ALBERT

Description: Chemoresistance represents a considerable barrier to improving outcomes for patients with acute myeloid leukemia (AML) and therapeutic approaches using multiple lines of therapy have been unsuccessful as cancer cells acquire resistance to the chemotherapeutic agents to which they are exposed. This vulnerable patient group needs individualizing therapy through careful selection of appropriate agents based on specific signaling pathways. The chemokine receptor CXCR4 mediates cell anchorage in the bone marrow microenvironment, is highly expressed in 25-30% of patients with AML and its expression is correlated with poor prognosis and drug resistance. The purpose of this study was to investigate a new humanized monoclonal IgG1 antibody to CXCR4 (PF-06747143) and its effects as a monotherapy in AML primary patient samples and in chemotherapy resistant patient-derived xenotransplantation (PDX) models. This antibody was previously shown to be able to induce cell death through its effector function (CDC and ADCC) and to be efficacious in cell-based xenograft models of AML, NHL, CLL and MM. Here we have shown that PF-06747143 binds strongly and specifically to AML cell lines and to AML primary cells, by flow cytometry. Of 16 samples evaluated, 7 displayed low CXCR4 expression (CXCR4neg/low) whereas 9 displayed high expression (CXCR4high). A good correlation was observed between 12G5, a commercially available CXCR4 Ab, and PF-06747143 staining, indicating that PF-06747143 can be used to stratify AML patients. Chemotaxis in response to CXCL12 was significantly inhibited in all AML patient primary samples analyzed. Administration of PF-06747143 to mice engrafted with AML patient cells (PDX models) induced rapid malignant cell mobilization into the peripheral blood at 4 hrs after a single antibody dose, with mobilized cell levels going back down to baseline at 24 hrs post-dose. This is in line with the ability of the antibody to block malignant cell homing to the bone marrow, inducing cell mobilization, as well as induction of cell death through effector function. To characterize the effects of PF-06747143 on leukemia progression, we used two different models: 1) P15 model characterized by high CXCR4 expression, inducing aggressive disease, with rapid progression of leukemia and widespread dissemination and chemoresistance; 2) P17 model characterized by a low CXCR4 expression, a less aggressive disease and limited dissemination. Weekly administration of PF-06747143 to leukemic mice previously engrafted with P17 or P15 malignant cells induced a sharp reduction of leukemia cells in the bone marrow, spleen and blood leading to increased survival of leukemic mice in both models. Activity of the antibody as monotherapy was superior to daunorubicin in the P15 chemoresistant model. Secondary transplantation of bone marrow cells from PF-06747143-treated or IgG1 control-treated animals showed that leukemic progenitors were also targeted by PF-06747143 treatment, with slower tumor growth in mice transplanted with PF-06747143-treated cells. In summary, PF-06747143, a CXCR4 IgG1 antibody, is significantly efficacious as a monotherapy and superior to daunorubicin in AML chemoresistant PDX models. These findings support evaluation of this antibody in AML therapy, with particular appeal to patients resistant to chemotherapy and to unfit patients, unable to tolerate intensive chemotherapy. Disclosures No relevant conflicts of interest to declare.

Description: CXCR4 is a chemokine receptor that belongs to the G-coupled protein receptor (GPCR) family. It is over-expressed in various cancers, including solid tumors and hematological malignancies, and correlates with poor prognosis. CXCR4 expressing cells actively respond to CXCL12 (SDF-1), a chemokine constitutively secreted by stromal cells in bone marrow. Activation of CXCR4 induces cell trafficking and homing to the marrow microenvironment, where CXCL12 retains these cells in close contact with marrow stromal cells that provide growth signals, promote self-renewal, and contribute to drug resistance, leading to poor prognosis and relapse. Here we describe the generation of a highly potent and selective anti-CXCR4 humanized IgG1 antagonist Ab (PF-06747143) that binds to human CXCR4 with high affinity and blocks SDF-1-induced Calcium flux and cAMP signaling. We have also characterized the ability of PF-06747143 to induce cell death through three different mechanisms: a) mobilization of cells from CXCL12-rich niches, making them more sensitive to chemotherapy b) direct cell-death through a mechanism dependent on the antibody’s bivalency; c) ADCC- and CDC-dependent cell death through the Fc-region in IgG1 backbone, when in the presence of effector cells or serum proteins. Weekly administration of PF-06747143 at 10 mg/kg, as a monotherapy, significantly improved survival, induced sustained regression and reduced bone marrow tumor burden in various patient population relevant murine disseminated tumor models of Acute Myeloid Leukemia (MV4-11, PDXs), Non Hodgkin Lymphoma (Raji and Ramos), Chronic Lymphocytic Leukemia (JVM-13) and Multiple Myeloma (OPM-2). The CXCR4 IgG1 antibody was also shown to be similar or more efficacious than approved standards of care agents currently employed for treatment of hematological malignancies. The safety and PK/PD profile of PF-06747143 were evaluated in a Non-Human Primate (NHP) exploratory toxicology study. Results from this study indicate that the CXCR4 IgG1 Ab was well tolerated in a two-week exploratory study at pharmacologically relevant doses. Upon treatment with PF-06747143, egression of white blood cells (WBC) from bone marrow (leukocytosis) was noted, which is consistent with target (CXCR4) modulation. Following the peak of leukocytosis between 1-6 hours post antibody administration, the number of circulating WBCs rapidly decreased back to baseline levels at 24 hrs. These results are likely explained by the direct cell killing through the effector function of this IgG1 CXCR4 antibody. Altogether, the promising preclinical efficacy and safety data support clinical evaluation of PF-06747143 in hematological malignacies. Disclosures Pernasetti: Pfizer: Employment. Liu:Pfizer: Employment. Hallin:Pfizer: Employment. Gu:Pfizer: Employment. Ho:Pfizer: Employment. Zhang:Pfizer: Employment. Pascual:Pfizer: Employment. Simmons:Pfizer: Employment. Yan:Pfizer: Employment. Huser:Pfizer: Employment. Wang:Pfizer: Employment. Lam:Pfizer: Employment. Spilker:Pfizer: Employment. Blasi:Pfizer: Employment. Tran:Pfizer: Employment. Kudaravalli:Pfizer: Employment. Ma:Pfizer: Employment. Chin:Pfizer: Employment. Shelton:Pfizer: Employment. Smeal:Pfizer: Employment. Fantin:Pfizer: Employment.

Description: Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults in the Western world. This leukemia is not curable and resistance to therapy is promoted by factors present in the tumor microenvironment including the chemokine CXCL12 (SDF-1), which interacts with its receptor CXCR4 and is thought to promote cancer cell survival. Here we explored the therapeutic potential of blocking CXCL12-CXCR4 interactions using PF-06747143, a humanized IgG1 antibody specific for CXCR4, which is expressed at high levels by CLL cells. Using primary leukemia cells from CLL patients, we found that PF-06747143 inhibited CXCL12-induced cell migration and blocked cytoskeletal changes via F-actin polymerization similar to AMD-3100 (Mozobil, a small molecule inhibitor of CXCR4). In addition, PF-06747143 induced apoptosis on CLL cells cultured alone or in the presence of human bone marrow-derived stromal cells (stroma-NK-tert). The pro-apoptotic activity of PF-06747143 was independent of high-risk prognostic factors including IGHV mutation status, ZAP-70 expression or TP53 mutation / 17p-deletion. Interestingly, AMD-3100, which binds and inhibits signaling through CXCR4, did not induce cell death in CLL or any of the cell lines tested. PF-06747143 did not induce apoptosis on normal B and T cells, and the ability of this anti-CXCR4 antibody to induce cell death on CLL cells appeared to be dependent on the crosslinking of CXCR4. This was supported by the fact that a Fab only fragment derived from PF-06747143 did not induce apoptosis despite of its high binding affinity for CXCR4. We observed that PF-06747143 induced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in CLL cells. However, this antibody did not induce caspase activation but rather its cell death activity appeared to be dependent on the production of reactive oxygen species (ROS) in leukemia cells. This effect was similar to that observed with other ROS dependent antibodies such as obinutuzumab (Gazyva). ROS induction was observed with PF-06747143, but not its Fab derived fragment and preceded apoptosis suggesting that this is critical component of its mechanism of action. We evaluated synergism of PF-06747143 with other CLL therapeutic agents and observed that this antibody synergized with fludarabine, bendamustine, ibrutinib and rituximab in the majority of CLL patient samples tested. In summary, our studies showed that PF-06747143, a CXCR4 IgG1 antibody is a potent inhibitor of the CXCR4-CXCL12 pathway and induces cell death primarily in CLL cells but not in normal lymphocytes. The cytotoxic effect of PF-06747143 was similar in CLL cells cultured alone or with stromal cells, suggesting that this antibody has the potential to overcome the protective effect of the tumor microenvironment. We also showed that PF-06747143 induced programmed cell death on CLL cells was dependent on ROS production and that this antibody synergized with agents currently used for the treatment of CLL patients. Overall, these findings highlight the biological relevance of the CXCR4-CXCL12 pathway in CLL, and provide rationale for clinical evaluation of PF-06747143 in CLL and other cancers. Disclosures Choi: Gilead: Consultancy, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Other: Advisory Board, Research Funding. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.