HYBRIDOMA Volume 19, Number 3, 2000 Mary Ann Liebert, Inc. A Mouse IgG 1 Monoclonal Antibody Specific for N-Glycolyl GM3 Ganglioside Recognized Breast and Melanoma Tumors ADRIANA CARR, AILETTE MULLET, ZAIMA MAZORRA, ANA MARIA VÁZQUEZ, MAURO ALFONSO, CIRCE MESA, ENRIQUE RENGIFO, ROLANDO PÉREZ, and LUIS ENRIQUE FERNÁNDEZ ABSTRACT 14F7 murine monoclonal antibody (MAb) is an IgG1 immunoglobulin that is generated by immunizing Balb/c mice with GM3(NeuGc) ganglioside hydrophobically conjugated with human very-low-density lipoproteins and in the presence of Freund’s adjuvants. 14F7 MAb binds specifically to GM3(NeuGc), whereas neither N-glycolyl or N-acetyl gangliosides, nor a sulfated glycolipid, are recognized as assessed by enzyme-linked im- munosorbent assay or immunostaining on thin layer chromatograms. Immunohistochemical studies in fresh tumor tissues showed that 14F7 MAb strongly recognized in antigen expressed in human breast and melanoma tumors. 241 INTRODUCTION G ANGLIOSIDES are sialic acid–containing glycosphingolipids that are present in the plasma membranes of vertebrates. (1) Some of these molecules have been reported as tumor-associ- ated antigens or tumor markers, (2) leading to the use of anti- ganglioside monoclonal antibodies (MAbs) in the diagnosis and therapy of cancer. (3,4) N-acetyl (NeuAc) and N-glycolyl (NeuGc) neuraminic acids are the most common types of sialic acids found in animals. (5) In general, the NeuGc residue is not expressed in human and chicken normal tissues, but is widely present in other vertebrate. (6,7) In contrast, it has been reported that Anti-NeuGc–containing ganglioside antibodies (8,9) recog- nize some human tumors and cancerous cell lines. These stud- ies have been carried out using polyclonal or monoclonal anti- bodies of chicken, (10) human, (11) or murine (12) origin, but in all these tumors minimal levels of NeuGc were reported. On the other hand, recently, we found increased levels of GM3(NeuGc) ganglioside in human breast cancer, (13) a finding that certainly makes this molecule an attractive target for cancer therapy. Several MAbs recognizing NeuGc-containing gangliosides showing different binding specificity have been obtained, while some of them react with an epitope shared by more than one of these gangliosides, as GMR8 or GMR3, (14) YK3, (15) and P3, (16,17) other MAbs are highly specific against a particular ganglioside, as Y-2-HD1, (18) MK-2-34, (19) or SHS1. (20) A com- mon characteristic of all these MAbs is that they are IgM anti- bodies. Here, we describe for the first time the generation and char- acterization of an IgG1 highly specific anti-GM3(NeuGc) MAb that has been designated as 14F7. This MAb was generated by immunization of mice with a vaccine formulation containing GM3(NeuGc) hydrophobically conjugated with human very- low-density lipoproteins (VLDL). (21) Noteworthy is the fact that a preliminary immunohistochemical study with 14F7 MAb showed a strong recognition of breast and melanoma tumor tis- sues. MATERIALS AND METHODS Animals Six- to 8-week-old female Balb/c mice were purchased from CENPALAB, Havana, Cuba. Glycolipids GM3(NeuAc) and GM3(NeuGc) were obtained from dog and horse erythrocytes using a modification of the Folch method. (22) GM1, GM2, GD1a, GD1b, and GT1b were purified from bovine brain, (23) and GD3 was purchased from Sigma. GM2(NeuGc) was obtained from the liver of Balb/c mice, (24) and (NeuGc-NeuGc)GD3, from bear erythrocytes, was kindly supplied by Y. Hashimoto and S. Suzuki (Tokyo Metropolitan Institute of Medical Science, Japan). S. Sonnino (Milan Uni- versity, Italy) and J. Portoukalian (Faculty of Medicine Lyon- Center of Molecular Immunology, Havana, Cuba.