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Introduction: High levels of immunosuppressive adenosine are found in the tumor microenvironment, reaching 50-100 μM in experimental models. Adenosine exerts its effects on immune cells primarily through the adenosine receptors A2aR/A2bR, which increase intracellular levels of cyclic AMP, leading to CREB phosphorylation (pCREB). We have previously shown that the dual A2aR/A2bR antagonist AB928 is capable of inhibiting adenosine-induced pCREB in healthy human volunteer (HV) blood lymphocytes. AB928 has also been shown to relieve adenosine-mediated T-cell suppression in vitro and exhibit combinatorial effects with standard-of-care chemotherapeutics in mouse syngeneic tumor models. Herein, we show that AB928 is capable of inhibiting NECA-induced gene expression changes and CREB phosphorylation in non-small cell lung carcinoma (NSCLC) patient whole blood (WB). Additionally, observations from our in vitro human studies showing the combinatorial effect of AB928 and α-PD-1 were reproduced in B16F10 syngeneic tumors. Methods: Human WB was stimulated with 5 μM of NECA and flow cytometry was used to quantify AB928-mediated inhibition of pCREB and CD3ζ phosphorylation. B16F10 tumors were treated with α-PD-1 +/- AB928 and gene expression was determined from excised mouse tumors using the nCounter PanCancer panel. Results: To ensure AB928 can successfully inhibit the high levels of intratumoral adenosine, we found that 5 μM NECA provides maximal stimulation and is significantly more potent (>20 fold) than adenosine in the pCREB assay. Additional experiments demonstrated that AB928 has comparable potency in NECA-stimulated WB from both HV and NSCLC patients. In addition to blocking downstream signaling, NanoString analysis showed that AB928 could prevent NECA-stimulated gene expression changes in NSCLC WB. We also found that NECA stimulation, alone or in combination with PD-1 inhibition, significantly reduced proximal TCR signaling, leading to reduced levels of CD3ζ phosphorylation at TYR142 (pTYR142). These reduced pTYR142 levels, with and without α-PD-1, could be significantly rescued by AB928, suggesting that blocking adenosine immunosuppression may provide additional benefit to PD-1 inhibition in tumors. Consistent with these results, AB928 was capable of suppressing growth (volume in mm3) of B16-F10 tumors both as a single agent (vehicle: 462 +/- 58; AB928: 292 +/- 55; p Conclusions: Collectively, these results support a role for AB928 in relieving adenosine-mediated immunosuppression by blocking A2aR/A2bR-induced signaling events, gene expression changes, and suppressing tumor growth in vivo. Citation Format: Daniel M. DiRenzo, Nell Narasappa, Dana Piovesan, Devika Ashok, Park Adam, Jenna L. Jeffrey, Manmohan R. Leleti, Joanne B.L. Tan, Lisa Seitz, Steve W. Young, Jay P. Powers, Matthew J. Walters. The dual A2aR/A2bR antagonist AB928 reverses adenosine-mediated immune suppression and inhibits tumor growth in vivo [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A10.

Background: In patients undergoing chemotherapy and/or immune therapy, endogenous T cells within the tumor micro-environment (TME) and in the periphery are subject to dynamic changes. Particularly, exhausted T cells in the TME and peripheral blood mononuclear cells (PBMCs) isolated from whole blood express the inhibitory receptor, Programmed Cell Death Receptor 1 (PD-1). A small but significant number of these T cells can recognize neo-epitopes (neoE) arising from patient specific mutations. We determined receptor occupancy (RO) in patients treated with monoclonal anti-PD-1 antibody AB122, which is currently in clinical development. Methods: Multi-color flow cytometry was used to determine RO post-AB122 administration using a directly-conjugated competitive antibody to AB122. In addition, we determined RO values using the established method for nivolumab using a biotinylated anti-human IgG4 to detect bound anti-PD-1. The assays were also optimized to determine RO in whole blood from patients with solid tumors treated with AB122. Receptor occupancy using both methods were compared. Further, whole exome sequencing from archival formalin-fixed, paraffin-embedded (FFPE) tissue was performed and T cells that recognized patient-specific neoE resulting from tumor-specific mutations were identified in patient blood. Results: In the dose-escalation study for AB122 monotherapy arm, a variety of dosing regimens have been evaluated, including Q2W, Q3W and Q4W. The number of doses received ranged from 1 to 20. Data from patients in the 80 and 240 mg Q2W cohorts showed that AB122 serum concentrations ≥1.5 μg/mL (equivalent to 10 nM) are associated with full receptor occupancy. The RO associated with AB122 dosing is maintained when dosed in combination with the potent A2aR/A2bR dual adenosine receptor antagonist AB928 in an ongoing clinical trial. Conclusions: Full RO is observed with both cohorts of AB122 monotherapy (80 and 240 mg Q2W) and in the 240 mg Q2W combination with AB928. Study of peripheral target engagement and expansion of neoE-specific T cell populations in a longitudinal study of solid tumor patients treated with AB122 could provide insight towards designing successful combination immune therapy regimens in future clinical trials with AB122. Citation Format: Devika Ashok, Songming Peng, Benjamin Yuen, Matt Walters, Stephen W. Young, Lisa Seitz. Receptor occupancy and neo-epitope specific T-cell repertoires in patients with solid tissue tumors following anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4100.

Background: Adenosine production mediated by CD73 and/or TNAP is a potential mechanism of immune suppression across many cancer types. We have previously shown that AB928, a dual A2aR/A2bR antagonist, in combination with anti-PD-1 or chemotherapy, rescues the immunosuppressive effects of adenosine in experimental tumor models. Oncogene-driven cancers are targeted with specific tyrosine kinase inhibitors, typically leading to the development of several resistance mechanisms that bypass the kinase signaling. These oncogene-driven cancers tend to be non-responsive to PD(L)-1 inhibition. We hypothesized that oncogenic drivers might be regulating adenosine production machinery as a mechanism to suppress and evade the immune system. Here we show that pan-RAS, BRAF and EGFR alterations drive the expression of CD73, which may contribute to suppressed anti-tumor immunity. Methods and Results: We used linear models to evaluate the ability of 299 pan-cancer consensus oncogenic drivers (Bailey et al, 2018) to predict CD73 expression independent of tumor type in PanCanAtlas TCGA dataset. We defined a given gene as either wild type or altered if it exhibited a SNV/copy number/fusion event in a given patient. Out of the 299 oncogenes, we identified 20 oncogenes that upregulated and 26 that downregulated CD73 expression (FDR < 0.05). Alterations in KRAS, BRAF and EGFR were the top 3 oncogenes upregulating CD73 expression, followed by other kinases such as RASA1, MET, RET, SMAD4 and CDK4. In KRAS/BRAF/EGFR altered tumors, CD73 expression was significantly higher compared to respective wild-type patients in all cancers combined, as well as in individual tumor types (BRCA, LUAD, LUSC, CRC, PAAD, HNSCC, UBC, CESC, ESCA, STAD, UCEC and LGG). Furthermore, the pan-cancer Ras activation classifier score (Way et.al 2018) was highly predictive of CD73 expression independent of tumor type (p-value < 2e-16). KRAS, HRAS, BRAF, EGFR as well as pan-Ras mutant cancer cell lines exhibited a significantly higher level of CD73 expression than wild-type cell lines, independent of cancer type. Conclusions: Activating mutations in KRAS and HRAS that result in deregulation of the Ras pathway serve as oncogenic drivers in a variety of cancer types such as CRC, NSCLC, gastric-esophageal, cervical, bladder and HNSCC and upregulate CD73. BRAF mutations are enriched in breast, CRC, NSCLC, and low-grade gliomas that exhibit higher CD73 expression. Finally, EGFR mutations are prominent in RCC, low-grade gliomas and NSCLC, which drive CD73 expression. Oncogene-driven cancers are largely unresponsive to PD-(L)1 inhibition and are targeted with kinase inhibitors resulting in significant but not durable clinical responses. Our data strongly suggests that CD73 inhibition with AB680 and/or A2R antagonism with AB928 might be effective in Ras pathway mutant and EGFR mutant cancers, either in TKI-naïve or relapsed settings. Citation Format: Akshata R. Udyavar, Daniel DiRenzo, Devika Ashok, Amy E. Anderson, Stephen W. Young, Matt J. Walters, Joanne Bl Tan. Altered pan-Ras pathway and activating mutations in EGFR result in elevated CD73 in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4980.

Adenosine suppresses antitumor immune responses via A2a and A2b receptors expressed on intratumoral immune cells. This effect is mediated by increased cyclic adenosine 5′-monophosphate (AMP) levels and phosphorylation of cyclic AMP response element binding protein (CREB). We conducted a phase 1, placebo-controlled, single-ascending-dose (SAD) and multiple-ascending-dose (MAD) study to assess the safety, tolerability, pharmacokinetics (PK), including food effect (FE), and pharmacodynamics (PD) of oral AB928, a novel dual A2aR/A2bR antagonist, in healthy volunteers. AB928 doses between 10 and 200 mg once daily and 100 mg twice daily were evaluated. The study enrolled 85 subjects (randomized 3:1, AB928:placebo), 40 each in the SAD and MAD cohorts, and 5 in the FE cohort. AB928 was well tolerated up to the highest dose tested and did not affect any physiologic parameters potentially sensitive to adenosine inhibition. No safety concern was identified. The PK profile of AB928 was linear and dose-proportional, and a clear PK/PD correlation was demonstrated. Significant inhibition of adenosine receptor-mediated phosphorylated CREB was observed at peak plasma concentrations in all dose cohorts and at trough plasma concentrations in the higher-dose cohorts. AB928 plasma levels ≥1 μM were associated with ≥90% adenosine receptor inhibition. In the postprandial state, the rate of AB928 absorption decreased but the extent of absorption was unchanged. Together, these data support further clinical development of oral AB928 in cancer patients.

Introduction: Extracellular adenosine (ADO), present at high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T-cell, natural killer (NK) cell, and dendritic cell (DC) activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases CD39 (ATP→AMP) and CD73 (AMP→ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Here we present the preclinical characterization of AB680, a novel, highly potent, reversible and selective small-molecule inhibitor of CD73, currently in preclinical development as a potential antitumor agent. The link between CD73 levels present in different tissues, efficacy in mouse tumor models, plasma and tumor exposure, and projected human pharmacokinetic (PK) profile can be combined to provide an expected AB680 dosing strategy for the upcoming first-in-human clinical trial. Methods: The potency of AB680 against human CD73 was determined using CHO-CD73 cells, blood CD8+ T-cells, recombinant human CD73, and serum/plasma using either malachite green assay, AMP-Glo assay, or LCMS/MS. The selectivity of AB680 against related ecto-nucleotidases was also assessed using similar methods. Quantitation of soluble CD73 in mouse and human serum, and mouse tumor homogenates, was performed via in-house developed and validated ELISA or Western blot methods. Syngeneic mouse tumor models were established to assess the efficacy of AB680 at multiple doses. AB680 levels in plasma and tumor associated with each dosing regimen were determined via LCMS/MS. The potency of AB680 in an intratumoral setting was determined using various biochemical methods. The effects of AB680 on syngeneic tumor volumes were assessed in prophylactic and therapeutic settings. The PK properties of AB680 were evaluated in multiple preclinical species and a projected human dosing schedule for AB680 was determined via allometric scaling. Results: AB680 is a highly potent, reversible and selective inhibitor of CD73 activity (IC50 < 0.01 nM on human CD8+ T-cells), which retains its high potency in the presence of human serum. Inhibition of AMP hydrolysis by AB680 completely reversed ADO-mediated suppression of CD4+ and CD8+ T-cell effector function, as measured by cytokine secretion and proliferation. The in vivo efficacy of AB680, as measured by its ability to restrict tumor growth at doses as low as 10 mg/kg once daily, is reflective of the following parameters: 1) plasma half-life, 2) partitioning from blood to tumor compartment, 3) potency against soluble CD73 in plasma or serum, and 4) potency against membrane-bound CD73. The PK properties of AB680 in rodent and non-rodent species are characterized by very low clearance and long half-lives. The combination of in vitro potency against mouse and human soluble and membrane-bound CD73, in vivo tumor growth regulation observed in mouse models, quantification of mouse and human CD73 levels, and the projected human PK profile for AB680 have resulted in predicted human PK properties (projected half life: 4-14 days) suitable for intravenous dosing on a schedule consistent with typical monoclonal antibody dosing cycles. Conclusions: AB680 is a highly potent and selective small-molecule inhibitor of CD73 which can mitigate AMP and ADO-mediated tumor immunosuppression by potently blocking the production of ADO. AB680 exhibits a unique projected human PK profile suitable for parental administration and is expected to enter clinical development in 2018. Citation Format: Joanne B.L. Tan, Jie Chen, Elaine Ginn, Devika Ashok, Amy E Anderson, Jesus Banuelos, Kristen Zhang, Amber Pham, Timothy Park, Ada Chen, Xiaoning Zhao, Kenneth K.V. Lawson, Jenna Jeffreys, Jarek Kalisiak, Manmohan R. Leleti, Matthew J. Walters, Jay P. Powers. Preclinical pharmacokinetic and pharmacodynamic characterization of AB680, a small-molecule CD73 inhibitor for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A157.

INTRODUCTION: CD73 converts extracellular adenosine monophosphate (AMP), derived from adenosine triphosphate, into adenosine (ADO). ADO suppresses immune responses, including those of T cells, natural killer (NK) cells and dendritic cells, via A2aR and A2bR receptors. Activation of these receptors results in increased intracellular levels of cyclic AMP (cAMP) and phosphorylation of the cAMP response element-binding protein (CREB). Monitoring the degree of pCREB induced by activation of the Aa2R and A2bR receptors in peripheral blood allows for a specific measurement of target engagement by AB928. In mice, the pCREB assay, coupled with changes in immune cell infiltrate and tumor growth rates, allows for a fuller understanding of the drug's PK/PD relationship. METHODS: Mouse and human whole blood was stimulated with the adenosine agonist NECA (5'-N-Ethylcarboxamidoadenosine) following in vitro spike in or in vivo dosing with AB928. Multi-color phospho-flow cytometry was used to assess levels of pCREB on immune cells. AB928 levels in plasma samples were determined using LC-MS-MS after protein precipitation. RESULTS: A2aR mRNA was the most prevalent adenosine receptor in CD4+ and CD8+ T cells. NK cells also express primarily A2aR, although A2bR was also detected. Dendritic cells and CD14+ monocytes expressed both A2aR and A2bR. NECA stimulation of mouse whole blood resulted in a significant increase in the levels of pCREB within CD8+ T cells with an EC50 of 100 nM, while in human whole blood the EC50 was higher at 700 nM. AB928 (100 nM) exhibited comparable shifts in the NECA dose response between human and murine whole blood, 15 and 16-fold, respectively. Due to the high level of adenosine in the tumor microenvironment, doses of AB928 associated with plasma levels that significantly inhibited 5 μM NECA in the whole-blood pCREB assay were selected for mouse efficacy studies. At these doses, significant reductions in tumor growth were noted. In human whole blood, approximately 90 nM AB928 was required to inhibit 50% of the 5 μM NECA-induced pCREB signal. Inhibition was observed on both CD4+ and CD8+ T cells. NECA-induced pCREB elevations were observed in CD14+ cells less frequently than T cells; however, when the signal was observed, AB928 inhibited it. The human pCREB assay is being utilized to monitor the PD responses in an ongoing clinical trial. Preliminary PK/PD results from this study will be presented. CONCLUSIONS: Systemic extent of receptor (A2aR and A2bR) occupancy by the novel dual antagonist AB928 can be assessed in humans and mice based on the extent of receptor-mediated CREB phosphorylation in blood lymphocytes. AB928 retains much of its inherent potency when competing against high concentrations of adenosine under physiologically relevant conditions (i.e., whole blood). Citation Format: Lisa Seitz, Devika Ashok, Manmohan R. Leleti, Jay P. Powers, Brandon Rosen, Dillon Miles, Lixia Jin, Adam Park, Tim Park, Steve Young, Ferdie Soriano, Aimee Rieger, Ulrike Schindler, Joyson Karakunnel, Matt J. Walters. Pharmacokinetic-pharmacodynamic relationship for AB928, a dual antagonist of the A2aR and A2bR adenosine receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3769.

Significance Group 3 innate lymphoid cells (ILC3s) play decisive roles in mammalian physiology including tissue repair, lymphoid tissue development, and immune regulation. So far, the functions of ILC3s in the adult immune system have been mainly linked to their capacity to release cytokines in response to microbial or inflammatory signals. The results presented here show that activated ILC3s can alter the outcome of adaptive immune responses by directly stimulating CD4 + T cells. Indeed, IL-1β–activated ILC3s express costimulatory molecules and induce cognate CD4 + T-cell responses. More importantly, antigen-driven T- and B-cell responses are impaired in vivo when this cellular interaction is disrupted. Overall, our data show that peripheral ILC3s play a yet unappreciated role in T-cell–mediated immunity.

Leishmania major infection induces self-healing cutaneous lesions in C57BL/6 mice. Both IL-12 and IFN-γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3(-/-) ) mice (C57BL/6 background) that lack the major IL-12 producing and cross-presenting CD8α(+) and CD103(+) DC subsets. Batf3(-/-) mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3(-/-) mice secreted less IFN-γ, but more Th2- and Th17-type cytokines, mirrored by increased serum IgE and Leishmania-specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α(+) DCs isolated from lymph nodes of L. major-infected mice induced significantly more IFN-γ secretion by L. major-stimulated immune T cells than CD103(+) DCs. We next developed CD11c-diptheria toxin receptor: Batf3(-/-) mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf-3-dependent DCs from day 17 or wild-type mice depleted of cross-presenting DCs from 17-19 days after infection maintained significantly larger lesions similar to mice whose Batf-3-dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf-3-dependent DCs in protection against L. major.

2604 Background: AB928, a selective, small-molecule A2aR/A2bR antagonist, potently blocks the immunosuppressive effects of high adenosine concentrations in the tumor microenvironment. Preclinically, combining adenosine receptor inhibition with either chemotherapy or anti-PD-1 resulted in greater tumor control, suggesting AB928 may have additive activity when paired with either of these agents in cancer pts. Methods: Three dose-escalation (3+3 design) studies are assessing the safety, pharmacokinetics (PK), pharmacodynamics, and clinical activity of increasing doses of AB928 (75, 150, 200 mg orally once daily) in combination with: standard pegylated liposomal doxorubicin in triple-negative breast cancer (TNBC) and ovarian cancer (OC); standard mFOLFOX6 in gastroesophageal cancer (GEC) and colorectal cancer (CRC); and AB122 (240 mg every 2 weeks) in various advanced tumors. Following identification of the recommended phase 2 dose of AB928 in combination with chemotherapy or AB122 in dose escalation, the following tumor cohorts may be expanded (15-40 pts/cohort) to further test the combinations: TNBC and OC, GEC and CRC, and renal cell carcinoma. Results: As of 01Feb2019, 9 pts were treated across the 3 studies, and time on treatment ranged from 1-182 days (table). Overall, AB928 combination therapy was well tolerated. Two pts underwent post-baseline disease assessment; both had stable disease. Preliminary data indicate that AB928 PK and adenosine receptor coverage in cancer pts are similar to what was previously assessed in healthy volunteers. AB122 PK and PD-1 coverage are equally unaffected by AB928 co-administration. Updated data, including biomarker data, will be presented at the meeting. Conclusions: Early results showed a favorable safety profile of AB928 combination therapy. All 3 studies are actively recruiting pts. Clinical trial information: NCT03719326; NCT03720678; NCT03629756. [Table: see text]

The renaissance of cancer immunotherapies and the positive clinical responses observed with chimeric antigen receptor modified T cells in the hematomalignancy setting has stimulated substantial interest in monitoring immune cell activation and suppression to determine efficacy, prognosis and safety in new agent investigational trials. However, immunophenotyping capacity has been limited and required multi-test tube panels. To address the growing needs of clinical trials, we have developed three fit-for-purpose high complexity (10 or more markers) T-cell phenotyping flow cytometry panels on a qualified LSR FortessaTM platform that enables detection of up to 15 markers in a single test tube. The first panel allows identification of multiple phenotypes along the T-cell differentiation pathway, namely, T-naive (TN), T-stem cell memory (TSCM), T-effector memory (TEM) and T-central memory (TCM) and T-effector memory RA+ (TEMRA). The second panel identifies the most common helper T-cell phenotypes such as Th1, Th2, Treg, and Th17. A third panel determines the functional status of T-cells (e.g., activation vs. suppression) but also enables quantitation of important checkpoint inhibitor expression (e.g., PD-1) on T-cells of interest. These high complexity flow cytometry panels can serve as powerful tools for comprehensive examination of T-cells in a small volume of patient specimen. We believe these new flow cytometry panels will have a substantial impact on the determination of efficacy and safety correlates of immunomodulating agents administered alone or in combination to patients with leukemia. Citation Format: Ghanashyam Sarikonda, Devika Ashok, Anil Pahuja, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. High complexity flow cytometry panels to monitor target expression, T-cell activation and suppresssion by novel immunotherapies in hematomalignancy clinical trials. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1302. doi:10.1158/1538-7445.AM2015-1302

Research in vitro facilitates discovery screening and pilot experiments often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging including the low frequency of DC in vivo thorough isolation requirements and the vulnerability of DC ex vivo. Critically there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study we report the generation of new murine DC lines named MutuDC originating from cultures of splenic CD8a conventional DC (cDC) tumors. By direct comparison to normalWT splenic cDC subsets we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo the splenic CD8a cDC. These features include expression of surface markers Clec9A DEC205 and CD24 positive response to TLR3 and TLR9 but not TLR7 stimuli secretion of cytokines and chemokines upon activation as well as cross presentation capacity. In addition to the close resemblance to normal splenic CD8a cDC a major advantage is the ease of derivation and maintenance of the MutuDC lines using standard culture medium and conditions importantly without adding supplementary growth factors or maturation inducing stimuli to the medium. Furthermore genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research. © 2012 Fuertes Marraco Grosjean Duval Rosa Lavanchy Ashok Haller Otten Steiner Descombes Luber Meissner Mann Szeles Reith and Acha Orbea.