CN106215179A - Immunogenic vaccine - Google Patents

Abstract

The invention discloses be used as treatment or vaccine comprise carbohydrate ingredient, lipid composition and the glycolipidpeptide of MUC1 peptide composition, its induction humoral immunoresponse(HI) and cellullar immunologic response.

Description

Immunogenic vaccine

The application is filing date on June 10th, 2011, Application No. 201180039067.0, invention entitled " immunity Originality vaccine " the divisional application of patent of invention.This application claims in the U.S. Provisional Application sequence that on June 11st, 2010 submits to Number 61/354,076 and the interests of U.S. Patent Application Serial Number 13/002,180 submitted to for 30th in December in 2010, described specially Profit is integrally incorporated herein with it each via quoting.

Government rights statement

The present invention is at the National Cancer by NIH (National Institutes of Health) Carried out by governmental support under the subsidy R01 CA088986 that institute (National Cancer Institute) is authorized.Beautiful Government of state has certain right in the present invention.

Background

Massive tumor related carbohydrate antigen (TACA) is expressed on human cancer cell with the form of glycolipid and glycoprotein.Carcinogenic The common trait converting cell is oligosaccharide such as Globo-H, Lewis^YProcess LAN with Tn antigen.Numerous researchs have shown this Aberrant glycosylation can promote transfer, and therefore its weak survival rate strong association with cancer patient.

Differential expression as the feature of these tumor related carbohydrate antigens causes to become for immunotherapy Attractive target with cancer vaccine exploitation.Recently, several first-class researchs are attempted utilizing tumor related carbohydrate Differential expression is used for developing cancer vaccine (such as Raghupathi, 1996, Cancer Immunol；43:152-157； Musselli et al., 2001, J Cancer Res Clin Oncol；127:R20-R26；Sabbatini et al., 2000, Int J Cancer；87:79-85；Lo-Man et al., 2004, Cancer Res；64:4987-4994；Kagan et al., 2005, Immunol Immunother；54:424-430).

Carbohydrate Antigens is at the one-tenth of human immunodeficiency virus (HIV) acquired immune deficiency syndrome (AIDS) (AIDS) Because being also abundant on the surface of agent.Hepatitis C virus (HCV) is it is also known that contain Carbohydrate Antigens.

For most of immunogens (including carbohydrate), antibody produces and depends on that the lymphocyte B of two types is thin Born of the same parents and the cooperative interaction of helper T cell.But, carbohydrate individually can not activate helper T cell, and therefore feature It is weak immunogenicity.Formation and not the existing of IgG antibody of low-affinity IgM antibody embody this limited immunogenicity. It is proved to be difficult to overcome the immunologic tolerance characterizing these antigens.

In the effort activating helper T cell, researcher has made Carbohydrate Antigens put together with exogenous carrier proteins matter, Described exogenous carrier proteins matter such as keyhole limpet hemocyanin (KLH) or detoxification tetanus toxoid (TT).Carrier protein strengthens Carbohydrate is presented for immune, and supply can activate T epi-position (generally 12-15 the amino of t helper cell The fragments of peptides of acid).

But, carbohydrate with puting together of carrier protein there is several new problem.Conjugation chemistry is difficult to control to, and causes Produce the conjugate of the ambiquity in terms of the Nomenclature Composition and Structure of Complexes with the repeatability that can affect immunne response.Additionally, external source Carrier protein can cause strong B cell response, and it can cause the most again the antibody response for carbohydrate epitope Suppression.When using autoantigen such as tumor related carbohydrate, the latter is a problem especially.Additionally, be used for making carbon aquation Compound can be immunogenic with self with the joint of protein-conjugate, causes epi-position to suppress.About in vaccine based on lipid With the summary of the adjuvant/carrier of carbohydrate, referring further to McGeary et al. (J. Peptide Sci. 9 (7): 405-418, 2003)。

Not surprisingly, fail with several clinical trials of carbohydrate-protein conjugate cancer vaccine All patients induce sufficiently strong helper T cell response.It is used for presenting tumor associated carbon accordingly, it would be desirable to develop alternative strategy Hydrate epi-position, it is more effectively changed causing to the classification of IgG antibody.These strategies may certify that for based on other The vaccine development of carbohydrate epitope (especially from those of Causative virus such as HIV and HCV) is also useful.

Summary of the invention

The method that present invention resides in the cytotoxicity (ADCC) generating antibody dependent cellular mediation in experimenter, the method bag Including and use glycolipidpeptide (glycolipopeptide) immunizing subjects, described glycolipidpeptide includes: include B cell epi-position at least A kind of glycosylation MUC1 glycopeptide component；At least one peptide composition including the MHC restricted helper T cell epitope of II class；At least A kind of lipid composition.In some respects, ADCC is that NK cell (NK) is cell-mediated.In some respects, ADCC cracks tumor Cell.In some respects, tumor cell is breast cancer cell or epithelial cancer cells.In some respects, MUC1 is expressed in ADCC cracking The cell of peptide sequence.In some respects, MUC1 peptide is Aberrant glycosylation.

The present invention includes the method treating the cancer in experimenter, and the method includes using glycolipidpeptide immunizing subjects, Described glycolipidpeptide includes: include at least one glycosylation MUC1 glycopeptide component of B cell epi-position；Restricted auxiliary including MHC II class Help at least one peptide composition of t cell epitope；With at least one lipid composition.

The present invention includes the method reducing the tumor load in experimenter, and the method includes by glycolipidpeptide immunity inoculation tested Person, described glycolipidpeptide includes: include at least one glycosylation MUC1 glycopeptide component of B cell epi-position；Limit including MHC II class At least one peptide composition of property helper T cell epitope；With at least one lipid composition.

The present invention includes the method preventing the tumor recurrence in experimenter, and the method includes by glycolipidpeptide immunity inoculation tested Person, described glycolipidpeptide includes: include at least one glycosylation MUC1 glycopeptide component of B cell epi-position；Limit including MHC II class At least one peptide composition of property helper T cell epitope；With at least one lipid composition.

The present invention includes the method preventing the cancer in experimenter, and the method includes using glycolipidpeptide immunizing subjects, Described glycolipidpeptide includes: include at least one glycosylation MUC1 glycopeptide component of B cell epi-position；Restricted auxiliary including MHC II class Help at least one peptide composition of t cell epitope；With at least one lipid composition.

At some aspects of the method for the present invention, cancer or tumor are breast carcinoma or epithelial cancer.

At some aspects of the method for the present invention, cancer or the glycosylated MUC1 of tumor abnormal expression.

Present invention resides in the method generating the cytotoxic T cell response for MUC1 express cell in experimenter, should Method includes using glycolipidpeptide immunizing subjects, described glycolipidpeptide to include: include at least one glycosylation of B cell epi-position MUC1 glycopeptide component；At least one peptide composition including the MHC restricted helper T cell epitope of II class；With at least one iipidomic Point.In some respects, MUC1 express cell is tumor cell.

The present invention includes the method promoting the anti-MUC1 antibody isotype conversion in experimenter, and the method includes using glycolipidpeptide Immunizing subjects, described glycolipidpeptide includes: include at least one glycosylation MUC1 glycopeptide component of B cell epi-position；Including At least one peptide composition of the restricted helper T cell epitope of MHC II class；With at least one lipid composition.

The present invention includes the method for immunizing subjects, and the method includes using glycolipidpeptide immunizing subjects, described Glycolipidpeptide includes: include at least one glycosylation MUC1 glycopeptide component of B cell epi-position；Including MHC II class restricted auxiliary T At least one peptide composition of cell epitope；With at least one lipid composition；Wherein in experimenter, inducing specific is combined in swollen The IgG subclass antibodies of the MUC1 protein expressed on oncocyte.

At some aspects of the method for the present invention, the glycosylation MUC1 glycopeptide component including B cell epi-position is included in one Or the glycosylation in multiple serine and/or threonine residues.

At some aspects of the method for the present invention, the glycosylation MUC1 glycopeptide component including B cell epi-position includes with being selected from The glycosylation that following saccharide residue is carried out: GalNAc, GlcNAc, Gal, NANA, NGNA, fucose, mannose and glucose.

At some aspects of the method for the present invention, glycolipidpeptide includes one of those shown in Figure 19.

At some aspects of the method for the present invention, the glycosylation MUC1 glycopeptide component including B cell epi-position is I class MHC limit Property epi-position processed.

At some aspects of the method for the present invention, including the glycosylation MUC1 glycopeptide component of B cell epi-position and/or include The peptide composition of the restricted helper T cell epitope of MHC II class includes people's MUC1 peptide sequence.

At some aspects of the method for the present invention, including the glycosylation MUC1 glycopeptide component of B cell epi-position and/or include The peptide composition of the restricted helper T cell epitope of MHC II class includes the aminoacid sequence of the endogenous MUC1 sequence homology with experimenter Row.

At some aspects of the method for the present invention, including the glycosylation MUC1 glycopeptide component of B cell epi-position and/or include The peptide composition of the restricted helper T cell epitope of MHC II class includes about 5 30 aminoacid of MUC1 protein sequence, described MUC1 protein sequence includes the extracellular region of MUC1 protein, and includes that glycosylated one or more serine or threonine are residual Base.

At some aspects of the method for the present invention, the MUC1 glycopeptide component including B cell peptide epitopes includes and SAPDTRPAP (SEQ ID NO:20), TSAPDTRPAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO:22) or TSAPDTRPL (SEQ ID NO:23) has the aminoacid sequence of at least about 50% sequence iden.In some respects, aminoacid sequence is included in Glycosylation on one or more serines and/or threonine residues.At some aspects of the method for the present invention, including B cell The MUC1 glycopeptide component of peptide epitopes include SAPDTRPAP (SEQ ID NO:20), TSAPDTRPAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO:22) or TSAPDTRPL (SEQ ID NO:23).In some respects, aminoacid sequence is included in Glycosylation on one or more serines and/or threonine residues.

At some aspects of the method for the present invention, lipid composition includes one or more lipid chain, one or more half Guang Histidine residue and one or more lysine residue.

At some aspects of the method for the present invention, lipid composition includes Toll-like receptor (TLR) part.In some respects, Toll-like receptor (TLR) part includes TLR2 part.In some respects, TLR2 part includes Pam3CysSK4.

At some aspects of the method for the present invention, lipid composition includes TLR9 agonist Pam3CysSK4.The present invention's Some aspects of method, lipid composition includes lipid adjuvant.In some respects, lipid adjuvant includes lipidated amino acid (LAA).

At some aspects of the method for the present invention, the peptide composition including the MHC restricted helper T cell epitope of II class includes Poliovirus sequence KLFAVWKITYKDT (SEQ ID NO:3).

At some aspects of the method for the present invention, the peptide composition including the MHC restricted helper T cell epitope of II class includes T Cell general DR epi-position PADRE sequence AKFVAAWTLKAAA (SEQ ID NO:24) or FVAAWTLKAAA (SEQ ID NO: 25)。

At some aspects of the method for the present invention, the peptide composition including the MHC restricted helper T cell epitope of II class includes The MHC II class restricted helper T cell peptide epitopes that MUC1 is derivative.In some respects, derivative for MUC1 B cell peptide epitopes and MHC II class restricted helper T cell peptide epitopes derivative for MUC1 includes adjacent aminoacid sequence.In some respects, adjacent amino Acid sequence is glycosylated on one or more threonine and/or serine residue.

At some aspects of the method for the present invention, B cell peptide epitopes that MUC1 is derivative and the derivative MHC II class limit of MUC1 Property helper T cell peptide epitopes processed includes adjacent aminoacid sequence.In some respects, adjacent aminoacid sequence includes and aminoacid sequence Row APGSTAPPAHGVTSA (SEQ ID NO:26), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27), APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:29) tool There is the sequence of at least about 50% sequence iden.In some respects, adjacent aminoacid sequence includes aminoacid sequence APGSTAPPAHGVTSA (SEQ ID NO:26)、APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27)、 APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:29).? Some aspects, adjacent aminoacid sequence is glycosylated on one or more threonine and/or serine residue.In the present invention Some aspects of method, glycolipidpeptide is used as liposome.In some respects, the lipid composition of glycolipidpeptide promotes the lipid bodily form Become.

At some aspects of the method for the present invention, the method includes using further immunomodulator.In some respects, execute By the compositions comprising glycolipidpeptide and immunomodulator.In some respects, immunomodulator is covalently attached to glycolipidpeptide.At some Aspect, immunomodulator includes TLR agonist.In some respects, TLR agonist includes TLR9 agonist.In some respects, TLR9 agonist includes CpG.In some respects, immunomodulator is TLR9 agonist, cox 2 inhibitor, GM-CSF, indole amine Inhibitor, chemotherapeutant or a combination thereof of 2,3-dioxygenase (IDO).

The present invention includes glycolipidpeptide, and it comprises: include at least one glycosylation MUC1 glycopeptide component of B cell epi-position；Bag Include at least one peptide composition of the MHC restricted helper T cell epitope of II class derivative for MUC1；With at least one lipid composition.? Some aspects of the glycolipidpeptide of the present invention, the glycosylated MUC1 glycopeptide component including B cell epi-position is included in one or more Glycosylation on serine and/or threonine residues.

At some aspects of the glycolipidpeptide of the present invention, the glycosylation MUC1 glycopeptide component including B cell epi-position includes with sugar The glycosylation that residue is carried out, described saccharide residue includes GAlNAc, GlcNAc, Gal, NANA, NGNA, fucose, mannose or Portugal Grape sugar.

At some aspects of the glycolipidpeptide of the present invention, the glycosylation MUC1 glycopeptide component including B cell epi-position is I class MHC Restricted epitope.

At some aspects of the glycolipidpeptide of the present invention, including the glycosylation MUC1 glycopeptide component of B cell epi-position and/or include The peptide composition of the restricted helper T cell epitope of MHC II class includes people's MUC1 peptide sequence.

At some aspects of the glycolipidpeptide of the present invention, including the glycolipidpeptide of B cell epi-position and/or include that MHC II class limits The peptide composition of property helper T cell epitope includes about 5 30 aminoacid of MUC1 protein sequence, described MUC1 protein sequence Including the extracellular region of MUC1 protein, and include glycosylated one or more serine or threonine residues.

At some aspects of the glycolipidpeptide of the present invention, including the glycosylation MUC1 glycopeptide component of B cell epi-position include with SAPDTRPAP (SEQ ID NO:20), TSAPDTRPAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO:22) or TSAPDTRPL (SEQ ID NO:23) has the aminoacid sequence of at least about 50% sequence iden.In some respects, thin including B The glycosylation MUC1 glycopeptide component of born of the same parents' epi-position is included in the glycosylation in one or more serine and/or threonine residues.

At some aspects of the glycolipidpeptide of the present invention, the glycosylation MUC1 glycopeptide component including B cell epi-position includes SAPDTRPAP (SEQ ID NO:20), TSAPDTRPAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO:22) or TSAPDTRPL(SEQ ID NO:23).In some respects, the glycosylation MUC1 glycopeptide component including B cell epi-position is included in one Glycosylation on individual or multiple serine and/or threonine residues.

At some aspects of the glycolipidpeptide of the present invention, lipid composition include one or more lipid chain, one or more half Cystine residue and one or more lysine residue.

At some aspects of the glycolipidpeptide of the present invention, lipid composition includes Toll-like receptor (TLR) part.Some sides Face, Toll-like receptor (TLR) part includes TLR2 part.In some respects, TLR2 part includes Pam₃CysSK₄。

At some aspects of the glycolipidpeptide of the present invention, lipid composition includes lipid adjuvant.In some respects, lipid adjuvant bag Include lipidated amino acid (LAA).

At some aspects of the glycolipidpeptide of the present invention, B cell peptide epitopes that MUC1 is derivative and the derivative MHC II class of MUC1 Restricted helper T cell peptide epitopes includes adjacent aminoacid sequence.

At some aspects of the glycolipidpeptide of the present invention, adjacent aminoacid sequence includes and aminoacid sequence APGSTAPPAHGVTSA (SEQ ID NO:26)、APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27)、 APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:29) tool There is the sequence of at least 50% sequence iden.In some respects, adjacent aminoacid sequence be at one or more threonine and/or On serine residue glycosylated.

At some aspects of the glycolipidpeptide of the present invention, adjacent aminoacid sequence includes aminoacid sequence APGSTAPPAHGVTSA (SEQ ID NO:26)、APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27)、 APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:29).? Some aspects, adjacent aminoacid sequence is glycosylated on one or more threonine and/or serine residue.

At some aspects of the glycolipidpeptide of the present invention, glycolipidpeptide include shown in Figure 19 in those of any one.One A little aspects, aminoacid sequence is glycosylated on one or more threonine and/or serine residue.

In some respects, glycolipidpeptide farther includes covalently bound immunomodulator.In some respects, immunomodulator Including TLR9 agonist, cox 2 inhibitor, GM-CSF, the inhibitor of indole amine 2,3-dioxygenase (IDO), chemotherapeutant Or a combination thereof.

The present invention includes pharmaceutical composition, and it comprises: glycolipidpeptide and pharmaceutically acceptable carrier as described herein.

The present invention includes the compositions comprising liposome, and described liposome includes glycolipidpeptide as described herein.At some Aspect, the lipid composition of glycolipidpeptide promotes that liposome is formed.In some respects, compositions comprises immunomodulator further.? Some aspects, immunomodulator includes TLR agonist.In some respects, TLR agonist includes TLR9 agonist.Some sides Face, TLR9 agonist includes CpG.

In some respects, immunomodulator includes TLR9 agonist, cox 2 inhibitor, GM-CSF, indoleamine 2, and 3-is double to be added Inhibitor, chemotherapeutant or a combination thereof of oxygenase (IDO).

The present invention includes immunogenic vaccine, and it includes glycolipidpeptide as described herein or compositions as described herein.

The present invention includes that glycolipidpeptide as described herein or compositions as described herein are for manufacturing treatment or prevention sense The purposes of the medicine of dye, disease or disease.

The present invention includes test kit, and it comprises: glycolipidpeptide as described herein；Packaging；And operation instructions.

Except as otherwise noted, " one ", " a kind of ", " described " and " at least one " are used interchangeably and mean one or super Cross one.

Accompanying drawing is sketched

Fig. 1 shows the exemplary glycolipidpeptide of the present invention.

Fig. 2 shows the flow cytometry for specificity anti-MUC-1 antibody.To MCF-7 (A) and SK-MEL-28 (B) cell tests is reactive.With (serum control before 3 immunity inoculations；Hollow peak) and rear (solid peak) assesses serum, and (1:50 is dilute Release) fluorescence intensity.

Fig. 3 shows after stimulating with LPS and synthesis compound, is produced by the TNF-α of mouse macrophage.Make Mus RAW γ NO (-) cell as shown with increase concentration escherichia coli (E. coli) LPS (■), 1 (●), Pam₂CysSK₄ (), 2 (), Pam₃CysSK₄ (▲) or 3 () incubation 5.5 hours together.

Fig. 4 shows the effect to cellular uptake of the TLR part.

Fig. 5 shows the chemical constitution of synthetic antigen.

Fig. 6 shows after stimulating with synthesis compound 21-24, Escherichia coli LPS and escherichia coli lipid A, passes through Mus The TNF-α of macrophage and IFN-β produce.Make Mus 264.7 RAW γ NO (-) cell as shown with increase concentration 21- 24, Escherichia coli LPS or escherichia coli lipid A incubation 5.5 hours together.ELISA is used to measure the TNF-α in cell supernatant And IFN-β (B) (A).Data represent meansigma methods ± SD (n=3).

Fig. 7 shows the cell recognition analysis about specificity anti-MUC1 antibody.To MCF7 cell tests serum reactivity. With the serial dilutions of the blood serum sample after 21 (A), 22/23 (B) or 4 immunity inoculations of 22/24 (C) and MCF7 cell Incubation together.After incubation together with the anti-mouse IgG antibody of FITC labelling, product of cell lysis is assessed fluorescence intensity. With serum before immunity inoculation and together with compareing SK-MEL-28 cell the blood serum sample of incubation do not observe relative to background glimmering Light (shown in Fig. 9).AU indicates any flat fluorescent.

Fig. 8 shows with after 4 immunity inoculations of 21,22,22/23,22/24 and 25/26, the anti-MUC1 of ELISA and anti-T Epitope antibodies titre.By elisa plate BSA-MI-MUC-1 conjugate (A-F) or neutravidin (neutravidin)-biotin-T epi-position (G) is coated, and by linear regression analysis, draws the dilution compared with absorbance Spend and measure titre.Titre be defined as obtain for normal control mice serum optical density be 0.1 or bigger the highest Dilution factor.Each data point represents the titre of individual mice after 4 immunity inoculations, and the group of horizontal line five mices of instruction Meansigma methods.

Fig. 9 shows the cell recognition analysis about specificity anti-MUC-1 antibody.MCF7 and SK-MEL-28 cell is surveyed Examination serum reactivity.With the blood serum sample (1:30 dilution) after 4 immunity inoculations of 21,22/23 or 22/24 and MCF7 and SK-MEL-28 cell incubation together.After incubation together with the anti-mouse IgG antibody of FITC labelling, in product of cell lysis Assessment fluorescence intensity.Also displayed is culture medium, conjugate and mice (normal control mice serum) comparison.Data represent average Value ± SD (n=3).AU indicates any flat fluorescent.

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 three completely synthetic immunogenic structures of component.

The chemical constitution of Figure 16: synthetic antigen 1,2,3,4 and 5.

Figure 17: reduced the MMT tumor load of MUC1.Tg mice by three component vaccines.MUC1.Tg mice is used as comparison Empty liposome (EL) or carry out immunity containing the liposome of 1,2,3,4+5 or 5 (25 μ g, containing 3 μ g carbohydrates) Inoculation.The chemical constitution of synthetic antigen 1,2,3,4 and 5 is as shown in Figure 16.With MUC1 express MMT tumor cell (1 × 10⁶Individual cell) tumor challenge before, give three immunity inoculations biweekly, be the once reinforcement after a week subsequently.? Within after a rear shot 7 days, put to death animal, and measure tumor weight in wet base.Data are rendered as comparison (with empty liposome vaccination Mice) percentage ratio.Each data point represents individual mice, and the meansigma methods of horizontal line instruction mice group.

The induction of the cytotoxicity (ADCC) of Figure 18 A and 18B: antibody dependent cellular mediation.Tumor cell Yac-MUC1 (Figure 18 A) and C57mg.MUC1 (Figure 18 B) chromium labelling 2 hours, and subsequently with the serum (1:50 dilution) one deriving from mice Rising 37 DEG C of incubations 30 minutes, described mice is as shown with empty liposome (EL) or containing 1,2,3,4+5 or the liposome of 5 Immunity inoculation in the case of being with or without (NT) tumor inducing.The chemical constitution such as Figure 16 of synthetic antigen 1,2,3,4 and 5 shows Show.Make tumor cell incubation 4 hours together with effector lymphocyte (NK cell KY-1 clone) subsequently.Effector is 50 with target ratio: 1.Spontaneous release is less than 15% discharged completely.Each data point represents individual mice, and horizontal line indicates the average of mice group Value.

Figure 19 A to 19C: cell response.Figure 19 A measures the CD8 producing IFN-γ in MUC1.Tg mice⁺T cell. CD8 is analyzed for MUC1 specificity IFN-γ spot formation⁺T cell, described CD8⁺T cell uses empty liposome from as shown (EL) or containing 1,2,3,4+5 or 5 liposome mice of immunity inoculation in the case of being with or without (NT) tumor inducing Lymph node in separate.The chemical constitution of synthetic antigen 1,2,3,4 and 5 is as shown in Figure 16.Each data point represents individuality Mice, and the meansigma methods of horizontal line instruction mice group.Figure 19 B measures the CD8 in MUC1.Tg mice⁺Cytotoxic T cell Induction.It is being with or without (NT) tumor with empty liposome (EL) or the liposome containing 1,2,3,4+5 or 5 from as shown The lymph node of the mice of immunity inoculation separates in the case of induction CD8⁺T cell, and make CD8⁺T cell is without any body Experience in the case of external stimulus⁵¹Cr discharges mensuration.Will be with MUC1 (Tn) peptide 6 (SAPDT for 1 (NT), 1,3,4+5 and 5 (Tn) RPAP) DC of (SEQ ID NO:26) pulse is used as target, and will be with MUC1 peptide 7 (SAPDTRPAP) (SEQ ID for 2 NO:20) DC of pulse is used as target or will be used as target with the DC of empty liposome pulse for EL.Effector is 100:1 with target ratio, because of Do not stimulate for CTL.Spontaneous release is less than 15% discharged completely.Each data point represents individual mice, and water The meansigma methods of horizontal line instruction mice group.Figure 19 C measures CD8⁺The epi-position demand of T cell.The mice liposome containing 1 or 2 enters Row immunity inoculation.Obtained by cell sorting and express the T cell that the lymph node of low-level CD62L derives, and using sugar for 1 Cultivate 14 days in the presence of peptide 6 or the DC for 2 use peptide 7 pulses.After the DC being exposed to the 6-9 pulse of use (sugared) peptide, analyze institute Obtain the CD8 of cell⁺IFNγ⁺The existence situation of T cell.Peptide 6 is SEQ ID NO:26, and peptide 7 is SEQ ID NO:20, and peptide 8 is SEQ ID NO:27, and peptide 9 is SEQ ID NO:29.

Figure 20 A to 20H: as shown in the case of being with or without (NT) tumor inducing with 1,2,3,4+5 or 5 The anti-MUC1 of ELISA after three times (Figure 20 A) or four (Figure 20 B-H) immunity inoculations and anti-auxiliary T epi-position (helper T- Epitope) antibody titer.The chemical constitution of synthetic antigen 1,2,3,4 and 5 is as shown in Figure 16.By elisa plate BSA- MI-MUC-1 (Tn) conjugate (Figure 20 A-G) or neutravidin-biotin-auxiliary T epi-position (Figure 20 H) are coated, and And by linear regression analysis drafting dilution factor compared with absorbance, and measure titre.Titre is defined as obtaining relative to just Often for control mice serum, optical density is the highest dilution of 0.1 or bigger.Each time point represents four immunity inoculations The titre of rear individual mice, and the meansigma methods of horizontal line instruction mice group.

Figure 21 A and 21B: by MUC1 (Tn) 6, the most glycosylated MUC1 7 and Tn monomer, with BSA-MI-MUC1 (Tn) The competitive inhibition of the antibodies of conjugate.

The sequence of compound 6 and 7 is shown in Figure 19.Elisa plate BSA-MI-MUC1 (Tn) conjugate is coated.Will Dilute in the case of there is not inhibitor, obtain exempting from 1 (Figure 21 A) and 2 (Figure 21 B) of in the ELISA OD of about 1 The postvaccinal blood serum sample of epidemic disease, first mixes with 6,7 or Tn monomer (0-500 μM of final concentration), and is subsequently applied to coated Microtitration plate.Optical density value is for the optical density value standardization obtained with single serum (0 μM of inhibitor, 100%).Data It is reported as the meansigma methods ± s.e.m of mice group (n=7).

Figure 22 A to 22J: stimulating 24 hours with the Liposomal formulation being mounted with compound 1,2 or 3 or Escherichia coli LPS After, produced by the cytokine of dendritic cell.The chemical constitution of synthetic antigen 1,2 or 3 is shown in Figure 16.Make primary tree The as directed Liposomal formulation one being mounted with compound 1,2 or 3 or Escherichia coli LPS with increase concentration of prominent shape mouse cell Play incubation 24 hours.ELISA is used to measure the TNF-α (Figure 22 A) in cell supernatant, IFN-β (Figure 22 B), RANTES (Figure 22 C), IL-6 (Figure 22 D), extracellular IL-1 β (Figure 22 E and Figure 22 F), IL-10 (Figure 22 G), IP-10 (Figure 22 H), IL-12 p70 (Figure 22 I) and IL-12/23 p40 (Figure 22 J).For ATP process after IL-1 β secretion estimation, with lure Lead thing incubation together after 24 hours, by cell incubation 30 minutes together with ATP (5 mM).Data report is triplicate process Meansigma methods ± SD.

Figure 23: with compound 2 (Pam₃CysSK₄-t helper cell epi-position (T helper ep.) (poliomyelitis)- MUC1 (the most glycosylated))；Compound 1 (Pam₃CysSK₄-t helper cell epi-position (poliomyelitis)-MUC1 (Tn))；Change Compound 1 is plus CpG (CpG oligodeoxynucleotide (CpG ODN)))；Compound 5 (Pam₃CysSK₄) plus compound 4, (T is auxiliary Synergidae epi-position (poliomyelitis)-MUC1 (Tn))；Compound 5；Compound 3 (Pam₃CysSK₄-t helper cell epi-position (ridge Tephromyelitis))；Compound 3 is plus CpG；EL (empty liposome) adds CpG；Or the MUC1.Tg mice of the preparation immunity inoculation of EL In with gram tumor weight that (gm) represents.The chemical constitution of synthetic antigen 1,2,3,4 and 5 is as shown in Figure 16 and 26.

Figure 24: derive from the cellular cytoxicity activity of the CD8+ cell of MUC1.Tg mice, described MUC1.Tg mice compound 2 (Pam₃CysSK₄-t helper cell epi-position (poliomyelitis)-MUC1 (the most glycosylated))；Compound 1 (Pam₃CysSK₄- T helper cell epi-position (poliomyelitis)-MUC1 (Tn))；Compound 1 is plus CpG (CpG oligodeoxynucleotide (CpG ODN)))；Compound 5 (Pam₃CysSK₄) plus compound 4 (t helper cell epi-position (poliomyelitis)-MUC1 (Tn))； Compound 5；Compound 3 (Pam₃CysSK₄-t helper cell epi-position (poliomyelitis))；Compound 3 is plus CpG；EL (empty fat Plastid) plus CpG；Or the preparation immunity inoculation of EL.The chemical constitution such as Figure 16 and 26 of synthetic antigen 1,2,3,4 and 5 shows 's.

Figure 25: the mensuration that the IFN-γ of the CD8+ T cell deriving from MUC1.Tg mice produces, described MUC1.Tg mice is used Compound 2 (Pam₃CysSK₄-t helper cell epi-position (poliomyelitis)-MUC1 (the most glycosylated))；Compound 1 (Pam₃CysSK₄-t helper cell epi-position (poliomyelitis)-MUC1 (Tn))；Compound 1 is plus CpG (CpG widow's deoxidation core Thuja acid (CpG ODN)))；Compound 5 (Pam₃CysSK₄) plus compound 4 (t helper cell epi-position (poliomyelitis)- MUC1(Tn))；Compound 5；Compound 3 (Pam₃CysSK₄-t helper cell epi-position (poliomyelitis))；Compound 3 adds CpG；EL (empty liposome) adds CpG；Or the preparation immunity inoculation of EL.Chemical constitution such as Figure 16 of synthetic antigen 1,2,3,4 and 5 With 26 in display.

Figure 26: compound 1 (Pam₃CysSK₄-t helper cell (T-helper)-MUC1), compound 2 (Pam₃CysSK₄-t helper cell), LAA-T auxiliary cell-MUC1 and LAA-T auxiliary cell structure.

Figure 27: immunization scheme.

Figure 28: three component vaccines reduce tumor load.With containing compound 1 (Pam₃CysSK₄-t helper cell-MUC1), Compound 2 (Pam₃CysSK₄-t helper cell), LAA-T auxiliary cell-MUC1 or LAA-T auxiliary cell (25 μ g, containing 3 μ G carbohydrate) liposome or be used as comparison empty liposome immunity inoculation MUC1.Tg mice.MMT is being expressed with MUC1 Tumor cell (1 × 10⁶Individual cell) tumor challenge before, give three immunity inoculations biweekly, after being one week subsequently Once strengthen.Inject the last time latter 7 days and put to death animal, and measure tumor weight in wet base.Data are rendered as comparison (with empty lipid The mice of body vaccination) percentage ratio.Each data point represents individual mice, and horizontal line indicates the average of mice group Value.

Figure 29: by vaccine-induced MUC1 specific cytotoxicity CD8 T cell.From as shown with empty liposome or contain There is compound 1 (Pam₃CysSK₄-t helper cell-MUC1), compound 2 (Pam₃CysSK₄-t helper cell), LAA-T auxiliary The liposome of cell-MUC1 or LAA-T auxiliary cell divides in the lymph node of the mice of immunity inoculation in the case of tumor inducing From CD8⁺T cell, and make CD8⁺T cell experiences 51Cr release in the case of without any stimulated in vitro and measures.Use Tn- The DC of MUC1 peptide (SAPDT (Tn) RPAP) (SEQ ID NO:26) or empty liposome pulse is used as target.Effector with target ratio is 100:1, because CTL does not stimulates.Spontaneous release is less than 15% discharged completely.Each data point represents individuality.Close Become the chemical constitution of antigen 1,2,3,4 and 5 as shown in Figure 16 and 26.

Figure 30: three component vaccines cause powerful antibody titre.The anti-MUC1 of ELISA after four immunity inoculations and anti-T epi-position Antibody titer.Anti-MUC1 and anti-T epitope antibodies titre present as the intermediate value of the group of four to seven mices.For anti-MUC1 antibody Titre, is coated elisa plate BSA-MI-MUC-1 (Tn) conjugate, or for anti-t helper cell epitope antibodies titre, will Elisa plate neutravidin-biotin-T epi-position is coated.Utilize and the dilution plotting compared with absorbance is led to Cross linear regression analysis and measure titre.Titre be defined as obtain for normal control mice serum optical density be 0.1 or Bigger highest dilution.

Figure 31: antibody is effective at antibody dependent cellular cytotoxicity (ADCC) aspect.C57mg.MUC1 mammary neoplasms is thin Born of the same parents with chromium labelling two hours, and subsequently with control serum (MUC1.Tg) or derive from the serum (1:50 that lotus has the mice of MMT tumor Dilution) together at 37 DEG C of incubations 30 minutes (min), described mice is as shown with empty liposome or containing compound 1 (Pam₃CysSK₄-t helper cell-MUC1), compound 2 (Pam₃CysSK₄-t helper cell), LAA-T assist cell-MUC1 Liposome immunization inoculation with LAA-T auxiliary cell.Make tumor cell subsequently together with effector lymphocyte (KY-1 cell-NK clone) Incubation four hours.Effector is 50:1 with target ratio.Spontaneous release is less than 15% discharged completely.Each data point represents individual little Mus, and the meansigma methods of horizontal line instruction mice group.The chemical constitution such as Figure 16 and 26 of synthetic antigen 1,2,3,4 and 5 shows 's.

Figure 32: the targeting sequencing of people MUC1, wherein Rankpep result of study highlights.The homing sequence of tandem sequence repeats There is underscore.Dotted line shows display and I-A^b15 aggressiveness of RANKPEP score of combination.Specify display and H2-D^b Or H2-K (dddd)^b (kkkk) combination or the non-germline selectivity (promiscuous) with both (bbbb) combine 9 aggressiveness of RANKPEP score.

Figure 33 A and 33B: by mice peptide immunity inoculation described in Figure 33 A, and it is low to obtain expression by cell sorting The T cell that the lymph node of horizontal CD62L is derivative, and cultivate 14 days in the presence of the DC with immunity inoculation peptide pulse.Cruelly After the DC of peptide pulse listed in dew y-axis, for CD4⁺IFNγ⁺And CD8⁺IFNγ⁺The existence situation of T cell is by thin Cell (Figure 33 B) obtained by intracellular cytokine analysis.

Figure 34: utilize the synthesis construct of people's MUC1 t helper cell sequence.

Figure 35 shows completely synthetic three component immunogens 52 and 53 and structure for its reagent 63-65 prepared.

Figure 36 show with after 4 immunity inoculations of 52 and 53 the anti-GSTPVS of ELISA (β-O-GlcNAc)SANM (68) antibody titer.By elisa plate with BSA-MI-GSTPVS (β-O-GlcNAc) SANM (BSA-MI-66) conjugate is coated, and And by linear regression analysis drafting dilution factor compared with absorbance, measure (a) total IgG, (b) IgG1, (c) IgG2a, (d) IgG2b, (e) IgG3 and (f) IgM titre.It is close that titre is defined as obtaining light for normal control mice serum Degree is the highest dilution of 0.1 or bigger.Each data point represents the titre of individual mice after 4 immunity inoculations, and water The meansigma methods of the group of horizontal line five mices of instruction.

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.

The description of illustrative embodiment

The glycolipidpeptide of the present invention comprises at least one B epitope, at least one T epi-position and lipid composition.In preferred embodiments, Glycolipidpeptide is substantially made up of three kinds of key components: containing at least one carbohydrate ingredient of B epitope；Containing auxiliary T table At least one peptide composition of position；With at least one lipid composition.Exemplary carbon hydrate, peptide and lipid composition in this article with And described in such as references cited herein, described list of references includes Koganty et al., public on March 30th, 2006 The United States Patent (USP) opened discloses 20060069238；Referring further to Koganty et al., 1996, Drug Disc Today；1(5):190- 198.Three kinds of components are the most covalently bound, to form single glycolipidpeptide molecule.It is indirectly connected with relating to optional connecing The use of head component " L ", to link together two or more key components.Three kinds of key components can be in any order Link together (directly or indirectly).Such as, lipid and carbohydrate ingredient can each be covalently attached to peptide composition, with Form glycolipidpeptide.Alternately, lipid composition and peptide composition can each be covalently attached to carbohydrate ingredient.Similarly, Carbohydrate ingredient and peptide composition can each be covalently attached to lipid composition.Or, all three component can so connect Connect so that three kinds of components each of are each covalently attached in other two kinds of components.Intermolecular cross-linking is also possible, as follows Literary composition is more fully described.

In preferred embodiments, the glycolipidpeptide of the present invention contains a kind of carbohydrate ingredient, a kind of peptide composition and Plant lipid composition.In another embodiment, glycolipidpeptide contains multiple kinds of carbohydrate component, and it can be identical or can Being different.Similarly, in another embodiment, glycolipidpeptide contains multiple peptide composition, and it can be identical or can Being different.Further, in another embodiment, glycolipidpeptide contains multiple lipid composition, and it can be identical Can be maybe different.Therefore, multiple embodiments of the glycolipidpeptide of the present invention can contain one or more carbohydrates Component, one or more peptide compositions and/or one or more lipid compositions.Such as, " multiple antigenicity glycopeptide (multiple Antigenic glycopeptides) " idea (Bay et al., U.S. Patent number 6,676, on January 13rd, 946,2004, Bay Et al.；WO is open on October 8th, 98/43677,1998, Bay et al.) may adapt to use in the present invention.High antigen is close Degree can use the core such as polylysine core that the peptide " arm " (peptide composition of the glycolipidpeptide of the present invention) of extension adheres to therewith Realizing, described peptide arm presents the Carbohydrate Antigens component showing glycolipidpeptide to cluster.The lipid composition of glycolipidpeptide is permissible Extending from lysine core equally, peptide composition is via the enforcement of non-end aminoacid with lysine core attachment the most wherein In scheme.High antigen density can also reach as delivery vector, as illustrated in embodiment 2 and 3 by using liposome. Additionally or alternatively, optionally crosslinkable glycolipidpeptide, to form multi-molecular complex, thus increase antigen density.

The multiple kinds of carbohydrate of glycolipidpeptide, peptide and lipid composition can structurally derived from or based on naturally occurring Biological molecule in find those and/or can simulate in naturally occurring biological molecule find those.Glycolipid Peptide composition preferably comprises the part of the molecular structure of those or structure that are equal to or are similar in living organism discovery and (includes table Position).Typically, although the component of glycolipidpeptide derived from, structurally based on and/or simulate naturally occurring structure, but they Use the most chemical or external enzymatic method synthetically prepared.In some embodiments, can be same or similar by being formed The different chemical structures (having different bonding order or pattern) of epi-position, is formed by molecular element in the glycolipidpeptide of the present invention In naturally occurring antigen formed epi-position, described molecular element be close in space but for chemical bonding each other Away from.

The three component sugars lipopeptids of the present invention can be considered as box, and wherein carbohydrate ingredient, peptide composition and lipid composition are each From being selected independently for being included in glycolipidpeptide.Form the carbohydrate ingredient as described herein of glycolipidpeptide, peptide composition Any combination (being i.e. mixed and matched) with lipid composition is included by the present invention.

Carbohydrate ingredient

The carbohydrate ingredient of glycolipidpeptide can be any component containing carbohydrate.Suitable carbohydrates component Example includes oligosaccharide, polysaccharide and monosaccharide, and glycosylation biomolecule (glycoconjugate), such as glycoprotein, glycopeptide, glycolipid, glycosyl Change aminoacid, DNA or RNA.Containing one or more carbohydrate portions and peptide or amino acid whose glycosylated peptide (glycopeptide) It is particularly preferred with glycosylated amino acid as the carbohydrate ingredient of the glycolipidpeptide of the present invention.The example of glycopeptide is CD52, it is expressed on essentially all human lymphocyte and it is believed that and plays an important role in human immune system.Glycosylation amino The example of acid is Tn antigen.Be to be understood that when carbohydrate ingredient is glycopeptide, the peptide moiety of glycopeptide optionally include T epi-position with And B epitope, and therefore can serve as the peptide composition of glycolipidpeptide.Glycopeptide containing T epi-position and B epitope is sometimes referred to as having " B- T " epi-position or " T-B " epi-position.The B epitope being present on the glycolipidpeptide of the present invention and T epi-position can be overlapping or can not be overlapping.? In preferred embodiment, T epi-position, B epitope and/or T-B epi-position, derived from MUC1 peptide sequence, include but not limited to that people MUC1 is many Peptide sequence.

The carbohydrate ingredient of the glycolipidpeptide of the present invention includes the carbohydrate containing one or more sugar monomers.Example As, carbohydrate can include monosaccharide, disaccharide or trisaccharide；It can include oligosaccharide or polysaccharide.Oligosaccharide is containing two or more Multiple sugar and be characterised by the oligosaccharide of the structure being able adequately determines.The structure being able adequately determines is characterised by the specific body of monomer Part, order, link position (including branch point) and connect spatial chemistry (α, β), and the structure being therefore able adequately determines has really Fixed molecular weight and composition.Oligosaccharide usually contains about 2-about 20 or more sugar monomer.On the other hand, polysaccharide is not have to fill Divide the polysaccharide of the structure determined；Identity, order, link position (including branch point) and/or connection spatial chemistry can be at molecules Between different.Polysaccharide usually contains more greater number of monomer component than oligosaccharide, and therefore has higher molecular weight.As made herein Term " polysaccharide " including oligosaccharide and polysaccharide, and include branch and non-branched polymer.Work as carbohydrate ingredient Containing when there is the carbohydrate of three or more sugar monomers, carbohydrate can be straight chain or it can be side chain. In preferred embodiments, carbohydrate ingredient contains less than about 15 sugar monomers；More preferably less than about 10 sugar are single Body.

The carbohydrate ingredient of glycolipidpeptide includes the carbohydrate containing B epitope.It is to be understood that carbohydrate can Being coextensive with B epitope, or carbohydrate can be including B epitope, or carbohydrate can only include B epitope Partly (i.e. B epitope can additionally comprise other parts of glycolipidpeptide, such as peptide composition, lipid composition and/or linker component).Bag The example of the glycopeptide including B epitope is glycosylated peptide MUC-1 (referred to herein as MUC1).Therefore, the carbon of B epitope " is comprised " Hydrate or carbohydrate ingredient are understood to mean that all or part of carbon comprising the B epitope being present on glycolipidpeptide Hydrate or carbohydrate ingredient.

Epi-position that B epitope can be naturally-occurring or the epi-position of non-naturally-occurring.Preferably, two of carbohydrate or More sugar monomers interact, to form the comformational epitope serving as B epitope.B epitope is by the epi-position of B cell identification.Containing B Any antigenicity carbohydrate of epi-position can serve as carbohydrate ingredient, and unrestricted.In preferred embodiments, B Epi-position, derived from MUC1 peptide sequence, includes but not limited to people's MUC1 peptide sequence.

The carbohydrate of the non-naturally-occurring that can serve as the component of the glycolipidpeptide of the present invention includes sugar analogies (glycomimetics), its be simulation sugar such as monosaccharide, disaccharide or the shape of oligosaccharide and feature molecule (see for example, 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).Sugar analogies can transform as the required B epitope of supply and The metabolic stability that potential offer is bigger.

In another embodiment, carbohydrate ingredient contains all or part of of autoantigen.Autoantigen is The antigen being typically found in animal body.They can be considered as " self-molecules present ", be such as present in zooblast or on point Son, or the protein such as insulin of circulation in animal blood.The example of autoantigen is derived from containing of the cancerous cell of animal Carbohydrate ingredient, such as tumor related carbohydrate antigen (TACA).Generally, this type of autoantigen demonstrates low immunity Originality.Example includes tumor related carbohydrate B epitope such as Le^yAntigen (be correlated with tetrose by cancer；Such as Fuc α (1,2)- Galβ(1,4)-[Fucα(1,3)]-GlcNAc)；Globo-H antigen (such as L-Fuc α (1,2)-Gal β (1,3)-GalNAc β (1,3)-Galα(1,4)-Galβ(1,4)-Glu)；T antigen (such as Gal β (1,3)-GalNAc α-O-Ser/Thr)；STn antigen (saliva acidic group Tn, such as NeuAc α (2,6)-GalNAc α-O-Ser/Thr)；With Tn antigen (such as α-GalNAc-O-Ser/ Thr).Another example of autoantigen is derived from the breast tumor of people's Polymorphic epithelial mucin (PEM) and is correlated with MUC-1's The glycopeptide of tandem sequence repeats, described PEM be mucins (Baldus et al., Crit. Rev. Clin. Lab. Sci., 41 (2):189-231(2004)).MUC-1 glycopeptide comprises at least one Tn and/or saliva acidic group Tn (saliva acidic group α-6 GalNAc Or " STn ") epi-position, it is preferably connected to threonine (T-Tn or T-STn).

In preferred embodiments, carbohydrate ingredient includes glycosylation MUC1 glycopeptide, and it is at amino derivative for MUC1 Glycosylation on one or more serines of acid peptide sequence and/or threonine residues.The aminoacid sequence bag that this type of MUC1 is derivative Include but be not limited to any MUC1 sequence described herein.

The structure of exemplary oncologic related carbohydrate antigen (TACA) that can serve as the component of glycolipidpeptide include but It is not limited to the structure in the scheme that is shown in 1 and 2.

Scheme 1

Should be understood that Tn, STn and TF structure shown in scheme 1 (monomer, trimer, cluster) all shows have threonine Residue.Corresponding serine analogs is also suitable construction.In the case of Tn3, STn3, TF3 and respective bunch, it is included in master The threonine of chain/serine composition has discrepant all possible same and different analog.

Scheme 2

Comprise for the another kind of autoantigen of use in the carbohydrate ingredient of glycolipidpeptide and be covalently attached to monosaccharide Aminoacid or the glycopeptide of peptide.Preferably, monosaccharide isN-acetylglucosamine (GlcNAc) orN-acetylgalactosamine (GalNAc).Excellent Choosing glycopeptide autoantigen be β-N-acetylglucosamine (β-O-GlcNAc) peptide modified.Preferably, monosaccharideOIt is coupled to the silk of polypeptide Propylhomoserin or threonine.Be also adapted to as autoantigen be relevant mercaptan (SConnection) and amino (NConnection) analog.Monosaccharide is preferred It is connected to peptide via β, but it can be α connection.In particularly preferred embodiments, the carbon water of the glycolipidpeptide of the present invention Compound component (when preparing as glycopeptide, it can be co-extensive with peptide composition) containing byOTPVSS (the SEQ that-GlcNAc modifies ID NO:10) aminoacid sequence.Glycopeptide containing β-GlcNAc modification is shown as figure as the example of the carbohydrate of B epitope Compound in 15 52 (OConnection) and 53 (SConnection).

In another embodiment, carbohydrate ingredient contains the Carbohydrate Antigens from microorganism (generally For polysaccharide) all or part of, the preferred pathogenic microorganism of described microorganism, such as virus (the carbon water being such as present on gp120 Compound, derived from the glycoprotein of inhibition of HIV), Gram-negative or gram positive bacteria be (such as derived from Haemophilus influenzae (Haemophilus influenzae), streptococcus pneumoniae (Streptococcus pneumoniae) or Neisseria meningitdis ball Bacterium (Neisseria meningitides) carbohydrate), fungus (such as 1,3-β joins polysaccharide), Parasitic protozoa (such as protozoon parasite such as leishmaniasis (Leishmania) and T. brucei (Trypanosoma bruceiThe GPI anchor found in)) or anthelmintic.Preferably, microorganism is pathogenic microorganism.

From the exemplary polysaccharide of viral pathogen from the Man9 of HIV-1 gp120, it is shown in scheme 3.

Scheme 3

Exemplary HIV carbohydrate and glycopeptide antigen are at Wang et al., Current Opinion in Drug Disc. & Develop., described in 9 (2): 194-206 (2006), and include naturally occurring HIV carbohydrate and glycopeptide, and Based on naturally occurring HIV carbohydrate and the synthetic carbohydrate of glycopeptide and glycopeptide.

Exemplary HCV carbohydrate and glycopeptide antigen are at Koppel et al.Cellular Microbiology 2005；7(2): 157-165 and Goffard et al.J. of Virology2005；79: described in 8400-8409, and include (13) Naturally occurring HCV carbohydrate and glycopeptide, and synthesis carbon water based on naturally occurring HCV carbohydrate and glycopeptide Compound and glycopeptide.

Exemplary polysaccharide from bacterial pathogens is shown in scheme 4.

Scheme 4

Exemplary polysaccharide from protozoal pathogens is shown in scheme 5.

Scheme 5

Exemplary polysaccharide from fungal pathogens is shown in scheme 6.

Scheme 6

Exemplary polysaccharide from anthelmintic substance is shown in scheme 7.

Scheme 7

Although skilled artisan would appreciate that numerous antigenicity carbohydrate structure is known, but exist many more Antigenicity carbohydrate structure because the most only identifying fraction antigenicity or immunogenicity carbohydrate. The example of the many Carbohydrate Antigens found so far can be found in: Kuberan et al., Curr. Org. Chem, and 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.Derived from mammal or any antigenicity carbohydrate of infectious organisms Can serve as the carbohydrate ingredient of the glycolipidpeptide of the present invention, and unrestricted.

Peptide composition

The peptide composition of glycolipidpeptide includes T epi-position, preferably auxiliary T epi-position.Peptide composition can be any containing peptide structure, and can contain There are the naturally occurring and/or aminoacid of non-naturally-occurring and/or amino acid analogue (such as D-aminoacid).Peptide composition is permissible From microorganism, such as virus, antibacterial, fungus and protozoacide.Therefore T epi-position may be constructed the whole of virus antigen or portion Point.Alternatively or additionally, T epi-position can come from mammal, and optionally forms all or part of of autoantigen.Such as, T Epi-position can be the part of the glycopeptide of process LAN on cancerous cell.When the peptide composition of the glycolipidpeptide of the present invention is glycopeptide, peptide group Divide and can also include all or part of of B epitope, as described elsewhere herein.More generally, it should be understood that glycolipidpeptide Peptide composition can be coextensive with T epi-position, or peptide composition can be including T epi-position, or peptide composition can include only part T table (that is, T epi-position can additionally comprise other parts of glycolipidpeptide, such as carbohydrate ingredient, lipid composition and/or joint in position Component).Therefore, " comprise " peptide of T epi-position or peptide composition is understood to mean that and comprises the whole of the T epi-position that is present on glycolipidpeptide Part peptide or peptide composition.

Peptide composition can be containing for example, less than about 50 aminoacid and/or amino acid analogue, less than about 40 aminoacid And/or amino acid analogue, less than about 30 aminoacid and/or amino acid analogue or less than about 20 aminoacid and/or ammonia Base acid-like substance.Peptide composition can contain about 50 aminoacid of e.g., from about 9-and/or about 40 amino of amino acid analogue, about 9- Acid and/or about 30 aminoacid of amino acid analogue, about 9-and/or amino acid analogue or about 20 aminoacid of about 9-and/or Amino acid analogue.Peptide composition can containing e.g., from 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 aminoacid and/or amino acid analogues, or these quote any scope of size.

The example of peptide composition includes general auxiliary T peptide QYIKANSKFIGITEL (" QYI ") (SEQ ID NO:1), general Auxiliary T p277 AFKYARHANVGRNAFELFL (" YAF ") (SEQ ID NO:2), auxiliary derived from the Mus of poliovirus T peptide KLFAVWKITYKDT (" KLF ") (SEQ ID NO:3) and general DR is helped to combine (PADRE) peptide (PCT WO 95/07707； Alexander et al., Immunity 1:751-761 (1994)；Alexander et al., J. Immunol. February 1 in 2000 Day；164(3):1625-33；U.S. Patent number 6,413,935 (Sette et al., on July 2nd, 2002)).

Immunogenicity peptide composition for using in the glycolipidpeptide of the present invention includes general (degeneracy or " non-germline selection Property ") helper T cell peptide, it is to be immunogenic in the individuality of many MHC (MHC) haplotypes Peptide.Numerous general helper T cell peptide structures are known；It is, however, to be understood that other promiscuous T epitopes will be identified in future, Including having some promiscuous T epitopes similar or even more efficient, and this type of peptide is very suitable as the sugar of the present invention The peptide composition of lipopeptid.

Example T cell peptide for using in glycolipidpeptide includes but not limited to:

The non-natural PADRE PEPD Ala-Lys-Cha-Val-Ala-Ala-Trp-Thr-Leu-Lys-Ala-Ala-DAla of synthesis, Including by Alexander et al. at Immunity, volume 1,751-761, all analog described in 1994；

Derived from the peptide of tetanus toxin, such as (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)；

Derived from the peptide of poliovirus, such as KLFAVWKITYKDT (SEQ ID NO:3)；

Derived from the peptide of Neisseria meningitidis, such as YAFKYARHANVGRNAFELFL (SEQ ID NO:8)；With

Derived from Plasmodium falciparum (P. falsiparum) peptide of CSP, such as EKKIAKMEKASSVFNVNN (SEQ ID NO:9)。

The peptide composition of glycolipidpeptide contains T epi-position.T epi-position is by the epi-position of T cell identification.T epi-position can cause CD4+ to answer Answer, thus stimulate the generation of helper T cell；And/or it can cause CD8+ response, thus stimulate cytotoxic lymphocyte Produce.Preferably, T epi-position be stimulate helper T cell produce epi-position (such as helper T cell epitope or Th epi-position), itself and then Make to be possibly realized for the humoral response of the B epitope by the carbohydrate ingredient supply of glycolipidpeptide of the present invention.

Being to be understood that the glycolipidpeptide of the present invention can contain multiple T epi-positions, it can be identical or different.Further Ground, T epi-position may reside in and (such as, including glycopeptide and/or glycolipid conduct on carbohydrate ingredient and/or lipid composition In the embodiment of carbohydrate and/or lipid composition), it is additional to or replaces peptide composition.

In some embodiments, B epitope and T epi-position are homologies；That is, they are derived from identical biology.Such as, suitable Together in being used as in the glycolipidpeptide of the vaccine of microbial pathogens, T epi-position can be present in microorganism cause of disease plus B epitope Epi-position in body.In another embodiment, B epitope and T epi-position are allos；That is, they are not derived from identical biology.Example As, the glycolipidpeptide being adapted for use as anti-cancer vaccine can have the B cell epi-position from cancerous cell, but has from antibacterial or virus T cell epitope.

In the preferred embodiment of the immunogenic vaccine of the present invention, T epi-position or B epitope can be many derived from MUC1 Peptide.In some embodiments, T epi-position and B epitope are derived from MUC1 polypeptide.MUC1 (MUC1 in people and non-personage Muc1 in kind) be in the epithelial cell and hematopoietic cell of multiple mucomembranous surface lining express weight glycosylated I type across Memebrane protein.People MUC1 is by Cytoplasm signal peptide, 28 transmembrane amino acid domains with by 20 amino acid whose variable numbers The ectodomain composition of tandem sequence repeats composition.Each repeat containing 5 potential O-glycosylation sites.MUC1 with at mucosal site On several adenocarcinoma be correlated with, and process LAN in more than the breast carcinoma of 90%, and relevant to ovary, lung, colon and cancer of pancreas. The tumor MUC1 that is correlated with is Aberrant glycosylation, produces the carbohydrate structure of truncate.

MUC1 peptide sequence can include people or Mouse MUC1 sequence.MUC1 peptide sequence can include MUC1 tandem sequence repeats sequence Row.This type of MUC1 tandem repetitive sequence can be containing B epitope and auxiliary T epi-position.

MUC1 sequence can be homology, from but autoantigen.MUC1 sequence can include from people or Mouse MUC1 One of peptide, two, three, four, five, six or more amino acid change.MUC1 sequence can be irregular change , including one, two, three, four or more amino acid changes, to strengthen MUC1 peptide in I class and/or II class mainly group Knit the combination on histocmpatibility (MHC) protein.People MHC is referred to herein as HLA complex.MUC1 sequence can be wrapped Include the sequence of extracellular region from MUC1 protein.MUC1 sequence can include being responsible for the sequence that I class MHC limits.MUC1 sequence Can include being responsible for the sequence that II class MHC limits and/or combines.In some embodiments, this type of I class and II class limit sequence It can be the adjacent aminoacid sequence in immunogenic vaccine construct.MHC limits sequence and includes but not limited to such as herein Any one in those of representing in any one in those of description and Figure 16,19 and 32-34.

MUC1 sequence can include one or more serine or threonine residues, and it is glycosylated, such as one, Two, three, be glycosylated on four or more these type of residues.This type of glycosylation can represent the glycosylation of normal structure Pattern, or this type of glycosylation can reflect Aberrant glycosylation.MUC1 sequence can be containing one or more B epitope and/or auxiliary T Epi-position.

MUC1 sequence can include about 30 aminoacid of about 5-of MUC1 protein sequence.MUC1 sequence can include MUC1 Less than about 50 aminoacid of protein sequence and/or amino acid analogue, less than about 40 aminoacid and/or amino acids are seemingly Thing, less than about 30 aminoacid and/or amino acid analogue or less than about 20 aminoacid and/or amino acid analogue.MUC1 Sequence can include about 50 aminoacid of e.g., from about 9-and/or amino acid analogue, about 9-about 40 aminoacid and/or aminoacid About 30 aminoacid of analog, about 9-and/or amino acid analogue or about 9-about 20 aminoacid and/or amino acid analogues. Peptide composition can containing about the 9 of such as MUC1 protein sequence, 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 aminoacid and/or amino acid analogues, or these quote any scope of size.

MUC1 sequence can include display with people's MUC1 sequence 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 the sequence of about 99% sequence iden.

MUC1 sequence can include any MUC1 sequence described herein, such as include but not limited to Figure 16,19,33A, Any one in those of representing in 33B and 34.Such as MUC1 sequence can include SAPDTRPAP (SEQ ID NO:20), TSAPDTRPAP (SEQ ID NO:21)、SAPDTRPL (SEQ ID NO:22)、TSAPDTRPL (SEQ ID NO:23)、 APGSTAPPAHGVTSA (SEQ ID NO:26)、APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27)、 APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:29), its Middle X be L, A or AP SKKKKGAPGSTAPPAHGVTSAPDTRPX (SEQ ID NO:30), SKKKKGSTAPPAHGVTSAPDTRPAP (SEQ ID NO:31)、SKKKKGSLSYTNPAVAAATASNL (SEQ ID NO: 32)、SKKKKGCKLFAVWKITYKDTGTSAPDTRPAP (SEQ ID NO:33)、SKKKKGCKLFAVWKITYKDT (SEQ ID NO:34), GGKLFAVWKITYKDTGTSAPDTRPAP (SEQ ID NO:35) or APGSTAPPAHGVTSAPDTRPAP (SEQ ID NO:28).Also included is such MUC1 sequence, and it has about 50% with these sequences, 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 iden. Also included is glycosylated in any combination of, two, three, four or more serines or threonine residues MUC1 sequence.

Lipid composition

It is assumed initially that the glycopeptide with the only two kinds i.e. carbohydrate ingredients of key component and peptide composition can effectively cause in animal Immunne response.Helper T cell epitope expection inducing T cell dependent immune response, causes for tumor associated carbon hydrate Thing B epitope such as Le^yGeneration with the IgG antibody of Tn.But, in some applications, do not find that two component vaccines are to have very much Effect.Speculate that B cell and helper T cell epitope lack and suitable " danger signal " for dendritic cell (DC) maturation are provided Ability.In order to remedy this problem, lipid composition is included in compound, results in the glycolipidpeptide of the present invention.

Lipid composition can be any containing lipid composition, such as lipopeptid, fatty acid, phospholipid, steroid or lipidization amino Acid and glycolipid such as lipid A derivative.Preferably, lipid composition is nonantigenic；That is, it does not cause for lipid composition The antibody of given zone.But, lipid composition can be and the most really serve as immunological adjuvant.Lipid composition can serve as multi-epitope The carrier of glycolipidpeptide or delivery system.It helps glycolipidpeptide to mix in vesicle or liposome, thin to promote that glycolipidpeptide is delivered to target Born of the same parents, and the picked-up of its intensifier target cell such as dendritic cell.The generation of lipid composition stimulating cytokine further.

A preferred lipid composition of class for using in the glycolipidpeptide of the present invention comprises multiple Toll-like receptor (TLR) Molecule ligand.Exist Toll-like receptor the known subclass of many (such as TLR1, TLR2, TRL3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, TLR15 and TLR16).About between Toll-like receptor Relation and the summary of evolution, see Roach et al., PNAS 2005,102:9577-9582；About the TLR in vaccination The discussion of signal transduction, sees Duin et al., TRENDS Immunol., and 2006,27:49-55.

TLR is the pattern recognition receptors family activated by the specific components of microorganism and some host molecule.They are constituted For the First Line defence of many pathogen, and play a crucial role in congenital immune function.Mammal In TLR first identified in 1997, and estimated that most of mammalian species has ten to ten five class Toll-like receptor. Known TLR includes: TLR1 (TLR1 part includes three acyl group lipoproteins)；(TLR2 part includes lipoprotein, gram sun to TLR2 Property Peptidoglycan, lipoteichoic acid, fungus and viral glycoprotein)；(TLR3 part includes as double in find in some virus TLR3 Chain RNA, and poly-I:C)；TLR4 (TLR4 part includes lipopolysaccharide and viral glycoprotein)；(TLR5 part includes flagellum egg to TLR5 In vain)；TLR6 (TLR6 part includes diacyl lipid albumen)；(TLR7 part includes little synthesis immunity modifying agent (such as to TLR7 Imiquimod, R-848, loxoribine and bropirimine) and single stranded RNA)；(TLR8 part includes little synthesis compound to TLR8 And single stranded RNA)；With TLR9 (TLR9 part includes unmethylated CpG DNA motif).See for example, 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；The summary of 85 (2): 85-95.

The lipid composition particularly preferably interacted with TLR2 and TLR4.TLR2 relates to from Gram-positive and leather The identification of the huge variety of microbial molecules of Lan Shi negative bacterium and mycoplasma and yeast.TLR2 part includes lipopolysaccharide, fat Polysaccharide, lipoteichoic acid and Peptidoglycan.TLR4 identifies Gram-negative lipopolysaccharide (LPS) and its toxin part of lipid A.TLR Part is such as extensively obtained commercially from Apotech and InvivoGen.Preferably, lipid composition is to promote that glycolipidpeptide passes through The TLR part (seeing embodiment 3) of the picked-up of antigen-presenting cell.

For including PamCys type lipid conformation as the suitable lipid of the lipid composition of the glycolipidpeptide of the present invention, such as, spread out It is conigenous Pam₃Cys (S-[(R)-2,3-bis-palmityl epoxide-propyl group]-N-palmityl-(R)-cysteine) and Pam₂Cys (S- [(R)-2,3-bis-palmityl epoxide-propyl group]-(R)-cysteine) those, Pam₂Cys lacks Pam₃The N-palmityl of Cys Base.Pam₃Cys and Pam₂Cys is derived from the immunocompetence N-terminal sequence of colibacillary major lipoprotein.This lipoids also wraps Include Pam₃CysSK₄ (N-palmityl-S-[(R) double (palmityl the epoxide)-propyl group of-2,3-]-(R) cysteinyl-(S)-silk ammonia Acyl-(S)-lysine-(S)-lysine-(S)-lysine-(S)-lysine) and Pam₂CysSK₄ (S-[(R) double (palm fibre of-2,3- Palmitic acid acyloxy)-propyl group]-(R) cysteinyl-(S)-seryl-(S)-lysine-(S)-lysine-(S)-lysine-(S)- Lysine (lysyne)), it lacks Pam₃The N-palmityl of CysSK4；It is to be understood that lysine number in these structures Can be 0,1,2,3,4,5 or more (i.e. K_n, wherein n=0,1,2,3,4,5 or more).In some embodiments, lipid Component includes one or more lipid chain, one or more cysteine residues and one or more lysine residue.

The lipid of another preferred classes includes the lipid of lipid A (LpA) type, such as derived from escherichia coli, Mus wound Cold Salmonella (S. typhimurium) and the lipid A of Neisseria meningitidis.Lipid A can be attached to sugar by joint The carbohydrate ingredient (containing B epitope) of lipopeptid and/or peptide composition (containing T epi-position), described joint is such as connected to different head Center or different head phosphate ester, C-4 ' phosphate ester or C-6 ' position.Phosphate ester can be modified such as to include one or more phosphoric acid Ethanolamine diester.Exemplary lipid A derivative is at such as Caroff et al., 2002, Microbes Infect；4:915-926； Raetz et al., 2002, Annu Rev Biochem；71:635-700；With Dixon et al., 2005, J Dent Res；84: Described in 584-595.

In some embodiments, lipid composition is lipidated amino acid.In some embodiments, lipid TLR2 is exciting The lipid aspect of agent component is replaced by different classes of adjuvant compound, and such as TLR4 agonist, TLR7 agonist, TLR8 are exciting Agent or TLR9 agonist.In some embodiments, agonist is TLR9 agonist CpG.

It it is hereafter the illustrative immunogenic lipid in the glycolipidpeptide for mixing the present invention in scheme 8.In the first row In first structure be Pam₃CysSK_n；Second structure in the first row is Pam₂CysSK_n；And last 4 structures It it is lipid A derivative.

Scheme 8

Structurally based on Pam₃The lipid of Cys is for being particularly preferred as lipid composition.Pam₃Cys is derived from large intestine bar The immunocompetence of the major lipoprotein of bacteriumNEnd sequence.These lipopeptids are strong immunological adjuvants.Recent research shows Pam₃Cys is by playing its activity with the interaction of Toll-like receptor 2 (TLR2).

Without being bound by theory, it is believed that the interaction between lipid composition and TLR causes proinflammatory cytokine and chemotactic The generation of the factor, itself and then stimulator antigen are delivery cell (APC), thus initial helper T cell is grown and activates.TLR part and B Covalently bound with T epi-position guarantees to produce on the site that cytokine vaccine wherein and immunocyte interact.This causes The high local concentrations of cytokine, promotes the maturation about immunocyte.Lipopeptid promotes antigen-presenting cell and bone-marrow-derived lymphocyte Selectivity targeting and picked-up.It addition, lipopeptid promotes that glycolipidpeptide mixes in liposome.Liposome is as the carrier in vaccine design Attracting to pay close attention to, this is due to its low inherent immunity originality, thus avoids the immunne response that undesirable carrier induces.

The immunogenic vaccine of the present invention can such as be connected by chemo-selective, more particularly native chemical connects (NCL) synthesis, as described in WO 2007/146070 and the open 2009/0196916A1 of United States Patent (USP).In short, vaccine One or more individual components embed in lipid conformation or dissolve, described lipid conformation such as lipid monolayer, double-layer of lipoid, fat Plastid, micelle, thin film, emulsion, substrate or gel.The reactant used in coupled reaction can include carbohydrate group Point, peptide composition, lipid composition or its conjugate or combination.Design or select these reactants to comprise required antigenicity or immunity The T epi-position of the immunogenic vaccine of originality feature, the such as present invention or B epitope.

Optionally joint

One or more joints (" L ") are optionally used for the assembling of three kinds of components of glycolipidpeptide.In one embodiment, joint is Bifunctional linker, it has the functional group (preferably on first and second end) in two diverse locations, in order to will Two kinds in three kinds of components are covalently joined together.Bifunctional linker can be that congenerous (i.e. contains the sense of two equivalents Group) or (i.e. the containing two different functional groups) of exclusive-OR function.In another embodiment, joint is three functions (different merits Energy or congenerous), and all three component of glycolipidpeptide can be linked together.Suitably functional group has for following In the reactivity of any one or comprise following in any one: amino, alcohol, carboxylic acid, sulfydryl, olefine, alkynes, Azide Thing, thioesters, ketone, aldehyde or hydrazine.Aminoacid such as cysteine may be constructed joint.

Bifunctional linker illustrates in scheme 9.

Scheme 9

Fig. 1 shows the glycolipidpeptide of the exemplary completely synthetic present invention, and it contains B epitope based on carbohydrate, peptide T table Position and lipopeptid.Compound shown in Fig. 1 contains the L-glycero-D-manno-heptose serving as B epitope, has been identified as human T-cell's MHC II class limits recognition site and the peptide sequence of the external membrane protein derived from Neisseria meningitidis YAFKYARHANVGRNAFELFL (SEQ ID NO:2), and lipopeptid S-2-3 [two palmityl epoxides]-(R/S)-propyl group-N-palm fibre Palmitic acid acyl-R-cysteine (Pam₃Cys).As elsewhere herein is pointed out, lipopeptid Pam₃Cys and have related compounds Pam₃CysSK₄It is highly effective B cell and macrophage activation thing.

As illustrated in embodiment, the method preparing glycolipidpeptide is also included by the present invention.Preferably, it is used for preparing glycolipid The method of peptide utilizes chemosynthesis, results in completely synthetic glycolipidpeptide.In the embodiment utilizing one or more joint In, optional linker component is functionalization, in order to promote alternative covalently bound with key component of one of key component. Such as, joint can use thiol reactant group such as maleimide or acetyl bromide functionalization on each end, and will Component to be connected is modified to include reactive mercaptan.For connect chemistry other options include native chemical connect, Staudinger connects and Huisgen connects (also referred to as " click chemistry ").Embodiment 2 illustrates carbohydrate ingredient How (being oligosaccharide in that case) and peptide composition are can be with containing thiol linker functionalization.Preferably, if you are using, then Linker component is nonantigenic.

The glycolipidpeptide of the present invention can generate immunne response in mammal.Glycolipidpeptide is antigenic, because it can To generate humoral response, cause activation and the generation of antibody (immunoglobulin) such as IgM of B cell.It addition, glycolipidpeptide is to exempt from Epidemic focus, because it can be with cellulation response；Such as, it promotes the activation of T cell particularly helper T cell, and T cell exists The generation of the more complicated antibody response including the generation of IgG is also helpful to.Finally, the immunity caused in animal Response includes the generation of anti-carbohydrate antibodies.

In another embodiment of the present invention, immunogenic vaccine is two component vaccines, and it comprises covalently bound At least one peptide composition and at least one adjuvant component.Peptide composition includes T epi-position, the auxiliary T epi-position that preferably MUC1 originates from, including But be not limited to described herein in those of any one.Although this embodiment of vaccine may not generate for specific B The specific immunity of epi-position, but it demonstrates antitumor character.The example of two component vaccines is to be covalently attached to auxiliary T epi-position Pam3CysSK4；See for example, the compound 3 in embodiment 8.In one embodiment, the adjuvant of immunogenic vaccine Component comprises toll sample receptor (TLR) part.At least 15 kinds of different mammal TLR be known (such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14 and TLR15), and Its part demonstrates that significant structure changes.Some TLR parts are described herein as, it is to be understood that this type of list is not with any Mode limits the present invention.In some embodiments, two component immunogenic vaccine can be formulated for using as compositions, Described compositions comprises other reagent, such as immunomodulator, adjuvant, TLR agonist and/or excipient further.TLR joins Body is that technical staff is well-known.They can take lipopeptid, glycolipid, lipoprotein, carbohydrate, little organic molecule, core Acid the most single or double chain DNA or the form of RNA, and known many TLR parts serve as immunostimulant.Immunostimulating TLR One example of part is TLR2 part, include but not limited to described herein in those of any one.Another example is logical It is frequently referred to the TLR9 part of " CpG ".This compound is the immunostimulatory oligodeoxynucleotides (ODN) containing CpG motif. CpG motif is identified as part (Rothenfusser et al., 2002, Human immunology 63 (12): 1111-by TLR9 1119).Preferably, CpG ODN is unmethylated.CpG ODN is short single strand dna, and it contains cytosine (" C " core Thuja acid) it is guanine (" G " nucleotide) subsequently." p " refers generally to the phosphodiester backbone of DNA.Optionally, CpG motif can be repaiied Decorations are for containing thiophosphate (PS) main chain, in order to protection ODN not by the degraded of nuclease such as DNA enzymatic (Dalpke et al., 2002, Immunology 106 (1): 102-12).The common length range of CpG ODN is about 18 nucleotide to about 28 core Thuja acid.Optionally, they contain palindrome.One example of the CpG for using in the present invention is 5'- TCCATGACGTTCCTGACGTT-3' (SEQ ID NO:36).The CpG motif being present in vertebrates DNA is owing to transcribing tune The most methylated (Sulewska et al., 2007, Folia Histochemica et caused by joint mechanism Cytobiologica 45(3):149-158).Unmethylated CpG motif has shown and has served as immunostimulant (Weiner etc. People, 1997, Proc. Natl. Acad. Sci. USA, 94:10833-10837).CpG has been used in research to strengthen 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., 2005, J. Clin. Oncol. 2005 ASCO Annual Meeting Proceedings. 23(16S):7039)。

Many CpG ODN are obtained commercially.Such as, CpG ODN can pass through as A type, Type B or c-type molecule InvivoGen (San Diego, CA) buys.These classifications based on architectural difference and immunostimulatory activity thereof (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., 2006, Immunology 120:526-535).

In another embodiment, the adjuvant component of immunogenic vaccine is that lipid composition as described herein (is also joined See that WO 2007/079448, United States Patent (USP) disclose 2009/0041836 A1 and WO 2010/002478).Some TLR parts are such as The part of TLR2 also constitutes lipid composition, but the lipid composition of immunogenic vaccine is not limited to TLR part；I.e. lipid composition can May function as any suitable immunogenicity or the antigenic lipids of adjuvant, such as lipidated amino acid (" LAA ").

In yet another aspect, the glycolipidpeptide of the present invention is used for producing polyclone or monoclonal antibody, and it identifies carbon hydrate Any one or both in thing component and peptide composition.The present invention comprises the method preparing described antibody, and antibody self and producing The hybridoma of the monoclonal antibody of the raw present invention.

The immunogenicity glycolipidpeptide of the present invention for using in producing antibody can contain any carbon described herein Hydrate component, and unrestricted.Preferably, it contains glycopeptide as its carbohydrate ingredient.Glycopeptide includes glycosylated peptide Sequence, it includes that carbohydrate portions is the most sugared.Sugar can be monosaccharide, oligosaccharide or polysaccharide.Preferably, for generating antibody The carbohydrate ingredient of glycolipidpeptide contains autoantigen as above.Advantageously, even if carbohydrate ingredient is the most sugared Peptide is poor antigen (such as autoantigen), and carbohydrate ingredient also produces with peptide composition and the covalently bound of lipid composition Notable immunogenic glycolipidpeptide.

In conjunction with the antibody of the present invention of glycolipidpeptide preferably in combination with B epitope, it includes sugar moieties, and in preferred embodiment Include peptide at least some of forming glycopeptide.Preferably antibody is combined with the glycopeptide as carbohydrate ingredient, but not Be combined with single de-glycosylation peptide or saccharide residue.

When being used for generating antibody, the glycolipidpeptide of the present invention is successfully generated high-affinity IgG antibody.This is weak anti-for having The embodiment of the glycolipidpeptide of immunogenic carbohydrate component such as autoantigen is the most astonishing and unexpected.Many Therefore clone or monoclonal antibody are preferably IgG isotype antibody.Without being bound by theory, it is believed that the glycolipidpeptide of the present invention is excellent Antigen (compared with non-lipid glycopeptide), because the local of its stimulating cytokine produces, raises stimulating protein altogether, strengthens By macrophage and the picked-up of dendritic cell and/or avoid epi-position to suppress.

The antibody of the present invention includes but not limited to those of identification B epitope, and described B epitope containsO-GlcNAc、O- GalNAc、O-mannose or other are sugar-modified.Can be included containing glycosaminoglycans by other B epitope of the antibody recognition of the present invention Those of fragment, glycosaminoglycans such as heparin, heparitin sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, acetyl Hyaluronic acid and general any glycosaminoglycans.In the case of by repeating the glycosaminoglycans that disaccharide unit is formed, B epitope can With containing one or more disaccharide unit.Pentose, hexose can be contained by the B epitope of the antibody recognition of the present invention or include acid Other sugar moieties, include but not limited to glucuronic acid, iduronic acid, hyaluronic acid, glucose, galactose, galactosamine, Portugal Osamine etc..The antibody of the present invention is preferably used the glycolipidpeptide of the present invention and produces as immunogen, and wherein carbohydrate ingredient contains There is purpose B epitope.The analog of naturally occurring B epitope such as contain N connection or S connection structure those or sugar analogies, permissible As carbohydrate ingredient, such as so that glycolipidpeptide immunogen more metabolic stability.

The antibody using the glycolipidpeptide of the present invention to produce advantageously comprises high-affinity IgG antibody, and it identifies wide spectrum sugar egg In vain.Therefore, even if using the antibody that the glycolipidpeptide of the present invention produces as immunogen for the sugar as carbohydrate ingredient Peptide is specific, and they can also be in conjunction with wide spectrum glycoprotein.For the glycosylated peptide containing purpose B epitope component or protein There is relatively extensively antibody selective and be referred to herein as " general specificity " antibody.The polyclone of the present invention or monoclonal anti Body can be general specificity or site-specific.Such as this term it is used in the present context, general specific antibody is such anti- Body, B epitope selected by its specific recognition such as containsOThe B epitope of-GlcNAc, but for the protein containing this B epitope and peptide There is relatively wide selectivity.General specific antibody is therefore, it is possible to combine the multiple different glycosylation containing purpose B epitope Protein or peptide, although it not necessarily combines all glycosylated proteins containing selected B epitope or peptide.

Do not expect bound by theory, glycosyl can be shared in by the different sugar albumen of the general specific antibody identification of the present invention Change essentially similar or (sugared) peptide sequence (i.e. primary sequence) of equivalent on site or two grades or three grades essentially similar knots Structure, thus cause wide spectrum to combine target by this antibody recognition.In combination by antibodyOThe glycoprotein that-GlcNAc modifies is shared Two grades or three grades of epitopic structures can be advantageously maintained in glycolipidpeptide immunogen, as by the IgG antibody identifying wide spectrum glycoprotein Successful generation prove.

Preferably, the antibodies of the present invention has and comprisesO-GlcNAc、O-GalNAc or other sugar-modified epi-positions Multiple glycosylated protein or peptide, but can not detectably combine protein or the peptide not containing this sugar.It is highly preferred that antibody knot Conjunction has and comprisesO-GlcNAc、O-GalNAc or the protein of other sugar-modified epi-positions or peptide, but will not detectably combine Do not containO-GlcNAc、O-GalNAc or other sugar-modified same protein or peptide.

The preferably example of polyclone or monoclonal antibody is to combine to containOThe polyclone of the glycopeptide of-GlcNAc monosaccharide residue Or monoclonal antibody.In particularly preferred embodiments, antibody have forOThe protein that-GlcNAc modifies is the most extensive Selectivity.Such as, many destination protein matter have byOTPVSS (the SEQ ID NO:10) sequence that-GlcNAc modifies, and Preferably this and/or similar glycosylated peptide sequence of monoclonal antibody identification.RightOIt is sequence-specific excellent that-GlcNAc modifies The example of selected monoclonal antibodies includes the Dan Ke produced by hybridoma cell line 1F5.D6,9D1.E4,18B10.C7 and 5H11.H6 Grand antibody.These monoclonal antibodies use compound 52 and/or 53 to produce as immunogen.Therefore, in one embodiment, The antibodies compound 52 of the present invention or the carbohydrate ingredient of compound 53.Hybridoma cell line 1F5.D6,9D1.E4 It is preserved in American type culture collection (ATCC), 10801 University on July 1st, 2008 with 18B10.C7 Blvd., Manassas, VA, 20110-2209, USA, and respectively specify that ATCC preserving number PTA-9339, PTA-9340 and PTA-9341.The present invention includes hybridoma cell line and monoclonal antibody produced by them.

Preferably another example of polyclone or monoclonal antibody is combine heparitin sulfate fragment that.

It is to be understood that there is any carbohydrate of clinical meaning or interest or glycopeptide can be as the glycolipid of the present invention The carbohydrate of peptide and/or peptide composition mix, and generate polyclone and monoclonal antibody for the method according to the invention.This Class carbohydrate and peptide include those with medical science and veterinary's interest, and have other business or research application that A bit.Be to be understood that the monoclonal of the present invention and polyclonal antibody are not limited to identify those of any particular ligand, but include and It is not limited to and is only used as example, for any kind of tumor related carbohydrate antigen (TACA) with derived from any micro-life The antibody of any sugar of thing.

In general, the monoclonal antibody using the glycolipidpeptide of the present invention to prepare the present invention is producing for its carbohydrate Or glycopeptide antigen to have in the monoclonal IgG antibody of high-affinity be effective surprisingly, even if when antigen is poor antigen Property time.This opens and produces for research, diagnose and treat immune related disorders or have autoimmune or inflammatory component The gate of the antibody that disease is useful, described disease includes cancer, type ii diabetes, allergy, asthma, Crow grace (Crohn' S) disease, Alzheimer, muscular dystrophy, microorganism infection etc..Such as identifyOThe basis of the glycoprotein that-GlcNAc modifies The monoclonal antibody of invention is far superior to the antibody being obtained commercially, such as CTD110.6 (Covance Research Products, Inc.).The glycolipidpeptide of the present invention can use module synthesis to assemble, wherein lipid, peptide and carbohydrate Component selects according to required application.Additionally, the glycolipidpeptide of the present invention is for producing the most general spy of general specific antibody The notable effective antigen of specific monoclonal IgG antibody, described antibody recognition containsOReceipts or other documents in duplicate sugar is such asOThe glycosylation of-GlcNAc Peptide and protein.

The antibody of the present invention and produced by the method for the present invention those be for identify and characterize protein, peptide and with The important research instrument of the other biological molecule that various disease states is relevant.Such as, the general specific antibody of the present invention can be used In from acquisition (pull down) glycoprotein of complex biological sample.This method may be used for detection and identify heretofore unknown with The protein that particular condition or morbid state are correlated with, thus identify potential treatment or diagnosis target.In one embodiment, originally The antibody of invention can make the antibody can be in conjunction with multiple glycosylated protein or glycosylated peptide and detect antibody-protein knot Contact with biological sample under conditions of conjunction.Optionally, the method can include separating glycosylated protein or glycosylated peptide.The party Method may further include one or more protein or peptide identified in multiple glycosylated protein or glycosylated peptide.Glycosyl Change the qualification of protein and peptide to provide and probe into glycosylated effect and machine that the biology in multiple bioprocess involves Meeting.Such as, the glycosylation of protein or peptide can relate to many bioprocesss, includes but not limited to transcribe, translates, signal turns Lead, the direct motion transport of ubiquitin approach, intracellular vesicles and post translational modification (such as SUMOization and phosphorylation).For identifying The method of protein or peptide is well-known in the art, and can include but not limited to such as mass spectrography and Ai Deman (Edman) technology such as degraded.

The general specific antibody of the present invention can be used for identifying the glycosylated albumen in morbid state with change Matter or peptide.O-GlcNAc modifies relevant to various disease states.Such as, in skeletal muscle and pancreas glycopeptideO-GlcNAc modifies Increase and associate with the development of type ii diabetes, and in neural glycopeptideOThe minimizing that-GlcNAc modifies and Alzheimer Outbreak association (Dias and Hart；Mol. BioSyst. 3:766-772(2007)).Therefore, existOThe level that-GlcNAc modifies The detection of the change of aspect can serve as diagnosis or prognostic tool.It addition, the glycosylation state of this proteinoid or peptide can be with Morbid state associates.For identifying that having the method changing glycosylated protein or peptide associated with morbid state includes making this Antibody incubation together with first biological sample with known morbid state of invention, and making antibody can be combined in first Under conditions of multiple glycosylated protein in sample and peptide and the multiple glycosylated protein in the second sample and peptide, make to resist Body is incubation together with second biological sample with non-diseased state, independently from described sample separate glycosylated protein and Glycosylated peptide, and identify described glycosylated protein and glycosylated peptide.The method may further include and compares at the first sample The glycosylated protein identified in product and glycosylated peptide and glycosylated protein in the second sample and glycosylated peptide, Qi Zhongxian Show that protein or the peptide of the change in the glycosylation state between the first and second samples indicate glycosylated protein or glycosyl Change peptide relevant to morbid state.Association between glycosylation and morbid state include relative to non-diseased state have increase or The glycosylated morbid state reduced.Additionally, morbid state can demonstrate glycosylation, and non-disease conditions shows glycosylated It is completely absent, or on the contrary, morbid state can show glycosylated being completely absent, and demonstrate glycosylated without disease Exist.In each example, protein or peptide are considered as the glycosylation in morbid state with difference or change.Use the present invention The method of the glycosylated change existed in situation or process LAN and detection level of glycosylation of antibody test the most retouched State.

The antibody of the present invention is widely useful in diagnosis or treatment use, as elsewhere herein is more fully described 's.Two or more different biological samples can be carried out by comparative analysis.Such as, mass immunization precipitation can be to treatment The sample intervening front and rear performs, or performs as time go by, with the progress of monitoring disease, or compare normal specimens with from Suspect the sample of the patient suffering from the disease of the change being characterised by protein glycosylation, infection or disease.

In one embodiment, the method that the present invention includes diagnosing the existence situation of the morbid state in experimenter.Should Method includes making the biological sample incubation together with the antibody of the present invention from experimenter, and detects antibody and at morbid state In there is the protein of differential glycosylation or the combination of peptide.The method of detection antibodies is described the most.Sugar wherein In the case of base is completely absent in morbid state, antibody indicates experimenter to have with the shortage of protein or the combination of peptide Morbid state.During glycosylation is present in morbid state wherein but in the case of being completely absent in non-disease conditions, antibody Combination with protein or peptide indicates the existence of morbid state in experimenter.Optionally, the method may further include and makes The biological sample of two non-diseased incubation together with the antibody of the present invention, detection antibody and protein or the combination of peptide, and compare The relatively antibodies in first and second sample.

It addition, be present in morbid state and non-disease conditions for wherein glycosylation, but change in morbid state Becoming protein and the peptide of (being i.e. increased or decreased), the method may further include the antibodies water in quantitative first sample Flat, the antibodies level in quantitative second non-diseased sample, and relatively described combination level.Compared with non-diseased sample Relatively, infection in the change instruction experimenter of the antibodies in first sample, disease or the existence situation of disease.

Preparation for the antibody of the present invention, it is possible to use provide and produce antibody molecule by the continuous cell line in cultivating Any technology.It is, for example possible to use initially developed by Kohler and Milstein (256 Nature 495-497 (1975)) Hybridoma technology.Referring further to Ausubel et al., Antibodies:a Laboratory Manual, (Harlow & Lane Editor, Cold Spring Harbor Lab. 1988)；Current Protocols in Immunology,(Colligan Et al., editor, Greene Pub. Assoc. & Wiley Interscience N.Y., 1992-1996).

Present invention also offers generation monoclonal antibody, preferred pin and its antigen is had the list of high degree of specificity and affinity The hybridoma cell line of clonal antibody.The present invention farther includes variant and the mutant of hybridoma cell line.This type of cell line Known method can be used artificially generated and still there are raw-material ins and outs.Such as, they can remain able to produce root According to the antibody or derivatives thereof of the present invention, and it is secreted in surrounding medium.Optionally, hybridoma cell line can send certainly Existing.The clone and subclone of hybridoma cell line is interpreted as hybridoma, and it is produced by initial clone by repeatedly cloning, and still There is the principal character of initial clone.

Antibody can be by causing in animal reservoir by the glycolipidpeptide immunity inoculation of the present invention, maybe can be thin by immunity Ion vitro immunization inoculation (sensitization) of born of the same parents is formed.Antibody can also produce in recombination system, and wherein suitably cell line is with suitable Antibody coding DNA carry out converting, transfect, infect or transduceing.Alternately, antibody can be by the heavy chain of purification and light chain Biochemistry builds.

Once antibody molecule is by animal generation, chemosynthesis or recombinant expressed, and it just can be by use known in the art Any method in purifying immunoglobulin molecule is purified, and such as by chromatography, (such as ion exchange, affinity are particularly By to the affinity of specific antigen and sub-sieve column chromatography after protein A), centrifugal, differential solubility or by for purification egg Any other standard technique of white matter.Additionally, the antibody of the present invention or its fragment can be fused to allos known in the art many Peptide sequence, to promote purification.

In preferred embodiments, monoclonal antibody identification and/or combination are present in the carbon aquation of glycolipidpeptide of the present invention Antigen on polymer component or peptide composition.In particularly preferred embodiments, monoclonal antibody combines and is present in carbon hydrate Antigen in the selected feature of thing component.The modification that the example of selected feature is included on glycopeptide is such asO-GlcNAc.Other are repaiied Decorations include but not limited to GalNAc and other are sugar-modified.

Term " antibody " uses with broadest sense, and contains monoclonal antibody (including full length monoclonal antibodies) especially And antibody fragment, as long as they demonstrate required biological activity." antibody fragment " comprises the part of full length antibody, and generally it resists Former land or variable region.The example of antibody fragment includes but not limited to Fab, Fab' and Fv fragment；Double antibody；Linear antibodies； And single-chain antibody molecules.As used herein term " monoclonal antibody " refer to high degree of specificity, for single antigen site Antibody.Term " antibody " also includes the antibody of naturally occurring antibody and non-naturally-occurring as used herein, including such as Single-chain antibody, chimeric, difunctional and humanized antibody, and its Fab.The antibody of this type of non-naturally-occurring is permissible Use Solid phase peptide synthesis to build, can recombinate and produce and can be such as made up of variable heavy chain and variable light by screening Combinatorial library obtain, as described in by Huse et al. (Science 246:1275-1281 (1989)).These and other prepare The method of function antibody be well known to the skilled person (Winter and Harris, Immunol. Today 14: 243-246(1993)；Ward et al., Nature 341:544-546 (1989)；Harlow and Lane, ibid, 1988)； Hilyard et al., Protein Engineering:A practical approach (IRL Press 1992)； Borrabeck, Antibody Engineering, second edition (Oxford University Press 1995)).

In all mammalian species, antibody peptide contains constant (i.e. high conservative) district and variable region, and rear In person, there is complementary determining region (CDR), and be made up of the aminoacid sequence in the variable region of weight or light chain but outside CDR So-called " framework region ".Preferably, the monoclonal antibody of the present invention has been humanized.As used herein, term " people source Change " antibody refers to that the most inhuman (usually from mice or rat) CDR transfers to from weight and the light variable chains of non-human immunoglobulin Antibody in following variable region, many aminoacid that described variable region is designed as containing finding in the framework region in human IgG are residual Base.The similarly transformation of mice/people's chimeric antibody to humanized antibody is described the most.Can for cloning murine immunoglobulin The general technology in structure changes territory is such as by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989) Publication describe, described document is integrally incorporated with it by quoting.For the technology of generating humanized MAbs such as 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., J. Immun. 150:2844 (1993) describes, and described document is each It is incorporated herein by reference.

The method using the monoclonal antibody of the component identifying and/or combining glycolipidpeptide is also comprised by the present invention.About this The purposes of the monoclonal antibody of invention includes but not limited to diagnosis, treats and study purposes.In preferred embodiments, monoclonal Antibody may be used for diagnostic purpose.BecauseO-GlcNAc modifies relevant to various disease states, soOThe water that-GlcNAc modifies The detection of the change in Ping can be construed to the early stage indicant of this type of seizure of disease.Such as, in skeletal muscle and pancreas glycopeptideOThe increase that-GlcNAc modifies associates with the development of type ii diabetes, and in neural glycopeptideOThe minimizing of-GlcNAc modification and Ah Outbreak association (Dias and Hart, the Mol. BioSyst. 3:766-772 (2007) of Er Cihai Mo's disease；Lefebvre et al., Exp. Rev. Proteomics 2(2):265-275(2005)).Accordingly, with respect to non-diseases control sample, identify at skeleton In the sample of muscular tissueOThe increase of-GlcNAc amount may indicate that the development of type ii diabetes.

It is to be understood that the monoclonal of the present invention and polyclonal antibody are not limited to identify those of any particular ligand, but bag Include and be not limited to and be only used as example, for any kind of tumor related carbohydrate antigen (TACA) with derived from any The antibody of any sugar of microorganism.The antibody of the present invention is widely useful in diagnosis or treatment use.

The antibody of the present invention may be used for detecting specified protein or the existence situation of specific modification or process LAN.For examining Survey technology be it known in the art, and include but not limited to Western blotting, Dot blot, immunoprecipitation, coagulation, ELISA measures, immunity ELISA mensuration, imaging of tissue, mass spectrography, immunohistochemistry and to Various Tissues or the streaming of body fluid Cell art, and multiple sandwich assay.See for example, the U.S. Patent number 5,876,949 being incorporated herein by reference.

In order to detectOThe change of the level of the glycopeptide that-GlcNAc modifies, the monoclonal antibody of the present invention can by many Know the covalently or non-covalently labelling of any one in detectable label, described detectable label such as fluorescence, radioactivity or enzymatic thing Matter, as known in the art.Alternately, secondary antibodies special for the monoclonal antibody to the present invention is detected with known Labelling is marked, and for detecting in the above-described techniquesO-GlcNAc specific antibody.

Preferably detectable label includes colored dye.AEC (AEC) is had in those of the most frequently used With 3,3 '-diaminobenzidine four hydrochlorate (DAB).These can use light microscopy to detect.Further preferably It it is fluorescent labeling.Fluorescein isothiocyanate (such as FITC and TRITC), indole three is had in the most frequently used fluorescent labelling compound Carbocyanine (Idotricarbocyanines) (such as Cy5 and Cy7), rhodamine, phycoerythrin, phycocyanin, other algae indigo plant egg In vain, phthalic aldehyde and fluorescamine.Can also use CL and BL compound, such as luminol, different luminol, Theromatic acridineEster, imidazoles, acridineSalt, oxalate, luciferin, luciferase and aequorin.When glimmering When the antibody of signal is exposed to the light of suitable wavelength, owing to its fluorescence can detect its existence.Further preferably put Penetrating property labelling.The useful especially radiosiotope of antibody for the labelling present invention includes³H、¹²⁵I、¹³¹I、³⁵S、³²P and¹⁴C。 Radiosiotope can be detected by this type of method, as used gamma counter, scintillation counter or the most aobvious by radiation Shadow art.May be used for detectable label antibody and can such as pass through spectrophotometer measurement, fluoremetry or visual means inspection The enzyme surveyed includes but not limited to malic dehydrogenase, staphylococcal nuclease, δ-5-steroid isomeras, yeast-alcohol dehydrogenation Enzyme, α-glycerophosphate dehydrogenase, phosphotriose isomerase, horseradish peroxidase, alkali phosphatase, asparaginase, Fructus Vitis viniferae Carbohydrate oxidase, beta galactosidase, ribonuclease, urease, catalase, zwischen-ferment, glucose starch Enzyme and acetylcholinesterase.The additive method of labelling and detection antibody is known in the art and within the scope of the invention.

Including the present invention of TLR agonist, t helper cell epi-position and glycosylation MUC1 epi-position (B/T cell epitope) three Component immunogenic vaccine demonstrates many advantages.Glycosylation B/T cell epitope can be more more effective than non-glycosylated epi-position. The strong cytolytic T cell response that vaccine causes, the cell of MUC1 is expressed in cracking.Generally thin by CD4+ and CD8+ T The activation of the interferon gamma secretion instruction T cell cell response of born of the same parents.Further, the activation of B cell response is turned by Ig classification Change and ADCC (antibody dependent cellular mediation thin of cell (tumor cell and YAC cell) at abduction delivering MUC1 Cellular toxicity) generation of the effective antibody of aspect indicates.Therefore, three component immunogenicity cancer vaccines based on MUC1 are dual draws Send out humoral and cellular immune response response, produce and cellular cytoxicity activity including antibody formation, interferon gamma, it is thus achieved that excellent treatment knot Really.In some embodiments, the interpolation of the second TLR agonist increases effectiveness further, such as, demonstrate the swollen of minimizing The cytotoxicity that tumor load, the IFN-γ generation of increase and the T cell increased mediate.

The present invention includes by with one or more immunogenic vaccine construct immunizing subjects described herein, The method generating the cytotoxicity (ADCC) of antibody dependent cellular mediation in experimenter.In some respects, ADCC is natural Kill (NK) cell-mediated.In some respects, ADCC cracks tumor cell.In some respects, tumor cell is that breast carcinoma is thin Born of the same parents or epithelial cancer cells.In some respects, the cell of MUC1 peptide sequence is expressed in ADCC cracking.In some respects, MUC1 peptide is different The most glycosylated.

The present invention includes by with one or more immunogenic vaccine construct immunizing subjects described herein, In experimenter, treat cancer, reduce tumor load, prophylaxis of tumours recurrence and/or the method for prophylaxis of cancer.Side in the present invention Some aspects of method, cancer or tumor are breast carcinoma or epithelial cancer.At some aspects of the method for the present invention, cancer or tumor table Reach the MUC1 of Aberrant glycosylation.

The present invention includes by with one or more immunogenic vaccine construct immunizing subjects described herein, In experimenter, generate the cytotoxic T cell response for MUC1 express cell, generate anti-MUC 1 antibody, and/or promote anti- The method of MUC1 antibody isotype conversion.In some respects, MUC1 express cell is tumor cell.Some of method in the present invention Aspect, cancer or the glycosylated MUC1 of tumor abnormal expression.

The present invention includes the method using glycolipidpeptide immunizing subjects, and described glycolipidpeptide includes comprising B cell epi-position At least one glycosylation MUC1 glycopeptide component；At least one peptide composition including the MHC restricted helper T cell epitope of II class；With At least one lipid composition.In some respects, in experimenter, inducing specific is combined on tumor cell the MUC1 egg expressed The antibody of the IgG hypotype of white matter.Because it is antigenicity and immunogenic, so the glycolipidpeptide of the present invention is very suitable for Immunotherapy medicaments compositions uses.Present invention accordingly comprises pharmaceutical composition, its glycolipidpeptide comprising the present invention and medicine Learn acceptable carrier.In preferred embodiments, pharmaceutical composition contains liposome, such as based on phospholipid liposome, and And glycolipidpeptide mixes in liposome caused by noncovalent interaction such as hydrophobic interaction.Alternately, glycolipidpeptide is permissible It is covalently attached to the component of liposome.Liposomal formulation can include having identical or different B epitope, identical or different T cell Epi-position and/or the glycolipidpeptide of identical or different lipid composition.

The three component immunogenic vaccine of the present invention have covalently bound at least one carbohydrate ingredient, at least one Plant peptide composition and at least one adjuvant component.Three component immunogenic vaccine contain B epitope and T epi-position, preferably auxiliary T epi-position. Normally, carbohydrate ingredient includes B epitope, and peptide composition contains T epi-position.B epitope may further include T epi-position. But, these epi-positions can be overlapping, and single glycopeptide such as MUC-1 glycopeptide can include B epitope and T epi-position.

The glycolipidpeptide of the present invention is easily formulated as veterinary or the pharmaceutical composition of people's purposes.Pharmaceutical composition is optional Comprising excipient or diluent, it is pharmaceutically acceptable as carrier and compatible with glycolipidpeptide.Term is " pharmaceutically acceptable Carrier " refer to one or more carriers such, it is compatible with other compositions of compositions and for its receiver or glycolipidpeptide It is " acceptable " in the sense that harmless.Suitably excipient includes such as water, saline, dextrose, glycerol, ethanol etc. and group thereof Close.Furthermore, it is necessary to time, pharmaceutical composition can contain trace auxiliary substance such as wetting agent or emulsifying agent, pH buffer agent, salt And/or adjuvant, it strengthens the effectiveness of immunostimulatory compositions.For oral administration, glycolipidpeptide can rise with plant or animal The protein in source or oil mixing.Prepare and use the method for this type of pharmaceutical composition to be also included in the present invention.

The pharmaceutical composition of the present invention can be applied to any experimenter, including people and performing animal (such as cat and dog). In preferred embodiments, pharmaceutical composition can be used as vaccine, and containing the amount inducing immunne response effective in experimenter Glycolipidpeptide.The dosage of the glycolipidpeptide vaccine of the present invention, timetable etc. for vaccination can be held by those skilled in the art Change places mensuration.Vaccine can use any convenient method to be applied to experimenter, and preferably parenteral is (such as via intramuscular, corium In or subcutaneous injection) or use via oral or nasal.Depend on treating the type of animal of vaccination, its age and weight, attenuation The immunogenicity of virus and method of application, useful dosage to be administered will change.

Three components or the two component immunogenic vaccine of the present invention can be used individually or together.It addition, because two components Vaccine is useful as adjuvant, so it can use to strengthen other treatments of cancer, such as chemotherapy, radiotherapy or Other kinds of immunization therapy.

In a kind of Therapeutic Method, at least one TLR part and the three component immunogenic vaccine of the present invention and/or two groups Immunogenic vaccine is divided to use the most altogether.The TLR part used altogether is used as other adjuvant.Example T LR part is at this Described in literary composition.Any TLR part can be used altogether with immunogenic vaccine.Preferably, TLR2 or TLR9 part such as CpG ODN Use altogether with immunogenic vaccine.When immunogenic vaccine contains the most covalently bound TLR2 part of TLR part as covalency During the adjuvant component connected, it should be understood that the TLR9 part that the TLR part used altogether is used the most altogether can be differently configured from covalency even The TLR part connect.

Therapeutic Method can relate to any combination of of three component vaccines, two component vaccines and/or the TLR part used altogether Use, as according to needed for the patient's condition to be treated or as according to health care professional indicate.

It is optional for being included in pharmaceutical composition by adjuvant.Adjuvant includes such as Alumen, QS-21 and TLR agonist. TLR agonist includes but not limited to any TLR agonist described herein.Preferably TLR agonist include TLR2 agonist, TLR4 agonist, TLR7 agonist, TLR8 agonist and TLR9 agonist.TLR9 passes through unmethylated sequence-activated containing CpG, Described sequence is included in DNA of bacteria or synthetic oligonucleotide (ODN) those found.This type of is unmethylated containing CpG sequence It is present in DNA of bacteria with altofrequency, but is rare in mammalian DNA.Therefore, unmethylated CpG sequence makes micro- Biological DNA distinguishes with mammalian DNA.See for example, 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；With Dunne and O'Neill, 2003, Sci. STKE 2003:re3。

TLR9 agonist can be the preparation of microbial DNA, and described DNA includes but not limited to e. coli dna, without endogenous toxin Element e. coli dna or the DNA of bacteria without endotoxin from e. coli k12.TLR9 agonist can be to separate from antibacterial , such as separate with bacterial origin；Synthesis, such as produced by the standard method of the chemosynthesis for polynucleotide 's；Produced by Standard recombinant methods, separate from bacterial origin subsequently；Or aforesaid combination.In many embodiments, TLR agonist is purification, and be the most 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% are pure or higher purity.

TLR9 agonist can be the synthetic oligonucleotide containing unmethylated CpG motif, referred to herein as " CpG oligodeoxynucleotide ", " CpGODN " or " ODN " (see for example, Hemmi et al. " A Toll-like receptor recognizes bacterial DNA," Nature 2000；408:740-745).The immunostimulating of at least three type CpG-ODN is described.A (or D) type ODN priority activation plasmacytoid dendritic shape cell (pDC), to produce IFN?, and B The propagation of (or K) type ODN induction B cell and the secretion of IgM and IL-6.Generate the feature of merging referred to as A and Type B Another type, and it is referred to as c-type.At least three types that the TLR9 agonist of the present invention can include obtaining describing Any one in zest ODN (A type, Type B and c-type).

CpG-oligodeoxynucleotide TLR9 agonist comprises CpG motif.CpG motif comprises the 5' side of CpG dinucleotide Two bases and two bases of 3' side.CpG-oligodeoxynucleotide can be by the standard of the chemosynthesis for polynucleotide Method produces.CpG-oligodeoxynucleotide can the most commercially be purchased from Coley Pharmaceuticals (Wellesley, MA), Axxora, LLC (San Diego, CA) or InVivogen, (San Diego, CA).CpG-oligodeoxynucleotide TLR9 swashs Dynamic agent can include broad range of DNA backbone, modifies and replace.

At some aspects of the present invention, TLR9 agonist is the nucleic acid comprising nucleotide sequence 5'CG 3'.In the present invention Some aspects, TLR9 agonist is to comprise nucleotide sequence 5'-purine-purine-Cytosine-guanine-pyrimidine-pyrimidine-3' Nucleic acid.In other aspects of the present invention, TLR9 agonist is to comprise nucleotide sequence 5'-purine-TCG-pyrimidine-pyrimidine-3' Nucleic acid.At some aspects of the present invention, TLR9 agonist is the nucleic acid comprising nucleotide sequence 5'-(TGC) n-3'.At this Other bright aspects, TLR9 agonist is the nucleic acid comprising sequence 5'-TCGNN-3', and wherein N is any nucleotide.

In some respects, TLR9 agonist can have length about 5-about 200, about 10-about 100, about 12-about 50, about 15- About 25, about 5-about 15, the sequence of about 10 or about 5-about 7 nucleotide of about 5-.In some respects, the length of TLR9 agonist can be little In about 15, less than about 12, less than about 10 or less than about 8 nucleotide.

TLR9 agonist includes but not limited to U.S. Patent number 6,194,388；6,207,646；6,239,116；6,339, 068；With 6,406,705,6,426,334 and 6,476,000 and disclosed U.S. Patent application US 2002/0086295, US Any one in those of described in 2003/0212028 and US 2004/0248837.

In some respects, TLR agonist can be bigger constructs (such as plasmid vector, viral vector or Other these type of constructs) part.Huge variety of plasmid and viral vector are to it known in the art, and without the most detailed Thin elaboration.A large amount of examples of such carriers are described in the multiple publication.See for example, Current Protocols in Molecular Biology, (F. M. Ausubel, et al., editor, 1987 and more new edition).Many examples of such carriers are that be purchased can ?.

The immunogenic vaccine of the present invention can be used together with the therapeutic agent other with one or more.At treatment additionally Reason include but not limited to excision, radiotherapy, chemotherapy, hormone therapy, anti-tumor vaccine, treatment based on antibody, Total irradiation, bone marrow transplantation, autologous peripheral blood stemcell transplant and chemotherapeutant are (referred to herein as " antitumor chemotherapy Agent ") use.Antitumor chemotherapeutant include but not limited to cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, Vincristine, ifosfamide, cisplatin, gemcitabine, busulfan (also referred to as 1,4-butanediol bismethane sulphonic acid ester or BU), Ara-C (also referred to as 1-β-D-R furanose cytosine or cytosine arabinoside), amycin, mitomycin, cyclophosphamide, first ammonia Pterin and combinations thereof.TLR agonist use can before other chemotherapeutant is used, during and/or after occur.Separately Outer therapeutic agent include such as one or more cytokines, antibiotic, antimicrobial, antiviral agent such as AZT, ddI or DdC, and combinations thereof.Use cytokine include but not limited to IL-1 α, IL-1 β, 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), conversion Growth factor-beta (TGF-β), granulocyte colony-stimulating factor (G-CSF), M-CSF (M-CSF), grain are thin Born of the same parents-M-CSF (GM-CSF)) (U.S. Patent number 5,478,556,5,837,231 and 5,861,159) or Flt-3 part (Shurin et al., Cell Immunol. 1997；179:174-184).Anti-tumor vaccine includes but not limited to peptide Vaccine, whole-cell vaccines, the whole-cell vaccines of genetic modification, recombinant protein vaccine or based on by recombinant viral vector to swollen The vaccine of the expression of tumor related antigen.Other therapeutic agent can be immunomodulator, and such as TLR4 agonist, TLR 8 are exciting Agent, TLR9 agonist, cox 2 inhibitor, GM-CSF, the inhibitor of indole amine 2,3-dioxygenase (IDO), chemotherapeutant or A combination thereof.

As noted, pharmaceutical composition is useful as vaccine.Vaccine can be prevention or protectiveness vaccine.Equally Ground, vaccine can be the treatment vaccine used after disease or disease such as formation of cancer.Therefore, the present invention comprise include as this The vaccine of the glycolipidpeptide that literary composition describes, including antimicrobial (such as antiviral or antibacterial) and anti-cancer vaccine.

The cancer can effectively treated or prevent includes but not limited to carcinoma of prostate, bladder cancer, colon cancer, breast carcinoma, black Element tumor, cancer of pancreas, pulmonary carcinoma, leukemia, lymphoma, sarcoma, ovarian cancer, Kaposi sarcoma, Hodgkin (Hodgkin's Disease), non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, idiopathic thrombocythemia Disease, primary macroglobulinaemia, small cell lung cancer, primary brain tumor, gastric cancer, malignant pancreatic insulinoma (malignant Pancreatic insulanoma), carcinoid malignant, skin lesion before deterioration, carcinoma of testis, lymphoma, thyroid carcinoma, one-tenth neural Glucagonoma, the esophageal carcinoma, genitourinary cancer, pernicious hypercalcemia, cervical cancer, carcinoma of endometrium, adrenocortical carcinoma and epithelium The cancer of origin of cell.As used herein, " tumor " refer in mammal find all types of cancers, vegetation or Malignant tumor.

The therapeutic efficiency of tumor can be estimated by any one in many kinds of parameters well-known in the art.This bag Include but be not limited to measure tumor size minimizing, measure the growth of tumor, propagation, aggressivity, vascularization, angiogenesis and/or The suppression of transfer, measures the suppression of the growth of any metastatic lesion, propagation, aggressivity and/or vascularization, and/or measure for The delayed hypersensitivity of the increase of tumor antigen.Therapeutic efficiency can also be estimated by following manner: measures experimenter The delay of middle recurrence or the delay of tumour progression, or measure experimenter survival rate the most after the treatment one or five year time increase Survival rate.As used herein, recurrence is tumor or excrescent recovery after it substantially stops, the most leukemic recovery.

The glycolipidpeptide of the present invention can be used in passive immunization method.Such as, glycolipidpeptide can be applied to host Animal, such as rabbit, mice, rat, chicken or goat, to generate antibody generation in host animal.For producing in host animal The scheme of raw polyclonal antibody is well-known.One or more the T epi-positions being included in glycolipidpeptide are optionally chosen as with anti- The corresponding T epi-position of the host animal that body produces wherein is same or similar.Separation antibody from animal, prevent subsequently or treat on It is applied to mammalian subject, preferably people experimenter, with treatment or prevention disease or infection.Glycolipidpeptide for the present invention Monoclonal antibody can separate from the hybridoma prepared according to standard laboratory protocols；They can also use recombinant technique example As phage display produces.This antibody-like is also useful for passive immunization.Optionally, anti-glycolipidpeptide monoclonal antibody It is people's antibody or humanized antibody.One or more B being included in the glycolipidpeptide for producing polyclone or monoclonal antibody Epi-position selects according to expection therapeutic purposes.The present invention comprises polyclone and monoclonal anti glycolipidpeptide antibody, and it is prepared And using method.

Correspondingly, the most provided by the present invention is pharmaceutical composition, its monoclonal comprising the present invention or polyclonal antibody And pharmaceutically acceptable carrier.Preferably, monoclonal antibody is humanized antibody.Humanized antibody is more preferably used in people's disease The treatment of disease or disease uses, because when introducing in human host, humanized antibody unlikely induces immunne response, especially It it is allergic response.As noted, pharmaceutical composition optionally comprises excipient or diluent, and it is pharmaceutically acceptable as carrier And compatible with monoclonal antibody, any experimenter can be applied to, including people and performing animal (such as cat and dog).Preparation and The method using this type of pharmaceutical composition is also included in the present invention.

The common trait of oncogenic transformation cell is oligosaccharide such as Globo-H, Lewis^YProcess LAN with Tn antigen.Optionally Ground, comprises the monoclonal of the present invention or polyclonal antibody and the pharmaceutical composition of the present invention of pharmaceutically acceptable carrier, permissible The tumor of the oncogenic transformation cell of this type of oligosaccharide of process LAN is comprised for targeting.Such as, the antibody of chemotherapeutic molecule it is conjugated to May be used for chemotherapeutic molecule is delivered to tumor.

The another kind of pharmaceutical composition of the present invention can comprise can affect the compound of protein active (such as antibody, part, Little molecule or peptide) and pharmaceutically acceptable carrier.The effect of compound for protein matter may include but be not limited to excitement, antagonism, Suppression or the normal biological processes of enhancing protein.Preferably, compound is the antibody of the epi-position on conjugated protein, described egg White matter comprisesO-glycosylation site.Preferably,O-glycosylation site isO-GlcNAc site.Numerous researchs have shown this exception Glycosylation can promote transfer, therefore its weak survival rate strong association with cancer patient.Therefore, Aberrant glycosylation albumen is affected The ability of the activity of matter is so that abnormal activity can be prevented.

Treatment valid density and amount can by known in vitro and in vivo system (include but not limited to described herein those In any one) in test compound, by rule of thumb every kind of application described herein is measured, subsequently can by its extrapolation use In people or the dosage of other animals.Therapeutic efficiency can be commented by any one in many kinds of parameters well-known in the art Estimate.This includes but not limited to the minimizing of tumor size, CD8⁺The increase of T cell activity, and/or the time-to-live increased.

As elsewhere herein is pointed out, have surprisingly found that Toll-like receptor (TLR) part is covalently attached to and comprise carbon Hydrate component (containing B epitope) and the glycopeptide of peptide composition (containing T epi-position), strengthen glycopeptide by the picked-up of target cell and interior Changing (seeing embodiment 3).Therefore the TLR part being characterized as lipid identified is for using in the glycolipidpeptide of the present invention Preferably lipid composition.Therefore the present invention further provides the method for identifying TLR part preferred lipid part, and it includes Make candidate compound contact with the target cell containing Toll-like receptor (TLR), and measure whether candidate compound combines TLR (i.e., Whether it is TLR part).Preferably, candidate compound passes through TLR by target cell internalization.By with the combination of TLR and optionally Expect it is immunogenic by the TLR part containing lipid identified in internalization to target cell, and be very suitable as this The lipid composition of the glycolipidpeptide of invention.The present invention the most also comprises glycolipidpeptide, and it includes use the method for the present invention to identify one Plant or the multiple TLR part containing lipid is as lipid composition.

Present invention additionally comprises diagnostic kit.Test kit provided by the present invention can antibody containing the present invention, preferably Monoclonal antibody, and suitably buffer (such as Tris, phosphate, carbonate etc.), thus cause test kit user to reflect FixedO-GlcNAc modifies.User subsequently can the most detectably traget antibody.Alternately, examination provided by the present invention Agent box can containing antibody in the solution, preferably in quencher buffer freezing or in powder type (as passed through lyophilization) Antibody.Detectable label or unconjugated antibody and buffer can be conjugated to included together in test kit, described buffering Liquid can the most also include stabilizer, Biocide, inert protein such as serum albumin etc..Usually, these materials will Existing less than 5 weight % with amount based on active antibodies, and generally to be again based at least about 0.001 weight % of antibody concentration Total amount exist.Optionally, test kit can include inertia extender or excipient, to dilute active component, and wherein excipient Can exist with about 1 weight %-99 weight % of total composition.In preferred embodiments, test kit the antibody provided is can Detection labelling so that the antibody of combination is detectable.Detectable label can be radioactive label, enzymatic labelling, fluorescence Labelling etc..Optionally, test kit can be containing the unconjugated monoclonal antibody of the present invention, and contain further can be in conjunction with one The secondary antibodies of level antibody.When using in mensuration in conjunction with the secondary antibodies of Primary antibodies, this is typically found in separately Bottle in.Secondary antibodies is generally conjugated to detectable label and prepares with in the way of similar with above-described antibody preparation. Test kit the most also includes packaging and one group of operation instructions.

As used herein, term " experimenter " includes but not limited to people and non-human vertebrate.Non-human vertebrate bag Include livestock animals, companion animals and laboratory animal.Non-human subject also includes non-human primate and rodent, Such as but not limited to rat or mice.Non-human subject also includes but not limited to chicken, horse, cattle, pig, goat, dog, cat, Cavia porcellus, storehouse Mus, ermine and rabbit.As used herein, term " experimenter ", " individual ", " patient " and " host " is used interchangeably.The most real Executing in scheme, experimenter is mammal, particularly people.

As used herein, " in vitro " is in cell is cultivated, and " in vivo " is internal experimenter.

As used herein, " process " or " treatment " includes that treatment and prevention process.Treatment disease or the patient's condition should mean to do This type of disease pre-or the patient's condition, in order to prevent or slow down disease or the development of the patient's condition, prevent or slow down disease or the progress of the patient's condition, stop Stop disease or the progress of the patient's condition or eliminate a disease or the patient's condition.

As used herein, term " pharmaceutically acceptable carrier " refers to that one or more compatible solids or liquid are filled Agent, diluent or encapsulating substance, it is suitable for being applied to people or other vertebratess.

As used herein, when being used for describing compound, term " separation " should mean from compound nature Come across in natural surroundings therein and take out.In one embodiment, separation mean take from the non-nucleic acid molecule of cell Go out.

When the scope of offer value, it should be understood that unless the context, over this range limit and lower limit it Between each intervening value to 1/10th of lower limit unit, and any other described value or intervening value in scope this described It is all contained in the present invention.The upper and lower bound of these less scopes can be independently include in less scope, and also wraps It is contained in the present invention, is limited by any concrete eliminating in described scope and administer.When described scope includes in limiting Or when two, get rid of those included limit in arbitrary or both scope be also included in the present invention.

In some embodiments, " effective dose " is the amount causing at least one pathological parameters to reduce.It is therefoie, for example, Compared with reducing with the expection of the parameter not connect in subject individuality, effectively reach 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%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, extremely Few 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% amount reduced.

Embodiment

The present invention is illustrated by following embodiment.It is to be understood that object lesson, material, amount and program should be according to such as originally The scope and spirit of the present invention broad interpretation that literary composition illustrates.

Embodiment 1

For anti-cancer vaccine based on completely synthetic carbohydrate: the conjunction of Lipidated glycopeptide of Tn antigen of being correlated with containing tumor Become and immunological evaluation

In this embodiment, it is prepared for completely synthetic candidate cancer epidemic disease by the combination of polymer support and solution phase chemistry Seedling, it is correlated with Tn antigen, peptide T epi-position and lipopeptid Pam by tumor₃Cys forms.Glycolipidpeptide is given in mixing liposome can be little Mus causes the preparation of T cell dependency antibody response.

The common trait of oncogenic transformation cell is oligosaccharide such as Globo-H, Lewis^YWith the process LAN of Tn antigen (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 researchs shown this Aberrant glycosylation can promote transfer (Sanders et al., Mol. Pathol. 1999,52, 174-178), the weak survival rate strong association of therefore its expression and cancer patient.

Several first-class researchs have utilized the differential expression of tumor related carbohydrate for developing cancer vaccine (Ragupathi, Cancer Immunol. 1996,43,152-157；Musselli et al., J Cancer Res. Clin. Oncol. 2001,127, R20-R26).But, carbohydrate can not activate helper T lymphocyte has made it as vaccine Purposes complicates (Kuberan et al., Current Organic Chemistry 2000,4,653-677).Great majority are exempted from Epidemic focus (includes carbohydrate), and antibody produces and depends on two quasi-lymphocyte B cell and the cooperative interaction of helper T cell (Jennings et al., Neoglycoconjugates, preparation and application, Academic, San Diego, 1994).Sugar individually can not activate helper T cell, therefore has limited immunogenicity.Low-affinity IgM antibody Formed and not existing of IgG antibody manifests this limited immunogenicity.

In order to overcome the T cell stand-alone nature of carbohydrate, past research has concentrated on sugar and exogenous carrier proteins matter Puting together of (such as keyhole limpet hemocyanin (KLH), detoxification tetanus toxoid).In this approach, carrier protein strengthens carbon Hydrate is presented to immune, and provides T epi-position (12-15 the amino acid whose peptide sheet that can activate t helper cell Section).

But, carbohydrate with puting together of carrier protein there is Railway Project.It is said that in general, conjugation chemistry is difficult to control System, results in the conjugate of the ambiquity in terms of the Nomenclature Composition and Structure of Complexes with the repeatability that can affect immunne response (Anderson et al., J. Immunol. 1989,142,2464-2468).Additionally, exogenous carrier proteins matter can cause strong B Cell response, it can cause the suppression of the antibody response for carbohydrate epitope.When using autoantigen such as tumor During related carbohydrate, the latter is bigger problem.Additionally, it is permissible with the joint puted together of protein for carbohydrate It is immunogenic, causes epi-position to suppress (Buskas et al., Chem. Eur. J. 2004,10,3517-3523).Do not make People surprisingly, fails to induce in all patients with several clinical trials of carbohydrate-protein conjugate cancer vaccine Sufficiently strong helper T cell response (Sabbatini et al., Int. J. Cancer 2000,87,79-85).Accordingly, it would be desirable to open Sending out strategy alternative is used for presenting tumor related carbohydrate epi-position, and it more effectively turns causing to the classification of IgG antibody Change (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., Cancer Immunol. Immunother. 2005,54,424-430； Reichel et al., Chem. Commun. 1997,21,2087-2088).

Herein, we report the conjunction of the completely synthetic anti-cancer vaccine material standed for (compound 9) being able adequately determines in structure Becoming and immunological evaluation, described anti-cancer vaccine material standed for constitutes collection and neutralizes needed for effective T cell dependent immune response Low limit architectural feature.Vaccine candidate object is correlated with Tn antigen, peptide T epi-position YAFKYARHANVGRNAFELFL (YAF) by tumor (SEQ ID NO:2) and lipopeptid S-［ (R)-2,3-two palmityl epoxide-propyl group ］-N-palmityl-(R)-cysteine (Pam₃Cys) composition.Serve as Tn antigen mistake on the surface of mammary gland, colon and prostatic people's epithelial tumor cell of B epitope Express.This antigen is not present on normal cell, and therefore causes to become the splendid target for immunization therapy.In order to gram Take the T cell stand-alone nature of Carbohydrate Antigens, mix YAF peptide.This 20 amino acid peptide sequence is derived from meningitis Neisser The external membrane protein of family name coccus, and have been identified as MHC II class restriction site (Wiertz et al., the J. Exp. for human T-cell Med. 1992,176,79-88).Imagine this helper T cell epitope can inducing T cell dependent immune response, cause for The generation of the IgG antibody of Tn antigen.The B cell merged lacks offer with helper T cell epitope and becomes for dendritic cell (DC) The ability (Medzhitov et al., Science 2002,296,298-300) of ripe suitable " danger signal ".Therefore, incorporation is spread out It is conigenous the immunocompetence of colibacillary major lipoproteinNThe lipopeptid Pam of end sequence₃Cys (Braun, Biochim. Biophys. Acta 1975,415,335-377).This lipopeptid has been identified as strong immunological adjuvant (Weismuller etc. People, Physiol. Chem. 1983,364,593), and recent research shown it by with Toll-like receptor-2 (TLR-2) Interaction play its activity (Aliprantis et al., Science 1999,285,736-73).This interaction causes Proinflammatory cytokine and the generation of chemotactic factor, itself and then stimulator antigen are delivery cell (APC), thus initial helper T cell is sent out Educate and activate (Werling et al., Vet. Immunol. Immunopathol. 2003,91,1-12).Lipopeptid also promotes antigen In incorporation liposome.Liposome has attracted as the carrier in vaccine design to pay close attention to (Kersten et al., Biochim. Biophys. Acta 1995,1241,117-138), this is due to its low inherent immunity originality, thus avoid without hope there is Carrier induction immunne response.

The synthesis of target compound 9 needs to use the synthesis strategy of the high concentration of following chemical operation, described chemical operation Existence with carbohydrate, peptide and lipid part is compatible.Imagination 9 can be combined by the Tn antigen 7 containing sept, polymer Peptide 1 HeS-［ double (palmityl epoxide) propyl group of 2,3-］-N-Fmoc-Cys (Pam₂FmocCys, 2, (Metzger et al., Int. J. Peptide Protein Res. 1991,38,545-554)) it is prepared.Use and activate mixture (scheme with conduct 10) the HMPB-MBHA resin of high acid-sensitive and 2-(1H-benzotriazole-1-base)-Oxy-1,1,3,3-tetramethylureaSix Fluorophosphate/I-hydroxybenzotriazole (HBTU/HOBt) (Knorr et al., Tetrahedron Lett. 1989,30,1927- 1930) the Fmoc protected amino acid combined, by the peptide 1 of automatization's Solid phase peptide synthesis assembling resin-bonded.Select HMPB-MBHA Resin, because it allows compound to crack from resin, and the adjoint removal of unprotected side chain blocking group.This feature is important , because the side chain functionalities of aspartic acid, glutamic acid and lysine otherwise can disturb the incorporation of Tn antigenic derivant 7.Connect down Come, in the mixture of DMF and dichloromethane in the presence of DIPEA, by Pam₂FmocCys derivant 2 uses PyBOP The manual N-terminal amine being coupled to peptide 1 of (Martinez et al., J. Med. Chem. 1988,28,1874-1879) and HOBt, with Provide the lipopeptid 3 of resin-bonded.The Fmoc group of 3 is removed at the standard conditions, and the unhindered amina of obtained compound 4 exists With Palmic acid coupling in the presence of PyBOP and HOBt, to provide protection completely and the lipopeptid 5 of resin-bonded.Pam₂Cys part Amine with 1 coupling after palmitoylation, to avoid the racemization of cysteine portion.The compound 5 cracking from resin is with 2% The dichloromethane solution of TFA reaches, and neutralizes immediately with the methanol solution of 5% pyridine subsequently.By LH-20 size exclusion Analysis after purification, uses DIC/HOAt/DIPEA (Carpino, J. Am. Chem. Soc 1993,115,4397-4398) conduct Coupling agent, makes the C-terminal carboxylic acid of lipopeptid 6 and the amine coupling of Tn derivant 7, with by Sephadex LH-20 size exclusion Analysis after purification, provides the Lipidated glycopeptide 8 protected completely with 79% yield.Shown by the analytical reagent composition of MALDI-TOFm/zSignal at 5239.6 and 5263.0, corresponds respectively to ［ M+H ］⁺［ M+Na ］⁺.Finally, 1，2-ethandithiol is utilized (EDT) as scavenger, the side chain protecting group by 8 is removed by processing with the aqueous solution of 95% TFA.Find triisopropyl silicon The alternative use of alkane (TIS) causes the formation of unidentified by-product.Target compound 9 is by size exclusion chromatography and subsequently The RP-HPLC purification of use Synchropak C4 post.The MALDI quality analysis of 9 shows the signal at m/z 3760.3, Corresponding to ［ M+Na ］⁺。

Scheme 10. a) PyBOP, HOBt, DIPEA, DMF/DCM (5/1, v/v)；B) piperidines/DMF (1/5, v/v)；c) CH₃(CH₂)₁₄COOH, PyBOP, HOBt, DMF/DCM (1/5, v/v)；D) the DCM solution of 2% TFA；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%.

It follows that compound 9 is mixed in liposome based on phospholipid.Therefore, containing 9, cholesterol, phosphatidylcholine and After the hydration of the lipid membrane of PHOSPHATIDYL ETHANOLAMINE, by preparing little via 100 nm Nuclepore polycarbonate membrane extrusions Type unilamellar vesicle (SUV).Confirm that liposome has evenly sized by the transmission electronic microscope checking (TEM) of negative staining, The expection diameter with about 100 nm (sees Buskas et al., Angew. Chem. Int. Ed. 2005,44,5985-5988 Fig. 1).It is the quantitative, just of high pH anion-exchange chromatography subsequently by hydrolyzing with TFAN-acetylgalactosamine content analysis fat Liposome preparation.Determine the concentration of about 30 μ g/mL GalNAc, its corresponding to about 10% the incorporation of initial compounds 9.

With the liposome of the fresh preparation containing 0.6 μ g carbohydrate, with weekly interval subcutaneous immunizations The group of five female BAl BIc/c mices.In order to probe into the lipopeptid Pam of embedding₃The adjuvant character of Cys, the liposome containing antigen together with Or do not use together together with effective saponin immunological adjuvant QS-21 (Antigenics Inc., Lexington, MA).Anti-Tn resists Body titre is measured by being coated microtitration plate with BSA-Tn conjugate, and with being marked with the anti-mouse of alkali phosphatase IgM or IgG antibody complete detection.As seen in Table 1, mice is used Liposomal formulation immunity inoculation, cause for Tn antigen IgM and IgG antibody (table 1, entry 1 and 2).It is thin that the existence of IgG antibody points out that the auxiliary T epitope peptide of 9 has activated T-helper Born of the same parents.Additionally, to the adjuvant only pointing out embedding by the observed result of mice (group 1) the generation IgG antibody of liposome immunization inoculation Pam₃Cys has triggered for DC maturation and the appropriate signals of the subsequent activation to helper T cell thereof.But, accept and QS-21 group The mice (group 2) of the liposome closed causes the anti-Tn antibody of higher titre.This higher immunne response is likely due to from mixed Close the transformation (Moore et al., Vaccine 1999,17,2517-2527) of Th1/Th2 to Th1 response.

Table 1. is with the ELISA anti-Tn antibody titer after 4 immunity inoculations of glycolipidpeptide/Liposomal formulation^{［ a ］}。

^{［ a ］}Elisa plate BSA-BrAc-Tn conjugate is coated.All titres are the meansigma methodss of the group about five mices. By regression analysis measure titre, mark and draw with dulling luminosity ratio compared with log10 dilution factor and measure titre.Titre is calculated as providing ratio Absorbance a height of 0.1 or higher highest dilution relative to the normal saline mice serum of 1:100 dilution.

Result presented herein provides former about use Lipidated glycopeptide as bottom line subunit vaccine first Reason proves (proof-of-principle).Expection may be made that several improvement.For instance, it has been found that the clustering to present and be of Tn antigen Mucinous more suitable analog thing, the antibody therefore produced for this structure more preferably identifies that the Tn expressed on cancerous cell resists Former (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 epi-position used in this is studied is known which are for people's MHC II class restricted epitope.Therefore, when using Mus Th epi-position, it is contemplated that change to the more effective classification of IgG antibody. On the other hand, compound 9 is the more suitably vaccine candidate object for using in people.Recent report points out Pam₂Cys is ratio Pam₃The more effective immunological adjuvant of Cys (Jackson et al., Proc. Nat. Acad. Sci. USA 2004,101,15440- 15445).Pam is had also been proposed₂Cys adjuvant have improvement solvable character (Zeng et al., J. Immunol. 2002,169, 4905-4912), it is the debatable feature of compound 9.The research solving these problems is underway.

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

Support information

Reagent and general experimental procedure.Aminoacid and resin derive from Applied Biosystems and NovaBiochem；DMF obtains From EM science；And NMP derives from Applied Biosystems.PHOSPHATIDYL ETHANOLAMINE (PE), cholesterol, phosphatidylcholine (PC；Egg yolk) and phosphatidyl glycerol (PG；Egg yolk) purchased from Sigma-Aldrich and Fluka.Every other chemicals is purchased from Aldrich, Acros and Fluka, and use without being further purified.The all solvents used all have reagent grade, and logical Cross and be dried through the backflow of suitable desiccant.TLC uses Kieselgel 60 F₂₅₄ (Merck) plate performs, by UV light (254 nm) and/or by with the ethanol solution carbonization of 8% sulphuric acid or being detected by 1,2,3-indantrione monohydrate.Column chromatography is at silicon dioxide Gel (Merck, sieve mesh 70-230) is upper to be performed.Size exclusion column chromatography performs on Sephadex LH-20.Extract is existed Concentrate≤40 DEG C (water-bath) under decompression.It is equipped with automatic sampler, UV detector and fraction collector and there is 1 mL/ minute stream Agilent 1100 Series HPLC System of the Synchropak C4 post 100x4.6 mm RP of speed is for analyzing and purification.Use HP-MALDI instrument uses gentisic acid as substrate record cation substance assistant laser desorpted ionized flight time (MALDI- TOF) mass spectrum.¹H NMR and¹³C H NMR spectroscopy Varian Inova300 spectrometer, Varian Inova500 spectrometer and Record on Varian Inova600 spectrometer, described spectrometer is all equipped with sun station.At CDCl₃Middle record¹H composes reference Residue CHCl at 7.26 ppm₃Or TMS, and¹³C spectrum is with reference to CDCl₃Central peak at 77.0 ppm.Use standard 1D Distribution is made in experiment and gCOSY/DQCOSY, gHSQC and TOCSY 2D experiment.

Lipopeptid6.Compound 1 is in the upper synthesis of HMPB-MBHA resin (peak load, 0.1 mmol).The synthesis of peptide 1 is being equipped with Hold on the ABI 433A peptide synthesizer of UV detector, use aminoacid and 2-(1H-benzotriazole-1-the base)-oxygen of Fmoc protection Base-1,1,3,3-tetramethylureaHexafluorophosphate (HBTU)/I-hydroxybenzotriazole (HOBt) is carried out as coupling agent.Single Individual coupling step caps with conditionality as required and carries out.After peptide 1 has synthesized, manual execution remaining step.WillN-fluorenes first (2,3-double (palmityl epoxide)-(2R-propyl group)-(R)-Cys2s (120 mg, 0.13 mmol) are dissolved in oxygen carbonyl-S- In DMF (5 mL), and add PyBOP (0.13 mmol), HOBt (0.13 mmol) and DIPEA (0.27 mmol).? After premixing 2 minutes, add DCM (1 mL), and feed the mixture in resin.Coupling step is performed twice.In coupling After completing (as by Kaiser measurements determination), use DMF (5 mL) the solution cracking N-Fmoc group of 20% piperidines.Use PyBop (0.3 mmol), HOBt (0.3 mmol) and the DMF solution of DIPEA (0.6 mmol), make Palmic acid as mentioned above (77 mg, 0.3 mmol) and unhindered amina coupling.Resin DMF and DCM is fully washed, and is dried under vacuum 4 hours.Logical Cross DCM (2.5 mL) solution with 2% trifluoroacetic acid to process 2 minutes, from resin, release the lipopeptid 6 of full guard.Will mixing Thing is filled in the methanol solution (5 mL) of 5% pyridine.This program is repeated, and the flow point merging containing lipopeptid is concentrated into It is dried.By size exclusion chromatography (LH-20, DCM/MeOH, 1:1) purification of crude product, to provide the lipopeptid 6 as white solid (275 mg, 0.057 mmol): R_f= 0.57 (DCM/MeOH 9:1)；Selected NMR data (CDCl₃/CD₃OD 1/1 v/v 600MHz): ¹H, δ 0.48-0.90 (m, 27 H, Pam CH₃, Leu CH₃, Val CH₃), 0.96-1.61 (m, Leu CH₂, Leu CH, Lys CH₂, ^tBu CH₃, Boc CH₃, Ala CH₃, Arg CH₂), 1.18 (br s, 72H, Pam CH₂), 1.95, 1.99 (s, 4x3H, Pbf CH₃C), 2.36, 2.41, 2.44 (s, 6x3H, Pbf CH₃), 2.48 (s, 2x2H, Pbf CH₂) 2.65-2.73 (m, 6H, S-CH ₂-glyceryl, His CH₂, Cys^β), 3.47 (m, 2H, Gly^α), 3.57 (m, 2H, Gly^α), 4.06 (m, 1H, S-glyceryl-CH ₂ ^bO), 4.32 (m, 1H, S-glyceryl-CH ₂ ^aO), 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, (aromat) of His CH, Tyr aromatics, Phe aromatics, Trt aromatics), 7.48-8.29 (m, NH).For C₂₆₉H₃₇₃N₃₃O₄₂S₃The MALDI-MS ［ M+Na ］ calculatedm/z=4860.22: measured value 4860.31.

Shielded glycolipidpeptide8.By lipopeptid 6 (22 mg, 4.6 μm ol), HOAt (6.3 mg, 46 μm ol) and DIC DCM/DMF (2/1 v/v, the 1.5 mL) solution of (7 μ L, 46 μm ol) stirs 15 minutes in ambient temperature under an argon.Will DMF (1.5 mL) solution of compound 7 (8 mg, 19 μm ol) and DIPEA (14 μ L, 92 μm ol) adds the stirring of lipopeptid In mixture, and reaction is kept 18 hours in room temperature.By mixture dilution with toluene and under reduced pressure it is concentrated to dryness.Logical Cross size exclusion (LH-20, DCM/MeOH 1:1) purification residue be given compound 8 as white solid (19 mg, 79%): selected NMR data (CDCl₃/CD₃OD 1/1 v/v 600MHz): ¹H,δ 0.60-0.90 (m, 27 H, Pam CH₃, Leu CH₃, Val CH₃), 0.96-1.61 (m, Leu CH₂, Leu CH, Lys CH₂, ^tBu CH₃, Boc CH₃, Ala CH₃, Arg CH₂), 1.18 (br s, 72H, Pam CH₂), 1.94, 1.98, 1.99, 2.00 (s, 6x3H, Pbf CH₃C, HNAc CH₃), 2.36, 2.41, 2.45 (s, 6x3H, Pbf CH₃), 2.48 (s, 2x2H, Pbf CH₂), 3.42-4.31 (m, Phe^α, Ala, Lys, Val, Asp, Glu, Tyr, Arg, Gly, Leu, His, Asn CH₂, Tyr CH₂, Phe CH₂, Arg CH₂), 3.71 (H-3), 3.88 (H-4) 4.06 (S-glycerol Base-CH ₂ ^βO), 4.20 (t, 1H, H-2), 4.32 (m, 1H, S-glyceryl-CH ₂ ^αO), 4.42 (m, 1H, Cys^α), 4.82 (d, 1H, H-1, J=3.68Hz), 5.06 (m, 1H, S-glyceryl-CH), 6.72-7.39 (m, 70H, His CH, Tyr aromatics, Phe aromatics, Trt aromatics), 7.48-8.29 (m, NH).For C₂₈₆H₄₀₃N₃₇O₄₉S₃ The MALDI-MS ［ M+Na ］ calculatedm/z=5262.67: measured value 5262.99.

Glycolipidpeptide9.Will be at TFA/H₂O/ ethane-1, in the deprotection mixture of 2-bis-mercaptan (95:2.5:2.5,3 mL) Compound 8 (12 mg, 2.3 μm ol) be stirred at room temperature 1 hour.Under reduced pressure remove solvent, then first pass through short chi Very little exclusion LH-20 post (DCM/MeOH 1:1) purification of crude compound, subsequently by using the aqueous solution (0.1% of 0-100% acetonitrile TFA) HPLC of gradient carrys out purification of crude compound, using be given after freeze drying compound 9 as white solid (6.8 mg, 79%): selected NMR data (CDCl₃/CD₃OD 600MHz): ¹H, δ 0.74-0.96 (m, 27H, Pam CH₃, Leu CH₃, Val CH₃), 1.11-2.35 (Leu CH₂, Leu CH, sp CH₂, Lys CH₂, Glu CH₂, Ala CH₃, Val CH, Asp CH₂), 1.29 (br S, 72H, Pam CH₂), 2.43-3.87 (Ala^α, Gly^α, S-glyceryl- OCH₂, 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 aromatics , Phe aromatics, NH).For C₁₈₆H₂₉₇N₃₇O₄₁The HR-MALDI-MS ［ M+Na ］ that S calculatesm/z=3760.1911: Measured value 3760.3384.

Tn derivant11.Compound 10 is dissolved in DMF (10 mL), and adds Diisopropylcarbodiimide (DIC) (82 μ L, 0.53 mmol) and HOAt (216 mg, 1.58 mmol).After stirring 15 minutes, add 3-(N-(tertiary fourth Epoxide carbonyl)-amino) propanol (111 mg, 0.63 mmol), and reaction is maintained ambient temperature 15 hours.By mixture Under reduced pressure it is concentrated to dryness, and by silica gel column chromatography (the DCM solution of 0-5%MeOH) and LH-20 size exclusion chromatography (DCM/MeOH 1:1) purification residue, to provide compound 11 (363 mg, 83%).R_f = 0.63 (DCM/MeOH 9: 1)； [α]_D + 4.4 (c 1.0 mg/mL, CH₂Cl₂)；NMR data (CDCl₃, 500MHz): ¹H, δ 1.27 (d, 3H, CH₃ Thr), 1.43 (s, 9H, ^tBu CH₃), 1.46-1.61 (m, 2H, CH₂), 1.99 (s, 3H, CH₃ Ac), 2.05 (s, 6H, CH₃ Ac), 2.06 (s, 3H, CH₃ Ac), 2.17 (s, 3H, CH₃ Ac), 3.17-3.27 (m, 3H, CH₂, 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, CH₂ 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, NH T), 6.81 (br d, 1H, NH GalNAc), 7.17-7.79 (m, 8H, the H of aromatics);¹³C (CDCl₃,75MHz)δ 17.19, 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。

For C₄₁H₅₄N₄O₁₄The HR-MALDI-MS ［ M+Na ］ calculatedm/z=849.3535: measured value 849.3391.

Tn derivant7。By molten in the DMF (5 mL) containing 20% piperidines of compound 11 (194 mg, 0.24 mmol) Liquid stirs 1 hour in ambient temperature.Mixture is concentrated to dryness, and by residue pyridine/acetic anhydride (3:1,5 mL) place Manage 2 hours.By reactant mixture dilution with toluene and be concentrated to dryness.Residue is dissolved in dichloromethane, and uses 1M HCl and saturated NaHCO₃Solution washing, uses MgSO₄It is dried, filters and concentrate.By size exclusion chromatography (LH-20, DCM/ MeOH 1:1) purification residue offer compound 12 (167 mg, 91%): NMR data (CDCl₃, 300MHz): ¹H, δ 1.24 (d, 1H, Thr CH₃), 1.42 (s, 9H, ^tBu CH₃), 1.55-1.59 (m, 2H, NHCH₂CH ₂CH₂NH), 1.95, 2.02, 2.03, 2.12, 2.14 (s, 15H, CH₃ Ac), 3.13-3.23 (m, 3H, CH₂ + 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).To C₂₈H₄₆N₄O₁₃ The MALDI-MS ［ M+Na ］ calculatedm/z=669.296: measured value 669.323.By the methanol (5 mL) with 5% hydrazine hydrate Solution is stirred at room temperature 35 minutes together, makes compound 12 deprotection.By reactant mixture dilution with toluene and concentrate.By remnants Thing is coevaporation twice together with toluene.By purification acquisition 13 (119 mg, 89%) of silica gel column chromatography (DCM/MeOH 5:1): NMR data (CD₃OD, 300MHz): ¹H, δ 1.26 (d, 3H, Thr CH₃), 1.43 (s, 9H, ^tBu CH₃), 1.57-1.63 (m, 2H, NHCH₂CH ₂CH₂NH), 2.06, 2.10 (s, 2x3H NHAc), 2.12-3.09 (m, 2H, CH₂), 3.15 (m, 2H, CH₂), 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).For C₂₂H₄₀N₄O₁₀The MALDI-calculated MS ［ M+Na ］m/z=543.264: measured value 543.301.Trifluoroacetic acid (4 mL) solution by 13 is under an argon at ring Border temperature stirs 45 minutes.Subsequently reactant mixture DCM diluted and be concentrated to dryness.By column chromatography (Iatro pearl, EtOAc/MeOH/H₂O 2:2:1 → MeOH/H₂O 1:1) purification of crude product.After the flow point merged concentrates, by solid from H₂O Lyophilizing, to provide the compound compound 7 (91 mg, 0.21 mmol, 95%) as white powder.R_f = 0.17(EtOAc/ MeOH/H₂O 6:3:1)； [α]_D -37 (c 1.0 mg/mL, H₂O)；NMR data (D₂O, 300MHz): ¹H, δ 1.15 (d, 3H, J = 6.3 Hz, Thr CH₃), 1.73-1.77 (m, 2H, CH₂), 1.95 (s, 3H, NHAc), 2.04 (s, 3H, NHAc), 2.82-2.87 (m, 2H, CH₂), 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 = 3.56 Hz, J = 3.9 Hz, H-1), 7.97 (br d, 1H, NH), 8.17 (br t, 1H, NH), 8.27 (br d, 1H, NH); ¹³C (D₂O, 75MHz), δ 18.17 Thr CH₃), 21.93, 22.33 (2xNAc) 26.98 (CH₂), 36.55 (CH₂), 37.22 (CH₂), 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).For C₁₇H₃₂N₄O₈The HR-MALDI-MS ［ M+Na ］ calculatedm/z=443.2118: real Measured value 443.2489.

Prepared by liposome.Liposome is by PC, PG, cholesterol and glycolipidpeptide 9 (15 μm ol, mol ratio 65:25:50:10) Preparation.Lipid is dissolved under an argon in DCM/MeOH (3/1, v/v).Subsequently by making dry nitrogen stream through with subsequently Be further dried under a high vacuum one hour and remove solvent.Obtained lipid membrane is suspended in containing 145 mM The 1 mL 10 mM Hepes buffer of NaCl, in pH 6.5.By solution on agitator (250 rpm) under an ar atmosphere 41 DEG C vortex 3 hours.By Liposomal suspensions 50 DEG C by 0.6 μm, 0.2 μm and 0.1 μm polycarbonate membrane (Whatman, Nuclepore, Track-Etch Membrane) extrude ten times, to obtain SUV.

Immunity inoculation.And added each with the liposome containing carbohydrate of 0.6 μ g when the 0th, 7,14 and 21 days The group of 10 μ g five mices of adjuvant QS-21 subcutaneous immunizations (female BAl BIc/c, 6 weeks) of persistent erection of the penis.Mice is made when the 28th day Blood-letting (lower limb vein), and with regard to the existence situation test sera of antibody.

ELISA.By 96 orifice plates with being dissolved in the Tn in the 0.2 M borate buffer solution (pH 8.5) containing 75 mM sodium chloride BSA (2.5 μg mL^-1) it is coated overnight (100 μ L/ hole) at 4 DEG C.By plate with containing 0.5% Tween 20% and 0.02% nitrine 0.01 M Tris buffer solution of change sodium three times.By making plate and being dissolved in the 0.01 M phosphate containing 0.14 M sodium chloride 1% BSA in buffer reaches to close at incubation at room temperature together for 1 hour.It follows that plate is washed, subsequently in room temperature and at phosphorus Serum dilutions in acid buffering saline incubation 2 hours together.Remove excessive antibodies, and plate is washed three times.By plate and rabbit Antibody (the Jackson ImmunoResearch that anti-mouse IgM and IgG Fc γ fragments specific alkali phosphatase are puted together Laboratories Inc., West Grove, PA) together incubation at room temperature 2 hours.Subsequently, after being washed by plate, add enzyme Substrate (p-nitrophenyl phosphate ester), and allow to react 30 minutes, then anti-by adding 3 M aqueous NaOH quencher enzymatics Should, then the dual wavelength at 405 and 490 nm reads absorbance.Antibody titer is measured, wherein for suction by regression analysis Luminosity marks and draws log₁₀Dilution factor.Titre is calculated as providing the highest dilute of the twice of the Normal Mouse Serum absorbance of 1:120 dilution Degree of releasing.

Embodiment 2

Two epi-position Liposomal formulations of non-covalent linking

In testing at first group, tumor related carbohydrate B epitope and promiscuous T epitopes peptide are separately mixed the lipid being prefabricated into Internal, to form two multi-epitope construct.It addition, by lipopeptid Pam₃Cys mixes in liposome, it is contemplated that it will serve as embedding adjuvant, Thus avoid using the necessity of other outside adjuvant such as QS-21.

Two kinds of different thiol reactant functional groups (functionality) maleimides and bromine second is carried from its surface Liposome prepared by the lipid anchor of acyl.Also by Pam₃Cys adjuvant mixes in the liposome being prefabricated into, and includes maleimide official Can group.Easily, maleimide and acetyl bromide group show significant difference at it to the reactive aspect of sulfydryl.Maleoyl Imines is in pH 6.5 and mercapto compound fast reaction, and acetyl bromide needs slightly greater pH 8-9, with mercaptan chemical combination Thing effecting reaction.

By utilizing this species diversity in terms of reactivity, it is prepared for carrying cancer and is correlated with Le^yTetrose and general T assist peptide The two epi-position liposome constructs (scheme 11) of QYIKANSKFIGITEL (QYI) (SEQ ID NO:1).For with thiol reactant Puting together of property anchor, oligosaccharide and peptide are all with containing thiol linker functionalization.Put together continuously with two steps of the liposome being prefabricated into and there is pole Big advantage: it is so that the very flexible approach of the liposome of multiple different carbohydrate B epitope is carried in easily preparation.As Based on to being covalently coupled to the carbohydrate of vesicle and the quantitative of peptide, it is the highest to put together yield, is 70-80% and right for oligosaccharide It is 65-70% in peptide, and result can highly reproduce.

It is important to note that at these in the first two epi-position liposome constructs, carbohydrate B epitope and peptide T Epi-position self is not linked together by covalent bond, but its respective lipid anchor puted together therewith by them and by hydrophobic Interaction remains close to.Several reports in the document about the vaccine candidate object with pathogen related peptides B epitope are Show that this method is successful, cause the good titre of IgM and specific IgG antibodies.These researchs also indicate that embedding adjuvant Pam₃Cys be enough to induce suitable immunne response.

But, use tumor related carbohydrate B epitope Le at us^yResearch in, describe in this embodiment The two epi-position Liposomal formulations of the use non-covalent linking immunity inoculation to mice, only results in the IgM antibody of extremely low titre.Do not examine Measure the anti-Le of IgG^yAntibody.Even more surprisingly, use liposome bacterin material standed for and strong outside adjuvant QS-21 altogether The most do not improve result.Additionally, it was found that (the most only carry maleimide and bromine second with the most coated control liposome The liposome of acyl functional group) mice of immunity inoculation, causes the high titre IgG antibody as detected by ELISA.Use multiple The sero-fast more detailed ELISA research from this group mice of protein conjugate discloses mice in response to maleimide Joint also causes the antibody for maleimide joint.Additionally, use by oneself for anti-joint antibody screening be coated with Le^yAntigen With the antiserum of the mice that the liposome immunization of QYI peptide is inoculated, find that these mices have caused the most for maleimide joint IgG antibody.

Scheme 11

Owing to it is at the high response close to neutral pH, maleimide joint is widely used in conjugation chemistry and reaches in immunity The sugar used further in inoculation study-and peptide-protein conjugate.There is the protein-conjugate test kit being obtained commercially (Pierce Endogen Inc.), it utilizes maleimide joint for antigen conjugate and detection conjugate.We Data display use these test kits can cause false positive results, especially when working together with the antigen of reduced immunogenicity (seeing T. Buskas, Y. Li and G-J. Boons, Chem. Eur. J., 10:3517-3523,2004).

In order to whether test height immunogenic maleimide joint suppresses for Le^yThe immunne response of tetrose, I Only use acetyl bromide joint prepare non-covalent two epi-position liposomees.In this experiment, the Le containing mercaptan^yTetrose and general T helper cell peptide is conjugated to the lipid containing acetyl bromide joint in separately reaction.The lipid puted together mixes subsequently, with Form lipid vesicle.By this new liposome preparation together with or be not applied to mice together with outside adjuvant QS-21, only produce The anti-Le of low titre^yAntibody.Therefore, for Le^yThe shortage of effective immunne response of tetrose is not only due to immunogenic horse Carry out acid imide joint.

The Le since it is known tumor is correlated with^yTetrose is only weak immunogenic, so we are prepared for another kind of two epi-position fat Plastid construct, the most more immunogenic Tn (bunch) antigens are used as target B epitope.But, obtain the moon equally with this antigen Property result.Again, the immunity inoculation of mice only results in anti-Tn (c) IgM antibody of extremely low titre.With the QS-as outside adjuvant 21 use the most altogether entirely without strengthening immunne response.

According to these results, we conclude that: when the tumor related carbohydrate antigen with reduced immunogenicity is used When making B epitope, a series of peptide antigens are proved that two epi-position liposome method of successful non-covalent linking have failed.Therefore, We need difference to be presented to immune system, to induce T cell with auxiliary T epi-position by inference tumor related carbohydrate B epitope Dependent immune response.

Embodiment 3

Two covalently bound epi-position Liposomal formulations

It is presumed that in order to reach more preferably presenting of carbohydrate B epitope and peptide T epi-position, possible they need covalently bound Together.In order to test this idea, we have synthesized construct 1 (scheme 12), and it is to neutralize effective T cell containing collection to depend on The anti-cancer vaccine material standed for being able adequately determines in the structure of the architectural feature needed for relying property immunne response.This vaccine candidate object is by tumor Relevant Tn antigen, peptide T epi-position YAFKYARHANVGRNAFELFL (YAF) (SEQ ID NO:2) (Neisseria meningitidis) and Lipopeptid Pam₃Cys forms.Owing to using the difficulty of original auxiliary T epitope peptide QYI in synthesis, use in this is studied and show The different promiscuous T epitopes (YAF) of the most solvable character.

The combination being combined to by solid phase and solution synthesizes compound 1 in high concentration mode.

Scheme 12

Subsequently construct is mixed in liposome based on phospholipid.Compound 1 has the low solubility in a series of solvents Shortcoming, its incorporation being probably in liposome is only the main cause of 10%.

By this construct with weekly Immunity at intervals Mice Inoculated.In order to probe into the lipopeptid Pam of embedding₃The adjuvant of Cys Character, will contain antigen liposome and together with (group 2) or not use together with (group 1) adjuvant QS-21.

As visible in table 1 (embodiment 1), mice is used Liposomal formulation immunity inoculation, cause for Tn antigen IgM and IgG antibody (table 1, entry 1 and 2).It is thin that the existence of IgG antibody points out that the auxiliary T epitope peptide of 1 has activated T-helper Born of the same parents.Additionally, the mice (group 1) inoculated with liposome immunization in the case of there is not outside adjuvant QS-21 is produced IgG antibody Observed result point out embed adjuvant Pam₃Cys has triggered for DC maturation and the conjunction of the subsequent activation to helper T cell thereof Suitable signal.But, the mice (group 2) accepting the liposome with QS-21 combination causes the anti-Tn antibody of higher titre.This higher Immunne response be likely due to from mixing Th1/Th2 to Th1 tilt response transformation.

This result provides first about using the Lipidated glycopeptide principle as MIN self-sustaining subunit vaccine Proving, described Lipidated glycopeptide contains carbohydrate B epitope, helper T cell epitope and lipopeptid adjuvant.Conclude further that: for Induction is for the T cell dependent immune response of tumor related carbohydrate antigen, carbohydrate B epitope and peptide T table It is inadequate that position is presented on the surface containing adjuvant liposome together with non-covalent fashion；On the contrary, entity preferably covalently is connected to Together.Finally, it was observed that when three kinds of components (carbohydrate B epitope, helper T cell epitope and lipopeptid) are covalently bound to be formed During Lipidated glycopeptide, it is not necessary to outside adjuvant (QS-21).

Alternative glycolipidpeptide component

Compound 1 can be made several improvement.For instance, it has been found that the antibody weak identification cancerous cell caused for Tn antigen.So And, cluster (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 Tn antigen is presented initiation as the part of MUC-1 glycopeptide have The antibody (Snijdewint et al., Int. J. Cancer 2001,93,97-106) of the combination feature improved.At compound 1 The T epi-position of middle employing is the MHC II class restricted epitope for people.Therefore, when using Mus T epi-position, it is contemplated that to IgG The more effective classification conversion of antibody.Moreover, it has been discovered that lipopeptid Pam₂Cys or Pam₃CysSK₄It is to compare Pam₃Cys more effectively exempts from Epidemic disease adjuvant (Spohn et al., Vaccine 2004,22,2494-2499).But, unknown Pam₂Cys or Pam₃CysSK₄It is connected to Whether T and B epitope can affect its effect and effect.Therefore, consider based on these, devise compound 2 and 3 (scheme 12), its Containing MUC-1 glycopeptide as B epitope, containing the Mus helper T cell epitope fully proved derived from poliovirus KLFAVWKITYKDT (KLF) (SEQ ID NO:3) (Leclerc et al., J. Virol. 1991,65,711-718) is as T table Position and contain lipopeptid Pam respectively₂Cys or Pam₃CysSK₄。

To as described in compound 1, glycolipidpeptide 2 and 3 mixed in liposome based on phospholipid.Surprisingly, puzzlementization The solubility of compound 1 is not problem for compound 2 and 3.By female BAl BIc/c mice with Liposomal formulation together with or not Together with outside adjuvant QS-21 with weekly Immunity at intervals inoculate four times (Kensil et al., J. Immunol. 1991, 146,431-437).Resist by being coated the microtitration plate anti-Muc1 of mensuration with CTSAPDT (α GalNAc) RPAP being conjugated to BSA Body titre, and complete detection with by the anti-mouse IgG antibody of alkali phosphatase enzyme mark.Result is summarized in table 2 and 3.

Table 2. is with the ELISA anti-MUC-1 antibody titer * after 4 immunity inoculations of glycolipidpeptide/Liposomal formulation.

* elisa plate BSA-BrAc-MUC-1 conjugate is coated.Anti-MUC1 antibody titer is as the group of five mices Meansigma methods presents.Titre be defined as obtaining optical density for the background of blank mice serum be 0.1 or bigger the highest Dilution factor.

Table 3. is with the ELISA anti-MUC-1 antibody titer * after 4 immunity inoculations of glycolipidpeptide/Liposomal formulation.

* elisa plate BSA-BrAc-MUC-1 conjugate is coated.Anti-MUC1 antibody titer is as the group of five mices Meansigma methods presents.Titre be defined as obtaining optical density for the background of blank mice serum be 0.1 or bigger the highest Dilution factor.

As seen in Table 2, the anti-MUC-1 of high titre is caused with the mice of the Liposomal formulation immunity inoculation of compound 2 and 3 IgG antibody.Surprisingly, with based on Pam₃CysSK₄Vaccine virus immunization mice cause ratio Pam₂Cys derivant is exempted from The antibody of the higher titre of mice of epidemic disease inoculation.These results with compare Pam₂Cys and Pam₃CysSK₄Adjuvanticity (adjuvancy) report contradicts.The sub-typing (IgG1, IgG2a, IgG2b and IgG3) of IgG antibody is pointed out to exempt from towards Th2 The deflection (entry 1 and 3, table 3) of epidemic disease response.Using altogether of adjuvant QS-21 is not resulted in dramatically increasing of IgG antibody, but, at this In the case of Xie, it was observed that mixing Th1/Th2 response (entry 2 and 4, table 3).

It is capable of identify that at MUC-1 glycopeptide natural present on cancerous cell in order to ensure mice serum, checks serum and expression The combination of the MCF-7 MCF-7 of MUC-1.Therefore, the serum that cell dilutes with 1:50 is processed 30 minutes, after this Add by the goat anti-mouse IgG antibody of FITC labelling.Positive cell percentage is measured with average by flow cytometry Fluorescence.As visible in (Fig. 2), antiserum and MUC-1 positive tumor cell kickback, and for deriving from first for testing The serum of mice do not observe combination.Additionally, when using SK-MEL 28 cell not expressing MUC-1 glycopeptide, do not observe In conjunction with.These results confirm the native antigen on human cancer cell of the anti-MUC-1 antibody recognition by 3 inductions.Further ELISA Research display is extremely low for the titre of T epi-position, and display does not occurs that significant epi-position suppresses.

The lipopeptid part of three component vaccines is initial to produce required cytokine and chemotactic factor (danger signal) is musted (Bevan, the Nat. Rev. Immunol. 2004,4,595-602 needed；Eisen et al., Curr. Drug Targets 2004,5,89-105；Akira et al., Nat. Immunol. 2001,2,675-680；Pasare et al., Immunity 2004,21,733-741；Dabbagh et al., Curr. Opin. Infect. Dis. 2003,16,199-204；Beutler, Mol. Immunol. 2004,40,845-859).The result of recent research point out lipopeptid by with on mononuclear phagocyte surface Toll-like receptor 2 interact and initial innate immune response.Upon activation, the intracellular domain of TLR-2 raises rank Meet protein MyD88, cause the activation of kinase cascade, cause many cytokines and the generation of chemotactic factor.On the other hand, fat Polysaccharide is by interacting and cellular response with Toll-like receptor 4 (TLR4)/MD2, and this causes adapter protein MyD88 With raising of TRIF, result in the cytokine of more complicated pattern.TNF-α secretion is the former of MyD88 dependent pathway activation Type is measured, and the secretion of IFN-β is typically used as indicant (Akira et al., the Nat. that TRIF dependent cell activates Immunol. 2001,2,675-680；Beutler, Mol. Immunol. 2004,40,845-859).

In order to check whether the connection of the glycopeptide containing T epi-position and B epitope and TLR part affects cytokine and produce, and surveys The TNF-α by compound 1,2 and 3 induction and effect (EC of IFN-β secretion are determined₅₀) and effect (maximum responsiveness), will knot Fruit and Pam₂CysSK₄、Pam₃CysSK₄Compare with those of LPS.Therefore, RAW NO is made^-Mouse macrophage is exposed to extensively The compound 1,2 and 3 of range of concentrations, Pam₂CysSK₄、Pam₃CysSK₄With escherichia coli 055:B5 LPS.After 5h, receive Obtain supernatant, use the capture ELISA mensuration of business or internal exploitation to be respectively directed to mice TNF-α and IFN-β checks this supernatant Liquid.

Table 4. Escherichia coli LPS and synthesis compound are for by mouse macrophage (RAW γ NO (-) cell) The EC of concentration-response curve that TNF-α produces₅₀And E_maxValue.

* the value of EC50 and Emax is reported as the best-fit values according to Prism (GraphPad Software, Inc).Make With the nonlinear least square method curve fitting analysis concentration-reply data in Prism.

As seen in Figures 3 and Table 4, glycolipidpeptide 3 and Pam₃CysSK₄The secretion of TNF-α, table is induced with similar effect and effect Cytokine and chemotactic factor response are not acted on by the connection of bright B epitope and T epi-position.Surprisingly, B epitope and T epi-position With Pam₂CysSK₄Connection cause substantially reducing of effect, the connection of B epitope and T epi-position causes living the most in this case The minimizing of property.Containing Pam₃The compound 1 of Cys part is lower than compound 2 and 3 remarkable activity, this possible explanation compound 1 Poor antigen.Compound 1,2 and 3 does not induce the generation of IFN-β.Surprisingly, with compound 1,2,3 and Pam₃CysSK₄ Comparing, escherichia coli 055:B5 shows for the TNF-α much bigger effect of induction and effect.Additionally, it can stimulate cell Produce IFN-β.Escherichia coli LPS is the most active, causes the excessive activation of innate immune system, causes the disease of septic shock Shape.

Speculating in addition to the generation of starter cytokine and chemotactic factor, lipopeptid can also promote in TLR2 dependency mode By selectivity targeting and the picked-up of antigen-presenting cell.In order to test this it is assumed that will execute containing fluorescently-labeled compound 4 For RAW NO^-Mouse macrophage, and harvesting after 30 minutes, crack and measure fluorescence.In order in view of possible Cell surface combine and without internalization, cell, the most before cracking by trypsin acting, is then checked for fluorescence.As can in Fig. 4 Seeing, significant amount of 4 is internalization, and is attached to cell surface on a small quantity.In order to measure whether picked-up is mediated by TLR2, use Natural HEK297 cell and the HEK297 cell with TLR2 or TLR4/MD2 transfection repeat picked-up research.It is essential that only when carefully When born of the same parents transfect with TLR2, it was observed that significantly absorb, show that picked-up is by this receptor-mediated.These research display TLR2 promotes antigen Picked-up, this be antigen processing and immunne response in important step.

Embodiment 4

Lipid composition covalently bound

In order to determine the covalently bound importance of TLR part and vaccine candidate object, design and synthesize and contained only B epitope and T The compound 5 (scheme 13) of epi-position.At PAM₃CysSK₄In the presence of, mice is exempted from weekly interval with this compound Epidemic disease is inoculated four times.It is interesting that glycopeptide 5 and adjuvant Pam₃CysSK₄Mixture do not cause IgG antibody or cause extremely low titre IgG antibody, it was demonstrated that Pam₃CysSK₄It is crucial with B epitope and T epi-position covalently bound for strong immune response.

Scheme 13

Embodiment 5

Lipid composition

In order to measure the importance lipidization with the part of Toll-like receptor, design and synthesize compound 6 (scheme 14).This Plant compound to be made up of the B epitope and T epi-position being connected to non-immunogenic lipidated amino acid.By the mice fat of compound 6 Liposome preparation immunity inoculation, is similarly to the program used for compound 1 and 2.Containing compound 6 liposome-induced significantly Less than the titre of those caused by compound 3, it was demonstrated that the TLR part of three component vaccines is important for optimal immunne response 's.

Scheme 14

Conclusion

The vaccine based on carbohydrate of three components has the many particular advantages relative to tradition conjugate vaccine.Such as, The shortcoming that bottom line subunit vaccine does not have the suppression of carbohydrate-protein conjugate distinctive epi-position.Except providing Outside danger signal, lipopeptid Pam₃CysSK₄Also promote that antigen mixes in liposome.Liposomal formulation is attractive, because it Antigen is effectively presented to immune system.The specific characteristic of vaccine is Pam₃CysSK₄Promote to be assisted by antigen-presenting cell, T The selectivity targeting of cell and bone-marrow-derived lymphocyte and picked-up, described cell expresses Toll loll sample receptor (embodiment 3).Finally, Completely synthetic compound has the advantage that it can be characterized completely, and this promotes that it is producing by playback system.

Embodiment 6

The antigenicity of synthesis tumor related carbohydrate antigen is increased by targeting Toll-like receptor

In this embodiment, design, the many completely synthetic vaccine candidate objects of chemosynthesis and immunological evaluation, to set up The weak immunogenic strategy and the research TLR in detail that overcome tumor related carbohydrate and glycopeptide participate in for antigen response Importance.TLR2 agonist, non-germline selectivity peptide t helper cell epi-position glycopeptide relevant with tumor covalently bound be given at little Mus causes the compound of the IgG antibody of the highest titre, the cancer of described IgG antibody recognition expression tumor related carbohydrate Cell.

The process LAN of oligosaccharide such as Globo-H, LewisY and Tn antigen is the common trait of oncogenic transformation cell (Springer, Mol. Med. 1997,75,594-602；Hakomori, Acta Anat. 1998,161,79-90；Dube, Nat. Rev. Drug Discov. 2005,4,477-488).Numerous researchs have shown that this Aberrant glycosylation can promote to turn Move (Sanders, J. Clin. Pathol. Mol. Pathol.1999,52, 174-178), the therefore table of these compounds Reach the weak survival rate strong association with cancer patient.Extensive and extensive magnitude clinic is front and clinical research confirmation is for carbon hydrate The natural acquisition antibody of thing associated tumor antigen, the antibody passively giving antibody or actively induction can eliminate in cancer patient Circulating tumor cell and small transfer (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).By being conjugated to exogenous carrier proteins matter (such as KLH and BSA) tumor related carbohydrate (Globo-H, Lewis^YAnd Tn) the conventional cancer vaccine candidate object that forms fails The IgG antibody of sufficiently high titre is caused in Most patients.Seem IgG antibody for tumor related carbohydrate Induction ratio causes the similar antibodies for virus and bacterial carbohydrates much more difficult.This observed result does not make us frightened It is surprised, because tumor associated sugars is autoantigen, thus is tolerated by immune system.The antigen of the tumor in growth comes off (shedding) this toleration is reinforced.Additionally, exogenous carrier proteins matter such as KLH can cause strong B cell response, this is permissible Cause the suppression of the antibody response for carbohydrate epitope.When using autoantigen such as tumor related carbohydrate Time, the latter is bigger problem.Additionally, can be immunogenic for carbohydrate with the joint puted together of protein, Epi-position is caused to suppress (Buskas, Chem. Eur. J. 2004,10,3517-3524；Ni, Bioconjug. Chem. 2006, 17,493-500).It is clear that the successful exploitation of cancer vaccine based on carbohydrate needs for more having to immune system Effect presents tumor related carbohydrate epi-position, causes New Policy (Reichel, the J. changed to the more effective classification of IgG antibody 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. 2004,64,4987- 4994；Buskas, Angew. Chem. Int. Ed. 2005,44,5985-5988 (embodiment 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).

At congenital and in the knowledge of the cooperation of adaptive immune response progress (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) provide for disease such as cancer The new way of vaccine design, for described disease, traditional vaccine method is the most failed.The quick responding to height of innate immune system is protected Keeping the family of compound, this high conservative compound is the major part of pathogen and is discovered for danger signal by host.These Identifying of molecular pattern is mediated by the set group of the receptor such as Toll-like receptor (TLR) of high conservative, and the activation of this receptor causes Acute inflammation response, such as direct local assault and the generation of different group cytokine invading pathogen.Except anti-micro- Outside biological property, cytokine and chemotactic factor also activate and regulate immune adaptability component (Lin, J Clin Invest 2007,117,1175-83).In this respect, cytokine stimulates the expression of many stimulating proteins altogether, at T Optimal interaction between auxiliary cell and B cell and antigen-presenting cell (APC).Additionally, some cytokines and chemotactic The factor is responsible for overcoming the suppression mediated by regulatory T cells.Other cytokines for guiding T auxiliary by effector T cell response Cell-1 (Th-1) or t helper cell-2 (Th-2) phenotype are important (Dabbagh, Curr. Opin. Infect. Dis. 2003,16,199-204).

Recently, we describe by tumor be correlated with MUC-1 glycopeptide B epitope, non-germline selectivity helper T cell epitope and Completely synthetic three component vaccine material standed fors (compound 21, Fig. 5) (Buskas, the Angew. Chem. of TLR2 part composition Int. Ed. 2005,44,5985-5988 (embodiments 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 property of three component vaccines is owing to as antigenicity and the possible induction any unnecessary spy of immunosuppressant That levies does not exists.But, it contains the All Media needed for causing about IgG immunne response.Additionally, TLR2 agonist Pam₃CysSK₄Connection with B epitope and T epi-position guarantees that is produced from the site that cytokine vaccine wherein and immunocyte interact Raw.This causes the cytokine of high local concentrations, promotes the maturation about immunocyte.In addition to danger signal are provided, lipopeptid Pam₃CysSK₄Also promote that antigen mixes in liposome, promote the selectivity targeting by antigen-presenting cell and bone-marrow-derived lymphocyte and Picked-up.

Optimal architecture and research TLR in detail in order to set up three completely synthetic component cancer vaccines participate in for anti- The importance of former response, our many completely synthetic vaccine candidate objects of chemosynthesis immunological evaluation.Have been found that by The Liposomal formulation of the compound 22 of the reticent lipopeptid of immunity, non-germline selectivity peptide t helper cell epi-position and MUC-1 glycopeptide composition Ratio is by TLR2 part (Pam₃CysSK₄) the notable antigenicity of compound 21 modified is lower.But, there is respectively TLR2 and TLR4 The Pam of agonist₃CysSK₄ (23) or monophosphoryl lipid A (24) compound 22 Liposomal formulation cause can be with compound 21 titres compared.But, the antiserum that the mixture by 22 and 23 or 24 causes has the impaired ability identifying cancerous cell. Surprisingly, by being connected to the MUC-1 glycopeptide B epitope of lipidated amino acid and being connected to Pam₃CysSK₄Auxiliary T epi-position group The mixture of the compound 25 and 26 become does not produces the antibody for MUC-1 glycopeptide.In a word, this result confirms that TLR participates in not being Required, but greatly strengthen the antigen response of the glycopeptide MUC-1 that is correlated with for tumor.TLR agonist and B epitope and auxiliary T epi-position Covalently bound be important for the Antibody maturation of cancer cell identification for improving.

Result and discussion.

Chemosynthesis.

Containing derived from the tumor of MUC-1 be correlated with glycopeptide (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 B epitope, derived from the Mus MHC restricted helper T cell of II class fully proved of poliovirus Epi-position KLFAVWKITYKDT (SEQ ID NO:3) (Leclerc, J. Virol. 1991,65,711-718) and lipopeptid Pam₃CysSK₄The compound 21 (Fig. 5) of (TLR2 agonist) (Spohn, Vaccine 2004,22,2494-2499), previously showed Show the IgG antibody (Ingale, Nat. Chem. Biol. 2007,3,663-667) causing the highest titre in mice.Change Compound 22 has the architecture similar to 21, but, TLR2 part have been replaced with lipidated amino acid (Toth, Tetrahedron Lett. 1993,34,3925-3928).The generation of lipidated amino acid not inducing cytokine, but, it Cause compound can mix in liposome.Therefore, glycolipidpeptide 22 is preferably suited for determining that TLR participates in for for tumor The importance of the antigen response of relevant glycopeptide.In order to measure the covalently bound importance of TLR part, use compound 22 and divide Wei the Pam of TLR2 and TRL4 agonist₃CysSK₄ (23) or monophosphoryl lipid A (24) Liposomal formulation (Spohn, Vaccine 2004,22,2494-2499；Chow, J. Biol. Chem. 1999,274,10689-10692).Finally, by even It is connected to the MUC-1 glycopeptide B epitope of lipidated amino acid and is connected to Pam₃CysSK₄Auxiliary T epi-position composition compound 25 He 26 for determining B epitope and the covalently bound importance of auxiliary T epi-position.Compound 21 prepare as previously described (Ingale, Nat. Chem. Biol. 2007,3,663-667；Ingale, Org. Lett. 2006,8,5785-5788).Use Rink The aminoacid of amide resin, Fmoc protection, Fmoc-Thr-(AcO₃-α-D-GalNAc) (Cato, J. Carbohydr. Chem. 2005,24,503-516) and Fmoc protection lipidated amino acid (Gibbons, Liebigs Ann. Chem. 1990, 1175-1183；Koppitz, Helv. Chim. Acta 1997,80,1280-1300), synthesize compound 22 by SPPS.Make With 2-(1H-benzotriazole-1-base)-Oxy-1,1,3,3-tetramethyl hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt) (Knorr, Tetrahedron Lett. 1989,30,1927-1930) introduces standard amino acid as activating reagent, Glycosylated amino acid is usedO-(7-azepine benzo triazol-1-yl)-N,N,N’,N'-tetramethylureaHexafluorophosphate (HATU)/ 1-hydroxyl-7-azepine benzotriazole (HOAt) loads, and lipidated amino acid is by benzotriazole-1-base-epoxide-tripyrrole alkane subbase PhosphorusHexafluorophosphate (PyBOP)/HOBt loads.After glycolipidpeptide has assembled, standard conditions are used to removeNEnd Fmoc Blocking group, obtained amine acetic anhydride and diisopropylethylamine (DIPEA)N-methyl pyrrolidone (NMP) solution passes through Acetylation caps.It follows that the acetonyl ester of the sugar moieties MeOH solution of 60% hydrazine cracks, with reagent B (TFA, H₂O, phenol, Triethyl silicane, 88/5/5/2, v/v/v/v) process and cause the removal of side chain protecting group and glycopeptide to be released from solid support Put.

By with after ice-cold diethyl ether precipitation and the HPLC purification of crude product on the semi-preparative post of C-4 subsequently, Obtain pure compound 22.Similar scheme is used for synthesizing compound 25.Derivant 26 is synthesized on Rink amide resin by SPPS, And after peptide assembles, obtained product is usedN-fluorenylmethyloxycarbonyl-R-(2,3-double (palmityl epoxide)-(2R-propyl group)- (R)-cysteine (Fmoc-Pam₂Cys-OH) manual coupling (Metzger, Int. J. Pept. Protein Res. 1991,38,545-554).Product is removed with the DMF solution of 20% piperidinesN-Fmoc group, and use PyBOB, HOBt and The DMF solution of DIPEA, makes obtained amine and Palmic acid coupling.By lipopeptid reagent B process, so that it is cracked from resin With removal side chain protecting group.By purifying by ice-cold diethyl ether precipitation and the HPLC on the semi-preparative post of C-4 subsequently Crude product.

Immunity inoculation and immunology.

By PC (PC), phosphatidyl glycerol (PG), cholesterol (Chol) and compound 21 or 22 (mole Ratio: 65/25/50/10) thin film hydration in the HEPES buffer (10 mM, pH 6.5) containing NaCl (145 mM), It is by extruding via 100 nm Nuclepore polycarbonate membranes subsequently, compound 21 and 22 is mixed based on phospholipid little In type unilamellar vesicle (SUV).By the group of five female BAl BIc/c mice with the liposome containing 3 μ g sugar with weekly interval Subcutaneous immunizations four times.Additionally, preparation has the glycopeptide 22 similar lipid to the mixture of 23 or 24 in HEPES buffer Body (mol ratio: PC/PG/Chol/22/23 or 24,65/25/5/5/5), uses four with weekly interval before serum is gathered in the crops Secondary.Finally, standardization program is used, by mice Liposomal formulation (mol ratio: the PC/PG/Chol/25/ of compound 25 and 26 26,65/25/5/5/5) immunity inoculation.

By being coated microtitration plate with glycopeptide TSAPDT (α-D-GalNAc) RPAP derivative for MUC-1 being conjugated to BSA Measure sero-fast anti-MUC-1 antibody titer, and complete with by anti-mouse IgM or the IgG antibody of alkali phosphatase enzyme mark Detection.The anti-MUC-1 IgG antibody (table 5) of the highest titre is caused with the mice of 21 immunity inoculations.The sub-typing of IgG antibody (IgG1, IgG2a, IgG2b and IgG3) points out the deflection towards Th2 immunne response.It was furthermore observed that high IgG3 titre be anti- The typical case of carbohydrate response.Cause significantly with replacing glycolipidpeptide 22 immunity inoculation of TLR2 part containing lipidated amino acid The IgG antibody of lower titre, it was demonstrated that it is very important that TLR participates in for optimal antigen response.But, there is Pam₃CysSK₄ Or the Liposomal formulation of compound 22 of monophosphoryl lipid A (24) causes IgG (always) titre similar to 21 (23).22 with In the case of the mixture of 23, as by high IgG1 and low IgG 2a, b titre proves, immunne response is partial to towards Th2 response. On the other hand, the use of monophosphoryl lipid A causes significant IgG1 and IG2a, b response, and the most this preparation causes mixing Th1/ Th2 response.Finally, the liposome containing compound 25 and 26 does not induce the anti-MUC-1 antibody that can measure titre, shows for anti- Former response, B epitope and T epi-position need covalently bound.

Table 5.By the anti-MUC1 of ELISA after 4 immunity inoculations of several formulations and anti-T epitope antibodies titre^a。

^aAnti-MUC1 and anti-T epitope antibodies titre present as the intermediate value of the group about five mices.For anti-MUC1 antibody Titre, is coated elisa plate BSA-MI-MUC1 conjugate, or for anti-T epitope antibodies titre, elisa plate is anti-by neutrality Biotin protein-biotin-T epi-position is coated.By linear regression analysis, mark and draw with dulling luminosity ratio compared with dilution factor and measure and drip Degree.Titre is defined as obtaining relative to the highest dilution that normal control mice serum optical density is 0.1 or bigger.

^bUse Liposomal formulation.

The anti-T epi-position of total IgG, the indivedual anti-MUC1 titre of IgG1, IgG2a, IgG2b, IgG3 and IgM and total IgG is at table 8 Middle report.

It follows that research is for the possible antigen response of auxiliary T epi-position.Therefore, Succ-PEG-DSPE is coated Microtitration plate processes with by the auxiliary T epi-position of biotin modification.After the serial dilutions adding serum, with by alkaline phosphatase Anti-mouse IgM of enzyme labelling or IgG antibody complete detection.It is interesting that compound 21 causes low resisting for auxiliary T epi-position Body, and the mixture of 22 and 23 or 24 does not causes the antibody for auxiliary T epi-position.

Respectively by interacting with TLR2 or TLR4 on mononuclear phagocyte surface, Pam₃CysSK₄Or monophosphoryl lipid Matter A produces (Kawai, Semin. Immunol. 2007,19,24-32) for starter cytokine.With Pam₃CysSK₄Swash After work, the intracellular domain of TLR2 raises adapter protein MyD88, causes the activation of kinase cascade, thus causes many thin Intracellular cytokine and the generation of chemotactic factor.On the other hand, lipopolysaccharide (LPS) and lipid A by with TLR4/MD2 complex phase interaction With and cellular response, this causes raising of adapter protein MyD88 and TRIF, thus cause the cell of more complicated pattern because of The induction of son.TNF-α secretion is that the prototype that MyD88 dependent pathway activates is measured, and the secretion of IFN-β is typically used as TRIF and depends on Rely the indicant that sexual cell activates.

In order to check that cytokine produces, make mouse macrophage (RAW γ NO (-) cell) to be exposed to broad range dense Compound 21-24, the escherichia coli 055:B5 LPS of degree and prototype escherichia coli two monophosphoryl lipid A (Zhang, J. Am. Chem. Soc. 2007,129,5200-5216).After 5.5 hours, gather in the crops catching of supernatant, use business or internal exploitation Obtain ELISA and be respectively directed to mice TNF-α and IFN-β checks this supernatant (Fig. 6).By using PRISM software to make dose response Curve matching, to logistic equation, measures effect (EC₅₀, produce the concentration of 50% activity) and effect (largest production level).Sugar Lipopeptid 21 and Pam₃CysSK₄ (23) induce TNF-α secretion with similar effect and effect, show that the connection of B epitope and T epi-position is right Cytokine response does not act on.As expected, the generation of none induction IFN-β of compound.Additionally, compound 22 is not induced TNF-α and IFN-β secretion, show that its lipid part is that immunity is reticent.Compound 24 stimulates cell to produce TNF-α and INF- β, but much smaller than escherichia coli 055:B5 LPS of its effect.Compared with compound 21 and 23, it shows much bigger TNF-α produces effect.Effect minimizing of compound 21 and 23 is probably favorable property, because LPS can be congenital with excessive activation Immune system, causes the symptom of septic shock.

It is next determined that the ability of natural MUC-1 antigen that mouse resisting anteserum identification is present on cancerous cell.Therefore, will The serial dilutions of blood serum sample add express MUC-1 MCF-7 human breast cancer cell (Horwitz, Steroids 1975, 26,785-95), and use FITC labelling anti-mouse IgG antibody set up identify.As shown in Figure 7, personal three component epidemic diseases are obtained Splendid MUC-1 tumor cell identification shown by the antiserum of Seedling 1 immunity inoculation, and when using the SK-not expressing MUC-1 antigen During MEL 28 cell, do not observe combination (Fig. 9).

Lipidization T-B epi-position (22) and Pam although must use by oneself₃CysSK₄ (23) blood of the mice of mixture immunity inoculation The high IgG antibody titre (table 5) that clear initiation is equal with 21, but observe the MCF-7 cell recognition of much less.This result is pointed out Adjuvant PamsCysSK₄ (23) being important with the covalently bound of B-T epi-position for correct Antibody maturation, correct antibody becomes The ripe cancer cell identification causing improving.Variable knot is caused by the mixture immunity inoculation of compound 22 and monophosphoryl lipid A (24) Really, two mices show that splendid MCF-7 cell recognition, three mices show medium MCF-7 cell recognition.

Discuss

The great majority being intended to develop cancer vaccine based on carbohydrate make great efforts to have concentrated on to use to be connected by manual splice Tumor related carbohydrate (Springer, Mol. Med. 1997,75,594-to the chemosynthesis of carrier protein 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 has been determined that use KLH conduct Carrier protein provides optimum with strong adjuvant QS-21 combination.But, the shortcoming of this method be KLH be very big and numb Tired protein, it can cause anti-KLH antibody (Cappello, the Cancer Immunol Immunother of high titre 1999,48,483-492) immunosuppressant of tumor related carbohydrate epi-position, is caused.Additionally, conjugation chemistry is often difficult to control System, because it results in the puting together of the ambiquity in terms of the Nomenclature Composition and Structure of Complexes with the repeatability that can affect immunne response Thing.Additionally, blank area can cause strong B cell response (Buskas, Chem. Eur. J. 2004,10,3517-3524； Ni, Bioconjug. Chem. 2006,17,493-500).Not surprisingly, carbohydrate-protein conjugate is used Clinical before and clinical research caused the result that mixes advantage.Such as, in the presence of adjuvant QS-21, with being conjugated to KLH's Trimer bunch (Tn (c)-KLH) immunized mice of Tn antigen causes IgG antibody (Kuduk, the J. Am. of medium titre Chem. Soc. 1998,120,12474-12485).Vaccine candidate is checked in the clinical trial of the patients with prostate cancer of recurrence Thing provides low intermediate value IgG and IgM antibody titre (Slovin, J. Clin. Oncol. 2003,21,4292-4298).

The research reported herein shows that wherein be correlated with glycopeptide B epitope, non-germline selectivity Mus MHC II class of MUC-1 is restricted Helper T cell epitope and covalently bound three component vaccines of TLR2 agonist (21), can cause strong IgG antibody response.Although Needed for TLR2 part and T-B glycopeptide epi-position covalently bound is not high IgG antibody titre, but find that it is for optimal cancerous cell Identification is very important.In this respect, containing compound 21 or the lipid of the mixture of compound 22 and TLR2 agonist 23 Body causes similar height anti-MUC-1 IgG antibody titre.But, obtain the antiserum of personal 21 immunity inoculations than 22 Hes of must using by oneself The cancerous cell of recognition expression MUC-1 under the serum dilution that the antiserum of the mixture immunity inoculation of 23 is much lower.Seem to use Three component vaccine 21 immunity inoculations cause more effective Antibody maturation, thus cause the cancer cell identification improved.

It was additionally observed that the difference in terms of the antigen response for auxiliary T epi-position.Therefore, 21 cause for auxiliary T epi-position The IgG antibody of low titre, and the mixture of 22 and 23 does not induce the antigen response of this part for candidate vaccine.Therefore, The covalently bound of TLR2 part makes the more antigenicity of compound 21, causes the low antibody response for auxiliary T epi-position.

Observe that compound 22 induces the total IgG antibody of similar high titre to the mixture of 23 or 24.But, work as employing TLR2 agonist Pam₃CysSK₄ (23) time, it was observed that towards the deflection of Th2 response (IgG1), and when using TLR4 agonist list During monophosphoryl lipid A (24), it is thus achieved that and mixing Th1/Th2 response (IgG2a, b).Difference in terms of the polarization of helper T cell is probably Owing to the cytokine of different mode is induced by TLR2 or TLR4.In this respect, Pam is previously observed₃Cys induction ratio large intestine bar Bacterium LPS lower level Th1 inducible for cytokine Il-12 (p70), and Th2 inductivity IL-10 of significantly higher level (Dillon, B. J Immunol 2004,172,4733-43).Difference is likely due to TLR4 and raises adapter protein MyD88 With the ability of Trif, and TLR2 only can raise MyD88.This result shows that immune system can suitably selecting with spy by adjuvant Determining direction customization, this is meaningful, because different IgG isotype performs different effect subfunction.

Result described herein also shows with compared with compound 21 ligand modified for TLR2, contains the reticent lipopeptid of immunity Compound 22 individually cause much lower IgG titre.Especially, compound 22 causes the ability of IgG2 antibody to be impaired. Use the recent research of the mice of defect in terms of TLR signal transduction that these innate immunity receptors are answered for adaptive immunity The importance answered throws doubt upon (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 respect, with the research of MyD88 deficient mice show IgM and IgG1 to a great extent but Not be completely dependent on TLR signal transduction, and IgG2 isotype to be complete TLR dependent (Blander, Nature 2006, 440,808-812).These observed results consistent with the result reported herein need TLR signal to turn owing to B cell maturation Lead.But, another research finds when under the presence or absence of several adjuvants, with trinitrophenol-hemocyanin (TNP- Hy) or during TNP-KLH immunity inoculation, MyD88^-/-/Trif^lps/lpsDouble KO mice causes titre similar to wild-type mice Antibody (Gavin, Science 2006,314,1936-1938).Conclude that and may need from adjuvant, get rid of TLR excitement Agent.Have been noted that the importance of adjuvant possibly relies on immunogenic antigenicity (Meyer-Bahlburg, J Exp Med 2007,204,3095-101；Pulendran, N Engl J Med 2007,356,1776-8).In this respect, the albumen of TNP Matter conjugate be high antigenicity and perhaps without adjuvant for optimal response.But, autoantigen such as tumor phase Close carbohydrate and there is low intrinsic antigenicity, and the result herein reported is explicitly shown when using TLR part altogether, it is thus achieved that Much better than antibody response.Additionally, confirm that the architecture of candidate vaccine is very important for optimal antigen response herein, TLR part and the covalently bound cancer cell identification causing improving of T-B epi-position especially.

The mixture of compound 25 and 26 can not cause anti-MUC-1 glycopeptide antibody to point out the covalently bound of T epi-position and B epitope Needed for being initiation antigen response.In this respect, B cell is needed by the activation of helper T cell and passes through antigen-presenting cell Helper T cell activate similar type cell-cell interaction.Therefore, the antigen containing protein or peptide needs by B cell Changing to be transported to endosomal vesicle, at this protease by digesting protein, some obtained fragments of peptides will be with II class MHC albumen Matter is combined.II class MHC-peptide complexes is transported to the cell surface of bone-marrow-derived lymphocyte subsequently, with mutual with helper T cell of mediation Effect, causes the classification conversion produced from low-affinity IgM to high-affinity IgG antibody.Different from antigen-presenting cell, B is thin Born of the same parents have weak phagocytosis character, and only internalization can combine the molecule of B-cell receptor.It is therefore contemplated that the internalization of auxiliary T epi-position By covalently bound being promoted with B epitope (MUC-1 glycopeptide), therefore the covalently bound of two kinds of epi-positions will cause higher resisting Former response.

In a word, the antigenic property having proven to completely synthetic cancer vaccine can be optimal by structure-activity relation research Change.In this respect, it has been determined that wherein tumor is correlated with MUC-1 glycopeptide B epitope, non-germline selectivity helper T cell epitope and TLR2 Three component vaccines that part is covalently bound, can cause extra high IgG antibody response, and it has the ability identifying cancerous cell. It is very important that auxiliary T epi-position is covalently attached to B epitope, it may be possible to because auxiliary T epi-position is needed by the internalization of B cell The existence of B epitope.The most shown TLR agonist mixes for being important for the be correlated with strong antigen response of glycopeptide antigen of tumor 's.In this respect, TLR2 part the cytokine induced is important for immune cell maturation, and immune cell maturation causes Powerful antibody response.It is surprisingly found that when TLR2 epi-position is covalently attached to glycopeptide T-B epi-position, it was observed that the cancer of improvement is thin Born of the same parents identify.The result herein presented provides the optimal important information constituted of three component vaccines, and will instruct based on carbon aquation The successful exploitation of the cancer vaccine of compound.

Experiment

Peptide symthesis:The scheme that peptide passes through to set up is at the ABI 433A peptide synthesizer (Applied being equipped with UV detector Biosystems) upper useN ^α The aminoacid of-Fmoc protection and 2-(1H-benzotriazole-1-base)-Oxy-1,1,3,3-tetramethyl Base hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt) (Knorr, Tetrahedron Lett. 1989,30,1927- 1930) synthesize as activating reagent.Single coupling step conditionality caps execution.Use following shielded aminoacid:N ^α -Fmoc-Arg(Pbf)-OH、N ^α -Fmoc-Asp(O ^t Bu)-OH、N ^α -Fmoc-Asp-Thr(ψ^Me,Mepro)-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( ^t Bu)- OH andN ^α-Fmoc-Tyr( ^t Bu)-OH.UseO-(7-azepine benzo triazol-1-yl)-N,N,N’,N'-tetramethylureaHexafluoro Phosphate (HATU)/1-hydroxyl-7-azepine benzotriazole (HOAt), as coupling agent, performs glycosylated amino acid by handN ^α- Fmoc-Thr-(AcO₃-α-D-GalNAc) idol of 1S (Cato, J. Carbohydr. Chem. 2005,24,503-516) Connection.Use benzotriazole-1-base-epoxide-tripyrrole alkane subbase-phosphorusHexafluorophosphate (PyBOP)/HOBt is as coupling agent (seeing support information), performsN ^α -Fmoc-lipophilic amino-acid (N ^α -Fmoc-D, L-tetradecanoic acid) 2S (Gibbons, Liebigs Ann. Chem. 1990,1175-1183；Koppitz, Helv. Chim. Acta 1997,80,1280-1300) WithN ^α -Fmoc-S-(double (the palmityl epoxide)-(2 of 2,3-R-propyl group)-(R)-cysteine 3S (Metzger, Int. J. Pept. Protein Res. 1991,38,545-554；Roth, Bioconj. Chem. 2004,15,541-553) (its by(R)-Prepared by (+)-2,3-Epoxy-1-propanol) coupling.Progress (Kaiser, Anal. by the test monitoring craft coupling of standard K aiser Biochem. 1970,34,595).

Prepared by liposome:By PC (PC), phosphatidyl glycerol (PG), cholesterol (Chol) and compound 21 or 22 (15 mmol, mol ratios 65:25:50:10) or PC/PG/Chol/22/23 or 24 (15 mmol, mol ratio 60:25:50: 10:5) or PC/PG/Chol/25/26 (15 mmol, mol ratio 65:25:50:5:5) be dissolved in trifluoroethanol and MeOH (1: 1, v/v, 5 mL) mixture in.Being removed in a vacuum by solvent, to provide thin lipid membrane, it is by existing under an argon 41 DEG C of vibration hydrations in 3 hours in the HEPES buffer (10 mM, pH 6.5) (1 mL) containing NaCl (145 mM).By capsule Bubble suspension supersound process 1 minute, subsequently 50 DEG C pass in succession through 1.0,0.4,0.2 and 0.1 μm polycarbonate membrane (Whatman, Nuclepore Track-Etch Membrane) extrusion, to obtain SUV.By heating SUV in sealing pipe at 100 DEG C The mixture of (50 μ L) and aqueous TFA (2 M, 200 μ L) measures GalNAc content in 4 hours.Subsequently that solution is the denseest Contracting, and by high pH anion-exchange chromatography, use pulsed amperometry (HPAEC-PAD；Methrome) and CarboPac post PA-10 and PA-20 (Dionex) is analyzed.

Dosage and immunity inoculation timetable:By group (female BAl BIc/c, the age 8-10 week of five mices；Jackson Laboratories) inoculate four times with weekly Immunity at intervals.Strengthen in Liposomal formulation, comprise 3 g sugar every time.Serum Sample acquisition in a week after immunity inoculation (blood-letting in advance) front and last immunity inoculation.Heart is passed through in last blood-letting Blood-letting completes.

Determination of serology:As previously described (Buskas, Chem. Eur. J. 2004,10,3517-3524), pass through Enzyme-linked immunosorbent assay (ELISA) measures anti-MUC-1 IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM antibody titre.Letter Yan Zhi, by elisa plate (Thermo Electron Corp.) the MUC-1 glycopeptide being conjugated to BSA by maleimide joint Conjugate (BSA-MI-MUC-1) is coated.The serial dilutions allowing serum combines fixing MUC-1.Sew by adding phosphate ester Anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.) of conjunction, IgG1 (Zymed), IgG2a (Zymed), IgG2b (Zymed), IgG3 (BD Biosciences Pharmingen) or IgM (Jackson ImmunoResearch Laboratories Inc.) antibody complete detection.Adding p-nitrophenyl phosphate ester (Sigma) After, use microplate (BMG Labtech) to measure absorbance at 405 nm, wherein wavelength calibration is located at 490 nm. Following mensuration is for the antibody titer of T (poliomyelitis) epi-position.Make Reacti-bind NeutrAvidin be coated and seal in advance The plate (Pierce) closed is incubation 2 hours together with biotin labeled T epi-position (10 g/mL).It follows that allow serum is Row diluent combines fixing T epi-position.It is completed as previously described detection.Antibody titer is defined as obtaining relative to normal control mice Serum optical density is the highest dilution of 0.1 or bigger.

Cell is cultivated:RAW 264.7 γ NO derived from RAW 264.7 mouse monokaryon cell/macrophage cell line (-) cell derives from ATCC.Cell is maintained in RPMI 1640 culture medium with L-glutaminate (2 mM), described RPMI 1640 culture medium are adjusted to containing sodium bicarbonate (1.5 g L^-1), glucose (4.5 g L^-1), HEPES (10 mM) and acetone Acid sodium (1.0 mM), and it is supplemented with penicillin (100 u mL^-1)/streptomycin (100 g mL^-1；And FBS Mediatech) (10%；Hyclone).Derive from people's breast adenocarcinoma cell (MCF7) (Horwitz, Steroids 1975,26,785-95) of ATCC Have L-glutaminate (2 mM) and Earle ' s BSS Iger minimum essential medium (Eagle ' s minimum Essential medium) middle cultivation, described Iger minimum essential medium is modified to containing sodium bicarbonate (1.5 g L^-1), non essential amino acid (0.1 mM) and Sodium Pyruvate (1 mM), and be supplemented with bovine insulin (0.01 mg mL^-1；Sigma) With FBS (10%).Application on human skin malignant melanoma cell (SK-MEL-28) derives from ATCC, and have L-glutaminate (2 mM) and Growing in the Iger minimum essential medium of Earle ' s BSS, described Iger minimum essential medium is adjusted to containing carbon Acid hydrogen sodium (1.5 g L^-1), non essential amino acid (0.1 mM) and Sodium Pyruvate (1 mM), and be supplemented with FBS (10%).All Cell maintains moist 5% CO at 37 DEG C₂In air.

TNF-α and IFN-β measure.Measure the same day exposing, by RAW 264.7 γ NO (-) cell is with 2 x 10⁵Individual carefully Born of the same parents/hole is plated in 96 orifice plates (Nunc), under the presence or absence of polymyxin B, and incubation 5.5 together with different stimulated thing Hour.Culture supernatant being collected and freezing (-80 DEG C) storage, producing until measuring cytokine.Use from R&D The TNF-α DuoSet ELISA Development kit measurement TNF-α concentration of Systems.Measure the dense of IFN-β as follows Degree.By ELISA MaxiSorp plate rabbit polyclonal antibody (the PBL Biomedical for mice IFN-β Laboratories) it is coated.The IFN-β in standard substance and sample is allowed to combine immobilized antibody.It is subsequently added big mouse-anti little Mus IFN-β antibody (USBiological), produces antibody-antigen-antibody " sandwich ".It follows that addition horseradish peroxidase (HRP) mountain goat anti rat IgG (H+L) antibody (Pierce) puted together and chromogenic substrate 3,3 ', the 5,5 '-tetramethyl biphenyl of HRP Amine (TMB；Pierce).After reaction stops, measuring absorbance at 450 nm, wherein wavelength calibration sets to 540 nm.Use Nonlinear least square method curve fitting analysis concentration-reply data in Prism (GraphPad Software, Inc.).With Following four these data of parameter logistic equation matching: Y=E_max /(1 +(EC₅₀/X)^{Hill slope}), wherein Y is cytokine Response, X is the concentration of stimulus object, E_maxIt is maximum response, and EC₅₀It is the stimulus object concentration producing 50% stimulation.Hill slope It is set to 1, so that the EC of different inducer can be compared₅₀Value.All cells factor values is as the triplicate meansigma methods ± SD measured Present, wherein test in triplicate every time.

For LPS pollution evaluation material:In order to ensure the aborning any increase of cytokine not by containing multiple stimulation The LPS of the solution of thing pollutes and causes, and affinity combines the lipid A region of LPS, thus the cytokine stoping LPS to induce produces (Tsubery, Biochemistry 2000,39,11837-44).In incubation 5.5 hours together with escherichia coli O55:B5 LPS Before with polymyxin B (30 g mL^-1；Bedford Laboratories) together in the precincubation cell supernatant of 30 minutes TNF-α and IFN-β concentration show and completely inhibit, and together with polymyxin B precincubation pair and synthesis compound 21 and 23 1 Play the TNF-α synthesis not effect of the cell of incubation.Therefore, the LPS of latter preparation pollutes is inessential.

By the cell recognition analysis of fluorescence measurement:Make the serum of immunity inoculation front and rear serial dilutions and MCF7 and SK-MEL-28 single cell suspension is together incubated on ice 30 minutes.It follows that cell is washed and glimmering with being conjugated to isothiocyanic acid Light element (FITC；Sigma) goat anti-mouse IgG γ-chain specific antibody is together incubated on ice 20 minutes.Wash at three times Wash after cracking with cell, use microplate (BMG Labtech), fluorescence intensity (485 ex/520 em) is analyzed thin Cellular lysate product.Three independent experiments are collected and represented to data point in triplicate.

Embodiment 7

The synthesis of compound

Conventional method:Fmoc-L-amino acid derivativges and resin are purchased from NovaBioChem and Applied Biosystems；Peptide Synthesis rankN, N-dimethylformamide (DMF) is purchased from EM Science；N-methyl pyrrolidone (NMP) is purchased from Applied Biosystems.PC (PC), phosphatidyl glycerol (PG), cholesterol (Chol) and monophosphoryl lipid A (MPL-A) Derive from Avanti Polar Lipids.EZ-Link NHS-biotin reagent (succinimido-6-(biotin amide) Alkyl caproate) derive from Pierce.Every other chemical reagent is purchased from Aldrich, Acros, Alfa Aesar and Fisher Scientific, and use without being further purified.The all solvents used are all SILVER REAGENT.Use with 1 mL/ minute The Agilent Zorbax Eclipse of flow velocity^TMC8 analytical column (5 μm, 4.6 x 150 mm), with 3 mL/ minute flow velocity Agilent Zorbax Eclipse^TMThe semi-preparative post of C8 (5 μm, 10 x 250 mm) or with 2mL/ minute flow velocity Phenomenex Jupiter^TMThe semi-preparative post of C4 (5 μm, 10 x 250 mm), is being equipped with automatic injector, Fraction collection RPHPLC (reversed-phase high-performance liquid chromatography) (RP-is performed on Agilent 1100 serial system of device and UV detector (detecting at 214 nm) HPLC).All operations all use linear gradient (solvent orange 2 A=5% acetonitrile, 0.1% trifluoro through the 0-100% solvent B of 40 minutes The aqueous solution of acetic acid (TFA), solvent B=5% water, the acetonitrile solution of 0.1%TFA) perform.Divide in ABI 4700 proteomics Matrix-assisted laser desorption ionization method (MALDI-TOF) mass spectrum is recorded in analyzer.

The synthesis of glycolipidpeptide 22:As described under peptide symthesis in an experiment, synthesis 22 is at Rink amide resin (28,0.1 Mmol) upper execution.Front four aminoacid Arg-Pro-Ala-Pro use standard scheme coupling on peptide synthesizer, to obtain 29. After completion of the synthesis, the manual coupling of 1S (0.2 mmol, 134 mg) is performed.WillN ^α-Fmoc-Thr-(AcO₃-α-D- GalNAc) 1S (Cato, J. Carbohydr. Chem. 2005,24,503-516) is dissolved in NMP (5 mL), willO- (7-azepine benzo triazol-1-yl)-N,N,N’,N'-tetramethylureaHexafluorophosphate (HATU；0.2 mmol, 76 mg), 1- Hydroxyl-7-azepine benzotriazole (HOAt；0.2 mmol, 27 mg) and diisopropylethylamine (DIPEA；0.4 mmol, 70 μ L) Add in solution, then obtained mixture is added in resin.By standard K aiser test monitoring coupling reaction.12 After hour, by resin NMP (6 mL) and dichloromethane (DCM；6 mL) washing, experience identical coupling condition the most again, with really Protect complete coupling.Glycopeptide 30 is extended subsequently on peptide synthesizer.After completion of the synthesis, by resin NMP (6 mL), DCM (6 And methanol (MeOH mL)；6 mL) fully wash, and be vacuum dried.Subsequently by resin in DCM (5 mL) swelling 1 hour, and And perform the remainder of coupling by hand.It follows that NMP (5 mL), benzotriazole-1-base-epoxide-tripyrrole alkane will be dissolved in Subbase-phosphorusHexafluorophosphate (PyBOP；0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 Mmol, 67 μ L) inN ^α -Fmoc-lipophilic amino-acid (N ^α -Fmoc-D, L-tetradecanoic acid) 2S (Gibbons, Liebigs Ann. Chem. 1990,1175-1183；Koppitz, Helv. Chim. Acta 1997,80,1280-1300) (0.3 Mmol, 139 mg) premixing 2 minutes, it is subsequently added in resin.By Kaiser test monitoring coupling reaction, then standing 8 Complete after hour.Use DMF (6 mL) the solution cracking of piperidines (20%)N ^α -Fmoc group.To be dissolved in NMP (5 mL), In PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μ L)N ^α - Fmoc-Gly-OH (0.3 mmol, 90 mg) premixing 2 minutes, is subsequently added in resin.By Kaiser test monitoring coupling Reaction, then completes after standing 4 hours.Use DMF (6 mL) the solution cracking of piperidines (20%)N ^α -Fmoc group.Use PyBOP (0.3 mmol, 156 mg), the NMP (5 mL) of HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μ L) Solution, another coupling cycle of 2S executed as described above (0.3 mmol, 139 mg).Finally, the DMF of piperidines (20%) is used (6 mL) solution cracksN ^α -Fmoc group, and by with Ac₂NMP (5mL) solution of O (10%) and DIPEA (5%) processes resin 10 minutes, make obtained free amine group acetylation.By resin NMP (5 mL x 2), DCM (5 mL x 2) and MeOH (5 ML x 2) fully wash, and be dried in a vacuum.By resin in DCM (5 mL) swelling 1 hour, with hydrazine (60%) MeOH^4,5(10 mL) solution processes 2 hours, abundant with NMP (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2) Washing, is then dried in a vacuum.By resin in DCM (5 mL) swelling 1 hour, subsequently with reagent B (TFA (88%), water (5%), phenol (5%) and TIS (2%), 10 mL) process 2 hours.By resin filter, wash with pure TFA (2 mL), subsequently will Filtrate is concentrated into about the 1/3 of its initial volume in a vacuum.Use diethyl ether (0 DEG C, 40 mL) precipitation glycolipidpeptide, by with 3, 000 rpm is centrifuged recovery in 15 minutes.Use is through the linear gradient of the solution A of the 0-95% solvent B of 40 minutes, semi-preparative By RP-HPLC purification of crude glycolipidpeptide on C-4 post, and by suitable flow point lyophilizing with provide 22 (Figure 10) (57 mg, 16%). C₁₆₅H₂₆₇N₃₇O₄₄, MALDI-ToF MS: observe, [M+] 3473.4900Da；Calculate, [M+] 3473.1070Da。

Scheme 15.Reagent and condition: a) use Fmoc chemistry SPPS, in the presence of the nmp solution of DIPEA with HBTU/HOBt coupling；B) 1S, HATU/HOAt, DIPEA, NMP, overnight；C) i. is in the presence of the nmp solution of DIPEA, The manual coupling of 2S Yu PyBOP/HOBt；Ii. the DMF solution of 20% piperidines；Iii. in the presence of the nmp solution of DIPEA, The manual coupling of 1S Yu PyBOP/HOBt；Iv. the DMF solution of 20% piperidines；V. in the presence of the nmp solution of DIPEA, 2S Manual coupling with PyBOP/HOBt；Vi. the DMF solution of 20% piperidines；vii. 10%Ac₂The nmp solution of O, 5%DIPEA reaches 10 Minute；The MeOH solution of (d) 60% hydrazine, 2 hours；E) reagent B, TFA (88%), phenol (5%), water (5%), TIS (2%), 2 is little Time.

The synthesis of lipopeptid 23:As described under peptide symthesis in an experiment, the synthesis of 23 is at Rink amide resin (28,0.1 Mmol) upper execution.After first five amino acid whose coupling, the lipid part of manual coupling molecule.WillN ^α -Fmoc-S-(2,3-is double (palmityl epoxide)-(2R-propyl group)-(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) be dissolved in In DMF (5 mL), by PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μ L) add in solution.After the 2 minutes, reactant mixture is added in resin.By Kaiser test monitoring coupling reaction, so After complete standing after 12 hours.It follows that use DMF (6 mL) the solution cracking of piperidines (20%)N ^α -Fmoc group, to obtain Obtain 36.Use PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μ L) DMF solution, makes the unhindered amina coupling of Palmic acid (0.3 mmol, 77 mg) and 36 as mentioned above.By resin DMF (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2) fully wash, be then dried in a vacuum.By resin in DCM (5 mL) Swelling 1 hour, subsequently with TFA (95%), water (2.5%) and TIS (2.5%) (10 mL) room temperature treatment 2 hours.By resin mistake Filter, washs with pure TFA (2 mL).Subsequently filtrate is concentrated in a vacuum about the 1/3 of its initial volume.Use diethyl ether (0 DEG C, 30 mL) precipitation lipopeptid, by being centrifuged recovery in 15 minutes with 3000 rpm.Use the 0-95% solvent B through 40 minute period The linear gradient of solvent orange 2 A solution, by RP-HPLC purification of crude lipopeptid on semi-preparative C-4 post, and by suitable flow point Lyophilizing is to provide 23 (Figure 11) (40 mg, 26%).C₈₁H₁₅₆N₁₁O₁₂S, MALDI-ToF MS: [M+Na] observed, 1531.2240Da；[M+Na] calculated, 1531.1734Da.

Scheme 16.Reagent and condition: a) use Fmoc chemistry SPPS, in the presence of the nmp solution of DIPEA with HBTU/HOBt coupling；B) in the presence of the DMF solution of DIPEA, the 3S craft coupling activated by PyBOP/HOBt；C) piperazine The DMF solution of pyridine (20%)；D) in the presence of the DMF solution of DIPEA, the Palmic acid coupling activated by PyBOP/HOBt；e) TFA (95%), water (2.5%), TIS (2.5%), 2 hours.

The synthesis of glycolipidpeptide 25:As described under peptide symthesis in an experiment, synthesis 25 Rink amide resin (28, 0.1 mmol) upper execution.Front four aminoacid Arg-Pro-Ala-Pro use standard scheme coupling on peptide synthesizer, to obtain Obtain 29.After completion of the synthesis, 1S (0.2 mmol, 134 mg) is used to perform manual coupling.1S is dissolved in NMP (5 mL), Add HATU (0.2 mmol, 76 mg), HOAt (0.2 mmol, 27 mg) and DIPEA (0.4 mmol, 70 μ L), then by institute The mixture obtained adds in resin.By standard K aiser test monitoring coupling reaction.After 12h, by resin NMP (6 mL) and DCM (6 mL) wash, and experience identical coupling condition the most again, to guarantee complete coupling.Subsequently on peptide synthesizer Extend glycopeptide 30.After completion of the synthesis, resin NMP (6 mL), DCM (6 mL) and MeOH (6 mL) are fully washed, then It is dried in a vacuum.Subsequently by resin in DCM (5 mL) swelling 1 hour, and have been manually done the remainder of peptide sequence.Will 2S (0.3 mmol, 139 mg) is dissolved in NMP (5 mL), by PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 Mg) add in solution with DIPEA (0.4 mmol, 67 μ L).After the 2 minutes, feed the mixture in resin.Pass through standard Kaiser test monitoring coupling reaction, and complete after standing 8 hours.It follows that use the DMF (6 mL) of piperidines (20%) Solution cracksN ^α -Fmoc group.WillN ^α -Fmoc-L-glycine (0.3 mmol, 90 mg) is dissolved in NMP (5 mL), and is inciting somebody to action Before reactant mixture adds in resin, with PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μ L) premixing 2 minutes.By Kaiser test monitoring coupling reaction, and complete after standing 4 hours. Use DMF (6 mL) the solution cracking of piperidines (20%)N ^α -Fmoc group.Use PyBOP (0.3 mmol, 156 mg), HOBt NMP (5 mL) solution of (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μ L), 2S (0.3 executed as described above Mmol, 139 mg) another coupling cycle.Finally, DMF (6 mL) the solution cracking of piperidines (20%) is usedN ^α -Fmoc base Group, and use Ac₂NMP (5 mL) solution of O (10%) and DIPEA (5%), makes obtained free amine group acetylation 10 points Clock.Resin NMP (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2) are fully washed, and does in a vacuum Dry.By resin in DCM (5 mL) swelling 1 hour, process 2 hours with MeOH (10 mL) solution of hydrazine (60%), with NMP (5 ML x 2), DCM (5 mL x 2) and MeOH (5 mL x 2) fully washs, and is dried in a vacuum.By resin at DCM (5 ML) in swelling 1 hour, by it with reagent B (TFA (88%), water (5%), phenol (5%) and TIS (2%), 10 mL) process after this 2 hours.By resin filter, wash with pure TFA (2 mL), subsequently filtrate is concentrated in a vacuum about the 1/ of its initial volume 3.Use diethyl ether (0 DEG C；40 mL) precipitation glycolipidpeptide, and by with 3,000 rpm is centrifuged recovery in 15 minutes.Use and pass through The linear gradient of the solution A of the 0-95% solvent B of 40 minutes, by RP-HPLC purification of crude glycolipid on semi-preparative C-4 post Peptide, by suitable flow point lyophilizing to provide 5 (Figure 12) (35 mg, 19%).C₈₄H₁₄₅N₁₉O₂₅, MALDI-ToF MS: observe, [M+] 1821.1991Da；Calculate, [M+] 1821.1624Da.

Scheme 17.Reagent and condition: a) use Fmoc chemistry SPPS, in the presence of the nmp solution of DIPEA with HBTU/HOBt coupling；B) 1S, HATU/HOAt, DIPEA, NMP, overnight；C) i. is in the presence of the nmp solution of DIPEA, The manual coupling of 2S Yu PyBOP/HOBt；Ii. the DMF solution of 20% piperidines；Iii. in the presence of the nmp solution of DIPEA,N ^α The manual coupling of-Fmoc-Gly-OH and PyBOP/HOBt；Iv. the DMF solution of 20% piperidines；V. at the nmp solution of DIPEA In the presence of, the manual coupling of 2S Yu PyBOP/HOBt；Vi. the DMF solution of 20% piperidines；vii. 10% Ac₂O, 5% DIPEA Nmp solution reach 10 minutes；The MeOH solution of (d) 60% hydrazine, 2 hours；E) reagent B, TFA (88%), phenol (5%), water (5%), TIS (2%), 2 hours.

The synthesis of lipopeptid 26:The synthesis of 26 is in the upper execution of Rink amide resin (28,0.1 mmol).By using standard After SPPS secretory piece, the lipid part of manual coupling molecule.3S (0.3 mmol, 267 mg) is dissolved in DMF (5 mL), PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μ L) are added solution In.After 3S activates 2 minutes, reactant mixture is added in resin.By Kaiser test monitoring coupling reaction, and quiet Complete after putting 12 hours.Use DMF (6 mL) the solution cracking of piperidines (20%)N-Fmoc group, to obtain 43.Use PyBOP (0.3 mmol, 156 mg), the DMF solution of HOBt (0.3 mmol, 40 mg) and DIPEA (0.4 mmol, 67 μ L), as above institute State the unhindered amina coupling making Palmic acid (77 mg, 0.3 mmol) and 43.By resin DMF (5 mL x 2), DCM (5 mL x 2) fully wash with MeOH (5 mL x 2), be then dried in a vacuum.By resin in DCM (5 mL) swelling 1 hour, with examination Agent B (TFA (88%), water (5%), phenol (5%) and TIS (2%), 10 mL) processes 2 hours, filters, and with pure TFA (2 mL) Washing.Subsequently filtrate is concentrated in a vacuum about the 1/3 of its initial volume, uses diethyl ether (0 DEG C, 30 mL) precipitation lipopeptid, Then by being centrifuged recovery in 15 minutes with 3000 rpm.Use the linear gradient of solution A through the 0-95% solvent B of 40 minutes, By RP-HPLC purification of crude lipopeptid on semi-preparative C-4 post, and by suitable flow point lyophilizing to provide 26 (Figure 13) (57 Mg, 18%).C₁₆₂H₂₇₈N₂₉O₃₁S, MALDI-ToF MS: observe, [M+] 3160.9423Da；Calculate, [M+] 3160.1814Da。

Scheme 18.Reagent and condition: a) use Fmoc chemistry SPPS, in the presence of the nmp solution of DIPEA with HBTU/HOBt coupling；B) in the presence of the DMF solution of DIPEA, the manual coupling of 3S, PyBOP, HOBt；C) 20% piperidines DMF solution；D) in the presence of the DMF solution of DIPEA, Palmic acid, the manual coupling of PyBOP, HOBt；E) reagent B, TFA (88%), phenol (5%), water (5%), TIS (2%), 2 hours.

The synthesis of biotin-T epitope peptide 27:As described in General Method, the synthesis of 27 Rink amide resin (28, 0.1 mmol) upper execution.After completion of the synthesis, by resin DMF (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2) fully wash, be dried in a vacuum subsequently.By resin in DCM (5 mL) swelling 1 hour.It follows that by EZ-Link NHS-biotin reagent (succinimido-6-(biotin amide) alkyl caproate) (0.2 mmol, 90 mg) and DIPEA (0.2 Mmol, 36 μ L) mixture in DMF (5 mL) adds in resin.By the test monitoring coupling of standard K aiser and even Complete in being associated in 8 hours.Resin DMF (5 mL x 2), DCM (5 mL x 2) and MeOH (5 mL x 2) are fully washed, Then it is dried in a vacuum.Resin is expanded 1 hour in DCM (5 mL), with reagent B (TFA (88%), water (5%), phenol (5%) and TIS (2%), 15 mL) room temperature treatment 2 hours.By resin filter, wash with pure TFA (2 mL).By filtrate very It is concentrated into about the 1/3 of its initial volume in the air.Use diethyl ether (0 DEG C, 30 mL) to precipitate this peptide, and pass through with 3,000 rpm Within centrifugal 15 minutes, reclaim.Use is through the linear gradient of the solvent orange 2 A solution of the 0-95% solvent B in 40 minute period, semi-preparative By RP-HPLC purification of crude peptide on C-8 post, and by suitable flow point lyophilizing with provide 27 (Figure 14) (60%, fill based on resin Loading capability).C₉₅H₁₄₇N₂₁O₂₁S, MALDI-ToF MS: [M+] observed, 1951.2966Da；[M+] calculated, 1951.3768Da。

Scheme 19.Reagent and condition: a) use the SPPS of Fmoc chemistry, in the presence of the nmp solution of DIPEA, with HBTU/HOBt coupling；B) in the presence of the DMF solution of DIPEA, succinimido-6-(biotin amide) alkyl caproate Manual coupling；C) reagent B, TFA (88%), phenol (5%), water (5%), TIS (2%), 2 hours.

Embodiment 8

Completely synthetic multicomponent cancer vaccine causes multi-mode immunne response (multi-model immune response)

The glycopeptide that this embodiment confirms to be covalently attached to glycosylation MUC1 of Toll-like receptor (TLR) agonist derivative can draw Send out humoral and cellular immune response response effective, and reversing tolerance and to generate in treatment response be effective.Many comparisonizations The curative effect checking confirmation three component vaccines of compound is the non-specific antitumor response owing to being caused by adjuvant, and passes through The specificity humoral of glycopeptide initiation derivative for MUC1 and cellullar immunologic response.Have been found that the glycosylation of MUC1 peptide is optimal for induction Response is crucial, additionally, it is required that auxiliary T epi-position and B epitope are covalently attached to TLR part.

Result

ANTIGEN DESIGNThe and tumor challenge research.Fully set up breast cancer mouse model (Akporiaye et al., 2007,Vaccine；The mixture of compound 1,2,3,4 and 5 and the merit of the Liposomal formulation of single 5 is checked in 25:6965-6974) Effect (Figure 16).Multicomponent vaccine material standed for 1 containing the tumor derived from MUC1 be correlated with glycopeptide (Baldus et al., 2004,Crit. Rev Clin Lab Sci；41:189-231；And Springer, 1997,J Mol Med；75:594-602), derived from spinal cord Mus MHC restricted helper T cell epitope KLFAVWKITYKDT of II class (SEQ ID NO:1) fully proved of poliovirus (Leclerc et al., 1991,J Virol；65:711-718) with as effective agonist of Toll-like receptor-2 (TLR2) Lipopeptid Pam3CysSK4 (Spohn et al., 2004,Vaccine；22:2494-2499).Previously, derivative for MUC1 glycopeptide SAPDT (α-GalNAc) RPAP be accredited as the tandem sequence repeats of MUC1 antigenicity dominance structure territory (Baldus et al., 2004,Crit. Rev Clin Lab Sci；41:189-231；And Springer, 1997,J Mol Med；75:594-602).Additionally, this table Position can also with MHC I class (K^b) complex present, cause the activation of cytotoxic T lymphocyte (CTL) (Apostolopoulos et al., 2003,Proc Natl Acad Sci USA；100:15029-15034).

As illustrated in this embodiment, the MHC II class restricted helper T cell epitope induction of 1 is raw from IgM to IgG antibody Classification conversion (Figure 20) produced, and promote external source glycopeptide presenting in MHC 1 class.Finally, the Pam3CysSK4 part of 1 is passed through Cause relevant cytokine and chemotactic factor serve as embedding adjuvant (Spohn et al., 2004,Vaccine；22:2494-2499). The importance of the carbohydrate portions in order to measure 1, checks construct 2, and it has the structure similar to 1, simply MUC1 peptide Threonine be not glycosylated.Compound 3 lacks the MUC1 glycopeptide epi-position of 1 and 2, and examined to explain owing to passing through adjuvant The possible curative effect of immune activation.Finally, check glycopeptide 4 and the mixture of adjuvant Pam3CysSK4 5, with determine adjuvant with MUC1 glycopeptide and the covalently bound importance of auxiliary T epi-position.

Multicomponent vaccine 1 by liposome-mediated native chemical connect (Ingale et al., 2006,Org Lett；8: 5785-5788) it is prepared.Compound 2,3,4, by SPPS scheme, uses the aminoacid of Rink amide resin, Fmoc protection Fmoc-Thr-(AcO₃-α-D-GalNAc) synthesis.By synthesis compound, PC, phosphatidyl glycerol and cholesterol Thin film in the HEPES buffer (10 mM, pH 6.5) containing NaCl (145 mM) hydration and subsequently via 100 nm Nuclepore^®The extrusion of polycarbonate membrane, mixes obtained compound in small unilamellar vesicles (SUV) based on phospholipid. With mixture and the Liposomal formulation of single 5 of compound 1,2,3,4 and 5, the MUC1.Tg mice of people MUC1 will be expressed (C57BL/6；H-2^b) group with once every two weeks Immunity at intervals inoculate three times.After 35 days, mice is used MMT mammary neoplasms Cell (being positive MUC1 and Tn) is attacked, and is the reinforcement again after a week subsequently.Immunity inoculation two weeks after the last time, By sacrifice, and measured effect of vaccine by tumor weight.Additionally, pass through the titre of MUC1 specific antibody and anti-blood Clear cracking lotus has the ability of the tumor cell of MUC1, assesses the robustness of humoral immunoresponse(HI).Additionally, produced by mensuration The CD8 of IFN-γ⁺The number of T cell and the ability of these cells cracking tumor cell, evaluate cellullar immunologic response.

With empty liposome or with do not contain MUC1 glycopeptide epi-position compound 3 process compared with, use multicomponent vaccine candidate What thing 1 immunity inoculation caused tumor load substantially reduces (Figure 17).It is interesting that compared with the application of empty liposome, with change Compound 3 immunity inoculation causes marginally smaller tumor, shows the antitumor character caused by nonspecific adjuvant effect.With right Comparing according to immunity inoculation, the mixture of glycosylation multicomponent vaccine material standed for 2 and compound 4 and 5 does not demonstrates cancer resistance Significantly improving of matter.In these cases, it was observed that the big dispersion in terms of tumor weight, lead by compound 1 immunity inoculation Cause the essence of tumor weight in all mices to reduce.

Humoral immunization.By being coated trace with CTSAPDT (α-D-GalNAc) RPAP being conjugated to the BSA that acetyl bromide is modified Titer plate measures anti-MUC1 antibody titer.Compound 1 has caused strong IgG antibody response, and the sub-typing of antibody points out mixing Th1/Th2 response (table 6).The mice attacked by 1 immunity inoculation but unused MMT tumor cell causes the antibody of similar titre, refers to The immunosuppressant going out cancerous cell is likely to be obtained reverse.Suppression ELISA display polyclonal serum has for glycosylation MUC1 epi-position Slightly greater affinity (table 7).Additionally, measure the low titer antibody for auxiliary T epi-position, it is indicated that candidate vaccine does not has Immunosuppressant shortcoming.Although compound 2 does not contains carbohydrate portions, but obtained antiserum can identify CTSAPDT (α-D-GalNAc) RPAP epi-position.But, in this case, it is not detected by IgG3 antibody.It is interesting that chemical combination The mixture of thing 4 and 5 has caused the antibody of low titre, highlights the covalently bound for by force of Pam3CysSK4 and glycopeptide epi-position The importance of antigen response.As expected, do not contain MUC1 to derive the comparison (3 and 5) of epi-position and do not cause anti-MUC1 antibody response.

By with⁵¹Two MUC1 of Cr labelling express cancer cell-types and antiserum subsequently and cytotoxic effect cell The interpolation of (NK cell) and release⁵¹The measurement of Cr, checks the cytotoxicity (ADCC) that antibody dependent cellular mediates.Such as figure In 18A and 18B visible, compared with control compound 3, by with 1 immunity inoculation obtain antiserum can dramatically increase cancer Cell cracks.It is essential that compared with compound 1, the antibody caused by compound 2 is substantially less had in cell cracks Effect, highlights glycosylation for the importance about antigen response.As expected, derived from 4 and 5 mixture and shortage The antiserum of the comparison derivant of MUC1 glycopeptide does not induce notable cell to crack.

Table 6. is by the anti-MUC1 of ELISA after 4 immunity inoculations of several formulations and anti-T epitope antibodies titre^[a]。

[a] anti-MUC1 and anti-T epitope antibodies titre present as the intermediate value of the group of four to ten three mices.For anti-MUC1 Antibody titer, is coated elisa plate BSA-MI-MUC1 (Tn) conjugate, or for anti-T epitope antibodies titre, by elisa plate It is coated by neutravidin-biotin-T epi-position.Titre is measured, wherein compared with absorbance by linear regression analysis Relatively dilution factor is mapped.It is 0.1 or bigger that titre is defined as obtaining optical density for normal control mice serum High dilution.[b] uses Liposomal formulation.MTT tumor is induced between the 3rd time and the 4th immunity inoculation.[c] EL=sky Liposome.[d] non-induced tumor.

Table 7. passes through ELISA^[a]MUC1 (Tn) and MUC1 (the most glycosylated) and BSA-MI-MUC1 (Tn) conjugate The competitive inhibition IC of antibodies₅₀Value.

Elisa plate BSA-MI-MUC1 (Tn) conjugate is coated by [a].By dilution with in the situation that there is not inhibitor Under obtain in ELISA about 1 OD with the blood serum sample of the group of 7 mices after 1 or 2 immunity inoculations, first with MUC1 (Tn) or the most glycosylated MUC1 (0-500 M final concentration) mixing, be subsequently applied to coated microtitration plate.By optical density Value is for the optical density value standardization obtained with single serum (0 M inhibitor, 100%).Intend with following logistic equation Close and suppress data: Y=bottom+(top base)/(1+10^{(X – Log IC50)}), wherein Y is standardized optical density, X It is the logarithm of inhibitor concentration, and IC₅₀It it is the inhibitor concentration making response reduce half.IC₅₀Value is reported as best-fit values With 95% confidence interval.

Cellular immunization.In order to assess the ability of vaccine candidate object activating cytotoxic T-lymphocyte, divided by magnetic cell Choosing separates CD8 from the lymph node of the mice by multiple compounds immunity inoculation⁺T cell, and on ELLISPOT plate with The illuminated DC incubation together of immunity inoculation peptide pulse.Being compared with a control, vaccine candidate object 1 and 2 demonstrates strong CD8⁺Response (Figure 19 A, 1 and 2 compare with 3).It is interesting that the mixture induction lesser number of glycopeptide 4 and adjuvant 5 (Pam3CysSK4) CD8⁺Activation, it is indicated that covalently bound optimal activation for CTL of MUC1 and auxiliary T epi-position and adjuvant is important.

Pass through⁵¹Cr release measure check the CD8+ cell separated without the lytic activity in the case of stimulated in vitro, wherein Glycopeptide SAPDT (Tn) RPAP (SEQ ID NO:26) that DC MUC1 is derivative or use peptide in the case of immunity inoculation 2 SAPDTRPAP (SEQ ID NO:20) pulse.As seen in Figure 19 B, it is compared with a control, is activated by compound 1 and 2 CTL demonstrates the biggest cytotoxicity.Additionally, demonstrate the cracking of minimizing with the mice of the mixture immunity inoculation of 4 and 5 Activity, is further characterized by the covalently bound importance of multiple epi-position.

In order to study CD8 in detail⁺The epi-position demand of cell, by the group of five MUC1.tg liposome of compound 1 and 2 Preparation immunity inoculation, gathers in the crops subsequently and merges CD8⁺Cell, is passed through respectively with glycopeptide SAPDT (Tn) RPAP (6) (SEQ ID NO:26) and the DC of peptide SAPDTRPAP (7) (SEQ ID NO:20) pulse stimulate 1 day in vitro, subsequently by with IL-2 Cultivate together with IL-7 and allow expanding propagation 14 days.After the glycopeptide 6-9 dendritic cells pulsed derivative with MUC1, determine generation The CD8 of IFN-γ⁺The percentage ratio of cell.Compound 1 has activated the not homotype that can be activated by glycosylation and non-glycosylated structure The CTL enclosed, and only shown and the responsiveness of not glycosyafated peptide 7 by those obtained by 2 immunity inoculations.Additionally, must use by oneself, 1 exempts from The CD8 of epidemic disease inoculation⁺Cell can crack with glycosylation and the DC (Figure 20) of not glycosyafated structure pulse.

These results are pointed out to identify that the structure of wider scope (includes glycosylation and nothing by the CTL activated by 1 immunity inoculation The peptide that glycosylation MUC1 is derivative), and not glycosyafated peptide is demonstrated strong preferential by the CTL obtained by compound 2.

Cytokine induction.Need the lipopeptid part of three component vaccines by with the TLR2 phase on mononuclear phagocyte surface Interaction, with the generation of initial required cytokine and chemotactic factor (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；With Spohn et al., 2004,Vaccine；22:2494-2499).Activating After, the intracellular domain of TLR2 raises adapter protein MyD88, causes the activation of kinase cascade, thus causes many cells The factor and the generation of chemotactic factor.On the other hand, lipopolysaccharide is induced thin by interacting with TLR4/MD2/CD14 complex Born of the same parents' response, this causes raising of adapter protein MyD88 and TRIF, thus causes the cytokine induction of more complicated pattern. TNF-γ secretion is that the prototype that MyD88 dependent pathway activates is measured, and the secretion of IFN-γ to be typically used as TRIF dependency thin Born of the same parents activate indicant (Akira et al., 2001,Nat Immunol；2:675-680；With van Amersfoort et al., 2003,Clin Microbiol Rev；16:379-414).

In order to check the pattern produced by the cytokine of multicomponent vaccine 1 and determine whether glycosylation affects response Property, check the TNF-α by compound 1,2 and 5 induction, IFN-β, Rantes, IL-6, IL-1, IL-10, IP-10, IL- Effect (EC of 12p70 and IL-12/23p40 secretion₅₀) and effect (maximum responsiveness).Therefore, the method being determined by is made to obtain Primary dendritic cell be exposed to compound 1,2,5 and the escherichia coli 055:B5 LPS of broad range concentration, use capture ELISA checks supernatant with regard to multiple mouse cytokine.Glycolipidpeptide 1, lipopeptid 2 with Pam3CysSK4 (5) with similar effect and effect Power induction TNF-α, Rantes, IL-6, IL-1 and IL-12/23p40 secretion, it is indicated that the connection of B and T epi-position and glycosylation are to carefully Intracellular cytokine response does not act on.See Figure 22, table 8 and table 9.As expected, two kinds of compounds the most do not induce the generation of IFN-β. It is interesting that compared with compound 1,2 and 5, escherichia coli 055:B5 LPS shows the effect much bigger for TNF-α induction And effect.Additionally, it can stimulate cell to produce IFN-β, IL-10, IP10 and IL-12p70.1, effect and effect of 2 and 5 subtracts It is favorable property less, because it is known that LPS can cause the symptom of septic shock with excessive activation innate immune system.

Produce in order to ensure cytokine and initiate in TLR2 dependency mode, make compound 1 and 5 be exposed to HEK 293T thin Born of the same parents, it is with Mus TLR2 stable transfection, and with plasmid (the NF-B dependency Lampyridea luciferin containing reporter gene pELAM-Luc Enzyme report carrier) and plasmid (sea pansy (Renilla) luciferase control report carrier) containing crt gene pRL-TK instantaneous Transfection.After 4 hours incubative times, use business dual luciferase to measure and measure activity, find that compound 1 and 5 can be with TLR2 dependency mode activates NF-B.

What table 8. obtained after primary dendritic cell incubation 24 hours is mounted with compound 1,2 or 3 and escherichia coli The cytokine plateau value of the dose response curve of the Liposomal formulation of LPS^[a](pg/mL)。

[a] uses nonlinear least square method curve matching according to the pick's cells factor/μ g gross protein, passes through Prism It is reported as the plateau value of best-fit values ± standard error.

[b] nd represents and is not detected by.

Table 9. is mounted with the Liposomal formulation of compound 1,2 or 3 and Escherichia coli LPS in primary dendritic cell Cytokine log EC₅₀Value^[a](nM)。

[a] uses nonlinear least square method curve matching, is reported as best-fit values ± standard error by Prism Log EC₅₀Value.

[b] nd represents for accurate EC₅₀It is not detected by the level measured.

Discuss

Manifesting successfully the evidence that cancer vaccine exploitation needs to affect at once the multi-mode treatment of immune several aspect. Although having observed the cell for MUC1 and humoral immunoresponse(HI) in certain cancers patient, but design can cause both The cancer vaccine material standed for of response is difficult.This embodiment confirms by derived from the glycopeptide of MUC1, non-germline selectivity peptide The multicomponent vaccine of auxiliary T epi-position and TLR2 agonist composition can cause IgG antibody, and its cancer that can crack expression MUC1 is thin Born of the same parents also stimulate cytotoxic T lymphocyte, thus reverse tolerance in the mouse model of breast carcinoma and generate treatment response.

Carefully analyzing of control compound discloses the minimizing of the tumor load mediated by multicomponent vaccine by for MUC1 Specific immunity and by by embed TLR2 agonist mediation nonspecific adjuvant effect cause.Manifesting TLR by swelling Oncocyte wide expression and its activation can cause suppression or the evidence of promotion of tumorigenicity.Additionally, produce after TLR activates Raw cytokine and chemotactic factor can stimulate the expression of many stimulating proteins altogether, in t helper cell and B cell and Optimal interaction between antigen-presenting cell.Recent research is pointed out that TLR1/2 agonist has and is reduced in vitro and in vivo Foxp3⁺The suppression function of regulatory T cells (Tregs) and the Cytotoxic unique ability of enhancing tumor-specific CTL, and And there is the Graft Versus Tumor potentially more favourable than other TLR agonist.

This embodiment also confirms that the covalently bound for causing antibody and optimal CTL of TLR2 agonist and glycolipidpeptide epi-position Function is crucial.With TLR2 agonist carry out lipidization make it possible in Liposomal formulation prepare multicomponent vaccine, This may strengthen its circulation time.Additionally, Liposomal formulation presents glycopeptide epi-position in multivalence mode, thus provide for B cell The chance effectively clustered of epi-position, this is necessary to initial B cell signal transduction and antibody generation.Such as institute in preceding embodiment Show, the covalently bound promotion of TLR2 agonist Pam3CysSK4 by express TLR2 immunocyte such as B cell and antigen in The selectivity internalization of delivery cell (APC).The picked-up of antigen and processing and T epi-position exist as the complex with MHC I or II class Follow-up on APC cell surface is presented, and is crucial for causing IgG antibody.Past 10 years, numerous researchs showed antigen Selectivity targeting APC will cause the immunne response improved.Such as, oxidation mannan, heat shock protein, bacteriotoxin and targeting The antibody of the cell surface receptor of dendritic cell has been connected to antigen, to increase the picked-up by dendritic cell.Although this A little picked-up strategies are attractive, but they have disadvantages in that targeting device is antigenic, and this may cause tumor The immunosuppressant of related carbohydrate.Pam3CysSK4 is for promoting to be that it is low intrinsic by the captivation of the picked-up of APC Immunity.Therefore, promotion is absorbed by three component vaccines, and does not have immunosuppressant shortcoming.

Finally, the present embodiment confirms that the glycosylation of MUC1 epi-position is crucial for most preferably reducing of tumor load.Mechanism Study and provide ultimate principle for these observed results, find compared with using the compound 2 lacking Tn antigen, with compound 1 Immunity inoculation causes the antibody of slightly greater titre, and it is substantially more to have cracking performance.By the NMR supplemented with light scattering measurement Conformation research have been pointed out the deglycosylation of MUC1 and cause less extending and more spherical structure.MUC1 is used to be correlated with O-glycopeptide Similar research shown that carbohydrate portions plays conformational effect, this can provide the most former of difference in immunne response Reason.Additionally, the use of glycosylation 1 causes effective activation of CTL, this CTL is capable of identify that glycosylation and the most glycosylated structure, its Before in, one is preferred.On the other hand, cause mainly identifying non-glycosylated structure by non-glycosylated compound 2 immunity inoculation CTL.Known short O on MUC1 tandem sequence repeats joins polysaccharide such as Tn and the STn DC course of processing in MHC II classpath Middle holding is complete, it is therefore possible to cause glycopeptide selectivity CTL response.Additionally, there are compared with corresponding non-glycosylated peptide, MUC1 glycopeptide can more strongly combine MHC I class mice allele H2K^bEvidence.Additionally, the progress of cancer not only with have The MUC1 of truncate sugar such as Tn antigen modifies relevant, and these structures also exist with much higher density, therefore effectively immunity Treatment needs to cause the response for this class formation.

Experimental section

Conventional method for automatization's Solid phase peptide synthesis (SPPS): use N^αThe aminoacid of-Fmoc protection and 2-(1H-benzo Triazol-1-yl)-1,1,3,3-tetramethylureaHexafluorophosphate (HBTU)/I-hydroxybenzotriazole (HOBt) is as activating examination Agent, by the scheme synthetic peptide on Applied Biosystems, the ABI 433A peptide synthesizer being equipped with UV detector set up. Single coupling step conditionality caps execution.

The conventional method prepared for the liposome of native chemical connection: preparation is containing 2 mM tri-(2-carboxyethyl) phosphines (TCEP) the pH 7.8 200 mM sodium phosphate buffer of and 0.3% EDTA.Buffer is deaerated 1 hour.WPH will be contained (1 equivalent), thioesters (2 equivalent) and dodecylphosphoric acid choline (dodecylposphocholine) (13 equivalent) are dissolved in 1:1 CHCl3: in trifluoroethanol, removes solvent.Subsequently lipid/peptide thin film is hydrated 4 hours at 41 DEG C in calorstat.By mixture Supersound process, by peptide/liquid suspension at 50 DEG C by 1.0 μm polycarbonate membrane (Whatman, Nucleopore, Track- Etch Membrane) extrusion, to obtain uniform vesicle.

The synthesis of glycosylation three component vaccine material standed for 1: by peptide thioesters X (1.1 mg, 0.674 μm ol), 1-9Nac MBP (1.0 Mg, 0.337 μm ol) and dodecylphosphoric acid choline (1.5 mg, 4.38 μm ol) be dissolved in 1:1 CHCl3: trifluoroethanol In mixture (5 mL).Solvent is under reduced pressure removed, to provide lipid/peptide thin film.Use containing 2 mM TCEP and 0.3% The 200 mM sodium phosphate buffers of EDTA, are hydrated lipid/peptide thin film 4 hours at 41 DEG C.By mixture supersound process, by peptide/ Liquid suspension is squeezed by 1.0 μm polycarbonate membranes (Whatman, Nucleopore, Track-Etch Membrane) at 50 DEG C Go out, to obtain uniform vesicle.Adding mistabrom sodium (1 mM) in vesicle suspension, with initial action, (1.5 mM are final Peptide concentration).After 20 minutes, use is through the solution A gradient of the 0-100% B in 40 minute period, by anti-at analytical type C-4 RP-HPLC purification reaction mixture on phase post.The lyophilizing of suitable flow point provides 1 (1.2 mg, 80%).C₂₁₇H₃₆₇N₄₅O₅₃S₂ HR MALDI-ToF MS: observe；4515.685 [M+] calculated.

The conventional method prepared for the liposome of immunity inoculation: by synthesis compound, PC, phosphatidyl The hydration in the HEPES buffer (10 mM, pH 7.4) containing NaCl (145 mM) of the thin film of glycerol and cholesterol and with After the extrusion via 0.1 m Nucleopore polycarbonate membrane, every kind of glycolipidpeptide is mixed small-sized list based on phospholipid In layer vesicle (SUV).

Immunity inoculation: with the Liposomal formulation of three component vaccine constructs (25 μ g, containing 3 μ g carbohydrates) with scarce Weary tumor is correlated with the respective comparison of MUC1 epi-position, will express 8 to the 12 week old MUC1.Tg mice (C57BL/6 of people MUC1；H-2b) Inoculate three times with the bottom intradermal immunization being spaced in tail once every two weeks.After 35 days, by mice with expressing MUC1's and Tn MMT breast tumor cell (1 × 10⁶Individual cell) attack.When the 42nd day, in tumor cell injection one week after, give again Immunity inoculation.When the 49th day, immunity inoculation one week after the last time, by sacrifice, and measure effect of vaccine.

Tumor palpation: after third time immunity inoculation 7 days, is used in 1 × 10 in 100 L PBS⁶Individual cancerous cell is in left flank Middle subcutaneous injection MUC1.Tg mice.Tangibly tumor is measured by caliper, and according to following formula calculating tumor weight: gram= [(length) X (width) 2]/2, wherein length and width is with a centimetre measurement.When terminal, by swollen to total surgical resection measurement Tumor weight.

⁵¹Chromium (Cr) release measures: use with the action effect cell of 100:1 ratio without any stimulated in vitro from TDLN CD8⁺ T cell and as target cell with respective peptide pulse⁵¹The DC of Cr labelling, passes through standard⁵¹Cr method for releasing continues 6 Hour and measure cell lysis activity.Before incubation together with effector, target cell is used 100 Ci ⁵¹Cr (Amersham Biosciences)/10⁶Individual target cell carries 2 hours.Use Topcount Microscintillation Counter (Packard Biosciences) measures radioactivity⁵¹Cr discharges, and calculates Specific lytic: (cpms is spontaneous in experiment The spontaneous cpms of the complete cpms of cpms/) x 100.Spontaneous lysis is total cracking < 15%.

The mensuration of cytotoxicity (ADCC) of antibody dependent cellular mediation: by tumor cell (Yac-MUC1 or C57mg.MUC1) 100 Ci are used⁵¹Cr 37 DEG C of labellings 2 hours, washing and with the control antibodies (mouse IgG) of 5 g/mL or Derive from the serum (1 in 25 dilution factors) of vaccinated mice together 37 DEG C of incubations 30 minutes.There is high expressed (KY-1 clones the NK cell of CD16 receptor, from Dr. Wayne M. Yokoyama, Washington University, St. The generous present of Louis) it is used as effector.These cells IL-2 (200 units/mL) stimulates 24 hours before the assay.With The effector of 50:1: target cell ratio, is inoculated in 96 well culture plates together with the tumor cell of antibody labeling by effector lymphocyte In (Costar height board) 4 hours.Measured in supernatant by Top Count⁵¹Cr discharges.Will⁵¹The spontaneous release of Cr Measure with maximum release, and less than 20%.The percentage ratio of mensuration specificity release: (release-spontaneous release/maximum release-spontaneous Release) x100.

IFN-γ ELISPOT measures: when putting to death, and separates the MAC from TDLN and divide from the MUC1.Tg mice processed The CD4 of choosing⁺And CD8⁺T cell, and it is used as respondent in IFN-γ ELISPOT measures.Pass through ZellNet Consulting, Inc. (Fort Lee, NJ), use computer assisted video image analysis to measure number of spots.Use by oneself The splenocyte of the C57BL/6 mice that Concavalin A stimulates is used as positive control.

Determination of serology: as previously described (Buskas et al., 2004,Chemistry, 10 (14): 3517-24), logical Cross enzyme-linked immunosorbent assay (ELISA) and measure anti-MUC-1 IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM antibody titre. In short, by elisa plate (Thermo Electron Corp.) the MUC-1 sugar being conjugated to BSA by maleimide joint Peptide conjugate (BSA-MI-MUC-1) is coated.The serial dilutions allowing serum combines fixing MUC-1.By adding phosphate ester Anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.) puted together, IgG1 (Zymed), IgG2a (Zymed), IgG2b (Zymed), IgG3 (BD Biosciences Pharmingen) or IgM (Jackson ImmunoResearch Laboratories Inc.) antibody complete detection.Adding p-nitrophenyl phosphate ester (Sigma) After, use microplate (BMG Labtech) to measure absorbance at 405 nm, wherein wavelength calibration is located at 490 nm. Following mensuration is for the antibody titer of T (poliomyelitis) epi-position.Make Reacti-bind NeutrAvidin be coated and seal in advance The plate (Pierce) closed and biotin labeled T epi-position (10 g/mL；100 μ L/ holes) incubation 2 hours together.It follows that permit The serial dilutions being permitted serum combines fixing T epi-position.It is completed as previously described detection.Antibody titer is defined as obtaining relative to just Often control mice serum optical density is the highest dilution of 0.1 or bigger.

Suppression ELISA: in order to probe into corresponding glycopeptide, peptide and the sugar competitive inhibition to the combination of MAb Yu MUC (Tn), will Blood serum sample dilutes by this way in dilution buffer, and described mode makes in the case of not having inhibitor, it is contemplated that Final optical density value be about 1.For each hole, by the blood serum sample of 60 L dilutions in the most coated microtitration plate with 60 L dilution buffers, the glycopeptide 6 (MUC (Tn)) being dissolved in dilution buffer with the final concentration of 0-500 M, peptide 7 (sugar The MUC1 of base) or the mixing of Tn-monomer.After incubation at room temperature 30 minutes, 100 l mixture are transferred to by BSA-MI-MUC1 (Tn) coated plate.The detection antibody that total IgG use alkali phosphatase is puted together, as mentioned above by microtitration plate incubation also Colour developing.By optical density value for the optical density value standardization obtained by single monoclonal antibody (0 M inhibitor, 100%).

Prepared by dendritic cell (DC): as previously described (Inaba et al., 1992,J Exp Med；176(6):1693- 702 and Mukherjee, 2003, J Immunother；26:47-62), mouse bone marrow cells culture DC is prepared.

Cytokine assay: when exposure measures the same day, using maturation DC as 4 × 10⁶Individual cells/well with 1.8 mL 24 Bed board in the tissue culturing plate of hole.Subsequently by cell and different stimulated thing (200 μ L, 10X) together at the final volume in 2 mL/ holes Middle incubation 24 hours.Stimulus object with concentration range widely (for 1 in liposome, 5 or 6, corresponding to 0.1 ng/mL-100 µg/mL PAM₃CysSK₄Final concentration, with for Escherichia coli LPS, corresponding to the final concentration of 0.001 ng/mL-10 g/mL) Give.Collect supernatant.For the ATP evaluation to the effect that IL-1 β secretes, by DC in same volume containing ATP (5 mM； Sigma) incubation 30 minutes again in culture medium, gather in the crops supernatant after this.By freezing for the culture supernatant of all collections (negative 80 DEG C) storage.

Cytokine ELISA performs in 96 hole MaxiSorp plates (Nalge Nunc International).According to system Make the description of business, by cytokine DuoSet ELISA Development test kit (R&D Systems) for mice TNF-α, RANTES, IL-6, IL-1 β, IL-10, IP-10, IL-12 p70 and IL-12/23 p40 cytokine quantitative.Make With microplate (BMG Labtech), measuring absorbance at 450 nm, wherein wavelength calibration sets to 540 nm.Following survey The mice IFN-β concentration being scheduled in culture supernatant.By the plate rabbit polyclonal antibody (PBL for mice IFN-β Biomedical Laboratories) it is coated.Allow at standard substance (PBL Biomedical Laboratories) and sample In IFN-β combine immobilized antibody.It is subsequently added rat anti-mouse IFN-β antibody (USBiological).It follows that add Mountain goat anti rat IgG (H+L) antibody (Pierce) that HRP puts together and chromogenic substrate 3,3 ', the 5,5 '-tetramethyl benzidine of HRP (Pierce).After reaction stops, measuring absorbance at 450 nm, wherein wavelength calibration sets to 540 nm.Cytokine values It is expressed as pg cytokine/mL.Use nonlinear least square method bent in Prism (GraphPad Software, Inc.) Line Fitting Analysis concentration-reply data.With following four these data of parameter logistic equation matching: Y=E_max /(1 + (EC₅₀/X)^{Hill slope}), wherein Y is cytokine response, and X is the concentration of stimulus object, E_maxIt is maximum response (plateau value), and EC₅₀ It is the stimulus object concentration producing 50% stimulation.Hill slope is set to 1, can compare the EC of different inducer₅₀Value.

Statistical analysis: use one way analysis of variance (ANOVA) to perform multiple comparisons with Bonferroni multiple comparative test. WhenP< when 0.05, difference is considered as significantly.For the comparison between two groups, use double tail Si ShitInspection and 95% confidence Interval analysis data.P-value < 0.05 be considered as statistically evident.

Embodiment 9

The interpolation of the second TLR agonist

This embodiment measures the effect adding the effectiveness to immunity inoculation of the second TLR agonist CpG.Follow embodiment The program being more fully described in 8, will express the old MUC1.Tg mice (C57BL/6 of people MUC1；H-2b) by following substances Preparation immunity inoculation: compound 2 (Pam₃CysSK₄-t helper cell epi-position (poliomyelitis)-MUC1 (the most glycosylated))； Compound 1 (Pam₃CysSK₄-t helper cell epi-position (poliomyelitis)-MUC1 (Tn))；(CpG is few plus CpG for compound 1 Deoxydization nucleotide (CpG ODN)))；Compound 5 (Pam₃CysSK₄) plus compound 4 (t helper cell epi-position (gray nucleus Scorching)-MUC1 (Tn))；Compound 5；Compound 3 (Pam₃CysSK₄-t helper cell epi-position (poliomyelitis))；Compound 3 Plus CpG；EL (empty liposome) adds CpG；Or EL.The structure of compound 1,2,3,4 and 5 is shown in Figure 16.Use standard Immunity inoculation timetable, uses together with TLR9 agonist CpG altogether by compound.As shown in Figure 23-25, TLR9 part CpG Combination use the antitumor character improving further three component vaccines 1.Specifically, the interpolation of the second agonist causes swelling The minimizing (Figure 23) the most further of tumor weight, and induce more effective immunne response (Figure 24 and 25).

Embodiment 10

In the MUC1.Tg mice with MMT tumor, both three component MUC1 glycopeptides vaccine-induced humoral and cellular immune response responses

The cell for tumor antigen and humoral immunoresponse(HI) are depended in effective immunization therapy for cancer.In breast carcinoma On also show that the glycosylation of change increasing the MUC1 of horizontal expression, it facilitates the formation of neoantigen.It is correlated with derived from tumor The qualification of MHC I and the II class binding peptide of MUC1 has promoted the exploitation of cancer vaccine based on MUC1.When along with carrying out with adjuvant Emulsifying when giving, the MHC I class from MUC1 tandem sequence repeats combines epi-position and causes strong cell response, and without antibody response.Have It is possible that use the individual glycopeptide for its more representative new model MUC1 as seen by cancer that may more do not tolerate, Obtain more preferably immunogenicity, and vaccine component be directly connected to ratio can be caused to deliver more preferably immunity as mixture Response.This embodiment shows that (it is also derived from the B cell table of MUC1 by TLR2 agonist, auxiliary epi-position and t cell epitope Position) three component vaccines that form, can destroy tolerance and cause body fluid and cellullar immunologic response.With only with adjuvant and sky fat The mice that plastid processes compares, and causes substantially reducing of tumor load with MUC1 glycopeptide vaccine virus immunization.Three component vaccines Activate MUC1 glycopeptide specific cytotoxicity CD8+ T cell, and cause the mediation of strong titre to crack relevant tumor by ADCC The IgG antibody of cell.

With three component vaccine compound 1 (Pam₃CysSK₄-t helper cell-MUC1) and LAA-T auxiliary cell-MUC1 (its Containing the reticent lipid of immunity) and as the compound 2 ((Pam compareed₃CysSK₄-t helper cell) and LAA-T auxiliary cell (this Both lack tumor to be correlated with MUC1 epi-position) Liposomal formulation, the MUC1.Tg mice (C57BL/6 of people MUC1 will be expressed；H- 2b) inoculate three times with Immunity at intervals once every two weeks.The structure of compound is shown in Figure 26.After 35 days, mice is used The MMT breast tumor cell expressing MUC1 and Tn is attacked.Immunity inoculation timetable is shown in Figure 27.Immunity connects the last time After Zhong three weeks, by sacrifice, and surveyed by tumor load, cell-mediated immunne response and antibody-mediated immunne response Determine effect of vaccine.With LAA-T auxiliary cell-MUC1 (lacking the compound of TLR2 agonist) and respective control compound 2 (TLR2 agonist t helper cell epi-position) compares with empty liposome, by three component glycosylation vaccination compound 1 immunity inoculations Cause tumor quality substantially reduces (seeing Figure 28).Three component glycosylation vaccination compound 1 cause the mediation of strong titre to pass through ADCC cracking is about the IgG antibody (seeing Figure 30 and 31) of tumor cell.As measured by chromium release assay, from immunity The lotus of inoculation has the cracking potentiality of the sorted CD8+ T cell of the mice of MMT tumor to show: with respective control compound 2 ((Pam₃CysSK₄-t helper cell) compare with the LAA-T auxiliary cell-MUC1 compound of EL and shortage TLR2 part, use Three component glycosylation vaccine virus immunization show the biggest cracking (seeing Figure 29).This is trigger cell and humoral response two The first bacterin preparation of person.

Embodiment 11

Utilize the synthesis three component construct of people's MUC1 t helper cell sequence

For I-A^b、H2-K^bAnd H2-D^bIn conjunction with the prediction of epi-position, dominant query Rankpe (Harvard, MA) location specific Score matrix (Position Specific Scoring Matrices, PSSM) program.Reverse inquiry the second program SYPEITHI (Institute for Cell Biology, Heidelberg, Germany), with cross validation H2-K^bAnd H2-D^bKnot Close epi-position.Figure 32 shows about people's MUC1 derived peptide and mice I-A^b15 aggressiveness and H2-D^bCombination with H2-Kb 9 aggressiveness Analyze.Many inspirer predictions are obvious.Dotted line show display about with I-A^bIn conjunction with RANKPEP score 15 Aggressiveness.Specify display about with H2-D^bOr H2-K (dddd)^b (kkkk) combination or the non-germline with two kinds (bbbb) are selected 9 aggressiveness of the RANKPEP score that selecting property combines.

For the induction of the interferon gamma generation to CD4 and cd8 cell, test the compound by this Analysis and Identification. By mice peptide immunity inoculation described in Figure 33 A, and spread out by the lymph node of cell sorting acquisition expression low-level CD62L Raw T cell, and cultivate 14 days in the presence of the DC with immunity inoculation peptide pulse.Peptide listed in exposure y-axis After the DC of pulse, with regard to CD4⁺IFNγ⁺And CD8⁺IFNγ⁺The existence situation of T cell passes through intracellular cytokine assay gained The cell (Figure 33 B) arrived.Cause for its own and non-glycosylated 15 aggressiveness by glycosylation 21 aggressiveness (PEPC) immunity inoculation (peptide A) and the strong specific C D4 of 21 aggressiveness (peptide B)⁺And CD8⁺Response.

Create the multiple synthesis construct utilizing the people's MUC1 t helper cell sequence shown in Figure 34.Follow enforcement The program being more fully described in example 8-10, will express the MUC1.Tg mice (C57BL/6 of people MUC1；H-2 ^b) use Figure 33 and 34 Shown in construct immunity inoculation.Follow the program being more fully described in embodiment 8-10, measure construct swollen in minimizing Tumor weight, initiation IgG antibody, mediation are by ADCC cracking tumor cell, initiation CD8+ cellular cytoxicity activity and generation IFN-γ And the effectiveness in terms of other cytokines.

Embodiment 12

By use that completely synthetic three component immunogens produce for carbohydrate and the monoclonal antibody of glycopeptide

Glycoconjugate is molecule the most various in function and structure in nature, and is able adequately determines protein and fat at present The sugar that matter combines plays basic role in affecting many molecular process of eukaryote and disease.The example of this class process includes Fertilization, embryo's generation, neuronal development, hormonal activity, cell proliferation and group thereof constitute particular organization.Cell-surface carbon aquation Notable change in compound occurs along with tumour progression, and this seems closely related with transfer.Additionally, carbohydrate can lure Leading protection antibody response, this immunoreation is the biological significant contributor of survival in course of infection.

Sugar can not activate helper T lymphocyte has made it complicate as the exploitation of vaccine.For most of immunogens (bag Include carbohydrate), antibody produces and depends on two quasi-lymphocyte B cell and the cooperative interaction of helper T cell (Jennings, Neoglyconjugates:Preparation and Applications 325-371 (Academic Press, Inc., 1994)；Kuberan, Curr. Org. Chem. 2000,4,653-677).Sugar individually can not activate auxiliary T Cell, therefore has limited immunogenicity, as embodied by not existing of low-affinity IgM antibody and IgG antibody.For The T cell stand-alone nature of carbohydrate, past research is overcome to concentrate on sugar and exogenous carrier proteins matter (such as keyhole blood Azurin (KLH), detoxification tetanus toxoid) put together (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, carrier protein Matter strengthens carbohydrate and presents to immune, and provides T epi-position (12-15 the aminoacid that can activate t helper cell Fragments of peptides).Therefore, complete to change from low-affinity IgM to the classification of high-affinity IgG antibody.This method is the most successfully It is applied to develop conjugate vaccine, with flu-prevention haemophilus infection.

The carbohydrate that such as tumor related carbohydrate and glycopeptide form by more overcritical Carbohydrate Antigens- Protein conjugate candidate vaccine fails to cause the IgG antibody of high titre.These results are the most astonishing, because tumor is correlated with Sugar has low antigenicity, because they are autoantigens, thus is tolerated by immune system.Taken off by the antigen of the tumor in growth Fall and reinforce this toleration.Additionally, exogenous carrier proteins matter such as KLH and BSA and sugar is connected to the joint of carrier protein Can cause strong B cell response, this can cause suppression (Buskas, the Chem. of the antibody response for carbohydrate epitope Eur. J. 2004,10,3517-3524；Ni, Bioconjug. Chem. 2006,17,493-500).It is clear that based on The successful exploitation of the cancer vaccine of carbohydrate needs for more effectively presenting tumor related carbohydrate to immune system Epi-position, the New Policy causing more effectively changing to the classification of IgG antibody (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. 2004,64,4987-4994；Buskas, Angew. Chem. Int. Ed. 2005,44,5985-5988 (embodiments I)；Dziadek, Angew. Chem. Int. Ed. 2005, 44,7624-7630；Krikorian, Bioconjug. Chem. 2005,16,812-819；Pan, J. Med. Chem. 2005,48,875-883).

As shown in preceding embodiment, relevant with tumor by TLR2 agonist, non-germline selectivity peptide t helper cell epi-position Three component vaccines of glycopeptide composition, can cause the IgG antibody of the highest titre in mice, and it can be with recognition expression tumor phase Close cancerous cell (seeing compound 21, Fig. 5, embodiment 6 and compound 51, Figure 15) (Ingale, the Nat. of carbohydrate Chem. Biol. 2007,3,663-667).The advantageous property of vaccine candidate object produces owing to the local of cytokine, stings altogether The rise of shock protein matter, picked-up by macrophage and dendritic cell strengthen and the avoiding of epi-position suppression.

The three component immunogen technology of the present invention can be used for generating for poor antigen carbohydrate and the Dan Ke of glycopeptide Grand antibody (MAb).We the most concentrated on for β-N-acetylglucosamine (β-O-GlcNAc) modified peptides MAb (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 cores in metazoa and cytoplasmic protein be on Ser and Thr residue by monosaccharide β-O-GlcNAc modifies 's.Response extracellular stimulusOHint quickly and is dynamically changed in-GlcNAc levelO-GlcNAc is at signal transduction pathway In pivotal role.OThe regulation of-GlcNAc level has remarkable effect to cell function, this partially byO-GlcNAc WithOComplexity between-phosphate ester influences each other mediation.Recently,O-GlcNAc involved type ii diabetes etiology, stress be anti- Answer the regulation of approach and the regulation of proteasome.Progress in this infusive research field is by the such as suitable MAbs of reagent Shortage seriously hinder.In this respect, only one has the most extensive specific poor performance IgM MAb (Comer, Anal. Biochem. 2001,293,169-177) it is (the Covance Research Products Inc) being obtained commercially.

We have designed and synthesized compound 52 (Figure 15), its contain the β derived from casein kinase i I (CKII)- GlcNAc modify glycopeptide (Kreppel, J. Biol. Chem. 1999,274,32015-32022) as B epitope, derived from Mus MHC II class restricted helper T cell epitope KLFAVWKITYKDT (the SEQ ID fully proved of poliovirus NO:3) and embed adjuvant Pam₃CysSK₄.Additionally, be prepared for the compound 53 with the GlcNAc part that artificial sulfur connects, Expect that it has more preferably metabolic stability.By synthesizing the thin of compound, PC, phosphatidyl glycerol and cholesterol Film in the HEPES buffer (10 mM, pH 6.5) containing NaCl (145 mM) hydration and subsequently via 100 nm The extrusion of Nucleopore polycarbonate membrane, mixes compound 52 and 53 in small unilamellar vesicles (SUV) based on phospholipid. With the liposome containing 3 μ g sugar, by the group of five female BAl BIc/c mice with weekly interval Intraperitoneal immunization inoculation four Secondary.

By be conjugated to the BSA that maleimide (MI) is modified CGSTPVS (β-O-GlcNAc) SANM is coated trace Titer plate, measures anti-glycopeptide antibody titer, then realizes detection with by the anti-mouse IgG antibody of alkali phosphatase enzyme mark.Such as table In 10 visible, compound 52 and 53 cause splendid titre anti-MUC1 IgG antibody.Additionally,OConnection andSBetween connection sugar derivatives Do not observe the significant difference of titre.

Table 10. with after 4 immunity inoculations of two kinds of different preparations the anti-GSTPVS of ELISA (β-O-GlcNAc)SANM (68) titre^a

^aAnti-GSTPVS (β-O-GlcNAc) SANM (68) antibody titer presents as the meansigma methods of the group of five mices.Will Elisa plate with BSA-MI-GSTPVS (β-O-GlcNAc) SANM (BSA-MI-66) conjugate is coated, and passes through linear regression Analyze, mark and draw with dulling luminosity ratio compared with dilution factor measure titre.Titre is defined as obtaining relative to normal control mice serum Optical density is the highest dilution of 0.1 or bigger.

^bUse Liposomal formulation.

^c O-GlcNAc 52；Pam₃CysSK₄G-C-KLFAVWKITYKDT-G-GSTPVS(β-O-GluNAc)SANM。

^d S-GlcNAc 53；Pam₃CysSK₄G-C-KLFAVWKITYKDT-G-GSTPVS(β-S-GluNAc)SANM。

For IgM titre, observe between 52 and 53 statistically evident difference (P = 0.0327)。

Indivedual titres about total IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM are reported in Figure 36.

It follows that results are usedOThe spleen of two mices of connection glycolipidpeptide 52 immunity inoculation, Standard hybridoma culture technique is given Go out seven IgG1, seven IgG2a, two IgG2b and 14 IgG3 and produce hybridoma cell line.Use ELISA and suppression The ligand specificity of the MAb that ELISA institute obtains.All MAb all identify be connected to BSA CGSTPVS (β-O-GlcNAc) SANM, and only minority identification is conjugated to the PEPC GSTPVSSANM (SEQ ID NO:12) of BSA.Additionally, 19 kinds of MAb and BSA- MI-CGSTPVS(β-O-GlcNAc) SANM interaction can by glycopeptide GSTPVS (β-O-GlcNAc) SANM suppression.

It is more fully described in WO 2010/002478 (" Glycopeptide and Uses Thereof "), miscellaneous Oncocyte system 1F5.D6,9D1.E4 and 18B10.C7 is handed over to be preserved in American type culture collection on July 1st, 2008 (ATCC), 10801 University Blvd., Manassas, VA, 20110-2209, USA, and respectively specify that ATCC preservation Number PTA-9339, PTA-9340 and PTA-9341.It is, however, to be understood that printed instructions herein is considered as being enough to cause this area Technical staff can put into practice the present invention completely.Additionally, the expection of preservation embodiment is as the single act of one aspect of the present invention Example illustrates, and should not be construed in any way as limiting the scope of claim.

Follow similar programs, can prepare for any MUC1 construct described herein have specific polyclone and Monoclonal antibody.

Embodiment 13

New synthetic immunogen is used to generate O-GlcNAc monoclonal antibody specific

Make three completely synthetic component immunogens cause having wide spectrum with hybridoma technology combination and be combined targetO-GlcNAc is special The generation of property IgG MAb.Extensive air gun proteomics causes identifying 254 kinds of mammalsOThe albumen that-GlcNAc modifies Matter, including a large amount of neoglycoproteins.These data implyO-GlcNAc transcribing/translational regulation, signal transduction, ubiquitin approach, Effect in the direct motion transport of intracellular vesicles and post translational modification.

Core and the serine of cytoplasmic protein and threonine by single β-N-Acetyl group-GLUCOSAMINE part (β- GlcNAc)OGlycosylation be all over post translational modification, it is highly dynamic and passes through cyclophoraseOConnection GlcNAc transferring enzyme (OGT) andOThe action response cytositimulation of-GlcNAcase (OGA) and fluctuate.This kind of glycosylation has involved many cell processes, This is generally carried out via with the influencing each other of phosphorylation that can occur on same amino acid residue.It is essential thatO- The change of GlcNAc level is correlated with the etiology of popular human disease's (including type ii diabetes and Alzheimer) (Hart et al., 2007 Nature 446,1017-1022).

It is different from the phosphorylation that can obtain broad range of general specificity and locus specificity phosphoric acid-antibody for it,O- The research of GlcNAc modification is lacked to be hindered for its detection, quantitative and location, site effective tool.Especially, only two kinds General-O-GlcNAc specific antibody is described: IgM is general-O-GlcNAc antibody (CTD 110.6；Comer et al., 2001 Anal. Biochem. 293,169-177) and forOIgG antibody (the RL-2 that the nucleopore component that-GlcNAc modifies produces； Snow et al., 1987 J. Cell Biol. 104,1143-1156), this IgG antibody show withOThe albumen that-GlcNAc modifies The limited cross reactivity of matter.It is true that multiple researchs showOThe glycoconjugate that-GlcNAc modifies does not causes relevant IgG same Plant type antibody, therefore causeOThe challenge of-GlcNAc specific IgG antibodies is sizable.Our inferenceO-GlcNAc specificity Antibody can cause by using three component immunogens (compound 52, Figure 35), and described three component immunogens are studied by this In containing derived from tyrosine kinase II (CKII) α subunitO-GlcNAc peptide (Kreppel and Hart, 1999 J. Biol. Chem. 274,32015-32022) the Mus MHC restricted helper T cell epitope of II class that, fully proves and as embedding adjuvant Toll-like receptor-2 (TLR2) agonist composition.The expection of this compounds is avoided by the carrier protein of typical case's conjugate vaccine The immunosuppressant that matter or connector area cause；But, it contains the All Media caused needed for strong and relevant IgG immunne response (Ingale et al., 2007 Nat. Chem. Biol. 3,663-667).Additionally, be prepared for having what artificial sulfur connected The compound 53 of GlcNAc part, with itOConnection counter pair compares, and described part has the metabolic stability of improvement, thus carries For causingOThe other chance of-GlcNAc specific antibody.

By liposome-mediated C-terminal lipopeptid thioesters 63, native chemical with glycopeptide 64 and 65 is connected (Ingale respectively Et al., 2006 Org. Lett. 8,5785-5788), it is readily available compound 52 and 53 (Figure 35).Initial thioesters 63 exists Assemble on sulfonamide " lock (safety-catch) " joint, then pass through and discharge with iodoacetonitrile alkylation and use benzyl mercaptan Process, to provide the compound using standard conditions deprotection.Be respectively adopted Rink amide resin, Fmoc protection aminoacid and Fmoc-Ser-(AcO3-α-D-GluNAc) or Fmoc-Ser-(1-sulfur generation-AcO3-α-D-GluNAc) prepare compound 64 He 65.After assembling completes, crack acetonyl ester by processing with the MeOH solution of 60% hydrazine, and by processing from tree with reagent K Fat cracks obtained compound, then by reverse HPLC-purified.Compound 52 and 53 is mixed based on phospholipid small-sized In unilamellar vesicle (SUV), then pass through 100 nm Nuclepore polycarbonate membrane extrusions.By five female BAl BIc/c mice Group with containing 3 μ g sugar liposome with interval biweekly Intraperitoneal immunization inoculate four times.By with being conjugated to maleoyl The CGSTPVS of the BSA that imines (MI) is modified (β-O-GlcNAc) SANM (66) is coated microtitration plate, measures anti-glycopeptide antibody Titre, and complete detection with by the anti-mouse IgG antibody of alkali phosphatase enzyme mark.Compound 52 and 53 causes splendid titre IgG antibody (table 10；Figure 36).Additionally,OConnection andSThe significant difference of IgG titre is not observed, therefore between connection sugar derivatives Further pay close attention to and concentrate on the mice by 52 immunity inoculations.

Gather in the crops with the spleen of two mices of 52 immunity inoculations, Standard hybridoma culture technique be given seven IgG1, seven IgG2a, two IgG2b and 14 IgG3 produce hybridoma cell line.By the part spy of the MAb that ELISA institute obtains The opposite sex.All MAb all identify be connected to BSA CGSTPVS (β-O-GlcNAc) SANM (BSA-MI-66), and only minority identification It is conjugated to the PEPC GSTPVSSANM (SEQ ID NO:12) (BSA-MI-67) of BSA.Additionally, the interaction of 20 kinds of MAb Can by glycopeptide GSTPVS (β-O-GlcNAc) SANM (68) suppression, but can not be by peptide GSTPVSSANM (SEQ ID NO:13) (69) or-O-GlcNAc-Ser (70) suppresses, and this confirms glycopeptide specificity.

By three kinds of hybridomas (18B10.C7 (3), 9D1.E4 (10), 1F5.D6 (14)) with one liter of scale evaluation, by full With the antibody obtained by ammonium sulfate precipitation and Protein G column chromatography purification subsequently, to obtain about 10 mg's in each case IgG.Suppression ELISA confirms that MAb needs carbohydrate and peptide (glycopeptide) to be used for combining.

In a word, three component immunogen methods are successfully used to generate the experimental subject group of general GlcNAc specificity MAb, its There is provided for probing into the strong new tool involved the biology of this kind of protein glycosylation.Recently identifyO-GlcNAc modifies Protein opens the new way probing into this kind of post translational modification for the importance of multiple bioprocess.Expect that three components are immune Inoculation technique is having and be widely used in terms of generating for the MAb of the protein glycosylation of other forms.

Method

Reagent and general procedure for synthesis.Fmoc-L-amino acid derivativges and resin purchased from NovaBioChem and Applied Biosystems, peptide symthesis rankN, N-dimethylformamide (DMF) is purchased from EM Science,N-methylpyrrole Alkanone (NMP) is purchased from Applied Biosystems.PC (PC), EPG (PG), cholesterol (Chol), monophosphoryl lipid A (MPL-A) and dodecylphosphoric acid choline (DPC) derive from Avanti Polar Lipids.All Other chemical reagent are purchased from Aldrich, Acros, Alfa Aesar and Fisher, and use without being further purified.Use All solvents all there is reagent grade.Use with 1 ml minute^-1The Agilent ZorbaxEclipse of flow velocity^TMC8 analyzes Post (5 μm, 4.6 x 150 mm), with 3 ml minute^-1The Agilent Zorbax semi-preparative post of EclipseTM C8 of flow velocity (5 μm, 10 x 250 mm) or with 2 ml minute^-1The Phenomenex Jupiter of flow velocity^TMThe semi-preparative post of C4 (5 μm, 10 x 250 mm), it is being equipped with automatic injector, fraction collector and the Agilent of UV detector (detecting at 214 nm) RPHPLC (reversed-phase high-performance liquid chromatography) (RP-HPLC) is performed on 1100 serial systems.All operations all use the 0-100% through 40 minutes Linear gradient (solvent orange 2 A=5% acetonitrile, the aqueous solution of 0.1% trifluoroacetic acid (TFA), solvent B=5% water, the 0.1%TFA of solvent B Acetonitrile solution) perform.ABI 4700 proteome analysis instrument records the substance assistant laser desorpted ionized flight time Mass spectrography (MALDI-TOF) mass spectrum.

Conventional method for Solid phase peptide synthesis (SPPS): useN ^αThe aminoacid of-Fmoc protection and 2-(1H-benzo three Azoles-1-base)-Oxy-1,1,3,3-tetraethyl hexafluorophosphate (HBTU)/I-hydroxybenzotriazole (HOBt；Knorr et al., 1989 Tetrahedron Lett. 30,1927-1930) as activating reagent, it is being equipped with UV detector by the scheme set up The upper synthetic peptide of ABI 433A peptide synthesizer (Applied Biosystems).Single coupling step conditionality caps execution. Use following shielded aminoacid:N ^o-Fmoc-Arg(Pbf)-OH、N ^α-Fmoc-Asp(O ^t Bu)-OH、N ^α-Fmoc-Asp- Thr(Ψ^Me,Mepro)-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( ^t Bu)-OH、N ^α-Fmoc-Tyr( ^t Bu)-OH.UseO-(7-azepine benzotriazole-1- Base)-N,N,N’,N'-tetramethylureaHexafluorophosphate (HATU)/1-hydroxyl-7-azepine benzotriazole (HOAt) is as coupling Agent, manual execution glycosylated amino acidN ^α-FmocSer-(AcO3-α-D-O-GlcNAc)OH、N ^α-FmocSer-(AcO3-α-D-S-GlcNAc) coupling of OH.Use benzotriazole-1-base-epoxide-tripyrrole alkane subbase-phosphorusHexafluorophosphate (PyBOP)/ HOBt, as coupling agent, performsN ^α-Fmoc-S-(double (the palmityl epoxide)-(2 of 2,3-R-propyl group)-(R)-cysteine (Metzger et al., 1991 Int. J. Pept. Protein Res. 38,545-554；Roth et al., 2004 Bioconjugate Chem. 15,541-553) (its by(R)-Prepared by (+)-2,3-Epoxy-1-propanol) coupling.Tested by standard K aiser The progress (Kaiser et al., 1970 Anal. Biochem. 34,595) of the manual coupling of monitoring.

The synthesis of lipopeptid 63:As described in the conventional method part for peptide symthesis, the synthesis of 63 is at H-Gly-ammonia Perform on sulphonyl butyryl Novasyn TG resin.After first five amino acid whose coupling, manual execution remaining step.WillN-α- Fmoc-S-(double (the palmityl epoxide)-(2 of 2,3-R-propyl group)-(R)-cysteine (267 mg, 0.3 mmol) is dissolved in DMF (5 ML) in, by PyBOP (156.12 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol) and DIPEA (67 μ l, 0.4 mmol) Premixing 2 minutes, is subsequently adding in resin.By Kaiser test monitoring coupling reaction, and complete after standing 12 hours. After coupling completes, DMF (6 mL) solution of 20% piperidines is used to crackN-Fmoc group, and use PyBOP (156.12 Mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol) and the DMF solution of DIPEA (67 μ l, 0.4 mmol), make Palmic acid (77 Mg, 0.3 mmol) and unhindered amina coupling as above.By resin DMF (10 ml), DCM (10 ml) and MeOH (10 ml) Fully washing, is dried in a vacuum subsequently.By resin in DCM (5 mL) swelling 1 hour, and with DIPEA (0.5 ml, 3 Mmol), NMP (6 ml) solution of iodoacetonitrile (0.36 ml, 5 mmol) processes.It is important to note that add resin it Before, filter iodoacetonitrile by the plug of alkali alumino.By resin lucifuge stir 24 hours, filter and with NMP (5 ml x 4), DCM (5 ml x 4) and THF (5 ml x 4) washing.By activateN-acyl sulfonamides resin is swelling 1 little in DCM (5 ml) Time, drain and be transferred to 50 ml round-bottomed flasks.In resiniferous flask add THF (4 ml), benzyl mercaptan (0.64 ml, 5 And phenylmercaptan. sodium (sodium thiophenate) (27 mg, 0.2 mmol) mmol).After agitation 24 hours, by resin mistake Filter, and wash with hexane (5 ml x 2).Filtrate and the cleaning mixture of merging are collected and be concentrated into its initial volume in a vacuum About 1/3.Subsequently by adding methyl tertiary butyl ether(MTBE) (0 DEG C；60 mL) precipitation raw product, by being centrifuged 15 with 3000 rpm Minute reclaim, and after ether decant, peptide precipitate is dissolved in mixture D CM and MeOH (1.5 ml/1.5 ml).Logical Cross and pass it through LH-20 size exclusion post, remove mercaptan impurities present in peptide precipitate.Collect the flow point containing product and remove Solvent, to provide complete shielded peptide thioesters.By shielded peptide reagent B (TFA 88%, phenol 5%, H2O 5%, TIS 2%；5 ml) room temperature treatment 4 hours.Subsequently TFA solution is added dropwise over containing ice-cold methyl tertiary butyl ether(MTBE) (40 ml) In screw-cap centrifuge tube, and obtained suspension is stood overnight at 4 DEG C, after this by with 3000 rpm (20 minutes) from The heart collects precipitate, and after ether decant, is resuspended to by peptide precipitate in ice-cold methyl tertiary butyl ether(MTBE) (40 ml), Washing process is repeated twice.Use is through the linear gradient of the solution A of the 0-100% solvent B of 40 minutes, at semi-preparative C-4 By HPLC purification of crude peptide on reversed-phase column, and by suitable flow point lyophilizing to provide 63 (110 mg, 65%). C₉₀H₁₆₅N₁₁O₁₃S₂, MALDI-ToF MS: observe, [M+Na] 1695.2335Da；Calculate, [M+Na] 1695.4714Da (Figure 39).

The synthesis of glycolipidpeptide 64: as described in general procedure, above perform at Rink amide resin (0.1 mmol) SPPS.Front four aminoacid Ser-Ala-Asn-Met use standard scheme coupling on peptide synthesizer.After completion of the synthesis, make WithNα-FmocSer-(AcO₃-α-D-O-GlcNAc) OH (0.2 mmol, 131 mg) withO-(7-azepine benzo triazol-1-yl)-N,N,N’,N'-tetramethylureaHexafluorophosphate (HATU；0.2 mmol, 76 mg), 1-hydroxyl-7-azepine benzotriazole (HOAt；0.2 mmol, 27 mg) and diisopropylethylamine (DIPEA；0.4 mmol, 70 μ l) NMP (5 ml) solution, perform Manual coupling 12 hours.By standard K aiser test monitoring coupling reaction.Subsequently by resin NMP (6 ml) and dichloromethane (DCM；6 ml) washing, experience identical coupling condition the most again, to guarantee that coupling completes.Sugar is extended subsequently on peptide synthesizer Peptide, fully washs resin NMP (6 ml), DCM (6 ml) and MeOH (6 ml) after this, is dried in a vacuum.By resin In DCM (5 ml) swelling 1 hour, process 2 hours, with NMP (5 ml x with MeOH (10 ml) solution of hydrazine (60%) subsequently 2), DCM (5 ml x 2) and MeOH (5 ml x 2) fully wash, be dried in a vacuum.By resin in DCM (5 ml) swelling 1 hour, after this, it is used reagent K (TFA (81.5%), phenol (5%), THIOANISOLE (5%), water (5%), EDT (2.5%), TIS (1%)) (30 ml) was room temperature treatment 2 hours.By resin filter, wash with pure TFA (2 ml).Subsequently by filtrate in a vacuum It is concentrated into about the 1/3 of its initial volume.Use diethyl ether (0 DEG C) (30 ml) precipitation of peptides, and by being centrifuged 15 with 3000 rpm Minute reclaim.Use is through the linear gradient of the solvent orange 2 A solution of the 0-100% solvent B in 40 minute period, at semi-preparative C-8 post On by RP-HPLC purification of crude peptide, by suitable flow point lyophilizing to provide 64 (118 mg, 40%).C₁₂₉H₂₀₄N₃₂O₄₀S₂, MALDI-ToF MS: [M+] observed, 2907.5916Da；[M+] calculated, 2905.4354 Da (Figure 40).

The synthesis of glycolipidpeptide 65: as described in general procedure, above perform at Rink amide resin (0.1 mmol) SPPS.Front four aminoacid Ser-Ala-Asn-Met use standard scheme coupling on peptide synthesizer.After completion of the synthesis, make WithNα-FmocSer-(AcO₃-α-D-S-GlcNAc) OH (0.2 mmol, 134 mg) withO-(7-azepine benzo triazol-1-yl)-N,N,N’,N'-tetramethylureaHexafluorophosphate (HATU；0.2 mmol, 76 mg), 1-hydroxyl-7-azepine benzotriazole (HOAt；0.2 mmol, 27 mg) and diisopropylethylamine (DIPEA；0.4 mmol, 70 μ l) NMP (5 ml) solution, perform Manual coupling 12 hours.By standard K aiser test monitoring coupling reaction.Subsequently by resin NMP (6 ml) and dichloromethane (DCM；6 ml) washing, experience identical coupling condition the most again, to guarantee complete coupling.Gained is extended subsequently on peptide synthesizer The glycopeptide arrived.After completion of the synthesis, resin NMP (6 ml), DCM (6 ml) and MeOH (6 ml) are fully washed, in vacuum In be dried.By resin in DCM (5 ml) swelling 1 hour, process 2 hours with MeOH (10 ml) solution of hydrazine (60%) subsequently, Fully wash with NMP (5 ml x 2), DCM (5 ml x 2) and MeOH (5 ml x 2), be dried in a vacuum.Resin is existed DCM (5 ml) expands 1 hour, after this, it is used TFA (81.5%), phenol (5%), THIOANISOLE (5%), water (5%), EDT (2.5%), TIS (1%) (30 ml) was room temperature treatment 2 hours.By resin filter, and wash with pure TFA (2 ml).Subsequently will Filtrate is concentrated into about the 1/3 of its initial volume in a vacuum.Use diethyl ether (30 ml, 0 DEG C) precipitation of peptides, by with 3000 Rpm is centrifuged recovery in 15 minutes.Use is through the linear gradient of the solvent orange 2 A solution of the 0-100% solvent B in 40 minute period, in half system By RP-HPLC purification of crude peptide on standby type C-8 post, by suitable flow point lyophilizing to provide 65 (95 mg, 34%). C₁₂₉H₂₀₄N₃₂O₃₉S₃, MALDI-ToF MS: [M+] observed, 2923.6716Da；[M+] calculated, 2923.3861Da (figure 41)。

The synthesis of glycolipidpeptide 52: by lipopeptid thioesters 63 (4.3 mg, 2.5 μm ol), glycopeptide 64 (5.0 mg, 1.7 μ And dodecylphosphoric acid choline (6.0 mg, 17.0 μm ol) is dissolved in trifluoroethanol and CHCl mol)₃ (2.5 ml/2.5 ml) Mixture in.Solvent is under reduced pressure removed, to provide lipid/peptide thin film, at three (carboxyethyl) phosphine (2% w/v, 40.0 μ G) and EDTA (0.1% w/v, 20.0 μ g) in the presence of, use 200 mM phosphate buffers (pH 7.5,3 ml), by described Lipid/peptide thin film is hydrated 4 hours at 37 DEG C.By mixture ultrasonic Treatment 1 minute.2-sulfydryl second sulphur is added in vesicle suspension Acid sodium (2%w/v, 40.0 μ g), with initial coupled reaction.In calorstat, perform reaction at 37 DEG C, and pass through MALDI-ToF Regular monitoring reaction progress, MALDI-ToF display glycopeptide 64 disappeared in 2 hours.Subsequently by reaction with being dissolved in connection buffer 2 mercapto ethanol (20%) dilution in (2 ml), use, through the solution A linear gradient of the 0-100% solvent B of 40 minutes, is passed through Semi-preparative C-4 reversed-phase column purification of crude peptide, the lyophilizing of suitable flow point provides 52 (4.3 mg, 57%).C₂₁₂H₃₆₀N₄₃O₅₃S₃ , MALDI-ToF MS: observe, 4461.9177Da, calculating, 4455.578Da (Figure 37).

The synthesis of glycolipidpeptide 53: by lipopeptid thioesters 63 (2.5 mg, 1.5 μm ol), glycopeptide 65 (3.0 mg, 1.0 μ And dodecylphosphoric acid choline (3.5 mg, 10 μm ol) is dissolved in trifluoroethanol and CHCl mol)₃ (2.5 ml/2.5 ml's) In mixture.Solvent is under reduced pressure removed, to provide lipid/peptide thin film, at three (carboxyethyl) phosphine (2%w/v, 40.0 μ g) and In the presence of EDTA (0.1%w/v, 20.0 μ g), use 200 mM phosphate buffers (pH 7.5,2 ml), by described fat Matter/peptide thin film is hydrated 4 hours at 37 DEG C.By mixture ultrasonic Treatment 1 minute.Mistabrom is added in vesicle suspension Sodium (2%w/v, 40.0 μ g), with initial coupled reaction.Reaction is performed at 37 DEG C in calorstat, and fixed by MALDI-ToF Phase monitoring reaction progress, MALDI-ToF display glycopeptide disappeared in 2 hours.Subsequently by reaction with being dissolved in connection buffer (2 Ml) 2 mercapto ethanol (20%) dilution in.Use is through the solution A linear gradient of the 0-100% solvent B of 40 minutes, by half Preparative C-4 reversed-phase column purification of crude peptide, the lyophilizing of suitable flow point provides 53 (2.8 mg, 64%).C₂₁₂H₃₆₀N₄₃O₅₂S₄ , MALDI-ToF MS: observe, 4469.9112Da, calculating, 4471.6437Da (Figure 38).

It is prepared on Rink amide resin (0.1 mmol) described in compound 66-70 such as standardization program part. Glycopeptide 66 (78 mg, 61 %)；C₄₈H₈₂N₁₄O₂₁S₂, MALDI-ToF MS: [M+Na] observed, 1277.4746Da；Calculate [M+Na], 1277.5220 Da (Figure 43).Peptide 67 (89 mg, 83%)；C₄₀H₆₉N₁₃O₁₆S₂, MALDI-ToF MS: observe [M+Na], 1074.4789 Da；[M+Na] calculated, 1074.4427 Da (Figure 44).Glycopeptide 68 (57 mg, 48%)； C₄₅H₇₇N₁₃O₂₀S, MALDI-ToF MS: [M+Na] observed, 1174.4740Da；[M+Na] calculated, 1174.5129 Da (Figure 45).Peptide 69 (76 mg, 78%).C₃₇H₆₄N₁₂O₁₅S, MALDI-ToF MS: [M+Na] observed, 969.8162Da；Meter [M+Na] calculated, 970.8657 Da (Figure 46).Glycosylated amino acid 70 (12 mg, 33%), C₁₄H₂₅N₃O₈, MALDI-ToF MS: [M+Na] observed, 386.2749 Da；[M+Na] 386.3636 Da (Figure 46) calculated.

For being conjugated to the general procedure of BSA-MI:Execution is instructed to put together according to Pierce Endogen Inc.In short, (sugared) peptide 66 or 67 (for 2.5 equivalent excess of the available MI group on BSA) of purification is dissolved in conjugate buffer (contain There are the sodium phosphate of EDTA and Hydrazoic acid,sodium salt, pH 7.2；100 μ l) in, and add the BSA (2.4 mg) that maleimide activates In solution in conjugate buffer (200 μ l).By mixture incubation at room temperature 2 hours, subsequently by D-Salt dextrose Acid anhydride desalting column (Pierce Endogen, Inc.) purification, balances and with the sodium phosphate buffer (pH containing 0.15 M sodium chloride 7.4) eluting.BCA protein determination is used to identify the flow point containing conjugate.Divided by the quantitative monosaccharide via HPAEC/PAD Analysis measures carbohydrate content.

For preparing the general procedure of liposome.By ovum PC, ovum PG, cholesterol, MPL-A and compound 52 or 53 (15 μ Mol, mol ratio 60:25:50:5:10) it is dissolved in the mixture of trifluoroethanol and MeOH (1:1, v/v, 5 ml).By solvent Removing in a vacuum, to produce thin lipid membrane, it is by being suspended in containing NaCl (145 mM at 41 DEG C under an argon；1 Ml) in HEPES buffer (10 mM, pH 6.5) 3 hours and be hydrated.By vesicle suspension supersound process 1 minute, subsequently 50 DEG C pass in succession through 1.0,0.6,0.4,0.2 and 0.1 μm polycarbonate membrane (Whatman, Nuclepore Track-Etch Membrane) extrusion, to obtain SUV.By 100 DEG C seal pipe heats SUV (50 μ L) and aqueous TFA (2 M, 200 μ L) mixture 4 hours, measure the sugar content of liposome.Subsequently solution is concentrated in a vacuum, and use pulse Ampere detector (HPAEC-PAD；Methrome) and CarboPac post PA-10 and PA-20 (Dionex), by high pH anion Displacement chromatography is analyzed.

Dosage and immunity inoculation timetable:By five mices, (female BAl BIc/c, age 8-10 week, from Jackson Laboratories) group is with two weekly interval immunity inoculation four times.Strengthen including 3 g sugar at Liposomal formulation every time.Serum Sample obtains for 1 week after immunity inoculation (blood-letting in advance) front and last immunity inoculation.Last blood-letting is put by heart Blood completes.

Hybridoma is cultivated and antibody produces: gathering in the crops the spleen with two mices of 52 immunity inoculations, Standard hybridoma is cultivated Technology provides 30 hybridoma cell lines producing IgG.Three kinds of hybridomas (18B10.C7 (3), 9D1.E4 (10), 1F5.D6 (14)) with one liter of scale evaluation, and by resisting obtained by saturated ammonium sulphate and Protein G column chromatography purification subsequently Body, to obtain the IgG of about 10 mg in each case.

Reagent for biotic experiment: protease inhibitor cocktail derives from Roche (Indianapolis, IN). PUGNAc O-(2-acetamide-2-deoxidation-D-Glucopyranose. subunit (glucopyranosylidene)) amino N-phenylamino Carbamate is ordered from Toronto Research Chemicals, Inc (Ontario, Canada).The anti-O-of Mouse IgM GlcNAc(CTD110.6；Comer et al., 2001 Anal. Biochem. 293,169-177) and the anti-OGT of rabbit polyclonal (AL28) antibody is previously at laboratory (the Johns Hopkins University School of of Dr. Gerald W. Hart Medicine, Baltimore, MD) generate.Rabbit polyclonal anti-OGA antibody is from Dr. Sidney W. Whiteheart The friendship of (University of Kentucky College of Medicine) is granted.Rabbit polyclonal anti-CKII Alpha antibodies (NB100-377 for immunoblotting and the NB100-378 for immunoprecipitation) is purchased from Novus Biologicals (Littleton, CO).Mouse monoclonal antibody and anti-mouse IgM (μ chain)-agarose for alpha-tubulin derive from Sigma (St. Louis, Missouri).Normal rabbit igg agarose, normal rabbit igg agarose and protein A/G PLUS agarose are certainly Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) order.

Determination of serology:(Buskas and Boons, 2004 Chem. Eur. J. 10,3517-as previously described 3524；Ingale et al., 2007 Nat. Chem. Biol. 3,663-66), surveyed by enzyme-linked immunosorbent assay (ELISA) Fixed anti-GSTPVS (β-O-GlcNAc) SANM (68) IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM antibody titre.Letter speech It, dense with 2.5 μ g ml-1 in being coated buffer (containing 0.2 M borate buffer solution of 75 mM sodium chloride, pH 8.5) Degree, by flat for Immulon II-HB 96 hole microtitration plates (Thermo Electron Corp.) passing through with 100 μ l/ holes Maleimide joint be conjugated to BSA glycopeptide conjugate (BSA-MI-GSTPVS (β-O-GlcNAc) SANM；BSA-MI-66) It is coated overnight at 4 DEG C.The serial dilutions allowing serum or the cell supernatant containing MAb combines fixing GSTPVS in room temperature (β-O-GlcNAc) SANM totally 2 hours.By adding the anti-mouse IgG (Jackson that alkali phosphatase is puted together ImmunoResearch Laboratories Inc.)、IgG1 (Zymed)、IgG2a (Zymed)、IgG2b (Zymed)、 IgG3 (BD Biosciences Pharmingen) or IgM (Jacksons ImmunoResearch Laboratories) Antibody completes detection.After adding p-nitrophenyl phosphate ester (Sigma), use microplate (BMG Labtech) 405 Measuring absorbance at nm, wherein wavelength calibration is located at 490 nm.Antibody titer be defined as obtain relative to optical density be 0.1 or Bigger highest dilution.

In order to probe into corresponding glycopeptide, peptide and sugar to MAb Yu GSTPVS (β-O-GlcNAc) competition of combination of SANM (68) Property suppression, by MAb by this way in dilution buffer dilute, if described mode makes in the case of inhibitor, Intended final OD value is about 1.For each hole, by the MAb of 60 l dilutions in the most coated microtitration plate with 60 l Dilution buffer, the glycopeptide 68 being dissolved in dilution buffer with the final concentration of 0-500 M (GSTPVS (β-O-GlcNAc) SANM), peptide 69 (GSTPVSSANM；SEQ ID NO:11) or sugar 70 (β-O-GlcNAc-Ser) mixing.At incubation at room temperature 30 After minute, 100 l mixture are transferred to by BSA-MICGSTPVS (β-O-GlcNAc) SANM (BSA-MI-66) is coated Plate.The detection antibody that suitable alkali phosphatase used as discussed above is puted together, by microtitration plate incubation and develop the color.

Plasmid construction: by people OGT and OGA cDNA with two step approach PCR expand, with 5' end introduce attB1 site and HA epi-position and 3' end introduce attB2 site, with promote entrance (Gateway) Strategies For The Cloning (Invitrogen, Carlsbad, CA).Primer includes that (1) draws with the justice that has in the ogt after HA epi-position is mixed start codon for a PCR Thing: 5 '-CCCCATGTATCCATATGACGTCCCAGACTATGCCGCGTCTTCCGTGGGCAACGT-3 ' (SEQ ID NO: 13)；(2) use HA-ogt PCR primer as the sense primer containing attB1 site of template: 5 '- GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGATGTATCCATATGACGTCCCAGACTATGCCGCGTCTTCCG-3’ (SEQ ID NO:14)；(3) for the antisense primer with 3 ' attB2 sites of two ogt PCR: 5 '- GGGGACCACTTTGTACAAGAAAGCTGGGTTCTATGCTGACTCAGTGACTTCAACGGGCTTAATCATGTGG-3’ (SEQ ID NO:15)；(4) for a PCR with by the sense primer in the oga after HA epi-position incorporation start codon: 5 '- CCCCATGTATCCATATGACGTCCCAGACTATGCCGTGCAGAAGGAGAGTCAAGC-3 ' (SEQ ID NO:16)；(5) Use HA-oga PCR primer as the sense primer containing attB1 site of template: 5 '- GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGATGTATCCATATGACGTCCCAGACTATGCCGTGCAGAAGG-3’ (SEQ ID NO:17)；(6) for the antisense primer with 3 ' attB2 sites of two oga PCR: 5 '- GGGGACCACTTTGTACAAGAAAGCTGGGTTCACAGGCTCCGACCAAGTAT-3 ' (SEQ ID NO:18).Basis subsequently The description of manufacturer, implements entrance clone to the DNA fragmentation of purification, it is thus achieved that final expression construct pDEST26/HA-OGT and pDEST26/HA-OGA。

Cell is cultivated, is transfected and process: HEK 293T cell derives from ATCC (Manassas, VA), and maintains by 5% CO₂The Da Er being supplemented with 10% hyclone (GIBCO/Invitrogen, Carlsbad, CA) in 37 DEG C of calorstats of humidification Bai Ke (Dulbecco ' s) MEM (4.5 g l-1 glucoses, Cellgro/Mediatech, Inc., Herndon, VA) in.According to the description of manufacturer, with 8 μ g DNA and Lipofectamine 2000 reagent (Invitrogen Carlsbad, CA)/10 cm cell plates perform transfection.Simulation transfection is performed in the case of dna not depositing. Transfect latter 48 hours harvestings.For immunoprecipitation experiment, with the cell of ice-cold PBS wash plate, and as precipitate (pellet)-80 DEG C it are stored in until using.For immunoblot experiment, cell is washed twice with ice-cold PBS, and scrapes To 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) in, by pyrolysis product at 4 DEG C at microfuge In with 16,000 g clarify 25 minutes.With Bradford protein determination BITBUS program (Bio-Rad, Hercules, CA) With in sample buffer, boil 5 minutes, quantifying protein concentration.For mass spectrometry experiment, by 2 X 15 cm of 293T cell Plate processes 24 hours with 50 μMs of PUGNAc, cell is formed precipitation and as above stores.

Immunoprecipitation and Western blotting: in order to prepare the core kytoplasm for CKII immunoprecipitation (nucleocytosolic) fraction, is resuspended to the low of 4 volumes by the HEK293T cell precipitate with simulation or OGT transfection Ooze in buffer (5 mM Tris-HCl, pH 7.5, protease inhibitor cocktail), and transfer in 2 ml homogenizers.? After incubated on ice 10 minutes, cell suspension is implemented Dounce homogenization, is the other 5 minutes incubations on ice subsequently.Subsequently By the high osmotic buffer of a volume (0.1 M Tris-HCl, pH 7.5,2 M NaCl, 5 mM EDTA, 5 mM DTT, protease / inhibitor mixture) add in pyrolysis product.By pyrolysis product incubated on ice 5 minutes, take turns Dounce homogenate for another subsequently Change.Obtained pyrolysis product is transferred to containing PUGNAc (final concentration 10 μMs) microfuge pipe, and 4 DEG C with 18,000g is centrifuged 25 minutes.Bradford protein determination (Bio-Rad, Hercules, CA) is used to measure protein concentration. Before IP, pyrolysis product 1% Igepal CA-630 and 0.1% SDS supplements, and with normal rabbit or mouse IgG AC and egg The mixture of white A/G PLUS agarose was 4 DEG C of presettlings 30 minutes.After clarification, in the presence of purpose antibody, by pre-clear Clear supernatant is at 4 DEG C of incubations 4.Adding after protein A/G PLUS agarose, by sample other 2 hours of 4 DEG C of incubations, and Wide with IP lavation buffer solution (10 mM Tris-HCl, pH 7.5,150 mM NaCl, 1% Igepal CA-630,0.1% SDS) General washing.Finally, SDSPAGE sample buffer is added in IP complex, and boils 3 minutes.By supernatant by 10% or The prefabricated little gel of 4-15% Tris-HCl (Bio-Rad, Hercules, CA) differentiates, and is transferred to Immobilon-P transfer membrane (Millipore, Bedford, MA).By film with 3% BSA (O-GlcNAc trace) or the TBST of 5% breast (Western blotting) (there is the TBS of 0.1% Tween 20) solution close, then with every kind of antibody (forO-GlcNAc trace is 1:1000 dilution Degree, is 1:8000 dilution factor for CKII, OGT and OGA trace, and is 1:10 for alpha-tubulin trace, 000 dilution factor) 4 DEG C of detections overnight, subsequently in room temperature incubation 2 hours together with the secondary antibodies being conjugated to HRP.Used as described below SuperSignal chemical luminous substrate (Thermo Scientific, Rockford, IL) performs the final of HRP activity and detects: MAb 18B10.C7 (3), 9D1.E4 (10) and 1F5.D6 (14) use Femto；CKII, OGT, OGA and tubulin use PICO.Thin film is made to be exposed to CL-XPosure film (Thermo Scientific, Rockford, IL).Thin film manifests figure After Xiang, trace 0.1 M glycine (pH 2.5) is peeled off 1 hour in room temperature, with ddH2O washing with as mentioned above for loading Comparison (CKII or alpha-tubulin) detects again.

MAb is prepared with the sample puted together and analyze for LC-MS/MS of agarose:According to the description of manufacturer, via Two butanimide substrates (DSS, Thermo Scientific, Rockford, IL), by MAb 18B10.C7 (3), 9D1.E4 (10) and 1F5.D6 (14) or CTD110.6 covalency are conjugated to protein A/G PULS agarose or anti-mouse IgM agar Sugar.As above prepare, with larger-scale, the HEK293T core cytoplasm fraction that PUGNAc processes, by it together with the agarose of antibody conjugate Incubation, and as above wash.For eluted protein matter from agarose, add 0.1 M glycine (pH 2.5), and by eluate Neutralize with 1 M Tris-HCl (pH 8.0) immediately.Subsequently sample is reduced as previously described and alkylation, and at 37 DEG C Implement LysC to digest overnight.After digestion, processed sample (Lim et al., 2008 J. Proteome Res. as previously described 7,1251-1263).

Mass spectrography: by sample with 19.5 μ l 0.1% formic acid (in water) and 0.5 μ l 80% acetonitrile/0.1% formic acid (at water In) resuspension, and filter with 0.2 μm filter (Nanosep, PALL).Subsequently sample off line is loaded to nanospray On C18 post, use Finnigan LTQ/XL mass spectrograph (ThermoFisher, San Jose, CA), (Lim etc. as described previously People, 2008 J. Proteome Res. 7,1251-1263) separate by 160 minutes linear gradients.Every kind of sample enforcement is had Different 3 operations arranged: (1) ETD (electron transfer dissociation) pattern, wherein collect full MS spectrum, are at highest peak subsequently 6 MS/MS spectrums after ETD (the supplementary activation (enabled supplemental activation) of activation).It is right dynamically to get rid of It was set to 1 in 30 second persistent period.(2) the CID-NL (-false neutral loss of dissociating of collision-induced) pattern, wherein collects full MS Spectrum, is 8 MS/MS spectrums after the CID of highest peak subsequently.Running into false neutral loss event (loss of GlcNAc, 203.08) After, produce MS8 spectrum based on MS/MS spectrum.Dynamically get rid of and there is the setting identical with ETD method.(3) DDNL-ETD is (under CID Data dependency neutrality lose MS8, be that the ETD after neutral loss event each time activates subsequently), wherein use CID (35% Normalized collision energy) collect the MS/MS spectrum from the peak, the highest 5 that MS scans the most completely, and monitor 203.08 Neutral loss, produces MS8 spectrum in this process.The ETD activated by supplementary activation is used to perform the repetition with neutral loss Scan event.Dynamically get rid of and arrange the most in the same manner as described above.

Data analysis and checking: use TurboSequest algorithm (Bioworks 3.3, Thermo Finnigan), pin To from Swiss-Prot human protein group data base extract people (homo sapiens (Homo sapiens), 32876 entries, 2007 Deliver on August 13) forward and reverse data library searching MS spectrum.With the threshold value of 15 kinds of ions and the TIC of 1e3, spectrum is generated DTA File.For oxidation methionine (methione), alkylated cysteine andOThe serine/threonine that-GlcNAc modifies, Consider that the dynamic mass of 15.99,57.02 and 203.08 Da increases respectively.Every kind that search forward and reverse data storehouse are obtained The gained OUT file of sample dissects with ProtoeIQ (Bioinquire) further, and with the 1% FDR (tolerance of use: F- Value) and be in 5 ProFDR initial peptide cover filter.

Statistical analysis: by double tails, Si Shi t inspection does not measures statistical significance between the groups in pairs.WhenP <0.05 Time, difference is considered as significantly.

The full disclosure content of The disclosures of all patents, patent application and publication and can the material that obtains of electronics (nucleotide sequence that includes such as in such as GenBank and RefSeq is submitted to, and such as SwissProt, PIR, PRF, Aminoacid sequence in PDB is submitted to, and the translation from the annotation coding region in GenBank and RefSeq) all by quoting also Enter.Aforementioned specification and embodiment are given only for being expressly understood that.Ying Youqi is not interpreted as unnecessary restriction.The present invention is also With described exact details shown in being not limited to, because the variation that will be apparent to those skilled in the art is included within being wanted by right In seeking the present invention of restriction.

Claims (37)

1. the medicine of the cytotoxicity (ADCC) that glycolipidpeptide mediates for generation antibody dependent cellular in experimenter in preparation In purposes, described glycolipidpeptide comprises:

Comprise at least one glycosylation MUC1 glycopeptide component of B cell epi-position；

Comprise at least one peptide composition of the MHC restricted helper T cell epitope of II class；With

At least one lipid composition.

2. the purposes of claim 1, wherein said ADCC is that NK cell (NK) is cell-mediated.

3. the purposes of claim 1 or 2, wherein said ADCC cracks tumor cell.

4. the purposes of claim 3, wherein said tumor cell is breast cancer cell.

5. the purposes of claim 1, the cell of MUC1 peptide sequence is expressed in wherein said ADCC cracking.

6. a glycolipidpeptide, it comprises:

Comprise at least one glycosylation MUC1 glycopeptide component of B cell epi-position；

Comprise at least one peptide composition of the MHC restricted helper T cell epitope of II class derivative for MUC1；With

At least one lipid composition.

7. the glycolipidpeptide of claim 6, the wherein said glycosylation MUC1 glycopeptide component comprising B cell epi-position be included in one or Glycosylation on multiple serines and/or threonine residues.

8. the glycolipidpeptide of claim 7, the wherein said glycosylation MUC1 glycopeptide component comprising B cell epi-position comprises with being selected from down The glycosylation that the saccharide residue stated is carried out: GAlNAc, GlcNAc, Gal, NANA, NGNA, fucose, mannose and glucose.

9. the glycolipidpeptide any one of claim 6-8, wherein said comprise B cell epi-position glycosylation MUC1 glycopeptide component be I class MHC restricted epitope.

10. the glycolipidpeptide of claim 6, the wherein said glycosylation MUC1 glycopeptide component comprising B cell epi-position and/or described bag Peptide composition containing the MHC restricted helper T cell epitope of II class comprises people's MUC1 peptide sequence.

The glycolipidpeptide of 11. claim 6, the wherein said glycolipidpeptide comprising B cell epi-position and/or described in comprise MHC II class limit The peptide composition of property helper T cell epitope processed comprises about 5-30 aminoacid of MUC1 protein sequence, described MUC1 protein sequence Row comprise the extracellular region of MUC1 protein, and comprise glycosylated one or more serine or threonine residues.

The glycolipidpeptide of 12. claim 6, the wherein said glycosylation MUC1 glycopeptide component comprising B cell epi-position comprise with SAPDTRPAP (SEQ ID NO:20), TSAPDTRPAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO:22) or TSAPDTRPL (SEQ ID NO:23) has the aminoacid sequence of at least about 50% sequence iden.

The glycolipidpeptide of 13. claim 6, the wherein said glycosylation MUC1 glycopeptide component comprising B cell epi-position comprises SAPDTRPAP (SEQ ID NO:20), TSAPDTRPAP (SEQ ID NO:21), SAPDTRPL (SEQ ID NO:22) or TSAPDTRPL (SEQ ID NO:23)。

The glycolipidpeptide of 14. claim 12 or 13, the wherein said glycosylation MUC1 glycopeptide component comprising B cell epi-position is included in Glycosylation on one or more serines and/or threonine residues.

Glycolipidpeptide any one of 15. claim 6-14, wherein said lipid composition comprise one or more lipid chain, one Or multiple cysteine residues and one or more lysine residue.

Glycolipidpeptide any one of 16. claim 6-15, wherein said lipid composition comprises Toll-like receptor (TLR) part.

The glycolipidpeptide of 17. claim 16, wherein said Toll-like receptor (TLR) part comprises TLR2 part.

The glycolipidpeptide of 18. claim 17, wherein said TLR2 part comprises Pam₃CysSK₄。

The glycolipidpeptide of one of 19. claim 6-15, wherein said lipid composition comprises lipid adjuvant.

The glycolipidpeptide of 20. claim 19, wherein said lipid adjuvant comprises lipidated amino acid (LAA).

The MHC II that the glycolipidpeptide of 21. claim 6, B cell peptide epitopes that wherein said MUC1 is derivative and described MUC1 are derivative Class restricted helper T cell peptide epitopes comprises adjacent aminoacid sequence.

The glycolipidpeptide of 22. claim 21, wherein said adjacent aminoacid sequence comprises and aminoacid sequence APGSTAPPAHGVTSA (SEQ ID NO:26)、APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27)、 APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:29) tool There is the sequence of at least 50% sequence iden.

The glycolipidpeptide of 23. claim 21, wherein said adjacent aminoacid sequence comprises aminoacid sequence APGSTAPPAHGVTSA (SEQ ID NO:26)、APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:27)、APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:28) or APGSTAPPAHGVTSAPDTRPL (SEQ ID NO:29).

The glycolipidpeptide of 24. claim 22 or 23, wherein said adjacent aminoacid sequence be at one or more threonine and/or On serine residue glycosylated.

Glycolipidpeptide any one of 25. claim 6-24, it comprises covalently bound immunomodulator further.

The glycolipidpeptide of 26. claim 25, wherein said immunomodulator is selected from TLR9 agonist, cox 2 inhibitor, GM- CSF, the inhibitor of indole amine 2,3-dioxygenase (IDO), chemotherapeutant and combinations thereof.

27. 1 kinds of pharmaceutical compositions, it comprises:

According to the glycolipidpeptide any one of claim 6-26；With

Pharmaceutically acceptable carrier.

28. 1 kinds of compositionss, it comprises the liposome containing the glycolipidpeptide any one of with good grounds claim 6-26.

The compositions of 29. claim 28, the lipid composition of wherein said glycolipidpeptide promotes that liposome is formed.

The compositions of 30. claim 27 or 28, it comprises immunomodulator further.

The compositions of 31. claim 30, wherein said immunomodulator comprises TLR agonist.

The compositions of 32. claim 31, wherein said TLR agonist comprises TLR9 agonist.

The compositions of 33. claim 32, wherein said TLR9 agonist comprises CpG.

The compositions of 34. claim 30, wherein said immunomodulator is selected from TLR9 agonist, cox 2 inhibitor, GM- CSF, the inhibitor of indole amine 2,3-dioxygenase (IDO), chemotherapeutant and combinations thereof.

35. 1 kinds of immunogenic vaccine, it comprises according in the glycopeptide any one of claim 6-26 or claim 27-34 The compositions of any one.

Glycolipidpeptide any one of 36. claim 6-26 or the compositions any one of claim 27-35 are controlled for manufacture The purposes of the medicine for the treatment of or prevention infection, disease or disease.

37. 1 kinds of test kits, it comprises:

Glycolipidpeptide any one of claim 6-26；

Packaging；With

Operation instructions.