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Gangliosides have been considered as potential targets for immunotherapy because they are overexpressed on the surface of melanoma cells. However, immunization with purified gangliosides results in a very poor immune response, usually mediated by IgM antibodies. To overcome this limitation, we immunized mice with R24, a monoclonal antibody (mAb) that recognizes the most tumor-restricted ganglioside (GD3); our goal was to obtain anti-idiotype (Id) antibodies bearing the internal image of GD3. Animals produced anti-Id and anti-anti-Id antibodies. Both anti-Id and anti-anti-Id antibodies were able to inhibit mAb R24 binding to GD3. In addition, the anti-anti-Id antibodies were shown to recognize GD3 directly. Anti-Id and anti-anti-Id mAb were then selected from two fusion experiments for evaluation. The most interesting finding emerged from the characterization of the anti-anti-Id mAb 5.G8. It was shown to recognize two different GD3-expressing human melanoma cell lines in vitro and to mediate tumor cell cytotoxicity by complement activation and antibody-dependent cellular cytotoxicity. The biological activity of the anti-anti-Id mAb was also tested in a mouse tumor model, in which it was shown to be a powerful growth inhibitor of melanoma cells. Thus, activity of the anti-anti-Id mAb 5.G8 matched that of the prototypic anti-GD3 mAb R24 both in vitro and in vivo. Altogether, our results indicate that the idiotype approach might produce high affinity, specific and very efficient antitumor immune responses. (Cancer Sci 2011; 102: 64–70)
Melanomas and other tumors of neuroectodermal origin have a distinct profile of cell-surface ganglioside expression.(1) The relevance of these carbohydrate antigens as immune targets in cancer cells can be inferred from earlier studies that described the ability of monoclonal antibodies (mAb) raised against gangliosides to induce complement-dependent cytotoxicity (CDC) in melanoma, neuroblastoma, sarcoma and astrocytoma cell lines.(2) Generally, immunization with whole tumor cells or cell lysates induces low titer IgM antibodies to carbohydrate antigens, but the use of conjugated vaccines can produce high titers of IgM and IgG antibodies.(3) The best vaccine design involves the conjugation of the antigen to keyhole limpet hemocyanin (KLH) and the use of saponins QS-21 and GPI-0100 as adjuvants.(4)
In contrast to normal cells, transformed melanocytes abundantly express disialoganglioside 3 (GD3). R24 is a mouse mAb that specifically recognizes GD3 and mediates in vitro effector functions such as CDC and antibody-dependent cellular cytotoxicity (ADCC).(5) It also stimulates proliferation of GD3-expressing T cells derived from human peripheral blood(6) and was shown to enhance lymphocyte RNA expression of IL-4, IL-10 and IFN-γ.(7) Normal melanocytes are not lysed by the R24-directed immune response due to their low GD3 expression. In contrast to the potent in vitro activity of R24, its effect in nu/nu mice bearing human melanoma grafts is much more modest; tumor inhibition was observed only when R24 treatment started within 3 days of tumor cell inoculation and no effect was shown on established tumors.(8) As a single agent, R24 was shown to induce clinical responses in patients with metastatic melanoma, including complete remissions.(9,10) Nevertheless, the dose-dependent toxicity of R24 can be substantial and constitutes a serious limitation for its clinical use.(11–14)
Anti-idiotype (anti-Id) antibodies that mimic a defined antigenic epitope are a relatively unexplored albeit potentially useful therapeutic tool. We hypothesized that an anti-Id antibody, being a protein, should be more immunogenic than GD3, a thymus-independent antigen. In support of this assumption, promising results have been reported with anti-Id antibodies in the context of several experimental models based on pathogen-derived(15–17) and tumor-associated(18–27)antigens. We have also previously described an anti-Id monoclonal antibody that was able to mimic glycoprotein carcinoembryonic antigen (CEA) and elicit an anti-anti-Id mAb that recognized the antigen in vitro and in vivo.(28–30) In the present study, we immunized BALB/c mice with the mAb R24 with the expectation of obtaining anti-Id antibodies bearing the internal image of GD3. Animals were shown to produce anti-Id and anti-anti-Id antibodies of IgM and IgG classes. One anti-Id antibody clone was selected for detailed evaluation. However, the most interesting finding emerged from the characterization of the anti-anti-Id antibody. The well-defined prototypic anti-GD3 monoclonal antibody R24 was used as a control in the assays of binding, cytotoxic activity in vitro and for the ability to protect against tumor challenge. We found that this anti-anti-Id mAb is a powerful growth inhibitor of melanoma cells in vitro and in vivo.
- Top of page
- Materials and Methods
- Disclosure Statement
- Supporting Information
Immunological tolerance is a major issue in cancer immunotherapy. To break the inability of the host’s immune system to recognize and react properly against malignant cells is particularly challenging given that tumor-associated antigens are self, poorly immunogenic molecules. Normal melanocytes express the mono-sialo GM3 as their major ganglioside, whereas the growth and metastatic potential of malignant melanoma correlate with excessive synthesis of GD3.(39) Although little is known about the precise composition of many gangliosides that are expressed in tumors, shedding of GD3 into the circulation has been observed in patients bearing certain malignancies.(39) It has been shown by several groups that this ganglioside might promote tumor growth and cell–cell adhesion.(40,41) Although GD3 is present in melanoma cells, its expression in normal tissues is limited and not related to altered cellular behavior. Altogether, these reports support the idea that GD3 might be a good target for the antitumor immune response and suggest the potential usefulness of an anti-GD3 antibody-based strategy in cancer therapy.(9,14,42,43) However, as the main antigenic epitopes in GD3 are carbohydrates, only a very weak and T-cell-independent immune response can be stimulated when it is used as an immunogen. Several attempts have been made to address the therapeutic potential of the anti-GD3-specific immune response.(14,42–50) Nevertheless, the anti-GD3 immune response was shown to be short-lived and mainly constituted by IgM antibodies.(1)
The anti-idiotypic antibody approach represents an alternative to circumvent the poor immunogenicity of GD3 and the toxicity of the mAb R24(11–14) in cancer therapy. The remarkable ability of anti-Id antibodies to mimic the original antigen has been shown in several models.(15–28) Anti-Id antibodies might induce a specific immune response against tumor-associated antigens, such as the carcinoembryonic antigen(29,51) and the human high molecular weight melanoma-associated antigen(52) among others.(22–27) In addition, two anti-idiotype R24 antibodies (BEC2 and BEC3) have been previously isolated.(53) Although BEC3 was incapable of eliciting anti-GD3 responses, BEC2 was shown to induce IgM but no IgG anti-GD3 antibodies in a fraction (22%) of immunized melanoma patients.(54–56) The application of these antigen surrogates in cancer immunotherapy is particularly appealing when the antigen is not a protein, as in the case of gangliosides. However, the fine specificity profile of anti-GD3 antibodies and of their anti-idiotypic counterparts might vary considerably with significant differences in binding, affinity and immunogenicity.(44–50,53) Thus, the search for novel anti-Id and anti-anti-Id antibodies, such as the ones described in this paper, might lead to the identification of molecules with the ability to induce high affinity IgG responses with increased antitumor activity in a larger fraction of immunized individuals.
In the present report, we have used the mAb R24 coupled to KLH to immunize BALB/c mice. An anti-Id immune response was identified after the first immunization as the serum inhibited binding of the mAb R24 to GD3. Anti-anti-Id antibodies capable of binding GD3 could also be detected in the third immunization. In view of these results, both anti-Id and anti-anti-Id mAb were produced in mice in order to evaluate the efficiency of the GD3-associated idiotypic network. We found that the anti-Id and anti-anti-Id antibody secreting clones belonged to different Ig classes and subclasses. The observed Ig isotype diversity suggests an effective T cell cooperation in the development of the anti-Id and anti-anti-Id immune responses; it is also likely to contribute to a higher efficiency of the immune response because it might augment the cytotoxic effect triggered and/or mediated by antibodies.
The monoclonal anti-Id antibody selected for detailed analysis in the present study, clone 5.E3, was able to inhibit the binding of the mAb R24 to GD3 in a dose-dependent fashion. When used as an immunogen, the mAb 5.E3 also elicited a GD3-binding anti-anti-Id humoral immune response in mice. The participation of the mAb 5.E3 in a cascade of idiotype–anti-idiotype interactions is supported by its ability to bind the mAb R24 as well as the anti-anti-Id mAb 5.G8.
The mAb 5.G8 and the mAb R24 were capable of recognizing the GD3 ganglioside in the polar lipid fraction of SKMel-28 cell extracts. Reactivity of the anti-anti-Id antibody was also tested in intact cells; five cell lines, including two human GD3-expressing melanoma cell lines, were tested for antibody binding. The mAb 5.G8 was shown to recognize both melanoma cell lines at comparable levels to those obtained with the original anti-GD3 mAb R24. It is noteworthy that both antibodies recognized SKMel-28 cells somewhat better than MeWo cells (25% higher reactivity); this finding is in agreement with the reported higher expression of GD3 in SKMel-28 cells.(8)
Antibody-dependent cellular cytotoxicity and CDC are important immune effector mechanisms and the ability to promote target lysis might be a valuable asset of antibodies considered for therapeutic use. Our analysis of the mAb 5.G8 revealed that it was highly effective in mediating lysis of target cells either by ADCC or by fixing complement. The ADCC activity was observed when activated peritoneal macrophages were used as effector cells, but not when splenocytes were the source of the effector cells (data not shown). At first sight, the mAb 5.G8 seemed to be less efficient than the mAb R24 in ADCC and CDC. However, the mAb R24 exhibits direct cytotoxic activity as evidenced by the aggregation of melanoma cells in culture, which usually leads to detachment and subsequent death.(8) In addition, the mAb R24 is an IgG3 that might form noncovalent molecular aggregates at a high concentration, thereby increasing the lysis of target cells in vitro.(57–60) If one accounts for this direct cytotoxicity, the mAb R24 and the mAb 5.G8 have fairly similar activities in ADCC and CDC. Indeed, our results indicate that the mAb 5.G8 was even slightly more efficient than the mAb R24 in its ability to fix complement (59.83 ± 12.29% for the mAb 5.G8 and 52.66 ± 0.82% for the mAb R24), as well as to promote ADCC (58.34 ± 1.57% and 49.20 ± 2.78%, respectively) if one discounts in each case the lysis obtained by the antibody alone.
In our in vivo tumor model, animals were preconditioned by sublethal irradiation and inoculated with the human melanoma cell line SKMel-28. All animals in the negative control groups developed tumors that were approximately 0.5 cm3 on day 28. This was in sharp contrast with groups that were treated with the anti-anti-Id or GD3-specific antibodies in which there were tumor-free animals at the end of the observation period and the average volume of the lesions when present was under 1 mm3. Thus, our data indicate that passive transfer of the anti-anti-Id mAb 5.G8 confers protection in the experimental model described here by delaying tumor growth.
We present evidence that immunization with the anti-GD3 mAb R24 induces a cascade of idiotype–anti-idiotype interactions. While anti-Id interactions are likely to occur in the context of natural immune responses, a definitive picture of their immune-regulatory importance remains to be established. However, our results demonstrate that the anti-Id approach works even in a T-cell-independent system and suggests a potential role to play in cancer immunotherapy. The isotype diversity also constitutes an advantage of the anti-Id strategy; this is particularly relevant if we consider that such diversity occurred in a system where the original antigen is a glycolipid that is unlikely to stimulate an efficient immune response on its own. To our knowledge, this is the first report on a melanoma-specific anti-anti-Id mAb that shows direct antitumor activity in vivo in a T-cell-independent way; this anti-anti-Id mAb might represent an alternative for cancer treatment if further studies identify a favorable profile in terms of toxicity, inflammatory reaction and tumor killing.