ALBERT

Description: Abstract 1543 BMS-936564/MDX-1338 is a fully human monoclonal antibody that specifically recognizes human CXCR4 and is currently in phase 1 studies in patients with relapsed/refractory acute myeloid leukemia (AML) and multiple myeloma (MM). CXCR4 has been identified as a prognostic indicator for AML and other malignancies, in which greater expression of CXCR4 correlates with disease severity. CXCR4 is a seven-transmembrane, G-protein-coupled receptor in the CXC chemokine receptor family. In response to stimulation by its ligand, the chemokine CXCL12, CXCR4 activates calcium flux, chemotaxis and mediates directional migration of hematopoietic cells. In healthy adults, the receptor is predominantly expressed on B and T cells, monocytes, macrophages, NK and dendritic cells, as well as lymphoid and myeloid precursor cells. Expression of CXCR4 is elevated in a variety of cancers and the interaction of CXCR4 on tumor cells with CXCL12 in the bone marrow promotes tumor cell survival and growth. An antagonist of this pathway is predicted to be efficacious in a variety of hematologic malignancies. In vitro studies demonstrate that BMS-936564/MDX-1338 binds to CXCR4expressing cells with low nanomolar affinity. The antibody blocks CXCL12 binding to CXCR4 expressing cells and inhibits CXCL12 induced migration and calcium flux with low nanomolar EC50 values. When given as monotherapy on established tumors, the antibody exhibits anti-tumor activity in multiple AML, NHL and MM xenograft models. BMS-936564/MDX-1338 is an IgG4 and thus does not elicit complement dependent cytotoxicity (CDC) or antibody dependent cell mediated cytotoxicity (ADCC). In vitro and in vivo studies suggest that BMS-936564/MDX-1338 induces apoptosis as one mechanism of tumor growth inhibition. Here we describe the in vitro and in vivo characterization and activities of BMS-936564/MDX-1338. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Mulvey:Bristol-Myers Squibb: Employment. Chen:Bristol-Myers Squibb: Employment. Pan:Bristol-Myers Squibb: Employment. Chong:Bristol-Myers Squibb: Employment. Niekro:Bristol-Myers Squibb: Employment. Kempe:Bristol-Myers Squibb: Employment. Henning:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Korman:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment.

Description: Background The C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating the biology of B-cell lymphoproliferative disorders. Recent whole genome sequencing studies have identified unique CXCR4 mutations in 29% of the 55 evaluated patients with Waldenstrom Macroglobulinemia (WM). In this study, we sought to better define the mutation status of CXCR4 in B-cell malignancies and define the functional role of this mutation in the progression of WM in vivo. Methods Allele-specific(AS) PCR has been performed on bone marrow (BM)-derived tumor cells of patients with WM (n: 131); IgM monoclonal gammopathy of undetermined significance (MGUS; n: 40); as well as in patients with diffuse large cell lymphomas (DLBCL; n: 75), splenic marginal zone lymphoma (SMZL; n: 14), B-chronic lymphocytic leukemia (B-CLL; n: 37), hairy cell leukemia (HCL; n: 35), multiple myeloma (MM; n: 36), IgA/IgG MGUS (n: 22), lymphoplasmacytic lymphoma without WM criteria (n: 13), and amyloidosis (n: 6). CXCR4-loss and -gain of function studies have been performed on WM cells stably expressing either shRNA-CXCR4, CXCR4-ORF-GFP-tagged or scramble-RFP-tagged (generated via lentivirus-based infection). A mutagenesis kit has been used to generate the C1013GCXCR4 mutant protein (C1013GCXCR4) in WM cells, via lentivirus-based infection. CXCR4-knock-in or C1013GCXCR4-mutated cells and the corresponding controls have been injected i.v. into SCID/Bg mice and tumor dissemination has been evaluated ex vivo by immunohistochemistry IHC (human-CD20; -CXCR4). C1013GCXCR4-mutated cells have been characterized at mRNA levels (U133 plus2) using GSEA. A novel human anti-CXCR4 mAb (BMS-936564/MDX-1338; Bristol Myers Squibb, NY) has been tested in vitro (cell proliferation, MTT, adhesion and migration to primary WM BM mesenchymal stromal cells) and in vivo (10mg/kg i.p. x3-4/week). Tumor growth has been evaluated by IHC ex vivo (hCD20; hCXCR4) and by immunofluorescence. Results We examined the mutational status of C1013GCXCR4 and confirmed the presence of this specific mutation in 28% of the 131 cases evaluated. The mutation was also detected at the stage of IgM-MGUS (20%); while it was present in a minority of patients with DLBCL (1%) and SMZL (7%). Remarkably, it was absent in all MM (n=36) and IgA/IgG MGUS patients (n=22), and it was not detected in healthy subjects (n=32). The functional relevance of the C1013G-CXCR4 variant was next examined in vivo. Mice injected with C1013GCXCR4-cells presented with a significant dissemination of tumor cells, demonstrating involvement of liver, bone marrow, lymph nodes, kidney and lung. IHC showed the presence of CXCR4+ and CD20+ cells in all the tissues examined; and quantification of CXCR4 and CD20 positivity was higher in C1013GCXCR4-cells-, compared to parental(p)-WM cell-injected mice (NIS Elements software, Nikon, Melville, NY; P

Description: Abstract 2887 Chronic lymphocytic leukemia (CLL) remains incurable despite advances in the biology and treatment of this disease. Current data support the notion that resistance to therapy is promoted by a “protective” tumor microenvironment in which non-leukemia cells produce factors that enhance the resistance of CLL cells to spontaneous or drug-induced apoptosis. One such factor is the chemokine CXCL12, which interacts with its receptor CXCR4 on CLL cells to promote cancer cell survival. To examine the therapeutic potential of blocking CXCL12-CXCR4 interactions, we studied the effect of BMS-936564, a fully human IgG4 anti-CXCR4 antibody, using an in vitro co-culture model of human bone marrow derived stomal-NKter cells – leukemia cell interaction. Such stromal-NKter cells secrete CXCL12 and enhance the resistance of CLL cells to apoptosis in vitro. We observed that primary CLL cells co-cultured with stromal-NKter cells had significantly greater viability than CLL cells cultured alone (20–60% above baseline at 48 hours). Moreover, CLL cells co-cultured with stromal cells had enhanced resistance to drug-induced apoptosis. We found that BMS-936564 antibody at concentrations of 2–200nM could enhance the rate of apoptosis of CLL cells cultured alone or in the presence of stromal cells. CLL cells that expressed unmutated IgVH genes or ZAP-70 appeared equally susceptible to treatment with BMS-936564 as did CLL cells that lack these adverse prognostic markers, as did CLL cells that harbored deletions in 17p13.2 and that were resistant to chemotherapeutic agents, such a fludarabine monophosphate. BMS-936564 antibody inhibited CXCL12 mediated F-Actin polymerization in CLL cells at lower concentrations (20–200nM) compared to AMD-3100 (Mozobil), a small molecule CXCR4 inhibitor (50–150μM). In addition, AMD-3100 did not induce apoptosis in CLL cells (10–300μM). In summary, we observed that the anti-CXCR4 antibody BMS-936564 inhibited CXCL12 mediated activation of the CXCR4 receptor in CLL cells and induced apoptosis in leukemia cells. The pro-apoptotic activity of BMS-936564 was observed in cells cultured alone or together with stromal cells suggesting that this antibody had direct cytotoxic effect on leukemia cells and that it can overcome the protective tumor microenvironment. More over, the activity of BMS-936564 was independent of the presence of poor prognostic factors such as del(17p) suggesting that its mechanism of action is P53 independent. These findings show evidence that the CXCR4-CXCL12 pathway is a valid therapeutic target in CLL and provide additional biological rationale for ongoing clinical trials in CLL and other hematological malignancies using BMS-936564. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Kipps:Abbott: Consultancy, Research Funding.

Description: Background. Presence of multiple osteolytic lesions is one of the main clinical features of patients with active multiple myeloma (MM), thus suggesting the ability of clonal plasma cells to disseminate from bone to bone. The bone marrow (BM) homing process of MM cells has been shown to be supported by the activation of the CXCR4/CXCL12 axis. Nevertheless, the role of CXCR4 in mediating MM cell bone metastasis, and the role of CXCR4-targeted therapy in inhibiting MM cell dissemination from bone-to-bone has not been previously reported. Methods. We dissected the in vivo functional relevance of CXCR4 in mediating MM cell dissemination, tumor growth and survival, by using gain- and loss-of function approaches. Tumor growth was monitored by bioluminescence imaging (BLI) and MM cell homing and engraftment within the BM niches was evaluated by performing intravital confocal microscopy. CXCR4-oeverexpressing (CXCR4+) MM cells were tested in vivo using a model of bone-to-bone dissemination. Empty vector or CXCR4+ cells were loaded into murine femurs and subsequently implanted s.q. into recipient mice. The ability of MM cells to disseminate from the implanted bone to the host bones was evaluted by flow cytometry and immunohistochemistry (IHC; CD138; hematoxylin-eosin) on the harvested host bones. Modulation of EMT-related genes (twist, snail, slug, E-cadherin, vimentin) was evaluated ex vivo by qRT-PCR. A novel anti-CXCR4 monoclonal antibody (BMS-936564) was tested both in vivo and in vitro to define the possible role in modulating MM cell dissemination and growth. Results. CXCR4-overexpression led to changes in actin cytoskeleton reorganization of MM cells with protrusion of cell pseudopodia, compared to empty vector-transfected cells, sustained by modulation of EMT-related markers, as shown by up-regulation of Slug, Snail and Twist, together with down-regulation of E-cadherin, in CXCR4-overexpressing (CXCR4+) MM.1S cells, compared to empty vector-transfected cells. CXCR4+ MM cells presented with enhanced invasive properties, compared to control cells. These findings were next recapitulated in vivo using an in vivo model of MM cell dissemination: CXCR4+ MM cells presented with higher ability to metastasize from bone to bone compared to control cells, as confirmed on harvested host femurs by using IHC and flow cytometry. Lower expression of human (h)-E-cadherin, and higher mRNA expression of h-Twist, h-Snail, h-Slug was confirmed within the BM of the host femurs. We next tested the novel monoclonal antibody, anti-CXCR4 (BMS-936564), in vivo. BMS-936564 exerted an anti-MM activity in situ, within the s.q. implanted bones; and also reduced MM cell dissemination from the implanted bone to the host bone. We examined whether BMS-936564 might have modulated EMT-related genes in the MM cells metastasized to the host bones, and found higher mRNA expression of human (h)-E-cadherin, together with reduced mRNA expression of h-Twist, h-Snail, h-Slug, h-vimentin and h-CXCR4 in the BM cells harvested from the host bones of mice treated with BMS-936564. Similar findings were confirmed in a model of breast cancer in vivo. CXCR4 silencing in MM cells led to inhibition of MM cell homing to the bone marrow, as shown by using intra-vital confocal microscopy and inhibition of MM tumor growth in vivo, as shown by BLI. Prolonged survival was documented in those mice that were injected with CXCR4-silenced MM cells compared to control mice injected with scramble probe-infected MM cells. Conclusion. CXCR4 enhanced the acquisition of an EMT-like phenotype in MM cells, leading to higher bone-to-bone dissemination of clonal plasma cells in vivo; while CXCR4-silencing in MM cells led to inhibited tumor growth and improved survival. BMS-936564 led to inhibited MM cell bone-to-bone dissemination, supported by suppression of the CXCR4-driven EMT-like phenotype. These studies suggest that targeting CXCR4 may represent a novel therapeutical strategy to prevent MM cell dissemination. Disclosures Cardarelli: BMS: Employment. Cohen:BMS: Employment. Kuhne:BMS: Employment. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 4009 Background. The SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. Also, hypoxia leads to EMT activation as well as CXCR4 up-regulation in MM cells. We therefore hypothesized that CXCR4 may represent a crucial regulator of EMT in MM and an important target for preventing MM disease dissemination. Methods. Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Dissemination of MM.1S/GFP+ cells to distant bone marrow niches was evaluated in vivo, by using in vivo confocal microscopy. CXCR4-loss of function studies were performed by transfecting MM cells with either a scrambled probe or CXCR4-siRNA. A novel HuMAb anti-CXCR4 (BMS-936564; Bristol Myers Squibb, NY) was used. Migration towards SDF-1 and BMSCs was evaluated. Cytotoxicity and DNA synthesis were measured by MTT and 3H-thymidine uptake, respectively. Cell signaling, apoptotic- and EMT-related pathways were studied by Western Blot. Synergism was calculated by using the Chou-Talalay method. In vivo, MM tumor growth was evaluated by using xenograft mouse models and a melanoma xenograft mouse model was used to validate the effect of anti-CXCR4 antibody on modulating tumor cell metastasis. Results. We demonstrated down-regulation of Twist, Snail and Slug, together with up-regulation of E-Cadherin in CXCR4-siRNA-transfected cells, compared to scrambled probe-transfected cells. These findings were next validated by using the new selective CXCR4 antibody (BMS-936564); and confirmed that BMS-936564-dependent inhibition of CXCR4 led to inhibition of Twist, Snail, and Slug; with up-regulation of E-Cadherin. These data were further corroborated in vivo, by using in vivo confocal microscopy: mice treated with BMS-936564 presented with less MM cell dissemination to distant bone marrow niches, compared to vehicle-treated mice, supporting the hypothesis that CXCR4 may represent a crucial modulator of tumor cell dissemination. These data were also confirmed in vivo, by using a xenograft melanoma model, where BMS-936564-treated mice presented with a reduced number of metastasis, compared to vehicle-treated mice. These in vivo data were supported by in vitro evidence showing the ability of BMS-936564 to functionally target MM cells in terms of migration, adhesion and survival. BMS-936564 inhibited migration of MM cells towards SDF-1a and primary MM BMSCs, in a dose-dependent manner. In addition, survival and adhesion of primary MM cells to BMSCs were inhibited by BMS-936564 in a dose-dependent manner. BMS-936564 targeted MM cells in the context of BM milieu, by overcoming BMSCs-induced proliferation of tumor cells. Moreover, BMS-936564 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS-936564-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorylation were inhibited by BMS-936564 in a dose-dependent manner. Importantly, BMS936564 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion. These findings indicate that CXCR4 represents a valid therapeutic target due to its ability to modulate EMT, and that BMS-936564 functionally targets MM cell migration, adhesion and survival; thus providing evidence for using the anti-CXCR4 antibody, BMS-936564, as a therapeutic modality for MM. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Novartis: Advisory Board Other; Onyx: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Bristol Myers Squibb: Advisory Board, Advisory Board Other.