Phase I Trial of Plerixafor and Bortezomib as a Chemosensitization Strategy In Relapsed or Relapsed/Refractory Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1943-1943 ◽  
Author(s):  
Irene Ghobrial ◽  
Abdel Kareem A. Azab ◽  
Jacob P. Laubach ◽  
Ranjit Banwait ◽  
Meghan Rourke ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Accessory Cells ◽  
Dose Levels ◽  
Higher Doses ◽  
Active Disease

Abstract Abstract 1943 Introduction: Plerixafor (Mozobil®), a potent CXCR4 inhibitor, is approved in combination with G-CSF to mobilize hematopoietic stem cells (HSCs) for autologous transplantation in multiple myeloma (MM) and non Hodgkin's lymphoma (NHL). Another area of investigation consists of exploring whether disruption of the CXCR4 pathway by plerixafor could potentiate the effect of chemotherapy in active disease. This study aimed to establish the maximum tolerated dose (MTD) of plerixafor in combination with bortezomib in patients who have active relapse/refractory MM. This was informed by preclinical studies showing that plerixafor induces de-adhesion of MM cells with sensitization to combination therapy with bortezomib in pre-clinical animal models. Methods: Eligibility criteria include: 1) patients with relapsed or relapsed/refractory MM with any prior lines of therapy including bortezomib, 2) measurable disease, 3) not receiving chemotherapy> 3weeks, or biological/novel therapy for MM > 2 weeks. Patients with active disease received plerixafor at the recommended dose sc on days 1–6 of every cycle. Dose levels include 0.16, 0.24, 0.32, 0.40, and 0.48 mg/kg. Bortezomib was given at the recommended dose twice a week on days 3, 6, 10, and 13 every 21 days. Dose levels include 1.0 and 1.3 mg/m2. Bortezomib was given 60–90 minutes after plerixafor. Patients were assessed after every cycle. Patients who had response or stable disease went on to receive a total of 8 cycles without planned maintenance therapy. 4 dose levels were initially planned at a maximum of 0.24 mg/kg plerixafor. The protocol was then modified to include 3 higher doses of plerixafor, to further evaluate the hypothesis that higher doses may induce better chemosensitization. To examine the in vivo effect of plerixafor and bortezomib on de-adhesion of MM cells and other accessory cells of bone marrow, blood samples were obtained from patients at 0, 2, 4 and 24 hours post-plerixafor injection on days 1 and 3, and time points 0, 2, and 4 hours on days 6, 10 and 13 of cycle 1 and examined for the presence of plasma cells or CD34+ cells using flow cytometry. Results: Thirteen patients have been treated to date, three in each cohort with cohort 5 currently enrolling. The median age is 60, the median lines of prior therapy is 2. All of the patients received prior bortezomib. Three patients were assessed by light chain, two patients had extramedullary disease. The median number of cycles on therapy was 5 (1-8). None of the patients came off study due to toxicity. To date, there have been no dose-limiting toxicities. Overall, the combination is very well tolerated. Grade 3 possibly related toxicities include lymphopenia (30%), hypophosphatemia (15%), anemia (8%), and hyponatremia (8%). No grade 2 or higher neuropathy has been noted in these patients. Twelve patients are evaluable for response, including 1 (8%) complete remission (CR) and 1 (8%) minimal response (MR), with an overall response rate including MR of 2 (16%) in this relapsed/refractory population. In addition, 8 (66%) patients had stable disease (SD), and 2 (18%) had progressive disease (PD). We also examined the number of plasma cells, CD34+ HSCs, and other accessory bone marrow cells (including endothelial progenitor cells and plasmacytoid dendritic cells) in the peripheral blood. Analysis of these samples is ongoing, but preliminary data indicate de-adhesion of plasma cells. Conclusions: : The combination of plerixafor and bortezomib is very well tolerated with minimal neuropathy or other toxicities. The responses observed are encouraging in this relapsed/refractory population. The ability to demonstrate transient de-adhesion of MM cells and accessory cells in vivo indicates that these cells can be separated from their protective stromal environment which may make them more sensitive to chemotherapy. This study was supported by R01CA133799-01, and by Genzyme. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees.

Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 133-133 ◽  
Author(s):  
Patricia Maiso ◽  
AbdelKareem Azab ◽  
Yang Liu ◽  
Yong Zhang ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Bone Marrow Microenvironment ◽  
Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Selectin Inhibition Disrupts Multiple Myeloma Cells Interaction with the Bone Marrow Microenvironment and Sensitizes Them to Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 453-453
Author(s):  
Abdel Kareem A. Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
John Magnani ◽  
John Patton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Bioluminescence Imaging ◽  
Plasma Cells ◽  
Tumor Burden ◽  
Advisory Committees ◽  
Normal Plasma

Abstract Abstract 453 Introduction: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment, which includes stromal (BMSCs) and endothelial cells (ECs), plays a crucial role in MM pathogenesis and drug resistance. We have previously shown that the chemokine stromal cell-derived factor-1 (SDF-1), its receptor-CXCR4, and GTPases in the downstream signaling of the receptor regulate this interaction. Selectins are adhesion molecules which are involved in the primary interaction of lymphocytes with the endothelium. In this study, we have tested the expression of selectins and their ligands in MM, and explored their role in the interaction with the BM milieu and its potential therapeutic applications. Methods AND RESULTS: Flow cytometry and immunohistochemical (IHC) staining of tissue microarrays revealed that P-selectin glycoprotein ligand-1 (PSGL-1, CD162) was over expressed in MM cells from patients (n=20) and cell lines (MM1s, H929, RPMI, OPM1 and OPM2) compared to normal plasma cells (n=3). Gene expression profiling (GEP) analysis showed that the expression of PSGL-1 was directly correlated with MM stage of progression (normal plasma cells, n=15 < MGUS, n=20 < smoldering MM, n=23 < MM, n=68 p<0.01). Moreover, it was shown that both BMSCs (isolated from MM patients and HS5 cell line) and ECs (isolated from MM patients and HUVECs) had high expression of P-selectin. SDF1 treatment increased the expression of P-selectin on ECs but it had no effect on the expression of PSGL on MM cells. The interaction of PSGL and P-selectin played a major role in the adhesion of MM cell to both BMSCs and ECs, and the inhibition of this interaction either by the pan-selectin inhibitor GMI-1070 (500uM, 3hrs) or by knock-down of P-selectin expression significantly decreased (50-60%) the adhesion of MM cells to BMSCs and ECs. The CXCR4 inhibitor AMD3100 (25uM, 3hrs) similarly induced similar inhibition of adhesion, and the combination of AMD3100 and GM1070 had more profound inhibition of MM adhesion to BMSCs and ECs (p = 0.006). Both AMD3100 and GMI1070 induced MM cell de-adhesion from BMSCs and ECs, but the combination of both drugs was not additive. AMD3100, GMI1070 or their combination prevented BMSCs or ECs mediated induction of proliferation of MM cells. Moreover, it was shown that the co-culture of MM cells with BMSCs and ECs reduced their sensitivity to bortezomib (5nM, 24hrs) and dexamethasone (25nm, 24hrs) compared to MM cells cultured alone. Importantly, GMI1070 restored the sensitivity of MM cells to bortezomib and dexamethasone to the level observed without co-culture with BMSCs or ECs. These effects were next tested in vivo using an orthotopic xenograft model of MM. SCID-beige mice were injected with luciferase-expressing MM1S cells, and tumor burden was assessed bioluminescence imaging. Mice with established disease were divided into treatment groups (n=10 per group) treated with vehicle, GMI1070 by osmotic pump, velcade at 1.5 mg/kg IP weekly, or a combination of GMI1070 and bortezomib. Tumor burden was determined by bioluminescence imaging. Treatment with GMI1070 alone was not different from vehicle treated control mice. While treatment with bortezomib alone had a minimal delay in tumor progression, the combined treatment of bortezomib and GMI1070 resulted in synergistic anti-tumor efficacy (p=0.0017). Conclusion: We have shown that PSGL-1 is highly expressed in MM cells as compared to normal plasma cells, and that it plays a major role in the interaction of MM cells with the BM microenvironment in relation with the SDF1/CXCR4 axis in vitro, an effect which was inhibited by the pan-selectin inhibitor GMI1070. We also demonstrated that selectin inhibition by GMI1070 reduced MM cell proliferation induced by BMSCs and ECs sensitized MM cells to bortezomib and dexamethasone in vitro, and significantly increased the sensitivity of MM tumors to bortezomib in vivo. This information provides the rationale for future clinical trials for increasing efficacy of existing therapies through a combination with selectin inhibitors for the treatment of myeloma. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Dynamic Regulation of the Level of Hypoxia In the Bone Marrow Regulates Cell Dissemination In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4035-4035
Author(s):  
Abdel Kareem A. Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
Brian Thompson ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Progression ◽  
Board Of Directors ◽  
Tumor Burden ◽  
Secondary Antibody ◽  
Advisory Committees ◽  
Hypoxic Conditions ◽  
Mild Hypoxia

Abstract Abstract 4035 INTRODUCTION: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis, implying that progression of MM occurs through continuous interaction between the BM and MM cells, which controls the ability of MM cells to egress out of the BM and home into new BM niches. We have previously shown that the CXCR4/SDF1 axis as well as Rho GTPases downstream of the receptor was important for chemotaxis, adhesion, homing and egress of MM cells. However, the driving force for MM cells to leave the BM and metastasize to other BM sites is not well understood. Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and are associated with poor patient prognosis, cell motility, associated angiogenesis and metastasis. In this study, we tested the role of hypoxia in the dissemination of MM cells in vivo, as well as regulation of the retention/egress of MM cells in and out of the BM. METHODS: To test the effect of hypoxia on induction of MM egress, MM1s-GFP+/Luc+ cells were injected into 12 SCID mice, and then mice with different stages of tumor development (based on the tumor size detected by bioluminescence) were treated with the hypoxia marker pimonidazole. Blood was drawn and BM was obtained from the femur. Mononuclear cells were then fixed, permeabilized, and stained with antibodies against pimonidazole, followed with an APC- secondary antibody, PE-mouse-anti-human CXCR4, and anti-cadherin antibody followed by an Alexa-Fluor-594 secondary antibody. MM cells in BM and peripheral blood were identified by gating on cells with high GFP signal. To confirm the effects of severe hypoxia found in vivo compared to physiologic mild hypoxia found in the BM, we tested the effect of mild hypoxic conditions (6% O2) and severe hypoxic conditions (0.5% O2) on MM expression of cadherins and CXCR4, as well on functional adhesion of MM cells to stromal cells and chemotaxis. RESULTS: Twelve mice with different stages of MM tumor progression were used. A bi-phasic correlation between tumor progression and the percent of hypoxic cells in BM was found, showing that severe hypoxic conditions in the BM correlated with tumor burden. The correlation between the tumor burden and the number of circulating cells was not linear; however, a direct linear correlation was observed between the number of circulating MM cells and hypoxia in the BM. Moreover, hypoxia in BM correlated directly with the expression of CXCR4 and negatively correlated with the expression of cadherins in MM cells isolated from the BM. To test the effect of the severe hypoxic conditions induced by tumor progression compared to mild hypoxic conditions found physiologically in the BM, we tested the effect of 0.5% O2 (severe hypoxia) and 6% O2 (mild hypoxia) compared to normoxia (21%) on MM cell adhesion to BMSCs, as well as on chemotaxis in response to SDF1, as well as expression of CXCR4 and cadherins. We found that severe hypoxic conditions decreased MM expression of cadherins and adhesion to BMSCs, as well as increased expression of CXCR4 and chemotaxis to SDF1 compared to cells in normoxia. In contrast, mild hypoxic conditions did not alter the expression of CXCR4 and cadherins, adhesion of MM cells to BMSCs, or chemotaxis of MM to SDF1 compared to normoxic cells. CONCLUSION: Hypoxia in the BM directly correlates with the number of circulating MM cells, and with changes in expression of cadherins and CXCR4 in vivo. Severe hypoxic conditions, but not mild hypoxic conditions, induce hypoxic responses in MM cells. Based on these findings, further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy in MM are warranted. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 311-311 ◽  
Author(s):  
Laurie Herviou ◽  
Alboukadel Kassambara ◽  
Stephanie Boireau ◽  
Nicolas Robert ◽  
Guilhem Requirand ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Ezh2 Expression ◽  
Bone Marrow Plasma

Abstract Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

Role of Selectins in the Pathogenesis of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 951-951 ◽  
Author(s):  
Abdel Kareem Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
Costas M Pitsillides ◽  
John T Patton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Cell Adhesion ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees

Abstract Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p<0.05). Moreover, we found that blockade of P-selectin on endothelial cells reduced their interaction with MM cells (p<0.01), while blockade of E and L-selectin did not show any effect. Treating endothelial cells with GMI-1070 mimicked the effect of blocking P-selectin. Moreover, we found that treating endothelial cells with the chemokine stroma cell-derived factor-1-alpha (SDF1) increased their expression of P but not E or L-selectin detected by flow cytometry. Neither the blockade of each of the selectins and their ligands nor the GMI-1070 inhibited the trans-well chemotaxis of MM cells towards SDF1-alpha. However, blockade of P-selectin (p<0.001) on endothelial cells by GMI-1070 inhibited the trans-endothelial chemotaxis of MM cells towards SDF1-alpha. Both adhesion to endothelial cells and activation with recombinant P-selectin induced phosphorylation of cell adhesion related molecules including FAK, SRC, Cadherins, Cofilin, AKT and GSK3. GMI-1070 decreased the activation of cell adhesion molecules induced by both recombinant P-selectin and endothelial cells. Using in vivo flow cytometry we found that both anti P-selectin antibody and GMI-1070 prevented the extravasation of MM cells out of blood vessels into the bone marrow in mice. Moreover, we found that, in a co-culture system, endothelial cells protected MM cells from bortezomib induced apoptosis, an effect which was reversed by using GMI-1070, showing synergistic effect with bortezomib. CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Phase I Trial of Plerixafor and Bortezomib As a Chemosensitization Strategy in Relapsed or Relapsed/Refractory Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1874-1874 ◽  
Author(s):  
Irene M. Ghobrial ◽  
Ranjit Banwait ◽  
Abdel Kareem Azab ◽  
Phong Quang ◽  
Jacob P. Laubach ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Recommended Dose ◽  
Advisory Committees ◽  
Post Dose ◽  
Hematopoietic Stem ◽  
Genzyme Corporation ◽  
Accessory Cells

Abstract Abstract 1874 INTRODUCTION: This study aimed to determine the safety and activity of plerixafor (CXCR4 inhibitor) in combination with bortezomib as a chemosensitization strategy in multiple myeloma (MM). This was based on our preclinical studies showing that plerixafor (Mozobil, Genzyme Corporation, MA) induces de-adhesion of MM cells and sensitization to bortezomib in preclinical animal models. METHODS: Eligibility criteria included: 1) patients with relapsed or relapsed/refractory MM with 1–5 prior lines of therapy including bortezomib (unless patients were refractory to bortezomib), 2) measurable disease, 3) not receiving chemotherapy > 3 weeks, or biological therapy for MM > 2 weeks prior to study treatment. Eight cohorts with different doses and two treatment schedules were planned. In cohorts 1–5, patients received plerixafor at the recommended dose sq on days 1–6 of each cycle and bortezomib at the recommended dose twice a week on days 3, 6, 10, and 13 every 21 days. To test the hypothesis that higher doses and a different schedule might induce better chemosensitization, in cohort 5b–7 plerixafor was given at the recommended dose sq on days 1, 3, 6, 10, and 13 and bortezomib was given at the recommended dose twice a week on days 3, 6, 10, and 13 every 21 days. Bortezomib was given 60–90 minutes after plerixafor. Patients were assessed after every cycle by modified EBMT/UC criteria. Patients who had a response or stable disease went on to receive a total of 8 cycles with maintenance therapy for patients with at least a minimal response (MR). To examine the in vivo effect of plerixafor and bortezomib on de-adhesion of MM cells and other accessory cells of the bone marrow, peripheral blood samples were obtained from the patients at 0, 1, 2, 3, 4 and 24 hours post-dose on days 1 and 3, and time points 0, 2, and 4 hours post-dose on days 6, 10 and 13 of cycle 1. RESULTS: Twenty-five patients were enrolled in this phase I trial from June 2009 to May 2011. Median age was 60 years (range, 44–85) and median lines of prior therapy were 2 (range, 1–4) with all but 3 patients receiving prior bortezomib. The median number of cycles on therapy was 3 (1–11). Dose limiting toxicities including insomnia, restlessness, and psychosis were observed in two patients at dose level 6 (plerixafor 0.40 mg/kg and bortezomib 1.3 mg/m2). To further explore the safety of maximum tolerated dose, three additional patients were enrolled at dose level 5b (plerixafor 0.32 mg/kg and bortezomib 1.3 mg/m2). Overall, the combination proved to be well tolerated. There were no grade 4 toxicities. Grade 3 toxicities included lymphopenia (40%), hypophosphatemia (20%), anemia (10%), hyponatremia (10%), hypercalcemia (10%), and bone fracture due to myeloma bone disease (10%). One patient came off treatment due to grade 2 painful neuropathy at cycle 5. Twenty-three patients were evaluable for response, including 1 (4%) complete response (CR), 1 (4%) very good partial response (VGPR) and 3 (13%) MR, with an overall response rate (including MR) of 5 (22%) in this relapsed and refractory population. In addition, 15 (65%) patients achieved stable disease (SD), with just 3 (13%) having progressive disease (PD) as their best response. We also examined in vivo mobilization of plasma cells, CD34+ hematopoietic stem cells and other accessory bone marrow cells. Analysis of these samples showed rapid mobilization of plasma cells at 2 hours post-plerixafor with a rapid return to normal levels at 4 and 24 hours post plerixafor. Similar results were observed on days 1 and 3, but less mobilization occurred on the following days. Hematopoietic stem cell mobilization occurred at 4 hours on days 1 and 3, and was less observed with subsequent doses of plerixafor consistent with prior studies. CONCLUSIONS: The combination of plerixafor and bortezomib is generally well tolerated with minimal neuropathy or other toxicities seen to date. The responses observed are encouraging in this relapsed and refractory population. The ability to demonstrate transient de-adhesion of MM cells and accessory cells in vivo in most of the patients indicates that chemosensitization can potentially be achieved in patients with MM using this approach. Further studies are warranted and a phase 2 trial is underway. This study was supported by R01CA133799-01, and by Genzyme Corporation. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Plerixafor in myeloma. Munshi:Millennium: 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; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees. Schlossman:Millennium: Consultancy; Celgene: Consultancy. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Role of TORC1 and TORC2 in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1815-1815
Author(s):  
Patricia Maiso ◽  
Yi Liu ◽  
Abdel Kareem Azab ◽  
Brittany Morgan ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Stromal Cells ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Cycle Arrest

Abstract Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, immunochemistry, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Daratumumab, a CD38 Monoclonal Antibody in Patients with Multiple Myeloma - Data From a Dose-Escalation Phase I/II Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 73-73 ◽  
Author(s):  
Torben Plesner ◽  
Henk Lokhorst ◽  
Peter Gimsing ◽  
Hareth Nahi ◽  
Steen Lisby ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Dose Escalation ◽  
Plasma Cells ◽  
Plasma Concentrations ◽  
Efficacy Data ◽  
Advisory Committees ◽  
Bone Marrow Plasma

Abstract Abstract 73 Background Daratumumab (HuMax™-CD38) is a human CD38 monoclonal antibody with broad-spectrum killing activity and effectively mediates killing of CD38-expressing tumor cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis. In this present ongoing first-in-human (FIH) dose-escalation study of daratumumab in pts with multiple myeloma (MM) (ClinicalTrials.gov CT00574288), the safety profile has been acceptable and preliminary efficacy data have already been published1,2. Here we present data from the dose escalation part of the study. Objectives The primary objective was to establish the safety profile. The secondary objectives were to establish the maximum tolerated dose (MTD), assessment of efficacy, pharmacokinetics (PK) and immunogenicity – Anti-Drug-Antibodies (ADA). Methods Pts ≥18 years and diagnosed with MM requiring systemic therapy and considered relapsed or refractory to at least two different prior lines of therapy and ineligible for ASCT were enrolled. The study was based on a 3+3 dose-escalation design. Daratumumab was administered over a 9-week period consisting of 2 pre- and 7 full doses. The doses ranged from 0.005 mg/kg to 24 mg/kg. Prednisolone/methylprednisolone was administered to prevent infusion related events (IREs) in a maximum dose equivalent to 27mg dexamethasone per week. Daratumumab plasma concentrations were measured by ELISA. Evaluation of efficacy data was according to IMGW guidelines3. A bridging ElectroChemiLuminesence (ELC) method on the MesoScale Discovery platform was used to detect ADA responses in pts to daratumumab. The results presented are based on data analyzed before database lock. Results Data from 32 pts including 2 pts in the ongoing 24mg/kg cohort were collected until now. Median age was 61 years (42–76). The median number of prior treatment lines was: 6.3 (2–12). PK analysis showed plasma peak levels as expected, but relatively rapid clearance at low dose levels. The clearance rate decreased with increasing dose suggesting an effect of target binding on the PK. At doses ≥ 4 mg/kg, daratumumab trough levels were consistent ≥ 10 μg/ml and observed PK values approximately estimated PK values (Figure 1). Preliminary efficacy evaluation in this abstract was based on best response to paraprotein as reflected by change in serum or urine M-component or free light chains (FLC) according to IMGW guidelines3. For groups ≤ 2 mg/kg, 4/20 pts achieved a reduction in paraprotein (12%, 14%, 19%, 55%); in the 4 mg/kg group, 3/3 pts had a reduction in paraprotein of 49%, 100%, and 64%, respectively. In the 8 mg/kg group, 2/3 pts had a reduction in paraprotein of 39%, and 100%, respectively whereas in the 16mg/kg cohort, 2/3 pts had a reduction in paraprotein of 50%, and 33%, respectively. A reduction of 80%-100% in the bone marrow plasma cells was seen in the 4 mg/kg group and onwards. No detectable ADA responses were found in the pts. No DLTs were reported in the 2, 4, 8 and 16mg/kg cohorts. The most common adverse events reported were infusion related events. The observed IREs occurred predominantly during the initial infusions, 10% of pts reported IREs during the pre-dose, 30% during the first full infusion with a gradual decrease in frequency after the 2nd full infusion. No dose relationship was observed. Most IREs had onset within 3–4 hours of infusion, two of the IREs were grade 3 and the remaining grade 1–2. Four IREs were SAEs; however, since implementation of steroids before all infusions and dilution of trial drug, no serious IREs were reported in the 4, 8 and 16mg/kg cohorts. Conclusion In pts with relapsed or refractory MM treated with daratumumab, a marked reduction in paraprotein and bone marrow plasma cells was observed in the higher dose cohorts. This has not previously been demonstrated with a single-agent monoclonal antibody in MM. No unexpected buildup of daratumumab was seen and in pts treated with 4mg/kg and upwards the observed plasma concentrations were close to those predicted. No ADA responses were detected. The MTD was not yet established and the toxicity was manageable. All data available from part 1 will be presented at the meeting. Disclosures: Plesner: Genmab: Consultancy; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Lokhorst:Genmab: Consultancy. Lisby:Genmab: Employment. Richardson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Vascular Endothelial Growth Factor Expression in CD138+/CD19- and CD138+/CD19+ Plasma Cells in Monoclonal Gammopathies and Impact on Multiple Myeloma Prognosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5342-5342
Author(s):  
Catarina Geraldes ◽  
Ana Cristina Gonçalves ◽  
Raquel Alves ◽  
Emília Cortesão ◽  
Maria Leticia Ribeiro ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Vascular Endothelial Growth Factor ◽  
Board Of Directors ◽  
Plasma Cells ◽  
Vascular Endothelial ◽  
Vegf Expression ◽  
Advisory Committees ◽  
Expression Levels ◽  
Endothelial Growth Factor

Abstract INTRODUCTION: Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of extracellular matrix remodeling and inflammatory cytokine generation with an important role in the bone marrow microenvironment of multiple myeloma (MM). Angiogenesis is enhanced in the bone marrow of MM patients in parallel with tumor progression. Myeloma and stromal cells secrete angiogenic factors that include VEGF. Previous studies showed heterogeneity in the expression of VEGF between plasma cells (PCs) from the same MM patient. However, no clear association with expression levels, phenotypic subtypes of PCs and prognosis was demonstrated. MATHERIALS AND METHODS: Bone marrow PCs from 128 patients with monoclonal gammopathies, 60 patients with newly diagnosed symptomatic MM and 68 with monoclonal gammopathy of uncertain significance (MGUS) and also from 11 non-neoplastic controls (Ctr) were analysed between April 2010 and July 2013. We evaluated the expression of cytoplasmic VEGF with monoclonal antibodies by flow cytometry in the two populations of PCs, identified by gating CD138+/CD19- (clonal PCs) and CD138+/CD19+ (non-clonal PCs). The results are presented as percentage of PCs expressing VEGF and as expression levels of VEGF in mean intensity of fluorescence (MIF). The effects of VEGF expression on progression-free survival (PFS) and overall survival (OS) were analysed. For statistical analysis, software IBM SPSS Statistics v22 was used. Survival was estimated according to the Kaplan-Meier method. RESULTS: In our cohort of patients, median age was 70 (39-86) years, 52% were male. We found increased expression levels of VEGF in CD138+/CD19- PCs from MM (80 ± 7,5 MIF) compared to MGUS patients (61,7 ± 6,2 MIF) (p=0,011), as well as superior to CD138+/CD19+ PCs expression (39,92 ± 1,74 MIF) in both populations of patients (p<0,001 and p=0,02, respectively). No diferences were observed in the expression levels of VEGF in CD138+/CD19+ PCs from MM (39,92 ± 1,74 MIF), MGUS patients (41,18 ± 1,92 MIF) and controls (32,8±1,5 MIF). However, the percentage of CD138+/CD19+ expressing VEGF was significantly higher in MGUS (39,4±4%) and in MM patients (46,7±4,5%) compared to Ctr (13,5±0,5%)(p=0,019 and p=0,003, respectively). In MM patients, we also found an association between increased VEGF expression levels in CD138+/CD19- PCs (superior or equal to 175 MIF) and inferior PFS (p=0,002) and OS (p=0,003), irrespective of first line therapy (bortezomib-based regimens for fit patients or alkylating-based treatments for unfit patients). Interestingly, we also observed an incresed percentage of CD138+/CD19+ PCs (higher or equal to 21%) expressing VEGF in MM patients with a more favorable PFS (p= 0,04) and OS (p=0,008). CONCLUSIONS: The results of our investigation showed that CD138+/CD19- and CD138+/CD19+ PCs have diferences in what concerns VEGF expression, not only in MM patients, but also in MGUS patients. The increased expression of VEGF in clonal PCs from MM compared to MGUS patients evidences the relevance of VEGF in myelomagenesis. We also demonstrated a negative prognostic impact of an increased VEGF expression in CD138+/CD19- PCs, highlighting the role of VEGF in the survival and maintenance of clonal PCs and as a predictor of outcome in MM progression. The association between the percentage of CD138+/CD19+ PCs and survival supports the suggestion that these cells may not be neutral players in the complex pathogenesis of MM. The results of our study should be further investigated in larger series of patients. Disclosures Geraldes: Celgene: Employment, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Bion-1301: A Novel Fully Blocking APRIL Antibody for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2112-2112 ◽  
Author(s):  
John Dulos ◽  
Driessen Lilian ◽  
Marc Snippert ◽  
Marco Guadagnoli ◽  
Astrid Bertens ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cell ◽  
Board Of Directors ◽  
Clinical Development ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Parental Antibody

Abstract A PRoliferation Inducing Ligand (APRIL, TNFSF13), is a ligand for the receptors BCMA and TACI. APRIL serum levels are enhanced in patients diagnosed with Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), and Colorectal Carcinoma correlated with poor prognosis. Our anti-APRIL antibody blocked CLL survival and inhibited mouse B1 hyperplasia in vivo (Guadagnoli et al., 2011). APRIL is produced by cells in the bone marrow niche, including myeloid-derived cells, osteoclasts and plasmacytoid dendritic cells. APRIL critically triggers BCMA in vitro and in vivoto drive proliferation and survival of human MM cells (Tai et al., 2016). Importantly, APRIL induces resistance to lenalidomide, bortezomib and other standard-of-care drugs. Furthermore, APRIL drives expression of PD-L1, IL-10, VEGF and TGFβ forcing an immunosuppressive phenotype on BCMA+ cells. As MM survival, resistance to treatment and the immunosuppressive phenotype can be blocked by neutralizing APRIL (Tai et al., 2016), development of an antibody blocking APRIL provides a novel avenue for the treatment of MM. A novel mouse anti-human APRIL antibody hAPRIL.01A (Guadagnoli et al., 2011) initially discovered using Aduro's B-Select platform, was humanized and further engineered enhancing its stability (designated as BION-1301). The antibody binds to recombinant human APRIL with a KDof 0.4 ± 0.15 nM determined by BioLayer Interferometry and an EC50 of 0.29 ± 0.05 nM by ELISA. The epitope of BION-1301 was mapped to the BCMA and TACI binding site explaining its fully blocking capacity. Blocking potency (IC50) was 1.61 ± 0.78 nM (BCMA) and 1.29 ± 0.89 nM (TACI) respectively, corroborated by potent and complete blockade of APRIL-induced cytotoxicity of BCMA-Fas and TACI-Fas Jurkat transfectants. In vitro, BION-1301 suppressed APRIL-induced B-cell IgA and IgG class switching in a dose-dependent fashion. In vivo, BION-1301 was shown to suppress human APRIL induced T cell-independent B cell responses to NP-Ficoll. Biophysical and functional experiments indicated that BION-1301 recapitulated all characteristics of the mouse parental antibody hAPRIL.01A. To support the clinical development of BION-1301, quantitative assays were developed using several mouse-anti-human APRIL antibodies and shown to detect free and complexed APRIL in human blood samples. Results obtained with assays demonstrate that APRIL can be quantified reproducibly in human sera and overcome the drawbacks of previous assays, such as requirement of polyclonal sera, Ig adsorption, interference by human serum and reduced sensitivity. In conclusion, we have generated and functionally characterized a novel humanized APRIL neutralizing antibody, designated BION-1301. The mechanism-of-action and anti-tumor activity described for the parental antibody hAPRIL.01A in vitro and in vivo strongly support the development of BION-1301 as a single agent or in combination with lenalidomide, bortezomib, and suggest a rationale for combination with checkpoint inhibitors. BION-1301 is expected to enter clinical development in 2017. References:Guadagnoli M, Kimberley FC, Phan U, Cameron K, Vink PM, Rodermond H, Eldering E, Kater AP, van Eenennaam H, Medema JP. Development and characterization of APRIL antagonistic monoclonal antibodies for treatment of B-cell lymphomas. Blood. 2011 Jun 23;117(25):6856-65Tai YT, Acharya C, An G, Moschetta M, Zhong MY, Feng X, Cea M, Cagnetta A, Wen K, van Eenennaam H, van Elsas A, Qiu L, Richardson P, Munshi N, Anderson KC. APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment. Blood. 2016 Jun 23;127(25):3225-36 Disclosures Dulos: Aduro Biotech Inc.: Equity Ownership. Lilian:Aduro Biotech Inc.: Equity Ownership. Snippert:Aduro Biotech Inc.: Equity Ownership. Guadagnoli:Aduro Biotech Inc.: Equity Ownership. Bertens:Aduro Biotech Inc.: Equity Ownership. David:Aduro Biotech Inc.: Equity Ownership. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Eenennaam:Aduro Biotech Inc.: Equity Ownership. Elsas:Aduro Biotech Inc.: Equity Ownership.

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