Vainstein, Abi

Abstract 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<0.0001). Furthermore, 88.8% of patients with M+G collected ≥6x10 6 CD34+ cells/kg in 1 apheresis day vs 9.5% with P+G (OR 118.0, 95% CI 25.36-549.35, p<0.0001); and 96.3% with M+G collected ≥2x10 6 CD34+ cells/kg within 1 apheresis day vs 64.3% with P+G (OR 18.9, 95% CI 4.47-80.04, p<0.0001). The PEP and SEPs were confirmed as statistically significant by central laboratory (all respective p-values <0.0001). The median # of HSCs mobilized in 1 apheresis day with M+G was 10.80x10 6 CD34+ cells/kg vs 2.14x10 6 CD34+ cells/kg with P+G. The # of cells infused was determined independently by each investigator according to local practice. Median time to neutrophil engraftment was 12 days in both arms (HR 0.94, 95% CI 0.62-1.41, p=0.75). Median time to platelet engraftment was 18 days (range: 17-19) with M+G and 17 days (range: 17-18) with P+G (HR 0.89, 95% CI 0.59-1.34, p=0.57). Graft durability at day 100 post-ASCT was 92.2% in the M+G arm and 91.9% in the P+G arm (OR 1.04, 95% CI 0.2-4.5, p=0.96). Overall, adverse events were reported in 98.8% (Grade 3/4: 68.8%) of patients with M+G vs 95.2% (Grade 3/4: 42.9%) with P+G, with cytopenias in the post-ASCT period prior to engraftment accounting for the majority of Grade 3/4 AEs in both arms, as expected. The most common AEs related to M included: local injection site reactions (any grade: 70.0%, Grade 3/4: 11.3%); and systemic reactions such as pruritis (33.8%), flushing (32.5%) and urticaria (12.5%). Additionally, mobilization with M+G resulted in a 10.5x increase in mean absolute # of CD34+CD45RA-CD123loCD38-CD90+CD49f+ primitive HSCs collected vs P+G (p<0.0001). Multicolor FACS and scRNA sequencing of CD34+ HSCs from both arms will be reported in a separate ASH abstract. 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<0.0001), while enabling 88.8% to collect ≥6x10 6 CD34+ cells/kg in just 1 apheresis (p<0.0001, Figure 1A). Despite the higher # of cells mobilized by M+G, the # of CD34+ cells/kg infused was determined independently by each investigator according to local practice with comparable engraftment kinetics and graft durability between the 2 arms. Finally, M+G mobilized 10.5x more immunophenotypically primitive CD34+ HSCs capable of durable multilineage hematopoietic engraftment vs P+G (p<0.0001, Figure 1B). 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.

Abstract 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 (p<0.0001). M+G also resulted in mobilization of higher #s of relevant CD34+ HSPC subsets vs P+G, including 5.6 fold more HSCs (CD45RA-CD123loCD38+CD90+CD49f+) (p<0.0001), 3.5 fold more multipotent progenitors (MPPs) and common myeloid progenitors (CMPs) (CD45RA-CD123loCD38+CD90-CD49f+) (p=0.0004) and 5.6 fold more granulocyte and macrophage progenitors (GMPs) (CD45RA+CD123loCD38+/-CD10-) (<0.0001). The median # of infused HSPCs in both arms was <6x10 6 CD34+ cells/kg (Table 1), with similar TPE of 17-18 days (HR 0.89, 95% CI 0.59-1.34, p=0.57) and TNE of 12 days (HR 0.94, 95% CI 0.62-1.41, p=0.75). However, pooled analysis of all patients (N=114) revealed infusion of ³6x10 6 CD34+ cells/kg was associated with faster median TPE of 16 days vs 18 days with <6x10 6 CD34+ cells/kg (HR 0.63, 95% CI 0.41-0.96, p=0.03) (Figure 1A). Further analysis demonstrated an inverse correlation between increasing CD34+ cells/kg infused and TPE (Pearson r=-0.2789, p=0.0027). In addition, pooled analysis of 36 patients (M+G N=24; P+G N=12) using extended multicolor FACS revealed infusion of higher #s (>75 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 <0.0004). This high # of CD34+ cells/kg mobilized with M+G enables the potential infusion of ≥6x10 6 CD34+ cells/kg and cryopreservation of cells for later use. A dose response was observed with significant correlation between faster TPE and infusion of higher #s of total CD34+ HSPC doses (³6x10 6 CD34+ cells/kg) and combined HSC, MPP, CMP and GMP subsets. Additionally, infusion of higher #s of CD34+ GMP subsets was independently associated with faster TPE, suggesting these more committed progenitors may play a critical role in early engraftment. 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.

Abstract 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 <60) per day on days 3-7. Six dose levels of BL-8040 (0.5 - 2.0 mg/kg) were tested. PD parameters such as mobilization of leukemic blasts and induction of differentiation and apoptosis were assessed after monotherapy with BL-8040 using frequent PB sampling and BM aspirates at baseline and on day 3 prior to Ara-C administration. 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.

Abstract 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 <60) on days 3-7. Five dose levels of BL-8040 (0.5, 0.75, 1, 1.25 and 1.5 mg/kg) have been tested. Extensive pharmacodynamic parameters such as CXCR4 expression on the leukemic cells, receptor occupancy, mobilization of leukemic cells and induction of apoptosis are being assessed after monotherapy with BL-8040 using peripheral blood sampling (PB) and BM aspirates at baseline and on day 3 prior to Ara-C administration. Clinical response to treatment is evaluated by BM biopsy on day 30 (-/+ 2). 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.