Immunotherapy based on carbohydrate antigens for the treatment of cancers has been intensively examined. Although they are self-antigens and the antibodies raised against these antigens may react to normal tissues, such therapy is still a viable option. However, the drawbacks of self-antigen-based vaccines are obvious; poor immunogenicity and potential autoimmune responses are two major concerns (33). By biochemically engineering cell surface antigens, specifically the sialic acid-containing antigens, we are able to temporarily remodel the cell surface and render it susceptible to targeted antibody responses. Such antibodies can be elicited by using a glycoconjugate vaccine in which a specifically modified antigen is covalently linked to a protein carrier.
In this study, we first synthesized four disialolactoside-KLH conjugates as vaccines in order to raise antibodies against the GD3 and its analogs on the cell surface. Two N-Ac groups of disialolactoside (GD3 tetrasaccharide) were substituted by N-Pr, N-Bu, and N-Bz groups, respectively, in order to optimize their immunogenicities and specificities. In contrast to its human response, GD3 is immunogenic in mice. However, it has been demonstrated that replacement of N-Ac groups of PSA by N-Pr groups overcomes the immune tolerance in animals (51, 52), indicating that a similar modification on GD3 may achieve similar effects in man. As expected GD3Pr-KLH and GD3Bu-KLH were good immunogens, and only a weak cross-reactivity between GD3Pr sera and GD3Bu was observed. The fact that neither of these antisera was cross-reactive with native GD3 confirms our supposition that it is possible to raise antibodies by using structurally modified immunogens to avoid generating autoantibodies. Sub-typing analysis revealed that the majority of the antigen-specific antibodies was of the IgG rather than the IgM subclass, suggesting the effective recruitment of T-helper cells in the induction of the immune response.
In order to use these specific antibodies to immunotarget SK-MEL-28 cells, one has to assume that modified GD3 is expressed on the cell surface, and many previous studies have succeeded in modifying cell surface sialic acids by the introduction of chemically modified precursors in the biosynthetic pathway (53). Thus, SK-MEL-28 cells treated with exogenous precursors, such as ManNPr, ManNBu, and ManNBz, express the corresponding N-acyl-modified sialic acid residues on the cell surfaces. Specific modifications of GD3 on the cells were identified by strong binding to their homologously modified N-acyl GD3-conjugate antisera.
Two parameters were considered in the remodeling of cell surface antigens by using metabolic precursors: 1) the incorporation efficiency, and 2) the metabolic rate. The effective expression of modified GD3 occurred at 1 mg/ml of precursor, and the fact that increasing precursor concentration did not improve the expression suggests that N-acyl mannosamines in the N-acyl sialic acid biosynthesis can be transferred efficiently by the sialyltransferases to lactosyl ceramide, forming modified GD3. The low concentration of precursor required and the relatively fast expression of modified GD3 (less than 24 h) in comparison with previous results on the sialic acid engineering of PSA (32) may add therapeutic advantages in clinical settings. Moreover, in comparison to the cell surface PSA antigen, the turnover rate of cell surface GD3 was slow. GD3Bu expression was still detectable on most cells 5 days after removal of ManNBu in vitro, indicating that modified GD3 could be a favorable immunotarget. Although Bertozzi and co-workers (54, 55) observed that ManNBu is able to block the biosynthesis of PSA of neural cell adhesion molecule, its impact on the biosynthesis of GD3 seems less significant. The identification of GD3Bu on Bu-SK-MEL-28 cell surface by flow cytometric and mass spectroscopic (electro-spray-MS) analyses suggests NeuNBu is a good substrate for α2–3siaT (SAT-I) and α2–8 siaT (SAT-II), both involved in the GD3 biosynthesis (56).
Although SK-MEL-28 cells expressed the modified GD3 epitope well in vitro, the expression of such an epitope on the tumor surface in vivo is a prerequisite for immunotargeting. Clearly, SK-MEL-28 cells grown in nude mice accepted Man-NBu as a metabolic precursor and incorporated it into GD3 molecules as evidenced by flow cytometric analysis on cells obtained from tumors grafted on the nude mice. But although the tumor cells did express GD3Bu, significant amounts of GD3 still remained on the cell surface. These results could be attributed to an insufficient concentration of precursor and/or that the biosynthesis in vivo favors using ManNAc as substrate. Nevertheless, the successful bioengineering of modified GD3 in vivo does provide a means to target SK-MEL-28 cells for immune destruction.
Similar to the function of mAb KM871 reported previously (27), both mAb 2A and GD3Bu antisera were capable of stimulating the complement-mediated lysis of Bu-SK-MEL-28 cells that express GD3Bu, and such CDC activity depended on the mAb and antibody concentrations. Without modification, SK-MEL-28 cells were affected by neither mAb 2A nor GD3Bu antiserum, which further indicates that expression of GD3Bu may serve as a trigger (switch) for the immune response. This result is significant because we may be able to achieve augmentation of immunogenicity without risk of evoking the autoantibodies, which has always been a dilemma in the use of common cancer vaccines.
Unfortunately, there are other limitations to the in vivo application of this technology when it was shown that the treatment of mice having established tumors, with mAb 2A and ManNBu, could arrest further tumor growth but not eliminate the tumor. Although disappointing, our results are not surprising because Livingston and co-workers (9, 12) also obtained similar results with GD2 antibodies, whereby the homologous antibodies were only able to remove microtumors and prevent the metastasis but not remove solid tumors. However, when the above treatment was applied to mice soon after tumor grafting, it was able to prevent establishment of the tumor, providing evidence that an active vaccination strategy using the GD3Bu-KLH conjugate in conjunction with ManNBu might be applicable to the prevention of tumor metastasis. Unfortunately, the nude mouse model that we employed was not a good model to further investigate the potential of this strategy.
Examination of tumor tissues from large size tumors after the above treatment failed to identify GD3Bu-expressing tumor cells by flow cytometric analysis. This observation indicates the limitation of immunotargeting a single epitope, because it is possible that sub-populations of tumor cells without this epitope could overgrow those with it and thus render the treatment ineffective. Similarly, it is known that tumors can down-regulate specific antigens such as sialyl Lewisx (57), and it is of interest to note that the down-regulation of GD3 expression has also been implicated in the reduction of both cellular proliferation and metastasis of neuroblastoma F-11 cells in animals (58–60).
In conclusion, we have demonstrated that synthetic disialolactoside conjugates can accurately imitate homologous epitopes expressed on the cell surface. The fact that no cross-reactivity was observed between SK-MEL-28 cells and GD3Bu-KLH antiserum indicates that the immunodominant epitope should minimize the risk of unwanted autoimmune responses. We have also demonstrated that GD3Bu-specific antibodies became potent cytotoxic reagents against Bu-SK-MEL-28 cells when GD3Bu molecules were incorporated. Studies in vivo indicated that the treatment using a combination of precursor and specific antibodies can prevent mice from tumor grafting but was ineffective for eliminating solid tumors.