Recent advances in the target-oriented synthesis of saponin immunostimulating agents have enabled the completion of the first syntheses of the adjuvant QS-21-Api and QS-21-Xyl. These efforts have resulted in a highly modular synthesis via assembly of the four principle substructures of the molecules. The successful results from these synthetic efforts provides samples of QS-saponins with a heretofore unparalleled degree of purity. With these SQS-21-Api/Xyl products, there is no chance of trace saponin contaminants derived from purification protocols from the natural source, thereby eliminating uncertainties associated with uncontrolled heteogeneity in QS-adjuvants derived from traditional isolation protocols. Notably, the synthetic process has also been successfully adapted to produce GMP-grade SQS-adjuvant for upcomimg clinical evaluation.
Herein, we describe in detail the adjuvanticity and toxicity of the synthetic apiose and xylose isomers that comprise QS-21, and compare this to the synthetic 65:35 mixture that simulates QS-21 obtained from the South American soap bark tree Quillaja saponaria
. Our findings are that 10, 20 or 50 µg of either isomer or the 65:35 mixtures is equally potent as immunological adjuvants in mice. The antibody responses induced against the ganglioside GD3 and the highly immunogenic carrier protein KLH after immunization with GD3-KLH conjugates plus synthetic adjuvant (SQS-21-Api or SQS-21-Xyl) or the mixture were comparable to each other and to PQS-21, which is QS-21 fractionated from Quillaja saponaria
]. The SQS-21-Xyl isomer, however, appeared to be slightly more toxic than either the corresponding apiose isomer, or the reconstituted SQS-21 mixture. The results described here, both adjuvanticity and toxicity, are quite similar to those that we have previously described with the same GD3-KLH conjugate and QS-21 isolated from Quillaja saponaria
These data demonstrate that the two synthetic isomers, SQS-21-Api and SQS-21-Xyl, are equally potent whether administered individually or as a mixture comparable to that of the QS-21 isomeric ratio reported to be obtained from natural sources [28
]. This leaves no doubt that the highly purified synthetically derived isomer constituents of the QS-21 fraction are competent adjuvants in ganglioside-KLH conjugate vaccines. These findings lay the groundwork for investigation and imminent establishment of a thorough structure-activity profile for this potent adjuvant, for which concrete mechanism-of-action studies are sorely lacking. For example, Marciani has described three components of QS-21 that are required for optimal immunogenicity [26
]. These include: (1) the hydrophobic chain, postulated to facilitate binding to the cell surface lipid bilayer; (2) the oligosaccharide moieties, postulated to mediate the interaction of saponin (or associated antigen complexes) to cell-surface lectins on antigen presenting cells; and (3) the saponin aldehyde, postulated to engage in covalent (Schiff-base) conjugation with certain T-cell surface-NH2
groups and evoking a co-stimulatory second signal leading to Th1 responses. It is this proposed combination of immunological effects on the macrophage, dendritic cell and T-cell arms of the immune system that may be important for the potency of saponins as immunological adjuvants. While SQS-21 has the academic advantage of unambiguously defining the structures of the active components in QS-21, it does not compete on a cost basis with QS-21 obtained by extraction. However, the advantages of the synthetic SQS products are evident when considering the high level of purity of these synthetic entities relative to naturally isolated QS-saponins, often incorporating variable quantities of trace natural impurities. Moreover, having demonstrated the potency and relative safety of the SQS-21 isomers, we are now uniquely poised to determine the contribution of these and additional QS-saponin molecular components to adjuvant potency and toxicity as we systematically probe structure/function correlations. This indeed highlights the true value of the SQS-chemical synthesis technologies – the ability to engage in unfettered systematic design and synthesis of novel SQS-analogues. The need for these advances arises as a result of some drawbacks associated with the use of naturally derived QS-21, including local and systemic toxicity [27
], as well as the rapid loss of adjuvant activity when stored at room temperature as a consequence of spontaneous chemical degradation arising from acyl chain hydrolysis[12
]. Using the immunological evaluation protocols described herein in combination with the chemistry innovations in SQS-saponin synthesis, the design and preparation of novel saponin adjuvants with increased potency, enhanced stability, and attenuated toxicity are currently underway, and will be reported in due course.
Based on these results, a clinical trial with SQS-21 containing a 65:35 ratio mix of the two synthetic isomers has recently been initiated with a GD3-KLH and GD2-KLH bivalent vaccine to confirm the safety and potency of SQS-21 in melanoma patients.