Genetic variants in KCNQ2 are associated with a range of epilepsies, from self- limited (familial) neonatal-infantile epilepsy to developmental and epileptic encephalopathy (DEE). We retrospectively reviewed clinical data from eight patients with KCNQ2-related DEE who were treated with ezogabine. Treatment was initiated at a median age of 8 months (range, 7 weeks to 2.5 years) and continued for a median of 2.6 years (range, 7 months to 4.5 years). Five individuals had daily seizures at baseline and experienced at least 50% seizure reduction with treatment, sustained in four. One individual with two to four yearly seizures improved to rare events. Two individuals were seizure-free; treatment targeted cognition and development. Developmental improvements were reported in all eight patients. Weaning of ezogabine was associated with increased seizure frequency (N = 4), agitation and irritability (N = 2), poor sleep (N = 1), and developmental regression (N = 2). These data suggest that treatment with ezogabine is effective at reducing seizure burden and is associated with improved development. Minimal side effects were observed. Weaning was associated with increased seizures and behavioral disturbances in a subset. An approach targeting potassium channel dysfunction with ezogabine is warranted in patients with KCNQ2-related DEE.

We evaluated the occurrence and distribution of patterns of catamenial epilepsy in a heterogenous cohort of women with epilepsy on no hormonal therapies, enrolled in a prospective, observational study. The primary aim of the study was pregnancy rate in women with epilepsy with no prior reproductive problems. In this analysis, we included women who recorded one or more menstrual cycles with one or more seizures. We measured progesterone concentrations for one to three cycles. We defined catamenial patterns as twofold or greater average daily seizure frequency around menstruation (C1), ovulation (C2), and for anovulatory cycles, from midcycle through menstruation (C3). Twenty-three of the 89 enrolled women with epilepsy were eligible for this analysis; 12 of 23 met criteria for catamenial epilepsy; five of 23 demonstrated only a C1 pattern, two of 23 only a C2 pattern, five of 23 a combined C1/C2 pattern, and the one woman with anovulatory cycles did not demonstrate a C3 pattern. There were no differences in likelihood of demonstrating a catamenial pattern between those who reported a prior catamenial pattern and those who did not (p = .855). This analysis demonstrates the utility of app-based tracking to determine a catamenial pattern. Larger prospective studies could confirm these findings and inform potential therapeutic trial designs for catamenial epilepsy.

7054 Background: Magrolimab is a monoclonal antibody that blocks CD47, a “don’t eat me” signal expressed on cancer cells to escape immune surveillance and macrophage-mediated clearance. Prior preclinical studies have shown that CD47 is critical to RBC homeostasis, with CD47 deficiency decreasing RBC half-life. Fc-mediated opsonization also depletes RBCs, raising concerns for potential on-target anemia from anti-CD47 agents via multiple mechanisms. Notwithstanding, several clinical trials have demonstrated that magrolimab can be safely administered as a monotherapy with initial lower “priming” dose yielding transient anemia with compensatory reticulocytosis, with anemia not observed at subsequent higher maintenance doses. However, the mechanism underlying this observed protection has not been fully defined. Here we describe manageable anemia in patients (pts) with HR-MDS treated with magrolimab in combination with azacitidine (AZA) (NCT03248479) and further investigate these underlying mechanisms in preclinical models. Methods: In a multicenter prospective study, CBCs, peripheral blood, and bone marrow (BM) were collected at prespecified timepoints from HR-MDS pts (n = 57) treated with magrolimab in combination with AZA. CBCs were measured, and blood and BM samples were analyzed by flow cytometry for expression of CD47 on RBCs and WBCs. Magrolimab was initially dosed with a priming dose (1mg/kg) followed by an initial weekly maintenance dosing (30mg/kg) before transitioning to every 2 weeks maintenance dosing. AZA 75mg/m2 was administered on days 1-7 of the 28-day cycle. Preclinical modeling studies were conducted with intact and Fc-deficient anti-mouse CD47 (MIAP410) and anti-human CD47 (magrolimab) antibodies in murine models, including C57BL/6J B-hSIRPA/hCD47 mice. Results: Combination treatment of magrolimab with AZA resulted in a tolerable anemia that correlated with rapid, near complete loss of CD47 from RBCs, but not WBCs. The initial 1mg/kg priming dose was sufficient for this CD47 loss, which persisted under subsequent 30mg/kg maintenance doses. Both findings are consistent with prior clinical observations in solid tumor pts with magrolimab monotherapy and lymphoma pts in combination with rituximab. Our preclinical studies with mouse models revealed that the CD47 removal is mechanistically independent of previously described RBC antigen modulation mechanisms and cellular compartments. Instead, this CD47 loss requires anti-CD47 crosslinking between RBCs and non-RBCs. Conclusions: Overall, these results support that on-target magrolimab mediated anemia is mitigated by a near complete loss of RBC CD47. HR-MDS patients treated with magrolimab in combination with AZA exhibit a tolerable anemia through priming and maintenance doses. Clinical trial information: NCT03248479.

SignificanceThis study demonstrates the efficacy of combining macrophage-checkpoint inhibition with tumor-specific antibodies for cancer immunotherapy. The combination of anti-CD47 (magrolimab) and anti-HER2 (trastuzumab) antibodies eliminated HER2+breast cancer cells with increased efficacy due to the enhancement of antibody-dependent cellular phagocytosis by macrophages, even when the cancer cells were tolerant to trastuzumab-induced antibody-dependent cellular cytotoxicity by natural killer cells. We believe these findings present a promising therapeutic approach for treating HER2+breast cancer patients whose tumors are either sensitive or resistant to trastuzumab treatment, as long as the cells harbor the HER2 trastuzumab-binding epitope. This study supports the notion that combining CD47 blockade with existing macrophage FcR-engaging tumor-specific antibodies may be an effective approach for treating a wide range of cancers.

Abstract Background: Cancer cells overexpress CD47 to evade phagocytic programmed cell removal (PrCR) by macrophages of the innate immune system. Blocking CD47 using magrolimab (Hu5F9-G4), an anti-CD47 humanized monoclonal antibody, works via preventing CD47 signaling via macrophage Sirpα to allow PrCR, as well as using an Fc-receptor mechanism to encourage macrophage-mediated phagocytosis. We have previously shown that magrolimab in combination with tumor-targeting antibodies (e.g. rituximab) further enhances magrolimab’s anti-cancer effects in preclinical models and in the clinic [Advani R, et al. N Engl J Med 2018; 379:1711-1721]. We therefore hypothesized that magrolimab combined with anti-HER2 monoclonal antibody would synergize to promote ADCP in vitro, and HER2+ breast xenograft growth inhibition in vivo. Methods: To test this hypothesis, 1 x 105 CFSE-labeled HER2+ BT474 and SKBR3 cells were plated in 96-well ultra-low attachment plates in serum-free media. Isotype control antibody, trastuzumab, magrolimab, and trastuzumab + magrolimab combination (10µg/ml) were added, and allowed to incubate at 37°C for 30 mins. 5 x 104 human macrophages were then added each well, and were co-cultured for 2 hours. Human macrophages were stained with anti-CD11b, and phagocytosis was determined as the percentage of cells that were CD11b+ and CSFE+ using a BD LSR Fortessa Analyzer. For in vivo tumor xenograft growth kinetics, GFP+/Luciferase+ BT474 cells (1X105) were implanted with 25% Matrigel into the mammary fat pads of 4-8 week-old NOD scid gamma (NSG) female mice. Twenty-five days after engraftment, trastuzumab (100ug) was administered via intraperitoneal (IP) injection weekly, magrolimab 250ug IP was administered every other day, and PBS control was administered IP at 100uL once weekly, and tumor growth was monitored for 17 weeks by bioluminescence (IVIS) after D-luciferin injection, and quantified using Image 4.0. Results: SKBR3 and BT474 parental and Trastuzumab-resistant lines demonstrated significantly increased susceptibility to ADCP when opsonized with combination treatment of trastuzumab + magrolimab (15.34, 95%CI= 12.2 to 16.6) compared to trastuzumab (9.66, 95%CI=1.861 to 7.7579, p = 0.034), or magrolimab alone (10.23, 95%CI=1.254 to 7.037, p = 0.0083). Treatment with magrolimab maintained tumor burden within the starting range but further progressed upon treatment cessation (2.09x109, CI95% = 2801531434 to 15343840962, p = 0.0006). Trastuzumab treatment resulted in lower starting range tumor burden, but also demonstrated progression once treatment was stopped (1.72x106, CI95% = -695208489 to -86402608, p = 0.0052). However, in the combinatorial treatment arm, tumors were significantly below the starting range and did not show signs of tumor progression within the 10-week non-treatment period (8.51x105, CI95%= -311406356 to -66914472, p = < 0.0001). Conclusion: We conclude magrolimab plus trastuzumab cooperate to inhibit HER2+ xenograft growth in vivo, and that treatment effect persists even after treatment is stopped. Our ex vivo data suggests one mechanism to explain the observed tumor growth inhibition is increased susceptibility to ADCP when HER2+ tumors are opsonized by the combination of trastuzumab + magrolimab. Future clinical translation of this combination is warranted. Citation Format: Rosalynd Upton, Dongdong Feng, Allison M Banuelos, Tanuka Biswas, Stephen Willingham, Kevin S Kao, Kelly McKenna, Benyamin Rosenthal, Michal C Tal, Jens-Peter Volkmer, Mark D Pegram, Irving L Weissman. Humanized anti-CD47 monoclonal antibody magrolimab (Hu5F9-G4) plus trastuzumab potentiates antibody-dependent cellular phagocytosis (ADCP), and cooperate to inhibit human HER2+ breast cancer (BC) xenografts growth in vivo [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-06.

ObjectiveWe aimed to characterize the phenotypic spectrum and functional consequences associated with variants in the gene GABRB2, coding for the γ‐aminobutyric acid type A (GABAA) receptor subunit β2.MethodsWe recruited and systematically evaluated 25 individuals with variants in GABRB2, 17 of whom are newly described and 8 previously reported with additional clinical data. Functional analysis was performed using a Xenopus laevis oocyte model system.ResultsOur cohort of 25 individuals from 22 families with variants in GABRB2 demonstrated a range of epilepsy phenotypes from genetic generalized epilepsy to developmental and epileptic encephalopathy. Fifty‐eight percent of individuals had pharmacoresistant epilepsy; response to medications targeting the GABAergic pathway was inconsistent. Developmental disability (present in 84%) ranged from mild intellectual disability to severe global disability; movement disorders (present in 44%) included choreoathetosis, dystonia, and ataxia. Disease‐associated variants cluster in the extracellular N‐terminus and transmembrane domains 1–3, with more severe phenotypes seen in association with variants in transmembrane domains 1 and 2 and the allosteric binding site between transmembrane domains 2 and 3. Functional analysis of 4 variants in transmembrane domains 1 or 2 (p.Ile246Thr, p.Pro252Leu, p.Ile288Ser, p.Val282Ala) revealed strongly reduced amplitudes of GABA‐evoked anionic currents.InterpretationGABRB2‐related epilepsy ranges broadly in severity from genetic generalized epilepsy to developmental and epileptic encephalopathies. Developmental disability and movement disorder are key features. The phenotypic spectrum is comparable to other GABAA receptor‐encoding genes. Phenotypic severity varies by protein domain. Experimental evidence supports loss of GABAergic inhibition as the mechanism underlying GABRB2‐associated neurodevelopmental disorders. ANN NEUROL 2021;89:573–586

Objective We evaluated the yield of systematic analysis and/or reanalysis of whole exome sequencing (WES) data from a cohort of well-phenotyped pediatric patients with epilepsy and suspected but previously undetermined genetic etiology. Methods We identified and phenotyped 125 participants with pediatric epilepsy. Etiology was unexplained at the time of enrollment despite clinical testing, which included chromosomal microarray (57 patients), epilepsy gene panel (n = 48), both (n = 28), or WES (n = 8). Clinical epilepsy diagnoses included developmental and epileptic encephalopathy (DEE), febrile infection-related epilepsy syndrome, Rasmussen encephalitis, and other focal and generalized epilepsies. We analyzed WES data and compared the yield in participants with and without prior clinical genetic testing. Results Overall, we identified pathogenic or likely pathogenic variants in 40% (50/125) of our study participants. Nine patients with DEE had genetic variants in recently published genes that had not been recognized as epilepsy-related at the time of clinical testing (FGF12, GABBR1, GABBR2, ITPA, KAT6A, PTPN23, RHOBTB2, SATB2), and eight patients had genetic variants in candidate epilepsy genes (CAMTA1, FAT3, GABRA6, HUWE1, PTCHD1). Ninety participants had concomitant or subsequent clinical genetic testing, which was ultimately explanatory for 26% (23/90). Of the 67 participants whose molecular diagnoses were "unsolved" through clinical genetic testing, we identified pathogenic or likely pathogenic variants in 17 (25%). Significance Our data argue for early consideration of WES with iterative reanalysis for patients with epilepsy, particularly those with DEE or epilepsy with intellectual disability. Rigorous analysis of WES data of well-phenotyped patients with epilepsy leads to a broader understanding of gene-specific phenotypic spectra as well as candidate disease gene identification. We illustrate the dynamic nature of genetic diagnosis over time, with analysis and in some cases reanalysis of exome data leading to the identification of disease-associated variants among participants with previously nondiagnostic results from a variety of clinical testing strategies.

In recent years, immunotherapies have been clinically investigated in AML and other myeloid malignancies. While most of these are focused on stimulating the adaptive immune system (including T cell checkpoint inhibitors), several key approaches targeting the innate immune system have been identified. Macrophages are a key cell type in the innate immune response with CD47 being identified as a dominant macrophage checkpoint. CD47 is a “do not eat me” signal, overexpressed in myeloid malignancies that leads to tumor evasion of phagocytosis by macrophages. Blockade of CD47 leads to engulfment of leukemic cells and therapeutic elimination. Pre-clinical data has demonstrated robust anti-cancer activity in multiple hematologic malignancies including AML and myelodysplastic syndrome (MDS). In addition, clinical studies have been underway with CD47 targeting agents in both AML and MDS as monotherapy and in combination. This review will describe the role of CD47 in myeloid malignancies and pre-clinical data supporting CD47 targeting. In addition, initial clinical data of CD47 targeting in AML/MDS will be reviewed, and including the first-in-class anti-CD47 antibody magrolimab.

Objective To characterize the phenotypic spectrum associated with GNAO1 variants and establish genotype-protein structure-phenotype relationships. Methods We evaluated the phenotypes of 14 patients with GNAO1 variants, analyzed their variants for potential pathogenicity, and mapped them, along with those in the literature, on a three-dimensional structural protein model. Results The 14 patients in our cohort, including one sibling pair, had 13 distinct, heterozygous GNAO1 variants classified as pathogenic or likely pathogenic. We attributed the same variant in two siblings to parental mosaicism. Patients initially presented with seizures beginning in the first 3 months of life (8/14), developmental delay (4/14), hypotonia (1/14), or movement disorder (1/14). All patients had hypotonia and developmental delay ranging from mild to severe. Nine had epilepsy, and nine had movement disorders, including dystonia, ataxia, chorea, and dyskinesia. The 13 GNAO1 variants in our patients are predicted to result in amino acid substitutions or deletions in the GNAO1 guanosine triphosphate (GTP)-binding region, analogous to those in previous publications. Patients with variants affecting amino acids 207-221 had only movement disorder and hypotonia. Patients with variants affecting the C-terminal region had the mildest phenotypes. <

May 6, 2019April 9, 2019Free AccessCatamenial epilepsy: prevalence in a heterogenous cohort of women with epilepsy (P2.5-002)McKenna Kelly, Page Pennell, Jacqueline French, Cynthia Harden, Anne Davis, Connie Lau, Alexa Ehlert, Stephanie Allien, and Sarah BarnardAuthors Info & AffiliationsApril 9, 2019 issue92 (15_supplement)https://doi.org/10.1212/WNL.92.15_supplement.P2.5-002 Letters to the Editor

AbstractPurpose: Near-infrared photoimmunotherapy (NIR-PIT) is a localized molecular cancer therapy combining a photosensitizer-conjugated mAb and light energy. CD47 is an innate immune checkpoint widely expressed on bladder cancer cells, but absent from luminal normal urothelium. Targeting CD47 for NIR-PIT has the potential to selectively induce cancer cell death and minimize damage to normal urothelium. Experimental Design: The cytotoxic effect of NIR-PIT with anti-CD47-IR700 was investigated in human bladder cancer cell lines and primary human bladder cancer cells derived from fresh surgical samples. Phagocytosis assays were performed to evaluate macrophage activity after NIR-PIT. Anti-CD47-IR700 was administered to murine xenograft tumor models of human bladder cancer for in vivo molecular imaging and NIR-PIT. Results: Cytotoxicity in cell lines and primary bladder cancer cells significantly increased in a light-dose–dependent manner with CD47-targeted NIR-PIT. Phagocytosis of cancer cells significantly increased with NIR-PIT compared with antibody alone (P = 0.0002). In vivo fluorescence intensity of anti-CD47-IR700 in tumors reached a peak 24-hour postinjection and was detectable for at least 14 days. After a single round of CD47-targeted NIR-PIT, treated animals showed significantly slower tumor growth compared with controls (P < 0.0001). Repeated CD47-targeted NIR-PIT treatment further slowed tumor growth (P = 0.0104) and improved survival compared with controls. Conclusions: CD47-targeted NIR-PIT increased direct cancer cell death and phagocytosis resulting in inhibited tumor growth and improved survival in a murine xenograft model of human bladder cancer.

CD47 is a cell surface molecule that inhibits phagocytosis of cells that express it by binding to its receptor, SIRPα, on macrophages and other immune cells. CD47 is expressed at different levels by neoplastic and normal cells. Here, to reveal mechanisms by which different neoplastic cells generate this dominant ‘don’t eat me’ signal, we analyse the CD47 regulatory genomic landscape. We identify two distinct super-enhancers (SEs) associated with CD47 in certain cancer cell types. We show that a set of active constituent enhancers, located within the two CD47 SEs, regulate CD47 expression in different cancer cell types and that disruption of CD47 SEs reduces CD47 gene expression. Finally we report that the TNF-NFKB1 signalling pathway directly regulates CD47 by interacting with a constituent enhancer located within a CD47 -associated SE specific to breast cancer. These results suggest that cancers can evolve SE to drive CD47 overexpression to escape immune surveillance.

Recent technological advances in gene sequencing have led to a rapid increase in gene discovery in epilepsy. However, the ability to assess pathogenicity of variants, provide functional analysis, and develop targeted therapies has not kept pace with rapid advances in sequencing technology. Thus, although clinical genetic testing may lead to a specific molecular diagnosis for some patients, test results often lead to more questions than answers. As the field begins to focus on therapeutic applications of genetic diagnoses using precision medicine, developing processes that offer more than equivocal test results is essential. The success of precision medicine in epilepsy relies on establishing a correct genetic diagnosis, analyzing functional consequences of genetic variants, screening potential therapeutics in the preclinical laboratory setting, and initiating targeted therapy trials for patients. The authors describe the structure of a comprehensive, pediatric Epilepsy Genetics Program that can serve as a model for translational medicine in epilepsy.

ObjectiveWe sought to identify genetic causes of early onset epileptic encephalopathies with burst suppression (Ohtahara syndrome and early myoclonic encephalopathy) and evaluate genotype–phenotype correlations.MethodsWe enrolled 33 patients with a referral diagnosis of Ohtahara syndrome or early myoclonic encephalopathy without malformations of cortical development. We performed detailed phenotypic assessment including seizure presentation, electroencephalography, and magnetic resonance imaging. We confirmed burst suppression in 28 of 33 patients. Research‐based exome sequencing was performed for patients without a previously identified molecular diagnosis from clinical evaluation or a research‐based epilepsy gene panel.ResultsIn 17 of 28 (61%) patients with confirmed early burst suppression, we identified variants predicted to be pathogenic inKCNQ2(n = 10),STXBP1(n = 2),SCN2A(n = 2),PNPO(n = 1),PIGA(n = 1), andSEPSECS(n = 1). In 3 of 5 (60%) patients without confirmed early burst suppression, we identified variants predicted to be pathogenic inSTXBP1(n = 2) andSCN2A(n = 1). The patient with the homozygousPNPOvariant had a low cerebrospinal fluid pyridoxal‐5‐phosphate level. Otherwise, no early laboratory or clinical features distinguished the cases associated with pathogenic variants in specific genes from each other or from those with no prior genetic cause identified.InterpretationWe characterize the genetic landscape of epileptic encephalopathy with burst suppression, without brain malformations, and demonstrate feasibility of genetic diagnosis with clinically available testing in >60% of our cohort, withKCNQ2implicated in one‐third. This electroclinical syndrome is associated with pathogenic variation inSEPSECS. Ann Neurol 2017;81:419–429

Atherosclerosis is the disease process that underlies heart attack and stroke. Advanced lesions at risk of rupture are characterized by the pathological accumulation of diseased vascular cells and apoptotic cellular debris. Why these cells are not cleared remains unknown. Here we show that atherogenesis is associated with upregulation of CD47, a key anti-phagocytic molecule that is known to render malignant cells resistant to programmed cell removal, or 'efferocytosis'. We find that administration of CD47-blocking antibodies reverses this defect in efferocytosis, normalizes the clearance of diseased vascular tissue, and ameliorates atherosclerosis in multiple mouse models. Mechanistic studies implicate the pro-atherosclerotic factor TNF-α as a fundamental driver of impaired programmed cell removal, explaining why this process is compromised in vascular disease. Similar to recent observations in cancer, impaired efferocytosis appears to play a pathogenic role in cardiovascular disease, but is not a fixed defect and may represent a novel therapeutic target.