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Targeting CD56 by the maytansinoid immunoconjugate IMGN901 (huN901‐DM1): a potential therapeutic modality implication against natural killer/T cell malignancy

2008; Wiley; Volume: 141; Issue: 1 Linguagem: Inglês

10.1111/j.1365-2141.2008.07000.x

1365-2141

Kenji Ishitsuka, Shiro Jimi, Victor S. Goldmacher, Olga Ab, Kazuo Tamura,

Immune Cell Function and Interaction

Natural killer (NK)/T cell malignancy, a rare disease showing geographic predilection in Asian countries, is subclassified into blastic NK cell lymphoma, aggressive NK cell leukaemia, and extranodal NK/T cell lymphoma in the World Health Organization classification (Oshimi et al, 2005). NK/T cell malignancies are derived from bi-potential T/NK cells or committed NK progenitor cells that are positive for cytoplasmic CD3ε and surface CD56 antigens, but negative for surface CD3 antigen. The tumour is closely associated with Eptein–Barr virus infection. The clinical course of NK/T cell malignancies is fulminant with disseminated organ involvement. Conventional chemotherapy accompanied with radiation therapy for limited disease and systemic chemotherapy for extended disease, are currently used as therapeutic modalities. However, the prognosis has been extremely poor, and no targeted therapeutic options are currently available (Oshimi et al, 2005; Lee et al, 2006). IMGN901 (huN901-DM1; ImmunoGen, Cambridge, MA, USA) is an immunoconjugate composed of the cytotoxic derivative of N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), conjugated to the antibody, huN901, which binds CD56 with high affinity. IMGN901 is a tumour-activated prodrug because the conjugation of DM1 to huN901 renders the cytotoxic drug inactive until it reaches the target site. The conjugate is then internalized and releases DM1, which inhibits tubulin polymerization and causes cell death. Maytansine is a natural product, originally derived from the Ethiopian shrub Maytenus serrata (Kupchan et al, 1972). The activity of maytansine is approximately 200 to 1000-fold greater than that of the vinca alkaloids, which act through a similar mechanism(Tassone et al, 2004; Wang et al, 2005). IMGN901 has been demonstrated to be safe and show promising efficacy in small cell lung cancer, CD56-positive small cell carcinoma and multiple myeloma in phase I/II clinical studies (Chanan-Khan et al, 2006; McCann et al, 2007). The present study demonstrated the cytotoxicity of IMGN901 against a CD56-positive NK cell line and fresh tumour cells derived from a patient with NK cell malignancy in vitro. NK-92MI, a CD56-positive NK cell line derived from a patient with malignant lymphoma presenting with typical features of NK/T cell malignancy (Gong et al, 1994), and CD56 negative cell lines RAJI, MOLT3, HL60 and K562, were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). These cell lines were cultured in media recommended by ATCC. Fresh peripheral blood mononuclear cells (PBMNCs) obtained from a patient with extranodal NK/T cell lymphoma after informed consent were separated from heparinized peripheral blood by Ficoll–Hipaque density sedimentation. IMGN901 and huN901-antibody (Immunogen) were supplied in phosphate-buffered saline at 1 mg/ml and 2·64 mg/ml stock solution, respectively. First, we confirmed the expression of CD56 on the cell surface of NK-92MI cell line. NK-92MI, RAJI, MOLT3, HL60 and K562 cell lines were cultured with either anti-CD56 antibody (BD Biosciences, San Diego, CA, USA) or huN901 for 30 min on ice and washed, then analyzed by EPICS XL flow cytometer (Beckman Coulter, Hialeah, FL, USA). huN901 showed selective affinity to the CD56-positive NK cell line, NK-92MI, but not to CD56-negative cell lines, RAJI, MOLP3, HL60 and K562 (data not shown). Next, we evaluated the cytotoxic effects of IMGN901 on the NK cell line. NK-92MI cells were pre-incubated with either huN901 or culture media for 5 h, and then treated with IMGN901 for 6 days. Cellular growth was determined by colorimetric assay using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). IMGN901 significantly inhibited growth of the cells and, more importantly, pre-treatment with huN901 blocked the cytotoxic effects at a concentration of less than 4 μg/ml IMGN901 (Fig 1A). These results suggest that IMGN901 specifically binds to CD56 on the NK-92MI cells at a concentration of less than 4 μg/ml. (A) NK-92MI cells were pre-incubated with either of huN901 or culture media for 5 h, and then treated with IMGN901 for 6 days. Cellular growth was determined by colorimetric assay. (B) Fresh PBMNCs derived from a patient with NK/T cell lymphoma were treated with IMGN901 for 6 days. Tumour cells express both CD56 and CD34, thereby the percentage of CD34 positive cells after treatment was determined by flow cytometric analysis to ascertain the toxic effect of IMGN901 against tumour cells. Finally, fresh PBMNCs derived from a patient with heavily treated, refractory and leukemic phase-extranodal NK/T cell lymphoma were exposed to IMGN901 for 6 days. After treatment, cells were harvested and incubated with fluorescein isothiocyanate-conjugated anti-CD34 antibody (BD Biosciences) for 30 min, followed by analysis using an EPICS XL flow cytometer. As the patient tumour cells particularly co-expressed CD56 and CD34 on their surface, a reduction in the percentage of CD34 positive cells was observed when the tumour cells were killed by IMGN901. This procedure enables the evaluation of the specific toxicity of IMGN901 on CD56-positive tumour cells co-expressing CD34. Indeed, IMGN901 reduced the percentage of CD34 positive tumour cells (Fig 1B). In conclusion, this study has shown the potential of IMGN901 to suppress the growth and viability of a NK cell line and fresh tumour cells derived from a patient with NK/T cell lymphoma. These results suggest that IMGN901 represents a promising novel targeted approach to improve patient outcome in NK cell malignancies. The activity of IMGN901 should be further validated in clinical trials. This study was funded by the Central Research Institute of Fukuoka University, #076003 (KI). Victor S. Goldmacher Ph.D. and Olga Ab Ph.D. are employees of ImmunoGen Inc.