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.
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