A fully synthetic anticancer vaccine 2 has been prepared via bio-conjugation of unimolecular pentavalent construct 1 – containing five prostate and breast cancer associated carbohydrate antigens, Globo-H, GM2, STn, TF and Tn – to maleimide-modified carrier protein KLH. An improved conjugation protocol has been developed, which allowed us to obtain a higher epitope ratio of the unimolecular pentavalent glycopeptide antigen to the carrier protein (505/1 versus 228/1 for the previous version). KLH conjugate 2 has been subsequently submitted to preclinical immunogenic evaluation in mice in the presence of QS-21 as an adjuvant. Through standard ELISA assay, this vaccine candidate showed high promise in inducing IgG and IgM antibodies against each of the five individual carbohydrate antigens. In addition, FACS analysis indicated that these antibodies were able to react with MCF-7 breast cancer cell lines expressing these five carbohydrate antigens.

QS-21 is a potent immunostimulatory saponin that is currently under clinical investigation as an adjuvant in various vaccines to treat infectious diseases, cancers, and congnitive disorders. Herein we report the design, synthesis, and preclinical evaluation of simplified QS-21 congeners to define key structural features that are critical for adjuvant activity. Truncation of the linear tetrasaccharide domain revealed that a trisaccharide variant is equipotent to QS-21 while the corresponding disaccharide and monosaccharide congeners are more toxic or less potent, respectively. Modification of the acyl domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in reduced adjuvant activity. Acylation of the terminal amine can restore adjuvant activity and enables the synthesis of fluorescently-labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the most highly adjuvant active of these fluorescently-labeled saponins does not simply associate with the plasma membrane, but rather is internalized by dendritic cells.

The success of antitumor and antiviral vaccines often requires the use of an adjuvant, a substance that significantly enhances the immune response to a co-administered antigen. Only a handful of adjuvants have both sufficient potency and acceptable toxicity for clinical investigation. One promising adjuvant is QS-21, a saponin natural product that is the immunopotentiator of choice in many cancer and infectious disease vaccine clinical trials. However, the therapeutic promise of QS-21 adjuvant is curtailed by several factors, including its scarcity, difficulty in purification to homogeneity, dose-limiting toxicity, and chemical instability. Here we report the design, synthesis, and evaluation of chemically stable synthetic saponins. These novel, amide-modified, non-natural substances exhibit immunopotentiating effects in vivo that rival or exceed that of QS-21 in evaluations with the GD3-KLH melanoma conjugate vaccine. The highly convergent synthetic preparation of these novel saponins establishes new avenues for discovering improved molecular adjuvants for specifically tailored vaccine therapies.

The success of antitumor and antiviral vaccines often requires the use of an adjuvant, a substance that is itself not necessarily immunogenic but significantly enhances the immune response of a patient to a coadministered antigen.[1] The adjuvant of choice in many recent immunotherapeutic advances against cancer, [2] human immunodeficiency virus, [3,4] and malaria [5,6] is the natural product saponin QS-21. [7] Extensive studies in the development of conjugate cancer vaccines demonstrated the superiority of QS-21 over 18 competing adjuvants in preclinical models. [8] Isolated in minute quantities from the cortex of the Quillaja saponaria (QS) tree, QS-21 is not a homogeneous substance; rather, it is the 21st of 22 fractions in an early reversed-phase (RP) HPLC trace of semipurified QS-bark extracts. [7,9] This adjuvant-active fraction comprises at least two principal constituents (Scheme 1), QS-21-Xyl (1) and QS-21-Api (2), which differ in the terminal sugar residue within the linear tetrasaccharide segment. [10,11] Although the multi-component QS-21 fraction has fueled numerous previous and ongoing vaccine clinical trials, the challenge to procure samples of consistent composition from natural sources is formidable. Indeed, high variability in saponin composition is seen even among QS trees of similar age and local environment (e.g., soil, season).[12] Furthermore, recent metabolomic analyses of semipurified QS-bark extracts revealed a complex mixture of over 100 distinct saponins, [13] considerably more than what the original 22-fraction RP HPLC profile might imply. [7,9] Because of the variability and heterogeneity of QS extracts, it is difficult to establish the adjuvant activity and toxicity of the component saponins. The possibility that trace quantities of additional saponins may be present in the QS-21 fraction impacts not only on efficacy and formulation aspects, but also on regulatory hurdles in clinical vaccine development in humans.Efforts to further advance QS-21 in the clinic, as well as to illuminate its unknown mechanism of action, require access to adjuvant-active samples of known composition. The recent chemical synthesis of the isomer QS-21-Api (2) enabled independent access to homogenous samples of this particular saponin. [14] We now report the chemical synthesis of the second

Sialyl Lewis a (sLe a ), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe a is known to be the ligand for endothelial cell selectins suggesting a role for sLe a in cancer metastases and adhesion. For these reasons, sLe a may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe a is structurally similar to sLe x and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe a will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe a hexasaccharide and its conjugation to KLH to construct a sLe a -KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe a -HSA by ELISA and against sLe a positive human cell lines adenocarcinoma SW626 and small cell lung NIH Public Access

The saponin fraction QS-21 from Quillaja saponaria has been demonstrated to be a potent immunological adjuvant when mixed with keyhole limpet hemocyanin conjugate vaccines, as well as with other classes of subunit antigen vaccines. QS-21 adjuvant is composed of two isomers that include the apiose and xylose forms in a ratio of 65:35, respectively. The chemical syntheses of these two isomers in pure form have recently been disclosed. Herein we describe detailed in vivo immunological evaluations of these synthetic QS-21 isomeric constituents, employing the GD3-KLH melanoma antigen. With this vaccine construct, high antibody titers against GD3 ganglioside and KLH were elicited when GD3-KLH was co-administered with adjuvant, either as the individual separate synthetic QS-21 isomers (SQS-21-Api or SQS-21-Xyl), or as its reconstituted 65:35 isomeric mixture (SQS-21). These antibody titer levels were comparable to that elicited by vaccinations employing naturally derived QS-21 (PQS-21). Moreover, toxicities of the synthetic saponin adjuvants were also found to be comparable to that of naturally derived PQS-21. These findings demonstrate unequivocally that the adjuvant activity of QS-21 resides in these two principal isomeric forms, and not in trace contaminants within the natural extracts. This lays the foundation for future exploration of structure-function correlations to enable the discovery of novel saponins with increased potency, enhanced stability, and attenuated toxicity.

In reiner Form wurde das komplexe Saponin QS‐21‐Xylose (der Xyloserest ist rot gezeigt) durch chemische Synthese erhalten. Die Verbindung wurde mit ihrem synthetischen Apioseisomer zum QS‐21(sQS‐21)‐Adjuvans kombiniert, das die Antikörperproduktion in Mäusen nach Injektion mit einer Melanomvakzine steigert. Die Herstellung von sQS‐21 eröffnet einen Zugang zu funktionellen Saponin‐Adjuvantien mit definierter Molekülzusammensetzung und ohne Verunreinigungen aus Naturstoff‐Isolierungsschritten.

We have tested a variety of naturally isolated and synthetic carbohydrate and peptide cancer antigens conjugated to keyhole limpet hemocyanin (KLH) plus an immunological adjuvant in pre clinical studies and clinical trials. The strength of the IgM and IgG antibody response was in each case determined by the potency of the immunological adjuvant, and the saponin fraction QS-21 was consistently the optimal adjuvant. However, there are draw backs to the use of QS-21 including local and systemic toxicity at immunologically relevant doses and poor stability at room temperature. Recent achievements in complex carbohydrate synthesis have enabled us to chemically synthesize a variety of saponin analogues. The effect of several of these synthetic saponin analogues were evaluated in mice in combination with GD3 ganglioside conjugated to KLH (GD3-KLH). Mice were immunized weekly for three weeks with GD3-KLH containing 10μg of GD3 alone, mixed with synthetic QS-21 (SQS-21) as positive control, or with 10, 20 or 50μg of synthetic QS-21 analogues SQS-101, SQS-102 or SQS-103 designed for improved stability as a consequence of amide modification. Toxicity (loss of weight) was monitored at 0h, 24h, 48h and 72h after each injection. Mice were bled 7 days after the third weekly vaccination and after the fourth (booster) vaccination on week 8. The sera were tested for the presence of antibody against GD3 or KLH by ELISA and against a tumor cell-line expressing GD3 antigen by FACS. We demonstrate that: 1) After vaccination with GD3-KLH alone, no IgM or IgG antibodies against GD3 were detected and the median titer of anti-KLH antibodies was 1/3200. 2) After vaccination with GD3-KLH plus 10 μg SQS-101, SQS-102 or SQS-103, or with positive control SQS-21, the boost antibody responses were similar with median anti-GD3 IgM titers of varies from 1/320 to 640, and anti-KLH IgG titers of 1/409,600 to 1,638,400. 3) SQS-21, SQS-101, SQS-102 and SQS-103, at the 50μg dose were each toxic with weight loss greater than 10% detected in most mice, but this was most extreme with SQS-102. The 10μg and 20μg dose of SQS-101 and 103, however, were well tolerated with no mice losing more than 10% weight. The 10μg and 20μg dose of SQS-102 mice lost more than 10% weight. 4) As expected anti-GD3 antibodies in each group were shown to react strongly with the cell surface of GD3 positive cancer cell lines by FACS. 5) These novel, amide-modified, non-natural synthetic saponin adjuvants selected for improved stability demonstrated adjuvant effects in vivo that equaled or exceeded that of SQS-21 and had comparable toxicity when mixed with a GD3-KLH conjugate vaccine. Supported by grants from the NIH (PO1 CA 52477) and Mr. William H. and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research” and “The Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center” and the Geoffrey Beene Cancer Research Center grant Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2417.