ALBERT

Description: Abstract 2260 Background: BKT140 is a high affinity CXCR4 inhibitor with an extended K off-rate. Pre-clinical studies in animal models with BKT140 showed a robust mobilization of white blood cells (WBC) and hematopoietic stem cells (HSC). Furthermore, BKT140 also showed a direct anti-tumor effect against human-derived multiple myeloma (MM), lymphoma and primary leukemia cells and cell lines in vitro and in vivo, causing significant apoptosis. Aims: To assess BKT140 toxicity (primary endpoints), the mobilization capacity of CD34+ hematopoietic progenitors and CD138 MM cells, and pharmacokinetic (PK) and pharmacodynamic (PD) (secondary endpoints). Methods: 16 MM patients in first CR/PR were included in a phase I/IIa study, in which escalating doses of BKT140 (30, 100,300,900 μg/kg) were administered together with a high-dose cyclophosphamide (Cy) (2 g/m2) and G-CSF (5 μg/Kg) for stem cell mobilization. G-CSF was started on day 5 post Cy and BKT140 was injected subcutaneously (SC) once on day 10. Toxicity, PK, and mobilization capacity (assessed by serial measurements of number of WBC and CD34+ and CD138+ cells) were measured pre- and post BKT140 administration. Results: BKT140 was well tolerated at all doses and none of the patients developed grade II-IV toxicity. BKT140 was rapidly absorbed with no observed lag time, with peak plasma concentrations occurring 0.5h after administration. Clearance was rapid, with a median terminal half-life of 0.69h. BKT140 administration resulted in a significant dose-dependent increase in the number of peripheral blood neutrophils, monocytes, lymphocytes, and CD34+ cells compared to the G-CSF/Cy individual patient baseline. The maximum increase in the number of WBC from baseline was observed within 8h following BKT140 injection, 2.5-, and 3.0-, 4.1- and 4.8-fold, for the 4 BKT140 doses, respectively. Furthermore, BKT140 administration resulted in a significant increase in the mean absolute PB CD34+ cells mobilized (6.6, 7.5, 11.2 and 20.6 ×106/kg) for the 4 BKT140 administered doses, respectively. Moreover, the number of aphaeresis was reduced from 2.25 procedures at the first two BKT140 doses to 1.25 and 1 aphaeresis at the highest BKT140 doses, respectively. An increase in the number of CD138+ cells was observed in 6 out of 6 pts that had CD138+ cells in their blood and were treated with lower doses of BKT140 (30 and 100 μg/kg). Interestingly, in pts that were treated with the highest doses of BKT140 (300 and 900 μg/kg) a reduced number of CD138+ cells was observed in 3 out of 7 pts that had CD138+ cells in their blood, whereas in 4 pts, an increase in the number of CD138+ cells was shown. Three pts who did not have CD138+ cells in their blood were not affected by BKT140. The BKT140 mobilized grafts were used for AutoSCT following 200 mg/m2 melphalan conditioning. Pts received an average of 5.3×106 CD34+ cells/kg. All transplanted pts rapidly engrafted (n=15). The median day for neutrophil (〉500/mm3) and platelet (〉20,000/mm3, 〉50,000/mm3,) was on day 11 (range, 0–13), day 11 (range, 0–14), and day 14 (range, 0–23), respectively. Conclusions: The current data suggests that BKT140 can safely be added to G-CSF-based harvesting regimens, can increase CD34+ cell mobilization and reduce the number of collection days. Furthermore, due to its ability to release MM cells from the bone marrow and stimulate their cell death, additional studies are warranted to further evaluate the effect of BKT140 as an anti-MM agent. Disclosures: Nagler: Biokine Therapeutics Ltd: Consultancy. Abraham: Biokine Therapeutics Ltd: Employment, Equity Ownership, Patents & Royalties. Wald: Biokine Therapeutics Ltd: Employment. Shaw: Biokine Therapeutics Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Eizenberg: Biokine Therapeutics Ltd: Employment, Equity Ownership, Patents & Royalties. Peled: Biokine Therapeutics Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Description: Abstract 2431 Poster Board II-408 Nicotinamide (NAM), is a form of VitB3 that recognized and inhibits SIRT1, the human ortholog of the yeast Sir2 class III NAD+-dependent histone deacetylase. We have previously demonstrated that NAM inhibits in vitro differentiation and enhances expansion, migration, homing and NOD/SCID engraftment efficacy of cord blood (CB)-derived CD34+ cells cultured with cytokines. In the current study, the in vivo function of ex vivo cultured cells with NAM was tested in a congenic mice model (BALB/C, CD45.1/CD45.2) for BM transplantation. Purified CD117+ BM cells from BALB/C CD45.1 mice were cultured with a combination of 4 cytokines (FLT3, SCF, TPO, IL-6, 50 ng/ml each), with and without 0.5mM NAM for three weeks. Numbers of CFUc, CD117+ and CD117+Lin- cells were significantly (p 〈 0.05) higher in cultures treated with NAM as compared with cultured treated with cytokines alone. Non-cultured, freshly purified CD117+ cells (1000 and 2500 cells/mice) and their total progeny following expansion with or without NAM were transplanted into ablated (1000 Rad) CD45.2 mice (n = 10/cohort), 24h post irradiation (Fig 1). Three months post transplantation, all the mice in the control group (non-transplanted) died. The percent survival of mice transplanted with cells cultured with cytokines and NAM was remarkably higher over the survival of mice in the cohort transplanted with cells cultured with cytokines alone or non-cultured cells (Fig 1). FACS analysis (CD45.1-donor / CD45.2-host) of peripheral blood from mice transplanted with NAM cultured cells show 80% donor cell chimerism (CD45.1), 3 and 6 months post transplantation. Percent of donor derived Gr-1+ and CD3+ cells were similar in mice transplanted with non-cultured or NAM cultured cells. Percentages of donor cell chimerism (CD45.1) in secondary mice transplanted with total BM cells derived from primary recipients originally transplanted with non-cultured and NAM cultured cells were 47 and 73, respectively, 6 weeks after the secondary transplantation. In a different experiment, to follow time to engraftment during the first month post transplantation, mice transplanted with non-cultured cells or cells cultured with cytokines and NAM (n = 10/cohort) were bled at weekly intervals and peripheral blood samples were counted for WBC and analyzed by the FACS to determine donor cell chimerism and lineage engraftment. The results show accelerated engraftment (Fig 2) and higher levels of donor cell chimerism (Fig 3) in the cohort transplanted with NAM cultured cells relative to the cohort transplanted with non-cultured cells. Number of granulocytes, T, NK and B cells during the first month post transplant were also significantly (p

Description: 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, and high levels of CXCR4 expression correlate with poor survival in AML patients. It is postulated that blocking the CXCL12/CXCR4 axis with a potent CXCR4 antagonist will disrupt the interaction of the malignant blasts with the BM and augment the anti-leukemic effect of chemotherapy. BL-8040 (BKT140) is a 14-residue, cyclic, synthetic peptide capped with an aromatic ring that acts as a selective inhibitor of the CXCR4 chemokine receptor. BL-8040 has strong affinity for the CXCR4 receptor and long receptor occupancy, resulting in a prolonged pharmacodynamic effect. BL-8040 has superior mobilization capacity, inverse agonism activity and direct pro-apoptotic activity against leukemia cells. Preclinical studies have shown that in addition to its activity as a mobilizer of hematopoietic cells, BL-8040 exhibits a CXCR4-dependent selective anti-tumor effect against malignant cells. A clinical trial for the treatment of adult relapsed/refractory AML patients using a combination of BL-8040 and cytarabine (Ara-C) is currently ongoing (NCT01838395). Method: The phase II clinical trial in relapsed/refractory AML patients includes a dose escalation phase (3+3 design) followed by an expansion phase using the highest safe and efficacious dose group. 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 (1.5g/m2 for patients ≥60; 3g/m2 for patients ≤60) on days 3-7. For each of the BL-8040 tested doses (0.5, 0.75, 1, 1.25 and 1.5 mg/kg) the safety and tolerability, pharmacokinetic profile and clinical responses are being evaluated. Additionally, CXCR4 expression on leukemia cells, receptor occupancy, timing of mobilization of leukemic blasts and stem/progenitor cells and induction of apoptosis are being followed using frequent peripheral blood sampling (PB) and BM aspirates at baseline and on day 3 prior to Ara-C administration. Results: Preliminary results from the ongoing study suggest that administration of BL-8040, in combination with Ara-C, is safe and well tolerated at all doses tested to date (0.5-1 mg/kg). There have been no DLTs or SAEs and no delays in count recovery attributable to BL-8040. The primary treatment related AE has been a transient injection site reaction. FACS analysis revealed that 2 days of BL-8040 monotherapy triggered substantial mobilization of AML blasts (CD45+Low/CD34+/CD117+/HLA-DR+) from the BM to the PB. Data from the first 9 subjects revealed a 5.14 fold (range 1.0-8.09) median increase in AML blasts in the PB. Moreover, a dramatic decrease in the amount of AML blasts in the BM following 2 days of BL-8040 monotherapy was noted. FACS data from 7 subjects, for which BM aspirate samples were evaluable for analysis, revealed a median decrease of 70.65% [range(-16.32%)-(-87.68%)] in the number of AML blasts (CD45+Low/CD34+/CD117+/HLA-DR+ out of total CD45+/CD34+ normal/progenitor cells) compared with baseline. Importantly, this effect was specific to the AML blasts as the levels of normal/progenitor cells remained stable post BL-8040 monotherapy (5/7 patients). This decrease of BM AML blasts was evident regardless of whether the patient mobilized AML blasts or not. In support of induction of apoptosis by CXCR4 inhibition, an increase in the number of blasts with cleaved caspase3 was ascertained in day 3 BM biopsies from 6/8 patients. Long receptor occupancy (up to 24 hours post dosing) was observed by FACS analysis measuring surface CXCR4 staining. Pharmacokinetics analysis confirmed increase in BL-8040 exposure over doses with a short plasma half-life. Conclusion: The current data demonstrate that BL-8040 induces mobilization of AML blasts from the BM and has sustained receptor occupancy. In addition, a direct effect on AML blast viability has been observed in samples obtained during BL-8040 monotherapy. Importantly, the data suggest a differential effect of BL-8040 monotherapy on AML blasts vs. normal progenitors. BL-8040 was found to be safe and well tolerated at all doses tested to date. The updated results of the dose escalation phase of this ongoing study will be presented. Disclosures Borthakur: BioLineRx: Research Funding. Nagler:BioLineRx: Research Funding. Peled:BioLineRx: Research Funding. Pereg:BioLineRx: Employment. Foley-Comer:BioLineRx: Consultancy. Russovsky:BioLineRx: Employment. Aharon:BioLineRx: Employment. Cortes:BioLineRx: Research Funding. Andreeff:BioLineRx: Research Funding.

Description: The mechanisms governing migration and extramedullary dissemination of leukemic cells remain obscure. In this study the migration and in vivo homing to the bone marrow of nonobese diabetic severe combined immunodeficient (NOD/SCID) mice injected with human precursor-B acute lymphoblastic leukemia (ALL) cells in comparison to normal CD34+ progenitors (both cord blood and mobilized peripheral blood) was investigated. Although migration and homing of both cell populations was dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions, major differences in receptor expression as well as the migratory capacity toward various concentrations of SDF-1 were found. Furthermore, unlike normal CD34+ progenitors, in vivo homing of the leukemic cells was superior when recipient NOD/SCID mice were not irradiated prior to transplantation. In addition, we report differences in the adhesion molecules activated following SDF-1 stimulation, documenting a major role for very late antigen 4 (VLA-4), but not VLA-5 and lymphocyte function-associated antigen-1 (LFA-1), in homing of precursor-B ALL cells. Interestingly, Toxin-B and pertussis toxin inhibited the homing of the leukemic cells but not that of normal CD34+ progenitors or normal CD10+/CD19+ precursor-B cells, revealing differences in CXCR4 signaling pathways that are based on changes that acquired by the leukemic cells. Altogether, our data provide new insights into different SDF-1–induced signaling, activation, and consequent motility between normal CD34+ and precursor-B ALL progenitors, which may lead to improved clinical protocols. (Blood. 2004;103: 2900-2907)

Description: Background The chemokine CXCL12 and its receptor CXCR4 are key players in mediating the interactions between the bone marrow (BM) microenvironment and Acute Myeloid Leukemia (AML) cells. CXCL12, which is constitutively secreted from the BM stroma and AML cells, is critical for the survival and retention of AML cells within the BM. CXCR4 expression is associated with poor prognosis in AML patients with or without a mutated FLT3 gene. Antagonists to CXCR4 inhibit migration of AML cells, induce mobilization of AML cells into the circulation and enhance anti-leukemic effects of chemotherapy in mice models. The hypothesis that CXCL12/CXCR4 interactions contribute to the resistance of AML cells to signal transduction inhibitor, and chemotherapy-induced apoptosis, is currently being tested in a series of clinical trials in humans. Methods In the present study, the effect of the high affinity CXCR4 antagonist BL-8040 (BKT140) and AMD3100 (Mozobil) alone and in combination with ARA-C or the FLT-3 inhibitor AC220 on the survival and proliferation of AML cells in-vitro was examined. In the in-vitro study HL60 (FLT3-WT), MV4-11 (FLT3-ITD) cell lines and human primary AML cells from patients with FLT3-ITD mutations and FLT3-WT genewere used. Cells were incubated for 48 hrs in the presence of BL-8040 (8µM-20µM), AMD3100 (20µM), ARA-C (10-200 ng/ml) and AC220 (0.5-50nM). The level of viable cells, percentage of apoptosis and cell cycle were evaluated by FACS using propidium iodide and 7-AAD. In-vivo, we used NOD scid gamma (NSG) mice engrafted with human primary AML blasts and explored the effects of single injection of BL-8040 on the mobilization and survival of the blasts in the blood and the BM of the engrafted mice. Results In-vitro, treatment of MV4-11 cells (FLT3-ITD) with BL-8040, unlike treatment with AMD3100, directly inhibited cell growth by 35% and increased cell death by 39%. Furthermore, in-vitro, treatment of primary AML cells (FLT3-ITD) with BL-8040 directly inhibited cell growth by 28-47% and increased cell death by 75-100%. A combination of BL-8040 with AC220 or ARA-C further increased the apoptotic effect of these agents achieving a 96% reduction in cell viability and inducing cell death by 70- 90% of AML cells. When we studied the in-vitro effect of these agents on FLT3-WT cells (HL-60 cell line and primary AML cells), we found that BL-8040 inhibits cell growth by 16-50%. Unlike the FLT3-ITD cells, in the FLT3-WTcells we did not observe additive effects on cell growth for the combined treatments of BL-8040 with AC220. The combined treatment of BL-8040 with ARA-C was found to further increase the percentage of AML cell death. Moreover, BL-8040 decreases the percent of cycling cells by reducing the number of cells in G2/M+S phase while increasing the number of apoptotic cells in sub-G0 phase. It is interesting to mention that the migration of all tested AML cells toward CXCL12 was entirely inhibited by BL-8040. In-vivo, we found that a single injection of BL-8040 (100μg/mice) into NSG mice engrafted with human AML cells, induces rapid mobilization of AML cells to the periphery within 4 hrs after injection (an 8 fold increase from the control). When mice were administered with 5 consecutive injections of BL-8040 (400μg/mice), a reduction in the number of AML blasts in the blood was observed (40-60% reduction) with induction of AML cell apoptosis within the BM and in the blood by 10-20-fold, compared to the control. Conclusions The CXCR4 antagonist BL-8040 was found to rapidly and efficiently induces cell death of AML cells both in-vitro and in-vivo. These results suggest potential therapeutic advantages of BL-8040 in both FLT3-positive and negative AML patients by targeting not only AML anchorage in the BM but AML survival as well. Furthermore, it could provide a rational basis for BL-8040 therapy in combination with ARA-C and the FLT3 inhibitor AC220. Disclosures: Eizenberg: Biokine: Employment. Pereg:BioLineRx LTD: Employment. Klapper:BioLineRx LTD: Employment. Abraham:Biokine: Employment.

Description: Abstract 2962 Background: Multiple myeloma (MM) is characterized by clonal proliferation of malignant plasma cells (PCs) in the bone marrow (BM) compartment. Interaction of plasma cells with the BM stromal cells (BMSCs) is critical for homing, growth and drug resistance acquisition of the malignant PCs. However, the functional significance of other cellular components of the MM milieu, which includes osteoclasts and immune effector cells, is less clear. Both MM-derived and stromal cell-produced factors, including cytokines and chemokines, are believed to participate in the cross-talk between the MM and stroma leading to disease progression. Aim and Results: We hypothesized an important role for CXCL12 (SDF-1) chemokine and its receptor CXCR4 in MM-stroma interactions and microenvironment formation. We now show that MM cell lines ARH77 and RPMI8226 and primary MM cells may produce high amounts of CXCL12 and co-express CXCR4 receptor. Co-culture of the MM cells with BMSCs significantly up-regulated both CXCR4 cell-surface expression and CXCL12 secretion by the MM cells. Enhanced CXCR4 signaling in the MM cells upon the interaction with BMSCs promoted the survival and proliferation of the cells in an autocrine way. Moreover, the paracrine effect of increased CXCL12 production on immune cell migration was tested. We found, that conditioned medium (CM) produced by MM cells cultured with BMSCs specifically attracted increased numbers of CXCR4-expressing PB CD14+ cells. Furthermore, CXCR4 inhibition, using neutralizing antibodies toward CXCR4, inhibited the MM-induced migration of CD14+ monocytes, suggesting the possible role of CXCR4/CXCL12 axis in monocyte recruitment to the site of the disease. We next examined the functional consequence of MM-macrophage interaction. We saw that PB-generated macrophages induced the proliferation of MM cells, even more effectively than BMSCs. Furthermore, co-culture with macrophages strongly increased the expression of various pro-inflammatory and pro-angiogenic factors by MM cells, including CCL2 (MCP-1), CCL4 (MIP1a), IL-1b, IL-8 and VEGF. Interestingly, expression of IL-10 by MM cells was also up-regulated following the interaction with macrophages, suggesting the possible reciprocal effect of MM-produced factors on macrophage phenotype polarization. Conclusion: Taken together, our findings demonstrate that interaction of MM with BM stromal cells positively regulates the expression of CXCR4 and CXCL12 by MM cells, affecting both MM proliferation and CXCR4-dependent monocyte recruitment. The migrated monocytes may in turn interact with MM cells, support their growth and activate cytokine release, therefore producing favorable pro-inflammatory and pro-angiogenic environment and promoting disease progression. Overall, our data provide the basis for future targeting MM-BMSCs and MM-macrophage interactions with anti-CXCR4 agents as a therapeutic strategy to improve the outcome of patients with MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1760 Hairy cell leukemia (HCL) has a unique pattern of tissue infiltration by the HCL cells, causing splenomegaly and marrow infiltration, but rarely affecting the lymph nodes, or causing a significant leukemic involvement. Chemokine receptors and adhesion molecules play key roles in normal B cell trafficking and homing to different tissue microenvironments, and expression of these molecules on hairy cells is thought to function in a similar fashion, regulating the trafficking of hairy cells between the blood, spleen, and bone marrow. In this study, we profiled the expression and function of chemokine receptors (CXCR4, CXCR5, CXCR3, CCR7) and adhesion molecules (CD49d/VLA-4, CD54/ICAM-1, CD44, and CD62L) in primary HCL cells and HCL cell lines (Bonna-12, HC-1 and ESKOL). Primary HCL cells from 9 different patients displayed high levels of CD49d/VLA-4, CD54/ICAM-1, CXCR4, CD44, CD103 and CD11c; only in few cases we found a small sub-population of cells expressing CXCR5. Cell adhesion molecules like CD62L and the chemokine receptors CCR7 that are critical for entry and positioning in lymph nodes were not expressed in any of the analyzed samples. Similar immunophenotypic profiles were found in the HCL cell lines, with the exception of CD103 and CD11c, which were not expressed on the HCL cell lines. Bone marrow stroma cells (BMSC) constitutively secrete CXCL12 and provide ligands for VLA-4 integrins, and thereby may attract and retain HCL cells in the marrow. Also, these two molecules can be clinically targeted using small molecule antagonists or monoclonal antibodies (mAbs). Therefore, we analyzed HCL cell migration and adhesion towards BMSC (R-15C and Tst-4) after 4 hours of co-culture. As documented via phase contrast microscopy and quantified by flow cytometry, HCL cell lines and primary HCL cells showed abundant adhesion and migration beneath BMSC. Next, we examined whether blocking of CXCR4 using the CXCR4 antagonists AMD3100 and BKT-140 or blocking of VLA-4 integrins using CS-1 peptides affects this adhesion of HCL cells to BMSC. Pre-incubation with AMD3100 significantly reduced BMSC adhesion of hairy cells to levels that were 70.6 ±7.8% (p

Description: Introduction: Multiple myeloma (MM) is an incurable hematological malignancy characterized by proliferation of malignant plasma cells in the bone marrow (BM). Interactions between MM cells and BM milieu facilitate disease progression and therapy resistance. Chemokine receptor CXCR4 and its cognate ligand CXCL12 are implicated in these processes and are associated with poor prognosis. Sphingosine-1-phosphate (S1P) pathway is involved in cancer progression, including oncogenesis, cell survival and cell migration, therefore representing an attractive target for anti-cancer therapy. FTY720 (fingolimod) is a modulator of S1P signaling system that exhibit immunosuppressive and anti-cancer properties. The role of S1P system and FTY720 modulator in MM is less defined. The aim of this study was to explore the functional consequences of possible cross-talk between the CXCR4/CXCL12 and the S1P axes in MM cells and to evaluate the effect of S1P targeting with FTY720 as potential anti-MM therapeutic strategy. Results: The partners of the S1P pathway (S1P receptor 1 and sphingosine kinase 1 (SPHK1)) and CXCL12 chemokine were found to be co-expressed in MM cell lines and primary BM samples from MM patients. Increased mRNA levels of SPHK1 and CXCL12 were detected in MM BM samples (n=24) comparing to BM from healthy donors (n=7) (p