significant toxicities and short-term effects of most antivirals.5,6,8,9,10 Is it time, therefore, to begin the transfer of T-cell manufacturing from academic phase I/II clinical trials into hospital or industry-supported facilities so that virus-specific T-cells can be made available to all high-risk HSCT recipients? Several barriers prevent the broader use of virus-specific T-cell therapies after stem cell transplantation. While T-cell therapies for EBV, CMV, and AdV have clearly demonstrated their safety and efficacy both as prophylaxis and as therapy, for many other viruses, the antigens that induce protective T-cells have yet to be identified. Moreover, these infections may occur in <5% of patients making it difficult to perform the rigorous comparative effectiveness studies that will be required to show lower overall cost, fewer adverse effects, and equivalent or superior efficacy. Before any of these barriers can be breached effectively by the academic institutions who are the major developers of these T-cells therapeutics, we must select and optimize manufacturing strategies that are robust and scalable and have the lowest possible cost. Finally, the leap to late phase trials cannot be accomplished by academic institutions alone, but requires partnership with industry. This article will largely deal with selection and optimization of virus-specific T-cell manufacturing strategies.

Induction of Antiviral Cytotoxic T Cells by Plasmacytoid Dendritic Cells for Adoptive Immunotherapy of Posttransplant Diseases

Virus-associated hematologic malignancies (EBV lymphoproliferative disease) and opportunistic infections (CMV) represent a major cause of hematopoietic stem cell and solid organ transplantation failure. Adoptive transfer of antigen-specific T lymphocytes appears to be a major and successful immunotherapeutic strategy, but improvements are needed to reliably produce high numbers of virus-specific T cells with appropriate requirements for adoptive immunotherapy that would allow extensive clinical use. Since plasmacytoid dendritic cells (pDCs) are crucial in launching antiviral responses, we investigated their capacity to elicit functional antiviral T-cell responses for adoptive cellular immunotherapy using a unique pDC line and antigens derived from Influenza, CMV and EBV viruses. Stimulation of peripheral blood mononuclear cells from HLA-A*0201(+) donors by HLA-A0201 matched pDCs pulsed with viral-derived peptides triggered high levels of multi-specific and functional cytotoxic T-cell responses (up to 99% tetramer(+) CD8 T cells) in vitro. Furthermore, the central/effector memory cytotoxic T cells elicited by the pDCs strongly display antiviral activity upon adoptive transfer into a humanized mouse model that mimics a virus-induced malignancy. We provide a simple and potent method to generate virus-specific CTL with the required properties for adoptive cellular immunotherapy of post-transplant diseases.