bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vitro and In Vivo Characterization of CD19/CD3 Tandab AFM11 and CD19/CD16A Tandab AFM12 Targeting NHL
Authors: Zhao Xing , Rajasekaran Narendiran , Reusch Uwe , Weichel Michael , Ellwanger Kristina , Marschner Jens-Peter , Treder Martin , Kohrt Holbrook 
Published: October 2019

Abstract
Introduction: CD19 is expressed by B cells from early development through differentiation into plasma cells, and represents a validated target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Different CD19-targeting T-cell engagers are investigated in clinical studies for the treatment of NHL or ALL, including Affimed's AFM11, a bispecific CD19/CD3 TandAb antibody, which is currently investigated in a phase 1 dose escalation study. Indeed, Affimed's bispecific tetravalent platform comprises not only T-cell engaging TandAbs with two binding sites for CD3, but also NK-cell recruiting TandAbs with two binding sites for CD16A. In the present study, Affimed's AFM11, was characterized and compared in in vitro and in vivo studies with the CD19/CD16A TandAb AFM12.
Methods: Analogous to the CD19/CD3 TandAb AFM11, a bispecific tetravalent TandAb AFM12 was constructed with two binding sites for CD19 and two sites for CD16A. Both TandAbs were characterized side by side for their biophysical properties, binding affinities to CD19+ tumor target cells and to their respective effector cells by flow cytometry. Kinetics and dose-response characteristics were evaluated in in vitro cytotoxicity assays. Potency and efficacy of both TandAbs were compared on different CD19+ tumor target cell lines using primary human effector cells. To compare the efficacy of AFM11 and AFM12 a patient-derived tumor xenograft model was developed.
Results: AFM12 mediated efficacious target cell lysis with a very fast on-set in vitro. Lysis induced by AFM11 was less efficacious (lower specific lysis than AFM12) but reproducibly more potent (lower EC50 value). In addition to the potency and efficacy of AFM11 and AFM12, different aspects of safety, such as effector cell activation in the presence and absence of target cells were investigated and will be described.
Conclusions:
Affimed's CD19/CD3 and CD19/CD16A TandAbs with identical anti-CD19 tumor-targeting domains but different effector cell-recruiting domains represent interesting molecules to study T-cell- or NK-cell-based immunotherapeutic approaches. The comparison of AFM11 and AFM12 demonstrated that AFM12-mediated lysis was fast and efficacious, whereas AFM11 showed a higher potency. In summary, the NK-cell recruiting TandAb AFM12 represents an alternative to T-cell recruiting molecules, as it may offer a different side effect profile, comparable to that of AFM13, the first NK-cell TandAb clinically investigated.

Disclosures
No relevant conflicts of interest to declare.
Preclinical Analysis of Linsitinib for the Treatment of Polycythemia Vera
Authors: Basu Titiksha , Gruender Albert , Dierks Christine , Pahl Heike L 
Published: October 2019

Abstract
Even after the discovery of JAK2V617F in majority of Polycythemia Vera (PV) patients and the generation of JAK2 inhibitors, the treatment of PV remains to be improved. An increased understanding of aberrant signaling in PV cells may yield novel drug targets for this patient population. The formation of erythroid colonies in the absence of exogenous erythropoietin (Epo), so called Epo-independent erythroid colonies (EECs), is a pathognomonic hallmark of PV. Over 20 years ago, Axelrad and colleagues proposed that EEC formation is not due to Epo-independence. Rather, they postulated that PV cells are hypersensitive to Insulin Growth Factor-1 (IGF-1). Subsequently, increased IGF-1 receptor (IGF-1R) phosphorylation was demonstrated in PV MNCs. In addition, serum concentrations of IGF-1 binding protein (IGF-1BP) are significantly elevated in PV patients. It has been shown that IGF-1 cooperates with JAK2V617F to increase JAK/STAT signaling, elevating constitutive phosphorylation of STAT factors and thereby augmenting aberrant proliferation. We therefore hypothesized that inhibition of IGF-1 signaling would ameliorate the PV phenotype, even in the presence of JAK2V617F.
The dual IGF-1R and insulin receptor (IR) kinase inhibitor linsitinib (formerly called OSI-906) was used to investigate the contribution of the IGF-1 pathway to PV pathophysiology. In methylcellulose colony assays, linsitinib significantly reduced the EEC formation from PV MNCs. In contrast, in the presence of EPO, colony formation from either PV or healthy control MNCs was not affected. These data suggest that linsitinib specifically inhibits growth of the PV clone. Linsitinib likewise significantly reduced the cytokine independent proliferation of Ba/F3 cells expressing JAK2V617F but did not affect proliferation of Ba/F3 cells expressing either JAKWT or JAK2V617F in the presence of cytokine (mIL3). These data again argue that IGF-1 signalling is required for aberrant, cytokine independent growth. In addition to reducing proliferation, Linsitinib also induced apoptosis in IL3-independent JAK2V617F transduced Ba/F3 cells. Using the JAK2V617F bone marrow transplant (BMT) model, we investigated the effect of linsitinib on the PV phenotype in vivo. Linsitinib treatment significantly reduced the elevated RBC count, hematocrit and hemoglobin in this PV mouse model. In addition, the white blood cell count was decreased.
The data presented here provide a pre-clinical rationale for expanding the investigation of linsitinib as well as similar IGF-1 pathway inhibitors for the treatment of PV. The effect of combining JAK2 and IGF-1 inhibition may prove especially promising in this patient population and is currently being investigated.

Disclosures
No relevant conflicts of interest to declare.
A Novel Erythroid Regulator MLF1IP Promotes Proliferation in Mice By Regulating Cell Cycle
Authors: Zhang Tianjiao , Ma Xiaotang , Li Bing , Huang Gang , Gale Robert Peter , Zhang Yue , Xu Zefeng , Qin Tiejun , Prchal Josef T. , Xiao Zhijian 
Published: October 2019

Abstract
Purpose: MLF1IP is a novel constitutive kinetochore component essential in physiological progress of cell cycle. Mouse homologue appears to be lineage-specifically expressed, but little is known about how it functions in hematopoiesis and whether it is involved in hematopoietic disorders. The goal of the study was to confirm biological function of MLF1IP in human K562 erythroleukemic cells, analyzed hematological abnormalities in MLF1IP transgenic mice and potential mechanisms underlying these abnormalities, and evaluated MLF1IP expression in erythroid colonies from PV and MDS.
Methods: MLF1IP was down-regulated by lentivirus in K562 cell line. The proliferation of K562 cells was analyzed by cell colony formation assay. VP16 was used to induce apoptosis of K562 cells. The entire coding region of human MLF1IP was cloned into the HS21/45-vav plasmid to generate transgenic mice, which were crossed with wild-type C57BL/6J mice, negative siblings of transgenic mice were used as controls. Genotypes of the transgenic mice were identified by PCR of tail genomic DNA with primers specific for transgenic MLF1IP sequence. Relative quantitative real-time PCR was used to detect mRNA level in BM cells. Western blot were carried out to check the expression of cell cycle associated proteins. Flow cytometry analyses were used to assess cell cycles and proportions of different stages of erythroblast populations. Absolute quantitative real-time PCR were used to examine MLF1IP expression in colonies from healthy donors and subjects.
Results: MLF1IP knock-down K562 cells are arrested in G2/M and have reduced colony-forming activity. MLF1IP knockdown had no detectable effect on apoptosis in K562 cells, while significantly enhanced etoposide-induced apoptosis was seen in MLF1IP-knockdown K562 cells at 48 h after treatment compared with that in same treated control cells. MLF1IP transgenic mice show increased hemoglobin, with normal levels of platelets and neutrophils. Erythroid progenitors and early-stage erythroblasts in bone marrow are markedly increased. These mice have increased cyclin D2, reduced p27 and p21 and increased cycling, suggesting that enhanced MLF1IP expression leads to erythroid expansion via accelerating cell cycle progression in bone marrow cells. Interestingly, MLF1IP expression in colony forming unit-erythropoiesis (CFU-E) is increased in polycythemia vera (PV) and decreased in low-risk myelodysplastic syndrome (MDS), consistent with erythropoiesis characteristics of these disorders.
Conclusion: Our data demonstrate that MLF1IP expression promotes erythroid proliferation via regulating cell cycle status of erythroid cells, implicating abnormal MLF1IP levels in erythropoiesis disorders. Correction of MLF1IP expression could be a novel therapy target in diverse dysfunctional erythropoiesis conditions.

Disclosures
No relevant conflicts of interest to declare.
Regulation of Transcription Factors Foxp1, E2A and Egr-1 By microRNA-191 Constrains B Lymphocyte Development and Suppresses Development of Diffuse Large B Cell Lymphoma
Authors: Lyszkiewicz Marcin , Blume Jonas , Zietara Natalia , Witzlau Katrin , Puchalka Jacek , Saran Namita , Duber Sandra , Bishnudeo Roy , Weiss Siegfried , Krueger Andreas 
Published: October 2019

Abstract
Development of B-cells in the bone marrow is a tightly controlled, multi-step process. Whereas its major transcriptional regulators are well characterized, less is known about its post-transcriptional regulation. It has been demonstrated that B cell development critically depends on microRNAs. Yet, the role of individual members still remains to be uncovered.
Relatively subtle regulatory effects of miRNAs are well-visible in the pathways which require tight control. Disruption of such a pathway, where the protein function (i.e. transcription factor) highly depends on the gene dosage, is major cause of malignant transformation.
We identified transcription factors Foxp1, E2A and Egr1 as direct targets of microRNA miR-191. Reduced expression of these transcription factors through ectopic expression of miR-191 in B-cell progenitors resulted in numerous secondary effects, such as downregulation of Rag1, Rag2, Dntt and Il7r alpha genes. In consequence, we observed impaired immunoglobulin heavy chain rearrangement and limited responsiveness to IL-7 of B cell progenitors resulting in partial developmental arrest and predisposition of immature B-cells to death during negative selection. Finally, we showed that co-overexpression of E2A or FoxP1 and miR-191 restored normal B cell development, supporting the hypothesis that both transcription factors are functionally relevant targets of miR-191.
Having established a regulatory cascade comprising miR-191 at the top of hierarchy and the transcription factors Foxp1, E2A and Egr1 as its direct targets we sought to identify a pathophysiological scenario in which this cascade might be explored therapeutically. Given that the most aggressive forms of human diffuse large B cell lymphoma (DLBCL) are FOXP1 dependent, we assessed whether miR-191 was able to limit growth of DLBCL. Of note, in vivo ectopic expression of miR-191 prevented the growth of DLBCL. Consistent with murine data, expression of the three target genes FOXP1, EGR1 and TCF3 (encoding E2A) was decreased upon miR-191 overexpression. Interestingly, treatment with the histone deacetylase inhibitor vorinostat revealed decreased resistance to cell death of DLBCL lines overexpressing miR-191 in vitro.
Thus, our data describe a new regulatory pathway, where miR-191 by fine-tuning key B-lineage transcription factors acts as modulator of B-cell development as well as tumor suppressor in B-lineage malignancy.

Disclosures
No relevant conflicts of interest to declare.
Belimumab, a Neutralizing BAFF (B Cell Activating Factor) Antibody, Enhances the Susceptibility of Chronic Lymphoid Leukemia (CLL) Cells to Abt-199 Treatment
Authors: Schmidt Jonas , Wild Julia , Strunz Benedikt , Kanz Lothar , Salih Helmut R 
Published: October 2019

Abstract
Abt-199 is a small molecule BCL-2 inhibitor which has recently been approved for treatment of CLL and mediates its effects by induction of apoptosis in the leukemic cells. The TNF family member B cell activating factor (BAFF) contributes to disease pathophysiology in mature B cell malignancies including CLL by sustaining survival and preventing apoptosis of the malignant B cells. BAFF also plays a prominent role in autoimmune diseases, and accordingly a BAFF-neutralizing antibody termed Belimumab (Benlysta®) has been developed and is approved for treatment of systemic lupus erythematosus. Notably, elevated levels of BAFF have also been detected in sera of B cell lymphoma patients. We demonstrated recently that BAFF protects CLL cells from NK cell cytotoxicity including Rituximab-induced antibody-dependent cellular cytotoxicity (ADCC), the major mechanism by which this CD20-antibody mediates its therapeutic effects. In turn, susceptibility of CLL cells to both direct and Rituximab-induced NK cell killing could be restored by BAFF neutralization with Belimumab (Wild et al, Leukemia 2015). Here we studied whether Belimumab could also serve to increase the susceptibility of CLL cells to Abt-199 treatment. As no information on Abt-199 plasma peak levels in CLL patients is available, we first exposed primary CLL cells of leukemia patients to varying concentrations of Abt-199 and identified 5 nMas in vitro LC50 concentration in analyses of leukemia cell metabolic activity. Next we studied how BAFF influenced the therapeutic efficacy of Abt-199. Analyses of CLL cell metabolic activity and apoptosis/cell death revealed that BAFF concentration-dependently protected primary CLL cells from the therapeutic effects of Abt-199. Notably, CLL cell susceptibility to Abt-199 could be restored by neutralization of BAFF by Belimumab. Together, our findings demonstrate that BAFF contributes to CLL cell resistance to Abt-199 treatment. BAFF neutralization by Belimumab may in turn constitute a promising strategy to increase/restore the therapeutic efficacy of Abt-199, which warrants future clinical studies of combinatorial approaches to improve CLL treatment.

Disclosures
No relevant conflicts of interest to declare.
CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization
Authors: Fooksman David R , Mazumder Amitabha , McCarron Mark 
Published: October 2019

Abstract
Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations.

Disclosures
No relevant conflicts of interest to declare.
Influence of CD34+ and CD3+ Graft Content on Gvhd Development after Haploidentical Allogeneic Transplantation with Post-Transplant Cyclophosphamide
Authors: Bastos-Oreiro Mariana , Balsalobre Pascual , Champ Diana , Churruca Juan , Pascual Maria Jesus , Herrera Pilar , Bento Leyre , Piñana Jose Luis , Jimenez Santiago , Humala Karem , Montesinos Pau , Heras Inmaculada , Iglesias Almudena , Perez-Corral Ana , Pascual Cristina , Martinez-Laperche Carolina , Kwon Mi , Gayoso Jorge , Diez-Martin Jose L. 
Published: October 2019

Abstract
Introduction:
CD34+ and CD3+ cell dose in peripheral blood stem cell (PBSC) graft have been related with increased incidence of graft versus host disease (GVHD) in some transplant settings. Our aim in this study was to evaluate the impact of CD34+ and CD3+ cells graft composition on GVHD incidence in the setting of haploidentical transplant (haplo-HSCT) with post-transplant cyclophosphamide (PT-Cy), in a multicenter analysis.
Materials (or patients) and methods:
We retrospectively evaluated 175 patients with hematologic malignancies who were treated with a haplo-HSCT from 2011 to 2014 with PBSC in GETH centers. All patients received Busulfan-Fludarabine as conditioning regimen and GVHD prophylaxis was performed with PT-Cy (50 mg/kg on days +3 and +4) and a calcineurin inhibitor plus mycophenolate from day +5. We analyzed the impact of CD34+ and CD3+ cell doses (low vs. high with cut-off at the median value, and the 25-75 percentiles) on the development of acute and chronic graft versus receptor disease. Death before GVHD development was considered as a competitive event. The analysis was adjusted for conditioning intensity. STATA software was used for data analysis
Results:
We analyzed 175 patients. Patient characteristics are resumed in table 1. Median CD34+ and CD3+ cell dose was 5.31x10e6/kg (p 25-75: 4.31-6.1) and 2.07x10e8/kg (p25-75: 1.19-2.94), respectively. Cumulative incidences of grade II-IV acute GVHD and all grades chronic GVHD was 32.6% and 30% respectively. The median follow-up was 12.3 months (r:6-46). No difference in terms of GVHD (acute and chronic), was found between low and high CD34+ cell doses. However, if we focus on T cells, high doses of CD3+, for values above the median, have been has been associated with increased incidence of acute GVHD II-IV (day 150: 47% vs 26% p=0.01, figure 2), as well as cGVHD (30 month: 39% vs 16%, p= 0.01 figure 1). If we focus the analyses regarding the intensity of conditioning regimen, the influence of CD3+ doses remains for the group of reduced-intensity conditioning (RIC) (aGVHD, day 150: 54% vs 24%, p=0.003; cGVHD, 30 month: 47% vs 16%, p=0.006 figure 2), but lost for patients who receive myeloablative intensity (aGVHD, day 150: 33% vs 33% p=0-8; cGVHD, 30 month: 21% vs 16%, p=0.81)
Conclusion:
In our series, in the setting of haplo-HSCT with Pt-Cy, the highest dose of lymphocytes was associated with a higher incidence of aGHVD II-IV and cGV HD, especially for the RIC procedures.

Disclosures
No relevant conflicts of interest to declare.
Natural Toll-like Receptor Agonists for in Situ Vaccination Lymphoma Immunotherapy
Authors: Peng Paul , Brody Joshua , Hammerich Linda 
Published: October 2019

Abstract
Introduction: There is an unmet need for novel and effective treatments for lymphoma, the fifth most common malignancy in the US. In situ vaccination-an immunotherapeutic maneuver involving local irradiation, intratumoral (i.t.) injections of Flt3L and Toll-like receptor (TLR) agonist-has been shown in recent clinical trials (NCT00185965, NCT00880581, NCT00226993) to induce partial and complete remissions in patients with low-grade lymphoma. The strength of anti-tumor response correlates with the potency of immunogenic dendritic cells (DCs) to efficiently uptake and present tumor antigens to T cells. While the latest clinical trial NCT01976585 employs Poly-ICLC-a synthetic TLR3 agonist-to activate DCs, we hypothesize that "natural" TLR agonists (nTLRa) contained within prophylactic vaccines (e.g. live/attenuated bacteria or viruses) could simultaneously target multiple TLRs and be repurposed as clinical-grade DC activators for the in situ vaccination maneuver.
Methods: Twenty-four prophylactic vaccines from our hospital's pharmacy were screened using in vitro assays for TLR ligand activity. DC subsets CD11c+ MHC-II+, CD11c+ CD11b+, and CD11c+ CD103+ were gated for flow-cytometric readouts of: (i.) activation markers (e.g. CD40, CD80, CD86, MHC class I and II), (ii.) ability to co-stimulate T cells in the context of TCR activation, as assessed by T cell activation markers (e.g. CD69, intracellular IFNγ), and (iii.) ability of nTLRa-activated DCs to induce T cell proliferation. TLR knock-out cell lines were used for dissecting the mechanistically-distinct properties of each prophylactic vaccine. Best single and combination nTLRa candidates were evaluated with the in situ vaccination maneuver in an in vivo A20 murine lymphoma model.
Results: We identify a combination of vaccines Typhim, BCG-TICE, and MMR that demonstrates the ability to induce high expression of costimulatory molecules on DCs (Figure A) in vitro, as well as possessing the ability to co-stimulate in vitro T cell activation and proliferation. DC costimulation of T cell activation and proliferation is highly dependent on the "live" status of several vaccines, including BCG, MMR, Zostavax, which suggests the involvement of other pattern-recognition receptors in addition to TLRs. Cohorts of mice implanted with A20 lymphoma tumors that received i.t. injections of Typhim, BCG, MMR demonstrated slower A20 growth status post in situ vaccination maneuver, as compared to the control cohort receiving only Flt3L and irradiation (XRT).
Conclusion: Prophylactic vaccines contain natural ligands to TLRs and are immediately translatable as sources of clinical-grade stimulators of dendritic cells. Combinations of the best nTLRa candidates display synergistic activation of DCs and demonstrate in vivo anti-A20 murine lymphoma immunity.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.