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Regression of cutaneous metastatic melanoma by intralesional injection with human monoclonal antibody to ganglioside GD 2 ( immunotherapy )

In this study we used human monoclonal antibody (Hu-mAb) L72 as an intratumoral injection of cutaneous metastasis of melanoma to study its anti-tumor effects in human patients. Hu-mAb L72 was developed by transforming peripheral blood lymphocytes from a melanoma patient in vitro with the Epstein-Barr virus, forming a human lymphoblastoid cell line that produces 2-5 gig ofIgM per ml. This IgM Hu-mAb was shown to react specifically with ganglioside GD2 and have a strong cytotoxic effect on human melanoma cells in the presence of complement. Patients with cutaneous metastatic melanoma were given intralesional injections on a daily or weekly injection schedule. Regression was seen in all tumors except in those of two patients whose tumors were shown to have low antigenicity. Histopathological data showed tumor degeneration, fibrosis, free melanin, and some degree of lymphocyte or macrophage infiltration. One patient with melanoma satellitosis treated with Hu-mAb showed complete regression with no sign of recurrence 20 months after the initial treatment. With the exception of mild erythema, no side effects were observed in any patient. Immunotherapy has been a fast-evolving area of cancer research since the development of the first murine monoclonal antibody. These highly specific tools have permitted specific lysis of malignant cells without the destruction of normal cells. However, the clinical application of the murine monoclonal antibody has limitations. Patients inoculated with mouse monoclonal antibody may develop anti-mouse Ig antibodies and side effects, including hypotension, dyspnea, fever, nausea, chills, diarrhea, bronchospasm, tachycardia, pruritus and urticaria, have been observed. Although some patients, particularly those with advanced disease, have not developed antibodies to murine monoclonal antibodies, these side effects have made prolonged treatment with this antibody difficult. Considering these limitations of murine monoclonal antibodies it is only a natural progression to develop anti-tumor monoclonal antibodies of human origin and investigate these antibodies in passive immunotherapy. We have successfully developed a human monoclonal antibody (Hu-mAb) (L72) of the IgM class that specifically reacts with ganglioside GD2 (1, 2). This antibody is secreted by human lymphoblastoid cells that were derived from peripheral blood B lymphocytes (PBL). The PBL were transformed in vitro by Epstein-Barr virus (EBV) and produce 2-5 ,ug/ofIgM per ml. The antibody has a strong cytotoxic effect on human melanoma cells in the presence of complement (3). Its in vivo anti-tumor effects were demonstrated by using the athymic nude mouse model (4, 5). In this study Hu-mAb L72 was tested as an intralesional injection into cutaneous nodules of recurrent malignant melanoma to study its anti-tumor effect in patients. Eight patients were so tested. Cutaneous melanoma was chosen because these tumors are easily measurable and 70% of melanoma patients have tumors expressing GD2 antigens (6). For patients with multiple tumors of similar size and depth, control injections were also given consisting of proteinadjusted human serum albumin or fresh autologous serum. Remarkable regression was observed in some Hu-mAbtreated tumors, without noticeable side effects. To the best of our knowledge, the clinical application of Hu-mAb for the treatment of cancer in humans has not been reported previously. MATERIALS AND METHODS Hu-mAb to GD2. The human IgM monoclonal antibody producing cell line L72 had been cultured in RPMI 1640 medium containing 2-3% fetal bovine serum and antibiotics (streptomycin, penicillin, and Fungizone). Cell numbers were adjusted to 2 x 105 per ml every 3-5 days upon collection of spent medium. After testing the antibody titer by the immune adherence assay (1), spent medium from each culture was pooled and centrifuged at 10,000 x g for 20 min to remove cell debris. Ten liters of cell-free spent medium from the L72 culture was processed at one time for antibody purification as described (3). The purified human IgM was concentrated to 5 mg/ml, filtrated through a 0.4-,um filter, and stored at -190TC until used. Sterility was tested by clinical bacteriology at the University of California, Los Angeles Medical Center. Patient Selection. Eight patients participated in the project. All patients had been diagnosed as recurrent melanoma with cutaneous metastasis, had not received any other treatment 14 days prior to their first injection, and had a performance status of 10 (Karnofsky scale). The study sample consisted of three males and five females, ranging in age from 31 to 89 years. Tumor size ranged from 0.2 to 4.0 cm in diameter. With the exception of patients 1 and 4, all patients had received some therapy or combination of therapy at some period before the trials. No previous therapy was effective in any case. Administration of Hu-mAb. Variable amounts of Hu-mAb (5 mg of human IgM per ml in 0.9% saline) were injected intralesionally. The antibody doses were determined by the size of the tumor: 100 Ag of antibody per 1 mm of tumor (diameter). Injections were given daily or weekly with 26-gauge needles and clinical response was determined 3 or 4 weeks after the last injection. Detection of GD2 on Biopsied Melanoma. The amount of GD2 expressed by biopsied melanoma cells was measured Abbreviations: Hu-mAb, human monoclonal antibody; EBV, Epstein-Barr virus; ShE, sheep erythrocyte(s). *To whom reprint requests should be addressed at: the Division of Surgical Oncology, 9th Floor, Louis Factor Building, UCLA School of Medicine, Los Angeles, CA 90024. 8694 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 83 (1986) 8695 before and/or after treatment by thin-layer chromatography (TLC) and TLC scanner of purified gangliosides from melanoma tissues. The ganglioside fraction was isolated and purified by the method described previously (6). The quantity of GD2 in the purified fraction was determined by photodensity metric scanning (high-speed TLC scanner) at 580 nm (7). The quantity of sialic acid in the entire ganglioside fraction was determined by the resorcinol HCl method (Roseman's