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T cell-based immunotherapy has significantly improved treatment options for many malignancies. However, despite these and other therapeutic improvements over the last decades, gastrointestinal cancers, in particular pancreatic, hepatic and gastric cancer, are still characterized by high relapse rates and dismal prognosis, with an accordingly high unmet medical need for novel treatment strategies. We here report on the preclinical characterization of a novel bispecific antibody in an IgG-based format termed CC-3 with B7-H3xCD3 specificity. In many cancer entities including pancreatic, hepatic and gastric cancers, B7-H3 (CD276) is overexpressed on tumor cells and also on the tumor vasculature, the latter allowing for improved access of immune effector cells into the tumor site upon therapeutic targeting. We demonstrate that CC-3 induces profound T cell reactivity against various pancreatic, hepatic and gastric cancer cell lines as revealed by analysis of activation, degranulation and secretion of IL2, IFNγ as well as perforin, resulting in potent target cell lysis. Moreover, CC-3 induced efficient T cell proliferation and formation of T cell memory subsets. Together, our results emphasize the potential of CC-3, which is presently being GMP-produced to enable clinical evaluation for treatment of pancreatic, hepatic and gastric cancer.

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins1,2, combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18–80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4+ and CD8+ T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency., A phase I open-label trial evaluating the immunogencity, reactogenicity and safety of a peptide-based SARS-CoV-2 vaccine candidate to induce SARS-CoV-2-specific T cell responses.

Both B cells and T cells are involved in an effective immune response to SARS‐CoV‐2, the disease‐causing virus of COVID‐19. While B cells—with the indispensable help of CD4+ T cells—are essential to generate neutralizing antibodies, T cells on their own have been recognized as another major player in effective anti‐SARS‐CoV‐2 immunity. In this report, we provide insights into the characteristics of individual HLA‐A*02:01‐ and HLA‐A*24:02‐restricted SARS‐CoV‐2‐reactive TCRs, isolated from convalescent COVID‐19 patients. We observed that SARS‐CoV‐2‐reactive T‐cell populations were clearly detectable in convalescent samples and that TCRs isolated from these T cell clones were highly functional upon ectopic re‐expression. The SARS‐CoV‐2‐reactive TCRs described in this report mediated potent TCR signaling in reporter assays with low nanomolar EC50 values. We further demonstrate that these SARS‐CoV‐2‐reactive TCRs conferred powerful T‐cell effector function to primary CD8+ T cells as evident by a robust anti‐SARS‐CoV‐2 IFN‐γ response and in vitro cytotoxicity. We also provide an example of a long‐lasting anti‐SARS‐CoV‐2 memory response by reisolation of one of the retrieved TCRs 5 months after initial sampling. Taken together, these findings contribute to a better understanding of anti‐SARS‐CoV‐2 T‐cell immunity and may contribute to paving the way toward immunotherapeutics approaches targeting SARS‐CoV‐2., T cell receptors (TCRs) directed against previously identified SARS‐CoV‐2 target epitopes were isolated from convalescent COVID‐19 patients by single‐cell sequencing. Upon ectopic re‐expression, these SARS‐CoV‐2 TCRs conferred potent effector functions such as cytotoxicity and Interferon‐gamma production.

T cell-based immunotherapy, for example, with T cell-recruiting bispecific antibody (bsAb), has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions are still rare. Several tumor immune evasion mechanisms have been reported to counteract efficiency of T cell-engaging therapeutics. Platelets largely affect cancer pathophysiology by mediating tumor invasion, metastasis, and immune evasion. On treatment of patients in a clinical trial with a PSMA×CD3 bsAb (NCT04104607), we observed profound treatment-associated platelet activation, mirrored by a decrease of total platelet count. On modeling the treatment setting, we found that platelet activation significantly reduced bsAb-mediated CD4+ and CD8+ T-cell reactivity as revealed by impaired T-cell degranulation, secretion of perforin, and ultimately, inhibition of target cell lysis. This effect occurred in a transforming growth factor beta (TGF-β)-dependent manner and was not restricted to PSMA×CD3 bsAb, but rather observed with various CD3-directed bispecific constructs, including the approved CD19×CD3 bsAb blinatumomab. BsAb-mediated T-cell reactivity could be restored by platelet inhibition and specifically by blocking the TGF-β axis. Together, our findings demonstrate that platelets undermine the efficacy of T cell-recruiting bsAb and identify modulation of platelet function as a means to reinforce the effectiveness of bsAb treatment.

While several genetic and morphological markers are established and serve to guide therapy of acute myeloid leukaemia (AML), there is still profound need to identify additional markers to better stratify patients. CD105 (Endoglin) is a type I transmembrane protein reported to induce activation and proliferation of endothelial cells. In addition, CD105 is expressed in haematological malignancies and the vessels of solid tumours. Here, CD105 associates with unfavourable disease course, but so far no data are available on the prognostic relevance of CD105 in haematological malignancies. We here generated a novel CD105 antibody for analysis of expression and prognostic relevance of CD105 in a cohort of 62 AML patients. Flow cytometric analysis revealed substantial expression in the various AML FAB types, with FAB M3 type displaying significantly lower surface levels. Next we established a cut-off specific fluorescence level of 5.22 using receiver-operating characteristics, which allowed to group patients in cases with CD105lo and CD105hi surface expression and revealed that high CD105 expression correlated significantly with poor overall and progression free survival. In conclusion, we here identify CD105 expression as a novel prognostic marker in AML, which may serve to optimize follow up and treatment decisions for AML patients.

Antigen-specific immunotherapies, in particular peptide vaccines, depend on the recognition of naturally presented antigens derived from mutated and unmutated gene products on human leukocyte antigens, and represent a promising low-side-effect concept for cancer treatment. So far, the broad application of peptide vaccines in cancer patients is hampered by challenges of time- and cost-intensive personalized vaccine design, and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. In this study, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics. Comparative mass spectrometry-based immunopeptidome analyses of primary chronic lymphocytic leukemia (CLL) samples, as representative example of low-mutational burden tumor entities, and a dataset of benign tissue samples enabled the identification of high-frequent non-mutated CLL-associated antigens. These antigens were further shown to be recognized by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, and were evaluated as pre-manufactured warehouse for the construction of personalized multi-peptide vaccines in a first clinical trial for CLL (NCT04688385). This workflow for the design of peptide warehouses is easily transferable to other tumor entities and can provide the foundation for the development of broad personalized T cell-based immunotherapy approaches.

Soft tissue sarcoma (STS) constitutes a rare group of heterogeneous malignancies. Effective treatment options for most subtypes of STS are still limited. As a result, especially in metastatic disease, prognosis is still dismal. The ligands for the activating immunoreceptor NKG2D (NKG2DL) are commonly expressed in STS, but generally absent in healthy tissues. This provides the rationale for utilization of NKG2DL as targets for immunotherapeutic approaches. We here report on the preclinical characterization of bispecific fusion proteins (BFP) consisting of the extracellular domain of the NKG2D receptor fused to Fab-fragments directed against CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) for treatment of STS. After characterization of NKG2DL expression patterns on various STS cell lines, we demonstrated that both NKG2D-CD16 and NKG2D-CD3 induce profound T and NK cell reactivity as revealed by analysis of activation, degranulation and secretion of IFNγ as well as granule associated proteins, resulting in potent target cell lysis. In addition, the stimulatory capacity of the constructs to induce T and NK cell activation was analyzed in heavily pretreated STS patients and found to be comparable to healthy donors. Our results emphasize the potential of NKG2D-CD3 and NKG2D-CD16 BFP to target STS even in an advanced disease.

Long-term immunological memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for the development of population-level immunity, which is the aim of vaccination approaches. Reports on rapidly decreasing antibody titers have led to questions regarding the efficacy of humoral immunity alone. The relevance of T cell memory after coronavirus disease 2019 (COVID-19) remains unclear. Here, we investigated SARS-CoV-2 antibody and T cell responses in matched samples of COVID-19 convalescent individuals up to six months post-infection. Longitudinal analysis revealed decreasing and stable spike- and nucleocapsid-specific antibody responses, respectively. In contrast, functional T cell responses remained robust, and even increased, in both frequency and intensity. Single peptide mapping of T cell diversity over time identified open reading frame-independent, dominant T cell epitopes mediating long-term SARS-CoV-2 T cell responses. Identification of these epitopes may be fundamental for COVID-19 vaccine design., Longitudinal analysis identifies SARS-CoV-2 peptide targets of durable memory T cell responses in COVID-19 convalescent individuals.

Cancer patients, in particular patients with hematological malignancies, are at increased risk for critical illness upon COVID-19. We here assessed antibody as well as CD4+ and CD8+ T cell responses in unexposed and SARS-CoV-2-infected cancer patients to characterize SARS-CoV‑2 immunity and to identify immunological parameters contributing to COVID-19 outcome. Unexposed patients with hematological malignancies presented with reduced prevalence of pre-existing SARS-CoV-2 cross-reactive CD4+ T cell responses and signs of T cell exhaustion when compared to solid tumor patients and healthy volunteers. Whereas SARS-CoV-2 antibody responses did not differ between COVID-19 cancer patients and healthy volunteers, intensity, expandability, and diversity of SARS-CoV-2 T cell responses were profoundly reduced in cancer patients, and the latter associated with a severe course of COVID-19. This identifies impaired SARS-CoV-2 T cell immunity as determinant for dismal outcome of COVID-19 in cancer patients.

The prostate‐specific membrane antigen (PSMA) has been demonstrated in numerous studies to be expressed specifically on prostate carcinoma cells and on the neovasculature of several other cancer entities. However, the simultaneous expression of PSMA on both, tumor cells as well as tumor vessels remains unclear, even if such “dual” expression would constitute an important asset to facilitate sufficient influx of effector cells to a given tumor site. We report here on the generation of a PSMA antibody, termed 10B3, which exerts superior dual reactivity on sections of prostate carcinoma and squamous cell carcinoma of the lung. 10B3 was used for the construction of T‐cell recruiting bispecific PSMAxCD3 antibodies in Fab‐ and IgG‐based formats, designated Fabsc and IgGsc, respectively. In vitro, both molecules exhibited comparable activity. In contrast, only the larger IgGsc molecule induced complete and durable elimination of established tumors in humanized mice due to favorable pharmacokinetic properties. Upon treatment of three patients with metastasized prostate carcinoma with the IgGsc reagent, marked activation of T cells and rapid reduction of elevated PSA levels were observed., Insufficient penetration of immune cells and therapeutic antibodies into the tumor core is a major limitation in the immunotherapy field. This study reports the development of a novel bispecific antibody, named CC‐1, for improved dual targeting of tumor‐ and vascular cells in PSMA positive tumors.

Long-term immunity to SARS-CoV-2 is crucial for the development of herd immunity and the aim of vaccination approaches. Reports on rapidly decreasing antibody titers question the efficacy of humoral immunity. The relevance of T cell memory after COVID-19 is yet unclear. Longitudinal analysis of SARS-CoV-2 immunity in convalescents up to six months post-infection revealed decreasing and stable spike and nucleocapsid antibody responses, respectively. In contrast, T cell responses remained robust and even increased in frequency and intensity. Single epitope mapping of T cell diversity over time identified ORF-independent, dominant T cell epitopes mediating long-term SARS-CoV-2 T cell responses and may be fundamental for vaccine design. Funding: This work was supported by the Bundesministerium fur Bildung und Forschung (BMBF, FKZ:01KI20130; J.W.), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, Grant WA 4608/1-2; J.W.), the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy (Grant EXC2180-390900677; S.S., H.-G.R., H.R.S., J.W.), the German Cancer Consortium (DKTK; S.S., H.-G.R., H.R.S.), the Wilhelm Sander Stiftung (Grant 2016.177.2; J.W.), the Jose Carreras Leukamie-Stiftung (Grant DJCLS 05 R/2017; J.W.) and the Fortune Program of the University of Tubingen (Fortune number 2451-0-0 and 2581-0-0; J.W.). Conflict of Interest: H.-G.R. is shareholder of Immatics Biotechnologies GmbH and Curevac AG. A.N., T.B., H.-G.R., and J.S.W. hold patents on peptides described in this manuscript secured under the numbers 20 169 047.6 and 20 190 070.1. The other authors declare no competing interests. Ethical Approval: The study was approved by and performed according to the guidelines of the local ethics committees (179/2020/BO2).

Owing to their key role in several diseases including cancer, activating and inhibitory immune checkpoint molecules are increasingly exploited as targets for immunotherapy. Recently, we demonstrated that platelets, which largely influence tumor progression and immune evasion, functionally express the ligand of the checkpoint molecule GITR. This immunoreceptor modulates effector functions of T cells and NK cells with its function varying dependent on cellular context and activation state. Here, we provide a comparative analysis of platelet-derived GITRL (pGITRL) in breast cancer patients and healthy volunteers. The levels of pGITRL were found to be higher on platelets derived from cancer patients and appeared to be specifically regulated during tumor progression as exemplified by several clinical parameters including tumor stage/grade, the occurrence of metastases and tumor proliferation (Ki67) index. In addition, we report that pGITRL is upregulated during platelet maturation and particularly induced upon exposure to tumor-derived soluble factors. Our data indicate that platelets modulate the GITR/GITRL immune checkpoint in the context of malignant disease and provide a rationale to further study the GITR/GITRL axis for exploitation for immunotherapeutic intervention in cancer patients. Supplementary Information The online version of this article (10.1007/s00262-021-02866-y) contains supplementary material, which is available to authorized users.

The SARS-CoV-2 pandemic calls for the rapid development of diagnostic, preventive, and therapeutic approaches. CD4+ and CD8+ T cell-mediated immunity is central for control of and protection from viral infections[1-3]. A prerequisite to characterize T-cell immunity, but also for the development of vaccines and immunotherapies, is the identification of the exact viral T-cell epitopes presented on human leukocyte antigens (HLA)[2-8]. This is the first work identifying and characterizing SARS-CoV-2-specific and cross-reactive HLA class I and HLA-DR T-cell epitopes in SARS-CoV-2 convalescents (n = 180) as well as unexposed individuals (n = 185) and confirming their relevance for immunity and COVID-19 disease course. SARS-CoV-2-specific T-cell epitopes enabled detection of post-infectious T-cell immunity, even in seronegative convalescents. Cross-reactive SARS-CoV-2 T-cell epitopes revealed preexisting T-cell responses in 81% of unexposed individuals, and validation of similarity to common cold human coronaviruses provided a functional basis for postulated heterologous immunity[9] in SARS-CoV-2 infection[10,11]. Intensity of T-cell responses and recognition rate of T-cell epitopes was significantly higher in the convalescent donors compared to unexposed individuals, suggesting that not only expansion, but also diversity spread of SARS-CoV-2 T-cell responses occur upon active infection. Whereas anti-SARS-CoV-2 antibody levels were associated with severity of symptoms in our SARS-CoV-2 donors, intensity of T-cell responses did not negatively affect COVID-19 severity. Rather, diversity of SARS-CoV-2 T-cell responses was increased in case of mild symptoms of COVID-19, providing evidence that development of immunity requires recognition of multiple SARS-CoV-2 epitopes. Together, the specific and cross-reactive SARS-CoV-2 T-cell epitopes identified in this work enable the identification of heterologous and post-infectious T-cell immunity and facilitate the development of diagnostic, preventive, and therapeutic measures for COVID-19.

T cell immunity is central for the control of viral infections. To characterize T cell immunity, but also for the development of vaccines, identification of exact viral T cell epitopes is fundamental. Here we identify and characterize multiple dominant and subdominant SARS-CoV-2 HLA class I and HLA-DR peptides as potential T cell epitopes in COVID-19 convalescent and unexposed individuals. SARS-CoV-2-specific peptides enabled detection of post-infectious T cell immunity, even in seronegative convalescent individuals. Cross-reactive SARS-CoV-2 peptides revealed pre-existing T cell responses in 81% of unexposed individuals and validated similarity with common cold coronaviruses, providing a functional basis for heterologous immunity in SARS-CoV-2 infection. Diversity of SARS-CoV-2 T cell responses was associated with mild symptoms of COVID-19, providing evidence that immunity requires recognition of multiple epitopes. Together, the proposed SARS-CoV-2 T cell epitopes enable identification of heterologous and post-infectious T cell immunity and facilitate development of diagnostic, preventive and therapeutic measures for COVID-19.

Bispecific antibodies (bsAb) and chimeric antigen receptor (CAR) T cells allow for antibody guided recruitment of T cells against tumors. Both are successfully used for treatment of CD19 expressing leukemias, but may cause cytokine release syndrome (CRS) as a major dose-limiting side effect. For CRS prevention, steroids are recommended prior to bsAb treatment, despite their well-known lymphotoxic activity. The IL-6 receptor antibody tocilizumab is established for treatment of CRS induced by CAR T cells, but was not considered for CRS prevention in bsAb therapy. We here compared the influence of dexamethasone and tocilizumab on bsAb-mediated T cell proliferation and tumor lysis in vitro and in vivo and found that dexamethasone profoundly inhibited T cell proliferation and antitumor activity as induced by two different bsAb, particularly at low effector:target ratios, whereas tocilizumab did not affect efficacy. When we applied tocilizumab early during treatment of three patients with a newly developed PSMAxCD3 bsAb, significant CRS attenuation despite high IL-6 serum levels was observed. Thus, early IL-6 blockade may reduce the undesired sequelae of CRS upon bsAb therapy without affecting therapeutic activity, allowing in turn for safe application of effective doses.

BackgroundIn lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns. In B-cell acute lymphoblastic leukemia (B-ALL), bsAbs and CAR-T therapy targeting CD19 and CD22 have demonstrated clinical success, but resistance via antigen loss is common, motivating the development of agents focused on alternative targets. An attractive emerging target is FLT3, a proto-oncogene expressed in both AML and B-ALL, with low and limited expression on myeloid dendritic cells and HSCs.MethodsWe developed and characterized CLN-049, a T cell-activating bsAb targeting CD3 and FLT3, constructed as an IgG heavy chain/scFv fusion. CLN-049 binds the membrane proximal extracellular domain of the FLT3 protein tyrosine kinase, which facilitates the targeting of leukemic blasts regardless of FLT3 mutational status. CLN-049 was evaluated for preclinical safety and efficacy in vitro and in vivo.ResultsCLN-049 induced target-restricted activation of CD4+ and CD8+ T cells. AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed on treatment with subnanomolar concentrations of CLN-049, whereas FLT3-expressing hematopoietic progenitor cells and dendritic cells were not sensitive to CLN-049 killing. Treatment with CLN-049 also induced lysis of AML and B-ALL patient blasts by autologous T cells at the low effector-to-target ratios typically observed in patients with overt disease. Lysis of leukemic cells was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft models, CLN-049 was highly active against human leukemic cell lines and patient-derived AML and B-ALL blasts.ConclusionsCLN-049 has a favorable efficacy and safety profile in preclinical models, warranting evaluation of its antileukemic activity in the clinic.

Chronic lymphocytic leukemia (CLL) can be defined as a clonal expansion of B cells with stereotypic BCRs. Somatic hypermutation of the BCR heavy chains (IGVH) defines a subgroup of patients with a better prognosis. In up to 10% of CLL cases, a transformation to an aggressive B cell lymphoma (Richter’s syndrome) with a dismal prognosis can be observed over time. NFAT proteins are transcription factors originally identified in T cells, which also play an important role in B cells. The TCL1 transgenic mouse is a well-accepted model of CLL. Upon B cell-specific deletion of NFAT2, TCL1 transgenic mice develop a disease resembling human Richter’s syndrome. Whereas TCL1 B cells exhibit tonic anergic BCR signaling characteristic of human CLL, loss of NFAT2 expression leads to readily activated BCRs indicating different BCR usage with altered downstream signaling. Here, we analyzed BCR usage in wild-type and TCL1 transgenic mice with and without NFAT2 deletion employing conventional molecular biology techniques and next-generation sequencing (NGS). We demonstrate that the loss of NFAT2 in CLL precipitates the selection of unmutated BCRs and the preferential usage of certain VDJ recombinations, which subsequently results in the accelerated development of oligoclonal disease.

Background:There is renewed interest in the role of autoantibodies to predict outcomes in systemic sclerosis (SSc). Among the newly identified subsets, patients with limited cutaneous form (lcSSc) but anti-topoisomerase I antibodies (Scl70) positivity draw particular attention, and namely, assessing the risk of developing interstitial lung disease (ILD) —the main cause of death in SSc—to improve the management of Scl70-lcSSc patients.Objectives:We aimed to characterize patients with Scl70-lcSSc in the large multicenter European Scleroderma Trial and Research (EUSTAR) cohort.Methods:The EUSTAR database was locked in July 2019. We included all patients fulfilling 1980 ACR and/or 2013 ACR/EULAR criteria for SSc, with disease duration at database entry ≤3 yrs and known and stable skin form during the first 3 yrs. Patients with lcSSc were compared: Scl70-lcSSc (target group) vs. ACA-lcSSc and ANA-lcSSc (Step 1); and Scl70-lcSSc vs. Scl70-dcSSc (Step 2). In the ANA subgroup we included ANA+ patients with negative SSc-specific antibodies (Scl70, ACA, RNA polymerase III). In each step, we performed 5 generalized mixed models (GMM) for the risk of the new onset of ILD (defined by imaging), primary myocardial involvement (PMI), pulmonary hypertension (PH), “any severe” (ILD+PMI+PH+scleroderma renal crisis) and all-cause-mortality. An additional GMM assessed the risk of forced vital capacity (FVC) decline ≥10% vs. FVC value at ILD onset. Each GMM was adjusted for age, sex and confounders.Results:Overall, 1285 SSc patients were included: 1068 (83%) females, 860 (67%) lcSSc and 425 (33%) dcSSc. Among patients with lcSSc, 537 (62%) had ACA+, 194 (23%) Scl70+ and 129 (15%) ANA+; 425 patients had dcSSc and Scl70+. Median follow-up was similar in all 4 groups: 7.2 to 8.1 yrs.Step 1: At baseline, Scl70-lcSSc patients had significantly shorter time from Raynaud’s phenomenon (RP) to SSc onset, higher mRSS (5.8±4.8 vs. 4.3±4, p=0.001), and higher rate of articular and muscular involvement vs. ACA-lcSSc patients (Figure 1). No differences were found between Scl70-lcSSc and ANA-lcSSc comparing the aforementioned variables. ILD was more frequent in Scl70-lcSSc (46%) than in ACA-lcSSc (10%) and ANA-lcSSc (25%), as well as restrictive lung disease. GMM showed that Scl70-lcSSc carries a higher risk of ILD than both ACA-lcSSc (HR 4.55, 95%CI 3.23-6.67) and ANA-lcSSc (HR 2.17, 95%CI 1.39-3.45), with a rate of FVC decline ≥10% over time similar to the other limited forms. In Scl70-lcSSc patients the risk of “any severe” organ involvement was similar to ANA-lcSSc and higher than ACA-lcSSc (HR 1.89, 95%CI 1.40-2.50). In particular, Scl70-lcSSc shows a risk of PMI similar to ANA-lcSSc and lower than ACA-lcSSc; no differences regarding PH risk. The mortality risk in patients with Scl70-lcSSc was similar to the other limited forms’.Step 2: At baseline, time from RP to SSc onset was longer in patients with Scl70-lcSSc, with less frequent joint synovitis and tendon friction rubs vs. patients with Scl70-dcSSc. Conversely, the frequency of muscular, cardiac and pulmonary involvement was similar. The risk of ILD in Scl70-lcSSc patients was similar to Scl70-dcSSc, with a lower risk of FVC decline ≥10% over time. The risk of “any severe” involvement (HR 0.66, 95%CI 0.49-0.83), PMI and PH was lower and the mortality risk tended to be lower (HR 0.57, 95%CI 0.33-1.01, p=0.053) vs. Scl70-dcSSc.Conclusion:In our large multicenter EUSTAR cohort one quarter of lcSSc patients were Scl70+. We show a ranking for major organ involvement within lcSSc: Scl70 the most severe, ANA+ intermediate and ACA the milder form. Scl70-dcSSc patients present the most severe phenotype, and Scl70 positivity, more than the cutaneous subset, is strongly predictive of ILD, whereas other variables may influence progression. These results may provide new insight to improve the management of Scl70-lcSSc patients.Disclosure of Interests:Elisabetta Zanatta: None declared, Dörte Huscher: None declared, Paolo Airò: None declared, Alexandra Balbir-Gurman: None declared, Elise Siegert: None declared, Augusta Ortolan: None declared, Marco Matucci-Cerinic: None declared, Franco Cozzi: None declared, Gabriela Riemekasten: None declared, Anna-Maria Hoffmann-Vold: None declared, Oliver Distler Speakers bureau: has/had consultancy relationship and/or has received research funding in the area of potential treatments for systemic sclerosis and its complications from (last three years): Abbvie, Acceleron Pharma, Amgen, AnaMar, Arxx Therapeutics, Baecon Discovery, Blade Therapeutics, Bayer, Boehringer Ingelheim, ChemomAb, Corbus Pharmaceuticals, CSL Behring, Galapagos NV, Glenmark Pharmaceuticals, GSK, Horizon (Curzion) Pharmaceuticals, Inventiva, iQvia, Italfarmaco, iQone, Kymera Therapeutics, Lilly, Medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Novartis, Pfizer, Roche, Sanofi, Serodapharm, Topadur, Target Bioscience and UCB. Patent issued “mir-29 for the treatment of systemic sclerosis” (US8247389, EP2331143)., Consultant of: has/had consultancy relationship and/or has received research funding in the area of potential treatments for systemic sclerosis and its complications from (last three years): Abbvie, Acceleron Pharma, Amgen, AnaMar, Arxx Therapeutics, Baecon Discovery, Blade Therapeutics, Bayer, Boehringer Ingelheim, ChemomAb, Corbus Pharmaceuticals, CSL Behring, Galapagos NV, Glenmark Pharmaceuticals, GSK, Horizon (Curzion) Pharmaceuticals, Inventiva, iQvia, Italfarmaco, iQone, Kymera Therapeutics, Lilly, Medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Novartis, Pfizer, Roche, Sanofi, Serodapharm, Topadur, Target Bioscience and UCB. Patent issued “mir-29 for the treatment of systemic sclerosis” (US8247389, EP2331143)., Grant/research support from: Kymera Therapeutics, Mitsubishi Tanabe, Armando Gabrielli: None declared, Stefan Heitmann: None declared, Nicolas Hunzelmann: None declared, Carlomaurizio Montecucco: None declared, Jadranka Morovic-Vergles: None declared, Camillo Ribi: None declared, Andrea Doria: None declared, Yannick Allanore: None declared

Chronic lymphocytic leukaemia (CLL) is a clonal disorder of mature B cells. Most patients are characterised by an indolent disease course and an anergic phenotype of their leukaemia cells, which refers to a state of unresponsiveness to B cell receptor stimulation. Up to 10% of CLL patients transform from an indolent subtype to an aggressive form of B cell lymphoma over time (Richter´s syndrome) and show a significantly worse treatment outcome. Here we show that B cell-specific ablation of Nfat2 leads to the loss of the anergic phenotype culminating in a significantly compromised life expectancy and transformation to aggressive disease. We further define a gene expression signature of anergic CLL cells consisting of several NFAT2-dependent genes including Cbl-b, Grail, Egr2 and Lck. In summary, this study identifies NFAT2 as a crucial regulator of the anergic phenotype in CLL., NFAT2 is a transcription factor that has been linked with chronic lymphocytic leukaemia (CLL), but its functions in CLL manifestation are still unclear. Here the authors show, by analysing mouse CLL models and characterising biopsies from CLL patients, that NFAT2 is an important regulator for the anergic phenotype of CLL.

Even when achieving morphological complete remission (CR) after induction therapy, roughly half of acute myeloid leukemia (AML) patients display measurable residual disease (MRD) and eventually relapse. The surface receptor FLT3/CD135 is expressed on AML cells in almost all patients and constitutes a highly selective target antigen for immunotherapy, as expression on healthy tissues is limited to low levels on dendritic cells, monocytes and hematopoietic progenitor cells. FLYSYN is a chimeric Fc-optimized IgG1 antibody that binds specifically and with high avidity to human FLT3 (CD135). Here we report updated results of an open-label, single-arm, first in man multicenter trial (recruitment March 2017 to March 2020) evaluating safety/tolerability and preliminary efficacy of FLYSYN in patients with AML (NCT02789254). Morphological CR with stable or increasing MRD in two sequential measurements using central RT-qPCR and/or next generation sequencing (NGS) constituted the main inclusion criterion. FLYSYN was administered i.v. over 3 h as single application in cohorts 1-5 (0.5 mg/m², 1.5 mg/m², 5 mg/m², 15 mg/m², 45 mg/m²); in cohort 6, 15 mg/m² were applied on day 1, 15 and 29. Three patients were treated per cohort except for cohorts 4 and 6, which were expanded to comprise 9 and 10 patients, respectively. Molecular response was defined as >1 log MRD reduction or negativity in bone marrow (BM). In total, 31 patients (median age 58 years; range, 21-80 years; male:female ratio: 1:1.8) were enrolled, of which 27, 3 and 1 were MRD-positive for mutated NPM1, mutated IDH2 and RUNX1-RUNX1T1, respectively. Based on pharmacokinetic analysis, the half-life of FLYSYN was estimated to be 6.5 days. In 8 patients (26%), a transient decrease of neutrophil count (2 adverse events (AEs) grade 3, others ≤ grade 2) was observed. No relevant effect on stem cell reserve as assessed by colony forming unit assays was detected in the so far analyzed 26 patients. No other AE > grade 2 or dose-limiting toxicity were observed. The most frequent AEs were unspecific and comprised fatigue and flu like symptoms (12%), musculoskeletal symptoms (8%) and laboratory abnormalities (42%). With regard to efficacy, molecular response to treatment was achieved in 11/31 patients (35%), with so far two patients achieving MRD negativity documented one year after treatment. Among the patients receiving 45 mg/m2 FLYSYN (in total, upon single or repetitive dosing), objective responses were achieved in 46% (6/13) cases, whereas 28% (5/18) responded to treatment with lower doses. Together, the results of our phase I trial demonstrate that FLYSYN is safe and very well tolerated as monotherapy in AML patients with molecular MRD. Early efficacy data are promising and warrant further evaluation in an up-coming phase II clinical trial. Disclosures Heuser: PriME Oncology: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Stemline Therapeutics: Consultancy; Janssen: Consultancy; Astellas: Research Funding; Roche: Research Funding; BerGenBio ASA: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Amgen: Research Funding; Karyopharm: Research Funding; Abbvie: Consultancy. Thol:Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kapp-Schwoerer:Jazz Pharmaceuticals: Honoraria, Research Funding. Grosse-Hovest:Synimmune: Current Employment. Steiner:Synimmune: Current Employment. Schlenk:Roche: Research Funding; Novartis: Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses, Research Funding, Speakers Bureau; PharmaMar: Research Funding; AstraZeneca: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Salih:Pfizer: Consultancy; Medigene: Consultancy; Novartis: Consultancy; Philogen: Consultancy; Synimmune: Consultancy, Research Funding.

NFAT2 activity was shown to be of critical importance in B cell receptor signaling, development and proliferation; however its role in B cell development in the periphery is still not completely understood. We confirmed that NFAT2 deletion leads to impaired B1 B cell development, supported by our finding of limited B1 progenitors in the bone marrow and spleen of NFAT2 deficient mice. Moreover, we show for the first time that loss of NFAT2 increases immature B cells in particular transitional T2 and T3 as well as mature follicular B cells while marginal zone B cells are decreased. We further demonstrate that NFAT2 regulates the expression of B220, CD23, CD38, IgM/IgD and ZAP70 in murine B cells. In vivo analyses revealed decreased proliferation and increased apoptosis of NFAT2 deficient B cells. In summary, this study provides an extensive analysis of the role of NFAT2 in peripheral B lymphocyte development.

Background: Substantial surface expression of the FLT3 receptor can be measured on blast cells in 70% to 100% of acute myeloid leukemia (AML) patients, while no or only low levels are expressed on healthy cells like monocytes and progenitor stem cells. Thus, FLT3 is a suitable and highly selective target for therapeutic antibodies. FLYSYN is a chimeric Fc-optimized IgG1 antibody and binds specifically and with high avidity to human FLT3 (CD135). Despite achievement of complete remission, roughly half of AML patients display minimal residual disease (MRD) after end of therapy and relapse. Methods: We perform an open-label, single-arm, first in man multicenter trial to assess safety and tolerability as well as preliminary efficacy of FLYSYN as monotherapy in adult (≥18 years) AML patients in complete remission with MRD (NCT02789254). FLYSYN is administered as IV infusion over a 3 hour period. Recruitment started in March 2017 with an estimated maximum number of 31 patients and estimated recruitment until January 2020. The main inclusion criterion was confirmed stable or increasing MRD positivity in two sequential measurements. MRD was measured by central RT-qPCR and/or next generation sequencing (NGS). Sensitivity with RT-qPCR (for NPM1 only) was 10-6 and 10-4 with NGS. Patients with acute promyelocytic leukemia as well as prior hematopoietic stem cell transplantation were excluded. Using a "3 + 3" dose escalation design, five of the planned six cohorts with escalating doses have currently completed treatment (cohort 1: 0.5 mg/m² BSA; cohort 2: 1.5 mg/m² BSA; cohort 3: 5 mg/m² BSA; cohort 4: 15 mg/m² BSA; cohort 5: 45 mg/m²; cohort 6: in total 45 mg/m²; 15 mg/m² on day 1, 15 and 29). Three patients were treated per cohort, except for cohort 4, which was expanded to nine patients. The interim analysis for preliminary efficacy was performed after 18 patients were treated in cohorts 1-4. Response is defined as 1 log MRD reduction. Results: Median age was 60 years (range, 21-80 years). Sixteen patients were MRD positive for NPM1 and one patient each for RUNX1-RUNX1T1 and IDH2. So far, FLYSYN was well tolerated. Only one temporary grade 3 adverse event (AE) occurred (neutrophil decrease on day 3 only) in a patient of cohort 3, which was suspected to be related to FLYSYN treatment. There were no reported dose-limiting toxicities. The most frequently reported AEs were grade 1 and 2 gastrointestinal toxicities and laboratory abnormalities, which all were manageable with supportive care. After treatment, neither human anti-mouse nor anti-human antibodies were detected in any of the patients. Preliminary pharmacokinetic analysis was performed in the first 12 patients of the study and revealed a half-life of FLYSYN of roughly 6.5 days. Regarding preliminary efficacy, 1 patient of cohort 1 achieved permanent MRD negativity in bone marrow (BM; lasting until day 545) and 1 patient a temporary BM MRD reduction (at day 15). One patient of cohort 2 achieved BM MRD negativity (day 22) with MRD progression on day 365, whereas the other 2 patients were BM MRD progressive. None of the patients of cohort 3 achieved a MRD response in BM, but 2 patients achieved a temporary MRD response in peripheral blood. Four patients of cohort 4 achieved non-permanent BM MRD negativity. Overall, 6 patients achieved BM MRD negativity (33 %, 6/18), enduring more than 1 year in 1 patient. Conclusions: Our data suggest that FLYSYN is safe and very well tolerated as monotherapy in MRD positive AML patients. Preliminary efficacy data are promising, and recruiting is ongoing in cohort 6 in which 15 mg/m² FLYSYN is given for three times. Up-dated results will be presented at the ASH meeting. Disclosures Heuser: Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Steiner:Synimmune: Employment, Other: shareholder interest. Grosse-Hovest:SYNIMMUNE: Employment, Other: shareholder interest. Jung:Synimmune: Other: shareholder interest. Salih:SYNIMMUNE: Consultancy, Research Funding.

NFAT2 is a highly phosphorylated transcription factor which regulates developmental and activation programs in diverse cell types. We and others have previously described a significant overexpression of NFAT2 in CLL cells as compared to physiological B cells. Three major isoforms of NFAT2 with different regulatory properties have been described (700aa short isoform, 800aa intermediate isoform, 900 aa long isoform). Here, we analyzed the role of different NFAT2 transcripts in CLL with respect to disease phenotype and cell proliferation. We investigated primary samples from CLL patients (n=30) for their expression profile of different NFAT2 isoforms using RT-PCR. Applying an shRNA approach, we generated stable knock-down cells of the CLL cell line MEC-1 for the long and intermediate isoforms and for the entire NFAT2 gene resulting in the complete ablation of all isoforms. The proliferation properties of the different MEC-1 cell lines was subsequently assessed in xeno-transplant experiments into NSG mice. While physiological B cells express comparable levels of the short and intermediate/long isoforms, we could detect a five fold overexpression of the intermediate/long isoforms in primary CLL samples. To further analyze the differential regulation of the different NFAT2 transcripts on tumor cell proliferation and cell cycle regulation, we injected NSG mice with MEC-1 cells with intact NFAT2 (n=6), MEC-1 cells with a knock-down of the intermediate and long isoforms (n=6) and MEC-1 cells with a complete NFAT2 knock-down (n=6). MEC-1 cells with selective ablation of the intermediate and long NFAT2 isoforms grew significantly faster in NSG mice than MEC-1 cells with intact NFAT2 expression or MEC-1 cells with a complete NFAT2 knock-down. MEC-1 cells selectively lacking the intermediate and long isoforms led to accelerated tumor proliferation upon subcutaneous injection. Cell cycle analysis as assessed by flow cytometry showed a significantly increased number of cells in the G1/S-Phase for the group without expression of the short isoform, while the group with complete NFAT knock-down exhibited a compromised growth pattern as compared to wild-type MEC-1 cells. In summary, our data demonstrate that genetic loss of the intermediate and long isoforms of NFAT2 leads to CLL acceleration Disclosures No relevant conflicts of interest to declare.

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. While some patients exhibit enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggressive disease, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype referring to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Up to 10% of CLL patients transform to an aggressive form of lymphoma over time (Richter´s syndrome) and show a significantly worse treatment outcome. Here, we analyzed the role of NFAT2 in CLL with respect to maintenance of the anergic phenotype and the development of Richter´s syndrome. For this purpose, we generated conditional NFAT2fl/fl CD19-Cre knock out mice on the Eµ-TCL1 transgenic background. These mice exhibit NFAT2 deletion limited to the B cell lineage and develop a human-like CLL due to the B cell specific expression of the TCL1 oncogene. Conditional TCL1+NFAT2fl/fl CD19-Cre mice with B cell specific deletion of NFAT2 served as the experimental cohort while TCL1 transgenic mice without an NFAT2 deletion served as controls. To extend our observations do the human disease, we further analyzed primary blood samples from patients with CLL (n=30) and Richter´s syndrome (n=5). Mice with genetic loss of NFAT2 in their B cell compartment showed a significantly more aggressive disease course with accelerated accumulation of CD5+ CD19+ CLL cells in different organs, significantly higher proliferation rates and a dramatically reduced life expectancy (200 vs. 325 days) as compared to TCL1 control animals. Immunohistochemical analysis of spleen and lymph nodes upon necropsy showed a transformed disease phenotype in the NFAT2 ko cohort with diffuse distortion of lymph node and spleen architecture by large B cells with blastic chromatin while animals with intact NFAT2 expression showed small lymphocytic infiltrates with conserved B and T cell areas as expected. Using calcium imaging technology, we were able to show that CLL cells with regular NFAT2 expression were essentially unable to mobilize calcium upon IgM ligation and thus exhibited the typical anergic phenotype. CLL cells with NFAT2 deletion, on the contrary, showed a highly IgM-responsive phenotype with inducible activation of downstream signaling via AKT and ERK. We subsequently defined a genetic signature of anergy in CLL cells employing Affymetrix microarrays consisting of Lck, Pacsin1 and the E3 ligases Cbl and Grail, which were highly expressed in anergic CLL cells with normal NFAT2 expression and virtually absent upon NFAT2 ablation. We could further show by western blotting with phospho-specific antibodies that Lck was constitutively activated in anergic CLL cells while it was essentially inactive in NFAT2 ko cells suggesting an important role for Lck in mediating the anergic phenotype. Primary human CLL samples (n=30) showed a significant overexpression of NFAT2 and Lck, predominantly in its active conformation, as assessed by western blotting and RT-PCR. Tissue samples (lymph nodes, bone marrow) from patients with Richter's Syndrome (n=4) on the other hand demonstrated a significantly reduced expression of NFAT2 and complete loss of Lck expression. Calcium imaging revealed that a subset of human CLL cells showed an anergic phenotype and were unable to flux calcium while tumor cells from Richter patients exhibited an intensive spontaneous calcium flux as well as hyperresponsiveness upon IgM ligation. In summary, our data provide strong evidence that NFAT2 and its target gene Lck are important mediators of anergy induction in CLL and that their loss leads to disease transformation to aggressive B cell lymphoma (Richter´s syndrome). Taken together, our results demonstrate that the NFAT2-LCK axis plays an important role in the pathogenesis of CLL and implicate it as a potential target in its treatment. Disclosures No relevant conflicts of interest to declare.

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. A defined subgroup of patients shows enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggressive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype referring to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL and in anergy induction in CLL cells. For this purpose, we generated conditional CD19-Cre NFAT2 knock out mice, which exhibit NFAT2 deletion limited to the B cell lineage. To investigate the role of NFAT2 in the pathogenesis of CLL, we used the Eµ-TCL1 transgenic mouse model. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 weeks to which the animals eventually succumb at an average age of 10 months. We generated TCL1+NFAT2 ko mice with TCL1 transgenic mice without an NFAT2 deletion serving as controls. To identify novel NFAT2 target genes in CLL cells, we also performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays. In order to asses the anergic phenotype in CLL cells and the role of NFAT2 in its induction, we performed Ca2+ mobilization assays using a flow cytometric approach and performed Western Blots for multiple downstream signaling molecules. Mice with NFAT2 ko exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells in different organs, significantly higher proliferation rates and a dramatically reduced life expectancy (200 vs. 325 days) as compared to TCL1 control animals. To identify NFAT2 target genes responsible for the observed alterations in the disease phenotype, we subsequently performed a gene expression analysis with CLL cells from both leukemic cohorts. Here, we detected a substantially altered expression profile of genes associated with B cell anergy in the TCL1+NFAT2 ko mice. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1 and the E3 ligase Cbl representing the biggest hits. To further delineate the anergic phenotype and the role of NFAT2 in its induction, we subsequently performed Ca2+ mobilization assays. While anergic CLL cells from TCL1 mice exhibited an unresponsive phenotype with respect to Ca2+ flux upon IgM ligation, TCL1+NFAT2 ko mice showed an entirely normal capacity to mobilize intracellular Ca2+. Furthermore, IgM stimulation did not activate normal phosphotyrosine induction (phosphorylation of AKT and ERK kinases) in TCL1 mice while NAFT2-deficient CLL cells exhibited an unremarkable activation pattern with respect to AKT and ERK as assessed by Western Blotting. NFAT2-deficient CLL cells on the contrary exhibited compromised activation of the anergy regulator Lck as assessed by Y394 phosphorylation. Bypassing the BCR by antigen-independent stimulation with CD40 and LPS demonstrated slightly increased proliferation in anergic TCL1 CLL cells while NFAT2-deficient CLL cells exhibited massive proliferation. In summary, our data provide strong evidence that genetic loss of NFAT2 leads to more aggressive disease in CLL which is associated with the loss of the anergic phenotype. We could show that NFAT2 controls the expression of several important anergy-associated genes and identified Lck as a critical target of NFAT2 in this context. Taken together, our data demonstrate that the NFAT2-Lck axis plays an essential role in the pathogenesis of CLL and implicate it as a potential target in its treatment. Disclosures No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of mature B cells and constitutes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. The clinical course of CLL can be predicted by serveral prognostic markers like CD38, ZAP70 and cytogenetic abnormalities. While the treatment of CLL has significantly improved during recent years, it remains an essentially incurable disease and the molecular events that lead to its development are still largely elusive. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated by a network of several kinases. Several recent studies have demonstrated that Ca2+/NFAT signaling is involved in the pathogenesis of a wide array of different tumor types including pancreatic adenocarcinoma, breast cancer and Non Hodgkin´s lymphoma. In this study we investigated the significance of the Ca2+/NFAT signaling pathway in B-CLL. For this purpose, we analyzed CLL cell lines (MEC-1, JVM-3) as well as primary blood samples from patients with CLL (n=30). The analyzed patient population exhibited a representative distribution of age, sex, Binet stage, WBC count, cytogenetics and IGVH mutational status. We detected a profound overexpression of NFAT2 mRNA as well as NFAT2 protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited an at least three fold overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors. In one case, NFAT2 expression in CLL cells was 200 times higher than in the corresponding controls. This profound overexpression of NFAT2 in CLL cells could be confirmed on the protein level using Western Blotting and Immunocytochemistry. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in healthy B cells. NFAT2 nuclear translocation could be inhibited using pretreatment with Cyclosporin A demonstrating that this process was still calcineurin-dependent in CLL cells. We could further show that nuclear NFAT2 in CLL cells was able to bind DNA using electrophoretic mobility shift assays (EMSA). To assess the transcriptional activity of NFAT2 in human CLL we determined the expression of the apoptosis regulators OX40L, osteopontin and PD-L2, which we previously identified as NFAT2 target genes in a gene expression analysis with CD19+CD5+ CLL cells from TCL1 transgenic mice with intact NFAT2 and NFAT2 deletion, respectively. Interestingly, qRT-PCR revealed a tremendous reduction of all three target genes in the analyzed CLL samples as compared to control B cells from healthy donors. This is particularly remarkable, since in the TCL1 mouse model we observed a similar reduction of the expression of these genes in CLL cells with NFAT2 ablation. In summary, these results provide strong evidence that the Ca2+/NFAT signaling axis is constitutively activated in CD19+CD5+ CLL cells. Our data suggest that the profound overexpression of NFAT2 in CLL cells leads to its targeting to aberrant genetic loci different from its phsiological target genes resulting in a consecutive knock out phenotype with respect to the expression of the apoptosis regulators OX40, osteopontin and PD-L2 in CLL. Further investigation is therefore warranted to decipher the therapeutic potential of modulating the Ca2+/Calcineurin/NFAT signaling pathway in this disease. Disclosures: No relevant conflicts of interest to declare.

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. Expression of CD38 and ZAP70 define a subgroup of patients with enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggessive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype refering to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL and in anergy induction in CLL cells. For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eµ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eµ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 wks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). To identify novel NFAT2 target genes in CLL cells, we performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays. Mice with NFAT2 knock out exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells and a significantly reduced life expectancy (200 vs. 325 days) as compared to control animals. Flow cytometric analysis at distinct time points showed a pronounced infiltration by CD5+ B cells of the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko exhibited significantly higher rates of proliferation than control animals. To identify NFAT2 target genes resonsible for the observed alterations in the disease phenotype, we subsequently peformed a gene expression analysis with CD5+CD19+ CLL cells from TCL1+NFAT2 ko mice with CLL cells from TCL1+ mice serving as controls. Here, we detected a significantly altered expression of 22 genes associated with B cell anergy in the TCL1+NFAT2 ko cohort. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1, Hspa14 and CD166 constituting the strongest hits with up to 10fold reduced gene expression. Downregulation of the identified target genes was subsequently confirmed using RT-PCR and Western Blotting. In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells. In addition, we could show that NFAT2 controls the expression of several anergy-associated genes and that its absence prevents the acquisition of an anergic phenotype by the CLL cells correlating with a significantly more aggressive course of the disease. Taken together, our data demonstrate that NFAT2 plays an essential role in the pathogenesis of CLL and implicate this transcription factor as a potential target in its treatment. Disclosures: No relevant conflicts of interest to declare.

7019 Background: CLL is a malignancy of mature B cells and constiututes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the significance of NFAT signaling in B-CLL. Methods: NFAT2 expression and aberrant nuclear translocation in CLL cells (n=30) was assessed by Western Blotting and immunofluorescence. In addition, NFAT2 mRNA levels were measured by qRT-PCR and its DNA binding capacity was assessed using an electrophoretic mobility shift assay. Transcriptional activity of NFAT2 proteins in CLL cells was further analyzed by determining the expression of several well characterized NFAT target genes. Results: We detected a profound overexpression of NFAT2 mRNA and protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited a significant overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors (8-200fold). This overexpression of NFAT2 in CLL cells could also be confirmed on the protein level. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in non-malignant B cells. Nuclear NFAT2 in CLL cells was able to bind DNA but its transcriptional activity with respect to several apoptosis-regulating genes (i.e. Spp1, Pdcd1) was severely compromised. Conclusions: These results provide strong evidence that the Ca2+/NFAT signalling axis is constitutively activated in CD5+CD19+ CLL cells. Reduced expression of several apoptosis regulators which are known target genes of NFAT2 links deregulation of this signaling cascade to CLL progression. Further investigation is warranted to investigate the therapeutic potential of modulating Ca2+/Calcineurin/ NFAT signaling in CLL.

Abstract 862 NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated and relocated to the cytoplasm by a network of several kinases. Although identified originally as a major transcriptional regulator in T cells, it is now clear that NFAT transcription factors also possess important roles in other cells of the hematopoietic system including dendritic cells, mast cells, megakaryocytes and B cells. Several recent studies have demonstrated that Calcineurin/NFAT signaling is involved in the pathogenesis of a wide array of hematological malignancies including diffuse large B cell lymphoma, CLL as well as Burkitt and Burkitt-like lymphomas. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL. For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice, in which the Cre recombinase is expressed under the control of the B cell-specific CD19 promoter. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eμ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eμ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 weeks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). Mice with NFAT2 knock out exhibited a significantly accelerated accumulation of CD5+CD19+ CLL cells as compared to control animals. Flow cytometric analysis at distinct time points showed a tremendous infiltration by CD5+ B cells in the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. At approximately 26 weeks of age, NFAT2 ko mice showed an approximately 40 fold increased lymphocyte count in the peripheral blood than their litter mate controls (1500/μL vs. 60000/μL). Splenomegaly and lymphatic adenopathy was also significantly increased in the NFAT ko population. Furthermore, NFAT2 ko mice showed a dramatically reduced median survival (200 vs. 325 days) and maximum survival (265 vs. 398 days) in comparison to regular TCL1 transgenic mice. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko at an age of approximately 7 months exhibited significantly higher rates of proliferation than control animals. In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells implicating Ca2+/NFAT signaling as a potential target for the treatment of this disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 3909 Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of mature B cells and constitutes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. The clinical course of CLL can be predicted by serveral prognostic markers like CD38, ZAP70 and cytogenetic abnormalities. While the treatment of CLL has significantly improved during recent years, it remains an essentially uncurable disease and the molecular events that lead to its development are still largely ellusive. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated by a network of several kinases. Several recent studies have demonstrated that Ca2+/NFAT signaling is involved in the pathogenesis of a wide array of different tumor types including pancreatic adenocarcinoma, breast cancer and Non Hodgkin′s lymphoma. In this study we investigated the significance of the Ca2+/NFAT signaling pathway in B-CLL. For this purpose, we analyzed CLL cell lines (MEC-1, JVM-3) as well as primary blood samples from patients with CLL (n=30). The analyzed patient population exhibited a representative distribution of age, sex, Binet stage, WBC count, cytogenetics and IGVH mutational status. PBMC were obtained by ficoll density gradient centrifugation and B cells were subsequently isolated using magnetic bead technology. NFAT2 expression and aberrant nuclear translocation was then assessed by Western Blotting and Immunofluorescence staining. In addition, NFAT2 mRNA levels were measured by qRT-PCR and its DNA binding capacity was assessed using an electrophoretic mobility shift assay (EMSA). Transcriptional activity of NFAT2 proteins in CLL cells was further analyzed by determining the expression of several well characterized NFAT target genes by qRT-PCR and Western Blotting. In our analysis, we detected a profound overexpression of NFAT2 mRNA as well as NFAT2 protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited an at least three fold overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors. In one case, NFAT2 expression in CLL cells was 200 times higher than in the corresponding control cell population. This profound overexpression of NFAT2 in CLL cells could be confirmed on the protein level using Western Blotting and Immunocytochemistry. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in healthy B cells. NFAT2 nuclear translocation could be inhibited using pretreatment with Cyclosporin A demonstrating that this process was still calcineurin-dependent in CLL cells. We could further show that nuclear NFAT2 in CLL cells was able to bind DNA but that its transcriptional activity with respect to several apoptosis and cell cycle regulating genes was severely compromised when compared with healthy CD19+CD5+ B cells. In summary, these results provide strong evidence that the Ca2+/NFAT signaling axis is con-stitutively activated in CD19+CD5+ CLL cells. Reduced expression of several apoptosis and cell cycle regulating proteins which are known target genes of NFAT2 potentially links deregulation of this signaling cascade to CLL progression. This is in line with our observation that genetic loss of NFAT2 can induce acceleration of CLL in TCL1 mouse model. Further investigation is warranted to decipher the therapeutic potential of modulating Ca2+/Calcineurin/NFAT signaling in CLL. Disclosures: No relevant conflicts of interest to declare.