Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses.

Abstract

The induction of CD8+ cytotoxic T lymphocytes (CTLs) is desirable for immunization against many diseases, and recombinant-synthetic peptide antigens are now favored agents to use. However, a major problem is how to induce CTLs, which requires a Ti-type response to such synthetic antigens. We (refs. 4 and 5; unpublished data). The unusual finding is that if the conjugation is done under oxidizing conditions, then cell-mediated immunity is selectively stimulated, compared with using reducing conditions for conjugation when antibody responses occur.A successful vaccine for cancer immunotherapy requires a suitable target antigen and production of cytotoxic T lymphocyte (CTL) responses (6). In adenocarcinomas, it appears that mucins, particularly MUC1, may provide a suitable target as there is a 10-fold increase in mucin expression on the cell surface, and altered glycosylation leads to exposure of normally hidden peptide sequences (e.g., APDTR). These changes in cancer generate a potential new target for immunotherapy that is apparently absent in normal mucin (7-10). The APDTR sequence [from the variable number of tandem repeats (VNTR) region] is immunogenic in mice, leading to antibody formation, whether the antigen is administered as MUC1+ cancer cells, purified mucin (HMFG), or as peptides (11,12). Such studies of immunogenicity in mice would be of little relevance to humans were it not for the findings that tumorspecific CTL precursors (CTLp) exist in the lymph nodes of patients with cancer of breast, ovary, or pancreas, or in multiple myeloma patients (13-16). The CTLp can be stimulated by antigen and interleukin 2 (IL-2) in vitro to become functional CD8+ CTLs, the target antigen being the APDTR sequence of MUC1 (13-16 MATERIALS AND METHODSChemical Studies. MUC1 FP containing 5 VNTR of human MUC1 and peptide (Cpl3-32) were produced as described (2). FP was conjugated to mannan in two ways. (i) Ox-M-FP: Mannan (Sigma), at 14 mg/ml in 0.1 M phosphate buffer (pH 6.0), was oxidized with sodium periodate (0.01 M) for 60 min at 40C. Ethanediol (10 ,ul) was added and incubated for a further 30 min at 4°C, and the mixture was passed through a Sephadex-G25 column equilibrated in bicarbonate buffer (pH 6.0-9.0). The oxidized mannan that eluted in the void volume (2 ml) was mixed with 900 gg of MUC1 FP, incubated overnight at room temperature, and used without further purification. (ii) Reduced (Red)-M-FP: The ox-M-FP mixture was treated with sodium borohydride (1 mg/ml) for 3 hr and used without further purification.Immunizations 10128The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The Adjuvanticity of a Mannosylated Antigen Reveals TLR4 Functionality Essential for Subset Specialization and Functional Maturation of Mouse Dendritic Cells

“…The functionality of TLR4 in MDO presentation may also elucidate the adjuvant mechanism of oxidized mannan-conjugated fusion mucin 1 protein (MFP), of which the adjuvanticity is also TLR4-mediated (24,34). MFP has been used for immunotherapy in phase III cancer clinical trails (35).…”

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo

“…Extensive O-mannosylation of proteins greatly enhanced antigen delivery, and deglycosylation profoundly inhibited T cell responses [7]. The role of mannose residues in antigen delivery was also shown by the mannose polymer, mannan, which enhanced antigen-specific Th1/CTL and Th2/antibody responses in oxidized and reduced forms, respectively [9][10][11][12][13][14][15]. Oxidized mannan conjugated to recombinant MUC1 fusion protein has been used in Phase II/III cancer clinical trials [16][17][18][19].…”

Antigenic Targeting of the Human Mannose Receptor Induces Tumor Immunity

“…In vitro studies have shown that MR targeting can enhance the efficiency of MHC class II presentation to Ag-specific T cells by several orders of magnitude, relative to nontargeted Ag (17,18). In addition, conjugation of the human tumor Ag (mucin 1) to oxidized-mannan induced Th1 and CTL responses and complete tumor protection in mice (19,20). However, the overlapping pattern recognition of mannose-type ligands between MR and other CLRs, such as DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), makes the interpretation of the MR contribution to the observed immune responses difficult (3,21).…”