CN106215179A - Immunogenic vaccine - Google Patents

Abstract

The present invention discloses glycolipopeptides comprising carbohydrate components, lipid components and MUCl peptide components for use as therapeutic or prophylactic vaccines, which induce both humoral and cellular immune responses.

Description

immunogenic vaccine

This application is a divisional application of an invention patent with an application date of June 10, 2011, an application number of 201180039067.0, and an invention title of "immunogenic vaccine". This application claims the benefit of U.S. Provisional Application Serial No. 61/354,076, filed June 11, 2010, and U.S. Patent Application Serial No. 13/002,180, filed December 30, 2010, each of which is incorporated by reference in its entirety Incorporated into this article.

Statement of Government Rights

This invention was made with Government support under Grant No. R01 CA088986 awarded by the National Cancer Institute, National Institutes of Health. The US Government has certain rights in this invention.

background

A large number of tumor-associated carbohydrate antigens (TACAs) are expressed on human cancer cells in the form of glycolipids and glycoproteins. A common feature of oncogenically transformed cells is the overexpression of oligosaccharides such as Globo-H, Lewis ^Y and Tn antigens. Numerous studies have shown that this aberrant glycosylation can promote metastasis, and thus it is strongly associated with poor survival of cancer patients.

The differential expression that characterizes these tumor-associated carbohydrate antigens makes them attractive targets for immunotherapy and cancer vaccine development. Recently, several leading studies have attempted to exploit the differential expression of tumor-associated carbohydrates for the development of cancer vaccines (e.g. Raghupathi, 1996, Cancer Immunol; 43:152-157; Musselli et al., 2001, J Cancer Res Clin Oncol; 127: R20-R26; Sabbatini et al., 2000, IntJ Cancer; 87:79-85; Lo-Man et al., 2004, Cancer Res; 64:4987-4994; Kagan et al., 2005, Immunol Immunother; ).

Carbohydrate antigens are also abundant on the surface of the human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS). Hepatitis C virus (HCV) is also known to contain carbohydrate antigens.

For most immunogens (including carbohydrates), antibody production depends on the cooperative interaction of two types of lymphocytes, B cells and helper T cells. However, carbohydrates alone are unable to activate helper T cells and are therefore characterized as weakly immunogenic. This limited immunogenicity is reflected in the formation of low-affinity IgM antibodies and the absence of IgG antibodies. The immune tolerance that characterizes these antigens has proven difficult to overcome.

In efforts to activate helper T cells, researchers have conjugated carbohydrate antigens to exogenous carrier proteins such as keyhole limpet hemocyanin (KLH) or detoxified tetanus toxoid (TT). The carrier protein enhances the presentation of carbohydrates to the immune system and supplies T epitopes (typically 12-15 amino acid peptide fragments) that can activate T helper cells.

However, the conjugation of carbohydrates to carrier proteins presents several new problems. Conjugation chemistry is difficult to control, resulting in conjugates with compositional and structural ambiguities that can affect the reproducibility of immune responses. Furthermore, exogenous carrier proteins can elicit strong B cell responses, which in turn can lead to suppression of antibody responses to carbohydrate epitopes. The latter is particularly problematic when employing self-antigens such as tumor-associated carbohydrates. Furthermore, linkers used to conjugate carbohydrates to proteins may themselves be immunogenic, leading to epitope suppression. See also McGeary et al. (J. Peptide Sci. 9(7):405-418, 2003) for a review of lipid and carbohydrate based adjuvants/carriers in vaccines.

Not surprisingly, several clinical trials with carbohydrate-protein conjugate cancer vaccines have failed to induce sufficiently strong helper T cell responses in all patients. Therefore, there is a need to develop alternative strategies for presenting tumor-associated carbohydrate epitopes that will lead to more efficient class switching to IgG antibodies. These strategies may prove useful for the development of vaccines based on other carbohydrate epitopes as well, especially those from pathogenic viruses such as HIV and HCV.

Summary of the invention

The invention includes a method of generating antibody-dependent cell-mediated cytotoxicity (ADCC) in a subject, the method comprising immunizing the subject with a glycolipepeptide comprising: B cells at least one glycosylated MUCl glycopeptide component of the epitope; at least one peptide component comprising an MHC class II-restricted helper T cell epitope; and at least one lipid component. In some aspects, ADCC is natural killer (NK) cell mediated. In some aspects, ADCC lyses tumor cells. In some aspects, the tumor cells are breast cancer cells or epithelial cancer cells. In some aspects, ADCC lyses cells expressing the MUCl peptide sequence. In some aspects, the MUCl peptide is abnormally glycosylated.

The present invention includes a method of treating cancer in a subject comprising immunizing the subject with a glycolipopeptide comprising: at least one glycosylated MUC1 glycopeptide component comprising a B cell epitope ; at least one peptide component comprising an MHC class II restricted helper T cell epitope; and at least one lipid component.

The present invention includes a method of reducing tumor burden in a subject, the method comprising immunizing the subject with a glycolipid peptide comprising: at least one glycosylated MUC1 glycopeptide set comprising a B cell epitope components; at least one peptide component comprising an MHC class II restricted helper T cell epitope; and at least one lipid component.

The present invention includes a method of preventing tumor recurrence in a subject, the method comprising immunizing the subject with a glycolipid peptide comprising: at least one glycosylated MUC1 glycopeptide panel comprising a B cell epitope components; at least one peptide component comprising an MHC class II restricted helper T cell epitope; and at least one lipid component.

The present invention includes a method of preventing cancer in a subject comprising immunizing the subject with a glycolipid peptide comprising: at least one glycosylated MUC1 glycopeptide component comprising a B cell epitope ; at least one peptide component comprising an MHC class II restricted helper T cell epitope; and at least one lipid component.

In some aspects of the methods of the invention, the cancer or tumor is breast cancer or epithelial cancer.

In some aspects of the methods of the invention, the cancer or tumor expresses aberrantly glycosylated MUCl.

The present invention includes a method of generating a cytotoxic T cell response against MUCl expressing cells in a subject, the method comprising immunizing the subject with a glycolipid peptide comprising at least one B cell epitope comprising: a glycosylated MUCl glycopeptide component; at least one peptide component comprising an MHC class II-restricted helper T cell epitope; and at least one lipid component. In some aspects, the MUCl expressing cells are tumor cells.

The invention includes a method of promoting class switching of an anti-MUCl antibody in a subject comprising immunizing the subject with a glycolipid peptide comprising at least one glycosylation comprising a B cell epitope a MUCl glycopeptide component; at least one peptide component comprising an MHC class II-restricted helper T cell epitope; and at least one lipid component.

The present invention includes a method of immunizing a subject comprising immunizing the subject with a glycolipopeptide comprising: at least one glycosylated MUC1 glycopeptide component comprising a B cell epitope; comprising at least one peptide component of an MHC class II-restricted helper T cell epitope; and at least one lipid component; wherein antibodies of the IgG subtype that specifically bind to a MUC1 protein expressed on tumor cells are induced in the subject .

In some aspects of the methods of the invention, the glycosylated MUCl glycopeptide component comprising the B cell epitope comprises glycosylation on one or more serine and/or threonine residues.

In some aspects of the methods of the invention, the glycosylated MUCl glycopeptide component comprising a B cell epitope comprises glycosylation with a sugar residue selected from the group consisting of: GalNAc, GlcNAc, Gal, NANA, NGNA, Rock Alcohol, Mannose and Glucose.

In some aspects of the methods of the invention, the glycolipopeptide comprises one of those shown in FIG. 19 .

In some aspects of the methods of the invention, the glycosylated MUCl glycopeptide component comprising the B cell epitope is an MHC class I restricted epitope.

In some aspects of the methods of the invention, the glycosylated MUCl glycopeptide component comprising B cell epitopes and/or the peptide component comprising MHC class II restricted helper T cell epitopes comprises human MUCl peptide sequences.

In some aspects of the methods of the invention, the glycosylated MUCl glycopeptide component comprising B-cell epitopes and/or the peptide component comprising MHC class II-restricted helper T-cell epitopes is included in a mixture with the subject's endogenous MUCl Amino acid sequences with sequence homology.

In some aspects of the methods of the invention, the glycosylated MUCl glycopeptide component comprising B cell epitopes and/or the peptide component comprising MHC class II restricted helper T cell epitopes comprises about 5-30% of the MUCl protein sequence amino acids, the MUCl protein sequence includes the extracellular region of the MUCl protein, and includes one or more glycosylated serine or threonine residues.

In some aspects of the methods of the invention, the MUCl glycopeptide component comprising a B-cell peptide epitope is comprised of a combination with SAPDTRPAP (SEQ ID NO: 20), TSAPDRPAP (SEQ ID NO: 21), SAPDTRPL (SEQ ID NO: 22) or TSAPDTRPL (SEQ ID NO: 23) has an amino acid sequence with at least about 50% sequence identity. In some aspects, the amino acid sequence includes glycosylation on one or more serine and/or threonine residues. In some aspects of the methods of the invention, the MUCl glycopeptide component comprising a B cell peptide epitope comprises SAPDTRPAP (SEQ ID NO:20), TSAPDTRAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO:22) or TSAPDTRPL (SEQ ID NO: 23). In some aspects, the amino acid sequence includes glycosylation on one or more serine and/or threonine residues.

In some aspects of the methods of the invention, the lipid component comprises one or more lipid chains, one or more cysteine residues, and one or more lysine residues.

In some aspects of the methods of the invention, the lipid component includes a Toll-like receptor (TLR) ligand. In some aspects, Toll-like receptor (TLR) ligands include TLR2 ligands. In some aspects, the TLR2 ligand includes Pam3CysSK4.

In some aspects of the methods of the invention, the lipid component includes the TLR9 agonist Pam3CysSK4. In some aspects of the methods of the invention, the lipid component includes a lipid adjuvant. In some aspects, lipid adjuvants include lipidated amino acids (LAAs).

In some aspects of the methods of the invention, the peptide component comprising an MHC class II restricted helper T cell epitope comprises the poliovirus sequence KLFAVWKITYKDT (SEQ ID NO:3).

In some aspects of the methods of the invention, the peptide component comprising an MHC class II restricted helper T cell epitope comprises a T cell pan DR epitope PADRE sequence AKFVAAWTLKAAA (SEQ ID NO:24) or FVAAWTLKAAA (SEQ ID NO:25) .

In some aspects of the methods of the invention, the peptide component comprising an MHC class II restricted helper T cell epitope comprises a MUCl derived MHC class II restricted helper T cell peptide epitope. In some aspects, the MUCl-derived B-cell peptide epitope and the MUCl-derived MHC class II-restricted helper T-cell peptide epitope comprise contiguous amino acid sequences. In some aspects, the contiguous amino acid sequence is glycosylated on one or more threonine and/or serine residues.

In some aspects of the methods of the invention, the MUCl-derived B-cell peptide epitope and the MUCl-derived MHC class II-restricted helper T-cell peptide epitope comprise contiguous amino acid sequences. In some aspects, the contiguous amino acid sequence comprises at least about 50 Sequences with % sequence identity. In some aspects, the contiguous amino acid sequence comprises the amino acid sequence APGSTAPPAHGVTSA (SEQ ID NO: 26), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO: 27), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO: 28), or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO: 29). In some aspects, the contiguous amino acid sequence is glycosylated at one or more threonine and/or serine residues. In some aspects of the methods of the invention, the glycolipopeptide is administered as liposomes. In some aspects, the lipid component of the glycolipopeptide promotes liposome formation.

In some aspects of the methods of the invention, the methods comprise further administering an immunomodulator. In some aspects, a composition comprising a glycolipopeptide and an immunomodulator is administered. In some aspects, the immunomodulatory agent is covalently linked to the glycolipopeptide. In some aspects, immunomodulators include TLR agonists. In some aspects, TLR agonists include TLR9 agonists. In some aspects, the TLR9 agonist comprises CpG. In some aspects, the immunomodulator is a TLR9 agonist, a COX-2 inhibitor, GM-CSF, an inhibitor of indoleamine 2,3-dioxygenase (IDO), a chemotherapeutic agent, or a combination thereof.

The invention includes glycolipopeptides comprising: at least one glycosylated MUCl glycopeptide component comprising a B-cell epitope; at least one peptide component comprising a MUCl-derived MHC class II-restricted helper T-cell epitope; and at least one lipid component. In some aspects of the glycolipopeptides of the invention, the glycosylated MUCl glycopeptide component comprising the B-cell epitope comprises glycosylation on one or more serine and/or threonine residues.

In some aspects of the glycolipopeptides of the invention, the glycosylated MUC1 glycopeptide component comprising the B cell epitope comprises glycosylation with sugar residues including GAlNAc, GlcNAc, Gal, NANA, NGNA, fucose, mannose or glucose.

In some aspects of the glycolipopeptides of the invention, the glycosylated MUCl glycopeptide component comprising the B cell epitope is an MHC class I restricted epitope.

In some aspects of the glycolipopeptides of the invention, the glycosylated MUCl glycopeptide component comprising B-cell epitopes and/or the peptide component comprising MHC class II-restricted helper T-cell epitopes comprises human MUCl peptide sequences.

In some aspects of the glycolipopeptides of the invention, the glycolipopeptide comprising a B-cell epitope and/or the peptide component comprising an MHC class II-restricted helper T-cell epitope comprises about 5-30 amino acids of the MUC1 protein sequence, The MUCl protein sequence includes the extracellular region of the MUCl protein and includes glycosylated one or more serine or threonine residues.

In some aspects of the glycolipopeptides of the invention, the glycosylated MUC1 glycopeptide component comprising a B-cell epitope comprises a combination with SAPDTRPAP (SEQ ID NO:20), TSAPDTRAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO :22) or TSAPDTRPL (SEQ ID NO: 23) amino acid sequences having at least about 50% sequence identity. In some aspects, a glycosylated MUCl glycopeptide component comprising a B cell epitope comprises glycosylation on one or more serine and/or threonine residues.

In some aspects of the glycolipopeptides of the invention, the glycosylated MUC1 glycopeptide component comprising a B cell epitope comprises SAPDTRPAP (SEQ ID NO:20), TSAPDTRAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO: 22) or TSAPDTRPL (SEQ ID NO: 23). In some aspects, a glycosylated MUCl glycopeptide component comprising a B cell epitope comprises glycosylation on one or more serine and/or threonine residues.

In some aspects of the glycolipopeptides of the invention, the lipid component includes one or more lipid chains, one or more cysteine residues, and one or more lysine residues.

In some aspects of the glycolipopeptides of the invention, the lipid component includes a Toll-like receptor (TLR) ligand. In some aspects, Toll-like receptor (TLR) ligands include TLR2 ligands. In some aspects, the TLR2 ligand includes _{Pam3CysSK4 .}

In some aspects of the glycolipopeptides of the invention, the lipid component includes a lipid adjuvant. In some aspects, lipid adjuvants include lipidated amino acids (LAAs).

In some aspects of the glycolipopeptides of the invention, the MUCl-derived B-cell peptide epitope and the MUCl-derived MHC class II-restricted helper T-cell peptide epitope comprise contiguous amino acid sequences.

In some aspects of the glycolipopeptides of the invention, the contiguous amino acid sequence comprises the amino acid sequence APGSTAPPAHGVTSA (SEQ ID NO:26), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28). :29) Sequences having at least 50% sequence identity. In some aspects, the contiguous amino acid sequence is glycosylated on one or more threonine and/or serine residues.

In some aspects of the glycolipopeptides of the invention, the contiguous amino acid sequence comprises the amino acid sequence APGSTAPPAHGVTSA (SEQ ID NO:26), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO: 29). In some aspects, the contiguous amino acid sequence is glycosylated on one or more threonine and/or serine residues.

In some aspects of the glycolipopeptides of the invention, the glycolipopeptides include any of those shown in FIG. 19 . In some aspects, the amino acid sequence is glycosylated on one or more threonine and/or serine residues.

In some aspects, the glycolipopeptide further includes a covalently linked immunomodulator. In some aspects, immunomodulators include TLR9 agonists, COX-2 inhibitors, GM-CSF, inhibitors of indoleamine 2,3-dioxygenase (IDO), chemotherapeutic agents, or combinations thereof.

The present invention includes pharmaceutical compositions comprising: a glycolipopeptide as described herein and a pharmaceutically acceptable carrier.

The invention includes compositions comprising liposomes comprising a glycolipopeptide as described herein. In some aspects, the lipid component of the glycolipopeptide promotes liposome formation. In some aspects, the composition further comprises an immunomodulator. In some aspects, immunomodulators include TLR agonists. In some aspects, TLR agonists include TLR9 agonists. In some aspects, the TLR9 agonist comprises CpG.

In some aspects, immunomodulators include TLR9 agonists, COX-2 inhibitors, GM-CSF, inhibitors of indoleamine 2,3-dioxygenase (IDO), chemotherapeutic agents, or combinations thereof.

The invention includes an immunogenic vaccine comprising a glycolipopeptide as described herein or a composition as described herein.

The invention includes the use of a glycolipopeptide as described herein or a composition as described herein for the manufacture of a medicament for treating or preventing an infection, disease or condition.

The invention includes a kit comprising: a glycolipopeptide as described herein; packaging; and instructions for use.

"A", "an", "the" and "at least one" are used interchangeably and mean one or more than one unless stated otherwise.

Brief description of the drawings

Figure 1 shows exemplary glycolipopeptides of the invention.

Figure 2 shows flow cytometric analysis for specific anti-MUC-1 antibodies. Reactivity was tested on MCF-7 (A) and SK-MEL-28 (B) cells. The fluorescence intensity of sera (diluted 1:50) was assessed before (serum control; open peaks) and after (closed peaks) immunization with 3.

Figure 3 shows TNF-[alpha] production by murine macrophages after stimulation with LPS and synthetic compounds. Mouse RAWγNO(-) cells were incubated with increasing concentrations of Escherichia coli ( E. coli ) LPS (■), 1 (●), Pam ₂ CysSK ₄ (▼), 2 (♦), Pam ₃ CysSK ₄ ( ▲) or 3 (□) were incubated together for 5.5 hours.

Figure 4 shows the effect of TLR ligands on cellular uptake.

Figure 5 shows the chemical structure of the synthetic antigen.

Figure 6 shows TNF-[alpha] and IFN-[beta] production by murine macrophages after stimulation with synthetic compounds 21-24, E. coli LPS and E. coli lipid A. Murine 264.7 RAW γNO(-) cells were incubated for 5.5 hours with increasing concentrations of 21-24, E. coli LPS or E. coli lipid A as indicated. TNF-α (A) and IFN-β (B) in cell supernatants were measured using ELISA. Data represent mean ± SD (n=3).

Figure 7 shows the analysis of cell recognition for specific anti-MUCl antibodies. Serum reactivity was tested on MCF7 cells. Serial dilutions of serum samples after four immunizations with 21 (A), 22/23 (B) or 22/24 (C) were incubated with MCF7 cells. Fluorescence intensity was assessed in cell lysates after incubation with FITC-labeled anti-mouse IgG antibody. No fluorescence relative to background was observed with pre-immunization serum and serum samples incubated with control SK-MEL-28 cells (shown in Figure 9). AU indicates arbitrary fluorescence units.

Figure 8 shows ELISA anti-MUCl and anti-T epitope antibody titers after 4 immunizations with 21, 22, 22/23, 22/24 and 25/26. ELISA plates were coated with BSA-MI-MUC-1 conjugates (A-F) or neutravidin-biotin-T epitope (G) and plotted against absorbance by linear regression analysis. The titer was determined by the ratio of the dilution. Titer was defined as the highest dilution to obtain an optical density of 0.1 or greater relative to normal control mouse serum. Each data point represents the titer of an individual mouse after 4 immunizations, and horizontal lines indicate the mean of a group of five mice.

Figure 9 shows cell recognition assays for specific anti-MUC-1 antibodies. Serum reactivity was tested on MCF7 and SK-MEL-28 cells. Serum samples (diluted 1:30) after 4 immunizations with 21, 22/23 or 22/24 were incubated with MCF7 and SK-MEL-28 cells. Fluorescence intensity was assessed in cell lysates after incubation with FITC-labeled anti-mouse IgG antibody. Also shown are media, conjugate and mouse (normal control mouse serum) controls. Data represent mean ± SD (n=3). AU indicates arbitrary fluorescence units.

Figure 10 shows compound 22.

Figure 11 shows compound 23.

Figure 12 shows compound 25.

Figure 13 shows compound 26.

Figure 14 shows compound 27.

Figure 15 shows the structure of the fully synthetic three-component immunogen.

Figure 16: Chemical structures of synthetic antigens 1, 2, 3, 4 and 5.

Figure 17: Reduction of MMT tumor burden in MUC1.Tg mice by three-component vaccine. MUC1.Tg mice were immunized with empty liposomes (EL) or liposomes containing 1, 2, 3, 4 + 5 or 5 (25 μg with 3 μg carbohydrate) as controls. The chemical structures of synthetic antigens 1, 2, 3, 4 and 5 are shown in FIG. 16 . Three biweekly immunizations were given before tumor challenge with MUCl expressing MMT tumor cells ( ¹ x 106 cells), followed by a booster one week later. Animals were sacrificed 7 days after the last injection, and tumor wet weights were determined. Data are presented as percentage of control (mice vaccinated with empty liposomes). Each data point represents an individual mouse, and the horizontal line indicates the mean of the group of mice.

Figures 18A and 18B: Induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Tumor cells Yac-MUC1 ( FIG. 18A ) and C57mg.MUC1 ( FIG. 18B ) were labeled with chromium for 2 hours and then incubated with serum from mice (diluted 1:50) at 37° C. for 30 minutes. Mice were immunized with empty liposomes (EL) or liposomes containing 1, 2, 3, 4+5 or 5 with or without (NT) tumor induction as indicated. The chemical structures of synthetic antigens 1, 2, 3, 4 and 5 are shown in FIG. 16 . Tumor cells were then incubated with effector cells (NK cell KY-1 clone) for 4 hours. The effector to target ratio was 50:1. Spontaneous release was less than 15% of complete release. Each data point represents an individual mouse, and the horizontal line indicates the mean of the group of mice.

Figures 19A to 19C: Cellular responses. Figure 19A measures IFN-γ producing CD8 ⁺ T cells in MUCl.Tg mice. CD8 ⁺ T cells from either empty liposomes (EL) or lipids containing 1, 2, 3, 4+5, or 5 were analyzed for MUCl-specific IFN-γ puncta formation ^. Bodies were isolated from lymph nodes of mice immunized with or without (NT) tumor induction. The chemical structures of synthetic antigens 1, 2, 3, 4 and 5 are shown in FIG. 16 . Each data point represents an individual mouse, and the horizontal line indicates the mean of the group of mice. Figure 19B measures the induction of CD8 ⁺ cytotoxic T cells in MUCl.Tg mice. From lymph nodes of mice immunized with empty liposomes (EL) or liposomes containing 1, 2, 3, 4 + 5, or 5 with or without (NT) tumor induction as indicated CD8 ⁺ T cells were isolated ^and subjected ^to51Cr release assay without any in vitro stimulation. DCs pulsed with MUCl(Tn) peptide 6 (SAPDT(Tn)RPAP) (SEQ ID NO: 26) were used as targets for 1 (NT), 1, 3, 4+5 and 5, and for 2 with MUCl Peptide 7 (SAPDTRPAP) (SEQ ID NO:20) pulsed DCs were used as targets or DCs pulsed with empty liposomes for EL were used as targets. The effector to target ratio was 100:1 because CTLs were not stimulated in vitro. Spontaneous release was less than 15% of complete release. Each data point represents an individual mouse, and the horizontal line indicates the mean of the group of mice. Figure 19C determines the epitope requirement of CD8 ⁺ T cells. Mice were immunized with liposomes containing 1 or 2. Lymph node-derived T cells expressing low levels of CD62L were obtained by cell sorting and cultured for 14 days in the presence of DC pulsed with glycopeptide 6 for 1 or peptide 7 for 2. After exposure to DC pulsed with (glyco)peptide 6-9, the resulting cells were analyzed for the presence of CD8 ⁺ IFNγ ⁺ T cells. Peptide 6 is SEQ ID NO:26, Peptide 7 is SEQ ID NO:20, Peptide 8 is SEQ ID NO:27, and Peptide 9 is SEQ ID NO:29.

Figures 20A to 20H: Three (Figure 20A) or four (Figure 20B-H) immunizations with 1, 2, 3, 4+5 or 5 with or without (NT) tumor induction as indicated ELISA anti-MUC1 and anti-helper T-epitope antibody titers after inoculation. The chemical structures of synthetic antigens 1, 2, 3, 4 and 5 are shown in FIG. 16 . ELISA plates were coated with BSA-MI-MUC-1(Tn) conjugate (Figure 20A-G) or neutravidin-biotin-helper T epitope (Figure 20H) and analyzed by linear regression The titer is determined by plotting the dilution compared to the absorbance. Titer was defined as the highest dilution to obtain an optical density of 0.1 or greater relative to normal control mouse serum. Each time point represents the titer of an individual mouse after four immunizations, and the horizontal line indicates the mean of the group of mice.

Figures 21A and 21B: Competitive inhibition of antibody binding to BSA-MI-MUCl(Tn) conjugates by MUCl(Tn) 6, unglycosylated MUCl 7 and Tn monomer.

The sequences of compounds 6 and 7 are shown in FIG. 19 . ELISA plates were coated with BSA-MI-MUCl(Tn) conjugate. Serum samples after immunization with 1 ( FIG. 21A ) and 2 ( FIG. 21B ) diluted to obtain an OD of about 1 in ELISA in the absence of inhibitors were first reacted with 6, 7 or Tn monomer ( 0-500 μM final concentration) and then applied to the coated microtiter plate. Optical density values were normalized to those obtained with serum alone (0 μM inhibitor, 100%). Data are reported as mean±s.e.m for groups of mice (n=7).

Figures 22A to 22J: Cytokine production by dendritic cells after 24 hours of stimulation with liposome formulations loaded with compound 1, 2 or 3 or E. coli LPS. The chemical structures of synthetic antigens 1, 2 or 3 are shown in FIG. 16 . Primary dendritic mouse cells were incubated for 24 hours with increasing concentrations of liposome formulations loaded with Compound 1, 2 or 3 or E. coli LPS as indicated. TNF-α (Fig. 22A), IFN-β (Fig. 22B), RANTES (Fig. 22C), IL-6 (Fig. 22D), extracellular IL-1β (Fig. 22E and Fig. 22F), IL-10 (FIG. 22G), IP-10 (FIG. 22H), IL-12 p70 (FIG. 22I) and IL-12/23 p40 (FIG. 22J). For estimation of IL-1β secretion after ATP treatment, cells were incubated with ATP (5 mM) for 30 min after 24 h incubation with inducer. Data are reported as mean ± SD of triplicate treatments.

Figure 23: Using compound 2 (Pam ₃ CysSK ₄ -T helper epitope (T helper ep.) (polio)-MUC1 (unglycosylated)); compound 1 (Pam ₃ CysSK ₄ -T helper Cellular epitope (polio) - MUC1(Tn)); compound 1 plus CpG (CpG oligodeoxynucleotide (CpG ODN))); compound 5 (Pam ₃ CysSK ₄ ) plus compound 4 (T helper Epitope (Polio) - MUC1(Tn)); Compound 5; Compound 3 (Pam ₃ CysSK ₄ -T helper epitope (Polio)); Compound 3 plus CpG; EL (empty liposome) Tumor weight in grams (gm) in MUCl.Tg mice immunized with formulations plus CpG; or EL. The chemical structures of synthetic antigens 1, 2, 3, 4 and 5 are shown in Figures 16 and 26 .

Figure 24: Cytotoxic activity of CD8+ cells from MUC1.Tg mice treated with compound 2 (Pam ₃ CysSK ₄ -T helper cell epitope (poliomyelitis)-MUC1 (unglycosylated Compound 1 (Pam ₃ CysSK ₄ -T helper epitope (polio)-MUC1(Tn)); Compound 1 plus CpG (CpG oligodeoxynucleotide (CpGODN))); Compound 5 (Pam ₃ CysSK ₄ ) plus compound 4 (T helper epitope (polio)-MUC1(Tn)); compound 5; compound 3 (Pam ₃ CysSK ₄ -T helper epitope (polio)) ; compound 3 plus CpG; EL (empty liposome) plus CpG; or EL formulation immunizations. The chemical structures of synthetic antigens 1, 2, 3, 4 and 5 are shown in Figures 16 and 26 .

Figure 25: Determination of IFN-γ production by CD8+ T cells from MUC1.Tg mice treated with compound 2 (Pam ₃ CysSK ₄ -T helper cell epitope (poliomyelitis)-MUC1 (unglycosylated)); compound 1 (Pam ₃ CysSK ₄ -T helper epitope (polio)-MUC1(Tn)); compound 1 plus CpG (CpG oligodeoxynucleotide (CpG ODN) )); compound 5 (Pam ₃ CysSK ₄ ) plus compound 4 (T helper epitope (polio)-MUC1(Tn)); compound 5; compound 3 (Pam ₃ CysSK ₄ -T helper epitope ( Polio)); Compound 3 plus CpG; EL (empty liposome) plus CpG; or EL for formulation immunization. The chemical structures of synthetic antigens 1, 2, 3, 4 and 5 are shown in Figures 16 and 26 .

Figure 26: Compound 1 (Pam ₃ CysSK ₄ -T helper cell (T-helper)-MUC1), compound 2 (Pam ₃ CysSK ₄ -T helper cell), LAA-T helper cell-MUC1 and LAA-T helper cell structure.

Figure 27: Immunization scheme.

Figure 28: Three-component vaccine reduces tumor burden. Cells containing compound 1 (Pam ₃ CysSK ₄ -T helper-MUC1), compound 2 (Pam ₃ CysSK ₄ -T helper), LAA-T helper-MUC1 or LAA-T helper (25 μg, containing 3 μg Carbohydrate) liposomes or MUCl.Tg mice were immunized with empty liposomes as a control. Three biweekly immunizations were given before tumor challenge with MUCl expressing MMT tumor cells ( ¹ x 106 cells), followed by a booster one week later. Animals were sacrificed 7 days after the last injection, and tumor wet weights were determined. Data are presented as percentage of control (mice vaccinated with empty liposomes). Each data point represents an individual mouse, and the horizontal line indicates the mean of the group of mice.

Figure 29: Induction of MUCl-specific cytotoxic CD8 T cells by vaccine. Liposomes were prepared with empty liposomes as indicated or containing compound 1 (Pam ₃ CysSK ₄ -T helper-MUC1), compound 2 (Pam ₃ CysSK ₄ -T helper), LAA-T helper-MUC1 or LAA- Liposomes of T Helper Cells CD8 ⁺ T cells were isolated from the lymph nodes of mice immunized with tumor induction, and the CD8 ⁺ T cells were subjected to 51Cr release assays without any in vitro stimulation. DCs pulsed with Tn-MUCl peptide (SAPDT(Tn)RPAP) (SEQ ID NO:26) or empty liposomes were used as targets. The effector to target ratio was 100:1 because CTLs were not stimulated in vitro. Spontaneous release was less than 15% of complete release. Each data point represents an individual. The chemical structures of synthetic antigens 1, 2, 3, 4 and 5 are shown in Figures 16 and 26 .

Figure 30: Three-component vaccine elicits strong antibody titers. ELISA anti-MUCl and anti-T epitope antibody titers after four immunizations. Anti-MUCl and anti-T epitope antibody titers are presented as median values for groups of four to seven mice. For anti-MUC1 antibody titers, ELISA plates were coated with BSA-MI-MUC-1(Tn) conjugate, or for anti-T helper epitope antibody titers, ELISA plates were coated with neutravidin- Biotin-T epitope coating. Titers were determined by linear regression analysis using a plot of dilution versus absorbance. Titer was defined as the highest dilution to obtain an optical density of 0.1 or greater relative to normal control mouse serum.

Figure 31 : Antibodies are effective in antibody-dependent cellular cytotoxicity (ADCC). C57 mg.MUC1 mammary tumor cells were labeled with chromium for two hours and then incubated with control serum (MUC1.Tg) or serum from MMT tumor-bearing mice (diluted 1:50) at 37°C for 30 minutes ( min), the mice were treated with empty liposomes as indicated or containing compound 1 (Pam ₃ CysSK ₄ -T helper-MUC1), compound 2 (Pam ₃ CysSK ₄ -T helper), LAA-T helper Liposome immunization of cells-MUC1 and LAA-T helper cells. Tumor cells were then incubated with effector cells (KY-1 cells-NK clones) for four hours. The effector to target ratio was 50:1. Spontaneous release was less than 15% of complete release. Each data point represents an individual mouse, and the horizontal line indicates the mean of the group of mice. The chemical structures of synthetic antigens 1, 2, 3, 4 and 5 are shown in Figures 16 and 26 .

Figure 32: Leader sequence of human MUC1 with Rankpep study results highlighted. The starting sequence of the tandem repeat is underlined. Dashed lines show 15mers showing ^RANKPEP scores for binding to IAb. 9mers showing ^RANKPEP scores for binding to H2- ^Db (dddd) or H2-Kb (kkkk) or non-germline-selective (promiscuous) binding to both (bbbb) are assigned.

Figures 33A and 33B: Mice were immunized with the peptides described in Figure 33A, and lymph node-derived T cells expressing low levels of CD62L were obtained by cell sorting and cultured in the presence of DC pulsed with the immunization peptide for 14 sky. After exposure to DCs pulsed with the peptides listed on the y-axis, the resulting cells were analyzed by intracellular cytokines for the presence of CD4 ⁺ IFNγ ⁺ and CD8 ⁺ IFNγ ⁺ T cells ( FIG. 33B ).

Figure 34: Synthetic constructs utilizing human MUCl T helper cell sequences.

Figure 35 shows the structures of the fully synthetic three-component immunogens 52 and 53 and the reagents 63-65 used for their preparation.

Figure 36 shows ELISA anti-GSTPVS(β- O -GlcNAc)SANM (68) antibody titers after 4 immunizations with 52 and 53. (a) Total IgG was determined by coating ELISA plates with BSA-MI-GSTPVS(β- O- GlcNAc)SANM(BSA-MI-66) conjugate and plotting dilutions versus absorbance by linear regression analysis , (b) IgG1, (c) IgG2a, (d) IgG2b, (e) IgG3 and (f) IgM titers. Titer was defined as the highest dilution to obtain an optical density of 0.1 or greater relative to normal control mouse serum. Each data point represents the titer of an individual mouse after 4 immunizations, and horizontal lines indicate the mean of a group of five mice.

Figure 37 shows compound 52.

Figure 38 shows compound 53.

Figure 39 shows compound 63.

Figure 40 shows compound 64.

Figure 41 shows compound 65.

Figure 42 shows compound 66.

Figure 43 shows compound 67; SEQ ID NO:12.

Figure 44 shows compound 68.

Figure 45 shows Compound 69; SEQ ID NO:11.

Figure 46 shows compound 70.

Description of Illustrative Embodiments

The glycolipopeptide of the present invention comprises at least one B epitope, at least one T epitope and a lipid component. In a preferred embodiment, the glycolipopeptide consists essentially of three major components: at least one carbohydrate component containing a B epitope; at least one peptide component containing a helper T epitope; and at least one lipid qualitative components. Exemplary carbohydrate, peptide, and lipid components are described herein and, for example, in references cited herein, including Koganty et al., US Patent Publication 20060069238, published March 30, 2006; see also Koganty et al., 1996, Drug Disc Today; 1(5):190-198. The three components are directly or indirectly covalently linked to form a single glycolipeptide molecule. Indirect linkage involves the use of an optional linker component "L" to link two or more major components together. The three main components can be linked together (directly or indirectly) in any order. For example, lipid and carbohydrate components can each be covalently linked to a peptide component to form a glycolipopeptide. Alternatively, the lipid component and the peptide component may each be covalently linked to the carbohydrate component. Likewise, the carbohydrate component and the peptide component can each be covalently linked to the lipid component. Alternatively, all three components may be linked such that each of the three components is covalently linked to each of the other two components. Intermolecular crosslinking is also possible, as described in more detail below.

In preferred embodiments, the glycolipid peptides of the invention contain a carbohydrate component, a peptide component and a lipid component. In another embodiment, the glycolipopeptide contains multiple carbohydrate components, which may be the same or may be different. Likewise, in another embodiment, the glycolipopeptide contains multiple peptide components, which may be the same or may be different. Further, in another embodiment, the glycolipopeptide contains multiple lipid components, which may be the same or may be different. Accordingly, various embodiments of the glycolipopeptides of the invention may contain one or more carbohydrate components, one or more peptide components, and/or one or more lipid components. For example, the concept of "multiple antigenic glycopeptides" (Bay et al., U.S. Patent No. 6,676,946, January 13, 2004, Bay et al.; WO 98/43677, published October 8, 1998 Bay et al) may be suitable for use in the present invention. High antigen density can be achieved using a core, such as a polylysine core, to which are attached extended peptide "arms" (peptide components of the glycolipopeptides of the invention), which are presented in clusters to display the Carbohydrate Antigen Component. The lipid component of the glycolipopeptide may also extend from the lysine core, particularly in embodiments where the peptide component is attached to the lysine core via a non-terminal amino acid. High antigen density can also be achieved by using liposomes as delivery vehicles, as exemplified in Examples 2 and 3. Additionally or alternatively, glycolipopeptides may optionally be cross-linked to form multimolecular complexes, thereby increasing antigen density.

The various carbohydrate, peptide and lipid components of glycolipopeptides may be structurally derived from or based on and/or may mimic those found in naturally occurring biological molecules . The glycolipopeptide fraction preferably contains molecular structures or portions of structures (including epitopes) identical or similar to those found in living organisms. Typically, although components of glycolipopeptides are derived from, structurally based on and/or mimic naturally occurring structures, they are prepared synthetically using, for example, chemical or in vitro enzymatic methods. In some embodiments, epitopes formed by molecular elements in naturally occurring antigens can be formed in glycolipopeptides of the invention by forming different chemical structures (with different bonding orders or patterns) of the same or similar epitopes. position, the molecular elements are close together in space but distant from each other in terms of chemical bonding.

The three-component glycolipopeptides of the invention can be viewed as cassettes, wherein the carbohydrate component, the peptide component, and the lipid component are each independently selected for inclusion in the glycolipeptide. Any combination (ie, mixing and matching) of carbohydrate components, peptide components, and lipid components as described herein to form glycolipopeptides is encompassed by the present invention.

carbohydrate composition

The carbohydrate component of the glycolipopeptide can be any component that contains carbohydrates. Examples of suitable carbohydrate components include oligosaccharides, polysaccharides and monosaccharides, and glycosylated biomolecules (glycoconjugates), such as glycoproteins, glycopeptides, glycolipids, glycosylated amino acids, DNA or RNA. Glycosylated peptides (glycopeptides) and glycosylated amino acids containing one or more carbohydrate moieties as well as peptides or amino acids are particularly preferred as the carbohydrate component of the glycolipopeptides of the invention. An example of a glycopeptide is CD52, which is expressed on lymphocytes of essentially all humans and is believed to play an important role in the human immune system. An example of a glycosylated amino acid is the Tn antigen. It is understood that when the carbohydrate component is a glycopeptide, the peptide portion of the glycopeptide optionally includes a T epitope as well as a B epitope, and thus may serve as the peptide component of the glycolipopeptide. Glycopeptides containing a T epitope and a B epitope are sometimes referred to as having a "B-T" epitope or a "T-B" epitope. The B epitopes and T epitopes present on the glycolipopeptides of the present invention may or may not overlap. In preferred embodiments, the T epitope, B epitope and/or T-B epitope is derived from a MUCl polypeptide sequence, including but not limited to a human MUCl polypeptide sequence.

The carbohydrate component of the glycolipopeptides of the invention includes carbohydrates containing one or more sugar monomers. For example, a carbohydrate may include monosaccharides, disaccharides, or trisaccharides; it may include oligosaccharides or polysaccharides. Oligosaccharides are oligosaccharides that contain two or more sugars and are characterized by a well-defined structure. A well-defined structure is characterized by the specific identity, order, location of attachment (including branch points), and attachment stereochemistry (α, β) of the monomers, and thus has a defined molecular weight and composition. Oligosaccharides generally contain from about 2 to about 20 or more sugar monomers. A polysaccharide, on the other hand, is a polysaccharide that does not have a well-defined structure; the identity, sequence, position of attachment (including branch points), and/or attachment stereochemistry may vary between molecules. Polysaccharides generally contain a greater number of monomer components than oligosaccharides, and thus have a higher molecular weight. The term "glycan" as used herein is inclusive of oligosaccharides and polysaccharides, and includes branched and unbranched polymers. When the carbohydrate component contains carbohydrates having three or more sugar monomers, the carbohydrate may be straight chain or it may be branched. In preferred embodiments, the carbohydrate component contains less than about 15 sugar monomers; more preferably less than about 10 sugar monomers.

The carbohydrate component of the glycolipopeptide includes carbohydrates containing B epitopes. It will be appreciated that the carbohydrate may be coextensive with the B epitope, or that the carbohydrate may include the B epitope, or that the carbohydrate may include only part of the B epitope (i.e., the B epitope may additionally include other portions of the glycolipopeptide , such as peptide components, lipid components and/or linker components). An example of a glycopeptide comprising a B epitope is the glycosylated peptide MUC-1 (also referred to herein as MUC1). Thus, a carbohydrate or carbohydrate component "comprising" a B epitope is understood to mean a carbohydrate or carbohydrate component comprising all or part of the B epitope present on the glycolipopeptide.

B epitopes may be naturally occurring epitopes or non-naturally occurring epitopes. Preferably, two or more sugar monomers of a carbohydrate interact to form a conformational epitope that acts as a B epitope. B epitopes are epitopes recognized by B cells. Any antigenic carbohydrate containing a B epitope can be used as the carbohydrate component without limitation. In preferred embodiments, the B epitope is derived from a MUCl polypeptide sequence, including but not limited to a human MUCl polypeptide sequence.

Non-naturally occurring carbohydrates that may be used as components of the glycolipopeptides of the invention include glycomimetics, which are molecules that mimic the shape and characteristics of sugars such as monosaccharides, disaccharides, or oligosaccharides (see, e.g., Barchi, 2000, Current Pharmaceutical Design; 6(4):485-501; Martinez-Grau et al., 1998, Chemical Society Reviews; 27(2):155-162; Schweizer, 2002, Angewandte Chemie-International Edition; 41(2 ):230-253). Glycomimetics can be engineered to supply desired B epitopes and potentially provide greater metabolic stability.

In another embodiment, the carbohydrate component contains all or part of an autoantigen. Autoantigens are antigens that are normally present in the body of an animal. They can be considered "self molecules," such as those present in or on an animal's cells, or proteins such as insulin that circulate in an animal's blood. Examples of self-antigens are carbohydrate-containing components of cancer cells derived from animals, such as tumor-associated carbohydrate antigens (TACA). Typically, such self-antigens show low immunogenicity. Examples include tumor-associated carbohydrate B epitopes such as Le ^y antigen (cancer-associated tetrasaccharide; e.g. Fucα(1,2)-Galβ(1,4)-[Fucα(1,3)]-GlcNAc); Globo-H antigen (e.g. L-Fucα(1,2)-Galβ(1,3)-GalNAcβ(1,3)-Galα(1,4)-Galβ(1,4)-Glu); T antigen (e.g. Galβ(1, 3)-GalNAcα-O-Ser/Thr); STn antigen (sialyl Tn, such as NeuAcα(2,6)-GalNAcα-O-Ser/Thr); and Tn antigen (such as α-GalNAc-O-Ser/ Thr). Another example of an autoantigen is a glycopeptide derived from the tandem repeats of breast tumor-associated MUC-1 derived from human polymorphic epithelial mucin (PEM) (Baldus et al., Crit. Rev. Clin . Lab. Sci., 41(2):189-231(2004)). The MUC-1 glycopeptide comprises at least one Tn and/or sialyl Tn (sialyl alpha-6 GalNAc or "STn") epitope, preferably linked to threonine (T-Tn or T-STn).

In a preferred embodiment, the carbohydrate component comprises a glycosylated MUCl glycopeptide which is glycosylated on one or more serine and/or threonine residues of the MUCl derived amino acid peptide sequence. Such MUCl-derived amino acid sequences include, but are not limited to, any of the MUCl sequences described herein.

Structures of exemplary tumor-associated carbohydrate antigens (TACAs) that can be used as components of glycolipopeptides include, but are not limited to, those shown in Schemes 1 and 2.

plan 1

It should be noted that the Tn, STn and TF structures (monomer, trimer, clustered) shown in Scheme 1 are all shown with threonine residues. The corresponding serine analogs are also suitable structures. In the case of Tn3, STn3, TF3 and their respective clusters, all possible homo- and iso-analogues that differ in the threonine/serine composition of the backbone are included.

Scenario 2

Another type of autoantigen for use in the carbohydrate component of glycolipopeptides is a glycopeptide comprising an amino acid or peptide covalently linked to a monosaccharide. Preferably, the monosaccharide is N -acetylglucosamine (GlcNAc) or N -acetylgalactosamine (GalNAc). Preferred glycopeptide autoantigens are β- N -acetylglucosamine (β- O -GlcNAc) modified peptides. Preferably, the monosaccharide is O -linked to a serine or threonine of the polypeptide. Also suitable for use as self-antigens are related thiol ( S -link) and amino ( N -link) analogs. The monosaccharide is preferably attached to the peptide via a beta linkage, but it could be an alpha linkage. In a particularly preferred embodiment, the carbohydrate component of the glycolipopeptide of the invention (which may be coextensive with the peptide component when formulated as a glycopeptide) contains TPVSS (SEQ ID NO: 10) modified by O -GlcNAc amino acid sequence. Examples of carbohydrates containing β-GlcNAc modified glycopeptides as B epitopes are shown as compounds 52 ( O -link) and 53 ( S -link) in FIG. 15 .

In another embodiment, the carbohydrate component contains all or part of carbohydrate antigens (usually glycans) from microorganisms, preferably pathogenic microorganisms, such as viruses (such as carbohydrates present on gp120, derived from Glycoproteins from HIV virus), Gram-negative or Gram-positive bacteria (such as those derived from Haemophilus influenzae , Streptococcus pneumoniae or Neisseria meningitides ) carbohydrates), fungi (e.g. 1,3-β-linked glycans), parasitic protozoa (e.g. GPI anchors found in protozoan parasites such as Leishmania and Trypanosoma brucei ) or worms. Preferably, the microorganism is a pathogenic microorganism.

An exemplary glycan from a viral pathogen is shown in Scheme 3 from Man9 of HIV-1 gp120.

Option 3

Exemplary HIV carbohydrate and glycopeptide antigens are described in Wang et al., Current Opinion in Drug Disc. & Develop., 9(2):194-206 (2006), and include naturally occurring HIV carbohydrates and glycopeptides, and Synthetic carbohydrates and glycopeptides based on naturally occurring HIV carbohydrates and glycopeptides.

Exemplary HCV carbohydrate and glycopeptide antigens are described in Koppel et al. Cellular Microbiology 2005; 7 (2):157-165 and Goffard et al . J. of Virology 2005; 79 (13):8400-8409 and include naturally occurring HCV carbohydrates and glycopeptides, and synthetic carbohydrates and glycopeptides based on naturally occurring HCV carbohydrates and glycopeptides.

Exemplary glycans from bacterial pathogens are shown in Scheme 4.

Option 4

Exemplary glycans from protozoan pathogens are shown in Scheme 5.

Option 5

Exemplary glycans from fungal pathogens are shown in Scheme 6.

Option 6

Exemplary glycans from helminth pathogens are shown in Scheme 7.

Option 7

Those skilled in the art will appreciate that while numerous antigenic carbohydrate structures are known, many more exist because only a small fraction of antigenic or immunogenic carbohydrates have been identified to date. Examples of many carbohydrate antigens discovered so far can be found in: Kuberan et al., Curr. Org. Chem, 4, 653-677 (2000); Ouerfelli et al., Expert Rev. Vaccines 4(5):677-685 (2005) ; Hakomori et al., Chem. Biol. 4, 97-104 (1997); Hakomori, Acta Anat. 161, 79-90 (1998); Croce and Segal-Eiras, Drugs of Today 38 (11): 759-768 ( 2002); Mendonca-Previato et al., Curr Opin. Struct. Biol. 15(5):499-505(2005); Jones, Anais da Academia Brasileira de Ciencias 77(2):293-324(2005); Goldblatt, J .Med.Microbiol.47(7):563-567(1998); Diekman et al., Immunol. Rev., 171:203-211, 1999; Nyame et al., Arch.Biochem.Biophys., 426(2): 182-200, 2004; Pier, Expert Rev. Vaccines, 4(5):645-656, 2005; Vliegenthart, FEBS Lett., 580(12):2945-2950, Sp. Iss., 2006; Ada et al., Clin. Microbiol. Inf., 9(2): 79-85, 2003; Fox et al., J. Microbiol. Meth., 54(2): 143-152, 2003; Barber et al., J. Reprod. Immunol. , 46(2): 103-124, 2000; and Sorensen, Persp. Drug Disc. Design, 5: 154-160, 1996. Any antigenic carbohydrate derived from mammals or infectious organisms can be used as the carbohydrate component of the glycolipopeptide of the present invention without limitation.

peptide component

The peptide component of the glycolipopeptide includes a T epitope, preferably a helper T epitope. The peptide component may be any peptide-containing structure, and may contain naturally occurring and/or non-naturally occurring amino acids and/or amino acid analogs (eg, D-amino acids). The peptide component can be derived from microorganisms such as viruses, bacteria, fungi and protozoa. T epitopes may thus constitute all or part of the viral antigen. Alternatively or additionally, the T epitope may be from a mammal and optionally constitutes all or part of an autoantigen. For example, a T epitope can be part of a glycopeptide that is overexpressed on cancer cells. When the peptide component of the glycolipopeptide of the invention is a glycopeptide, the peptide component may also include all or part of the B epitope, as described elsewhere herein. More generally, it is understood that the peptide component of the glycolipopeptide may be coextensive with the T epitope, or that the peptide component may include the T epitope, or that the peptide component may include only part of the T epitope (i.e., T An epitope may additionally comprise other parts of a glycolipopeptide, such as a carbohydrate component, a lipid component and/or a linker component). Thus, a peptide or peptide component "comprising" a T-epitope is understood to mean a peptide or peptide component comprising all or part of the T-epitope present on the glycolipopeptide.

The peptide component may contain, for example, less than about 50 amino acids and/or amino acid analogs, less than about 40 amino acids and/or amino acid analogs, less than about 30 amino acids and/or amino acid analogs, or less than about 20 amino acids and/or or amino acid analogs. The peptide component may contain, for example, about 9 to about 50 amino acids and/or amino acid analogs, about 9 to about 40 amino acids and/or amino acid analogs, about 9 to about 30 amino acids and/or amino acid analogs, or about 9 to about 20 amino acids and/or amino acid analogs. The peptide component may contain, for example, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23 , about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, or about 80 amino acids and/or amino acid analogs, or these quoted sizes any range of .

Examples of peptide components include the universal helper T peptide QYIKANSKFIGITEL ("QYI") (SEQ ID NO:1), the universal helper T peptide YAFKYARHANVGRNAFELFL ("YAF") (SEQ ID NO:2), murine Helper T peptide KLFAVWKITYKDT ("KLF") (SEQ ID NO:3) and pan DR binding (PADRE) peptide (PCT WO 95/07707; Alexander et al., Immunity 1:751-761 (1994); Alexander et al., J . Immunol. 2000 Feb 1;164(3):1625-33; US Patent No. 6,413,935 (Sette et al., 2002 Jul 2)).

Immunogenic peptide components for use in the glycolipopeptides of the invention include universal (degenerate or "non-germline-selective") helper T cell peptides, which are found in many major histocompatibility complex (MHC ) haplotype are immunogenic peptides. Numerous universal helper T cell peptide structures are known; however, it is understood that additional universal T epitopes will be identified in the future, including some with similar or even higher potency, and that such peptides are well suited for use in As the peptide component of the glycolipopeptide of the present invention.

Exemplary T cell peptides for use in glycolipopeptides include, but are not limited to:

Synthetic non-natural PADRE peptide DAla-Lys-Cha-Val-Ala-Ala-Trp-Thr-Leu-Lys-Ala-Ala-DAla, included by Alexander et al. in Immunity, Vol. 1, 751-761, 1994 All analogs described;

Peptides derived from tetanus toxin, for example (TT830-843) QYIKANSKFIGITEL (SEQ ID NO:1), (TT1084-1099) VSIDKFRIFCKANPK (SEQ ID NO:4), (TT1174-1189) LKFIIKRYTPNNEIDS (SEQ ID NO:5) , (TT1064-1079) IREDNNITLKLDRCNN (SEQ ID NO:6) and (TT947-967)FNNFTVSFWLRVPKVSASHLE (SEQ ID NO:7);

A peptide derived from poliovirus, such as KLFAVWKITYKDT (SEQ ID NO:3);

A peptide derived from Neisseria meningitidis, such as YAFKYARHANVGRNAFELFL (SEQ ID NO: 8); and

A peptide derived from the CSP of P. falsiparum , eg, EKKIAKMEKASSVFNVNN (SEQ ID NO:9).

The peptide component of the glycolipopeptide contains a T epitope. T epitopes are epitopes recognized by T cells. A T epitope can elicit a CD4+ response, thereby stimulating the generation of helper T cells; and/or it can elicit a CD8+ response, thereby stimulating the generation of cytotoxic lymphocytes. Preferably, the T epitope is an epitope that stimulates helper T cell production (e.g. helper T cell epitope or Th epitope), which in turn causes a humoral response to the B epitope supplied by the carbohydrate component of the glycolipopeptide of the invention become possible.

It should be understood that the glycolipopeptides of the invention may contain multiple T epitopes, which may be the same or different. Further, the T epitope may be present on the carbohydrate component and/or the lipid component (e.g., in embodiments comprising glycopeptides and/or glycolipids as the carbohydrate and/or lipid component), additionally in or in place of the peptide component.

In some embodiments, the B and T epitopes are homologous; that is, they are derived from the same organism. For example, in a glycolipopeptide suitable for use as a vaccine against a microbial pathogen, the T epitope plus the B epitope may be the epitopes present in the microbial pathogen. In another embodiment, the B and T epitopes are heterologous; that is, they are not derived from the same organism. For example, a glycolipopeptide suitable for use as an anticancer vaccine may have B-cell epitopes from cancer cells, but T-cell epitopes from bacteria or viruses.

In a preferred embodiment of the immunogenic vaccine of the invention, the T epitope or B epitope may be derived from a MUCl polypeptide. In some embodiments, both the T epitope and the B epitope are derived from a MUCl polypeptide. MUCl (MUCl in humans and Mucl in non-human species) is a heavily glycosylated type I transmembrane protein expressed in epithelial cells lining various mucosal surfaces, as well as in hematopoietic cells. Human MUCl consists of a cytoplasmic signal peptide, a 28 amino acid transmembrane domain, and an extracellular domain consisting of a variable number of tandem repeats of twenty amino acids. Each repeat contains 5 potential O-glycosylation sites. MUCl is associated with several adenocarcinomas at mucosal sites, is overexpressed in more than 90% of breast cancers, and is associated with ovarian, lung, colon and pancreatic cancers. Tumor-associated MUCl is aberrantly glycosylated, producing truncated carbohydrate structures.

The MUCl peptide sequence may comprise a human or mouse MUCl sequence. The MUCl peptide sequence may comprise a MUCl tandem repeat. Such MUCl tandem repeats may contain both B and helper T epitopes.

The MUCl sequence may be homologous and thus be an autoantigen. The MUCl sequence may include one, two, three, four, five, six or more amino acid changes from the human or mouse MUCl peptide. The MUCl sequence can be irregularly varied, including one, two, three, four or more amino acid changes to enhance the MUCl peptide in class I and/or class II major histocompatibility complex (MHC) proteins on the combination. Human MHC is also referred to herein as the HLA complex. The MUCl sequence may include sequence from the extracellular region of the MUCl protein. The MUCl sequence may include sequences responsible for MHC class I restriction. The MUCl sequence may include sequences responsible for MHC class II restriction and/or binding. In some embodiments, such Class I and Class II restriction sequences may be contiguous amino acid sequences in an immunogenic vaccine construct. MHC restriction sequences include, but are not limited to, eg, any of those described herein and any of those represented in Figures 16, 19, and 32-34.

The MUCl sequence may comprise one or more serine or threonine residues which are glycosylated, for example at one, two, three, four or more such residues. Such glycosylation may represent normal tissue glycosylation patterns, or such glycosylation may reflect abnormal glycosylation. The MUCl sequence may contain one or more B epitopes and/or helper T epitopes.

The MUCl sequence can include about 5 to about 30 amino acids of the MUCl protein sequence. The MUCl sequence may comprise less than about 50 amino acids and/or amino acid analogs, less than about 40 amino acids and/or amino acid analogs, less than about 30 amino acids and/or amino acid analogs, or less than about 20 amino acids of the MUCl protein sequence and/or amino acid analogs. The MUCl sequence may comprise, for example, about 9 to about 50 amino acids and/or amino acid analogs, about 9 to about 40 amino acids and/or amino acid analogs, about 9 to about 30 amino acids and/or amino acid analogs or about 9- About 20 amino acids and/or amino acid analogs. The peptide component may contain, for example, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 80 amino acids and/or amino acid analogs, or Any range of sizes for these references.

The MUCl sequence can comprise about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about A sequence of 96%, about 97%, about 98%, or about 99% sequence identity.

The MUCl sequence may comprise any MUCl sequence described herein, for example including but not limited to any of those represented in FIGS. 16 , 19 , 33A, 33B and 34 . For example a MUCl sequence may include SAPDTRPAP (SEQ ID NO:20), TSAPDRPAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO:22), TSAPDTRPL (SEQ ID NO:23), APGSTAPPAHGVTSA (SEQ ID NO:26), APGSTAPPAHGVTSAPPDTRPL (SEQ ID NO:27), APGSTAPPAHGVTSAPPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPPDTRPL (SEQ ID NO:29), SKKKKGAPGSTAPPAHGVTSAPDTRPX (SEQ ID NO:30) where X is L, A or AP, SKKKKGSEAPPAHGVTSAPPDAP (NO:30) : 31), SKKKKGSLSYTNPAVAAATASNL (SEQ ID NO: 32), SKKKKGCKLFAVWKITYKDTGTSAPDTRPAP (SEQ ID NO: 33), SKKKKGCKLFAVWKITYKDT (SEQ ID NO: 34), GGKLFAVWKITYKDTGTSAPDTRPAP (SEQ ID NO: 35) or APGVTSAPDTRPAP (SEQ ID NO: 35). Also included are MUCl sequences that share about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, About 95%, or about 96%, about 97%, about 98%, or about 99% sequence identity. Also included are MUCl sequences that are glycosylated at any combination of one, two, three, four or more serine or threonine residues.

Lipid composition

It was initially assumed that glycopeptides with only two major components, carbohydrate and peptide, would effectively elicit an immune response in animals. Helper T-cell epitopes are expected to induce T-cell-dependent immune responses, resulting in the production of IgG antibodies directed against tumor-associated carbohydrate B epitopes such as Le ^y and Tn. However, in some applications, two-component vaccines have not been found to be very effective. It is speculated that B-cell and T-helper epitopes lack the ability to provide suitable "danger signals" for dendritic cell (DC) maturation. To remedy this problem, a lipid component is included in the compounds, resulting in the glycolipopeptides of the invention.

The lipid component can be any lipid-containing component, such as lipopeptides, fatty acids, phospholipids, steroids or lipidated amino acids and glycolipids such as lipid A derivatives. Preferably, the lipid component is non-antigenic; that is, it does not elicit antibodies against specific regions of the lipid component. However, the lipid component can and preferably does act as an immune adjuvant. The lipid component can serve as a carrier or delivery system for the polyepitopic glycolipopeptide. It facilitates the incorporation of glycolipopeptides into vesicles or liposomes to facilitate delivery of glycolipopeptides to target cells, and it enhances uptake by target cells such as dendritic cells. Further lipid components stimulate cytokine production.

One class of preferred lipid components for use in the glycolipopeptides of the invention comprises various molecular ligands for Toll-like receptors (TLRs). There are many known subclasses of Toll-like receptors (eg, TLR1, TLR2, TRL3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, TLR15, and TLR16). For a review of the relationship and evolution among Toll-like receptors, see Roach et al., PNAS 2005, 102:9577-9582; for a discussion of TLR signaling in vaccination, see Duin et al., TRENDS Immunol. , 2006, 27:49-55.

TLRs are a family of pattern recognition receptors activated by specific components of microorganisms and certain host molecules. They constitute the first line of defense against many pathogens and play a key role in the function of the innate immune system. TLRs in mammals were first identified in 1997, and it has been estimated that most mammalian species have ten to fifteen classes of Toll-like receptors. Known TLRs include: TLR1 (TLR1 ligands include triacyl lipoproteins); TLR2 (TLR2 ligands include lipoproteins, Gram-positive peptidoglycan, lipoteichoic acid, fungal and viral glycoproteins); TLR3 (TLR3 ligands TLR4 (TLR4 ligands include lipopolysaccharide and viral glycoproteins); TLR5 (TLR5 ligands include flagellin); TLR6 (TLR6 ligands including diacyl lipoproteins); TLR7 (TLR7 ligands include small synthetic immune modifiers (such as imiquimod, R-848, loxoribine, and bropirimine) and single-stranded RNA); TLR8 (TLR8 Ligands include small synthetic compounds and single-stranded RNA); and TLR9 (TLR9 ligands include unmethylated CpG DNA motifs). See, e.g., by Akira, "Mammalian Toll-like receptors," Curr Opin Immunol 2003;15(1):5-11 and Akira and Hemmi, "Recognition of pathogen-associated molecular patterns by TLR family," Immunol Lett 2003;85 (2): A review of 85-95.

Particularly preferred are lipid components that interact with TLR2 and TLR4. TLR2 is involved in the recognition of a wide variety of microbial molecules from Gram-positive and Gram-negative bacteria, as well as Mycoplasma and yeast. TLR2 ligands include lipoglycans, lipopolysaccharides, lipoteichoic acid, and peptidoglycan. TLR4 recognizes Gram-negative lipopolysaccharide (LPS) and lipid A, its toxic moiety. TLR ligands are widely commercially available eg from Apotech and InvivoGen. Preferably, the lipid component is a TLR ligand that facilitates the uptake of glycolipopeptides by antigen presenting cells (see Example 3).

Suitable lipids for use as the lipid component of the glycolipopeptides of the invention include PamCys-type lipid structures, for example derived from Pam ₃ Cys ( S -[( R )-2,3-dipalmitoyloxy- Propyl] -N -palmitoyl-( R )-cysteine) and Pam ₂ Cys ( S -[( R )-2,3-dipalmitoyloxy-propyl]-( R )-cysteine amino acid), Pam ₂ Cys lacks the N-palmitoyl group of Pam ₃ Cys. Pam ₃ Cys and Pam ₂ Cys are derived from the immunologically active N-terminal sequence of the major lipoprotein of E. coli. This class of lipids also includes Pam ₃ CysSK ₄ ( N -palmitoyl- S -[( R )-2,3-bis(palmitoyloxy)-propyl]-( R )–cysteinyl-( S )-seryl-( S )-lysine-( S )-lysine-( S )-lysine-( S )-lysine) and Pam ₂ CysSK ₄ ( S -[( R ) -2,3-bis(palmitoyloxy)-propyl]-( R )-cysteinyl-( S )-seryl-( S )-lysine-( S )-lysine- ( S )-lysine-( S )-lysine (lysine)), which lacks the N-palmitoyl group of Pam ₃ CysSK4; it is understood that the number of lysines in these structures can be 0, 1, 2, 3, 4, 5 or more (ie K _n , where n = 0, 1, 2, 3, 4, 5 or more). In some embodiments, the lipid component includes one or more lipid chains, one or more cysteine residues, and one or more lysine residues.

Another preferred class of lipids includes lipids of the lipid A (LpA) type, such as lipid A derived from Escherichia coli, S. typhimurium and Neisseria meningitidis. Lipid A can be attached to the carbohydrate component (containing the B epitope) and/or the peptide component (containing the T epitope) of the glycolipopeptide via a linker, for example linked to the anomeric center or to the anomeric phosphate, C-4' phosphate or C-6' position. Phosphate esters can be modified, for example, to include one or more phosphoethanolamine diesters. Exemplary lipid A derivatives are described, for example, in Caroff et al., 2002, Microbes Infect; 4:915-926; Raetz et al., 2002, Annu Rev Biochem; 71:635-700; and Dixon et al., 2005, J Dent Res ;84:584-595 described in.

In some embodiments, the lipid component is a lipidated amino acid. In some embodiments, the lipid aspect of the lipid TLR2 agonist component is replaced by a different class of adjuvant compounds, such as TLR4 agonists, TLR7 agonists, TLR8 agonists, or TLR9 agonists. In some embodiments, the agonist is the TLR9 agonist CpG.

Below in Scheme 8 are exemplary immunogenic lipids for incorporation into the glycolipid peptides of the invention. The first structure in the first row is _Pam3CysSKn ; the _second structure in the first row _{is Pam2CysSKn} ; and the last 4 structures are lipid A derivatives.

Option 8

Lipids based on _Pam3Cys in structure are particularly preferred for use as lipid components. Pam ₃ Cys is derived from the immunologically active N -terminal sequence of the major lipoprotein of E. coli. These lipopeptides are potent immune adjuvants. Recent studies have shown that Pam ₃ Cys exerts its activity through the interaction with Toll-like receptor 2 (TLR2).

Without being bound by theory, it is believed that interactions between lipid components and TLRs result in the production of proinflammatory cytokines and chemokines, which in turn stimulate antigen presenting cells (APCs), thereby initiating helper T cell development and activation. Covalent attachment of TLR ligands to B and T epitopes ensures that cytokines are produced at the sites where the vaccine interacts with immune cells. This results in high local concentrations of cytokines, promoting maturation of the involved immune cells. Lipopeptides facilitate selective targeting and uptake by antigen-presenting cells and B lymphocytes. In addition, lipopeptides facilitate the incorporation of glycolipopeptides into liposomes. Liposomes have attracted attention as carriers in vaccine design due to their low intrinsic immunogenicity, thereby avoiding undesired carrier-induced immune responses.

Immunogenic vaccines of the invention can be synthesized, for example, by chemoselective ligation, more particularly native chemical ligation (NCL), as described in WO 2007/146070 and US Patent Publication 2009/0196916A1. Briefly, one or more individual components of the vaccine are entrapped or dissolved within lipid structures such as lipid monolayers, lipid bilayers, liposomes, micelles, films, milky liquid, matrix or gel. Reactants used in the ligation reaction may include carbohydrate components, peptide components, lipid components, or conjugates or combinations thereof. These reactants are designed or selected to contain the desired antigenic or immunogenic characteristics, eg, T epitopes or B epitopes of the immunogenic vaccines of the invention.

Optional connector

One or more linkers ("L") are optionally used for assembly of the three components of the glycolipopeptide. In one embodiment, the linker is a bifunctional linker having functional groups in two different positions (preferably on the first and second ends) to covalently link two of the three components in Together. Bifunctional linkers can be homofunctional (ie, contain two equivalent functional groups) or heterofunctional (ie, contain two different functional groups). In another embodiment, the linker is trifunctional (heterofunctional or homofunctional) and can link together all three components of the glycolipopeptide. Suitable functional groups are reactive toward or comprise any of the following: amino, alcohol, carboxylic acid, sulfhydryl, alkene, alkyne, azide, thioester, ketone , aldehyde or hydrazine. Amino acids such as cysteine may constitute linkers.

A bifunctional linker is exemplified in Scheme 9.

Option 9

Figure 1 shows an exemplary fully synthetic glycolipopeptide of the invention containing a carbohydrate-based B epitope, a peptide T epitope, and a lipopeptide. The compound shown in Figure 1 contains L-glycerol-D-mannose-heptose serving as the B epitope, identified as an MHC class II restricted recognition site for human T cells and derived from the outer membrane protein of Neisseria meningitidis The peptide sequence YAFKYARHANVGRNAFELFL (SEQ ID NO:2), and the lipopeptide S-2-3[dipalmitoyloxy]-(R/S)-propyl-N-palmitoyl-R-cysteine (Pam ₃ Cys). As noted elsewhere herein, the lipopeptide _Pam3Cys and the related compound _Pam3CysSK4 are highly potent activators of B cells and macrophages _.

As exemplified in the Examples, methods of preparing glycolipopeptides are also encompassed by the present invention. Preferably, the method for preparing the glycolipopeptide utilizes chemical synthesis, resulting in a fully synthetic glycolipeptide. In embodiments utilizing one or more linkers, optionally the linker components are functionalized so as to facilitate covalent attachment of one of the main components to another of the main components. For example, the linker can be functionalized on each end with a thiol-reactive group, such as maleimide or bromoacetyl, and the components to be attached are modified to include the reactive thiol. Other options for ligation chemistry include native chemical ligation, Staudinger ligation, and Huisgen ligation (also known as "click chemistry"). Example 2 illustrates how carbohydrate components (in that case oligosaccharides) and peptide components can be functionalized with thiol-containing linkers. Preferably, the linker component, if used, is non-antigenic.

The glycolipopeptides of the invention are capable of generating an immune response in a mammal. Glycolipid peptides are antigenic because they generate a humoral response leading to activation of B cells and production of antibodies (immunoglobulins) such as IgM. In addition, glycolipopeptides are immunogenic because they generate cellular responses; for example, they promote the activation of T cells, especially helper T cells, which are also involved in the generation of more complex antibody responses including IgG production helpful. Finally, the immune response elicited in animals includes the production of anti-carbohydrate antibodies.

In another embodiment of the invention, the immunogenic vaccine is a two-component vaccine comprising at least one peptide component and at least one adjuvant component covalently linked. The peptide component includes a T epitope, preferably a helper T epitope of MUCl origin, including but not limited to any of those described herein. Although this embodiment of the vaccine may not be able to generate specific immunity against a particular B epitope, it exhibits antitumor properties. An example of a two-component vaccine is Pam3CysSK4 covalently linked to a helper T epitope; see eg, Compound 3 in Example 8. In one embodiment, the adjuvant component of the immunogenic vaccine comprises a toll-like receptor (TLR) ligand. At least 15 different mammalian TLRs are known (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, and TLR15), and their ligands show significant structural changes. Some TLR ligands are described herein, but it is understood that such listing does not limit the invention in any way. In some embodiments, two-component immunogenic vaccines may be formulated for administration as compositions further comprising additional agents such as immunomodulators, adjuvants, TLR agonists, and/or excipients. TLR ligands are well known to the skilled person. They can take the form of lipopeptides, glycolipids, lipoproteins, carbohydrates, small organic molecules, nucleic acids such as single- or double-stranded DNA or RNA, and many TLR ligands are known to act as immunostimulants. An example of an immunostimulatory TLR ligand is a TLR2 ligand, including but not limited to any of those described herein. Another example is the TLR9 ligand commonly referred to as "CpG". This compound is an immunostimulatory oligodeoxynucleotide (ODN) containing a CpG motif. CpG motifs are recognized by TLR9 as ligands (Rothenfusser et al., 2002, Human immunology 63(12):1111-1119). Preferably, the CpG ODN is unmethylated. CpG ODNs are short single-stranded DNA molecules that contain cytosine ("C" nucleotide) followed by guanine ("G" nucleotide). "p" generally refers to the phosphodiester backbone of DNA. Optionally, the CpG motif can be modified to contain a phosphorothioate (PS) backbone in order to protect the ODN from degradation by nucleases such as DNase (Dalpke et al., 2002, Immunology 106(1):102-12). CpG ODNs typically range in length from about 18 nucleotides to about 28 nucleotides. Optionally, they contain palindromic sequences. An example of a CpG for use in the present invention is 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 36). CpG motifs present in vertebrate DNA are often methylated due to transcriptional regulatory mechanisms (Sulewska et al., 2007, Folia Histochemica et Cytobiologica 45(3):149-158). Unmethylated CpG motifs have been shown to act as immunostimulants (Weiner et al., 1997, Proc. Natl. Acad. Sci. USA, 94:10833-10837). CpGs have been used in research to enhance tumor immunity (Nierkens et al., 2009, PLoS One. 4(12):e8368; Cooper et al., 2004, J. Clin. Immunol. 24(6):693-701; Leichman et al. People, 2005, J. Clin. Oncol. 2005 ASCO Annual Meeting Proceedings. 23(16S):7039).

Many CpG ODNs are commercially available. For example, CpG ODNs can be purchased through InvivoGen (San Diego, CA) as Type A, Type B, or Type C molecules. These categories are based on structural differences and their immunostimulatory activity (Krug et al., 2001. Eur J Immunol, 31(7):2154-63; Marshall et al., 2005 DNA Cell Biol. 24(2):63-72; Martinson et al. People, 2006, Immunology 120:526-535).

In another embodiment, the adjuvant component of the immunogenic vaccine is a lipid component as described herein (see also WO 2007/079448, US Patent Publication 2009/0041836 Al and WO 2010/002478). Some TLR ligands, such as those of TLR2, also constitute the lipid component, but the lipid component of an immunogenic vaccine is not limited to TLR ligands; i.e., the lipid component can be any suitable immunogen that can act as an adjuvant Antigenic or antigenic lipids, such as lipidated amino acids ("LAA").

In another aspect, the glycolipopeptides of the invention are used to generate polyclonal or monoclonal antibodies that recognize either or both of the carbohydrate component and the peptide component. The invention includes methods of making the antibodies, as well as the antibodies themselves and hybridomas that produce the monoclonal antibodies of the invention.

The immunogenic glycolipopeptides of the invention for use in the production of antibodies may contain any of the carbohydrate components described herein without limitation. Preferably, it contains glycopeptides as its carbohydrate component. Glycopeptides include glycosylated peptide sequences that include carbohydrate moieties such as sugars. Sugars can be monosaccharides, oligosaccharides or polysaccharides. Preferably, the carbohydrate component of the glycolipopeptide used to generate antibodies contains an autoantigen as described above. Advantageously, covalent attachment of carbohydrate components to peptide and lipid components results in significantly immunogenic glycolipopeptides even if the carbohydrate components, such as glycopeptides, are weakly antigenic (eg, self-antigens).

Antibodies of the invention that bind glycolipopeptides preferably bind a B epitope, which includes a sugar moiety and, in a preferred embodiment, at least a portion of a peptide forming a glycopeptide. Preferred antibodies bind to glycopeptides used as carbohydrate components, but not to individual deglycosylated peptides or sugar residues.

When used to generate antibodies, the glycolipopeptides of the present invention successfully generate high affinity IgG antibodies. This is especially surprising and unexpected for embodiments of glycolipopeptides with weakly antigenic carbohydrate components such as self-antigens. Polyclonal or monoclonal antibodies are therefore preferably IgG isotype antibodies. Without being bound by theory, it is believed that the glycolipopeptides of the present invention are superior antigens (compared to non-lipidated glycopeptides) because they stimulate local production of cytokines, upregulate co-stimulatory proteins, and enhance passage through macrophages and dendritic cells. Cellular uptake and/or avoidance of epitope inhibition.

Antibodies of the invention include, but are not limited to, those that recognize B epitopes that contain O -GlcNAc, O -GalNAc, O -mannose, or other sugar modifications. Other B epitopes that may be recognized by the antibodies of the invention include those containing fragments of glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronan and any glycosaminoglycan in general. In the case of glycosaminoglycans formed by repeating disaccharide units, the B epitope may contain one or more disaccharide units. B epitopes recognized by the antibodies of the invention may contain pentoses, hexoses, or other sugar moieties including acids, including but not limited to glucuronic acid, iduronic acid, hyaluronic acid, glucose, galactose, galactose, Lactosamine, Glucosamine, etc. The antibodies of the invention are preferably produced using the glycolipopeptides of the invention as immunogens, wherein the carbohydrate component contains the B epitope of interest. Analogs of naturally occurring B epitopes, such as those containing N-linked or S-linked structures, or glycomimetics, may be used as carbohydrate components, for example, to render the glycolipopeptide immunogen more metabolically stable.

Antibodies generated using the glycolipopeptides of the invention advantageously include high affinity IgG antibodies that recognize a broad spectrum of glycoproteins. Therefore, even though antibodies raised using the glycolipopeptides of the present invention as immunogens are specific for the glycopeptide used as the carbohydrate component, they can bind a broad spectrum of glycoproteins. Antibodies with relatively broad selectivity for glycosylated peptides or proteins containing the B-epitope component of interest are referred to herein as "pan-specific" antibodies. The polyclonal or monoclonal antibodies of the invention can be pan-specific or site-specific. As the term is used herein, a pan-specific antibody is an antibody that specifically recognizes a selected B epitope, such as a B epitope containing O -GlcNAc, but has relative specificity for proteins and peptides containing that B epitope. Wide range of options. A pan-specific antibody is thus capable of binding a variety of different glycosylated proteins or peptides containing the B epitope of interest, although it does not necessarily bind all glycosylated proteins or peptides containing the B epitope of choice.

Without intending to be bound by theory, different glycoproteins recognized by pan-specific antibodies of the invention may share substantially similar or identical (glyco)peptide sequences (i.e. primary sequences) or substantially similar secondary or tertiary structure, resulting in a broad spectrum of binding targets recognized by the antibody. Secondary or tertiary epitope structures shared by O -GlcNAc-modified glycoproteins to which antibodies bind can be favorably maintained in glycolipopeptide immunogens, as demonstrated by the successful generation of IgG antibodies that recognize a broad spectrum of glycoproteins .

Preferably, the antibodies of the invention bind various glycosylated proteins or peptides with epitopes comprising O -GlcNAc, O -GalNAc, or other sugar modifications, but cannot detectably bind proteins or peptides that do not contain such sugars. More preferably, the antibody binds to a protein or peptide having an epitope comprising O -GlcNAc, O -GalNAc, or other sugar modification, but does not detectably bind to the same protein that does not contain O -GlcNAc, O -GalNAc, or other sugar modification or peptides.

Examples of preferred polyclonal or monoclonal antibodies are polyclonal or monoclonal antibodies that bind to glycopeptides containing O -GlcNAc monosaccharide residues. In particularly preferred embodiments, the antibody has a relatively broad selectivity for O -GlcNAc modified proteins. For example, many proteins of interest have the sequence TPVSS (SEQ ID NO: 10) modified with O -GlcNAc, and preferred monoclonal antibodies recognize this and/or similar glycosylated peptide sequences. Examples of preferred monoclonal antibodies specific for O -GlcNAc modified sequences include monoclonal antibodies produced by the hybridoma cell lines 1F5.D6, 9D1.E4, 18B10.C7 and 5H11.H6. These monoclonal antibodies were raised using compounds 52 and/or 53 as immunogens. Thus, in one embodiment, an antibody of the invention binds the carbohydrate component of Compound 52 or Compound 53. The hybridoma cell lines 1F5.D6, 9D1.E4 and 18B10.C7 were deposited in the American Type Culture Collection (ATCC) on July 1, 2008, 10801 University Blvd., Manassas, VA, 20110-2209, USA, and respectively Assigned ATCC deposit numbers PTA-9339, PTA-9340 and PTA-9341. The invention includes hybridoma cell lines and the monoclonal antibodies they produce.

Another example of a preferred polyclonal or monoclonal antibody is one that binds a heparan sulfate fragment.

It is understood that any carbohydrate or glycopeptide of clinical significance or interest may be incorporated as the carbohydrate and/or peptide component of the glycolipopeptides of the invention and used to generate polyclonal and monoclonal antibodies according to the methods of the invention. Such carbohydrates and peptides include those of medical and veterinary interest, as well as those of other commercial or research applications. It should be understood that the monoclonal and polyclonal antibodies of the invention are not limited to those recognizing any particular ligand, but include, without limitation and by way of example only, directed against any type of tumor-associated carbohydrate antigen (TACA) and derived from any microorganism. Antibodies to any sugar.

In summary, the preparation of the monoclonal antibodies of the present invention using the glycolipopeptides of the present invention is surprisingly effective in producing monoclonal IgG antibodies with high affinity for their carbohydrate or glycopeptide antigens, even when the antigens are weak when antigenic. This opens the door to the generation of antibodies useful for the study, diagnosis and treatment of immune-related diseases or diseases with an autoimmune or inflammatory component, including cancer, type II diabetes, allergies, asthma, Crohn's ) disease, Alzheimer's disease, muscular dystrophy, microbial infection, etc. For example, monoclonal antibodies of the invention that recognize O -GlcNAc modified glycoproteins are far superior to commercially available antibodies, such as CTD110.6 (Covance Research Products, Inc.). The glycolipopeptides of the invention can be assembled using modular synthesis in which lipid, peptide and carbohydrate components are selected according to the desired application. Furthermore, the glycolipopeptides of the present invention are remarkably effective antigens for the generation of pan-specific antibodies, particularly pan-specific monoclonal IgG antibodies, which recognize glycosylated peptides containing O -linked monosaccharides such as O -GlcNAc and protein.

The antibodies of the invention and those produced by the methods of the invention are important research tools for the identification and characterization of proteins, peptides and other biomolecules associated with various disease states. For example, pan-specific antibodies of the invention can be used to pull down glycoproteins from complex biological samples. This approach can be used to detect and identify proteins that have not been hitherto associated with a particular disorder or disease state, thereby identifying potential therapeutic or diagnostic targets. In one embodiment, an antibody of the invention can be contacted with a biological sample under conditions such that the antibody is capable of binding a variety of glycosylated proteins or glycosylated peptides and detecting antibody-protein binding. Optionally, the method may include isolating glycosylated proteins or glycosylated peptides. The method may further comprise identifying one or more proteins or peptides within the plurality of glycosylated proteins or glycosylated peptides. The identification of glycosylated proteins and peptides can provide an opportunity to investigate the role of glycosylation and its biological involvement in a variety of biological processes. For example, glycosylation of proteins or peptides can be involved in many biological processes including but not limited to transcription, translation, signal transduction, ubiquitin pathway, anterograde trafficking of intracellular vesicles, and post-translational modifications such as SUMOylation and phosphorylation change). Methods for identifying proteins or peptides are well known in the art and may include, but are not limited to, techniques such as mass spectrometry and Edman degradation.

The pan-specific antibodies of the invention can also be used to identify proteins or peptides with altered glycosylation in disease states. O -GlcNAc modification is associated with various disease states. For example, increased O -GlcNAc modification in skeletal muscle and pancreatic glycopeptides is associated with the development of type II diabetes, whereas decreased O -GlcNAc modification in neuroglycopeptides is associated with the onset of Alzheimer's disease (Dias and Hart ; Mol. BioSyst. 3:766-772 (2007)). Therefore, detection of changes in the level of O -GlcNAc modification can be used as a diagnostic or prognostic tool. Additionally, the glycosylation state of such proteins or peptides can be correlated with disease states. The method for identifying a protein or peptide having altered glycosylation associated with a disease state comprises incubating an antibody of the invention with a first biological sample having a known disease state, and, after enabling the antibody to bind to the first biological sample, Incubating the antibody with a second biological sample having a non-diseased state, independently from Glycosylated proteins and glycosylated peptides are isolated from the sample, and the glycosylated proteins and glycosylated peptides are identified. The method can further comprise comparing the glycosylated proteins and glycosylated peptides identified in the first sample to the glycosylated proteins and glycosylated peptides in the second sample, wherein the glycosylated proteins and glycosylated peptides identified between the first and second samples A protein or peptide with a change in glycosylation state between indicates that the glycosylated protein or glycosylated peptide is associated with the disease state. An association between glycosylation and a disease state includes a disease state having increased or decreased glycosylation relative to a non-diseased state. Furthermore, a disease state can show glycosylation while a non-disease state shows the complete absence of glycosylation, or conversely a disease state can show the complete absence of glycosylation while a non-disease state shows the presence of glycosylation . In each instance, the protein or peptide was considered to have differential or altered glycosylation in the disease state. Methods for detecting the presence or overexpression of glycosylation and detecting changes in glycosylation levels using the antibodies of the invention have been described previously.

The antibodies of the invention are broadly useful in diagnostic or therapeutic applications, as described in more detail elsewhere herein. Comparative analysis can be performed on two or more different biological samples. For example, large-scale immunoprecipitation can be performed on samples before and after therapeutic intervention, or over time, to monitor disease progression, or to compare normal samples with those from a disease suspected to be characterized by changes in protein glycosylation. , a sample from a patient with an infection or disorder.

In one embodiment, the invention includes a method of diagnosing the presence of a disease state in a subject. The method comprises incubating a biological sample from a subject with an antibody of the invention, and detecting binding of the antibody to a protein or peptide having differential glycosylation in a disease state. Methods for detecting antibody binding have been described previously. In cases where glycosylation is completely absent in the disease state, the lack of binding of the antibody to the protein or peptide indicates that the subject has the disease state. In cases where glycosylation is present in the disease state but completely absent in the non-disease state, binding of the antibody to the protein or peptide is indicative of the presence of the disease state in the subject. Optionally, the method may further comprise incubating a second non-diseased biological sample with the antibody of the invention, detecting binding of the antibody to the protein or peptide, and comparing the binding of the antibody to the protein or peptide in the first and second sample. Antibody binding.

Additionally, for proteins and peptides in which glycosylation is present in both disease states and non-disease states, but is altered (i.e., increased or decreased) in the disease state, the method may further comprise quantifying antibody binding in the first sample level, quantify the level of antibody binding in a second non-diseased sample, and compare the binding levels. A change in antibody binding in the first sample compared to a non-diseased sample is indicative of the presence of an infection, disease or condition in the subject.

For the production of the antibodies of the invention, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technology originally developed by Kohler and Milstein (256 Nature 495-497 (1975)) can be used. See also Ausubel et al., Antibodies: a Laboratory Manual, (Harlow & Lane ed., Cold Spring Harbor Lab. 1988); Current Protocols in Immunology, (Colligan et al., ed., Greene Pub. Assoc. & Wiley Interscience N.Y., 1992- 1996).

The invention also provides hybridoma cell lines that produce monoclonal antibodies, preferably monoclonal antibodies with high specificity and affinity for their antigens. The invention further includes variants and mutants of the hybridoma cell lines. Such cell lines can be artificially produced using known methods and still possess the characteristic properties of the starting material. For example, they may still be capable of producing antibodies according to the invention or derivatives thereof and secreting them into the surrounding medium. Optionally, hybridoma cell lines can arise spontaneously. Clones and subclones of hybridoma cell lines are to be understood as hybridomas which have been produced by iterative cloning from a starting clone and which still possess the main characteristics of the starting clone.

Antibodies can be elicited in animal hosts by immunization with the glycolipopeptides of the invention, or can be formed by in vitro immunization (sensitization) of immune cells. Antibodies can also be produced in recombinant systems, where appropriate cell lines are transformed, transfected, infected or transduced with appropriate antibody-encoding DNA. Alternatively, antibodies can be constructed by biochemical remodeling of purified heavy and light chains.

Once an antibody molecule has been produced in an animal, chemically synthesized, or recombinantly expressed, it can be purified by any method known in the art for the purification of immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, especially protein A followed by affinity for a specific antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique used to purify proteins. In addition, antibodies or fragments thereof of the invention may be fused to heterologous polypeptide sequences known in the art to facilitate purification.

In a preferred embodiment, the monoclonal antibody recognizes and/or binds an antigen present on the carbohydrate component or the peptide component of the glycolipopeptide of the invention. In particularly preferred embodiments, the monoclonal antibody binds an antigen present on a selected feature of the carbohydrate component. Examples of selected features include modifications on glycopeptides such as O -GlcNAc. Other modifications include, but are not limited to, GalNAc and other sugar modifications.

The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies) and antibody fragments so long as they exhibit the desired biological activity. "Antibody fragment" comprises a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', and Fv fragments; diabodies; linear antibodies; and single-chain antibody molecules. The term "monoclonal antibody" as used herein refers to a highly specific antibody directed against a single antigenic site. The term "antibody" as used herein also includes naturally occurring antibodies as well as non-naturally occurring antibodies including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, and antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be produced recombinantly and can be obtained, for example, by screening combinatorial libraries consisting of variable heavy and variable light chains, as described by Huse et al. (Science 246:1275 -1281 (1989)) described. These and other methods of making functional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, Id., 1988); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995)).

In all mammalian species, antibody peptides contain constant (i.e., highly conserved) and variable regions, and within the latter, there are complementarity determining regions (CDRs), and The amino acid sequences outside the CDRs constitute the so-called "framework regions". Preferably, the monoclonal antibodies of the invention have been humanized. As used herein, the term "humanized" antibody refers to an antibody in which non-human (usually from mouse or rat) CDRs have been transferred from the heavy and light variable chains of a non-human immunoglobulin into the following variable regions, so The variable regions are designed to contain many of the amino acid residues found within the framework regions of human IgG. Similar conversion of mouse/human chimeric antibodies to humanized antibodies has been described previously. General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989), which is incorporated by reference in its entirety. enter. Techniques for generating humanized MAbs are described, for example, by Jones et al., Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988), Verhoeyen et al., Science 239:1534 (1988), and Singer et al. Al, J. Immun. 150:2844 (1993), each of which is hereby incorporated by reference.

Methods using monoclonal antibodies that recognize and/or bind components of the glycolipopeptide are also encompassed by the invention. Uses for the monoclonal antibodies of the invention include, but are not limited to, diagnostic, therapeutic, and research uses. In preferred embodiments, monoclonal antibodies may be used for diagnostic purposes. Because O -GlcNAc modification is associated with various disease states, detection of changes in the level of O -GlcNAc modification can be interpreted as an early indicator of the onset of such diseases. For example, increased O -GlcNAc modification in skeletal muscle and pancreatic glycopeptides is associated with the development of type II diabetes, whereas decreased O -GlcNAc modification in neuroglycopeptides is associated with the onset of Alzheimer's disease (Dias and Hart , Mol. BioSyst. 3:766-772 (2007); Lefebvre et al., Exp. Rev. Proteomics 2(2):265-275 (2005)). Therefore, identifying an increase in the amount of O -GlcNAc in a sample of skeletal muscle tissue relative to a non-disease control sample can be indicative of the development of type II diabetes.

It should be understood that the monoclonal and polyclonal antibodies of the invention are not limited to those recognizing any particular ligand, but include, without limitation and by way of example only, directed against any type of tumor-associated carbohydrate antigen (TACA) and derived from any microorganism. Antibodies to any sugar. The antibodies of the invention are broadly useful in diagnostic or therapeutic applications.

Antibodies of the invention can be used to detect the presence or overexpression of specific proteins or specific modifications. Techniques for detection are known in the art and include, but are not limited to, Western blot, dot blot, immunoprecipitation, agglutination, ELISA assay, immunoELISA assay, tissue imaging, mass spectrometry, immunohistochemistry, and detection of various tissue or flow cytometry of body fluids, and a variety of sandwich assays. See, eg, US Patent No. 5,876,949, incorporated herein by reference.

To detect changes in the levels of O -GlcNAc modified glycopeptides, the monoclonal antibodies of the invention can be covalently or non-covalently labeled with any of a number of known detectable labels, such as fluorescent, radioactive or enzymatic substances, as known in the art. Alternatively, secondary antibodies specific for the monoclonal antibodies of the invention are labeled with a known detectable label and used to detect O -GlcNAc specific antibodies in the techniques described above.

Preferred detectable labels include chromogenic dyes. Among the most commonly used are 3-amino-9-ethylcarbazole (AEC) and 3,3'-diaminobenzidine tetrahydrochloride (DAB). These can be detected using light microscopy. Also preferred are fluorescent labels. Among the most commonly used fluorescently labeled compounds are fluorescein isothiocyanate (such as FITC and TRITC), indoletricarbocyanines (such as Cy5 and Cy7), rhodamine, phycoerythrin, phycocyanin, Phycocyanin, o-phthalaldehyde and fluorescamine. Chemiluminescent and bioluminescent compounds such as luminol, isoluminol, theromatic acridine can also be used Ester, imidazole, acridine Salt, Oxalate, Luciferin, Luciferase and Aequorin. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected due to its fluorescence. Also preferred are radioactive labels. Radioisotopes that are particularly useful for labeling the antibodies of the invention ^include3H , ^125I , ^131I , ^35S , ^{32P and14C} . Radioactive isotopes can be detected by such methods as using gamma counters, scintillation counters or by autoradiography. Enzymes that can be used to detectably label antibodies and can be detected, for example, by spectrophotometric measurements, fluorometric assays, or visual methods include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast Alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonucleic acid enzymes, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Other methods of labeling and detecting antibodies are known in the art and are within the scope of the present invention.

The three-component immunogenic vaccine of the present invention comprising a TLR agonist, a T helper epitope and a glycosylated MUCl epitope (B/T cell epitope) shows many advantages. Glycosylated B/T cell epitopes may be more potent than non-glycosylated epitopes. The vaccine elicited a strong cytolytic T-cell response that lysed MUCl-expressing cells. Activation of T cell cellular responses is usually indicated by interferon gamma secretion by CD4+ and CD8+ T cells. Further, activation of B cell responses was indicated by Ig class switching and the production of antibodies effective in inducing ADCC (antibody-dependent cell-mediated cytotoxicity) of MUCl-expressing cells (both tumor cells and YAC cells). Thus, the MUC1-based three-component immunogenic cancer vaccine dually elicited humoral and cellular immune responses, including antibody formation, interferon-γ production, and cytotoxic activity, leading to excellent therapeutic outcomes. In some embodiments, the addition of a second TLR agonist further increases effectiveness, eg, exhibits reduced tumor burden, increased IFN-γ production, and increased T cell-mediated cytotoxicity.

The invention includes methods of generating antibody-dependent cell-mediated cytotoxicity (ADCC) in a subject by immunizing the subject with one or more immunogenic vaccine constructs described herein. In some aspects, ADCC is natural killer (NK) cell mediated. In some aspects, ADCC lyses tumor cells. In some aspects, the tumor cells are breast cancer cells or epithelial cancer cells. In some aspects, ADCC lyses cells expressing the MUCl peptide sequence. In some aspects, the MUCl peptide is abnormally glycosylated.

The invention includes methods of treating cancer, reducing tumor burden, preventing tumor recurrence, and/or preventing cancer in a subject by immunizing the subject with one or more immunogenic vaccine constructs described herein. In some aspects of the methods of the invention, the cancer or tumor is breast cancer or epithelial cancer. In some aspects of the methods of the invention, the cancer or tumor expresses aberrantly glycosylated MUCl.

The invention comprises generating in a subject a cytotoxic T cell response against MUCl expressing cells, generating anti-MUCl antibodies, and/or by immunizing the subject with one or more immunogenic vaccine constructs described herein, and/or Or a method of promoting class switching of anti-MUC1 antibodies. In some aspects, the MUCl expressing cells are tumor cells. In some aspects of the methods of the invention, the cancer or tumor expresses aberrantly glycosylated MUCl.

The present invention includes methods of immunizing a subject with a glycolipid peptide comprising at least one glycosylated MUCl glycopeptide component comprising a B cell epitope; including an MHC class II restricted helper T cell epitope at least one peptide component; and at least one lipid component. In some aspects, antibodies of the IgG subtype that specifically bind to the MUCl protein expressed on the tumor cells are induced in the subject. Because it is antigenic and immunogenic, the glycolipopeptide of the invention is well suited for use in immunotherapeutic pharmaceutical compositions. The present invention therefore includes pharmaceutical compositions comprising the glycolipopeptides of the present invention together with a pharmaceutically acceptable carrier. In a preferred embodiment, the pharmaceutical composition comprises liposomes, such as phospholipid-based liposomes, and the glycolipid peptides are incorporated within the liposomes due to non-covalent interactions, such as hydrophobic interactions. Alternatively, glycolipopeptides can be covalently linked to liposome components. Liposomal formulations may include glycolipopeptides with the same or different B epitopes, the same or different T cell epitopes, and/or the same or different lipid components.

The three-component immunogenic vaccines of the invention have covalently linked at least one carbohydrate component, at least one peptide component and at least one adjuvant component. The three-component immunogenic vaccine contains a B epitope and a T epitope, preferably a helper T epitope. Typically, the carbohydrate component includes the B epitope and the peptide component contains the T epitope. B epitopes may further include T epitopes. However, these epitopes may overlap, and a single glycopeptide, such as the MUC-1 glycopeptide, may include both B and T epitopes.

The glycolipopeptides of the invention are readily formulated as pharmaceutical compositions for veterinary or human use. The pharmaceutical composition optionally comprises excipients or diluents which are pharmaceutically acceptable as carriers and compatible with the glycolipopeptide. The term "pharmaceutically acceptable carrier" refers to one or more carriers that are "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof or the glycolipopeptide. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Furthermore, if desired, the pharmaceutical compositions can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, salts and/or adjuvants, which enhance the effectiveness of the immunostimulatory composition. For oral administration, the glycolipopeptides may be mixed with proteins or oils of vegetable or animal origin. Methods of making and using such pharmaceutical compositions are also included in the invention.

The pharmaceutical compositions of the present invention can be administered to any subject, including humans and domesticated animals (eg, cats and dogs). In a preferred embodiment, the pharmaceutical composition is useful as a vaccine and contains a glycolipopeptide in an amount effective to induce an immune response in a subject. The dose of the glycolipopeptide vaccine of the present invention, the schedule for vaccination, etc. can be easily determined by those skilled in the art. The vaccine may be administered to a subject using any convenient method, preferably parenterally (eg, via intramuscular, intradermal or subcutaneous injection) or via oral or nasal administration. The useful dose to be administered will vary depending on the type of animal to be vaccinated, its age and weight, the immunogenicity of the attenuated virus and the mode of administration.

The three-component or two-component immunogenic vaccines of the invention can be administered alone or together. Additionally, because the bicomponent vaccine is useful as an adjuvant, it can be administered to enhance other cancer treatments, such as chemotherapy, radiation therapy, or other types of immunotherapy.

In one method of treatment, at least one TLR ligand is co-administered with a three-component immunogenic vaccine and/or a two-component immunogenic vaccine of the invention. Co-administered TLR ligands are administered as additional adjuvants. Exemplary TLR ligands are described herein. Any TLR ligand can be co-administered with the immunogenic vaccine. Preferably, a TLR2 or TLR9 ligand such as a CpG ODN is co-administered with the immunogenic vaccine. When an immunogenic vaccine contains a TLR ligand, such as a covalently linked TLR2 ligand, as a covalently linked adjuvant component, it is understood that the co-administered TLR ligand, such as a co-administered TLR9 ligand, may be different from the covalently linked TLR ligands.

The method of treatment may involve the administration of any combination of three-component vaccines, two-component vaccines, and/or co-administered TLR ligands, as required by the condition to be treated or as directed by a healthcare professional.

The inclusion of adjuvants in pharmaceutical compositions is optional. Adjuvants include, for example, alum, QS-21 and TLR agonists. TLR agonists include, but are not limited to, any of the TLR agonists described herein. Preferred TLR agonists include TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR8 agonists and TLR9 agonists. TLR9 is activated by unmethylated CpG-containing sequences, including those found in bacterial DNA or synthetic oligonucleotides (ODNs). Such unmethylated CpG-containing sequences are present at high frequency in bacterial DNA but are rare in mammalian DNA. Thus, unmethylated CpG sequences distinguish microbial DNA from mammalian DNA. See, eg, Janeway and Medzhitov, 2002, Ann Rev Immunol; 20:197; Barton and Medzhitov, 2002, Curr Top Microbiol Immunol; 270:81; Medzhitov, 2001, Nat Rev Immunol; 1:135; Heine and Lein, 2003, Int Arch Allergy Immunol; 130:180; Modlin, 2002, Ann Allergy Asthma Immunol; 88:543; and Dunne and O'Neill, 2003, Sci. STKE2003:re3.

The TLR9 agonist may be a preparation of microbial DNA including, but not limited to, E. coli DNA, endotoxin-free E. coli DNA, or endotoxin-free bacterial DNA from E. coli K12. TLR9 agonists can be isolated from bacteria, e.g., from a bacterial source; synthetic, e.g., produced by standard methods for chemical synthesis of polynucleotides; produced by standard recombinant methods, and subsequently isolated from a bacterial source ; or a combination of the foregoing. In many embodiments, the TLR agonist is purified and is, for example, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% , at least about 99% pure or higher.

A TLR9 agonist can be a synthetic oligonucleotide containing an unmethylated CpG motif, also referred to herein as a "CpG oligodeoxynucleotide", "CpGODN" or "ODN" (see, e.g., Hemmi et al. "A Toll-like receptor recognizes bacterial DNA," Nature 2000; 408: 740-745). At least three types of immunostimulatory CpG-ODNs have been described. A (or D) type ODN preferentially activates plasmacytoid dendritic cells (pDC) to produce IFN?, while B (or K) type ODN induces the proliferation of B cells and the secretion of IgM and IL-6. Another type that incorporates features called both Type A and Type B has been generated and called Type C. The TLR9 agonists of the present invention may comprise any of at least three types of stimulatory ODNs (Type A, Type B and Type C) that have been described.

CpG-oligodeoxynucleotide TLR9 agonists comprise a CpG motif. A CpG motif comprises two bases on the 5' side and two bases on the 3' side of a CpG dinucleotide. CpG-oligodeoxynucleotides can be produced by standard methods for chemical synthesis of polynucleotides. CpG-oligodeoxynucleotides can be purchased commercially, eg, from Coley Pharmaceuticals (Wellesley, MA), Axxora, LLC (San Diego, CA) or InVivogen, (San Diego, CA). CpG-oligodeoxynucleotide TLR9 agonists can include a wide range of DNA backbones, modifications and substitutions.

In some aspects of the invention, the TLR9 agonist is a nucleic acid comprising the nucleotide sequence 5' CG 3'. In some aspects of the invention, the TLR9 agonist is a nucleic acid comprising the nucleotide sequence 5'-purine-purine-cytosine-guanine-pyrimidine-pyrimidine-3'. In other aspects of the invention, the TLR9 agonist is a nucleic acid comprising the nucleotide sequence 5'-purine-TCG-pyrimidine-pyrimidine-3'. In some aspects of the invention, the TLR9 agonist is a nucleic acid comprising the nucleotide sequence 5'-(TGC)n-3'. In other aspects of the invention, the TLR9 agonist is a nucleic acid comprising the sequence 5'-TCGNN-3', where N is any nucleotide.

In some aspects, the TLR9 agonist can have a length of about 5 to about 200, about 10 to about 100, about 12 to about 50, about 15 to about 25, about 5 to about 15, about 5 to about 10, or about 5 to about A sequence of 7 nucleotides. In some aspects, the TLR9 agonist can be less than about 15, less than about 12, less than about 10, or less than about 8 nucleotides in length.

TLR9 agonists include, but are not limited to, those described in any of U.S. Patent Nos. 6,194,388; 6,207,646; 6,239,116; 6,339,068; A sort of.

In some aspects, a TLR agonist can be part of a larger nucleotide construct such as a plasmid vector, viral vector, or other such construct. A wide variety of plasmid and viral vectors are known in the art and need not be elaborated here. A large number of such vectors have been described in various publications. See, eg, Current Protocols in Molecular Biology, (F. M. Ausubel, et al., eds., 1987 and later). Many such vectors are commercially available.

Immunogenic vaccines of the invention may be administered with one or more additional therapeutic agents. Additional therapeutic treatments include, but are not limited to, surgical resection, radiation therapy, chemotherapy, hormone therapy, antitumor vaccines, antibody-based therapy, total body irradiation, bone marrow transplantation, peripheral blood stem cell transplantation, and chemotherapeutic agents (also referred to herein as Administration of "anti-tumor chemotherapeutic agents"). Antineoplastic chemotherapeutic agents include, but are not limited to, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, vincristine, ifosfamide, cisplatin, gemcitabine, busulfan (also known as 1, 4-Butanediol dimethanesulfonate or BU), ara-C (also known as 1-β-D-arabinofuranosylcytosine or cytarabine), doxorubicin, mitomycin, cyclic Phosphamide, methotrexate, and combinations thereof. Administration of the TLR agonist can occur before, during and/or after administration of the additional chemotherapeutic agent. Additional therapeutic agents include, for example, one or more cytokines, antibiotics, antimicrobials, antivirals such as AZT, ddI or ddC, and combinations thereof. Cytokines used include, but are not limited to, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL- 18. IL-19, IL-20, IFN-α, IFN-β, IFN-γ, tumor necrosis factor (TNF), transforming growth factor-β (TGF-β), granulocyte colony-stimulating factor (G-CSF) , macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF)) (US Pat. Nos. 5,478,556, 5,837,231 and 5,861,159), or Flt-3 ligand (Shurin et al., Cell Immunol. 1997; 179:174-184). Anti-tumor vaccines include, but are not limited to, peptide vaccines, whole cell vaccines, genetically modified whole cell vaccines, recombinant protein vaccines, or vaccines based on the expression of tumor-associated antigens by recombinant viral vectors. Additional therapeutic agents may be immunomodulators such as TLR4 agonists, TLR8 agonists, TLR9 agonists, COX-2 inhibitors, GM-CSF, inhibition of indoleamine 2,3-dioxygenase (IDO) agents, chemotherapeutic agents, or combinations thereof.

As noted, the pharmaceutical compositions are useful as vaccines. Vaccines can be prophylactic or protective. Likewise, a vaccine may be a therapeutic vaccine administered after a disease or condition, such as cancer, has developed. Accordingly, the invention encompasses vaccines comprising glycolipopeptides as described herein, including antimicrobial (eg, antiviral or antibacterial) and anticancer vaccines.

Cancers that can be effectively treated or prevented include, but are not limited to, prostate cancer, bladder cancer, colon cancer, breast cancer, melanoma, pancreatic cancer, lung cancer, leukemia, lymphoma, sarcoma, ovarian cancer, Kaposi's sarcoma, Hodgkin's Hodgkin's Disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, essential thrombocythemia, primary macroglobulinemia, small cell lung cancer, primary Brain tumor, gastric cancer, malignant pancreatic insulinoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia Cancers of cervical, endometrial, adrenocortical, and epithelial origin. As used herein, "tumor" refers to all types of cancer, neoplasm or malignancy found in mammals.

The efficacy of treatment of a tumor can be assessed by any of a variety of parameters well known in the art. This includes, but is not limited to, measuring reduction in tumor size, measuring tumor growth, dissemination, invasiveness, vascularization, angiogenesis, and/or inhibition of metastasis, measuring the growth, dissemination, invasiveness, and/or vascularization of any metastatic lesions Inhibiting, and/or measuring increased delayed-type hypersensitivity to tumor antigens. Efficacy of treatment can also be assessed by determining delay in relapse or delay in tumor progression in a subject, or determining survival of a subject, eg, increased survival at one or five years following treatment. As used herein, relapse is the return of a tumor or neoplasm after its apparent cessation, such as the return of leukemia.

The glycolipopeptides of the invention can also be used in passive immunization methods. For example, a glycolipopeptide can be administered to a host animal, such as a rabbit, mouse, rat, chicken, or goat, to generate antibody production in the host animal. Protocols for the production of polyclonal antibodies in host animals are well known. The one or more T epitopes contained in the glycolipopeptide are optionally selected to be identical or similar to the corresponding T epitopes of the host animal in which the antibody was raised. Antibodies are isolated from animals and then administered prophylactically or therapeutically to a mammalian subject, preferably a human subject, to treat or prevent a disease or infection. Monoclonal antibodies directed against glycolipopeptides of the invention can be isolated from hybridomas prepared according to standard laboratory protocols; they can also be produced using recombinant techniques such as phage display. Such antibodies are also useful for passive immunization. Optionally, the anti-glycolipid peptide monoclonal antibody is a human antibody or a humanized antibody. The one or more B epitopes contained in the glycolipopeptides used to generate polyclonal or monoclonal antibodies are selected according to the intended therapeutic purpose. The invention encompasses polyclonal and monoclonal anti-glycolipid peptide antibodies, and methods of making and using them.

Accordingly, also provided by the present invention are pharmaceutical compositions comprising a monoclonal or polyclonal antibody of the present invention and a pharmaceutically acceptable carrier. Preferably, the monoclonal antibodies are humanized antibodies. Humanized antibodies are more preferred for use in the treatment of human diseases or disorders because humanized antibodies are less likely to induce an immune response, especially an allergic response, when introduced into a human host. As noted, the pharmaceutical compositions optionally comprise excipients or diluents that are pharmaceutically acceptable as carriers and compatible with the monoclonal antibodies, and can be administered to any subject, including humans and domesticated animals (e.g., cats and dog). Methods of making and using such pharmaceutical compositions are also included in the invention.

A common feature of oncogenically transformed cells is the overexpression of oligosaccharides such as Globo-H, Lewis ^Y and Tn antigens. Optionally, a pharmaceutical composition of the invention comprising a monoclonal or polyclonal antibody of the invention and a pharmaceutically acceptable carrier can be used to target tumors comprising oncogenically transformed cells overexpressing such oligosaccharides. For example, antibodies conjugated to chemotherapeutic molecules can be used to deliver chemotherapeutic molecules to tumors.

Another pharmaceutical composition of the present invention may comprise a compound (such as an antibody, ligand, small molecule or peptide) that can affect the activity of a protein and a pharmaceutically acceptable carrier. The effect of a compound on a protein may include, but is not limited to, agonizing, antagonizing, inhibiting or enhancing the normal biological process of the protein. Preferably, the compound is an antibody that binds an epitope on a protein comprising O -glycosylation sites. Preferably, the O -glycosylation site is an O -GlcNAc site. Numerous studies have shown that this aberrant glycosylation can promote metastasis and thus it is strongly associated with poor survival of cancer patients. Thus, the ability to affect the activity of abnormally glycosylated proteins may enable the prevention of abnormal activity.

Therapeutically effective concentrations and amounts can be determined empirically for each application described herein by testing the compound in known in vitro and in vivo systems, including but not limited to any of those described herein, and can then be determined from its in vitro Dosages for use in humans or other animals. The efficacy of treatment can be assessed by any of a variety of parameters well known in the art. This includes, but is not limited to, reduction in tumor size, increase in CD8 ⁺ T cell activity, and/or increased survival time.

As noted elsewhere herein, it has surprisingly been found that Toll-like receptor (TLR) ligands are covalently linked to glycopeptides comprising a carbohydrate component (containing a B epitope) and a peptide component (containing a T epitope), enhancing Uptake and internalization of glycopeptides by target cells (see Example 3). Thus identified TLR ligands characterized as lipids are preferred lipid components for use in the glycolipopeptides of the invention. The present invention thus further provides a method for identifying a TLR ligand, preferably a lipid ligand, comprising contacting a candidate compound with a target cell containing a Toll-like receptor (TLR), and determining whether the candidate compound binds the TLR (i.e., whether are TLR ligands). Preferably, the candidate compound is internalized by the target cell via TLRs. Lipid-containing TLR ligands identified by binding to TLRs and optionally by internalization into target cells are expected to be immunogenic and are well suited for use as the lipid component of the glycolipeptides of the invention. The present invention thus also encompasses glycolipopeptides comprising, as a lipid component, one or more lipid-containing TLR ligands identified using the methods of the present invention.

The invention also includes diagnostic kits. Kits provided by the invention may contain antibodies of the invention, preferably monoclonal antibodies, and suitable buffers (eg Tris, phosphate, carbonate, etc.), thereby enabling kit users to identify O -GlcNAc modifications. The user can then detectably label the antibody as desired. Alternatively, the kits provided by the invention may contain the antibody in solution, preferably frozen in quenching buffer or in powder form (eg by lyophilization). Antibodies, which may be conjugated to a detectable label or unconjugated, are included in the kit together with a buffer which may optionally further include stabilizers, biocides, inert proteins such as serum albumin, and the like. Generally, these materials will be present in a total amount of less than 5% by weight based on the amount of active antibody, and usually at least about 0.001% by weight, again based on the concentration of the antibody. Optionally, the kit may include inert extenders or excipients to dilute the active ingredients, wherein the excipients may be present at about 1% to 99% by weight of the total composition. In preferred embodiments, the antibodies provided by the kit are detectably labeled such that bound antibodies are detectable. Detectable labels can be radioactive labels, enzymatic labels, fluorescent labels, and the like. Optionally, the kit may contain an unconjugated monoclonal antibody of the invention, and further contain a secondary antibody capable of binding the primary antibody. When a secondary antibody capable of binding the primary antibody is employed in the assay, this is usually present in a separate vial. Secondary antibodies are typically conjugated to a detectable label and formulated in a manner similar to the antibody preparations described above. Kits generally also include packaging and a set of instructions for use.

As used herein, the term "subject" includes, but is not limited to, humans and non-human vertebrates. Non-human vertebrates include livestock animals, companion animals, and laboratory animals. Non-human subjects also include non-human primates as well as rodents such as, but not limited to, rats or mice. Non-human subjects also include, but are not limited to, chickens, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink, and rabbits. As used herein, the terms "subject", "individual", "patient" and "host" are used interchangeably. In a preferred embodiment, the subject is a mammal, especially a human.

As used herein, "in vitro" is in cell culture and "in vivo" is within the body of a subject.

As used herein, "treatment" or "treatment" includes both curative and prophylactic treatments. Treating a disease or condition shall mean interfering with such a disease or condition in order to prevent or slow the development of the disease or condition, prevent or slow the progression of the disease or condition, halt the progression of the disease or condition, or eliminate the disease or condition.

As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to humans or other vertebrates.

As used herein, the term "isolated" when used to describe a compound shall mean removed from the natural environment in which the compound occurs in nature. In one embodiment, isolated means removed from cells other than nucleic acid molecules.

When a range of values is provided, it is understood that unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated value in that stated range Values or intermediate values are included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

In some embodiments, an "effective amount" is an amount that results in a reduction of at least one pathological parameter. Thus, for example, effective to achieve at least about 10%, at least about 15%, at least about 20%, or at least about 25%, at least about 30%, at least about 35% compared to the expected reduction of the parameter in an individual not receiving treatment , at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% , at least about 90% or at least about 95% reduced amount.

Example

The invention is illustrated by the following examples. It should be understood that specific examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Example 1

Targeting fully synthetic carbohydrate-based anticancer vaccines: synthesis and immunological evaluation of lipidated glycopeptides containing tumor-associated Tn antigens

In this example, a fully synthetic cancer vaccine candidate consisting of tumor-associated Tn antigen, peptide T epitope and lipopeptide Pam ₃ Cys was prepared by a combination of polymer support and solution phase chemistry. Incorporation of glycolipid peptides into liposomes gives formulations capable of eliciting T cell-dependent antibody responses in mice.

A common feature of oncogenically transformed cells is the overexpression of oligosaccharides such as Globo-H, Lewis ^Y and Tn antigens (Lloyd, Am. J Clin. Pathol. 1987, 87, 129; Feizi et al., Trends in Biochem. Sci. 1985, 10, 24-29; Springer, J. Mol. Med. 1997, 75, 594-602; Hakomori, Acta Anat. 1998, 161, 79-90). Numerous studies have shown that this aberrant glycosylation can promote metastasis (Sanders et al., Mol. Pathol. 1999, 52, 174-178), so its expression is strongly correlated with poor survival of cancer patients.

Several first-rate studies have exploited differential expression of tumor-associated carbohydrates for the development of cancer vaccines (Ragupathi, Cancer Immunol. 1996, 43, 152-157; Musselli et al., J Cancer Res. Clin. Oncol. 2001, 127, R20- R26). However, the inability of carbohydrates to activate helper T lymphocytes has complicated their use as vaccines (Kuberan et al., Current Organic Chemistry 2000, 4, 653-677). For most immunogens, including carbohydrates, antibody production relies on the cooperative interaction of two types of lymphocytes, B cells and helper T cells (Jennings et al., Neoglycoconjugates, preparation and application, Academic, San Diego, 1994). Sugar alone cannot activate helper T cells and therefore has limited immunogenicity. The formation of low affinity IgM antibodies and the absence of IgG antibodies manifests this limited immunogenicity.

To overcome the T cell independent nature of carbohydrates, past studies have focused on conjugation of carbohydrates to exogenous carrier proteins (eg keyhole limpet hemocyanin (KLH), detoxified tetanus toxoid). In this approach, the carrier protein enhances the presentation of carbohydrates to the immune system and provides T epitopes (peptide fragments of 12-15 amino acids) that can activate T helper cells.

However, the conjugation of carbohydrates to carrier proteins has several problems. In general, conjugation chemistry is difficult to control, leading to conjugates with compositional and structural ambiguities that can affect the reproducibility of the immune response (Anderson et al., J. Immunol. 1989, 142, 2464-2468) . Furthermore, exogenous carrier proteins can elicit strong B cell responses, which can lead to suppression of antibody responses to carbohydrate epitopes. The latter is a greater problem when employing self-antigens such as tumor-associated carbohydrates. Furthermore, linkers used for the conjugation of carbohydrates to proteins can be immunogenic, leading to epitope suppression (Buskas et al., Chem. Eur. J. 2004, 10, 3517-3523). Not surprisingly, several clinical trials with carbohydrate-protein conjugate cancer vaccines failed to induce sufficiently strong helper T cell responses in all patients (Sabbatini et al., Int. J. Cancer 2000, 87, 79-85). Therefore, there is a need to develop alternative strategies for the presentation of tumor-associated carbohydrate epitopes that will lead to more efficient class switching to IgG antibodies (Keil et al., Angew. Chem. Int. Ed. 2001, 40, 366-369; Angew. Chem. 2001, 113, 379-382; Toyokuni et al., Bioorg. & Med. Chem. 1994, 2, 1119-1132; Lo-Man et al., Cancer Res. 2004, 64, 4987-4994; Kagan et al. Human, Cancer Immunol. Immunother. 2005, 54, 424-430; Reichel et al., Chem. Commun. 1997, 21, 2087-2088).

Here, we report the synthesis and immunological evaluation of a structurally well-defined, fully synthetic anticancer vaccine candidate (compound 9) that constitutes the complex required for a focused and efficient T cell-dependent immune response. minimum structural features. The vaccine candidate consists of tumor-associated Tn antigen, peptide T epitope YAFKYARHANVGRNAFELFL (YAF) (SEQ ID NO: 2) and lipopeptide S -[(R)-2,3-dipalmitoyloxy-propyl]-N- Palmitoyl-(R)-cysteine (Pam ₃ Cys) composition. The Tn antigen serving as the B epitope is overexpressed on the surface of human epithelial tumor cells of the breast, colon and prostate. This antigen is not present on normal cells and thus makes it an excellent target for immunotherapy. To overcome the T cell independent nature of carbohydrate antigens, YAF peptides were incorporated. This 20 amino acid peptide sequence is derived from the outer membrane protein of Neisseria meningitidis and has been identified as an MHC class II restriction site for human T cells (Wiertz et al., J. Exp. Med. 1992, 176, 79 -88). It is hypothesized that such helper T-cell epitopes induce a T-cell-dependent immune response, leading to the production of IgG antibodies against Tn antigens. Combined B-cell and T-helper epitopes lack the ability to provide a suitable "danger signal" for dendritic cell (DC) maturation (Medzhitov et al., Science 2002, 296, 298-300). Thus, the lipopeptide Pam ₃ Cys was incorporated from the immunologically active N -terminal sequence of the major lipoprotein derived from E. coli (Braun, Biochim. Biophys. Acta 1975, 415, 335-377). This lipopeptide has been recognized as a potent immune adjuvant (Weismuller et al., Physiol. Chem. 1983, 364, 593), and recent studies have shown that it interacts with Toll-like receptor-2 (TLR-2) exert its activity (Aliprantis et al., Science 1999, 285, 736-73). This interaction leads to the production of proinflammatory cytokines and chemokines, which in turn stimulate antigen-presenting cells (APCs) to initiate helper T cell development and activation (Werling et al., Vet. Immunol. Immunopathol. 2003, 91, 1-12). Lipopeptides also facilitate the incorporation of antigen into liposomes. Liposomes have attracted attention as carriers in vaccine design (Kersten et al., Biochim. Biophys. Acta 1995, 1241, 117-138), due to their low inherent immunogenicity, thereby avoiding unwanted carrier-induced immune response.

The synthesis of target compound 9 required a highly convergent synthetic strategy employing chemical manipulations compatible with the presence of carbohydrate, peptide and lipid moieties. Scenario 9 can be composed of spacer-containing Tn antigen 7, polymer-conjugated peptide 1, and S- [2,3-bis(palmitoyloxy)propyl] -N -Fmoc-Cys (Pam ₂ FmocCys, 2,( Metzger et al., Int. J. Peptide Protein Res. 1991, 38, 545-554)) for preparation. Using highly acid-sensitive HMPB-MBHA resin and 2-(1H-benzotriazol-1-yl)-oxy-1,1,3,3-tetramethylurea as the activation mixture (Scheme 10) Hexafluorophosphate/1-hydroxybenzotriazole (HBTU/HOBt) (Knorr et al., Tetrahedron Lett. 1989, 30, 1927-1930) combined Fmoc-protected amino acids for assembly of resin-bound peptides by automated solid-phase peptide synthesis 1. HMPB-MBHA resin was chosen because it allows cleavage of the compound from the resin without concomitant removal of side chain protecting groups. This feature is important because the side chain functional groups of aspartic acid, glutamic acid and lysine would otherwise interfere with the incorporation of the Tn antigen derivative7. Next, the Pam ₂ FmocCys derivative 2 was manually coupled using PyBOP (Martinez et al., J. Med. Chem. 1988, 28, 1874-1879) and HOBt in the presence of DIPEA in a mixture of DMF and dichloromethane to the N-terminal amine of peptide 1 to give resin-bound lipopeptide 3. The Fmoc group of 3 was removed under standard conditions and the resulting free amine of compound 4 was coupled to palmitic acid in the presence of PyBOP and HOBt, to give fully protected and resin-bound lipopeptide 5. The amine of the Pam ₂ Cys moiety is palmitoylated after coupling with 1 to avoid racemization of the cysteine moiety. Cleavage of compound 5 from the resin was achieved with 2% TFA in dichloromethane, followed by immediate neutralization with 5% pyridine in methanol. After purification by LH-20 size exclusion chromatography, the C-terminal carboxylate of lipopeptide 6 was made The acid was coupled to the amine of the Tn derivative 7 to give the fully protected lipidated glycopeptide 8 in 79% yield after purification by Sephadex LH-20 size exclusion chromatography. Mass spectrometry analysis by MALDI-TOF showed signals at m / z 5239.6 and 5263.0, corresponding to [M+H] ⁺ and [M+Na] ⁺ , respectively. Finally, the side chain protecting group of 8 was removed by treatment with 95% TFA in water using 1,2-ethanedithiol (EDT) as a scavenger. An alternative use of triisopropylsilane (TIS) was found to result in the formation of unidentified by-products. Target compound 9 was purified by size exclusion chromatography followed by RP-HPLC using a Synchropak C4 column. MALDI mass analysis of 9 showed a signal at m/z 3760.3, corresponding to [M+Na] ⁺ .

Scheme 10. a) PyBOP, HOBt, DIPEA, DMF/DCM (5/1, v/v); b) piperidine/DMF (1/5, v/v); c) CH ₃ (CH ₂ ) ₁₄ COOH , PyBOP, HOBt, DMF/DCM (1/5, v/v); d) 2% TFA in DCM; e) 7, DIC, HOAt, DIPEA, DMF/DCM (2/1, v/v), 79%; f) TFA/ _H2O /EDT (95/2.5/2.5, v/v/v), 79%.

Next, compound 9 was incorporated into phospholipid-based liposomes. Therefore, after hydration of lipid films containing 9, cholesterol, phosphatidylcholine, and phosphatidylethanolamine, small unilamellar vesicles (SUVs) were prepared by extrusion through a 100 nm Nuclepore® polycarbonate membrane. Liposomes were uniformly sized by negative-stained transmission electron microscopy (TEM), with an expected diameter of about 100 nm (see Buskas et al., Angew. Chem. Int. Ed. 2005, 44, 5985-5988). figure 1). Liposome formulations were analyzed for N -acetylgalactosamine content by hydrolysis with TFA followed by quantification by high pH anion exchange chromatography. A concentration of about 30 μg/mL GalNAc, corresponding to about 10% incorporation of starting compound 9, was determined.

Groups of five female BALB/c mice were immunized subcutaneously at weekly intervals with freshly prepared liposomes containing 0.6 μg carbohydrate. To explore the adjuvant properties of the embedded lipopeptide _Pam3Cys , antigen-containing liposomes were administered with or without the potent saponin immunoadjuvant QS-21 (Antigenics Inc., Lexington, MA). Anti-Tn antibody titers were determined by coating microtiter plates with BSA-Tn conjugate and detection was accomplished with anti-mouse IgM or IgG antibodies labeled with alkaline phosphatase. As can be seen in Table 1, mice were immunized with liposome formulations, eliciting IgM and IgG antibodies against the Tn antigen (Table 1, entries 1 and 2). The presence of IgG antibodies indicated that the helper T epitope peptide of 9 had activated helper T lymphocytes. Furthermore, the observation that IgG antibodies were produced in mice immunized with liposomes only (Group 1 ) indicated that the embedded adjuvant Pam ₃ Cys had triggered the appropriate signals for DC maturation and their subsequent activation of helper T cells. However, mice receiving liposomes in combination with QS-21 (Group 2) elicited higher titers of anti-Tn antibodies. This stronger immune response may be due to a shift from a mixed Th1/Th2 to a Th1 response (Moore et al., Vaccine 1999, 17, 2517-2527).

Table 1. ELISA anti-Tn antibody titers ^[a] after 4 immunizations with glycolipopeptide/liposome formulations.

^[a] ELISA plates were coated with BSA-BrAc-Tn conjugate. All titers are means for groups of five mice. Titers were determined by regression analysis plotting log10 dilutions compared to absorbance. Titers were calculated as the highest dilution that gave an absorbance greater than 0.1 or greater relative to a 1:100 dilution of saline mouse serum.

The results presented here provide for the first time a proof-of-principle for the use of lipidated glycopeptides as minimal subunit vaccines. Several improvements are expected to be made. For example, the clustered presentation of the Tn antigen has been found to be a more suitable mimic of mucin, and thus antibodies raised against this structure better recognize the Tn antigen expressed on cancer cells (Nakada et al., J. Biol. Chem. 1991 , 266, 12402-12405; Nakada et al., Proc. Natl. Acad. Sci. USA 1993, 90, 2495-2499; Reddish et al., Glycoconj. J. 1997, 14, 549-560; Reis et al., Glycoconj. J. 1998, 15, 51-62). The Tn epitope used in this study is a known MHC class II restricted epitope for humans. Therefore, more efficient class switching to IgG antibodies can be expected when murine Th epitopes are employed. Compound 9, on the other hand, is a more suitable vaccine candidate for use in humans. A recent report indicated that Pam ₂ Cys is a more effective immune adjuvant than Pam ₃ Cys (Jackson et al., Proc. Nat. Acad. Sci. USA 2004, 101, 15440-15445). The Pam ₂ Cys adjuvant has also been suggested to have improved solubility properties (Zeng et al., J. Immunol. 2002, 169, 4905-4912), which is a problematic feature of compound 9. Research to address these issues is ongoing.

This work is reported in Buskas et al., Angew. Chem. Int. Ed . 2005, 44, 5985-5988.

support information

Reagents and general experimental procedures. Amino acids and resins were obtained from Applied Biosystems and NovaBiochem; DMF was obtained from EM science; and NMP was obtained from Applied Biosystems. Phosphatidylethanolamine (PE), cholesterol, phosphatidylcholine (PC; egg yolk) and phosphatidylglycerol (PG; egg yolk) were purchased from Sigma-Aldrich and Fluka. All other chemicals were purchased from Aldrich, Acros and Fluka and used without further purification. All solvents employed were of reagent grade and dried by refluxing through a suitable desiccant. TLC was performed using Kieselgel 60 F ₂₅₄ (Merck) plates with detection by UV light (254 nm) and/or by charring with 8% sulfuric acid in ethanol or by ninhydrin. Column chromatography was performed on silica gel (Merck, mesh 70-230). Size exclusion column chromatography was performed on Sephadex LH-20. The extract was concentrated under reduced pressure at < 40°C (water bath). An Agilent 1100 series HPLC system equipped with an autosampler, UV detector and fraction collector and a Synchropak C4 column 100x4.6 mm RP with a flow rate of 1 mL/min was used for analysis and purification. Cationic matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were recorded using an HP-MALDI instrument using gentisic acid as the matrix. ¹ H NMR and ¹³ C NMR spectra were recorded on a Varian Inova300 spectrometer, a Varian Inova500 spectrometer and a Varian Inova600 spectrometer, all equipped with a Sun workstation. The ¹ H spectrum recorded in CDCl ₃ was referenced to the residue CHCl ₃ or TMS at 7.26 ppm, and the ¹³ C spectrum was referenced to the central peak of CDCl ₃ at 77.0 ppm. Assignments were made using standard 1D experiments as well as gCOSY/DQCOSY, gHSQC and TOCSY 2D experiments.

lipopeptide 6. Compound 1 was synthesized on HMPB-MBHA resin (maximum load, 0.1 mmol). The synthesis of peptide 1 was performed on an ABI 433A peptide synthesizer equipped with a UV detector, using Fmoc-protected amino acids and 2-(1H-benzotriazol-1-yl)-oxyl-1,1,3,3- Tetramethylurea Hexafluorophosphate (HBTU)/1-hydroxybenzotriazole (HOBt) was performed as coupling agent. Individual coupling steps were performed with conditional capping as needed. After peptide 1 synthesis was complete, the remaining steps were performed manually. N -Fmoxy-S-(2,3-bis(palmitoyloxy)-(2R-propyl)-(R)-cysteine 2 (120 mg, 0.13 mmol) was dissolved in DMF ( 5 mL), and added PyBOP (0.13 mmol), HOBt (0.13 mmol) and DIPEA (0.27 mmol). After premixing for 2 minutes, DCM (1 mL) was added, and the mixture was added to the resin. The coupling step This was performed twice. After the coupling was complete (as determined by the Kaiser test), the N-Fmoc group was cleaved using 20% piperidine in DMF (5 mL). Using PyBop (0.3 mmol), HOBt (0.3 mmol) and DIPEA (0.6 mmol) in DMF, palmitic acid (77 mg, 0.3 mmol) was coupled with the free amine as described above. The resin was washed well with DMF and DCM and dried under vacuum for 4 hours. Treatment with fluoroacetic acid in DCM (2.5 mL) for 2 min released the fully protected lipopeptide 6 from the resin. The mixture was filtered into 5% pyridine in methanol (5 mL). This procedure was repeated and the lipopeptide containing Fractions were combined and concentrated to dryness. The crude product was purified by size exclusion chromatography (LH-20, DCM/MeOH, 1:1) to give lipopeptide 6 (275 mg, 0.057 mmol) as a white solid: R _f = 0.57 (DCM/MeOH 9:1); selected NMR data (CDCl ₃ /CD ₃ OD 1/1 v/v 600MHz): ¹ H, δ 0.48-0.90 (m, 27 H, Pam CH ₃ , Leu CH ₃ , Val CH ₃ ), 0.96-1.61 (m,Leu CH ₂ , Leu CH, Lys CH ₂ , ^t Bu CH ₃ , Boc CH ₃ , Ala CH ₃ , Arg CH ₂ ), 1.18 (br s, 72H, Pam CH ₂ ), 1.95, 1.99 (s, 4x3H, Pbf CH ₃ C), 2.36, 2.41, 2.44 (s, 6x3H, Pbf CH ₃ ), 2.48 (s, 2x2H, Pbf CH ₂ ) 2.65-2.73 (m, 6H, SC H ₂ -Glyceryl, His CH ₂ , Cys ^β ), 3.47(m, 2H, Gly ^α ), 3.57 (m, 2H, Gly ^α ), 4.06 (m, 1H, S-Glyceryl- CH ₂ ^b O), 4.32 (m, 1H,S-glyceryl- CH ₂ ^a O), 3.65-4.39 (m, 17H, Phe ^α , Ala ^α , His ^α , Lys ^α , Val ^α , Asn ^α , Glu ^α ,Tyr ^α , Arg ^α ), 4.45 (m, 1H, Cys ^α ), 5.06 (m, 1H, S-glyceryl- CH ), 6.72-7.39 (m, 70H, His CH, Tyr aromatic (aromat), Phe aromatic, Trt aromatic), 7.48-8.29 (m , NH). MALDI-MS [M+Na] m/z _{= 4860.22 calculated} for _{C269H373N33O42S3} : found _4860.31 .

Protected Glycolipidin 8. A solution of lipopeptide 6 (22 mg, 4.6 μmol), HOAt (6.3 mg, 46 μmol) and DIC (7 μL, 46 μmol) in DCM/DMF (2/1 v/v, 1.5 mL) was prepared under argon atmosphere at Stir at ambient temperature for 15 minutes. A solution of compound 7 (8 mg, 19 μmol) and DIPEA (14 μL, 92 μmol) in DMF (1.5 mL) was added to the stirred mixture of lipopeptides, and the reaction was maintained at room temperature for 18 hours. The mixture was diluted with toluene and concentrated to dryness under reduced pressure. Purification of the residue by size exclusion (LH-20, DCM/MeOH 1:1) gave compound 8 (19 mg, 79%) as a white solid: Selected NMR data (CDCl ₃ /CD ₃ OD 1/1 v/v 600MHz): ¹ H,δ 0.60-0.90 (m, 27 H, PamCH ₃ , Leu CH ₃ , Val CH ₃ ), 0.96-1.61 (m, Leu CH ₂ , Leu CH, Lys CH ₂ , ^t Bu CH ₃ , BocCH ₃ , Ala CH ₃ , Arg CH ₂ ), 1.18 (br s, 72H, Pam CH ₂ ), 1.94, 1.98, 1.99, 2.00 (s,6x3H, Pbf CH ₃ C, HNAc CH ₃ ), 2.36 , 2.41, 2.45 (s, 6x3H, Pbf CH ₃ ), 2.48 (s, 2x2H,Pbf CH ₂ ), 3.42-4.31 (m, Phe ^α , Ala, Lys, Val, Asp, Glu, Tyr, Arg, Gly, Leu,His, Asn CH ₂ , Tyr CH ₂ , Phe CH ₂ , Arg CH ₂ ), 3.71 (H-3), 3.88 (H-4) 4.06 (S-glyceryl- CH ₂ ^β O), 4.20 ( t, 1H, H-2), 4.32 (m, 1H, S-glyceryl- CH ₂ ^α O), 4.42 (m, 1H, Cys ^α ), 4.82 (d, 1H, H-1, J =3.68 Hz), 5.06 (m, 1H, S-glyceryl- CH ), 6.72-7.39 (m, 70H, His CH, Tyr aromatic, Phe aromatic, Trt aromatic), 7.48-8.29 (m , NH). MALDI-MS [M+Na] m/z = 5262.67 calculated for C ₂₈₆ H ₄₀₃ N ₃₇ O ₄₉ S ₃ : found 5262.99.

Glycolipidin 9. Compound 8 (12 mg, 2.3 μmol) in a deprotection mixture of TFA/H ₂ O/ethane-1,2-dithiol (95:2.5:2.5, 3 mL) was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure and the crude compound was purified first by a short size-exclusion LH-20 column (DCM/MeOH 1:1) followed by HPLC using a gradient of 0-100% acetonitrile in water (0.1% TFA) The crude compound was purified to give compound 9 (6.8 mg, 79%) as a white solid after lyophilization: selected NMR data (CDCl ₃ /CD ₃ OD 600 MHz): ¹ H, δ 0.74-0.96 (m , 27H, Pam CH ₃ , LeuCH ₃ , Val CH ₃ ), 1.11-2.35 (Leu CH ₂ , Leu CH, sp CH ₂ , Lys CH ₂ , Glu CH ₂ , Ala CH ₃ ,Val CH, Asp CH ₂ ), 1.29 (br S, 72H, Pam CH ₂ ), 2.43-3.87 (Ala ^α , Gly ^α , S-glyceryl-OCH ₂ , Cys ^β , H-2, H-3, H-4, H-5, H -6), 4.05-4.73 (m, Cys ^α , Phe ^α , Tyr ^α , His ^α , Leu ^α , Lys ^α , Asp ^α , Val ^α , Arg ^α , Glu ^α , H-1), 5.12 (m, 1H , S-glyceryl- CH ), 6.64-6.71 (dd+dd, 2H, His CH, NH), 6.86-7.12 (dd+dd 2H, His CH, NH) 7.16-8.23 (m, Tyr aromatic , Phe aromatic, NH). HR-MALDI-MS calculated for C ₁₈₆ H ₂₉₇ N ₃₇ O ₄₁ S [M+Na] m/z = 3760.1911 : found 3760.3384.

Tn derivatives 11. Compound 10 was dissolved in DMF (10 mL), and diisopropylcarbodiimide (DIC) (82 μL, 0.53 mmol) and HOAt (216 mg, 1.58 mmol) were added. After stirring for 15 minutes, 3-(N-(tert-butoxycarbonyl)-amino)propanol (111 mg, 0.63 mmol) was added and the reaction was maintained at ambient temperature for 15 hours. The mixture was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (0-5% MeOH in DCM) and LH-20 size exclusion chromatography (DCM/MeOH 1:1) to give Compound 11 (363 mg, 83%). R _f = 0.63 (DCM/MeOH 9:1); [α] _D +4.4 (c 1.0 mg/mL, CH ₂ Cl ₂ ); NMR data (CDCl ₃ , 500MHz): ¹ H, δ 1.27 (d, 3H ,CH ₃ Thr), 1.43 (s, 9H, ^t Bu CH ₃ ), 1.46-1.61 (m, 2H, CH ₂ ), 1.99 (s, 3H, CH ₃ Ac), 2.05 (s, 6H, CH ₃ Ac ), 2.06 (s, 3H, CH ₃ Ac), 2.17 (s, 3H, CH ₃ Ac), 3.17-3.27(m, 3H, CH ₂ , CH _2a ), 3.48-3.50 (m, 1H, CH _2b ) , 4.07-4.28 (m, 6H, H-6, H-5, Thr ^α ,Thr ^β , CH Fmoc), 4.43-4.51 (m, 2H, CH ₂ Fmoc), 4.62 (dd, 1H, H-2) , 4.89 (br t,1H, NH), 5.04-5.11 (m, 2H, H-1, H-3), 5.41 (d, 1H, H-4), 5.75 (br d, 1H, NHT), 6.81 (br d, 1H, NH GalNAc), 7.17-7.79 (m, 8H, aromatic H); ¹³ C (CDCl ₃ , 75MHz)δ17.19, 20.92, 20.99, 21.09, 23.30, 28.55, 30.69, 35.87, 36.92, 47.43, 47.77,58.57, 62.36, 67.47, 68.68, 77.46, 80.08, 99.88, 120.25, 125.34, 127.35,128.00, 128.76, 129.13, 141.55, 143.94, 144.01, 156.51, 157.52, 169.68,170.66, 170.94, 170.99。

HR-MALDI-MS [M+Na] m/z = 849.3535 calculated for C ₄₁ H ₅₄ N ₄ O ₁₄ : found 849.3391.

Tn derivatives 7 . A solution of compound 11 (194 mg, 0.24 mmol) in 20% piperidine in DMF (5 mL) was stirred at ambient temperature for 1 hour. The mixture was concentrated to dryness, and the residue was treated with pyridine/acetic anhydride (3:1, 5 mL) for 2 hours. The reaction mixture was diluted with toluene and concentrated to dryness. The residue was dissolved in dichloromethane and washed with 1M HCl and saturated aqueous NaHCO ₃ , dried over MgSO ₄ , filtered and concentrated. Purification of the residue by size exclusion chromatography (LH-20, DCM/MeOH 1:1) provided compound 12 (167 mg, 91%): NMR data (CDCl ₃ , 300 MHz): ¹ H, δ1.24 (d, 1H, Thr CH ₃ ), 1.42 ( ^s , 9H, tBu CH ₃ ), 1.55-1.59 (m, 2H, NHCH ₂ CH ₂ CH ₂ NH), 1.95, 2.02, 2.03, 2.12, 2.14 (s, 15H , CH ₃ Ac), 3.13-3.23 (m, 3H, CH ₂ + CH _2a ),3.36-3.41 (m, 1H, CH _2b ), 4.03-4.12 (m, 2H), 4.19-4.23 (m, 2H, Thr ^β ), 4.54-4.61(m, H-2, Thr ^α ), 4.88 (m, 1H, NH), 4.96 (s, 1H, J =3.57 Hz, H-1), 5.07 (dd, 1H,H -3), 5.35 (d, 1H, H-4), 6.43 (br S, 1H, NH), 6.72 (br S, 1H, NH). MALDI-MS [ _M +Na] m/z = _669.296 calculated for _C28H46N4O13 : found _669.323 . Compound 12 was deprotected by stirring with 5% hydrazine hydrate in methanol (5 mL) for 35 minutes at room temperature. The reaction mixture was diluted with toluene and concentrated. The residue was co-evaporated twice with toluene. Purification by silica gel column chromatography (DCM/MeOH 5:1) afforded 13 (119 mg, 89%): NMR data (CD ₃ OD, 300 MHz): ¹ H, δ 1.26 (d, 3H, Thr CH ₃ ), 1.43 (s, 9H, ^t Bu CH ₃ ), 1.57-1.63 (m, 2H, NHCH ₂ CH ₂ CH ₂ NH), 2.06, 2.10 (s, 2x3H NHAc), 2.12-3.09 (m, 2H, CH ₂ ), 3.15 (m, 2H, CH ₂ ), 3.31 (br s, 2H, H-6), 3.68-3.76 (m, 2H, H-3, H-5), 3.88 (d, 1H, H-4 ), 4.22-4.26 (m, 2H, H-2, Thr ^β ), 4.46 (m, 1H, Thr ^α ), 4.84 (d,1H, H-1), 6.60 (br m, 1H, NH), 7.50 (br d, 1H, NH). MALDI-MS [M+Na] m/z = 543.264 calculated for C ₂₂ H ₄₀ N ₄ O ₁₀ : found 543.301. A solution of 13 in trifluoroacetic acid (4 mL) was stirred at ambient temperature for 45 min under an atmosphere of argon. The reaction mixture was then diluted with DCM and concentrated to dryness. The crude product was purified by column chromatography (Iatro beads, EtOAc/MeOH/H2O ₂ :2:1 → MeOH/ _H2O 1:1). After concentration of the combined fractions, the solid was lyophilized from H ₂ O to give compound 7 (91 mg, 0.21 mmol, 95%) as a white powder. R _f = 0.17 (EtOAc/MeOH/H ₂ O 6:3:1); [α] _D -37 (c 1.0 mg/mL, H ₂ O); NMR data (D ₂ O, 300MHz): ¹ H, δ 1.15 (d,3H, J = 6.3 Hz, Thr CH ₃ ), 1.73-1.77 (m, 2H, CH ₂ ), 1.95 (s, 3H, NHAc), 2.04 (s,3H, NHAc), 2.82-2.87 (m, 2H, CH ₂ ), 3.11-3.15 (m, 1H, CH _2a ), 3.22-3.26 (m, 1H, CH _2b ), 3.65 (m, 2H, H-6), 3.76 (dd, 1H, J = 2.9, 11.2 Hz, H-3), 3.87 (d, 1H, J = 2.9 Hz, H-4), 3.92 (t, 1H, H-5), 3.99 (dd, 1H, J = 3.41, 11.2 Hz, H-2),4.28-4.30 (m, 1H, Thr ^β ), 4.32 (d, 1H, J = 2.4 Hz, Thr ^α ) 4.78 (d, 1H, J = 3.56Hz, J = 3.9 Hz, H -1), 7.97 (br d, 1H, NH), 8.17 (br t, 1H, NH), 8.27 (br d,1H, NH); ¹³ C (D ₂ O, 75MHz), δ 18.17 Thr CH ₃ ) , 21.93, 22.33 (2xNAc) 26.98 (CH ₂ ),36.55 (CH ₂ ), 37.22 (CH ₂ ), 49.98 (C-6), 58.30 (C-3), 61.46 (C-4), 67.76 (C- 5), 68.65 (C-2), 71.54 (C-Thr ^β ), 74.60 (C-Thr ^α ), 98.60 (C-1), 172.09, 174.37, 175.18 (3x C=O, NHAc). HR-MALDI-MS [M+Na] m/z = _{443.2118 calculated} for _C17H32N4O8 : found _443.2489 .

Liposome preparation. Liposomes were prepared from PC, PG, cholesterol and glycolipeptide 9 (15 μmol, molar ratio 65:25:50:10). Lipids were dissolved in DCM/MeOH (3/1, v/v) under argon atmosphere. The solvent was then removed by passing a stream of dry nitrogen and subsequent further drying under high vacuum for one hour. The resulting lipid film was suspended in 1 mL of 10 mM Hepes buffer, pH 6.5, containing 145 mM NaCl. The solution was vortexed at 41 °C for 3 hours on a shaker (250 rpm) under Ar atmosphere. The liposome suspension was extruded ten times at 50°C through 0.6 μm, 0.2 μm and 0.1 μm polycarbonate membranes (Whatman, Nuclepore®, Track-Etch Membrane) to obtain SUVs.

immunization . Five mice (female BALB/c, 6-week ) group. Mice were bled (leg vein) on day 28 and sera were tested for the presence of antibodies.

ELISA . A 96-well plate was coated with Tn–BSA (2.5 μg mL ⁻¹ ) dissolved in 0.2 M borate buffer (pH 8.5) containing 75 mM sodium chloride overnight at 4° C. (100 μL/well). Plates were washed three times with 0.01 M Tris buffer containing 0.5% Tween 20% and 0.02% sodium azide. Blocking was achieved by incubating the plate with 1% BSA in 0.01 M phosphate buffer containing 0.14 M sodium chloride for 1 hour at room temperature. Next, plates were washed, followed by incubation with serum dilutions in phosphate-buffered saline for 2 hours at room temperature. Excess antibody was removed and plates were washed three times. Plates were incubated with rabbit anti-mouse IgM and IgG Fcγ fragment-specific alkaline phosphatase-conjugated antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 2 hours at room temperature. Subsequently, after the plate was washed, the enzyme substrate (p-nitrophenyl phosphate) was added and allowed to react for 30 minutes, then the enzymatic reaction was quenched by adding 3 M aqueous sodium hydroxide, followed by a reaction at 405 and 490 nm. Dual wavelength reads absorbance. Antibody titers were determined by regression analysis in which log ₁₀ dilutions were plotted against absorbance. Titers were calculated as the highest dilution giving twice the absorbance of normal mouse serum at a 1:120 dilution.

Example 2

Noncovalently Linked Diepitope Liposome Formulations

In the first set of experiments, tumor-associated carbohydrate B-epitope and universal T-epitope peptides were incorporated separately into preformed liposomes to form di-epitope constructs. Additionally, the lipopeptide Pam ₃ Cys was incorporated into liposomes, where it is expected to act as an intercalation adjuvant, thus avoiding the necessity of using additional external adjuvants such as QS-21.

Liposomes were prepared from lipid anchors bearing two different thiol-reactive functionalities, maleimide and bromoacetyl, on their surface. The Pam ₃ Cys adjuvant was also incorporated into preformed liposomes and included maleimide functionality. Conveniently, maleimide and bromoacetyl groups display marked differences in their reactivity toward sulfhydryl groups. Maleimide reacts rapidly with sulfhydryl compounds at pH 6.5, while bromoacetyl requires a slightly higher pH of 8-9 to react efficiently with thiol compounds.

By exploiting this difference in reactivity, a dipetopic liposome construct carrying the cancer-associated Le ^y tetrasaccharide and the universal T helper peptide QYIKANSKFIGITEL (QYI) (SEQ ID NO: 1 ) was prepared (Scheme 11). For conjugation to thiol-reactive anchors, both oligosaccharides and peptides were functionalized with thiol-containing linkers. The two-step sequential conjugation to preformed liposomes has a great advantage: it is a very flexible method that allows easy preparation of liposomes carrying a variety of different carbohydrate B epitopes. As based on quantification of carbohydrates and peptides covalently coupled to vesicles, conjugation yields were high, 70-80% for oligosaccharides and 65-70% for peptides, and the results were highly reproducible.

It is important to note that in these first di-epitope liposome constructs, the carbohydrate B epitope and the peptide T epitope are not themselves covalently linked together, but rather via their conjugated Their respective lipid anchors are kept in proximity by hydrophobic interactions. Several reports in the literature on vaccine candidates with pathogen-associated peptide B epitopes have shown that this approach is successful, leading to good titers of IgM and specific IgG antibodies. These studies also indicate that the intercalation adjuvant Pam ₃ Cys is sufficient to induce an appropriate immune response.

However, in our studies with the tumor-associated carbohydrate B epitope Le ^y , the immunization of mice described in this Example using a non-covalently linked dipetope liposome formulation resulted in only very low titer Degree of IgM antibody. IgG anti-Le ^y antibodies were not detected. Even more surprisingly, co-administration of the liposomal vaccine candidate with the potent external adjuvant QS-21 did not improve the outcome. In addition, it was found that mice that had been immunized with uncoated liposome controls (i.e. liposomes carrying only maleimide and bromoacetyl functional groups on the surface) elicited high titers as detected by ELISA IgG antibodies. More detailed ELISA studies of antisera from this group of mice using various protein conjugates revealed that the mice had responded to and elicited antibodies against the maleimide linker. Furthermore, antisera from mice immunized with liposomes coated with Le ^y antigen and QYI peptide were screened for anti-linker antibodies and found that these mice had also elicited IgG antibodies against the maleimide linker.

Scheme 11

Due to their high reactivity at near neutral pH, maleimide linkers are widely used in conjugation chemistry to reach sugar- and peptide-protein conjugates for further use in immunization studies. There are commercially available protein conjugation kits (Pierce Endogen Inc.) that utilize maleimide linkers for both antigen conjugates and detection conjugates. Our data show that use of these kits can lead to false positive results, especially when working with antigens of low immunogenicity (see T. Buskas, Y. Li and G-J. Boons, Chem. Eur. J., 10:3517 -3523, 2004).

To test whether the highly immunogenic maleimide linker suppresses the immune response against the Le ^y tetrasaccharide, we prepared non-covalent diepitopic liposomes using only the bromoacetyl linker. In this experiment, a thiol-containing Le ^y tetrasaccharide and a universal T helper peptide were conjugated to a lipid containing a bromoacetyl linker in separate reactions. The conjugated lipids are then mixed together to form lipid vesicles. Administration of this new liposomal formulation to mice with or without the external adjuvant QS-21 produced only low titers of anti-Le ^y antibodies. Thus, the lack of an effective immune response against the Le ^y tetrasaccharide is not solely due to the immunogenic maleimide linker.

Because the tumor-associated Le ^y tetrasaccharide is known to be only weakly immunogenic, we prepared another bi-epitope liposome construct in which the more immunogenic Tn (cluster) antigen was used as the target B-sheet bit. However, negative results were also obtained with this antigen. Again, immunization of mice resulted in only very low titers of anti-Tn(c) IgM antibodies. Co-administration with QS-21 as an external adjuvant did not enhance the immune response at all.

From these results, we conclude that the non-covalently linked bi-epitope liposome approach, which has proven successful for a range of peptide antigens, fails when tumor-associated carbohydrate antigens with low immunogenicity are used as B epitopes up. Therefore, we reasoned that tumor-associated carbohydrate B epitopes and helper T epitopes require differential presentation to the immune system to elicit T cell-dependent immune responses.

Example 3

Covalently Linked Diepitope Liposome Formulations

We speculate that in order to achieve better presentation of the carbohydrate B epitope and the peptide T epitope, perhaps they need to be covalently linked together. To test this idea, we synthesized Construct 1 (Scheme 12), a structurally well-defined anticancer vaccine candidate containing the structural features required for a focused and efficient T cell-dependent immune response. This vaccine candidate consists of the tumor-associated Tn antigen, the peptide T epitope YAFKYARHANVGRNAFELFL (YAF) (SEQ ID NO: 2) (Neisseria meningitidis), and the lipopeptide Pam ₃ Cys. Due to the difficulty in using the original helper T-epitope peptide QYI in the synthesis, a different universal T-epitope (YAF) displaying better solubility properties was used in this study.

Compound 1 was synthesized in a highly convergent manner by a combination of solid-phase and solution-phase syntheses.

Scheme 12

The constructs are then incorporated into phospholipid-based liposomes. Compound 1 has the disadvantage of low solubility in a range of solvents, which may be the main reason why incorporation in liposomes is only 10%.

Mice were immunized with this construct at weekly intervals. To explore the adjuvant properties of the embedded lipopeptide Pam ₃ Cys, antigen-containing liposomes were administered with (group 2) or without (group 1) adjuvant QS-21.

As can be seen in Table 1 (Example 1), immunization of mice with the liposome formulation elicited IgM and IgG antibodies against the Tn antigen (Table 1, entries 1 and 2). The presence of IgG antibodies indicates that the helper T epitope peptide of 1 has activated helper T lymphocytes. Furthermore, the observation that IgG antibodies were produced in mice immunized with liposomes in the absence of the external adjuvant QS-21 (Group 1) indicated that the embedded adjuvant Pam ₃ Cys has triggered a process for DC maturation and its Appropriate signal for subsequent activation of helper T cells. However, mice receiving liposomes in combination with QS-21 (Group 2) elicited higher titers of anti-Tn antibodies. This stronger immune response may be due to a shift from a mixed Th1/Th2 to a Th1 skewed response.

The results provide the first proof of principle for the use of lipidated glycopeptides containing carbohydrate B epitopes, helper T cell epitopes and lipopeptide adjuvants as minimally self-contained subunit vaccines. It was also concluded that in order to induce a T cell-dependent immune response against tumor-associated carbohydrate antigens, it is not sufficient that the carbohydrate B epitope and the peptide T epitope are presented together in a non-covalent manner on the surface of adjuvanted liposomes instead, the entities are preferably covalently linked together. Finally, it was observed that when the three components (carbohydrate B epitope, helper T cell epitope and lipopeptide) were covalently linked to form lipidated glycopeptides, no external adjuvant was required (QS-21).

Alternative Glycolipidin Components

Several improvements can be made to compound 1. For example, it has been found that antibodies elicited against the Tn antigen weakly recognize cancer cells. However, clustering (Nakada et al., Proc. Natl. Acad. Sci. USA 1993, 90, 2495-2499; Reddish et al., 1997, 14, 549-560; Zhang et al., Cancer Res. 1995, 55, 3364 -3368; Adluri et al., Cancer Immunol. Immunother 1995, 41, 185-192) or presentation of the Tn antigen as part of the MUC-1 glycopeptide elicits antibodies with improved binding characteristics (Snijdewint et al., Int. J. Cancer 2001 , 93, 97-106). The T epitope employed in compound 1 is an MHC class II restricted epitope for humans. Therefore, a more efficient class switch to IgG antibodies can be expected when murine T epitopes are employed. Furthermore, the lipopeptides Pam ₂ Cys or Pam ₃ Cys SK ₄ have been found to be more effective immune adjuvants than Pam ₃ Cys (Spohn et al., Vaccine 2004, 22, 2494-2499). However, it is unknown whether attachment of Pam ₂ Cys or Pam ₃ CysSK ₄ to T and B epitopes affects their efficacy and potency. Therefore, based on these considerations, compounds 2 and 3 were designed (Scheme 12), which contain the MUC-1 glycopeptide as the B epitope, containing the well-documented murine helper T cell epitope KLFAVWKITYKDT (KLF) derived from poliovirus (SEQ ID NO:3) (Leclerc et al., J. Virol. 1991, 65, 711-718) as T epitope and containing lipopeptides Pam ₂ Cys or Pam ₃ CysSK ₄ , respectively.

Glycolipidins 2 and 3 were incorporated into phospholipid-based liposomes as described for compound 1 . Surprisingly, the solubility issues that plagued compound 1 were not an issue for compounds 2 and 3. Female BALB/c mice were immunized four times at weekly intervals with the liposomal formulation with or without the external adjuvant QS-21 (Kensil et al., J. Immunol. 1991, 146, 431-437). Anti-Muc1 antibody titers were determined by coating microtiter plates with CTSAPDT (αGalNAc) RPAP conjugated to BSA, and detection was accomplished with an anti-mouse IgG antibody labeled with alkaline phosphatase. The results are summarized in Tables 2 and 3.

Table 2. ELISA anti-MUC-1 antibody titers* after 4 immunizations with glycolipopeptide/liposome formulations.

*ELISA plates were coated with BSA-BrAc-MUC-1 conjugate. Anti-MUCl antibody titers are presented as mean values of groups of five mice. Titer was defined as the highest dilution to obtain an optical density of 0.1 or greater relative to the background of blank mouse serum.

Table 3. ELISA anti-MUC-1 antibody titers* after 4 immunizations with glycolipopeptide/liposome formulations.

*ELISA plates were coated with BSA-BrAc-MUC-1 conjugate. Anti-MUCl antibody titers are presented as mean values of groups of five mice. Titer was defined as the highest dilution to obtain an optical density of 0.1 or greater relative to the background of blank mouse serum.

As can be seen in Table 2, mice immunized with liposomal formulations of Compounds 2 and 3 elicited high titers of anti-MUC-1 IgG antibodies. Surprisingly, mice immunized with Pam ₃ CysSK ₄ -based vaccines elicited higher titers of antibodies than mice immunized with Pam ₂ Cys derivatives. These results contradict reports that have compared the adjuvancy of Pam ₂ Cys and Pam ₃ CysSK ₄ . The subtypes of IgG antibodies (IgG1, IgG2a, IgG2b and IgG3) indicate a bias towards Th2 immune responses (entries 1 and 3, Table 3). Co-administration of the adjuvant QS-21 did not lead to a significant increase in IgG antibodies, however, in these cases mixed Th1/Th2 responses were observed (entries 2 and 4, Table 3).

To ensure that mouse sera were able to recognize native MUC-1 glycopeptides present on cancer cells, sera were checked for binding to the MCF-7 human breast cancer cell line expressing MUC-1. Therefore, cells were treated with 1:50 diluted serum for 30 minutes, after which FITC-labeled goat anti-mouse IgG antibody was added. The percentage of positive cells and mean fluorescence were determined by flow cytometry analysis. As can be seen in ( FIG. 2 ), the antiserum reacted strongly with MUC-1 positive tumor cells, while no binding was observed for sera from naive mice. Furthermore, no binding was observed when using SK-MEL 28 cells that do not express the MUC-1 glycopeptide. These results demonstrate that anti-MUC-1 antibodies induced by 3 recognize native antigens on human cancer cells. Further ELISA studies showed extremely low titers against the T epitope, showing no significant epitope suppression.

The lipopeptide portion of the three-component vaccine is required to initiate the production of essential cytokines and chemokines (danger signals) (Bevan, Nat. Rev. Immunol. 2004, 4, 595-602; Eisen et al., Curr. Drug Targets2004, 5, 89-105; Akira et al., Nat. Immunol. 2001, 2, 675-680; Pasare et al., Immunity2004, 21, 733-741; Dabbagh et al., Curr. Opin. Infect. Dis. 2003 , 16, 199-204; Beutler, Mol. Immunol. 2004, 40, 845-859). Results of recent studies indicate that lipopeptides initiate innate immune responses by interacting with Toll-like receptor 2 on the surface of mononuclear phagocytes. Upon activation, the intracellular domain of TLR-2 recruits the adapter protein MyD88, leading to the activation of a kinase cascade leading to the production of a number of cytokines and chemokines. On the other hand, LPS induces cellular responses by interacting with Toll-like receptor 4 (TLR4)/MD2, which leads to the recruitment of the adapter proteins MyD88 and TRIF, resulting in a more complex pattern of cytokine production. TNF-α secretion is a prototypical measure of MyD88-dependent pathway activation, whereas IFN-β secretion is commonly used as an indicator of TRIF-dependent cellular activation (Akira et al., Nat. Immunol. 2001, 2, 675-680; Beutler, Mol. Immunol. 2004, 40, 845-859).

To examine whether the linkage of glycopeptides containing T- and B-epitopes to TLR ligands affects cytokine production, the potency (EC ₅₀ ) of TNF-α and IFN-β secretion induced by compounds 1, 2 and 3 was determined and potency (maximal responsiveness), the results were compared to those of Pam ₂ CysSK ₄ , Pam ₃ CysSK ₄ and LPS. Therefore, RAW NO ⁻ mouse macrophages were exposed to a wide range of concentrations of Compounds 1, 2 and 3, Pam ₂ CysSK ₄ , Pam ₃ CysSK ₄ and E. coli 055:B5 LPS. After 5 hours, supernatants were harvested and examined for mouse TNF-α and IFN-β, respectively, using commercial or in-house developed capture ELISA assays.

Table 4. _EC50 and _Emax values of concentration-response curves of E. coli LPS and synthetic compounds for TNF-α production by mouse macrophages (RAW γNO(-) cells).

* EC50 and Emax values are reported as best fit values according to Prism (GraphPad Software, Inc). Concentration-response data were analyzed using nonlinear least squares curve fitting in Prism.

As can be seen in Figure 3 and Table 4, GLP 3 and Pam ₃ CysSK ₄ induced the secretion of TNF-α with similar efficacy and potency, indicating that linkage of B and T epitopes has no effect on cytokine and chemokine responses . Surprisingly, linking of B and T epitopes to Pam ₂ CysSK ₄ resulted in a significant reduction in potency, thus in this case linking of B and T epitopes resulted in a decrease in activity. Compound 1 containing the Pam ₃ Cys moiety was significantly less active than compounds 2 and 3, which may explain the weak antigenicity of compound 1. Compounds 1, 2 and 3 did not induce IFN-β production. Surprisingly, E. coli 055:B5 displayed much greater potency and efficacy for TNF-α induction compared to compounds 1, 2, 3 and Pam ₃ CysSK ₄ . In addition, it is able to stimulate cells to produce IFN-β. E. coli LPS is too active, leading to hyperactivation of the innate immune system, leading to symptoms of septic shock.

It is hypothesized that in addition to initiating cytokine and chemokine production, lipopeptides may also facilitate selective targeting and uptake by antigen-presenting cells in a TLR2-dependent manner. To test this hypothesis, compound 4 containing a fluorescent label was administered to RAW NO ^- mouse macrophages and after 30 min the cells were harvested, lysed and fluorescence was measured. To allow for possible cell surface binding without internalization, cells were also trypsinized prior to lysis and subsequently checked for fluorescence. As can be seen in Figure 4, a significant amount of 4 was internalized, while a small amount was attached to the cell surface. To determine whether uptake is mediated by TLR2, uptake studies were repeated using native HEK297 cells and HEK297 cells transfected with TLR2 or TLR4/MD2. Importantly, significant uptake was observed only when cells were transfected with TLR2, suggesting that uptake is mediated by this receptor. These studies show that TLR2 promotes antigen uptake, an important step in antigen processing and immune response.

Example 4

Covalent attachment of lipid components

To determine the importance of covalent attachment of TLR ligands to vaccine candidates, compound 5 containing only B and T epitopes was designed and synthesized (Scheme 13). Mice were immunized with this compound _four times at weekly intervals in the presence of _PAM3CysSK4 . Interestingly, the mixture of glycopeptide 5 and adjuvant Pam ₃ CysSK ₄ did not elicit IgG antibodies or elicited very low titers of IgG antibodies, confirming that covalent attachment of Pam ₃ CysSK ₄ to B and T epitopes is essential for strong immune responses is critical.

Scheme 13

Example 5

Lipid composition

To determine the importance of lipidation with ligands for Toll-like receptors, compound 6 was designed and synthesized (Scheme 14). This compound consists of a B epitope and a T epitope linked to a non-immunogenic lipidated amino acid. Mice were immunized with a liposomal formulation of Compound 6, similar to the procedure employed for Compounds 1 and 2. Liposomes containing Compound 6 induced significantly lower titers than those elicited by Compound 3, demonstrating that the TLR ligands of the three-component vaccine are important for optimal immune responses.

Scheme 14

in conclusion

Three-component carbohydrate-based vaccines have many unique advantages over traditional conjugate vaccines. For example, minimal subunit vaccines do not suffer from the disadvantages of epitope suppression typical of carbohydrate-protein conjugates. In addition to providing a danger signal, the lipopeptide Pam ₃ CysSK ₄ also facilitates antigen incorporation into liposomes. Liposomal formulations are attractive because of their efficient presentation of antigens to the immune system. A unique feature of the vaccine is that Pam ₃ CysSK ₄ facilitates selective targeting and uptake by antigen presenting cells, T helper cells and B lymphocytes expressing Toll loll-like receptors (Example 3). Finally, a fully synthetic compound has the advantage that it can be fully characterized, which facilitates its production in a reproducible manner.

Example 6

Increased antigenicity of synthetic tumor-associated carbohydrate antigens by targeting Toll-like receptors

In this example, a number of fully synthetic vaccine candidates have been designed, chemically synthesized, and immunologically evaluated to establish strategies to overcome the weak immunogenicity of tumor-associated carbohydrates and glycopeptides and to study in detail the importance of TLR engagement for antigen response sex. Covalent linkage of a TLR2 agonist, a non-germline-selective peptide T helper epitope, and a tumor-associated glycopeptide gives a compound that elicits exceptionally high titers of IgG antibodies that recognize expressed tumor-associated carbohydrates in mice Compounds of cancer cells.

Overexpression of oligosaccharides such as Globo-H, LewisY and Tn antigens is a common feature of oncogenically transformed cells (Springer, Mol. Med. 1997, 75, 594-602; Hakomori, Acta Anat. 1998, 161, 79-90; Dube , Nat. Rev. Drug Discov. 2005, 4, 477-488). Numerous studies have shown that this aberrant glycosylation can promote metastasis (Sanders, J. Clin. Pathol. Mol. Pathol . 1999, 52 , 174-178), so the expression of these compounds is strongly correlated with poor survival of cancer patients. Extensive and expanded preclinical and clinical studies have demonstrated that naturally acquired, passively administered or actively induced antibodies against carbohydrate-associated tumor antigens can eliminate circulating tumor cells and micrometastases in cancer patients (Livingston, Cancer Immunol. 1997, 45, 10-19; Ragupathi, Cancer Immunol. 1996, 43, 152-157; von Mensdorff-Pouilly, Int. J. Cancer 2000, 86, 702-712; Finn, Nat. Rev. Immunol. 2003, 3, 630 -641). Traditional cancer vaccine candidates consisting of tumor-associated carbohydrates (Globo-H, Lewis ^Y , and Tn) conjugated to exogenous carrier proteins (e.g., KLH and BSA) fail to elicit sufficiently high titers of IgG in most patients Antibody. It appears that induction of IgG antibodies against tumor-associated carbohydrates is much more difficult than eliciting similar antibodies against viral and bacterial carbohydrates. This observation is not surprising since tumor-associated sugars are self-antigens and thus tolerated by the immune system. Antigen shedding by growing tumors reinforces this tolerance. Furthermore, exogenous carrier proteins such as KLH can elicit strong B cell responses, which can lead to suppression of antibody responses to carbohydrate epitopes. The latter is a greater problem when employing self-antigens such as tumor-associated carbohydrates. Furthermore, linkers used for the conjugation of carbohydrates to proteins can be immunogenic, leading to epitope suppression (Buskas, Chem. Eur. J. 2004, 10, 3517-3524; Ni, Bioconjug. Chem. 2006, 17 , 493-500). It is clear that the successful development of carbohydrate-based cancer vaccines requires new strategies for more efficient presentation of tumor-associated carbohydrate epitopes to the immune system, resulting in more efficient class switching to IgG antibodies (Reichel, J. Chem. Commun. 1997, 21, 2087-2088; Alexander, J. Immunol. 2000, 164, 1625-1633; Kudryashov, Proc. Natl. Acad. Sci. USA 2001, 98, 3264-3269; Lo-Man, J. Immunol.2001 , 166, 2849-2854; Jiang, Curr. Med. Chem. 2003, 10, 1423-1439; Jackson, Proc. Natl. Acad. Sci. USA 2004, 101, 15440-5; Lo-Man, Cancer Res. 2004 , 64,4987-4994; Buskas, Angew.Chem.Int.Ed.2005,44,5985-5988 (Example 1); Dziadek, Angew.Chem.Int.Ed.2005,44,7630-7635; Krikorian, Bioconjug. Chem. 2005, 16, 812-819; Pan, J. Med. Chem. 2005, 48, 875-883).

Advances in knowledge of the cooperation of innate and adaptive immune responses (Pasare, Semin. Immunol. 2004, 16, 23-26; Pashine, Nat. Med. 2005, 11, S63-S68; Akira, Nat. Rev. Immunol.2004, 4, 499-511; O'Neill, Curr Opin Immunol 2006, 18, 3-9; Lee, Semin Immunol 2007, 19, 48-55; Ghiringhelli, Curr Opin Immunol 2007, 19, 224-31) Provides new avenues for vaccine design for diseases, such as cancer, for which traditional vaccine approaches have failed. The innate immune system responds rapidly to a family of highly conserved compounds that are an integral part of pathogens and are perceived by the host as danger signals. Recognition of these molecular patterns is mediated by sets of highly conserved receptors such as Toll-like receptors (TLRs), the activation of which leads to acute inflammatory responses such as direct local attack against invading pathogens and production of diverse sets of cytokines . In addition to antimicrobial properties, cytokines and chemokines also activate and regulate adaptive components of the immune system (Lin, J ClinInvest 2007, 117, 1175-83). In this regard, cytokines stimulate the expression of a number of co-stimulatory proteins for optimal interaction between T helper cells and B cells and antigen presenting cells (APCs). Furthermore, several cytokines and chemokines are responsible for overcoming suppression mediated by regulatory T cells. Other cytokines are important for directing effector T cell responses towards a T helper-1 (Th-1) or T helper-2 (Th-2) phenotype (Dabbagh, Curr. Opin. Infect. Dis. 2003, 16 , 199-204).

Recently, we described a fully synthetic three-component vaccine candidate (compound 21, Fig. Buskas, Angew. Chem. Int. Ed. 2005, 44, 5985-5988 (Example 1); Ingale, Nat. Chem. Biol. 2007, 3, 663-667; Ingale, J. Org. Lett. 2006, 8 , 5785-5788; Bundle, Nat. Chem. Biol. 2007, 3, 604-606). The superior antigenic properties of the three-component vaccine are due to the absence of any unnecessary features that are antigenic and may induce immunosuppression. However, it contains all the mediators required for eliciting an IgG-related immune response. In addition, linkage of the TLR2 agonist Pam ₃ CysSK ₄ to the B and T epitopes ensures cytokine production at the sites where the vaccine interacts with immune cells. This results in high local concentrations of cytokines that promote maturation of the involved immune cells. In addition to providing a danger signal, the lipopeptide Pam ₃ CysSK ₄ also facilitates antigen incorporation into liposomes, facilitating selective targeting and uptake by antigen-presenting cells and B lymphocytes.

In order to establish the optimal architecture of a fully synthetic three-component cancer vaccine and to study in detail the importance of TLR engagement for antigen response, we have chemically synthesized and immunologically evaluated a number of fully synthetic vaccine candidates. A liposomal formulation of compound 22 consisting of an immunosilencing lipopeptide, a non-germline-selective peptide T helper epitope, and a MUC-1 glycopeptide was found to be significantly more potent than compound 21 modified with a TLR2 ligand (Pam ₃ CysSK ₄ ). less antigenic. However, liposomal formulations of Compound 22 with Pam ₃ CysSK ₄ (23) or monophosphoryl lipid A (24), which are TLR2 and TLR4 agonists, respectively, elicited titers comparable to Compound 21. However, antisera raised with a mixture of 22 and 23 or 24 had impaired ability to recognize cancer cells. Surprisingly, the mixture of compounds 25 and 26 consisting of the MUC-1 glycopeptide B epitope linked to lipidated amino acids and the helper T epitope linked to Pam ₃ CysSK ₄ did not produce a reaction against the MUC-1 glycopeptide Antibody. Taken together, the results demonstrate that TLR involvement is not essential but greatly enhances the antigenic response against the tumor-associated glycopeptide MUC-1. Covalent attachment of TLR agonists to B and helper T epitopes is important for antibody maturation for improved cancer cell recognition.

Results and discussion.

chemical synthesis.

Contains tumor-associated glycopeptides derived from MUC-1 (Berzofsky, Nat. Rev. Immunol. 2001, 1, 209-219; Baldus, Crit. Rev. Clin. Lab. Sci. 2004, 41, 189-231; Apostolopoulos, Curr. Opin. Mol. Ther. 1999, 1, 98-103; Hang, Bioorg. Med. Chem. Lett. 2005, 13, 5021-5034) as a well-documented mouse B epitope, derived from poliovirus MHC class II restricted helper T cell epitope KLFAVWKITYKDT (SEQ ID NO:3) (Leclerc, J. Virol. 1991, 65, 711-718) and lipopeptide Pam ₃ CysSK ₄ (TLR2 agonist) (Spohn, Vaccine 2004 , 22, 2494-2499), compound 21 ( FIG. 5 ), was previously shown to elicit particularly high titers of IgG antibodies in mice (Ingale, Nat. Chem. Biol. 2007, 3, 663-667). Compound 22 has a similar architecture to 21, however, the TLR2 ligand has been replaced by a lipidated amino acid (Toth, Tetrahedron Lett. 1993, 34, 3925-3928). Lipidated amino acids do not induce cytokine production, however, they enable compound incorporation into liposomes. Glycolipid22 is therefore ideally suited to determine the importance of TLR engagement for antigenic responses against tumor-associated glycopeptides. To determine the importance of covalent attachment of TLR ligands, liposomal formulations of compound 22 and Pam ₃ CysSK ₄ (23) or monophosphoryl lipid A (24), which are TLR2 and TRL4 agonists, respectively (Spohn, Vaccine 2004, 22, 2494-2499; Chow, J. Biol. Chem. 1999, 274, 10689-10692). Finally, compounds 25 and 26, consisting of the MUC-1 glycopeptide B epitope linked to a lipidated amino acid and the helper T epitope linked to Pam ₃ CysSK ₄ , were used to determine the covalency of the B epitope and helper T epitope The importance of connections. Compound 21 was prepared as previously described (Ingale, Nat. Chem. Biol. 2007, 3, 663-667; Ingale, Org. Lett. 2006, 8, 5785-5788). Using Rink amide resin, Fmoc-protected amino acid, Fmoc-Thr-(AcO ₃ -α-D-GalNAc) (Cato, J. Carbohydr. Chem. 2005, 24, 503-516) and Fmoc-protected lipidated amino acid ( Gibbons, Liebigs Ann. Chem. 1990, 1175-1183; Koppitz, Helv. Chim. Acta 1997, 80, 1280-1300), compound 22 was synthesized by SPPS. Using 2-(1H-benzotriazol-1-yl)-oxy-1,1,3,3-tetramethylhexafluorophosphate (HBTU)/ N -hydroxybenzotriazole (HOBt) (Knorr , Tetrahedron Lett. 1989, 30, 1927-1930) as an activation reagent to introduce standard amino acids, glycosylated amino acids with O -(7-azabenzotriazol-1-yl) -N , N , N',N' -Tetramethylurea Hexafluorophosphate (HATU)/1-hydroxy-7-azabenzotriazole (HOAt) loaded, lipidated amino acid composed of benzotriazol-1-yl-oxy-tripyrrolidinophosphorus Hexafluorophosphate (PyBOP)/HOBt loading. After the glycolipopeptide assembly is complete, the N -terminal Fmoc protecting group is removed using standard conditions, and the resulting amine is added by acetylation with acetic anhydride and diisopropylethylamine (DIPEA) in N -methylpyrrolidone (NMP). cap. Next, the acetyl ester of the sugar moiety was cleaved with 60% hydrazine in MeOH with reagent B (TFA, _H2O , phenol, triethylsilane, 88/5/5/2, v/v/v/v) Treatment results in removal of the side chain protecting groups and release of the glycopeptide from the solid support.

Pure compound 22 was obtained after purification of the crude product by precipitation with ice-cold diethyl ether followed by HPLC on a C-4 semi-preparative column. A similar scheme was used to synthesize compound 25. Derivative 26 was synthesized by SPPS on Rink amide resin, and after peptide assembly, the resulting product was treated with N -fluorenylmethoxycarbonyl- R- (2,3-bis(palmitoyloxy)-( 2R -propyl )-( R )-cysteine (Fmoc-Pam ₂ Cys-OH) manual coupling (Metzger, Int. J. Pept. Protein Res. 1991, 38, 545-554). DMF with 20% piperidine Solution removes the N -Fmoc group of the product and the resulting amine is coupled to palmitic acid using PyBOB, HOBt and DIPEA in DMF. The lipopeptide is treated with Reagent B to cleave it from the resin and remove the side Chain protecting group. The crude product was purified by precipitation with ice-cold diethyl ether followed by HPLC on a C-4 semi-preparative column.

Immunization and Immunology.

Pass the membrane of egg phosphatidylcholine (PC), phosphatidylglycerol (PG), cholesterol (Chol) and compound 21 or 22 (molar ratio: 65/25/50/10) in HEPES buffer containing NaCl (145 mM) Compounds 21 and 22 were incorporated into phospholipid-based small unilamellar vesicles (SUVs) by hydration in solution (10 mM, pH 6.5) followed by extrusion through a 100 nm Nuclepore® polycarbonate membrane. Groups of five female BALB/c mice were immunized subcutaneously four times at weekly intervals with liposomes containing 3 μg of sugar. In addition, similar liposomes with a mixture of glycopeptides 22 and 23 or 24 were prepared in HEPES buffer (molar ratio: PC/PG/Chol/22/23 or 24, 65/25/5/5/5), Four doses were administered at weekly intervals prior to serum harvest. Finally, mice were immunized with liposomal formulations of compounds 25 and 26 (molar ratio: PC/PG/Chol/25/26, 65/25/5/5/5) using standard procedures.

Anti-MUC-1 antibody titers of antisera were determined by coating microtiter plates with the MUC-1-derived glycopeptide TSAPDT (α-D-GalNAc) RPAP conjugated to BSA, and labeled with alkaline phosphatase Anti-mouse IgM or IgG antibodies complete the detection. Mice immunized with 21 elicited particularly high titers of anti-MUC-1 IgG antibodies (Table 5). The subtypes of IgG antibodies (IgGl, IgG2a, IgG2b and IgG3) indicate a bias towards Th2 immune responses. Furthermore, the high IgG3 titers observed were typical of an anticarbohydrate response. Immunization with glycolipopeptide 22 containing lipidated amino acids instead of TLR2 ligands resulted in significantly lower titers of IgG antibodies, demonstrating that TLR engagement is critical for optimal antigen responses. However, liposomal formulations of compound 22 with Pam ₃ CysSK ₄ (23) or monophosphoryl lipid A (24) elicited IgG (total) titers similar to 21. In the case of a mixture of 22 and 23, the immune response was skewed towards a Th2 response as evidenced by high IgGl and low IgG2a,b titers. On the other hand, the use of monophosphoryl lipid A resulted in significant IgG1 and IG2a,b responses, thus this formulation elicited a mixed Th1/Th2 response. Finally, liposomes containing compounds 25 and 26 did not induce measurable titers of anti-MUC-1 antibodies, indicating that covalent linkage of B and T epitopes is required for antigen response.

Table 5. ELISA anti-MUCl and anti-T epitope antibody titers ^a after 4 immunizations with various formulations.

^a Anti-MUCl and anti-T epitope antibody titers are presented as median values for a group of five mice. For anti-MUC1 antibody titers, coat ELISA plates with BSA-MI-MUC1 conjugate, or for anti-T epitope antibody titers, coat ELISA plates with neutravidin-biotin-T epitope quilt. Titers were determined by linear regression analysis, plotting dilutions compared to absorbance. Titer was defined as the highest dilution to obtain an optical density of 0.1 or greater relative to normal control mouse serum.

^b using liposome formulations.

Individual anti-MUCl titers for total IgG, IgGl, IgG2a, IgG2b, IgG3 and IgM and anti-T epitopes for total IgG are reported in Table 8.

Next, possible antigenic responses to helper T epitopes were investigated. Therefore, streptavidin-coated microtiter plates were treated with biotin-modified helper T epitopes. Detection was accomplished with anti-mouse IgM or IgG antibodies labeled with alkaline phosphatase after addition of serial dilutions of sera. Interestingly, compound 21 elicited low antibodies against the helper T epitope, whereas a mixture of 22 with 23 or 24 elicited no antibodies against the helper T epitope.

Pam ₃ CysSK ₄ or monophosphoryl lipid A are used to initiate cytokine production by interacting with TLR2 or TLR4 on the surface of mononuclear phagocytes, respectively (Kawai, Semin. Immunol. 2007, 19, 24-32). Upon activation with Pam ₃ CysSK ₄ , the intracellular domain of TLR2 recruits the adapter protein MyD88, leading to the activation of a kinase cascade leading to the production of a number of cytokines and chemokines. On the other hand, lipopolysaccharide (LPS) and lipid A induce cellular responses by interacting with the TLR4/MD2 complex, which leads to the recruitment of the adapter proteins MyD88 and TRIF, leading to the induction of a more complex pattern of cytokines. TNF-α secretion is a prototypical measure of MyD88-dependent pathway activation, whereas IFN-β secretion is commonly used as an indicator of TRIF-dependent cellular activation.

To examine cytokine production, mouse macrophages (RAW γNO(-) cells) were exposed to a wide range of concentrations of compounds 21-24, E. coli 055:B5 LPS, and prototype E. coli diphosphoryl lipid A (Zhang, J. Am. Chem. Soc. 2007, 129, 5200-5216). After 5.5 hours, supernatants were harvested and examined for mouse TNF-α and IFN-β, respectively, using commercial or in-house developed capture ELISAs ( FIG. 6 ). Potency ( _EC50 , concentration giving 50% activity) and efficacy (maximal production level) were determined by fitting dose response curves to the logistic equation using PRISM software. Glycolipidin 21 and Pam ₃ CysSK ₄ (23) induced TNF-α secretion with similar potency and potency, suggesting that linkage of B and T epitopes has no effect on cytokine responses. As expected, none of the compounds induced IFN-[beta] production. Furthermore, compound 22 did not induce TNF-α and IFN-β secretion, suggesting that its lipid fraction is immunosilencing. Compound 24 stimulated cells to produce TNF-α and INF-β, but it was much less potent than E. coli 055:B5 LPS. Compared to compounds 21 and 23, it exhibited much greater efficacy in TNF-[alpha] production. The reduced efficacy of compounds 21 and 23 may be a favorable property because LPS can overactivate the innate immune system, leading to symptoms of septic shock.

Next, the ability of the mouse antisera to recognize the native MUC-1 antigen present on cancer cells was determined. Therefore, serial dilutions of serum samples were added to MCF-7 human breast cancer cells expressing MUC-1 (Horwitz, Steroids 1975, 26, 785-95), and recognition was established using FITC-labeled anti-mouse IgG antibody. As can be seen in Figure 7, antisera obtained from immunization with the three-component vaccine 1 exhibited excellent recognition of MUC-1 tumor cells, whereas no binding was observed when using SK-MEL 28 cells that do not express the MUC-1 antigen (Figure 9).

Although sera from mice immunized with a mixture of lipidated TB epitopes ( ₂₂ ) and _Pam3CysSK4 (23) elicited high IgG antibody titers equivalent to 21 (Table 5), much less was observed Recognition of MCF-7 cells. This result indicates that covalent attachment of the adjuvant PamsCysSK ₄ (23) to the BT epitope is important for proper antibody maturation, which leads to improved cancer cell recognition. Immunization with a mixture of compound 22 and monophosphoryl lipid A (24) resulted in variable results, with two mice showing excellent MCF-7 cell recognition and three mice showing moderate MCF-7 cell recognition.

discuss

Most efforts aimed at developing carbohydrate-based cancer vaccines have focused on the use of chemically synthesized tumor-associated carbohydrates linked to carrier proteins by artificial linkers (Springer, Mol. Med. 1997, 75, 594-602; Dube, Nat. . Rev. Drug Discov. 2005, 4, 477-488; Ouerfelli, Expert Rev. Vaccines 2005, 4, 677-685; Slovin, Immunol. Cell Biol. 2005, 83, 418-428). It was determined that the use of KLH as a carrier protein in combination with the potent adjuvant QS-21 gave the best results. However, the disadvantage of this approach is that KLH is a very large and troublesome protein that can elicit high titers of anti-KLH antibodies (Cappello, Cancer Immunol Immunother 1999, 48, 483-492), resulting in immunosuppression of tumor-associated carbohydrate epitopes . Furthermore, conjugation chemistry is often difficult to control as it results in conjugates with compositional and structural ambiguities that can affect the reproducibility of the immune response. Furthermore, linker moieties can elicit strong B cell responses (Buskas, Chem. Eur. J. 2004, 10, 3517-3524; Ni, Bioconjug. Chem. 2006, 17, 493-500). Not surprisingly, preclinical and clinical studies with carbohydrate-protein conjugates have resulted in results of mixed merit. For example, immunization of mice with trimeric clusters of Tn antigen conjugated to KLH (Tn(c)-KLH) in the presence of the adjuvant QS-21 elicited moderate titers of IgG antibodies (Kuduk, J. Am. Chem. Soc. 1998, 120, 12474-12485). Examination of vaccine candidates in clinical trials of recurrent prostate cancer patients gave low median IgG and IgM antibody titers (Slovin, J. Clin. Oncol. 2003, 21, 4292-4298).

Studies reported here show that a three-component vaccine in which a MUC-1-associated glycopeptide B epitope, a non-germline-selective murine MHC class II-restricted helper T-cell epitope, and a TLR2 agonist (21) are covalently linked can elicit Strong IgG antibody response. Although covalent attachment of TLR2 ligands to T-B glycopeptide epitopes is not required for high IgG antibody titers, it was found to be very important for optimal cancer cell recognition. In this regard, liposomes containing compound 21 or a mixture of compound 22 and TLR2 agonist 23 elicited similarly high anti-MUC-1 IgG antibody titers. However, antisera from immunization with 21 recognized MUC-1 expressing cancer cells at much lower serum dilutions than antisera from immunization with a mixture of 22 and 23. It appears that immunization with the three-component vaccine21 resulted in more efficient antibody maturation, resulting in improved cancer cell recognition.

Differences in antigenic responses to helper T epitopes were also observed. Thus, 21 elicited low titers of IgG antibodies against the helper T epitope, whereas the mixture of 22 and 23 did not induce an antigenic response against this part of the vaccine candidate. Thus, covalent attachment of TLR2 ligands made compound 21 more antigenic, resulting in low antibody responses against the helper T epitope.

Mixtures of compound 22 and 23 or 24 were observed to induce similarly high titers of total IgG antibodies. However, a bias towards a Th2 response (IgG1) was observed when the TLR2 agonist Pam ₃ CysSK ₄ (23) was used, whereas a mixed Th1/Th2 response was obtained when the TLR4 agonist monophosphoryl lipid A (24) was used (IgG2a,b). Differences in the polarization of helper T cells may be due to different patterns of cytokine induction by TLR2 or TLR4. In this regard, it was previously observed that Pam ₃ Cys induces lower levels of the Th1-inducible cytokine Il-12 (p70) than E. coli LPS, and much higher levels of Th2-inducible IL-10 (Dillon, B. J Immunol 2004, 172, 4733-43). The difference may be due to the ability of TLR4 to recruit the adapter proteins MyD88 and Trif, whereas TLR2 can only recruit MyD88. This result suggests that the immune system can be tailored in specific directions by appropriate choice of adjuvants, which is meaningful since different IgG isotypes perform different effector functions.

The results described herein also show that compound 22 containing the immunosilencing lipopeptide alone elicits much lower IgG titers compared to compound 21 modified by TLR2 ligand. In particular, the ability of compound 22 to elicit IgG2 antibodies was impaired. Recent studies using mice deficient in TLR signaling cast doubt on the importance of these innate immune receptors for the adaptive immune response (Blander, Nature 2006, 440, 808-812; Gavin, Science 2006, 314, 1936-1938; Meyer-Bahlburg, J Exp Med 2007, 204, 3095-101; Pulendran, N Engl J Med 2007, 356, 1776-8). In this regard, studies with MyD88-deficient mice showed that IgM and IgG1 are largely but not completely dependent on TLR signaling, whereas the IgG2 isotype is completely TLR-dependent (Blander, Nature 2006, 440, 808 -812). These observations, consistent with the results reported here, have been attributed to the requirement of TLR signaling for B cell maturation. However, another study found that MyD88 ^-/- /Trif ^lps/lps Double knockout mice elicited similar titers of antibodies to wild type mice (Gavin, Science 2006, 314, 1936-1938). It was concluded that TLR agonists may need to be excluded from adjuvants. It has been noted that the importance of adjuvants may depend on the antigenicity of the immunogen (Meyer-Bahlburg, J Exp Med 2007, 204, 3095-101; Pulendran, N Engl J Med 2007, 356, 1776-8). In this regard, protein conjugates of TNP are highly antigenic and may not require adjuvants for an optimal response. However, self-antigens such as tumor-associated carbohydrates have low intrinsic antigenicity, and the results reported here clearly show that a much stronger antibody response is obtained when TLR ligands are co-administered. Furthermore, it is demonstrated here that the architecture of a vaccine candidate is very important for optimal antigen response, in particular covalent attachment of TLR ligands to TB epitopes leads to improved cancer cell recognition.

The inability of the mixture of compounds 25 and 26 to elicit anti-MUC-1 glycopeptide antibodies indicates that covalent attachment of the T epitope to the B epitope is required to elicit an antigenic response. In this regard, activation of B cells by helper T cells requires similar types of intercellular interactions as activation of helper T cells by antigen-presenting cells. Thus, protein or peptide-containing antigens need to be internalized by B cells for transport to endosomal vesicles where proteases will digest the protein and some of the resulting peptide fragments will complex with MHC class II proteins. Class II MHC-peptide complexes are then transported to the cell surface of B lymphocytes to mediate interactions with helper T cells, resulting in a class switch from low-affinity IgM to high-affinity IgG antibody production. Unlike antigen presenting cells, B cells have weak phagocytic properties and can only internalize molecules that bind to the B cell receptor. Therefore, it is expected that the internalization of the helper T epitope is facilitated by covalent linkage to the B epitope (MUC-1 glycopeptide), and thus covalent linkage of the two epitopes will lead to a stronger antigenic response.

In conclusion, it has been demonstrated that the antigenic properties of fully synthetic cancer vaccines can be optimized through structure-activity relationship studies. In this regard, it has been determined that a three-component vaccine in which tumor-associated MUC-1 glycopeptide B epitopes, non-germline-selective helper T-cell epitopes, and TLR2 ligands are covalently linked elicits particularly high IgG antibody responses, It has the ability to recognize cancer cells. The covalent linkage of the helper T epitope to the B epitope is very important, probably because the internalization of the helper T epitope by B cells requires the presence of the B epitope. The incorporation of TLR agonists has also been shown to be important for strong antigenic responses against tumor-associated glycopeptide antigens. In this regard, cytokines induced by TLR2 ligands are important for immune cell maturation, which leads to strong antibody responses. A surprising finding was that improved cancer cell recognition was observed when the TLR2 epitope was covalently linked to the glycopeptide T-B epitope. The results presented here provide important information on the optimal composition of three-component vaccines and will guide the successful development of carbohydrate-based cancer vaccines.

experiment

Peptide synthesis: Peptides were processed by established protocols on an ABI 433A peptide synthesizer (Applied Biosystems) equipped with a UV detector using Nα -Fmoc protected amino acids and 2-(1H-benzotriazol-1-yl) ^-oxy- 1,1,3,3-Tetramethylhexafluorophosphate (HBTU)/ N -hydroxybenzotriazole (HOBt) (Knorr, Tetrahedron Lett. 1989, 30, 1927-1930) was synthesized as an activating reagent. Individual coupling steps are performed with conditional capping. The following protected amino acids were used: N ^α -Fmoc-Arg(Pbf)-OH, N ^α -Fmoc-Asp( O ^t Bu ) -OH, N ^α -Fmoc-Asp-Thr(ψMe ^,Me pro)- OH, N ^α -Fmoc-Ile-Thr(ψ ^Me,Me pro)-OH, N ^α -Fmoc-Lys(Boc)-OH, N ^α -Fmoc-Ser( ^t Bu)-OH, N ^α -Fmoc- Thr( ^tBu )-OH and Nα - ^Fmoc -Tyr( ^tBu )-OH. Using O -(7-azabenzotriazol-1-yl) -N , N , N',N' -tetramethylurea Glycosylated amino acid N ^α -Fmoc-Thr-(AcO ₃ -α-D-GalNAc ) 1S (Cato, J. Carbohydr. Chem. 2005, 24, 503-516). Using benzotriazol-1-yl-oxy-tripyrrolidino-phosphorus Hexafluorophosphate (PyBOP)/HOBt was used as ^a coupling agent (see Supporting Information) to perform Nα -Fmoc-lipophilic amino acid ( Nα - ^Fmoc -D,L-tetradecanoic acid) 2S (Gibbons, Liebigs Ann. Chem. 1990, 1175-1183; Koppitz, Helv. Chim. Acta 1997, 80, 1280-1300) and N ^α -Fmoc- S -(2,3-bis(palmitoyloxy)-(2 R -propyl )-( R )-cysteine 3S (Metzger, Int. J. Pept. Protein Res. 1991, 38, 545-554; Roth, Bioconj. Chem. 2004, 15, 541-553) (which is derived from (R ) - Coupling of glycidol preparation). The progress of the manual coupling was monitored by the standard Kaiser test (Kaiser, Anal. Biochem. 1970, 34, 595).

Liposome preparation: egg phosphatidylcholine (PC), phosphatidylglycerol (PG), cholesterol (Chol) and compound 21 or 22 (15 mmol, molar ratio 65:25:50:10) or PC/PG/ Chol/22/23 or 24 (15 mmol, molar ratio 60:25:50:10:5) or PC/PG/Chol/25/26 (15 mmol, molar ratio 65:25:50:5:5) dissolved in a mixture of trifluoroethanol and MeOH (1:1, v/v, 5 mL). The solvent was removed in vacuo to give a thin lipid film by shaking in HEPES buffer (10 mM, pH 6.5) (1 mL) containing NaCl (145 mM) for 3 h at 41 °C under an argon atmosphere. Hydrate. The vesicle suspension was sonicated for 1 min and subsequently extruded sequentially through 1.0, 0.4, 0.2 and 0.1 μm polycarbonate membranes (Whatman, Nuclepore® Track-Etch Membrane) at 50°C to obtain SUVs. GalNAc content was determined by heating a mixture of SUV (50 μL) and aqueous TFA (2 M, 200 μL) in a sealed tube at 100 °C for 4 hours. The solution was then concentrated in vacuo and analyzed by high pH anion exchange chromatography using a pulsed amperometric detector (HPAEC-PAD; Methrome) and CarboPac columns PA-10 and PA-20 (Dionex).

Dosage and Immunization Schedule: Groups of five mice (female BALB/c, age 8-10 weeks; Jackson Laboratories) were immunized four times at weekly intervals. Each booster contains 3 µg of sugar in the liposomal formulation. Serum samples were obtained before immunization (pre-bleeding) and one week after the last immunization. The final phlebotomy is done by exsanguination from the heart.

Serological assays: Anti-MUC-1 IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM antibody titer. Briefly, ELISA plates (Thermo Electron Corp.) were coated with MUC-1 glycopeptide conjugate (BSA-MI-MUC-1 ) conjugated to BSA via a maleimide linker. Serial dilutions of sera were allowed to bind immobilized MUC-1. By adding phosphate-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.), IgG1 (Zymed), IgG2a (Zymed), IgG2b (Zymed), IgG3 (BD Biosciences Pharmingen) or IgM (Jackson ImmunoResearch Laboratories Inc.) antibodies detection. After addition of p-nitrophenyl phosphate (Sigma), absorbance was measured at 405 nm using a microplate reader (BMG Labtech) with wavelength correction set at 490 nm. Antibody titers against T (polio) epitopes were determined as follows. Reacti-bind NeutrAvidin coated and pre-blocked plates (Pierce) were incubated with biotin-labeled T epitope (10 µg/mL) for 2 hours. Next, serial dilutions of sera are allowed to bind the immobilized T epitopes. Assays were done as described above. Antibody titers were defined as the highest dilution that achieved an optical density of 0.1 or greater relative to normal control mouse serum.

Cell culture: RAW 264.7 γNO(-) cells derived from the RAW 264.7 mouse monocyte/macrophage cell line were obtained from ATCC. Cells were maintained in RPMI 1640 medium with L-glutamine (2 mM) adjusted to contain sodium bicarbonate (1.5 g L ^-1 ), glucose (4.5 g L ^-1 ), HEPES (10 mM) and sodium pyruvate (1.0 mM), supplemented with penicillin (100 u mL ^-1 )/streptomycin (100 µg mL ^-1 ; Mediatech) and FBS (10%; Hyclone). Human breast adenocarcinoma cells (MCF7) from ATCC (Horwitz, Steroids 1975, 26, 785-95) were cultured in Eagle's minimal essential medium with L-glutamine (2 mM) and Earle's BSS ) in Eagle's minimal basal medium modified to contain sodium bicarbonate (1.5 g L ^-1 ), non-essential amino acids (0.1 mM) and sodium pyruvate (1 mM), supplemented with bovine insulin ( 0.01 mg mL ⁻¹ ; Sigma) and FBS (10%). Human cutaneous malignant melanoma cells (SK-MEL-28) were obtained from ATCC and grown in Eagle's minimal basal medium with L-glutamine (2 mM) and Earle's BSS, which Basal medium was adjusted to contain sodium bicarbonate (1.5 g L ⁻¹ ), non-essential amino acids (0.1 mM) and sodium pyruvate (1 mM), supplemented with FBS (10%). All cells were maintained at 37°C in a humidified 5% _CO2 atmosphere.

TNF-α and IFN-β assays. On the day of the exposure assay, RAW 264.7 γNO(-) cells were plated at ² x 105 cells/well in 96-well plates (Nunc) in the presence or absence of polymyxin B, together with different stimuli Incubate for 5.5 hours. Culture supernatants were harvested and stored frozen (-80°C) until assayed for cytokine production. TNF-α concentrations were determined using the TNF-α DuoSet ELISA Development kit from R&D Systems. The concentration of IFN-[beta] was determined as follows. ELISA MaxiSorp plates were coated with rabbit polyclonal antibody against mouse IFN-β (PBL Biomedical Laboratories). Immobilized antibodies allowed for binding of IFN-β in standards and samples. Rat anti-mouse IFN-β antibody (US Biological) was then added to create an antibody-antigen-antibody "sandwich". Next, horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (H+L) antibody (Pierce) and the chromogenic substrate for HRP, 3,3',5,5'-tetramethyl Benzidine (TMB; Pierce). After the reaction was stopped, absorbance was measured at 450 nm with wavelength correction set to 540 nm. Concentration-response data were analyzed using nonlinear least squares curve fitting in Prism (GraphPad Software, Inc.). These data were fitted with the following four-parameter logistic equation: Y= _Emax /(1+( _EC50 /X) ^{Hill slope} ), where Y is the cytokine response, X is the concentration of the stimulus, and _Emax is the maximum response, and the _EC50 is the concentration of the stimulus that produces 50% stimulation. Hill slope was set at 1 to enable comparison of _EC50 values of different inducers. All cytokine values are presented as mean ± SD of triplicate measurements, where each experiment was repeated three times.

Materials were evaluated for LPS contamination: To ensure that any increase in cytokine production was not caused by LPS contamination of solutions containing various stimuli, the lipid A region of LPS was affinity bound, thereby preventing LPS-induced cytokine production (Tsubery, Biochemistry 2000, 39, 11837-44). TNF-α and IFN-β in supernatants of cells preincubated with polymyxin B (30 µg mL ^-1 ; Bedford Laboratories) for 30 minutes prior to incubation with E. coli O55:B5 LPS for 5.5 hours concentrations showed complete inhibition, whereas pre-incubation with polymyxin B had no effect on TNF-α synthesis in cells incubated with synthetic compounds 21 and 23. Therefore, LPS contamination of the latter formulation was insignificant.

Cell recognition assay by fluorescence measurement: Serial dilutions of pre- and post-immunization sera were incubated with MCF7 and SK-MEL-28 single cell suspensions for 30 minutes on ice. Next, cells were washed and incubated for 20 minutes on ice with goat anti-mouse IgG γ-chain specific antibody conjugated to fluorescein isothiocyanate (FITC; Sigma). After three washes and cell lysis, cell lysates were analyzed for fluorescence intensity (485 ex/520 em) using a microplate reader (BMG Labtech). Data points were collected in triplicate and represent three independent experiments.

Example 7

compound synthesis

General methods: Fmoc-L-amino acid derivatives and resins were purchased from NovaBioChem and Applied Biosystems; peptide synthesis grade N,N -dimethylformamide (DMF) was purchased from EM Science; N -methylpyrrolidone (NMP) was purchased from Applied Biosystems . Egg phosphatidylcholine (PC), phosphatidylglycerol (PG), cholesterol (Chol) and monophosphoryl lipid A (MPL-A) were obtained from Avanti Polar Lipids. EZ-Link® NHS-Biotin reagent (succinimidyl-6-(biotinamide)hexanoate) was obtained from Pierce. All other chemical reagents were purchased from Aldrich, Acros, Alfa Aesar and Fisher Scientific and used without further purification. All solvents employed were of reagent grade. Agilent Zorbax Eclipse ^™ C8 analytical column (5 μm, 4.6 x 150 mm) at 1 mL/min flow rate, Agilent Zorbax Eclipse ^™ C8 semi-preparative column (5 μm, 10 x 250 mm) at 3 mL/min flow rate , or a Phenomenex Jupiter ^TM C4 semi-preparative column (5 μm, 10 x 250 mm) at a flow rate of 2 mL/min on an Agilent 1100 series equipped with an autoinjector, fraction collector and UV detector (detection at 214 nm) Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed on the system. All runs used a linear gradient of 0-100% solvent B over 40 minutes (solvent A = 5% acetonitrile, 0.1% trifluoroacetic acid (TFA) in water, solvent B = 5% water, 0.1% TFA in acetonitrile) implement. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectra were recorded on an ABI 4700 proteomics analyzer.

Synthesis of glycolipopeptide 22: Synthesis 22 was performed on Rink amide resin (28, 0.1 mmol) as described under Peptide Synthesis in Experiments. The first four amino acids Arg-Pro-Ala-Pro were coupled on a peptide synthesizer using standard protocols to obtain 29. After the synthesis was complete, manual coupling of 1S (0.2 mmol, 134 mg) was performed. N ^α -Fmoc-Thr-(AcO ₃ -α-D-GalNAc) 1S (Cato, J. Carbohydr. Chem. 2005, 24, 503-516) was dissolved in NMP (5 mL), O- (7 -Azabenzotriazol-1-yl)- N , N , N',N' -tetramethylurea Hexafluorophosphate (HATU; 0.2 mmol, 76 mg), 1-hydroxy-7-azabenzotriazole (HOAt; 0.2 mmol, 27 mg) and diisopropylethylamine (DIPEA; 0.4 mmol, 70 μL ) was added to the solution, and the resulting mixture was then added to the resin. Coupling reactions were monitored by standard Kaiser tests. After 12 hours, the resin was washed with NMP (6 mL) and dichloromethane (DCM; 6 mL) before being subjected to the same coupling conditions again to ensure complete coupling. Glycopeptide 30 was subsequently elongated on a peptide synthesizer. After the synthesis was complete, the resin was washed well with NMP (6 mL), DCM (6 mL) and methanol (MeOH; 6 mL), and dried in vacuo. The resin was then swelled in DCM (5 mL) for 1 hour and the remainder of the coupling was performed manually. Next, dissolve in NMP (5 mL), benzotriazol-1-yl-oxy-tripyrrolidino-phosphorus N ^α -Fmoc-lipophilic amino acid ( N ^α -Fmoc - D,L -tetradecanoic acid) 2S (Gibbons, LiebigsAnn. Chem. 1990, 1175-1183; Koppitz, Helv. Chim. Acta 1997, 80, 1280-1300) (0.3mmol, 139 mg) pre-mixed for 2 minutes, then added to the resin middle. The coupling reaction was monitored by Kaiser test and then completed after standing for 8 hours. The N ^α -Fmoc group was cleaved using piperidine (20%) in DMF (6 mL). N ^α -Fmoc - Gly-OH (0.3 mmol, 90 mg) was pre-mixed for 2 minutes and then added to the resin. The coupling reaction was monitored by Kaiser test and then completed after 4 hours of standing. The N ^α -Fmoc group was cleaved using piperidine (20%) in DMF (6 mL). Using PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μL) in NMP (5 mL), another 2S (0.3 mmol, 139 mg) was performed as described above. Coupling cycle. Finally, the N ^α -Fmoc group was cleaved using piperidine (20%) in DMF (6 mL) and the resin was treated with Ac ₂ O (10%) and DIPEA (5%) in NMP (5 mL) for 10 min , to acetylate the resulting free amino group. The resin was washed well with NMP (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2), and dried in vacuo. The resin was swollen in DCM (5 mL) for 1 h, treated with hydrazine (60%) in MeOH ^4,5 (10 mL) for 2 h, washed with NMP (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2) was washed well, then dried in vacuo. The resin was swelled in DCM (5 mL) for 1 hour, then treated with reagent B (TFA (88%), water (5%), phenol (5%) and TIS (2%), 10 mL) for 2 hours. The resin was filtered, washed with neat TFA (2 mL), and the filtrate was concentrated in vacuo to about 1/3 of its original volume. Glycolipid peptides were precipitated using diethyl ether (0°C, 40 mL) and recovered by centrifugation at 3,000 rpm for 15 minutes. The crude glycolipopeptide was purified by RP-HPLC on a semi-preparative C-4 column using a linear gradient of 0-95% solvent B in A over 40 minutes, and the appropriate fractions were lyophilized to provide 22 (Fig. 10) (57 mg, 16%). C ₁₆₅ H ₂₆₇ N ₃₇ O ₄₄ , MALDI-ToF MS: Observed, [M+] 3473.4900 Da; Calculated, [M+] 3473.1070 Da.

Program 15. Reagents and conditions: a) SPPS using Fmoc chemistry, coupled to HBTU/HOBt in the presence of DIPEA in NMP; b) 1S, HATU/HOAt, DIPEA, NMP, overnight; c) i. in DIPEA in NMP ii. 20% piperidine in DMF; iii. manual coupling of 1S to PyBOP/HOBt in the presence of DIPEA in NMP; iv. 20% piperidine Pyridine in DMF; v. Manual coupling of 2S to PyBOP/HOBt in the presence of DIPEA in NMP; vi. 20% piperidine in DMF; vii. 10% _Ac2O , 5% DIPEA in NMP Up to 10 minutes; (d) 60% hydrazine in MeOH, 2 hours; e) Reagent B, TFA (88%), phenol (5%), water (5%), TIS (2%), 2 hours.

Synthesis of lipopeptide 23: Synthesis of 23 was performed on Rink amide resin (28, 0.1 mmol) as described under Peptide Synthesis in Experiments. After coupling of the first five amino acids, the lipid portion of the molecule was manually coupled. N ^α -Fmoc- S -(2,3-bis(palmitoyloxy)-(2 R -propyl)-( R )-cysteine, 3S (Metzger, Int. J. Pept. Protein Res .1991,38,545-554; Roth, Bioconj.Chem.2004,15,541-553) (0.3 mmol, 267 mg) was dissolved in DMF (5 mL), PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μL) were added to the solution. After 2 minutes, the reaction mixture was added to the resin. The coupling reaction was monitored by Kaiser test and then completed after standing for 12 hours. Next , using piperidine (20%) in DMF (6 mL) to cleave the N ^α -Fmoc group to obtain 36. Using PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol , 67 μL) in DMF, palmitic acid (0.3 mmol, 77 mg) was coupled with the free amine of 36 as described above. The resin was washed with DMF (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2) was washed thoroughly, and then dried in vacuo. The resin was swelled in DCM (5 mL) for 1 hour, followed by TFA (95%), water (2.5%) and TIS (2.5%) (10 mL) in Treated at room temperature for 2 hours. The resin was filtered and washed with pure TFA (2 mL). The filtrate was then concentrated in vacuo to about 1/3 of its original volume. The lipopeptide was precipitated using diethyl ether (0°C, 30 mL) and passed Recovered by centrifugation at 3000 rpm for 15 minutes. Crude lipopeptides were purified by RP-HPLC on a semi-preparative C-4 column using a linear gradient of 0-95% solvent B in solvent A over a 40 minute period and appropriate Fractions were lyophilized to provide 23 ( FIG. 11 ) (40 mg, 26%). C ₈₁ H ₁₅₆ N ₁₁ O ₁₂ S, MALDI-ToF MS: Observed [M+Na], 1531.2240 Da; Calculated [M + Na], 1531.1734 Da.

Program 16. Reagents and conditions: a) SPPS using Fmoc chemistry coupled to HBTU/HOBt in the presence of DIPEA in NMP; b) manual coupling by 3S activated by PyBOP/HOBt in the presence of DIPEA in DMF; c ) piperidine (20%) in DMF; d) palmitic acid coupling activated by PyBOP/HOBt in the presence of DIPEA in DMF; e) TFA (95%), water (2.5%), TIS (2.5 %),2 hours.

Synthesis of glycolipopeptide 25: Synthesis of 25 was performed on Rink amide resin (28, 0.1 mmol) as described under Peptide Synthesis in Experiments. The first four amino acids Arg-Pro-Ala-Pro were coupled on a peptide synthesizer using standard protocols to obtain 29. After the synthesis was complete, manual coupling was performed using 1S (0.2 mmol, 134 mg). 1S was dissolved in NMP (5 mL), HATU (0.2 mmol, 76 mg), HOAt (0.2 mmol, 27 mg) and DIPEA (0.4 mmol, 70 μL) were added, and the resulting mixture was added to the resin. Coupling reactions were monitored by standard Kaiser tests. After 12 hours, the resin was washed with NMP (6 mL) and DCM (6 mL) before being subjected to the same coupling conditions again to ensure complete coupling. Glycopeptide 30 was subsequently elongated on a peptide synthesizer. After the synthesis was complete, the resin was washed well with NMP (6 mL), DCM (6 mL) and MeOH (6 mL), then dried in vacuo. The resin was then swollen in DCM (5 mL) for 1 hour, and the remainder of the peptide sequence was completed by hand. 2S (0.3 mmol, 139 mg) was dissolved in NMP (5 mL), and PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μL) were added to the solution. After 2 minutes, the mixture was added to the resin. The coupling reaction was monitored by standard Kaiser test and was complete after 8 hours of standing. Next , the N ^α -Fmoc group was cleaved using piperidine (20%) in DMF (6 mL). N ^α -Fmoc - L-glycine (0.3 mmol, 90 mg) was dissolved in NMP (5 mL), and before adding the reaction mixture to the resin, mixed with PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μL) were premixed for 2 minutes. The coupling reaction was monitored by Kaiser test and was complete after 4 hours of standing. The N ^α -Fmoc group was cleaved using piperidine (20%) in DMF (6 mL). Using PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μL) in NMP (5 mL), another 2S (0.3 mmol, 139 mg) was performed as described above. Coupling cycle. Finally, the N ^α -Fmoc group was cleaved using piperidine ( ₂₀ %) in DMF (6 mL) , and the resulting Acetylation of the free amino group for 10 min. The resin was washed well with NMP (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2), and dried in vacuo. The resin was swelled in DCM (5 mL) for 1 h, treated with hydrazine (60%) in MeOH (10 mL) for 2 h, washed with NMP (5 mL x 2 ), DCM (5 mL x 2 ) and MeOH (5 mL x 2) Wash well and dry in vacuum. The resin was swollen in DCM (5 mL) for 1 hour after which it was treated with reagent B (TFA (88%), water (5%), phenol (5%) and TIS (2%), 10 mL) for 2 hours . The resin was filtered, washed with neat TFA (2 mL), and the filtrate was concentrated in vacuo to about 1/3 of its original volume. Glycolipid peptides were precipitated using diethyl ether (0°C; 40 mL) and recovered by centrifugation at 3,000 rpm for 15 minutes. Crude glycolipopeptides were purified by RP-HPLC on a semi-preparative C-4 column using a linear gradient of 0-95% solvent B in A over 40 minutes, and the appropriate fractions were lyophilized to provide 5 (Figure 12 ) (35 mg, 19%). C ₈₄ H ₁₄₅ N ₁₉ O ₂₅ , MALDI-ToF MS: Observed, [M+] 1821.1991 Da; Calculated, [M+] 1821.1624 Da.

Program 17. Reagents and conditions: a) SPPS using Fmoc chemistry, coupled to HBTU/HOBt in the presence of DIPEA in NMP; b) 1S, HATU/HOAt, DIPEA, NMP, overnight; c) i. in DIPEA in NMP In the presence of 2S and PyBOP/HOBt; ii. 20% piperidine in DMF; iii. In the presence of DIPEA in NMP, the manual coupling of N ^α -Fmoc-Gly-OH and PyBOP/HOBt iv. 20% piperidine in DMF; v. manual coupling of 2S to PyBOP/HOBt in the presence of DIPEA in NMP; vi. 20% piperidine in DMF; vii. 10% Ac ₂ O , 5% DIPEA in NMP for 10 minutes; (d) 60% hydrazine in MeOH for 2 hours; e) Reagent B, TFA (88%), phenol (5%), water (5%), TIS (2 %),2 hours.

Synthesis of lipopeptide 26: Synthesis of 26 was performed on Rink amide resin (28, 0.1 mmol). After assembling the peptides by using standard SPPS, the lipid portion of the molecule was manually coupled. 3S (0.3 mmol, 267 mg) was dissolved in DMF (5 mL), and PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μL) were added to the solution. After 2 min of 3S activation, the reaction mixture was added to the resin. The coupling reaction was monitored by Kaiser test and was complete after 12 hours of standing. The N- Fmoc group was cleaved using piperidine (20%) in DMF (6 mL) to obtain 43 . Palmitic acid (77 mg, 0.3 mmol) was coupled with the free amine of 43 as described above using PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μL) in DMF. couplet. The resin was washed well with DMF (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2), then dried in vacuo. The resin was swelled in DCM (5 mL) for 1 h, treated with reagent B (TFA (88%), water (5%), phenol (5%) and TIS (2%), 10 mL) for 2 h, filtered, And washed with pure TFA (2 mL). The filtrate was then concentrated in vacuo to about 1/3 of its original volume, the lipopeptide was precipitated using diethyl ether (0°C, 30 mL), and recovered by centrifugation at 3000 rpm for 15 minutes. The crude lipopeptide was purified by RP-HPLC on a semi-preparative C-4 column using a linear gradient of 0-95% solvent B in A over 40 minutes, and the appropriate fractions were lyophilized to provide 26 (Figure 13 ) (57 mg, 18%). _{C162H278N29O31S} , MALDI- _ToF MS: observed, [M+] _3160.9423 Da; calculated, [M+] _3160.1814 Da.

Program 18. Reagents and conditions: a) SPPS using Fmoc chemistry, coupling to HBTU/HOBt in the presence of DIPEA in NMP; b) manual coupling of 3S, PyBOP, HOBt in the presence of DIPEA in DMF; c) 20% piperidine in DMF; d) manual coupling of palmitic acid, PyBOP, HOBt in the presence of DIPEA in DMF; e) reagent B, TFA (88%), phenol (5%), water (5%) %), TIS (2%), 2 hours.

Synthesis of biotin-T epitope peptide 27: Synthesis of 27 was performed on Rink amide resin (28, 0.1 mmol) as described in General Methods. After the synthesis was completed, the resin was washed well with DMF (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2), followed by drying in vacuo. The resin was swelled in DCM (5 mL) for 1 h. Next, EZ-Link® NHS-Biotin Reagent (succinimidyl-6-(biotinamide) hexanoate) (0.2 mmol, 90 mg) and DIPEA (0.2 mmol, 36 μL) were dissolved in DMF ( 5 mL) was added to the resin. Coupling was monitored by standard Kaiser test and was complete within 8 hours. The resin was washed well with DMF (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2), then dried in vacuo. The resin was swelled in DCM (5 mL) for 1 h and treated with reagent B (TFA (88%), water (5%), phenol (5%) and TIS (2%), 15 mL) at room temperature for 2 h. The resin was filtered, washed with neat TFA (2 mL). The filtrate was concentrated in vacuo to about 1/3 of its original volume. The peptide was precipitated using diethyl ether (0°C, 30 mL) and recovered by centrifugation at 3,000 rpm for 15 minutes. The crude peptide was purified by RP-HPLC on a semi-preparative C-8 column using a linear gradient of 0-95% solvent B in solvent A over a 40 minute period, and the appropriate fractions were lyophilized to provide 27 (Fig. 14) (60%, based on resin loading capacity). _{C95H147N21O21S} , MALDI- _ToF MS: observed [M+], _1951.2966 Da; calculated [M+], _1951.3768 Da.

Program 19. Reagents and conditions: a) SPPS using Fmoc chemistry, in the presence of DIPEA in NMP, was coupled to HBTU/HOBt; b) in the presence of DIPEA in DMF, succinimidyl-6-(biotin amide) manual coupling of hexanoate; c) Reagent B, TFA (88%), phenol (5%), water (5%), TIS (2%), 2 hours.

Example 8

A fully synthetic multicomponent cancer vaccine elicits a multi-model immune response

This example demonstrates that glycosylated MUCl -derived glycopeptides covalently linked to Toll-like receptor (TLR) agonists can elicit potent humoral and cellular immune responses and are effective in reversing tolerance and generating therapeutic responses. Examination of a number of control compounds confirmed that the efficacy of the three-component vaccine was due to a non-specific antitumor response elicited by the adjuvant, and a specific humoral and cellular immune response elicited by the MUCl-derived glycopeptide. Glycosylation of the MUCl peptide was found to be critical for the induction of an optimal response and, moreover, covalent attachment of helper T and B epitopes to TLR ligands was required.

result

Antigen design and tumor challenge studies. The efficacy of a mixture of compounds 1, 2, 3, 4 and 5 and a liposomal formulation of 5 alone was examined in a well-established mouse model of breast cancer (Akporiaye et al., 2007, Vaccine ; 25:6965-6974) ( Figure 16). Multicomponent vaccine candidate 1 contains tumor-associated glycopeptides derived from MUC1 (Baldus et al., 2004, Crit. Rev Clin Lab Sci ; 41:189-231; and Springer, 1997, J Mol Med ; 75:594-602 ), the well documented murine MHC class II restricted helper T cell epitope KLFAVWKITYKDT (SEQ ID NO:1) derived from poliovirus (Leclerc et al., 1991, J Virol ; 65:711-718) and as Toll The lipopeptide Pam3CysSK4, a potent agonist of TLR2-like receptor-2 (TLR2) (Spohn et al., 2004, Vaccine ; 22:2494-2499). Previously, the MUCl-derived glycopeptide SAPDT (α-GalNAc) RPAP was identified as the tandem repeat antigenically dominant domain of MUCl (Baldus et al., 2004, Crit. Rev Clin Lab Sci ; 41:189-231; and Springer, 1997, J Mol Med ; 75:594-602). Furthermore, this epitope can also be presented in complex with MHC class I (K ^b ), leading to the activation of cytotoxic T lymphocytes (CTL) (Apostolopoulos et al., 2003, Proc Natl Acad Sci USA ; 100:15029-15034 ).

As shown in this example, the MHC class II-restricted helper T cell epitope of 1 induces a class switch from IgM to IgG antibody production ( FIG. 20 ) and promotes the presentation of exogenous glycopeptides on MHC class 1. Finally, the Pam3CysSK4 portion of 1 acts as an intercalation adjuvant by priming associated cytokines and chemokines (Spohn et al., 2004, Vaccine ; 22:2494-2499). To determine the importance of the carbohydrate moiety of 1, construct 2, which has a similar structure to 1 except that the threonine of the MUCl peptide is not glycosylated, was examined. Compound 3 lacks the MUC1 glycopeptide epitope of 1 and 2 and was examined to account for possible efficacy due to immune activation by adjuvants. Finally, the mixture of glycopeptide 4 and adjuvant Pam3CysSK4 5 was examined to determine the importance of covalent attachment of the adjuvant to the MUC1 glycopeptide and helper T epitope.

Multicomponent Vaccine 1 was prepared by liposome-mediated native chemical ligation (Ingale et al., 2006, Org Lett ; 8:5785-5788). Compounds 2, 3, and 4 were synthesized by SPPS protocol using Rink amide resin, Fmoc-protected amino acid Fmoc-Thr-(AcO ₃ -α-D-GalNAc). Hydration of thin films of synthetic compounds, egg phosphatidylcholine, phosphatidylglycerol, and cholesterol in HEPES buffer (10 mM, pH 6.5) containing NaCl (145 mM) and subsequent hydration via a 100 nm Nuclepore ^® polycarbonate membrane Extrusion incorporates the resulting compounds into small phospholipid-based unilamellar vesicles (SUVs). Groups of MUCl.Tg mice (C57BL/6; H-2 ^b ) expressing human MUCl were treated every two weeks with liposomal formulations of compounds 1, 2, 3, 4, and 5 and 5 alone. immunized three times at intervals. After 35 days, mice were challenged with MMT mammary tumor cells (positive for MUCl and Tn), followed by another boost one week later. Two weeks after the last immunization, mice were sacrificed and vaccine efficacy was determined by tumor weight. In addition, the robustness of the humoral immune response was assessed by the titers of MUCl-specific antibodies and the ability of the antiserum to lyse MUCl-bearing tumor cells. In addition, cellular immune responses were assessed by measuring the number of IFN-γ producing CD8 ⁺ T cells and the ability of these cells to lyse tumor cells.

Immunization with multicomponent vaccine candidate 1 resulted in a significant reduction in tumor burden compared to empty liposomes or treatment with compound 3 not containing the MUCl glycopeptide epitope (Figure 17). Interestingly, immunization with compound 3 resulted in slightly smaller tumors compared to application of empty liposomes, indicating antitumor properties due to nonspecific adjuvant effects. Glycosylated multicomponent vaccine candidate 2 and the mixture of compounds 4 and 5 did not show significant improvement in anticancer properties compared to control immunizations. In these cases, a large spread in tumor weight was observed, whereas immunization with compound 1 resulted in a substantial reduction in tumor weight in all mice.

Humoral immunity. Anti-MUCl antibody titers were determined by coating microtiter plates with CTSAPDT (α-D-GalNAc) RPAP conjugated to bromoacetyl-modified BSA. Compound 1 had elicited a strong IgG antibody response, and subtyping of the antibodies indicated a mixed Th1/Th2 response (Table 6). Mice immunized with 1 but not challenged with MMT tumor cells elicited similar titers of antibody, indicating that the immunosuppression of cancer cells may be reversed. Inhibition ELISA showed polyclonal sera to have slightly higher affinity for glycosylated MUCl epitopes (Table 7). Furthermore, low titers of antibodies against the helper T epitope were measured, indicating that the candidate vaccine does not have the disadvantage of immunosuppression. Although Compound 2 does not contain a carbohydrate moiety, the resulting antiserum recognizes the CTSAPDT (α-D-GalNAc) RPAP epitope. However, in this case, IgG3 antibodies were not detected. Interestingly, a mixture of compounds 4 and 5 had elicited low titers of antibodies, highlighting the importance of covalent attachment of Pam3CysSK4 to glycopeptide epitopes for strong antigenic responses. Controls (3 and 5) that did not contain MUCl-derived epitopes did not elicit anti-MUCl antibody responses, as expected.

Examination of antibody-dependent cell-mediated cytotoxicity (ADCC) by labeling two ^MUCl -expressing cancer cell types with Cr and subsequent addition and release of ^antisera and cytotoxic effector cells (NK cells) . As can be seen in Figures 18A and 18B, the antiserum obtained by immunization with 1 was able to significantly increase cancer cell lysis compared to the control compound 3. Importantly, antibodies elicited by compound 2 were significantly less effective in cell lysis compared to compound 1, highlighting the importance of glycosylation for relevant antigen responses. Antisera derived from the mixture of 4 and 5 and the control derivative lacking the MUCl glycopeptide did not induce significant cell lysis, as expected.

Table 6. ELISA anti-MUCl and anti-T epitope antibody titers after 4 immunizations with various formulations ^[a] .

[a] Anti-MUCl and anti-T epitope antibody titers are presented as median values for groups of four to thirteen mice. For anti-MUC1 antibody titers, ELISA plates were coated with BSA-MI-MUC1(Tn) conjugate, or for anti-T epitope antibody titers, ELISA plates were coated with Neutravidin-Biotin-T Epitope coating. Titers were determined by linear regression analysis in which dilution was plotted against absorbance. Titer was defined as the highest dilution to obtain an optical density of 0.1 or greater relative to normal control mouse serum. [b] Using a liposome formulation. MTT tumors were induced between the 3rd and 4th immunization. [c] EL = empty liposomes. [d] No tumor was induced.

Table 7. Competitive inhibition _IC50 values of antibody binding of MUCl(Tn) and MUCl (unglycosylated) to BSA-MI-MUCl(Tn) conjugate by ELISA ^[a] .

[a] ELISA plates were coated with BSA-MI-MUCl(Tn) conjugate. Serum samples from groups of 7 mice after immunization with 1 or 2, diluted to obtain an OD of about 1 in ELISA in the absence of inhibitors, were first reacted with MUCl(Tn) or unglycosylated MUC1 (0-500 µM final concentration) was mixed and then applied to the coated microtiter plate. Optical density values were normalized to those obtained with serum alone (0 µM inhibitor, 100%). Inhibition data were fitted with the following logistic equation: Y = Bottom + (Top - Bottom)/(1 + 10 ^{(X - Log IC50)} ), where Y is the normalized optical density and X is the logarithm of the inhibitor concentration , and the _IC50 is the concentration of inhibitor that halve the response. _IC50 values are reported as best fit values with 95% confidence intervals.

cellular immunity. To assess the ability of vaccine candidates to activate cytotoxic T lymphocytes, CD8 ⁺ T cells were isolated by magnetic cell sorting from the lymph nodes of mice immunized with various compounds and plated on ELLISPOT plates with The irradiated DCs were incubated together. Vaccine candidates 1 and 2 showed strong CD8 ⁺ responses compared to controls (Figure 19A, compare 1 and 2 with 3). Interestingly, the mixture of glycopeptide 4 and adjuvant 5 (Pam3CysSK4) induced the activation of a smaller number of CD8 ⁺ , indicating that the covalent attachment of MUCl and helper T epitopes to the adjuvant is important for optimal activation of CTLs.

The lytic activity of isolated CD8+ cells in the absence of in vitro stimulation was examined by Cr release assay in which DCs were treated with the MUCl-derived glycopeptide ^SAPDT (Tn)RPAP (SEQ ID NO:26) or in the case of immunization with 2 Peptide SAPDTRPAP (SEQ ID NO:20) pulsed. As can be seen in Figure 19B, CTLs activated by compounds 1 and 2 showed significantly greater cytotoxicity compared to controls. Furthermore, mice immunized with a mixture of 4 and 5 showed reduced lytic activity, further confirming the importance of covalent linkage of multiple epitopes.

To study the epitope requirement of CD8 ⁺ cells in detail, groups of five MUC1.tg were immunized with liposomal formulations of compounds 1 and 2, and CD8 ⁺ cells were subsequently harvested and pooled, which were treated separately with the glycopeptide SAPDT(Tn ) RPAP (6) (SEQ ID NO:26) and peptide SAPDTRPAP (7) (SEQ ID NO:20) pulsed DCs were stimulated in vitro for 1 day and then allowed to expand by incubation with IL-2 and IL-7 for 14 sky. The percentage of IFN-γ-producing CD8 ⁺ cells was determined after dendritic cells were pulsed with MUCl-derived glycopeptides 6–9. Compound 1 had activated a diverse range of CTLs that could be activated by glycosylated and aglycosylated structures, whereas those obtained by immunization with 2 showed responsiveness only to aglycosylated peptide 7. Furthermore, CD8 ⁺ cells obtained from immunization with 1 could lyse DCs pulsed with glycosylated and aglycosylated constructs ( FIG. 20 ).

These results indicate that CTLs activated by immunization with 1 recognize a wider range of structures (including glycosylated and aglycosylated MUC1-derived peptides), whereas CTLs obtained from compound 2 show a strong response to aglycosylated peptides. priority.

Cytokine induction. The lipopeptide portion of the three-component vaccine is required to initiate the production of essential cytokines and chemokines by interacting with TLR2 on the surface of mononuclear phagocytes (Akira et al., 2001, Nat Immunol ; 2:675-680 ; Finlay and Hancock, 2004, Nat Rev Microbiol ; 2:497-504; van Amersfoort et al., 2003, Clin Microbiol Rev ; 16:379-414; and Spohn et al., 2004, Vaccine ; 22:2494-2499). Upon activation, the intracellular domain of TLR2 recruits the adapter protein MyD88, leading to the activation of a kinase cascade leading to the production of a number of cytokines and chemokines. On the other hand, LPS induces cellular responses by interacting with the TLR4/MD2/CD14 complex, which leads to the recruitment of the adapter proteins MyD88 and TRIF, leading to a more complex pattern of cytokine induction. TNF-γ secretion is a prototypical measure of MyD88-dependent pathway activation, whereas IFN-γ secretion is commonly used as an indicator of TRIF-dependent cellular activation (Akira et al., 2001, Nat Immunol ; 2:675-680; and van Amersfoort et al., 2003, Clin Microbiol Rev ; 16:379-414).

To examine the pattern of cytokine production by multicomponent vaccine 1 and determine whether glycosylation affects responsiveness, TNF-α, IFN-β, Rantes, IL-6, IL- 1. Efficacy ( _EC50 ) and potency (maximal responsiveness) of IL-10, IP-10, IL-12p70 and IL-12/23p40 secretion. Therefore, primary dendritic cells obtained by established methods were exposed to a wide range of concentrations of compounds 1, 2, 5 and E. coli 055:B5 LPS, and supernatants were examined for various mouse cytokines using capture ELISA. Glycolipeptide 1, lipopeptide 2, and Pam3CysSK4 (5) induce TNF-α, Rantes, IL-6, IL-1, and IL-12/23p40 secretion with similar potency and potency, pointing to linkage of B and T epitopes and sugar Kylation had no effect on cytokine responses. See Figure 22, Table 8 and Table 9. As expected, neither compound induced IFN-[beta] production. Interestingly, compared to compounds 1, 2 and 5, E. coli 055:B5 LPS displayed much greater potency and efficacy for TNF-α induction. In addition, it is able to stimulate cells to produce IFN-β, IL-10, IP10, and IL-12p70. The reduced potency and efficacy of 1, 2, and 5 are favorable properties because LPS is known to overactivate the innate immune system, leading to sepsis Symptoms of shock.

To ensure that cytokine production is initiated in a TLR2-dependent manner, compounds 1 and 5 were exposed to HEK 293T cells stably transfected with murine TLR2 and treated with a plasmid containing the reporter gene pELAM-Luc (NF-B-dependent firefly firefly luciferase reporter vector) and a plasmid containing the control gene pRL-TK (Renilla luciferase control reporter vector) was transiently transfected. After a 4 h incubation time, activity was measured using a commercial dual luciferase assay and it was found that compounds 1 and 5 were able to activate NF-B in a TLR2-dependent manner.

Table 8. Cytokine Plateau ^[a] (pg/mL) for Dose Response Curves of Liposome Formulations Loaded with Compound 1, 2 or 3 and E. coli LPS Obtained After 24 Hours of Incubation of Primary Dendritic Cells .

[a] Curve fitting using non-linear least squares in pg cytokine/[mu]g total protein, reported as plateau of best fit ± standard error by Prism.

[b]nd means not detected.

Table 9. Cytokine log _EC50 values ^[a] (nM) of liposome formulations loaded with compound 1, 2 or 3 and E. coli LPS in primary dendritic cells.

[a] Log _EC50 values reported as best fit ± standard error by Prism using nonlinear least squares curve fitting.

[b] nd means not detected at the level used for accurate _EC50 determination.

discuss

Evidence is emerging that successful cancer vaccine development requires multimodal treatments that affect several aspects of the immune system at once. Although cellular and humoral immune responses against MUCl have been observed in some cancer patients, designing cancer vaccine candidates that can elicit both responses has been difficult. This example demonstrates that a multicomponent vaccine consisting of a glycopeptide derived from MUCl, a non-germline-selective peptide helper T epitope, and a TLR2 agonist can elicit IgG antibodies that can lyse MUC1-expressing cancer cells and stimulate cytotoxic T lymphocytes, thereby reversing tolerance and generating a therapeutic response in a mouse model of breast cancer.

Careful analysis of control compounds revealed that the reduction in tumor burden mediated by the multicomponent vaccine was caused by specific immunity against MUCl and by nonspecific adjuvant effects mediated by intercalating TLR2 agonists. Evidence is emerging that TLRs are ubiquitously expressed by tumor cells and that their activation can lead to inhibition or promotion of tumorigenicity. Furthermore, cytokines and chemokines produced following TLR activation can stimulate the expression of many co-stimulatory proteins for optimal interaction between T helper cells and B cells and antigen-presenting cells. Recent studies have pointed to the unique ability of TLR1/2 agonists to reduce the suppressive function of Foxp3 ⁺ regulatory T cells (Tregs) and enhance the cytotoxicity of tumor-specific CTLs in vitro and in vivo, and potentially have more favorable effects than other TLR agonists. antitumor effect.

This example also demonstrates that covalent attachment of TLR2 agonists to glycolipopeptide epitopes is critical for eliciting antibodies and optimal CTL function. Lipidation with TLR2 agonists makes it possible to formulate multicomponent vaccines in liposomal formulations, which may enhance their circulation time. Furthermore, liposomal formulations present glycopeptide epitopes in a multivalent manner, thereby providing an opportunity for efficient clustering of B-cell epitopes, which are required for initiating B-cell signaling and antibody production. As shown in previous examples, covalent attachment of the TLR2 agonist Pam3CysSK4 facilitates selective internalization by TLR2-expressing immune cells such as B cells and antigen presenting cells (APCs). The uptake and processing of antigen and the subsequent presentation of T epitopes as complexes with MHC class I or II on the surface of APC cells are critical for eliciting IgG antibodies. Over the past decade, numerous studies have shown that selective targeting of antigens to APCs will lead to improved immune responses. For example, oxidized mannan, heat shock proteins, bacterial toxins, and antibodies targeting cell surface receptors of dendritic cells have been linked to antigens to increase uptake by dendritic cells. Although these uptake strategies are attractive, they have the disadvantage that the targeting device is antigenic, which may lead to immunosuppression of tumor-associated carbohydrates. The attractiveness of Pam3CysSK4 for facilitating uptake by APCs is its low innate immunity. Thus, a three-component vaccine would facilitate uptake without the disadvantage of immunosuppression.

Finally, this example demonstrates that glycosylation of the MUCl epitope is critical for optimal reduction of tumor burden. Mechanistic studies provided a rationale for these observations, finding that immunization with Compound 1 resulted in slightly higher titers of antibodies, which were significantly more lytic, compared with Compound 2 lacking the Tn antigen. Conformational studies by NMR supplemented by light scattering measurements have indicated that deglycosylation of MUCl leads to a less extended and more globular structure. Similar studies using MUCl-associated O-glycopeptides have shown that carbohydrate moieties exert conformational effects, which may provide a rationale for differences in immune responses. Furthermore, the use of glycosylation 1 resulted in efficient activation of CTLs capable of recognizing glycosylated and unglycosylated structures, with the former being preferred. On the other hand, immunization with aglycosylated compound 2 resulted in CTLs predominantly recognizing aglycosylated structures. Short O-linked glycans such as Tn and STn on MUCl tandem repeats are known to remain intact during DC processing in the MHC class II pathway, thus potentially eliciting glycopeptide-selective CTL responses. Furthermore, there is evidence that MUC1 glycopeptides can bind MHC class I mouse allele H2K ^b more strongly than the corresponding non-glycosylated peptides. Furthermore, not only is cancer progression associated with MUCl modifications with truncated sugars such as the Tn antigen, but these structures are also present in much higher densities, thus eliciting responses against such structures is required for effective immunotherapy.

Experimental part

General Method for Automated Solid-Phase Peptide Synthesis (SPPS): Using N ^α -Fmoc-Protected Amino Acids and 2-(1H-Benzotriazol-1-yl)-1,1,3,3-Tetramethylurea Hexafluorophosphate (HBTU)/1-hydroxybenzotriazole (HOBt) was used as the activation reagent, and peptides were synthesized by an established protocol on an Applied Biosystems, ABI 433A peptide synthesizer equipped with a UV detector. Individual coupling steps are performed with conditional capping.

General method for liposome preparation for native chemical attachment: Prepare pH 7.8 200 mM sodium phosphate buffer containing 2 mM tris(2-carboxyethyl)phosphine (TCEP) and 0.3% EDTA. The buffer was degassed for 1 h. Cysteine-containing peptides (1 equiv), thioesters (2 equiv), and dodecylposphocholine (13 equiv) were dissolved in 1:1 CHCl3:trifluoroethanol and the solvent removed. The lipid/peptide films were subsequently hydrated in an incubator at 41 °C for 4 hours. The mixture was sonicated and the peptide/lipid suspension was extruded through a 1.0 μm polycarbonate membrane (Whatman, Nucleopore, Track-Etch Membrane) at 50 °C to obtain homogeneous vesicles.

Synthesis of Glycosylated Three-Component Vaccine Candidate 1: Peptide Thioester X (1.1 mg, 0.674 μmol), Peptide X (1.0 mg, 0.337 μmol) and Dodecylphosphocholine (1.5 mg, 4.38 μmol) Dissolve in a 1:1 mixture of CHCl3:trifluoroethanol (5 mL). The solvent was removed under reduced pressure to give lipid/peptide films. Lipid/peptide films were hydrated at 41°C for 4 hr using 200 mM sodium phosphate buffer containing 2 mM TCEP and 0.3% EDTA. The mixture was sonicated and the peptide/lipid suspension was extruded through a 1.0 μm polycarbonate membrane (Whatman, Nucleopore, Track-Etch Membrane) at 50 °C to obtain homogeneous vesicles. Sodium 2-mercaptoethanesulfonate (1 mM) was added to the vesicle suspension to initiate the reaction (1.5 mM final peptide concentration). After 20 minutes, the reaction mixture was purified by RP-HPLC on an analytical C-4 reverse phase column using a gradient of 0-100% B in A over a 40 minute period. Lyophilization of the appropriate fractions provided 1 (1.2 mg, 80%). _{C217H367N45O53S2HR MALDI} - _ToF MS: Observed; Calculated _4515.685 [M ₊ ].

General method for the preparation of liposomes for immunization: Thin films of synthetic compounds, egg phosphatidylcholine, phosphatidylglycerol and cholesterol in HEPES buffer (10 mM, pH 7.4) containing NaCl (145 mM) Hydration and subsequent extrusion through a 0.1 µm Nucleopore® polycarbonate membrane incorporates each glycolipopeptide into small phospholipid-based unilamellar vesicles (SUVs).

Immunization: 8- to 12-week-old MUC1.Tg mice expressing human MUC1 were challenged with liposomal formulations of the three-component vaccine construct (25 μg, containing 3 μg carbohydrate) and respective controls lacking tumor-associated MUC1 epitopes. Mice (C57BL/6; H-2b) were immunized three times intradermally at the base of the tail at biweekly intervals. After 35 days, mice were challenged with MMT breast tumor cells ( ¹ x 106 cells) expressing MUCl and Tn. On day 42, one week after tumor cell injection, another immunization was given. On day 49, one week after the last immunization, mice were sacrificed and the efficacy of the vaccine was determined.

Tumor palpation: 7 days after the third immunization, MUC1.Tg mice were injected subcutaneously in the left flank with ¹ x 106 cancer cells in 100 µL of PBS. Palpable tumors were measured by calipers, and tumor weights were calculated according to the formula: grams = [(length) x (width)2]/2, where length and width are measured in centimeters. At endpoint, tumors were surgically excised and tumor weights were measured.

⁵¹ Chromium (Cr) release assay: Using CD8 ⁺ T cells from TDLN without any in vitro stimulation as effector cells at a ratio of 100:1 and ⁵¹ Cr-labeled DCs pulsed with the respective peptides as target cells, passed standard ⁵¹ The Cr release method lasted 6 hours to measure the cell lytic activity. Target cells were loaded with 100 µCi ⁵¹ Cr (Amersham Biosciences)/10 ⁶ target cells for 2 hours prior to incubation with effectors. Radioactive51Cr release was measured using a Topcount Microscintillation Counter (Packard Biosciences) and specific cleavage was calculated: (Experimental ^cpms - Spontaneous cpms/Complete cpms - Spontaneous cpms) x 100. Spontaneous lysis was <15% of total lysis.

Determination of antibody-dependent cell-mediated cytotoxicity (ADCC): Tumor cells (Yac-MUC1 or C57mg.MUC1) were labeled with 100 µCi ⁵¹ Cr at 37°C for 2 hours, washed and mixed with 5 µg/mL of a control antibody ( Mouse IgG) or sera from vaccinated mice (1 out of 25 dilutions) were incubated at 37°C for 30 minutes. NK cells with high expression of the CD16 receptor (KY-1 clone, a generous gift from Dr. Wayne M. Yokoyama, Washington University, St. Louis) were used as effectors. These cells were stimulated with IL-2 (200 units/mL) for 24 hours before the assay. Effector cells were seeded together with antibody-labeled tumor cells in 96-well culture plates (Costar High Binding Plates) at an effector:target cell ratio of 50:1 for 4 hours. ⁵¹ Cr release in the supernatant was determined by Top Count. The spontaneous release and maximum release of ⁵¹ Cr were measured and were less than 20%. The percentage of specific release was determined: (release-spontaneous release/maximum release-spontaneous release) x 100.

IFN-γ ELISPOT Assay: At sacrifice, MAC-sorted CD4 ⁺ and CD8 ⁺ T cells from TDLN were isolated from treated MUC1.Tg mice and used as responders in the IFN-γ ELISPOT assay. The number of spots was determined using computer-aided video image analysis by ZellNet Consulting, Inc. (Fort Lee, NJ). Splenocytes from C57BL/6 mice stimulated with Concavalin A were used as positive controls.

Serological assays: Anti-MUC-1 IgG, IgG1, IgG2a, IgG2b, IgG3 by enzyme-linked immunosorbent assay (ELISA) as previously described (Buskas et al., 2004, Chemistry , 10(14):3517-24) and IgM antibody titers. Briefly, ELISA plates (Thermo Electron Corp.) were coated with MUC-1 glycopeptide conjugate (BSA-MI-MUC-1 ) conjugated to BSA via a maleimide linker. Serial dilutions of sera were allowed to bind immobilized MUC-1. By adding phosphate-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.), IgG1 (Zymed), IgG2a (Zymed), IgG2b (Zymed), IgG3 (BD Biosciences Pharmingen), or IgM (Jackson ImmunoResearch Laboratories Inc. ) antibodies Complete the test. After addition of p-nitrophenyl phosphate (Sigma), absorbance was measured at 405 nm using a microplate reader (BMG Labtech) with wavelength correction set at 490 nm. Antibody titers against T (polio) epitopes were determined as follows. Reacti-bind NeutrAvidin coated and pre-blocked plates (Pierce) were incubated with biotinylated T-epitopes (10 µg/mL; 100 µL/well) for 2 hours. Next, serial dilutions of sera are allowed to bind the immobilized T epitopes. Assays were done as described above. Antibody titers were defined as the highest dilution that achieved an optical density of 0.1 or greater relative to normal control mouse serum.

Inhibition ELISA: To investigate the competitive inhibition of MAb binding to MUC(Tn) by corresponding glycopeptides, peptides and sugars, serum samples were diluted in dilution buffer in such a way that in the absence of inhibitor Below, the expected final optical density value is about 1. For each well, mix 60 µL of diluted serum samples in an uncoated microtiter plate with 60 µL of dilution buffer, glycopeptide 6 (MUC(Tn )), peptide 7 (unglycosylated MUC1) or Tn-monomer mix. After 30 minutes of incubation at room temperature, 100 µl of the mixture was transferred to a plate coated with BSA-MI-MUC1(Tn). For total IgG using an alkaline phosphatase-conjugated detection antibody, microtiter plates were incubated and developed as described above. Optical density values were normalized to those obtained with monoclonal antibodies alone (0 µM inhibitor, 100%).

Dendritic cells (DC) preparation: as previously described (Inaba et al., 1992, J Exp Med ; 176(6):1693-702 and Mukherjee, 2003 , J Immunother ; 26:47-62), from mice DCs were prepared from bone marrow cultures.

Cytokine assays: On the day of the exposure assay, mature DCs were plated as ⁴ x 106 cells/well in 1.8 mL in 24-well tissue culture plates. Cells were then incubated with different stimuli (200 μL, 10X) for 24 hours in a final volume of 2 mL/well. The stimuli were prepared at a wide concentration range (corresponding to a final concentration of 0.1 ng/mL-100 µg/mL PAM ₃ CysSK ₄ for 1, 5, or 6 in liposomes, and 0.001 ng/mL for E. coli LPS -10 µg/mL final concentration) was administered. Collect the supernatant. For the evaluation of the effect of ATP on IL-1β secretion, DCs were incubated for an additional 30 minutes in the same volume of medium containing ATP (5 mM; Sigma), after which supernatants were harvested. All collected culture supernatants were stored frozen (minus 80°C).

Cytokine ELISA was performed in 96-well MaxiSorp plates (Nalge Nunc International). Cytokine DuoSet ELISA Development Kit (R&D Systems) was used for mouse TNF-α, RANTES, IL-6, IL-1β, IL-10, IP-10, IL-12 p70 and IL according to the manufacturer’s instructions Cytokine quantification of -12/23 p40. Absorbance was measured at 450 nm using a microplate reader (BMG Labtech) with wavelength correction set to 540 nm. The concentration of mouse IFN-β in the culture supernatant was determined as follows. Plates were coated with rabbit polyclonal antibody against mouse IFN-[beta] (PBL Biomedical Laboratories). Antibodies were immobilized to allow IFN-β binding in standards (PBL Biomedical Laboratories) and samples. Rat anti-mouse IFN-β antibody (US Biological) was then added. Next, HRP-conjugated goat anti-rat IgG (H+L) antibody (Pierce) and the chromogenic substrate for HRP, 3,3',5,5'-tetramethylbenzidine (Pierce), were added. After the reaction was stopped, absorbance was measured at 450 nm with wavelength correction set to 540 nm. Cytokine values are expressed as pg cytokine/mL. Concentration-response data were analyzed using nonlinear least squares curve fitting in Prism (GraphPad Software, Inc.). These data were fitted with the following four-parameter logistic equation: Y= _Emax /(1+( _EC50 /X) ^{Hill slope} ), where Y is the cytokine response, X is the concentration of the stimulus, and _Emax is the maximum Response (plateau), and _EC50 is the stimulus concentration that produces 50% stimulation. Hill slope was set at 1 to enable comparison of _EC50 values of different inducers.

Statistical analysis: Multiple comparisons were performed using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test. Differences were considered significant when P < 0.05. For comparisons between two groups, data were analyzed using a two-tailed Student's t -test with 95% confidence intervals. P -values <0.05 were considered statistically significant.

Example 9

Addition of a second TLR agonist

This example determines the effect of the addition of a second TLR agonist, CpG, on the effectiveness of immunization. Following the procedure described in more detail in Example 8, aged MUCl.Tg mice (C57BL/6; H-2b) expressing human MUCl were immunized with a preparation of Compound 2 (Pam ₃ CysSK ₄ -T Helper Epitope (Polio) - MUC1(unglycosylated)); Compound 1 (Pam ₃ CysSK ₄ -T Helper Epitope (Polio) - MUC1(Tn)); Compound 1 plus CpG (CpG oligodeoxynucleotide (CpG ODN))); compound 5 (Pam ₃ CysSK ₄ ) plus compound 4 (T helper cell epitope (polio)-MUC1(Tn)); compound 5; compound 3 ( Pam ₃ CysSK ₄ -T helper epitope (polio)); Compound 3 plus CpG; EL (empty liposome) plus CpG; or EL. The structures of compounds 1, 2, 3, 4 and 5 are shown in FIG. 16 . Compounds were co-administered with the TLR9 agonist CpG using a standard immunization schedule. As shown in Figures 23-25, administration of the combination of TLR9 ligands CpGs further improved the anti-tumor properties of the three-component vaccine 1 . Specifically, the addition of the second agonist resulted in a significant further reduction in tumor weight (Figure 23) and induced a more potent immune response (Figures 24 and 25).

Example 10

In MUC1.Tg mice bearing MMT tumors, a three-component MUC1 glycopeptide vaccine induces both humoral and cellular immune responses

Effective immunotherapy for cancer depends on both cellular and humoral immune responses to tumor antigens. MUCl, expressed at increased levels on breast cancer, also exhibits altered glycosylation that contributes to the formation of neoantigens. The identification of MHC class I and II binding peptides derived from tumor-associated MUCl has facilitated the development of MUCl-based cancer vaccines. MHC class I binding epitopes from MUCl tandem repeats resulted in strong cellular responses, but no antibody responses, when administered with emulsification with adjuvant. It is possible that using a glycopeptide more representative of the new form of MUCl as seen in cancer, to which an individual may be less tolerant, would result in better immunogenicity, and the direct linking of vaccine components would result in a better immunogenicity than its Delivers a better immune response as a mixture. This example shows that a three-component vaccine consisting of a TLR2 agonist, a helper epitope, and a T-cell epitope (which is also a B-cell epitope derived from MUCl ) can break tolerance and elicit both humoral and cellular immune responses. Immunization with the MUC1 glycopeptide vaccine resulted in a significant reduction in tumor burden compared to mice treated with adjuvant and empty liposomes only. The three-component vaccine activated MUC1 glycopeptide-specific cytotoxic CD8+ T cells and elicited strong titers of IgG antibodies that mediated lysis of associated tumor cells via ADCC.

A three-component vaccine compound 1 (Pam ₃ CysSK ₄ -T helper-MUC1) and LAA-T helper-MUC1 (which contains immune silencing lipids) and compound 2 ((Pam ₃ CysSK ₄ -T helper cells) and LAA-T helper cells (both of which lack tumor-associated MUC1 epitopes), MUC1.Tg mice (C57BL/6; H-2b) expressing human MUC1 were administered biweekly The interval of immunization was three times. The structure of the compound is shown in Figure 26. After 35 days, the mice were challenged with MMT mammary tumor cells expressing MUC1 and Tn. The immunization schedule is shown in Figure 27. After the last immunization Three weeks, the mice were sacrificed, and the efficacy of the vaccine was determined by tumor burden, cell-mediated immune response and antibody-mediated immune response.Comparison with LAA-T helper cells-MUC1 (compound lacking TLR2 agonist) and respective Immunization with the three-component glycosylated vaccine compound 1 resulted in a significant reduction in tumor mass compared to the control compound 2 (TLR2 agonist – T helper epitope) and empty liposomes (see Figure 28). Compound 1 elicited strong titers of IgG antibodies mediating lysis of associated tumor cells by ADCC (see Figures 30 and 31). As determined by chromium release assay, MMT tumor-bearing mice from immunized The lytic potential of sorted CD8+ T cells was shown: compared with the respective control compound 2 ((Pam ₃ CysSK ₄ -T helper) and EL and the LAA-T helper-MUC1 compound lacking TLR2 ligands, with three groups Glycosylated vaccine immunizations showed significantly greater lysis (see Figure 29). This is the first vaccine formulation to elicit both cellular and humoral responses.

Example 11

Synthetic three-component constructs utilizing human MUC1 T helper cell sequences

For the prediction of IA ^b , H2-K ^b and H2-D ^b binding epitopes, the Rankpe (Harvard, MA) Position Specific Scoring Matrices (PSSM) program was primarily queried. A second program, ^{SYPEITHI (Institute for Cell Biology, Heidelberg, Germany), was reverse-queried to cross-validate H2-Kb and H2-Db binding} epitopes. Figure 32 shows an analysis of the binding of human MUCl -derived peptides to mouse IA ^b 15mers and H2- ^Db and H2-Kb 9mers. Many encouraging predictions are evident. Dashed lines show 15mers showing RANKPEP scores for binding to ^IAb . 9mers showing ^RANKPEP scores for binding to H2- ^Db (dddd) or H2-Kb (kkkk) or non-germline selective binding to both (bbbb) are assigned.

Compounds identified by this assay were tested for induction of interferon gamma production on CD4 and CD8 cells. Mice were immunized with the peptides described in Figure 33A, and lymph node-derived T cells expressing low levels of CD62L were obtained by cell sorting and cultured for 14 days in the presence of DC pulsed with the immunization peptides. After exposure to DCs pulsed with the peptides listed on the y-axis, the resulting cells were analyzed by intracellular cytokines for the presence of CD4 ⁺ IFNγ ⁺ and CD8 ⁺ IFNγ ⁺ T cells ( FIG. 33B ). Immunization with the glycosylated 21mer (peptide C) elicited strong specific CD4 ⁺ and CD8 ⁺ responses against itself as well as the aglycosylated 15mer (peptide A) and 21mer (peptide B).

Various synthetic constructs utilizing the human MUCl T helper sequence shown in Figure 34 have been generated. Following the procedures described in more detail in Examples 8-10, MUCl.Tg mice expressing human MUCl (C57BL/6; H- ^2b ) were immunized with the constructs shown in Figures 33 and 34 . Following the procedures described in more detail in Examples 8-10, the constructs were assayed for reducing tumor weight, eliciting IgG antibodies, mediating lysis of tumor cells by ADCC, eliciting CD8+ cytotoxic activity, and producing IFN-γ and other cytokines. effectiveness.

Example 12

Monoclonal antibodies against carbohydrates and glycopeptides generated by using a fully synthetic three-component immunogen

Glycoconjugates are the most functionally and structurally diverse molecules in nature, and it is now well established that protein- and lipid-bound sugars play fundamental roles in many molecular processes affecting eukaryotic biology and disease. Examples of such processes include fertilization, embryogenesis, neuronal development, hormonal activity, cell proliferation and their organization into specific tissues. Significant changes in cell-surface carbohydrates occur with tumor progression, which appear to correlate strongly with metastasis. Furthermore, carbohydrates are able to induce protective antibody responses, an immune response that is a major contributor to the survival of organisms during infection.

The inability of sugar to activate helper T lymphocytes has complicated its development as a vaccine. For most immunogens, including carbohydrates, antibody production relies on the cooperative interaction of two classes of lymphocytes, B cells and helper T cells (Jennings, Neoglyconjugates: Preparation and Applications 325-371 (Academic Press, Inc., 1994); Kuberan , Curr. Org. Chem. 2000, 4, 653-677). Sugars alone are unable to activate helper T cells and therefore have limited immunogenicity, as manifested by the absence of low affinity IgM and IgG antibodies. To overcome the T cell-independent nature of carbohydrates, past studies have focused on conjugation of carbohydrates to exogenous carrier proteins such as keyhole limpet hemocyanin (KLH), detoxified tetanus toxoid (Jennings, Neoglyconjugates: Preparation and Applications 325 -371 (Academic Press, Inc., 1994); Kuberan, Curr. Org. Chem. 2000, 4, 653-677; Jones, An. Acad. Bras. Cienc. 2005, 77, 293-324). In this approach, the carrier protein enhances the presentation of carbohydrates to the immune system and provides T epitopes (peptide fragments of 12-15 amino acids) that can activate T helper cells. Thus, a class switch from low-affinity IgM to high-affinity IgG antibodies is accomplished. This approach has been successfully applied to the development of conjugate vaccines to prevent H. influenzae infection.

Carbohydrate-protein conjugate vaccine candidates consisting of more demanding carbohydrate antigens such as tumor-associated carbohydrates and glycopeptides failed to elicit high titers of IgG antibodies. These results are not surprising since tumor-associated sugars are low antigenic because they are self-antigens and thus tolerated by the immune system. This tolerance is reinforced by antigen shedding by growing tumors. Furthermore, exogenous carrier proteins such as KLH and BSA and linkers linking sugars to carrier proteins can elicit strong B cell responses, which can lead to suppression of antibody responses against carbohydrate epitopes (Buskas, Chem. Eur. J. 2004, 10, 3517-3524; Ni, Bioconjug. Chem. 2006, 17, 493-500). It is clear that the successful development of carbohydrate-based cancer vaccines requires new strategies for more efficient presentation of tumor-associated carbohydrate epitopes to the immune system, resulting in more efficient class switching to IgG antibodies (Reichel, Chem. Commun. 1997 , 21, 2087-2088; Alexander, J. Immunol. 2000, 164, 1625-1633; Kudryashov, Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3264-3269; Lo-Man, J. Immunol. 2001, 166, 2849-2854; Jiang, Curr. Med. Chem. 2003, 10, 1423-1439; Jackson, Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15440-5; Lo-Man, Cancer Res. 64, 4987-4994; Buskas, Angew. Chem. Int. Ed. 2005, 44, 5985-5988 (Example 1); Dziadek, Angew. Chem. Int. Ed. 2005, 44, 7624-7630; Krikorian, Bioconjug 2005, 16, 812-819; Pan, J. Med. Chem. 2005, 48, 875-883).

As shown in previous examples, a three-component vaccine consisting of a TLR2 agonist, a non-germline-selective peptide T helper epitope, and a tumor-associated glycopeptide elicited particularly high titers of IgG antibodies in mice, It can recognize cancer cells expressing tumor-associated carbohydrates (see Compound 21, Figure 5, Example 6 and Compound 51, Figure 15) (Ingale, Nat. Chem. Biol. 2007, 3, 663-667). The superior properties of vaccine candidates have been attributed to local production of cytokines, upregulation of co-stimulatory proteins, enhanced uptake by macrophages and dendritic cells, and avoidance of epitope suppression.

The three-component immunogen technology of the present invention can be used to generate monoclonal antibodies (MAbs) against weakly antigenic carbohydrates and glycopeptides. We have initially focused on MAbs directed against β- N -acetylglucosamine (β- O -GlcNAc) modified peptides (Wells, Science 2001, 291, 2376-2378; Whelan, Methods Enzymol. 2006, 415, 113-133; Zachara, Biochim. Biophys. Acta, 2006, 1761, 599-617; Dias and Hart, Mol. Biosyst. 2007, 3, 766-772; Hart, Nature 2007, 446, 1017-1022; Lefebvre, Exp. Rev. Proteomics 2005, 2, 265-275). Numerous nuclear and cytoplasmic proteins in metazoans are modified at Ser and Thr residues by the monosaccharide β- O -GlcNAc. Rapid and dynamic changes in O -GlcNAc levels in response to extracellular stimuli imply a critical role for O -GlcNAc in signal transduction pathways. Regulation of O -GlcNAc levels has profound effects on cellular function, mediated in part through complex interplay between O -GlcNAc and O -phosphate. Recently, O -GlcNAc has been implicated in the etiology of type II diabetes, regulation of stress response pathways and regulation of the proteasome. Progress in this exciting field of research is severely hampered by the lack of reagents such as suitable MAbs. In this regard, only one poorly performing IgM MAb with relatively broad specificity (Comer, Anal. Biochem. 2001, 293, 169-177) is commercially available (Covance Research Products Inc).

We have designed and synthesized compound 52 (Figure 15), which contains a β-GlcNAc modified glycopeptide derived from casein kinase II (CKII) (Kreppel, J. Biol. Chem. 1999, 274, 32015-32022) as B epitope, the well documented murine MHC class II restricted helper T cell epitope KLFAVWKITYKDT (SEQ ID NO:3) derived from poliovirus, and the intercalating adjuvant Pam ₃ CysSK ₄ . In addition, compound 53 was prepared with an artificial sulfur-linked GlcNAc moiety, which is expected to have better metabolic stability. Hydration of thin films of synthetic compounds, egg phosphatidylcholine, phosphatidylglycerol, and cholesterol in HEPES buffer (10 mM, pH 6.5) containing NaCl (145 mM) and subsequent hydration via a 100 nm Nucleopore® polycarbonate membrane Compounds 52 and 53 were incorporated into phospholipid-based small unilamellar vesicles (SUVs) by extrusion. Groups of five female BALB/c mice were immunized four times intraperitoneally at weekly intervals with liposomes containing 3 μg of sugar.

Anti-glycopeptide antibody titers were determined by coating microtiter plates with CGSTPVS(β- O -GlcNAc)SANM conjugated to maleimide (MI)-modified BSA, followed by anti-glycopeptide labeled with alkaline phosphatase. Detection of mouse IgG antibody. As can be seen in Table 10, compounds 52 and 53 elicited excellent titers of anti-MUCl IgG antibodies. Furthermore, no significant differences in titers were observed between O -linked and S -linked saccharide derivatives.

Table 10. ELISA anti- ^GSTPVS (β- O -GlcNAc) SANM(68) titers after 4 immunizations with two different formulationsa

^a Anti-GSTPVS(β- O -GlcNAc)SANM (68) antibody titers are presented as the mean of a group of five mice. ELISA plates were coated with BSA-MI-GSTPVS(β- O -GlcNAc)SANM(BSA-MI-66) conjugate and titers were determined by linear regression analysis plotting dilutions compared to absorbance. Titer was defined as the highest dilution to obtain an optical density of 0.1 or greater relative to normal control mouse serum.

^b using liposome formulations.

cO- ^GlcNAc52 ; Pam3CysSK4GC _- KLFAVWKITYKDT _- G-GSTPVS(β- O - GluNAc )SANM.

dS- ^GlcNAc53 ; Pam3CysSK4GC _- KLFAVWKITYKDT _- G-GSTPVS(β- S - GluNAc )SANM.

For IgM titers, a statistically significant difference was observed between 52 and 53 ( P =0.0327).

Individual titers for total IgG, IgGl, IgG2a, IgG2b, IgG3 and IgM are reported in Figure 36.

Next, spleens from two mice immunized with O -linked glycolipopeptide 52 were harvested, and standard hybridoma culture techniques gave seven IgG1, seven IgG2a, two IgG2b, and fourteen IgG3 producing hybridoma cell lines. The ligand specificity of the resulting MAbs was investigated using ELISA and inhibition ELISA. All MAbs recognized CGSTPVS(β- O -GlcNAc)SANM linked to BSA, while only a few recognized the peptide CGSTPVSSANM (SEQ ID NO: 12) conjugated to BSA. In addition, the interaction of nineteen MAbs with BSA-MI-CGSTPVS(β- O -GlcNAc)SANM could be inhibited by the glycopeptide GSTPVS(β- O -GlcNAc)SANM.

The hybridoma cell lines 1F5.D6, 9D1.E4 and 18B10.C7 were deposited with the American Type Culture Collection on July 1, 2008 as described in more detail in WO 2010/002478 ("Glycopeptide and Uses Thereof") (ATCC), 10801 University Blvd., Manassas, VA, 20110-2209, USA, and assigned ATCC Deposit Nos. PTA-9339, PTA-9340, and PTA-9341, respectively. However, it should be understood that the written description herein is considered sufficient to enable those skilled in the art to fully practice the invention. Furthermore, the deposited embodiments are intended as single illustrations of one aspect of the invention and should not be construed as limiting the scope of the claims in any way.

Following similar procedures, polyclonal and monoclonal antibodies specific for any of the MUCl constructs described herein can be prepared.

Example 13

Generation of O-GlcNAc-specific monoclonal antibodies using a novel synthetic immunogen

Combining a fully synthetic three-component immunogen with hybridoma technology resulted in the generation of O -GlcNAc-specific IgG MAbs with a broad spectrum of binding targets. Large-scale shotgun proteomics led to the identification of 254 mammalian O -GlcNAc-modified proteins, including a large number of new glycoproteins. The data implicate a role for O -GlcNAc in transcriptional/translational regulation, signal transduction, ubiquitin pathway, anterograde trafficking of intracellular vesicles, and post-translational modification.

O -glycosylation of serine and threonine of nuclear and cytoplasmic proteins by a single β- N- acetyl-D-glucosamine moiety (β-GlcNAc) is a ubiquitous post-translational modification that is highly dynamic and occurs through The action of the cycling enzymes O -linked GlcNActransferase (OGT) and O -GlcNAcase (OGA) fluctuates in response to cellular stimuli. Such glycosylation has been implicated in many cellular processes, often via interaction with phosphorylation which may occur on the same amino acid residue. Importantly, alterations in O- GlcNAc levels have been linked to the etiology of prevalent human diseases, including type II diabetes and Alzheimer's disease (Hart et al., 2007 Nature 446, 1017-1022).

Unlike phosphorylation of a broad range of pan-specific and site-specific phospho-antibodies for which a wide range of pan-specific and site-specific phospho-antibodies are available, the study of O -GlcNAc modification is hampered by the lack of efficient tools for its detection, quantification and site localization. In particular, only two pan- O -GlcNAc-specific antibodies have been described: the IgM pan- O -GlcNAc antibody (CTD 110.6; Comer et al., 2001 Anal. Biochem. 293, 169-177), and the anti- O -GlcNAc modified IgG antibodies produced by the nuclear pore component of the β-Na (RL-2; Snow et al., 1987 J. Cell Biol. 104, 1143-1156), which show limited cross-reactivity with O -GlcNAc-modified proteins. In fact, multiple studies have shown that O -GlcNAc-modified glycoconjugates do not elicit antibodies of the relevant IgG isotype, so the challenge of eliciting O -GlcNAc-specific IgG antibodies is considerable. We reasoned that O -GlcNAc-specific antibodies could be elicited by using a three-component immunogen (compound 52, Figure 35) derived from the tyrosine kinase II (CKII) α subunit in this study. Contains O -GlcNAc peptide (Kreppel and Hart, 1999 J. Biol. Chem. 274, 32015-32022), well-documented murine MHC class II-restricted helper T-cell epitopes, and Toll-like receptor-2 as an intercalating adjuvant (TLR2) agonist composition. This class of compound is expected to avoid immunosuppression caused by carrier proteins or linker regions of typical conjugate vaccines; however, it contains all the mediators required to elicit a strong and relevant IgG immune response (Ingale et al., 2007 Nat. Chem. Biol. 3, 663-667). Furthermore, compound 53 was prepared with an artificial sulfur-linked GlcNAc moiety that has improved metabolic stability compared to its O -linked counterpart, providing an additional opportunity to elicit O -GlcNAc-specific antibodies.

Compounds 52 and 53 ( Figure 35). The starting thioester 63 was assembled on the sulfonamide "safety-catch" linker, followed by release by alkylation with iodoacetonitrile and treatment with benzylthiol to give the compound deprotected using standard conditions. Compounds 64 and 65 were prepared using Rink amide resin, Fmoc-protected amino acid and Fmoc-Ser-(AcO3-α-D-GluNAc) or Fmoc-Ser-(1-thio-AcO3-α- D -GluNAc), respectively. After assembly was complete, the acetyl ester was cleaved by treatment with 60% hydrazine in MeOH, and the resulting compound was cleaved from the resin by treatment with reagent K, followed by purification by reverse phase HPLC. Compounds 52 and 53 were incorporated into phospholipid-based small unilamellar vesicles (SUVs) followed by extrusion through a 100 nm Nuclepore® polycarbonate membrane. Groups of five female BALB/c mice were immunized four times intraperitoneally at biweekly intervals with liposomes containing 3 μg of sugar. Anti-glycopeptide antibody titers were determined by coating microtiter plates with CGSTPVS(β- O- GlcNAc)SANM (66) conjugated to maleimide (MI)-modified BSA, and assayed with alkaline phosphatase Labeled anti-mouse IgG antibodies complete the detection. Compounds 52 and 53 elicited excellent titers of IgG antibodies (Table 10; Figure 36). Furthermore, no significant differences in IgG titers were observed between O -linked and S -linked saccharide derivatives, so further attention was focused on mice immunized with 52.

Spleens from two mice immunized with 52 were harvested and standard hybridoma culture techniques gave seven IgG1, seven IgG2a, two IgG2b and fourteen IgG3 producing hybridoma cell lines. The ligand specificity of the resulting MAbs was studied by ELISA. All MAbs recognized CGSTPVS(β- O -GlcNAc)SANM linked to BSA (BSA-MI-66), while only a few recognized the peptide CGSTPVSSANM (SEQ ID NO:12) (BSA-MI-67) conjugated to BSA . Furthermore, the interaction of twenty MAbs was inhibited by the glycopeptide GSTPVS(β- O -GlcNAc)SANM (68), but not by the peptide GSTPVSSANM (SEQ ID NO: 13) (69) or -O -GlcNAc-Ser ( 70) Inhibition, which confirms glycopeptide specificity.

Three hybridomas (18B10.C7(3), 9D1.E4(10), 1F5.D6(14)) were cultured on a one-liter scale, and the resulting hybridomas were purified by saturated ammonium sulfate precipitation followed by protein G column chromatography Antibodies, to obtain about 10 mg of IgG in each case. Inhibition ELISA confirmed that MAbs require carbohydrates and peptides (glycopeptides) for binding.

In conclusion, the three-component immunogen approach has been successfully used to generate a panel of pan-GlcNAc-specific MAbs, which provides a powerful new tool for exploring the biological implications of this class of protein glycosylation. The newly identified O -GlcNAc-modified proteins open new avenues to explore the importance of such post-translational modifications for a variety of biological processes. The three-component immunization technique is expected to have broad application for generating MAbs against other forms of protein glycosylation.

method

Reagents and general procedures used in the synthesis. Fmoc- L -amino acid derivatives and resins were purchased from NovaBioChem and Applied Biosystems, peptide synthesis grade N,N -dimethylformamide (DMF) was purchased from EM Science, and N -methylpyrrolidone (NMP) was purchased from Applied Biosystems. Egg phosphatidylcholine (PC), egg phosphatidylglycerol (PG), cholesterol (Chol), monophosphoryl lipid A (MPL-A) and dodecylphosphocholine (DPC) were obtained from Avanti Polar Lipids. All other chemical reagents were purchased from Aldrich, Acros, Alfa Aesar and Fisher and used without further purification. All solvents used were of reagent grade. Agilent Zorbax Eclipse ^™ C8 analytical column (5 μm, 4.6 x 150 mm) at a flow rate of ^{1 ml min-1 ,} Agilent Zorbax Eclipse™ C8 semi-preparative column (5 μm, 10 x 250 mm) at a flow rate of 3 ml min-1 was used , or a Phenomenex Jupiter ^TM C4 semi-preparative column (5 μm, 10 x 250 mm) at a flow rate of 2 ml min ^-1 , on an Agilent 1100 equipped with an autoinjector, fraction collector and UV detector (detected at 214 nm) Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed on a series system. All runs used a linear gradient of 0-100% solvent B over 40 minutes (solvent A = 5% acetonitrile, 0.1% trifluoroacetic acid (TFA) in water, solvent B = 5% water, 0.1% TFA in acetonitrile) implement. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectra were recorded on an ABI 4700 proteomics analyzer.

General method for solid-phase peptide synthesis (SPPS) : using N ^α -Fmoc protected amino acids and 2-(1H-benzotriazol-1-yl)-oxy-1,1,3,3-tetraethyl Hexafluorophosphate (HBTU)/1-hydroxybenzotriazole (HOBt; Knorr et al., 1989 Tetrahedron Lett. 30, 1927-1930) was used as an activation reagent by an established protocol on ABI 433A peptide equipped with a UV detector. Peptides were synthesized on a synthesizer (Applied Biosystems). Individual coupling steps are performed with conditional capping. The following protected amino acids were used: N ^o -Fmoc-Arg(Pbf)-OH, N ^α -Fmoc-Asp( O ^t Bu ) -OH, N ^α -Fmoc-Asp-Thr(Ψ ^Me,Me pro)- OH, N ^α -Fmoc-Ile-Thr(Ψ ^Me ,Mepro)-OH, N ^α -Fmoc-Lys(Boc)-OH, N ^α -Fmoc-Ser( ^t Bu)-OH, N ^α -Fmoc-Thr ( ^tBu )-OH, Nα - ^Fmoc -Tyr( ^tBu )-OH. Using O -(7-azabenzotriazol-1-yl) -N , N , N',N' -tetramethylurea Glycosylated amino acid Nα -FmocSer-(AcO3- ^α -D- O -GlcNAc) was performed manually using hexafluorophosphate (HATU)/1-hydroxy-7-azabenzotriazole (HOAt) as a coupling agent Coupling of OH, N ^α -FmocSer-(AcO3-α-D- S -GlcNAc)OH. Using benzotriazol-1-yl-oxy-tripyrrolidino-phosphorus Hexafluorophosphate (PyBOP)/HOBt was used as a coupling agent to perform N ^α -Fmoc- S -(2,3-bis(palmitoyloxy)-(2 R -propyl)-( R )-cysteine Coupling of acids (Metzger et al., 1991 Int. J. Pept. Protein Res. 38, 545-554; Roth et al., 2004 Bioconjugate Chem. 15, 541-553) prepared from (R) -glycidol. The progress of the manual coupling was monitored by the standard Kaiser test (Kaiser et al., 1970 Anal. Biochem. 34, 595).

Synthesis of lipopeptide 63: Synthesis of 63 was performed on H-Gly-sulfamoylbutyryl Novasyn TG resin as described in the General Methods section for peptide synthesis. After coupling of the first five amino acids, the remaining steps were performed manually. Dissolve N -α-Fmoc- S- (2,3-bis(palmitoyloxy)-( 2R -propyl)-( R )-cysteine (267 mg, 0.3 mmol) in DMF (5 mL ), PyBOP (156.12 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol) and DIPEA (67 μl, 0.4 mmol) were premixed for 2 minutes before adding to the resin. The coupling reaction was monitored by Kaiser test, and at It was completed after standing for 12 hours. After the coupling was completed, the N-Fmoc group was cleaved using 20% piperidine in DMF (6 mL), and the N- Fmoc group was cleaved using PyBOP (156.12 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol ) and DIPEA (67 μl, 0.4 mmol) in DMF to couple palmitic acid (77 mg, 0.3 mmol) to the free amine as described above. The resin was washed with DMF (10 ml), DCM (10 ml) and MeOH ( 10 ml) was washed thoroughly, followed by drying in vacuo. The resin was swelled in DCM (5 mL) for 1 hour and washed with DIPEA (0.5 ml, 3 mmol), iodoacetonitrile (0.36 ml, 5 mmol) in NMP (6 ml) Solution handling. It is important to note that iodoacetonitrile was filtered through a plug of basic alumina prior to addition to the resin. The resin was agitated in the dark for 24 hours, filtered and washed with NMP (5 ml x 4), DCM (5 ml x 4) and THF (5 ml x 4). Swell the activated N -acylsulfonamide resin in DCM (5 ml) for 1 hour, drain and transfer to a 50 ml round bottom flask. Add to the flask containing the resin THF (4 ml), benzylthiol (0.64 ml, 5 mmol) and sodium thiophenate (27 mg, 0.2 mmol). After stirring for 24 hours, the resin was filtered and washed with hexane (5 ml x 2 ) wash. The combined filtrate and washings were collected and concentrated in vacuo to about 1/3 of its original volume. The crude product was then precipitated by adding methyl tert-butyl ether (0°C; 60 mL) It was recovered by centrifugation at rpm for 15 minutes, and after ether decantation, the peptide precipitate was dissolved in the mixture DCM and MeOH (1.5 ml/1.5 ml).By passing it through the LH-20 size exclusion column, the peptide precipitate was removed. thiol impurities. Fractions containing product were collected and solvent removed to give the fully protected peptide thioester. The protected peptide was treated with reagent B (TFA 88%, phenol 5%, H2O 5%, TIS 2%; 5 ml) at room temperature for 4 hours. Then the TFA solution was added dropwise to a screw-cap centrifuge tube containing ice-cold methyl tert-butyl ether (40 ml), and the resulting The suspension was left standing overnight at 4°C, after which the precipitate was collected by centrifugation at 3000 rpm (20 min), and after decanting from ether, the peptide precipitate was resuspended in ice-cold methyl tert-butyl ether (40 ml) , the washing process was repeated twice. The crude peptide was purified by HPLC on a semi-preparative C-4 reverse phase column using a linear gradient of 0-100% solvent B in A over 40 minutes, and the appropriate fractions were lyophilized to provide 63 (110 mg, 65%). C ₉₀ H ₁₆₅ N ₁₁ O ₁₃ S ₂ , MALDI-ToF MS: Observed, [M+Na] 1695.2335 Da; Calculated, [M+Na] 1695.4714 Da ( FIG. 39 ).

Synthesis of Glycolipeptide 64: SPPS was performed on Rink amide resin (0.1 mmol) as described in the general procedure. The first four amino acids Ser-Ala-Asn-Met were coupled on a peptide synthesizer using standard protocols. After completion of the synthesis, use N α-FmocSer-(AcO ₃ -α-D- O -GlcNAc)OH (0.2 mmol, 131 mg) with O -(7-azabenzotriazol-1-yl) -N , N , N',N' -tetramethylurea Hexafluorophosphate (HATU; 0.2 mmol, 76 mg), 1-hydroxy-7-azabenzotriazole (HOAt; 0.2 mmol, 27 mg) and diisopropylethylamine (DIPEA; 0.4 mmol, 70 μl ) in NMP (5 ml), a manual coupling was performed for 12 hours. Coupling reactions were monitored by standard Kaiser tests. The resin was subsequently washed with NMP (6 ml) and dichloromethane (DCM; 6 ml) before being subjected to the same coupling conditions again to ensure completion of the coupling. Glycopeptides were then elongated on a peptide synthesizer, after which the resin was washed extensively with NMP (6 ml), DCM (6 ml) and MeOH (6 ml), dried in vacuo. The resin was swollen in DCM (5 ml) for 1 h, then treated with hydrazine (60%) in MeOH (10 ml) for 2 h, and treated with NMP (5 ml x 2), DCM (5 ml x 2) and MeOH (5 ml x 2) Wash thoroughly and dry in vacuum. The resin was swollen in DCM (5 ml) for 1 hour after which it was treated with reagent K (TFA (81.5%), phenol (5%), thioanisole (5%), water (5%), EDT (2.5 %), TIS (1%)) (30 ml) at room temperature for 2 hours. The resin was filtered, washed with neat TFA (2 ml). The filtrate was then concentrated in vacuo to about 1/3 of its original volume. Peptides were precipitated using diethyl ether (0°C) (30 ml) and recovered by centrifugation at 3000 rpm for 15 minutes. The crude peptide was purified by RP-HPLC on a semi-preparative C-8 column using a linear gradient of 0-100% solvent B in solvent A over a 40 minute period and the appropriate fractions were lyophilized to afford 64 (118 mg , 40%). _{C129H204N32O40S2} , MALDI-ToF MS: observed [M ₊ ], _2907.5916 Da; calculated [M+], _2905.4354 Da (Figure ₄₀ ).

Synthesis of Glycolipeptide 65: SPPS was performed on Rink amide resin (0.1 mmol) as described in the general procedure. The first four amino acids Ser-Ala-Asn-Met were coupled on a peptide synthesizer using standard protocols. After the synthesis was completed, use N α-FmocSer-(AcO ₃ -α-D- S -GlcNAc)OH (0.2 mmol, 134 mg) with O- (7-azabenzotriazol-1-yl) -N , N , N',N' -tetramethylurea Hexafluorophosphate (HATU; 0.2 mmol, 76 mg), 1-hydroxy-7-azabenzotriazole (HOAt; 0.2 mmol, 27 mg) and diisopropylethylamine (DIPEA; 0.4 mmol, 70 μl ) in NMP (5 ml), a manual coupling was performed for 12 hours. Coupling reactions were monitored by standard Kaiser tests. The resin was subsequently washed with NMP (6 ml) and dichloromethane (DCM; 6 ml) before being subjected to the same coupling conditions again to ensure complete coupling. The resulting glycopeptides are then elongated on a peptide synthesizer. After the synthesis was complete, the resin was washed well with NMP (6 ml), DCM (6 ml) and MeOH (6 ml) and dried in vacuo. The resin was swelled in DCM (5 ml) for 1 h, then treated with hydrazine (60%) in MeOH (10 ml) for 2 h, and treated with NMP (5 ml x 2), DCM (5 ml x 2) and MeOH ( 5 ml x 2) Wash well and dry in vacuum. The resin was swelled in DCM (5 ml) for 1 hour after which it was treated with TFA (81.5%), phenol (5%), thioanisole (5%), water (5%), EDT (2.5%), TIS (1%) (30 ml) was treated at room temperature for 2 hours. The resin was filtered and washed with neat TFA (2 ml). The filtrate was then concentrated in vacuo to about 1/3 of its original volume. Peptides were precipitated using diethyl ether (30 ml , 0° C.) and recovered by centrifugation at 3000 rpm for 15 minutes. The crude peptide was purified by RP-HPLC on a semi-preparative C-8 column using a linear gradient of 0-100% solvent B in solvent A over a 40 minute period and the appropriate fractions were lyophilized to provide 65 (95 mg , 34%). _{C129H204N32O39S3} , MALDI _- ToF MS: observed [M+], _2923.6716 Da; calculated [M+], _2923.3861 Da (Figure ₄₁ ).

Synthesis of glycolipeptide 52: lipopeptide thioester 63 (4.3 mg, 2.5 μmol), glycopeptide 64 (5.0 mg, 1.7 μmol) and dodecylphosphocholine (6.0 mg, 17.0 μmol) were dissolved in trifluoro In a mixture of ethanol and CHCl ₃ (2.5 ml/2.5 ml). The solvent was removed under reduced pressure to give lipid/peptide films in the presence of tris(carboxyethyl)phosphine (2% w/v, 40.0 μg) and EDTA (0.1% w/v, 20.0 μg) , using 200 mM phosphate buffer (pH 7.5, 3 ml), the lipid/peptide film was hydrated at 37°C for 4 hours. The mixture was sonicated for 1 min. Sodium 2-mercaptoethanesulfonate (2% w/v, 40.0 μg) was added to the vesicle suspension to initiate the ligation reaction. The reaction was performed at 37°C in an incubator, and the progress of the reaction was regularly monitored by MALDI-ToF, which showed that glycopeptide 64 disappeared within 2 hours. The reaction was then diluted with 2-mercaptoethanol (20%) in ligation buffer (2 ml) and run through a semi-preparative C-4 reaction using a linear gradient of 0-100% solvent B in A over 40 minutes. The crude peptide was column purified and lyophilization of the appropriate fractions provided 52 (4.3 mg, 57%). _{C212H360N43O53S3} , MALDI _{- ToF} MS: observed, _4461.9177 Da, calculated, _4455.578 Da (Figure 37).

Synthesis of glycolipeptide 53: lipopeptide thioester 63 (2.5 mg, 1.5 μmol), glycopeptide 65 (3.0 mg, 1.0 μmol) and dodecylphosphocholine (3.5 mg, 10 μmol) were dissolved in trifluoro In a mixture of ethanol and CHCl ₃ (2.5 ml/2.5 ml). The solvent was removed under reduced pressure to give lipid/peptide films in the presence of tris(carboxyethyl)phosphine (2% w/v, 40.0 μg) and EDTA (0.1% w/v, 20.0 μg) , using 200 mM phosphate buffer (pH 7.5, 2 ml), the lipid/peptide film was hydrated at 37° C. for 4 hours. The mixture was sonicated for 1 min. Sodium 2-mercaptoethanesulfonate (2% w/v, 40.0 μg) was added to the vesicle suspension to initiate the ligation reaction. The reaction was performed at 37°C in an incubator, and the progress of the reaction was monitored regularly by MALDI-ToF, which showed that the glycopeptide disappeared within 2 hours. The reaction was then diluted with 2-mercaptoethanol (20%) in ligation buffer (2ml). The crude peptide was purified by a semi-preparative C-4 reverse phase column using a linear gradient of 0-100% solvent B in A over 40 minutes, and lyophilization of the appropriate fractions provided 53 (2.8 mg, 64%). _{C212H360N43O52S4} , MALDI _{- ToF} MS: observed, _4469.9112 Da, calculated, _4471.6437 Da (Figure 38).

Compounds 66-70 were prepared on Rink amide resin (0.1 mmol) as described in the standard procedures section. Glycopeptide 66 (78 mg, 61 %); C ₄₈ H ₈₂ N ₁₄ O ₂₁ S ₂ , MALDI-ToF MS: Observed [M+Na], 1277.4746 Da; Calculated [M+Na], 1277.5220 Da ( Figure 43). Peptide 67 (89 mg, 83%); C ₄₀ H ₆₉ N ₁₃ O ₁₆ S ₂ , MALDI-ToF MS: Observed [M+Na], 1074.4789 Da; Calculated [M+Na], 1074.4427 Da (Fig. 44). Glycopeptide 68 (57 mg, 48%); C ₄₅ H ₇₇ N ₁₃ O ₂₀ S, MALDI-ToF MS: Observed [M+Na], 1174.4740 Da; Calculated [M+Na], 1174.5129 Da (Fig. 45). Peptide 69 (76 mg, 78%). _{C37H64N12O15S} , MALDI- _ToF MS: observed [M+Na], _969.8162 Da; calculated [M+Na], _970.8657 Da (Figure 46). Glycosylated amino acid 70 (12 mg, 33%), C ₁₄ H ₂₅ N ₃ O ₈ , MALDI-ToFMS: observed [M+Na], 386.2749 Da; calculated [M+Na] 386.3636 Da (Figure 46 ).

General procedure for conjugation to BSA-MI: Conjugation was performed following Pierce Endogen Inc directions. Briefly, purified (glyco)peptide 66 or 67 (2.5 equivalent excess to available MI groups on BSA) was dissolved in conjugation buffer (sodium phosphate containing EDTA and sodium azide, pH 7.2; 100 μl), and a solution of maleimide-activated BSA (2.4 mg) in conjugation buffer (200 μl) was added. The mixture was incubated at room temperature for 2 hours, then purified by D-Salt™ dextran desalting column (Pierce Endogen, Inc.), equilibrated and eluted with sodium phosphate buffer (pH 7.4) containing 0.15 M sodium chloride. Fractions containing the conjugate were identified using the BCA protein assay. Carbohydrate content was determined by quantitative monosaccharide analysis via HPAEC/PAD.

General procedure for the preparation of liposomes . Egg PC, egg PG, cholesterol, MPL-A, and compound 52 or 53 (15 μmol, molar ratio 60:25:50:5:10) were dissolved in trifluoroethanol and MeOH (1:1, v/v, 5 ml) in the mixture. The solvent was removed in vacuo to yield a thin lipid film which was hydrated by suspending in HEPES buffer (10 mM, pH 6.5) containing NaCl (145 mM; 1 ml) for 3 hours at 41 °C under an argon atmosphere. The vesicle suspension was sonicated for 1 min and then extruded sequentially through 1.0, 0.6, 0.4, 0.2 and 0.1 μm polycarbonate membranes (Whatman, Nuclepore Track-EtchMembrane) at 50°C to obtain SUVs. The sugar content of liposomes was determined by heating a mixture of SUV (50 μL) and aqueous TFA (2 M, 200 μL) in a sealed tube at 100 °C for 4 h. The solution was then concentrated in vacuo and analyzed by high pH anion exchange chromatography using a pulsed amperometric detector (HPAEC-PAD; Methrome) and CarboPac columns PA-10 and PA-20 (Dionex).

Dosage and immunization schedule: Groups of five mice (female BALB/c, age 8-10 weeks, from Jackson Laboratories) were immunized four times at two-week intervals. Each booster included 3 µg of sugar in the liposomal formulation. Serum samples were obtained before immunization (pre-bleeding) and 1 week after the last immunization. The final phlebotomy is done by exsanguination from the heart.

Hybridoma culture and antibody production : Spleens from two mice immunized with 52 were harvested, and standard hybridoma culture techniques gave 30 IgG-producing hybridoma lines. Three hybridomas (18B10.C7(3), 9D1.E4(10), 1F5.D6(14)) were cultured on a one-liter scale, and the resulting samples were purified by saturated ammonium sulfate precipitation followed by protein G column chromatography. Antibodies, to obtain about 10 mg of IgG in each case.

Reagents for biological experiments : Protease inhibitor cocktail was obtained from Roche (Indianapolis, IN). PUGNAc O-(2-acetamide-2-deoxy-D-glucopyranosylidene)amino N-phenylcarbamate was purchased from Toronto Research Chemicals, Inc (Ontario, Canada). Mouse IgM anti-O-GlcNAc (CTD110.6; Comer et al., 2001 Anal. Biochem. 293, 169-177) and rabbit polyclonal anti-OGT (AL28) antibodies were previously obtained in the laboratory of Dr. Gerald W. Hart (Johns Hopkins University School of Medicine, Baltimore, MD). Rabbit polyclonal anti-OGA antibody was a kind gift from Dr. Sidney W. Whiteheart (University of Kentucky College of Medicine). Rabbit polyclonal anti-CKII alpha antibodies (NB100-377 for immunoblotting and NB100-378 for immunoprecipitation) were purchased from Novus Biologicals (Littleton, CO). Mouse monoclonal antibody against α-tubulin and anti-mouse IgM (μ chain)-Sepharose were obtained from Sigma (St. Louis, Missouri). Normal Rabbit IgG Sepharose, Normal Rabbit IgG Sepharose and Protein A/G PLUS Sepharose were ordered from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Serological assays: by ELISA as previously described (Buskas and Boons, 2004 Chem. Eur. J. 10, 3517-3524; Ingale et al., 2007 Nat. Chem. Biol. 3, 663-66) (ELISA) Determination of anti-GSTPVS (β- O -GlcNAc) SANM (68) IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM antibody titers. Briefly, Immulon II-HB flat-bottom 96-well microtiter plates ( Thermo Electron Corp.) with 100 μl/well of a glycopeptide conjugate conjugated to BSA via a maleimide linker (BSA-MI-GSTPVS(β- O- GlcNAc) SANM; BSA-MI-66) in Coating overnight at 4°C. Serial dilutions of serum or MAb-containing cell supernatants were allowed to bind to immobilized GSTPVS(β- O -GlcNAc) SANM for 2 hours at room temperature. By adding alkaline phosphatase-conjugated anti-mouse IgG (JacksonImmunoResearch Laboratories Inc.), IgG1 (Zymed), IgG2a (Zymed), IgG2b (Zymed), IgG3 (BD Biosciences Pharmingen), or IgM (Jacksons ImmunoResearch Laboratories) antibodies Complete the test. After addition of p-nitrophenyl phosphate (Sigma), absorbance was measured at 405 nm using a microplate reader (BMG Labtech) with wavelength correction set at 490 nm. Antibody titer was defined as the highest dilution to obtain a relative optical density of 0.1 or greater.

To investigate the competitive inhibition of MAb binding to GSTPVS(β- O -GlcNAc)SANM (68) by the corresponding glycopeptides, peptides and sugars, MAbs were diluted in dilution buffer in such a way that if In the case of inhibitors, the expected final OD value is about 1. For each well, mix 60 µl of diluted MAb in an uncoated microtiter plate with 60 µl of dilution buffer, Glycopeptide 68 (GSTPVS(β- O -GlcNAc)SANM), peptide 69 (GSTPVSSANM; SEQ ID NO: 11) or sugar 70 (β- O- GlcNAc-Ser) mixed. After 30 minutes of incubation at room temperature, 100 µl of the mixture was transferred to a plate coated with BSA-MICGSTPVS(β- O -GlcNAc)SANM (BSA-MI-66). Microtiter plates were incubated and developed as described above using the appropriate alkaline phosphatase-conjugated detection antibody.

Plasmid construction : Human OGT and OGA cDNAs were PCR amplified in two steps to introduce an attB1 site and an HA epitope at the 5' end and an attB2 site at the 3' end to facilitate the Gateway cloning strategy (Invitrogen , Carlsbad, CA). Primers included (1) a sense primer for the first PCR to incorporate the HA epitope into the ogt after the start codon: 5'-CCCCATGTATCCATATGACGTCCCAGACTATGCCGCGTCTTCCGTGGGCAACGT-3' (SEQ ID NO: 13); (2) using HA - ogt PCR product as template with sense primer containing attB1 site: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGATGTATCCATATGACGTCCCAGACTATGCCGCGTCTTCCG-3' (SEQ ID NO: 14); (3) antisense with 3' attB2 site for both ogt PCRs Primer: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTCTATGCTGACTCAGTGACTTCAACGGGCTTAATCATGTGG-3' (SEQ ID NO: 15); (4) Sense primer for the first PCR to incorporate the HA epitope into oga after the start codon: 5'-CCCCATGTATCCATATGACGTCCCAGACTATGCCGTGCAGAAGGAGAGTCAAGC- 3' (SEQ ID NO: 16); (5) A sense primer containing an attB1 site using HA-oga PCR product as a template: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGATGTATCCATATGACGTCCCAGACTATGCCGTGCAGAAGG-3' (SEQ ID NO: 17); (6) with Antisense primer with 3' attB2 site for two oga PCRs: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTCACAGGCTCCGACCAAGTAT-3' (SEQ ID NO: 18). Purified DNA fragments were then subjected to entry cloning according to the manufacturer's instructions to obtain the final expression constructs pDEST26/HA-OGT and pDEST26/HA-OGA.

Cell culture, transfection and treatment : HEK 293T cells were obtained from ATCC (Manassas, VA) and maintained in a 37°C incubator humidified with 5% CO ₂ supplemented with 10% fetal bovine serum (GIBCO/Invitrogen, Carlsbad, CA) in Dulbecco's Modified Eagle's Medium (4.5 g l-1 glucose, Cellgro/Mediatech, Inc., Herndon, VA). Transfections were performed with 8 μg of DNA and Lipofectamine 2000 reagent (Invitrogen Carlsbad, CA) per 10 cm cell plate according to the manufacturer's instructions. Mock transfection was performed in the absence of DNA. Cells were harvested 48 hours after transfection. For immunoprecipitation experiments, plated cells were washed with ice-cold PBS and stored as pellets at -80°C until use. For immunoblotting experiments, cells were washed twice with ice-cold PBS and scraped into lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 0.1% SDS, 4 mM EDTA, 1 mM DTT, 0.1 mM PUGNAc, protease inhibitor cocktail), the lysate was clarified in a microfuge at 16,000 g for 25 min at 4°C. Protein concentrations were quantified using the Bradford protein assay with standard procedures (Bio-Rad, Hercules, CA) and boiling in sample buffer for 5 minutes. For mass spectrometry experiments, 2 X 15 cm plates of 293T cells were treated with 50 μM PUGNAc for 24 hours, cells were pelleted and stored as above.

Immunoprecipitation and Western blotting : To prepare nucleocytosolic fractions for CKII immunoprecipitation, HEK293T cell pellets with mock or OGT transfection were resuspended in 4 volumes of hypotonic buffer (5 mM Tris- HCl, pH 7.5, Protease Inhibitor Cocktail) and transferred to a 2 ml homogenizer. After a 10 min incubation on ice, Dawns homogenization was performed on the cell suspension, followed by an additional 5 min incubation on ice. A volume of hypertonic buffer (0.1 M Tris-HCl, pH 7.5, 2 M NaCl, 5 mM EDTA, 5 mM DTT, protease inhibitor cocktail) was then added to the lysate. Lysates were incubated on ice for 5 min, followed by another round of Dawns homogenization. The resulting lysate was transferred to a microfuge tube containing PUGNAc (final concentration 10 μΜ) and centrifuged at 18,000 g for 25 minutes at 4°C. Protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA). Before IP, lysates were supplemented with 1% Igepal CA-630 and 0.1% SDS, and pre-cleared with a mixture of normal rabbit or mouse IgG AC and protein A/G PLUS agarose at 4°C for 30 minutes. After clarification, the pre-cleared supernatant was incubated at 4 °C for 4 in the presence of the antibody of interest. After adding protein A/G PLUS agarose, samples were incubated at 4°C for another 2 hours and washed with IP wash buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 0.1 % SDS) wash extensively. Finally, SDS PAGE sample buffer was added to the IP complex and boiled for 3 minutes. Supernatants were resolved by 10% or 4-15% Tris-HCl precast mini-gels (Bio-Rad, Hercules, CA) and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). Membranes were blocked with 3% BSA ( O -GlcNAc blotting) or 5% milk (Western blotting) in TBST (TBS with 0.1% Tween 20) and then diluted 1:1000 with each antibody (for O -GlcNAc blotting) 1:8000 dilution for CKII, OGT, and OGA blots, and 1:10,000 dilution for α-tubulin blots) probed at 4°C overnight, followed by secondary antibody conjugated to HRP at room temperature Incubate together for 2 hours. Final detection of HRP activity was performed using SuperSignal chemiluminescent substrates (Thermo Scientific, Rockford, IL) as follows: MAb 18B10.C7(3), 9D1.E4(10) and 1F5.D6(14) using Femto; CKII, OGT, OGA and tubulin use PICO. Films were exposed to CL-XPosure film (Thermo Scientific, Rockford, IL). After developing the image on the film, the blot was stripped with 0.1 M glycine (pH 2.5) for 1 hour at room temperature, washed with ddH2O and reprobed for loading controls (CKII or α-tubulin) as described above.

Conjugation of MAb to agarose and sample preparation for LC-MS/MS analysis: MAb 18B10.C7 was prepared via a disuccinimide substrate (DSS, Thermo Scientific, Rockford, IL) according to the manufacturer's instructions. (3), 9D1.E4(10) and 1F5.D6(14) or CTD110.6 were covalently conjugated to protein A/G PULS agarose or anti-mouse IgM agarose. PUGNAc-treated HEK293T nucleocytoplasmic fractions were prepared on a larger scale as above, incubated with antibody-conjugated agarose, and washed as above. For elution of proteins from agarose, 0.1 M glycine (pH 2.5) was added and the eluate was immediately neutralized with 1 M Tris-HCl (pH 8.0). Samples were then reduced and alkylated as previously described, and LysC digestion was performed overnight at 37°C. After digestion, samples were processed as previously described (Lim et al., 2008 J. Proteome Res. 7, 1251-1263).

Mass Spectrometry : Samples were resuspended with 19.5 μl 0.1% formic acid (in water) and 0.5 μl 80% acetonitrile/0.1% formic acid (in water) and filtered with 0.2 μm filter (Nanosep, PALL). The samples were then loaded offline onto a nanospray C18 column using a Finnigan LTQ/XL mass spectrometer (ThermoFisher, San Jose, CA) as previously described (Lim et al., 2008 J. Proteome Res. 7, 1251-1263) with 160 minute linear gradient separation. 3 runs with different settings were performed for each sample: (1) ETD (electron transfer dissociation) mode, where full MS spectra were collected, followed by ETD at the most intense peak (enabled supplemental activation) 6 MS/MS spectra after. Dynamic exclusions are set to 1 for a duration of 30 seconds. (2) CID-NL (Collision-Induced Dissociation-Pseudo-Neutral Loss) mode, where a full MS spectrum is collected followed by 8 MS/MS spectra after the CID of the strongest peak. MS8 spectra were generated based on MS/MS spectra after encountering a false neutral loss event (loss of GlcNAc, 203.08). Dynamic exclusion has the same settings as the ETD method. (3) DDNL-ETD (data-dependent neutral loss MS8 under CID, followed by ETD activation after each neutral loss event), where data from each complete MS is collected with CID (35% normalized collision energy) The MS/MS spectrum of the top 5 peaks was scanned and monitored for neutral loss at 203.08, generating an MS8 spectrum in the process. Repeat scan events with neutral loss were performed using ETD activated by complement activation. Dynamic exclusions are also set in the same way as above.

Data analysis and verification : using the TurboSequest algorithm (Bioworks 3.3, Thermo Finnigan), the human (Homo sapiens ( Homo sapiens ), 32876 entries, published on August 13, 2007) extracted from the Swiss-Prot human proteome database was positively analyzed. Searches MS spectra to and from databases. DTA files were generated with a threshold of 15 ions and a TIC pair spectrum of 1e3. Dynamic mass gains of 15.99, 57.02 and 203.08 Da were considered for oxidized methionine, alkylated cysteine and O -GlcNAc modified serine/threonine, respectively. The resulting OUT files for each sample obtained by searching the forward and reverse databases were further parsed with ProtoeIQ (Bioinquire) and filtered with a starting peptide coverage of 1% FDR (metric used: F-value) and a ProFDR at 5.

Statistical Analysis : Statistical significance between groups was determined by two-tailed, unpaired Student's t-test. Differences were considered significant when P < 0.05.

Full disclosure and electronically available material of all patents, patent applications, and publications cited herein (including, for example, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequences in, e.g., SwissProt, PIR, PRF, PDB Sequence submissions, and translations of annotated coding regions from GenBank and RefSeq) are all incorporated by reference. The foregoing description and examples have been given for clarity of understanding only. It should not be construed as unnecessary limitations. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be encompassed within the invention defined by the claims.

Claims (37)

1. Use of a glycolipopeptide in the preparation of a drug for generating antibody-dependent cell-mediated cytotoxicity (ADCC) in a subject, the glycolipopeptide comprising: at least one glycosylated MUC1 glycopeptide component comprising a B-cell epitope; at least one peptide component comprising an MHC class II restricted helper T cell epitope; and at least one lipid component. 2. The use of claim 1, wherein the ADCC is natural killer (NK) cell mediated. 3. The use according to claim 1 or 2, wherein the ADCC lyses tumor cells. 4. The use of claim 3, wherein the tumor cells are breast cancer cells. 5. The use of claim 1, wherein the ADCC lyses cells expressing the MUCl peptide sequence. 6. A glycolipopeptide comprising: at least one glycosylated MUC1 glycopeptide component comprising a B-cell epitope; at least one peptide component comprising a MUCl-derived MHC class II-restricted helper T-cell epitope; and at least one lipid component. 7. The glycolipopeptide of claim 6, wherein said glycosylated MUCl glycopeptide component comprising a B-cell epitope comprises glycosylation on one or more serine and/or threonine residues. 8. The glycolipopeptide of claim 7, wherein the glycosylated MUC1 glycopeptide component comprising a B-cell epitope comprises glycosylation with a sugar residue selected from the group consisting of: GAlNAc, GlcNAc, Gal, NANA , NGNA, fucose, mannose and glucose. 9. The glycolipopeptide of any one of claims 6-8, wherein the glycosylated MUCl glycopeptide component comprising a B cell epitope is an MHC class I restricted epitope. 10. The glycolipopeptide of claim 6, wherein said glycosylated MUC1 glycopeptide component comprising a B cell epitope and/or said peptide component comprising an MHC class II restricted helper T cell epitope comprises human MUC1 peptide sequence. 11. The glycolipopeptide of claim 6, wherein said glycolipopeptide comprising a B cell epitope and/or said peptide component comprising an MHC class II restricted helper T cell epitope comprises about 5- 30 amino acids, the MUCl protein sequence includes the extracellular region of the MUCl protein, and includes one or more glycosylated serine or threonine residues. 12. The glycolipopeptide of claim 6, wherein the glycosylated MUC1 glycopeptide component comprising a B cell epitope comprises a combination with SAPDTRPAP (SEQ ID NO:20), TSAPDTRAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO: 22) or TSAPDTRPL (SEQ ID NO: 23) amino acid sequences having at least about 50% sequence identity. 13. The glycolipopeptide of claim 6, wherein the glycosylated MUC1 glycopeptide component comprising a B cell epitope comprises SAPDTRPAP (SEQ ID NO:20), TSAPDTRAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO:22) or TSAPDTRPL (SEQ ID NO:23). 14. The glycolipopeptide of claim 12 or 13, wherein the glycosylated MUCl glycopeptide component comprising a B cell epitope comprises glycosylation on one or more serine and/or threonine residues. 15. The glycolipopeptide of any one of claims 6-14, wherein the lipid component comprises one or more lipid chains, one or more cysteine residues, and one or more lysine Residues. 16. The glycolipopeptide of any one of claims 6-15, wherein the lipid component comprises a Toll-like receptor (TLR) ligand. 17. The glycolipopeptide of claim 16, wherein the Toll-like receptor (TLR) ligand comprises a TLR2 ligand. 18. The glycolipopeptide of claim 17, wherein the TLR2 ligand comprises _{Pam3CysSK4 .} 19. The glycolipopeptide of any one of claims 6-15, wherein the lipid component comprises a lipid adjuvant. 20. The glycolipopeptide of claim 19, wherein the lipid adjuvant comprises lipidated amino acids (LAA). 21. The glycolipopeptide of claim 6, wherein said MUCl-derived B-cell peptide epitope and said MUCl-derived MHC class II-restricted helper T-cell peptide epitope comprise contiguous amino acid sequences. 22. The glycolipopeptide of claim 21 , wherein the contiguous amino acid sequence comprises the amino acid sequence APGSTAPPAHGVTSA (SEQ ID NO:26), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) ID NO:29) sequences having at least 50% sequence identity. 23. The glycolipopeptide of claim 21, wherein the contiguous amino acid sequence comprises the amino acid sequence APGSTAPPAHGVTSA (SEQ ID NO:26), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) NO: 29). 24. The glycolipopeptide of claim 22 or 23, wherein the contiguous amino acid sequence is glycosylated at one or more threonine and/or serine residues. 25. The glycolipopeptide of any one of claims 6-24, further comprising a covalently linked immunomodulator. 26. The glycolipopeptide of claim 25, wherein the immunomodulator is selected from the group consisting of TLR9 agonists, COX-2 inhibitors, GM-CSF, inhibitors of indoleamine 2,3-dioxygenase (IDO), Chemotherapeutic agents and combinations thereof. 27. A pharmaceutical composition comprising: A glycolipopeptide according to any one of claims 6-26; and pharmaceutically acceptable carrier. 28. A composition comprising liposomes comprising a glycolipopeptide according to any one of claims 6-26. 29. The composition of claim 28, wherein the lipid component of the glycolipopeptide promotes liposome formation. 30. The composition of claim 27 or 28, further comprising an immunomodulator. 31. The composition of claim 30, wherein said immunomodulator comprises a TLR agonist. 32. The composition of claim 31, wherein the TLR agonist comprises a TLR9 agonist. 33. The composition of claim 32, wherein the TLR9 agonist comprises CpG. 34. The composition of claim 30, wherein the immunomodulator is selected from the group consisting of TLR9 agonists, COX-2 inhibitors, GM-CSF, inhibitors of indoleamine 2,3-dioxygenase (IDO), chemical Therapeutic agents and combinations thereof. 35. An immunogenic vaccine comprising a glycopeptide according to any one of claims 6-26 or a composition according to any one of claims 27-34. 36. Use of the glycolipopeptide of any one of claims 6-26 or the composition of any one of claims 27-35 for the manufacture of a medicament for the treatment or prevention of an infection, disease or condition. 37. A kit comprising: The glycolipopeptide of any one of claims 6-26; packaging; and user's manual.