SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition.

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.

Targeting MDM2 enhances antileukemia immunity after allogeneic transplantation via MHC-II and TRAIL-R1/2 upregulation.

Patients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell-mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2 inhibition.

Soluble NKG2DLs Are Elevated in Breast Cancer Patients and Associate with Disease Outcome.

Ligands of the natural killer group 2D (NKG2DL) family are expressed on malignant cells and are usually absent from healthy tissues. Recognition of NKG2DLs such as MICA/B and ULBP1-3 by the activating immunoreceptor NKG2D, expressed by NK and cytotoxic T cells, stimulates anti-tumor immunity in breast cancer. Upregulation of membrane-bound NKG2DLs in breast cancer has been demonstrated by immunohistochemistry. Tumor cells release NKG2DLs via proteolytic cleavage as soluble (s)NKG2DLs, which allows for effective immune escape and is associated with poor prognosis. In this study, we collected serum from 140 breast cancer (BC) and 20 ductal carcinoma in situ (DCIS) patients at the time of initial diagnosis and 20 healthy volunteers (HVs). Serum levels of sNKG2DLs were quantified through the use of ELISA and correlated with clinical data. The analyzed sNKG2DLs were low to absent in HVs and significantly higher in BC patients. For some of the ligands analyzed, higher sNKG2DLs serum levels were associated with the classification of malignant tumor (TNM) stage and grading. Low sMICA serum levels were associated with significantly longer progression-free (PFS) and overall survival (OS). In conclusion, we provide the first insights into sNKG2DLs in BC patients and suggest their potential role in tumor immune escape in breast cancer. Furthermore, our observations suggest that serum sMICA levels may serve as a prognostic parameter in the patients analyzed in this study.

Targeting NKG2DL with Bispecific NKG2D-CD16 and NKG2D-CD3 Fusion Proteins on Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer with a poor response rate to conventional systemic treatment and high relapse rates. Members of the natural killer group 2D ligand (NKG2DL) family are expressed on cancer cells but are typically absent from healthy tissues; thus, they are promising tumor antigens for novel immunotherapeutic approaches. We developed bispecific fusion proteins (BFPs) consisting of the NKG2D receptor domain targeting multiple NKG2DLs, fused to either anti-CD3 (NKG2D-CD3) or anti-CD16 (NKG2D-CD16) Fab fragments. First, we characterized the expression of the NKG2DLs (MICA, MICB, ULBP1-4) on TNBC cell lines and observed the highest surface expression for MICA and ULBP2. Targeting TNBC cells with NKG2D-CD3/CD16 efficiently activated both NK and T cells, leading to their degranulation and cytokine release and lysis of TNBC cells. Furthermore, PBMCs from TNBC patients currently undergoing chemotherapy showed significantly higher NK and T cell activation and tumor cell lysis when stimulated with NKG2D-CD3/CD16. In conclusions, BFPs activate and direct the NK and T cells of healthy and TNBC patients against TNBC cells, leading to efficient eradication of tumor cells. Therefore, NKG2D-based NK and T cell engagers could be a valuable addition to the treatment options for TNBC patients.

Identification of CD105 (endoglin) as novel risk marker in CLL.

Several genetic and clinical markers are established as prognostic factors in chronic lymphocytic leukemia (CLL). However, additional markers are needed for risk stratification. Flow cytometric analysis is a mainstay of CLL diagnostics, thus identification of novel prognostic surface markers can improve risk assessment without increasing burden for patients and physicians. Furthermore, surface molecules preferentially expressed in high-risk cases could serve as therapeutic targets for immunotherapy. CD105 (endoglin) is a TGF-beta coreceptor and activates endothelial cells in healthy tissues and cancer. In addition, it is expressed on healthy hematopoietic precursors as well as lymphoid and myeloid leukemias. In acute myeloid leukemia (AML), a CD105 antibody is successfully applied in clinical studies. In CLL, mRNA expression of the CD105 gene ENG reportedly correlates with other risk factors but failed to show significant correlation with overall survival. However, CD105 protein expression in CLL has never been studied. We here analyzed CD105 surface expression on CLL cells from 71 patients by flow cytometry and report for the first time that substantial levels of CD105 are detectable on CLL cells in 70.4% of patients. Using receiver operating characteristics, we established a cutoff of 5.99% positive cells to distinguish between low and high CD105 levels, the latter correlating with decreased time to first treatment and overall survival. High CD105 expression further correlates with CD38 expression. Our study identified membrane expression of CD105 as a potential risk marker and therapeutic target in high-risk CLL. However, multivariant analyses of large cohorts should be performed in confirmatory studies.

Pivotal role of PDK1 in megakaryocyte cytoskeletal dynamics and polarization during platelet biogenesis.

During thrombopoiesis, megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Phosphoinositide-dependent protein kinase-1 (PDK1) is required for Ca2+-dependent platelet activation, but its role in MK development and regulation of platelet production remained elusive. The present study explored the role of PDK1 in the regulation of MK maturation and polarization during thrombopoiesis using a MK/platelet-specific knockout approach. Pdk1-deficient mice (Pdk1-/-) developed a significant macrothrombocytopenia as compared with wild-type mice (Pdk1fl/fl). Pdk1 deficiency further dramatically increased the number of MKs without sinusoidal contact within the BM hematopoietic compartment, resulting in a pronounced MK hyperplasia and a significantly increased extramedullary thrombopoiesis. Cultured Pdk1-/- BM-MKs showed impaired spreading on collagen, associated with an altered actin cytoskeleton structure with less filamentous actin (F-actin) and diminished podosome formation, whereas the tubulin cytoskeleton remained unaffected. This phenotype was associated with abrogated phosphorylation of p21-activated kinase (PAK) as well as its substrates LIM domain kinase and cofilin, supporting the hypothesis that the defective F-actin assembly results from increased cofilin activity in Pdk1-deficient MKs. Pdk1-/- BM-MKs developed increased ploidy and exhibited an abnormal ultrastructure with disrupted demarcation membrane system (DMS). Strikingly, Pdk1-/- BM-MKs displayed a pronounced defect in DMS polarization and produced significantly less proplatelets, indicating that PDK1 is critically required for proplatelet formation. In human MKs, genetic PDK1 knockdown resulted in increased maturity but reduced platelet-like particles formation. The present observations reveal a pivotal role of PDK1 in the regulation of MK cytoskeletal dynamics and polarization, proplatelet formation, and thrombopoiesis.

The HLA ligandome landscape of chronic myeloid leukemia delineates novel T-cell epitopes for immunotherapy.

Antileukemia immunity plays an important role in disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Thus, antigen-specific immunotherapy holds promise for strengthening immune control in CML but requires the identification of CML-associated targets. In this study, we used a mass spectrometry-based approach to identify naturally presented HLA class I- and class II-restricted peptides in primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimens and samples from CML patients in deep molecular remission delineated a panel of novel frequently presented CML-exclusive peptides. These nonmutated target antigens are of particular relevance because our extensive data-mining approach suggests the absence of naturally presented BCR-ABL- and ABL-BCR-derived HLA-restricted peptides and the lack of frequent tumor-exclusive presentation of known cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells de novo in samples from healthy volunteers and CML patients. Thus, these antigens are prime candidates for T-cell-based immunotherapeutic approaches that may prolong TKI-free survival and even mediate cure of CML patients.

C-Cbl regulates c-MPL receptor trafficking and its internalization.

Thrombocyte formation from megakaryocyte and their progenitor cells is tightly regulated by thrombopoietin (TPO) and its receptor c-MPL, thereby maintaining physiological functionality and numbers of circulating platelets. In patients, dysfunction of this regulation could cause thrombocytopenia or myeloproliferative syndromes. Since regulation of this pathway is still not completely understood, we investigated the role of the ubiquitin ligase c-Cbl which was previously shown to negatively regulated c-MPL signalling. We developed a new conditional mouse model using c-Cblfl/fl Pf4Cre mice and demonstrated that platelet-specific knockout of c-Cbl led to severe microthrombocytosis and impaired uptake of TPO and c-MPL receptor internalization. Furthermore, we characterized a constitutive STAT5 activation c-Cbl KO platelets. This study identified c-Cbl as a potential player in causing megakaryocytic and thrombocytic disorders.

Genetic loss of NFAT2 (NFATc1) impairs B cell development of B1 and B2 B cells.

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.

Identification of CD318 (CDCP1) as novel prognostic marker in AML.

Genetic and morphological markers are well-established prognostic factors in acute myeloid leukemia (AML). However, further reliable markers are urgently needed to improve risk stratification in AML. CD318 (CDCP1) is a transmembrane protein which in solid tumors promotes formation of metastasis and correlates with poor survival. Despite its broad expression on hematological precursor cells, its prognostic significance in hematological malignancies so far remains unclear. Here, we evaluated the role of CD318 as novel prognostic marker in AML by immunophenotyping of leukemic blasts. Flow cytometric evaluation of CD318 on leukemic cells in 70 AML patients revealed a substantial expression in 40/70 (57%) of all cases. CD318 surface levels were significantly correlated with overall survival in patients receiving anthracycline-based induction therapy or best available alternative therapy. Using receiver-operating characteristics, we established a cut-off value to define CD318lo and CD318hi expression in both cohorts. Notably, high CD318 expression correlated inversely as prognostic marker in both treatment cohorts: as poor prognostic marker in patients receiving intense therapy, whereas upon palliative care it correlated with better outcome. In conclusion, FACS-based determination of CD318 expression may serve as novel prognostic factor depending on implemented therapy in AML patients.

CK2ß regulates thrombopoiesis and Ca2+-triggered platelet activation in arterial thrombosis.

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.

Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling.

UNLABELLED: Polymorphonuclear neutrophil granulocytes (neutrophils) are tightly controlled by an incompletely understood homeostatic feedback loop adjusting the marrow's supply to peripheral needs. Although it has long been known that marrow cellularity is inversely correlated with G-CSF levels, the mechanism linking peripheral clearance to production remains unknown. Herein, the feedback response to antibody induced neutropenia is characterized to consist of G-CSF­dependent shifts of marrow hematopoietic progenitor populations including expansion of the lin-/Sca-1/c-kit (LSK) and granulocyte macrophage progenitor (GMP) compartments at the expense of thrombopoietic and red cell precursors. Evidence is provided that positive feedback regulation is independent from commensal germs as well as T, B, and NK cells. However, in vivo feedback is impaired in TLR4-/- and TRIF-/-, but not MyD88-/- animals. In conclusion, steady-state neutrophil homeostasis is G-CSF­dependent and regulated through pattern-recognition receptors,thereby directly linking TLR-triggering to granulopoiesis. KEY POINTS: Steady-state and emergency granulopoiesis are both dependent on TLR signaling.

The bispecific B7H3xCD3 antibody CC-3 induces T cell immunity against bone and soft tissue sarcomas.

Sarcomas are rare and heterogeneous malignancies that are difficult to treat. Approximately 50% of patients diagnosed with sarcoma develop metastatic disease with so far very limited treatment options. The transmembrane protein B7-H3 reportedly is expressed in various malignancies, including different sarcoma subtypes. In several cancer entities B7-H3 expression is associated with poor prognosis. In turn, B7-H3 is considered a promising target for immunotherapeutic approaches. We here report on the preclinical characterization of a B7-H3xCD3 bispecific antibody in an IgG-based format, termed CC-3, for treatment of different sarcoma subtypes. We found B7-H3 to be expressed on all sarcoma cells tested and expression on sarcoma patients correlated with decreased progression-free and overall survival. CC-3 was found to elicit robust T cell responses against multiple sarcoma subtypes, resulting in significant activation, release of cytokines and effector molecules. In addition, CC-3 promoted T cell proliferation and differentiation, resulting in the generation of memory T cell subsets. Finally, CC-3 induced potent target cell lysis in a target cell restricted manner. Based on these results, a clinical trial evaluating CC-3 in soft tissue sarcoma is currently in preparation.

Asciminib Maintains Antibody-Dependent Cellular Cytotoxicity against Leukemic Blasts.

B cell acute lymphoblastic leukemia (B-ALL) is characterized by an accumulation of malignant precursor cells. Treatment consists of multiagent chemotherapy followed by allogeneic stem cell transplantation in high-risk patients. In addition, patients bearing the BCR-ABL1 fusion gene receive concomitant tyrosine kinase inhibitor (TKI) therapy. On the other hand, monoclonal antibody therapy is increasingly used in both clinical trials and real-world settings. The introduction of rituximab has improved the outcomes in CD20 positive cases. Other monoclonal antibodies, such as tafasitamab (anti-CD19), obinutuzumab (anti-CD20) and epratuzumab (anti-CD22) have been tested in trials (NCT05366218, NCT04920968, NCT00098839). The efficacy of monoclonal antibodies is based, at least in part, on their ability to induce antibody-dependent cellular cytotoxicity (ADCC). Combination treatments, e.g., chemotherapy and TKI, should therefore be screened for potential interference with ADCC. Here, we report on in vitro data using BCR-ABL1 positive and negative B-ALL cell lines treated with rituximab and TKI. NK cell activation, proliferation, degranulation, cytokine release and tumor cell lysis were analyzed. In contrast to ATP site inhibitors such as dasatinib and ponatinib, the novel first-in-class selective allosteric ABL myristoyl pocket (STAMP) inhibitor asciminib did not significantly impact ADCC in our settings. Our results suggest that asciminib should be considered in clinical trials.

Expression and Prognostic Value of a Novel B7-H3 (CD276) Antibody in Acute Myeloid Leukemia.

Despite recent advances in immunophenotyping, the prognosis of acute myeloid leukemia (AML) is still mainly estimated using age and genetic markers. As the genetic heterogeneity of AML patients is high, flow cytometry-based classification with appropriate biomarkers can efficiently complement risk stratification and treatment selection. An increased expression of B7-H3 (CD276), an immune checkpoint protein, has been reported and associated with poor prognosis. However, the available data are limited and heterogeneous. Here, we used a novel, proprietary murine anti-B7-H3 8H8 antibody for the flow cytometric analysis of B7-H3 expression in AML blasts from 77 patients. Our antibody reliably detected substantial B7-H3 expression in 62.3% of AML patients. B7-H3 expression was higher in the monocytic French-American-British (FAB) M5 group and in intermediate and poor risk patients according to the European Leukemia Network. Using receiver operating characteristics (ROCs), we identified a specific fluorescence intensity cut-off of 4.45 to discriminate between B7-H3high and B7-H3low expression. High B7-H3 expression was associated with shorter overall survival (OS) and progression-free survival (PFS). In conclusion, we have developed a novel B7-H3 antibody that serves as a new tool for the detection of B7-H3 expression in AML and may help to facilitate risk stratification and treatment selection in AML patients.

Induction of NK cell reactivity against acute myeloid leukemia by Fc-optimized CD276 (B7-H3) antibody.

Acute myeloid leukemia (AML) remains a therapeutic challenge despite recent therapeutic advances. Although monoclonal antibodies (mAbs) engaging natural killer (NK) cells via antibody-dependent cellular cytotoxicity (ADCC) hold promise in cancer therapy, almost none have received clinical approval for AML, so far. Recently, CD276 (B7-H3) has emerged as a promising target for AML immunotherapy, due to its high expression on leukemic blasts of AML patients. Here, we present the preclinical development of the Fc-optimized CD276 mAb 8H8_SDIE with enhanced CD16 affinity. We demonstrate that 8H8_SDIE specifically binds to CD276 on AML cell lines and primary AML cells and induces pronounced NK cell activation and degranulation as measured by CD69, CD25, and CD107a. Secretion of IFNγ, TNF, granzyme B, granulysin, and perforin, which mediate NK cell effector functions, was induced by 8H8_SDIE. A pronounced target cell-restricted lysis of AML cell lines and primary AML cells was observed in cytotoxicity assays using 8H8_SDIE. Finally, xenograft models with 8H8_SDIE did not cause off-target immune activation and effectively inhibited leukemia growth in vivo. We here present a novel attractive immunotherapeutic compound that potently induces anti-leukemic NK cell reactivity in vitro and in vivo as treatment option for AML.

PD-1 checkpoint inhibition enhances the antilymphoma activity of CD19-CAR-iNKT cells that retain their ability to prevent alloreactivity.

BACKGROUND: Relapse and graft-versus-host disease (GVHD) are the main causes of death after allogeneic hematopoietic cell transplantation (HCT). Preclinical murine models and clinical data suggest that invariant natural killer T (iNKT) cells prevent acute and chronic GVHD. In addition, iNKT cells are crucial for efficient immune responses against malignancies and contribute to reduced relapse rates after transplantation. Chimeric antigen receptors (CAR) redirect effector cells to cell surface antigens and enhance killing of target cells. With this study, we aimed to combine enhanced cytotoxicity of CD19-CAR-iNKT cells against lymphoma cells with their tolerogenic properties. METHODS: iNKT cells were isolated from peripheral blood mononuclear cells and transduced with an anti-CD19-CAR retrovirus. After in vitro expansion, the functionality of CD19-CAR-iNKT cells was assessed by flow cytometry, image stream analysis and multiplex analysis in single-stimulation or repeated-stimulation assays. Moreover, the immunoregulatory properties of CD19-CAR-iNKT cells were analyzed in apoptosis assays and in mixed lymphocyte reactions. The effect of checkpoint inhibition through nivolumab was analyzed in these settings. RESULTS: In this study, we could show that the cytotoxicity of CD19-CAR-iNKT cells was mediated either through engagement of their CAR or their invariant T-cell receptor, which may circumvent loss of response through antigen escape. However, encounter of CD19-CAR-iNKT cells with their target induced a phenotype of exhaustion. Consequently, checkpoint inhibition increased cytokine release, cytotoxicity and survival of CD19-CAR-iNKT cells. Additionally, they showed robust suppression of alloreactive immune responses. CONCLUSION: In this work, we demonstrate that CAR-iNKT cells are a powerful cytotherapeutic option to prevent or treat relapse while potentially reducing the risk of GVHD after allogeneic HCT.

Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies.

Despite the advances in cancer treatment achieved, for example, by the CD20 antibody rituximab, an urgent medical need remains to optimize the capacity of such antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) that determines therapeutic efficacy. The cytokine IL-15 stimulates proliferation, activation, and cytolytic capacity of NK cells, but broad clinical use is prevented by short half-life, poor accumulation at the tumor site, and severe toxicity due to unspecific immune activation. We here report modified immunocytokines consisting of Fc-optimized CD19 and CD20 antibodies fused to an IL-15 moiety comprising an L45E-E46K double mutation (MIC+ format). The E46K mutation abrogated binding to IL-15Rα, thereby enabling substitution of physiological trans-presentation by target binding and thus conditional IL-15Rßγ stimulation, whereas the L45E mutation optimized IL-15Rßγ agonism and producibility. In vitro analysis of NK activation, anti-leukemia reactivity, and toxicity using autologous and allogeneic B cells confirmed target-dependent function of MIC+ constructs. Compared with Fc-optimized CD19 and CD20 antibodies, MIC+ constructs mediated superior target cell killing and NK cell proliferation. Mouse models using luciferase-expressing human NALM-6 lymphoma cells, patient acute lymphoblastic leukemia (ALL) cells, and murine EL-4 lymphoma cells transduced with human CD19/CD20 as targets and human and murine NK cells as effectors, respectively, confirmed superior and target-dependent anti-leukemic activity. In summary, MIC+ constructs combine the benefits of Fc-optimized antibodies and IL-15 cytokine activity and mediate superior NK cell immunity with potentially reduced side effects. They thus constitute a promising new immunotherapeutic approach shown here for B cell malignancies.

Phase I study evaluating the Fc-optimized FLT3 antibody FLYSYN in AML patients with measurable residual disease.

BACKGROUND: About half of AML patients achieving complete remission (CR) display measurable residual disease (MRD) and eventually relapse. FLYSYN is an Fc-optimized antibody for eradication of MRD directed to FLT3/CD135, which is abundantly expressed on AML cells. METHODS: This first-in-human, open-label, single-arm, multicenter trial included AML patients in CR with persisting or increasing MRD and evaluated safety/tolerability, pharmacokinetics and preliminary efficacy of FLYSYN at different dose levels administered intravenously (cohort 1-5: single dose of 0.5 mg/m2, 1.5 mg/m2, 5 mg/m2, 15 mg/m2, 45 mg/m2; cohort 6: 15 mg/m2 on day 1, 15 and 29). Three patients were treated per cohort except for cohorts 4 and 6, which were expanded to nine and ten patients, respectively. Primary objective was safety, and secondary efficacy objective was ≥ 1 log MRD reduction or negativity in bone marrow. RESULTS: Overall, 31 patients were treated, of whom seven patients (22.6%) experienced a transient decrease in neutrophil count (two grade 3, others ≤ grade 2). No infusion-related reaction or dose-limiting toxicity was observed. Adverse events (AEs) were mostly mild to moderate, with the most frequent AEs being hematologic events and laboratory abnormalities. Response per predefined criteria was documented in 35% of patients, and two patients maintained MRD negativity until end of study. Application of 45 mg/m2 FLYSYN as single or cumulative dose achieved objective responses in 46% of patients, whereas 28% responded at lower doses. CONCLUSIONS: FLYSYN monotherapy is safe and well-tolerated in AML patients with MRD. Early efficacy data are promising and warrant further evaluation in an up-coming phase II trial. Trial registration This clinical is registered on clinicaltrials.gov (NCT02789254).

DNMT and HDAC inhibition induces immunogenic neoantigens from human endogenous retroviral element-derived transcripts.

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.