ALBERT

Description: Key Points NOTCH2 activation confers a marked increase in BCR responsiveness by cGVHD patient B cells that associates with increased BLNK. ATRA increases the IRF4-to-IRF8 ratio and blocks aberrant NOTCH2-BCR activation without affecting cGVHD patient B-cell viability/function.

Description: Key Points A preclinical model system was developed to define the molecular landscape dictating lymphoma resistance to immunotherapy. This system revealed that Gal-1 significantly inhibits CD20 immunotherapy within the lymphoma microenvironment in vivo.

Description: While Notch signaling is being well studied with regard to T cell pathology and graft-versus host disease (GVHD) (Tran IT et al., 2013. J. Clin. Invest.), the role of Notch receptors in the development and activation of B cell subsets in chronic GVHD (cGVHD) genesis remains unknown. We previously identified a subset of Ôpre-germinal centerÕ B cells within the peripheral blood of cGVHD patients that is largely absent in patients without cGVHD. In addition to cell surface characteristics, this extrafollicular B cell subset has potential functional characteristics of marginal zone (MZ)-like B cells, including increased responsiveness to surrogate antigen stimulation. Along with increased proliferative responses to BCR stimulation, B cells from patients with active cGVHD had significantly increased signaling via proximal B cell receptor (BCR) molecules, including Syk and BLNK. In murine models with lymphopenic environments, Notch 2 binds the ligand Delta-like 1 (DLL1/Dll1) and drives maturation of MZ-like B cells. Also, healthy human B cells have increased Notch receptor responsiveness after BCR stimulation. Together previous studies allowed us to hypothesize that a Notch 2 signaling axis underpins B cell hyper-responsiveness in human cGVHD. We found that limiting dose BCR stimulation with surrogate antigen in the presence of Notch ligand over-expressing cells (OP9-DL1) resulted in maintenance of cell surface Notch 2 expression at significantly higher levels on B cells from patients with active cGVHD compared to patients without cGVHD, as assessed by flow cytometry analysis (P 〈 0.01). We also found that in the presence of Notch ligand, B cells from patients with active cGVHD responded to minimal BCR stimulation with surrogate antigen. Using nearly 100x less surrogate antigen than was required to induce proliferation without Notch ligand, cGVHD B cells proliferated to a significantly greater degree than B cells from patients with no cGVHD, as evaluated by Ki-67 staining using flow cytometry (P 〈 0.001 in a two-sided t-test, Figure 1A). Likewise, concomitant BCR- Notch activation of active cGVHD patient B cells was associated with significantly increased B-cell size compared to patients without disease (P 〈 0.01). BLNK expression in active cGVHD B cells was also maintained at higher levels under these conditions, suggesting a mechanistic link between the BCR and Notch pathways in cGVHD. Strikingly, targeting Notch 2 with an antagonistic monoclonal antibody (mAb) (Wu Y et al., 2010. Nature; kindly provided by Genentech, Inc.) completely abrogated the BCR-Notch axis hyper-responsiveness of active cGVHD B cells without affecting B-cell survival (P 〈 0.001, Figure 1B). In this in vitro system, using nanoString Technologies¨ gene profiling, we found that two, well-defined effector genes downstream of Notch signaling were significantly decreased in active cGVHD B cells after exposure to the anti-Notch 2 mAb (P = 0.0006 and P 〈 0.02, respectively, compared to isotype control mAb). Also consistent with a Notch 2-driven activation pathway, the expression of multiple genes involved in homeostasis/cell cycle regulation were altered in active cGVHD B cells exposed to anti-Notch 2 mAb (P 〈 0.01). Finally, ongoing in vivo analyses of the Notch 2 mAb in a pre-clinical mouse model of cGVHD indicates that Notch 2 blockade does not negatively impact early B cell recovery following bone marrow transplantation. These results may reveal that therapeutic targeting of Notch 2 alone would be sufficient to quell B cell hyper-responsiveness in active cGVHD, while preserving protective humoral immunity. In summary, our data suggest a working model in which Notch-mediated aberrant B cell maturation contributes to cGVHD pathophysiology. In this model, Notch 2 stimulation along with a combination of complex B-cell selection and tolerance mechanisms afford production of pathological B cells. Given that Notch 2 is a cell surface receptor expressed by activated B cell subsets of pathological relevance, and Notch 2 blockade has been shown to be well-tolerated in pre-clinical models, our findings support an important clinical opportunity: Targeting Notch 2 on B cells in active cGVHD represents a viable future therapeutic strategy worthy of continued investigation. This work was supported by National Institutes of Health grant 5K08-HL107756, and a Translational Research Program grant from the Leukemia & Lymphoma Society. Figure 1. Figure 1. Disclosures Rizzieri: Teva: Other: ad board, Speakers Bureau; Celgene: Other: ad board, Speakers Bureau.

Description: Despite the demonstrated clinical efficacy of CD20 monoclonal antibody (mAb) for lymphoma therapy, the in vivo mechanisms of tumor depletion remain controversial and variable. To identify the molecular mechanisms responsible for lymphoma killing by CD20 mAb in a homologous system amenable to mechanistic studies and genetic manipulation, a mouse lymphoma model was developed using primary tumor cells from a C57BL/6 Eμ-cMyc transgenic mouse and mouse antimouse CD20 mAbs. CD20 mAb treatment of syngeneic mice with adoptively transferred lymphomas prevented tumor development or significantly prolonged mouse survival depending on tumor volume, mAb dose, and treatment timing. Cooperative FcγRIV, FcγRIII, and FcγRI interactions mediated optimal lymphoma depletion by CD20 mAb in vivo, whereas clodronate-mediated depletion of macrophages eliminated the therapeutic benefit of CD20 mAb. Although CD20 mAbs activated complement in vitro and in vivo, normal and malignant B-cell depletion was induced through C1q- and C3-independent mechanisms. Thus, the ability of CD20 mAbs to deplete malignant B cells in vivo required FcγR-dependent use of the innate mononuclear cell immune system. These findings allow for mechanism-based predictions of the biologic outcome of CD20 mAb therapy and treatment optimization.

Description: Our group previously showed that B cells signal aberrantly through the B cell receptor (BCR) in allogeneic hematopoietic stem cell transplant (HCT) patients with active chronic graft-versus-host disease (cGVHD). Preclinical mouse studies have demonstrated the importance of the proximal BCR molecule, spleen tyrosine kinase (SYK), in cGVHD development. Hypothesizing that the oral small molecule SYK inhibitor, fostamatinib, would safely target aberrant BCR-activated B cells in HCT patients, we are conducting a single-center, investigator-initiated phase 1 trial (NCT02611063). Our primary objective is to evaluate the safety and tolerability of fostamatinib in patients early after HCT and in those with refractory active cGVHD. Secondary objectives include assessment of cGVHD manifestations, B cell activation, and immune recovery. Methods: All patients receive HCT treatment per program standards at Duke University. Prophylaxis (P-cGVHD) subjects enroll 80-150 days after HCT and have no evidence of cGVHD. P-cGVHD subjects receive drug for up to 1 year post-transplant (215-285 fostamatinib days). Steroid-refractory cGVHD (SR-cGVHD) subjects enroll with active cGVHD that persists despite systemic high-dose steroids. SR-cGVHD subjects receive drug for up to 365 days total. For all enrollees, modified continual reassessment criteria are used to determine starting dose (100mg daily, 150mg daily, or 100mg twice BID) and any needed dose modifications. We monitor for drug-limiting toxicities (DLTs), adverse events (AEs), and cGVHD manifestations using NIH cGVHD consensus criteria at up to 12 follow-up visits. Results: 15 of a planned 18 total patients have enrolled. In the P-cGVHD group (n=5), of the 4 patients who completed treatment (mean 239 fostamatinib days), 1 patient developed cGVHD while enrolled and 2 patients subsequently developed cGVHD, 4 and 6 weeks after study completion. The fifth P-cGVHD subject discontinued therapy on study day 155 (provider decision to initiate donor lymphocyte infusion for low CD3+ chimerism). In the SR-cGVHD group (n=10), 2 patients completed treatment (mean 365 fostamatinib days); 3 patients withdrew (mean 132 fostamatinib days), for non-cardiac chest pain, progression of cGVHD, and moved away; and 5 patients are actively enrolled (mean 207 fostamatinib days). Both SR-cGVHD patients who completed the study clinically improved while on fostamatinib and requested continuation of drug. A total of 2, 9, and 4 patients have been initiated on 100mg daily, 150mg daily, and 100mg BID, respectively. At the 100mg daily dose, no DLTs were noted. At the 150mg daily dose, 1 patient developed liver function test (LFT) elevation. At the 100mg BID dose, 2 patients developed LFT elevation and 1 patient developed non-cardiac chest pain. One patient required dose adjustment: 100mg BID to 150mg daily, for LFT elevation. Two serious AEs possibly related to fostamatinib occurred: 1 patient developed non-cardiac chest pain and 1 patient developed a deep venous thrombosis. No probably- or definitely-related serious AEs occurred. To assess whether fostamatinib effectively targets aberrantly activated B cells, we examined subjects' whole blood using flow cytometry. When comparing CD19+ B cells on study day 1 versus study day 60 in the SR-cGVHD group (n=7), we found the relative proportion of CD19+CD38+IgDlow plasmablast-like cells was decreased (p=0.03, Fig 1A-B), suggesting fostamatinib 'hit target.' Importantly, in the P-cGVHD group, total lymphocyte and B cell counts did not decrease during day 1 to day 225 (Fig 1C-D), suggesting fostamatinib did not affect immune recovery when given early after HCT. Further investigations with functional assays are underway. Conclusions: This study demonstrates for the first time that fostamatinib is safe and tolerated in HCT recipients both early after transplant and in those with active cGVHD. Importantly, fostamatinib does not appear to hinder lymphocyte or B cell recovery when initiated between days 80-150 after HCT. Additionally, fostamatinib may effectively target aberrantly activated B cells in patients with active SR-cGVHD. Fostamatinib, now FDA-approved for treatment of immune thrombocytopenia, merits a phase 2, randomized controlled trial to assess efficacy as a prophylactic agent against cGVHD. This work was supported by a National Institutes of Health grant, NIH (NHLBI) R01 HL 129061. Fostamatinib was supplied by Rigel. Disclosures Horwitz: Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Gasparetto:BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Sung:Novartis: Research Funding; Merck: Research Funding; Seres: Research Funding. Rizzieri:Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Stemline: Research Funding.

Description: Allogeneic hematopoietic stem cell transplantation (HCT)-related immune pathology severely limits patient (Pt) survival, largely due to infections. Chronic graft versus host disease (cGVHD) Pts are particularly immune incompetent with impaired memory B cell responses. Paradoxically, cGVHD Pts are hypogammaglobulinemic and yet are variably immune tolerant with potentially pathogenic alloantibodies frequently produced. B cells with marginal zone (MZ)-like properties are expanded in cGVHD Pts with high BAFF/B-cell ratios (rev. Sarantopoulos, Blood 2015), similar to findings in BAFF-overexpressing mice. We previously found that the increased BCR responsiveness of cGVHD B cells depended on NOTCH2 signaling, since a specific monoclonal antibody (Genentech) blocked hyperactivation (ASH 2015 oral abstract: Blood. 2015. 126:145). Our current objective is to define the molecular defects underlying this aberrant NOTCH2-BCR axis. Increased NOTCH2 expression/activity is found in mice genetically deficient in the transcription factor IRF4 (J. Exp. Med. 2013. 210:2887-2902). Likewise, IRF4 levels are subnormal in unstimulated B cells from active cGVHD Pts relative to healthy donors (Blood. 2014. 123: 2108-15). Thus, we hypothesized that a B cell-intrinsic IRF4 deficiency contributes to the NOTCH2-BCR axis in cGVHD. We studied multi-center (Duke Univ., NCI-NIH, Dana Farber Cancer Inst.) de-identified clinical samples from 38 Pts with or without NIH diagnostic criteria of cGVHD who were 〉12 months post-HCT and not receiving high dose steroids. We employed our in vitro culture system utilizing NOTCH ligand-expressing feeder cells and measured proliferative responses (Ki-67% by flow cytometry) to surrogate Ag. First, adding to our previous findings, B cells from active cGVHD Pts (n=8) proliferated robustly to minimal BCR ligation when NOTCH was activated compared to B cells from Pts without cGVHD (n=7) (38.1% vs 15.3%, P =0.006). To test our new hypothesis, IRF4 levels were determined by quantitative PCR analysis in B cells isolated from HCT Pts with active cGVHD and with no cGVHD stimulated ex vivo for 24 hours (h) with a limiting amount of surrogate Ag (anti-IgM). BCR-engaged B cells from active cGVHD Pts (Figure 1A, open bars) maintained significantly lower IRF4 levels compared to B cells from Pts with no cGVHD (Figure 1A, filled bars; n=4 in each group, P =0.01 in a two-sided t-test). Importantly, this IRF4 deficiency may be correctable therapeutically in cGVHD Pts, since the ex vivo treatment of human B cells with all-trans retinoic acid (ATRA) increases IRF4 expression (J. Immunol. 2015. 195:2601-11), and ATRA has demonstrated efficacy in a mouse model of cGVHD (Blood. 2012. 119:285-95). When ATRA (100 nM) was added to cultures of surrogate Ag-stimulated B cells (Figure 1A, right), IRF4 levels increased significantly in both active cGVHD B cells (n = 4, P

Description: Increased B cell-activating factor (BAFF) and aberrant B cell survival and activation are associated with chronic graft versus host disease (cGVHD) in patients. Whether excessive BAFF production has a pathologic role in the development of cGVHD after allogeneic hematopoietic stem cell transplantation (allo-HCT) remains unknown. Herein, we address this hypothesis by employing BAFF knockout (KO) and BAFF overexpressing (Tg) donor mice in the major MHC mismatched C57BL/6 (B6)-to-BALB/c cGVHD model (Wu et al J Immunol 2013;191:488), except we increased the T cell depleted bone marrow (TCDBM) dose to 1x107 per recipient to improve engraftment. BALB/c recipients receiving TCDBM plus 1x106 splenocytes (Sp) are the disease group, and those receiving TCDBM only are controls. After verifying cGVHD phenotypic manifestations (including longevity, weight loss, eye and lung findings), we examined whether cGVHD mice had the increased BAFF and B cell findings as occur in human cGVHD (Sarantopoulos et al, Blood 2009; 113: 3865). We observed that cGVHD mice had higher BAFF/B cell ratios compared to controls (Fig. 1A, n=10 each). We fully characterized the peripheral B cell compartment in these mice, and found that the relative composition of B cell subsets was significantly altered in diseased mice. CD93 cell surface expression has been used in mice to define B cells that emigrate from BM and circulate through secondary lymphoid organs. In cGVHD mice, we found a significant increase in the relative proportion of CD93+ B cells and a significant decrease in the proportion of CD93- B cells (Fig1B). Also, a significant increase in the frequency of cells positive for the germinal center and activation marker GL7 was found only within CD93- B cell subset of cGVHD mice (Fig 1C). When stimulated via BCR ex vivo, this GL7+ mature B cell subset had significantly increased Syk and BLNK activation, measured by phosphoflow (n=5, p=0.008). We next aimed to determine whether high BAFF alone or high BAFF and alloantigen together lead to altered B cell homeostasis and to the promotion of GL7+ BCR-activated B cells. C57BL/6 (B6) BAFF Tg TCDBM cells were used as donor to afford excessive and persistent BAFF levels after HCT in either syngeneic or BALB/c recipients. After B6 BAFF Tg TCDBM to BALB/c HCT, body weight decreased significantly. By contrast, B6 BAFF Tg TCDBM to B6 syngeneic HCT recipients remained healthy (Fig.1D, left panel,*p=0.004). Notably, the frequency of the CD93-GL7+ B cell subset was increased in BALB/c mice that received B6 BAFF Tg TCDBM only, compared to recipients of syngeneic BAFF Tg or WT TCDBM only (Fig.1D, right panel, p=0.008). Thus, both BAFF and alloantigen are required for B cell activation after HCT. Together, our data also suggest that GL7+ B cells identify aberrantly BCR-activated B cells in murine cGVHD, arising in the setting of BAFF-driven altered B cell homeostasis. While polymorphisms in either donor or recipient BAFF genes are associated with human GVHD (Clark et al Blood 2011; 118: 1140), the source of excess BAFF after allo-HCT remains unknown. We addressed whether pathologic BAFF was derived from donor or recipient hematopoietic cells using TCDBM from B6 BAFF KO mice vs WT as donor cells for HCT to BALB/c recipients. Consistent with the known requirement of recipient BAFF for recovery of the B cell compartment after syngeneic transplant (Gorelik et al JEM 2003; 198:937), recovery of a donor peripheral B cell pool was not significantly different between groups. Remarkably, plasma BAFF levels were also not different between BALB/c recipients of B6 BAFF KO TCDBM +Sp vs recipients of WT TCDBM +Sp, suggesting that radioresistant recipient cells produced BAFF in cGVHD. Chronic GVHD-associated weight loss was also not different between groups (Fig.1E, representative of 3 separate experiments). We found that BAFF production in the engrafted KO recipient BM was uniformly low. Thus, we have now determined that non-BM, radioresistant recipient cells are the principal source of soluble pathologic BAFF in murine cGVHD. Further characterization of BAFF-producing cells is underway in order to identify therapeutic targets. In summary, we have now demonstrated that recipient-derived BAFF has a mechanistic role in aberrant activation of B cells in cGVHD. Our findings will lead to the development of anti-BAFF and BCR targeting agents for patients. This work was supported the National Institutes of Health (NHLBI R01 HL 129061-01). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: While B cells are known to contribute to the pathogenesis of chronic graft-versus-host disease (cGVHD) in mice, it has been challenging to elucidate intrinsic mechanisms of tolerance loss in patients. To identify distinct and potentially targetable B-cell subsets in cGVHD, we employed single-cell RNA-Seq along with an unsupervised hierarchical clustering analysis, targeting 10,000 single B cells from each of eight patients who were 〉12 months post-allogeneic hematopoietic stem cell transplantation (HCT) and either had active cGVHD manifestations (n=4) or never developed cGVHD (n=4). Bioinformatics analysis of pooled cell data (using R with Seurat extension package) identified 6 major B cell clusters common to all patients (Figure 1A). "Intra-cluster" gene comparison (using R package DESeq2, false-discovery rate 0.05) revealed numerous differentially expressed genes between patient groups. The greatest number of differentially-expressed genes occurred in a cluster referred to herein as 'Cluster 6' (Figure 1A, in yellow with asterisk). Within Cluster 6, B cells from active cGVHD patients expressed significantly increased ITGAX (CD11c, Padj =0.007), TNFRSF13B (TACI, a receptor for BAFF, Padj =0.003), IGHG1 (IgG1, Padj =9.3e-06) and IGHG3 (IgG3, Padj =1.7e-12), along with 44 additional genes (to be discussed). Thus, Cluster 6 in cGVHD patients may represent a CD11cpos, BAFF-responsive B cell subset primed to undergo isotype switching in response to alloantigen. Flow cytometry analysis on PBMCs from an independent HCT patient cohort (n=10) confirmed that CD11cpos B cells were indeed significantly expanded in cGVHD (P 〈 0.01, Figure 1B), and revealed these B cells were also TACIpos, CD19high, forward scatter high (FSChigh) blast-like cells (Figure 1C). We found that these CD11cpos B cells had mixed expression of CD21, CD27, IgD and CD24 (Figure 1C). Remarkably, other recent studies on bulk patient B cells have suggested that similar CD11cposCD21negCD19highT-BETpos cells are critical drivers of humoral autoimmunity in diseases including systemic lupus erythematosus (SLE; Scharer et al. 2019; Rubtsova et al. 2017; Rubtsov et al. 2011). This subset now identified by single-cell RNA-Seq is consistent with a population of TACIhigh B cells that produced IgG in response to BAFF treatment ex vivo (Sarantopoulos 2009). Data suggest we have identified functionally distinct and potentially targetable B cell subpopulations. We are employing functional assays to determine whether the additional molecular pathways now elucidated account for our previous work showing greater ex vivo B cell survival rates and hyper-responsiveness to surrogate antigen (Allen et al. 2012, 2014), certain TLR agonists (Suthers et al. 2017), and NOTCH ligand (Poe et al. 2017). In addition to more deeply characterizing B-cell subsets in cGVHD, our single-cell RNA-Seq analyses identified several genes significantly altered across multiple B cell clusters in the cGVHD group, implicating more broad alterations of some genes in this disease. Among these is CKS2, a critical cell cycle regulator, which was significantly increased in cGVHD B cells (Padj 1.0e-10 to 0.018, depending on the cluster evaluated). Increased CKS2 expression was validated by qPCR analysis on B cells from a separate HCT patient cohort with or without cGVHD (P 〈 0.001, Figure 1D), suggesting that the majority of cGVHD B cells are primed to enter the cell cycle at multiple stages of differentiation when exposed to the proper stimuli. In summary, we used an unbiased approach to identify and further characterize an extrafollicular CD11cposTACIposCD19high B cell population in cGVHD patients that appears to be activated and undergoing active IgG isotype switching. This plasmablast-like B cell population is potentially amenable to therapeutic intervention to prevent pathogenic antibody production. Importantly, we also identify gene alterations across the cGVHD peripheral B cell compartment that potentially underpin promotion of hyperactivated B cells in this disease. Therapeutic strategies to target these pathways will also be discussed. This work was supported by a National Institutes of Health grant, R01HL129061. Disclosures Ho: Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Horwitz:Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Rizzieri:Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Stemline: Research Funding.