Vaccines prepared with sialyl-Tn and sialyl-Tn trimers using the 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide linker group result in optimal antibody titers against ovine submaxillary mucin and sialyl-Tn-positive tumor cells

Abstract

Sialyl-Tn (STn) is an O-serine- or O-threonine-linked disaccharide [NeuAcalpha(2-->6)GalNAcalpha-O-Ser/Thr) expressed on mucins of most types of adenocarcinoma as single STn or clustered STn [STn(c)] epitopes. Though STn is expressed on some normal tissues it is relatively tumor-specific, especially in the clustered conformation. Clinical trials with STn-keyhole limpet hemocyanin (KLH) conjugate vaccines, prepared using reductive amination with a two-carbon linker group, have resulted in high titers against STn but lower titers against natural forms of STn (ovine submaxillary mucin, or tumor cells). To obtain antibodies of more appropriate specificity, we attempted to prepare STn(c)-KLH conjugates to establish their immunogenicity in mice in preparation for clinical trials; however, conjugation efficiency was poor when the same two-carbon linker was used, presumably because of steric hindrance. STn-KLH and STn(c)-KLH conjugates were prepared using the regular two-carbon or the recently developed more efficient longer heterobifunctional 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) linkers, and the resulting immunogenicities in mice were compared. The highest titers against STn were seen with the STn-KLH conjugate with the two-carbon linker, and the highest titers against STn(c) were seen with STn(c)-KLH with the MMCCH linker. Conjugation with MMCCH resulted in the highest conjugation efficiency (yield) and the highest titers against ovine submaxillary mucin and STn-positive tumor cells, and is the method of choice for the preparation of STn(c) vaccine for clinical trials.

Immunogenicity in a Mouse Model of a Conjugate Vaccine Made with a Synthetic Single Repeating Unit of Type 14 Pneumococcal Polysaccharide Coupled to CRM197

“…The results reported here show that synthetically accessible OSs can be used to prepare immunogenic conjugates, in accordance with the findings with synthetic S. pneumoniae type 6B di-, tri-, and tetrasaccharide-protein conjugates (14), synthetic S. pneumoniae type 3 di-, tri-, and tetrasaccharide-CRM197 conjugates (4), or antitumor therapeutics (11,28,29,34,37). This approach will eventually allow the preparation of well-defined conjugates using OSs related to bacterial PSs for which no simple degradative scheme can be devised and the stimulation of immune responses against the most important protective epitopes while avoiding potentially damaging autoimmune responses.…”

Improving the Antigenicity of sTn Antigen by Modification of Its Sialic Acid Residue for Development of Glycoconjugate Cancer Vaccines

“…Second, the expression of sTn has been identified as an independent indicator for poor prognosis of cancer (11)(12)(13)(14). Consequently, sTn antigen and relevant mucins that are aberrantly glycosylated with sTn have been extensively investigated for the immunotherapy of cancer (15)(16)(17)(18)(19)(20). The protein conjugates of sTn antigen could indeed elicit humoral immune response that showed remarkable specificity for cancer cells (21).…”

A Fully Synthetic Therapeutic Vaccine Candidate Targeting Carcinoma-Associated Tn Carbohydrate Antigen Induces Tumor-Specific Antibodies in Nonhuman Primates

“…For instance, the N-acetylgalactosaminyltransferase T3, responsible for the glycosylation of consecutive threonine residues, is overexpressed in adenocarcinomas leading to the expression of Tn clusters (20). Therefore, the strategy of carbohydrate clustering has greatly improved the immunogenicity of these short haptenic molecules allowing the recognition of native carbohydrate structures on tumor cells (21)(22)(23).…”