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Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here, we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIC long isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform–specific inactivation of Cic selectively increased CD4+CD25+Foxp3+ T cells in vivo. We also found recurrent (13%) 3′-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, REL truncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell–like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors, suggesting their activities in ATL. By combining the analyses for coding and noncoding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into 2 molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery.

Increased macrophage phagocytosis of antibody-coated platelets, as well as decreased numbers and/or impaired function of CD4+CD25+Foxp3+ regulatory T (Treg) cells, has been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Low-dose histone deacetylase inhibitors (HDACi’s) are anti-inflammatory and immunomodulatory agents that can enhance immunosuppression in graft-versus-host disease by increasing the number and function of Foxp3+ Treg cells, but it is unclear whether they have the potential to promote immune tolerance and platelet release in ITP. In this study, we performed in vitro and in vivo experiments and found that a low-dose HDACi (chidamide) alleviated thrombocytopenia in passive and active murine models of ITP. Further, low-dose HDACi’s attenuated macrophage phagocytosis of antibody-coated platelets, stimulated the production of natural Foxp3+ Treg cells, promoted the peripheral conversion of T cells into Treg cells, and restored Treg cell suppression in vivo and in vitro. Finally, we confirmed that low-dose HDACi’s could regulate CTLA4 expression in peripheral blood mononuclear cells through modulation of histone H3K27 acetylation. Low-dose HDACi treatment in ITP could be offset by blocking the effect of CTLA4. Therefore, we propose that low-dose chidamide administration has potential as a novel treatment for ITP in the clinic.

Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Leukemia relapse remains the major cause of allogeneic hematopoietic stem cell transplantation (HCT) failure, and the prognosis for patients with post-HCT relapse is poor. There is compelling evidence that potent selective antileukemic effects can be delivered by donor T cells specific for particular minor histocompatibility (H) antigens. Thus, T-cell receptors (TCRs) isolated from minor H antigen–specific T cells represent an untapped resource for developing targeted T-cell immunotherapy to manage post-HCT leukemic relapse. Recognizing that several elements may be crucial to the efficacy and safety of engineered T-cell immunotherapy, we developed a therapeutic transgene with 4 components: (1) a TCR specific for the hematopoietic-restricted, leukemia-associated minor H antigen, HA-1; (2) a CD8 coreceptor to promote function of the class I–restricted TCR in CD4+ T cells; (3) an inducible caspase 9 safety switch to enable elimination of the HA-1 TCR T cells in case of toxicity; and (4) a CD34-CD20 epitope to facilitate selection of the engineered cell product and tracking of transferred HA-1 TCR T cells. The T-cell product includes HA-1 TCR CD4+ T cells to augment the persistence and function of the HA-1 TCR CD8+ T cells and includes only memory T cells; naive T cells are excluded to limit the potential for alloreactivity mediated by native TCR coexpressed by HA-1 TCR T cells. We describe the development of this unique immunotherapy and demonstrate functional responses to primary leukemia by CD4+ and CD8+ T cells transduced with a lentiviral vector incorporating the HA-1 TCR transgene construct.

Adoptively transferred T-cell receptor (TCR)-engineered T cells depend on host-derived costimulation and cytokine signals for their full and sustained activation. However, in patients with cancer, both signals are frequently impaired. Hence, we developed a novel strategy that combines both essential signals in 1 transgene by expressing the nonlymphoid hematopoietic growth factor receptor c-MPL (myeloproliferative leukemia), the receptor for thrombopoietin (TPO), in T cells. c-MPL signaling activates pathways shared with conventional costimulatory and cytokine receptor signaling. Thus, we hypothesized that host-derived TPO, present in the tumor microenvironment, or pharmacological c-MPL agonists approved by the US Food and Drug Administration could deliver both signals to c-MPL-engineered TCR-transgenic T cells. We found that c-MPL+ polyclonal T cells expand and proliferate in response to TPO, and persist longer after adoptive transfer in immunodeficient human TPO-transgenic mice. In TCR-transgenic T cells, c-MPL activation enhances antitumor function, T-cell expansion, and cytokine production and preserves a central memory phenotype. c-MPL signaling also enables sequential tumor cell killing, enhances the formation of effective immune synapses, and improves antileukemic activity in vivo in a leukemia xenograft model. We identify the type 1 interferon pathway as a molecular mechanism by which c-MPL mediates immune stimulation in T cells. In conclusion, we present a novel immunotherapeutic strategy using c-MPL-enhanced transgenic T cells responding to either endogenously produced TPO (a microenvironment factor in hematologic malignancies) or c-MPL-targeted pharmacological agents.

INTRODUCTION Steroid-resistant (SR) acute graft-versus-host disease (aGVHD) lacks standard second-line treatment. Mesenchymal stromal cells (MSCs) have potential efficacy in SR aGVHD. To assess efficacy and safety of MSCs combined with anti-CD25 antibody and calcineurin inhibitors as second-line therapy for SR aGVHD. METHORDS A randomized phase 3 trial involved 203 SR aGVHD patients at 10 centers in China (September 2014-March 2019). Final follow-up was on June 30, 2020. Participants were randomized in a 1:1 ratio to receive second-line therapy (anti-CD25 antibody combined with calcineurin inhibitors) with MSCs (n=101) or without MSCs (n=102). The primary endpoint was overall response (OR) at day 28 with a predefined threshold of -20%. Secondary and safety endpoints included durable OR at day 56, failure-free survival, overall survival (OS), chronic GVHD (cGVHD), infection, haematological toxicity and relapse. RESULTS Of 203 patients, 198 (97.5%; mean age, 30.1years; 40.4% women) completed the study. OR at day 28 was higher in the MSCs group than in the control group (82.8% [82 patients] vs 70.7% [70]; odds ratio, 2.00; 95% confidence interval [CI], 1.01-3.94; P=.043). Durable OR at day 56 was also higher in the MSCs group (78.8% [78 patients] vs. 64.6% [64]; odds ratio, 2.02; 95% CI, 1.08-3.83; P=.027). The median failure-free survival was longer in the MSCs group compared with control group (11.3 months vs. 6.0 month; hazard ratio (HR) 0.68; 95% CI, 0.48-0.95, P=.024). The 2-year cumulative incidence of cGVHD was 39.5% (95% CI, 29.3%-49.4%) and 62.7% (51.4%-72.1%) in the MSCs and control groups (HR 0·55, 95% CI, 0·36-0·84; P=0·005). Within 180 days after study treatments, the most common grade 3 and 4 adverse events were infections (65 [65.7%] in the MSCs group vs 78 [78.8%] in the control group), haematological toxicity (37 [37.4%] vs 53 [53.5%]). CONCLLUSIONS MSCs plus second-line treatments may increase the efficacy of SR aGVHD, decrease drug toxicity of second-line therapy and cGVHD development, and are well tolerated. Disclosures No relevant conflicts of interest to declare.

A number of studies have recently demonstrated that super-enhancers, which are large cluster of enhancers typically marked by a high level of acetylation of histone H3 lysine 27 and mediator bindings, are frequently associated with genes that control and define cell identity during normal development. Super-enhancers are also often enriched at cancer genes in various malignancies. The identification of such enhancers would pinpoint critical factors that directly contribute to pathogenesis. In this study, we performed enhancer profiling using primary leukemia samples from adult T-cell leukemia/lymphoma (ATL), which is a genetically heterogeneous intractable cancer. Super-enhancers were enriched at genes involved in the T-cell activation pathway, including IL2RA/CD25, CD30, and FYN, in both ATL and normal mature T cells, which reflected the origin of the leukemic cells. Super-enhancers were found at several known cancer gene loci, including CCR4, PIK3R1, and TP73, in multiple ATL samples, but not in normal mature T cells, which implicated those genes in ATL pathogenesis. A small-molecule CDK7 inhibitor, THZ1, efficiently inhibited cell growth, induced apoptosis, and downregulated the expression of super-enhancer–associated genes in ATL cells. Furthermore, enhancer profiling combined with gene expression analysis identified a previously uncharacterized gene, TIAM2, that was associated with super-enhancers in all ATL samples, but not in normal T cells. Knockdown of TIAM2 induced apoptosis in ATL cell lines, whereas overexpression of this gene promoted cell growth. Our study provides a novel strategy for identifying critical cancer genes.

We have recently described a low-affinity second-generation anti-CD19 Chimeric Antigen Receptor (CAR) (CAT), characterized by faster antigen dissociation rate which showed enhanced expansion, cytotoxicity and anti-tumour efficacy compared with the high affinity (FMC63 based) CAR used in Tisagenlecleucel in pre-clinical models. Furthermore, CAT CAR T cells showed an excellent toxicity profile, enhanced in vivo expansion and long-term persistence in a Phase I clinical study (Ghorashian et al Nature Med 2019). However the molecular mechanisms behind the improved properties of CAT CAR T cells remain unknown. Herein, we performed a systematic in vitro characterization of the transcriptomic (bulk RNA-seq) and protein (CyTOF) changes occurring in CAR T cells expressing a low-affinity (CAT) vs high affinity (FMC63) anti-CD19 CARs following stimulation with CD19 expressing targets. Untransduced (UT) controls and T cells lentivirally transduced to express CAT or FMC63 CD19 CARs were compared both at baseline and following stimulation with CD19+ Acute Lymphoblastic Leukaemia cell line NALM6. In Principal Component Analysis for both RNA-seq and protein results, we found that the major variance across conditions was explained by CD19-mediated CAR T activation. Strikingly, unstimulated CAT CAR T cells showed an intermediate degree of activation between UT T cells and antigen stimulated CAR T cells. Indeed, when comparing RNA-seq results of unstimulated CAT vs FMC63, we found enhanced expression (FDR To identify the mechanisms underlying the stronger basal activation of CAT CAR T cells, we analysed cytokine expression at the single cell level by mass cytometry. Interestingly, rather than an increment in the expression of individual cytokines, we found that the distinctive feature of CAT CAR T cells was a shift toward a cytokine polyfunctional phenotype, with a marked increase in the proportion of cells co-expressing 3 or more cytokines (17.50% CAT vs 7.33% FMC63) (Figure 1b). Of note, cytokine polyfunctionality (expression of more than 1 cytokine/cell) in pre-infusion CAR T cell products has been associated to improved clinical efficacy. The functional phenotype observed in CAT CAR T cells was linked to the preferential activation of the p38 MAPK phospo-signalling, which is activated downstream of TCR CD3ζ chain (present in the CARs) but is also central to cytokine-dependent T cell activation in memory T cells. Interestingly, cytokine polyfunctional CAT CAR T cells were enriched in the CD3+CD19+ trogocytic (trog+) population, found at higher proportion in CAT vs FMC63 at 24h post antigen stimulation. Although trogocytosis has been associated to CAR T cell fratricide killing, trog+ CAT CAR T cells displayed higher levels of proliferation (pRB), activation (CD25, NFAT1) and cytotoxic (Granzyme B, Perforin B) markers, pointing at a stimulatory role of trogocytosis over fratricide killing, potentially due to the low-affinity CAR T cells distinctive property of better discriminating between low (trog+ CAR T cells) and high (tumour cells) target expression levels. In conclusion, we described the molecular mechanisms underlying the low affinity CAT CAR T cells functional phenotype. Our results show that the potent and long-term anti-tumour responses observed with CAT may be sustained by the establishment of CAR T cells self-reinforcing circuits activated through polyfunctional cytokine crosstalk. This work may inform the future design of versatile CAR T cells, capable of balancing safety, efficacy and long-term persistence. Disclosures Ghorashian: Amgen: Honoraria; UCLB: Patents & Royalties; Novartis: Honoraria. Pule:Autolus: Current Employment, Other: owns stock in and receives royalties, Patents & Royalties; UCLB: Patents & Royalties; Mana Therapeutics: Other: entitled to share of revenue from patents filed by UCL.

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding:Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

Chimeric antigen receptor (CAR) T-cells targeting CD19 has become a promising treatment option for relapsed/refractory B cell acute lymphoblastic leukemia and diffuse large B Cell lymphoma. For acute myeloid leukemia (AML), CAR T-cells targeting molecules such as CD33 and CD123 are under clinical evaluation. Regardless of target diseases or antigens, it is essential to understand mechanisms underlying both on- and off-target effects of CAR T-cells such as cytokine release syndrome. In the study, we aimed to develop a CAR T-cell treatment for poor prognosis AML. To this end, we analyzed gene expression of patient-derived AML-initiating cells with demonstrated capacity for in vivo AML development in a NOD/SCID/Il2rgKO (NSG) xenogeneic transplantation assay. CD25 (IL-2 receptor alpha chain), previously reported as a marker for poor prognosis in AML, was over-represented in AML-initiating cells as compared with normal CD34+CD38- hematopoietic stem/progenitor cells (HSPCs) (Saito et al., Science Translational Medicine 2010). In addition, the antigen is expressed in other hematologic malignancies such as CML, adult T cell leukemia/lymphoma, and Hodgkin's lymphoma. We therefore engineered lentiviral vector containing TCR sequence and Fab antigen recognition sites for human CD25 antigen. Following transduction of CD25-CAR lentiviral particles into cord blood-derived human T cells, we achieved in vitro CD25-CAR T-cell expansion to more than 2x107 cells. In vivo treatment of human AML patient-derived xenotransplantation (PDX) mice with 5x106 CD25-CAR (25CAR) T-cells (patient n=3, PDX n=3 for each patient) resulted in reduction of patient-derived leukemic cells in the peripheral blood (PB) of PDX mice, but abundant leukemic cells remained in the bone marrow (BM). To improve homing and targeting of AML cells in the BM, we engineered CAR construct with mouse CXCR4 expression (CXCR4-25CAR). Injection of 5x106 CXCR4-25CAR T-cells resulted in complete elimination of human AML cells in PB of PDX mice (0.0+/-0.0 hCD33+ cells/ml, n=3), while patient-derived AML cells remained in peripheral blood of PDX treated with 5x106 non-CXCR4-expressing 25CAR T-cells (1416.2+/-661.0 hCD33+ cells/ml PB, n=3) (Untreated PDX: 15677 hCD33+ cells/ml PB, n=1). At 4 weeks post-CAR T-cell injection, we found complete eradication of hCD33+ AML cells only in mice treated with CXCR4-25CAR T (CXCR4-25CAR T-cell treated: BM 0.0+/-0.0 cells, spleen 0.0+/-0.0 cells spleen, n=3; 25CAR T-cell treated: BM 1.9+/-0.4x107 cells, spleen 6.2+/-2.8x107 cells, n=3). In addition, histopathological examination demonstrated no xenogeneic GVHD in liver, lung, and intestine of the CXCR4 25 CAR-treated mice. In one CXCR4-25CAR T-cell-treated PDX mouse with longer-term observation, the number of CXCR4-25 CAR T-cells decreased and murine CD45+ hematopoietic cells increased in PB without evidence of AML relapse after 3 months (2 weeks post-injection: hCD33+AML 1040.3 +/-159.7 cells/ml, hCD3+T cells 474.7 +/-85.8 cells/ml, mouse CD45+ cells 751 +/-31.0 cells/ml, n=3; 3 months post-injection: hCD33+AML 0.0+/-0.0 cells/ml, hCD3+T cells 136.3 +/-77.6 cells/ml, mCD45+ cells 1864 +/-428 cells/ml, n=3). These findings indicate that elimination of human AML cells is mediated by specific targeting of CD25 by CAR T-cells. Injection of CXCR4-expressing CD25-CAR T-cells in NSG mice engrafted with normal CB CD34+CD38- HPSCs resulted in no change in the percentage of human CD3+Foxp3+ cells among PB CD4+ T cells (Pre-injection 10.5+/-1.4%, n=2; 4 weeks post-injection: 14.8+/-0.9% n=2). Additionally, CXCR4-25CAR did not affect to cell numbers of CD3+CD4+FoxP3+ cells, total CD3+ cells, CD19+ cells, CD33+ cells, and CD56+ cells in PB, BM, and spleen. CXCR4-expressing CD25-CAR T-cells is a promising treatment strategy for poor prognosis AML. Disclosures No relevant conflicts of interest to declare.

The advent of chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL). CD19 directed CAR T cells have demonstrated the ability to induce complete remissions in up to 90% of r/r ALL patients. Despite this remarkable upfront success, relapse after CAR T cell therapy remains a major obstacle to long term remissions. A major mechanism for relapse after CD19-directed CAR T cell therapy is the recurrence of antigen-negative ALL cells. In recent years, CD22 CAR T cell therapy has emerged as an effective salvage therapy for patients with CD19-negative ALL. In a phase I clinical trial, CD22 CAR T cells were able to induce remission in up to 80% of patients with CD19-negative ALL. Patients achieving remission, who did not undergo a consolidative hematopoietic stem cell transplant, were found to be at high risk of relapse due to downregulation of the CD22 antigen below the threshold required for effective CD22 CAR T cell activity. Thus, strategies to increase the antigen-sensitivity of CD22 CAR T cells have the potential to enhance the induction and duration of remission in ALL patients. As the properties of a CAR that influence sensitivity to antigen are not well defined, we began by testing the impact of increasing the affinity of the single-chain fragment variable (scFv) for the CD22 antigen. T cells from healthy donors were activated and transduced with a second-generation, 4-1BB CAR containing either the standard affinity (SA)-m971 scFv used in the prior clinical trial, or a high affinity (HA) scFv generated by affinity maturation of the m971 scFv. SA- and HA-CD22 CAR T cells were evaluated in vitro and in vivo against clones of the pre-B ALL cell line, NALM6, which express CD22 at wild type levels (CD22WT), sub-physiologic levels (CD22Lo), supra-physiologic levels (CD22Hi) or in which CD22 was deleted (CD22Neg). We found that the amount of CD22 expressed on the leukemia cells resulted in dose-dependent expression of activation markers, such as CD69 and CD25 (p Previous clinical experience has demonstrated the importance of using a short linker (consisting of a single G4S sequence) between the heavy and light chains of the m971 scFv, therefore we next evaluated the impact of linker length on the activity of the HA-CD22 CAR. HA-CD22 CARs were generated with either a short- or long-linker (G4S x1 vs G4S x3, respectively) and evaluated in vitro and in vivo. While the short linker improved proliferation in vitro, there was no significant impact of linker length on cytokine production or lysis of CD22Lo NALM6. In a xenograft model, HA-CD22 CAR T cells with the long-linker demonstrated slower progression of CD22Lo leukemia and significantly prolonged survival of NSG mice with CD22WT leukemia relative to HA-CD22 CAR T cells with the short-linker (p Taken together, these studies suggest that increasing the affinity of a scFv is a promising strategy for enhancing CAR sensitivity to low levels of target antigen, with the potential to decrease post-CAR T cell relapses due to antigen downregulation. Disclosures No relevant conflicts of interest to declare.

The LEOPARD trial evaluated lenalidomide and alternate day prednisolone (RAP) as post ASCT maintenance in newly diagnosed transplant eligible MM patients (TE NDMM). 60 patients were recruited. Estimated median potential follow-up was 44 months (IQR 26m - 52m). Median PFS from time of commencing RAP was 38.3m (95% CI, 25.8 to 54.8); median OS was not reached (71.4% of patients were alive at 36 months). Here we present the findings from correlative immune studies of this trial. Aims: To undertake mass cytometry (CyTOF) based immune profiling in patients with TE NDMM treated with RAP maintenance post ASCT. Methods: The LEOPARD trial was a phase II, multi centre, open label, single arm study of RAP maintenance after a single melphalan conditioned (200mg/m2) ASCT as part of up-front therapy. Patients were restaged at D+42 ASCT, and if eligible, were commenced on RAP maintenance (LEN 10mg daily increasing to 15mg daily after 8 weeks and alternate day prednisolone 50mg) within 8 weeks of D+0 of the ASCT. Therapy continued until toxicity/progression. CyTOF was performed in sequential samples in two selected groups of patients: long runners (LR, n=7), defined as those with PFS > 36 months (median) and early relapsers (ER, n=8), defined as those who progressed/died before reaching the lower quartile of PFS. [All patients had peripheral blood collected at baseline (pre-ASCT), 6w post-ASCT and weeks 4, 8, 12, 20, 28 and 40 of RAP]. Cells were barcoded using the Cell-ID 20-Plex Pd barcoding kit (Fluidigm) followed by staining with sub-set/function defining antibodies (targeting myeloid, B, T and NK cells: CD45, CD3, CD19, CD5, CD1c, CD226. CD8, CD11c, CD16, CD127, CD138, CD123, NKG2A, TIGIT, TIM3, CD45RA. CD274, CD27, CD197, CD28, Ki67, CD66b, CD183, KLRG1, CD43, NKG2D, CD38, CD278/ICOS, CD25, HLA-DR, CD4, CD57, GramB, PD-1, CD14, CD56, CD11b, Tbet, CD33). Samples were acquired on the Helios instrument. Supervised analysis was performed to determine differences in canonical immune cell populations. Unsupervised analysis was then performed: data were clustered in the VORTEX package. Significant differences in cluster frequency were assessed by Mann-Whitney test for statistical significance. Cluster phenotypes were determined and validated via multiple visualisation approaches. Results: Median age was 56yrs for LR versus 63yrs for ER. Median PFS for LR was 46.3m (38.4 - 51.5m) versus 10.2 m (2.1 - 21.3m) for ER. Supervised analysis was performed on all samples, dichotomized into baseline and last time point sampled for each patient. At baseline, Ki67+CD8+ T cells, ICOS+CD8+ T cells, HLA-DR+CD4+ T cells and CD11c+ myeloid cells were enriched in LR compared to ER. At the last timepoint sampled, Ki67+CD8+ T cells and ICOS+CD8+ T cells were again enriched in LR compared to ER. Conversely, B-reg-like cells (CD19+CD5+CD43-) were enriched in ER compared to LR at the last timepoint sampled. Unsupervised analysis was performed on all samples (all timepoints were pooled). Five clusters were significantly enriched in LR compared to ER. Four of these clusters represented activated/cytotoxic NK cells: CD56 dim, CD16-, NKG2A(CD159a)+, NKG2D(CD314)+, Granzyme B+ and CD38+, and additional expression of CD57 on one cluster; one cluster represented a mature myeloid population, with high expression of HLA-DR, CD11b and CD11c and low expression of CD33. One cluster was significantly enriched in ER compared to LR, representing activated neutrophils, with high expression of CD66b, CD11b and CD16. The clusters that were enriched were then assessed longitudinally over all time points. There was no difference in the kinetics of these populations between groups. Conclusions Significant differences in both T-cell and NK cell populations were demonstrable at baseline in LR versus ER patients. Subsequently, durable responses to post-ASCT lenalidomide maintenance were associated with a cytotoxic, controlled immune response whereas early relapse was characterised by a more uncontrolled inflammatory response and the emergence of B-reg-like cells prior to relapse. We conclude that immune profiling at baseline and after initiation of therapy may help to predict a more sustained response to lenalidomide maintenance enabling pre-emptive tailored treatment decisions. Disclosures Kalff: Roche: Honoraria; Janssen: Honoraria; Amgen: Honoraria; CSL: Honoraria; Celgene: Honoraria. Young:Bristol Meyers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierceall:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company; Oxford University: Other: visiting professor. Oppermann:Bristol Meyers Squibb: Research Funding. Guo:Bristol Meyers Squibb: Research Funding. Reynolds:Novartis AG: Current equity holder in publicly-traded company. Spencer:AbbVie, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Consultancy; AbbVie, Amgen, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Honoraria; Amgen, Celgene, Haemalogix, Janssen, Servier and Takeda: Research Funding; Celgene, Janssen and Takeda: Speakers Bureau.

Background Immune checkpoint (IC) inhibitors (ICI) is a promising group of agents with potential effect in myelodysplastic syndrome (MDS). However, there is not enough data on the pattern of IC expression in MDS bone marrow, though this can guide future therapies with ICI. Methods We prospectively included 57 patients with high risk MDS during 2019-2020 years after signing informed consent for participation in research. We used 7 eight-colored panels in each patient to define IC expression on T- and NK-cells (CD3, CD8, CD4, CD56, CD16), myeloid precursors (CD117, CD34), T-regulators (CD25, CD4), myeloid-derived suppressor cells (CD15, CD11b, CD14, HLA-DR, CD33). In each population we assessed expression of CD279, CD152, CD223, TIM3, CD273, CD274, CD275, CD80. Results In lymphoid populations we observed an extremely high percentage of PD-1 positive lymphocytes (10.5 ± 8.0% of all nucleated cells), including PD-1 positive CD4 lymphocytes (6.3 ± 3.7%) and CD8 lymphocytes (4.7 ± 3.5%). Interestingly, on average 63 ± 34% of all lymphocytes were PD-1 positive (Figure 1A), which indicates an extreme degree of T-cell exhaustion and significant exploitation of the PD-1 system by a malignant tumor. Among the PD-1 ligands PD-1L predominated, however it was present only on a small percentage of myeloid precursors (2.2 ± 0.39% of nucleated cells), but most of the PD-1L ligand expression was observed on granulocytes in the process of their differentiation from myeloid precursors (5.8 ± 4.1% of nucleated cells), and in some patients the proportion of such granulocytes reached more than 20%. Moreover, on the cells of the monocytic series, PD-1L is practically not expressed, but CD80 is expressed, a ligand for CTLA4 (1.6 ± 0.7% of nucleated cells). Interestingly, despite the presence of expression of CD80 on monocytic and dendritic cells, the receptor for this ligand on T-lymphocytes was practically absent. The proportion of such cells was only 0.02 ± 0.01%, i.e. the expression of CD80 facilitates the activation of lymphocytes through interaction with CD28 rather than the suppression of the immune response through CD152. Thus, it can be suggested that CTLA4 does not have a clinically significant role in MDS. At the same time, a certain number of TIM3 positive lymphocytes were observed, the proportion of which was 0.42 ± 0.16%, and the percentage of lymphocytes carrying this receptor was 3.6 ± 1.2% (Figure 1A). However, significantly greater TIM3 expression was detected on NK cells. The proportion of such cells was 1.3 ± 0.6% of all nucleated cells, while 30% of all NK cells carried the TIM3 receptor (Figure 1A). Only 0.1% of cells from early myeloid progenitors that expressed TIM3 receptor were detected during flow cytometry, while the proportion of TIM3 positive myeloid cells increased during maturation just in the same way as the PD-1L. Expression of Gal9 was negligible, as it is a protein secreted into the intercellular space. Only a small percentage is associated with cell membranes. The analysis of the correlation matrix showed synchronous changes in the number of most bone marrow T-cell populations. A negative correlation of the level of myeloid tumor precursors actively expressing IC ligands and activated T cells, expressing CD223, NK cells, NKT was observed. The total number of myeloid tumor precursors positively correlated with the number of PD-1 positive lymphocytes (Figure 1B). Conclusion It was found that IC system plays a role in high-risk MDS. Moreover, the expression of ligands is mainly realized during maturation of blast cells into granulocytes, which are the main population expressing these inhibitory molecules. The leading role in the suppression of the T-cell response in MDS is played by the PD-1-PD-1L system, the CTLA4 system plays a minor role in our study cohort. The second most important system is GAL9-TIM3, effecting predominantly NK cells, while T cell are less affected. Thus, the data obtained justify the trials of dual blockade with anti-PD-1 and anti-TIM3 agents in high-risk MDS. Figure 1 Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy in adults with dismal outcomes. γδ T cells have been reported to exert effective anti-tumor activities on various types of solid tumors and hematologic malignancies, and thus become a potential weapon used in the treatment of cancer. However, compared to the striking results demonstrated in vitro and in mouse models, the clinical efficacy of γδ T cells-based immunotherapy is disappointing. Meanwhile, recent studies identified a pro-tumor role of γδ T cells in some types of solid tumors. Whether γδ T cells could be transformed from warriors to foe in the environment of leukemia is unclear. In the current study, we aim to dissect the abnormal changes and functions of intrinsic γδ T cells in the context of AML. Considering bone marrows (BM) are the primary tumor foci of AML and likely provide more representative immune characteristics during leukemia development, the immunophenotyping analyses of γδ T cells in BM from newly diagnosed AML patients (n = 21) were detected using flow cytometry, compared with healthy individual controls (n = 21). We observed that the proportion of γδ T cells and the composition of Vδ1+ and Vδ2+ subgroups were comparable between AML patients and healthy controls. Interestingly, a dramatically elevated subpopulation in γδ T cells expressing CD25 and dim CD127 was observed in AML patients compared with that in healthy controls (3.2% versus 0.8%, P < 0.001). Since their phenotypes are akin to the conventional CD4+ regulatory T cells (Tregs), these CD25+CD127dim γδ T cells are called γδ Tregs here. Paired comparison was also performed on AML patients at diagnosis (n = 13) and completed remission (CR) after chemotherapy (n = 13). A significant decrease in proportion of γδ Tregs was observed at CR, compared with that at newly diagnoses (3.1% versus 0.4% (P < 0.001). These results suggest that this aberrant γδ T subset, γδ Tregs, is induced in the microenvironment of AML and can be reversed after effective therapy for elimination of leukemia blasts. To further explore the direct effect of AML blasts on γδ T cells, primary malignant cells were sorted from AML patients, and co-cultured with peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. As expected, γδ Tregs was significantly induced and the frequency was continuously elevated from 0.2% to 4.0% following the increased ratios of AML cells (P < 0.001). These results confirmed that AML cells were capable to trigger the transformation of normal γδ T cells into CD25+CD127dim γδ Tregs. To determine the function of this special γδ T subset, CD25+CD127dim γδ Tregs were expanded in vitro and co-cultured with AML cell lines, U937 cells. Unlike pamidronate-expanded cytotoxic Vδ2+ T cells, γδ Tregs lost the ability of killing AML cells. Furthermore, we explored the impact of γδ Tregs on the anti-AML activity of normal αβ T cells. γδ T cell-depleted PBMCs (more than 90% are αβ T cells) of healthy donors were co-cultured with U937 cells, with or without autologous γδ Tregs. Flow cytometry analysis showed that 7-AAD-positive fraction in U937 cells was dramatically increased from 0.3% to 7.6% after co-culture with PBMCs and without γδ Tregs. Whereas the mortality of U937 cells was gradually declined (from 7.6% to 4%) in the presence of γδ Tregs at increasing ratios (P = 0.031). During this process, γδ Tregs remarkably downregulated the expressions of HLA-DR and CD38, and productions of TNF-α and IFN-γ in both CD4+ and CD8+ αβ T cells. These findings indicated that γδ Tregs significantly impaired the cytotoxic ability of effector αβ T cells on AML cells, suggesting γδ Tregs play a suppressive role in the anti-leukemia immunity. In summary, we first reported the significant induction of CD25+CD127dim γδ Tregs in the primary BM of AML patients. These γδ Tregs did not exert anti-leukemia activity and attenuated the cytotoxic effect of normal αβ T cells. Our findings will help advance the current understanding of immune mechanism associated with leukemia cell evasion. It also proposes that boosting the cytotoxic γδ T cells should be coupled with inhibition of the suppressive γδ Tregs, for future improvement of γδ T-cell-based immunotherapy. Disclosures No relevant conflicts of interest to declare.

Introduction We present preclinical evidence confirming the expression of ITK in double negative (DN) T cells of ALPS patients and demonstrate that CPI-818 inhibits the TCR-induced activation of these cells ex vivo. Furthermore, CPI-818 effectively suppressed the accumulation of DN T cells in the MRL/lpr mice, leading to dramatic improvements in lymphadenopathy, splenomegaly and other pathologies associated with lymphocyte accumulation due to Fas-deficiency. ALPS is a disorder of lymphocyte apoptosis resulting in childhood onset lymphadenopathy, multilineage cytopenias, hypersplenism and autoimmune peripheral destruction. Most ALPS cases are associated with a loss of function mutation in the Fas gene resulting in immune dysregulation such as a marked elevation of abnormal TCR+CD4-CD8-double negative (DN) T cells. ALPS patients are currently treated with immunosuppressive and cytotoxic agents, hence targeted, less toxic treatments are imperative. The critical role of Fas protein in maintaining lymphocyte homeostasis and peripheral immune tolerance to prevent autoimmunity was initially discovered with studies in Fas-deficient MRL/LpJ-Tnfrsf6lpr (MRL/lpr) mice. These mice have dramatically elevated levels of DN T cells and develop many clinical manifestations similar to those observed in ALPS patients. Thus, MRL/lpr mice have been a useful model to assess compounds and therapeutic strategies for the treatment of ALPS. Interleukin-2-inducible T cell kinase (ITK) is a tyrosine kinase critical to T cell receptor (TCR) signal transduction, which modulates T cell proliferation and differentiation. We sought to test whether the effect of selective inhibition of TCR signaling through ITK inhibition could be a strategy to suppress the DN T cell expansion and disease manifestation in MRL/lpr mice. CPI-818 is an orally bioavailable, covalent inhibitor of ITK that potently inhibits TCR signal transduction. It is currently being evaluated in a phase 1/1b clinical trial in T-cell lymphoma (NCT03952078) where it has been well tolerated and resulted in anti-tumor activity. Methods PBMC samples were obtained from healthy donors or ALPS patients collected under NIAID IRB-approved protocol 93-I-0063. Cells were cultured with or without anti-CD3/CD28 activation. The impact of CPI-818 on viability, expression of ITK, and expression of the surface markers CD 25 and CD69 was assessed by flow cytometry. For in vivo studies, MRL/lpr mice received a control diet or a CPI-818 formulated diet (300 mg/kg/day). Lymph nodes were calipered weekly over the course of 22 weeks of disease development. Spleens, lungs and kidneys were harvested at 22 weeks to assess the impact of CPI-818 treatment on T cell subsets and histology. Results Ex vivo studies with PBMC preparations from normal donors and ALPS patients confirmed the expression of ITK in CD4+, CD8+, and DN T cells from ALPS patients. When these T cells were stimulated with anti-CD3/CD28, CPI-818 potently inhibited the upregulation of the activation markers CD25 and CD69 in DN T cells assessed by flow cytometry. In companion experiments, CPI-818 was not cytotoxic to DN T cells when activated through the TCR signaling pathway. Treatment of MRL/lpr mice with CPI-818 prevented the onset or, in a therapeutic design, led to the regression of lymphadenopathy and splenomegaly comparable to the treatment effects observed with cyclophosphamide. Unlike the effects seen with the cytotoxic agent cyclophosphamide which suppressed all T cells, as well as B, NK and myeloid cells in the spleen, CPI-818 selectively depleted DN T cells (see Figure). CPI-818 treatment also resulted in decreased levels of DN T cells in the kidneys and lungs of these mice without impacting the level of normal CD4 and CD8 T cells. CPI-818 limited the increased proteinuria measured in the control animals during the 22-week treatment window. Histological assessment of the kidney revealed reduced inflammatory damage in CPI-818 treated animals. Conclusions The selective, covalent ITK inhibitor, CPI-818, significantly reduced DN T cell numbers, lymphadenopathy and other disease end points in the MRL/lpr murine model of ALPS. This suggests that the dysregulated growth of Fas-deficient DN T cells requires functional TCR signaling. Further, our data suggests that blocking TCR signaling by targeting ITK may be an effective strategy for treating adult and pediatric ALPS. Figure Disclosures Ng: Corvus Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Hill:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Miller:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Buggy:Corvus Pharmaceuticals: Consultancy, Current Employment, Current equity holder in publicly-traded company. Janc:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

Introduction: Cami is an antibody (Ab)-drug conjugate composed of the human monoclonal Ab, HuMax®-TAC, directed against human CD25, and conjugated through a cleavable linker to SG3199, a pyrrolobenzodiazepine (PBD) dimer cytotoxin. Cami has shown notable single-agent antitumor activity and manageable toxicity in a phase 1 trial in patients (pts) with relapsed or refractory (R/R) Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) (Collins et al, ICML Jun 18-22, 2019;https://onlinelibrary.wiley.com/doi/10.1002/hon.61_2629; Collins et al, ASH Dec 1-4, 2018;https://doi.org/10.1182/blood-2018-99-115986). Methods: This open-label, single-arm, dose escalation and expansion trial (NCT04052997) enrolled pts aged ≥18 years with histologically confirmed R/R HL or R/R NHL. The primary objectives included characterizing the safety and tolerability of Cami. The secondary objectives addressed the pharmacokinetic (PK) profile of Cami, PBD-conjugated Ab (cAb, drug-to-Ab ratio [DAR] ≥1), total Ab (tAb, DAR≥0) and unconjugated warhead SG3199, and assessed the anti-drug Ab (ADA) response in serum. Also reported are exploratory PK/pharmacodynamic correlates to concentrations of soluble CD25 (sCD25), time course of peripheral blood B cells, NK cells, and T-cell subsets: Treg(CD25+/CD127low/FoxP3+[CD3+/CD4+]), Teff (CD8+), and Teff:Treg ratio, using TruCount flow cytometry (Covance, Indianapolis, IN, USA), circulating cytokines, and immunohistochemistry (IHC) on archival or pre-treatment tumor biopsies for CD25 expression. Results: Data from 133 pts with lymphoma (HL and NHL) were available for PK analysis following Cami doses of 3-300 µg/kg administered every 3 weeks. Mean exposures increased with dose. Apparent clearance for doses of 30-80 µg/kg was relatively constant (for cAb, 1.66 L/day [CV=64%] in the 45 µg/kg dose group during Cycle 1). Good linker stability of the immunoconjugate was demonstrated by close similarity of profiles between cAb and tAb. By Cycle 2, similar respective exposures with lower inter-patient variability were noted as relative to Cycle 1. Apparent half-life of cAb was 2.7 days (CV=40%), with no accumulation observed by the end of the 3-week cycle. Levels of SG3199 were generally too low for characterization. There were no instances of positive pre- or post-dose ADA response. For pts receiving the 30-60 µg/kg doses, preferential decreases over time for IL-6, IL-8, and IL-10 were noted for pts achieving clinical response, with decreases most evident by end of first cycle. For total lymphocytes, CD19+ B cells, CD3+ and CD4+ T cells, CD8+ Teff and CD16+/CD56+ NK cell subsets, similar modulations by time were observed at 30 and 45 µg/kg doses, comprising transient increases following dosing (Figure 1). Small absolute decreases in Treg cell concentrations over time were seen following the 30 and 45 µg/kg doses, but with no distinction by best overall clinical response (complete response [CR] + partial response vs. non-responders). However, most pts had clear increases in Teff:Treg ratios over time, with greater effects in Cycle 2 relative to Cycle 1. Mean sCD25 concentrations displayed apparent decrease from baseline over time following the 45 µg/kg dose. For pts with HL, higher baseline levels of sCD25 were associated with lower Cami exposure in both responders and non-responders. Particularly for pts exhibiting CR, clustering was apparent with sCD25 concentrations Conclusion: While Treg modulation was seen with all populations, these data support further study, particularly of the Cami 45 µg/kg dose for pts with HL. Given that CD25 is the cognate target of Cami, these data suggest pts with HL achieving CR have higher drug exposure, resulting from lower baseline sCD25, and potential association with lower tumor burden. Although exposure at the 45 µg/kg dose showed moderate inter-patient variability, it was associated with noteworthy, cycle-related modulation in circulating Treg and clear increases in Teff:Treg ratios, thought to favor clinical response. Funding: Study funded by ADC Therapeutics SA. Disclosures Boni: ADC Therapeutics America, Inc: Current Employment, Current equity holder in publicly-traded company. Havenith:ADC Therapeutics (UK) Ltd: Current Employment, Current equity holder in publicly-traded company. Hamadani:Janssen R&D; Incyte Corporation; ADC Therapeutics; Celgene Corporation; Pharmacyclics, Omeros, AbGenomics, Verastem, TeneoBio: Consultancy; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Company; Spectrum Pharmaceuticals; Astellas Pharma: Research Funding; Sanofi Genzyme, AstraZeneca: Speakers Bureau. Caimi:Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Kite Pharma: Other: Advisory Board; ADC Therapeutics: Other: Advisory Board, Research Funding; Verastem: Other: Advisory Board; Celgene: Speakers Bureau. Anderson:ADC Therapeutics (UK) Ltd: Current Employment, Current equity holder in publicly-traded company. Kopotsha:ADC Therapeutics (UK) Ltd: Current Employment, Current equity holder in publicly-traded company. Cruz:ADC Therapeutics SA: Current Employment, Current equity holder in publicly-traded company. Wuerthner:ADC Therapeutics SA: Current Employment, Current equity holder in publicly-traded company.

Camidanlumab tesirine (ADCT-301) is an antibody-drug conjugate (ADC) comprised of HuMax®-TAC, a monoclonal antibody directed against human CD25, conjugated to the pyrrolobenzodiazepine dimer payload tesirine[1]. Currently, camidanlumab tesirine is being evaluated in a pivotal Phase 2 clinical trial in patients with relapsed or refractory Hodgkin lymphoma (HL) (NCT04052997) and in a Phase 1b clinical trial in patients with advanced solid tumors (NCT03621982). In pre-clinical studies, camidanlumab tesirine demonstrated strong and durable single agent activity in CD25-expressing lymphoma xenograft models[1] and in vitro it synergised with selected targeted agents[2]. Moreover, CD25-ADC, a mouse CD25 cross-reactive surrogate of camidanlumab tesirine, induced potent anti-tumor immunity against established syngeneic solid tumor models by depleting CD25-positive tumor-infiltrating T regulatory cells (Tregs) and it showed synergistic activity when combined with PD-1 blockade[3]. Here, we investigated the in vitro and in vivo anti-tumor activity of camidanlumab tesirine combined with gemcitabine, a common standard-of-care chemotherapeutic agent used both in a hematological and solid tumor clinical setting. In vitro, the combination of camidanlumab tesirine and gemcitabine was evaluated in three human-derived cancer cell lines (two HL and one anaplastic large cell lymphoma, ALCL) and resulted in synergistic activity as determined by the Chou-Talalay method. In vivo, camidanlumab tesirine was tested either alone (0.05 or 0.1 mg/kg, single dose) or in combination with gemcitabine (80 mg/kg, q3dx4) in the CD25-expressing ALCL Karpas299 xenograft model. At both ADC dose levels, combination with gemcitabine resulted in synergistic anti-tumor activity (coefficient of drug interaction (CDI) 0.51 and 0.17, respectively), better response rates and increased survival compared to monotherapy with camidanlumab tesirine. In order to extend the investigation to solid tumor models, CD25-ADC was tested in the CT26 syngeneic model, a colorectal cancer model with CD25-expressing tumor-infiltrating Tregs. CD25-ADC was administered either alone (0.1, 0.5 or 1 mg/kg, single dose) or in combination with gemcitabine (80 mg/kg, q3dx4). At the 0.1 mg/kg dose of CD25-ADC, combination with gemcitabine resulted in synergistic anti-tumor activity (CDI 0.68). Moreover, at 0.5 and 1 mg/kg, the combination of CD25-ADC and gemcitabine resulted in more durable anti-tumor activity and better response rates compared to both monotherapy treatments. In conclusion, the combination of camidanlumab tesirine and gemcitabine was synergistic both in vitro and in vivo in lymphoma preclinical models. Synergistic anti-tumor activity was also demonstrated in a colorectal cancer model using CD25-ADC, a mouse-cross-reactive version of camidanlumab tesirine, in combination with gemcitabine. Altogether, these novel pre-clinical data warrant translation of the combination between camidanlumab tesirine and gemcitabine into the clinic. 1.Flynn, M.J., et al., ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody-Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies. Mol Cancer Ther, 2016. 15(11): p. 2709-2721. 2.Spriano, F., et al., The anti-CD25 antibody-drug conjugate camidanlumab tesirine (ADCT-301) presents a strong preclinical activity both as single agent and in combination in lymphoma cell lines. Hematological Oncology, 2019. 37(S2): p. 323-324. 3.Zammarchi, F., et al., A CD25-targeted antibody-drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity. Journal for ImmunoTherapy of Cancer, 2020. In press. Disclosures Jabeen: ADC Therapeutics: Current Employment. Hartley:ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding. Van Berkel:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zammarchi:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Introduction: CD6 is a T-cell costimulatory receptor that has been implicated in the pathogenesis of multiple autoimmune and inflammatory (AI) diseases. In GVHD, CD6 is expressed on reconstituting T cells soon after transplant (Rambaldi et al., 2019), including Th1 and Th17 cells which are both implicated in the induction and pathogenesis of acute GVHD (aGVHD). CD6 is highly expressed on these cells and promotes immune synapse formation, T-cell activation, and T-cell migration via interaction with its ligand activated leukocyte cell adhesion molecule (ALCAM). Furthermore, studies have demonstrated that ex-vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998), highlighting the importance of CD6. While the contribution of CD6 to T cell activation has been well described, less is known regarding the expression levels and role of CD6 on effector and memory T cells (Teff) which are prominent in all diseases including aGVHD. Consequently, the aim of this study was to determine the role of CD6 specifically on effector T cells, and further illuminate the mechanism of itolizumab, an anti-CD6 monoclonal antibody. Methods: Naïve T cells were enriched from frozen PBMCs via a naïve T cell magnetic separation kit (Stemcell). Naïve T cells were polarized towards a Th1 phenotype for 6 days with a Th1 differentiation cocktail (Stemcell) and CD3/CD28 T cell activator (Stemcell) and rested overnight prior to entering restimulation conditions. Naïve T cells were polarized towards a Th17 phenotype for 8 days with IL-6, IL-1β, TGF-β, IL-23, anti-IL-4 and IFN-γ, the CD3/CD28 T cell activator was used to activate the T cells. To re-stimulate differentiated T cells, anti-CD3 mAb and ALCAM-Fc or anti-CD3 mAb alone were coated on 48-well plates overnight at 4oC. Th1 or Th17 T cells were labeled with CFSE and seeded with isotype control or itolizumab for 72hrs. Cells were collected for flow cytometry analysis and supernatant collected for relevant cytokine detection. To assess surface levels of CD6, cryopreserved PBMCs were thawed and incubated with itolizumab or isotype at 37oC for specific timepoints. Following incubation, cells were washed and stored at 4oC for subsequent staining. All samples were stained at the same time and surface levels of CD6 was detected using a monoclonal anti-CD6 antibody that does not compete with itolizumab. Results: Blockade of the CD6 pathway, using itolizumab during restimulation of differentiated Teff cells in the presence of ALCAM, inhibited multiple effector functions including proliferation and changes in cell size. An average of a 40% decrease in CFSE proliferation was observed across multiple donors. Furthermore, treatment of Teff cells with itolizumab resulted in a significant decrease in expression level of T cell markers of activation and exhaustion such as CD25, PD-1 and Tim3. This effect was exclusively in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. When levels of CD6 were assessed, CD45RO+ (Teff/mem) cells expressed higher levels than CD45RA+CD45RO- (Tnaive). Itolizumab treatment of in vitro generated CD45RO+ T cells inhibited this stimulation-induced increase in CD6 in a dose-dependent manner, suggesting that the drug may modulate surface CD6 expression as a mechanism separate from physical blockade of CD6. Conclusions: These findings are the first to characterize the CD6-ALCAM pathway as a key regulator of differentiated effector T-cell function. Modulation of activation markers and CD6 itself by itolizumab, suggest modulation of Teff activity by both direct and indirect inhibition of CD6 signaling. These data further support targeting the CD6-ALCAM pathway to inhibit both naïve and effector T cell populations in aGVHD. Disclosures Ampudia: Equillium: Current Employment, Current equity holder in publicly-traded company. Chu:Equillium: Current Employment, Current equity holder in publicly-traded company. Doherty:Equillium Inc.: Research Funding. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company.

Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 T cells, are enriched in B-cell non-Hodgkin lymphoma (NHL) and constitute a barrier to potent antitumor immune responses. Despite extensive studies, the significance of tumor-infiltrating Tregs on disease outcome is unclear and while Tregs may express co-inhibitory and co-stimulatory receptors, the role of intratumoral Tregs in the context of immune checkpoint therapy remains elusive. Emerging evidence suggests heterogeneity among Tregs and their suppressive capacities in cancer, emphasizing the need for additional markers to identify highly suppressive Tregs. Therefore, an in-depth characterization of Treg heterogeneity in NHL could provide important insight into the disease pathogenesis and have implications for rational drug design. Methods: Expression of checkpoint receptors in Tregs was characterized by fluorescence flow cytometry and mass cytometry analysis of single-cell suspensions from diffuse large B-cell lymphoma (DLBCL; n = 16), follicular lymphoma (FL; n = 8), mantle cell lymphoma (MCL; n = 10), marginal zone lymphoma (MZL; n = 2), chronic lymphocytic lymphoma (CLL; n = 7), as well as tonsils (n = 8) and peripheral blood (n = 4) from healthy donors. Functional characterization of intratumoral Tregs was performed by a proliferation assay using FACS-sorted Tregs as suppressor cells and autologous CellTrace Violet-labelled T effector cells as responder cells. Single-cell RNA sequencing (scRNA-seq) was performed on FACS-sorted CD4 T cells from 3 DLBCL, 3 FL and 3 healthy donor tonsils using the 10X Genomics single cell 5' based library construction and VDJ libraries for TCR-sequencing. Additionally, for simultaneous profiling of phenotypic features with the mRNA expression in single cells, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq) was applied. The Treg compartment was characterized by clustering into distinct transcriptional Treg states and differential expression of marker genes. Results: TIGIT and CTLA-4 were identified as common markers of intratumoral Tregs, in addition to FOXP3 and CD25. Unsupervised computational analysis revealed two distinct Treg subsets, based on contrasting expression of PD-1, OX40, CD226 and ICOS (Figure 1A). One subset displayed a checkpoint receptorlow phenotype that corresponded to peripheral blood Tregs. The second subset had a checkpoint receptorhigh phenotype with elevated levels of PD-1, OX40, ICOS, TIGIT, CTLA-4 and increased levels of activation markers CD28, CD69 and CD95/Fas. The frequency of checkpoint receptorhigh Tregs was significantly increased in NHL tumors, compared to PBMCs and tonsils from healthy donors. FL tumors had the highest frequency of Tregs with receptorhigh phenotype among the NHL entities (median frequency of 86%, range 71-92%) and DLBCL had the highest donor-to-donor variation (median frequency of 77%, range 35-98%) (Figure 1B). This phenotypic heterogeneity of the Treg compartment reflected different suppressive capacities of the two subsets. Checkpoint receptorhigh Tregs were more potent mediators of immunosuppression in terms of suppressing the proliferation of autologous effector CD4 and CD8 T cells (Figure 1C). Furthermore, transcriptomic analysis of CD4 T cells by scRNA-seq and CITEseq revealed distinct transcriptomic signatures of the checkpoint receptorhigh and -receptorlow subsets. In addition, a third subset of Tregs, characterized by increased expression of LAG3 and immunosuppression-associated genes (CTLA-4, IL10, CD38, KLRB1) but lack of FOXP3, was identified (Figure 1D-E). Analysis of scTCR-sequences to compare TCR repertoires and to identify developmental trajectories will further add to our knowledge of intratumoral Tregs. Conclusions: These results reveal heterogeneity within the Treg compartment in NHL based on expression of checkpoint receptors, transcriptional profiles and suppressive capacities. As intratumoral Treg phenotypes differ from peripheral blood Tregs, this presents new therapeutic opportunities. Specific targeting of intratumoral Tregs would lead to stronger antitumor effects while limiting immune-related adverse events. A deeper understanding of Treg heterogeneity within the tumor microenvironment could therefore open new paths for rational design of immune checkpoint therapy. Disclosures Kolstad: Merck: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen-presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway modulates T cell activation, proliferation and trafficking playing a central role in inflammation. Early studies by Soiffer and colleagues demonstrated that ex-vivo depletion of CD6+ donor T cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized IgG1 anti-CD6 monoclonal antibody, has been shown to block CD6 signaling, leading to a reduction in T cell activation and proliferation. The aim of this study was to characterize expression of CD6 early after HCT and to examine the effects of Itolizumab on T cell responses in the setting of aGVHD. We analyzed immune reconstitution in 95 adult patients who underwent allogeneic HCT for hematological malignancies. Patients received myeloablative (50.5%) or reduced intensity (49.5%) conditioning. Almost all patients received peripheral blood stem cell grafts from HLA-matched related (MRD; 27.4%), matched unrelated (MUD; 66.3%) or mismatched unrelated donors (MMUD; 6.3%). Tacrolimus and methotrexate were used for GVHD prophylaxis. Forty-two of 95 patients developed grade I-IV aGVHD at a median of 50 days (range 20-294) after HCT and systemic immunosuppression was started in 81% of these cases. aGVHD grade severity was 38.1%, 40.5%, 7.1% and 14.3% of grade I, II, III and IV, respectively. Patient samples were collected at 1, 2, 3 and 6 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of Itolizumab was tested in vitro using peripheral blood mononuclear cells (PBMCs) obtained from HD and patients before (preGVHD) and after aGvHD onset (postGVHD) after 72 hour stimulation with antiCD3/CD2/CD28 coated beads. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation were monitored by expression of CD25 and CD45RO, respectively. To test if Itolizumab activity was dependent on the presence of the CD6 cognate ligand, ALCAM, HD CD3+ T cells were stimulated with anti-CD3 antibody with or without ALCAM-Fc for 96 hours. CD6+ T cells reconstituted early after transplant, accounting for 77.8% (range, 69.9-93.7) of CD4Treg, 98.1% (range, 96.5-99.5) of CD4Tcon, 84% (range, 77.1-92.1) of CD8 T cells at 1 month. This pattern remained unchanged at 2, 3 and 6 months and in HD. CD6 expression (MFI) was the highest in Tcon and lowest in Treg in patients as well as in HD. Compared to HD, CD6 MFI in CD4 Treg was significantly lower in patients at 1,2,3 and 6 months after HCT (p Prior to GVHD onset, Itolizumab inhibited CD4 and CD8 T cell proliferation in a similar fashion to HD control. This effect was less prominent in T cells collected after GVHD onset, when patients were already receiving immunosuppressive medications, especially for the CD8 T cells (Fig 1D). Similar results were observed for CD25 and CD45RO expression (Fig 1D). Addition of ALCAM-Fc to anti-CD3 antibody enhanced T cell proliferation, activation and maturation. Itolizumab efficiently inhibited T cell proliferation, activation and maturation in the presence of ALCAM-Fc and anti-CD3 antibody, while no effect was observed in the presence of anti-CD3 antibody alone (Fig 2A). Finally, Itolizumab alone did not induce significant CDC, ADC or ADCC in vitro (Fig 2B). In conclusion, we demonstrate that CD6 positive T cells reconstitute rapidly after HCT with higher levels of expression in Tcon compared to Treg and CD8 T cells. Percentages of CD6+ T cells do not change during aGVHD, but levels of CD6 expression decrease in Tcon and Treg after aGVHD. Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMCs from patients with aGvHD. Functional inhibition of the CD6-ALCAM pathway without T cell depletion may be a novel therapeutic strategy for treating aGvHD. A phase I/II study using Itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Rambaldi: Equillium: Research Funding. Koreth:EMD Serono: Consultancy; Biolojic Design Inc: Consultancy; Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; BMS: Other: Research Support; Miltenyi: Other: Research Support; Clinigen: Other; Therakos: Membership on an entity's Board of Directors or advisory committees; Equillium: Consultancy; Moderna Therapeutics: Consultancy; Amgen: Consultancy. Cutler:Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Soiffer:Juno: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Cugene: Consultancy; alexion: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Consultancy; Mana Therapeutics: Consultancy; Rheos Therapeutics: Consultancy; Novartis: Consultancy; VOR Biopharma: Consultancy. Ampudia:Equillium: Current Employment, Current equity holder in publicly-traded company. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ritz:Rheos Medicines: Consultancy; Amgen: Research Funding; Avrobio: Consultancy; Talaris Therapeutics: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Equillium: Research Funding; Falcon Therapeutics: Consultancy; Kite Pharma: Research Funding; TScan Therapeutics: Consultancy.

Background Relapse of acute myeloid leukemia (AML) after allogeneic stem cell transplant has a poor prognosis with limited treatment options. Cytokine-induced memory like natural killer (CIML NK) cells are a novel therapy with enhanced cytotoxicity independent of KIR ligand interactions able to induce remission of relapsed/refractory AML (Romee et al, Science TM 2016). We are evaluating the safety and potential efficacy of donor-derived CIML NK cells in patients with relapsed myeloid malignancies after haploidentical donor transplant (haploSCT) in a phase 1/1b study (clinicaltrials.gov: NCT040247761). Here we report on manufacturing, safety, and in vivo correlative biology of the adoptively transferred CIML NK cells in the first 3 enrolled patients. Methods The primary endpoint is to identify the maximum-tolerated dose (MTD) of CIML NK cells in patients with MDS, MPN, or AML who relapsed after haplo-SCT. NK cells were enriched from donor-derived non-mobilized leukapheresis product via two-step CD3 depletion followed by selection of CD56+ cells using the CliniMACS reagent system (Miltenyi Biotec). The enriched NK cell product was cultured for 12-16 hours in X-VIVO 15 media containing rhIL-12, rhIL-15, and rhIL-18 to generate CIML NK cells (Figure 1A). Patients in the current cohort were lymphodepleted with fludarabine 25mg/m2 daily for 3-5 days and cyclophosphamide 60mg/kg daily for 2 days followed by CIML NK cells at a dose of 5-10 x 106 cells/kg and IL-2 106 IU/m2 QOD for 7 doses. Dose-limiting toxicities were evaluated for 6 weeks following NK cell infusion. Response to therapy was assessed at day+28 following CIML NK cell infusion. Results Patient #001 has FLT3-ITD AML that relapsed 5 months after a reduced intensity (RIC) haploSCT. She received 7.4x106 NK cells/kg followed by IL-2 106 IU/m2 QOD for 7 doses. Her day+28 bone marrow had no leukemia blasts although FLT3-ITD mutation remained detectable. Two months post-CIML NK the leukocyte and granulocyte chimerism were 88% and 89%, respectively. Patient #002 has AML with multiple pathogenic variants, including a potentially pathogenic variant in TP53. His disease relapsed 15 months post haplo-SCT with repeat marrow showing all the original mutations, including the TP53 variant (VAF 50.5%). He received 9.5x106 NK cells/kg followed by IL-2 106 IU/m2 QOD for 7 doses. His day+28 marrow had trilineage hematopoiesis without any mutations, including no TP53 mutation (Figure 1B). Two months post-CIML NK the leukocyte and granulocyte chimerism were both 99%. Patient #003 has MDS whose disease relapsed 8 months post RIC haploSCT with persistence of all her diagnostic pathogenic mutations. She received approximately 9.2x106 NK cells/kg and has only just completed IL-2 106 IU/m2 QOD for 7 doses. Among the 3 patients, the main toxicity was prolonged cytopenia requiring stem cell boost in one case. Donor NK cells demonstrated a dramatic shift from a predominantly CD56dimCD16hi (88% of NK cells) to a CD56dimCD16lo phenotype (49% of NK cells) as CIML NK cells. Infused CIML NK cells expanded massively, with approximately 50-fold, 10-fold, and 15-fold maximum in vivo expansion in the first three patients, respectively (Figure 1C). CIML NK cells were the major population in the day+28 marrows in both patients #001 and #002, with CD56+CD7+ cells constituting 89% of the cellularity in the former and 48% in the latter (Figure 1C). CIML NK cells persisted for 3 and 6 months post-infusion in the first two patients. The NK cells were predominantly mature, with most expressing CD16 and low levels of the inhibitory receptor NKG2A. PD-1 expression was much lower on the expanded NK cells vs the pre-infusion donor-derived NK cells. There was minimal concurrent expansion of CD4+CD25+ T-regulatory cells (Figure 1D). Conclusion We show with the first 3 patients in this trial that CIML NK cells can be generated and infused safely, can expand massively in the peripheral blood and bone marrow within the first 30 days post-infusion, and can persist for several months. In addition, CIML NK cell infusion can reduce the burden of pathogenic variant alleles to below the limit of detection, including the burden of high-risk mutations such as in TP53. Though our results are preliminary, the massive in vivo expansion and long-term persistence of adoptively transferred CIML NK cells underscores the unique biology of these cells that makes them an attractive option for cellular therapy protocols. Disclosures Nikiforow: Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Rambaldi:Equillium: Research Funding. Cutler:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees. Koreth:Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; Clinigen: Other; Miltenyi: Other: Research Support; BMS: Other: Research Support; Therakos: Membership on an entity's Board of Directors or advisory committees; Equillium: Consultancy; EMD Serono: Consultancy; Biolojic Design Inc: Consultancy; Amgen: Consultancy; Moderna Therapeutics: Consultancy. Wu:Pharmacyclics: Research Funding; BionTech: Current equity holder in publicly-traded company. Soiffer:Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; VOR Biopharma: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Rheos Therapeutics: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; alexion: Consultancy; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Ritz:TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; Falcon Therapeutics: Consultancy; Infinity Pharmaceuticals: Consultancy.

Introduction: New immunotherapies directed against CD38, SLAMF7 or BCMA have significantly improved the outcome of multiple myeloma (MM) patients. However, most patients eventually relapse, underscoring the need for additional immunotherapeutic targets. We have previously shown that expression levels of GPRC5D, an orphan G protein-coupled receptor, are significantly higher on MM cells, compared to normal plasma cells or other immune cells. We also showed that the novel GPRC5DxCD3 bispecific antibody (BsAb) JNJ-7564, has promising anti-MM activity in patient-derived BM samples (Verkleij et al., EHA 2019). To elucidate which factors contribute to the observed heterogeneity in ex vivo response, we analyzed the impact of tumor and patient characteristics on efficacy of JNJ-7564. We further investigated whether tumor-intrinsic factors may be determinants of response by also testing in these assays JNJ-7957, a BCMA-targeting BsAb that differs from JNJ-7564 only in the tumor-antigen-binding domain. Methods: Bone marrow (BM) samples obtained from 13 newly diagnosed (ND), 17 daratumumab-naive relapsed/refractory (DARA-naive RR; median of 3 prior therapies) and 15 daratumumab-refractory (DARA-R, median of 6 prior therapies) MM patients were analyzed for tumor- and immune cell composition, and subsequently incubated with JNJ-7564 (0.00128-4.0 µg/mL) or JNJ-7957 (0.8 µg/mL). After 48 hours, MM cell lysis was assessed by flow cytometry. Luciferase-transduced MM cell lines were incubated with JNJ-7564 (0.032-4.0 µg/mL) in the presence of healthy peripheral blood mononuclear cells (PBMCs), purified CD4+CD25- T-cells or regulatory T-cells (Tregs). After 48 hours, MM cell lysis was assessed by bioluminescence assay. Results: We found no difference in JNJ-7564 efficacy with respect to disease stage (NDMM vs DARA-naive RRMM vs DARA-R MM, P=0.48). Importantly, the presence of high-risk cytogenetic abnormalities [del(17p), t(4;14) and t(14;16)] did not impair JNJ-7564 efficacy. The level of target expression was an important determinant of response, as evidenced by superior MM cell lysis in samples with higher than median GPRC5D expression, when compared to lower GPRC5D expression (Fig. 1A). Inferior MM cell lysis was observed in older patients (>67 years), in samples with low T-cell counts or low effector:target (E:T) ratios, and in those with a high frequency of PD-1+ T-cells, HLA-DR+ activated T-cells, or Tregs. These determinants of response also affected JNJ-7564-mediated T-cell activation and degranulation. To further analyze the impact of Tregs, we performed additional cell line experiments. Purified Tregs impaired T-cell proliferation, and were significantly less potent to kill MM cells when redirected by JNJ-7564, compared to CD4+CD25- T-cells (Fig. 1B). This was accompanied by reduced secretion of IFN-γ, TNF-α, IL-2 and granzyme B. To evaluate the impact of BM stromal cells (BMSCs) on JNJ-7564 activity, MM cell lines were co-incubated with PBMCs and patient-derived BMSCs. Direct cell-cell contact hampered MM cell lysis, while indirect contact (transwell) did not affect JNJ-7564 activity. Direct contact also decreased secretion of TNF-α and IL-2, and reduced GPRC5D expression on MM cells, contributing to BMSC-mediated resistance to JNJ-7564. Finally, we simultaneously evaluated the single agent activity of both JNJ-7564 and JNJ-7957 (0.8 µg/mL, dose whereby a plateau in MM cell lysis was observed with both BsAbs) in 40 BM samples. MM cell lysis induced by both agents was strongly correlated (Fig. 1C). In 6 samples, both agents exhibited poor activity (80% lysis). Comparison of characteristics between these groups showed that a low E:T ratio (Fig. 1D) and high frequency of Tregs (Fig. 1E) significantly impaired efficacy of both BsAbs, suggesting patient-specific factors can determine response to T-cell redirectors targeting different antigens. Conclusion: We show that tumor-related factors, such as GPRC5D expression, as well as differences in the composition of the BM microenvironment, including E:T ratio, frequency of PD-1+ or HLA-DR+ T-cells or immune-suppressing Tregs or BMSCs, contribute to the variability in response to JNJ-7564. Our data indicate that strategies aiming at optimizing E:T ratio (e.g. induction therapy) or Treg depletion, may improve response to T-cell redirecting antibodies in MM. Disclosures Wong: Jhonson & Jhonson: Current Employment. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Verona:Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Adams:Johnson & Johnson: Ended employment in the past 24 months. Mutis:Janssen Pharmaceuticals: Research Funding; Genmab: Research Funding; Takeda: Research Funding; Onkimmune: Research Funding; Gadeta: Research Funding. van de Donk:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Ferrer: Membership on an entity's Board of Directors or advisory committees.

Background: Acute lymphoblastic leukemia (ALL) is a molecularly heterogeneous disease originating from clonal proliferation of precursor B-lineage cells. In adults, ALL diagnosis is still associated with a dismal prognosis due to the lack of specific targeted therapies. This study was designed to investigate the expression of interleukin-2 receptor alpha chain CD25 in B-ALL and its biological significance, especially following the availability of specific CD25 targeting compounds. Methods:The expression of IL2RA (CD25 gene) was detected by flow cytometry (FC), immunohistochemistry and Western blot analysis, in 25 newly diagnosed ALL patients, both Philadelphia positive (12 patients) and Philadelphia negative (13 patients). Similarly, CD25 expression was assessed in four B-ALL commercially available cell lines. Infection with shRNA specifically directed against CD25 was used to evaluate apoptosis induction and cell cycle arrest in primary B-ALL cells established from two patients. Results:Our data suggest that ALL, and in particular Ph-positive ALL, aberrantly expresses the interleukin-2 receptor alpha chain, CD25. Whereas normal B cells display low amounts of CD25, primary ALL cells and ALL cell lines (over)-express CD25. While the high frequency of CD25 on the surface of many different hematological tumor cells has been established and confirmed in our study, there is little investigation focusing on the significance of CD25 expression. Indeed, CD25 may be present on ALL cells and enable oncogenic signaling pathways. In such respect, we observed that CD25 silencing in primary cells promotes cell cycle arrest and apoptosis induction. While these data support the rational to target CD25, ALL cells did not appear to be in-vitro sensitive to basiliximab, an antibody able to target the Il2RA, but in-vivo investigations are needed to better assess the effects of this therapeutic approach in ALL context. Conclusions:We concluded that CD25 expression is elevated in patients with B-ALL. Our results also demonstrate that CD25 silencing induces cell cycle arrest and apoptosis. The latter result has important implications from a therapeutic point of view. Targeting CD25 receptor with anti-CD25 antibodies or peptide mimetics could be an effective strategy for targeting leukemic cells. Additionally, high CD25 expression could be exploited for the development of CAR-T therapy Disclosures Saglio: Roche:Research Funding;Pfizer:Research Funding;Incyte:Research Funding;Novartis:Research Funding;Ariad:Research Funding;Bristol-Myers Squibb:Research Funding.

Chronic graft-versus-host disease (cGVHD) is associated with inadequate reconstitution of tolerogenic CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs). Previous phase 1 studies identified a low daily dose of interleukin-2 (IL-2) that was well tolerated, did not exacerbate alloimmunity, augmented Treg in vivo, and was associated with improvement of active cGVHD. In the current phase 2 study, 35 adults with steroid-refractory cGVHD received daily IL-2 (1 × 10(6) IU/m(2)) for 12 weeks. Median time from transplantation and cGVHD onset was 616 days (range, 270-2145 days) and 317 days (range, 28-1880 days), respectively. Two patients withdrew and 5 required IL-2 dose reductions due to side effects. Twenty of 33 evaluable patients (61%) had clinical responses at multiple cGVHD sites (liver, skin, gastrointestinal tract, lung, joint/muscle/fascia). Three patients (9%) had progressive cGVHD. Compared with pretreatment levels, Treg and natural killer cell counts rosefivefold (P.001) andfourfold (P.001), respectively, without significant change in conventional CD4 T cells (Tcons) or CD8 T cells. The Treg:Tcon ratio rosefivefold (P.001). Clinical responders initiated IL-2 earlier (508 vs 917 days after transplantation, P = .005; 249 vs 461 days after cGVHD onset; P = .03). Treg:Tcon ratios ≥0.07 at baseline and ≥0.2 at week 1 also predicted clinical response (P = .003; P = .0003, respectively). After a 4-week treatment hiatus, clinical responders were eligible to continue IL-2 therapy indefinitely. During 2 years of extended IL-2 therapy, clinical and Treg immune responses persisted, while Tcon count and Treg:Tcon ratio gradually normalized. Low-dose IL-2 provides durable clinical improvement in active cGVHD and extended therapy is well-tolerated.

BCMA-targeting bispecific T-cell engagers in clinical development have demonstrated encouraging preclinical efficacy. The most advanced of these is AMG 420, which showed significantly improved response rates in relapsed/refractory multiple myeloma (MM) patients. Nevertheless, median duration until relapse is currently limited to approximately 12 months, highlighting the need for new drugs with novel MoA. Recently, we reported on a Local Activator and T cell Engager (LocATE) antibody that targets BCMA and selectively blocks programmed death-ligand 1 (PD-L1) on malignant cells (ASCO, June 2019). LocATE induced superior T cell activation and cancer cell killing, in vitro and ex vivo, compared to a BCMAxCD3 BiTE alone or in combination with a PD-L1 inhibitor. Here, we sought to further characterize the novel MoA of our LocATE. To assess the therapeutic potential of the LocATE, we first investigated whether potent cytotoxicity is uncoupled from high levels of cytokine release. We evaluated three LocATE molecules with different PD-L1 affinities (low, medium, high). Using BCMA-expressing MM cell lines (U-266, MM.1S, RPMI-8226 and H929) with distinct PD-L1 surface expression levels (3 - 53%), we determined the cytokine profile (IL-2, IL-6, IFN-γ, TNF-α) and target cell lysis induced by each candidate in the presence of CD3-positive human T cells. All three candidates exhibited comparable killing potency, however, low-affinity PD-L1 LocATE antibodies induced significantly less cytokine release (up to 10-fold) than its higher PD-L1 affinity counterparts across all cell lines investigated. Notably, using the low-affinity PD-L1 LocATE, we observed a 2-fold increase in tumor cell killing compared to bispecific BCMAxCD3 targeting controls in cell lines expressing high PD-L1 levels (53%), underlining the contribution of PD-L1 inhibition. Accordingly, phenotypic profiling of effector cells showed that the LocATE more potently induced dose-dependent upregulation of the activation markers CD69, CD25 and HLA-DR compared to bispecific controls. Importantly, cytotoxic activity, T cell activation and cytokine release were not induced when BCMA-negative cells expressing high levels of PD-L1 were treated with LocATE, underlining the BCMA-selective killing mechanism. Since the superior efficacy of LocATE was found to correlate with the expression level of PD-L1 on MM cell lines and upregulation of PD-L1/PD-1 has been reported as one of the major myeloma cell escape mechanisms during treatment with BiTEs, we subsequently investigated the efficacy of LocATE using primary bone marrow samples and peripheral blood mononuclear cell (PBMCs) obtained from MM patients. Six bone marrow mononuclear cell (BMMC) and eight PBMC samples from MM donors of different disease stages were characterized for PD-1/PD-L1 expression levels; analysis of T cell frequency and level of activation/exhaustion was performed based on CD4, CD8, CD25, CD69, Tim-3, Lag-3 and PD-1 marker expression. Using patient samples with high frequencies of PD-1 expressing T cells prior to treatment, LocATE induced superior MM tumor cell lysis and T cell activation compared to BCMAxCD3 bispecific antibodies. No activity was observed on healthy cells, underlining the safe and selective killing mechanism through tumor-local reactivation of exhausted T cells. Collectively, these findings demonstrate that the simultaneous T cell redirection and tumor-specific checkpoint inhibition with the LocATE leads to an improved therapeutic index with robust tumor cell killing and low levels of cytokine release. Capable of counteracting adaptive immune resistance caused by increased PD-1/PD-L1 signaling, the LocATE antibody has the prospect to significantly improve survival for multiple myeloma patients. Disclosures Vrohlings: CDR-Life: Current Employment. Jungmichel:CDR-Life: Current Employment, Other: current option holder. Senn:CDR-Life: Current Employment. Schleier:CDR-Life: Current Employment, Current equity holder in private company. Scheifele:CDR-Life: Current Employment, Current equity holder in private company. Wendelspiess:CDR-Life: Current Employment. Leisner:CDR-Life: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Jaeger:CDR Life AG: Consultancy, Research Funding; Miltenyi: Consultancy, Honoraria; Karyopharm: Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria; F. Hoffmann-La Roche: Honoraria, Research Funding. Borras:CDR-Life: Current Employment, Current equity holder in private company.

The classical BCR-ABL1-negative myeloproliferative neoplasms (MPN) are characterized by over-production of myeloid cells, disease-related mutations in certain driver-genes (JAK2, CALR, MPL) and an increased risk to transform to secondary acute myeloid leukemia (sAML). Although considered stem cell-derived neoplasms, little is known about the phenotype and functional properties of disease-initiating neoplastic stem cells (NSC) in MPN and sAML. Recent data suggest that MPN NSC reside in a CD34+ fraction of the malignant clone. Therefore, these cells are considered most critical target populations to be examined for expression of molecular and immunological targets with the aim to develop improved or even curative NSC-eliminating therapies, such as antibody-based or CAR-T cell approaches. Using a panel of monoclonal antibodies (n=40) and multicolor flow cytometry, we established the immunological phenotype and target expression profiles of putative CD34+/CD38─ NSC and CD34+/CD38+ progenitor cells in patients with polycythemia vera (PV, n=18), essential thrombocythemia (ET, n=29), primary myelofibrosis (PMF, n=38) and post-MPN sAML (n=11). In almost all patients, the putative MPN stem cells expressed the stem cell invasion receptors Hermes (CD44) and ADGRE5 (CD97), C1qR1 (CD93), the migration/adhesion receptor MIC2 (CD99), and the stem cell antigen AC133 (CD133). Contrasting normal stem cells, MPN NCS and sAML stem cells failed to express Thy-1 (CD90). Among the cytokine receptors tested, MPN NSC invariably displayed the TGFßR-related antigen endoglin (CD105), TPOR (CD110), SCFR KIT (CD117), IL-3RA (CD123), CXCR4 (CD184) and IGF-1R (CD221). NSC expressed particularly high levels of KIT and low levels of TPOR and IGF-1R. The IL-2RA (CD25) was identified on NSC in most patients with PMF and sAML, and in a few with ET, but not in patients with PV. Similarly, the GM-CSFR (CD116) was found to be expressed on NSC in most patients with PMF, a few with ET and no with PV. MPN NSC did not exhibit substantial amounts of M-CSFR (CD115), IL-3RB (CD131), FLT3 (CD135), NGFR (CD271) VEGFR-2 KDR (CD309), EPOR, MET or OSMRB. The CD34+/CD38+ MPN progenitor cells displayed a similar profile of cytokine receptors. In addition, MPN and sAML progenitor cells expressed IL-1RAP and CLL-1 in most donors examined. We next examined the expression of various immunological targets and resistance-mediating immune checkpoint antigens on NSC and MPN progenitor cells. In all MPN patients and all sAML patients tested, NSC were found to express substantial amounts of Siglec-3 (CD33) and low levels of Campath-1 (CD52) and MDR-1 (CD243). In addition, MPN NSC and sAML stem cells invariably displayed the "don't eat" me checkpoint IAP (CD47) and the classical checkpoint PD-L1 (CD274). Exposure to interferon-gamma (200 U/ml, 24 hours) resulted in an upregulation of PD-L1 on NSC. In a subset of patients, MPN NSC expressed low levels of HB15 (CD83). In contrast, MPN NSC and sAML stem cells failed to express B7-1 (CD80), B7-2 (CD86), PD-L2 (CD273) and PD1 (CD279). MPN progenitor cells and sAML progenitors expressed an identical profile of cell surface targets and checkpoint antigens. Finally, we confirmed the disease-initiating capacity of MPN stem- and progenitor cells (CD34+ cells) using primary PMF cells in xenotransplantation experiments employing NSGS mice expressing human interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF). After 28 weeks post injection, engraftment of human CD45+ cells in the bone marrow of NSGS mice was found in 15/15 mice injected with bulk mononuclear cells (MNC) containing CD34+ cells and in 0/15 NSGS mice injected with MNC depleted of CD34+ cells. Together, MPN NSC reside in a CD34+ fraction of the malignant clone and display a unique phenotype, including cytokine receptors, immune checkpoint molecules and other target antigens. The phenotypic characterization of neoplastic stem cells should facilitate their enrichment and the development of NSC-eradicating treatment concepts in MPN. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

Background. On 11 March, 2020 the World Health Organization declared COVID-19, caused by SARS-CoV-2, a global pandemic with almost 17,000,000 confirmed cases worldwide by the end of July, of which 4,500,000 were in the US. Approximately 20% of patients develop severe disease that can evolve into acute respiratory distress syndrome leading to respiratory or multiorgan failure, with an overall mortality of up to 4%. Older age, comorbidities such as hypertension and diabetes, and immune compromise have been identified as major risk factors associated with poor prognosis. For example, in immunocompromised HSCT patients mortality rates as high as 20% have been reported (www.cibmtr.org/COVID19). Furthermore, there is accumulating evidence regarding the protective role of T cells, with reduced counts and dysregulation seen more prominently in individuals with severe rather than mild COVID-19. Our group has previously demonstrated the feasibility, safety and clinical efficacy of administering allogeneic ex vivo expanded multivirus-specific T cells (multi-VSTs) as a banked, off-the-shelf product for the treatment of EBV, CMV, BKV, HHV6 and AdV infections/disease in immunocompromised individuals. Given the lack of preventative or therapeutic agents and the emerging evidence of the pivotal protective role of SARS-CoV-2-specific CD4+ and CD8+ T cells, we sought to explore the feasibility of developing a banked, SARS-CoV-2-specific VST product to treat those at highest risk of severe COVID-19 disease (i.e. HSCT recipients, elderly individuals, patients with comorbidities). Methods. To first identify immunogenic and protective SARS-CoV-2 antigens we screened PBMCs from convalescent individuals with mild COVID-19 (not requiring hospitalization) for T cell activity against overlapping peptide libraries (pepmixes) spanning 18 structural and non-structural SARS-CoV-2 proteins of which 8 [structural proteins: Spike (S), Membrane (M) and Nucleoprotein (N); non-structural proteins (Nsps): 3, 4, 6, 12; and the accessory protein (AP) 7a] were identified as immunodominant and advanced for VST manufacturing. We subsequently utilized our optimized VST manufacturing process and culture in a G-Rex device in medium supplemented with activating cytokines to generate SARS-CoV-2-specific T cells with activity against this combination of immunodominant targets. Results. We achieved a mean 29±7 fold expansion (mean±SEM; n=5) of cells that were comprised almost exclusively of CD3+ T cells (97.1±0.7%; mean±SEM), with a mixture of cytotoxic (CD8+; 10.2±1.2%) and helper (CD4+; 85.5±1.8%) T cells. These cells had a phenotype consistent with effector function and memory potential, as evidenced by upregulation of the activation markers CD25, CD69, and CD28 and expression of central (CD45RO+/CD62L+) and effector memory markers (CD45RO+/CD62L−), with minimal PD1 or Tim3 expression. To confirm the anti-viral activity of our expanded cells we performed an IFNγ ELIspot using each of the individual stimulating antigens as an immunogen and all lines proved to be reactive against the target antigens [S: 2,118±479 SFC/2x105; M: 1,084±182; N: 1,124±335; Nsp3: 71±48.6; Nsp4: 68±30; Nsp6: 23±6.7; AP7a: 65±43; and Nsp12: 29±9]. As demonstrated by intracellular cytokine staining (ICS), the immune response was mediated by both CD4+ and CD8+ T cell subsets, and the majority of IFNg-producing cells also produced TNFa. Reactive cells exhibited a primarily Th1-polarized profile as measured by Granzyme B production, luminex array and single-cell protein analysis. In addition, the expanded cells were able to kill viral pepmix-loaded autologous PHA blasts with minimal/no activity against non-antigen-expressing autologous and allogeneic targets (Figure 1). Conclusion. SARS-CoV-2 VSTs generated from convalescent individuals are Th1-polarized, polyfunctional and selectively able to kill viral antigen-expressing targets with no auto- or alloreactivity, indicative of both their selectivity and safety for clinical use. We are rapidly advancing this product to the clinic for administration in a randomized clinical trial (VSTs+SOC vs SOC) to prevent the development of severe disease in high risk hospitalized patients such as those post-transplant or therapy for hematologic malignancy. Figure 1: SARS-CoV-2 Specific T cells Demonstrate Selective Cytolytic Activity against viral antigen-expressing targets. Figure 1 Disclosures Vasileiou: AlloVir: Consultancy. Kuvalekar:AlloVir: Consultancy. Workineh:AlloVir: Current Employment. Watanabe:AlloVir: Consultancy. Heslop:Novartis: Consultancy; Gilead Biosciences: Consultancy; PACT Pharma: Consultancy; Kiadis: Consultancy; Tessa Therapeutics: Consultancy, Research Funding; AlloVir: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Hill:Incyte: Membership on an entity's Board of Directors or advisory committees. Leen:AlloVir: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Immune checkpoint therapy has revolutionized the treatment of patients with cancer. Checkpoint receptors and their ligands play an important role in T cell activation and exhaustion and are currently the focus in understanding the antitumoral immune responses. In patients with acute myeloid leukemia (AML) limited data have been published that comprehensively describe the expression of checkpoint receptors on different T cell subsets. We performed multicolor flow cytometry on peripheral blood mononuclear cells (PB, PBMCs) from patients with newly diagnosed AML (n=20) and PBMCs from age matched healthy donors (HDs; n=12), focusing on differentiation, the clinically actionable exhaustion receptor T cell immunoglobulin and ITIM domain (TIGIT), the two ectoenzymes ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73). Our studies included also analysis of interleukin-7 receptor-α (CD127) and the intracellular expression of the transcription factor T cell factor 1 (TCF-1). Both markers are known to be expressed in long living memory CD8+ T cells and harboring the ability for self-renewal. The thymocyte selection-associated high mobility group box protein (TOX) was also analyzed, since this molecule has recently been described as regulator of CD8+ T cell exhaustion. Comparison of PB from patients with newly diagnosed AML vs. HDs revealed that the frequency of CD8+, CD4con (CD4+CD127+CD25-) and CD4reg (CD4+CD127-CD25+) T cells was similar among both groups. However, the frequency of CD8+ EMRA T cells (CCR7- CD45RO- CD8+ CD3+) was increased in PB from patients with AML compared to PB from HDs (39,05 ± 4,38 vs. 14,29 ± 3,83; p TIGIT, CD39 and CD73 emerged as checkpoints of interest on CD8+ T cells. The frequency of TIGIT+ CD8+ T cells and CD39+ CD8+ T cells in PBs from patients with newly diagnosed AML was increased compared with that in HDs (42,60 ± 4,67 vs. 20,36 ± 3,68; p=0,00 and 6,54 ± 2,05 vs. 1,31 ± 0,32; p=0,05). Whereas reduced frequency of CD73+ CD8+ T cells occurred in PB from patients with AML vs. HD (41,35 ± 4,75 vs. 66,18 ± 7,28; p=0,01). Analysis between TIGIT and CD73 expression showed inverse correlation between both targets in AML (r=-0,53; p=0,01). The frequency of the TIGIT+ CD73- CD8+ T cell population was increased in AML (36,85 ± 5,17, vs. 16,23 ± 5,09 ;p=0,01). This increased frequency of TIGIT+ CD73- cells in AML was related to EM (CCR7- CD45RO+ CD8+ CD3+)T cells (42,89 ± 4,78 vs. 25,27 ± 3,39; p=0,01) and EMRA CD8+ T cells (56,01 ± 5,68 vs. 36,54 ± 4,85; p=0,03). Moreover, CD39 was aberrantly expressed on this population: we observed an increased frequency of CD39+ TIGIT+ CD73- CD8+ T cells in PB from patients with newly diagnosed AML compared to PB from HDs (13,47 ± 4,03 vs. 3,03 ± 1,21; p=0,02). Next we focused on CD127 and TCF-1 which are involved in creating long living antigen independent memory CD8+ T cells and ability to self-renewal while producing differentiated effector cells. Comparing expression of CD127 and TCF-1 on CD39+ TIGIT+ CD73- CD8+ T cells showed a significantly decreased frequency of CD127 (17,73 ± 2,16, vs. 30,72 ± 7,12; p=0,04) and TCF-1 (14,67 ± 2,90 vs. 39,74 ± 9,32; p=0,03) in PB from patients with newly diagnosed AML compared to HDs. Expression of TIGIT and TCF-1 inversely correlated in AML (r =-0,87; p To further evaluate the exhaustion status of TIGIT+ CD73- CD8+ T cells we examined the expression of TOX, recently described as one of the key regulators governing CD8+ T cell exhaustion, the frequency of TOX+ cells was increased in AML (50,57 ± 8,21 vs. 22,14 ± 4,86; p=0,01). Analysis of co-expression showed that the TOX+ CD39+ TIGIT+ CD73- CD8+ population was significantly increased in PB from patients with newly diagnosed AML compared to their counterparts in PB from HDs (21,81 ± 3,14 vs. 3,84 ± 1,10; p=0,04). In summary, we could show that in PB from patients with newly diagnosed AML an aberrant cell population of CD39+ TIGIT+ CD73- CD8+ T cells is prevalent in contrast to the PB from HDs. In this cell population we observed elevated expression of TOX which has recently described as one of the key regulators governing CD8+ T cell exhaustion. In contrast downregulation of CD127 and TCF-1 was found in these cells. These data might contribute to CD8+ T cell exhaustion in AML and support further functional analysis to investigate the relevance of combinatorial inhibition of TIGIT and CD39. Disclosures Brauneck: Daiichi Sankyo: Consultancy, Honoraria, Other: support for meeting attendance; Novartis: Other: support for meeting attendance; Jazz Pharmaceuticals: Other: support for meeting attendance. Bokemeyer:Merck KGaA: Honoraria; Janssen-Cilag: Research Funding; Roche: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Taiho Pharmaceutical: Research Funding; Pfizer: Other; Karyopharm Therapeutics: Research Funding; Millenium: Research Funding; MSD: Research Funding; Nektar: Research Funding; Novartis: Research Funding; Rafael Pharmaceuticals: Research Funding; Springworks Therapeutics: Research Funding; Sanofi: Consultancy, Honoraria, Other: travel accomodations; Bristol-Myers Squibb: Honoraria, Other: travel accomodations, Research Funding; AstraZeneca: Honoraria, Research Funding; Merck Sharp & Dohme: Consultancy, Honoraria; Lilly/ImClone: Consultancy, Research Funding; Merck Serono: Consultancy, Other: travel accomodations; Bayer Schering Pharma: Consultancy; GSO: Consultancy; AOK Health Insurance: Consultancy; Abbvie: Research Funding; ADC Therapeutics: Research Funding; Agile Therapeutics: Research Funding; Alexion Pharmaceuticals: Research Funding; Amgen: Research Funding; Apellis Pharmaceuticals: Research Funding; Astellas: Research Funding; BerGenBio: Research Funding; Blueprint Medicines: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Eisai: Research Funding; Gilead Sciences: Research Funding; Glycotope GmbH: Research Funding; GSK: Research Funding; Incyte: Research Funding; IO Biotech: Research Funding; Isofol Medical: Research Funding. Fiedler:Amgen: Consultancy, Honoraria, Other: support for meeting attendance, Patents & Royalties, Research Funding; ARIAD/Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Daiichi Sankyo: Other: support for meeting attendance.

Background: Mantle cell lymphoma (MCL) is an aggressive, incurable B cell malignancy. Major advances have been achieved with the development of clinically effective targeted agents including BTK and BCL-2 inhibitors. However, patients often relapse after these treatments. The emerging immune therapies combining immune checkpoint blockade and adoptive T-cell transfer are becoming increasingly desirable, but patient responses are heterogeneous, with a significant rate of failure. Tumor microenvironment (TME) plays an important role by inducing immune checkpoint and immune suppressive signaling or creating a metabolic barrier to antitumor immunity, particularly effector T-cells. To evade immune defenses, tumor and stromal cells cooperate and tactically express inhibitory ligands, such as PD-L1, while upregulating immune checkpoint receptors on effector T-cells and CAR T cells, resulting in negative signaling that leads to T-cell exhaustion, one of the major challenges in CAR T-cell therapy. Limited nutrient accessibility and a hostile immunosuppressive TME posts a strong barrier to effective immune therapies, with T-cell immunity being mostly affected. Impaired metabolism may also inhibit the function of effector T-cells while promoting suppressive activity of regulatory T-cells. Results: Our recent transcriptomic analysis of primary MCL clinical specimens comprised of microenvironment cell populations, revealed that genes related to immune function were dysregulated, particularly the genes responsible for IFNG-mediated anti-tumor immunity. Proliferation in response to TCR stimulation is an important measurement for T-cell effector function and potency. Reduced proliferation upon T-cell activation denotes T-cell dysfunction. Indeed, T-cells isolated from MCL specimens especially CAR T non-response patients exhibit diminished cell division upon stimulation with anti-CD3/CD28 beads (25-75% less expansion). To recapitulate the interplay between T-cells and TME, MCL cells were co-cultured with the non-MCL fraction from the purification, and T-cells were then phenotypically profiled. Intriguingly, MCL-derived T-cells mostly displayed exhaustion phenotypes, indicated by decreased expression of T-cell activation markers (CD25, CD28, CD38, and CD71), but increased expression of exhaustion and senescence molecules (CTLA4, LAG3 and PD-1). Moreover, MCL-derived T cells failed to induce the expression of cytokine, such as INF-γ ( Conclusion: We presented evidence that MCL cancer cells suppress immune cell function by upregulating immunosuppressive signaling and forging a metabolic barrier to effector T-cells and immunotherapy. Targeting the MCL TME while enhancing T-cell metabolic fitness would dramatically advance current immunotherapy. Despite the success of checkpoint blockade and adoptive cell transfer therapy, complimentary approaches to overcome the metabolic barrier of immunosuppressive TME holds promise to dramatically enhance the efficacy of immunotherapies. Further, identification of the precise pathways and targets that determine T-cell metabolic fitness would facilitate developing new approaches to bolster current immunotherapy. Disclosures Wang: AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Acerta Pharma: Research Funding; Oncternal: Consultancy, Research Funding; Guidepoint Global: Consultancy; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Dava Oncology: Honoraria; Molecular Templates: Research Funding; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Lu Daopei Medical Group: Honoraria; InnoCare: Consultancy; OncLive: Honoraria; Verastem: Research Funding; VelosBio: Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Beijing Medical Award Foundation: Honoraria; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; BioInvent: Research Funding; MoreHealth: Consultancy; Targeted Oncology: Honoraria.

Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third-party CD4(+) iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4(+) iNKT cells from third-party mice resulted in a significant survival benefit with retained graft-versus-tumor effects. In vivo expansion of alloreactive T cells was diminished while displaying a T helper cell 2-biased phenotype. Notably, CD4(+) iNKT cells from third-party mice were as protective as CD4(+) iNKT cells from donor mice although third-party CD4(+) iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third-party CD4(+) iNKT cells resulted in a robust expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third-party availability and feasibility of in vitro expansion provide the basis for clinical translation.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg) are critical elements for maintaining immune tolerance, for instance to exogenous antigens that are introduced during therapeutic interventions such as cell/organ transplant or gene/protein replacement therapy. Coadministration of antigen with rapamycin simultaneously promotes deletion of conventional CD4(+) T cells and induction of Treg. Here, we report that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin. This occurs via selective expansion of plasmacytoid dendritic cells (pDCs), which further augments the number of Treg. Whereas in conventional DCs, rapamycin effectively blocks mammalian target of rapamycin (mTOR) 1 signaling induced by Flt3L, increased mTOR1 activity renders pDCs more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDCs. This concept is supported by the finding that Treg induction is abrogated upon pDC depletion. The combination with pDCs and rapamycin is requisite for Flt3L/antigen-induced Treg induction because Flt3L/antigen by itself fails to induce Treg. As co-administering Flt3L, rapamycin, and antigen blocked CD8(+) T-cell and antibody responses in models of gene and protein therapy, we conclude that the differential effect of rapamycin on DC subsets can be exploited for improved tolerance induction.

Although information about the molecular pathogenesis of Waldenström macroglobulinemia (WM) has significantly advanced, the precise cell of origin and the mechanisms behind WM transformation from immunoglobulin-M (IgM) monoclonal gammopathy of undetermined significance (MGUS) remain undetermined. Here, we undertook an integrative phenotypic, molecular, and genomic approach to study clonal B cells from newly diagnosed patients with IgM MGUS (n = 22), smoldering (n = 16), and symptomatic WM (n = 11). Through principal component analysis of multidimensional flow cytometry data, we demonstrated highly overlapping phenotypic profiles for clonal B cells from IgM MGUS, smoldering, and symptomatic WM patients. Similarly, virtually no genes were significantly deregulated between fluorescence-activated cell sorter-sorted clonal B cells from the 3 disease groups. Interestingly, the transcriptome of the Waldenström B-cell clone was highly different than that of normal CD25-CD22+ B cells, whereas significantly less genes were differentially expressed and specific WM pathways normalized once the transcriptome of the Waldenström B-cell clone was compared with its normal phenotypic (CD25+CD22+low) B-cell counterpart. The frequency of specific copy number abnormalities [+4, del(6q23.3-6q25.3), +12, and +18q11-18q23] progressively increased from IgM MGUS and smoldering WM vs symptomatic WM (18% vs 20% and 73%, respectively; P = .008), suggesting a multistep transformation of clonal B cells that, albeit benign (ie, IgM MGUS and smoldering WM), already harbor the phenotypic and molecular signatures of the malignant Waldenström clone., This study was supported by Cooperative Research Thematic Network grants RD12/0036/0058 and RD12/0036/0048 of the Red de Cancer (Cancer Network of Excellence), Consejería de Sanidad, Junta de Castilla y Leon, Valladolid, Spain (557/A/10).

Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.

Hemophilia A is the X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). To address serious complications of inhibitory antibody formation in current replacement therapy, we created tobacco transplastomic lines expressing FVIII antigens, heavy chain (HC) and C2, fused with the transmucosal carrier, cholera toxin B subunit. Cholera toxin B-HC and cholera toxin B-C2 fusion proteins expressed up to 80 or 370 µg/g in fresh leaves, assembled into pentameric forms, and bound to GM1 receptors. Protection of FVIII antigen through bioencapsulation in plant cells and oral delivery to the gut immune system was confirmed by immunostaining. Feeding of HC/C2 mixture substantially suppressed T helper cell responses and inhibitor formation against FVIII in mice of 2 different strain backgrounds with hemophilia A. Prolonged oral delivery was required to control inhibitor formation long-term. Substantial reduction of inhibitor titers in preimmune mice demonstrated that the protocol could also reverse inhibitor formation. Gene expression and flow cytometry analyses showed upregulation of immune suppressive cytokines (transforming growth factor β and interleukin 10). Adoptive transfer experiments confirmed an active suppression mechanism and revealed induction of CD4+CD25+ and CD4+CD25− T cells that potently suppressed anti-FVIII formation. In sum, these data support plant cell-based oral tolerance for suppression of inhibitor formation against FVIII.

With the notable exception of B-cell malignancies, the efficacy of chimeric antigen receptor (CAR) T cells has been limited, and CAR T cells have not been shown to expand and persist in patients with nonlymphoid tumors. Here we demonstrate that redirection of primary human T cells with a CAR containing the inducible costimulator (ICOS) intracellular domain generates tumor-specific IL-17-producing effector cells that show enhanced persistence. Compared with CARs containing the CD3ζ chain alone, or in tandem with the CD28 or the 4-1BB intracellular domains, ICOS signaling increased IL-17A, IL-17F, and IL-22 following antigen recognition. In addition, T cells redirected with an ICOS-based CAR maintained a core molecular signature characteristic of TH17 cells and expressed higher levels of RORC, CD161, IL1R-1, and NCS1. Of note, ICOS signaling also induced the expression of IFN-γ and T-bet, consistent with a TH17/TH1 bipolarization. When transferred into mice with established tumors, TH17 cells that were redirected with ICOS-based CARs mediated efficient antitumor responses and showed enhanced persistence compared with CD28- or 4-1BB-based CAR T cells. Thus, redirection of TH17 cells with a CAR encoding the ICOS intracellular domain is a promising approach to augment the function and persistence of CAR T cells in hematologic malignancies.

Just as normal stem cells require niche cells for survival, leukemia-initiating cells (LICs) may also require niche cells for their maintenance. Chronic myeloid leukemia (CML) is caused by the activity of BCR-ABL, a constitutively active tyrosine kinase. CML therapy with tyrosine kinase inhibitors is highly effective; however, due to the persistence of residual LICs, it is not curative. Several factors are known to support CML LICs, but purification of LICs and a thorough understanding of their niche signals have not yet been achieved. Using a CML-like mouse model of myeloproliferative disease, we demonstrate that CML LICs can be divided into CD25(+)FcεRIα(-) Lineage marker (Lin)(-) Sca-1(+)c-Kit(+) (F(-)LSK) cells and CD25(-)F(-)LSK cells. The CD25(+)F(-)LSK cells had multilineage differentiation capacity, with a preference toward cytokine-producing mast cell commitment. Although cells interconverted between CD25(-)F(-)LSK and CD25(+)F(-)LSK status, the CD25(+)F(-)LSK cells exhibited higher LIC capacity. Our findings suggest that interleukin-2 derived from the microenvironment and CD25 expressed on CML LICs constitute a novel signaling axis. The high levels of CD25 expression in the CD34(+)CD38(-) fraction of human CML cells indicate that CD25(+) LICs constitute an "LIC-derived niche" that could be preferentially targeted in therapy for CML.

Fanconi anemia (FA) is a genetic disorder associated with bone marrow (BM) failure and leukemia. Recent studies demonstrate variable immune defects in FA. However, the cause for FA immunodeficiency is unknown. Here we report that deletion of Fanca or Fancd2 dysregulates the suppressive activity of regulatory T cells (Tregs), shown functionally as exacerbation of graft-vs-host disease (GVHD) in mice. Recipient mice of Fanca(-/-) or Fancd2(-/-) BM chimeras exhibited severe acute GVHD after allogeneic BM transplantation (BMT). T cells from Fanca(-/-) or Fancd2(-/-) mice induced higher GVHD lethality than those from wild-type (WT) littermates. FA Tregs possessed lower proliferative suppression potential compared with WT Tregs, as demonstrated by in vitro proliferation assay and BMT. Analysis of CD25(+)Foxp3(+) Tregs indicated that loss of Fanca or Fancd2 dysregulated Foxp3 target gene expression. Additionally, CD25(+)Foxp3(+) Tregs of Fanca(-/-) or Fancd2(-/-) mice were less efficient in suppressing the production of GVHD-associated inflammatory cytokines. Consistently, aberrant NF-κB activity was observed in infiltrated T cells from FA GVHD mice. Conditional deletion of p65 in FA Tregs decreased GVHD mortality. Our study uncovers an essential role for FA proteins in maintaining Treg homeostasis, possibly explaining, at least in part, the immune deficiency reported in some FA patients.

INTRODUCTION Adoptive immunotherapy with donor conventional T cells (Tcons) and CD4+CD25+FOXP3+regulatory T cells (Tregs) is used to enhance immune reconstitution in clinical trials of haploidentical transplantation. Donor Tregs are infused to prevent adverse alloreactions mediated by Tcons against normal tissues, which cause GvHD, in the absence of any post transplant immune suppression. This strategy proved to be effective in preventing leukemia relapse and chronic GvHD, allowing for ~70% event-free survival in patients with acute leukemia (Pierini et al. submitted). However, acute GvHD occurs in ~30% of the patients. We are investigating if ex-vivo stimulation of Tregs with a combination of low-dose IL-2 and TNF-α before adoptive transfer could improve acute GvHD prevention. Tregs express higher levels of the TNF receptor 2 (TNFR2) compared with Tcons and TNFR2 expression defines a Treg subpopulation with maximal suppressive capacity. Recent studies showed that activation of TNFR2 signaling in mouse Tregs reduced GvHD lethality in mouse models. METHODS Human CD4+or CD4+CD25+T cells were incubated for 60 hours with IL-2 (10 UI/ml) with or without TNF-α (20 ng/ml). Treg proliferation in the presence of allogeneic irradiated peripheral blood mononuclear cells (PBMC), and Treg-mediated suppression of Tcon proliferation were evaluated by CFSE assay. Colon biopsies from patients with suspected GvHD were stained with anti-CD3 and anti-FOXP3 antibodies. RESULTS We developed a translatable in vitro system for improving human Treg function by adding a short term stimulation with low-dose IL-2 and TNF-α (IL-2/TNF) in the selection process. CD4+T cells were primed with IL-2/TNF before CD25 positive selection. When total CD4+T cells were stimulated with IL-2/TNF, CD25 expression was selectively upregulated on CD4+FOXP3+Tregs, but not on CD4+FOXP3- T cells. IL-2/TNF-primed Tregs maintained TNFR2 expression and expressed higher levels of FOXP3 and CD25 compared with Tregs stimulated with IL-2 alone (p We analyzed GvHD clinical signs of the last 20 patients who were diagnosed with grade II-IV acute GvHD after Treg/Tcon-based haploidentical transplantation at our institution. Nineteen presented with early-onset gut GvHD and 40% of those patients did not have any skin involvement. We hypothesized that preferential targeting to the gut was the consequence of defective homing of Tregs to the gut. Compared with Tcons, Tregs express higher levels of skin homing receptors (such as CCR4 and CLA-4), but lower levels of the gut homing receptor α4β7-integrin. We analyzed the infiltration of Tregs and Tcons in 31 colon biopsies from patients with suspected GvHD. The number of Tregs was higher in patients who were not diagnosed with acute GvHD compared with those who were (158.3/mm2 vs 86.3/mm2, p CONCLUSION The present study shows that activation of TNFR2 signaling in human Tregs enhances their proliferation and suppressive function. It also suggests that Treg suppressive function exerted in periphery could be critical for tissue-specific GvHD control. Moreover, IL-2/TNF-priming could promote homing of Tregs to the gut and their capability to inhibit gut GvHD. Thus, IL-2/TNF-priming may be critical to optimize the ongoing clinical trial of adoptive immunotherapy with Tregs for GvHD prevention. Future studies will ascertain if IL-2/TNF-priming of Tregs can also allow for the infusion of higher doses of Tcons to further enhance immune reconstitution and leukemia killing without increasing the risk of GvHD. Disclosures No relevant conflicts of interest to declare.

Background: Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Tregs) effectively prevents conventional T cell (Tcons) mediated Graft versus Host Disease (GvHD) while it does not impair graft-versus-leukemia effect in haploidentical hematopoietic cell transplantation (haplo-HCT). Moreover Treg immunotherapy promotes fast donor T cell recovery after transplant. Recent studies showed that mouse bone marrow (BM) Tregs localize in the hematopoietic stem cell (HSC) niche, where they contribute to HSCs maintenance and promote donor engraftment and B cell lymphopoiesis. We are investigating if human Tregs promote B cell reconstitution and immunity in preclinical models and in haplo-HCT patients. Methods: Human sample analysis: B cell reconstitution was analysed monthly by FACS in BM and peripheral blood (PB) samples from 66 patients who underwent either Treg/Tcon haplo-HCT (45 patients), or T-cell depleted haplo-HCT (8 patients) or haplo-HCT with post-transplant cyclophosphamide (PTCy, 13 patients). Diagnosis was acute leukemia in 52 patients, lymphoma in 11 and multiple myeloma in 3. PB total immunoglobulin (Ig), anti-Cytomegalovirus (CMV) IgM and CMV viremia were monitored. Humanized mouse model: donor derived human Tregs and purified CD34+ HSCs were co-infused in sublethally irradiated (2 Gy) immune-deficient NSG mice. Donor engraftment and B cell reconstitution were analysed by FACS and histology. Results: B cell reconstitution was faster after Treg/Tcon haplo-HCT when compared to other haplo-HCT protocols. B cell counts were higher in PB of patients that received Treg/Tcon haplo-HCT (p To further assess whether HSC-derived B cells were functional in patients that received Treg/Tcon haplo-HCT, we analysed total Ig production and specific responses to CMV reactivation. Post-transplant hypogammaglobulinemia was rapidly corrected in Treg/Tcon haplo-HCT patients. Total IgM were higher compared to other haplo-HCT protocols and reached normal levels by 3 months after transplant (96±155 mg/dL, Fig.1B). CMV reactivation rate was lower (47% vs 79%) and it occurred later after Treg/Tcon immunotherapy (51±19 vs 40±48 days). New production of anti-CMV specific IgM was documented in 43% of CMV seropositive patients 98±48 days after Treg/Tcon haplo-HCT, while anti-CMV specific IgM were almost undetectable after other haplo-HCT protocols within the first 6 months after transplant (Fig.1C). Such potent B cell responses coupled with T cell reconstitution resulted in a reduction of CMV second reactivations (Fig.1D p Conclusions: Adoptive transfer of human Tregs boosts donor HSC engraftment and facilitates the reconstitution of functional HSC-derived donor B cells. Such results suggest that Treg/Tcon haplo-HCT patients could be early vaccinated after transplant. Treg/Tcon immunotherapy promotes control of infections and B cell immunity in patients undergoing haplo-HCT. Disclosures No relevant conflicts of interest to declare.

Introduction Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has demonstrated high success for B-cell acute lymphoblastic leukemia (B-ALL). Despite the initial high complete remission (CR) rate, about half of patients (pts) relapse at 1 year. CD19 antigen loss was observed in a significant number of relapsed patients. CD22 is another leukemic marker that often expressed on the surface of CD19- relapsed B-ALL blasts. We have developed a bispecific CAR construct targeting CD19 and CD22. Here we report results from a phase Ⅰ clinical trial of CD19/CD22 (GC022) dual CAR-T to evaluate the safety and feasibility of treating patients with relapsed/refractory B-ALL. Methods The CD19/CD22 dual CAR-T cells were manufactured in a cGMP facility. Patients' peripheral blood (PB) mononuclear cells were first collected, and CD3+ T cells were separated. The cells were then transfected by lentivirus encoded with CD19 CD22 bispecific scFv sequences. The CAR-T cells produced in this way contained a 4-1BB co-stimulatory signal domain. The CAR-T cells were then cultured for 8-14 days until sufficient cells were harvested for infusion. All pts received conditioning regimen of fludarabine and cyclophosphamide intravenously for 3 consecutive days with doses of 30 mg/m2/day and 250 mg/m2/day, respectively before a single infusion of CAR-T cells. The level of infused CAR-T cell proliferation in PB was analyzed by qPCR and flow cytometry. The primary end points were to evaluate feasibility and toxicity, and the secondary end points included disease response and engraftment/persistence of infused CD19/CD22 dual CAR-T cells. Results From Feb. 2019 to 23 July. 2019, 17 patients (pts) with relapsed/refractory B-ALL including 4 pts who previously treated with CD19 CAR-T cells were enrolled and pts were treated with CD19/CD22 dual CAR-T GC022 (US NIH Clinical#: NCT03825731). Four were adults, 13 pediatrics (age 1-45, Table 1). The median bone marrow (BM) blasts was 19.09 (0.36-87.82) %. Four patients received a low-dose (2.5-5×105/kg) dual CAR-T, 7 received a medium-dose (1-2.5×106/kg) and 5, a high-dose (3-5×106/kg). One patient withdrew immediately before CAR-T infusion due to his personal issue. Anti-leukemic efficacy was evaluated in 11/16 pts (5 pts have not yet reached D15). The 3/4 pts received low dose of GC022 had no response to treatment and 1 had MRD-positive CR. Seven patients who received medium dose achieved 100% CR on D15, highlighting the dose-dependent anti-leukemic activity. Six out of seven pts had MRD negative CR in this medium dose group. Five pts in high dose group have not reached the time for evaluation. No one relapsed with a median observation time of 60 (7-139) days. Cellular kinetic data was analyzed. Median peak of CAR-T copies was 1.09 (0.0022-4.98) x105 copy number/µg PB genomic DNA (Fig.1). The proliferation of medium or high dose groups was significantly better than the low dose group 3.47(0.43-4.98) x105 vs. 0.023(0.0022-0.81) x105(P=0.02) and 2.02(1.89-2.16) x105 vs. 0.023(0.0022-0.81) x105(P=0.004), (Fig.2). The peaks of IL-6, IFN-γ, IL-10, and CD25 were observed around day 7-10. Sixteen out of seventeen pts had grade 0-1 cytokine release syndrome (CRS) and only 1 patient experienced grade 2 CRS. None developed neurotoxicity. Conclusion Our study demonstrates safety and technical feasibility of CD19 and CD22 dual CAR-T in treating patients with CD19+CD22+ relapsed/refractory B-ALL. A low toxicity with dose-dependent high CR rate including pts who previously treated with CD19 CAR-T cells were observed. Longer observation time and more patients are needed to evaluate a beneficial advantage of the CD19/CD22 dual CAR-T over CD19 CAR-T product. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Bcr-abl1 oncogene targeted treatment with tyrosine kinase inhibitors (TKI) showed an impressive efficacy against proliferating chronic myeloid leukemia (CML) cells. However, rapid relapses in more than half of CML patients after discontinuation of the treatment suggest a presence of quiescent leukemic stem cells inherently resistant to BCR-ABL1 inhibition. Understanding the heterogeneity of CML stem cell compartment is crucial for preventing the treatment failure. Specificity of already established leukemic stem cell (LSC) markers has been tested mainly in bulk CD34+CD38- populations at diagnosis. Phenotypes and molecular signatures of therapy resistant BCR ABL1 positive stem cells is however yet to be established. AIMS: Identification of BCR-ABL1 dependent LSC markers at single cell level by direct comparison their surface and transcript expression with the levels and the presence of BCR-ABL1 transcript at diagnosis and after administration of TKI treatment. METHODS: Total number of 375 cells were obtained from bone marrow and peripheral blood of 4 chronic phase CML patients. Cells were collected prior any treatment and three months after TKI treatment initiation. Normal bone marrow cells and BCR-ABL1 positive K562 cell line were used as controls. Indexed immuno-phenotyping and sorting of CD34+CD38- single cells was performed using a panel of 11 specific surface markers. Collected single cells were lysed and cDNA was enriched for 11 targets using 22 cycle pre-amplification. Expression profiling was carried on SmartChip real-time PCR system (Takara Bio) detecting following genes: BCR-ABL1, CD26, CD25, IL1-Rap, CD56, CD90, CD93, CD69, KI67, and control genes GUS and HPRT. Unsupervised clustering was performed using principal component analysis (PCA). Correlations were measured by Spearman rank method. RESULTS: At diagnosis, majority of BCR-ABL1+ C34+CD38- stem cells co-express IL1-Rap, CD26, and CD69 on their surface (88%, 82%, 78% overlap). Only 56% of BCR-ABL1+ cells positive for aforementioned markers co-express CD25, 28% CD93 and 16% CD56. The expression of these markers could also be detected in 4-11% of BCR-ABL1- cell, although this could be technical inaccuracy caused by the single cell profiling. CD90 marker did not show any correlation with BCR-ABL1 expression. At transcript level the expression of IL-1Rap, CD26, CD25 and CD56 was observed in 62%, 52% 45% and 16% BCR-ABL1+ cells, and up to 7% of BCR-ABL1- cells. CD69 expression was observed in 90% of BCR-ABL+ cells at transcript level, but also in 71% BCR-ABL- cells. BCR-ABL1 independent expression was observed for cKIT. (60% vs. 76 % in positive vs negative). Finally proliferation marker KI67 was expressed only in 6% of the BCR-ABL1+ cells. PCA analysis divided cells into several distinct clusters with BCR-ABL1 as the main contributor, and cKIT, CD69 and CD26, IL-1RAP as other significant factors. Interestingly BCR-ABL1+ cells collected during TKI treatment showed persistent surface expression of IL-1Rap and CD26, while CD56, CD69 and CD93 were only on part of the BCR-ABL1+ cells. CD25 was significantly deregulated during TKI treatment. CONCLUSION: At diagnosis up to 80% of LSC co-express 3 specific surface markers - IL-1RAP, CD26 and CD69. Variable portion of LSC co-express additional markers such are CD25, CD56 and CD93. During TKI treatment the surface expression of majority of markers is decreased, where the best correlated LSC marker is IL-1Rap, followed by CD26 and CD69. CD56 marker seems to persist in the same proportion of cells while CD25 disappears. cKIT is highly expressed in normal BM and HSC from CML patients, but also in some LSC. CD34+CD38- cells show non-proliferating phenotype. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

Introduction: Recent advancements in the treatment of multiple myeloma (MM) have centered on engaging the immune system to target multiple myeloma cells. Although these therapies are being combined with immunomodulatory imide drugs (IMiDs) and corticosteroids, the individual contributions of these drugs on the immune system of MM patients has not been examined in the upfront setting. In this study, we examined the peripheral blood immunophenotypes of newly diagnosed multiple myeloma (NDMM) patients receiving the IMiD lenalidomide with or without the corticosteroid dexamethasone. Methods: To characterize immunophenotypes, we utilized flow cytometry to profile white blood cell populations from 35 patients enrolled in a clinical trial testing the efficacy of lenalidomide with and without dexamethasone in NDMM (NCT00772915). In this trial, all patients were initiated on single-agent lenalidomide. Dexamethasone was initiated in patients that did not meet desirable responses or for disease progression. At each cycle, peripheral blood was stained with a 17-marker antibody panel against several immune lineages and functional surface markers. We grouped patients into two groups: 1) lenalidomide alone or 2) lenalidomide with dexamethasone according to their treatment regimen at each cycle timepoint. Results: First we confirmed anti-myeloma cell activity for both groups by measuring a steady decline in circulating plasma cells in both groups. Examining peripheral blood immunophenotypes showed an expected decrease in T cells and a smaller decrease in B cells in both groups of patients. Closer inspection of B cell populations revealed a switch towards a more immature B cell phenotype in both treatment groups. This was measured as a switch from CD19-CD20+ cells to CD19+CD20- B cells. Inspection of T cell subsets revealed that patients receiving single-agent lenalidomide had a sustained decrease in the levels of CD4+ T cells and increase in the levels of CD8+ T cells. This was seen in both naïve and regulatory T cells evidenced by a decrease in the CD4/CD8 ratio among CD28+ T cells as well as CD25+ T cells. Importantly, this alteration did not lead to sustained alterations in the overall level of CD25+ or CD28+ T cells, and the addition of dexamethasone reverses this trend. In addition to the effects seen on T and B cell numbers, we detected expansions of NK cell populations in patients receiving lenalidomide alone. This expansion is detected as an overall increase in CD56+ mononuclear cells with the majority of cells being CD56+CD3- cells. Conclusions: Our data show that lenalidomide and dexamethasone therapy have shared but distinct effects on peripheral blood immunophenotypes in NDMM. Both drugs alter B cells numbers and populations leading to an expansion of CD19+CD20- B cells. However, lenalidomide alone decreases the CD4/CD8 T cell ratio; and, lenalidomide more strongly expands NK cell populations. The addition of dexamethasone reverses this trend and leads to a restoration of the CD4/CD8 ratio. This suggests that lenalidomide without dexamethasone might be counterproductive in immunotherapies intended to recruit CD4+ T cells. Conversely, lenalidomide alone could increase the efficacy of immunotherapies dependent on NK cell recruitment such as antibody-dependent cellular cytotoxicity (ADCC). This information may benefit future investigations of immune responses in MM patients and improve the adoption of immunotherapies to MM patients. Figure Disclosures Kumar: Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

Background Foxp3+ regulatory T cells (Tregs) comprise of natural (n) and induced (i) Treg subsets play an important role in immune system. Currently, isolation of nTregs and in vitro-expanded nTregs was shown to be an effective therapy to GVHD patients. However, shortage of nTregs in peripheral blood and time consumption of expansion in vitro may eventually limit the clinical application. Conversely, iTregs can be generated in vitro from naïve T cells and to a large number of iTregs in short time. As we known, regulatory T cells would decay after a period of time, in vivo or in vitro. Keeping a certain number of iTregs during the GVHD treatment is necessary, it should be the best to provide iTregs to the patient more than single usage. Aim Manipulated supplements of TGF-β1-induced Foxp3+ regulatory T cells should be a good way for prevention from acute graft-versus-host disease within a short time. Investigation was performed via animal model. Methods Splenocytes from C57BL/6 mice were used as a source of naïve T cells by a CD4+ naïve T cell isolation Kit. To induce Foxp3+ regulatory T cells (iTregs), the CD4+ naïve T cells were incubated with anti-CD3/CD28 coated 24-well plate in the presence of IL-2 (20U/ml) and TGF-b1 (50ng/ml) for 3 days. Foxp3+iTregs were harvested and identified as the expressions of CD4+/CD25+/FoxP3+/CD127- via flow cytometry (Fig.1). In this experiment, recipients (BALB/c) were irradiated with 800cGy and then infused with donor (C57BL/6) bone marrow cells with (TCD-BM+CD4T) or without donor T cells (TCD-BM) by intravenous injection. TCD-BM+CD4T cells mice would appear aGVHD phenotype. 8x106 Foxp3+ iTregs were injected into the TCD-BM with donor T cell mouse one or twice (TCD-BM+CD4 T +iTreg) for immunosuppression assay as shown in Fig.2. Mouse GVHD phenotype, body weights and survival rates were investigated lasting for over 90 days. Tissue sections were stained with haematoxylin-eosin. Results According to our preliminary data, it indicated the injection of iTregs in the prevention of aGVHD should be feasible (Fig.3). Consequently, we have tried to investigate preventative efficiency of repeated iTregs supplements in TCD-BM mice. First of all, we compared the single-dose of iTregs with the repetition-dose of iTregs in aGVHD prevention. The data showed in Fig.4. The data showed that the survival rate was 73.3% in repeated treatment in mice, however, the survival rate was only 45.8% in single-dose of iTregs mice within 24 days. As the TCD-BM survival rate was 76.1%. It indicated that the repetition-dose of iTregs would prevent the occurrence of aGVHD, and the survival rate was similar as the bone marrow transplantation mice. The BM-CD4T mice with aGVHD phenotype could survive no more than 10 days. Furthermore, we investigated the survival time of the continual iTreg supplements mice. The data showed in Fig.5. After 90 days later, the body weight of iTregs treated mice could maintain the recovery efficiency to 83.8±2.1% and the survival rate to 78%, comparing with the TCD-BM mice was 88.8±0.6% and 73%. All of these mice could keep alive more than 90 days. Using histographic staining, we confirmed the aGVHD prevention with repeated supplement of Foxp3+iTregs to the CD4T mice (Fig.6). The mice, administration of CD4T cells with bone marrow cells, failed to survival for the serious damage of intestine villi (Fig.6A) and Peyer's patches (Fig.6B). In contrast, CD4T mice with Foxp3+-iTregs (iTregs) could survival more than 90 days and intestine villi were recovered after 90 days (Fig.6A). Peyer's patches are an important gut associated lymphoid tissue in small intestine and play a crucial role in immune response. Therefore, we have investigated the changes of Peyer's patches (Fig.6B). As the recovery of mice with iTregs for twice, the Peyer's patches reappeared after 90 days later. It indicated that keeping more iTregs in vivo could more efficient on prevention of aGVHD. It indicated that more alive iTregs to prevent GVHD occurrence more efficient and may provide the information pre-clinically. Conclusion We showed that repetition supplement of iTreg cells to TCD-BM+CD4T-treated mice, could maintain the mice in high survival rate. Therefore, we may provide more of the functional iTregs to GVHD patients, continuously. It's a good way to prevent the occurrence of GVHD. The result should develop a novel-cell based approach for potentially reducing the risk of acute GVHD clinically. Disclosures No relevant conflicts of interest to declare.

Background: Regulatory T cells (Treg) play important roles in onset of graft-versus-host disease (GVHD) and chronic GVHD (cGVHD) in elderly people is more frequent than in younger people after allogeneic hematopoietic stem cell transplantation (HCT). However, little is known about the differences in the number and function of Treg and natural killer (NK) cells in elderly and young patients. In this study, we examined the cause of the difference in incidences of cGVHD in these patient populations by measuring Treg and NK cells over time after HCT. Patients and Methods: The subjects were 65 consecutive patients aged >18 years who underwent HCT for different hematologic diseases at our hospital between December 2008 and December 2018 and survived for >100 days after HCT. The median age was 57 years (range: 19-73 years), and those aged ≥58 (n=33) and ≤57 (n=32) were classified as elderly and younger patients, respectively. Evaluated variables included recipient and donor characteristics, and transplant-related factors, including conditioning regimen, donor type, degree of match, and GVHD prophylaxis. After obtaining informed consent, blood was drawn from all patients on day 100 and at 12-month intervals thereafter. Analysis of CD4+CD25+Foxp3+ Treg cells, CD3+CD4+ T cells, CD3+CD8+ T cells, CD19+ B cells, and CD3-CD56+ NK cells was performed using flow cytometry, and the absolute numbers of these cells were calculated. NK activity was measured by the standard 51Cr release assay at an effector:target (E:T) ratio of 20:1. Results: The incidence of grades II-IV acute GVHD was 29.2% at 100 days. There was no difference in the incidence of acute GVHD between elderly and younger patients [HR=0.85, 95% CI 0.34-2.09, p=0.72]. However, the overall incidence of cGVHD was 37.1%. In univariate analysis, the incidence of cGVHD in elderly patients was significantly higher than that in younger patients [HR=3.61, 95% CI 1.13-11.59, p=0.031]. Elderly patients had a significantly lower percentage of B cells than younger patients at 24 months (13.4±9.6% vs. 27.0±15.6%, p=0.024) and 36 months (16.4±10.3% vs. 31.1±11.2%, p Conclusion: The percentage and absolute number of Treg cells in elderly patients were significantly lower than those in younger patients, whereas CD8+ T cells were significantly higher in elderly patients after HCT. The absolute number of Treg cells tended to reconstitute one year after HCT in younger patients, but tended to decrease gradually in elderly patients, with no recovery even three years after HCT. In contrast, there was no change in NK cells. Similar results were obtained in an analysis excluding patients who developed cGVHD. These results suggest that a difference in immunological reconstitution between elderly and younger patients may explain the difference in the incidence of cGVHD between these patients. This may be useful in determining the choice of therapy with consideration of the immunologic mechanism. Disclosures Suzumiya: Celgene, Kyowa Kirin, Chugai-Roche, Eisai, Takeda, Celltrion, SymBio, Astellas, Ono, AstraZeneca, Ootsuka, Taiho, Mundi, Dainihon-Sumitomo: Research Funding.

Introduction: Aplastic anemia (AA) is an autoimmune-mediated disease with a complex mechanism. Innate as well as acquired immune system have been considered to participate in its pathogenesis. Although several studies have demonstrated the role of T and B cells in AA, there is limited research focusing on natural killer (NK) cells, which are important in innate and adaptive immune system. NK cells interact with T cells and alter their immune responses in a variety of diseases. Recent investigation suggests that NK cells serve immunoregulatory roles by attenuating autologous CD8+ T-cell response in AA (Chen et al. Cell Immunol 335:6-14, 2019). Here we assessed the kinetics of NK cells before and after immunosuppressive therapy (IST) in patients with AA to determine the association between NK cells and clinical courses. Methods: Patients with severe AA, in Japan, who required IST as first-line therapy were prospectively enrolled between May 2012 and October 2017. The IST regimen comprised rabbit anti-thymocyte globulin (ATG, thymoglobulin®, 2.5 or 3.5 mg/kg/day for 5 days), cyclosporine A (6 mg/kg/day for minimum 6 months), and methylprednisolone (2 mg/kg/day for 5 days) with subsequent tapering of the dose for 28 days. Flow cytometry was used to assess lymphocyte subsets, consisting of CD3+, CD4+, and CD8+ T cells, CD19+ B cells, CD56+ NK cells, and CD4+CD25+CD127+ regulatory T cells (Tregs), before and after ATG administration on days 0, 14, 28, 60, 90, 120, and 180. Plasma rabbit ATG levels were measured using a rabbit IgG ELISA kit on days 14 and 28 (Bethyl Laboratories, Montgomery, TX, USA). Receiver-operator characteristic (ROC) curves were generated to differentiate between response and no response to IST. Results: A total of 81 patients (aged 1.7-67.9 years) were randomized; 43 and 38 patients received 2.5 and 3.5 mg/kg of r-ATG, respectively. Median follow-up duration was 445 days (range; 183-2165 days) from first ATG infusion. After 6 months, 50 patients (58%) responded to the therapy, including 4 (5%) who achieved a complete response and 45 (56%) a partial response. The response rates did not differ significantly between the two dosages in ATG groups at 6 months (2.5 mg/kg, 63% vs. 3.5 mg/kg, 58%, P = 0.820). The median absolute number of NK cells before IST was 92/µL (0-1,085/µL). To judge patients' responses to IST, we defined 138/µL as the absolute number of NK cells count before IST as the cut-off value using ROC curves. The response rate of patients with higher NK cells (≥ 138 /mL) to IST at 6 months was significantly higher than those with lower NK cells (Figure 1, < 138 /mL, 78% vs. 52%, P = 0.031). Multivariate logistic regression analysis identified the absolute number of NK cells before IST and reticulocyte at diagnosis (odds ratio [OR] = 0.16; 95% confidence interval [CI], 0.04-0.62; P = 0.008 and OR = 0.20; 95% CI, 0.06-0.67; P = 0.009, respectively) and the r-ATG plasma concentration on day 28 (OR = 0.11; 95% CI, 0.03-0.39; P = 0.001) as independent predictors of response to IST. The median absolute number of NK cells on days 90, 120, and 180 after IST was higher in the responders (113/mL, 118/mL, and 159/mL, respectively) than in the non-responders (57/mL, 53/mL, and 64/mL, P = 0.001, P Conclusions: The current study indicates that NK cells play a role in the pathogenesis of AA and may be the predictors of response to IST. Recovery of NK cell count correlated with hematopoietic recovery following IST, indicating that they may be used as markers to assess disease activity, treatment response and relapse. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Ono Pharmaceutical: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Celgene: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria.

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an intractable T cell malignancy with regulatory T cell phenotype, which caused by long-term infection of human T-cell leukemia virus type-1 (HTLV-1). In addition to HTLV-1-derived oncogenic proteins such as Tax and HBZ, genomic aberrations including gain-of-function alterations in genes related to T-cell receptor (TCR) signaling pathway have been implicated in the pathogenesis of ATL. RLTPR is essential for CD28 co-stimulation in human T cells and loss-of-function mutations in RLTPR is reported to reduce proportions of regulatory T cells. The RLTPR p.Q575E has been reported as a recurrent mutation in cutaneous T cell lymphoma1. Here, we show that RLTPR p.Q575E is a novel recurrent gain-of-function mutation related to T-cell receptor co-stimulatory pathway in patients with acute type ATL. Methods To elucidate the genomic basis of ATL, we performed whole exome sequence (WES) in peripheral blood mononuclear cells (CD4/CD25 positive cells were sorted if ATL cell were not abundant) from 47 patients with acute-type ATL. Sequences were aligned against the reference genome (GRCh37/hg19) by using TMAP Alignment. Sanger sequencing was performed to confirm the variant obtained after WES genotyping for some genes including RLTPR. Clinical information regarding ATL cell phenotype, treatment and clinical course were also collected. To analyze the function of RLTPR Q575E, the human RLTPR isoform 3 wild type (WT) (most abundant isotype in lymphoid cells, RLTPR-WT hereafter) and RLTPR isoform 3 Q575E (RLTPR Q575E hereafter) expression retroviral vector were constructed. RLTPR isoform 1 WT and RLTPR isoform 1 Q575E were used as control. RLPTR WT or RLTPR Q575E transduced Jurkat cells were generated by retroviral transduction. NFAT, NF- κB or AP-1 promoter activity was measured as luciferase activity. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin calcium salt for TCR stimulation and CD86 Fc chimera for CD28 stimulation. IL-2 mRNA was quantitated by using Taqman Gene Expression Assays. Results Exome analysis from 47 acute-type ATL samples revealed gene alterations precipitated in TCR signaling pathway (CARD11;30.0%, PLCG1;27.7%, PRKCB;21.3%, STAT3;21.3%, VAV1;19.1%, NOTCH1;19.1%, RELA;12.8%, CNSK1A1;8.5%, IRF4;6.4%, FYN;2.1%, CBLB;2.1%, IKBKB;2.1%). In addition to these previously reported driver genes, a novel mutation, RLTPR Q575E was discovered in 4 out of 47 acute-type ATL samples (8.5%) with median variant allele frequency 0.52 (range 0.11-0.68). Although RLTPR Q575E has been reported in cutaneous T cell lymphoma, 3 out of 4 ATL patients carrying RLTPR Q575E mutation have no skin involvement by ATL. ATL patients carrying RLTPR Q575E were also harboring CARD11 (75%), PLCG1 (25%), PRKCB (25%), or IKBKB (25%) mutations, which are related to TCR/NF-κB signaling pathway. We next performed luciferase reporter assay to evaluate NF-κB activity in transfected 293T cells with RLTPR Q575E cDNA to explore its function. 293T cells transfected with RLTPR Q575E cDNA has higher NF-κB activity than those transfected with RLTPR wild type cDNA. No significant increased promoter activity of AP-1 or NFAT was observed with RLTPR Q575E. IL-2 mRNA was significantly increased in RLTPR Q575E transduced Jurkat cells under PMA/ionomycin and CD86 co-stimulation. We immunoprecipitated FLAG-tagged RLTPR WT or RLTPR Q575E and blotted for CARD11. We found that the RLTPR Q575E increases interaction of RLTPR with CARD11. Previously identified gene alterations such as CARD11 and PRKCB in ATL have been reported as Tax interactome, thus we further analyzed the interaction of RLTPR with Tax. We immunoprecipitated FLAG-tagged WT RLTPR or RLTPR Q575E and blotted for Tax. We found that the RLTPR WT and Q575E increases interaction of RLTPR with Tax. Conclusions In patients with acute-type ATL, we have identified RLTPR Q575E, a novel gain of function mutation, and functionally validated this mutation. This mutation has minimal effect without TCR co-stimulation, but patients with this mutation harboring gain-of-function mutations in TCR signaling molecules. We also found direct interaction between Tax and RLTPR. In the presence of TCR activation by Tax or gain-of-function mutations in this signaling pathway, RLTPR Q575E mutation selectively upregulates the NF-κB signaling pathway and confers the oncogenic effect on the pathogenesis of ATL. Reference 1 Joonhee Park, Jingyi Yang, Alexander T. Wenzel, Akshaya Ramachandran, Wung J. Lee, Jay C. Daniels, Juhyun Kim, Estela Martinez-Escala, Nduka Amankulor, Barbara Pro, Joan Guitart, Marc L. Mendillo, Jeffrey N. Savas, Titus J. Boggon, Jaehyuk Choi; Genomic analysis of 220 CTCLs identifies a novel recurrent gain-of-function alteration in RLTPR (p.Q575E). Blood 2017; 130 (12): 1430-1440. doi: https://doi.org/10.1182/blood-2017-02-768234 Disclosures Yoshimitsu: Novartis: Speakers Bureau; Bristol-Myer-Squibb,: Speakers Bureau; Shire: Speakers Bureau; Takeda: Speakers Bureau; Chugai: Speakers Bureau; Sanofi: Speakers Bureau. Ishitsuka:Novartis: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; Otsuka Pharmaceutical: Honoraria; Janssen Pharmaceutical: Honoraria; Taiho Pharmaceutical: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Shire: Honoraria; mundiharma: Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Teijin Pharma: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria; Teijin Pharma: Research Funding; Eli Lilly: Research Funding; Eisai: Honoraria, Research Funding; Mochida: Honoraria, Research Funding; sanofi: Honoraria; Pfizer: Honoraria; Alexion: Honoraria; Genzyme: Honoraria; Astellas Pharma: Honoraria, Research Funding; Asahi kasei: Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Yakult: Research Funding; MSD: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; MSD: Research Funding; Asahi kasei: Research Funding; Yakult: Research Funding; Bristol-Myers Squibb: Honoraria; Shire: Honoraria; Eli Lilly: Research Funding.

Despite the success of autologous anti-CD19 CAR T cell therapy in B-Acute lymphoblastic leukaemia (B-ALL) and Diffuse Large B Cell Lymphoma (DLBCL), treatment failures occur. One contributing factor may be the intrinsic T cell fitness of the CAR T cell product that is influenced by the underlying malignancy and prior treatments. With the advent of gene editing, 'off the shelf' non-HLA matched healthy donor (HD) CAR T cells are under investigation for the treatment of patients (pts) in clinical trials. UCART19 (S68587) is a first-in-class allogeneic CAR T cell product expressing a second generation anti-CD19 CAR with TALEN®-mediated gene knockouts of T cell receptor alpha chain (TRAC) and CD52 to prevent graft versus host disease and to render them resistant to anti-CD52 antibody used for lymphodepletion. Preliminary clinical trial data on the use of UCART19 in B-ALL was previously reported at ASH (Benjamin et al, 2018). The phenotypic and functional characteristics of CAR T cell products manufactured from B-ALL, Chronic Lymphocytic Leukaemia (CLL) and DLBCL pts were compared to young adult healthy donor (HD) CAR T cell products. In addition, potential effects related to knocking out TRAC in HD TCR-CAR T cells were examined. Thawed PBMCs from B-ALL, CLL, DLBCL pts and HDs underwent T cell enrichment, activation with anti-CD3/CD28 beads and IL-2, followed by transduction with anti-CD19 4-1BB CD3ζ lentiviral CAR construct and expansion. HD TCR- CAR T cells were manufactured by electroporation of HD CAR T cells with mRNA coding for TRAC TALEN® and residual TCRαβ+cells were removed by magnetic bead selection. CAR expression levels, T cell subsets, and exhaustion markers were examined by flow cytometry. Expression of activation markers CD25 and CD69 was measured in response to co-culture with the CD19+cell line NALM-6. Cytotoxicity against NALM-6 and Raji was assessed and antigen-mediated proliferation measured over 14 days. HD CAR T cells (n=11) expanded significantly more during manufacture than CAR T cells derived from B-ALL (n=9), CLL (n=8) or DLBCL (n=8) pts. As expected, the electroporation step resulted in a transient decrease in viability which recovered over time in culture (n=10). Median CAR expression level was higher on CLL CAR T cell products compared to those from B-ALL pts and HDs, thought to be due to a higher CD4:CD8 ratio in some CLL products. As a consequence of TCR knockout, CD3 expression was lost on HD TCR- CAR T cells (n=10), apart from a small population of γδ CAR T cells. CLL and DLBCL CD8+CAR+cells expressed higher levels of PD1 than HD CD8+CAR+cells. DLBCL CD4+CAR+cells also expressed significantly higher levels of PD1 than HD or HD TCR-CD4+CAR+T cells. CAR+CD8+CD27+PD1- T cells have been previously described as a functionally important population that correlated with clinical outcome in pts who received CLL CAR T cells (Fraietta et al, 2018). We found HD (n=13) and HD TCR- (n=10) CAR T cells had significantly more CD8+CD27+PD1- CAR T cells compared to those derived from CLL (n=8) and DLBCL (n=6) pts, but similar levels to B-ALL pts (n=10). In the absence of CD19 antigen, DLBCL CAR+CD8+ T cells (n=6) had greater expression of CD25 and CD69. However, in response to stimulation with CD19+ NALM-6 cells, HD (n=12), HD TCR- (n=10) and B-ALL (n=10) CAR T cells had higher fold increase in CD69+ cells compared to DLBCL (n=6) CAR T cells. On paired analysis (n=6), no difference was seen in activation in response to CD19 antigen on HD compared to HD TCR- CAR T cells. All CAR T cell products demonstrated comparable cytotoxicity against NALM-6 and Raji cell lines in short term in vitro assays. However, long-term cytotoxicity will be evaluated in a murine model. We performed a detailed comparison of the phenotypic and functional characteristics of CAR T cells derived from pts with B-cell malignancies and HDs. DLBCL CAR T cells showed lower antigen specific activation but higher baseline activation which could lead to more differentiated exhausted T cells. CAR T cells derived from HDs show a higher proportion of the therapeutically relevant CAR+CD8+CD27+PD1- cells compared to patients with mature B cell malignancies (CLL and DLBCL), which is maintained after TRAC knockout. This suggests allogeneic CAR T cells, such as UCART19, may provide a more effective product for pts with T cell dysfunction. Disclosures Graham: Gillead: Other: Funding to attend educational meeting; Servier: Research Funding. Jozwik:Servier: Research Funding. Metelo:Pfizer: Research Funding; Allogene: Research Funding. Almena-Carrasco:Servier: Employment. Peranzoni:Servier: Employment. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Dupouy:Servier: Employment. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Patten:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Roche: Honoraria, Research Funding. Benjamin:Amgen: Honoraria; Allogene: Research Funding; Gilead: Honoraria; Servier: Research Funding; Eusapharm: Consultancy; Pfizer: Research Funding; Takeda: Honoraria; Novartis: Honoraria.

Background: DNAM-1 (DNAX accessory molecule-1, CD226) is a costimulatory molecule that is constitutively expressed by NK cells and CD8+ T cells. Interaction between DNAM-1 on NK cells and CD8+ T cells with its ligands on target cells triggers cell-mediated cytotoxicity and cytokine production. Previous mouse model had showed that the expression of DNAM-1 is associated with NK education. Moreover, Monika's group found that DNAM-1 serves as an intrinsic, readout-independent marker for NK cell education in health donor. Our previous study had demonstrated that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells would achieve better functional education and contribute to least relapse for the patients post allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the roles of DNAM-1 in NK education post allo-HSCT were unknown. Aims: In this study, we have investigated the contribution of DNAM-1 expression to NK education post transplantation. Methods :We prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore the NK cell dynamic education at days 30, 90 and 180 post-transplant. Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry to evaluate of the phenotype as well as functional recovery of NK cells with different maturation expression levels of NKG2A, KIR, and CD57, as well as of activating receptors (NKp30, NKp46, NKG2D, DNAM-1) and CD25, CD122 as well as CD107a and IFN-gamma on NK cells. To study the correlation between the expression of DNAM-1 and effect of donor and host HLA on NK cell education, the expression of DNAM-1 on single-KIR+ NK cells (exhibiting NKG2A, KIR2DL1, KIR2DL2/KIR2DL3, or KIR3DL1 as their sole receptor) was compared based on the combination between donor/host HLA and donor inhibitory KIR. Results: The DNAM-1 expression on single-KIR+ NK cell ligated by sole donor HLA, or sole host HLA, or both donor and host HLA is higher compared to those single-KIR+ NK cells lacking ligands from donor or host or both. From the donor point of view, when donor only presented C1C1 ligand for KIR2DL2/L3, the MFI of DNAM-1 on single KIR2DL2/L3+ NK cells was significantly higher than KIR2DL1+ NK cells and KIR3DL1+ NK cells at 90day (P Summary: This study demonstrated that no matter from donor or/and host point of view, DNAM-1 expression contributes to optimal NK cells education post allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Introduction: Non-Hodgkin lymphoma (NHL), the most frequent hematological malignancy observed during pregnancy, is reported to be associated with advanced disease stage at diagnosis, essential involvement of reproductive organs, and a turbulent course. Immunosuppression is one of the key players in maternal-fetal tolerance that may have an impact on the tumor microenvironment (TME), thus contributing to cancer progression in this setting. Immunomodulatory cells, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), recruited by TME, are capable of inhibiting anti-tumor immune responses. The current study was designed to explore potential contribution of the pregnancy milieu to induction of immunosuppressive microenvironment supporting lymphoma growth and progression. Methods: A20 murine lymphoma B cells were injected subcutaneously to pregnant and non-pregnant female wild type Balb/C mice. The tumor volume was measured twice weekly and a multiple comparison was done with Bonferroni correction. Following delivery, mice from both groups were sacrificed and tumor cells were harvested. RNA was extracted from tumors of 5 control and 6 pregnant mice and RNAseq data were generated. Then, statistical analysis was performed for differentially expressed genes in these two groups. Tregs (CD4+CD25+) and MDSCs (CD11b+Gr1+) within tumors were estimated by FACS analysis. Results: Pregnant mice demonstrated significantly accelerated tumor growth compared to non-pregnant animals. The tumor volume in mice sacrificed after parturition reached 752.3 ± 492.7 mm3 versus 499.3 ± 373.5 mm3 in the control group (n = 41 and 29, respectively; p Conclusions: Pregnant mice demonstrated accelerated tumor growth following lymphoma cell inoculation. The tumor gene expression profile in pregnant mice is likely to reflect the cellular pathways that could be associated with differentiation and recruitment of immunosuppressive cells to the TME. In this regard, MDSCs emerge as potential candidates contributing to pregnancy-associated lymphoma progression. Further studies are warranted to define the kinetics of this cell subpopulation and identify biological effects of MDSCs in such setting. Disclosures No relevant conflicts of interest to declare.

Introduction: Novel therapies widely used in treatment of CLL and lymphoma, e.g. Bruton tyrosine kinase and phoshpoinotiside-3 kinase inhibitors, have complex immunomodulatory effects. Detailed understanding of the immune-modulatory effects of novel agents will help battle toxicities and inform the development of combination approaches in CLL and other lymphoid malignancies. Small ubiquitin-like modifier (SUMO) family proteins regulate target protein function by post-translational modification. Sumoylation regulates a variety of cellular processes, including nuclear import/export, transcriptional regulation, protein stability and cell cycle progression. While sumoylation has been shown to be deregulated in cancer and may contribute to carcinogenesis, it has also been implicated in T cell biology and function. Importantly, sumoylation may regulate NFκB signaling and PLCγ1-mediated NFAT activation, both indispensable for T-cell activation. Despite this, the role of sumoylation in broad aspects of T cell biology remain largely understudied. TAK-981 is a small molecule SAE inhibitor that forms an irreversible covalent adduct with SUMO molecules, thereby preventing transfer of SUMO from the E1 (SAE) to the E2 (Ubc9) enzyme, leading to a decrease in SUMO-conjugated proteins. Here, we investigated the immunomodulatory effects of TAK-981 in CLL ex vivo. Methods: Peripheral blood mononuclear cells were isolated from patients with CLL and T cells were purified using Dynabeads. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). For gene expression analysis, FACS-sorted naïve CD4+ T cells were pre-treated with TAK-981 for 1 hour and then subjected to concurrent T-cell receptor (TCR; αCD3/CD28) stimulation; RNA was harvested 3 or 24 hours after stimulation and analyzed on a Clariom S microarray chip. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for up to 7 days under Th1/2/17/Treg-polarizing conditions. Results: Protein sumoylation was induced within 15 minutes (SUMO1) and 24 hours (SUMO2/3) of TCR stimulation. Treatment with TAK-981 depleted nearly all polySUMO2/3-modified proteins but had less effect on SUMO1 conjugation in T cells at 24 hours. GSEA and Reactome pathway analysis of gene expression microarray data from TAK-981-treated CD4+ naïve TCR-stimulated T cells demonstrated minimal changes in NFκB- or NFAT-regulated genes. Significantly upregulated genes included those involved in transcriptional initiation/elongation (3 h), type I interferon response genes and PI3K/AKT signaling (24 h). Meanwhile, genes regulating cell cycle transition and DNA damage responses were downregulated. Activation (CD69, CD25, CD40L and HLA-DR) and survival of CD4+ andCD8+ T cell subpopulations was unimpeded by SAE inhibition within the first 24 hours of treatment, but was modestly reduced at 48 and 96 hours. T cell proliferation (CFSE, Edu) was reduced in a dose and time-dependent manner following exposure to TAK-981 (by 27.8% and 60.8% following treatment with 0.05 and 1.0 μM TAK-981 for 72 hours, respectively). Allogeneic T-cell cytotoxicity (using OCI-LY19 and OCI-LY3 lymphoma cells as target cells) was not disrupted by SAE inhibition. Remarkably, TCR-activated CD4+ naïve T cells treated with TAK-981 exhibited increased expression of CD38, a type I/II interferon response molecule [1]. Furthermore, sorted CD4+ naïve T cells showed enhanced IFNγ production, a type II IFN (as analyzed by flow cytometry), and increased TH1 differentiation in both TH1-polarized and non-polarized conditions. By contrast, differentiation of both TH17 and inducible regulatory T cells (iTregs) was reduced under the respective polarizing conditions. This was accompanied by diminished IL-2expression within the CD4+ T cell population. Conclusions: Our data suggest that targeting SAE may shift the T cell balance toward healthy immune cell subsets in CLL via induction of type I/II interferon response. TH1 polarization was accompanied by a reduction of immunosuppressive Treg phenotype. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. Bürgler, S., et al., Chronic Lymphocytic Leukemia Cells Express CD38 in Response to Th1 Cell-Derived IFN-γ by a T-bet-Dependent Mechanism. The Journal of Immunology, 2015. 194(2): p. 827-835. Disclosures Huszar: Takeda Pharmaceuticals: Employment, Equity Ownership. Danilov:Verastem Oncology: Consultancy, Other: Travel Reimbursement , Research Funding; Celgene: Consultancy; Aptose Biosciences: Research Funding; Seattle Genetics: Consultancy; Takeda Oncology: Research Funding; MEI: Research Funding; Pharmacyclics: Consultancy; AstraZeneca: Consultancy, Research Funding; Bristol-Meyers Squibb: Research Funding; Abbvie: Consultancy; Genentech: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Janssen: Consultancy; Gilead Sciences: Consultancy, Research Funding; TG Therapeutics: Consultancy; Curis: Consultancy.