Potent Cytotoxicity of an Auristatin-Containing Antibody-Drug Conjugate Targeting Melanoma Cells Expressing Melanotransferrin/P97
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1474 Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97 Leia M. Smith, Albina Nesterova, Stephen C. Alley, with normal tissue, in conjunction with the greater Michael Y. Torgov, and Paul J. Carter sensitivity of tumor cells to L49-vcMMAF, supports further evaluation of antibody-drug conjugates for target- Seattle Genetics, Inc., Bothell, Washington ing p97-overexpressing tumors. [Mol Cancer Ther 2006;5(6):1474–82] Abstract Identifying factors that determine the sensitivity or Introduction f resistance of cancer cells to cytotoxicity by antibody-drug Malignant melanoma is responsible for 79% of deaths f conjugates is essential in the development of such from skin cancer ( 7,900 deaths estimated in the United conjugates for therapy. Here the monoclonal antibody States in 2006; ref. 1). Surgery is often curative for early L49 is used to target melanotransferrin, a glycosylphos- stage melanoma but is not a treatment option for disease phatidylinositol-anchored glycoprotein first identified that has metastasized to distant organs such as lung or as p97, a cell-surface marker in melanomas. L49 was brain. Available treatments are limited to use of immuno- a conjugated via a proteolytically cleavable valine-citrulline therapy ( -IFN or interleukin-2) with or without combina- linker to the antimitotic drug, monomethylauristatin F tion chemotherapy drugs to delay recurrence of disease. (vcMMAF). Effective drug release from L49-vcMMAF Chemotherapy and radiation therapy for stage IV melano- likely requires cellular proteases most commonly located ma patients are not curative but mostly used to relieve in endosomes and lysosomes. Melanoma cell lines with symptoms or extend the life of patients. The 5-year relative the highest surface p97 expression (80,000–280,000 survival rate of stage III melanoma patients is poor (45%) sites per cell) were sensitive to L49-vcMMAF whereas and significantly worse in stage IV disease (10%). Unques- most other cancer cell lines with lower p97 expression tionably, there is an unmet need for a more effective were resistant, as were normal cells with low copy treatment for malignant melanoma. numbers (V20,000 sites per cell). Cell line sensitivity to Melanotransferrin or p97 was first identified as a 97-kDa L49-vcMMAF was found by immunofluorescence micros- cell-surface marker for malignant melanoma cells (2) and copy to correlate with intracellular fate of the conjugate. has extensively been studied (3, 4). The expression of p97 Specifically, L49-vcMMAF colocalized with the lysosomal is selective for, but not absolutely tumor restricted (4), as marker CD107a within sensitive cell lines such as observed with many tumor-associated differentiation SK-MEL-5 and A2058. In contrast, in resistant cells antigens (5). The p97 protein is highly expressed in expressing lower p97 levels (H3677; 72,000 sites per neoplastic cells and fetal tissues, including umbilical cord, cell), L49-vcMMAF colocalized with caveolin-1, a protein intestine, and sweat gland ducts, and is also present in prominent in caveolae, but not with CD107a. Thus, for some normal tissues such as salivary glands, sweat antibody-drug conjugates targeting p97, antigen level and glands, brain endothelial cells, smooth muscle, and liver trafficking to the lysosomes are important factors for sinusoidal cells (3, 6–10). This antigen belongs to the achieving robust in vitro cytotoxicity against cancer cells. transferrin family of iron-binding proteins and shows 37% Immunohistochemical analysis with L49 revealed that to 39% sequence homology with human serum transferrin, 62% of metastatic melanoma tumors had strong staining lactoferrin, and ovotransferrin (11, 12). There are two for p97. Overexpression of p97 in melanoma as compared forms of the p97 antigen: membrane-bound and soluble secreted protein. The membrane-bound form, which is attached to the cell membrane by a glycosylphosphatidyl inositol anchor (7, 13), binds iron and is internalized into cells independent of transferrin/transferrin receptor endo- Received 1/17/06; revised 4/17/06; accepted 4/27/06. cytosis. Recently, membrane-bound p97 has been reported The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked to be a potential cell-surface plasminogen receptor and may advertisement in accordance with 18 U.S.C. Section 1734 solely to play a key role in activation of cell invasion (14). The soluble indicate this fact. p97 protein is found at very low levels (3.2 ng/mL) in Note: The current address for M.Y. Torgov is CuraGen Corporation, the plasma of normal individuals and is actively secreted 15 Commercial Street, Branford, CT 06405. into the blood and cerebrospinal fluid in patients Requests for reprints: Leia M. Smith, Seattle Genetics, Inc., 21823 30th Drive Southeast, Bothell, WA 90021. Phone: 425-527-4654; with Alzheimer’s disease (median 15 ng/mL; ref. 15). Fax: 425-527-4609. E-mail: [email protected] Soluble p97 does not effectively donate iron to the brain Copyright C 2006 American Association for Cancer Research. and erythropoeitic tissue (16) and its function remains doi:10.1158/1535-7163.MCT-06-0026 unknown. Mol Cancer Ther 2006;5(6). June 2006 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Molecular Cancer Therapeutics 1475 L49 is a murine immunoglobulin G1(IgG1)monoclonal Biological Materials, Braunschweig, Germany). H3677 cells antibody (mAb) against human p97, which was derived were from Dr. K.E. Hellstro¨m (University of Washington, from BALB/c mice immunized with lung carcinoma Seattle, WA) and A375-M and A375-P cell lines were from (H2981and CH3) and melanoma (W56) cell lines (17). Dr. I. J. Fidler (M.D. Anderson Cancer Center, Houston, j Earlier studies showed that p97 is stably expressed on the TX). Cell lines were cultured at 37 C with 5% CO2 in cell surface and, on binding of mAb, does not detectably appropriate media. Melanoma cell lines were grown in internalize (17, 18). In one of these studies, a single-chain Fv RPMI 1640 with 10% fetal bovine serum supplemented fragment fused to h-lactamase was used for antibody- with 2 mmol/L L-glutamine. Normal human endothelial dependent enzyme-mediated prodrug therapy (17). More cells, smooth muscle cells, and hepatocytes were obtained recently, another mAb to p97, hup97, was used as a non- from Cambrex (Walkersville, MD) and grown in media internalizing control in combination with a cross-linking recommended by the vendor. secondary antibody conjugated to a highly potent anti- Antibodies tubulin drug, monomethyl auristatin E (MMAE; ref. 19). Hybridoma cell lines producing the two murine anti-p97 The anti-p97 antibody plus cross-linking antibody-drug IgG1mAbs, L235 (American Type Culture Collection) and conjugate showed significant cytotoxicity only at 100- to L49 (17), were grown as recommended and media collected 1,000-fold higher concentration than for antibodies to other for antibody purification. Antibodies were purified with antigens known to be efficiently internalized. As for MabSelect Protein A column (Amersham, Piscataway, NJ). previous studies, it was concluded that the p97 antigen is cAC10, a chimeric mAb against CD30 (25), and MOPC21, a not readily internalized by antibodies (19). mouse IgG1(Sigma, St. Louis, MO), were used as negative In this study, L49 was directly conjugated to the controls in the cytotoxicity assays. auristatin drugs MMAE (20), monomethylauristatin F Immunohistochemistry (MMAF; ref. 21), and auristatin F phenylenediamine Frozen tissues on slides and frozen melanoma tissue (AFP; ref. 22), using linkers that contain or lack a microarrays (TriStar, Rockville, MD) were air-dried then protease-cleavable dipeptide, valine-citrulline (20, 21). As fixed in 4% paraformaldehyde for 15 minutes at room previously reported, internalization of unconjugated L49 temperature. Endogenous peroxidase activity was blocked was not observed. However, in contrast to earlier studies, with 0.6% H2O2 for 15 minutes whereas endogenous L49-drug conjugates showed efficient internalization in biotin was blocked with an Avidin-Biotin Blocking kit cell lines that express high levels of p97. One of the goals (Vector Laboratories, Burlingame, CA). For primary anti- of this study was to understand the mechanism of drug bodies, mAbs against p97 (L49-biotinylated and L235) and delivery via antibody-drug conjugates to p97, a glycosyl- control mouse IgG were used at 2 Ag/mL for 1-hour phosphatidyl inositol–linked cell-surface target. Previous incubation at room temperature. Biotinylated secondary studies have shown that most glycosylphosphatidyl antimouse antibody was used for L235 (VectaStain Elite inositol–linked proteins are associated with caveolin-1 Kit, Vector Laboratories), followed by avidin conjugated and internalized via the caveolae pathway (23, 24). to horseradish peroxidase. 3,3¶-Diaminobenzidine was Another goal was to determine the factors necessary for used as substrate for horseradish peroxidase. Tissues selective and efficient killing of malignant melanoma cells. were counterstained with hematoxylin and slides were Briefly, tumor cell lines and normal cells were analyzed dehydrated and coverslipped. Two separate stainings for p97 antigen expression by quantitative fluorescence- were done for each mAb and slides were then scored activated cell sorting (FACS) and then compared for their manually using