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moDC stimulated NK cells retain surface expression of the gamma chain receptor despite the presence of JAK inhibitors.

TG101348 and ruxolitinib inhibit JAK2 mediated STAT5 and STAT3 phosphorylation.

Inhibition of JAK1/2 with ruxolitinib reduces NK cell numbers and function among treated patients with myeloproliferative neoplasm (MPN).

Purpose:Acute and chronic GVHD remain major causes of transplant-related morbidity and mortality (TRM) after allogeneic hematopoietic cell transplantation (alloHCT). We have shown CD83 chimeric antigen receptor (CAR) T cells prevent GVHD and kill myeloid leukemia cell lines. In this pilot study, we investigate CD83 expression on GVHD effector cells, correlate these discoveries with clinical outcomes, and evaluate critical therapeutic implications for transplant recipients.Experimental Design:CD83 expression was evaluated among circulating CD4+ T cells, B-cell subsets, T follicular helper (Tfh) cells, and monocytes from patients with/without acute or chronic GVHD (n = 48 for each group), respectively. CD83 expression was correlated with survival, TRM, and relapse after alloHCT. Differential effects of GVHD therapies on CD83 expression was determined.Results:CD83 overexpression on CD4+ T cells correlates with reduced survival and increased TRM. Increased CD83+ B cells and Tfh cells, but not monocytes, are associated with poor posttransplant survival. CD83 CAR T eliminate autoreactive CD83+ B cells isolated from patients with chronic GVHD, without B-cell aplasia as observed with CD19 CAR T. We demonstrate robust CD83 antigen density on human acute myeloid leukemia (AML), and confirm potent antileukemic activity of CD83 CAR T in vivo, without observed myeloablation.Conclusions:CD83 is a promising diagnostic marker of GVHD and warrants further investigation as a therapeutic target of both GVHD and AML relapse after alloHCT.

Human CD83 cell surface tissue microarray (TMA)

Key Reagents

Supplemental Tables

Impact of JAK2/mTOR blockade on human NK cell function and proliferation. A) Graph shows the cytolytic activity ({plus minus}SEM) of NK cells after 4 hours of culture with K562 target cells, while exposed to DMSO, ruxolitinib (lμM), pacritinib (1.25μM), sirolimus 10ng/ml, or a combination of pacritinib plus sirolimus. B) Human NK cells (105 ) were stimulated with a cytokine cocktail of recombinant human (rh) IL-2 (200 IU/ml) and rhll-15 (10 ng/ml), in the presence of DMSO, ruxolitinib, pacritinib, sirolimus, or pacritinib plus sirolimus. Drugs were added once on day 0. Cytokines were replenished on day +3. Bar graph shows NK cell proliferation ({plus minus}SEM) on day +5 of the culture using a colorimetric assay.

The table shows the pharmacokinetic analysis of pacritinib concentration over time.

The table shows pacritinib adherence during treatment period according to dose level on the PAC/SIR/TAC phase I trial.

Safety measures monitored on trial including (A) QTcF, (B) cardiac ejection fraction, (C) prothrombin time, (D) partial thromboplastin time, (E) and thrombin time

The table shows adverse events per CTCAE version 5 encountered during the PAC/SIR/TAC phase I trial.

Significant Gene List for RNA-Seq Data

CD4+ T cell Aurora kinase A activation as a GVHD resistance mechanism. A) Graph shows frequency of CD4+, pHistone 3 serine 10+ (pH3ser10) (downstream of CD28, and parallel to mTOR) T cells (mean{plus minus} SEM) at day +21 among patients treated on dose level 1 or dose level 2 of the phase I trial compared to baseline control values. B) Graph depicts the frequency (mean {plus minus} SEM) of CD4+, pH3ser10+ T cells recovered from NSG spleens at day +14, following transplantation with human PBMCs and then treated with vehicle, pacritinib, 531-201, sirolimus, pacritinib plus sirolimus, or 531-201 plus sirolimus for 2 weeks.

The table shows the maximum chronic GVHD organ involvement and overall score per NIH Diagnosis and Staging Criteria through full extent of study follow up on the PAC/SIR/TAC phase I trial.

Impact of JAK2 or STAT3 inhibition plus mTOR blockade on DC-allostimulated human T cell production of IFNγ, IL-13, and GM-CSF. 5-day alloMLRs were treated with DMSO vehicle control, pacritinib 1.25μM, S3I-201 5μM, sirolimus 10ng/ml, pacritinib plus sirolimus, or S3I-201 plus sirolimus. The graphs show the concentrations of A) IFNγ, B) IL-13, and C) GM-CSF (mean{plus minus}SEM) as measured in the supernatants of the culture medium by a cytokine multiplex assay.

Supplemental Figures with Legends

Extended Methods including information and details regarding flow cytometry, cell lines, allogeneic mixed leukocyte reactions, Treg Suppression Assays, Xenogeneic GVHD modeling, and key reagents.

The table shows donor chimerism in unsorted bone marrow, and peripheral blood CD3 and CD33 populations from patients treated on the PAC/SIR/TAC phase I trial.

Serial measures of absolute neutrophil count (ANC), hemoglobin (Hgb) and platelet counts (PLT) through complete pacritinib treatment period

The table shows donor/recipient CMV serostatus.

Impact of JAK2/mTOR blockade on human T cell responses toward CMV, EBV, Influenza, and tetanus stimulation. A, B) Graphs show the recall proliferation of CD4+ and CD8+ T cell proliferation in response to CEFT peptides. C, D) Graph shows the frequency of IFNy+, CD107a+ CD4+ or cos+ T cells after stimulation with CEFT peptides.

Amphiregulin (AREG) is increased in circulation in acute graft-versus-host disease (aGVHD) and is associated with poor steroid response and lower survival. The expression of AREG in aGVHD target organs and its association with clinical outcomes are unknown.We performed AREG immunohistochemical staining on skin specimens from 67 patients with aGVHD between the years 2010 and 2015. Two blinded reviewers assessed AREG expression and scored specimens with a semiquantitative scale ranging from 0 (absent) to 4 (most intense).Median AREG score of aGVHD cases was 3. Sixteen of 67 (23.9%) aGVHD cases had an AREG3. High skin AREG expression (3 vs. ≤3) was associated with increased overall clinical grade of aGVHD (52.9% vs. 33.4% clinical grade III-IV, p = 0.02), reduced 3-year overall survival (OS; 13% vs. 61%, p 0.01), and increased 3-year non-relapse mortality (NRM; 56% vs. 20%, p = 0.05).High skin AREG immunohistochemical expression is associated with high clinical grade aGVHD, poor OS, and increased NRM.

Prevention of allograft rejection often requires lifelong immune suppression, risking broad impairment of host immunity. Nonselective inhibition of host T cell function increases recipient risk of opportunistic infections and secondary malignancies. Here we demonstrate that AJI-100, a dual inhibitor of JAK2 and Aurora kinase A, ameliorates skin graft rejection by human T cells and provides durable allo-inactivation. AJI-100 significantly reduces the frequency of skin-homing CLA(+) donor T cells, limiting allograft invasion and tissue destruction by T effectors. AJI-100 also suppresses pathogenic Th1 and Th17 cells in the spleen yet spares beneficial regulatory T cells (Tregs). We show dual JAK2/Aurora kinase A blockade enhances human type 2 innate lymphoid cell (ILC2) responses, which are capable of tissue repair. ILC2 differentiation mediated by GATA3 requires STAT5 phosphorylation (pSTAT5) but is opposed by STAT3. Further, we demonstrate that Aurora kinase A activation correlates with low pSTAT5 in ILC2s. Importantly, AJI-100 maintains pSTAT5 levels in ILC2s by blocking Aurora kinase A and reduces interference by STAT3. Therefore, combined JAK2/Aurora kinase A inhibition is an innovative strategy to merge immune suppression with tissue repair after transplantation.

Background : Higher CD34 cell dose is associated with improved engraftment after peripheral blood allogeneic hematopoietic stem cell transplantation (alloHCT), but may also increase the risk of long-term complications, such as graft-versus-host disease (GVHD). Prior studies examining the relationship between CD34 cell dose and long-term survival outcomes have yielded conflicting results. Objective : This study sought to clarify the prognostic impact of CD34 cell dose by examining a large, contemporary cohort of patients undergoing alloHCT with matched sibling peripheral blood stem cell (PBSC) donors. Study Design : We retrospectively examined the impact of CD34 cell dose on overall survival (OS), neutrophil engraftment, platelet engraftment, treatment-related mortality (TRM), relapse, acute GVHD grades II-IV and III-IV, and chronic GVHD in 377 consecutive patients undergoing alloHCT with PBSC graft source from matched sibling donors at the University of Minnesota from 2002-2015. Patients were classified into three groups based on tertile (T) of CD34 cell dose received (T1, Results : Multivariable analysis demonstrated that high CD34 cell dose was associated with superior 5-year OS (hazard ratio [HR] 0.57, P = 0.01) and more rapid platelet engraftment (HR 1.70, P Conclusion : Higher CD34 cell dose (>7.5 × 106/kg) is associated with superior OS at 5 years and improved engraftment but does carry an increased risk of chronic GVHD. These data support a target CD34 cell dose goal of 7.5 × 106/kg for peripheral blood sibling donors.

Key Points Ofatumumab with glucocorticoid therapy for cGVHD resulted in 62.5% ORR at 6 months and 53% FFS at 12 months.Safety was observed with ofatumumab plus glucocorticoid for initial therapy., Visual Abstract, Standard initial therapy of chronic graft vs. host disease (cGVHD) with glucocorticoids results in suboptimal response. Safety and feasibility of therapy with ofatumumab (1000 mg IV on days 0 and 14) and prednisone (1 mg/kg/day) was previously established in our phase I trial (n = 12). We now report the mature results of the phase II expansion of the trial (n = 38). The overall NIH severity of cGVHD was moderate (63%) or severe (37%) with 74% of all patients affected by the overlap subtype of cGVHD and 82% by prior acute cGVHD. The observed 6 month clinician-reported and 2014 NIH-defined overall response rates (ORR = complete + partial response [CR/PR]) of 62.5% (1-sided lower 90% confidence interval=51.5%) were not superior to pre-specified historic benchmark of 60%. Post-hoc comparison of 6 month NIH response suggested benefit compared to more contemporaneous NIH-based benchmark of 48.6% with frontline sirolimus/prednisone (CTN 0801 trial). Baseline cGVHD features (organ involvement, severity, initial immune suppression agents) were not significantly associated with 6-month ORR. The median time to initiation of second-line therapy was 5.4 months (range 0.9-15.1 months). Failure-free survival (FFS) was 64.2% (95% CI 46.5-77.4%) at 6 months and 53.1% (95% CI 35.8-67.7%) at 12 months, whereas FFS with CR/PR at 12 months of 33.5% exceeded a benchmark of 15% in post-hoc analysis, and was associated with greater success in steroid discontinuation by 24 months (odds ratio 8 (95% CI 1.21-52.7). This single-arm phase II trial demonstrated acceptable safety and potential efficacy of the upfront use of ofatumumab in combination with prednisone in cGVHD. This trial was registered at www.clinicaltrials.gov as #NCT01680965.

Purpose:Acute and chronic GVHD remain major causes of transplant-related morbidity and mortality (TRM) after allogeneic hematopoietic cell transplantation (alloHCT). We have shown CD83 chimeric antigen receptor (CAR) T cells prevent GVHD and kill myeloid leukemia cell lines. In this pilot study, we investigate CD83 expression on GVHD effector cells, correlate these discoveries with clinical outcomes, and evaluate critical therapeutic implications for transplant recipients.Experimental Design:CD83 expression was evaluated among circulating CD4+ T cells, B-cell subsets, T follicular helper (Tfh) cells, and monocytes from patients with/without acute or chronic GVHD (n = 48 for each group), respectively. CD83 expression was correlated with survival, TRM, and relapse after alloHCT. Differential effects of GVHD therapies on CD83 expression was determined.Results:CD83 overexpression on CD4+ T cells correlates with reduced survival and increased TRM. Increased CD83+ B cells and Tfh cells, but not monocytes, are associated with poor posttransplant survival. CD83 CAR T eliminate autoreactive CD83+ B cells isolated from patients with chronic GVHD, without B-cell aplasia as observed with CD19 CAR T. We demonstrate robust CD83 antigen density on human acute myeloid leukemia (AML), and confirm potent antileukemic activity of CD83 CAR T in vivo, without observed myeloablation.Conclusions:CD83 is a promising diagnostic marker of GVHD and warrants further investigation as a therapeutic target of both GVHD and AML relapse after alloHCT.

Immunomodulatory properties of histone deacetylase inhibitors represent a reasonable approach for acute graft-versus-host disease (aGVHD) prevention. We report a phase 2 trial evaluating panobinostat (PANO) administered over 26 weeks, starting on day −5 (5 mg orally 3 times a week) with tacrolimus initiated on day −3 plus sirolimus on day −1, with a median patient age of 58 years (range, 19-72 years) (n = 38). Donor source consisted of HLA 8/8–matched donors, related (n = 13) or unrelated (n = 25), using granulocyte colony-stimulating factor–stimulated peripheral blood stem cells. Myeloablative (n = 18) or reduced-intensity (n = 20) conditioning regimens were used for patients with acute myeloid leukemia (n = 17), myelodysplastic syndrome (n = 13), or other malignancies (n = 8). The cumulative incidence of aGVHD II-IV by day 100 was 18.4% (90% confidence interval [CI], 9.4% to 29.9%). Cumulative incidence of chronic GVHD at 1 year was 31.6% (90% CI, 19.5% to 44.3%). Adverse events related to PANO were thrombocytopenia (n = 5), leukopenia (n = 6), gastrointestinal toxicity (n = 3), rash (n = 4), renal failure/peripheral edema (n = 1), and periorbital edema (n = 1). At 1 year, overall survival was 89.5% (90% CI, 81.6% to 98.0%), relapse-free survival was 78.9% (90% CI, 68.8% to 90.6%), nonrelapse mortality was 2.6% (90% CI, 0.3% to 9.9%), and GVHD relapse-free survival was 60.5% (90% CI, 48.8% to 75.1%). PANO hits histone 3 as early as day 15 in CD8, CD4 and T regs. In conclusion, PANO combination met the primary study end point for aGVHD prevention and warrants further testing. This trial was registered at www.clinicaltrials.gov as #NCT02588339.

We compared chronic graft-versus-host disease (cGvHD) following umbilical cord blood (UCBT) and matched sibling donor peripheral blood transplant (MSD). 145 patients (2010–2017) with cGvHD after MSD (n = 104) and UCBT (n = 41) were included. Prior acute GvHD was less frequent in MSD (55% vs. 85%; p = 0.01). Severe cGvHD (32% vs. 15%, p = 0.01) and de-novo onset (45% vs. 15%, p < 0.01) were more frequent following MSD. Liver was more frequently involved in MSD recipients (38% vs. 6%); and GI in UCBT (33% vs. 63%), both p < 0.01. Overall response (CR + PR) was similar between both cohorts. 2-year CR was higher in UCBT (14% vs 33%, p = 0.02). Karnofsky score (KPS) ≥ 90 at cGvHD diagnosis was associated with higher odds of response (95%CI: 1.42–10, p < 0.01). The cumulative incidence of durable discontinuation of immune-suppressive therapy, failure-free survival (FFS) and NRM at 2-years were similar between cohorts. KPS < 90 (95%CI: 3.1–24.9, p < 0.01) and platelets

Graft-versus-host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). For decades, GVHD prophylaxis has included calcineurin inhibitors, despite their incomplete efficacy and impairment of graft-versus-leukemia (GVL). Distinct from pharmacologic immune suppression, we have developed what we believe is a novel, human CD83-targeted chimeric antigen receptor (CAR) T cell for GVHD prevention. CD83 is expressed on allo-activated conventional CD4(+) T cells (Tconvs) and proinflammatory dendritic cells (DCs), which are both implicated in GVHD pathogenesis. Human CD83 CAR T cells eradicate pathogenic CD83(+) target cells, substantially increase the ratio of regulatory T cells (Tregs) to allo-activated Tconvs, and provide durable prevention of xenogeneic GVHD. CD83 CAR T cells are also capable of treating xenogeneic GVHD. We show that human acute myeloid leukemia (AML) expresses CD83 and that myeloid leukemia cell lines are readily killed by CD83 CAR T cells. Human CD83 CAR T cells are a promising cell-based approach to preventing 2 critical complications of allo-HCT — GVHD and relapse. Thus, the use of human CD83 CAR T cells for GVHD prevention and treatment, as well as for targeting CD83(+) AML, warrants clinical investigation.

Background: Patients with life-threatening acute graft-versus-host disease (aGVHD) often have severe symptoms related to organ/tissue damage, elevated blood biomarkers of aGVHD, or both. Monitoring such patients can be challenging due to unpredictable variations in symptoms that may not be related to aGVHD pathogenesis, such as dietary changes, medication side effects, or infections. In our previously published phase 1 study of urinary-derived human chorionic gonadotropin/epidermal growth factor (uhCG/EGF) for the treatment of life-threatening aGVHD (NCT02525029), we recognized amphiregulin (AREG) as a potential GVHD monitoring biomarker that is elevated at the start of treatment and decreases in patients who respond to treatment. Here we show the results of AREG as a monitoring biomarker in our larger uhCG/EGF treated cohort and validate them in an independent cohort of patients with steroid-refractory aGVHD receiving ruxolitinib as second-line therapy in the REACH1 study. Methods: Longitudinal plasma samples were cryopreserved at study baseline and on study Days 7, 14, 28, and 56 from patients enrolled in both NCT02525029 (N=51) and REACH1 (N=60). In samples collected during NCT02525029, AREG was measured using enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), and ST2 and REG3a were measured using a multiplex Luminex-based array (R&D Systems). Samples from REACH1 were analyzed for concentrations of AREG, ST2, and REG3a using the ProteinSimple Ella platform (Bio-Techne, San Jose, CA). Comparisons of biomarker concentrations between response groups were performed using nonparametric one-way analysis of variance (Kruskal-Wallis test). Analyses of change of biomarkers from baseline to subsequent study days were performed using nonparametric matched pairs analysis (Wilcoxon signed rank test). Biomarker cutoffs relevant for overall survival at study baseline were identified using recursive partitioning on the NCT02525029 dataset and tested in the REACH1 dataset. Results: In patients with a complete response (CR) at Day 28 to uhCG/EGF, AREG decreased 3-fold from baseline to Day 56 (mean, 98 vs 32 pg/mL, P=0.006, Figure 1A). AREG did not significantly change over time in patients with a partial response (PR) or no response (NR) to uhCG/EGF. Similarly, in patients with a Day 28 CR to ruxolitinib, AREG decreased 2.8-fold from baseline to Day 56 (mean, 174.7 vs 63.6 pg/mL, P=0.007, Figure 1B). AREG also decreased 2.0-fold over time in patients treated with ruxolitinib who had a PR to treatment (mean baseline 288.2 vs 146.1 pg/mL at Day 56, P=0.017) but no change in those with progressive disease (PD). ST2 declined with CR, but to a lesser extent, over time in patients with a CR in both studies (1.4-fold decrease in NCT02525029, P=0.04 and 2.2-fold decrease in REACH1, P=0.021). REG3a did not significantly change over time in responders in either clinical trial. Biomarker cutoffs associated with a rapidly fatal course were identified in NCT02525029 for AREG (>212 pg/mL associated with a median survival of 62 days, P=0.006, Figure 1C), ST2 (>292 ng/mL associated with a median survival of 239 days, P13.5 ng/mL associated with a median survival of 416 days, P=0.01). These cutoffs were not statistically significant in REACH1, although patients with a baseline AREG >212 pg/mL had a median survival of 165 days vs not reached (P=0.1, Figure 1D) in REACH1. Conclusions: Using samples collected during 2 prospective clinical trials on 2 different measurement platforms, we conclude that AREG is a useful longitudinal monitoring biomarker for patients with life-threatening aGVHD. AREG levels were generally higher in REACH1 than NCT02525029, which could reflect differences in assay, severity of illness, or both. Patients with a high baseline AREG are at high risk of early death and should be monitored on a consistent platform during serial assessments. Figure 1 Figure 1. Disclosures Pratta: Incyte: Current Employment. Betts: Patent Disclosures: Patents & Royalties: B.C.B. holds a patent (WO2015120436A2) related to CD4+ T cell pSTAT3 as a marker and therapeutic target of acute GVHD. B.C.B. additionally holds a provisional patent (WO2017058950A1) related to the use of JAK inhibitors for rejection and GVHD prevention. . Galvin: Incyte: Current Employment. MacMillan: Equilium: Other: DSMB member; Incyte: Consultancy; Jazz Pharmaceuticals: Consultancy. Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding. Holtan: Generon: Consultancy; Incyte: Consultancy, Research Funding. OffLabel Disclosure: uhCG/EGF has orphan drug designation for the treatment of acute GVHD