Cancer Therapy : Preclinical

Purpose: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need.We sought to identify melanoma-associated cell surface molecules and to develop aswell as preclinically test immunotherapeutic reagents designed to exploit such targets. Experimental Design and Results: By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma.The CR011monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011alone didnot inhibit the growthofmelanoma cells. However,when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positivemelanoma cells in vitro. Ectopic overexpression and small interfering RNAtransfection studies showed that GPNMBexpression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as1.25 mg/kg. Conclusion:These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma. Melanoma is a common neoplasm and its incidence is increasing worldwide at a dramatic rate (1). Melanoma accounts for only 4% of skin cancer cases yet causes f79% of all skin cancer deaths. In 2004, an estimated 55,100 Americans were diagnosed with melanoma and f7,910 would die of the disease (2). An increasing frequency of newly diagnosed melanomas, ranging from 3% to 8% annually, has also been observed worldwide (3, 4). Therapeutic options for patients with late-stage melanoma presenting with regional and/or distant metastases are limited. Dacarbazine is the only cytotoxic drug currently approved by the Food and Drug Administration for the treatment of stage IV metastatic melanoma, with a response rate of <15% and a median response duration of 4 to 5 months (5). The majority of polychemotherapy regimens failed to show significant survival benefits (6), nor did the use of adjuvant therapeutic agents such as IFN-a and interleukin 2, which pose severe toxicity (7, 8). The poor efficacy and adverse side effects of available therapies has led to a considerable interest in the development of alternative therapies, such as monoclonal antibodies (mAb), for the treatment of metastatic melanoma (9). Recent advances in genetic engineering have significantly decreased antibody immunogenicity and increased antibody half-life (10, 11). Antibody-based therapeutics, such as Rituxan, Herceptin, and Avastin, have recently enjoyed clinical success in the treatment of some hematopoietic malignancies and solid tumors. Although antibodies that target tumor or its vasculature may be useful in an unconjugated form, it is sometimes advantageous to couple a tumor-targeting antibody to an isotope (e.g., Zevalin and Bexxar) or to a cytotoxic compound (e.g., Mylotarg). This strategy allows for the selective delivery of cytotoxic agents to the tumor with the goal of reducing the toxicity that is often associated with the systemic administration of cytotoxic agents while preserving or enhancing the antitumor activity of these agents. Our genome-wide transcript expression profiling, coupled with a systems biology analysis of human melanoma clinical Cancer Therapy: Preclinical Authors’ Affiliations: CuraGen, Branford, Connecticut; Abgenix, Fremont, California; and Seattle Genetics, Bothell,Washington Received 9/16/05; revised12/9/05; accepted12/14/05. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: K.F.Tse andM. Jeffers contributed equally to this work. Requests for reprints:William J. LaRochelle, CuraGen Corporation, 322 East Main Street, Branford, CT 06405. Phone: 203-871-4288; Fax: 203-315-3301; E-mail: wlarochelle@curagen.com. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-2018 www.aacrjournals.org Clin Cancer Res 2006;12(4) February15, 2006 1373 Research. on May 29, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from specimens and cell lines, led to the identification of a tumorassociated protein, called glycoprotein NMB (GPNMB), as a potential target that can be exploited for the treatment of melanoma. GPNMB is predicted to be a 560-amino-acid type I transmembrane protein with closest homology (26% amino acid identity) to the melanocyte/melanoma–specific protein, pMEL17 (12). The normal function of human GPNMB is unknown, and orthologues have been isolated from mouse (DC-HIL; ref. 13), rat (Osteoactivin; ref. 14), and quail (QNR-71; ref. 15). Previous investigations have associated GPNMB expression and function with cancer. GPNMB was first identified as a gene that was differentially expressed among melanoma cell lines with high and low metastatic potential (12) and was subsequently identified as a candidate glioma tumor marker due to its high transcript expression in this tumor type and restricted normal tissue distribution (16). GPNMB expression has also been described in liver cancer, squamous cell lung carcinoma, and soft tissue tumors (17–19). Moreover, ectopic expression of GPNMB in cancer cells increased their in vitro invasiveness and promoted their metastasis in vivo (17, 20). Finally, GPNMB was shown to interact with the surface of endothelial cells (13), a finding that may have implications for GPNMB-expressing melanoma cell transendothelial migration and metastasis. To explore the potential utility of GPNMB as a target for melanoma therapy, fully-human mAbs were generated to this protein. The lead mAb, CR011, was characterized and coupled to the dolastatin-10-related cytotoxic drug monomethylauristatin E (MMAE), a potent inhibitor of mitotic spindle formation (21). The resulting antibody-drug conjugate, designated CR011-vcMMAE, was evaluated for growth-inhibitory activity on melanoma cell lines in vitro and for activity against melanoma xenografts in vivo . The results presented in this study suggest that GPNMB represents a promising target for the identification and treatment of advanced melanoma and that CR011-vcMMAE is worthy of continued therapeutic evaluation. Materials andMethods Cell lines and transfections. M14, UACC-257, and LOXIMVI cell lines were obtained from the National Cancer Institute (Bethesda, MD) and all others from the American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM or RPMI containing 10% fetal bovine serum and penicillin-streptomycin. To establish stable cell lines overexpressing GPNMB, HEK293 cells were transfected with either control vector (pcDNA3.1-V5-His) or this vector containing full-length GPNMB, using LipofectAMINE (Invitrogen, Carlsbad, CA) according to the protocol of the manufacturer. Following selection in medium containing G418 (0.8 mg/mL), individual clones were selected and propagated. Small interfering RNA (siRNA) was used to inhibit GPNMB expression in SK-Mel-2 cells. Cells were transfected with 50 nmol/L of siGENOME SMART pool reagents (Dharmacon, Inc., Chicago, IL), designed to specifically target GPNMB, or siRNA to thymidylate synthase as a negative control, using the OligofectAMINE transfection reagent (Invitrogen) following the instructions of the manufacturer. Reverse transcription-PCR and real-time quantitative PCR. Total RNA was isolated using the RNeasy kit with a DNase digestion step (Qiagen, Inc., Valencia CA). Reverse transcription-PCR (RT-PCR) was done using the OneStep RT-PCR kit (Qiagen) as follows. Reverse transcription: 50jC for 45 minutes and 95jC for 15 minutes for one cycle. PCR: 1 minute at 95jC, 1 minute at 50jC, and 2 minutes at 72jC for 30 cycles with final extension for 10 minutes at 72jC. Products were separated on a 2% agarose/0.33% low melting point agarose gel and visualized by ethidium bromide staining. The integrity of each RNA sample was verified via RT-PCR with primers designed to amplify glyceraldehyde-3-phosphate dehydrogenase. The primers used for amplification are as follows (5V-3V): Real-time quantitative PCR analysis was done with an ABI Prism 7700 Sequence Detection System using TaqMan reagents (PE Applied Biosystems, Foster City, CA). Equal quantities of normalized RNAs were used as a template in PCR reactions for 40 cycles with GPNMB-specific primers to obtain threshold cycle (CT) values. The primers used for amplification are as follows (5V-3V): Forward-TCAATGGAACCTTCAGCCTTA Reverse-GAAGGGGTGGGTTTTGAAG Probe-TET-CTCACTGTGAAAGCTGCAGCACCAG-TAMRA Production and purification of recombinant human GPNMB extracellular domain protein. Oligonucleotide primers were designed to amplify the cDNA encoding the GPNMB extracellular domain (GPNMB-ECD) using a human fetal brain cDNA template. The forward primer included an in-frame BamHI site and the reverse primer contained an in-frame SalI restriction site. The primers used for amplification are as follows (5V-3V): Forward-GGATCCAAACGATTTCATGATGTGCTGGGCAATGAA Reverse-GTCGACCGAGGCTGGGTCTCTGTCAGGAACAGAAAT The PCR product was cloned into the pCR2.1-Topo vector (Invitrogen). The cDNA insert was verified by sequencing and subcloned into the BamHI/XhoI sites of pCEP4 (Invitrogen), which was modified by inserting the murine Ign secretion signal upstream, and a V5-His tag downs