Donor whole blood DNA methylation is not a strong predictor of acute graft versus host disease in unrelated donor allogeneic haematopoietic cell transplantation.

Allogeneic hematopoietic cell transplantation (HCT) is used to treat many blood-based disorders and malignancies, however it can also result in serious adverse events, such as the development of acute graft-versus-host disease (aGVHD). This study aimed to develop a donor-specific epigenetic classifier to reduce incidence of aGVHD by improving donor selection. Genome-wide DNA methylation was assessed in a discovery cohort of 288 HCT donors selected based on recipient aGVHD outcome; this cohort consisted of 144 cases with aGVHD grades III-IV and 144 controls with no aGVHD. We applied a machine learning algorithm to identify CpG sites predictive of aGVHD. Receiver operating characteristic (ROC) curve analysis of these sites resulted in a classifier with an encouraging area under the ROC curve (AUC) of 0.91. To test this classifier, we used an independent validation cohort (n = 288) selected using the same criteria as the discovery cohort. Attempts to validate the classifier failed with the AUC falling to 0.51. These results indicate that donor DNA methylation may not be a suitable predictor of aGVHD in an HCT setting involving unrelated donors, despite the initial promising results in the discovery cohort. Our work highlights the importance of independent validation of machine learning classifiers, particularly when developing classifiers intended for clinical use.

Fcγ receptors and immunomodulatory antibodies in cancer.

The discovery of both cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) as negative regulators of antitumour immunity led to the development of numerous immunomodulatory antibodies as cancer treatments. Preclinical studies have demonstrated that the efficacy of immunoglobulin G (IgG)-based therapies depends not only on their ability to block or engage their targets but also on the antibody's constant region (Fc) and its interactions with Fcγ receptors (FcγRs). Fc-FcγR interactions are essential for the activity of tumour-targeting antibodies, such as rituximab, trastuzumab and cetuximab, where the killing of tumour cells occurs at least in part due to these mechanisms. However, our understanding of these interactions in the context of immunomodulatory antibodies designed to boost antitumour immunity remains less explored. In this Review, we discuss our current understanding of the contribution of FcγRs to the in vivo activity of immunomodulatory antibodies and the challenges of translating results from preclinical models into the clinic. In addition, we review the impact of genetic variability of human FcγRs on the activity of therapeutic antibodies and how antibody engineering is being utilized to develop the next generation of cancer immunotherapies.

Treatment for First Cytomegalovirus Infection Post-Hematopoietic Cell Transplant in the AURORA Trial: A Multicenter, Double-Blind, Randomized, Phase 3 Trial Comparing Maribavir With Valganciclovir.

BACKGROUND: Neutropenia may limit the use of valganciclovir treatment for cytomegalovirus (CMV) infection following hematopoietic cell transplant (HCT). A phase 2 study indicated efficacy of maribavir with fewer treatment-limiting toxicities than valganciclovir. METHODS: In this multicenter, double-blind, phase 3 study, patients with first asymptomatic CMV infection post-HCT were stratified and randomized 1:1 to maribavir 400 mg twice daily or valganciclovir (dose-adjusted for renal clearance) for 8 weeks with 12 weeks of follow-up. The primary endpoint was confirmed CMV viremia clearance at week 8 (primary hypothesis of noninferiority margin of 7.0%). The key secondary endpoint was a composite of the primary endpoint with no findings of CMV tissue-invasive disease at week 8 through week 16. Treatment-emergent adverse events (TEAEs) were assessed. RESULTS: Among patients treated (273 maribavir; 274 valganciclovir), the primary endpoint of noninferiority of maribavir was not met (maribavir, 69.6%; valganciclovir, 77.4%; adjusted difference: -7.7%; 95% confidence interval [CI]: -14.98, -.36; lower limit of 95% CI of treatment difference exceeded -7.0%). At week 16, 52.7% and 48.5% of patients treated (maribavir and valganciclovir, respectively) maintained CMV viremia clearance without tissue-invasive disease (adjusted difference: 4.4%; 95% CI: -3.91, 12.76). With maribavir (vs valganciclovir), fewer patients experienced neutropenia (16.1% and 52.9%) or discontinued due to TEAEs (27.8% and 41.2%). Discontinuations were mostly due to neutropenia (maribavir, 4.0%; valganciclovir, 17.5%). CONCLUSIONS: Although noninferiority of maribavir to valganciclovir for the primary endpoint was not achieved based on the prespecified noninferiority margin, maribavir demonstrated comparable CMV viremia clearance during post-treatment follow-up, with fewer discontinuations due to neutropenia. Clinical Trials Registration. NCT02927067 [AURORA].

AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival.

BACKGROUND: AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets. METHODS: VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale. RESULTS: AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP. CONCLUSION: Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse.

Impact of Baseline and Week 2 and Week 4 Posttransplant CMV Cell-Mediated Immunity on Risk of CMV Infections and Mortality in Recipients of Allogeneic Hematopoietic Cell Transplant.

Background: Cytomegalovirus (CMV) infection is a common opportunistic infection after allogeneic hematopoietic cell transplant (alloHCT). We explored whether a change in CMV cell-mediated immunity during the first month after transplant predicts the risk of development of CMV infection and all-cause mortality. Methods: This follow-up analysis is based on data from the REACT study, a multicenter prospective observational study of recipients of alloHCT who were CMV-seropositive. Production of interferon γ following ex vivo stimulation with CMV antigens IE1 (immediate early 1) and pp65 (phosphoprotein 65) was assessed by CMV ELISPOT assay at baseline and 2 and 4 weeks after transplant. Clinically significant CMV infection (CS-CMVi) was defined as CMV viremia and/or disease necessitating antiviral therapy. We evaluated the impact of CMV CMI changes on the risk of CS-CMVi and post transplant mortality. Results: The analysis included 226 recipients of alloHCT with CMV cell-mediated immunity data at baseline and 2 and/or 4 weeks after transplant. CS-CMVi occurred in 64 patients (28%). On Cox regression analyses, independent predictors of CS-CMVi included a negative Δ change from baseline to week 2 of pp65 spot counts (hazard ratio, 3.65 [95% CI, 1.65-8.04]; P = .001) to week 4 of IE1 spot counts (hazard ratio, 2.79 [95% CI, 1.46-5.35]; P = .002), anti-thymocyte globulin conditioning regimen, type of transplant, female sex, and corticosteroid use. Kaplan-Meir analysis showed a significant association of a negative IE1 change from baseline to week 4 and increased all-cause mortality after transplant (log rank test = 0.041). Conclusions: A decrease in CMV-specific T-cell responses during the first month after transplant may predict CS-CMVi and is associated with all-cause mortality in recipients of alloHCT.

Dual targeting of CD19 and CD22 with bicistronic CAR-T cells in patients with relapsed/refractory large B-cell lymphoma.

Relapse after CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy for large B-cell lymphoma (LBCL) is commonly ascribed to antigen loss or CAR-T exhaustion. Multiantigen targeting and programmed cell death protein-1 blockade are rational approaches to prevent relapse. Here, we test CD19/22 dual-targeting CAR-T (AUTO3) plus pembrolizumab in relapsed/refractory LBCL (NCT03289455). End points include toxicity (primary) and response rates (secondary). Fifty-two patients received AUTO3 and 48/52 received pembrolizumab. Median age was 59 years (range, 27-83), 46/52 had stage III/ IV disease and median follow-up was 21.6 months. AUTO3 was safe; grade 1-2 and grade 3 cytokine release syndrome affected 18/52 (34.6%) and 1/52 (1.9%) patients, neurotoxicity arose in 4 patients (2/4, grade 3-4), and hemophagocytic lymphohistiocytosis affected 2 patients. Outpatient administration was tested in 20 patients, saving a median of 14 hospital days per patient. Overall response rates were 66% (48.9%, complete response [CR]; 17%, partial response). Median duration of remission (DOR) for CR patients was not reached and for all responding patients was 8.3 months (95% confidence interval [CI]: 3.0-not evaluable). 54.4% (CI: 32.8-71.7) of CR patients and 42.6% of all responding patients were projected to remain progression-free at ≥12 months. AUTO3 ± pembrolizumab for relapsed/refractory LBCL was safe and delivered durable remissions in 54.4% of complete responders, associated with robust CAR-T expansion. Neither dual-targeting CAR-T nor pembrolizumab prevented relapse in a significant proportion of patients, and future developments include next-generation-AUTO3, engineered for superior expansion in vivo, and selection of CAR binders active at low antigen densities.

A novel predictive algorithm to personalize autologous T-cell harvest for chimeric antigen receptor T-cell manufacture.

BACKGROUND AIMS: The most widely accepted starting materials for chimeric antigen receptor T-cell manufacture are autologous CD3+ T cells obtained via the process of leukapheresis, also known as T-cell harvest. As this treatment modality gains momentum and apheresis units struggle to meet demand for harvest slots, strategies to streamline this critical step are warranted. METHODS: This retrospective review of 262 T-cell harvests, with a control cohort of healthy donors, analyzed the parameters impacting CD3+ T-cell yield in adults with B-cell malignancies. The overall aim was to design a novel predictive algorithm to guide the required processed blood volume (PBV) (L) on the apheresis machine to achieve a specific CD3+ target yield. RESULTS: Factors associated with CD3+ T-cell yield on multivariate analysis included peripheral blood CD3+ count (natural log, ×109/L), hematocrit (HCT) and PBV with coefficients of 0.86 (95% confidence interval [CI], 0.80-0.92, P < 0.001), 1.30 (95% CI, 0.51-2.08, P = 0.001) and 0.09 (95% CI, 0.07-0.11, P < 0.001), respectively. The authors' model, incorporating CD3+ cell count, HCT and PBV (L), with an adjusted R2 of 0.87 and root-mean-square error of 0.26 in the training dataset, was highly predictive of CD3+ cell yield in the testing dataset. An online application to estimate PBV using this algorithm can be accessed at https://cd3yield.shinyapps.io/cd3yield/. CONCLUSIONS: The authors propose a transferrable model that incorporates clinical and laboratory variables accessible pre-harvest for use across the field of T-cell therapy. Pending further validation, such a model may be used to generate an individual leukapheresis plan and streamline the process of cell harvest, a well-recognized bottleneck in the industry.

Immunotherapy with CD25/CD71-allodepleted T cells to improve T-cell reconstitution after matched unrelated donor hematopoietic stem cell transplant: a randomized trial.

BACKGROUND AIMS: Delayed immune reconstitution is a major challenge after matched unrelated donor (MUD) stem cell transplant (SCT). In this randomized phase 2 multi-center trial, Adoptive Immunotherapy with CD25/71 allodepleted donor T cells to improve immunity after unrelated donor stem cell transplant (NCT01827579), the authors tested whether allodepleted donor T cells (ADTs) can safely be used to improve immune reconstitution after alemtuzumab-based MUD SCT for hematological malignancies. METHODS: Patients received standard of care or up to three escalating doses of ADTs generated through CD25+/CD71+ immunomagnetic depletion. The primary endpoint of the study was circulating CD3+ T-cell count at 4 months post-SCT. Twenty-one patients were treated, 13 in the ADT arm and eight in the control arm. RESULTS: The authors observed a trend toward improved CD3+ T-cell count at 4 months in the ADT arm versus the control arm (230/µL versus 145/µL, P = 0.18), and three ADT patients achieved normal CD3+ T-cell count at 4 months (>700/µL). The rates of significant graft-versus-host disease (GVHD) were comparable in both cohorts, with grade ≥2 acute GVHD in seven of 13 and four of eight patients and chronic GVHD in three of 13 and three of eight patients in the ADT and control arms, respectively. CONCLUSIONS: These data suggest that adoptive transfer of ADTs is safe, but that in the MUD setting the benefit in terms of T-cell reconstitution is limited. This approach may be of more use in the context of more rigorous T-cell depletion.

Anti-CTLA-4 antibodies drive myeloid activation and reprogram the tumor microenvironment through FcγR engagement and type I interferon signaling.

Despite the clinical success of checkpoint inhibitors, a substantial gap still exists in our understanding of their mechanism of action. While antibodies to cytotoxic T lymphocyte-associated protein-4 (CTLA-4) were developed to block inhibitory signals in T cells, several recent studies have demonstrated that Fcγ receptor (FcγR)-dependent depletion of regulatory T cells (Treg) is critical for antitumor activity. Here, using single-cell RNA sequencing, we dissect the impact of anti-CTLA-4-blocking, Treg cell-depleting and FcR-engaging activity on the immune response within tumors. We observed a rapid remodeling of the innate immune landscape as early as 24 h after treatment. Using genetic Treg cell ablation models, we show that immune remodeling was not driven solely by Treg cell depletion or CTLA-4 blockade but mainly through FcγR engagement, downstream activation of type I interferon signaling and reduction of suppressive macrophages. Our findings indicate that FcγR engagement and innate immune remodeling are involved in successful anti-CTLA-4 treatment, supporting the development of optimized immunotherapy agents bearing these features.

Durable Responses and Low Toxicity After Fast Off-Rate CD19 Chimeric Antigen Receptor-T Therapy in Adults With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia.

PURPOSE: Prognosis for adult B-cell acute lymphoblastic leukemia (B-ALL) is poor, and there are currently no licensed CD19 chimeric antigen receptor (CAR) therapeutics. We developed a novel second-generation CD19-CAR (CAT19-41BB-Z) with a fast off rate, designed for more physiologic T-cell activation to reduce toxicity and improve engraftment. We describe the multicenter phase I ALLCAR19 (NCT02935257) study of autologous CAT19-41BB-Z CAR T cells (AUTO1) in relapsed or refractory (r/r) adult B-ALL. METHODS: Patients age ≥ 16 years with r/r B-ALL were eligible. Primary outcomes were toxicity and manufacturing feasibility. Secondary outcomes were depth of response at 1 and 3 months, persistence of CAR-T, incidence and duration of hypogammaglobulinemia and B-cell aplasia, and event-free survival and overall survival at 1 and 2 years. RESULTS: Twenty-five patients were leukapheresed, 24 products were manufactured, and 20 patients were infused with AUTO1. The median age was 41.5 years; 25% had prior blinatumomab, 50% prior inotuzumab ozogamicin, and 65% prior allogeneic stem-cell transplantation. At the time of preconditioning, 45% had ≥ 50% bone marrow blasts. No patients experienced ≥ grade 3 cytokine release syndrome; 3 of 20 (15%) experienced grade 3 neurotoxicity that resolved to ≤ grade 1 within 72 hours with steroids. Seventeen of 20 (85%) achieved minimal residual disease-negative complete response at month 1, and 3 of 17 underwent allogeneic stem-cell transplantation while in remission. The event-free survival at 6 and 12 months was 68.3% (42.4%-84.4%) and 48.3% (23.1%-69.7%), respectively. High-level expansion (Cmax 127,152 copies/µg genomic DNA) and durable CAR-T persistence were observed with B-cell aplasia ongoing in 15 of 20 patients at last follow-up. CONCLUSION: AUTO1 demonstrates a tolerable safety profile, high remission rates, and excellent persistence in r/r adult B-ALL. Preliminary data support further development of AUTO1 as a stand-alone treatment for r/r adult B-ALL.

Natural History of Epstein-Barr Virus Replication and Viral Load Dynamics after Alemtuzumab-Based Allogeneic Stem Cell Transplantation.

Epstein-Barr virus (EBV) load monitoring after allogeneic hematopoietic stem cell transplantation (HSCT) enables earlier detection of EBV replication and often serves as a trigger for preemptive therapies aimed at reducing EBV-related diseases. Our institutional strategy is to treat patients with clinical signs of EBV-related disease accompanied by a rising viral load, rather than to intervene based solely on viral load. This affords an opportunity to study the natural history of EBV replication and to assess whether our strategy reduces overtreatment without compromising outcomes. The objectives of the present study were to assess the natural history of untreated EBV replication in patients who underwent an alemtuzumab-based allogeneic HSCT and to examine whether our clinical strategy reduced overtreatment without compromising patient outcomes. In this retrospective single-center observational study of 515 consecutive patients (age ≥18 years) undergoing T cell-depleted allogeneic HSCT incorporating alemtuzumab, patients underwent surveillance monitoring for EBV by quantitative PCR in the peripheral blood at least weekly up to 100 days post-transplantation and longer if they remained on immunosuppressive therapy. The cumulative incidence of EBV detection and EBV-related disease were assessed. Among the 515 patients, 192 had EBV DNA detectable on ≥1 occasion, with a cumulative incidence of 35.8% (31.8% to 40.4%), although this remained below the limit of quantification in 93 patients. The median time to first detection was 89.5 days (range, 0 to 2254 days). The incidence was higher in recipients of sibling donor transplants (45.4% versus 30%; P = .00021) compared with recipients of unrelated donor transplants. Twenty patients developed EBV-related disease (cumulative incidence, 3.9%). Two patients had immunosuppression reduction alone, 18 received rituximab, and 5 required additional therapies. Five patients died from post-transplantation lymphoproliferative disorder, all of whom had received rituximab. The positive predictive value of EBV load for disease was higher in the unrelated donor cohort but remained <75% regardless of EBV threshold (57.1% to 72.7%). The cumulative incidence of EBV-related disease in our study (3.9%) is comparable to that reported in other studies incorporating alemtuzumab, and our clinical strategy reduced overtreatment in this patient population. PCR-based surveillance strategies have limitations, as reflected in the relatively low sensitivity of the assay coupled with the low positive predictive value, which may influence the potential choice of a threshold for preemptive intervention. We conclude that it remains unclear whether treatment based on a rising EBV viral load alone provides superior overall results to treatment based on the development of clinical signs of EBV-related disease in the context of a rising viral load.

Non-cryopreserved hematopoietic stem cells in autograft patients with lymphoma: a matched-pair analysis comparing a single center experience with the use of cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry.

BACKGROUND AIMS: Around 50 000 autologous stem cell transplantations are done each year worldwide using cryopreserved peripheral blood stem cells (PBSCs). Cryopreservation is time-consuming and expensive. Since 2007, several retrospective studies have shown that PBSCs can be stored at 4°C for 2-3 days, allowing autologous stem cell transplantation in patients with multiple myeloma receiving high-dose melphalan. Data with non-cryopreserved PBSCs in patients autografted for lymphoma following longer pre-conditioning regimens are limited. In addition, no controlled comparison has been able to detect unforeseen differences. METHODS: The authors compared outcomes of 94 consecutive adult patients with lymphoma (66 with Hodgkin lymphoma) autografted in our department in Oran (Algeria) using PBSCs stored at 4°C, from 2009 to 2018, with patients receiving cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry. Patients autografted in Oran were matched with patients receiving cryopreserved PBSCs in the registry (four controls per patient in Oran). RESULTS: Neutrophil engraftment was significantly faster with cryopreserved PBSCs (P = 0.003). By day 10, only 17% of patients receiving non-cryopreserved PBSCs engrafted versus 48% for cryopreserved PBSCs. Likewise, platelet recovery to 20 000/mm3 was significantly faster in patients receiving cryopreserved PBSCs (P = 0.01). However, all patients in both groups had recovered by day 20. There were no significant differences in non-relapse mortality (9% versus 7%, P = 0.4), relapse incidence (22% versus 32%, P = 0.13), progression-free survival (70% versus 61%, P = 0.4) or overall survival (85% versus 75%, P = 0.3). CONCLUSIONS: This analysis suggests that, in patients with lymphoma receiving pre-transplant regimens such as carmustine, etoposide, cytarabine and melphalan, PBSCs stored at 4°C for up to 6 days can be used safely in centers with no cryopreservation facility. However, the kinetics of hematopoietic recovery showed a significant, albeit small, delay in engraftment for both neutrophils and platelets, which favors the use of cryopreservation if available.

Pembrolizumab for the treatment of progressive multifocal leukoencephalopathy following anti-CD19 CAR-T therapy: a case report.

Progressive multifocal leukoencephalopathy (PML) is an opportunistic brain infection with few treatment options and poor survival when reversal of the underlying immune dysfunction is not achievable. JC polyomavirus reactivation resulting in PML can rarely complicate chimeric antigen receptor T-cell (CAR-T) therapy. We describe successful treatment of PML with Programmed death-1 (PD-1) blockade using pembrolizumab, 4 months following axicabtagene ciloleucel. Radiological features of immune reconstitution inflammatory syndrome without clinical deterioration were seen. Evidence of anti-viral immune reconstitution by in vitro detection of JC-specific T-cells and sustained neurological recovery in this patient suggest PD-1 blockade may be an effective treatment approach for PML post-CAR-T.