CN102215862B - GLOBO H and related anticancer vaccines containing novel glycolipid adjuvants - Google Patents

Abstract

The present invention provides immunogenic compositions, cancer vaccines, and methods for treating cancer. It provides a composition comprising: (a) a glycan, such as Globo? H, or an immunogenic fragment thereof, wherein the glycan is complexed to the carrier protein via a linker (e.g., p-nitrophenyl); and (b) an adjuvant comprising a glycolipid, such as an alpha-galactosyl-ceramide derivative, that binds to CD1d on dendritic cells, wherein the immunogenic composition induces an immune response that induces a higher relative content of IgG isoform antibodies compared to IgM isoform antibodies. It provides an immunogenic composition comprising the carrier protein diphtheria toxin cross-reactive material 197(DT-CRM197) and adjuvant C34. Antibodies produced by the immunogenic compositions disclosed herein can further neutralize the antigen Globo? H. At least one of stage-specific embryo antigen-3 (SSEA-3) and stage-specific embryo antigen-4 (SSEA-4). Also provided are therapeutic agents against breast cancer stem cells, comprising immunogenic compositions comprising Globo? H. SSEA-3, or SSEA-4.

Description

GLOBO H and related anticancer vaccines containing novel glycolipid adjuvants

Cross References to Related Applications

This application is a continuation-in-part of U.S. Patent Application Serial No. 12/485,546 (invention title "Compositions for inducing immune responses specific to Globo Hand SSEA-3 and use of cancer treatment", filed June 16, 2009), which claims U.S. Provisional Patent Application Serial No. 61 Priority of /061,968 (filed June 16, 2008). The contents of these patent applications are incorporated in this specification by reference data in their entirety.

technical field of invention

The present invention relates to the field of cancer vaccines. In particular, the application relates to a sugar-based vaccine containing the B-cell epitope GloboH complexed with the immunogenic carrier DT-CRM197. More specifically, the present invention relates to anti-cancer GloboH-DT vaccines co-administered with novel glycolipid adjuvants such as C34.

Background of the invention

To design therapies against cancer, it is desirable to seek molecular targets of cancer or cancer stem cells that are not present in normal cells. Aberrant glycosylation is often associated with tumor development and was first described by Meezan et al. in 1969, showing that cancer glycans differ from normal cells (Meezan E, et al. (1969) Biochemistry 8:2518-2524). Aberrant glycosylation includes loss or overexpression of certain structures, retention of truncated structures, and appearance of novel structures. These structural differences were later supported by much histological evidence using comparisons of lectin staining of healthy and malignant tissues (Turner GA (1992) Clin Chim Acta 208: 149-171; Gabius HJ (2000) Naturwissenschaften 87: 108-121).

Recently, tumor-associated carbohydrate antigens have been identified by monoclonal antibodies and mass spectrometry (ShriverZ, et al. (2004) NatRevDrugDisc3:863-873; PacinoG, et al. (1991) BrJCancer63:390-398). So far, many tumor-associated antigens expressed on cancer cells in the form of glycolipids or glycoproteins have been characterized and associated with certain types of cancer (Bertozzi CR, Dube DH (2005) Nat Rev Drug Discovery 4: 477-488). Although relatively little is known about the role of surface carbohydrates in malignant cells, antibodies against these antigens, either administered passively or elicited by vaccines, have been shown to correlate with better prognosis.

Among the reported tumor-associated glycans, the glycolipid antigen GloboH (Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glc) was first isolated and identified from breast cancer MCF-7 cells by Hakomori et al. in 1984 (BremerEG, et al. . (1984) J Biol Chem 259: 14773-14777). Further studies with anti-GloboH monoclonal antibodies have shown that GloboH is present in many other cancers, including prostate, stomach, pancreas, lung, ovary, and colon cancers, and that it is not readily accessible by the immune system There is only trace expression on the luminal surface of normal secretory tissue (RagupathiG, et al. (1997) Angew Chem Int Ed 36: 125-128). In addition, it has been confirmed that the serum of breast cancer patients contains high levels of anti-GloboH antibodies (Gilewski Telal. (2001) ProcNatlAcadSciUSA98: 3270-3275; HuangC-Y, et al. (2006) ProcNatlAcadSciUSA103: 15-20; WangC-C, et al. (2008) ProcNatlAcadSciUSA105(33):11661-11666), and patients with GloboH-positive tumors showed shorter survival compared with patients with GloboH-negative tumors (Chang, Y-J, et al. (2007) ProcNatlAcadSciUSA104 ( 25): 10299-10304). These findings make GloboH, a hexasaccharide epitope, an attractive tumor marker and a viable target for cancer vaccine development.

GloboH is a cancer antigen overexpressed in various epithelial cancers. Such antigens have been suggested as targets for cancer immunotherapy. Although a vaccine that induces an antibody response against GloboH has been developed, its anticancer efficacy is unsatisfactory due to the low antigenicity of GloboH. There is therefore still a need for new vaccines that can induce high levels of immune responses targeting GloboH.

Stem cells are defined as a cell population with the ability to self-renew and differentiate into different types of cells and tissues (Reya Te et al., (2001) Nature 414: 105-111). Since both malignant and normal tissues contain heterogeneous cell populations, cancer stem cells may play a key role in tumor growth and maintenance of tumor heterogeneity. Cancer stem cells have been identified from a variety of solid tumors, such as brain, breast, colon, and prostate cancers. Breast cancer stem cells (BCSCs) were first demonstrated by Al-Hajj et al. to be present in the CD24-CD44+ subpopulation of breast cancer based on their ability to generate phenotypically diverse tumors when xenografted into NOD/SCID mice (Al-HajjM , et al., (2003) Proc Natl Acad Sci USA 100: 3983-3988). Most of the early disseminated cancer cells in the bone marrow of breast cancer patients have a CD24-CD44+ phenotype (Balic Metal., (2006) Clin Cancer Res 12: 5615-5621), which suggests that BCSCs can metastasize. Based on their ability to grow, differentiate, and metastasize, as well as their resistance to radiation exposure, BCSCs have become a major target for breast cancer therapy (Tang C. et al., (2007) faseb J. 21: 1-9).

In breast cancer, GloboH expression is observed in >60% of ductal, lobular, and tubular carcinomas, but not in non-epithelial breast tumors (Mariani-Constantini Retal., (1984) Am. J. Pathol. 115:47-56). GloboH is not expressed in normal tissues, except for weak expression in apical epithelial cells at the luminal margins, which appear to be sites inaccessible to the immune system (Id.; Zhang S. et al., (1997) Int. J. Cancer 73:42-49).

GloboH is also expressed in breast cancer stem cells (BCSCs). Flow cytometry showed that GloboH was expressed in 25/41 breast cancer samples (61.0%). 25/25 non-BCSCs and 8/40 (20%) BCSCs expressed GloboH. Stage-specific embryonic antigen-3 (SSEA-3), the pentasaccharide precursor of GloboH, was expressed in 31/40 (77.5%) tumors. 29/31 non-BCSCs and 25/40 (62.5%) BCSCs expressed SSEA-3. (Chang W-W. et al., (2008) ProcNatlAcadSciUSA105(33): 11667-11672.)

Danishefsky and Livingston had previously reported the GloboH-KLH vaccine against various cancers (GilewskiTelal. (2001) ProcNatlAcadSciUSA98: 3270-3275; RagupathiG, et al. (1997) AngewChemIntEd36: 125-128; KudryashovV, et al. (1998) GlycoconjJ. 15:243-249; SlovinSFetal (1997) ProcNatlAcadSciUSA96:5710-5715) and heptavalent vaccine (comprising GM2, GloboH, LewisY, Tn, STn, TF, and Tn-MUC1 compounded with KLH individually; SabbatiniPJetal (2007) ClinCancerRes13: 4170-4177). However, patients immunized with the heptavalent vaccine only induced antibody responses against five of the seven antigens, except for GM2 and LewisY antibodies. Unlike ubiquitously expressed antigens such as GM2, GloboH is specifically expressed on tumor cells and only minimally expressed in normal secretory tissues, making it an ideal target for vaccine development. In their study, reductive amination with a KLH carrier protein after ozonolysis of the GloboH glycoside yielded approximately 150 sugar units per protein (Ragupathi G, et al. (1997) Angew Chem Int Ed 36: 125-128). Further refinement increased the glycocomplexation ratio to approximately 720:1 by using the MMCCH linker (Wang S-K, et al. (2008). ProcNatlAcadSciUSA105:3690-3695). However, the precise determination of this glycocomplexation is extremely difficult. Furthermore, the synthetic vaccine combined with the immune adjuvant QS-21 was shown to induce predominantly IgM only and to a lesser extent IgG antibodies in both prostate and metastatic breast cancer patients. In Phase 1 clinical trials, the vaccine also showed minimal toxicity, producing a transient local skin reaction at the vaccination site. (GilewskiTelal. (2001) ProcNatlAcadSciUSA98: 3270-3275; RagupathiG, et al. (1997) AngewChemIntEd36: 125-128; SlovinSFetal (1997) ProcNatlAcadSciUSA96: 5710-5715.) The mild influenza-like symptoms observed on some patients may Associated with side effects of QS-21. Pentavalent vaccines containing five prostate and breast cancer-associated carbohydrate antigens (Globo-H, GM2, STn, TF, and Tn) complexed with the maleamide-modified carrier protein KLH have been reported to produce higher than Anti-GloboH serum with IgG titer of IgM (Zhu J. et al. (2009) J. Am. Chem. Soc. 131(26):9298-9303).

Therefore, it would be desirable to identify alternative carriers and adjuvants to enhance antibody responses to GloboH, especially with higher IgG titers, and improve vaccine efficacy with minimal side effects.

Contents of the invention

The present invention relates to a sugar-based vaccine containing a diphtheria toxin cross-reactive material 197 (DT-CRM197) (Th epitope) chemically complexed via a p-nitrophenyl linker. GloboH (B cell epitope). The synthetic vaccine combined with a glycolipid adjuvant induced IgG, IgG1, and IgM antibodies in breast cancer models and provided excellent immunogenicity, which showed delayed tumorigenesis in xenograft studies. Glycan array analysis of the antibodies elicited by GloboH-DT and glycolipid C34 showed that these antibodies could not only identify GloboH, but also SSEA-3 (Gb5) and SSEA-4 (sialylated Gb5) polysaccharides. Sugars, which are specific to cancer cells and cancer stem cells.

The present invention relates to an immunogenic composition comprising: (a) a glycan consisting essentially of GloboH or an immunogenic fragment thereof, wherein the glycan is complexed to a carrier protein via a linker; and ( b) an adjuvant comprising glycolipids that bind to CD1d molecules on dendritic cells, wherein the immunogenic composition induces an immune response that induces a higher expression of antibodies compared to the IgM isotype Relative content of IgG isotype antibodies.

In some aspects, the carrier protein is diphtheria toxin cross-reactive material 197 (DT-CRM197). In some aspects, the linker is a p-nitrophenyl linker.

In some aspects, the adjuvant is a synthetic analog of α-galactosyl-ceramide (α-GalCer). In some embodiments, the adjuvant is C34, wherein C34 comprises the structure:

In some aspects, the immune response is preferably directed towards the production of antibodies of the IgG isotype. In some aspects, the immunogenic composition comprises at least one adjuvant that induces a humoral or cellular immune response.

In some aspects, antibodies generated by the immune response can neutralize antigens expressed on cancer cells or cancer stem cells. In some embodiments, the antibodies produced by the immune response can neutralize at least one of antigen Gb4, stage-specific embryonic antigen-3 (SSEA-3), and stage-specific embryonic antigen-4 (SSEA-4) kind. In some embodiments, the antibody that can neutralize at least one of antigen Gb4, stage-specific embryonic antigen-3 (SSEA-3), and stage-specific embryonic antigen-4 (SSEA-4) comprises Higher relative content of IgM isotype antibodies IgG isotype antibodies.

The present invention relates to a cancer vaccine comprising an immunogenic composition capable of inducing an anti-cancer immune response in a subject. In some aspects, the cancer vaccine is suitable for treating a cancer selected from the group consisting of breast cancer, lung cancer, liver cancer, buccal cancer, gastric cancer, colon cancer, nasopharyngeal cancer, skin cancer, kidney cancer, brain tumor, prostate cancer , ovarian cancer, cervical cancer, bowel cancer, and bladder cancer.

In some aspects, the cancer tissue expresses GloboH antigen on the cell surface. In some aspects, the GloboH antigen is expressed on epithelial cells of breast cancer tumors.

In some embodiments, the cancer vaccine can produce at least one of the neutralizing antigens GloboH, Gb4, stage-specific embryonic antigen-3 (SSEA-3), and stage-specific embryonic antigen-4 (SSEA-4). antibodies. In some aspects, the antigens are expressed on breast cancer stem cells.

The present invention relates to a method of treatment comprising inhibiting tumor growth comprising: (a) administering to a subject in need thereof an immunogenic composition comprising: a glycan consisting essentially of Globo

H or an immunogenic fragment thereof, wherein the glycan is complexed to a carrier protein via a linker; and an adjuvant comprising a glycolipid that binds to a CD1d molecule on a dendritic cell; and (b) induces a An immune response that induces a higher relative amount of IgG isotype antibodies compared to IgM isotype antibodies.

In some embodiments of the method, the linker is p-nitrophenol, the carrier protein is diphtheria toxin cross-reactive material 197 (DT-CRM197), and the adjuvant is α-galactosyl-ceramide ( α-GalCer) synthetic analogs. In a specific example, the adjuvant is C34.

In some embodiments of the method, the immunogenic composition further comprises a cancer vaccine, and wherein the one or more treatments with an effective amount of the cancer vaccine inhibits tumor growth. In some embodiments, administration of the cancer vaccine reduces tumor size.

In some embodiments of the method, the immune response is preferably directed towards the production of antibodies of the IgG isotype that neutralize the antigens GloboH, Gb4, stage-specific embryonic antigen-3 (SSEA-3), and stage At least one of specific embryonic antigen-4 (SSEA-4). In some aspects, at least one of the antigens GloboH, stage-specific embryonic antigen-3 (SSEA-3), and stage-specific embryonic antigen-4 (SSEA-4) is expressed on breast cancer stem cells. In some aspects, the GloboH antigen is expressed on epithelial cells of breast cancer tumors.

The present invention relates to a cancer vaccine comprising: (a) an immunogenic composition comprising: a glycan consisting essentially of GloboH or an immunogenic fragment thereof, wherein the glycan is linked via a complexed with a carrier protein; and an adjuvant comprising glycolipids that bind to CD1d molecules on dendritic cells, wherein the immunogenic composition induces an immune response that induces an immune response compared to IgM a higher relative content of IgG isotype antibodies; and (b) a pharmaceutically acceptable excipient.

In some aspects, the cancer vaccine comprises an immunogenic composition, wherein the linker is p-nitrophenol, the carrier protein is diphtheria toxin cross-reactive material 197 (DT-CRM197), and the adjuvant is α- Synthetic analogue of galactosyl-ceramide (α-GalCer). In a specific example, the adjuvant is C34.

In some aspects, the cancer vaccine is used to treat cancer, wherein the one or more treatments with an effective amount of the cancer vaccine can inhibit tumor growth. In some embodiments, administration of the cancer vaccine reduces tumor size. In some embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, liver cancer, buccal cancer, stomach cancer, colon cancer, nasopharyngeal cancer, skin cancer, kidney cancer, brain tumor, prostate cancer, ovarian cancer, Cervical cancer, bowel cancer, and bladder cancer.

The present invention relates to an immunogenic composition comprising: (a) a glycan consisting essentially of GloboH-related glycans or immunogenic fragments thereof, wherein the glycan is linked to a carrier via a linker protein complex; and (b) an adjuvant comprising glycolipids that bind to CD1d molecules on dendritic cells, wherein the GloboH-associated glycans are selected from the group consisting of SSEA-3 and SSEA-4, and Wherein the immunogenic composition induces an immune response that induces a higher relative level of IgG isotype antibodies compared to IgM isotype antibodies.

In some aspects of the immunogenic composition, the carrier protein is diphtheria toxin cross-reactive material 197 (DT-CRM197), and the adjuvant is a synthetic analog of α-galactosyl-ceramide (α-GalCer). and the linker is a p-nitrophenyl linker. In a specific example, the adjuvant is C34.

The present invention relates to a therapeutic agent against breast cancer stem cells, the therapeutic agent comprising: GloboH complexed with diphtheria toxin cross-reactive material 197 (DT-CRM197) carrier protein via a p-nitrophenyl linker; and an adjuvant , which contains glycolipids that bind to CDld molecules on dendritic cells. In some embodiments of the therapeutic agent, the adjuvant is C34.

The present invention relates to a therapeutic agent against breast cancer stem cells, the therapeutic agent comprising: SSEA-3 complexed with diphtheria toxin cross-reactive material 197 (DT-CRM197) carrier protein via a p-nitrophenyl linker; and a An adjuvant comprising glycolipid C34 that binds to CDld molecules on dendritic cells.

The present invention relates to a therapeutic agent against breast cancer stem cells, comprising: SSEA-4 complexed with diphtheria toxin cross-reactive material 197 (DT-CRM197) carrier protein via a p-nitrophenyl linker. In some embodiments, the therapeutic agent further comprises an adjuvant comprising a glycolipid that binds to CD1d molecules on dendritic cells.

Administration of the therapeutic agents of the invention to a subject induces the generation of antibodies that identify an antigen expressed on breast cancer stem cells (BCSC), wherein the antigen is selected from the group consisting of GloboH, SSEA-3, and SSEA-4 group. The present invention relates to a method of treating breast cancer comprising administering the therapeutic agent of the present invention.

Brief description of the drawings

The following drawings form a part of this specification and are included to further illustrate certain aspects of the disclosure in which the invention described will be useful with reference to one or more of these drawings in combination with specific embodiments presented herein. Examples are sometimes clearer. For purposes of illustrating the invention, a presently preferred embodiment is shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. This patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication (including color drawings) will be provided by the Office upon request and payment of the necessary fee.

Figure 1 shows the structures of GloboH and cleaved derivatives.

Figures 2A-2C show the binding specificities of monoclonal antibodies VK9 and Mbr1 (against GloboH) and anti-SSEA-3, respectively.

Figures 3A-3B show the serum responses of mice vaccinated with various GloboH complexes and [alpha]-GalCer. Groups of three C57BL/6 mice were vaccinated s.c. with 1 μg of glycoconjugate in the presence or absence of 2 μg of glycolipids. Mouse sera were diluted 1:60 and 1:240 for IgM (FIG. 3A) and IgG (FIG. 3B) antibody analysis, respectively. Fluorescence detection was performed at 532nm, PMT500 using Cy3-anti-mouse IgG or IgM secondary antibody. Data are expressed as mean fluorescence intensity ± SEM of three mice.

Figure 4 shows the structures of α-GalCer and its analogs.

Figure 5 shows the IgM content of mice vaccinated with GloboH complexes and α-GalCer derivatives. Mouse sera were collected and analyzed after the second and third vaccinations as indicated. Detection was performed using Cy3 secondary anti-mouse IgM at 532nm, PMT400. The results are expressed as the mean fluorescence intensity ± SEM of three mice.

Figure 6 shows good specificity of mouse polyclonal antibodies (anti-GloboH, anti-Gb5, anti-SSEA-4, and anti-Gb4) after vaccination. Mice sera (dams, Balb/c, i.m.) were obtained two weeks after the third vaccination with 1.6 μg GH-DT in the presence or absence of 2 μg sugar adjuvant. IgG titers were analyzed with glycan microarrays and defined as the highest dilution yielding an MFI greater than 1000 (10 times background), PMT400. Each point represents an individual mouse titer.

Figure 7 shows the IgM to IgG antibody titers produced by GloboH-DT with different adjuvants.

Figure 8 shows the evaluation of the activity of the adjuvants for the GH-KLH vaccine. Female Balb/c mice were vaccinated i.m. with 1.6 μg GH-KLH and 2 μg of the indicated adjuvants and bled every two weeks after vaccination. Serum was diluted and introduced into microarray analysis.

Figure 9 shows antibody isotype profiling after immunization. Mice were vaccinated as described. Sera (1:60 dilution) were introduced into microarrays for antibody subpopulation analysis (532nm, PMT300). Data are presented as mean fluorescence ± SEM of three mice.

Figure 10 shows the IgM versus IgG antibody titers induced by SSEA-3-DT or SSEA-4-DT and different types of glycolipid adjuvants.

Figure 11 shows the structures of 24 glycans on the cell surface.

Figures 12A-12C show cross-reactivity studies against IgG elicited by different vaccines. Figure 12A: Anti-GloboHIgG induced by GloboH-DT and C1 adjuvant; Figure 12B: Anti-Gb5 IgG induced by Gb5-DT and C1 adjuvant; Figure 12C: Induced by SSEA-4-DT and C1 adjuvant Induced anti-SSEA-4 IgG.

Figure 13 shows the mouse xenograft model. ^2x105 4T1 mouse metastatic mammary tumor seed cells were prepared in sterile PBS and injected subcutaneously to vaccinate Balb/c mice. Mouse tumor size was measured with a Vernier caliper and defined as (length x height x width)/2 (mm ³ ).

Figure 14 shows the process for the synthesis of GloboH half esters and glycoconjugates.

Figure 15 shows flow cytometric analysis of SSEA-4 expression in primitive breast cancer stem cells. SSEA-4 expression on the surface of BCSCs and non-BCSCs was analyzed by four-color immunofluorescent staining followed by flow cytometry. BCSCs were defined as CD45 ⁻ /CD24 ⁻ /CD44 ⁺ cells, while non-BCSCs were defined as the rest of the population of CD45 ⁻ cells, as shown in the left column. Target antigen expression on the surface of BCSCs and non-BCSCs is shown in the middle and right columns, respectively. Dotted lines represent the isoform control group, while numbers represent the percentage of positive cells.

Figure 16 shows restricted expression of SSEA-4 in normal tissues. SSEA-4 expression in the breast, small intestine, and rectum was examined using immunohistochemical staining of normal tissue arrays. Positive staining for SSEA-4 was limited to the apical surface of epithelial cells.

detailed description

The present invention relates to the surprising discovery that DT-CRM197 is a promising carrier protein for GloboH and SSEA-4 not only because it has been widely used for decades for human vaccination against diphtheria, but also because it is highly immunogenic sex. Most importantly, it has been approved by the FDA for use in various glycoconjugate vaccines. Diphtheria toxin cross-reactive material 197 (DT-CRM197) is an avirulent mutant (G52E) of DT that shares immunogenicity with the original molecule and its ability to bind heparin-binding epidermal growth factor (HB-EGF). ability, the heparin-binding epidermal growth factor is a specific cell membrane receptor for DT, which is often overexpressed in cancer (Buzzi S. et al., Cancer Immunology, Immunotherapy (2004), 53(11): 1041-1048).

Using C34 as an adjuvant, both GH-DT and SSEA-4-DT showed the most effective immune responses, eliciting more IgG antibodies than IgM antibodies against tumor antigens. GH-DT binding to C34 elicits antibodies that neutralize not only GloboH but also SSEA-3 (Gb5) and SSEA-4, both of which are specific for breast cancer cells and cancer stem cells.

At the same time, the glycan microarray disclosed in the present invention can provide a powerful platform for testing antibody specificity, and can be used to identify patients for vaccine trials and monitor their immune response after immunization.

In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which are shown by way of illustration specific embodiments that may be practiced. These embodiments are described in detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and structural, logical, and electrical changes may be made without departing from the scope of the invention. . The following detailed description of exemplified specific examples should therefore not be considered limiting, but the scope of the invention is defined by the scope of the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents mentioned herein are incorporated by reference for all purposes, including describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analyses, and method. All references cited in this patent specification are considered to be indicative of the state of the art in this field. Nothing herein is to be construed as an admission that the present invention is not incompatible with antedating such disclosure by antedating such disclosure.

Before the materials and methods of the present invention are described, it is to be understood that this invention is not limited to the particular methods, procedures, materials, and reagents described as such may vary. It should also be understood that the terminology used herein is only used to describe specific examples, and it is not intended to limit the scope of the present invention, which will be limited only by the scope of the appended claims.

definition

It must be noted that herein, and in the appended claims, the singular forms "a" and "the" include plural references unless the content clearly dictates otherwise. Meanwhile, "a", "one or more", and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising", "including", and "having" are used interchangeably.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature.参见，例如，MolecularCloningALaboratoryManual，2ndEd，Sambrooked.，FritschandManiatis(ColdSpringHarborLaboratoryPress，1989)；DNACloning，VolumesIandII(D.N.Glovered.，1985)；CultureOfAnimalCells(R.I.Freshney，AlanR.Liss，Inc.，1987)；ImmobilizedCellsAndEnzymes(IRLPress，1986) ；B.Perbal，APracticalGuideToMolecularCloning(1984)；thetreatise，MethodsInEnzymology(AcademicPress，Inc.，N.Y.)；GeneTransferVectorsForMammalianCells(J.H.MillerandM.P.Caloseds.，1987，ColdSpringHarborLaboratory)；MethodsInEnzymology，Vols.154and155(Wuetal.eds.)，ImmunochemicalMethodsInCellAndMolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, Harlow and Lanes (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

The term "lipid" as used herein refers to any fat-soluble (lipophilic) molecule involved in a cell signaling pathway.

The term "glycolipid" as used herein refers to lipids to which sugars are attached, which serve as markers for cell identification.

The term "α-galactosylceramide" or "α-GalCer" as used herein refers to a glycolipid that stimulates natural killer T cells to produce both T helper (TH)1 and TH2 cytokines. Glycolipid derivative C34 used herein has the following structure:

The α-GalCer analogs of the present disclosure include α-GalCer analogs of bacterial origin (Group I: C2, C3, and C14), α-GalCer analogs modified by sulfonation (Group II: C4, C5 , and C9), phenyl-alkyl chain α-GalCer analogs (Group III: C6-C8, C10-C11, C15-C16, C18-C34, C8-5, and C8-6), and plant sheaths Phytosphingosine cleaved α-GalCer analogs (Group IV: C12, C13, and C17). The structure of C34 and other α-galactosylceramide analogues and their use as adjuvants are disclosed in detail in PCT patent application PCT/US2008/060275 (filed April 14, 2008).

Synthetic α-GalCer analogs (including C34) can form complexes with CDld molecules. Synthetic α-GalCer analogs can be identified by NKTs T cell receptors. Synthetic α-GalCer analogs can elicit TH1-type, TH2-type, or TH1-type, as well as TH2-type responses. α-GalCer analogs activate NKTs in test tubes. α-GalCer analogs can activate NKTs in vivo.

The term "glycan" as used herein refers to polysaccharides or oligosaccharides. Glycans are also used herein to refer to sugar moieties of glycocomplexes, such as glycoproteins, glycolipids, glycopeptides, glycoproteomes, peptidoglycans, lipopolysaccharides, or proteoglycans. Glycans are generally composed only of O-glycosidic linkages between monosaccharides. For example, cellulose is a polysaccharide (or more specifically glucan) composed of β-1,4-linked D-glucose, while chitin is composed of β-1,4- Linked N-acetyl-D-glucosamine composition. Glycans may be homo- or heteropolymers of monosaccharide residues, and they may be linear or branched. Glycans can be found attached to proteins, such as in glycoproteins and proteoglycans. It is generally found on the outer surface of cells. O- and N-linked glycans are most commonly found in eukaryotic cells, but although less common, they can also be found in prokaryotic cells. N-linked glycans can be found attached to the R-group nitrogen atom (N) of the asparagine in the sequon. A sequon is an Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except proline.

The term "glycoprotein" as used herein refers to a protein covalently modified with glycans. There are four types of glycoproteins: 1) N-linked glycoproteins, 2) O-linked glycoproteins (mucins), 3) glycosaminoglycans (GAGs, which are also known as proteoglycans ), 4) GPI-anchored proteins. Most glycoproteins have structural microheterogeneity (multiple different glycan structures attached to the same glycosylation site) and macrostructural heterogeneity (multiple sites and types of glycan attachment).

The term "antigen" as used herein is defined as any substance capable of eliciting an immune response.

The term "immunogen" as used herein refers to an antigen or a substance that induces the production of an antigen, such as a DNA vaccine.

The term "immunogenicity" as used herein refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

The term "immunotherapy" as used herein refers to a group of therapeutic strategies based on the concept of modulating the immune system to achieve prophylactic and/or therapeutic goals.

The term "CDld" as used herein refers to a member of the CD1 (cluster of differentiation 1) glycoprotein family expressed on the surface of various human antigen-presenting cells. Lipid antigens presented by CD1d activate natural killer T cells. CD1d has a deep antigen-binding groove in which glycolipid antigens bind. CDld molecules expressed on dendritic cells can bind and present glycolipids, including α-GalCer analogs, such as C34.

The term "adaptive immune system" as used herein refers to a highly specialized, systemic set of cells and responses that can ward off pathogenic challenges. The cells of the adaptive immune system are a type of white blood cell called lymphocytes. B cells and T cells are the main types of lymphocytes.

The terms "T cells" and "Ts" as used herein refer to a group of white blood cells called lymphocytes, which play a central role in cell-mediated immunity. T cells can be distinguished from other lymphocyte types, such as B cells and NKs, by the presence of a specific receptor on their cell surface called the T cell receptor (TCR). Several different subsets of T cells have been described, each with unique functions. The helper T (TH) cell line is the "middle man" of the adaptive immune system. Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or "help" the immune response. Depending on the cytokine signal received, these cells differentiate into TH1, TH2, TH17, or one of other subpopulations, which secrete different cytokines.

The term "antigen presenting cell" (APC) as used herein refers to a cell displaying a foreign antigen complex with the major histocompatibility complex (MHC) on its surface. T cells can identify such complexes using their TCRs. Dendritic cells (DCs) belong to this type of action, and they can present antigens to T cells with CD1. In an exemplary implementation, the DCs used in the methods of the invention can be any of several DC subsets, which in one implementation arise from lymphoid differentiation, or, in another implementation, on myeloid progenitors. cell differentiation.

The term "naive cell" as used herein refers to an undifferentiated immune system cell, eg, a CD4 T cell, which has not been specialized to recognize a particular pathogen.

The terms "natural killer cells" and "NKs" as used herein refer to a type of lymphocyte that is activated by interferon to participate in the innate host defense against viruses and other intracellular pathogens.

The term "natural killer T cells" (NKTs) as used herein refers to a subset of T cells that share the same characteristics/receptors with both conventional Ts and NKs. Many of these cells identified the nonpleomorphic CD1d molecule, an antigen-presenting molecule that binds autologous and exogenous lipids and glycolipids. The TCRs of NKTs identify glycolipid molecules presented (accompanied) by CD1d molecules. One of the main responses of NKTs is the rapid secretion of cytokines after stimulation, including IL-4, IFN-γ, and IL-10, and thus affect various immune responses and pathogenic effects. NKTs can be homogeneous or heterogeneous populations. In an exemplary implementation, the population may be "non-invariant NKTs," which may include human and mouse bone marrow and human liver T cell populations that are, for example, CD1d-reactive non-invariant NKTs expressing various TCRs. Denatured T cells, which also produce large amounts of IL-4 and IFN-γ. The best known subset of CDld-dependent NKTs express an invariant TCR-α chain. These are called type I or invariant NKTs (iNKTs). These cells are conserved between humans (Vα24iNKTs) and mice (Vα14iNKTs) and are involved in many immune functions.

The term "cytokine" as used herein refers to any of a number of small, secreted proteins that can act by affecting the differentiation of immune cells, usually involving changes in gene expression that make precursor cells distinctly specialized type) to regulate the intensity and duration of the immune response. Cytokines have been named lymphokines, interleukins, and chemokines, respectively, according to their presumed functions, secreting cells, or targets of action. For example, some common interleukins include, but are not limited to, IL-12, IL-18, IL-2, IFN-γ, TNF, IL-4, IL-10, IL-13, IL-21, and TGF -β.

The term "chemokine" as used herein refers to any of a variety of small chemotactic cytokines released at the site of infection that provide a vehicle for the mobilization and activation of lymphocytes. Chemokines attract leukocytes to the site of infection. Chemokines have conserved cysteine residues that allow them to be distinguished into four groups. These groups and representative chemokines are C-C chemokines (RANTES, MCP-1, MIP-1α, and MIP-1β), C-X-C chemokines (IL-8), C chemokines (lymphocyte Chemokine (Lymphotactin), and CXXXC chemokine (Fractalkine).

The term "TH2-type response" as used herein refers to a pattern of cytokine expression that results in the production of certain types of cytokines, interferons, chemokines. Typical TH2 cytokines include, but are not limited to, IL-4, IL-5, IL-6, and IL-10.

The term "TH1-type response" as used herein refers to a pattern of cytokine expression that results in the production of certain types of cytokines, interferons, chemokines. Typical TH1 cytokines include, but are not limited to, IL-2, IFN-γ, GMCSF, and TNF-β.

The term "TH1 bias" as used herein refers to an immunogenic response in which the production of TH1 cytokines and/or chemokines is increased to a greater extent than the production of TH2 cytokines and/or chemokines.

The term "antigenic epitope" as used herein is defined as the portion of an antigenic molecule that contacts the antigen binding site of an antibody or T cell receptor.

The term "vaccine" as used herein refers to a preparation containing antigens derived from whole disease-causing organisms (killed or weakened) or components of such organisms (such as proteins, peptides, or polysaccharides) Constituted to develop immunity against diseases caused by that organism. Vaccine formulations may be natural, synthetic, or derived by recombinant DNA techniques.

The term "immune adjuvant" as used herein refers to a substance used in conjunction with an immunogen that enhances or modifies the immune response to that immunogen. The α-GalCer analogue of the present invention is used as an immune adjuvant to modify or enhance the effect of the vaccine, and stimulate the immune system of the patient administered the vaccine to make it react more violently to the vaccine. In an exemplary implementation, it uses the analogue C34 as an adjuvant.

The term "alum adjuvant" as used herein refers to an aluminum salt having immunoadjuvant activity. Such agents absorb and precipitate protein antigens in solution; the resulting precipitate improves the immunogenicity of the vaccine by assisting in the slow release of the antigen from the vaccine depot formed at the site of vaccination.

The term "anti-tumor immunotherapeutic active agent" as used herein refers to antibodies produced by the vaccine of the present invention, which can inhibit, reduce, or eliminate tumors.

The term "antigen specificity" as used herein refers to the property of a cell population such that the presentation of a specific antigen or fragment of an antigen results in the proliferation of specific cells.

The term "flow cytometry" or "FACS" as used herein means a technique for examining the physical or chemical properties of particles or cells suspended in a fluid flow by means of optical and electronic detection devices.

Amino acid residues in peptides will be abbreviated hereinafter as follows: Phe or F for phenylalanine; Leu or L for leucine; Ile or I for isoleucine; Met or M for methionine; Acid is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic acid is Asp or D; Glutamic acid is Glu or E; Cysteine is Cys or C; tryptophan is Trp or W; arginine is Arg or R; and glycine is Gly or G. For a further description of amino acids, see Proteins: Structure and Molecular Properties, Creighton, T.E., W.H. Freeman & Co., New York 1983.

The compositions disclosed herein may be included in pharmaceutical or nutraceutical compositions in combination with other active agents, carriers, vehicles, excipients, or adjuvants.

These pharmaceutical or nutraceutical compositions preferably comprise at least one pharmaceutically acceptable carrier. In these pharmaceutical compositions, the composition disclosed herein forms its "active compound", also referred to as the "active agent". The term "pharmaceutically acceptable carrier" as used herein includes solvents, dispersion matrices, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical compositions are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral (eg, intravenous, intradermal, subcutaneous), oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following compositions: sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid salt, citrate, or phosphate; and agents to adjust osmolarity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparations can be presented in glass or plastic ampoules, disposable syringes, or multiple dose vials.

As used herein, subjects refer to humans and non-human primates (e.g., gorillas, macaques, marmosets), livestock (e.g., sheep, cows, horses, donkeys, and pigs), pets (e.g., dogs, cats), experimental Laboratory animals (eg, mice, rabbits, rats, guinea pigs, hamsters), captive wild animals (eg, foxes, deer), and any other organism that would benefit from the agents of the invention. There is no limitation as to the type of animal that may benefit from the agents of the invention. A subject, whether it is a human or non-human organism, can be referred to as a patient, individual, animal, host, or recipient.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (which are water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, sterilized water, CremophorEL ^™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be in liquid form to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion base containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols (such as mannitol, sorbitol), or sodium chloride. Prolonged absorption of the injectable compositions is achieved by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion matrix and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying or freeze-drying which yield a powder of the active ingredient plus any other desired ingredient from a previously sterile-filtered solution thereof. .

Oral compositions generally include a blunt diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be mixed with excipients and used in the form of troches, tablets, or capsules (eg, gelatin capsules). Oral compositions can also be prepared using a liquid carrier as a mouthwash. Pharmaceutically compatible binders or adjuvant materials may be included as part of the composition. Troches, pills, capsules, tablets, and the like may contain any of the following ingredients, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as , starch or lactose; disintegrants, such as alginic acid, Primogel, or cornstarch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or citrus flavoring.

For administration by inhalation, the compounds are delivered as an aerosol spray from a pressurized container or dispenser containing a suitable propellant (eg, a gas such as carbon dioxide), or from a nebuliser.

Systemic administration may also be transmucosal or transdermal. For transmucosal or transdermal administration, the appropriate penetrant for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds may be formulated into ointments, ointments, gels, or creams as generally known in the art. The compounds may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

Depending on practice, the active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes that can be targeted to infected cells with monoclonal antibodies directed against cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These suspensions may be prepared according to methods known to those skilled in the art, for example, as described in US Patent No. 4,522,811, which is hereby incorporated by reference.

Oral or parenteral compositions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, combined with The desired pharmaceutical carrier.

The toxicity and therapeutic efficacy of these compounds can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose that is therapeutically effective in 50% of the population). dosage). The dose ratio between toxic and therapeutic efficacy is its therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds with high therapeutic indices are preferred. Although compounds with toxic side effects may be used, care should be taken in designing delivery systems that target these compounds to affected sites in order to minimize potential damage to uninfected cells and thus reduce side effects.

A range of dosage for use in humans can be formulated using the data obtained from cell culture assays and animal experimentation. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (ie, the concentration of test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount (i.e., an effective dose) of the active compound may range from about 0.001 to 100 g/kg body weight, or would be apparent and readily apparent to those skilled in the art without undue experimentation. other ranges. Those skilled in the art will appreciate that certain factors can affect the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the general health or age of the subject , and other existing diseases.

According to another aspect, one skilled in the art can contemplate one or more kits of parts capable of performing at least one of the methods disclosed herein, the kit of parts comprising two or more compositions, the combinations Compositions comprising, alone or in combination, an effective amount of a composition of the invention according to at least one of the methods described above.

Such kits may also include compositions comprising active agents, biological event identifiers, or other compounds that can be identified by those of skill in the art upon reading this disclosure. The kit may also comprise at least one composition comprising an effective amount of a composition or cell line of the invention. The composition of the kit of parts and the cell line can be used to perform at least one method disclosed herein according to procedures identifiable by those skilled in the art.

The term "polypeptide" as used herein refers to any polymer or polymer of amino acid residues. A polypeptide can be composed of two or more polypeptide chains. Polypeptides include proteins, peptides, or oligopeptides. Polypeptides may be linear or branched. A polypeptide may comprise modified amino acid residues, amino acid analogs, or non-naturally occurring amino acid residues, and may be interrupted by non-amino acid residues. Included within this definition are amino acid polymers that are modified, whether naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, methylation, acetylation, phosphorylation, or By manipulation. For example, composite marker composition components.

The term "specific binding" as used herein refers to the interaction between a binding pair (eg, antibody and antigen). In various instances, specific binding can be effected by an affinity constant of about 10-6 moles/liter, about ^10-7 moles/liter, or about ^10-8 moles/liter, or less.

Cancer vaccine of the present invention

One embodiment of the invention is a method of treating cancer by administering to a subject in need thereof an effective amount of an immunogenic composition comprising GloboH or a fragment thereof (e.g., stage-specific embryonic antigen-3 (SSEA-3, also known as Gb5) or SSEA-4) and an adjuvant. Types of cancers of interest include, but are not limited to, breast cancer (including stages 1-4), lung cancer (eg, small cell lung cancer), liver cancer (eg, hepatocellular carcinoma), oral cancer, gastric cancer (including T1-T4), colon cancer , nasopharyngeal carcinoma, skin cancer, kidney cancer, brain tumors (eg, astrocytoma, glioblastoma multiforme, and meningioma), prostate cancer, ovarian cancer, cervical cancer, bladder cancer, And endometrioma, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, and gastrointestinal stromal tumors.

Cancers by site include cancers of the oral cavity and pharynx (lips, tongue, salivary glands, floor of the mouth, gums, and other parts of the mouth, nasopharynx, tonsils, oropharynx, hypopharynx, other mouth/pharynx); Cancers of the system (esophagus; stomach; small intestine; colon and rectum; anus, anal canal, and anorectum; peritoneum, omentum, and mesentery; other digestive systems); cancers of the respiratory system (nasal cavity, middle ear, and sinuses; larynx ; lung and bronchus; pleura; trachea, mediastinum, and other respiratory systems); mesothelioma; bone and joint; and soft tissue (including heart) cancers; skin cancers, including melanoma and other non-epithelial skin cancers; Kaposi's sarcoma and breast cancer; cancers of the female reproductive system (cervix; uterus; uterus, not specified (NOS); ovary; vagina; vulva; and other female reproductive systems); cancers of the male reproductive system (prostate; testicles ; penis; and other male reproductive systems); cancers of the urinary system (bladder; kidneys and renal pelvis; ureters; and other urinary systems); cancers of the eye and orbit; cancers of the brain and nervous system (brain; and other nervous systems); Cancers of the endocrine system (thyroid and other endocrine systems, including the thymus); lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma), multiple myeloma, and leukemias (lymphoblastic leukemia; bone marrow leukemia; monocytic leukemia; and other leukemias).

Other cancers classified by histological type that may be suitable targets for cancer vaccines of the invention include, but are not limited to, neoplastic, malignant; carcinoma, NOS; carcinoma, undifferentiated, NOS; giant and spindle cell carcinomas; small cell carcinomas , NOS; papillary carcinoma, NOS; squamous cell carcinoma, NOS; lymphoepithelial carcinoma, NOS; basal cell carcinoma, NOS; Carcinoma, NOS; Gastrinoma, malignant; Cholangiocarcinoma; Hepatocellular carcinoma, NOS; Combined hepatocellular carcinoma and cholangiocarcinoma; Trabecular adenoma; Adenoid cystic carcinoma; Adenocarcinoma in adenomatous polyp; Carcinoma, familial polyposis of the colon; solid carcinoma, NOS; carcinoid, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma, NOS; chromophobe carcinoma; Cell carcinoma; clear cell adenocarcinoma, NOS; granulosa cell carcinoma; follicular adenocarcinoma, NOS; papillary and follicular adenocarcinoma, NOS; noncystic sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma ; Skin appendage carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Cerumen carcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma, NOS; Papillary cystadenocarcinoma, NOS; Adenocarcinoma, NOS; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma, NOS; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous adenocarcinoma with squamous cell metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor of the ovary, malignant; (Sertoli) cell tumor; Leydig cell tumor, malignant; lipocytoma, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomus sarcoma; malignant Melanoma, NOS; Amelanotic melanoma; Superficial disseminating melanoma; , NOS; Malignant melanoma in giant pigmented nevus; Epithelioid melanoma; Blue nevus, malignant; Sarcoma, NOS; Fibrosarcoma, NOS; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma, NOS; , malignant, NOS; Mullerian mixed tumors; Wilms tumor; hepatoblastoma; carcinosarcoma, NOS; mesenchymal tumor, malignant; Brenner's tumor, malignant; phyllodes tumor , malignant; synovial sarcoma, NOS; mesothelioma, malignant; embryonal malignant; embryonal carcinoma, NOS; teratoma, malignant, NOS; goiter-like ovarian tumor, malignant; choriocarcinoma; Hemangioma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Perivascular cell tumor, malignant; Lymphangiosarcoma, malignant; Osteosarcoma, NOS; Paracortical osteosarcoma; Chondrosarcoma, NOS; Chondroblastoma, malignant ; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's (E wing) sarcoma, malignant; odontogenic tumor, malignant; ameloblastoma, malignant; ameloblastoma, malignant; ameloblastoma, malignant; pineal tumor, malignant; chordoma; glioma, malignant; ventricular Angioma, NOS; Astrocytoma, NOS; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma, NOS; Oligodendroglioma, NOS; oligodendroglioblastoma; primitive neuroectodermal tumor; cerebellar sarcoma, NOS; Neoplasm, Malignant; Neurofibrosarcoma; Schwannomas, Malignant; Granulosa Cell Tumor, Malignant; Malignant Lymphoma, NOS; Hodgkin's Disease, NOS; Hodgkin's Paragranuloma, NOS; Malignant Lymphoma, Small Lymphoma cellular; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular, NOS; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; Mast cell sarcoma; immunoproliferative small bowel disease; leukemia, NOS; lymphocytic leukemia, NOS; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia, NOS; basophilic leukemia; eosinophilic leukemia ; monocytic leukemia, NOS; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma;

The term "treating" as used herein refers to administering or administering a composition comprising one or more active agents to a subject having cancer, symptoms of cancer, or a predisposition to cancer for the purpose of curing, treating , alleviate, lessen, alter, remedy, ameliorate, ameliorate, or affect the cancer, symptoms of cancer, or predisposing factors to cancer. As used herein, an "effective amount" refers to the amount of each active agent, alone or in combination with one or more other active agents, required to produce a therapeutic effect on the subject. Effective amounts will vary depending on the route of administration, the use of excipients, and the co-administration of other active agents, as known to those skilled in the art.

The immunological composition used in the above methods may contain a glycan (ie, a molecule containing a sugar moiety), which is GloboH or a fragment thereof, and an adjuvant. GloboH contains a hexasaccharide epitope (Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glc), and optionally contains a glycan that is not a sugar molecule part. Glycans whose fragments contain the hexasaccharide epitope and, if applicable, fragments of the non-saccharide moiety. These oligosaccharides can be prepared by conventional methods (see, Huang et al., Proc. Natl. Acad. Sci. USA103:15-20 (2006)). It can be linked to non-sugar molecular moieties if desired.

The parent application, U.S. Patent Application Serial No. 12/485,546, is based on the unforeseen discovery that (1) SSEA-3, an immediate precursor of GloboH, is highly expressed in breast cancer stem cells and thus may serve as an appropriate marker for breast cancer therapy target, and (2) α-galactosyl-ceramide (α-GalCer) is a potent adjuvant that promotes the production of anti-GloboH and anti-SSEA-3 antibodies.

US Patent Application Serial No. 12/485,546 proposes an immune composition comprising GloboH or a fragment thereof (eg, SSEA-3) and an adjuvant (eg, α-GalCer). GloboH or fragments thereof can be complexed with keyhole limpet hemocyanin (KLH). When administered to a subject (e.g., a human), such immune compositions elicit an immune response (e.g., antibody production) targeting GloboH or fragments thereof, and are thus effective in treating cancer (e.g., breast, prostate, ovarian cancer, and lung cancer).

U.S. Patent Application Serial No. 12/485,546 relates to a method for preparing an antibody specific for GloboH or a fragment thereof by administering the above-mentioned and isolating an antibody that binds GloboH or a fragment thereof from the mammal.

GloboH or other glycans described in this disclosure are complexed to a protein carrier, such as DT-CRM197. It can then be mixed with an adjuvant (such as C34) and optionally a pharmaceutically acceptable carrier (such as phosphate buffered saline, or bicarbonate solution) to form an immunological composition (such as a vaccine) by conventional methods. See, eg, US Patent Nos. 4,601,903; 4,599,231; 4,599,230; and 4,596,792. The composition can be prepared as injectables, liquid solutions, or emulsions, and the carrier is selected according to the mode and route of administration and according to standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, and pharmaceutical excipients for their use, are described in Remington's Pharmaceutical Sciences. The immune composition preferably contains α-GalCer as an adjuvant. Other examples of adjuvants include, but are not limited to, cholera toxin, E. coli heat-resistant enterotoxin (LT), liposomes, immunostimulatory complexes (ISCOMs), or immunostimulatory sequence oligodeoxynucleotides (ISS -ODN). The composition may also include polymers that may assist in in vivo delivery. See, Audran R. et al. Vaccine 21: 1250-5, 2003; and Denis-Mize et al. Cell Immunol., 225: 12-20, 2003. If necessary, it may also contain minor amounts of auxiliary substances, such as wetting and emulsifying agents, or pH buffering agents, to enhance the ability of the composition to elicit an immune response against the sugar moiety in GloboH or its fragments. The immunological compositions described herein can be administered parenterally (eg, intravenously, subcutaneously, or intramuscularly). Alternatively, other modes of administration may be required, including suppositories and oral formulations. In the case of suppositories, binders and carriers can include, for example, polyalkylene glycols or triglycerides. Oral formulations may include normally used excipients such as, for example, pharmaceutical grades of saccharine, cellulose, magnesium carbonate, and the like. These compositions may be in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders and contain 10-95% of the immunological compositions described herein.

The immune composition is administered in a manner compatible with the dosage formulation, and the amount administered is a therapeutically effective, protective, and immunogenic amount. The amount administered depends on the subject to be treated, including, for example, the ability of the individual's immune system to synthesize antibodies and, if necessary, generate a cell-mediated immune response. The precise amount of active ingredient required to be administered is at the discretion of the practicing physician. However, appropriate dosage ranges can be readily determined by those skilled in the art. Appropriate regimens of initial administration and booster are also variable, but may include an initial administration followed by subsequent administrations. The dose of the vaccine may also depend on the route of administration and vary according to the size of the host.

The immunological composition of the present invention can also be used to generate antibodies in animals for the production of antibodies that are useful both in the treatment and diagnosis of cancer. Methods for producing monoclonal and polyclonal antibodies and fragments thereof in animals (eg, mice, rabbits, goats, sheep, or horses) are known in the art. See, eg, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term "antibody" includes whole immunoglobulin molecules as well as fragments thereof, such as, Fab, F(ab')2, Fv, scFv (single chain antibody), and dAb (domain antibody; Ward, et. al. (1989) Nature, 341, 544).

GloboH-DT-CRM197 and related vaccines

GloboH(1) and fragments 2-10 thereof were synthesized as described herein. For protein complexation, purified GloboH half-ester 12 was co-located with individual carrier proteins, as shown in FIG. 14 .

These GloboH-proteins were characterized by MALDI-TOF analysis to determine the number of GloboH molecules on each carrier protein. The average number of GloboH inclusions is listed in Table 1.

Table 1. MALDI-TOF analysis of GloboH inclusion

^a Peak m/z in MALDI-TOF; ND: not determined; *GH-KLH provided by OptimerInc.

The GH-KLH complex showed the greatest number of GloboH incorporations, mostly due to the larger size and more Lys residues of KLH. The same protocol using the p-nitrophenyl linker was also applied to bamboo mosaic virus, which contains over 100,000 lysine residues on the viral coat. However, the instability of the virus when reacted in sodium phosphate buffer (pH=7.2) at 4°C is a major concern for further development. Furthermore, GH-BaMV16 limits its detection by MALDI-TOF analysis due to its large size.

Synthetic GloboH and truncated fragments (Figure 1) were attached to the reducing end with a pentylamine linker and covalently immobilized on NHS-coated glass slides. Of the eleven oligosaccharides, nine were selected to be imprinted on the microarray. Each microarray slide was spotted with 12 replicates of nine GloboH analogs (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, BB4, BB3, and BB2) at 50 μM each.

To validate the carbohydrates on the microarray, use mouse monoclonal antibodies (VK9 and Mbr1 against GloboH, and anti-SSEA-3) and use the corresponding secondary antibodies (goat anti-mouse IgG and IgM) to test the pool specificity, and these results are shown in Figures 2A-2C. The data suggest that both VK9 and Mbr1 can recognize GloboH as well as the outer tetrasaccharide BB4, but MBr1 can also slightly recognize BB3. Furthermore, anti-SSEA-3 antibody can specifically recognize SSEA-3 antigen (Gb5) without any cross-reactivity. These results indicate that GloboH microarrays can be used to profile the specificity and titer of polyclonal antibodies from immunized mice.

As previously reported, immunization of mice with the fully synthetic GloboH vaccine and co-administered QS-21 resulted in the production of antibodies against human breast cancer cells; however, despite several booster immunizations, these mouse antibodies remained predominantly IgM (Ragupathi G, et al. (1997) Angew Chem Int Ed 36: 125-128).

A population of mice was immunized subcutaneously with 1 μg of the synthetic GloboH-complex with or without the glycolipid adjuvant α-GalCer (C1). GH-KLH, GH-DT, and GH-BV were found to be the most effective immunogens induced by IgM, followed by GH-TT and GH-BSA, as summarized in Figure 3A, and α-GalCer can stimulate immune responses to induce high amount of IgM antibodies. A similar trend was also observed for mouse IgG antibodies (Fig. 3B), whereas the relative IgG content was higher than the IgM content. In brief, despite the lower sugar density of the synthetic glycoconjugate, GH-DT has similar immunogenicity to GH-KLH, and α-GalCer adjuvant was shown to enhance the immune response.

Since α-GalCer has been shown to be an effective adjuvant for GH-DT, other glycolipids with better adjuvant activity than C1 were examined, as shown in FIG. 4 . Groups of mice were immunized with GH-DT and GH-BV with or without glycolipids. Serum was obtained and introduced into glycan microarray analysis. Generally speaking, the mouse anti-GloboHIgG titer increased with the progress of the immunization process, but the IgM content was almost independent of the number of immunizations (Figure 5). In the GH-BV immunized group, there were no significant differences in IgM levels between the glycolipid-vaccine treatment and the vaccine alone treatment. Although these results suggest that GH-BV combined with glycolipids is not an effective immunization regimen, this poor immunogenicity may be caused by the instability of BaMV. However, α-GalCer analogs, especially 7DW8-5, work well with GH-DT to induce immune responses in mice.

Interestingly, mouse polyclonal IgG antibodies produced by GH-DT and various glycolipid adjuvants can not only neutralize GloboH, but also cross-react with Gb5, SSEA-4, and Gb4, and C34 seems to be the most Potent glycolipid adjuvant (Figure 6). In search of novel vaccine compositions that elicit IgG titers much higher than IgM, GloboH _- DT complexes were tested with glycolipids C1 or C34 or commercially available adjuvants AlPO4 (aluminum phosphate) or MF59.

Surprisingly, after the third vaccination, GloboH-DT and glycolipid C34 could almost completely and exclusively induce IgG antibodies (Fig. 7). In summary, the novel glycolipid adjuvant 7DW8-5 combined with the GH-DT complex enhanced both anti-GloboHIgG and IgM antibodies, while the combination of the glycolipid adjuvant C34 with GH-DT induced a much higher IgG antibody titer. It also has different binding affinities for SSEA-3 (Gb5) and SSEA-4 antigens, both of which are specifically expressed on the surface of breast cancer stem cells.

In order to further compare the effects of different glycolipid adjuvants on GloboH vaccine, seven groups of mice were immunized with GH-KLH. These results suggested that mice vaccinated with glycolipids induced higher levels of anti-GloboH antibodies (Figure 8). Although MF59 is a potent adjuvant, it fails to cooperate with GH-KLH to elicit antibodies against GloboH. AlPO ₄ (aluminum phosphate) also showed no significant effect on antibody induction. On the other hand, GH-KLH combined with C34 showed better immunogenicity after the first and second immunizations, but showed no significant difference from C1 after the third immunization. Taken together, these findings suggest the potential of these novel glycolipid derivatives as adjuvants for sugar-based vaccines.

The nature of cellular and humoral immune responses is influenced not only by the combination of antigen and adjuvant, but also by the carrier and the route of immunization. As described by Sesardic and colleagues, DT-CRM197, a nontoxic mutant toxin, induces antigen-specific T cell proliferation and increases splenocyte production of IL-2, IFN-γ, and IL-6, thus Suggested role in pathways driven by Th1 (MiyajiEN et al. (2001) InfectImmun69: 869-874; GodefroyS, et al. (2005) InfectImmun73: 4803-4809; StickingsP, et al. (2008) InfectImmun76: 1766-1773 ). Despite its predominantly Th1 cytokine content, the subgroup of the anti-CRM197 antibody was IgG1 with no detectable IgG2a, suggesting a mixed Th1/Th2 response. These results prompted the assessment of antibody isotype profiles against GloboH vaccines, and this study showed that GH-DT or GH-KLH in combination with glycolipid adjuvants induced predominantly IgG1 antibodies with trace amounts of IgG2a (Fig. 9).

Although glycolipid adjuvants enhanced TH1-biased cytokine secretion when administered intravenously (i.v.) alone, antibody class switching (IgG2a) was not observed. Overall, glycolipids play key roles in enhancing both cellular and humoral immune responses.

GloboH, SSEA-3, and SSEA-4 cancer vaccines

SSEA-3 (Gb5) and SSEA-4 complexed with DT were synthesized and tested. After the third vaccination, the antibody titers of IgM and IgG were compared, and it was found that SSEA-3-DT and SSEA-4-DT could also induce much higher IgG titers than IgM ( FIG. 10 ).

Since GH-DT and C34 can induce antibodies to identify GloboH, Gb5, and SSEA-4, using an array of 24 glycans, in the presence of adjuvants, specificity against SSEA-3-DT and SSEA-4-DT vaccines The tests were carried out, focusing on IgG studies (Figure 11).

As shown in Figure 12, mice immunized with GloboH-DT and C34 adjuvant can induce antibodies that can identify GloboH, SSEA-3 (Gb5), and SSEA-4 with high selectivity, while the vaccine SSEA-3-DT and The adjuvant MF59 can induce a high immune response with low selectivity. On the other hand, SSEA-3-DT combined with adjuvant C34 could only induce antibodies against GloboH, SSEA-3, and SSEA-4.

Interestingly, with or without adjuvant, SSEA-4-DT (sialylated-Gb5) elicited IgG and glycoproteins specifically identifying SSEA-4 and its truncated structure (SSEA-4 with front lactose deletion). IgM antibodies, without being bound by theory, it is hypothesized that sialic acid is highly immunogenic and can induce a highly specific immune response.

Immunization of mice with SSEA-3-DT-C34 induced antibodies reactive to GloboH, SSEA-3, and SSEA-4, suggesting that GloboH-based vaccines could target the expression of GloboH, SSEA-3, and SSEA- 4 tumor cells and breast cancer stem cells.

Immunization of mice with GloboH-DT-C34 induced antibodies reactive to GloboH, SSEA-3, and SSEA-4, suggesting that GloboH-based vaccines could target the expression of GloboH, SSEA-3, and SSEA-4 Tumor cells and breast cancer stem cells.

Immunization of mice with SSEA-4-DT induced SSEA-4-reactive antibodies, suggesting that SSEA-4-DT-based vaccines could target SSEA-4-expressing tumor cells and breast cancer stem cells. Tumor Size Reduction by Tumor Vaccines

To directly assess the efficacy of the synthetic glycoconjugate vaccine, tumor size was measured 3 times per week, as shown in FIG. 13 . In general, tumors were grown for 2 weeks after injection of 4T1, a breast cancer cell line with GloboH. At day 24, all vaccination groups combined with glycolipid adjuvants still showed smaller tumor progression compared to GH-DT alone and PBS control groups. The data suggest that vaccination with GH-DT and a glycolipid adjuvant may partially delay tumor progression in vivo.

Expression of SSEA-3 and SSEA-4 in breast cancer and BCSCs

It has been confirmed that GloboH is expressed in BCSCs, but its frequency is lower than that of non-BCSCs, and in breast cancer and BCSCs, SSEA-3 is expressed at a higher frequency than GloboH (ChangW-W.etal., (2008) ProcNatlAcadSciUSA105(33): 11667-11672, which is incorporated herein by reference in its entirety).

Table 2 summarizes the clinical characteristics of 35 breast cancer patients in whom SSEA-3 or SSEA-4 expression was measured. The median age was 48 (ranging from 31 to 82). It consists of one stage 0, 10 stage I, 19 stage II, and 5 stage III. The majority of tumor samples had pathology of invasive ductal carcinoma (80.0%), of which 51.4% were positive for ER and 65.7% were positive for lymph node metastasis. In Table 2, the range of SSEA-3 or SSEA-4 expression is expressed as the percentage of positive cells in the total cancer cells. Statistical analysis was performed for SSEA-3 or SSEA-4 expression relative to HER-2 or lymph node metastatic status using t-tests. HER-2 expression was determined by immunohistochemistry. There was no significant correlation between the expression of SSEA-3 or SSEA-4 on tumors and various clinicopathological factors, such as stage (SSEA-4: P=0.3498; SSEA-3:, P=0.9311), or HER-2 (SSEA-4: P=0.0142; SSEA-3: , P=0.0128) (Table 2).

Table 2. Clinical characteristics of breast cancer patients

Primitive tumor cells isolated by enzyme digestion from participating patients were stained with antibodies specific for CD45, CD24, and CD44, and CD45 ⁺ cells were first gated to remove leukocytes. To compare the expression of SSEA-3 or SSEA-4 between BCSCs and non ^- BCSCs, CD45- tumor cells were further separated into BCSCs and non-BCSCs according to the expression of their surface markers. BCSCs were identified as CD45 ⁻ /CD24 ⁻ /CD44 ⁺ cells; the rest of the CD45 ⁻ population were considered non-BCSCs.

Using this method, SSEA-3 or SSEA-4 expression in BCSCs and non-BCSCs was assessed in 35 tumor samples. Overall, SSEA-4 was detected in 34/35 (97.1%) of these tumors and SSEA-3 was detected in 27/35 (77.1%) of these tumors (Table 3). The expression of SSEA-3 or SSEA-4 was determined by flow cytometry. BCSCs were defined as CD45 ^- CD24 ^- CD44 ⁺ cells identified, while non-BCSCs were defined as the remaining population of this CD45- cell. Ranges are calculated as percent positive cells out of total cells.

As summarized in Table 3, 27/35 (77.1%) of the samples expressed SSEA-3, and the percentage of positive cells ranged from 1.4% to 66.4%. Non-BCSCs isolated from 25/35 tumors expressed SSEA-3, and the percentage of positive cells ranged from 24.3% to 70.4%. In comparison, BCSCs from 23 (65.7%) of 35 tumors showed positive staining for SSEA-3, with the percentage of positive cells ranging from 5.0% to 58.4%.

In 34/35 (97.1%) samples expressing SSEA-4, the percentage of positive cells ranged from 0.5% to 77.1%. Non-BCSCs isolated from 32/35 tumors expressed SSEA-4, and the percentage of positive cells ranged from 24.0% to 78.1%. In comparison, BCSCs from 31 (88.6%) of 35 tumors showed positive staining for SSEA-4, with the percentage of positive cells ranging from 5.6% to 83.6%.

Table 3. Comparison of SSEA4 and SSEA3 expression in BCSCs and non-BCSCs

Expression of SEA-4 in BCSCs

To compare the expression of SSEA-4 between BCSCs and non-BCSCs, CD45 ⁺ tumor cells were further separated into BCSCs and non-BCSCs according to the expression of their surface markers. BCSCs were identified as CD45 ⁻ /CD24 ⁻ /CD44 ⁺ cells; the rest of the CD45 ⁻ population were considered non-BCSCs. SSEA-4 expression in each of the two gated populations varied by tumor sample, as shown in FIG. 15 . For example, BCSCs from patient BC0264, which accounted for 5.7% of the total isolated tumor cells, were SSEA-4 negative, while 60.3% of non-BCSCs expressed SSEA-4. For patient BC0266, SSEA-4 expression was only detected in 59.4% of non-BCSCs and 55.7% of BCSCs. For patient BC0313, SSEA-4 expression was detected in 32.4% of non-BCSCs and 83.6% of BCSCs. In total, SSEA-4 was detected in 34/35 (97.1%) of the test samples, with the percentage of positive cells ranging from 0.5% to 77.1%. (table 3).

Expression of SSEA-3 and SSEA-4 in normal tissues

Using tissue microarrays, SSEA-4 expression was analyzed by immunohistochemical staining in 20 different organs, as shown in Table 4 (E, epithelium; C, connective tissue).

Table 4. Expression of SSEA4 in normal tissues

SSEA-4 is expressed on epithelial cells of several glandular tissues, such as breast, colon, gastrointestinal tract, kidney, lung, ovary, pancreas, rectum, stomach, testis, thymus, and cervix (Table 4). At the same time, in a manner similar to GloboH and SSEA-3 (ChangW-W.etal., (2008) ProcNatlAcadSciUSA105 (33): 11667-11672), the expression of SSEA-4 is mainly limited to the cytoplasm or apical surface of epithelial cells, and its basic The above is inaccessible to the immune system, as shown in Figure 16.

In contrast, GloboH is expressed on epithelial cells of several glandular tissues, such as breast, gastrointestinal tract, pancreas, prostate, and cervix. The distribution of SSEA3 is similar to GloboH, except that it does not exist in normal breast tissue, but exists in kidney, rectum, testis, and thymus, which are GloboH negative (ChangW-W.etal., (2008) ProcNatlAcadSciUSA105(33) : 11667-11672).

example

The following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in these examples represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

General Methods, Materials, and Apparatus

Material

Commercially available solvents and reagents were used as received without further purification and were purchased from Sigma-Aldrich, Acros, Merck, Echochemical, and Senn Chemical. Monoclonal antibody Mbr1 was purchased from ALEXIS biochemicals, Cy3-complexed anti-mouse IgG (IgG, IgG1, and IgG2a) and IgM antibodies were purchased from Jackson ImmunoResearch. DT-CRM197 protein and tetanus toxoid were purchased from Merck and Adimmune, respectively. Aluminum phosphate gel adjuvant (AlPO ₄ ) was purchased from Brenntag Biosector. Bamboo virus and VK9 monoclonal antibodies were prepared from the laboratories of Dr. Lin and Dr. Yu, respectively. Glycolipid derivatives were synthesized and provided by Dr. Wong's laboratory.

general approach

Molecular sieves (MS, AW-300) for glycosylation were ground and activated prior to use. Reactions were monitored with analytical TLC plates (PLC Silica Gel-60, F254, 2 mm, Merck) and visualized under UV (254 nm) or stained with p-anisaldehyde. Flash column chromatography was performed on silica gel (40-63 μm) or LiChroprep RP18 (40-63 μm). Dialysis membranes (CelluloseEster, _MCCO =10,000) were washed with ddH2O before use.

instrument

Proton nuclear magnetic resonance (1HNMR) spectrum and carbon nuclear magnetic resonance (13CNMR) spectrum were recorded with BrukerAdvance600 (600MHz/150MHz) NMR spectrometer. Chemical shifts for protons are reported in ppm units (delta scale) and are referenced to tetramethylsilane (delta = 0). Chemical shifts for carbon are also reported in parts per million (ppm units, delta scale). Multiplicity was determined using DEPT135 (Enhanced Distortionless Polarization Transfer). Data are expressed as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), integration and coupling coefficient (J) The unit is Hz. High-resolution mass spectra were obtained with BioTOFIII, and MALDI-TOFMS was used with UltraflexIITOF/TOF200.

Example 1: Synthesis of GloboH complexed with different carrier proteins

Using a process-controlled programmable one-pot strategy, GloboH (1; see Figure 11) and its fragments 2-10 were synthesized (Huang C-Y, et al. (2006) ProcNatlAcadSciUSA103: 15-20). The reaction of 1 is carried out with sufficient homobifunctional (homobifunctional) linkers in anhydrous DMF solution at room temperature (WuX, et al. (2004) Org Lett6: 4407-4410; WuX, BundleDR (2005) JOrgChem70 :7381-7388). The reaction can be easily monitored by TLC. Once the free amine disappeared and the larger Rf product appeared, the reaction mixture was evaporated to remove DMF and washed with dichloromethane and water to remove excess linker. Finally, the product was purified by reverse-phase (C18) column chromatography and gradually eluted with water containing 1% acetic acid to 40% methanol in water. The solution was then lyophilized to yield the pale yellow product 12. Finally, for protein complexation, the purified GloboH half-ester 12 (30-40 equivalents) and individual carrier proteins were co-located in phosphate buffer (10 mM, pH 7.2) at room temperature for 24 hours ( Figure 14). Importantly, the protein concentration must be adjusted to ~5 mg/mL to maximize the coupling of lysine residues to the GloboH half-ester. After 24 hours, the glycoconjugate was diluted and dialyzed against deionized water to remove remaining p-nitrophenyl groups. This solution was then lyophilized to a white powder to yield 13, 14, and 15.

These GloboH-protein complexes were characterized by MALDI-TOF analysis to determine the number of GloboH molecules on each carrier protein. The average number of GloboH incorporations are listed in Table 1 shown above.

Glycoplexes 13, 14, and 15 were dissolved in ddH2O to give a final concentration of approximately ₁ pmol/μL. Sinapinic acid was chosen as the matrix and mixed with freshly prepared acetonitrile and deionized water (1:1 v/v) to yield a final matrix concentration of 10 mg/mL with 0.1% TFA. Each sample was detected in linear positive ion mode to obtain m/z spectra. The molecular weight of each glycoconjugate was determined in m/z. Glycoplex 14 showed heterogeneity, indicating an average of 2-4 inclusions. The GH-KLH complex showed the greatest number of GloboH incorporations, mostly due to the larger size and more Lys residues of KLH. The same coupling procedure using a p-nitrophenyl linker was also applied to bamboo mosaic virus, which contains over 100,000 lysine residues on the viral coat. However, the instability of the virus when reacted in sodium phosphate buffer (pH=7.2) at 4°C is a major concern for further development. Furthermore, GH-BaMV16 limits its detection by MALDI-TOF analysis due to its large size. Finally, lyophilized glycoconjugates were stored at -30°C and reconstituted with sterile water before use.

Example 2: Fabrication and Validation of Glycan Microarrays

Synthetic GloboH and truncated fragments (Figure 1) were attached to the reducing end with a pentylamine linker and covalently immobilized on NHS-coated glass slides. Of the eleven oligosaccharides, nine were selected to be imprinted on the microarray. A series of oligosaccharide concentrations (1, 5, 10, 20, 40, 50, 80, 100 [mu]M) were tested to optimize binding affinity and fluorescence intensity. Each microarray slide was spotted with 12 replicates of nine GloboH analogs (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, BB4, BB3, and BB2) at 50 μM each. After reacting in an 80% humidity atmosphere, the slides were stored in a desiccator at room temperature until use.

To validate carbohydrates on microarrays, mouse monoclonal antibodies (VK9 and Mbr1 against GloboH, and anti-SSEA-3) and corresponding secondary antibodies (goat anti-mouse IgG and IgM) were used to test pool specificity , and these results are shown in Figures 2A-2C. The data suggest that both VK9 and Mbr1 can recognize GloboH as well as the outer tetrasaccharide BB4, but MBr1 can also slightly recognize BB3. (GilewskiTelal. (2001) ProcNatlAcadSciUSA98: 3270-3275; HuangC-Y, et al. (2006) ProcNatlAcadSciUSA103: 15-20.) In addition, anti-SSEA-3 antibody can specifically identify SSEA-3 antigen (Gb5) without any crossover reactivity. These results indicate that GloboH microarrays can be used to profile the specificity and titer of polyclonal antibodies from immunized mice.

Example 3: Mouse Immunization

In this experiment, a group of mice were immunized subcutaneously with 1 μg of the synthetic GloboH(GH)-complex in the presence or absence of the glycolipid adjuvant α-GalCer(C1). After 10 days of three immunizations at weekly intervals, mouse sera were collected and subsequently introduced into glycan microarrays to assess antibody levels. It is found that GH-KLH, GH-DT, and GH-BV are the most effective immunogens induced by IgM, followed by GH-TT and GH-BSA, as summarized in Figure 3A, and α-GalCer can stimulate immune responses to induce High levels of IgM antibodies. A similar trend was also observed for mouse IgG antibodies (Fig. 3B), whereas the relative IgG content was higher than the IgM content. In brief, despite the lower sugar density of the synthetic glycoconjugate, GH-DT has similar immunogenicity to GH-KLH, and α-GalCer adjuvant was shown to enhance the immune response.

Since C1 has been shown to be an effective adjuvant for GH-DT, other glycolipids with better adjuvant activity than C1 were examined, as shown in FIG. 4 . (Fujio M, et al. (2006) J Am Chem Soc 128:9022-9023.)

Groups of mice were immunized intramuscularly twice a week with 1.6 μg of GH-DT and GH-BV in the presence or absence of 2 μg of glycolipids. Sera were obtained two weeks after the third vaccination and included in glycan microarray analysis. Generally speaking, the mouse anti-GloboHIgG titer increased with the progress of the immunization process, but the IgM content was almost independent of the number of immunizations (Figure 5). In the GH-BV immunized group, there were no significant differences in IgM levels between the glycolipid-vaccine treatment and the vaccine alone treatment. Although these results suggest that GH-BV combined with glycolipids is not an effective immunization regimen, this poor immunogenicity may be caused by the instability of BaMV. However, α-GalCer analogs, especially 7DW8-5, work well with GH-DT to induce immune responses in mice.

Interestingly, mouse polyclonal IgG antibodies produced by GH-DT and various glycolipid adjuvants can not only neutralize GloboH, but also cross-react with Gb5, SSEA-4, and Gb4, and C34 seems to be the most effective (Figure 6). In search of novel vaccine compositions that elicit IgG titers much higher than IgM, GloboH _- DT complexes were tested with glycolipids C1 or C34 or commercially available adjuvants AlPO4 (aluminum phosphate) or MF59. Surprisingly, GloboH-DT with glycolipid C34 could almost induce IgG antibodies after the third vaccination (Fig. 7). In summary, the novel glycolipid adjuvant 7DW8-5 combined with the GH-DT complex enhanced both anti-GloboHIgG and IgM antibodies, while the combination of the glycolipid adjuvant C34 with GH-DT induced a much higher IgG antibody titers. It also has different binding affinities for Gb5 and SSEA-4 antigens, both of which are specifically expressed on the surface of breast cancer stem cells.

In order to further compare the effects of different glycolipid adjuvants on GloboH vaccine, seven groups of mice were immunized with GH-KLH. These results suggested that mice vaccinated with glycolipids induced higher levels of anti-GloboH antibodies (Figure 8). Although MF59 is a potent adjuvant, it fails to cooperate with GH-KLH to elicit antibodies against GloboH. AlPO ₄ (aluminum phosphate) also showed no significant effect on antibody induction. On the other hand, GH-KLH combined with C34 showed better immunogenicity after the first and second immunizations, but showed no significant difference from C1 after the third immunization.

DT-CRM197, a nontoxic mutant toxin, induces antigen-specific T cell proliferation and increases splenocyte production of IL-2, IFN-γ, and IL-6, thus suggesting its role in a Th1-driven pathway (MiyajiENetal. (2001) InfectImmun69: 869-874; GodefroyS, et al. (2005) InfectImmun73: 4803-4809; StickingsP, et al. (2008) InfectImmun76: 1766-1773). Despite its predominantly Th1 cytokine content, the subgroup of the anti-CRM197 antibody was IgG1 with no detectable IgG2a, suggesting a mixed Th1/Th2 response. These results prompted the assessment of antibody isotype profiles against GloboH vaccines, and this study showed that GH-DT or GH-KLH in combination with glycolipid adjuvants induced predominantly IgG1 antibodies with trace amounts of IgG2a (Fig. 9).

Although glycolipid adjuvants enhanced Th1-biased cytokine secretion when administered intravenously (i.v.) alone, antibody class switching (IgG2a) was not observed. Overall, glycolipids play key roles in enhancing both cellular and humoral immune responses.

Gb5 and SSEA-4 complexed with DT were also synthesized by the same strategy. After the third vaccination, the antibody titers of IgM and IgG were compared, and it was found that Gb5-DT and SSEA-4-DT could also induce much higher IgG titers than IgM ( FIG. 10 ).

Example 4: Specific studies of antibodies elicited by different vaccine compositions

Since GH-DT and C34 can induce antibodies to identify GloboH, Gb5 (SSEA-3), and SSEA-4, using an array of 24 glycans, in the presence of adjuvants, against SSEA-3-DT and SSEA-4-DT The specificity of the vaccine was examined, focusing on IgG studies (Fig. 11).

As shown in Figure 12, mice immunized with GloboH-DT and C34 adjuvant can induce antibodies that can identify GloboH, SSEA-3 (Gb5), and SSEA-4 with high selectivity, while the vaccine SSEA-3-DT and The adjuvant MF59 can induce a high immune response with low selectivity. On the other hand, SSEA-3-DT combined with adjuvant C34 could only induce antibodies against GloboH, SSEA-3, and SSEA-4.

Interestingly, SSEA-4-DT elicited IgG and IgM antibodies specifically recognizing SSEA-4 and its truncated structure (SSEA-4 with a front lactose deletion) with or without adjuvant. However, the source of this selectivity is not clear.

To directly assess the efficacy of the synthetic glycoconjugate vaccine, tumor size was measured 3 times per week, as shown in FIG. 13 . In general, tumors were grown for 2 weeks after injection of 4T1, a breast cancer cell line with GloboH. At day 24, all vaccination groups combined with glycolipid adjuvants still showed smaller tumor progression compared to GH-DT alone and PBS control groups. This preliminary data suggests that vaccination with GH-DT and a glycolipid adjuvant may partially delay tumor progression in vivo.

Example 5: Preparation of GloboH half esters

GloboH half esters were prepared as follows:

GloboH half esters (12)

GloboH amine 1 (5 mg, 4.54 μmol) was dissolved in dry DMF solution. Then p-nitrophenyl ether linker (8.8 mg, 22.7 μmol) was added and stirred at room temperature for 1-3 hours. The reaction was monitored by TLC (1% AcOH in methanol) and Ninhydrintest. Disappearance of the free amine and appearance of a larger Rf product indicated completion of the reaction. The reaction mixture was evaporated under reduced pressure without heating to remove DMF, then extracted twice with _CH2Cl2 and water containing ₁ % acetic acid. The aqueous solution was concentrated and purified by reverse phase (C18) column chromatography, and gradually eluted with H _{2 O containing 1% acetic acid to MeOH:H 2 O} =4:6. The solution was then lyophilized to a pale yellow solid product 12 (5.4 mg, 88% yield) 1HNMR (600MHz, D ₂ O) δ8.25 (d, 2H, J=9.0Hz), 7.28 (d, 2H, J=9.0Hz), 5.12(d, 1H, J=3.9Hz), 4.79(d, 1H, J=3.7Hz), 4.51(d, 1H, J=7.7Hz), 4.44(d, 1H, J=7.7Hz), 4.44(d, 1H, J= 7.7Hz), 4.39(d, 1H, J=7.7Hz), 4.31-4.28(t, 2H, J=7.7Hz), 4.15-4.11(m, 2H), 3.99(d, 1H, J=2.0Hz) , 3.92(d, 1H, J=2.8Hz), 3.89-3.44(m, 33H), 3.16(t, 1H, J=8.6Hz), 3.10(t, 2H, J=6.7Hz), 2.62(t, 2H, J=6.9Hz), 2.20(t, 2H, J=6.6Hz), 1.93(s, 3H), 1.62-1.49(m, 4H), 1.54-1.48(m, 2H), 1.45-1.40(m, 2H), 1.30-1.24 (m, 2H), 1.11 (d, 3H, J=6.5Hz) 13CNMR (150MHz, D ₂ O) δ178.0, 176.1, 176.0, 156.9, 147.1, 127.3, 124.5, 105.7, 105.0 , 103.7, 103.6, 102.2, 101.0, 80.5, 80.0, 78.9, 78.0, 77.8, 77.1, 76.7, 76.4, 76.3, 76.2, 75.2, 74.6, 73.8, 73.5, 72.5, 72.1, 71.8, 71.2, 70.9, 70.8, 7 , 69.7, 69.5, 68.5, 62.6, 62.6, 62.0, 62.0, 61.7, 53.3, 40.8, 37.1, 35.0, 30.0, 29.7, 26.4, 25.0, 24.1, 23.9, 17.0 HRMS: C ₅₅ H ₈₇ N ₃ O ₃₅ Na[ M+Na]+calculated: 1372.5018; found: 1372.5016.

Example 6: General procedure for producing glycoconjugates

Glycoplexes were prepared as follows:

Dissolve BSA, DT-CRM197, and tetanus toxoid (Adimmune, Taiwan) in 100 mM phosphate buffer (pH 7.2) (~5 mg/ml), and add 30 to 40 equivalents of GloboH half Ester 35. The mixture was stirred gently at room temperature for 24 hours. The mixture was then diluted with deionized water and dialyzed against 5 changes of deionized water. The solution was then lyophilized to a white powder. The obtained GloboH-protein complex was qualitatively analyzed by MALDI-TOF to determine the sugar incorporation rate. Produced 13, 14, and 15. 41 (GH-BSA), MALDI-TOF found 76029, 42 (GH-DT-CRM197) found 62138, 43 (GH-TT) found 162902, 44 (GH-BaMV) not determined .

Example 7:

Glycoplexes 41, 42, 43 and original carrier protein were reconstituted with ddH2O ( _-1 μg/μL). Matrix sinapinic acid was freshly prepared in acetonitrile and deionized water 1:1 to yield a final matrix concentration of 10 mg/mL including 0.1% TFA. Gently load and mix the matrix solution and glycoconjugate, then air dry the test plate. Calibration with bovine serum albumin is required prior to measurement. Detect individual glycoplexes and native carrier protein samples in linear positive ion mode. The average number of sugar incorporations on the carrier protein can be calculated using the average molecular weight.

Example 8: Preparation of glycan microarrays

With a machine needle (SMP3, TeleChem International Inc., USA), 0.7 nL of various concentrations of aminoglycans in imprint buffer (300 mM phosphate buffer containing 0.005% Tween-20, pH 8.5), from 96 wells were deposited onto NHS-coated glass slides to print microarrays (BioDot, Cartesian Technologies, USA). Each microarray slide was spotted with 12 replicates of nine GloboH analogs (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, BB4, BB3, and BB2) at 50 μM each. The printed slides were reacted in an 80% humidity atmosphere for one hour, followed by overnight drying. The slides were stored in a desiccator at room temperature until use.

Example 9: Serological Analysis (Glycan Microarray)

As a primary screen, mouse sera were diluted 1 :60 with 0.05% Tween20 in 3% BSA/PBS buffer (pH 7.4). The glycan microarray was blocked with 50 mM ethanolamine for 1 h, and washed twice with ddH ₂ O and PBS buffer before use. These serum dilutions were then introduced into the GloboH microarray and co-located for 1 h at room temperature. The microarray slides were further washed three times with PBST (0.05% Tween-20 in PBS buffer) and PBS buffer, respectively. Next, Cy3-affiniPure goat anti-mouse IgG (H+L), IgG1, IgG2a, or anti-mouse IgM were added to the microarray slides and then sealed for co-location at room temperature for 1 hour. Finally, the slides were washed three times with PBST, PBS, and ddH ₂ O sequentially. The microarray slides were dried, and then scanned at 532 nm with a microarray fluorescent chip reader (Genepix4000B). Data were analyzed with the software GenePixPro6.0 (AxonInstruments, Union City, CA, USA). For accurate measurement, adjust the photomultiplier tube gain value (PMTGain) to 400 to avoid fluorescence saturation. Local background values were subtracted from the signal for each glycan spot. Points with obvious defects or no detectable signal were omitted. The final fluorescence intensity was defined as the mean value of "F532nm-B532nm median" of repeated experimental points.

Example 10: Serological Analysis (ELISA)

0.2 μg Globo-H ceramide in 100 μl sodium bicarbonate buffer (pH 10) was coated in 96-well plates (NUNC) overnight at 4°C. Wash with PBS and block with 3% fetal bovine serum albumin for 30 minutes at room temperature. Serial dilutions of mouse serum were added to each well and allowed to stand at room temperature for 1 h, followed by washing with DPBST (Dulbecco's phosphate-buffered saline, 0.05% Tween20). Goat anti-mouse IgG-AP (1:200, SouthernBiotech., USA) was added and allowed to stand at room temperature for 45 minutes. Assay plates were washed five times with PBST, followed by co-incubation with alkaline phosphatase substrate p-nitrophenylphosphate (Sigma) for 8 minutes at 37°C. After incubation, 3M NaOH solution was added to stop the reaction and the assay plate was read visually at 405 nm on an ELISA reader (SpectraMax, Molecular Devices). Titer was defined as the highest dilution yielding an optical density greater than 0.1.

Example 11: Dosing and Immunization

(1) For three mouse groups (6-week-old female C57BL/6 mice, BioLASCO, Taiwan), GH-KLH (OptimerInc .), GH-BSA, GH-TT, GH-CRM197, and GH-BaMV were administered subcutaneously to the abdominal region three times at weekly intervals. Each vaccination contained 1 μg of GloboH with or without 2 μg of glycolipid adjuvant. Control mice were injected with phosphate-buffered saline (PBS) only. The mice were bled before the first immunization (before immunization) and 10 days after the third immunization. (2) For three mouse groups (8-week-old female Balb/c mice, BioLASCO, Taiwan), with or without C1, C23, or 7DW8-5, GH-BaMV or GH-CRM197 , three times by intramuscular immunization at two-week intervals. Each vaccination contained 1.6 μg of GloboH with or without 2 μg of adjuvant. Mice in the control group were injected with phosphate-buffered saline (PBS). Blood was collected from the mice before immunization and 2 weeks after each immunization. (3) For three groups of mice (8-week-old female Balb/c mice, BioLASCO, Taiwan), with or without adjuvant C1, C17, 7DW8-5, C30, AlPO ₄ , MF59 (1:1 mixture ), the immunization was carried out as described in (2). All sera were obtained by centrifugation at 4000g for 10 minutes. Serological responses were analyzed by glycan microarray or compared with conventional ELISA analysis.

Example 12: Xenograft Model

(1) For five groups of immunized female Balb/c mice (respectively PBS, alone or in combination with C1, C23, and GH-CRM197 of 7DW8-5), 8 weeks after the final immunization, 2x10 ^Five metastatic mouse mammary tumor cell lines 4T1 (in sterile PBS). (2) For seven groups of immunized female Balb/c mice (GH-KLH alone or in combination with C1, C17, 8-5, C30, AlPO ₄ , and MF59), 6 weeks after the final immunization , by subcutaneous injection of ^2x105 metastatic mouse mammary tumor cell line 4T1 (in sterile PBS). Mouse anti-GloboH sera were monitored before and after tumor xenografts. The mouse tumor size was measured three times a week with a Vernier caliper and defined as (length x height x width)/2 (mm ³ ).

Example 13: Isolation of primary tumor cells from human breast cancer samples

Human breast cancer samples were obtained from patients who had undergone primary surgery at the Tri-Service General Hospital (Taipei, Taiwan). Samples were fully coded to protect patient privacy, and were used under protocols approved by the Academia Sinica Medical Research Ethics Committee (Taipei, Taiwan). The tumor samples ^were sliced into 1mm square fragments, and then placed in RPMI1640 medium containing collagenase (1,000U/ml), hyaluronidase (300U/ml), and DNaseI (100μg/ml), at 37°C It was placed together for 2 hours, and it was subjected to enzymatic digestion. Primitive breast cancer cells were harvested by filtration through a 100-μm cell strainer (BD Biosciences), and resuspended in RPMI1640 medium supplemented with 5% FBS.

Example 14: Flow Cytometry Analysis

Primitive breast cancer cells were prepared as ¹ x 105 cells in PBS containing ₂ % FBS and 0.1% NaN3. Cells were labeled with an antibody mixture of anti-CD24-PE, anti-CD44-APC, and anti-CD45-PerCP-Cy5.5 (1 μl each). GloboH expression was detected by staining with monoclonal anti-GloboH antibody complexed with Alexa488 (VK-9). Analysis was performed on a FACSCanto flow cytometer (Becton Dickinson). BCSCs were defined as CD45- ^/ CD24- ^/ CD44 ⁺ cells, while non ^- BCSCs were defined as the rest of the population of CD45- cells. GloboH expression was further analyzed in the gated region.

Example 15: Cell Sorting

Human breast cancer-derived cells transplanted into mice were stained with an antibody mixture of anti-CD24-PE, anti-CD44-APC, and anti-H2K ^d -FITC (BD Biosciences). Fluorescence-activated cell sorting of antibody-labeled cells was performed on a FACSAria cell sorter (Becton Dickinson). H2K ^d- /CD24- ^/ CD44 ⁺ cells were sorted as BCSCs; while other H2K ^d- populations were sorted as non-BCSCs. Typical purities for BCSCs and non-BCSCs are >85% and >90%, respectively.

Example 16: Immunohistochemistry

For expression of SSEA-4 on normal tissues, tissue microarray slides (Biomax) containing 20 different organs, each derived from five individuals, were used. Slides were dried overnight at 56°C and rehydrated according to standard histopathology protocols followed by antigen retrieval with AR-10 solution pH 9.0 (BioGenex Laboratories). Expression of SSEA-4 was determined using an anti-SSEA-4 antibody (eBioscience). Staining of SSEA-4 was detected using anti-rat IgM as a secondary antibody and developed with DAB substrate. Slides were contrast stained with hematoxylin. Primitive breast cancer BC0145 and tumor xenografts from NOD/SCID mice were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Paraffin sections were cut at a thickness of 2 μM and placed on SuperFrostPlus microscope slides (Menzel- ), and then dried overnight at 55°C. Sections were deparaffinized in xylene, rehydrated according to standard histopathology protocols, and stained with hematoxylin and eosin (H&E). The slides were first placed in 10 mmol/L citrate buffer (pH 6.0) and microwaved for 15 minutes before immunostaining. The slides were then incubated overnight with anti-ER or anti-PR antibodies. Immunodetection was performed with SuperSensitive Polymer-HRPIHC Detection System (BioGenex).

All publications and patent documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail for the purpose of clarity by way of illustration and example, it will be readily apparent to those skilled in the art that changes and modifications may be made in accordance with the teachings of the invention without departing from the appended the spirit or scope of the claims.

In order to comply with 37 C.F.R. §1.72(b), the abstract of the invention is provided so that readers can quickly ascertain the nature and gist of the technical disclosure of the invention. It is presented with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

Claims (18)

1. An immunogenic composition comprising: (a) a glycan complex comprising a carrier protein and a glycan comprising GloboH, an immunogenic fragment thereof or stage-specific embryonic antigen-4, wherein the glycan is complexed to the carrier protein via a linker ;and (b) an adjuvant comprising an analog of α-galactosyl-ceramide which is C34, which has the structure: 2. The immunogenic composition according to claim 1, wherein the carrier protein is diphtheria toxoid cross-reactive material 197, diphtheria toxoid, tetanus toxoid or bovine serum albumin. 3. The immunogenic composition according to claim 1, wherein the linker is a p-nitrophenyl linker. 4. The immunogenic composition according to claim 3, wherein the carrier protein is diphtheria toxin cross-reactive material-197. 5. The immunogenic composition according to claim 1, wherein the glycan is GloboH, stage-specific embryonic antigen-4, Bb4, Gb4 or stage-specific embryonic antigen-3. 6. The immunogenic composition according to claim 5, wherein the glycan is GloboH. 7. The immunogenic composition according to claim 5, wherein the glycan is stage specific embryonic antigen-3. 8. The immunogenic composition according to claim 5, wherein the glycan is stage specific embryonic antigen-4. 9. The immunogenic composition according to claim 6, wherein the carrier protein is diphtheria toxin cross-reactive material-197. 10. The immunogenic composition according to claim 7, wherein the carrier protein is diphtheria toxin cross-reactive material-197. 11. The immunogenic composition according to claim 8, wherein the carrier protein is diphtheria toxin cross-reactive material-197. 12. The immunogenic composition according to claim 6, further comprising a pharmaceutically acceptable excipient. 13. The use of the immunogenic composition according to claim 1 in the preparation of a medicament for treating or inhibiting the growth of breast tumors, wherein administration of said composition induces an immune response to produce a lower antibody than that of the IgM isotype High relative amount of IgG isotype antibodies. 14. Use according to claim 13, wherein the glycan is GloboH, stage-specific embryonic antigen-3 or stage-specific embryonic antigen-4. 15. Use according to claim 14, the carrier protein being a diphtheria toxin cross-reactive material 197. 16. Use according to claim 15, wherein the glycan is GloboH. 17. Use according to claim 15, wherein the glycan is stage-specific embryonic antigen-3. 18. Use according to claim 15, wherein the glycan is stage-specific embryonic antigen-4.