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[CANCER RESEARCH 64, 4629–4636, July 1, 2004] -In Vitro and in Vivo Activity of the Maytansinoid Immunoconjugate huN901-N2 Deacetyl-N2-(3-Mercapto-1-Oxopropyl)-Maytansine against CD56؉ Multiple Myeloma Cells Pierfrancesco Tassone,1,2,3 Antonella Gozzini,1 Victor Goldmacher,4 Masood A. Shammas,2 Kathleen R. Whiteman,4 Daniel R. Carrasco,1 Cheng Li,1 Charles K. Allam,2 Salvatore Venuta,3 Kenneth C. Anderson,1 and Nikhil C. Munshi1,2 1Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, and 2VA Boston Healthcare System, Harvard Medical School, Boston, Massachusetts; 3University of “Magna Græcia”, Catanzaro, Italy; and 4ImmunoGen, Inc., Cambridge, Massachusetts ABSTRACT is now a resurgence of interest due to approval of gemtuzumab ozogamicin (Mylotarg; Ref. 11), a humanized anti-CD33 mAb linked HuN901 is a humanized monoclonal antibody that binds with high to a semisynthetic, highly cytotoxic calicheamicin ␥ derivative. affinity to CD56, the neuronal cell adhesion molecule. HuN901 conjugated 1 with the maytansinoid N2-deacetyl-N2-(3-mercapto-1-oxopropyl)-maytan- Gemtuzumab ozogamicin has demonstrated potent antitumor activity sine (DM1), a potent antimicrotubular cytotoxic agent, may provide tar- in tumor xenograft models of myeloid leukemia in athymic mice (12) ϩ geted delivery of the drug to CD56 expressing tumors. Based on gene and in patients with CD33 myeloid leukemia (13). Maytansine expression profiles of primary multiple myeloma (MM) cells showing derivatives represent a new class of highly cytotoxic agents suitable expression of CD56 in 10 out of 15 patients (66.6%) and flow cytometric for conjugation with mAbs. Maytansine (14) is a natural product, bright lo profiles of MM (CD38 CD45 ) cells showing CD56 expression in 22 originally derived from the Ethiopian shrub Maytenus serrata, which out of 28 patients (79%), we assessed the efficacy of huN901-DM1 for the inhibits tubulin polymerization, thereby resulting in mitotic block and treatment of MM. We first examined the in vitro cytotoxicity and speci- ficity of huN901-DM1 on a panel of CD56؉ and CD56؊ MM cell lines, as cell death. The activity of maytansine is approximately 200–1,000- -well as a CD56؊ Waldenstrom’s macroglobulinemia cell line. HuN901- fold greater than that of the Vinca alkaloids, which exert their cyto DM1 treatment selectively decreased survival of CD56؉ MM cell lines and toxic potential by a similar mechanism. Maytansine has shown some depleted CD56؉ MM cells from mixed cultures with a CD56؊ cell line or activity in clinical studies (15–18), but its narrow therapeutic window adherent bone marrow stromal cells. In vivo antitumor activity of huN901- precluded additional clinical development. A recently developed ؉ DM1 was then studied in a tumor xenograft model using a CD56 OPM2 highly cytotoxic maytansine derivative N2Ј-deacetyl-N2Ј-(3-mercapto-1- human MM cell line in SCID mice. We observed inhibition of serum oxopropyl)-maytansine (DM1; Ref. 19) has been linked to the mAb paraprotein secretion, inhibition of tumor growth, and increase in sur- huC242. This antibody targets a glycotope (CanAg) on MUC1 that is vival of mice treated with huN901-DM1. Our data therefore demonstrate that huN901-DM1 has significant in vitro and in vivo antimyeloma activity expressed on human colorectal and pancreatic tumors (20). After at doses that are well tolerated in a murine model. Taken together, these preclinical evaluation (21), a Phase I study of huC242-DM1 in chemo- data provide the framework for clinical trials of this agent to improve therapy-refractory patients confirmed biological activity in the ab- patient outcome in MM. sence of severe toxic effects (22). Based on these encouraging results, a maytansinoid immunoconjugate with the humanized anti-CD56 INTRODUCTION mAb, huN901, has been generated for the treatment of CD56-express- ing tumors. Recent advances in monoclonal antibody (mAb) technology includ- CD56 antigen, identified as neuronal cell adhesion molecule ing humanization and mAb engineering have facilitated the use of (NCAM; Ref. 23), is a membrane glycoprotein belonging to the mAbs to treat human cancers with enhanced antitumor activity (1, 2). immunoglobulin superfamily. In the hematopoietic compartment, These mAbs selectively target tumor tissues (3–5) and have been CD56 expression is restricted to NK cells and a subset of T lympho- safely administered in cancer patients. The recent approvals of mAbs cytes (24, 25) that specifically express the M 140,000 isoform of for clinical use (1, 2) have renewed interest in mAb-based therapies. r NCAM glycoprotein (26). The expression of CD56 has also been The direct linking of mAbs with drugs, toxins, or radionuclides to detected on a variety of cancer cells of hematopoietic and neuroen- specifically target cancer cells has been widely studied (6). However, docrine origin, including multiple myeloma (MM), leukemia, neuro- the use of mAbs to deliver therapeutic doses of conventional cytotoxic blastoma, and small cell lung carcinoma (SCLC; Refs. 27 and 28). drugs, including doxorubicin, methotrexate, and Vinca alcaloids, has led to only limited benefit (7–9). mAbs conjugated to protein toxins, Although normal plasma cells (PCs) do not express CD56 (29), it is such as ricin or Pseudomonas toxin, are highly active in vitro and are expressed by a subset of PCs in patients with monoclonal gammapa- selective, unlike corresponding unconjugated toxins (6, 10). However, thy of undetermined significance and strongly expressed on MM cells in vivo these immunotoxins have significant side effects and retain (29, 30) from a majority of MM (31, 32). In contrast, CD56 is absent their high immunogenicity. Despite initial disappointing results, there on malignant PCs from patients with PC leukemia and/or extramed- ullary plasmacytoma (33–36). Received 1/15/04; revised 3/5/04; accepted 4/26/04. The restricted expression profile of CD56 in the normal hematopoietic Grant support: Multiple Myeloma Research Foundation Awards (M. Shammas, N. compartment, coupled with its expression on malignant PCs, provided the Munshi, and K. Anderson); VA merit review, and the Leukemia and Lymphoma Society rationale for evaluating CD56 as a potential target for mAb-based therapy Scholar in Translational Research Award (N. Munshi); NIH Grants RO1-50947, P50- 100707, and PO1-78378 (N. Munshi and K. Anderson); the Doris Duke Distinguished in MM. Herein, we report selective in vitro cytotoxicity of huN901-DM1 Clinical Research Scientist Award (K. Anderson). against a panel of CD56ϩ MM cell lines, MM cells adherent to bone 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 with marrow stroma cells (BMSCs), and patient MM cells. We also demon- 18 U.S.C. Section 1734 solely to indicate this fact. strate in vivo antitumor activity of huN901-DM1 in a tumor xenograft Requests for reprints: Nikhil C. Munshi, Dana-Farber Cancer Institute, 44 Binney ϩ Street (D1B25), Boston, MA 02115. Phone: (617) 632-5607; Fax: (617) 582-7904; E- model with human CD56 MM cells in SCID mice, providing a rationale mail: [email protected]. for its evaluation in MM patients. 4629 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. ANTIMYELOMA ACTIVITY OF huN901-DM1 Fig. 1. Expression of CD56 in MM. A, CD56 gene expression profile in CD138ϩ purified (Ͼ 95%) normal (three samples) and malignant PCs (15 MM samples); B, percentage of patients expressing CD56ϩ MM cells, as determined by flow cytometry on fresh BM aspirate samples; C, percentage (the mean value) of CD56ϩ cells in fresh BM aspirates taken from 22 CD56ϩ patients as determined by flow cytometry; D, MFI of staining of CD56ϩ MM cells within CD38brightCD45lo population (a mean value for 22 patients); E, flow cytometric profiles of CD56 expression on primary MM cells from patients pt1, pt2, and pt3. CD38brightCD45lo population was assessed for CD56 or CD138 expression within the gate. MATERIALS AND METHODS by both flow cytometry (Becton-Dickinson FACSort) monitoring the expres- sion of CD38, CD45, and forward and side scatter characteristics of PC or Preparation of mAb-DM1 Conjugate. The thiol-containing maytansinoid morphological examination. DM1 was synthesized from the microbial fermentation product ansamitocin Total RNA was isolated from 5 ϫ 106 cells using RNeasy kit (Qiagen). Total P-3 as described previously (19). Humanized antibodies N901 (huN901) and RNA (10–15 g) was reverse-transcribed to get cDNA using Superscript II RT kit C242 (huC242) have been described previously (37). Antibody-drug conju- (Invitrogen). cDNA was used in an in vitro transcription reaction to synthesize gates were prepared as described elsewhere (21). An average of about 3.5 DM1 biotin-labeled cRNA using Enzo RNA labeling kit (Enzo Diagnostics). Labeled molecules were linked per antibody molecule. cRNA was purified with the RNeasy Mini-kit (Qiagen) and quantitated. Purified Cell Lines and Patient Cells. U266 MM cell line was obtained from the cRNA (15 g) was hybridized to human genome U133 GeneChip arrays (Af- American Type Culture Collection (Rockville, MD). OPM1 and OPM2 cell fymetrix) representing approximately 33,000 human genes. GeneChip arrays were lines were kindly provided by Dr. Edward I. B. Thompson (University of scanned on a GeneArray Scanner (Affymetrix). Normalization of arrays and Texas Medical Branch, Galveston, TX); LP1 MM cell line, WSU-Walden- calculation of expression values were performed using the DNA-Chip Analyzer strom’s macroglobulinemia (WM) cell line, and SUDHL4 lymphoma cell line (dChip) program. Arrays were normalized based on relative signal produced for an were kindly provided by Dr. P. Leif Bergsagel (Weill Medical College of Cornell University, New York, NY), Dr. Ayad Al-Katib (Wayne State Uni- invariant subset of genes. This model-based method was used for probe selection versity, Detroit, MI), and Dr. Margaret Shipp (Dana-Farber Cancer Institute, and computing expression values. Boston, MA), respectively. Cell lines were cultured in RPMI 1640 (Life Colorimetric Survival Assay.