anti-CD34 Antibody Research Articles

MDS-223: Response to Erythroid Stimulating Agents (ESA) and Flow Cytometric Characteristics of Bone Marrow Erythroid Precursors in IPSS-R Lower-Risk Myelodysplastic Syndrome Patients

Context: Standard treatment of anemia in lower-risk myelodysplastic syndromes (LR-MDS) is based on erythropoietic-stimulating agents (ESAs), but some patients are primary refractory (PR) or lose response (secondary refractory, SR). High serum erythropoietin (sEPO) is the main negative predictor, and treatment of ESA-resistant anemia is challenging, so more predictors are needed. EPO promotes differentiation of committed erythroid cells (COMe) into erythroid precursors (EP), constituted by early (EEP) and late (LEP) precursors. Flow cytometry (FC) can characterize COMe and EP. RED score is an FC parameter that assesses dyserythropoiesis. Objective: We aimed to correlate COMe, EP, and RED score with response to ESAs, WHO category, and sEPO in 44 anemic LR-MDS patients. Design: We evaluated 10 negative controls, 17 PR patients, 17 SR patients, and 10 long responders (LR, response duration >24 months). Fifty-nine percent had sEPO 500 mU/mL(H-EPO); 23% had single lineage dysplasia (MDS-SLD), 34% multilineage dysplasia (MDS-MLD), 23% ring sideroblasts (MDS-RS), and 20% deletion of 5q (MDS-5q-). FC analysis was performed on bone marrow aspirates for all subgroups at diagnosis and within SR subgroup at loss of response to ESA (SRII). We used anti-CD34, CD36, CD105, CD117, CD33, CD71, and CD45 antibodies to identify COMe, total erythroid cells (TOTe), and EP. Coefficients of variance of CD71 and CD36 were used to calculate RED score. Results: TOTe and COMe were increased in LR-MDS compared to controls (p=0.013 and p=0.04, respectively), with the exception of MDS-5q- cases. In the PR subgroup, LEP were increased and COMe were reduced compared to LR. In the SRII group, COMe were lower than at diagnosis (p=0.039). RED score was higher in PR compared to responders (p=0.011). MDS-5q- cases showed reduced TOTe (p=0.047), while MDS-RS cases had higher LEP and RED score (p=0.008 and p=0.012, respectively). Finally, LEP were increased in the H-EPO group compared to L-EPO (p=0.023). Conclusions: These data suggest that COMe seem to be positively associated with ESA response, whereas higher LEP and RED score are negatively associated and also correlate with MDS-RS and H-EPO. Finally, MDS-5q- cases displayed fewer TOTe and COMe, suggesting premature apoptosis of erythroid precursors.

5-Azacytidine depletes HSCs and synergizes with an anti-CD117 antibody to augment donor engraftment in immunocompetent mice

AbstractDepletion of hematopoietic stem cells (HSCs) is used therapeutically in many malignant and nonmalignant blood disorders in the setting of a hematopoietic cell transplantation (HCT) to eradicate diseased HSCs, thus allowing donor HSCs to engraft. Current treatments to eliminate HSCs rely on modalities that cause DNA strand breakage (ie, alkylators, radiation) resulting in multiple short-term and long-term toxicities and sometimes even death. These risks have severely limited the use of HCT to patients with few to no comorbidities and excluded many others with diseases that could be cured with an HCT. 5-Azacytidine (AZA) is a widely used hypomethylating agent that is thought to preferentially target leukemic cells in myeloid malignancies. Here, we reveal a previously unknown effect of AZA on HSCs. We show that AZA induces early HSC proliferation in vivo and exerts a direct cytotoxic effect on proliferating HSCs in vitro. When used to pretreat recipient mice for transplantation, AZA permitted low-level donor HSC engraftment. Moreover, by combining AZA with a monoclonal antibody (mAb) targeting CD117 (c-Kit) (a molecule expressed on HSCs), more robust HSC depletion and substantially higher levels of multilineage donor cell engraftment were achieved in immunocompetent mice. The enhanced effectiveness of this combined regimen correlated with increased apoptotic cell death in hematopoietic stem and progenitor cells. Together, these findings highlight a previously unknown therapeutic mechanism for AZA which may broaden its use in clinical practice. Moreover, the synergy we show between AZA and anti-CD117 mAb is a novel strategy to eradicate abnormal HSCs that can be rapidly tested in the clinical setting.

Anti-tumour necrosis factor α antibodies and circulating lymphocyte phenotypes in inflammatory bowel disease

ObjectiveCirculating lymphocyte subtypes are not fully explored parameters for monitoring chronic T cell activation during inflammatory bowel disease (IBD). Tumor necrosis factor α (TNFα), one of the main mediators of IBD related inflammation induces expression of CD70 on T cells. CD70 limits T cell expansion and controls CD27 receptor on activated B lymphocytes. Aim of this study was to assess the number and the frequency of CD70+ T cells and CD27+ B cells in IBD patients during inactive phase of the disease under or without anti-TNFα treatment. DesignWe studied 91 patients with inactive IBD, 31 untreated, 29 treated with infliximab (IFX), and 31 treated with adalimumab (ADA). Lymphocyte phenotypes were assessed by flow cytometry using anti-CD45, CD19, CD27, CD3, and CD70 monoclonal antibodies. IFX and ADA actual capacity of TNFα neutralization in serum was estimated by the recoveryELISA technique. ResultsWhereas CD3+ T cells were increased in treated compared to untreated patients, the percentage of the CD70+ T cells was significantly lower in treated patients indicating a ‘cooling’ effect of the biological therapy. This effect differs between samples according to the therapeutic range of the circulating drug. Although the CD19+ B-cell percentage tended to be lower in treated patients, CD19+27+ memory B cells did not show significant differences between groups. ConclusionsFrequency of peripheral blood CD70+ T cells was significantly reduced by treatment with anti-TNFα antibodies. Monitoring of this parameter of T cells can give better insight to the disease progression and therapy application in IBD patients.

Phage-Phenotype Imaging of Myeloma Plasma Cells by Phage Display

Multiple myeloma (MM) is a malignant disease based on differentiated plasma cells (PCs) in the bone marrow (BM). Flow cytometry and fluorescence microscopy, used to identify a large combination of clusters of differentiation (CDs), are applied for MM immunophenotyping. However, due to the heterogeneous MM immunophenotypes, more antibody panels are necessary for a preliminary diagnosis and for the monitoring of minimal residual disease (MRD). In this study, we evaluated the use of phage clones as probes for the identification of several PCs immunophenotypes from MM patients. First, A 9-mer M13-pVIII phage display library was screened against an MM.1 cells line to identify peptides that selectively recognize MM.1 cells. Then, the most representative phage clones, with amino acid sequences of foreign peptides closer to the consensus, were labelled with isothiocyanate of fluorescein (FITC) and were used to obtain a fluorescent signal on cells in ex-vivo samples by fluorescence microscopy. Selected phage clones were able to discriminate different MM immunophenotypes from patients related to CD45, CD38, CD56, and CD138. Our results highlight the possibility of using a phage-fluorescence probe for the simultaneous examination of the presence/absence of CDs associated with disease usually detected by combination of anti-CD antibodies. The design of a multi-phage imaging panel could represent a highly sensitive approach for the rapid detection of immunophenotype subtypes and the subsequent characterization of patient disease status.

Simultaneous enumeration of CD34+ and CD45+ cells using EasyCounter image cytometer

Accurate counting of CD34-positive cells is important for successful hematopoietic stem cell transplantation that is applied to various diseases. The aim of this study was simultaneous counting of viable CD34+ (vCD34+) and CD45+ (vCD45+) cells in apheresis samples by automatic immunofluorescence counter – EasyCounter BC. CD34+ and CD45+ cells were counted using two conjugates anti-CD34 antibody – dR110 and anti-CD45 antibody – ATTO620, respectively. The conjugates were prepared by carbodiimide method. Dead nuclear cells were counted by using monomethine cyanine dye PO-TEDM 1. The linearity and reproducibility of EasyCounter BC for CD34+ cell counting were determined (R2 = 0.99; CV values for vCD34+ cells were 6.8 ÷ 8.5% and for vCD45+ cells 4.1 ÷ 7.2%). The obtained results by EasyCounter BC were compared with those by other two standard methods – flow cytometry (Guava easyCyte 8HT) and fluorescence microscopic method (Olympus BX51) with the same conjugates. Passing–Bablok regression was performed to determine the relationship between the results of the three methods, analyzing 43 apheresis samples. Correlation coefficients for vCD45+ and vCD34+ between EasyCounter BC and Olympus microscope were 0.987 and 0.982, respectively (P < 0.0001). Better results were obtained between EasyCounter BC and flow cytometer Guava, 0.998 for vCD45+ and 0.998 for vCD34+ (P < 0.0001).

Single Dose of N-Acetylcysteine in Local Anesthesia Increases Expression of HIF1α, MAPK1, TGFβ1 and Growth Factors in Rat Wound Healing.

In this study, we aimed to investigate the influence of N-acetylcysteine (NAC) on the gene expression profile, neoangiogenesis, neutrophils and macrophages in a rat model of incisional wounds. Before creating wounds on the backs of 24 Sprague–Dawley rats, intradermal injections were made. Lidocaine–epinephrin solutions were supplemented with 0.015%, 0.03% or 0.045% solutions of NAC, or nothing (control group). Scars were harvested on the 3rd, 7th, 14th and 60th day post-surgery. We performed immunohistochemical staining in order to visualize macrophages (anti-CD68), neutrophils (anti-MPO) and newly formed blood vessels (anti-CD31). Additionally, RT-qPCR was used to measure the relative expression of 88 genes involved in the wound healing process. On the 14th day, the number of cells stained with anti-CD68 and anti-CD31 antibodies was significantly larger in the tissues treated with 0.03% NAC compared with the control. Among the selected genes, 52 were upregulated and six were downregulated at different time points. Interestingly, NAC exerted a significant effect on the expression of 45 genes 60 days after its administration. In summation, a 0.03% NAC addition to the pre-incisional anesthetic solution improves neovasculature and increases the macrophages’ concentration at the wound site on the 14th day, as well as altering the expression of numerous genes that are responsible for the regenerative processes.

A simple optical tissue clearing pipeline for 3D vasculature imaging of the mediastinal organs in mice.

Optical tissue clearing (OTC) methods render tissue transparent by matching the refractive index within a sample to enable three-dimensional (3D) imaging with advanced microscopes. The application of OTC method in mediastinal organs in mice remains poorly understand. Our aim was to establish a simple protocol pipeline for 3D imaging of the mediastinal organs in mice. Trachea, oesophagus, thymus and heart were harvested from mice after retrograde perfusion via the abdominal aorta. We combined and optimized antibody labelling of thick tissue samples, OTC with cheap and non-toxic solvent ethyl cinnamate (ECi), and light-sheet fluorescence microscopy (LSFM) or laser confocal fluorescence microscopy (LCFM) to visualize the vasculature of those tissues. A high degree of optical transparency of trachea, oesophagus, thymus and heart was achieved after ECi-based OTC. With anti-CD31 antibody immunofluorescence labelling before ECi-based OTC, the vasculature of these tissues with their natural morphology, location and organizational network was imaged using LSFM or LCFM. This simple protocol pipeline provides an easy-to-setup and comprehensive way to study the vasculature of mediastinal organs in 3D without any special equipment. We anticipate that it will facilitate diverse applications in biomedical research of thoracic diseases and even other organs.

A STAT5B–CD9 axis determines self-renewal in hematopoietic and leukemic stem cells

AbstractThe transcription factors signal transducer and activator of transcription 5A (STAT5A) and STAT5B are critical in hematopoiesis and leukemia. They are widely believed to have redundant functions, but we describe a unique role for STAT5B in driving the self-renewal of hematopoietic and leukemic stem cells (HSCs/LSCs). We find STAT5B to be specifically activated in HSCs and LSCs, where it induces many genes associated with quiescence and self-renewal, including the surface marker CD9. Levels of CD9 represent a prognostic marker for patients with STAT5-driven leukemia, and our findings suggest that anti-CD9 antibodies may be useful in their treatment to target and eliminate LSCs. We show that it is vital to consider STAT5A and STAT5B as distinct entities in normal and malignant hematopoiesis.

Contrast agent retention features in contrast-enhanced ultrasound: diagnostic performance for the prediction of papillary thyroid carcinoma

PurposeTo report a new feature of contrast-enhanced ultrasound (CEUS) and its diagnostic performance for the prediction of papillary thyroid carcinoma (PTC). MethodsThis retrospective study was conducted from October 2018 to March 2019, including 276 patients with 308 thyroid nodules who underwent CEUS examinations prior to surgery (90 patients, 122 nodules) or fine needle aspiration (186 patients, 186 nodules). Quantitative analysis of CEUS features was performed using time-intensity curves. After surgery, tissue sections stained with HE and an anti-CD34 primary antibody were used to characterize the cell number and microvessel density. The nodules were divided into retention and non-retention groups. ResultsThere were 168 malignant nodules and 140 benign nodules. The contrast-agent retention (CAR) feature was only observed in 52 papillary carcinomas. The CAR feature showed the sensitivity of 30.9% albeit the high specificity of 100%, for the diagnosis of thyroid cancers. The maximum slope coefficient of the washout index was significantly lower in the retention group than in the non-retention group (P < 0.001). The enhancement intensity during the late stage of enhancement index was significantly higher in the retention group than in the non-retention group (P < 0.001). The cell number and microvessel density in nodules with CAR features were higher (P < 0.001, P = 0.004). ConclusionThe combination of the retention pattern of the CEUS observed herein with other CEUS features may be a useful tool to improve the diagnostic of the PTC.

Rapid modification of antibodies on the surface of liposomes composed of high-affinity protein A-conjugated phospholipid for selective drug delivery

Antibody-modified liposomes, immuno-liposomes, can selectively deliver encapsulated drug ‘cargos’ to cells via the interaction of cell surface proteins with antibodies. However, chemical modification of both the antibodies and phospholipids is required for the preparation of immuno-liposomes for each target protein using conventional methods, which is time-consuming. In the present study, we demonstrated that high-affinity protein A- (Protein A-R28: PAR28) displaying liposomes prepared by the post-insertion of PAR28-conjugated phospholipid through polyethylene glycol (PEG)-linkers (PAR28-PEG-lipo) can undergo rapid modification of antibodies on their surface, and the liposomes can be delivered to cells based on their modified antibodies. Anti-CD147 and anti-CD31 antibodies could be modified with PAR28-PEG-lipo within 1 h, and each liposome was specifically taken up by CD147- and CD31-positive cells, respectively. The cellular amounts of doxorubicin delivered by anti-CD147 antibody-modified PAR28-PEG-lipo were significantly higher than those of isotype control antibody-modified liposomes. PAR28-PEG-lipo can easily and rapidly undergo modification of various antibodies on their surface, which then makes them capable of selective drug delivery dependent on the antibodies.

Abstract 2649: An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma

Abstract Introduction: Renal Cell Carcinoma (RCC) is highly resistant to systemic chemotherapy; however, targeting pro-angiogenic pathways has shown significant clinical benefit. The development of targeted anti-angiogenetic agents has been hampered by the lack of clinically relevant models for screening. We have previously reported that the generation of 3D-tumoroids containing the unaltered tumor stroma could effectively be used to detect tumor response to small molecule inhibitors as well as antibody-based therapeutic agents. In this study we described a novel ex vivo platform to develop anti-angiogenic therapeutic strategies using fresh patient tumoroids of hepatocellular carcinoma and renal cell carcinoma. Materials and Methods: All fresh tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays, 3D tumoroids measuring 150 µm in size were treated with tyrosine kinase inhibitors (TKIs) sunitinib, sorafenib or axitinib. Treatment-mediated changes in endothelial cell viability was assessed by the confocal-based high-content real time imaging platform using fluorescent labeled anti-CD31 and anti-VEGFR2 antibodies, combined with custom image analysis algorithms. Additionally, treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages, cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Results: Our data shows that endothelial cells and capillary structures could clearly be visualized within3D tumoroids by confocal microscopy. Furthermore, we were able to quantify apoptotic cell death in endothelial cells induced by TKI inhibitors. Flow cytometry analysis demonstrated significant changes in T-cell activation upon treatment with TKIs that closely correlated with increased proinflammatory cytokine release. Observations found that the combination of anti-angiogenic treatments and immuno-modulatory agents significantly improved treatment responses. Conclusion: The ex vivo platform described in this study allowed assessment of the effect of anti-angiogenetic TKIs on tumor capillary endothelial cells as well as on tumor resident immune cell populations in intact RCC tumoroids of fresh patient tumor samples. We believe this clinically relevant approach can be used to identify the most effective anti-angiogenic and immunotherapeutic drugs and drug combinations in RCC and may improve personalized therapy for individual patients in clinical studies. Citation Format: Jared Ehrhart, Mibel M. Pabón, Stephen Iwanowycz, Zhisong Tong, Tina Pastoor, Soner Altiok. An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2649.

Abstract 1719: OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages

Abstract Background: OR2805 is a fully human IgG1 antibody that binds to CD163, an immune-suppressive receptor highly expressed on tumor associated macrophages (TAMs). High numbers of CD163-expressing TAMs generally predict an unfavorable prognosis in solid tumors. CD163-expressing TAMs contribute to an immune-suppressive tumor microenvironment (TME) and inhibit an anti-tumor T-cell response by engaging immune checkpoints, producing immune-suppressive cytokines, and promoting T-cell skewing towards a pro-cancer Th2 phenotype. Relieving the immune suppression of CD163-expressing TAMs in the TME to improve T-cell-mediated responses is a rational adjunct to immune checkpoint inhibitor (CPI) therapy. Methods: OR2805 was discovered using OncoResponse's discovery platform by cell-based functional and phenotypic assays. B cells derived from CPI elite responders were cultured at clonal density, and IgG antibodies in supernatants were evaluated for binding to myeloid-derived suppressor cells. Variable-regions from positive hits were sequenced, cloned, and expressed as recombinant IgG1. OR2805 was identified as one of the top hits that relieved myeloid cell-mediated immune suppression. Co-cultures of immunosuppressive primary human M2 macrophages and autologous T cells were used to interrogate OR2805-dependent immunomodulatory responses in vitro. The anti-tumor activity of OR2805 in vivo was evaluated in a humanized mouse model. Results: OR2805 binds to CD163 expressed on TAMs and M2-like macrophages. OR2805 does not bind to other hematopoietic cells nor to a panel of human primary non-immune cells. OR2805 treatment reduces expression of cell-surface markers associated with tumor-promoting M2c-like macrophages. In co-culture assays, OR2805 relieves the suppressive effect of M2 macrophages and results in increased T-cell activation and proliferation, upregulation of T-cell activation markers, and enhanced T-cell-mediated tumor cell killing. Administration of OR2805 in humanized NSG-SGM3 mouse tumor models resulted in approximately 55% and 75% reduction in A549 tumor growth and NCI-H1975 tumor growth, respectively. In this model, OR2805 treatment significantly increased the proportions of human CD8+ T cells and human CD11b+ myeloid cells, as well as significantly enhanced expression of activation markers by human CD8+ T cells. Conclusions: OR2805 reduces M2 macrophage-mediated immunosuppression and enhances anti-tumor immune responses. OR2805 treatment induces anti-tumor activity in lung cancer xenograft models in humanized mice. These data support further development of OR2805 as an anti-cancer therapy, both as a monotherapy and an addition to current CPI therapy. Citation Format: Peter Probst, Randi Simmons, Valerie Wall, Meghan Zuck, Myriam Bouchlaka, Sam Lam, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri. OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1719.

Abstract 475: Improving liposarcoma diagnosis through cross-flow microfiltration

Abstract Introduction: Dedifferentiated liposarcoma (DDLPS) is frequently diagnosed late and patients typically respondpoorly to treatments bringing to an overall survival rate of only 10% at 10 years. Prognosis isenhanced by early detection of recurrent lesions, but current diagnostic methods rely on invasivetissue biopsies combined with whole or part-body imaging. These diagnostic and prognosticchallenges prompted us to explore the use of circulating biomolecules as potential biomarkersaccessible through bodily fluids. Extracellular Vesicles (EVs) are membrane vesicles containingproteins and nucleic acids that circulate in the blood, so they are an emerging class of easilyaccessible blood biomarkers. We recently identified extracellular vesicle EV-MDM2 as liquidbiopsy biomarker for disease identification in DDLPS. Unfortunately, due to these challenges forisolation and capture of EVs, despite the potential diagnostic and prognostic utility, the practicalrelevance of using EVs for routine analysis remains limited. Methods: A microfluidic device was fabricated via standard soft lithography in PDMS(polydimethylsiloxane). A 5 x 5 mm2 nanocapillary array membrane (nominal capillary diameterof 0.2 µm) was sandwiched between two microchannels for a vertically aligned configuration withan injection and separation channel (each 500 µm wide and 150 µm high) perpendicular to eachother. The separation channel surface was functionalized with the anti-CD63 antibody forcapturing EVs. Results: We present here a facile approach to fabricate and operate a micro-nanofluidic device that usescross-flow filtration to isolate and capture liposarcoma derived EVs. Our methodology integratesthe unit operations of size based separation with immunoaffinity-based capture of extracellularvesicles in the same device. The eluted media collected showed ~84% extracellular vesiclerecovery from the liposarcoma cell conditioned media and ~38% extracellular vesicle recoveryfrom dedifferentiated liposarcoma patient serum when compared to extracellular vesicle isolationand subsequent quantification by ultracentrifugation. The results reported here also show a five-fold increase in amount of critical liposarcoma relevant extracellular vesicle cargo obtained in30 minutes presenting a significant advance over existing state-of-art. Conclusion: In conclusion we developed a cross-flow filtration micro-nanofluidic device for isolation, selectivecapture and release of liposarcoma EVs. Citation Format: Lucia Casadei, Patricia Sarchet, Adarsh Choudhury, Shaurya Prakash, Raphael Pollock. Improving liposarcoma diagnosis through cross-flow microfiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 475.

Vincristine-Loaded Platelets Coated with Anti-CD41 MAbs: A New Proposal for the Treatment of Immune Thrombocytopenia

Objective: Immune thrombocytopenia (ITP) is an autoimmune disorder in which platelet-reactive autoantibodies accelerate the destruction of platelets. Macrophages play a crucial role in ITP through IgG-Fcγ receptor-mediated platelet clearance. Patients responding to initial therapies often troubled with frequent relapses, severe reverse reactions and poor life quantity. Therefore, searching new effective strategies for ITP treatment is in urgent need.Methods: In this study, platelets coated with anti-CD41 antibodies, which can mimic autoantibody-binding platelets in ITP patients, were used as a smart drug delivery system to load vincristine (VCR). Vincristine-loaded platelets coated with anti-CD41 mAbs (CD41-VLP) can selective release VCR in macrophages through IgG-Fcγ receptor-mediated phagocytosis. Scanning electron microscopy (SEM), dynamic light scattering (DLS), and platelet aggregation assays were performed to evaluate the morphological and functional changes of CD41-VLP. The encapsulation efficiency, drug loading and intracellular VCR concentration were determined using high performance liquid chromatography (HPLC). Macrophages were derived from THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The expression of cell differentiation antigen (CD11b and CD14) and was detected by flow cytometry (FCM). The growth inhibition of CD41-VLP on macrophages was detected by Cell Counting Kit-8 (CCK-8) assay and the macrophage apoptosis was quantified by FCM. The possible mechanism of CD41-VLP on macrophages was further investigated by western blotting.Results: platelets coated with anti-CD41 antibodies could load VCR with high drug loading and encapsulation efficiency. There is no significant difference in morphology and function between platelets and CD41-VLP. Meanwhile, VCR was released from CD41-VLP in a pH-triggered behavior and VCR would be rapidly released in an acidic condition. The THP-1 derived macrophages was detected with a strong ability of phagocytosis and a high expression of CD11b and CD14. In the group of macrophages treated with CD41-VLP, intracellular concentration of VCR was significant increased compared with other groups, which demonstrates that Cd41-VLP can selectively deliver VCR to macrophages. In the CD41-VLP group, the growth inhibition rate was also much higher than other groups and the apoptosis rate was significant increased compared with other groups. These results suggest that CD41-VLP can enhance the cytotoxicity of VCR against macrophages. Furthermore, In the CD41-VLP group, the expressions of pro-apoptotic proteins,including Bax, caspase-3 and CyclinB1, were significantly increased and the expression of Bcl-2, anti-apoptotic protein, was decreased. This finding indicates that CD41-VLP might improve the effects of VCR on macrophages through regulating the expression of apoptosis-related proteins.Conclusion: CD41-VLP can improve the cytotoxicity of VCR against macrophages, thus establishing a new targeted therapy for ITP treatment. DisclosuresNo relevant conflicts of interest to declare.

A Descriptive Study of Viral RNA Sequences Identified from Single-Cell Analysis of Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a hematopoietic disease responsible for a large number of cancer-related deaths, with an estimated 10,600 deaths in 2017. Viruses are known causative agents for murine and avian myeloid leukemias; however, a viral etiology has not been definitively established for human AML. Identifying an infectious etiology may lead to an improved understanding of the disease which in turn leads to potential better diagnostics and therapeutic strategies. In this study, single-cell RNA sequencing was employed to analyse the transcriptomic landscape of mononuclear cells found in AML patients in an unbiased way. The sequencing data obtained were then mapped to a viral nucleotide database to identify potential viral RNA transcripts in the cells.White blood cells or mononuclear cells were isolated from the bone marrow samples obtained from 3 AML patients by pre-staining with Hoescht, which selects for all white blood cells. Anti-CD34 antibody was also used to select for CD34 cells, which are known multipotent progenitor cells in the blood. Using the Automated Microfluidic C1 System, the isolated cells were lysed and the mRNA obtained was reverse transcribed and amplified. Using the Nextera XT DNA Sample preparation kit, cDNA library was prepared from the individual cells and processed on Illumina Next Sequencing (NexSeq) and HiSequencing 2000 (HiSeq) platform. Paired reads were generated for downstream analysis. The sequencing data were then mapped to a viral nucleotide database and mapped reads were analysed using an in-house virus identification script to determine the validity of the viral RNA transcripts found.Single-Cell RNA sequencing was performed on 212 mononuclear cells, comprising 132 CD34 cells and 80 non-CD34 cells. The sequencing data obtained from 73 cells were successfully mapped to 13 different viruses. 44 of these cells observed to have viral RNA transcripts were CD34 cells. Some of the viruses that were found included Moloney Murine Leukemia Virus (M-MuLV), Murine Osteosarcoma Virus (MSV), Feline Sarcoma Virus (FeSV), Friend Murine Leukemia Virus (F-MuLV), Fujinami Sarcoma Virus (FJSV), Hepatitis C Virus (HCV), HERV-K Provirus (HERV-K), Adenovirus (ADV), Moloney Murine Sarcoma Virus (M-MuSV), Rauscher Murine Leukemia Virus (R-MuLV) and Tomato Mosaic Virus (ToMV).Single-cell RNA sequencing was the method of choice as it enables us to analyze the transcriptomic landscapes at a single cell level, eliminating the likelihood of any minute viral RNA transcripts from being eclipsed as is the case with bulk sequencing. Through this, we have identified a variety of viral RNA sequences and it was observed that CD34 cells were found to harbor more viral RNA transcripts relative to non-CD34 cells. The presence of viruses in CD34 cells may cause genetic instability and lead to alteration of oncogenes or tumour-suppressor genes. These mutations in the progenitor cells could then be passed down to the cells it would differentiate to. However, further work is required to validate these observations and determine the properties of the identified viral sequences to be able to understand its role in tumorigenicity [Display omitted] DisclosuresChng:Janssen China R&D: Research Funding.

Anti-CD45 Radioimmunotherapy Followed By Haploidentical Allogeneic Hematopoietic Cell Transplantation for Advanced Acute Leukemia or High-Risk MDS

Background: Although hematopoietic cell transplantation (HCT) may offer the best chance for cure for patients with relapsed/refractory hematologic malignancies, many of these patients will relapse after HCT. Escalating doses of total body irradiation (TBI) or chemotherapy prior to HCT can decrease relapse, but incurs higher toxicity, resulting in no survival benefit. Therefore, there is a need to reduce relapse without increasing the toxicity of the conditioning regimen prior to HCT. In addition, many potentially curable candidates for HCT do not have a fully human leukocyte antigen (HLA) matched donor. This is an especially challenging hurdle for many ethnic minority patients, yet nearly all patients have a related donor identical for one HLA haplotype (i.e., haploidentical). Radioiodinated anti-CD45 radioimmunotherapy (RIT) has been shown to deliver high-doses of radiation specifically to hematolymphoid organs, while sparing normal tissues. We report a phase I/II study using iodine-131 (131I)-anti-CD45 antibody (BC8) combined with pre- and post-transplant cyclophosphamide, and reduced intensity conditioning prior to haploidentical HCT.Methods and Patients: Eligible patients over the age of 18 had relapsed or refractory acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), or high-risk (>5% active marrow blasts) myelodysplastic syndrome (MDS). Patients received a 131I-anti-CD45 dosimetry dose, followed by serial gamma-camera imaging for organ dosimetry estimates. One to two weeks later a therapeutic dose delivering escalating doses (12 to 26 Gy) to the liver (the normal organ that received the highest dose) was administered, followed by fludarabine (FLU; 30 mg/m2 on day -6 to -2), cyclophosphamide (CY; 14.5 mg/m2 on day -6 and -5), 200 cGy TBI on day -1, and subsequent haploidentical marrow HCT on day 0. For GVHD prophylaxis, patients received CY (50 mg/kg) on day+3 plus standard tacrolimus and mycophenolate mofetil.Results: A total of 25 patients have been treated (21 with advanced AML, 3 with ALL and 1 with high-risk MDS). Ten patients were in florid refractory relapse with 6-20% abnormal marrow blasts while 9 were in remission with minimal residual disease (MRD) 0.03-5.4% by flow cytometry, and only 6 were in remission and MRD negative by flow cytometry. All 24 patients who had marrow aspiration on day 28 after HCT achieved a morphologic remission, with only two patients showing MRD by flow cytometry (0.002 -1.8% abnormal blasts). There were no graft failures, and all patients with chimerism studies (n=15) had 100% donor CD3+ and CD33+ cell engraftment at day 28 after haploidentical HCT. Median time to neutrophil engraftment (ANC>500/ml for 3 consecutive days) was 15 (range, 12-19) days. Median time to platelet engraftment (platelets >20,000/ml for 7 consecutive days without transfusion support) was 23 (19-44) days. The maximum tolerated dose was not determined and there were no unexpected adverse events, as the most frequent grade ≥3 adverse events were infections/neutropenic fevers (32), gastrointestinal (22), and laboratory abnormalities (cytopenias 18; metabolic 16). Five patients had grades 3-4 acute GVHD. Twelve patients relapsed with median time to relapse of 2.4 months. Most deaths have been from relapse (11), and infections (2). Eight (32%) patients were alive at a median follow up of 4 years after HCT (range 1 to 9 years). Overall survival at one and three year was 42% and 26%, and progression-free survival was 40% and 25%, respectively (Figure).Conclusions: Although patients with active disease are not traditionally referred for HCT, the use of 131I-anti-CD45 RIT prior to a haploidentical HCT may facilitate improved survival for these patients. Given these results, anti-CD45 RIT prior to HCT using haploidentical donors warrants further investigation for patients with advanced leukemia and high-risk MDS. [Display omitted] DisclosuresOrozco:Actinium Pharmaceuticals: Other: Research Funding to Institution for sponsored Clinical Trials. Shadman:PLEXXIKON: Research Funding; AbbVie: Other: advisory board; Genentech: Consultancy, Research Funding; Emergent: Research Funding; TG Therapeutics: Research Funding; Gilead: Research Funding; Acerta Pharma: Research Funding; Celgene: Research Funding; Pharmacyclics: Other: advisory board, Research Funding; Merck: Research Funding. Gopal:Seattle Genetics: Consultancy, Research Funding. Pagel:Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Distinctive Platelet Features and Lymphocyte Sets Distribution in Immune Thrombocytopaenia Patients Responders to Thrombopoietin Receptor Agonists

Background: The heterogeneous pathophysiology of immune thrombocytopaenia (ITP) may explain the wide variation in the presentation of the disease, the clinical course and the response to therapeutic treatments. Among them, agonists of thrombopoietin receptor (TPO-RA) were incorporated as a second line agent for the management of ITP patients who do not respond to other treatments. We hypothesized that platelet and white cells from these patients might present some characteristics different from those observed in ITP patients who don't need to be treated and those responders to a first line therapeutic agent. In order to test this hypothesis we designed this study to evaluate platelet function and distribution of lymphocyte´s compartments in three ITP patient's groups: 1) untreated (UT) and treated with 2) TPO-RA or 3) corticoids.Materials and Methods: We recruited 33 UT ITP patients (30% male), 35 treated with TPO-RA (45% male), 15 with corticoids (25 % male) and 96 healthy controls (42% male).The La Paz Universitary Hospital Ethics Committee approved the experimental protocol.Platelet-activation, granule's content release, surface exposure of phosphatidylserine (PS) and activities of caspase-3, -8 or -9 were assessed by flow cytometry (respectively performed with PAC1, anti P-selectin and anti-CD63 antibodies, from BD; Annexin V from BD Pharmingen and Millipore kit).The frequency of T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes, natural killer (NK) CD56bright and CD56dim and regulatory T (Treg) [CD4+CD25+ forkhead box protein 3(FoxP3)+] were determined by multi-parametric flow cytometry panels (BD Biosciences).The statistical analysis of the experimental data was using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA).Results: Platelet count were similar in all ITP groups but lower than controls (p<0.001). Platelet activation, evaluated by binding of PAC1 (antibody that only recognizes the active form of fibrinogen receptor) after the stimulation with TRAP (an agonist for PAR-1 receptors), were equally reduced in all ITP groups. Platelet activation with ADP was only reduced in the TPO-RA ITP patients. A diminished exposure of P-selectin and CD63 was also observed in UT and TPO-RA ITP patients after platelet stimulation with TRAP (Table 1). No differences were observed in surface expression of CD41, CD61, CD42a and CD42b between ITP patients and healthy controls. Platelet apoptosis evaluation showed that ITP patients tended to have an increased activity of caspases -3,7,-8 and -9 activities, but this observation was significant only in the ITP group of treated with TPO-RA (Table 1).Regarding lymphocytes, no differences were observed in LT subsets among all groups. Intermediate (IM, CD14++CD16+), but not non-classical (NCM, CD14+CD16++) and classical (CM, CD14++CD16-) monocytes were increased in UT and TPO-RA ITP patients. CD56brightCD16- NKand CD56brightCD16+NK, abundant cytokine producers with a weak cytotoxic activity, were increased only in TPO-RA ITP patients. In this group a diminution was observed in memory LB cells. Regarding LTreg population, their count was reduced in UT and corticoid-treated patients, whereas their values reach normality in the TPO-RA treated group (Table 2).Conclusions: All ITP patients, untreated or responders to treatment with TPO-RA and corticoids recovered platelet count but not its function. TPO-RA-responders patients showed an increased apoptosis compared to the other ITP patients recruited. Nevertheless, TPO-RA seems to have a beneficial regulatory effect on immune system that might counteract platelet destruction.Work supported by grant from FIS-FEDER PI15/01457. NVB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresÁlvarez-Roman:Shire: Consultancy; Novo Nordisk: Consultancy. Jimenez-Yuste:Novo Nordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy.

Developing Live Imaging Strategies to Analyze the Influence of Cell Cycle Rate on MEP Fate Decisions at the Single Cell Level

Elucidating the mechanisms underlying hematopoietic cell fate decision is critical to our understanding of physiological and pathological hematopoiesis. Robust response to physiological stress requires rapid commitment and expansion of mature blood cells derived from less committed progenitors. The processes dictating the directed differentiation of these progenitors are poorly understood.Preliminary data from our lab suggest that cell cycle rate influences commitment of the Megakaryocyte/Erythroid Progenitor (MEP) to the megakaryocytic and erythroid (Er) lineages. To test whether slower cycling MEPs tend to be more committed to the megakaryocytic lineage, we have developed a time-lapse microscopy imaging approach to measure the number and duration of cell cycles of single cells and their progeny as they make their cell fate decision determined by their fluorescent marker expression in real-time.CD34-selected mobilized peripheral blood cells were stained with desired antibodies and sorted using our recently published scheme: Lin-CD34+CD38midCD45RA-FLT3-MPL+CD36-CD41- (Sanada and Xavier-Ferrucio, et al. Blood 2016) that is highly enriched for bipotent MEPs than populations previously reported. MEPs were plated at low density (25 cells/15μL) in MegaCult collagen-based medium containing SCF, IL3, IL6, TPO, and EPO in the center of a MatTek plate under a coverslip to minimize focal range. After the MegaCult polymerized, 1.5mL of additional MegaCult and cytokines were added to the dish to support growth and expansion of megakaryocytes and erythroid cells for 12 days. Cells were imaged every 2 hours in DIC for the first three days, and every 20 minutes for 8 additional days. On the 10th day, fluorescently conjugated anti-CD41 (to identify Mks) and anti-CD235a (to identify Er cells) antibodies were diluted in IMDM and added to the imaged dish. The antibodies permeated the MegaCult and labeled the committed cells. Imaging continued every 20 minutes in DIC, FITC, and Cy5 channels for an additional day. Using the Baxter Algorithm to analyze time-lapse images, we traced the progeny back to the parent cell, and calculated the number of cell cycles, as well as the duration of each cycle leading to lineage commitment. We have validated this method by measuring cell cycle rates of bi-potent MEPs, Mk-restricted progenitors, and Er-restricted progenitors, and found Er-restricted progenitors to have a significantly shortened cell cycle rate compared to Mk-restricted progenitors.To further investigate the role of cell cycle rate in MEP commitment, we utilized our live imaging strategy to collect and analyze cell cycle rate data for transduced MEPs expressing shRNA against Myb. Myb is a known regulator of the G2/M transition by binding the promoter and altering expression of Cyclin B1 in human blood cells (Nakata et al., MCB, 2007). Here we show that MEPs knocked down for Myb have no difference in survival compared to scramble controls. Interestingly, the rate of cell cycle divisions is significantly longer in MEP progeny lacking Myb, and this correlates with an increase in the ratio of CFU-Mk to BFU-E and CFU-Mk/E. This data suggests that by lengthening the cell cycle rate, fate decision of the bipotent MEP can be swayed towards the Mk lineage, which has a longer cell cycle rate compared to the Er lineage. In the future, live imaging analysis of single cells can be utilized to answer a plethora of questions regarding fate decisions and lineage commitment of hematopoietic stem and progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

A Novel Combination of Anti-CD3 x Anti-CD123 Monoclonal Antibodies to Redirect Activated T Cells to Target Acute Myelogenous Leukemia

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy of the bone marrow (BM) and blood, characterized by clonal expansion of neoplastic myeloid precursor cells, and impairment of normal hematopoiesis. AML accounts for the largest number of annual deaths from leukemias among adults in the United States. AML blasts as well as leukemic stem cells express high levels of interlekin-3 (IL-3) receptor-a(CD123). Our previous studies show the anti-CD3 activated T cells (ATC) can be redirected with bispecific antibodies (BiAb) to CD20 for non-Hodgkin's lymphoma after stem cell transplantation (SCT), Her2/ neu for breast cancer, EGFR for pancreatic cancer and GD2 for neuroblastoma. In this study, we investigated whether ATC armed with anti-CD3 x anti-CD123 bispecific antibody (CD123Bi) can target CD123 expressing targets. Methods: ATC were produced by stimulating peripheral blood mononuclear cells (PBMC) with anti-CD3 in the presence of IL-2. Bispecific antibody was produced by chemical hetero-conjugation of anti-CD3 (OKT3) with anti-CD123 monoclonal antibodies. We tested four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) as targets for cytotoxicity by CD123Bi armed ATC using 51Cr release assay. Cytokines and chemokine release during CD123Bi armed ATC mediated killing of targets were analyzed by multiplex luminex assay. Results: All four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) showed 70-90% CD123 expression and 10-70% CD123 expression on leukemic blasts. Arming dose titration ranging from 0.1 to 100 ng CD123Bi/106 ATC showed saturation at 5 ng dose, the mean specific cytotoxicity 20% plateaued at arming doses of 5, 50 and 100 ng/106 ATC at an effector to target ratio (E/T) of 10:1. ATC from 4 normal donors at 50 ng arming dose at E/T of 25:1 showed mean specific cytotoxicity of 22 and 40% directed at TF-1 and NoMo-1 cell lines, respectively. The cytotoxicity was also confirmed by analysis of IFNγ and TNFα release during cytotoxic activity directed at AML cell lines by CD123Bi armed ATC. Conclusions: ATC armed with CD123Bi at 50 ng/106 cells showed comparable cytotoxicity directed at either high or low leukemic blast populations. Since most of the CD33 positive cells were also CD123+, it is likely that CD123Bi armed ATC will be able to target CD33 positive leukemia stem cells. This approach may provide a non-toxic strategy to target leukemic blasts and leukemic stem cells. DisclosuresNo relevant conflicts of interest to declare.

Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplant Using a Human Antibody Drug Conjugate Targeting C-KIT

For patients undergoing hematopoietic stem cell transplantation (HSCT), conditioning remains a primary source of transplant related morbidity and mortality and limits the broader application of this life-saving therapy. Current conditioning regimens, including total body irradiation and high dose chemotherapy, cause acute toxicity, cytokine storm, increased graft-versus-host disease, and organ damage. In addition, these regimens are also associated with the development of secondary malignancies, autoimmunity, and infertility.To address this significant unmet medical need, we have developed a conditioning regimen using an antibody drug conjugate (ADC) targeting C-KIT (CD117) to specifically deplete host hematopoietic stem cells. CD117 is highly expressed on hematopoietic stem and progenitor cells (HSPCs) and binding to its ligand, stem cell factor, promotes hematopoiesis, self-renewal, and rapid internalization of the receptor. The rapid internalization and selective expression of CD117 on HSPCs make it an ideal target for an ADC-based approach to depleting HSPCs, while limiting systemic toxicity. In addition, CD117 is frequently overexpressed on malignant cells in patients with acute myelogenous leukemia. Thus, an ADC targeting CD117 may provide an opportunity for disease control prior to or concomitant with an HSCT. Previous studies have demonstrated that an antagonistic antibody against CD117 lead to high levels of HSPC depletion and donor engraftment in immunocompromised mice, but failed to facilitate this effect in immunocompetent hosts. To overcome the engraftment barrier, additional studies were conducted with an anti-CD117 antibody conjugated to the ribosome inactivating toxin, saporin, which allowed for robust HSPC depletion and high levels of congenic donor chimerism in immunocompetent mice (Czechowicz et.al., Blood 2016, 128:493).To advance this technology toward the clinic, we have developed a fully human anti-CD117 antibody conjugated to a small molecule toxin capable of depleting proliferating and quiescent cells (CD117-ADC), that demonstrated greater than 90% killing of the leukemia cell line Kasumi-1, and was equally effective at killing primary human CD34+ bone marrow cells during in vitro culture. Humanized NSG mice treated with CD117-ADC had greater than 90% depletion of human HSPCs in the bone marrow, 21 days following a single administration of the ADC. The specificity of CD117-ADC for HSPCs was confirmed by the presence of stable peripheral human lymphocyte populations, and absence of human myeloid cells; indicating a lack of stem and progenitor cells capable of replenishing these short-lived cells. In addition to the ability of CD117-ADC to deplete HSPCs in humanized mice, preliminary data suggests that CD117-ADC is also effective at reducing tumor burden in mice challenged with CD117 expressing tumor cells.Eliminating HSPCs from the stem cell niche with a targeted ADC has the promise to enable the engraftment of donor HSCs, while preventing the systemic toxicity associated with irradiation and chemotherapy. We have demonstrated that an ADC targeting CD117 is capable of depleting human HSPCs in vitro and in vivo and has the potential to reduce tumor burden in mice. Ultimately, this novel approach for safer conditioning prior to HSCT could greatly increase the number of patients eligible for transplant and significantly reduce the damaging side effects associated with current protocols and may provide effective non-genotoxic conditioning for all patients suffering from malignant or non-malignant diseases who require a HSCT. [Display omitted] DisclosuresHartigan:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Pearse:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Goncalves:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.