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Individual data of cytokine release and metabolic activity upon OX40 stimulation of AML cells.

The TNF receptor family member OX40 promotes activation and proliferation of T cells, which fuels efforts to modulate this immune checkpoint to reinforce antitumor immunity. Besides T cells, NK cells are a second cytotoxic lymphocyte subset that contributes to antitumor immunity, particularly in leukemia. Accordingly, these cells are being clinically evaluated for cancer treatment through multiple approaches, such as adoptive transfer of ex vivo expanded polyclonal NK cells (pNKC). Here, we analyzed whether and how OX40 and its ligand (OX40L) influence NK-cell function and antileukemia reactivity. We report that OX40 is expressed on leukemic blasts in a substantial percentage of patients with acute myeloid leukemia (AML) and that OX40 can, after stimulation with agonistic OX40 antibodies, mediate proliferation and release of cytokines that act as growth and survival factors for the leukemic cells. We also demonstrate that pNKC differentially express OX40L, depending on the protocol used for their generation. OX40L signaling promoted NK-cell activation, cytokine production, and cytotoxicity, and disruption of OX40–OX40L interaction impaired pNKC reactivity against primary AML cells. Together, our data implicate OX40/OX40L in disease pathophysiology of AML and in NK-cell immunosurveillance. Our findings indicate that effects of the OX40–OX40L receptor–ligand system in other immune cell subsets and also malignant cells should be taken into account when developing OX40-targeted approaches for cancer immunotherapy. Cancer Immunol Res; 6(2); 209–21. ©2018 AACR.

Antibodies against the B cell-specific antigens CD20 and CD19 have markedly improved the treatment of B cell-derived lymphoma and autoimmune diseases by depleting malignant and autoreactive B cells. However, since CD20 and CD19 are also expressed on healthy B cells, such antibodies lack disease specificity. Here, we optimize a previously developed concept that uses bispecific antibodies to induce apoptosis selectively in malignant and autoreactive B cells that express the death receptor CD95. We describe the development and characterization of bispecific antibodies with CD95xCD20 and CD95xCD19 specificity in a new IgG-based format. We could show that especially the CD95xCD20 antibody mediated a strong induction of apoptosis in malignant B cells in vitro. In vivo, the antibody was clearly superior to the previously used Fabsc format with identical specificities. In addition, both IgGsc antibodies depleted activated B cells in vitro, leading to a significant reduction in antibody production and cytokine secretion. The killing of resting B cells and hepatocytes that lack CD95 and CD20/CD19, respectively, was marginal. Thus, our results imply that bispecific anti-CD95 antibodies in the IgGsc format are an attractive tool for a more selective and efficient depletion of malignant as well as autoreactive B cells.

Antibodies against the B cell-specific antigens CD20 and CD19 have markedly improved the treatment of B cell-derived lymphoma and autoimmune diseases by depleting malignant and autoreactive B cells. However, since CD20 and CD19 are also expressed on healthy B cells, such antibodies lack disease specificity. Here, we optimize a previously developed concept that uses bispecific antibodies to induce apoptosis selectively in malignant and autoreactive B cells that express the death receptor CD95. We describe the development and characterization of bispecific antibodies with CD95xCD20 and CD95xCD19 specificity in a new IgG-based format. We could show that especially the CD95xCD20 antibody mediated a strong induction of apoptosis in malignant B cells in vitro. In vivo, the antibody was clearly superior to the previously used Fabsc format with identical specificities. In addition, both IgGsc antibodies depleted activated B cells in vitro, leading to a significant reduction in antibody production and cytokine secretion. The killing of resting B cells and hepatocytes that lack CD95 and CD20/CD19, respectively, was marginal. Thus, our results imply that bispecific anti-CD95 antibodies in the IgGsc format are an attractive tool for a more selective and efficient depletion of malignant as well as autoreactive B cells.

Risk assessment in acute myeloid leukemia (AML) mainly relies on (cyto-)genetic and morphologic features. Nonetheless, further markers are needed to allow for accurate risk stratification. Type I Fc gamma receptors (FcγRs) such as CD16, CD32, and CD64 play an important role in mediating immunomodulatory functions in different myeloid cell types as well as NK and B cells. We here evaluated expression of the three FcγR on peripheral blood AML blasts. Using flow cytometry, we found heterogeneous expression of the FcγR throughout the patient cohort. Correlation of expression levels with disease outcome revealed significantly shorter OS in patients with CD16+ blasts at first diagnosis. CD32 and CD64 expression showed no association with survival but correlated with a mature phenotype and FAB M6. Our data provide clear evidence for the value of immunophenotyping FcγR expression on leukemic cells using peripheral blood, which is rapidly available and improves risk stratification in AML.

Several genetic and molecular markers are general predictors of outcome in acute myeloid leukemia (AML), but only few are predictive of outcomes after allogeneic hematopoietic stem cell transplantation (HSCT). Novel markers are needed to improve treatment decisions regarding HSCT. CD105 (endoglin) is a type I transmembrane protein capable of activating endothelial cells. Moreover, CD105 mediates stem cell properties of hematopoietic stem cells and enables repopulation within the bone marrow. Expression of CD105 on vessels of solid tumors and on AML blasts is correlated with poor prognosis. We recently demonstrated that CD105 expression is an independent predictor of overall survival in AML. However, its role in patients receiving intensive treatment with consecutive allogenic transplantation has not been assessed. Using flow cytometry, we analyzed primary samples of 41 patients who underwent HSCT. Using the previously defined SFI cut-off of 5.2, we identified differences in CD105 expression regarding FAB classification and NCCN risk score. Moreover, we detected differences regarding transplant indication and WHO classification with regards to CD105 surface levels. In patients undergoing HSCT high CD105 expression correlated significantly with poor overall survival. We identify CD105 expression in AML as prognostic marker for outcome after HSCT in AML.

Antitumor antibodies like Rituximab and Trastuzumab activate the patient’s immune system to engage malignant cells. Despite the advances achieved by their introduction in cancer treatment, an urgent medical need remains to improve and optimize the efficacy of antibody therapy. This may be achievable using the cytokine IL-15 which induces proliferation and activation of both, NK cells and CD8 T cells, and enhances their cytolytic capacity. So far, short half-life, poor accumulation at the tumor site and severe toxicity upon systemic application due to unspecific immune activation prevent the broad use of IL-15 for treatment of cancer patients (Conlon et al, JCO 2015). IL-15 predominantly acts as part of an immunological synapse with IL-15Rα expressed on antigen-presenting cells, which present IL-15 in trans to IL-15Rβγ receptor expressing cytotoxic lymphocytes. We here report on the generation of so called “modified immunocytokines” (MICs) which consist of an Fc-optimized (SDIE modification) CD19 or CD20 antibody for targeting of (malignant) B cells fused to an IL-15 mutant with abolished binding to IL-15Rα termed MIC19 or MIC20. The abrogation of IL-15Rα binding by the IL-15 mutation enables substitution of the physiological trans-presentation by target binding of the antibody, allowing for target antigen-restricted stimulation of the IL-15Rβγ on NK cells and CD8 T cells. Analyses of activation and toxicity with MIC19 compared to a CD19 immunocytokine with an unmodified IL-15 (IC19) or immunocytokines with unrelated specificity as control confirmed target antigen-restricted function of the MIC proteins. Killing of autologous and allogeneic CD19+/CD20+ target cells was clearly superior as compared to the CD19-directed Fc optimized antibody (Tafasitamab) and Rituximab that bear no cytokine activity. Compared to antibody treatment, the MIC proteins induced a pronounced and long-lasting proliferation of effector cells and were effective to treat leukemia in a xenograft (Nalm-6) leukemia mouse model. Anti-leukemic activity was further confirmed in a murine NSG xenotransplantation model using patient ALL cells and human NK cells as effectors. In summary, MIC constructs consisting of an Fc-optimized antibody and mutated IL-15 with deficient IL-15Rα binding induce target cell restricted activation and proliferation of NK cells, resulting in efficient anti-leukemic activity. Our MICs thus combine the benefits of Fc-optimized antibodies and IL-15 activity and constitute a promising novel treatment modality for B cell malignancies with low side effects that allow the application of truly effective doses. Citation Format: Ilona Hagelstein, Latifa Zekri, Boris Klimovich, Martina S. Lutz, Carsten Greve, Stefanie Müller, Melanie Märklin, Bastian Schmied, Gundram Jung, Helmut R. Salih. Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2863.

Despite substantial improvements over the last decades, survival rates in metastatic gastrointestinal cancer are still far from satisfactory, with an accordingly high medical need for new treatment strategies. B7-H3 (CD276) is a member of the B7 immune checkpoint family. Initially thought to act as co-stimulator, recent studies revealed that B7-H3 rather has an inhibitory role for T cells and contributes to tumor immune evasion. Clinically, its overexpression has been linked to invasive and metastatic potential as well as poor prognosis. Due to its expression on both, tumor cells and tumor vasculature, in a variety of cancer entities including colorectal cancer, B7-H3 attracted our interest as therapeutic target for T cell-recruiting bispecific antibodies (bsAbs). We postulate that “dual targeting” of both, the cancer cells and the tumor vasculature may support the influx of T cells into the tumor site, a critical prerequisite for successful immunotherapy of solid tumors allowing for subsequent destruction of antigen-positive malignant cells. We generated a panel of monoclonal antibodies directed to different epitopes of the B7-H3 molecule. After biochemical characterization, we selected two antibodies with distinct binding proprieties and subsequently used them for the construction of Tcell-recruiting B7-H3xCD3 bsAbs in an IgG-based (IgGsc) format. To reduce side effects, constructs were cloned using a UCHT-1 derived low affinity anti-CD3 sequence. In vitro characterization using colorectal and other carcinoma cells allowed for selection of a construct with optimal functional properties (thereafter termed CC-3) as revealed by target cell-restricted induction of T cell activation, proliferation and tumor cell killing. In vivo, potent efficacy of CC-3 was documented in a lung metastasis model and by its ability to eliminate large established flank tumors using immunocompromised NSG mice adoptively transferred with human effector cells. Despite the high therapeutic efficacy of CC-3, no toxicity was observed in the absence of target cells. In summary, CC-3 is a bsAb with promising therapeutic activity against B7-H3 positive tumors. GMP compliant production of CC-3 is presently ongoing to enable evaluation in a clinical “first in human” study in patients with colorectal cancer. Citation Format: Latifa Zekri, Martina S. Lutz, Ilona Hagelstein, Timo Manz, Monika Engel, Boris Klimovich, Nisha Prakash, Anna Chashchina, Sebastian Hörner, Stefanie Müller, Melanie Märklin, Martin Pflügler, Gundram Jung, Helmut R. Salih. CC-3, an IgG-based B7-H3xCD3 bispecific antibody for targeting of gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2865.

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.

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.

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.

OX40 and its ligand are members of the TNF/TNF receptor superfamily, which includes various molecules influencing cellular signaling and function of both tumor and immune cells. The ability of OX40 to promote proliferation and differentiation of activated T cells fueled present attempts to modulate this immune checkpoint to reinforce antitumor immunity. While we recently found evidence for the involvement of OX40 in pathophysiology of acute myeloid leukemia including natural killer (NK) cell immunosurveillance, less is known on its role in acute lymphoblastic leukemia (ALL). In the present study, OX40 expression on ALL cells was significantly associated with positivity for the adverse risk factor BCR-ABL. In line, signaling via OX40 increased metabolic activity of primary ALL cells and resulted in release of cytokines involved in disease pathophysiology. Furthermore, interaction of ALL-expressed OX40 with its cognate ligand on NK cells stimulated ALL cell lysis. The data presented thus not only identify the yet unknown involvement of OX40/OX40L in ALL pathophysiology and NK cell immunosurveillance but also point to the necessity to thoroughly consider the consequences of modulating the OX40/OX40L molecule system beyond its effects on T cells when developing OX40-targeting approaches for cancer immunotherapy.

The TNF receptor family member OX40 promotes activation and proliferation of T cells, which fuels efforts to modulate this immune checkpoint to reinforce antitumor immunity. Besides T cells, NK cells are a second cytotoxic lymphocyte subset that contributes to antitumor immunity, particularly in leukemia. Accordingly, these cells are being clinically evaluated for cancer treatment through multiple approaches, such as adoptive transfer of ex vivo expanded polyclonal NK cells (pNKC). Here, we analyzed whether and how OX40 and its ligand (OX40L) influence NK-cell function and antileukemia reactivity. We report that OX40 is expressed on leukemic blasts in a substantial percentage of patients with acute myeloid leukemia (AML) and that OX40 can, after stimulation with agonistic OX40 antibodies, mediate proliferation and release of cytokines that act as growth and survival factors for the leukemic cells. We also demonstrate that pNKC differentially express OX40L, depending on the protocol used for their generation. OX40L signaling promoted NK-cell activation, cytokine production, and cytotoxicity, and disruption of OX40–OX40L interaction impaired pNKC reactivity against primary AML cells. Together, our data implicate OX40/OX40L in disease pathophysiology of AML and in NK-cell immunosurveillance. Our findings indicate that effects of the OX40–OX40L receptor–ligand system in other immune cell subsets and also malignant cells should be taken into account when developing OX40-targeted approaches for cancer immunotherapy. Cancer Immunol Res; 6(2); 209–21. ©2018 AACR.

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. Electronic supplementary material The online version of this article (10.1007/s00277-020-03907-9) contains supplementary material, which is available to authorized users.