Our data demonstrate that milatuzumab mediates direct cytotoxicity in CLL cells by a mechanism involving aggregation of CD74 on the cell surface. formulation for this mAb. Based on these data, future development of the milatuzumab-immunoliposome formulation as a therapeutic Salermide agent for CLL is warranted. Introduction Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, and is a progressive and incurable disease. CLL treatments include alkylating drugs, purine analogs, and more recently, monoclonal antibodies (mAbs). mAbs such as rituximab that target the CD20 antigen selectively expressed on CLL cells augment the cytotoxicity of traditional chemotherapy agents, and are associated with improved response and progression-free survival.1C4 However, nearly all patients eventually relapse after such treatments, indicating a need for novel and specific therapeutic agents. CD74 is a type II transmembrane protein expressed on B cells that has recently been pursued as a target for antibody-mediated therapy.5 It associates with the and chains of HLA-DR, and normally functions as a major histocompatibility complex class II chaperone. Signaling through CD74 is also implicated in B-cell proliferation, nuclear factor B activation, and cell survival.6,7 CD74 expression is increased on the surface of leukemic B cells, making it an attractive target for CLL and other B-cell malignancies. CD74 signaling is initiated after engagement with macrophage migration-inhibitory factor (MIF) and subsequent activation of survival pathways to inhibit apoptosis and stimulate proliferation.8,9 In addition, a recent study demonstrates that CD74 signaling induces TAp63 and VLA-4 to enhance CLL cell survival and homing to the bone marrow.10 Therefore, disruption of CD74 signaling represents a potential therapeutic option in CLL and other CD74-expressing malignancies.5 Here we describe an antagonistic humanized mAb to CD74, milatuzumab. Milatuzumab has demonstrated antiproliferative activity in non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) cell lines and extends the survival of severe combined immune-deficient (SCID) mice injected with NHL and MM cells.5,7,11 However, little is known about the efficacy of milatuzumab in CLL. Our data demonstrate that milatuzumab mediates direct cytotoxicity in CLL cells by a mechanism involving aggregation of CD74 on the cell surface. Furthermore, incorporation of milatuzumab into a liposome potentiates the cytotoxic effect of Salermide this antibody, suggesting a novel therapeutic formulation. Methods Patients, cell separation, culture conditions, and reagents For in vitro studies, written, informed consent was obtained in accordance with the Declaration of Helsinki to procure cells from patients with previously diagnosed CLL, as defined by the modified National Cancer Institute criteria, under an Institutional Review BoardCapproved protocol at The Ohio State University.12 Patient characteristics are available in supplemental Table 1 (available on the Web site; see the Supplemental Materials link at the top of the online article). Isolated mononuclear cells were negatively B-cell selected and placed in culture, as previously described by our group.13 HS-5 stromal cells were obtained Salermide from ATCC. CD40L was purchased from PeproTech. Milatuzumab was provided by Immunomedics Inc. Goat antiChuman IgG antibody (Fc gamma fragment-specific, anti-Fc) was purchased Rabbit polyclonal to PDK4 from Jackson ImmunoResearch Laboratories. Q-VD-OPH pan-caspase inhibitor was purchased from MP Biomedicals. Flow cytometric assays Viability was determined by flow cytometry using propidium iodide (PI). For surface staining, CLL cells were washed in phosphate-buffered saline and stained with antibodies to CD20 or CD74 (BD Biosciences). Immunoblot analysis Immunoblots were performed as described.14 Antibodies used included PARP (Calbiochem); caspase 3 and 9 (R&D Systems), caspase 2, 6 and 8 (Cell Signaling), and tubulin (Santa Cruz Biotechnology). Preparation of ILs Immunoliposomes (ILs) were prepared as previously described.15 A postinsertion method was used to incorporate milatuzumab into preformed liposomes, and targeted milatuzumab-IL was prepared with an antibody-to-lipid ratio of 1 1:1000. Further details are available in supplemental Methods. Statistical analysis All Salermide reported statistical evaluations were performed by the Center for Biostatistics at The Ohio State University. Because the observations from the same patient are correlated, linear mixed models were used for analysis to take account of this within patient correlation. Treatment differences were estimated and tested from these models. The Holm step-down procedure was used to adjust for multiple comparisons or multiple.
Our data demonstrate that milatuzumab mediates direct cytotoxicity in CLL cells by a mechanism involving aggregation of CD74 on the cell surface
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