SUMMARY Understanding how functional lipid domains in live cell membranes are generated has posed a challenge. Here, we show that transbilayer interactions are necessary for the generation of cholesteroldependent nanoclusters of GPI-anchored proteins mediated by membrane-adjacent dynamic actin filaments. We find that long saturated acyl-chains are required for forming GPI-anchor nanoclusters. Simultaneously, at the inner leaflet, long acyl-chaincontaining phosphatidylserine (PS) is necessary for transbilayer coupling. All-atom molecular dynamics simulations of asymmetric multicomponent-membrane bilayers in a mixed phase provide evidence that immobilization of long saturated acyl-chain lipids at either leaflet stabilizes cholesterol-dependent transbilayer interactions forming local domains with characteristics similar to a liquid-ordered (lo) phase. This is verified by experiments wherein immobilization of long acyl-chain lipids at one leaflet effects transbilayer interactions of corresponding lipids at the opposite leaflet. This suggests a general mechanism for the generation and stabilization of nanoscale cholesterol-dependent and actin-mediated lipid clusters in live cell membranes.
The problem of immunotolerance to GM3, an important tumor-associated trisaccharide antigen, seriously hinders its usage in cancer vaccine development. To solve this problem, the keyhole limpet hemocyanin (KLH) conjugates of a series of GM3 derivatives were synthesized and screened as therapeutic cancer vaccines. First, the β-linked anomeric azides of differently Nacylated GM3 analogs were prepared by a highly convergent procedure. Next, a pentenoyl group was linked to the reducing end of the carbohydrate antigens following selective reduction of the azido group. The linker was thereafter ozonolyzed to give an aldehyde functionality permitting the conjugation of the antigens to KLH via reductive amination. Finally, the immunological properties of the resultant glycoconjugates were studied in C57BL/6 mice by assessing the titers of specific antibodies induced by the GM3 analogs. While KLH-GM3 elicited low levels of immune response, the KLH conjugates of N-propionyl, N-butanoyl, N-iso-butanoyl and N-phenylacetyl GM3's induced robust immune reactions with antibodies of multiple isotypes, indicating significantly improved and T-cell dependent immune responses that lead to isotype switching, affinity maturation and the induction of immunological 'memory'. It was suggested that GM3PhAc-KLH is a promising vaccine candidate for glycoengineered immunotherapy of cancer with GM3 as the primary target.
Tumor-associated carbohydrate antigens (TACAs) are useful targets in the development of therapeutic cancer vaccines. However, a serious problem with them is the poor immunogenicity. To overcome the problem, a monophosphorylated derivative of Neisseria meningitidis lipid A was explored as a potential carrier molecule and built-in adjuvant for the construction of structurally defined fully synthetic glycoconjugate vaccines. Some paradigm-shifting discoveries about the monophosphoryl lipid A (MPLA)-TACA conjugates were that they elicited robust IgG antibody responses, indicating T cell-mediated immunity, without an external adjuvant and that an external adjuvant, e.g., Titermax Gold, actually reduced, instead of promoting, the immunological activity of the conjugates. The induced antibodies were proved to bind selectively to target tumor cells. MPLA was therefore demonstrated to be a powerful built-in immunostimulant and adjuvant for an all new design of fully synthetic glycoconjugate cancer vaccines.
Summary Tumor-associated carbohydrate antigens (TACAs) are important molecular markers on the cancer cell surface, useful for the development of therapeutic cancer vaccines or cancer immunotherapies. However, due to their poor immunogenicity and/or immunotolerance, most TACAs fail to induce T cell-mediated immunity that is critical for cancer therapy. This review summarizes the recent effort to overcome this problem via constructing TACA conjugates with improved immunogenicity, such as by covalently coupling TACAs to proper carrier molecules to form clustered or multi-epitopic conjugate vaccines, coupling TACAs to a T cell peptide epitope and/or an immunostimulant epitope to form fully synthetic multi-component glycoconjugate vaccines, and developing vaccines based on chemically modified TACAs, which is combined with metabolic engineering of cancer cells.
Fully synthetic carbohydrate-based cancer vaccine is an attractive concept, while an important topic in the area is to develop proper vaccine carriers that can improve the immunogenicity and other immunological properties of tumor-associated carbohydrate antigens (TACAs). In this context, four monophosphoryl derivatives of Neisseria meningitidis lipid A were synthesized via a highly convergent and effective strategy and evaluated as vaccine carriers and adjuvants. The conjugates of these monophosphoryl lipid A (MPLA) derivatives with a modified form of the sTn antigen were found to elicit high titers of antigen-specific IgG antibodies, indicating T cell-dependent immune response, in the absence of an external adjuvant. It was concluded that MPLA’s could be utilized as potent vaccine carriers and built-in adjuvants to create fully synthetic self-adjuvanting carbohydrate-based cancer vaccines. The lipid composition and structure of MPLA were shown to have a significant influence on its immunological activity, and among the MPLA’s examined, natural N. meningitidis MPLA exhibited the most promising properties. Moreover, Titermax Gold, a conventional vaccine adjuvant, was revealed to inhibit, rather than promote, the immunological activity of MPLA conjugates, maybe via interacting with MPLA text goes here.
Abnormal carbohydrates expressed on tumor cells, which are referred to as tumor-associated carbohydrate antigens (TACAs), are potential targets for development of cancer vaccines. However, immune tolerance to TACAs has severely hindered progress in this area. To overcome this problem, we have developed a novel immunotherapeutic strategy based on synthetic cancer vaccines and metabolic engineering of TACAs on tumor cells. One critical step of this new strategy is metabolic engineering of cancer, namely to induce expression of an artificial form of a TACA by supplying tumors with an artificial monosaccharide precursor. To identify the proper precursor for this application, N-propionyl, N-butanoyl, N-iso-butanoyl and N-phenylacetyl derivatives of Dmannosamine were synthesized, and their efficiency as biosynthetic precursors to modify sialic acid and induce expression of modified forms of GM3 antigen on tumor cells was investigated. For this purpose, tumor cells were incubated with different N-acyl-D-mannosamines, and modified forms of GM3 expressed on tumor cells were analyzed by flow cytometry using antigen-specific antisera. Nphenylacetyl-D-mannosamine was efficiently incorporated in a time and dose dependent manner to bioengineer GM3 expression by several tumor cell lines including K562, SKMEL-28 and B16-F0. Moreover, these tumor cell lines also showed ManPAc-dependent sensitivity to cytotoxicity medicated by anti-PAcGM3 immune serum and complement. These results provide an important validation for this novel therapeutic strategy. Because N-phenylacetyl GM3-protein conjugates are particularly immunogenic, the combination of an N-phenylacetyl GM3 conjugate vaccine with systemic N-phenylacetyl-D-mannosamine treatment is a promising immunotherapy for future development and application to melanoma and other GM3-bearing tumors. KeywordsMetabolic engineering; cancer vaccine; GM3; D-mannosamine; sialic acid Sialic acids are unique nine-carbon acidic monosaccharides ubiquitously distributed in high animals and human beings (1). The most common form of sialic acids is N-acetyl-D-neuraminic acid (Neu5Ac, also knows as N-acetyl sialic acid). Neu5Ac is a fascinating sugar in that it usually appears at the nonreducing end of cell surface glycans and thus plays forefront roles in various biological functions such as cell proliferation, immune recognition and pathogenic invasion (2-8).
A chemoenzymatic method was developed for the synthesis of macrocyclic peptides and glycopeptides. Sortase A was found to mediate either head to tail cyclization or oligomerization and then head to tail cyclization of peptides and glycopeptides, depending on the peptide length, to produce 15-mer or higher cyclic peptides and glycopeptides.
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