Background
CXCR4 mediates the retention and survival of acute myelogenous leukemia blasts in bone marrow and contributes to their resistance to chemotherapy. The authors evaluated a combination of ...the high‐affinity CXCR4 antagonist BL‐8040 with high‐dose cytarabine (HiDAC) chemotherapy in a phase 2a study of patients with relapsed and refractory AML.
Methods
Forty‐two patients received treatment with BL‐8040 monotherapy for 2 days followed by a combination of BL‐8040 with HiDAC for 5 days. Six escalating BL‐8040 dose levels were investigated (0.5, 0.75, 1.0, 1.25, 1.5, and 2.0 mg/kg), and 1.5 mg/kg was selected as the dose for the expansion phase (n = 23).
Results
BL‐8040 in combination with HiDAC was safe and well tolerated at all dose levels. Clinical response was observed with BL‐8040 doses ≥1.0 mg/kg. The composite response rate (complete remissions plus complete remissions with incomplete hematologic recovery of platelets or neutrophils) was 29% (12 of 42) in all patients and 39% (9 of 23) in the 1.5‐mg/kg phase. The median overall survival was 8.4 months for all patients, 10.8 months in the 1.5‐mg/kg phase, and 21.8 months for responding patients in the 1.5‐mg/kg cohort. Two days of BL‐8040 monotherapy triggered the mobilization of blasts into peripheral blood, with significantly higher mean fold‐changes in responders versus nonresponders. This was accompanied by a decrease in bone marrow blasts.
Conclusions
The current results demonstrate the efficacy of CXCR4 targeting with BL‐8040 and support continued clinical development in acute myelogenous leukemia.
BL‐8040 is a high‐affinity CXCR4 inhibitor. Combined with cytarabine, BL‐8040 treatment results in improved composite complete response rates and median overall survival in hard‐to‐treat patients with relapsed/refractory acute myelogenous leukemia.
The dual specificity (Tyr/Thr) phosphatase Cdc25A activates cyclin-dependent kinases (Cdks) to promote cell-cycle progression and has significant oncogenic potential. Cdc25A protein levels are ...regulated tightly in normal tissues, but many human cancers overexpress Cdc25A. The underlying mechanism for overexpression has been enigmatic. Here we show that Cdc25A is stabilized by the ubiquitin hydrolase Dub3. Upon binding Cdc25A, Dub3 removes the polyubiquitin modifications that mark Cdc25A for proteasomal degradation. Dub3 knockdown in cells increased Cdc25A ubiquitylation and degradation, resulting in reduced Cdk/Cyclin activity and arrest at G1/S and G2/M phases of the cell cycle. In contrast, acute Dub3 overexpression produced a signature response to oncogene induction: cells accumulated in S and G2 because of replication stress, and activated a DNA damage response. Dub3 also transformed NIH-3T3 cells and cooperated with activated H-Ras to promote growth in soft agar. Importantly, we show that Dub3 overexpression is responsible for an abnormally high level of Cdc25A in a subset of human breast cancers. Moreover, Dub3 knockdown significantly retarded the growth of breast tumour xenografts in nude mice. As a major regulator of Cdc25A, Dub3 is an example of a transforming ubiquitin hydrolase that subverts a key component of the cell cycle machinery.
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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 Treatment of Diffuse Large B Cell Lymphoma (DLBCL) patients with rituximab and the CHOP treatment regimen is associated with frequent intrinsic and acquired resistance. However, treatment ...with a CD47 monoclonal antibody in combination with rituximab yielded high objective response rates in patients with relapsed/refractory DLBCL in a phase I trial. Here, we report on a new bispecific and fully human fusion protein comprising the extracellular domains of SIRPalpha and 4-1BBL, termed DSP107, for the treatment of DLBCL. DSP107 blocks the CD47:SIRPalpha 'don't eat me' signaling axis on phagocytes and promotes innate anticancer immunity. At the same time, CD47-specific binding of DSP107 enables activation of the costimulatory receptor 4-1BB on activated T cells, thereby, augmenting anticancer T cell immunity. Methods Using macrophages, polymorphonuclear neutrophils (PMNs), and T cells of healthy donors and DLBCL patients, DSP107-mediated reactivation of immune cells against B cell lymphoma cell lines and primary patient-derived blasts was studied with phagocytosis assays, T cell activation and cytotoxicity assays. DSP107 anticancer activity was further evaluated in a DLBCL xenograft mouse model and safety was evaluated in cynomolgus monkey. Results Treatment with DSP107 alone or in combination with rituximab significantly increased macrophage- and PMN-mediated phagocytosis and trogocytosis, respectively, of DLBCL cell lines and primary patient-derived blasts. Further, prolonged treatment of in vitro macrophage/cancer cell co-cultures with DSP107 and rituximab decreased cancer cell number by up to 85%. DSP107 treatment activated 4-1BB-mediated costimulatory signaling by HT1080.4-1BB reporter cells, which was strictly dependent on the SIRPalpha-mediated binding of DSP107 to CD47. In mixed cultures with CD47-expressing cancer cells, DSP107 augmented T cell cytotoxicity in vitro in an effector-to-target ratio-dependent manner. In mice with established SUDHL6 xenografts, the treatment with human PBMCs and DSP107 strongly reduced tumor size compared to treatment with PBMCs alone and increased the number of tumor-infiltrated T cells. Finally, DSP107 had an excellent safety profile in cynomolgus monkeys. Conclusions DSP107 effectively (re)activated innate and adaptive anticancer immune responses and may be of therapeutic use alone and in combination with rituximab for the treatment of DLBCL patients. Keywords: B cell lymphoma, CD47-SIRPalpha 'don't eat me' signaling, Phagocytosis, T cell co-stimulation, 4-1BB
Introduction
GvHD is a threat to the wellbeing and survival of patients following hematopoietic stem cell transplantation (HSCT). In-vivo and ex-vivo T cell depletion are the most effective methods ...for GvHD prevention, but attendant risks of infection, graft failure and leukemic recurrence offset the advantages of these manipulations. Less extensive T cell depletion leaves many patients at risk for the ravages of treatment-resistant GvHD. Alternative approaches to eliminate GvHD-causing cells are needed. Fas ligand (FasL), a member of the tumor necrosis factor (TNF) family, can selectively induce apoptosis of mature T cells while sparing stem and progenitor cells. We have developed a novel FasL-mediated selection process which eliminates mature T cell subsets in hematopoietic stem cell grafts and effectively prevents GvHD in pre-clinical models without impairing engraftment and immune system recovery. We report here data from ex-vivo and in-vivo studies supporting the use of FasL-mediated depletion of mature T-cells, a process we have named ApoGraft, as a novel effective method for GvHD prophylaxis in the context of allogeneic HSCT.
Methods:
Aliquots of mobilized peripheral blood cells (MPBC) were collected from 104 consenting healthy donors and were used ex-vivo for optimization of the ApoGraft manufacturing process. ApoGraft is manufactured under GMP guidelines, and includes two washing steps, a 2 hr incubation of the graft at 37oC with human recombinant different concentrations of FasL (MegaFasL, Adipogen), and 2 additional washing steps to remove unbound FasL. Aliquots from 25 MPBC apheresis that had been subjected to the complete ApoGraft process were analyzed ex-vivo for cell yield, viability and cellular constitution by FACS and for clonogenicity potential by colony forming unit (CFU) assays. Additionally, engraftment and GvHD prevention of ApoGraft were evaluated with 7 aliquots of MPBC apheresis in NOD-SCID IL2-Rgamma-null (NSG) mice (n=349). Here we report results from representative studies were unfractionated ApoGraft (2.5x10^6 cells) or CD34+ purified cells (1.0x10^5 cells) from ApoGraft were transplanted into irradiated (2-2.75Gy) NSG mice. The mice were monitored clinically for GVHD occurrence post transplantation; engraftment and differentiation were evaluated using FACS analysis.
Results:
Cellular viability was 98% (±1%) in the ApoGraft samples analyzed. Comparison of ApoGraft and Starting Material (SM) showed a mean 19.9% (±4.3%) reduction of total CD3+ T cells; early apoptosis of CD3+ cell increased by 5-fold as compared to Starting Material. Importantly, the percentage of viable CD34+ stem and progenitor cells was not affected by ApoGraft processing. Proliferation and differentiation of hematopoietic progenitors in colony forming unit (CFU) assay was not affected. In NSG mice infused with unfractionated ApoGraft or CD34+ cells purified from it, chimerism level and differentiation were similar to those of mice infused with control MPBC (figure 1A-1B). Most importantly, ApoGraft infused mice had lower GvHD clinical score and prolonged survival as compared to mice infused with control MPBC (maximal score 1.7±0.7 vs. 8.8±0.8) (Figure 1C).
Conclusion:
ApoGraft is an apoptosis-based selection process that eliminates mature T cells from stem cells grafts, while preserving CD34+ cells quantity, quality and functionality. In our preclinical murine model, ApoGraft reduced GvHD without impairing engraftment. Ongoing animal studies are evaluating the graft vs. leukemic capabilities of ApoGraft and the preservation of anti-viral immunity after ApoGraft processing. Additionally, ApoGraft is currently being evaluated in a Phase I/IIa study (NCT02828878) in subjects with hematological malignancies undergoing matched related allogeneic HSCT.
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Zuckerman:BioSight Ltd.: Consultancy. Yarkoni:Cellect Biotechnology Ltd.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Nechushtan:Cellect Biotechnology Ltd.: Employment. Rodionov:Cellect Biotechnology Ltd.: Employment. Gez:Cellect Biotechnology Ltd.: Employment. Rosenzwaig:Cellect Biotechnology Ltd.: Employment. Rodin:Cellect Biotechnology Ltd.: Employment. Pereg:Cellect Biotechnology Ltd.: Employment.
Background: Acute Myeloid Leukemia (AML) is a heterogeneous group of diseases characterized by uncontrolled proliferation and survival of hematopoietic stem and progenitor cells. The chemokine CXCL12 ...and its receptor CXCR4 are key players in the survival, bone marrow (BM) retention and the maintenance of AML blasts in their stemness state. CXCR4 overexpression is associated with poor prognosis in AML patients. Signaling activated through CXCR4 was shown to be detrimental by increasing survival of tumor cells and promoting resistance to therapy.
Objective: To study the effect of the CXCR4-antagonist, BL-8040, on the survival of human AML blasts and to investigate the molecular mechanism by which inhibition of CXCR4 signaling leads to leukemia cell death.
Methods: Human AML cell lines and human primary AML samples were used for in vitro studies. The in-vivo effect of BL-8040 was tested using the MV4-11, U-937, THP-1 cells and human primary AML cells engrafted in NOD scid gamma (NSG) mice.
Results: We found that BL-8040 directly induced apoptosis of AML cells both in FLT3-ITD and FLT3-WT AML, in-vitro and in-vivo. BL-8040 treatment triggered mobilization of AML blasts from their protective BM microenvironment and induced their terminal differentiation, in-vitro and in-vivo. The apoptosis of AML cells induced by BL-8040 was attributed to miR-15a/miR-16-1 up-regulation resulting in down-regulation of their target genes BCL-2, MCL-1 and cyclin-D1. The increase in miR-15a/miR-16-1 levels directly induced AML cell death. Moreover, CXCR4 blockade by BL-8040 also inhibited survival signals by the ERK/AKT kinases enhancing the apoptosis effect. Survival of AML cells was found to be dependent on BCL-2 as demonstrated by the ability of the BCL-2 inhibitor, ABT-199, to induce dose dependent apoptosis in vitro. It was reported that the MCL-1 protein plays a key role in acquiring resistance to ABT-199. We found that BL-8040 synergizes with ABT-199 in inducing AML cell death. This could be attributed to the reduction of both, AKT/ERK and MCL-1 levels, by treatment with BL-8040. In addition, BL-8040 synergizes with the FLT3 inhibitor AC220 in the induction of AML cell death both in-vivo and in-vitro. The combined treatment of BL-8040 and AC220 was found to prolong survival and reduce minimal residual disease in-vivo. Interestingly, the combined treatment was also associated with a significant reduction in the expression of BCL-2 and ERK signaling.
Conclusions: BL-8040 can be a potential therapeutic option in AML by targeting not only AML anchorage in the BM but also AML survival and differentiation. Our results demonstrate that BL-8040 in AML regulates the expression of miR-15a/16-1 and their target genes BCL-2, MCL-1 and cyclin-D1. Furthermore, these results indicate that the CXCR4 antagonist, BL-8040 may tip the balance toward cell death by down- regulating survival signals through miR-15a/16-1 pathway and inhibition of the ERK/AKT survival signaling cascade in AML cells. Our results provide rational for combination of BL-8040 with ABT-199 to overcome potential acquired resistance to ABT-199 in AML patients. The synergistic effect of BL-8040 with AC220 could provide a rational basis for the combination of BL-8040 with FLT3 inhibitors in FLT3-ITD AML patient population.
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Abraham:Biokine Therapeutics Ltd: Employment. Bulvik:Biokine Therapeutics Ltd: Employment. Wald:Biokine Therapeutics Ltd: Employment. Eizenberg:Biokine Therapeutics Ltd: Employment. Pereg:BioLineRx Ltd: Employment. Peled:Biokine Therapeutics Ltd: Consultancy, Employment.
Abstract
Background: CXCR4 is overexpressed in the majority of tumor cells and its degree of expression often correlates with disease severity. Binding of CXCL12 to the CXCR4 receptor activates ...signaling pathways which are crucial for the interaction of hematopoietic cells with the microenvironment and cell survival. Signaling activated through CXCR4 was shown to be detrimental by increasing survival of tumor cells and promoting resistance to therapy in many types of cancer. CXCR4-antagonists, such as BL-8040, currently in phase II trials, were shown to selectively inhibit tumor cell growth and to induce apoptosis in vitro and in vivo. However, the molecular mechanism by which CXCR4 overexpression triggers tumor cell survival and its inhibition leads to cell death is not fully understood.
Objective: To study the mechanism by which the CXCR4 pathway controls malignant cell survival and death through regulating miR-15a/16-1 expression.
Method: We assessed the effect of CXCR4 overexpression, its activation and inhibition, on the expression of miR-15a/16-1 and their target genes, BCL-2, MCL-1 and cyclin D1, in a variety of tumor cells in vitro and in vivo.
Results:
We found that overexpression of CXCR4 in tumor cells or stimulation of cells with its ligand, CXCL12, lead to up-regulation of miR-15a/16-1, resulting in down-regulation of their target genes BCL-2, MCL-1 and cyclin D1. Furthermore, overexpression of CXCR4 in these cells increases tumorgenesis and shifts their oncogenic dependency from the BCL-2 to the CXCR4/ERK signaling pathway. Antagonists of CXCR4 such as BL-8040 were shown to induce apoptotic cell death of malignant cells. BL-8040 was found to increase the expression of miR-15a/16-1 and reduce the expression of BCL2, MCL1 and cyclin D1. Importantly, CXCR4 inhibition using BL-8040 induced apoptosis in vitro and in vivo in AML and neuroblastoma tumors. This was mediated by inhibition of survival signals by ERK and down-regulation of BCL-2 expression. In support of these results overexpression of miR-15a/16-1 in AML and neuroblastoma cells was shown to induce their apoptosis.
Conclusions:
Our results demonstrate, for the first time to the best of our knowledge, that CXCR4 signaling regulates the expression of miR-15a/16-1 and their target genes. Our results suggest that overexpression of CXCR4 may override the survival dependency of tumor cells on BCL-2, MCL-1, and cyclin D1 leading to resistance of tumor cells to inhibition of these pathways. Furthermore, these results indicate that ligands of CXCR4 may tip the balance toward cell death by down- regulating survival signals through miR-15a/16-1 suppression of BCL2, MCL1 and cyclin D1 expression.
Citation Format: Shiri Klein Silberman, Michal Abraham, Baruch Bulvik, Hanna Wald, Orly Eizenberg, Dvorah Olam, Lola Weiss, Katia Beider, Ori Wald, Shlomo Bulvik, Abraham Avigdor, Ohad Benjamini, Eithan Galun, Arnon Nagler, Yaron Pereg, Amnon Peled. CXCR4 controls BCL-2 expression and function by regulating miR-15a/16-1 expression in tumor cells. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3556.
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.
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.
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.
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.
Eizenberg:Biokine: Employment. Pereg:BioLineRx LTD: Employment. Klapper:BioLineRx LTD: Employment. Abraham:Biokine: Employment.
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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.
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 (re)activation of anticancer innate and adaptive immunity is at the forefront of developments in cancer therapy. Here, we report on a new immunotherapeutic fusion protein, ...termed Dual Signaling Protein 107 (DSP107). DSP107 was designed to combine activation of innate and adaptive immunity, by both blocking CD47/SIRPα interaction and activating 4-1BB. CD47 is overexpressed on cancer cells and upon binding to SIRPα on phagocytes transmits a “don’t eat me” signal, thereby suppressing innate immunity. 4-1BB is a costimulatory receptor that is transiently upregulated on tumor-infiltrating T-cells and is considered a surrogate marker for the tumor-reactive T-cell population. Activation of 4-1BB using its ligand or by agonistic antibodies reactivates anti-cancer T-cell immunity. In DSP107, the extracellular domains of SIRPα and 41BBL have been fused, yielding a dual function protein. DSP107 is produced as a homotrimer due to the trimerization property of 41BBL, an essential element for activating the 41BB receptor, a member of the TNF super-family of receptors. DSP107 was designed to bind to CD47 on tumor cells, thereby removing the inhibitory signal delivered to phagocytes. Simultaneously, CD47-mediated surface immobilization of DSP107 enables delivery of the 41BBL-4-1BB costimulatory signal to tumor localized T-cells. This dual immunomodulatory effect of DSP107 is designed to unleash both innate and adaptive immune responses targeted to the tumor site. Methods and Results: Trimeric DSP107 was successfully produced in a mammalian expression system. Both sides of DSP107 bound their cognate counterparts in kinetic Blitz binding assays and on human tumor and immune cell surfaces. The binding affinity of DSP107 was 1.6 nM for human CD47 and 0.69 nM for human 4-1BB as determined using BIAcore analysis. DSP107 blocked the interaction of SIRPα with CD47 in an ELISA-based competition assay (EC50 of 0.03 nM). DSP107 induced granulocyte- and macrophage-mediated phagocytosis of several lymphoma, leukemia and carcinoma cell lines in vitro. Further, DSP107 treatment triggered phagocytosis of primary AML cells by autologous macrophages. Co-treatment with DSP107 and therapeutic tumor-targeting antibodies, i.e., rituximab or cetuximab, resulted in enhanced phagocytosis of lymphoma or carcinoma cells, respectively. In a reporter assay measuring IL-8 secretion upon binding to/activation of 4-1BB, DSP107 activated 4-1BB signaling only in the presence of CD47-expressing cells. Further, DSP107 augmented the activation of purified T-cells activated by suboptimal concentrations of αCD3 + IL2 or αCD3/αCD28 Dynabeads in CD47 coated plates, as measured by percentage of CD25 expressing cells (up to 3-fold). When PBMCs were co-cultured with or without CD47-expressing cancer cells and stimulated with suboptimal concentrations of αCD3 + IL2, DSP107 treatment resulted in increased secretion of IFNg up to 2-fold), and increased T-cell proliferation (up to 2-fold). Conclusions: Here we demonstrate the feasibility and functional activity of DSP107, a novel therapeutic protein that combines activation of both the innate and adaptive immune system. Dual targeting, by the two functional sides of DSP107, offers multiple functionalities that act simultaneously and may result in a synergistic effect. DSP107 is now in IND-enabling studies and CMC development. The DSP platform can be designed for selective tumor site or microenvironment targeting and is adaptable to most checkpoint targets.
Citation Format: Yosi M. Gozlan, Susan Hilgendorf, Alexandra Aronin, Yehudith Sagiv, Liat Ben-gigi-Tamir, Shira Amsili, Ami Tamir, Iris Pecker, Shirley Greenwald, Ayelet Chajut, Adam Foley-Comer, Yaron Pereg, Amnon Peled, Michal Dranitzki-Elhalel, Edwin Bremer. DSP107—a novel SIRPα-4-1BBL dual signaling protein (DSP) for cancer immunotherapy abstract. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A076.
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