Inhibition of CXCR4 by LY2624587, a Fully Humanized Anti-CXCR4 Antibody Induces Apoptosis of Hematologic Malignancies.

SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies

Plasma from patients with bacterial sepsis or severe COVID-19 induces suppressive myeloid cell production from hematopoietic progenitors in vitro

Bacterial sepsis and severe COVID-19 share similar clinical manifestations and are both associated with dysregulation of the myeloid cell compartment. We previously reported an expanded CD14⁺ monocyte state, MS1, in patients with bacterial sepsis and validated expansion of this cell subpopulation in publicly available transcriptomics data. Here, using published datasets, we show that the gene expression program associated with MS1 correlated with sepsis severity and was up-regulated in monocytes from patients with severe COVID-19. To examine the ontogeny and function of MS1 cells, we developed a cellular model for inducing CD14⁺ MS1 monocytes from healthy bone marrow hematopoietic stem and progenitor cells (HSPCs). We found that plasma from patients with bacterial sepsis or COVID-19 induced myelopoiesis in HSPCs in vitro and expression of the MS1 gene program in monocytes and neutrophils that differentiated from these HSPCs. Furthermore, we found that plasma concentrations of IL-6, and to a lesser extent IL-10, correlated with increased myeloid cell output from HSPCs in vitro and enhanced expression of the MS1 gene program. We validated the requirement for these two cytokines to induce the MS1 gene program through CRISPR-Cas9 editing of their receptors in HSPCs. Using this cellular model system, we demonstrated that induced MS1 cells were broadly immunosuppressive and showed decreased responsiveness to stimulation with a synthetic RNA analog. Our in vitro study suggests a potential role for systemic cytokines in inducing myelopoiesis during severe bacterial or SARS-CoV-2 infection

LY2624587 induces CXCR4 receptor internalization and down-regulation of receptor density on the cell surface.

<p>LY2624587 was labeled with Alexa 488. The labeled antibody was then used to treat MDA-MB-435/CXCR4 stably transfected cells for 1 or 2 hours at 37°C. After these treatments, the cells were examined under a fluorescent microscope for localization of receptor. In one condition, cells were incubated with labeled LY2624587 for 1 or 2 hours first, then fixed with 2% formaldehyde for 10 min (A). In another condition, the cells was fixed with 2% formaldehyde for 10 min first, then incubated with Alexa 488-labeled LY2624587 for 1 or 2 hours (B). The scale bar is 10 nm. C. LY2624587 induces CXCR4 receptor down-regulation of Namalwa cells. D. Peptide antagonist LY2510924 does not induce CXCR4 receptor down-regulation. NHL Namalwa cells were treated by LY2624587 (C) or LY2510924 (D) for 4 days, and analyzed by flow cytometry with a PE-conjugated anti-CXCR4 antibody that is not competing with LY2624587 or LY2510924 for CXCR4 binding. Grey color was isotype IgG control, green color was no drug treatment; and red color was treated by LY2624587 (C) or LY2510924 (D).</p

LY2624587 inhibits SDF-1 and CXCR4-mediated cell signaling in tumor cells.

<p>Human leukemia CCRF-CEM (A) and NHL Namalwa cells (B)were treated with 100 ng/ml SDF-1 for 10 min in the presence of different concentrations of LY2624587 (CXCR4AB) or control isotype IgG (hIgG4). Western blot analysis of phospho-ERK, phospho-AKT, total ERK or actin was conducted as described under the “Materials and Methods.”</p

<i>In vitro</i> activities of anti-CXCR4 monoclonal antibody LY2624587.

<p>A. Inhibition of [¹²⁵I] SDF-1α binding to CXCR4 by LY2624587 (square) or control IgG (triangle). Ligand binding assay was developed with CCRF-CEM cells, and the CCPM in the Y axis stands for corrected counts per minute. B. Dose dependent inhibition of SDF-1 induced GTP binding by LY2624587 (solid circle) or control IgG (triangle). GTP binding assay was developed with CCRF-CEM membrane. C. Inhibition of SDF-1 induced cell migration (chemotaxis) in U937 cells by LY2624587. D. Inhibition of SDF-1 induced cell migration (chemotaxis) in CCRF-CEM cells by LY2624587. E and F. LY2624587 has no apparent agonist activity. LY2624587 was tested in GTPγS35 binding assay (E) or cell migration assay (F) in agonist mode. The GTPγS35 binding assay was developed with CCRF-CEM cell membrane, and the SDF-1-induced cell migration assay was developed with U937 cells as described under “Materials and Methods.”</p

LY2624587 induces apoptosis of hematologic tumor cells <i>in vitro</i>.

<p>A. Annexin V analysis by flow cytometry in NHL Namalwa cells. Namalwa cells were treated with isotype IgG (hIgG4) or LY2624587 (CXCR4 AB 93–6) for 48 h, then subjected to flow cytometry analysis utilizing FITC-conjugated annexin V. B. DNA fragmentation and cleaved caspase 3 analysis by cellomics analysis in NHL Namalwa cells. The cells were treated for 48 h in the growth medium with 10 μg/ml control IgG or LY2624587 (CXCR4 ab). C. DNA fragmentation and cleaved caspase 3 analysis by cellomics analysis in leukemia CCRF-CEM cells. The cells were treated for 96 h in growth medium with 10 μg/ml control IgG or LY2624587 (CXCR4 ab). The inserts are amplified single cells to show DNA fragmentation or caspase 3 staining.</p

CXCR4 expression in human primary chronic lymphocytic leukemia (CLL) cells and LY2624587 binds to the CXCR4 of CD19+ cells of CCL patients.

<p>The blood samples of 4 CLL patients, EL-1279, EL-1308, EL-1289 and EL-1278 were freshly collected from Methodist Hospital, Indiana University at Indianapolis. 20 ml of fresh blood sample from each patient was collected and subjected to flow cytometry analysis with a PE-conjugated CXCR4 antibody and a FITC-conjugated CD19 antibody. A. CXCR4 surface expression in CD19+ cells from a CLL patient. B. CXCR4 intensities in tumor cells of four CCL patients. Three patients express high levels of CXCR4, and one patient has relatively low CXCR4 expression. C-F. LY2624587 (93–6) binds to the CXCR4 of CD19+ cells from CLL patient of EL-1279 (B), EL-1308 (C), EL-1289 (D), and EL-1278 (E). Competitive binding of LY2624587 to CXCR4 was determined using 16 nM of a PE-conjugated CXCR4 antibody from R&D Systems as a tracer. This tracer CXCR4 antibody binds to the same CXCR4 epitope of LY2624587.</p

Anti-tumor growth activities of LY2624587 in NHL Namalwa xenograft models developed with SCID mice.

<p>A. NHL Namalwa cells were subcutaneously implanted in the rear flank side and grown as solid tumors. B. NHL Namalwa cells were intravenously injected into SCID mice via tail vein, and grown as disseminated lymphomas as described under the “Materials and Methods.”</p

Large birds travel farther in homogeneous environments

Aim: Animal movement is an important determinant of individual survival, population dynamics and ecosystem structure and function. Nonetheless, it is still unclear how local movements are related to resource availability and the spatial arrangement of resources. Using resident bird species and migratory bird species outside the migratory period, we examined how the distribution of resources affects the movement patterns of both large terrestrial birds (e.g., raptors, bustards and hornbills) and waterbirds (e.g., cranes, storks, ducks, geese and flamingos). Location: Global. Time period: 2003–2015. Major taxa studied: Birds. Methods: We compiled GPS tracking data for 386 individuals across 36 bird species. We calculated the straight‐line distance between GPS locations of each individual at the 1‐hr and 10‐day time‐scales. For each individual and time‐scale, we calculated the median and 0.95 quantile of displacement. We used linear mixed‐effects models to examine the effect of the spatial arrangement of resources, measured as enhanced vegetation index homogeneity, on avian movements, while accounting for mean resource availability, body mass, diet, flight type, migratory status and taxonomy and spatial autocorrelation. Results: We found a significant effect of resource spatial arrangement at the 1‐hr and 10‐day time‐scales. On average, individual movements were seven times longer in environments with homogeneously distributed resources compared with areas of low resource homogeneity. Contrary to previous work, we found no significant effect of resource availability, diet, flight type, migratory status or body mass on the non‐migratory movements of birds. Main conclusions: We suggest that longer movements in homogeneous environments might reflect the need for different habitat types associated with foraging and reproduction. This highlights the importance of landscape complementarity, where habitat patches within a landscape include a range of different, yet complementary resources. As habitat homogenization increases, it might force birds to travel increasingly longer distances to meet their diverse needs.National Trust for Scotland; Penguin Foundation; The U.S. Department of Energy, Grant/Award Number: DE-EE0005362; Australian Research Council; NASA's Arctic Boreal Vulnerability Experiment (ABoVE), Grant/Award Number: NNX15AV92A; Netherlands Organization for Scientific Research, Grant/Award Number: VIDI 864.10.006; BCC; NSF Award, Grant/Award Number: ABI-1458748; U.K. Department for Energy and Climate Change; ‘Juan de la Cierva ‐ Incorporación’ postdoctoral grant; Irish Research Council, Grant/Award Number: GOIPD/2015/81 ; DECC; Goethe International Postdoctoral Programme, People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007‐2013/ under REA grant agreement no [291776]; German Aerospace Center Award, Grant/Award Number: 50JR1601; Scottish Natural Heritage; Solway Coast AONB Sustainable Development Fund; COWRIE Ltd.; Heritage Lottery Fund; Robert Bosch Stiftung; NSF Division of Biological Infrastructure Award, Grant/Award Number: 1564380; Spanish Ministry of Economy and Competitiveness, Grant/Award Number: IJCI-2014-19190; Energinet.dk; NASA Award, Grant/Award Number: NNX15AV92A; MAVA Foundation; Fundação para a Ciência e Tecnologia, Grant/Award Number: SFRH/BPD/118635/2016; National Key R&D Program of China, Grant/Award Number: 2016YFC0500406; Green Fund of the Greek Ministry of Environmen