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Severe combined immunodeficiency (SCID) results from defects in the differentiation of hematopoietic stem cells into mature T lymphocytes, with additional lymphoid lineages affected in particular genotypes. In 2014, the Primary Immune Deficiency Treatment Consortium published criteria for diagnosing SCID, which are now revised to incorporate contemporary approaches. Patients with typical SCID must have less than 0.05 × 10

We established and characterized a bank of 138 CMVpp65 peptide-specific T-cell (CMVpp65CTLs) lines from healthy marrow transplant donors who consented to their use for treatment of individuals other than their transplant recipient. CMVpp65CTL lines included 131 containing predominantly CD8+ T cells and 7 CD4+ T cells. CD8+ CMVpp65CTLs were specific for 1 to 3 epitopes each presented by one of only 34 of the 148 class I alleles in the bank. Similarly, the 7 predominantly CD4+ CMVpp65CTL lines were each specific for epitopes presented by 14 of 40 HLA DR alleles in the bank. Although the number of HLA alleles presenting CMV epitopes is low, their prevalence is high, permitting selection of CMVpp65CTLs restricted by an HLA allele shared by transplant recipient and hematopoietic cell transplant donor for >90% of an ethnogeographically diverse population of hematopoietic cell transplant recipients. Within individuals, responses to CMVpp65 peptides presented by different HLA alleles are hierarchical. Furthermore, within groups, epitopes presented by HLA B*07:02 and HLA A*02:01 consistently elicit immunodominant CMVpp65CTLs, irrespective of other HLA alleles inherited. All dominant CMVpp65CTLs exhibited HLA-restricted cytotoxicity against epitope loaded targets and usually cleared CMV infections. However, immunodominant CMVpp65CTLs responding to epitopes presented by certain HLA B*35 alleles were ineffective in lysing CMV-infected cells in vitro or controlling CMV infections post adoptive therapy. Analysis of the hierarchy of T-cell responses to CMVpp65, the HLA alleles presenting immunodominant CMVpp65 epitopes, and the responses they induce may lead to detailed algorithms for optimal choice of third-party CMVpp65CTLs for effective adoptive therapy.

PDF file 96K, Pre-treatment of donor T-cells with a c-Rel antagonist promotes IL-2 secretion in vivo

PDF file 128KB, Treg depletion from c-Rel deficient donor T-cells results in exacerbated GVHD

PDF file 92K, Table 1. Characteristics of c-Rel inhibitor compounds; Table 2. Roles of c-Rel in T-cell responses and transplantation immunology

PDF file 135KB, Non-specific negative effect of DMSO on the GVT activity of T-cells

PDF file 128KB, c-Rel-deficient T-cells are polarized towards Th2 responses during GVHD

PDF file 191K, Recipients of c-Rel-deficient T-cells exhibit reduced damage of GVHD target organs

PDF file 121K, IL-2 secretion is enhanced in irradiated recipients transplanted with allogeneic T-cells

PDF file - 78KB, ESKM has a modified Fc glycosylation pattern, altering binding to FcgammaRs but not to the RMF/A2 target. Where applicable, data points are averages of each group, and error bars represent SEM. (A) Comparison of the oligosaccharide profile of ESK1 and ESKM. Peak assignment is based on the retention time and the monosaccharide composition analysis. G# indicates the number of terminal galactoses, F indicates presence of core fucose, Hex5GlcNAc2 denotes (GlcNAc)2 core with terminal Hexose 5 glycan structure (terminating in mannose and/or glucose). (B-C) Representative binding curves of ESK1 and ESKM against human FcgammaRI, FcgammaRIIa, FcgammaRIIb, FcgammaRIIIa - 158V, FcgammaRIIIa -158F, and FcRn (B), and mouse FcgammaRIIb and FcgammaRIV (C). (D) 125 I-labeled ESK1 and ESKM mAbs were titrated against JMN cells. All curves were fit with a non-linear single-site total binding saturation curve, and Kd was calculated using Prism software.

PDF file - 145KB, All human antibodies tested accumulated more in spleens of HLA-A2+ transgenic mice, but ESK1 did not bind specifically to isolated HLA-A2+ spleen, bone marrow or thymus cells. (A) Accumulation of 125 I-labeled antibodies in spleens of C57BL6/J or HLA-A2+ transgenic mice relative to antibody level in the blood. Mice were injected retroorbitally with 2microg indicated antibody, then sacrificed after 24 hours for blood and spleen collection. (B) Specific binding of 125I-labeled ESK1 to bone marrow, spleen, or thymus cells isolated from C57BL6/J or HLA-A2+ transgenic mice. Tissues were collected from 2 (C57) or 3 (HLA-A2+ transgenic) mice, then bound by 1microg/mL 125 I-labeled ESK1 either alone or after blocking with 50-fold excess unlabeled ESK1. Specific binding was determined, and #ESK1 bound per cell was calculated.

PDF file - 38KB, Histopathologic report from ESKM- or isotype control human IgG1-treated HLA-A*02:01+ transgenic mice.

Purpose: RMFPNAPYL (RMF), a Wilms' tumor gene 1 (WT1)–derived CD8 T-cell epitope presented by HLA-A*02:01, is a validated target for T-cell–based immunotherapy. We previously reported ESK1, a high avidity (Kd < 0.2 nmol/L), fully-human monoclonal antibody (mAb) specific for the WT1 RMF peptide/HLA-A*02:01 complex, which selectively bound and killed WT1+ and HLA-A*02:01+ leukemia and solid tumor cell lines.Experimental Design: We engineered a second-generation mAb, ESKM, to have enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) function due to altered Fc glycosylation. ESKM was compared with native ESK1 in binding assays, in vitro ADCC assays, and mesothelioma and leukemia therapeutic models and pharmacokinetic studies in mice. ESKM toxicity was assessed in HLA-A*02:01+ transgenic mice.Results: ESK antibodies mediated ADCC against hematopoietic and solid tumor cells at concentrations below 1 μg/mL, but ESKM was about 5- to 10-fold more potent in vitro against multiple cancer cell lines. ESKM was more potent in vivo against JMN mesothelioma, and effective against SET2 AML and fresh ALL xenografts. ESKM had a shortened half-life (4.9 days vs. 6.5 days), but an identical biodistribution pattern in C57BL/6J mice. At therapeutic doses of ESKM, there was no difference in half-life or biodistribution in HLA-A*02:01+ transgenic mice compared with the parent strain. Importantly, therapeutic doses of ESKM in these mice caused no depletion of total WBCs or hematopoetic stem cells, or pathologic tissue damage.Conclusions: The data provide proof of concept that an Fc-enhanced mAb can improve efficacy against a low-density, tumor-specific, peptide/MHC target, and support further development of this mAb against an important intracellular oncogenic protein. Clin Cancer Res; 20(15); 4036–46. ©2014 AACR.

PDF file - 183KB, (A) ESKM reduced individual tumor burden during the treatment course in 3 of 5 mice. Alternate representation of data in Figure 2A, where signal for each mouse is normalized to starting luminescence. (B) ESKM had no effect against JMN in a NOG mouse model. (C) Comparison of BV173 (Ph+ ALL) and SET2 (AML) cell lines in NSG mice.

PDF file - 142KB, Human PBMCs were separated into effector cell populations and tested for in vitro ADCC activity against BV173 cells, mediated by ESKM or isotype control mAb. Only fractionated NK cells showed activity relative to isotype control antibody. % Lysis is calculated based on spontaneous release (target cells, no effectors or mAb) as 0% and maximal release (target cells + SDS) as 100%.

IT-901 inhibits growth of human DLBCL cell lines.

c-Rel is constitutively active in a wide range of human lymphoma types.

ROS/RNS induction by IT-­901 treatement in normal tissues.

c-Rel inhibitor treatment is associated with partial recovery of lost body weight during severe acute GVHD.

NF-κB plays a variety of roles in oncogenesis and immunity that may be beneficial for therapeutic targeting, but strategies to selectively inhibit NF-κB to exert antitumor activity have been elusive. Here, we describe IT-901, a bioactive naphthalenethiobarbiturate derivative that potently inhibits the NF-κB subunit c-Rel. IT-901 suppressed graft-versus-host disease while preserving graft-versus-lymphoma activity during allogeneic transplantation. Further preclinical assessment of IT-901 for the treatment of human B-cell lymphoma revealed antitumor properties in vitro and in vivo without restriction to NF-κB–dependent lymphoma. This nondiscriminatory, antilymphoma effect was attributed to modulation of the redox homeostasis in lymphoma cells resulting in oxidative stress. Moreover, NF-κB inhibition by IT-901 resulted in reduced stimulation of the oxidative stress response gene heme oxygenase-1, and we demonstrated that NF-κB inhibition exacerbated oxidative stress induction to inhibit growth of lymphoma cells. Notably, IT-901 did not elicit increased levels of reactive oxygen species in normal leukocytes, illustrating its cancer selective properties. Taken together, our results provide mechanistic insight and preclinical proof of concept for IT-901 as a novel therapeutic agent to treat human lymphoid tumors and ameliorate graft-versus-host disease. Cancer Res; 76(2); 377–89. ©2016 AACR.

IT-901 is a more potent c-Rel inhibitor than IT-603.

Effects of IT-901 on lymphocyte subsets in the spleen.

IT-901 has a superior pharmacokinetic profile.

IT-901 is a more potent NF-κB inhibitor than IT-603.

IT-901 mediates anti-lymphoma activity in a xenograft model of EBV-induced lymphoma.

IT-901 has no significant adverse effects on major organ functions and blood cell counts.

Treatment with IT-901 for 14 days does not alter lymphocyte numbers.

BackgroundCertain phosphorylated peptides are differentially presented by MHC molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide a stability advantage over their nonphosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity. Whether tumor-associated phosphopeptides presented by other common alleles exhibit immunogenicity and structural characteristics similar to those presented by A*02:01 is unclear. Therefore, we determined the identity, structural features, and immunogenicity of phosphopeptides presented by the prevalent alleles HLA-A*03:01, -A*11:01, -C*07:01, and - C*07:02.MethodsWe isolated peptide-MHC complexes by immunoprecipitation from 10 healthy and neoplastic tissue samples using mass spectrometry, and then combined the resulting data with public immunopeptidomics datasets to assemble a curated set of phosphopeptides presented by 20 distinct healthy and neoplastic tissue types. We determined the biochemical features of selected phosphopeptides by in vitro binding assays and in silico docking, and their immunogenicity by analyzing healthy donor T cells for phosphopeptide-specific multimer binding and cytokine production.ResultsWe identified a subset of phosphopeptides presented by HLA-A*03:01, A*11:01, C*07:01 and C*07:02 on multiple tumor types, particularly lymphomas and leukemias, but not healthy tissues. These phosphopeptides are products of genes essential to lymphoma and leukemia survival. The presented phosphopeptides generally exhibited similar or worse binding to A*03:01 than their nonphosphorylated counterparts. HLA-C*07:01 generally presented phosphopeptides but not their unmodified counterparts. Phosphopeptide binding to HLA-C*07:01 was dependent on B- pocket interactions that were absent in HLA-C*07:02. While HLA-A*02:01 and -A*11:01 phosphopeptide-specific T cells could be readily detected in an autologous setting even when the nonphosphorylated peptide was co-presented, HLA-A*03:01 or -C*07:01 phosphopeptides were repeatedly nonimmunogenic, requiring use of allogeneic T cells to induce phosphopeptide- specific T cells.ConclusionsPhosphopeptides presented by multiple alleles that are differentially expressed on tumors constitute tumor-specific antigens that could be targeted for cancer immunotherapy, but the immunogenicity of such phosphopeptides is not a general feature. In particular, phosphopeptides presented by HLA-A*02:01 and A*11:01 exhibit consistent immunogenicity, while phosphopeptides presented by HLA-A*03:01 and C*07:01, although appropriately presented, are not immunogenic. Thus, to address an expanded patient population, phosphopeptide-targeted immunotherapies should be wary of allele-specific differences.What is already known on this topic –Phosphorylated peptides presented by the common HLA alleles A*02:01 and B*07:02 are differentially expressed by multiple tumor types, exhibit structural fitness due to phosphorylation, and are targets of healthy donor T cell surveillance, but it is not clear, however, whether such features apply to phosphopeptides presented by other common HLA alleles.What this study adds –We investigated the tumor presentation, binding, structural features, and immunogenicity of phosphopeptides to the prevalent alleles A*03:01, A*11:01, C*07:01, and C*07:02, selected on the basis of their presentation by malignant cells but not normal cells. We found tumor antigens derived from genetic dependencies in lymphomas and leukemias that bind HLA-A3, -A11, -C7 molecules. While we could detect circulating T cell responses in healthy individuals to A*02:01 and A*11:01 phosphopeptides, we did not find such responses to A*03:01 or C*07:01 phosphopeptides, except when utilizing allogeneic donor T cells, indicating that these phosphopeptides may not be immunogenic in an autologous setting but can still be targeted by other means.How this study might affect research, practice or policy– An expanded patient population expressing alleles other than A*02:01 can be addressed through the development of immunotherapies specific for phosphopeptides profiled in the present work, provided the nuances we describe between alleles are taken into consideration.

PURPOSE Calcineurin inhibitors (CNI) are standard components of graft-versus-host disease (GVHD) prophylaxis after hematopoietic cell transplantation (HCT). Prior data suggested that CNI-free approaches using donor T-cell depletion, either by ex vivo CD34 selection or in vivo post-transplant cyclophosphamide (PTCy) as a single agent, are associated with lower rates of chronic GVHD (cGVHD). METHODS This multicenter phase III trial randomly assigned patients with acute leukemia or myelodysplasia and an HLA-matched donor to receive CD34-selected peripheral blood stem cell, PTCy after a bone marrow (BM) graft, or tacrolimus and methotrexate after BM graft (control). The primary end point was cGVHD (moderate or severe) or relapse-free survival (CRFS). RESULTS Among 346 patients enrolled, 327 received HCT, 300 per protocol. Intent-to-treat rates of 2-year CRFS were 50.6% for CD34 selection (hazard ratio [HR] compared with control, 0.80; 95% CI, 0.56 to 1.15; P = .24), 48.1% for PTCy (HR, 0.86; 0.61 to 1.23; P = .41), and 41.0% for control. Corresponding rates of overall survival were 60.1% (HR, 1.74; 1.09 to 2.80; P = .02), 76.2% (HR, 1.02; 0.60 to 1.72; P = .95), and 76.1%. CD34 selection was associated with lower moderate to severe cGVHD (HR, 0.25; 0.12 to 0.52; P = .02) but higher transplant-related mortality (HR, 2.76; 1.26 to 6.06; P = .01). PTCy was associated with comparable cGVHD and survival outcomes to control, and a trend toward lower disease relapse (HR, 0.52; 0.28 to 0.96; P = .037). CONCLUSION CNI-free interventions as performed herein did not result in superior CRFS compared with tacrolimus and methotrexate with BM. Lower rates of moderate and severe cGVHD did not translate into improved survival.

Shearer et al in 2014 articulated well-defined criteria for the diagnosis and classification of severe combined immunodeficiency (SCID) as part of the Primary Immune Deficiency Treatment Consortium's (PIDTC's) prospective and retrospective studies of SCID.Because of the advent of newborn screening for SCID and expanded availability of genetic sequencing, revision of the PIDTC 2014 Criteria was needed.We developed and tested updated PIDTC 2022 SCID Definitions by analyzing 379 patients proposed for prospective enrollment into Protocol 6901, focusing on the ability to distinguish patients with various SCID subtypes.According to PIDTC 2022 Definitions, 18 of 353 patients eligible per 2014 Criteria were considered not to have SCID, whereas 11 of 26 patients ineligible per 2014 Criteria were determined to have SCID. Of note, very low numbers of autologous T cells (0.05 × 10The PIDTC 2022 Definitions describe SCID and its subtypes more precisely than before, facilitating analyses of SCID characteristics and outcomes.

T cells specific for major histocompatibility complex (MHC)-presented tumor antigens are capable of inducing durable remissions when adoptively transferred to patients with refractory cancers presenting such antigens. When such T cells are derived from healthy donors, they can be banked for off-the-shelf administration in appropriately tissue matched patients. Therefore, tumor antigen-specific, donor-derived T cells are expected to be a mainstay in the cancer immunotherapy armamentarium. In this chapter, we analyze clinical evidence that tumor antigen-specific donor-derived T cells can induce tumor regressions when administered to appropriately matched patients whose tumors are refractory to standard therapy. We also delineate the landscape of MHC-presented and unconventional tumor antigens recognized by T cells in healthy individuals that have been targeted for adoptive T cell therapy, as well as emerging antigens for which mounting evidence suggests their utility as targets for adoptive T cell therapy. We discuss the growing technological advancements that have facilitated sequence identification of such antigens and their cognate T cells, and applicability of such technologies in the pre-clinical and clinical settings.

Background: Diagnosis and remission status at the time of allogeneic haematopoietic stem-cell transplantation (HSCT) are the principal determinants of overall survival following transplantation. We sought to develop a contemporary disease-risk stratification system (DRSS) that accounts for heterogeneous transplantation indications. Methods: In this retrospective cohort study we included 55 histology and remission status combinations across haematological malignancies, including acute leukaemia, lymphoma, multiple myeloma, and myeloproliferative and myelodysplastic disorders. A total of 47 265 adult patients (aged ≥18 years) who received an allogeneic HSCT between Jan 1, 2012, and Dec 31, 2016, and were reported to the European Society for Blood and Marrow Transplantation registry were included. We divided EBMT patients into derivation (n=25 534), tuning (n=18 365), and geographical validation (n=3366) cohorts. Disease combinations were ranked in a multivariable Cox regression for overall survival in the derivation cohort, cutoff for risk groups were evaluated for the tuning cohort, and the selected system was tested on the geographical validation cohort. An independent single-centre US cohort of 660 patients transplanted between Jan 1, 2010, and Dec 31, 2015 was used to externally validate the results. Findings: The DRSS model stratified patients in the derivation cohort (median follow-up was 2·1 years [IQR 1·0–3·2]) into five risk groups with increasing mortality risk: low risk (reference group), intermediate-1 (hazard ratio for overall survival 1·26 [95% CI 1·17–1·36], p

Adenosine deaminase (ADA) deficiency causes ∼13% of cases of severe combined immune deficiency (SCID). Treatments include enzyme replacement therapy (ERT), hematopoietic cell transplant (HCT), and gene therapy (GT). We evaluated 131 patients with ADA-SCID diagnosed between 1982 and 2017 who were enrolled in the Primary Immune Deficiency Treatment Consortium SCID studies. Baseline clinical, immunologic, genetic characteristics, and treatment outcomes were analyzed. First definitive cellular therapy (FDCT) included 56 receiving HCT without preceding ERT (HCT); 31 HCT preceded by ERT (ERT-HCT); and 33 GT preceded by ERT (ERT-GT). Five-year event-free survival (EFS, alive, no need for further ERT or cellular therapy) was 49.5% (HCT), 73% (ERT-HCT), and 75.3% (ERT-GT; P < .01). Overall survival (OS) at 5 years after FDCT was 72.5% (HCT), 79.6% (ERT-HCT), and 100% (ERT-GT; P = .01). Five-year OS was superior for patients undergoing HCT at

An association between early CD4+ T cell immune reconstitution (CD4+ IR) and survival after T-replete allogeneic hematopoietic cell transplantation (HCT) has been previously reported. Here we report validation of this relationship in a separate cohort that included recipients of ex vivo T-cell-depleted (TCD) HCT. We studied the relationship between CD4+ IR and clinical outcomes.A retrospective analysis of children/young adults receiving their first allogeneic HCT for any indication between January 2008 and December 2017 was performed. We related early CD4+ IR (defined as achieving50 CD4+ T cells/µL on two consecutive measures within 100 days of HCT) to overall survival (OS), relapse, non-relapse mortality (NRM), event-free survival (EFS) and acute graft-versus-host disease (aGVHD). Fine and Gray competing risk models and Cox proportional hazard models were used.In this analysis, 315 patients with a median age of 10.4 years (interquartile range 5.0-16.5 years) were included. The cumulative incidence of CD4+ IR at 100 days was 66.7% in the entire cohort, 54.7% in TCD (N = 208, hazard ratio [HR] 0.47, P0.001), 90.0% in uCB (N = 40) and 89.6% in T-replete (N = 47) HCT recipients. In multi-variate analyses, not achieving early CD4+ IR was a predictor of inferior OS (HR 2.35, 95% confidence interval [CI] 1.46-3.79, P0.001) and EFS (HR 1.80, 95% CI 1.20-2.69, P = 0.004) and increased NRM (HR 6.58, 95% CI 2.82-15.38, P0.001). No impact of CD4+ IR on relapse or aGVHD was found. Within the TCD group, similar associations were observed.In this HCT cohort, including recipients of TCD HCT, we confirmed that early CD4+ IR was an excellent predictor of outcomes. Finding strategies to predict or improve CD4+ IR may influence outcomes.