Search

Introduction: Regulatory T (Treg) cells, an immunosuppressive subset of CD4+ T cells characterized by the expression of the master transcription factor forkhead box protein P3 (FOXP3), are a component of the immune system with essential roles in maintaining self-tolerance. Treg cells which are indispensable for preventing autoimmunity, also suppress effective tumor immunity (Togashi et al. Nat Rev Clin Oncol 2019) Treg cells abundantly infiltrate into tumor tissues, present in the tumor microenvironment where they promote tumor development and progression by dampening antitumor immune responses. The abundantly infiltrate of Treg cells into tumor tissues is often associated with poor prognosis in cancer patients (Tanaka et al Eur. J. Immunol. 2019). The chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1/CXCL12) are critically involved in immune cell trafficking. CXCR4 overexpression, which has been identified in multiple cancer types, also supports cancer metastasis, recurrence and therapeutic resistance. More importantly, CXCR4 was shown to enhance tumor immune evasion by recruiting Treg (Santagata et al. Oncotarget. 2017) Objective: To study the effect of the high affinity CXCR4 antagonist, BL-8040, on the biology of Treg cells. To study how BL-8040 affects the ability of these cells to penetrate into the tumors, their migratory ability, their survival and also the differentiation of naive T cells towards Treg. Methods:C57BL/6 mice bearing LivMet pancreatic tumors and control mice were used for in-vivo study. In-vitro study was done with CD4 +CD25 hiFOXP3 + (Treg) cells which were isolated from fresh whole blood. CD4 +CD25 - cells were served as T conventional cells (Tconv). Differentiation of Treg cells was done from Naïve CD4+ T cells which were isolated from cord blood and stimulated with anti-CD3/CD28, TGF-b, IL-2 with or without BL-8040 for 6 days. Results: When mice bearing pancreatic cancer were treated with BL-8040, we found a significant reduction in the number of infiltrating Treg into the tumor. Following treatment with BL-8040 there was no alteration in the number of Treg in the blood neither in control mice nor in mice bearing tumors. To further understand the mechanism by which BL-8040 effected Treg cells we isolated Treg and Tconv cells and found that Treg cells express lower level of CXCR4, as compared to Tconv (Figure1). Further to, when we compared their motility, we found that Treg migration less efficiently towards CXCL12. Despite this, BL8040 more efficiently suppressed CXCL12 induced migration of Treg compared to Tconv. 100 nM of BL8040 was found to inhibits the migration of 82 % from the Treg compared to only 56.6% of Tconv cells (Figure 2). CXCR4 involves classical pathways of cell survival. In order to study the role of CXCR4 in the viability of Treg, we incubated Treg and Tconv cells in the presence of BL-8040 for 24 hr. We found that Treg cells are more sensitive to BL-8040 treatment with 19.2% of cell death compared to only 3.5% of Tconv cell death (Figure 3). Treg are one of the lineages of T helper (Th) cells which differentiated from naïve CD4 T cells. We found that BL-8040 inhibits the differentiation of naive CD4 T cells toward Treg. 10uM of BL-8040 shows a 5-fold inhibition in Treg differentiation from naïve CD4 T cells (Figure 4). Conclusions: In this work we show that the CXCR4 antagonist, BL-8040, can act as an immunomodulator by affecting the biology of regulatory T cells. BL8040 reduce the amount of infiltrating Treg into the tumors, impaired the migratory capacity of Treg toward CXCL12 and induces their cell death. Furthermore, BL-8040 was found to inhibit the differentiation of naïve CD4 T cells toward Treg. Taking all these together, BL-8040 may therefore improve the anticancer immune response, without impairing the activity of Tconv and thus can potentially serve as an effective immunomodulatory agent for cancer. Figure 1 Figure 1. Disclosures Abraham: Biokine Therapeutics: Current Employment. Vainstein: BioLineRx LTD: Current Employment. Shemesh-Darvish: BioLineRx LTD: Current Employment. Sorani: BioLineRx LTD: Current Employment. Eizenberg: Biokine Therapeutics Ltd: Current Employment. Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding.

Background: Autologous stem cell transplantation (ASCT) in multiple myeloma (MM) has been shown to improve survival compared to conventional chemotherapy alone. However, the ability to perform ASCT relies, in part, on collecting a sufficient number (#) of CD34+ hematopoietic stem cells (HSCs), typically from peripheral blood. The ideal HSC mobilization regimen would enable collection of optimal #s of HSCs (5-6x10 6 CD34+ cells/kg) within the minimum # of apheresis sessions possible. Yet, despite currently available G-CSF (G) based mobilization regimens and multiple apheresis days, many remain unable to collect optimal #s of HSCs. Motixafortide (M) is a novel CXCR4 inhibitor, with high affinity (IC 50 0.54-4.5 nM) and long receptor occupancy (>48 hours). Methods: In this prospective, phase 3, double blind, placebo controlled, multicenter trial, 122 patients were randomized (2:1) to receive either M+G or placebo (P)+G for HSC mobilization prior to ASCT for MM. All patients received G (10 mcg/kg) on days 1-5 (and 6-8, if needed). Patients received either M (1.25 mg/kg, subcutaneous injection) or P on day 4 (and 6, if needed). Apheresis began day 5, with the primary (PEP) and secondary (SEP) endpoints of collecting ≥6x10 6 CD34+ cells/kg in up to 2 apheresis days or 1 day, respectively. Apheresis continued on days 6-8 if needed. Total CD34+ cells/kg were analyzed on site to determine if patients mobilized to the goal and all samples were subsequently sent for assessment by central laboratory. Patients that did not collect ≥2x10 6 CD34+ cells/kg by day 8 proceeded to rescue mobilization. The # of CD34+ cells infused was determined independently by each investigator according to local practice (minimum ≥2x10 6 CD34+ cells/kg). Analyses of the PEP/SEPs were performed on an intent-to-treat basis. Results: Demographics between the 2 treatment arms were similar. Mobilization with M+G resulted in 92.5% of patients collecting ≥6x10 6 CD34+ cells/kg within 2 apheresis days vs 26.2% with P+G (Odds Ratio (OR) 53.3, 95% CI 14.12-201.33, p Conclusions: A single injection of M on top of G significantly increased the proportion of patients mobilizing ≥6x10 6 CD34+ cells/kg for ASCT (92.5%) vs G (26.2%) in up to 2 apheresis days (p Figure 1 Figure 1. Disclosures Crees: BioLineRx Ltd.: Research Funding. Larson: TORL biotherapeutics: Current holder of individual stocks in a privately-held company; Bioline: Research Funding; Abbvie: Research Funding; BMS: Research Funding; Celgene: Research Funding; GSK: Research Funding; Janssen: Research Funding; Juno: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Takeda: Research Funding. Illés: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stiff: Incyte: Research Funding; Cellectar: Research Funding; Seagen: Research Funding; Gamida Cell: Research Funding; Cellectar: Research Funding; Actinium: Research Funding; Bristol Myers Squibb: Research Funding; BioLineRX: Research Funding; Macrogenics: Research Funding; CRISPR Therapeutics: Consultancy, Honoraria; Amgen: Research Funding; Janssen: Research Funding; Kite, a Gilead Company: Research Funding; Karyopharm: Consultancy, Honoraria; MorphoSys: Consultancy, Honoraria. Sborov: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; SkylineDx: Consultancy. Pereira: Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Mikala: Novartis: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Krka: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Holtick: Celgene: Honoraria; Sanofi: Honoraria. Qazilbash: Amgen: Research Funding; Oncopeptides: Other: Advisory Board; Bristol-Myers Squibb: Other: Advisory Board; Biolline: Research Funding; Angiocrine: Research Funding; NexImmune: Research Funding; Janssen: Research Funding. Hardy: American Gene Technologies, International: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; InCyte: Membership on an entity's Board of Directors or advisory committees. Vainstein: BioLineRx LTD: Current Employment. Sorani: BioLineRx LTD: Current Employment. Gliko-Kabir: BioLineRx Ltd.: Current Employment. Goldstein: BioLineRx Ltd.: Current Employment. Kadosh: BioLineRx Ltd.: Current Employment.

Background: CD34+ hematopoietic stem and progenitor cell (HSPC) dose during hematopoietic cell transplantation (HCT) remains one of the most reliable clinical parameters to predict quality of engraftment. A minimum HSPC dose of 2-2.5x10 6 CD34+ cells/kg is considered necessary for reliable engraftment, while optimal doses of 5-6x10 6 CD34+ cells/kg are associated with faster engraftment, as well as fewer transfusions, infections, and antibiotic days. CXCR4 inhibition significantly improves the number (#) of CD34+ HSPCs mobilized for HCT, when added to G-CSF (G). Motixafortide (M), a novel CXCR4 antagonist, is a potent mobilizer of HSPCs recently evaluated in the phase 3, double blind, placebo controlled, multicenter GENESIS Trial as a mobilizing agent prior to autologous HCT (ASCT) in multiple myeloma (MM). Methods: Patients received G (10 mcg/kg) on days 1-5 (and days 6-8, if needed). On day 4 (and day 6, if needed), patients received either M (1.25 mg/kg) or placebo (P). Apheresis began day 5, with up to 4 days of apheresis if needed. The primary and secondary endpoints were collection of ³6x10 6 CD34+ cells/kg in up to 2 days of apheresis or 1 day, respectively. The # of CD34+ cells/kg infused was determined independently by each investigator according to local practice, but a minimum of ³2x10 6 CD34+ cells/kg was required. A post-hoc analysis was performed pooling data from both arms to evaluate time to platelet engraftment (TPE) (≥20x10 9/L without transfusions x7 days) and neutrophil engraftment (TNE) (ANC ≥0.5x10 9/L x3 days) based on total # of CD34+ cells/kg and # of specific CD34+ HSPC subsets infused. CD34+ HSPC immunophenotyping was performed via multicolor fluorescence-activated cell sorting (FACS). TPE/TNE was analyzed using Kaplan-Meier curves and Cox proportional hazards model. Results: 114 MM patients underwent apheresis, ASCT and were evaluable (M+G N=77; P+G N=37). M+G mobilization yielded a median of 10.8x10 6 CD34+ cells/kg collected in 1 apheresis vs 2.3x10 6 CD34+ cells/kg with P+G (p75 th percentile) of combined CD34+ HSC, MPP, CMP and GMP subsets was associated with faster TPE of 12 days vs 19 days with lower #s of these subsets (p=0.003) (Figure 2A). Furthermore, higher #s (>75 th percentile) of GMPs was individually associated with faster TPE of 13 days vs 19 days with lower GMP cell doses (p=0.0116) (Figure 2C). TNE was not impacted by increasing doses of total CD34+ HSPCs or any specific CD34+ HSPC subset (all p>0.05) (Figures 1B, 2B and 2D). Conclusions: M+G mobilization enabled significantly more CD34+ cells to be collected in 1 apheresis (median 10.8x10 6 CD34+ cells/kg) vs P+G (2.3x10 6 CD34+ cells/kg), as well as 3.5-5.6 fold higher #s of HSCs, MPPs, CMPs and GMPs (all p-values Figure 1 Figure 1. Disclosures Crees: BioLineRx Ltd.: Research Funding. Retting: BioLineRx Ltd.: Research Funding. Larson: TORL biotherapeutics: Current holder of individual stocks in a privately-held company; Abbvie, Bioline, BMS, Celgene, GSK, Janssen, Juno, Novartis, Pfizer, Takeda: Research Funding. Illes: Novartis, Janssen, Pfizer, Roche: Other: Travel, Accommodations, Expenses; Takeda, Seattle Genetics: Research Funding; Janssen, Celgene, Novartis, Pfizer, Takeda, Roche: Consultancy. Stiff: CRISPR: Consultancy; Gamida-Cell, Atara, Amgen, Incyte, Takeda, Macrogenetics, Eisai: Research Funding. Sborov: SkylineDx: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Pereira: Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Mikala: Abbvie: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Krka: Consultancy; Novartis: Consultancy; Takeda: Consultancy. Holtick: Sanofi: Honoraria; Celgene: Honoraria. Qazilbash: Janssen: Research Funding; Oncopeptides: Other: Advisory Board; Biolline: Research Funding; Bristol-Myers Squibb: Other: Advisory Board; NexImmune: Research Funding; Amgen: Research Funding; Angiocrine: Research Funding. Hardy: Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; American Gene Technologies, International: Membership on an entity's Board of Directors or advisory committees; InCyte: Membership on an entity's Board of Directors or advisory committees. Sorani: BioLineRx LTD: Current Employment. Shemesh-Darvish: BioLineRx LTD: Current Employment. Vainstein: BioLineRx LTD: Current Employment; Enlivex: Consultancy. Kadosh: StatExcellence: Current holder of individual stocks in a privately-held company; BioLineRx: Honoraria.

Background: CD34 expression remains the most common immunophenotypic cell surface marker defining human hematopoietic stem and progenitor cells (HSPCs). Recently, use of multicolor fluorescence-activated cell sorting (mFACS) and single-cell RNA sequencing (scRNA seq) has illustrated the heterogenous nature of CD34+ HSPCs, with immunophenotypically and transcriptionally distinct subsets ranging from primitive hematopoietic stem cells (HSCs) capable of long-term multilineage potential to differentiated, lineage-committed progenitors. Meanwhile, the addition of CXCR4 inhibitors (CXCR4i) to G-CSF (G) has increased mobilization of CD34+ HSPCs for stem cell transplantation (SCT). Yet, the effect of CXCR4i +/- G on mobilization of specific immunophenotypic and transcriptional CD34+ HSPC subsets is not well-characterized. Motixafortide (M) is a novel cyclic peptide CXCR4i with a low receptor off rate and extended in vivo action vs plerixafor (Px). M was recently evaluated in the phase 3, double blind, placebo controlled GENESIS Trial as an HSPC mobilizer prior to autologous SCT (ASCT) in multiple myeloma (MM). Methods: GENESIS Trial patients were prospectively randomized (2:1) to receive either M+G or placebo (P)+G for HSPC mobilization. Demographically similar patients undergoing mobilization with Px+G prior to ASCT for MM were prospectively enrolled on a parallel tissue banking protocol. All patients received G (10 mcg/kg) on days 1-5 (and 6-8 if needed). Patients also received either M (1.25 mg/kg) or P on day 4 (and 6 if needed); or Px (0.24 mg/kg) on day 4 (and 5-7 if needed). Apheresis began day 5 (and 6-8 if needed). HSPCs were purified from apheresis product on day 5 via CD34+ immunomagnetic selection. CD34+ HSPC subset profiling was performed via mFACS and scRNA seq. CXCR4 expression and receptor occupancy was evaluated by antibody binding capacity of 12G5 and 1D9 clones. Results: Demographics were similar between the M+G (n=24), P+G (n=13) and Px+G (n=14) cohorts. By mFACS, M+G mobilized a 5.5 fold higher absolute number (#) of HSCs, multipotent progenitors (MPP) and common myeloid progenitors (CMP) vs P+G (p0.05). 1D9 binding to CXCR4 on CD34+ HSPCs was similar between all 3 arms (p-values 0.45-0.75). 12G5 binding (which competes with CXCR4i's for binding to CXCR4) was significantly lower with M+G (MFI:11) vs P+G (MFI:74; p By scRNA seq, UMAP clustering identified 3 transcriptionally similar HSC sub-clusters (HSC-I, -II and -III) mobilized by all 3 regimens; and 1 distinct HSC-PL cluster mobilized by P+G expressing heat shock protein genes (HSPA1 A/B) (Figure 1C). Differentially expressed genes (DEGs) of HSCs I-III included CD52, FTH1, HLA-E, KLF2 and LMNA. AVP was unique to HSC-I while EGR1, JUN and ZFP36 were unique to HSC-III. MPPs clustered into 3 sub-clusters (MPP-I, -II and -III). MPP-I clustered closely to HSC-II/III with low expression of genes of differentiation. MPP-II expressed DEGs (PLEK) on a continuum toward megakaryocyte-erythroid progenitors (MEP-I/II), which expressed DEGs of erythroid differentiation (HBD/HBB). MPP-I and -II contained cells from all 3 regimens. However, MPP-III was specific to M+G with DEGs (MT-ATP8, HIST1H1) associated with monocyte, lymphocyte and NK cell differentiation. Conclusions: Extended CXCR4i with M+G mobilized significantly higher #s of combined CD34+ HSCs, MPPs and CMPs vs Px+G and P+G (p-values Figure 1 Figure 1. Disclosures Crees: BioLineRx Ltd.: Research Funding. Retting: BioLineRx Ltd.: Research Funding. Jayasinghe: WUGEN: Consultancy; MMRF: Consultancy. Vainstein: BioLineRx LTD: Current Employment. Sorani: BioLineRx LTD: Current Employment. Ickowicz: BioLineRx Ltd.: Current Employment. Shemesh-Darvish: BioLineRx LTD: Current Employment. Kadosh: BioLineRx Ltd.: Current Employment.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. Prior studies have established that most cases pf T-ALL are addicted to CXCR4 signaling. Indeed, strong preclinical data demonstrating therapeutic activity of BL-8040, a potent CXCR4 antagonist, have led to a clinical trial of BL-8040 in combination with nelarabine for patients with relapsed/refractory T-ALL (NCT02763384). However, the molecular mechanisms by which CXCR4 blockade induces T-ALL cell death are unknown. Using a human T-ALL xenotransplantation model, we previously reported that treatment with BL-8040 killed T-ALL cells through a non-apoptotic mechanism. Transcriptome sequencing revealed that BL-8040 induced alterations in genes involved in oxidative phosphorylation and carbohydrate metabolism. Indeed, seahorse experiments show that BL-8040 markedly reduced both oxidative phosphorylation and glycolysis. However, metabolic tracing studies using 13C-labeled glucose show that BL-8040 treatment does not have a major effect on the contribution of glucose to either glycolysis or the citric acid cycle. Instead, the major alteration observed is the reduced entry of glucose into the pentose phosphate pathway (PPP). A major function of the PPP pathway is to generate NADPH, which regulates reactive oxygen species (ROS) by producing reduced glutathione (GSH). Indeed, BL-8040 treatment resulted in a significant decrease in the ratio of reduced glutathione to oxidized glutathione. Together, these data suggest that BL-8040 induces oxidative stress by inhibiting GSH production. One mechanism utilized by cancer cells to regulate GSH levels and oxidative stress is the system xc- amino acid antiporter that mediates the exchange of extracellular L-cystine and intracellular l-glutamate across the plasma membrane, resulting in the production of GSH and oxidative protection. We measured L-cystine levels in the media of T-ALL cells cultured for 24 hours with or without BL-8040. A significant decrease in L-cystine in the media was observed. These data, along with increased expression of the xc- transporter (SLC7A11), suggested that increased system xc- activity was compensating for the loss of GSH induced by BL-8040. To test this possibility, we cultured T-ALL cells in L-cystine deficient media. Loss of L-cystine in the media resulted in a modest decrease in T-ALL cell viability that was markedly increased, in a synergistic fashion, upon treatment with BL-8040. Interestingly, caspase 3 was not activated, suggesting that, similar to in vivo results, BL-8040 induces a non-apoptotic cell death. This observation, coupled with the reduction in GSH, suggested the hypothesis that BL-8040 induces ferroptosis. Consistent with the hypothesis, treatment of T-ALL cells with ACXT-3102, a novel system xc- inhibitor, significantly enhanced BL-8040 killing of T-ALL cells in vitro. Collectively, these data suggest that T-ALL cells are sensitive to perturbations of the glutathione axis. Combined inhibition of CXCR4 signaling and system xc- activity exploits this vulnerability and presents a promising new therapeutic approach for T-ALL. Disclosures Uy: Astellas Pharma: Honoraria; Jazz Pharmaceuticals: Consultancy; Genentech: Consultancy; Agios: Consultancy; Pfizer: Consultancy; Daiichi Sankyo: Consultancy. Sorani:BiolineRx Ltd: Current Employment. Vainstein:BiolineRx Ltd: Current Employment. Davish:BiolineRx Ltd: Current Employment. Hawkins:Accuronix Therapeutics: Membership on an entity's Board of Directors or advisory committees.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. CXCL12 is a CXC chemokine that is constitutively expressed at high levels in the bone marrow. CXCR4 is the major receptor for CXCL12 and is by far the most highly expressed chemokine receptor on T-ALL cells. Two groups recently showed that genetic loss of CXCR4 signaling in murine or human T-ALL cells markedly suppressed their growth in vivo. We previously reported that BL-8040, a potent new CXCR4 antagonist with sustained receptor occupancy, is active as monotherapy against T-ALL in mice. Indeed, a 2-week course of daily BL-8040 resulted in a median reduction in tumor burden of 32.1-fold (range 6.8 to 176) across 5 different T-ALL xenografts. Preliminary data from a clinical trial of BL-8040 plus nelarabine for relapsed T-ALL also suggest therapeutic activity, with a complete remission rate observed in 4/8 patients (50%), which compares favorably to published response rates of approximately 30% with single agent nelarabine. Here, we explore molecular mechanisms by which CXCR4 blockade induces T-ALL death. NOD-scid IL2Rgammanull (NSG) mice were injected with P12-Ichikawa cells, a T-ALL cell line modified to express click beetle red luciferase and GFP. Following T-ALL engraftment, mice were treated with a single dose of BL-8040, and then leukemic cells in the bone marrow harvested 24-48 hours later. Treatment with BL-8040 resulted in a marked suppression of Akt and Erk1/2 phosphorylation, suggesting that signaling through CXCR4 is the major source of PI3 kinase pathway activation in T-ALL cells. Surprisingly, treatment with BL-8040 did not affect cellular proliferation, as measured by Ki67/FxCycle Violet staining or by EdU labeling. Moreover, no increase in apoptosis, as measured by annexin V or activated caspase 3 expression, was observed. These data suggest that CXCR4 blockade induces a non-apoptotic cell death. To explore this possibility further, we performed transcriptome sequencing on T-ALL cells recovered from mice 24 hours after 1 dose of BL-8040. A total of 151 differentially expressed genes (FDR of < 0.05% and ≥ 2-fold change) were identified. Gene set enrichment analysis was strongly positive for alterations in oxidative phosphorylation, ribosome biogenesis, and carbohydrate metabolism. Ribosome function was assessed using O-propargyl-puromycin (OPP), which monitors global protein translation. No difference in global protein synthesis in T-ALL cells was observed after CXCR4 blockade in vivo. T-ALL cells are dependent on glutamine as a source of carbon, and PI3 kinase signaling positively regulates glutaminolysis. Thus, we hypothesized that CXCR4 blockade may induce T-ALL cell death by reducing glutamine metabolism. However, treatment of T-ALL cells in vitro with BL-8040 did not alter the cellular levels of glutamine or glutamate, as measured using a commercial bioluminescent assay. Confirmatory metabolic tracing studies using 13C-labeled glutamine and glucose are in progress. Finally, to explore the reduction in oxidative phosphorylation, we examined mitochondria function using Mitotracker Green. Treatment of T-ALL cells in vitro with BL-8040 for 24-48 hours induced a significant decrease in mitochondria number, suggesting induction of mitophagy. Collectively, these data suggest that T-ALL cells are addicted to CXCR4 signaling in vivo. CXCR4 blockade with BL-8040 induces a non-apoptotic cell death that is characterized by a loss of mitochondria. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership.

Background: The chemokine receptor, CXCR4, mediates the trafficking and retention of hematopoietic stem cells to the marrow through its interaction with the chemokine, CXCL12. CXCR4 is the most highly expressed chemokine receptor on T-ALL cells, and preclinical data demonstrate that CXCR4 is critical for the growth and survival of T-cell acute lymphoblastic lymphoma (T-ALL). Genetic loss or pharmacologic blockade of CXCR4 signaling suppresses T-ALL cell growth and leukemia initiating activity in murine models. BL-8040 is a novel synthetic peptide antagonist of the chemokine receptor, CXCR4. Compared to other CXCR4 inhibitors, BL-8040 is a more potent mobilizer of normal HSCs with sustained occupancy and inhibition of CXCR4. We previously reported that BL-8040 potently suppresses T-ALL cell growth in xenotransplantation models of T-ALL. To test the efficacy of BL-8040 in T-ALL by conducting a multicenter, open label pilot study of BL-8040 in combination with nelarabine in patients with relapsed or refractory disease (ClinicalTrials.gov: NCT0276338). Methods: Adults with relapsed or refractory T-ALL/LBL, age ≥18 years and ECOG PS ≤2, were eligible for the study. A peripheral blood lymphoblast count ≤50,000/mm3 was required prior to starting treatment. During cycle 1, subjects received daily subcutaneous administration of BL-8040 1.5 mg/kg from Day 1 to Day 6 with nelarabine 1,500 mg/m2 intravenously over 2 hours on Days 2, 4 and 6. In subsequent 21-day cycles, BL-8040 1.5 mg/kg was administered daily on days 1-5 with nelarabine 1,500 mg/m2 given on days 1, 3 and 5. Up to 4 cycles of treatment was allowed. Results: Nine patients with median age of 29 years (range 20-75) have been enrolled in the study out of which including fout patients were in untreated 1st relapse, one in 2nd relapse, two with relapsed and refractory disease, and two primary refractory patient. Two patients had failed prior allogeneic hematopoietic cell transplant. The rate of complete remission is 56% (5/9) with all responses occurring after 1 cycle of treatment. Adverse events occurred in 2 subjects requiring dose modification. Consistent with the known mechanism of BL-8040, one subject developed G3 leukocytosis after BL-8040 administration with the peripheral WBC increasing from 5.3 to 273 h/mm3 with evidence of spontaneous tumor lysis requiring interruption of BL-8040 and leukapheresis. A second patient developed injection site pain requiring omission of the final dose of BL-8040. Peripheral blood analyzed at baseline and 24 hours after the first dose of BL-8040 (prior to nelarabine) showed the following: 1) sustained blockade of CXCR4 on leukemic blasts; 2) mobilization of leukemic blasts into the blood; and 3) no induction of leukemia blast apoptosis as measured by activated caspase 3 expression. Conclusions: BL-8040 can be combined with nelarabine in subjects with relapsed or refractory T-ALL with encouraging CR rates. Evidence of on target effects on CXCR4 has been observed in this study with sustained CXCR4 receptor occupancy at 24 hours after administration along with tumor mobilization of ALL blasts into the peripheral circulation. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Kadia:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bioline RX: Research Funding; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding. Stock:Astellas: Membership on an entity's Board of Directors or advisory committees; Daiichi: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; UpToDate: Honoraria; Agios: Membership on an entity's Board of Directors or advisory committees; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Brammer:Verastem, Inc: Research Funding; Viracta Therapeutics, Inc.: Research Funding; Bioniz Therapeutics, Inc.: Research Funding. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Chen:BiolineRx: Employment. DiPersio:Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; NeoImmune Tech: Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Incyte: Consultancy, Research Funding; Karyopharm Therapeutics: Consultancy. OffLabel Disclosure: BL-8040 for ALL

Background. Mobilized hematopoietic stem/progenitor cells (HSPCs) are the most commonly used graft source for hematopoietic cell transplantation (HCT). Current practices for harvesting HSPCs with G-CSF and/or plerixafor are costly, involve a multi-day procedure with suboptimal HSPC yields in up to 30% of patients and are associated with some morbidity and mortality. Here, we sought to test BL-8040, a high affinity CXCR4 antagonist with rapid mobilizing kinetics and long receptor occupancy, in donors for allogeneic HCT (alloHCT). Methods. The primary endpoint was to assess the efficacy of a single injection of BL-8040 to mobilize ≥ 2 x 106 CD34+ cells/kg of recipient weight after no more than two leukapheresis (LP) collections. Donors received BL-8040 by subcutaneous (SC) injection 3 hours prior to LP on day 1. The study was conducted in 2 parts. Part 1 enrolled HLA-identical pairs and 14 donors were treated with 1 mg/kg BL-8040. Part 2 enrolled HLA-identical and haploidentical pairs and treated 11 donors with 1.25 mg/kg BL-8040. When < 5x106 CD34+ cells/kg recipient weight were collected, a second injection of BL-8040 and LP collection was performed the next day. Immunophenotyping of lymphoid, myeloid, and HSPC subsets in the blood and LPs was performed by flow cytometry. Results. 25 donor-recipient pairs were enrolled in the study. The median age of donors was 55 years (range 20-69); 18 were fully HLA-matched siblings, and 7 were haploidentical donors. One donor was replaced per protocol because of problems with vascular access. All 11 donors treated at the 1.25 mg/kg dose and 22/24 (92%) patients overall collected the minimum goal of 2 x 106 CD34+ cells/kg recipient weight in 2 LPs. 9/11 donors treated with 1.25 mg/kg BL-8040 collected the target of 5 x 106 CD34+ cells/kg recipient weight. The median CD34+ cell count was 15 cells/µL blood at 3-4 hours after BL-8040 and was maintained for > 24 hours. BL-8040-adverse related events consisted primarily of grade 1-2 injection site reactions and transient systemic reactions (hives). Recipients were a median of 58 years of age (26-71) with an ECOG PS of 0-2 undergoing alloHCT for AML (n=16, 64%), ALL (n=4, 16%), MDS (n=3, 12%), MPN (n=1, 4%) or Hodgkin lymphoma (n=1, 4%). 22 recipients were transplanted with BL-8040-mobilized grafts. Time to neutrophil engraftment occurred at a median of 13 days (n=22, range 11-26) and platelet engraftment occurred at a median of 20 days (n=20, range 15-41; no platelet nadir for 1 pt and no engraftment in 1 pt). With a median follow-up of 258 days, 5 deaths have been observed on study. These deaths were due to sepsis (Day +84), complications of sepsis in setting of grade 3 GVHD (Day+67), AML relapse (Day+143,+166), and complications of intrabdominal infection (Day+252). Grade II acute GVHD was observed in 2/22 recipients (9%) both of which resolved. Grade III-IV GVHD was observed in 5 out of 22 recipients (23%), of which 3 resolved. Flow cytometric evaluation of HSPCs purified from LPs by CD34 immunoselection revealed that BL-8040 preferentially mobilized CD34+ plasmacytoid dendritic cell (pDC) precursors (pre-pDCs) that express high levels of CXCR4 (Fig. 1A). These pre-pDCs are the immediate progenitors of pDCs; a distinct DC population capable of producing large amounts of type 1 interferons in response to viral infections. In contrast to plerixafor, BL-8040 remained bound (as determined by inhibition of anti-CXCR4 binding) to CXCR4 on all lymphoid, myeloid, and HSPCs after LP (Fig. 1B and data not shown). BL-8040 induced pan-mobilization of all major myeloid and lymphoid subsets, with maximum relative changes in the absolute numbers of circulating pDCs, B cells, basophils, myeloid DCs (mDCs), CD8+ T cells and CD14+HLA-DR+CD16lo classical monocytes (Fig. 1C). In general, the magnitude of mobilization of each subset correlated with the level of surface CXCR4 at baseline (Fig 1C, gray bars, R2 = 0.4; P = 0.02). Although there was pan mobilization of all CD8+ and CD4+ T cell subsets, BL-8040 preferentially mobilized naïve and central memory CD8+ T cells relative to the other T cell subsets (Fig. 1D). Conclusions. Treatment of normal donors with a single dose of BL-8040 results in rapid and sustained mobilization of HSPCs for use in allogeneic transplant. BL-8040 mobilized grafts result in rapid and reliable engraftment in transplant recipients. Additional data is required to assess the impact of BL-8040-mobilized grafts on rates of GVHD and relapse. Disclosures Rettig: Novimmune: Research Funding; Amphivena Therapeutics: Research Funding. Uy:Curis: Consultancy; GlycoMimetics: Consultancy. Devine:Kiadis Pharma: Consultancy. Jaglowski:Juno: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding. Vainstein:BioLineRx Ltd.: Employment. Sorani:BioLineRx Ltd.,: Employment. Chen:BioLineRx Ltd.,: Employment. Bohana-Kashtan:BioLineRx Ltd.,: Employment, Equity Ownership; Cell Cure Neurosciences: Equity Ownership. Shaw:BioLineRx Ltd.: Employment, Equity Ownership.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. Approximately 20-25% of pediatric and 50% of adult patients with T-ALL relapse following induction therapy, and the prognosis after relapse is dismal, with 3-year event-free survival of only 10-15%. Thus, there is a clear unmet clinical need for better therapies for T-ALL. CXCL12 (stromal-derived factor-1, SDF1) is a CXC chemokine that is constitutively expressed at high levels in the bone marrow. CXCR4 is the major receptor for CXCL12 and is by far the most highly expressed chemokine receptor on T-ALL cells. Two groups recently showed that genetic loss of CXCR4 signaling in murine or human T-ALL cells markedly suppressed their growth in vivo. Here we explore the hypothesis that inhibition of the CXCR4 signaling with a potent new CXCR4 antagonist, BL-8040 alone, or in combination with ABT263 (navitoclax), a BCL2/BCLXL antagonist, will have therapeutic activity against T-ALL. To test this hypothesis, we xenotransplanted a human T-ALL cell line (P12/Ichikawa cells) or 5 different patient-derived T-ALL xenografts into non-irradiated NSG mice. In each case, the T-ALL cells were allowed to engraft and then mice were randomly assigned to one of four treatment groups: 1) vehicle control; 2) BL-8040 alone; 3) ABT263 alone; 4) combined BL-8040 and ABT263. Treatment was given for 2 weeks, and leukemia burden monitored weekly by bioluminescent imaging or by flow cytometry to quantify human CD45+ T-ALL cells in the blood. A minimum of 10 mice in each cohort from two independent experiments were analyzed (Figure 1). A significant reduction in T-ALL burden was observed in all T ALL samples after treatment with BL8040 alone, with a mean fold-decrease compared with control mice after 2 weeks of treatment of 966 ± 651 (range 16-3,486). Consistent with a prior report, the response to ABT263 alone was more variable, with responses seen in 4 of 6 T-ALL samples. All T-ALL samples had a marked response to the combination of BL-8040 and ABT263, with a mean fold decrease of 4,389 ± 2,602 (range, 139-15,199). A previous study suggested that inhibition of CXCR4 signaling may suppress Myc expression (Pitt et al, Cancer Cell, 2015), which is relevant, since overexpression of Myc secondary to mutations in the Notch pathway are common in T-ALL. However, we observed no difference in Myc protein expression or expression of Myc target genes following BL-8040 treatment. Consistent with this observation, treatment with BL8040 had no impact on the cell cycle status of T-ALL cells in vivo. Of note, we observed a marked decrease in Akt and Erk phosphorylation following BL8040, suggesting that CXCR4 signaling may regulate T ALL survival in vivo by suppressing these pathways. Conclusion. Collectively, these data suggest that BL-8040, either alone or in combination with ABT263, is highly active in T-ALL and support our ongoing clinical trial of BL-8040 in combination with nelarabine for patients with relapsed T-ALL (NCT02763384). Figure 1. Figure 1. Disclosures Uy: GlycoMimetics: Consultancy; Curis: Consultancy. Vainstein:Biolinerx: Employment. Sorani:BioLineRx Ltd.,: Employment. Bohana-Kashtan:BioLineRx Ltd.,: Employment, Equity Ownership; Cell Cure Neurosciences: Equity Ownership. Shaw:BioLineRx Ltd.: Employment, Equity Ownership.

Introduction: Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. The bulk of HSCs are CD34+, however, most CD34+ cells are lineage-restricted progenitors and HSCs remain rare. HSCs can be enriched further based on CD45RA, Thy1 (CD90), and CD38 expression. Loss of CD90 expression was proposed to be sufficient to separate CD34+CD38−CD45RA− CD90+ HSCs from CD34+CD38−CD45RA− CD90- multipotent progenitors (MPPs). Recently, it was demonstrated that the expression CD49f is a specific HSC marker. Single CD49f(+) cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting MPPs. Results: CD34+ cells were purified from BL-8040 and G-CSF mobilized grafts and stained for CD38, CD45RA, CD90, and CD49f. The percentage of CD34+CD38- hematopoietic stem and progenitors was similar in both grafts (Figure 1A). However, whereas 23.2 % of BL-8040 mobilized CD34+ CD38- cells did not express CD45RA; only 1.6% of G-CSF mobilized CD34+ CD38- cells did not express CD45RA (Figure 1B). The percentages of CD34+CD38−CD45RA−CD49f+CD90+/-, CD34+CD38−CD45RA−CD49f+ CD90+, and CD34+CD38−CD45RA− CD90+ HPCS were increased significantly by 45, 25 and 12 -fold in the BL-8040 graft compared to G-CSF graft derived CD34+CD38- cells (Figure 1C). To assess the long-term engraftment potential of the BL-8040 mobilized CD34+ cells, engraftment was allowed for 4, 8, and 22 weeks after transplantation. Successful and robust long-term human engraftment of CD45+ and CD45+CD34+ cells was observed at week 22 (Figure 2A, 2B). The % of human CD45 cells remained stable in the BM whereas the percentage of CD45 cells in the blood and spleen increased at week 22 (Figure 2C). At 4 weeks, human CD3+CD4+ T cells were only observed at a low percentage in the spleen but not in the BM, whereas no significant percentage of CD3+CD8+ cells were found neither in the BM nor in the spleen (Figure 2D, 2E). 22 weeks after transplantation, the percentage of human CD3+CD4+ and CD3+CD8+ T cells was significantly increased in the spleen (30% vs. 5%, respectively) and to much lower levels in the BM (Figure 2D, 2E). Furthermore, successful and robust long-term human engraftment of secondary recipient was observed 14 weeks following the second transplantation (Figure 2A and 2B). Conclusion: In association with the high percentage of HPCs in the BL-8040-derived graft, we found a robust myeloid and lymphoid long-term engraftment (week 22) of BL-8040 mobilized human CD34+ cells in NSG mice. The ability of BL-8040 to collect high numbers of HPCs may be beneficial for a variety of HPCs dependent therapeutics. Disclosures Abraham: Biokine: Employment. Oberkovitz: BiolineRx: Employment. Eizenberg: Biokine: Employment. Vainstein: BiolineRx: Employment. Benami: BiolineRx: Employment. Golan: BiolineRx: Employment. Or: Bioline: Consultancy. Peled: Biokine: Consultancy; Biosight: Consultancy.

e14544 Background: Cancer cells affect their micro-environment by recruiting immune cells that support tumor growth, metastasis and inhibition of anti-tumor effector T and NK cell recruitment. In this study, we investigated the role of BL-8040, a CXCR4 antagonist in cancer immunotherapy and its ability to modulate the immunosuppressive tumor micro-environment. Methods: The effect of BL8040 on tumor micro-environment was tested in 3 different cancer mouse models: lung cancer, pancreatic cancer and melanoma. The mobilization of immune cells to the periphery in response to BL8040 was tested, as well as the accumulation of immune cells both within and surrounding the tumor in the pancreatic cancer mouse model. Results: BL8040 was found to be a potent and robust mobilizer of immune cells. Immunophenotyping of the mobilized cells revealed that the mobilization of CD4 and CD8 T lymphocytes, as well as of dendritic cells (DC), was significantly increased in the cancer-bearing mice compared to their naïve counterparts. Importantly, a significant mobilization of effector CD8 T cells and activated CD8 T cells in the cancer-bearing mice was also detected following BL8040 treatment. Concomitantly, in the pancreatic cancer mouse model, treatment with BL8040 increased CD8 T cell accumulation within the tumor and inhibited tumor growth. Conclusions: The immune cell population that is mobilized in response to BL8040 treatment is different in cancer mouse models and naïve mice. The ability of BL8040 to induce mobilization of leukocytes, cytotoxic and activated CD8 T cells and DCs is affected by the presence of a tumor. In our models of pancreatic cancer, mobilization of immune cells from the bone marrow into the circulation and their accumulation within the tumor and tumor microenvironment resulted in inhibition of tumor growth. These results indicate that BL8040 may affect the tumor microenvironment and therefore can potentially synergize with immunomodulatory agents. In vivo pre-clinical studies as well as clinical studies are currently ongoing for testing the combination of BL8040 with immunomodulatory agents in different cancer models.

Background: Persistence of minimal residual disease (MRD) after induction, consolidation and salvage therapies is an independent poor prognostic marker in AML. The bone marrow (BM) microenvironment is a protective niche that promotes chemotherapy resistance of leukemia cells. This residual subset of chemotherapy-resistant cells present during remission eventually contributes to the leukemia relapse/persistence. Adhesion of AML cells to BM stroma is dependent on the CXCR4 receptor binding to its ligand CXCL12. High levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis will dislodge AML cells from the protective microenvironment and, when combined with chemotherapy, may improve the response to therapy. BL-8040 (BKT140) is a short peptide which inhibits the binding of CXCR4 to CXCL12, resulting in mobilization of leukemic blasts from the BM to the peripheral blood (PB). BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy and as a single agent induces differentiation and apoptosis of leukemia cells in preclinical models. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of relapsed/refractory AML patients was recently completed (NCT01838395). Here we report results from correlative studies done on samples from patients who participated in this trial. Objective: To assess BL-8040 single agent anti leukemia effects in relapsed/refractory AML. Method: Patients received a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5g/m2 for patients ≥60; 3g/m2 for patients Results: 45 patients (including 3 receiving compassionate use BL-8040) with a median age of 61 yrs were treated. The majority of patients had poor risk disease with 24% secondary AML and 17% with prior allogeneic stem-cell transplantation. Most patients were heavily pretreated with 19% relapsing after a short first remission (CR1 ≤12 months), 17% had ≥2 relapses and 45% refractory to 1-2 inductions. The CR+CRi rate was 38% in subjects receiving BL-8040 dose ≥1.0 mg/kg (n=39). Ongoing follow-up (FU) of responding patients (expansion phase; N=19) shows median EFS of 9.3 months (range 4.3-12.8 months). At the time of this analysis 10/19 patients are alive with a FU range of 0.96 - 12.8 months. FACS analysis revealed that BL-8040 monotherapy triggered an average 4.3-fold increase of immature AML blasts (CD45+Low/CD34+/CD117+/HLA-DR+) in PB (i.e., mobilization). Response to treatment was associated with more robust mobilization of AML blasts following BL-8040 monotherapy (responders 9.5 vs non-responders 1.7 fold median). Preferential mobilization of AML blasts over normal cells (4.7 fold vs. 1.4 fold, respectively) was confirmed by FISH analysis in a subset of patients with informative cytogenetic abnormalities. Induction of granulocytic differentiation of immature leukemia progenitors (2.5 fold) in the BM following BL-8040 monotherapy was noted. Finally, induction of AML blasts apoptosis (40% increase) by BL-8040 alone was evident by FACS in day 3 (pre-araC) bone marrow biopsies. Conclusions: The data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 is safe and well tolerated and when given in combination with Ara-C improves the response rate achieved historically with Ara-C alone. In addition, treatment with BL-8040 as a single agent rapidly and efficiently induces mobilization, differentiation and cell death of AML blasts. This selective effect on chemotherapy-resistant cells may be translated into reduction of residual disease arguing for incorporation into front-line trials and such studies are ongoing. Disclosures Foran: Millennium Pharmaceuticals, Inc.: Research Funding; medscape: Honoraria; karyopharm: Honoraria; pfizer: Honoraria; novartis: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding. DiPersio:Incyte Corporation: Research Funding. Peled:Biokine Therapeutics: Employment. Abraham:Biokine Therapeutics Ltd: Employment. Pereg:BioLineRx Ltd: Employment. Vainstein:BioLineRx Ltd.: Employment. Oberkovitz:BioLineRx Ltd.: Employment. Aharon:BioLineRx Ltd.: Employment. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

Background: The bone marrow (BM) niche protects acute myeloid leukemia (AML) cells from chemotherapy. BM homing of AML cells is dependent on CXCR4 and its ligand CXCL12; high levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis with a potent CXCR4 antagonist will disrupt the interaction of the leukemic blasts with the BM and augment the anti-leukemic effect of chemotherapy. BL-8040 (BKT140) is a short synthetic peptide which inhibits the binding of CXCR4 to CXCL12, resulting in the mobilization of leukemic blasts from the bone marrow to the peripheral blood. BL-8040 also inhibits CXCR4/CXCL12 mediated pro-survival ERK/Akt signaling and alters balance of pro/anti-apoptotic Bcl-2 proteins. BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy, and, in contrast to other CXCR4 inhibitors, as a single agent induces apoptosis of leukemia cells ex-vivo in clinical samples. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of adult relapsed/refractory AML patients is currently ongoing (NCT01838395). Objective: To assess the safety, efficacy and pharmacodynamics/pharmacokinetic parameters of BL-8040 in combination with Ara-C in first or second relapse and refractory AML patients. Method: The study includes a dose escalation phase (3+3 design) followed by an expansion phase. Each patient receives a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5 g/m2 for patients ≥60; 3 g/m2 for patients Results: Twenty two patients (median age, 61 y; range, 43-74 y) with relapsed/refractory AML were treated during the escalation phase. BL-8040 was escalated up to 1.5 mg/kg (recommended phase 2 dose) without reaching the MTD. Combination of BL-8040 with Ara-C was safe and well tolerated at all doses tested. Only one SAE (Sweet Syndrome) was reported as possibly related to study drug. The primary treatment related AEs were transient injection site and systemic reactions. The composite complete remission including complete remission (CR) and complete remission with incomplete blood count recovery (CRi) rate was 38% in subjects receiving BL-8040 doses of 1 mg/kg and higher (n=16). Two days of BL-8040 monotherapy triggered an average 40.2-fold (range 1.0-350.9) mobilization of immature AML progenitors (CD45+Low/CD34+/CD117+/HLA-DR+) from the BM to the PB. Moreover, a significant apoptotic effect on the immature leukemia progenitors in the BM following 2 days of BL-8040 monotherapy was noted. Flow cytometric evidence of apoptosis by BL-8040 was corroborated by an increase in cleaved caspase 3 positive blasts in day 3 bone marrow biopsies. In addition, BL-8040 treatment resulted in a median decrease of 57.7% (range, -10.4% to -96.2%) in the number of BM Leukemia progenitor cells (CD45+Low/CD34+/CD117+/HLA-DR+ out of total CD45+/CD34+ normal/progenitor cells) compared with the baseline biopsy. This decrease was accompanied with a 3.1 fold increase in granulocytes in the day 3 BM biopsy supporting differentiation effect. Finally, long receptor occupancy (up to 24 hours post dosing) was confirmed by FACS analysis measuring surface CXCR4 staining. Pharmacokinetics analysis confirmed dose incremental BL-8040 exposure with a short plasma half-life. Conclusions: The current data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 has potent anti leukemic activity and in combination with Ara-C may improve the response typically achieved in this advanced AML population. Mechanistically, BL-8040 was able to induce apoptosis of leukemic progenitor cells, overcoming the stroma protection, inducing mobilization and differentiation. Disclosures Rowe: BioLineRx Ltd.: Consultancy; Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:Novartis: Research Funding. Altman:Seattle Genetics: Consultancy; Novartis: Consultancy; Spectrum: Consultancy; Astellas: Consultancy; Ariad: Consultancy; BMS: Consultancy. Peled:Biokine Ltd.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BiolineRX: Consultancy, Research Funding. Pereg:BioLineRx: Employment. Vainstein:BioLineRx: Employment. Aharon:BioLineRx: Employment. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Cortes:Teva: Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; BerGenBio AS: Research Funding; Novartis: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.