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The overexpression of aberrantly glycosylated tumor-associated mucin-1 (TA-MUC1) in human cancers makes it a major target for the development of anticancer vaccines derived from synthetic MUC1-(glyco)peptide antigens. However, glycopeptide-based subunit vaccines are weakly immunogenic, requiring adjuvants and/or additional immunopotentiating approaches to generate optimal immune responses. Among these strategies, unimolecular self-adjuvanting vaccine constructs that do not need coadministration of adjuvants or conjugation to carrier proteins emerge as a promising but still underexploited approach. Herein, we report the design, synthesis, immune-evaluation in mice, and NMR studies of new, self-adjuvanting and self-assembling vaccines based on our QS-21-derived minimal adjuvant platform covalently linked to TA-MUC1-(glyco)peptide antigens and a peptide helper T-cell epitope. We have developed a modular, chemoselective strategy that harnesses two distal attachment points on the saponin adjuvant to conjugate the respective components in unprotected form and high yields via orthogonal ligations. In mice, only tri-component candidates but not unconjugated or di-component combinations induced significant TA-MUC1-specific IgG antibodies able to recognize the TA-MUC1 on cancer cells. NMR studies revealed the formation of self-assembled aggregates, in which the more hydrophilic TA-MUC1 moiety gets exposed to the solvent, favoring B-cell recognition. While dilution of the di-component saponin–(Tn)MUC1 constructs resulted in partial aggregate disruption, this was not observed for the more stably-organized tri-component candidates. This higher structural stability in solution correlates with their increased immunogenicity and suggests a longer half-life of the construct in physiological media, which together with the enhanced antigen multivalent presentation enabled by the particulate self-assembly, points to this self-adjuvanting tri-component vaccine as a promising synthetic candidate for further development.
Vaccination represents one of the key achievements in the history of medicine, making it possible to reduce the burden of life-threatening viral and bacterial diseases, and having saved millions of lives to date. More recently, in the context of cancer immunotherapy, impressive advances have been made over the last decades, enabling the development of a variety of strategies devoted to the selective targeting and clearance of malignant cells. Among these approaches, anticancer vaccines are a type of antigen-specific active immunotherapy aimed at triggering the patient's immune system to mount a tumor-selective, adaptive response that can lead to eradication of the tumor with minimal impact on neighboring healthy cells. The vast majority of cancer vaccines reported to date have incorporated tumor-associated antigens (TAAs) in their formulations. TAAs include differentiation antigens expressed only on tumor cells and the tissue of origin (e.g. prostate-specific antigen "PSA"), overexpressed antigens found on normal cells only at low levels (e.g. breast and ovarian "HER2/neu"), and cancer testis antigens, which are aberrantly expressed in a wide variety of cancer types restricted to reproductive tissues (e.g. melanoma antigen gene "MAGE"). Because of their shared expression profiles across many tumors and their ability to elicit cancer-specific cellular and humoral immunity, TAAs represent attractive targets in cancer immunotherapy. In particular, the well-known tumor-associated mucin-1 (TA-MUC1) oncoprotein (and its glycoforms), regarded as a high-ranked antigen by the National Cancer Institute pilot project, is being included in a growing number of experimental vaccines. Thus, established antigens such as the TA-MUC1 glycoprotein represent a gold standard to design improved strategies for the development of potent and safe anticancer vaccines that can lead to long-lasting, adaptive immune responses. A widely used approach to generate subunit vaccines based on homogeneous antigenic fragments exploits the use of immunogenic carrier proteins to enhance the immunogenicity of the covalently attached hapten as well as to activate helper T lymphocytes. On the other hand, fully-synthetic vaccines rely on modular chemical approaches whereby subunit components can be obtained separately and then subsequently assembled. These subunit vaccines are molecularly-defined minimal constructs, devoid of unnecessary elements that could negatively influence the immunological outcome. They are tractable and monodisperse entities compared to protein conjugates, enabling a more reliable characterization via benchmark laboratory techniques (e.g. mass spectrometry, liquid chromatography, nuclear magnetic resonance), while drastically minimizing batch-to-batch variations. Leveraging on synthetic building blocks and orthogonal ligation chemistries, unimolecular multicomponent anticancer vaccines can benefit from efficient "plug-and-play" design approaches that facilitate in vivo evaluation of various B-cell antigens, T-cell epitopes, adjuvants, linkers/spacers, and scaffolds.
Over the last few years, growing attention has been paid to the role of vaccine adjuvants, not only as enhancers but also as "directors" of the immune response. In addition to increasing the immunogenicity of the antigen, adjuvants can indeed shape the fate of the immune response either by unspecific means (e.g. emulsions, controlled release, nanomedicine), or via receptor-mediated mechanisms involving the innate immune system. While the large majority of vaccines are simply coformulated with adjuvants, self-adjuvanting vaccines are state-of-the-art constructs in which the antigen and the adjuvant modules are covalently attached within the same molecule, thus enabling their simultaneous uptake by the same antigen-presenting cell (e.g. B lymphocytes, dendritic cells), ultimately leading to enhanced antigen-directed immune responses. Pioneering examples of such fully-synthetic self-adjuvanting vaccines featuring the MUC1 tumor-associated antigen include those reported by Boons and co-workers based on the TLR1/TLR2 agonist Pam 3 CysSK 4, a design that inspired other research groups to develop further MUC1-based synthetic vaccine candidates with mixed outcomes.
In the framework of our research program on adjuvant development, we have applied a versatile semisynthetic strategy to develop a streamlined saponin platform inspired by the potent QS-21 natural product adjuvant. This includes one of our saponin lead compounds (2) and its free amine containing analogue 3 ("QA"), which is amenable to late-stage chemoselective functionalization. QS-21 is a purified saponin fraction extracted from the bark of the Quillaja saponaria (QS) tree that consists of a ≈ 2 : 1 mixture of isomers sharing the four main structural domains but differing at the terminal apiose (QS-21 Api = 1a), or xylose (QS-21 Xyl = 1b) residue. Despite its potent adjuvant activity and clinical promise, the scarcity, heterogeneity and dose-limiting toxicity of natural QS-21 prompted us to develop optimized synthetic saponin adjuvants overcoming such constraints. In a recent report, we exploited our minimal saponin platform 3, featuring a 6-aminohexanoic acyl chain, to covalently link the Tn carbohydrate antigen as a preliminary glycoconjugate design. Herein, we have gone one step further with the development of di- and tri-component, self-adjuvanting vaccine candidates that incorporate both peptide (MUC1) and glycopeptide (TnMUC1) TAAs, investigating the immunological and structural properties of the synthetic conjugates. The target constructs (4–7) were assembled using an expedient and efficient synthetic strategy involving one (for 4 and 5) or two (for 6 and 7) late-stage conjugation steps for final coupling of advanced, unprotected saponin and peptide building blocks. The immunological evaluation in mice showed that administration of tri-component vaccines 6 and 7 alone induced significantly higher antibody levels than their respective di-component constructs and/or combinations of their non-conjugated admixed components, eliciting antibodies that recognized the native TA-MUC1 antigen on the cancer cell surface. NMR studies provided early insights into the structural features of the conjugates in solution, showing the presence of self-assembled aggregates with a solvent-exposed multivalent (Tn)MUC1 display, and different concentration-dependent aggregation behaviors that correlated with their in vivo immunogenicity.
(A) QS-21 is a mixture of isomers comprising four principal domains: a branched trisaccharide, a central triterpene core (quillaic acid), a linear tetrasaccharide terminating in either apiose (1a) or xylose (1b), and a glycosylated diester acyl chain. Selected modifications based on structure–activity relationships enabled the streamlined chemical synthesis of homogeneous minimal variants endowed with increased stability, potent adjuvant activity and reduced toxicity. (B) Minimal saponin adjuvant 2 was obtained upon key structural modifications, including: branched trisaccharide deletion, ester-to-amide replacement and backbone simplification of the acyl chain, and truncation of the fourth sugar residue on the "eastern" domain. Minimal saponin scaffold 3 ("QA") features a shorter acyl chain terminating with a primary amine, providing a more suitable chemical handle for chemoselective conjugation strategies.
Structure of di-component (4 and 5), and tri-component (6 and 7) constructs evaluated as lead compounds in the present study. Conjugations of TA-(Tn)MUC1 antigens were performed at the acyl chain terminal amine, while the C4 aldehyde of the quillaic acid triterpene was derivatized with an oxime-functionalized helper T cell (Th) peptide epitope (KLFAV
