Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 14, Num. 1, 1997, pp. 45-46

HUMAN ANTI-GANGLIOSIDE MONOCLONAL ANTIBODIES GENERATED BY in vitro IMMUNIZATION

Mauro Alfonso,^1 Boel Lanne,^2 Peter Ifversen,^3 Jacques Portoukalian,^4

Ana M Vazquez,^1 Josefa Lombardero,^1 Ernesto Moreno,^1 Rolando Perez^1 and Jesper Zeuthen^3

^1 Dept. of Research and Development, Center of Molecular Immunology,

P.O. Box 16040, Havana 11600, Cuba.
^2 Dept. of Medical Biochemistry, Iniversity of Goteborg, S-41390 Goteborg, Sweden.
^3 Dept. Tumor Cell Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark.
^4 Glycobiology Group, Faculty of Medicine Lyon-Sud, France.

Code Number:BA97009
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Introduction

Naturally ocurring antibodies against tumor-associated antigens can be found in humans, mainly in cancer patients (1), but in order to increase the proportion of specific antibody-producing cells, several primary in vitro immunization protocols have been succesfully developed.

Immunogenic preparations containing both T- and B-cell epitopes have been used for in vivo and in vitro immunization systems to stimulate a number of antigen-specific B cells in mice and humans to be further immortalized by fusion or Epstein-Barr virus transformation (2). It has recently been demonstrated that immunization with relevant T helper and B-cell epitopes co-entrapped in an apropriate delivery system such as liposomes is sufficient to overcome the nonresponsiveness to defined B-cell epitopes and even produce an active IgG response (3).

A specific goal addressed by our laboratory has been the production of human monoclonal antibodies (MAbs) to different monosialogangliosides of relevance as therapeutic targets in human tumors such as melanoma, lung and breast carcinomas.

Human MAbs against tumor-associated ganglioside antigens have been obtained by several groups using different approaches, but the generation of human anti-ganglioside MAbs by in vitro immunization of human B lymphocytes has not been previously reported.

Material and Methods

Several experiments on in vitro immunization of human B lymphocytes from normal donors or melanoma and breast cancer patients were performed using liposomes containing gangliosides as the immunizing antigen, with or without either complete tetanus toxoid or a synthetic T helper epitope derived from tetanus toxin (determinant 830-843). After 6 days in culture, the immunized B cells were immortalized by Epstein-Barr virus infection and the human anti-ganglioside antibody response was evaluated by an indirect ELISA using different mono and disialogangliosides (4). Further characterization of the specificity of the human MAbs was performed by High Performance Thin Layer Chromatography (HPTLC) using a large panel of glicolipids and extracts from different human tumors (5). Immunohistological staining of formalin-fixed paraffin-embedded sections of different malignant melanoma and invasive ductal carcinoma of the breast was performed using the avidin-biotin complex (ABC) technique. The variable region of the heavy and light chain genes were amplified by PCR, cloned into pCRII vector (Invitrogen) and sequenced by the dideoxynucleotide method.

Results and Discussion

Clones producing antigen-specific human antibodies of the IgM isotype against the gangliosides NeuAcGM3, NeuGcGM3 and NeuAcGM2 used as immunogen, were selected from different experiments.Our results indicated that the in vitro immunizations were antigen-driven, since the antibody responses specific for the ganglioside used for the immunization, were found. The cultures showing specific antibody production were predominant in the cases where the corresponding ganglioside and the T-helper epitope were incorporated into liposomes and no clones producing anti-ganglioside antibodies were obtained when the antigen was omitted from the cultures.

A number of initially selected clones after few weeks in culture ceased to produce human antibodies. To overcome this widely reported problem concerning the instability of the immunoglobulin production by antibody- secreting cell lines, a method of positive selection using ganglioside- coated magnetic beads (Dynal) has been developed which allowed us to rescue unstable clones.

One of the original clones showing a stable antibody production, identified as IM-11, was further characterized. The binding of the human antibody IM- 11 to a large panel of glycosphingolipids separated on high performance thin layer chromatography (HPTLC) plates was studied. The human MAb was found to bind strongly to NeuAcGM3, IV^3NeuAcnLc^4 and sulfate containing glycolipids and weakly to NeuGcGM3, suggesting that the binding epitope of IM-11 is the terminal sugar residue NeuAcalpha2-3Galbeta1-4Glc but also NeuAcalpha2-3Galbeta1-4GlcNAc present in IV^3NeuAcnLc^4. The HPTLC immunostaining of glycolipid extracts from different human tumors (lung, colon, liver and melanoma) with IM-11 revealed a specific recognition of NeuAcGM3 and sulfated glycolipids.

In addition, immunohistological staining of different melanoma and breast cancer biopsy sections has shown a positive reactivity of IM-11 with tumor cells with an intensity which varied among different tumors and when treatment of the tissue sections with neuraminidase from Vibrio cholerae was performed, a substantial reduction of the staining was observed in the melanoma sections analyzed, while immunostaining of the breast cancer cells was unaffected.

The variable region of the heavy chain gene was cloned and sequenced indicating that it belong to the heavy chain subgroup III, showing a high homology with previously reported germline sequences.

References

1. Lloyd KO, Old LJ. Cancer Res 1989; 49:3445-51.

2. Chin LT et al. Immunology 1994;81: 428-34.

3. Ifversen P et al. Immunology 1995; 84:111-16.

4. Alfonso M et al. Hybridoma 1995;14: 209-16.

5. Alfonso M et al. Hum Antibod Hybridomas 1995;6:102-12.

Copyright 1997 Elfos Scientiae