Kinetics of the Immune Response and Regression of Metastatic Lesions following Development of Humoral Anti-High Molecular Weight-Melanoma Associated Antigen Immunity in Three Patients with Advanced Malignant Melanoma Immunized with Mouse Antiidiotypic Monoclonal Antibody MK2-231

Active specific immunotherapy has been implemented in patients with advanced malignant melanoma, utilizing the mouse antiidiotypic (anti-id) monoclonal antibody (mAb) MK2-23 which bears the internal image of high molecular weight-melanoma associated antigen (HMW-MAA). In a previous study, development of anti-HMW-MAA immunity in patients with advanced malignant melanoma immunized with anti-id mAb MK2-23 was found to be associated with a statistically significant survival prolongation. Since no information is available about the relationship between development of immunity and clinical response in patients immunized with anti.id mAb, the present study has characterized the kinetics of the immune response in three patients with advanced malignant melanoma who experienced regression of metastatic lesions following immunization with the anti-id mAb MK2-23. The three patients developed anti-mouse IgG antibodies, anti-anti-id antibodies and anti-HMW-MAA antibodies. The anti-HMW-MAA antibodies are mainly IgG, suggesting that the immune response elicited by anti-id mAb MK2-23 is T-cell dependent. The development of anti-HMW-MAA immunity preceded the reduction in the size of metastatic lesions. This temporal relationship suggests but does not prove that the anti-HMW.MAA immunity elicited by anti-id mAb MK2-23 has a beneficial effect on the clinical course of the disease in patients with malignant melanoma. This finding in conjunction with minor side effects associated with repeated administrations of mouse anti-id mAb MK2.23 suggest that active specific immunotherapy with anti-id mAb which bear the internal image of melanoma-associated antigen represents a viable therapeutic approach to malignant melanoma. normal tissues (for review, see Ref. 2). Furthermore, HMW-MAA appears to play a role in the metastatic potential of melanoma cells (14-17). Therefore, inhibition of the functional properties of HMWMAA with antibodies elicited by anti-id mAb is expected to reduce or suppress the metastatic potential of HMW-MAA-bearing melanoma cells. In a recent study, we have shown that the mouse anti-id mAb MK2-23, which mimics the determinant defined by the anti-HMWMAA mAb 763.74 (18), induces humoral anti-HMW-MAA immunity in about 60% of patients with advanced malignant melanoma (13). The development of anti-HMW-MAA antibodies is associated with a statistically significant prolongation of survival and with regression of metastatic lesions in some of the immunized patients (13). No information is available in the literature about the kinetics of the immune response elicited by anti-id mAb in patients with malignant melanoma and about the relationship between development of immunity to a well-defined MAA and regression of metastatic lesions. Therefore, in the present study we have investigated the kinetics of the humoral immune response in three patients with advanced malignant melanoma who experienced regression of metastatic lesions following development of humoral anti-HMW-MAA immunity elicited by anti-id mAb MK2-23. M A T E R I A L S AND M E T H O D S I N T R O D U C T I O N The expression of MAA 3 by human melanoma cells (for review, see Refs. 1-3) provides targets for active specific immunotherapy. Interest in this type of therapy has been rekindled by the lack of any effective therapy in malignant melanoma, once the tumor has metastasized (4), and by the recent progress in biotechnology which has facilitated the preparation of reagents to be used as immunogens. During the last few years, several clinical trials have been implemented to evaluate the ability of the available immunogens to induce anti-MAA immunity and its effect on the clinical course of the disease (5-13). We have focused our investigations on active specific immunotherapy of malignant melanoma with anti-id mAb which bear the internal image of the human HMW-MAA. The latter has been selected as a target because of its expression in a high percentage of melanoma lesions with limited heterogeneity and its restricted distribution in Received 7/23/93; accepted 11/8/93. 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 18 U.S.C. Section 1734 solely to indicate this fact. 1 This investigation was supported by PHS Grants CA37959 and CA51814 awarded by the National Cancer Institute, Department of Health and Human Services, and by Grant EDT-71269 awarded by the American Cancer Society. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: MAA, melanoma-associated antigen; anti-id, anti-idiotypic; HMW-MAA, high molecular weight melanoma-associated antigen; mAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; KLH, keyhole lympet hemocyanin; BCG, Bacillus Calmette-Gudrin; CAT, computerized axial tomography; MRI, magnetic resonance imaging. Patients. Of the three patients investigated in this study, one had documented, measurable stage III and two had documented, measurable stage IV melanoma. Cell Lines. Cultured human melanoma cells Colo 38 and M14/13; cultured human B-lymphoid cells L14, which are autologous to M14/13 cells; and cultured human B-lymphoid cells LG-2 were grown in medium RPMI 1640 supplemented with 10% serum plus (JRH Bioscience, Lenexa, KS) and 2 mM e-glutamine. mAb and Conventional Antisera. Mouse mAbs 149.53 (an IgG1), 225.28 (an IgG2a), and 763.74 (an IgG1) to distinct and spatially distant determinants of HMW-MAA; the mouse anti-intercellular adhesion molecule-1 mAb CL207.14 (an IgG1) and the mouse anti-id mAbs MFll-30 and MK2-23 (both IgG1) to idiotopes within the antigen combinding sites of the immunizing mAbs 225.28 and 763.74, respectively; and the mouse anti-HLA-DQ3 mAb KS13 (an IgG2b) and the mouse anti-id mAb KO3-34 (an IgG1) to an idiotope within the antigen combining site of the immunizing mAb KS13 were developed as described (19-23). mAbs were purified from ascitic fluid by sequential precipitation with caprylic acid and ammonium sulfate (24). The purity of the mAbs was monitored by SDS-PAGE (25). Affinity-purified goat antibodies to human IgM, IgG, and IgG+M were purchased from Jackson ImmunoResearch Laboratories (Avondale, PA). Antibodies were labeled with 125I utilizing the Iodo-Gen method (26). Immunization Schedule. Patients were immunized on days 0 and 7 with s.c. injections of anti-id mAb MK2-23 (2 mg/injection) conjugated to KLH and mixed with 0.1 ml (1 • 10 7 organisms) of Tice BCG (Organon, West Orange, N J); then on day 28, patients were immunized with mAb MK2-23 conjugated to KLH. Additional injections of mAb MK2-23 conjugated to KLH but without BCG were given if, on day 35, the titer of the anti-anti-id antibodies which