ALBERT

Description: Acute graft-versus-host disease (aGVHD) develops in more than half of patients after allogeneic hematopoietic cell transplantation (allo-HCT) despite poly-pharmacy immunoprophylaxis. Importantly, the dysregulated pathways responsible for this breakthrough disease remain largely unidentified. Thus, the discovery of these pathways represents one of the critical challenges for the field of allo-HCT. To address these needs, we have developed a model of aGVHD in rhesus macaques, which allows us to study the mechanisms of aGVHD both in its untreated state and in a variety of immunoprophylactic settings. Using a systems-based approach, we have created both a multiparameter flow cytometric and transcriptomic map of the immune landscape of aGVHD in allo-HCT recipients, in comparison to two critical control groups: (1) healthy untransplanted controls, and (2) those receiving autologous transplantation. We find that recipients of allo-HCT receiving 1) no immunoprophylaxis 2) monotherapy with CTLA4Ig or 3) monotherapy with sirolimus develop early fulminant aGVHD with multi-organ disease (Figure 1a-grouped as 'Primary GVHD'). The immunophenotype of T cells from the Primary GVHD cohort exhibits an effector/memory phenotype with robust proliferation and acquisition of cytotoxic function. Transcriptomic analysis reveals enrichment of Th1-associated transcripts (IL12RB2, CCR5, CXCR3) as well as programs of proliferation early in the post transplant period (Figure 1b). Flow cytometric data confirms an increase in the number of CD4 and CD8 T cells producing the Th1 cytokine, IFN-g at this time-point (Figure 1c). In contrast, standard-of-care Tacrolimus/Methotrexate (Tac/Mtx) as well as novel CTLA4Ig/sirolimus combination immunoprophylaxis (CoBS) both significantly improved survival of animals after allo-HCT. However, similar to human patients undergoing allo-HCT, these recipients often developed clinical signs of breakthrough aGVHD (starting around day 30 post-transplant) characterized by both gastrointestinal and skin pathology. This cohort was thus termed the "Breakthrough GVHD" cohort (Figure 1a). Unexpectedly, despite the presence of breakthrough clinical aGVHD, the Tac/Mtx and CoBS cohorts were still able to control programs of T cell proliferation, effector phenotype acquisition and Th1 cytokine skewing. However, both transcriptional and flow cytometric profiles demonstrated enrichment for molecules that reflect Th17/Th22 skewing (RORC, IL17A, AHR, and IL22) (Figure 2a) and production of IL17a (Figure 2b). These results suggest that while current methods of immunoprophylaxis are able to limit both T cell proliferation and Th1 polarization, breakthrough Th17/Th22 pathway activation occurs despite these therapies. These data suggest that emphasis should be placed on exploration of pharmacologic inhibitors of IL17/IL22 for the prevention/treatment of breakthrough aGVHD. Disclosures No relevant conflicts of interest to declare.

Description: The Notch signaling pathway is an evolutionarily conserved, cell-cell communication system with critical functions in organogenesis and tissue homeostasis, including hemato- and immuno-poiesis. Recent data have revealed important roles for Notch in the regulation of mature T cell differentiation and function. Studies in mouse models have identified Notch as a critical regulator of pathogenic T-cell responses during acute GVHD (aGVHD) (Zhang Y, 2011, Blood). However, the exact biological effects and the therapeutic potential of Notch pathway manipulation in clinical settings remains unclear. To address this question, we tested the activity of Notch pathway blockade in a non-human primate (NHP) aGVHD model, previously shown to exhibit donor T cell-intrinsic activation of the Notch pathway during aGVHD (Furlan SN, 2015, Sci Transl Med). To inhibit the Notch pathway, we used a blocking mAb to the Notch ligand DLL4, identified as the dominant ligand in a mouse aGVHD model (Tran, 2013, JCI; Chung, 2017, JCI). Prophylactic treatment regimens with either a single administration of anti-DLL4 mAb on day 0 (3 mg/kg), or with 3 doses (3 mg/kg each) on days 0, 7 and 14 significantly improved GVHD-free survival of allo-HCT recipients (median survival time (MST) = 26.5 days for the single dose regimen, and MST = 26 days for the triple dose regimen) in comparison with unprophylaxed controls (MST = 8 days, p

Description: As the field of regulatory T cell (Treg) adoptive therapy develops, questions concerning how to best to deploy these cells in patients must be addressed. Prominent among these questions is which adjunctive therapies will pair most synergistically with the transferred cells. Most ideally, a therapy that could prolong persistence and stabilize function of the cells is sought. Prior work has shown that mechanistic target of rapamycin (mTOR) inhibition can afford both stability and a degree of increased persistence to ex-vivo expanded thymic Tregs (tTregs) upon adoptive transfer. In this study, we investigated whether the addition of low-dose interleukin-2 (IL2) to the mTOR inhibitor rapamycin could impart additional persistence to ex-vivoexpanded tTregs after adoptive transfer. Using a non-human primate model of CFSE-labeled autologous tTregs, we found that the addition of IL2 to rapamycin supported a near 10-fold increase in the half-life of adoptively transferred tTregs, effectively doubling the cells in the tTreg compartment for the first month after adoptive transfer. Using a combination of single cell approaches, we were then able to show that transferred tTregs, in the setting of IL2 and rapamycin adjunctive therapy, retain high levels of Treg-specific genes, including FOXP3, after adoptive transfer. Additionally, we found that adoptively transferred tTregs are remarkably homogenous and become more transcriptionally similar to endogenous tTregs with time in vivo. Together these preclinical data support the use of combination IL2 and rapamycin as adjunctive therapy forex-vivo expanded adoptively transferred tTregs. Disclosures Tkachev: Regeneron Pharmaceuticals, Inc.: Research Funding. Blazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Kean:Regeneron Pharmaceuticals, Inc.: Research Funding.

Description: Key PointsThe transcriptional networks controlling breakthrough acute GVHD can be mapped, and correlate closely with clinical disease. Breakthrough acute GVHD is transcriptionally controlled by T-cell persistence, inflammation, and Th/Tc17 skewing.

Description: While calcineurin inhibition (CNI)-based strategies remain the mainstay for GVHD prevention, CNI are notoriously antagonistic to immune tolerance induction. Rapamycin (Rapa) has been shown to be more pro-tolerogenic; however, the best agents to combine with Rapa are still undetermined, and it remains a second-line GVHD prevention strategy without clear superiority over CNI. Finding tolerogenic partners for Rapa, therefore, represents a critical unmet need in the field. Of the possible partners for Rapa, the OX40/OX40L pathway represents an important target. OX40 is a costimulatory receptor expressed on activated human T cells, which, upon interaction with OX40L delivers activation signals to conventional T cells (Tconv) promoting their proliferation, survival and clonal expansion. Notably, these same OX40/OX40L signals may either inhibit or promote Treg functions, depending on context, suggesting that blockade of this pathway may simultaneously control Tconv activation while permitting Treg homeostasis. During GVHD in non-human primates (NHP), we found OX40L upregulation on myeloid dendritic cells and OX40 upregulation on activated T cells in recipients treated with multiple immunosuppressive agents, including Rapa (Fig 1). These data provided strong rationale for testing KY1005, a novel human monoclonal antibody that binds to OX40L and blocks its interaction with OX40, as a potential partner with Rapa. We tested the outcomes of prophylactic blockade of this pathway on NHP GVHD, using KY1005 alone and in combination with Rapa. These experiments utilized our previously published NHP GVHD model, in which GVHD is studied after T cell-replete haplo-identical HCT. KY1005 was dosed at 10mg/kg weekly from days -2ˆ+54 and Rapa was continued through Day +100. Prophylaxis with KY1005 alone provided initial evidence for its in vivo activity, with control of CD4〉CD8 T cell proliferation and mitigation of the expansion of CD4〉CD8 T effector/memory cells. Consistent with the partial control of T cell activation, these recipients demonstrated improved GVHD-free survival versus unprophylaxed controls, but disease ultimately broke through (Median Survival Time (MST) = 19.5 days with KY1005 (n=4) compared to 8 days in unprophylaxed recipients (n= 10, Fig 2)). We next investigated the impact of OX40L blockade + Rapa. We have published that Rapa as a monotherapy minimally controlled both immunologic and clinical disease, with an MST = 14 days (n=6). Combined prophylaxis was striking: recipients given KY1005+Rapa (n=5) maintained robust health throughout the entire experiment (MST 〉100d), and demonstrated high levels of donor T cell chimerism (86 +/- 3% at Day 100), rapid hematopoietic reconstitution, and had a terminal GVHD Grade of 0, compared to a Grade of III-IV in both KY1005- and Rapa-monotherapy cohorts. Immunologic analysis demonstrated synergistic control of both CD4 and CD8 T cell proliferation, restoring it to the level observed during autologous immune reconstitution, and resulting in a concomitant abrogation of CD4 and CD8 memory/effector expansion while preserving T cells with a na•ve phenotype. In striking contrast to the inhibition of Tconv activation by KY1005+Rapa, recipients of dual therapy demonstrated intact Treg reconstitution post-HCT, which resulted in a favorable Treg:Tconv ratio of 5.4 vs 1.4:100 in KY1005+Rapa treated compared to untreated recipients (p 〈 0.05). Transcriptomic analysis confirmed the unique immunologic state conferred by KY1005+Rapa on purified T cells, with gene arrays from these recipients demonstrating separation from all other transplant cohorts in Principal Component space (Figure 3A) and Class Neighbor Analysis identifying unique expression modules that tracked with KY1005 + Rapa prophylaxis (Figure 3B red and blue boxes). These results underscore the critical role of OX40/OX40L signaling in the development of GVHD and demonstrate the striking control of GVHD in KY1005+Rapa recipients. They represent the first demonstration of uniform, long-term GVHD-free survival in the primate model of high-risk haplo-identical HCT, and the first therapeutic strategy that simultaneously controls Tconv activation while supporting Treg homeostasis in this model. They suggest that OX40L blockade + Rapa is a novel, evidence-based combinatorial strategy to control GVHD that is an exceptional candidate regimen for clinical translation. Disclosures Tkachev: Kymab Ltd: Patents & Royalties: US Patent 9,382,325, Research Funding. Casson:Kymab Ltd: Employment. Kirby:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Bland-Ward:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Kean:Juno Therapeutics, Inc: Research Funding.

Description: One of the major barriers to developing targeted therapies for aGVHD control is the difficulty in identifying T cell signatures specific for GVHD pathology while distinguishing these from the pathways essential for tissue-specific T cell immune reconstitution. To address this need we have interrogated the migration patterns, as well as the phenotypic and transcriptomic characteristics of allogeneic T cells infiltrating aGVHD target organs in non-human primates. To rigorously study T cell migration during aGVHD we tracked T cells labeled following in vivo infusion with fluorescently tagged anti-CD45 antibodies given to NHP transplant recipients with aGVHD on day 8 post-HCT, during active disease. Anti-CD45 antibodies with distinct fluorescent tags were given at 6 hours before necropsy (anti-CD45-AlexaFluor647) and 5 minutes before necropsy (anti-CD45-AlexaFluor488), in order to measure T cells that were in the circulation and those migrating to GVHD target tissues, based on their labeling with one or both fluorescently-tagged antibodies. These experiments identified increased migration of both allogeneic CD8 T cells (Figure 1A) and CD4 T cells (not shown) during aGVHD, with trafficking into secondary lymphoid organs as well as non-lymphoid GVHD target organs (intestine and kidney). While migration was increased during aGVHD, these T cells, which demonstrated some phenotypic similarities to CD8 T cells in the peripheral blood (Figure 1B), also adopted tissue-specific phenotypes as measured by flow cytometry (Figure 1C), including the expression of canonical markers of resident-memory T cells (CD69+CD103-/+). However, unlike the tissue-resident T cells in healthy controls during homeostasis, tissue-infiltrating T cells during aGVHD expressed multiple markers of activation, including Ki67 and Granzyme B (Figure 1D). These flow cytometric characteristics suggested that the phenotype of organ-infiltrating T cells during aGVHD included attributes of both tissue-residency and of pathogenic alloreactivity. To further identify aGVHD-specific signatures, we performed transcriptomic analysis of tissue-infiltrating T cells during aGVHD. Using an unsupervised weighted gene correlation network analysis (WGCNA) we characterized the gene sets associated with individual GVHD target organs (Figure 2). We found that tissue-infiltrating T cells during aGVHD could be characterized by divergent features: First, they maintained a core tissue localization signature, which included genes previously linked to tissue-resident T cells (e.g. RUNX3, IFNG, CXCR6). Importantly, however, they also acquired an aGVHD-specific transcriptional signature including expression of IL1RL1 (encoding ST2), ICOS, TNFRSF9 (CD137) and TNFRSF4 (OX40). This signature also included enrichment for transcripts encoding the cytotoxic mediators GRMB and GRMA, the proliferation markers MKI67 and AURKA, as well as cytokines and cytokine receptors (IL18, IL18R, IL21, IL21R). Proteins encoded by each of these transcripts have been linked to aGVHD-causing T cells, strengthening the inference that these constitute a robust transcriptomic signature of aGVHD pathogenesis. Thus, for the first time in a large-animal model, we have been able to directly measure both the kinetics and the protein and RNA expression signatures of T cells during their migration into aGVHD target organs, This study provides new evidence for the evolution of a phenotypic and transcriptomic dichotomy during aGVHD-mediated tissue infiltration, in which T cells take on both tissue- and aGVHD-specific characteristics. These data provide novel insights into the spatial organization of systemic alloimmunity during aGVHD, which should enable more precise targeting of pathogenic T cell populations while preserving normal tissue immune reconstitution after transplantation. Disclosures Tkachev: Regeneron Pharmaceuticals, Inc.: Research Funding. Blazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Kean:Regeneron Pharmaceuticals, Inc.: Research Funding.

Description: Although acute graft-versus-host-disease (AGVHD) is one of the major causes of non-relapse mortality after hematopoietic stem cell transplant (HCT), we are still unable to predict which patients will develop the most severe form of this disease, or which molecular pathways are dysregulated in the T cells that cause disease. Thus, understanding the molecular features of AGVHD is a critical unmet need. To address this, we have performed a companion mechanistic study as a part of our completed Phase 2 trial of abatacept, a CD28:CD80/86 costimulation blockade agent, for severe AGVHD prevention (Clinicaltrials.gov # NCT01743131, 'ABA2'). ABA2 has demonstrated significant improvement in AGVHD in patients prophylaxed with abatacept in addition to calcineurin inhibition (CNI) + Methotrexate (MTX) compared to controls receiving CNI/MTX alone. To begin to uncover mechanisms responsible for the control of AGVHD with abatacept, and given that CD4+ T cells have been consistently documented to be the main therapeutic target of this drug, we interrogated the transcriptome of CD4+ T cells reconstituting in patients prophylaxed with abatacept compared to CNI/MTX. To perform this analysis, we flow cytometrically sorted CD4+ T cells on Days 21-28 post-transplant from all patients on ABA2, as well as a cohort of 12 untransplanted healthy controls, and subsequently performed mRNA-sequencing on these cells. Weighted Gene Correlation Network Analysis (WGCNA) was performed on the top 6000 most variant transcripts from the resulting sequencing data. Hierarchical clustering of the WGCNA co-expression matrix enabled the identification of self-assembling modules (SAMs) that met a threshold of coexpression (Figure 1A). For the ABA2 dataset, we considered the following variables in the WGCNA model: patient cohort (7/8 patients, 8/8 patients, healthy controls), +/- prophylaxis with abatacept, CMV reactivation, EBV reactivation, Grade of GVHD (0-4), relapse, non-relapse mortality, and all-cause mortality. The WGCNA clustering analysis resulted in the identification of 4 discrete SAMs, which were highly correlated with clinical variable metamodules. This analysis revealed a strong positive correlation of a 476-gene SAM (the Turquoise module) in patients prophylaxed with CNI/MTX + placebo and anti-correlation of this module in patients prophylaxed with CNI/MTX + abatacept, as demonstrated in both the WGCNA heatmap and through Gene Set Enrichment Analysis (Figure 1 A-B). These opposing correlations suggested that interrogation of this module would reveal mechanistic correlates with standard prophylaxis that were decoupled by abatacept. Pathway analysis using the Reactome database (Figure 1C) revealed the turquoise SAM to be dominated by four types of pathways: (1) Those that define canonical cell-cycle pathways (2) Those involved in T cell metabolism (3) Those involved in apoptosis and (4) Those involved in T cell activation, consistent with upregulation of these transcripts in placebo versus abatacept patients. In addition to being highly correlated with patients receiving placebo, the expression of a subset of the transcripts in the Turquoise module were also directly correlated with the severity of AGVHD in these patients. Thus, linear regression analysis of the 476 transcripts in this module identified a subset of 93 genes for which transcript expression level was increased both in placebo compared to abatacept, and for which expression level also positively correlated with Grade of AGVHD. As with the Turquoise module as a whole, this subset of genes also formed a highly correlated network, linking transcripts involved in T cell proliferation, apoptosis, activation, metabolism as well as the T cell checkpoint (Figure 1D). This analysis represents the first comprehensive interrogation of the transcriptomic correlates of AGVHD. It identifies a novel set of transcripts which positively associate with the severity of AGVHD, and which costimulation blockade with abatacept down-regulates and de-couples from AGVHD severity. These results suggest a profound reprograming of T cell activation with abatacept that is correlated with the control of AGVHD. Disclosures Qayed: Bristol-Myers Squibb: Honoraria. Langston:Astellas Pharma: Other: Research Support; Incyte: Other: Research Support; Jazz Pharmaceuticals: Other: Research Support; Chimerix: Other: Research Support; Takeda: Other: Research Support; Kadmon Corporation: Other: Research Support; Novartis: Other: Research Support; Bristol Myers Squibb: Other: Research Support. Blazar:Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Kymab: Consultancy; Jazz: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding. OffLabel Disclosure: Abatacept: Approved for Rheumatoid Arthritis; used in this trial for prevention of GVHD.

Description: Background: Chronic graft-versus-host disease (CGVHD) is the leading cause of long-term morbidity and mortality following hematopoietic stem cell transplant (HCT) and occurs in over 50% of patients undergoing unrelated donor HCT. Despite its frequency, the mechanisms driving this disease remain incompletely understood, making its prevention and successful treatment challenging. To address this issue, we have undertaken a transcriptomic analysis of T cell reconstitution after unrelated donor HCT, to dissect mechanisms driving CGVHD. Methods: The patients studied were enrolled on a Phase 2, randomized, placebo-controlled trial of abatacept for GVHD prevention in patients receiving 8/8 unrelated-donor HCT for hematologic malignancies (NCT01743131). All immune analyses in the current study were performed on patients randomized to standard GVHD prophylaxis with calcineurin inhibition + methotrexate alone (placebo cohort, n =69), and thus provide insights into the drivers of CGVHD during standard unrelated donor HCT. On Day +100, CD4+ T cells were purified from the peripheral blood of these patients, and then analyzed by RNASeq. To determine the transcriptomic drivers of CGVHD without the confounder of significant prior acute GVHD (AGVHD) or exposure to steroids, we focused on profiling the CD4+ transcriptome of de novo CGVHD (CGVHD which develops in the absence of prior grade II-IV AGVHD, n = 7) and compared these patients to those who were 'operationally tolerant' and never developed either grade II-IV AGVHD or any CGVHD (n= 4). Gene expression from the resulting transcriptomes was quantified using kallisto. Differentially expressed (DE) genes were identified using DESeq2 (threshold for DE, adjusted (for multiple testing) p

Description: Allogeneic hematopoietic cell transplantation (HCT) may be curative for patients with marrow and immune disorders, but graft-vs-host-disease (aGVHD) and infections cause significant morbidity and non-relapse mortality. We have conducted a multicenter, double blind, placebo-controlled phase II trial of costimulation blockade with abatacept (Aba) combined with standard GVHD prophylaxis with a calcineurin inhibitor and methotrexate (CNI + MTX) following HLA matched unrelated donor transplant (n=142). In order to assess the effects of Aba on immune reconstitution, and to assess whether this reconstitution is influenced during CMV reactivation, we longitudinally evaluated post-transplant whole blood samples with multiparameter flow cytometry using markers for CD3, CD4, CD8, CD197 and CD45RA to measure reconstitution of CD4 and CD8 T cell populations and their respective memory subsets over time. Results: We observe that post-transplant CMV reactivation induces a marked expansion of CD8 effector memory (EM) cells, which is similar in magnitude for Aba vs placebo patients. We found that development of moderate (gr 2-4) or severe (gr 3-4) GVHD was not associated with an increased frequency of CMV reactivation, but patients with moderate GVHD showed a blunted expansion of CD8 EM cells compared to those without GVHD, and CD8 EM expansion was essentially absent among CMV reactivating patients with severe aGVHD. Clinical correlates will be presented. Conclusions: Our results suggest that adding abatacept to CNI/MTX does not materially affect reconstitution of T cell immunity in the presence or absence of CMV reactivation, but aGVHD remains a major driver of compromised immune recovery after HCT. Disclosures Watkins: Bristol Myers Squibb: Research Funding. Qayed:Mesoblast: Consultancy; Novartis: Consultancy. Horan:Bristol Myers Squib: Honoraria, Research Funding. Kean:gilead: Research Funding; bluebird bio: Research Funding; fortyseven: Consultancy; magenta: Research Funding; regeneron: Research Funding; hifibio: Consultancy; kymab: Consultancy; Bristol Meyers Squibb: Research Funding; novartis: Consultancy. Langston:Kadmon Corporation: Research Funding; Bristol Myers Squib: Research Funding; Incyte: Research Funding; Chimerix: Research Funding; Takeda: Research Funding; Astellas Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding.