Characterization of Human IgG1 Monoclonal Antibody Against Gangliosides Expressed on Tumor Cells

Abstract

A human IgG1.k monoclonal antibody (MAb) designated GMA1 was developed by fusing pooled lymph node lymphocytes from cancer patients with the human lymphoblastoid cell line, SHFP-1. The GMA1 MAb reacted with several melanoma and neuroblastoma cell lines. Normal tissue derived from human brain and tumor-cell lines derived from colon, ovary, and breast were not reactive. FACS analysis performed using live cells demonstrated that the antibody recognizes a cell-surface antigen. Enzyme immunoassay (EIA) and thin layer chromatography (TLC) immunostaining with purified gangliosides indicated that the antibody has specificity for the major tumor associated gangliosides GD3, GM3, and GD2. GMA1 heavy and light chain genes were isolated by RT-PCR and a recombinant derivative of this human antibody was expressed in Chinese hamster ovary (CHO) cells. High-level antibody synthesis and secretion was achieved using a vector designed to maximize expression. FACS analysis and TLC immunostaining indicated recombinant GMA1 reacted with human tumor cell lines and gangliosides GD3, GM3, GD2 in a manner similar to the antibody produced by the hybridoma cell line, demonstrating that the specificity of the antibody was not altered during molecular cloning.

Anti-ganglioside anti-idiotypic vaccination: more than molecular mimicry

“…Silent auto-reactive B clones have also been identified in cancer patients, from which human monoclonal antibodies (mAb) against gangliosides were generated. The mAb GMA1 reacted with the gangliosides GD3, GM3, and GD2 from melanoma and neuroblastoma cell lines and not normal tissues (Mukerjee et al, 1998). The human monoclonal IgM antibody 7c11.e8, also generated by fusing lymph node cells isolated from a surgical specimen of malignant melanoma reacted with GM4, GM3, and GD3.…”

Nature's best weapons to fight cancer. Revival of human monoclonal IgM antibodies

“…These IgM antibodies also exhibited characteristics common to conventional antisera, like cross-reactivity or poly-specificity and low affinity. The antibodies also reacted not only with membrane-bound structures, but also with cytoplasmic and nuclear proteins, and with carbohydrate structures and glycolipids [31][32][33][34][35][36]. Subsequent sequence analysis revealed that these human IgM antibodies with anti-tumour activity were part of the innate (natural) immunity, germ-line coded and not affinity maturated [37][38][39].…”

Lessons learned about the therapeutic potential of the natural human immune response to lung cancer

“…One will consist of known antigens such as the Ep-CAM antigen [23], the antigen to AE6F4 [20], or the ganglioside GD3 [24,25], and the other class will consist of novel antigens such as AgSK1 [18] or AgTB94 [21,22], which have not been previously described. This could be significant, especially in light of the fact that the human immune response recognises allotypic antigens immunologically blind to other species.…”

“…Prior studies [7,14,18,19,24,31] have demonstrated the clinical utility of obtaining antibody-secreting B-cells from regional draining lymph nodes and their applications in cancer diagnosis and therapy. The principles learned should be applicable to lung cancer.…”

“…Successful programmes, using both recombinant phage-display human antibodies [37,38] and those derived from xenomouse technology [23], are of interest. The amino acid sequences of the current crop of human antibodies are known (for example, see [24]) and, in aggregate, the variable region sequences are typical of antibody light chains, irrespective of the heavy chains which range from IgM, various IgG subclasses and IgA, in that appropriate variable region framework sequences are in place, as well as CDR regions. As an example, the primary sequence of the light chain variable region of the antilung cancer human antibody, TB94, is shown in Figure 3.…”