EBV-specific Cytotoxic T Lymphocytes Research Articles

EBV Reactivation and Disease in Allogeneic Hematopoietic Stem Cell Transplant (HSCT) Recipients and Its Impact on HSCT Outcomes.

The acquisition or reactivation of Epstein-Barr virus (EBV) after allogeneic Hematopoietic Stem Cell Transplant (HSCT) can be associated with complications including the development of post-transplant lymphoproliferative disorder (PTLD), which is associated with significant morbidity and mortality. A number of risk factors for PTLD have been defined, including T-cell depletion, and approaches to monitoring EBV, especially in high-risk patients, with the use of preemptive therapy upon viral activation have been described. Newer therapies for the preemption or treatment of PTLD, such as EBV-specific cytotoxic T-cells, hold promise. Further studies to help define risks, diagnosis, and treatment of EBV-related complications are needed in this at-risk population.

Establishment of a protocol for rapidly expanding Epstein–Barr-virus-specific cytotoxic T cells with enhanced cytotoxicity

BackgroundLytic Epstein–Barr virus (EBV) infection plays a major role in the pathogenesis of nasopharyngeal carcinoma (NPC). For patients with recurrent or metastatic NPC and resistant to conventional therapies, adoptive cell therapy using EBV-specific cytotoxic T cells (EBV-CTLs) is a promising option. However, the long production period (around 3 to 4 weeks) and low EBV-CTL purity (approximately 40% of total CD8 T cells) in the cell product limits the application of EBV-CTLs in clinics. Thus, this study aimed to establish a protocol for the rapid production of EBV-CTLs.MethodsBy culturing peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors with EBV-specific peptides and interleukin (IL)-2, IL-15, and interferon α (IFN-α) for 9 days, we identified that IL-15 can enhance IL-2-mediated CTL activation and significantly increase the yield of CTLs.ResultsWhen IFN-α was used in IL-2/IL-15-mediated CTL production from days 0 to 6, the productivity of EBV-CTLs and EBV-specific cytotoxicity significantly were reinforced relative to EBV-CTLs from IL-2/IL-15 treatment. Additionally, IFN-α-induced production improvement of virus-specific CTLs was not only the case for EBV-CTLs but also for cytomegalovirus-specific CTLs.ConclusionWe established a novel protocol to rapidly expand highly pure EBV-CTLs from PBMCs, which can produce EBV-CTLs in 9 days and does not require feeder cells during cultivation.

Abstract PO-013: Combined epigenetic therapy to induce latency II/III antigen expression in latency I EBV+ lymphoma

Abstract In EBV+ lymphomas the virus exists in one of three latent states. Cellular therapy with EBV cytotoxic T cells (CTLs) is effective in latency II/III tumors that express the viral proteins LMP1/2 and/or EBNA2-6 but not in latency I tumors. Previously, we reported that EBV latency restriction is regulated through DNA methyltransferase 1 (DNMT1, Guo at al, Nat Micrbiol 2020) and that treatment with decitabine (DCB) induces latency II/III antigen expression in latency I Burkitt lymphoma (BL), rendering BL sensitive to EBV-specific CTLs (Dalton et al, Blood 2020). We have also found that a DNMT1 specific inhibitor (DNMT1i) converts BL to latency II/III, highlighting the key role of DNMT1 in latency regulation. One limitation to DNMT1 inhibition as a therapeutic approach in latency I EBV+ lymphoma is that the induction of latency II/III is only observed in a subset of cells. Here we investigated mechanisms of resistance to latency conversion to develop rational combinations to maximize the conversion of latency I tumors to latency II/III thereby sensitizing them to viral-directed immunotherapy. To determine if epigenetic mechanisms are responsible for resistance to latency conversion, we performed ATACseq and ChIP-qPCR on sensitive vs resistant KemI cells. ATACseq revealed open chromatin at Cp, which controls expression of EBNA2-6, in DCB-sensitive cells, but closed-chromatin in DCB-resistant cells, suggesting chromatin compaction may play a role in suppression of EBNA2-6. We observed no difference in chromatin accessibility at the LMP1 promoter, suggesting an independent mechanism may regulate LMP1. We performed ChIP-qPCR to evaluate activating (H3K27ac, H3K4me3 and H3K36me2) and repressive (H2K27me3 and H3K9me3) marks at Cp and LMP1p. We observed an increase in H3K27me3 and H3K9me3 on Cp and LMP1p in resistant cells. H3K27me3 was increased 2-fold on Cp (adj-p=0.0009) and 2.45-fold on LMP1p (adj-p=0.0086) in DNMT1i resistant vs. sensitive cells. H3K9me3 was increased a 2.4-fold (adj-p<0.0001) and 2.3-fold (adj-p<0.0001) on Cp in DCB and DNMT1i resistant cell respectively. H3K9me3 was increased 3.46-fold (adj-p=0.0013) on LMP1p in DCB resistant cells. Given our observation of increased H3K27me3 resistant cells, we next evaluated the effect of the EZH2 inhibitor tazemetostat, which targets H3K27me3, alone and in combination with DCB or DNMT1i in KemI. Tazemetostat treatment alone did not induce expression of latency II/III. However, when combined with DCB or DNMT1i, tazemetostat increases the percentage of LMP1+ EBNA2+ cells compared to DCB or DNMT1i alone (DCB: 32.3% to 44.5% adj-p=0.0006; DNMT1i: 22.2% to 35.6% adj-p=0.0002). Our data suggests that repressive histone marks contribute to resistance to DNMT1 inhibition-induced latency switch and that the addition of tazemetostat to DCB or DNMT1i can improve latency switch, potentially resulting in a more immunogenic tumor. These findings will guide the development of therapies to improve the treatment of otherwise immune refractory EBV+ latency I lymphoma. Citation Format: Isabella Y Kong, Vicenta Trujillo Alonso, Sarah Clark, Suhong Sun, Alicia Alonso, Rebecca Whitehouse, Stephanie Trezise, Roberta Zappasodi, Ethel Cesarman, Lisa Giulino Roth. Combined epigenetic therapy to induce latency II/III antigen expression in latency I EBV+ lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-013.

Efficacy and Safety of Cell-based Immunotherapy in The Treatment of Recurrent or Metastatic Nasopharyngeal Carcinoma – A Systematic Review and Meta-analysis

BackgroundRecurrent/Metastatic Nasopharyngeal Carcinoma (RM-NPC) remains difficult to treat and contributes to considerable mortality. The first-line treatment for RM-NPC is Gemcitabine and Cisplatin and second-line treatment options differ. The endemic variant of NPC is associated with Epstein-Barr Virus (EBV). Therefore, Cell-based Immunotherapy (CBI) targeting EBV-specific RM-NPC may be effective. MethodsWe systematically searched PubMed, Embase and the Cochrane Library for randomised or observational studies investigating the efficacy and safety of CBI in the treatment of RM-NPC. We performed all meta-analyses using the random-effects model. Studies were further stratified by endemicity, nature of disease and drug type to investigate for potential between-study heterogeneity and additional pre-specified tests were employed to assess for publication bias. ResultsWe screened 1,671 studies and included 13 studies with 403 participants in the systematic review, of which nine studies were eligible for meta-analysis. The use of CBI monotherapy as second or subsequent line treatment for EBV-positive RM-NPC revealed an ORR of 10 % (95 %CI = 3 %–29 %), median PFS of 2.37 months (95 %CI = 1.23–3.51) and median OS of 10.16 months (95 %CI = 0.67–19.65). For EBV-specific Cytotoxic T-Lymphocyte monotherapy, the pooled PD rate was 54 % (95 %CI = 9 %–93 %), SD rate was 22 % (95 %CI = 2 %–75 %) and incidence rate of any grade adverse events was 45 %. For Dendritic Cell monotherapy, a PD rate of 80 % (95 % CI = 29 %–98 %), SD rate of 11 % (95 % CI = 0 %–82 %) and incidence rate of any grade adverse events of 29 % was achieved. ConclusionCBI monotherapy demonstrates some activity in pre-treated RM-NPC. More trials are needed to better understand how to integrate CBI into RM-NPC care.

Targeting Epstein-Barr virus in multiple sclerosis: when and how?

Epidemiological evidence implicates Epstein-Barr virus (EBV) as the cause of multiple sclerosis (MS). However, its biological role in the pathogenesis of MS is uncertain. The article provides an overview of the role of EBV in the pathogenesis of MS and makes a case for targeting EBV as a treatment strategy for MS. EBV potentially triggers autoimmunity via molecular mimicry or immune dysregulation. Another hypothesis, supported by immunological and virological data, indicates that active EBV infection via latent-lytic infection cycling within the central nervous system or periphery drives MS disease activity. This supports testing small molecule anti-EBV agents targeting both latent and lytic infection, central nervous system-penetrant B-cell therapies and EBV-targeted immunotherapies in MS. Immunotherapies may include EBV-specific cytotoxic or chimeric antigen receptors T-cells, therapeutic EBV vaccines and immune reconstitution therapies to boost endogenous EBV-targeted cytotoxic T-cell responses. EBV is the probable cause of MS and is likely to be driving MS disease activity via latent-lytic infection cycling. There is evidence that all licensed MS disease-modifying therapies target EBV, and there is a compelling case for testing other anti-EBV strategies as potential treatments for MS.

Membrane-bound CD95 ligand modulates CD19-mediated B cell receptor signaling and EBV activation.

Post-transplant lymphoproliferative disorders (PTLDs) are associated with Epstein-Barr virus (EBV) infection in transplant recipients. Most of lymphoblastoid cell lines (LCLs) derived from EBV-immortalized B cells or PTLDs are sensitive to CD95-mediated apoptosis and cytotoxic T cell (CTL) killing. CD95 ligand (CD95L) exists as a transmembrane ligand (mCD95L) or a soluble form (sCD95L). Using recombinant mCD95L and sCD95L, we observed that sCD95L does not affect LCLs. While high expression of mCD95L in CTLs promotes apoptosis of LCLs, low expression induces clathrin-dependent CD19 internalization, caspase-dependent CD19 cleavage, and proteasomal/lysosomal-dependent CD19 degradation. The CD95L/CD95-mediated CD19 degradation impairs B cell receptor (BCR) signaling and inhibits BCR-mediated EBV activation. Interestingly, although inhibition of the caspase activity restores CD19 expression and CD19-mediated BCR activation, it fails to rescue BCR-mediated EBV lytic gene expression. EBV-specific CTLs engineered to overexpress mCD95L exhibit a stronger killing activity against LCLs. This study highlights that engineering EBV-specific CTLs to express a higher level of mCD95L could represent an attractive therapeutic approach to improve T cell immunotherapy for PTLDs.

EBV Reactivation and Lymphomagenesis: More Questions than Answers.

Epstein-Barr Virus (EBV) is a ubiquitous herpesvirus that affects almost all humans and establishes lifelong infections by infecting B-lymphocytes leading to their immortalization. EBV has a discrete life cycle with latency and lytic reactivation phases. EBV can reactivate and cause lymphoproliferation in both immunocompetent and immunocompromised individuals. There is sparse literature on monitoring protocols for EBV reactivation and no standardized treatment protocols to treat EBV-driven lymphoproliferation. While there are no FDA-approved therapies to treat EBV, there are several strategies to inhibit EBV replication. These include immunosuppression reduction, nucleoside analogs, HDAC inhibitors, EBV-specific cytotoxic T-lymphocytes (CTLs), and monoclonal antibodies, such as rituximab. There is currently an open clinic trial combining the use of a HDAC inhibitor, nanatinostat, and ganciclovir to treat refractory/relapsed EBV lymphomas. Another novel therapy includes tabelecleucel, which is an allogenic EBV-directed T-cell immunotherapy that was approved by the European Medicines Agency, but is currently only available in the US for limited use in relapsed or refractory EBV-positive PTLD. Further research is needed to establish EBV monitoring protocols in high-risk populations, such as those with autoimmune disease, cancer, HIV, or receiving immunosuppressive therapy. Additionally, standardized treatments for both the prevention of EBV reactivation in high-risk populations and treatment of EBV reactivation and lymphoproliferation need to be established.

Long-lasting responses with chemotherapy followed by T-cell therapy in recurrent or metastatic EBV-related nasopharyngeal carcinoma.

Refractory or metastatic nasopharyngeal carcinoma (NPC) patients have a poor prognosis due to the lack of effective salvage treatments and prolonged survival by means of combination chemotherapy being described only for a minority of younger patients with oligometastatic disease. Targeting the Epstein - Barr virus (EBV) proteins expressed in NPC cells has been shown to be a feasible strategy that could help control systemic disease. Between 2011 and 2014, 16 patients with recurrent/metastatic EBV-NPC received first-line chemotherapy (CT) followed by 2 doses of autologous cytotoxic EBV specific T-lymphocytes (15-25 x 107 total cells/dose, 2 weeks apart), based on our previous studies showing the feasibility and efficacy of this infusion regimen. Cumulative overall survival (OS) and median OS were analysed in the whole population and according to specific clinical and biological parameters. All patients received the planned T-cell therapy schedule, 9 after reaching partial (n=5) or complete (n=4) disease remission with CT, and 7 after failing to obtain benefit from chemotherapy. No severe adverse events were recorded. Patients who received cytotoxic T-lymphocytes (CTLs) had a cumulative 10-year OS of 44%, with a median OS of 60 months (95% CI 42-62). Patients responding to CT, with oligometastatic disease (<3 disease sites), and plasma EBV-DNA <1000 copies/mL had a better outcome. Autologous EBV-specific CTLs transplanted following conventional first-line CT demonstrated promising efficacy with several patients obtaining long-lasting disease control. The rationale provided by this study, with the crucial role likely played by the timing of CTL administration when trying to induce synergy with conventional treatment needs to be confirmed in a prospective controlled trial.

Patient-tailored adoptive immunotherapy with EBV-specific T cells from related and unrelated donors.

BACKGROUNDAdoptive transfer of EBV-specific T cells can restore specific immunity in immunocompromised patients with EBV-associated complications.METHODSWe provide results of a personalized T cell manufacturing program evaluating donor, patient, T cell product, and outcome data. Patient-tailored clinical-grade EBV-specific cytotoxic T lymphocyte (EBV-CTL) products from stem cell donors (SCDs), related third-party donors (TPDs), or unrelated TPDs from the allogeneic T cell donor registry (alloCELL) at Hannover Medical School were manufactured by immunomagnetic selection using a CliniMACS Plus or Prodigy device and the EBV PepTivators EBNA-1 and Select. Consecutive manufacturing processes were evaluated, and patient outcome and side effects were retrieved by retrospective chart analysis.RESULTSForty clinical-grade EBV-CTL products from SCDs, related TPDs, or unrelated TPDs were generated for 37 patients with refractory EBV infections or EBV-associated malignancies with and without a history of transplantation, within 5 days (median) after donor identification. Thirty-four patients received 1-14 EBV-CTL products (fresh and cryopreserved). EBV-CTL transfer led to a complete response in 20 of 29 patients who were evaluated for clinical response. No infusion-related toxicity was reported. EBV-specific T cells in patients' blood were detectable in 16 of 18 monitored patients (89%) after transfer, and their presence correlated with clinical response.CONCLUSIONPersonalized clinical-grade manufacture of EBV-CTL products via immunomagnetic selection from SCDs, related TPDs, or unrelated TPDs in a timely manner is feasible. Overall, EBV-CTLs were clinically effective and well tolerated. Our data suggest EBV-CTL transfer as a promising therapeutic approach for immunocompromised patients with refractory EBV-associated diseases beyond HSCT, as well as patients with preexisting organ dysfunction.TRIAL REGISTRATIONNot applicable.FUNDINGThis study was funded in part by the German Research Foundation (DFG, 158989968/SFB 900), the Deutsche Kinderkrebsstiftung (DKS 2013.09), Wilhelm-Sander-Stiftung (reference 2015.097.1), Ellen-Schmidt-Program of Hannover Medical School, and German Federal Ministry of Education and Research (reference 01EO0802).

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.

Post-Transplant Lymphoproliferative Disease (PTLD) after Allogeneic Hematopoietic Stem Cell Transplantation: Biology and Treatment Options.

Post-transplant lymphoproliferative disease (PTLD) is a serious complication occurring as a consequence of immunosuppression in the setting of allogeneic hematopoietic stem cell transplantation (alloHSCT) or solid organ transplantation (SOT). The majority of PTLD arises from B-cells, and Epstein-Barr virus (EBV) infection is present in 60-80% of the cases, revealing the central role played by the latent infection in the pathogenesis of the disease. Therefore, EBV serological status is considered the most important risk factor associated with PTLDs, together with the depth of T-cell immunosuppression pre- and post-transplant. However, despite the advances in pathogenesis understanding and the introduction of novel treatment options, PTLD arising after alloHSCT remains a particularly challenging disease, and there is a need for consensus on how to treat rituximab-refractory cases. This review aims to explore the pathogenesis, risk factors, and treatment options of PTLD in the alloHSCT setting, finally focusing on adoptive immunotherapy options, namely EBV-specific cytotoxic T-lymphocytes (EBV-CTL) and chimeric antigen receptor T-cells (CAR T).

Extranodal natural killer/T-cell lymphoma: An overview on pathology and clinical management

Natural killer (NK)/T-cell lymphomas arise mainly from NK-cells and occasionally T-cells, and are universally infected with Epstein Barr virus (EBV). They are uncommon lymphomas more prevalent in Asian and Central/South American populations. NK/T-cell lymphomas are clinically aggressive and predominantly extranodal. The most commonly involved sites are the nasal cavity, followed by non-nasal sites including the skin, gastrointestinal tract and testis. The diagnosis of extranodal NK/T-cell lymphoma is established with histological and immunohistochemical examination, together with the demonstration of EBV in the tumour cells. Staging by positron emission tomography computed tomography is essential to inform the optimal management. Plasma EBV DNA quantification should be performed as it serves as a marker for prognostication and treatment response. Survival outcomes of patients with early-stage disease are good following treatment with nonanthracycline based chemotherapy, together with sequential/concurrent radiotherapy. For advanced-stage disease, asparaginase-containing regimens are mostly used and allogeneic haematopoietic stem cell transplantation should be considered for those at high risk of relapse. Salvage chemotherapy is largely ineffective for relapsed/refractory disease, which has a grave prognosis. Novel therapeutic approaches including immune check-point blockade, EBV-specific cytotoxic T-cells, and monoclonal antibodies are being investigated to improve outcomes for those with high risk and relapsed/refractory disease.

Viral-specific cytotoxic T-cell responses in HLA-sensitized kidney transplant patients maintained on everolimus and low-dose tacrolimus.

Maintenance with "everolimus + reduced dose tacrolimus" (Ev + Taclow ) was reported to reduce the risk of viral infections compared to "tacrolimus + mycophenolate mofetil" (Tac + MMF). Here we examined viremia and viral-specific T-cell (viral-Tc) responses in patients treated with Ev + Taclow versus Tac + MMF in highly-human leukocyte antigen (HLA)-sensitized patients. HLA-sensitized (HS) kidney transplant patients were monitored pre- and post-transplant for viremia (cytomegalovirus (CMV), BK, and Epstein-Barr virus (EBV)) by polymerase chain reaction (PCR) in 19 Ev + Taclow and 48 Tac + MMF patients. For CMV PCR analysis, we compared infection rates in 19 Ev + Taclow patients to 48 CMV D+/R- (#28) or CMV D-/R- (#20) Tac + MMF patients. CMV-specific cytotoxic T cell (CMV-Tc) and EBV-specific cytotoxic T cell (EBV-Tc) were evaluated by cytokine flow cytometry, and donor-specific antibody (DSA) levels by Luminex for selected patients in both groups. CMV and EBV viremia rates were similar in Ev + Taclow versus Tac + MMF patients, but BK virus (BKV) rates were significantly higher in Ev + Taclow patients. No patient in either group developed BK virus-associated allograft nephropathy (BKAN) or post-transplant lymphoproliferative disorders (PTLD). CMV-Tc and EBV-Tc decreased significantly after alemtuzumab induction but returned to pre-treatment levels 1-2 months post-transplant in most patients. de novo DSA was similar in both groups as were patient and graft survival and graft rejection. CMV-Tc and EBV-Tc were similar in Ev + Taclow and Tac + MMF patients. EBV and CMV viremia rates were similar over 1 year. BKV rates were significantly higher in Ev + Taclow patients suggesting no benefit for Ev + Taclow in enhancing viral-Tc effector functions or limiting viral infections.

Recent Advances in the Diagnosis and Treatment of Natural Killer Cell Malignancies.

Simple SummaryNatural killer (NK)/T-cell lymphomas are aggressive extranodal Epstein–Barr virus (EBV)-positive malignancies. They can be divided into three subtypes: nasal (involving the nose and upper aerodigestive tract), non-nasal (involving skin, gastrointestinal tract, testis and other organs) and disseminated (involving multiple organs). Lymphoma cells are positive for CD3ε, CD56, cytotoxic molecules and EBV-encoded small RNA. There is a predilection for Asian and Central/South American populations. Genome-wide association studies have identified lymphoma susceptibility loci in Asians. Positron emission tomography computed tomography and plasma EBV DNA quantification are crucial at diagnosis and follow-up. Stage I/II patients receive non-athracycline asparaginse-containing regimens, together with sequential/concurrent radiotherapy. Anthracycline-containing regimens are ineffective. Stage III/IV patients receive asparaginase-containing regimens, followed by allogeneic haematopoietic stem cell transplantation (HSCT). Autologous HSCT does not improve outcome. In relapsed/refractory patients, novel approaches include PD1/PD-L1 targeting, EBV-specific cytotoxic T-cells, and monoclonal antibodies. Small molecules including histone deacetylase inhibitors may be beneficial.Natural killer (NK)/T-cell lymphomas are aggressive malignancies. Epstein–Barr virus (EBV) infection in lymphoma cells is invariable. NK/T-cell lymphomas are divided into nasal, non-nasal, and disseminated subtypes. Nasal NK/T-cell lymphomas involve the nasal cavity and the upper aerodigestive tract. Non-nasal NK/T-cell lymphomas involve the skin, gastrointestinal tract, testis and other extranodal sites. Disseminated NK/T-cell lymphoma involves multiple organs, rarely presenting with a leukaemic phase. Lymphoma cells are positive for CD3ε (not surface CD3), CD56, cytotoxic molecules and EBV-encoded small RNA. There is a predilection for Asian and Central/South American populations. Genome-wide association studies have identified lymphoma susceptibility loci in Asian patients. Positron emission tomography computed tomography and plasma EBV DNA quantification are crucial evaluations at diagnosis and follow-up. Stage I/II patients typically receive non-athracycline regimens containing asparaginse, together with sequential/concurrent radiotherapy. Anthracycline-containing regimens are ineffective. Stage III/IV patients are treated with asparaginase-containing regimens, followed by allogeneic haematopoietic stem cell transplantation (HSCT) in suitable cases. Autologous HSCT does not improve outcome. In relapsed/refractory patients, novel approaches are needed, involving PD1/PD-L1 targeting, EBV-specific cytotoxic T-cells, and monoclonal antibodies. Small molecules including histone deacetylase inhibitors may be beneficial in selected patients. Future strategies may include targeting of signalling pathways and driver mutations.

Induced pluripotent stem cell-derived rejuvenated cytotoxic T lymphocyte therapy for Epstein-Barr virus-associated lymphomas: application to clinical practice

Epstein-Barr virus (EBV)-associated lymphomas are common in Asia and exhibit a poor prognosis. As EBV antigens LMP1 and LMP2 are often expressed in EBV-associated lymphomas, these lymphomas should be a good target for antigen-specific cytotoxic T lymphocyte (CTL) therapy. However, CTLs continuously exposed to viral or tumor antigens often become exhausted. Antigen-specific CTLs generated from induced pluripotent stem cells are functionally rejuvenated, showing a strong antitumor effect on EBV-associated lymphomas and persistence in vivo. For feasible "off-the-shelf" therapy, we generated allogeneic EBV-specific CTLs in the cell processing center and prepared them for actual use in clinical settings.

EBV‐specific cytotoxic T lymphocytes for refractory EBV‐associated post‐transplant lymphoproliferative disorder in solid organ transplant recipients: a systematic review

The use of Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) in adoptive immunotherapy in hematopoietic stem cell transplantation (HSCT) patients with post-transplantation lymphoproliferative disorder (PTLD) has demonstrated safety and effectiveness. EBV-CTLs might also be the effective treatment of refractory PTLD of solid organ transplantation (SOT) recipients. Two independent assessors searched Pubmed, Embase, Cochrane Library, and Web of Science from their inception to November 2020. Eleven studies with 76 patients (42, 55% male) were included. We extracted the data and completed the quality assessments. Most of the studies were from Europe and the USA. Liver and kidney transplantation accounted for most of the transplant types. Thirty-five (46.1%) patients were diagnosed with monomorphic PTLD, and B lymphocyte type was the most common. All the patients received primary treatment for PTLD while it was ineffective. CTLs included autologous EBV-CTLs (15/76, 22%) and HLA-matched third-party EBV-CTLs (61/76, 78%). The response rate for EBV-CTL treatment of refractory PTLD was 66%. Of 50 patients, 36 achieved complete remission and 14 achieved partial remission. EBV-DNA level decreased in 39 patients. Adverse reactions were rare and mild. We conclude that adoptive therapy with EBV-specific CTLs is safe, well-tolerated, and effective in PTLD.

Epstein-Barr virus posttransplant lymphoproliferative disorder: update on management and outcomes.

Management of Epstein-Barr virus posttransplant lymphoproliferative disorder (EBV PTLD) is complex, involving risk stratification, prevention and/or preemptive measures involving monitoring EBV DNAemia and balancing treatment options, using a combination of reduction of immune suppression, anti-B cell therapy, and cytotoxic T lymphocytes (CTLs). The highest risk factor for the development of EBV PTLD in hematopoietic cell transplant (HCT) remains T cell depletion, with increasing use of antithymocyte globulin (ATG) or alemtuzumab in conditioning. In solid organ transplantation (SOT), the incidence of PTLD is highest among EBV seronegative recipients who are at risk for primary EBV infection following transplant in the first 12 months. Prevention is a critical component of the management of EBV PTLD. Although preemptive therapy remains standard of care, there continues to be heterogenicity and debate over the optimal choice of EBV DNA quantification and the threshold to use. Novel therapies such as donor-derived multipathogen and EBV specific CTLs for the prevention and third party CTLs for the treatment of EBV PTLD are promising, with rapidly expanding evidence, including large scale Phase III trials currently underway. With an increasing number of risk groups for developing EBV PTLD in HCT and SOT, management strategies using prophylaxis or preemptive therapy remain standard of care, however the use of prophylactic or preemptive EBV specific or multipathogen CTLs show promising results and safety profiles.

Inducible MyD88/CD40 (iMC) Costimulation Enhances Polyclonal Epstein-Barr Virus-Specific Cytotoxic T Lymphocyte (EBV-CTL) Proliferation and Anti-Tumor Activity

Introduction: Adoptive T cell transfer with antigen-specific cytotoxic T lymphocytes (CTLs) have shown promise for the treatment of cancer. Antitumor efficacy correlates with T cell expansion and persistence following infusion, suggesting that enhancing T cell proliferation and survival may increase the potency of T cell therapies. Here, we demonstrate that genetic modification of EBV-specific T cells with inducible MyD88/CD40 (iMC) provides user-controlled costimulation that amplifies IL-2 production, CTL proliferation and cytotoxicity against EBV-transformed B cells, suggesting that iMC may be used to augment EBV-CTL against EBV-associated malignancies.Methods: Peripheral blood mononuclear cells (PBMCs) from healthy donors were used to generate EBV lymphoblastoid cells (LCLs) by transforming B cells with B95-8 virus. EBV-CTL were subsequently generated by stimulating PBMCs with irradiated (40 Gy) autologous LCLs at a T cell to LCL ratio of 40:1 (day 0) and then subsequently expanded for an additional 2 weeks by restimulation (4:1 CTL:LCL ratio) in the presence of 50 U/ml IL-2. EBV-CTL were then transduced with bicistronic retroviral vectors encoding iMC and truncated CD19 (ΔCD19) (SFG-iMC-ΔCD19) or a control vector lacking the MC costimulatory domains (SFG-FKBP-ΔCD19). EBV specificity and transduction efficiency of EBV-CTL was assessed by EBV-specific dextramers and CD3+CD19+ expression by flow cytometry, respectively. In vitro antitumor activity and T cell proliferation was measured using a 7 day live cell imaging (IncuCyte) coculture assays with transduced EBV-CTL and autologous or HLA-disparate LCL with or without 1 nM rimiducid to activate the iMC costimulatory molecule at effector to target (E:T) ratios of 40:1, 20:1, and 10:1. Cytokine production was measured by ELISA.Results: EBV-CTL were successfully generated from EBV-seropositive donors and demonstrated specificity against EBV lytic antigens (EBNA3A; BZLF1) and showed specific recognition of autologous EBV-LCL, but not HLA-mismatched LCL. EBV-CTL were also efficiently transduced with iMC and control (FKBP) retroviral vectors (93% and 82%, respectively). Cytokine production (IL-2 and IL-6) by gene-modified EBV-CTL targeting autologous LCL was significantly enhanced by iMC activation with rimiducid (48-fold increase compared to FKBP-ΔCD19-transduced EBV-CTL and iMC-ΔCD19-transduced EBV-CTLs without rimiducid stimulation). In vitro live cell imaging demonstrated at high E:T ratios (40:1) that EBV-CTLs efficiently killed matched LCLs. However, at lower E:T ratios (20:1 and 10:1), only EBV-CTLs transduced with iMC-ΔCD19 and stimulated with rimiducid were able to control LCL outgrowth over 7 days, showing a 10-fold increase inhibition in tumor growth.Conclusion: iMC provides a flexible, costimulatory module that can be used to enhance EBV-specific CTL to increase anti-tumor activity. Activation of iMC with rimiducid, in combination with recognition of cognate EBV antigens, induces IL-2 production and promotes CTL proliferation and survival. These data suggest that genetic modification of EBV-CTL, or other virus or tumor-associate antigen-specific T cells, including tumor infiltrating lymphocytes (TILs), with iMC may provide necessary costimulation on demand to increase T cell proliferation and persistence leading to increased anti-tumor efficacy. DisclosuresKhalil:Bellicum Pharmaceuticals: Employment. Yvon:Bellicum Pharmaceuticals: Equity Ownership. Mahendravada:Bellicum Pharmaceuticals: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders . Foster:Bellicum Pharmaceuticals: Employment, Other: stockholders .

Culture expansion of epstein-barr virus-specific cytotoxic T-lymphocytes (EBV-CTLs) using bioreactor with expandable culture area (BECA)

Culture expansion of epstein-barr virus-specific cytotoxic T-lymphocytes (EBV-CTLs) using bioreactor with expandable culture area (BECA)

Emerging Role of Adoptive T Cell Therapy for EBV Induced Nasopharyngeal Carcinoma (NPC) - a Systematic Review

Introduction: Nasopharyngeal carcinoma (NPC) is defined as the cancer of squamous epithelium lining nasopharynx. The single most common culprit of undifferentiated NPC is the Epstein-Barr virus (EBV). Recurrent local-regional or metastatic NPC cannot be treated with repeated chemo-radiotherapy because of poor overall survival and profound effect of these therapies on quality of life. One safer approach is immunotherapy with autologous EBV specific cytotoxic T lymphocytes (EBV-CTLS) targeted to the EBV antigens EBNA1, latent membrane protein LMP1, and LMP2 expressed by most NPC tumors. This study aims to review the efficacy and toxicity of adoptive immunotherapy with EBV-CTLS in patients with EBV induced NPC. Methods: A systematic search of PubMed, Embase, Clinicaltrials.gov, and Web of Science was performed for adoptive immunotherapy in EBV induced NPC patients from inception to May 28, 2020. Out of 604 studies, 07 phase I and II clinical trials were selected for the systematic review. Results: A total of 134 patients (pts) were evaluated out of 157 pts. 56 had a locoregional disease, 63 had distant metastasis, 15 had both locoregional disease as well as distant metastasis, 8 were in remission and disease status was unknown in 5 pts. Li et al. (2015) in their phase I clinical trial on 20 NPC pts with ECOG performance status of &amp;lt;3 after chemoradiotherapy (CCRT) showed overall response rate (ORR) of 95% with complete response (CR) in 19 patients. One patient showed progressive disease (PD). Median progression-free survival (PFS) was observed to be 16 months. Eighteen (90%) pts showed disease-free survival of greater than 12 months after adoptive cell therapy (ACT). Grade (G) ≥3 adverse events (AEs) included leukopenia (5%) and neutropenia (5%). Phase I/II dose-escalation trial by Louis et al. (2010) on 23 pts showed ORR of 48.7% (20% CR, 13.3% undetermined complete response [Cru], 15.4% PR) among pts with active disease. Eight pts remained in remission while 10 had metastatic disease at the time of infusion. PD was 21.7%, Stable Disease (SD) 13%, and 3 pts (13%) had recurrent disease. The median time to progression was 1059 days with PFS of 65% and 52% at 1 and 2 years respectively while the (Overall Survival) OS was 87% and 70% at 1 year and 2 years respectively. There was a higher risk of disease progression (HR: 3.91, P= 0.015) and decreased overall survival (HR: 5.55, P=0.022) in metastatic disease as compared to locoregional disease. Huang J. et al (2017) conducted a phase I/ II trial in 21 pts with a mean waiting period of 71 days after chemotherapy. Two CTL infusions were given 2 weeks apart. Two pts (9.5%) maintained SD but all other pts (85%) showed PD after 8 weeks follow- up. One patient achieved CR (4.8%). Hence, ORR was 4.8% while median PFS and OS were of 2.2 months and 16.7 months respectively. In a phase II trial, 24 patients completed 6 EBV- CTL therapy cycles after receiving chemotherapy cycles of Gemcitabine and Carboplatin. ORR was observed to be 42.9% (CR 5.7%, PR 31.7%). SD was 20% while PD was 31.4%. Median OS was 29.9 months (95% CI 20.8-39.3) with 1, 2, and 3-year rates being 77.1%, 62.9%, and 37.1 % respectively. Median PFS was 7.6 months (95% CI 7.4-8.4). All G≥3 AE occurred during chemotherapy. (Chia et al, 2014) Secondino et al. (2011) conducted a phase I/II study in 11 NPC patients who also received chemotherapy consisting of cyclophosphamide and fludarabine. After a mean follow-up of 4 weeks, ORR was 27% (PR 18%, Minor Response [MR] 9%). PD was reported to be 45% and SD 27%. Median PFS at 6 months was 54% (6/11 pts). Only G≥3 AE reported was neutropenia (36%). Phase I/II trial by Comoli et al. (2005) evaluated 10 EBV-related stage IV NPC in progression after CCRT. After receiving two to twenty-three EBV-specific CTLs infusions, 2 patients showed PR (20%), 40% of pts maintained SD and all others showed evidence of PD (40%) at 1-2 months follow up. Median PFS was 6.5 months. Smith et al (2012) in their phase I trial on 14 patients with locoregional and metastatic NPC reported SD 71.4% and PD 28.6% of patients at a median follow up of 1 month. Median OS and PFS were 17.4 months and 4.5 months respectively. No G≥3 AEs were reported. Conclusion : Adoptive Immunotherapy with EBV-CTLS has shown impressive efficacy with improvement in median PFS and OS and a favorable safety profile. Key Words: Adoptive cell therapy, Cytotoxic T lymphocytes, nasopharyngeal carcinoma, Phase I/II clinical trials, Epstein-Barr virus. Disclosures Anwer: Celgene: Research Funding; AbbVie Pharmaceuticals: Research Funding; Incyte Pharmaceuticals: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Astellas Pharma: Research Funding; Acetylon Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Millennium Pharmaceuticals: Research Funding.