EBV-specific T-cell Research Articles

Differential EBV protein-specific antibody response between responders and non-responders to EBVSTs immunotherapy.

Epstein-Barr virus (EBV) is associated with a diverse range of lymphomas. EBV-specific T-cell (EBVST) immunotherapies have shown promise in safety and clinical effectiveness in treating EBV-associated lymphomas, but not all patients respond to treatment. To identify the set of EBV-directed antibody responses associated with clinical response in patients with EBV-associated lymphomas, we comprehensively characterized the immune response to the complete EBV proteome using a custom protein microarray in 56 EBV-associated lymphoma patients who were treated with EBVST infusions enrolled in Phase I clinical trials. Significant differences in antibody profiles between responders and non-responders emerged at 3 months post-EBVST infusion. Twenty-five IgG antibodies were present at significantly higher levels in non-responders compared to responders at 3 months post-EBVST infusion, and 10 of these IgG antibody associations remained after adjustment for sex, age, and cancer diagnosis type. Random forest prediction analysis further confirmed that these 10 antibodies were important for predicting clinical response. Differential IgG antibody responses were directed against LMP2A (four fragments), BGRF1/BDRF1 (two fragments), LMP1, BKRF2, BKRF4, and BALF5. Paired analyses using blood samples collected at both pre-infusion and 3 months post-EBVST infusion indicated an increase in the mean antibody level for six other anti-EBV antibodies (IgG: BGLF2, LF1, BGLF3; IgA: BGLF3, BALF2, BBLF2/3) in non-responders. Overall, our results indicate that EBV-directed antibodies can be biomarkers for predicting the clinical response of individuals with EBV-associated lymphomas treated with EBVST infusions.

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.

Broader anti-EBV TCR repertoire in multiple sclerosis: disease specificity and treatment modulation.

Epstein-Barr virus (EBV) infection has long been associated with the development of multiple sclerosis (MS). MS patients have elevated titers of EBV-specific antibodies in serum and show signs of CNS damage only after EBV infection. Regarding CD8+ T-cells, an elevated but ineffective response to EBV was suggested in MS patients, who present with a broader MHC-I-restricted EBV-specific T-cell receptor beta chain (TRB) repertoire compared to controls. It is not known whether this altered EBV response could be subject to dynamic changes, e.g., by approved MS therapies, and whether it is specific for MS. 1317 peripheral blood TRB repertoire samples of healthy donors (n=409), patients with MS (n=710) before and after treatment, patients with neuromyelitis optica spectrum disorder (n=87), myelin-oligodendrocyte-glycoprotein antibody-associated disease (n=64) and Susac's syndrome (n=47) were analyzed. Apart from MS, none of the evaluated diseases presented with a broader anti-EBV TRB repertoire. In MS patients undergoing autologous hematopoietic stem-cell transplantation, EBV reactivation coincided with elevated MHC-I-restricted EBV-specific TRB sequence matches. Therapy with ocrelizumab, teriflunomide or dimethyl fumarate reduced EBV-specific, but not CMV-specific MHC-I-restricted TRB sequence matches. Together, this data suggests that the aberrant MHC-I-restricted T-cell response directed against EBV is specific to MS with regard to NMO, MOGAD and Susac's Syndrome and that it is specifically modified by MS treatments interfering with EBV host cells or activated lymphocytes.

Evaluation of novel Epstein-Barr virus-derived antigen formulations for monitoring virus-specific T cells in pediatric patients with infectious mononucleosis

BackgroundInfection with the Epstein-Barr virus (EBV) elicits a complex T-cell response against a broad range of viral proteins. Hence, identifying potential differences in the cellular immune response of patients with different EBV-associated diseases or different courses of the same disorder requires interrogation of a maximum number of EBV antigens. Here, we tested three novel EBV-derived antigen formulations for their ability to reactivate virus-specific T cells ex vivo in patients with EBV-associated infectious mononucleosis (IM).MethodsWe comparatively analyzed EBV-specific CD4+ and CD8+ T-cell responses to three EBV-derived antigen formulations in 20 pediatric patients during the early phase of IM: T-activated EBV proteins (BZLF1, EBNA3A) and EBV-like particles (EB-VLP), both able to induce CD4+ and CD8+ T-cell responses ex vivo, as well as an EBV-derived peptide pool (PP) covering 94 well-characterized CD8+ T-cell epitopes. We assessed the specificity, magnitude, kinetics, and functional characteristics of EBV-specific immune responses at two sequential time points (v1 and v2) within the first six weeks after IM symptom onset (Tonset).ResultsAll three tested EBV-derived antigen formulations enabled the detection of EBV-reactive T cells during the early phase of IM without prior T-cell expansion in vitro. EBV-reactive CD4+ and CD8+ T cells were mainly mono-functional (CD4+: mean 64.92%, range 56.15-71.71%; CD8+: mean 58.55%, range 11.79-85.22%) within the first two weeks after symptom onset (v1) with IFN-γ and TNF-secreting cells representing the majority of mono-functional EBV-reactive T cells. By contrast, PP-reactive CD8+ T cells were primarily bi-functional (>60% at v1 and v2), produced IFN-γ and TNF and had more tri-functional than mono-functional components. We observed a moderate correlation between viral load and EBNA3A, EB-VLP, and PP-reactive CD8+ T cells (rs = 0.345, 0.418, and 0.356, respectively) within the first two weeks after Tonset, but no correlation with the number of detectable EBV-reactive CD4+ T cells.ConclusionsAll three EBV-derived antigen formulations represent innovative and generic recall antigens suitable for monitoring EBV-specific T-cell responses ex vivo. Their combined use facilitates a thorough analysis of EBV-specific T-cell immunity and allows the identification of functional T-cell signatures linked to disease development and severity.

Safety of tabelecleucel with pembrolizumab in recurrent/metastatic Epstein–Barr virus-associated nasopharyngeal carcinoma.

6096 Background: Despite standard treatment with radiation and/or chemotherapy, approximately one in four patients (pts) with nasopharyngeal carcinoma (NPC) will develop recurrent/metastatic (RM) disease that is associated with poor prognosis. Programmed cell death ligand 1 (PD-L1) expression is upregulated in NPC due to Epstein–Barr virus (EBV) activation. Tabelecleucel (tab-cel), an off-the-shelf, allogeneic, EBV-specific T-cell immunotherapy, has shown promising antitumor activity in pts with RM EBV+NPC (Prockop, JCO 2016). The anti-PD-1 antibody pembrolizumab (pembro) is also active in NPC. To characterize the safety profile and preliminary efficacy of tab-cel combined with pembro in RM EBV+NPC, we initiated a phase 1b/2 study (NCT03769467). Here we report safety outcomes in 12 subjects treated in the phase 1b portion of the study. Methods: The multicenter, open-label, single-arm phase 1b portion of the study was designed to evaluate the safety of tab-cel in combination with pembro in pts with incurable RM EBV+NPC previously treated with platinum-containing therapy. Pts were either checkpoint-inhibitor naïve (n=6) or experienced to anti-PD-1/PD-L1 therapy (n=6). Tab-cel was administered intravenously (IV) on days 1, 8, and 15 of a 21-day cycle. Initial tab-cel dose was 2x106 cells/kg. Pembro was administered at 200 mg IV Q3W. Primary endpoints of phase 1b focused on assessing safety. Results: As of the final data cutoff, 12 subjects were treated as part of the phase 1b portion of the study. Subjects were 75% Asian and 50% male, with a median age (min, max) of 51 (20, 72) years and a median time from initial diagnosis to first dose of tab-cel (min, max) of 47.7 (8.0, 221.8) months. All subjects had received prior platinum-based chemotherapy/chemoradiation. No dose-limiting toxicities or fatal treatment-emergent adverse events (TEAEs) were identified. All 12 subjects reported ≥1 TEAE; the most common TEAEs reported by ≥4 subjects were disease progression (8 subjects, 66.7%), myalgia (5 subjects, 41.7%), pyrexia, arthralgia, and hyponatremia (4 subjects, 33.3%, each). No ≥ grade 3 TEAEs were considered related to tab-cel or pembro. No TEAEs considered related to tab-cel or pembro led to study discontinuation. No clinically significant abnormal trends were identified in laboratory parameters or vital signs. Although the phase 1b portion of the study was not designed to assess efficacy, the best response seen was stable disease in 6 of 12 pts (50%). Conclusions: Phase 1b data demonstrated combination therapy of tab-cel and pembro to be well tolerated and identified a recommended phase 2 dose in subjects with platinum-pretreated RM EBV+NPC. The study was discontinued after the phase Ib portion due to an evolving immunotherapy treatment landscape for this disease, which would need to be considered for further clinical development. Clinical trial information: NCT03769467 .

Rejection resistant CD30.CAR-modified Epstein-Barr virus-specific T cells as an off-the-shelf platform for CD30+ lymphoma

Off-the-shelf (OTS) adoptive T-cell therapies have many benefits such as immediate availability, improved access and reduced cost, but face the major challenges of graft-vs-host disease (GVHD) and graft rejection, mediated by alloreactive T-cells present in the graft and host, respectively. We have developed a platform for OTS T-cell therapies by using Epstein-Bar virus (EBV)-specific T-cells (EBVSTs) expressing a chimeric antigen receptor (CAR) targeting CD30. Allogeneic EBVSTs have not caused GVHD in several clinical trials, while the CD30.CAR, that is effective for the treatment of lymphoma, can also target alloreactive T-cells that upregulate CD30 on activation. Although EBVSTs express high levels of CD30, they were protected from fratricide in cis, by the CD30.CAR. Hence, they could proliferate extensively and maintained function both through their native EBV-specific T-cell receptor (TCR) and the CD30.CAR. The CD30.CAR enabled EBVSTs to persist in co-cultures with naïve and primed alloreactive T-cells and eliminate activated NK cells that can also be alloreactive. In conclusion, we show that CD30.CAR EBVSTs have the potential to be an effective OTS therapy against CD30+ tumors and if successful could then be used as a platform to target other tumor antigens.

Targeting latent viral infection in EBV-associated lymphomas.

Epstein-Barr virus (EBV) contributes to the development of a significant subset of human lymphomas. As a herpes virus, EBV can transition between a lytic state which is required to establish infection and a latent state where a limited number of viral antigens are expressed which allows infected cells to escape immune surveillance. Three broad latency programs have been described which are defined by the expression of viral proteins RNA, with latency I being the most restrictive expressing only EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) and latency III expressing the full panel of latent viral genes including the latent membrane proteins 1 and 2 (LMP1/2), and EBNA 2, 3, and leader protein (LP) which induce a robust T-cell response. The therapeutic use of EBV-specific T-cells has advanced the treatment of EBV-associated lymphoma, however this approach is only effective against EBV-associated lymphomas that express the latency II or III program. Latency I tumors such as Burkitt lymphoma (BL) and a subset of diffuse large B-cell lymphomas (DLBCL) evade the host immune response to EBV and are resistant to EBV-specific T-cell therapies. Thus, strategies for inducing a switch from the latency I to the latency II or III program in EBV+ tumors are being investigated as mechanisms to sensitize tumors to T-cell mediated killing. Here, we review what is known about the establishment and regulation of latency in EBV infected B-cells, the role of EBV-specific T-cells in lymphoma, and strategies to convert latency I tumors to latency II/III.

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.

Epstein-Barr virus-specific T-cell response in pediatric liver transplant recipients: a cross-sectional study by multiparametric flow cytometry.

Epstein-Barr virus (EBV) specific T-cell response measurement can help adjust immunosuppression in transplant patients with persistent infections. We aim to define T-cell responses against EBV in a cohort of pediatric liver-transplant patients. Thirty-eight immunosuppressed pediatric liver-transplant patients (IP) and 25 EBV-seropositive healthy-adult controls (HC) were included in our cross-sectional study. Based on their EBV serological (S) and viral load (VL) status, patients were categorized into IP-SNEG, IP-SPOSVLNEG and IP-SPOSVLPOS groups. T-cell response was assessed at two timepoints by stimulating cells with EBV peptides (PepTivator®) and performing intracellular-cytokine and activation-induced marker staining. Background subtraction was used to determine EBV-specific T-lymphocyte frequency. Polyfunctional CD8+ T cells indicated previous EBV contact (IP-SNEG 0.00% vs IP-SPOS 0.04% and HC 0.02%; p=0.001 and p=0.01, respectively). Polyfunctional CD8+CD107a+IFNɣ+IL2-TNFα- profile was increased in serology-positive (IP-SNEG 0.01% vs IP-SPOS 0.13% and HC 0.03%; p=0.01 and p=0.50, respectively) and viral-load positive (IP-SPOSVLPOS 0.43% vs IP-SPOSVLNEG 0.07% and HC 0.03%; p=0.03 and p=0.001, respectively) patients. Central-memory cells were increased among serology-positive adults (IP-SNEG 0.00% vs IP-SPOS 0.13% and HC 4.33%; p=0.58 and p=0.002, respectively). At the second timepoint, IP-SNEG patients remained negative (first visit 0.01% vs second visit 0.00%, p=0.44). On the other hand, IP-SPOSVLPOS patients had cleared viral loads and, subsequently, decreased polyfunctional CD8+CD107a+IFNɣ+IL2-TNFα- cells (first visit 0.43% vs second visit 0.10%, p=0.81). Polyfunctional CD8+ EBV-specific T-cell response allows detecting EBV previous contact in liver-transplant children. %CD8+CD107a+IFNɣ+IL2-TNFα- is increased in patients with positive viral loads. Central memory CD4+ T-cell population more effectively determines prior EBV-exposure in adults.

Ineffective control of Epstein-Barr-virus-induced autoimmunity increases the risk for multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease of the CNS. Epstein-Barr virus (EBV) contributes to the MS pathogenesis because high levels of EBV EBNA386-405-specific antibodies cross react with the CNS-derived GlialCAM370-389. However, it is unclear why only some individuals with such high autoreactive antibody titers develop MS. Here, we show that autoreactive cells are eliminated by distinct immune responses, which are determined by genetic variations of the host, as well as of the infecting EBV and human cytomegalovirus (HCMV). We demonstrate that potent cytotoxic NKG2C+ and NKG2D+ natural killer (NK) cells and distinct EBV-specific Tcell responses kill autoreactive GlialCAM370-389-specific cells. Furthermore, immune evasion of these autoreactive cells was induced by EBV-variant-specific upregulation of the immunomodulatory HLA-E. These defined virus and host genetic pre-dispositions are associated with an up to 260-fold increased risk of MS. Our findings thus allow the early identification of patients at risk for MS and suggest additional therapeutic options against MS.

Antibody-mediated delivery of viral epitopes to redirect EBV-specific CD8+ T-cell immunity towards cancer cells.

Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8+ T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo.

Immunology of THymectomy And childhood CArdiac transplant (ITHACA): protocol for a UK-wide prospective observational cohort study to identify immunological risk factors of post-transplant lymphoproliferative disease (PTLD) in thymectomised children

IntroductionPaediatric heart transplant patients are disproportionately affected by Epstein-Barr virus (EBV)-related post-transplant lymphoproliferative disease (PTLD) compared with other childhood solid organ recipients. The drivers for this disparity remain poorly understood....

Effectiveness and safety outcomes in patients with EBV+ PTLD treated with allogeneic EBV-specific T-cell immunotherapy (tabelecleucel) under an expanded access program (EAP) in Europe.

7521 Background: Pts with relapsed or refractory (r/r) EBV+ post-transplant lymphoproliferative disease (PTLD) in Europe have had historically poor overall response rates and median overall survival (OS) with no approved treatment options. Tabelecleucel, an off-the-shelf, allogeneic EBV-specific T-cell immunotherapy has shown clinical benefit and favorable safety profile in the treatment of EBV+ PTLD failing rituximab ± chemotherapy (Prockop EBMT 2021, ATC 2021, ASH 2022). Its recent European marketing authorization (EMA) represents the first approval of an allogenic T-cell immunotherapy globally. Methods: Atara Bio supported expanded access requests for tabelecleucel in Europe. Here we report updated effectiveness and safety data for r/r EBV+ PTLD pts who provided consent for research between Sep 2020 and Dec 2022. Results: 74 EAP requests were received from 10 countries for pts with r/r EBV+ diseases. 27 EBV+ PTLD pts consented to secondary use of data and 24 pts had received ≥1 dose of tabelecleucel, including 4 pts with primary central nervous system (PCNS) PTLD. 16 of 24 (66.7%) PTLD pts achieved a partial (PR) (33.3%) or complete (CR) (33.3%) response, with median time to response (TTR) of 1.0 mo (0.8–2.2). Response rate for PCNS PTLD pts was 75% (1 CR, 2 PR). 1-yr OS Kaplan–Meier (KM) estimate rates were 73.7% (95% CI: 47.3, 88.3) overall, 87.5% in allogeneic hematopoietic stem cell transplant (HCT) and 66.5% in solid organ transplant (SOT), with a median follow-up time of 9.9 (2.4–13.9) and 6.0 (0.7–18.0) mo, respectively. Serious treatment-emergent adverse events (TESAEs) were reported in 7 (29.2%) pts, including 1 fatal event of disease progression. Predefined risks for tabelecleucel were reported in 3 pts; 1 (4.2%) SOT pt had a TESAE of liver transplant rejection (grade 2) and 2 (8.3%) HCT pts had non-serious TEAEs of chronic graft-versus-host disease (grade 1 and 2). No cases of cytokine release syndrome or tumor flare reaction were reported. No AEs were reported as related to tabelecleucel by the treating physician. Conclusions: These updated real-world results for pts with r/r EBV+ PTLD post-HCT or post-SOT treated in the European EAP continue to reinforce the favorable risk:benefit profile of tabelecleucel and are in line with clinical study data supporting its recent EMA approval. [Table: see text]

Reinforcement of cell-mediated immunity driven by tumor-associated Epstein-Barr virus (EBV)-specific T cells during targeted B-cell therapy with rituximab.

In immunocompromised patients, Epstein-Barr virus (EBV) infection or reactivation is associated with increased morbidity and mortality, including the development of B-cell lymphomas. The first-line treatment consists of reduction of immunosuppression and administration of rituximab (anti-CD20 antibody). Furthermore, the presence of EBV-specific T cells against latent EBV proteins is crucial for the control of EBV-associated diseases. Therefore, in addition to effective treatment strategies, appropriate monitoring of T cells of high-risk patients is of great importance for improving clinical outcome. In this study, we hypothesized that rituximab-mediated lysis of malignant EBV-infected B cells leads to the release and presentation of EBV-associated antigens and results in an augmentation of EBV-specific effector memory T-cell responses. EBV-infected B lymphoblastoid cell lines (B-LCLs) were used as a model for EBV-associated lymphomas, which are capable of expressing latency stage II and III EBV proteins present in all known EBV-positive malignant cells. Rituximab was administered to obtain cell lysates containing EBV antigens (ACEBV). Efficiency of cross-presentation of EBV-antigen by B-LCLs compared to cross-presentation by professional antigen presenting cells (APCs) such as dendritic cells (DCs) and B cells was investigated by in vitro T-cell immunoassays. Deep T-cell profiling of the tumor-reactive EBV-specific T cells in terms of activation, exhaustion, target cell killing, and cytokine profile was performed, assessing the expression of T-cell differentiation and activation markers as well as regulatory and cytotoxic molecules by interferon-γ (IFN-γ) EliSpot assay, multicolor flow cytometry, and multiplex analyses. By inhibiting parts of the cross-presentation pathway, B-LCLs were shown to cross-present obtained exogenous ACEBV-derived antigens mainly through major histocompatibility complex (MHC) class I molecules. This mechanism is comparable to that for DCs and B cells and resulted in a strong EBV-specific CD8+ cytotoxic T-cell response. Stimulation with ACEBV-loaded APCs also led to the activation of CD4+ T helper cells, suggesting that longer peptide fragments are processed via the classical MHC class II pathway. In addition, B-LCLs were also found to be able to take up exogenous antigens from surrounding cells by endocytosis leading to induction of EBV-specific T-cell responses although to a much lesser extent than cross-presentation of ACEBV-derived antigens. Increased expression of activation markers CD25, CD71 and CD137 were detected on EBV-specific T cells stimulated with ACEBV-loaded APCs, which showed high proliferative and cytotoxic capacity as indicated by enhanced EBV-specific frequencies and increased secretion levels of cytotoxic effector molecules (e.g. IFN-γ, granzyme B, perforin, and granulysin). Expression of the regulatory proteins PD-1 and Tim-3 was induced but had no negative impact on effector T-cell functions. In this study, we showed for the first time that rituximab-mediated lysis of EBV-infected tumor cells can efficiently boost EBV-specific endogenous effector memory T-cell responses through cross-presentation of EBV-derived antigens. This promotes the restoration of antiviral cellular immunity and presents an efficient mechanism to improve the treatment of CD20+ EBV-associated malignancies. This effect is also conceivable for other therapeutic antibodies or even for therapeutically applied unmodified or genetically modified T cells, which lead to the release of tumor antigens after specific cell lysis.

Adoptive T-cell therapy targeting Epstein-Barr virus as a treatment for multiple sclerosis.

Emergence of a definitive link between Epstein-Barr virus (EBV) and multiple sclerosis has provided an impetus to develop immune-based therapies to target EBV-infected B cells. Initial studies with autologous EBV-specific T-cell therapy demonstrated that this therapy is safe with minimal side effects and more importantly multiple patients showed both symptomatic and objective neurological improvements including improved quality of life, reduction of fatigue and reduced intrathecal IgG production. These observations have been successfully extended to an 'off-the-shelf' allogeneic EBV-specific T-cell therapy manufactured using peripheral blood lymphocytes of healthy seropositive individuals. This adoptive immunotherapy has also been shown to be safe with encouraging clinical responses. Allogeneic EBV T-cell therapy overcomes some of the limitations of autologous therapy and can be rapidly delivered to patients with improved therapeutic potential.

Utilizing TCR-Seq to Detect Tabelecleucel, an Allogeneic Epstein-Barr Virus (EBV)-Specific T-Cell Immunotherapy, Post-Infusion

Background: Tabelecleucel is an off-the-shelf, allogeneic EBV-specific T-cell immunotherapy that targets EBV-antigen presenting cells in an HLA restricted manner. It is not genetically modified and therefore lacks a distinct universal molecular moiety to detect it post-infusion. To date, the most effective measure of EBV reactive T-cells in patients (pts) treated with tabelecleucel is the EBV-cytotoxic T lymphocyte precursor (CTLp) assay, which measures the number of EBV reactive CTL precursors in each sample but cannot distinguish between the pt's endogenous CTLs or the infused product CTLs. We have previously shown that EBV-CTLp peak correlates with pt response. Prior methods to identify circulating product CTLs have relied on differences in the genomic profiles of the pt and dosing product lot. However, the limit of detection for these assays to date has not been sensitive enough to reliably detect product CTLs post-infusion. One aspect of T-cell biology that could be utilized to track product specific CTL clones is the unique T cell receptors (TCR) which impart antigen specificity. TCRs are highly diverse and only a small minority of TCR clones are found to be shared across unrelated individuals. Therefore, we leveraged TCRβ sequencing of the CDR3 region to barcode unique TCR clones in tabelecleucel product lots and pt derived samples to detect product T cell clones post-infusion, and annotate likely EBV-targeting T cell clones based upon sequence homology to previously reported EBV-targeting TCR clones. Using this approach, we aimed to identify corollaries associated with clinical outcomes. Methods: Peripheral blood mononuclear cell samples from pts enrolled in the ALLELE (NCT03394365) study were obtained pre-dose on cycle (C) 1 day (D)1/D8/D15, C2D1/D8/D15 and at safety follow-up. EBV-CTLp was used to measure circulating EBV reactive T-cells, and both the pre-infusion baseline timepoint, and the timepoint of peak on-treatment EBV-CTL expansion were selected for downstream TCRβ sequencing. TCR repertoires for pt samples and the tabelecleucel dosing lot were assessed using TCRβ immunoSEQ assay. The captured TCR repertoires of pt samples were compared to the TCR repertoire of the dosing tabelecleucel lot to annotate product TCR clones and were compared against publicly available databases of previously identified EBV-specific TCRβ sequences to annotate putative EBV targeting TCR clones. Results: Analyses for an initial subset of 7 pts with EBV-positive post-transplant lymphoproliferative disease have been completed and are described here. Six unique tabelecleucel lots were used to treat these pts and were included in the analysis. On average 0.15 ± 0.04% of TCRs detected were shared between multiple lots, allowing the ability to leverage TCRβ sequences as a unique barcode to identify specific product lots. All 7 pts tested had TCRβ clones matching previously identified EBV-specific TCRs detected at baseline (median [min, max]: 3498 [1855, 12486]) and peak EBV-CTLp; clones detected at peak EBV-CTLp were either those previously detected at baseline (532 [129,1627]) or newly emerged (3622, [1801,5347]). TCRβ clones matching the dosing lot were identified in 6 out of 7 pts. Three pts had TCRβ clones matching the dosing lot present at baseline prior to tabelecleucel infusion (2 [1,5]). Post-infusion emergence of TCRs matching the dosing lot was detected in 5 pts (1 [1,3]). TCRβ sequencing on additional pts is currently underway. Association of post-infusion detection of dosing lot specific TCR clones with pt best overall response is ongoing and will be reported at the time of presentation. Conclusions: Utilizing TCRβ sequencing we can identify putative EBV-targeting TCR clones and dosing product TCR clones in pt TCR repertoires. Overall, an average of 12.5 ± 1.6% of captured TCR repertoires were annotated as EBV-targeting TCR clones. As publicly available reference datasets for EBV-specific TCRs are currently limited, this is likely an underrepresentation of the totality of EBV-specific TCRs present within the samples. Additionally, an average of 75.8% of the dosing product TCR clones detected were found exclusively after tabelecleucel infusion. Altogether, these results confirm that tracking TCRβ clones can be utilized as a novel assessment to detect post-infusion non-genetically modified allogenic cell therapies and future associations with clinical efficacy will be determined.

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.

Third-party CMV- and EBV-specific T-cells for first viral reactivation after allogeneic stem cell transplant

AbstractVirus-specific T-cells (VSTs) from third-party donors mediate short- and long-term antiviral effects in allogeneic hematopoietic stem cell transplant (HSCT) recipients with relapsed or refractory viral infections. We investigated early administration of third-party VSTs, together with antiviral therapy in patients requiring treatment for first cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. Thirty HSCT patients were treated with 1 to 4 VST infusions (2 × 107 cells/m2; CMV n=27, EBV n=3) at a median of 4 days after initiation of antiviral treatment. The overall viral response rate was 100%, with a complete response (CR) rate of 94%. Of the 28 patients who achieved a CR, 23 remained virus PCR negative (n=9) or below quantitation limit (n=14) for the duration of follow-up. Four patients had brief episodes of quantifiable reactivation not requiring additional therapy, and one required a second infusion after initial CR, remaining PCR negative thereafter. All 3 patients treated for EBV post-transplant lymphoproliferative disorder achieved sustained CR. Rates of aGVHD and cGVHD after infusion were 13% and 23%, respectively. There were no serious infusion-related adverse events. VST infusion was associated with rapid recovery of CD8+CD45RA−CD62L− and a slower recovery of CD4+CD45RA−CD62L− effector memory T-cells; CMV-specific T-cells comprised up to 13% of CD8+ cells. At 1 year post-transplant, non-relapse mortality was 10%, cumulative incidence of relapse was 7%, overall survival was 88% and 25 of 27 patients had ECOG status of 0 or 1. Early administration of third-party VSTs in conjunction with antiviral treatment appears safe and leads to excellent viral control and clinical outcomes. Registered on Australian New Zealand Clinical Trials Registry as #ACTRN12618000343202.

P1457: DEMOGRAPHICS AND TREATMENT OUTCOMES IN PATIENTS WITH EBV+ PTLD TREATED WITH OFF-THE-SHELF EBV-SPECIFIC CTL (TABELECLEUCEL) UNDER AN ONGOING EXPANDED ACCESS PROGRAM IN EUROPE: FIRST ANALYSES

Background: Patients undergoing allogeneic hematopoietic cell transplant (HCT) or solid organ transplant (SOT) are at risk of developing Epstein–Barr virus driven post-transplant lymphoproliferative disorder (EBV+ PTLD), a rare hematologic malignancy, which is often aggressive and life-threatening. Patients with relapsed or refractory (r/r) EBV+ PTLD have few treatment options with poor outcomes, demonstrating a clear unmet medical need. Tabelecleucel is an investigational, off-the-shelf, allogeneic EBV-specific T-cell immunotherapy being studied in patients with serious EBV+ diseases (NCT04554914 & NCT03394365) that has demonstrated clinical benefit and favorable safety profile in the treatment of EBV+ PTLD after failure of rituximab (R) ± chemotherapy (Prockop, EBMT 2021, ATC 2021, ASH 2021). Aims: First analyses of demographics, efficacy and safety results of r/r EBV+ PTLD patients following SOT or HCT who presented between Jul 2020 and Nov 2021 and consented to research. Methods: Atara Biotherapeutics supports an ongoing expanded access program (EAP) in Europe for patients with EBV+ diseases who have no other treatment options. Demographics, efficacy and safety data of r/r EBV+ PTLD patients following SOT or HCT who presented to the EAP between Jul 2020 and Nov 2021 and consented to research were analyzed. Results: A total of 48 EAP requests from 9 countries for patients with EBV+ diseases were received. Twenty-two patients from 7 countries consented to this research: 16 EBV+ PTLD and 6 EBV+ non-PTLD. Of the 16 PTLD patients 15 received at least one dose of tabelecleucel. Overall, 9 out of 15 (60%) patients achieved a response as assessed by the treating physician, with 6 complete responses and 3 partial responses. Eight out of nine responses were seen after the first cycle. No adverse events were reported as related to tabelecleucel by the treating physician. Image:Summary/Conclusion: The successful execution of this European EAP demonstrates the feasibility to deliver an off-the-shelf allogeneic EBV+ T-cell therapy in time-sensitive clinical situations when no other treatment options exist. These data show clinically meaningful outcomes for patients with r/r EBV+PTLD post-SOT or post-HCT treated with tabelecleucel consistent with previously reported favorable safety and efficacy profile (Prockop, ASH 2021).

Adoptive cellular immunotherapy for Epstein-Barr virus-associated lymphoproliferative disease

: This review focuses on the current state of adoptive cellular immunotherapy for Epstein-Barr virus-associated lymphoproliferative disease (EBV-LD). EBV-positive post-transplant lymphoproliferative disease (PTLD) is a major complication of hematopoietic stem cell and solid organ transplantation. First successes with adoptive immunotherapy were described using donor lymphocyte infusion for the treatment of EBV+ PTLD in hematopoietic stem cell transplantation (HSCT) recipients but carried an appreciable risk of graft-versus-host disease (GVHD). Subsequently, EBV-specific T-lymphocytes (EBVSTs) were developed and have been evaluated clinically for the treatment of EBV-LD over the last two decades predominantly in specialized centers. With impending commercialization, current state and knowledge is critically reviewed. The objectives of this review are: (I) to describe different manufacturing strategies of EBV specific T-lymphocytes (EBVSTs); (II) to review different EBVST donor options and their advantages and limitations; (III) to summarize current clinical experience with adoptive cellular therapy for EBV-LD and finally; (IV) to explore the potential for combination therapies with other immunotherapeutic strategies. This review is based on a review of published literature spanning 20 years from 2000 to 2020 in addition to personal experience and data from the authors. EBVSTs can be readily produced from seropositive donors using good-manufacturing-practice (GMP)-compliant protocols and cryopreserved for future use. In clinical trials, adoptive cellular therapy using EBVSTs has shown impressive results in immunocompromised patients with EBV-LD and there is the potential for wider use. Current research is focused on strategies to enhance EBVST potency in vivo through genetic engineering as well as combination therapies.