Antigen presentation frequently employs mucin-type glycopeptides since this antigen category delivers both innate immune benefits and structural advantages. MUC1 is a heavily O-glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on tumor cells. The short O-linked glycan, sialyl-Tn (STn) antigen, is also overexpressed on tumor cells. Due to their overexpression on tumors, MUC1 and the STn antigen have been well studied in the context of cancer immunotherapy. In particular, both MUC1-derived glycopeptides and STn glycopeptides have been used as antigens for immunization. As is the case for many vaccines, these antigens were conjugated to a carrier protein such as keyhole limpet hemocyanin (KLH) or CRM197. Mucin-type glycopeptides are one of the few glycopeptide-based antigens that can elicit a strong immune response and it is in part due to the conformational changes the peptides adopt. For instance, α-O-glycosylation of MUC1 forces the peptide into an extended conformational state that is different than the one adopted in the native protein. The natural conformation can therefore be taken advantage of in the design of antigen mimics of mucin-type proteins that have more potent immunological activity. Using a variety of modifications to a model mucin-type glycopeptide, it was shown that the binding to antibodies and antibody-induced immunogenicity could be improved. Simple changes, such as replacement of the glycosidic linkage with sulfur (S) or selenium (Se), were used to further optimize the peptide backbone for antibody binding. After using this optimized peptide, researchers showed an order of magnitude increase in immunogenicity when generating anti-MUC1 antibodies. Immunized mice were able to generate high titers of antibodies that recognize cancer cells in patient biopsy samples.
Fig. 1 Glycans in mammals.1,2
Synthetic Tn and STn-glycopeptides are under evaluation as cancer vaccines. Constructs using this approach are present in different stages of clinical development pipelines. For instance, immunization of preclinical models with MUC1 glycopeptides carrying the Tn antigen (defined here as sialyl-Tn carrying MUC1 peptides with a ThrGlySerSerSer sequence motif) induced very high levels of anti-MUC1 IgG (titers > 2,000,000) and the antibodies reacted with a panel of MUC1 glycopeptides that differ only by the peptide sequence (sialyl-Tn core is invariant). These antibodies could kill MUC1-expressing tumor cells in vitro and induced regression of established tumors in vivo. To increase the efficiency of carrier for the antigen, one strategy is the conjugation of the MUC1 glycopeptides to the bacteriophage Qβ, known to be an efficient carrier for many antigens. We showed that anti-MUC1 IgG responses after immunization with MUC1 conjugated to bacteriophage Qβ were stronger than those obtained with other carrier proteins such as KLH. The anti-MUC1 IgG antibodies obtained were able to mediate protection in an experimental metastatic model, where a dramatic decrease in the number of tumor foci was observed. In general, most of the synthetic Tn/STn-glycopeptides present in clinical development pipelines have been tested at the early-phase clinical trial stage.
Points to consider when designing immunogenic glycopeptides include epitope exposure and glycan positioning. An epitope (or antigenic determinant) is a portion of an antigen that is recognized by antibodies. Glycans attached to peptides can strongly influence the conformation and immunogenicity of the exposed epitope. Epitope accessibility to the immune system is an important parameter to consider in the design of glycopeptides that will result in the strongest immune response. The glycopeptide should be designed in a way that the epitope is exposed and will be effectively recognized by APCs and T cells. In some cases, the glycan can sterically shield the epitope. The binding of antibodies to the epitope should not be sterically hindered by the glycan. For mucin-type glycopeptides, the glycan may be able to adopt conformation that will either expose or shield the underlying peptide epitope. Design considerations include modifications to the glycan or peptide backbone to expose the epitope.
Scientists tested diverse recombinant proteins from various pathogens on mice as delivery systems for multiple fungal and meningococcal glycans with CRM197 serving as the standard measure. Some of them, like the pneumococcal recombinant spr 96/2021 and spr1875, and the Extra intestinal Pathogenic E. coli Upec-5211 and Orf3526 proteins, showed to be potential good carriers. A rationally designed recombinant protein, carrying strings of promiscuous human CD4+ T-cell epitopes from various pathogens (including tetanus, influenza virus, Plasmodium falciparum and hepatitis B virus), was demonstrated to be a very good carrier for Hib and meningococcal oligosaccharides. The recombinant tetanus toxin HC fragment has been used as carrier of synthetic fragments of O-PS of Vibrio cholerae O:1. In contrast to B cell epitopes, which are often of conformational nature, the T cell epitopes of proteins are linear sequences of a minimum of 8–12 amino acids that can bind to MHC class II and interact with T cell receptors on the surface of CD4+ T cells. Based on this consideration, many researchers have addressed the question if the complexity of protein carrier could be recapitulated by using synthetic peptides as carrier for carbohydrate antigens. A synthetic peptide representing the T-cell epitope of CRM197 was investigated as carrier of Hib carbohydrate antigen. A polio virus-derived synthetic peptide served as a T cell epitope in a synthetic three-component vaccine targeting cancer with tumor-associated glycopeptides. Synthetic peptides derived from Candida albicans were conjugated to a synthetic β-mannan trisaccharide and immunized in mice, eliciting antibodies specific for the carbohydrate and peptide moieties.
The initial proteins chosen as carrier for glycoconjugate vaccines were those already licensed for human vaccines and with a strong track record of safety in conjunction with availability at industrial scale, such as tetanus toxoid (TT) and diphtheria toxoid (DT). Safety and large-scale manufacturability are obviously key selection criteria also for new protein carriers as well as for all new vaccines intended for human use. Any toxic or enzymatic activity should be removed before testing a protein as new carrier. As learned from the history of tetanus and diphtheria toxoids, detoxification can be achieved by chemical treatment with formaldehyde, however this results in extensive modification and heterogeneity that renders more difficult product characterization and might limit the accessibility to some amino acid residues for conjugation. Protein carriers should contain a sufficient number of exposed amino acids targeted for the selected conjugation process, should be stable and have good solubility in the buffers and concentrations at which conjugation reactions take place. In the case of proteins engineered to insert one or more sites for saccharide attachment, these should be sufficiently surface exposed to allow efficient in vivo glycosylation or in vitro glyco-conjugation. In the case of the dual role carrier/protective antigen, the protein provides not only T cell helper epitopes, but also protective B cell epitopes; therefore, carrier selection and vaccine design become more complex and have to meet additional criteria as compared to the classical carrier role. Proper epitope mapping of the carrier protein during conjugation design helps preserve essential protective epitopes on the protein.
As a standard service in our glycopeptide cancer vaccine R&D package, we construct peptide-glycan libraries with the objective of screening and further optimizing tumor associated antigens of choice in a flexible and straightforward manner. They are comprised of mixtures of various glycopeptides featuring different glycans and peptides to increase the odds of getting a favorable response from the immune system to cancer cells. Custom library construction is based on techniques like solid-phase peptide synthesis (SPPS) and chemoenzymatic methods. Peptide-glycan libraries are built by incorporating protected glycosylserine/threonine residues into a growing peptide chain via the Fmoc-strategy SPPS followed by deprotection of the carbohydrates after cleavage from the resin. This enables the direct incorporation of tumor-associated carbohydrate antigens (TACAs) including TN, STn, T, and ST. In addition to classical SPPS, we also use the novel strategy of fragment condensation of suitably protected (glyco)peptide fragments that are activated at the C-termini as pentafluorophenyl esters. This has allowed us to successfully create self-adjuvanting vaccine candidates with good to excellent yields. Special attention is required in order to protect against undesirable reactions on protected lipopeptide fragments. Peptide-glycan libraries can be further expanded by using click chemistry which enables the simple and high-yield coupling of glycopeptides to lipopeptides or other functionalized molecules. This method also results in a higher purity of the end products.
The synthetic preparations of the tumor-specific glycopeptides represent an important part of our cancer vaccine services. The vaccine target is specifically identified and designed to mimic the natural antigen, thereby ensuring specificity and high efficacy. We design synthesis that ensures high purity and yield. Synthesis steps are precisely developed to decrease impurities while achieving the best possible yield for the target compound. The analytical quality control (QC) of the synthesis is done by high performance liquid chromatography (HPLC) and mass spectrometry (MS) to confirm the identity, purity, and homogeneity of the synthesized glycopeptides. The QC reports provide detailed information on the molecular weight, glycan composition, and peptide sequence of the glycopeptides, and they give an assurance of the quality of the synthesized glycopeptides. Customized analytical characterization services are available upon request. These services include glycan profiling, stability studies, and functional assays to determine the immunogenicity and efficacy of the glycopeptides. In addition to synthesis and QC, we also offer services for vaccine formulation and delivery, such as multivalent glycopeptide-lipopeptide vaccines and other delivery systems. These formulations are designed to enhance the immunogenicity and stability of the glycopeptides, and are optimized for use in preclinical and clinical settings.
Peptide Synthesis Services at Creative Peptides
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