Immunization of high-risk breast cancer patients with clustered sTn-KLH conjugate plus the immunologic adjuvant QS-21.

PURPOSE: To determine the clinical toxicities and antibody response against sTn and tumor cells expressing sTn following immunization of high-risk breast cancer patients with clustered sTn-KLH [sTn(c)-KLH] conjugate plus QS-21. EXPERIMENTAL DESIGN: Twenty-seven patients with no evidence of disease and with a history of either stage IV no evidence of disease, rising tumor markers, stage II (>or=4 positive axillary nodes), or stage III disease received a total of five injections each during weeks 1, 2, 3, 7, and 19. Immunizations consisted of sTn(c)-KLH conjugate containing 30, 10, 3, or 1 microg sTn(c) plus 100 microg QS-21. Induction of IgM and IgG antibodies against synthetic sTn(c) and natural sTn on ovine submaxillary mucin were measured before and after therapy. Fluorescence-activated cell sorting analyses assessed reactivity of antibodies to LSC and MCF-7 tumor cells. RESULTS: The most common toxicities were transient local skin reactions at the injection site and mild flu-like symptoms. All patients developed significant IgM and IgG antibody titers against sTn(c). Antibody titers against ovine submaxillary mucin were usually of lower titers. IgM reactivity with LSC tumor cells was observed in 21 patients and with MCF-7 cells in 13 patients. There was minimal IgG reactivity with LSC cells. CONCLUSION: Immunization with sTn(c)-KLH conjugate plus QS-21 is well tolerated and immunogenic in high-risk breast cancer patients. Future trials will incorporate sTn(c) as a component of a multiple antigen vaccine.

Monophosphoryl lipid A analogues with varying 3-O-substitution: synthesis and potent adjuvant activity.

Structurally defined immunostimulatory adjuvants play important roles in the development of new generation vaccines. Here described are the syntheses of three monophosphoryl lipid A analogues (1-3) with different substitution at 3-O-position of the reducing sugar and their potent immunostimulatory adjuvant activity. The syntheses involve the preparation of glycosylation acceptors benzyl 3,4-di-O-benzyl-2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-beta-D-glucopyranoside (16) and benzyl 3-O-allyl-4-O-benzyl-2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-beta-D-glucopyranoside (17). The glycosylation reactions between the donor 4,6-di-O-benzylidene-2-deoxy-2-(2',2',2'-trichloroethoxycarbonylamino)-alpha-d-glucopyranosyl trichloroacetimidate (21) and acceptors 16 and 17 provide the desired beta-(1-->6)-linked disaccharides 22 and 23, respectively. Selective reductive ring opening of the 4,6-di-O-benzylidene group, installation of a phosphate group to the 4'-hydroxyl group, and the final global debenzylation produce the designed monophosphoryl lipid A analogues 1-3. All three synthetic analogues induce antigen specific T-cell proliferation and interferon-gamma (IFN-gamma) production in ex vivo experiments with a totally synthetic liposomal vaccine system. The immunostimulatory potency of compound 1-3 is in the same order of magnitude as that of the detoxified natural lipid A product isolated from Salmonella minnesota R595 (R595 lipid A). The substituent at the 3-O-position of the reducing sugar does not have much effect on the adjuvant activity of monophosphoryl lipid A analogues. The preliminary lethal toxicity study indicates that the 3-O-acylated hepta-acyl monophosphoryl lipid A may not be more toxic than its 3-O-deacylated hexa-acyl analogue.

Synthetic vaccines: the role of adjuvants in immune targeting.

A clear understanding of the mechanism of function of immune stimulatory adjuvants, which commonly accompany vaccines, is beginning to emerge. Recent investigations have demonstrated that Toll-like receptors (TLRs) are the critical link between the innate and the adaptive immunity. This link, which is normally activated as a result of collaboration between adjuvants and TLRs in triggering adaptive immunity, has been a subject of several recent investigations. With the advent of well-defined synthetic small molecules, which are designed to either mimic the adjuvants or, as in many cases, to structurally represent pathogen associated molecular patterns, it is now possible to design reproducible experiments and to draw credible conclusions. An adjuvant alerts the host immune system through a mechanism similar to that of an infection by a pathogen, which involves interaction with a TLR followed by a lsqou;danger signal' to the immune system. Secretion of cytokines and regulation of the expression of co-stimulatory molecules induced by innate response shape the magnitude and quality of adaptive response. Synthetic vaccines containing specific epitopes to which immune responses are desired, are expected to be far superior in target specificity while the benefits may be long-lasting. The immune responses by therapeutic vaccines are generally adaptive in nature and such responses often require the participation of the components of innate immunity, most importantly the TLRs and their pathogen-associated binding compliments. Structurally well-defined synthetic molecules derived from lipid A, muramyl di-peptide (MDP), and CpG motifs from bacterial DNA offer a wide range of immune stimulants for the development of fully synthetic vaccines. Lipo-peptide and self-adjuvanted antigens, in combination with additional immune stimulatory adjuvants in liposome delivery system, may be important in vaccine design. Combinations of synthetic mimics of microbial products are known to display synergistic effects in stimulating the immune system. Either alone or in combination with chemotherapy, innate immune therapy using TLR ligands to stimulate the immune system may offer an alternate therapeutic approach against rapidly mutating viral infections-(HIV/AIDS), and cancers.

Lipid A structures containing novel lipid moieties: synthesis and adjuvant properties.

Structurally well-defined immune stimulatory molecules are important components of new generation molecular vaccines. In this paper, the design and synthesis of two lipid A analogues containing an unnatural tri-lipid acyl group are described. In a totally synthetic liposomal vaccine system, these re-designed lipid A analogues demonstrate potent immune stimulatory properties including antigen specific T-cell activation.

Effect of glycosylation on MUC1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions.

MUC1 mucin is a large transmembrane glycoprotein, of which the extracellular domain is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is underglycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above) as well as the normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc), and TF (Gal beta1-3 GalNAc) carbohydrates. In the present study, NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRP core epitope region to the recognition and binding of a monoclonal antibody, Mab B27.29, raised against the intact tumor-associated MUC1 mucin. Four peptides were studied: a MUC1 16mer peptide of the sequence Gly1-Val2-Thr3-Ser4-Ala5-Pro6-Asp7-Thr8-Arg9-Pro10-Ala11-Pro12-Gly13-Ser14-Thr15-Ala16, two singly Tn-glycosylated versions of this peptide at either Thr3 or Ser4, and a doubly Tn-glycosylated version at both Thr3 and Ser4. The results of these studies showed that the B27.29 MUC1 B-cell epitope maps to two separate parts of the glycopeptide, the core peptide epitope spanning the PDTRP sequence and a second (carbohydrate) epitope comprised of the Tn moieties attached at Thr3 and Ser4. The implications of these results are discussed within the framework of developing a glycosylated second-generation MUC1 glycopeptide vaccine.