CD45 Antibody Research Articles

AT1412, a Patient-Derived Antibody in Development for the Treatment of CD9 Positive Precursor B-Acute Lymphoblastic Leukemia

Despite rapid advances in immunotherapeutic options for precursor B-acute lymphoblastic leukemia (ALL), outcomes remain poor especially for adult ALL and relapsed pediatric ALL. With conventional chemotherapy, remission percentages in adult ALL range from 75 to 90%, but relapse rates are high and long-term leukemia-free survival ranges between 35-70% depending on age and risk group. The introduction of CD19 targeting immunotherapy has significantly improved patient outcomes in (relapsed) B-ALL. However, tumor escape via downregulation of CD19 occurs in a significant number of patients. Therefore an ongoing urgency remains for the identification of additional or alternative immunotherapeutic targets for the treatment of ALL. AT1412 is an antibody that was identified from the peripheral blood memory B cell pool of a patient cured of metastatic melanoma after adoptive T-cell therapy, using a B cell immortalization technology (AIMSelect) with ectopic Bcl-6 and Bcl-xL expression as described previously [Kwakkenbos et al. Nat. Med. 2010]. The antibody was selected based on differential binding to melanoma cells as compared to healthy melanocytes and was shown to be successful in killing melanoma cells in vitro and in vivo [manuscript submitted]. In addition to melanoma, AT1412 binds other tumor types including B-ALL, gastric, colon- and pancreatic cancer. The target of AT1412 is the tetraspanin CD9, which is expressed by more than half of all B-ALL. Expression of CD9 has been correlated with adverse prognosis [Liang et al. Cancer Biomark. 2018]. We assessed binding of this human CD9 antibody to a panel of ALL cell lines using flow cytometry. Binding of AT1412 to the B-ALL cell lines SUP-B15, MHH-CALL-2 and CCRF-SB varied as expected based on the CD9 levels that we detected using a commercial CD9 antibody. AT1412 induced antibody dependent cellular cytotoxicity (ADCC) on these cells, in line with the level of AT1412 binding. No binding was seen to the T-ALL cell line Jurkat. Importantly, these findings were confirmed in primary ALL samples, obtained prospectively at diagnosis from a cohort of patients with T- or B-ALL (n=30). AT1412 showed binding to 61% of B-ALL samples but not to T-ALL samples. The potential of AT1412 to induce ADCC was tested on patient samples from the same panel. Remarkably, AT1412 induced ADCC of all B-ALL samples it bound to (8 out of 14) and of none of the T-ALL samples. Cytotoxicity significantly correlated with the level of AT1412 binding. These findings were supported by the observation that AT1412 induced B-ALL cell death when a freshly drawn whole bone marrow sample from a patient with newly diagnosed B-ALL was cocultured with AT1412. AT1412-induced cell death of B-ALL blasts occurred without affecting the monocytic, granulocytic and lymphocytic populations. This cell death was not observed when this patient's ALL blasts were incubated with AML-targeting antibodies. Remarkably, AT1412 induced cell death in the absence of added effector cells or other (chemo)therapeutic agents, while the bone marrow sample contained over 80% blasts and as little as 3% lymphocytes. We are currently investigating the in vivo efficacy of the antibody in a humanized immune system mouse model with human B-ALL. Taken together, the majority of precursor B-ALL blasts express CD9 and expression of CD9 is associated with a dismal outcome. Our data demonstrate that CD9 can be successfully targeted by the human CD9 antibody AT1412, suggesting that AT1412 has the potential to be developed as a therapeutic antibody for B-ALL. AT1412 is currently being advanced through preclinical development. Disclosures De Jong: AIMM Therapeutics: Employment. Levie:AIMM Therapeutics: Employment. Schotte:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. Pos:AIMM Therapeutics: Patents & Royalties: Patent WO2017119811A1. Go:AIMM Therapeutics: Employment, Patents & Royalties: Patent WO2017119811A1. Yasuda:AIMM Therapeutics: Employment, Equity Ownership. Cercel:AIMM Therapeutics: Employment. van Hal-van Veen:AIMM Therapeutics: Employment. Frankin:AIMM Therapeutics: Employment. Villaudy:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. van Helden:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. van Eenennaam:AIMM Therapeutics: Employment. Spits:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. Hazenberg:AIMM Therapeutics: Other: Employment/equity of partner/spouse.

Engineering High Affinity and Cleavage Resistant CD16 to Augment ADCC of Placental Hematopoietic Stem Cells-Derived Natural Killer Cells

Background: Celularity, Inc. is developing human placental hematopoietic stem cells-derived, cryopreserved, off-the-shelf, ex-vivo expanded and allogenic natural killer (PNK) cells for various hematological malignancies and solid tumors. NK cells play a central role in antibody dependent cell mediated cytotoxicity (ADCC) through Fc receptor CD16 in monoclonal antibody mediated anti-tumor therapies. Two allelic forms of CD16 have been identified. The 158Val/Val form has shown to have higher IgG binding affinity compared to the 158Phe/Phe form.1 The high IgG binding allele are found in about 10-20% of the normal population.2,3 In addition, activation of NK cells induces CD16 shedding by matrix metalloprotease ADAM17 at 197Ser, thus limiting ADCC responses. A single mutation (Ser197Pro) prevents CD16 shedding and increases ADCC activity in NK cells.4 Since the antibody binding affinity and CD16 expression of PNK could vary with different donors, we hypothesize that expressing a high affinity (158Val) and proteinase cleavage resistant (197Pro) CD16 variant (CD16VP) augments anti-tumor ADCC activity. Methods: Lentivirus expressing CD16VP was used to transduce human placental CD34+ cells. After transduction, the cells were cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate PNK-CD16VP cells. Non-transduced PNK cells (NT) served as a control. Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage (CD16 shedding assay) followed by immunostaining with CD16 antibody and analyzed using flowcytometry. ADCC of PNK-CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E:T) ratios. IgG was used as ADCC control. In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3x106) were intravenously (IV) administered at day 0, followed by PNK-CD16VP cells (10x106) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI). Statistical differences between the groups were calculated using paired t-test using Prism. Results: Lentiviral transduction of CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells expanded [7095 ± 2998 folds (n=8)] and differentiated into PNK cells (>90% CD56+CD3-) at day 35. PNK-CD16VP expressed 64.6 ± 10.3% (n=8) of CD16, while the NT expressed 12.1 ± 3.3% (n=8) CD16. PMA/ionomycin induced >89% shedding of CD16 in NT cells, while significantly less (<11%) CD16 shedding was observed in PNK-CD16VP cells. These results indicated that CD16VP was expressed and maintained throughout the culture process. In vitro ADCC assay demonstrated improved anti-tumor activity of PNK-CD16VP cells over NT cells against Daratumumab or Rituximab opsonized lymphoma cell lines. At 10:1 E:T ratio PNK-CD16VP cells elicited higher cytotoxicity compared to NT: 47 ± 13% against Daratumumab opsonized Daudi cells versus 25 ± 5% (n=5; p<0.05); 30 ± 13% against Daratumumab opsonized HS-Sultan cells versus 21 ± 14% (n=3; p<0.05); 30 ± 7% against Daratumumab opsonized Sudhl6 cells versus 16 ± 10% (n=3; p<0.05). Improved ADCC activities in PNK-CD16VP were also observed in other cell lines including Raji and Sudhl4 with Daratumumab and Rituximab antibodies. PNK-CD16VP were used to test anti-tumor ADCC in vivo using a disseminated Daudi Xenograft model. The preliminary data demonstrated that PNK-CD16VP combined with Daratumumab reduced BLI signal (>50%) compared to vehicle or Daratumumab alone at day 10 after treatment. This observation suggested that PNK-CD16VP demonstrated in vivo ADCC anti-tumor activity. Conclusions: In this study, we genetically modified PNK to express high affinity and cleavage resistant CD16 variant using lentivirus. The PNK-CD16VP cells demonstrated enhanced ADCC function against lymphoma cell lines in vitro and in vivo. Further development of PNK-CD16VP for immune-oncology therapeutics is warranted.

Development of an Easily Accessible Patient-Derived Xenograft (PDX) Mouse Model in Multiple Myeloma

Preclinical mouse models are important tools to recapitulate human multiple myeloma (MM) disease. Different preclinical models allow for specific hypothesis-driven research and enables researchers to address multiple questions. Though the SCID-Hu and SCID-synth-hu mice, and a recently established humanized mouse model containing the knock-in of human cytokine genes permit the growth of primary pre-neoplastic and malignant plasma cells, the high-cost, long-term workflow, lack of access to genetically engineered mice are overwhelming disadvantages of these current humanized MM mouse models. Our objective is to establish a unique patient-derived-xenograft (PDX) MM mouse model as an easily accessible approach for prevention and therapy of human MM disease. Bone marrow aspirates from MM patients upon diagnosis were obtained from the Multiple Myeloma Molecular Epidemiology Resource (University of Iowa) and mononuclear cells were isolated. Groups of 7-8 weeks old NOD/SCID/IL2RΥgnull (NSG) mice were administrated with sub-lethal irradiation. 3-5×106 unsorted MM patient-derived bone marrow mononuclear cells were intravenously injected into each recipient NSG mouse after irradiation. In order to monitor engraftment, recipient mice were bled weekly from week 2 after inoculation and serial Serum Protein Electrophoresis (SPEP) tests of recipient mice were performed. Detection of distinct M-protein band by the SPEP test with weight loss and/or limited mobility of injected recipient mice were indicative of successful MM engraftment and the endpoint of this study. M protein was found in all 30 mice after 3 ~ 5 weeks of injection human MM mononuclear cells. To further confirm that the M protein was secreted from human MM cells, we performed flow cytometry to determine human MM cells using anti-human CD138 antibody from mouse tissues. About 10% human CD138+ MM cells were detected in spleen and bone marrow from these PDX-NSG mice by flow cytometry, whereas human CD138+ cells were absent in irradiated control mice without injection of human MM cells. We also performed immunohistochemistry on bone marrow sections of PDX-NSG mice. Human CD138 protein and human light chain protein were positively stained on these samples. We next examined MM related organ damage, which is part of the defining criteria of human MM disease. Elevated blood urea nitrogen (BUN) was detected in xenograft mouse serum compared to control mice, suggesting renal insufficiency rendered by MM engraftment. Meanwhile, xenograft mouse kidney sections were stained with PAS (Periodic acid-Schiff), which demonstrated protein and cellular cast nephropathy and inflammatory infiltration. We also performed TRAP staining on representative xenograft mouse bone sections. TRAP positive osteoclasts were increased in the distal portions of the femur bones derived from these PDX-NSG mice. We present robust data that a newly developed PDX-NSG model can grow primary human MM cells. Our hypothesis holds that cells from the same patient bone marrow microenvironment support tumor plasma cells survival and growth. These factors enables this new model to recapitulate more accurately the features of human MM. We will further investigate whether this new humanized PDX-NSG model provides a better tool for understanding MM development and for a personalized medicine. Disclosures Zhan: BIPHARM LLC: Consultancy, Other: % Allocation of Profit.

NEK2 Promotes Bortezomib Resistance through Enhancing Autophagy By Upregulation of Beclin1 in Multiple Myeloma Cells

Background: Multiple Myeloma(MM) is a neoplastic plasma-cell disorder that is characterized by clonal proliferation of malignant of plasma cells in the bone microenvironment, and monoclonal protein in the blood or urine. Drug resistance is one of the main causes of cancer relapse, thus exploring the molecular mechanisms of drug resistance is important for improving therapeutic effect in MM. Our previous publications have demonstrated NEK2 mediated drug resistance via up-regulation of multidrug resistance proteins. In this study, we found autophagy was involved in NEK2 induced Bortezomib resistance in MM cells. Materials and Methods: Primary CD138 positive cells derived from healthy donors(n=6), newly diagnosed MM patients(n=9) and relapsed MM patients(n=7) were isolated by using beads conjugated with human CD138 antibody. Immunofluorescence was performed to detect the expression of NEK2 and LC3B(LC3B-Ⅰand LC3B-Ⅱ) in primary CD138 positive cells. DALGreen was used to detect autolysosome in MM cells. TAP-MS and CO-IP were used to analyze NEK2 interacting proteins. Cell proliferation were examined with soft agar colony formation and cell count. Cell apoptosis was tested through detecting cleaved Caspase3 and cleaved PARP by western blot. Xenograft mouse model of MM were prepared by subcutaneous injection of MM cells(KMS11 NEK2 OE+Scramble, KMS11 NEK2 OE+Beclin1 sh) into immunodeficient B-NDG mouse(1×106 cell/mice). Results: Firstly, immunofluorescence results showed NEK2 expression and LC3B-Ⅱ labeled autophagosome were significantly increased in CD138 positive cells derived from relapsed MM patients as compared with newly diagnosed MM patients and HD. Moreover, over-expression of NEK2 enhanced antophagy, while knockdown of NEK2 suppressed autophagy in MM cells. To explore the underling mechanisms of NEK2 induces autophagy in MM cells, TAP-MS was performed. As a result, Beclin1, an important regulatory protein of autophagy, was identified as NEK2 interacting protein, which was further confirmed by CO-IP in MM cell lines KMS11 and RPMI 8226. Additionally, we found that NEK2 regulated the expression of Beclin1 at protein level, but not at transcriptional level. Subsequent mechanism study indicated NEK2 increased the stability of Beclin1 protein through USP7 mediated deubiquitination, thereby promoting the formation of the Beclin1-Vps15-Vps34 complex, and finally, enhancing autophagy in MM cells. Furthermore, knockdown of Beclin1 significantly suppressed NEK2 induced autophagy in MM cells. Alternatively, down-regulation of Beclin1 overcame NEK2 mediated Bortezomib resistance in xenograft mouse model of MM , suggesting that targeting Beclin1 is a promising approach to therapy MM patients with high NEK2 expression. Conclusion: Our findings revealed NEK2 induced autophagy through up-regulation of Beclin1 at protein level, and confirmed autophagy was involved in NEK2 mediated Bortezomib resistance in MM. Taken together, this study provided novel insight into treating relapsed MM patients. Disclosures No relevant conflicts of interest to declare.

The Necessary for Long-Term Follow-up of Mutated Genes and Clonal Evolutions in Multiple Myeloma Combined with cfDNA Assay

Introduction)Somatic mutations in multiple myeloma (MM) are strongly related to the clinical outcome and clonal evolution over the clinical course, and are a major problem. From a clinical viewpoint, although numerous novel drugs have been utilized, achieving long-lasting and complete remission remains difficult. Recent studies have elucidated the mutated genes using next-generation sequencing, and have examined how clonal change can be acquired in myeloma. In this study, we traced the transition of the somatic mutations of bone marrow tumor cells in patients with MM over a long-term follow-up. Furthermore, we compared the somatic mutations found in serum cell-free DNA (cfDNA) and mutated genes obtained from bone marrow myeloma cells. Material and Methods)Patients diagnosed with multiple myeloma who provided written informed consent to participate in the study were enrolled. Patients were treated by immuno-chemotherapy with or without radiation between 2000 and 2017 at our institute. Bone marrow aspiration and biopsy were performed at the time of diagnosis and upon disease progression. Around the time of bone marrow aspiration, serum was obtained from a peripheral blood sample for cfDNA analysis. Myeloma cells were separated from bone marrow samples with MicroBeads of CD138 antibody and genomic DNA was extracted. The peripheral blood samples derived from myeloma patients. The cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA and bone marrow, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 126 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research. Result)We followed 7 patients' long term-clinical course and the transition of mutations (8.5 year average). The expression of myeloma driver genes, such as RAS, BRAF, and MYC, were not critical. We did, however, detect a relationship between an increase in the dominant mutated gene, such as TP53, DIS3, FAM46C, KDM6B, and EGR1 and poor prognosis in patients with myeloma. Next, we calculated the cfDNA concentrations from 34 cases. The cfDNA concentrations were significantly higher than 10 control cases (average 62.0 ng/mL (0-200 ng/mL) and 8.18 ng/mL (4.3-14.1 ng/mL), P=0.0046). The 2.5 year-progression free survival (PFS) during the first treatment of MM were tend to be poorer in the group with cfDNA>50 ng/mL (72.9%) than the group with cfDNA<50 ng/mL(25.9%), however there are no statistical significance (P = 0.15).We caluculated concordance rate of derived mutations from bone marrow MM cells and cfDNA in 7 cases. The somatic mutations found in serum cell-free DNA (cfDNA) and bone marrow MM cells were determined the correlation coefficients. However, there are few difference expression pattern in each source. In cfDNA assay, CREEP, EGR1, HDAC4, HDAC6, and JMJD1C were highly expressed as 57.1% (4/7) - 85.7% (6/7), and these results were almost the same as those for bone marrow MM cells. On the other hand, KDM1A (85.7%), PI3KCD (71.4%), and KDM3B (57.1%) were highly detected in cfDNA, although those were not frequently expressed in bone marrow. Discussion)Our data demonstrate the importance of the long-term follow-up of somatic mutations during the clinical course of myeloma. Serum cfDNA is a useful alternative source for detecting somatic mutations in MM patients during long-term follow-up. Disclosures Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy; Celgene K.K.: Honoraria. Hatake:Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Honoraria. Terui:Bristol-Myers Squibb K.K.: Research Funding; Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria.

CD9 blockade suppresses disease progression of high-risk pediatric B-cell precursor acute lymphoblastic leukemia and enhances chemosensitivity.

CD9 has been implicated in cancer progression but its prognostic relevance and therapeutic potential in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are largely unknown. In a cohort of pediatric BCP-ALL patients, we found that CD9+ cases had a significantly lower 5-year relapse-free survival rate than CD9- cases. Multivariate analysis demonstrated that CD9 positivity independently predicted inferior survival outcomes, and could be applied with established prognostic features, including prednisone response and cytogenetic status, to refine patient stratification. Administration of CD9 antibody substantially suppressed disease progression in NOD/SCID mice xenografted with CD9+ cell lines and primary leukemic blasts from patients with high-risk and refractory BCP-ALL, without compromising hematopoietic stem cell engraftment. Combination of anti-CD9 with conventional chemotherapy further reduced leukemic burden and prolonged animal survival. Mechanistically, CD9 blockade inhibited leukemic cell proliferation, induced G0/G1 cell cycle arrest, activated p38, and enhanced chemotherapeutic agent-induced apoptosis. Further, CD9 physically interacted with integrin very late antigen-4, regulated affinity to vascular cell adhesion molecule-1, and was involved in leukemia-stroma interaction. Collectively, our study established CD9 as a new prognostic marker, validated the preclinical efficacy of CD9 antibody, and laid the foundation for clinical development of CD9-targeted therapy for high-risk and refractory pediatric BCP-ALL.

Surface Proteome of Plasma Extracellular Vesicles as Biomarkers for Pneumonia and Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

Community-acquired pneumonia (CAP) and acute exacerbation of chronic obstructive pulmonary disease (AECOPD) represent a major burden of disease and death and their differential diagnosis is critical. A potential source of relevant accessible biomarkers are blood-borne small extracellular vesicles (sEVs). We performed an extracellular vesicle array to find proteins on plasma sEVs that are differentially expressed and possibly allow the differential diagnosis between CAP and AECOPD. Plasma samples were analyzed from 21 healthy controls, 24 patients with CAP, and 10 with AECOPD . The array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. We detected significant differences in the protein decoration of sEVs between healthy controls and patients with CAP or AECOPD. We found CD45 and CD28 to be the best discrimination markers between CAP and AECOPD in receiver operating characteristic analyses, with an area under the curve >0.92. Additional ensemble feature selection revealed the possibility to distinguish between CAP and AECOPD even if the patient with CAP had COPD, with a panel of CD45, CD28, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), tumor necrosis factor-R-II, and CD16. The discrimination of sEV-associated proteins is a minimally invasive method with potential to discriminate between CAP and AECOPD.

P2557A novel tool for light sheet-guided analysis of myocardial injury in murine and human samples

Abstract Background Cardiovascular diseases are the most prominent global health threats. For optimal treatment, myocardial biopsies are an indispensable requirement. Current analysis includes histological and biochemical assays with particular limits. Here we present a first approach utilizing light sheet fluorescence microscopy (LSFM) to visualize the 3D vasculatory architecture in the murine heart after experimental myocardial infarction (MI) and in human cardiac biopsies with simultaneously imaging of various immunological infiltrates for disease investigation. Methods and results For murine hearts, cardiac vasculature was stained using CD31-AF790 antibody, where we found distinct spots of negative staining, which correlated in size and localisation with other markers of cardiac damage such as triphenyl tetratzolium chloride (TTC) staining, ejection fraction (EF) reduction and plasma cardiac troponin I levels. Using a Ly6G-AF647 antibody, we were able to visualize neutrophil infiltrates 24 h after MI, which localized at the border of the I/R-injury zone. 5 d after MI we imaged the healing vasculatory network using the CD31 marker as well as infiltrated macrophages using F4/80 staining. Human cardiac vasculature was stained using CD31-AF790 antibody in a whole-mount staining approach. In short, human samples were fixed in 4% PFA and permeabelized using saponin-based detergents. Antibody staining was conducted at 30 °C in the dark over several days. Immunological infiltrates were visualized using human CD66b, CD16, CD64, CD3 and CD19 antibodies in various, simultaneous approaches. After staining, cardiac samples were dehydrated and cleared using ethyl cinnamate (ECi) for light sheet microscopy. The spatial resolution of cardiac blood vessels was determined by CD31 staining, revealing a highly organized and structured vascular tree. Simultaneous staining for immunological infiltrates showed association with areas of enhanced CD31 staining, displaying activated endothelium at sites of inflammation. Ly6G events in a murine heart Conclusion Light sheet-guided analysis of cardiac samples shows promising results in terms of synchronous vascular and immunological investigation. We highlighted its applicability in identifying specific immune cells based on immune-phenotyping. This novel technique may enable future clinical heart disease investigation with a powerful tool for visualization of 3D cardiac structure and identification of multiple immune cell infiltrates.

6-Sulfo LacNAc (Slan) as a Marker for Non-classical Monocytes.

Monocytes are subdivided into three subsets, which have different phenotypic and functional characteristics and different roles in inflammation and malignancy. When in man CD14 and CD16 monoclonal antibodies are used to define these subsets, then the distinction of non-classical CD14low and intermediate CD14high monocytes requires setting a gate in what is a gradually changing level of CD14 expression. In the search for an additional marker to better dissect the two subsets we have explored the marker 6-sulfo LacNAc (slan). Slan is a carbohydrate residue originally described to be expressed on the cell surface of a type of dendritic cell in human blood. We elaborate herein that the features of slan+ cells are congruent with the features of CD16+ non-classical monocytes and that slan is a candidate marker for definition of non-classical monocytes. The use of this marker may help in studying the role of non-classical monocytes in health and in diagnosis and monitoring of disease.

A single-center investigational study of CD36 antigen deficiency and platelet alloantibody distribution in different populations in Northern China as well as platelet alloantibodies effect on pregnancy

BackgroundPlatelet antibodies can lead to clinical diseases such as platelet transfusion refractoriness (PTR), fetal/neonatal alloimmune thrombocytopenia (FNAIT), etc. This study is aimed at understanding CD36 expression, platelet alloantibody distribution in different populations in Northern China, and effects of platelet alloantibodies on pregnancy. Study design and methodsWhole blood samples of 612 subjects including hematological patients, pregnant women, and blood donors were collected at a single center, then CD36 expressions were determined, followed by platelet antibody screening and characterization of platelet antibody specificity. A retrospective analysis was performed in 1552 pregnant women admitted to Department of Obstetrics, in order to investigate FNAIT occurrence. ResultsRate of CD36 deficiency expression was 2.12% (13/612), all cases exhibited type II deficiency without type I deficiency being detected, and such rate is lower than that in Southern China (3.43%), Japanese (4.87%) and in the black people (4.18%), and higher than that in the White people (0.09%). Positive rates of platelet antibody screening in hematological patient group (6.86%, 14/204) and in pregnant women group (6.31%, 13/206) are higher than that in blood donor group (0.49%, 1/202), P < .01. Out of 1552 pregnant women, there were not children with FNAIT. ConclusionThe frequency of CD36 deficiency in northern China was low, all of them were type II deficiency, and no CD36 antibody was detected. It is speculated that the risk of immune-related thrombocytopenia caused by CD36 deficiency in this population is very low. Platelet antibodies should be monitored early in patients with hematological and multiple miscarriages pregnant.

Primary Cerebral Plasmacytoma: A Rare Case Report with Review of Literature

AbstractSolitary craniocerebral plasmacytoma is the least common form of extramedullary solitary plasmacytoma (SP). Cerebral SP is very rare. The world literature counts only seven intracranial cases. The authors report a case of cerebral tumor of SP in a 52-year-old female who presented to a hospital with headache and difficulty in walking for 6 months. Contrast-enhanced computed tomography (CECT) brain showed left occipital intracranial space occupying lesions (ICSOL) of size 26 mm × 14 mm adjacent to superior sagittal sinus with disproportionate perilesional edema and midline shift toward the right. She underwent gross total tumor excision with left fronto-temporo-parietal decompressive craniectomy. Microscopic sections showed highly cellular tumor infiltrating white matter and gray matter. Histopathology revealed plasmacytoma. These plasma cells were seen infiltrating the adjoining brain parenchyma. Immunohistochemistry study showed the following pattern of antigens: most of the cells were negative to CD138 antibody, and majority of cells were positive (++ to +++) to CD56 antibody, more groups of cells were positive to lambda antibody than KAPPA. Bone marrow biopsy showed only 1% polyclonal plasma cells. Whole body bone scan showed no evidence of any osteoblastic skeletal metastasis. The patient recovered well, and cranioplasty was done after 6 weeks. Conclusion Cerebral SP is rarely found in clinical practice and if diagnosed properly can save patients life. This case report would definitely address many unexplored facts about cerebral SP and set milestone in the field of clinical research.

Characterization of myeloid-derived suppressor cells and cytokines GM-CSF, IL-10 and MCP-1 in dogs with malignant melanoma receiving a GD3-based immunotherapy

Melanoma in humans and canines is an aggressive and highly metastatic cancer. The mucosal forms in both species share genetic and histopathologic features, making dogs a valuable spontaneous disease animal model. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin with immunosuppressive capabilities, which are increased in many human cancers and contribute to tumor immune evasion. They are a possible target to improve immunotherapy outcomes. Current information regarding MDSCs in canines is minimal, limiting their use as translational model for the study of MDSCs. The objective of this study was to characterize major MDSCs subsets (monocytic and polymorphonuclear) and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10) and monocyte chemoattractant protein-1 (MCP-1) in canines with malignant melanoma and to evaluate changes in MDSCs and the cytokines over time in response to a GD3-based active immunotherapy. Whole blood and serum collected from 30 healthy controls and 33 patients enrolled in the University of Florida melanoma vaccine trial were analyzed by flow cytometry with canine specific CD11b, MHCII and anti-human CD14 antibodies to assess ostensibly polymorphonuclear-MDSC (CD11b+ MHCII− CD14−) and monocytic-MDSC (CD11b+ MHCII− CD14+) subsets. IL-10, MCP-1 and both MDSCs subsets were significantly elevated in melanoma dogs versus controls. Both MDSCs subsets decreased significantly following GD3-based immunotherapy administration but no significant changes in cytokines were seen over time. To our knowledge, this is the first report documenting increased monocytic-MDSCs in canine melanoma. This is consistent with human malignant melanoma data, supporting dogs as a valuable model for therapeutic intervention studies.

PF164 CD9 IS AN ADVERSE PROGNOSTIC MARKER AND PROMISING THERAPEUTIC TARGET IN HIGH‐RISK PEDIATRIC B‐PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background:B‐precursor acute lymphoblastic leukemia (B‐ALL) is the commonest childhood malignancy. Despite advances in chemotherapy‐based treatment protocols, specific subtypes of high‐risk and relapsed/refractory B‐ALL have been associated with dismal outcomes, underscoring the need for development of novel targeted agents. CD9, a tetraspanin family protein, has been implicated in tumor progression but its prognostic relevance and therapeutic value in pediatric B‐ALL remain largely unknown.Aims:(1) To characterize the expression of CD9 in a pediatric B‐ALL patient cohort and its association with long‐term survival outcomes; (2) to assess the efficacy of targeting CD9 by a neutralizing antibody for treatment of pediatric B‐ALL in the NOD/SCID mouse xenograft model; and (3) to evaluate the potential toxicity of anti‐CD9 using a hematopoietic stem cell transplantation model.Methods:Primary B‐ALL cells were isolated from bone marrow (BM) aspirates of patients consecutively enrolled in the HKALL97, IC‐BFM‐ALL2002 and CCLG2008 studies. Immunophenotypes of leukemic blasts were characterized with CD9, CD19, CD34 and CD45 antibodies by flow cytometry. Patients were stratified into CD9+ and CD9‐ subgroups for comparison of overall survival (OS) and relapse‐free survival (RFS). NOD/SCID mice were infused with B‐ALL cell lines, patient‐derived leukemic blasts or cord blood CD34+ cells, and treated with IgG control or anti‐CD9 as a single‐agent or combined with standard chemotherapy. Leukemic load and stem cell engraftment in hematopoietic organs and CNS were measured by flow cytometry, bioluminescence imaging and immunohistochemistry.Results:Among 136 pediatric B‐ALL cases, blasts of 107 patients (78.7%) were CD9+. The 5‐year RFS rate of CD9+ patients was significantly lower than that of CD9‐ patients (65.4% vs.86.2%; P = 0.038). Subgroup analysis revealed significantly reduced RFS (33.3% vs.80.0%; P = 0.022) and a trend of lower OS (55.6% vs.90.0%; P = 0.055) in CD9+ patients of the high‐risk group. Multivariate analysis revealed that CD9 positivity independently predicted inferior survival (HR = 5.2; P = 0.004), and could be applied with established prognostic features, including prednisone response and cytogenetic status, for refinement of patient stratification. In NOD/SCID mice xenografted with CD9+ B‐ALL cell lines (n = 3) and primary leukemic blasts from patients with high‐risk and refractory B‐ALL (n = 5), administration of CD9 antibody substantially reduced leukemic burden in BM, spleen, blood, liver and CNS by 78.4–99.9% (P &lt; 0.01) and prolonged animal survival by 1.6–2.1‐fold (P &lt; 0.01). Anti‐CD9 did not compromise cord blood stem cell engraftment (n = 3), indicating leukemia‐selective activities. Combined treatment with anti‐CD9 and conventional chemotherapy (vincristine, dexamethasone and L‐asparaginase) further reduced leukemic load and extended survival, when compared with animals receiving anti‐CD9 (P = 0.043) or chemotherapy alone (P &lt; 0.001).Summary/Conclusion:Our study identified CD9 as a new prognostic marker for predicting survival outcomes in pediatric B‐ALL and validated the efficacy of anti‐CD9 for suppressing leukemia progression. We propose that inclusion of CD9 into the diagnostic marker panels would potentially identify patients who might benefit from targeted therapy. In addition, anti‐CD9 could be developed as an adjunct to standard chemotherapy for treatment of high‐risk or relapsed/refractory pediatric B‐ALL, and this strategy could be extended to adult B‐ALL and other malignancies that express the CD9 surface antigen.

PF222 EPIGENETIC SILENCING OF CD9 CONFERS POOR SURVIVAL IN PEDIATRIC ACUTE MYELOID LEUKEMIA AND AGGRAVATES DISEASE PROGRESSION

Background:Acute myeloid leukemia (AML) accounts for ∼5% of pediatric malignancies with inferior survival rates of 50–70% despite optimal application of risk‐directed, chemotherapy‐based treatment protocols. It is of prime importance to identify novel molecular targets that are amenable to therapeutic interventions, which relies critically on the dissection of key regulatory pathways governing disease progression and/or pathogenesis. CD9, a tetraspanin family protein, has been implicated in cancer progression but its expression, prognostic impact and functions in pediatric AML remain largely unknown.Aims:(1) To characterize the expression and epigenetic regulation of CD9 in a cohort of pediatric AML patients and its association with long‐term survival outcomes; and (2) to elucidate the multi‐faceted functions of CD9 in pediatric AML, focusing on the crucial cellular properties affecting leukemia progression.Methods:Bone marrow (BM) samples were collected from pediatric AML patients consecutively enrolled in the NOPHO‐AML 2004 and NOPHO‐DBH AML 2012 studies. Immunophenotypes of isolated leukemic blasts were characterized with CD9, CD33, CD34 and CD45 antibodies by flow cytometry. Patients were stratified into CD9+ and CD9‐ subgroups for comparison of overall survival (OS) and relapse‐free survival (RFS). Epigenetic control of CD9 in AML cell lines was investigated by bisulfite sequencing of CD9 promoter, and confirmed by decitabine and panobinostat treatment. The impact of CD9 on leukemic cell proliferation, cell division and chemosensitivity was measured by Trypan Blue exclusion, competition, colony formation, SNARF‐1 and Annexin V/7‐AAD assays, subsequent to transduction of a CD9‐expressing lentiviral vector. The influence of CD9 on leukemia progression was evaluated in the NOD/SCID mouse xenograft model.Results:The median CD9 expression on leukemic blasts of AML patients was significantly lower than that on CD34+hematopoietic stem cells of normal BM donors (11.6% vs.46.9%, P &lt; 0.001). Higher abundance of methylated CpG sites in CD9 promoter was detected in AML compared with B‐ALL cell lines (7.3% vs. 1.7%). Treatment of CD9‐ AML cells with the DNA methyltransferase inhibitor decitabine or histone deacetylase inhibitor panobinostat increased CD9 expression by 4.9–64.1‐fold (P &lt; 0.05). Of 58 cases of pediatric AML, blasts of 19 patients (32.8%) were CD9+ (≥20% CD9 expressing cells). Kaplan‐Meier analyses revealed that the 5‐year survival rates of CD9‐ patients were significantly lower than those of CD9+ patients (OS: 48.1% vs.88.2%, P = 0.004; RFS: 35.7% vs.66.7%, P = 0.009). Overexpression of CD9 in the AML cell line MV4–11 reduced proliferation, cell division and clonogenic potential, and enhanced cytarabine‐induced apoptosis (P &lt; 0.05). NOD/SCID mice xenografted with GFP‐tagged, CD9‐overexpressing MV4–11 cells exhibited significantly reduced leukemic load in bone marrow, spleen, blood and liver by ≥71.4% (P &lt; 0.01), and prolonged survival by 1.4‐fold (P = 0.002) when compared with animals transplanted with control, GFP‐only MV4–11 cells.Summary/Conclusion:CD9 was epigenetically silenced in pediatric AML and associated with dismal survival outcomes. Reexpression of CD9 substantially suppressed AML progression and enhanced sensitivity to conventional chemotherapy. Our study collectively established CD9 as a novel prognostic factor and tumor suppressor in pediatric AML, which could potentially be applied for refinement of clinical risk group stratification and derivation of new targeted therapies.

Circulating exosomes deliver free fatty acids from the bloodstream to cardiac cells: Possible role of CD36.

Regulation of circulating free fatty acid (FFA) levels and delivery is crucial to maintain tissue homeostasis. Exosomes are nanomembranous vesicles that are released from diverse cell types and mediate intercellular communication by delivering bioactive molecules. Here, we sought to investigate the uptake of FFAs by circulating exosomes, the delivery of FFA-loaded exosomes to cardiac cells and the possible role of the FFA transporter CD36 in these processes. Circulating exosomes were purified from the serum of healthy donors after an overnight fast (F) or 20 minutes after a high caloric breakfast (postprandial, PP). Western blotting, Immunogold Electron Microscopy and FACS analysis of circulating exosomes showed that CD36 was expressed under both states, but was higher in postprandial-derived exosomes. Flow cytometry analysis showed that circulating exosomes were able to take-up FFA directly from serum. Importantly, preincubation of exosomes with a blocking CD36 antibody significantly impeded uptake of the FFA analogue BODIPY, pointing to the role of CD36 in FFA exosomal uptake. Finally, we found that circulating exosomes could delivery FFA analogue BODIPY into cardiac cells ex vivo and in vivo in a mice model. Overall, our results suggest a novel mechanism in which circulating exosomes can delivery FFAs from the bloodstream to cardiac tissue. Further studies will be necessary to understand this mechanism and, in particular, its potential involvement in metabolic pathologies such as obesity, diabetes and atherosclerosis.

Preliminary effects on the innate immune system with the combination of cetuximab and durvalumab in metastatic/ relapsed head and neck squamous cell carcinoma in a phase II trial.

e14273 Background: Cetuximab is a recombinant chimeric monoclonal IgG1 antibody which binds specifically to the epidermal growth factor receptor (EGFR) and stimulates an innate immune response by promoting natural killer (NK) cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Cetuximab is approved as a single agent in relapsed/metastatic head and neck squamous cell carcinoma (R/M HNSCC). PD-1 check-point inhibitors which release the inhibition of the adaptive immune response, are also approved as single agents in this setting. However the response rates with these drugs, when used individually range from 10-20%. We hypothesized that adding a PD-L1 inhibitor, durvalumab to cetuximab would cause anti-tumor synergy by activating the innate as well as adaptive immune systems without compromising safety in this phase-2 trial in R/M HNSCC patients who have progressed on platinum based therapy. Methods: Blood samples were collected from the first six enrolled patients prior to starting treatment and 4 weeks after the first combined dose of cetuximab and durvalumab. PBMCs were isolated, stained with a live/dead stain as well as CD3, CD4, CD8, CD56, CD16 and NK2GD (natural killer group 2 member D activation receptor) antibodies and analyzed by flow cytometry. Cytokine levels in plasma were measured using standardized ELISA assay kits. Results: Compared to pre-treatment levels, post-treatment samples showed an increase in activated cytokine producing NK cells (CD56bright/CD16-) in all but one patient. Activated cytotoxic NK cell subpopulations (CD56dim/CD16+) showed variable results post-treatment. CD8+ T cells were similar pre and post-treatment in 5 patients. TGF-b levels increased in 5 patients and decreased in 1 patient post-treatment. Interestingly, the patient with decreased TGF-b levels post-treatment had an almost doubling of CD8+ T-cells and an increase in activated cytokine producing NK cells (CD56bright/CD16-). Conclusions: The clinical trial is ongoing and therefore, comparison to clinical response has not yet been analyzed. However, these findings support the combination of cetuximab and durvalumab in R/M HNSCC given the activation of an NK-cell mediated innate immune response in these patient samples. Clinical trial information: NCT03691714.

Optimizing the Method of Cell Separation from Bile of Patients with Cholangiocarcinoma for Flow Cytometry.

Analysis of the change of the cells in bile is an evolving research field in biliary pathophysiology and has potential value in diagnosis and therapy. However, laboratory studies of cell in bile across the world are scarce. Bile was collected from the clinical patients with cholangiocarcinoma (CC). To optimize the cell separation method in bile of patients with CC, we studied the factors that may affect cell vitality in bile including the dilution buffer, centrifugal force, centrifugal time, and store time and temperature. Then these factors were modified and performance was evaluated by flow cytometry with respect to the percentage and total yield of viable cells. The separated cells from bile were stained with CD3, CD4, CD8, CD56, TCRγ/δ, CD16, CD14, HLA-DR, CD33, CD15, CD11b, lineage cocktail (CD3, CD14, CD19, CD20, and CD56), CD66b, and CD45 antibodies. The different buffer solutions were joined in bile of patients with CC; experiment results show that the different dilutions have nearly no effect on the ratio of cells in bile by flow cytometry. The best centrifugal procedure was 300 g, 10 min. Bile should be stored at 4°C rather than at normal temperature. Our study further showed that the shorter time of the bile storage, the higher viability of the cell, and immune cells existed in cells isolated from bile. Evaluating bile cell viability is necessary to evaluate method performance.

Anti-atherogenic effects of CD36-targeted epigallocatechin gallate-loaded nanoparticles

Intimal macrophages play a critical role in atherosclerotic lesion initiation and progression by taking up oxidized low-density lipoprotein (oxLDL) and promoting inflammatory process. 1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), a major type of oxidized phosphatidylcholines (PC) found on oxLDL, has a high binding affinity to the macrophage scavenger receptor CD36 and participates in CD36-mediated recognition and uptake of oxLDL by intimal macrophages. We successfully synthesized epigallocatechin gallate (EGCG)-loaded nanoparticles (Enano), which were composed of EGCG, PC, (+) alpha-tocopherol acetate, and surfactant. We also incorporated KOdiA-PC on the surface of Enano to make ligand-coated Enano (L-Enano) to target intimal macrophages. The objectives of this study were to determine the anti-atherogenic effects of Enano and L-Enano in LDL receptor null (LDLr−/−) mice.Our in vitro data demonstrated that L-Enano had a higher binding affinity to mouse peritoneal macrophages than Enano. This high binding affinity was diminished by CD36 antibodies or knockdown of the CD36 receptor in mouse peritoneal macrophages, confirming the specific binding of L-Enano to the macrophage CD36 receptor. LDLr−/− mice were randomly divided to six groups and received weekly tail vein injection with PBS, EGCG, void nanoparticles (Vnano), Enano, ligand-coated Vnano (L-Vnano), or L-Enano once per week for 22 weeks. The dose of EGCG was 25 mg per kg body weight. L-Enano at 20 μg/mL significantly decreased production of monocyte chemoattractant protein-1, tumor necrosis factor alpha, and interleukin-6 from mouse macrophages, while having no effect on their plasma levels compared to the PBS control. There were no significant differences in blood lipid profiles among six treatment groups. Mice treated with L-Enano also had significantly smaller lesion surface areas on aortic arches compared to the PBS control. Liver EGCG content was decreased by treatments in the order of EGCG>Enano>L-Enano. Native EGCG had inhibitory effects on liver and body fat accumulation. This molecular target approach signals an important step towards inhibiting atherosclerosis development via targeted delivery of bioactive compounds to intimal macrophages.

Anti-human CD9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins.

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell‐derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV‐associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti‐CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9‐green fluorescent protein fusion protein and various melanoma cell lines and bone marrow‐derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab‐mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti‐melanoma therapeutic strategies.

A Mass-Ratiometry-Based CD45 Barcoding Method for Mass Cytometry Detection.

Mass cytometry (CyTOF) is a critical cell profiling tool in acquiring multiparameter proteome data at the single-cell level. A major challenge in CyTOF analysis is sample-to-sample variance arising from the pipetting process, staining variation, and instrument sensitivity. To reduce such variations, cell barcoding strategies that enable the combination of individual samples prior to antibody staining and data acquisition on CyTOF are often utilized. The most prevalent barcoding strategy is based on a binary scheme that cross-examines the existence or nonexistence of certain mass signals; however, it is limited by low barcoding efficiency and high cost, especially for large sample size. Herein, we present a novel barcoding method for CyTOF application based on mass ratiometry. Different mass tags with specific fixed ratios are used to label CD45 antibody to achieve sample barcoding. The presented method exponentially increases the number of possible barcoded samples with the same amount of mass tags compared with conventional methods. It also reduces the overall time for the labeling process to 40 min and avoids the need for expensive commercial barcoding buffer reagents. Moreover, unlike the conventional barcoding process, this strategy does not pre-permeabilize cells before the barcoding procedure, which offers additional benefits in preserving surface biomarker signals.