TW201100098A - Globo H and related anti-cancer vaccines with novel glycolipid adjuvants - Google Patents

Abstract

Immunogenic compositions, cancer vaccines and methods for treating cancer are provided. Compositions comprising: (a) a glycan such as Globo H or an immunogenic fragment thereof, wherein the glycan is conjugated with a carrier protein by a linker such as para-nitrophenyl; and (b) an adjuvant comprising glycolipid capable of binding CD1d on a dendritic cell, such as an α -galactosyl-ceramide derivative, wherein the immunogenic composition induces an immune response that induces a higher relative level of IgG isotype antibodies as compared to IgM isotype antibodies, are provided. Immunogenic compositions comprising the carrier protein diphtheria toxin cross-reacting material 197 (DT-CRM197) and the adjuvant C34 are provided. Antibodies generated by immunogenic compositions disclosed herein further neutralize at least one of the antigens Globo H, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4). Therapeutics against breast cancer stem cells comprising immunogenic compositions comprising Globo H, SSEA-3 or SSEA-4 conjugated with DT-CRM197.

Description

201100098 VI. INSTRUCTIONS: CROSS-REFERENCE TO RELATED APPLICATIONS RELATED APPLICATIONS RELATED APPLICATIONS RELATED APPLICATIONS RELATED APPLICATIONS RELATED APPLICATIONS Serial No. 12/485,546 (Inventor Name: Used to Evoke Specific to G1〇b〇H and SSEA-3 Composition of sexual immune response and its use in cancer treatment (c〇mp〇siti〇ns f〇r inducing immune responses specific to Globo H and SSEA-3 and uses thereof in cancer treatment), June 16, 2009 Application), the application is continued, and the case claims the priority of US Provisional Patent Application No. 61/〇61,968 (Application for the next day of June 2008). The full text of these patent applications is incorporated herein by reference. TECHNICAL FIELD OF THE INVENTION The present invention relates to the field of cancer vaccines. Specifically, the present application reads 帛 on the basis of a saccharide, which contains a B cell epitope G1〇b〇H conjugated to a nucleus carrier (10). More specific, it is related to Nalu _ (such as C34 fine-sentence; cancer 'ο Η-DT vaccine. [Previous technique] In order to design a sharp reduction in the disease, it is expected that there will be glycosylation in cancer abnormalities. The effect is usually different from that of normal J cells (Μ_ E^^W_8:2518-2524). Abnormal loss of glycosyl 1 j or excessive silk, extinction, existence, and composition ^Although the classification of the new works, the difference in the structure of the predecessor has been supported by the subsequent evidence of Xu Nianzhi. (_(10) 201100098

Acta 208.149-171, Gabius HJ (2000) Naturwissenschaften 87:108-121). Recently, various tumor-associated carbohydrate antigens (Shriver Z, α/· (2004) iVa/ and ev £) have been identified by monoclonal antibody and mass spectrometry 3: 863-873;

Pacino G’eia/. (1991) 63:390-398). At present, the characteristics of many tumor-associated antigens expressed on cancer cells in the form of glycolipids or glycoproteins have been described and their correlation with specific types of cancer has been known (Bertozzi CR, Dube DH (2005) Nat Rev Drug Discovery O 4:477-488). Despite the limited knowledge of the role of surface saccharides in malignant cells, it is known that anti-systems that are passively administered or induced by vaccines against these antigens are associated with improved prognosis. Among the reported tumor-associated glycans, the glycolipid antigen G1〇b〇Η (Fucal-2 Galpl-3 GalNAcpi-3 Galal~^4 Gaipi-4 Glc) was first introduced in 1984 by Hakomori et al. -7 cells were isolated and confirmed (Bremer EG, β α/. (1984) J 5/〇/ C/zem 259: 14773-14777). Further studies with anti-Globo H monoclonal antibodies have shown that 'GloboΗ is also present in many other cancers, including prostate cancer, gastric cancer, pancreatic cancer, lung cancer, ovarian cancer, and colon cancer, and it is not easily The luminal surface of the normal secretory tissue that enters has only a small amount of expression (Ragupathi G, et al. (1997) Angew Chem Int Ed 36: 125-128). In addition, serum of breast cancer patients has also been confirmed to contain high levels of anti-Globo Η antibody (Gilewski T e/α/. (2001) Proc AM Jed Fine USA 98:3270-3275; Huang CY, et al. (2006) Proc Natl Acad Sci USA 103:15-20; Wang CC, et al. (2008) Proc Natl Acad Sci USA 105(33): 11661-11666) 'The disease with Globo H-positive tumor compared to Globo Η - Negative tumor patients have a shorter survival period (chang, YJ, et al 104(25): 10299-10304). These hairs 201100098 became the eye-catching swollen age of various sputum shots of recorded cancer antigens. The purpose of this research has been to study the anti-cancer vaccine against GlGbGH, which can be used as an anti-cancer vaccine against GlGbGH. There is still a need for new vaccines that can induce high levels of immune response to GloboH. A stem cell line is defined as a cell population that has self-renewal and differentiation into forest-type cells and tissues (Reya τ et al" (2〇〇ι) 4Μ:1〇5-111). Due to both malignant and normal tissues They all contain heterogeneous cell populations. Therefore, cancer cells may play a key role in tumor growth and maintenance of tumor heterogeneity. Cancer stem cells have been selected from various non-solid tumors, such as ', brain tumor, breast cancer colon cancer, And prostate cancer. Breast cancer stem cells (BCSCs) were first confirmed by Al-Hajj et al. based on their ability to produce phenotypic diverse tumors when xenografted into 々n〇d/scid mice. CD44+^# t ( Al-Hajj M, et al., (2003) Proc Natl Acad Sci (4) 1 983-3988) m!, most of the early spread of cancer cells do not have CD24'CD44l type (Balic M et al., (2006) C7/« C 12:5615-5621), suggesting that BCSCs have metastatic ability. Based on the ability of BCSCs to grow, differentiate, and metastasize, and their resistance to radiation exposure, 'BCSCs became The main standard of breast cancer treatment (Tang c over al 〇〇FASEB丄2\· -9). In breast cancer, Globo sputum was observed in more than 60% of ductal carcinoma, breast cancer, and tubular cancer, but G1〇b〇Η was not observed in non-epithelial breast cancer tumors ( Manam-Constantini R et ai” (1984), JJ Ρί^/. 115:47-56). GloboH does not manifest in normal tissues except for the weak expression of epithelial cells at the apex of the luminal edge, which appears to be inaccessible to the immune system 201100098 (M; Zhang S. et al., 1997 ) / 73:42-49).

GloboH is also expressed in breast cancer stem cells (BCSCs). Flow cytometry showed a 25/41 breast cancer sample (61.0%) showing Globo Η. There are 25/25 non-BCSCs and 8/40 (20%) BCSCs showing Globo Η. 31/40 (77.5%) of the tumors showed stage-specific embryonic antigen-3 (SSEA-3) (Globo quinone pentose precursor). There are 29/31 non-BCSCs and 25/40 (62.5%) BCSCs ^ ^ SSEA-3 (Chang WW. et al., (2008) Proc Natl Acad O ^/(/5^ 105(33):11667- 11672).

Danishefsky and Livingston have previously reported Globo H-KLH vaccine against multiple cancers (Gilewski T e/α/· (2001) /Voc iVai/ Jcac/ USA 98:3270-3275; Ragupathi G, et al. (1997) Angew Chem Int Ed 36: 125-128; Kudryashov V, et al. (1998) Glycoconj J. 15: 243-249; Slovin SF et al (1997) Proc Natl Acad Sci USA 96: 5710-5715) and heptavalent vaccine ( Preparation of GM2, Globo®, Lewis Y, Τη, STn, TF, and Tn-MUCl conjugated individually with KLH; Sabbatini PJ a/(2007) C/, as 13:4170-4177). However, patients who received the seven-valent vaccine immunization only induced an antibody response against five of the seven antigens (except for the GM2 and Lewis Y antibodies). Unlike widely expressed antigens (such as GM2), Globo tethers are particularly expressed on tumor cells and exhibit minimal traces in normal secretory tissues, making them ideal for epidemiological research & development. In the study, after decomposing the Gi〇b〇η ligand by ozonation, reductive amination with KLH carrier protein produced about 150 saccharide units per protein (RagupathiG, deduction a/. (1997: M-wC) /zew/ I wish 36:125-128). By further preparation using the MMCCH linker, the ratio of sugar-conjugated linkages is increased to approximately 720:1 (Wang SK, β政(2〇〇8) iW Μ·· C/like 105:3690_3695). However, it is difficult to accurately distinguish the characteristics of the 7201100098 sugar joint vehicle. In addition, in the two cancer patients with prostate and metastatic breast cancer, the synthetic vaccine combined with the immunoadjuvant QS_21 showed mainly induced IgM, while the IgG antibody was low. In the first phase of the clinical trial, the vaccine also showed trace toxicity' and produced a transient local skin reaction at the vaccination site (Gilewski T e/ < 3/· (2001) gamma/like j/like 98:3270 -3275; Ragupathi G, et al (1997) Angew Chem Int Ed 36: 125-128; Slovin SF et al (1997) Proc Natl Acad Sci USA 96: 5710-5715). Mild flu-like symptoms have also been observed in some patients, which may be related to the side effects of QS-21. It has been reported that in EUSA analysis 1, five kinds of scorpion glands and breast cancer-associated biliary antigens (Gi〇b〇-H, GM2, STn, TF, and conjugated to the mammalian-modified carrier protein KLH are conjugated. The pentavalent vaccine of Τη) can produce anti-Gk)b blood/month with an IgG titer higher than IgM (Zhu J. ei α/· (2009) 1(26): 9298-9303). Therefore, there is a need to find alternative vectors and adjuvants to enhance the antibody response to G1〇b〇 ’, especially with higher IgG titers, and to be only highly eccentric. Fox [invention content]

The present invention is a kind of sugar-based vaccine, in which a phenyl-linked linker and a sputum-secreted Y ί 3 antigen (four) position are chemically coupled to each other> IgG cancer model induces IgG, IgG1 And IgM antibodies, and extractability, which showed that it can delay tumorigenesis in xenograft studies; the analysis of glycan arrays of stems and sputum is not obvious, and 5 scorpion corpses can not only recognize G1〇b〇 H, also 201100098 == sialylated Gb5) _, (4) in cancer cells and cancer stems The present invention relates to an immunogenic composition comprising: ( ί ίίΐ consisting of ?lGbG H or an immunological fragment thereof, wherein The glycan is linked to the carrier protein by a linker; and (b) an adjuvant comprising a glycolipid of a CDld molecule on the dendritic cell, the immunogenicity of the towel:

An immune response that induces a relative amount of IgG isotype antibody to her IgM car. a few-

In some aspects, the adjuvant is a synthetic analog of alpha-galactosyl-neuroquinone U-GalCer. In part of the concrete C34, where C34 contains the structure:

In XT, in a partial state, the immune response is preferably directed to the production of an IgG isotype antibody. In some aspects, the immunogenic composition comprises at least one adjuvant that induces a humoral or cellular immune response. x In a partial aspect, an antibody produced by the immune response neutralizes an antigen expressed on a ^ cell or a Hep cell. In some specific examples, the antibody produced by the immunoreversion can neutralize at least one of the antigen Gb4, the stage-specific embryo antigen_3 (SSEA-3), and the stage-specific embryo antigen_4 (SSEA_4). . In some specific examples, the towel and antigen Gb4, stage-specific embryo 9 201100098 fetal antigen-3 (SSEA-3), and stage-specific embryonic antigen 4 (ssEA-4)

The antibody of at least one of the antibodies comprises an IgG isotype antibody having a higher phase content than the IgM isotype antibody. 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 used to treat cancer 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, intestinal cancer, and bladder cancer. In some aspects, the cancerous tissue exhibits a Gl〇b〇η antigen on the cell surface. In the partial sample towel, the GloboH anti-, Wei Wei _ epithelial cells. In some embodiments, the cancer vaccine produces an antibody capable of neutralizing at least one of the antigens Globo(R), Gb4, stage-specific embryonic antigen_3 (SSEA_3), and stage-specific embryonic antigen-4 (SSEA_4). In some aspects, the sputum antigen system is expressed on breast cancer stem cells. The present invention relates to a method for treating a stimuli, which comprises administering an immunological composition to a subject in need thereof, which composition is substantially composed of G1°bGH or an immunogen thereof. Fragment composition, which = the "linking to the carrier protein via a linker; and adjuvant, ligating = binding to the glycolipid of the CDld molecule on the dendritic cells; and (7) inducing the phase immune response to induce phase Higher relative summer IgG isotype antibody compared to 1 gM isotype antibody. In some specific examples, the linker is a pair of nifedipine, a laryngone toxin cross-reactive material 197 (DT_CRM197), a synthetic analog of lactoalgglutination (a_Gaicer), in a specific example, The adjuvant is C34. 10 Ο

G 201100098 Cancer goes 'The immunogenic composition still contains one or more treatments that inhibit tumor growth and reduce tumor size. The specific embryonic antigen can be used in the vaccination of the vaccination vaccine. The specific embryonic antigen can be: -, -H, Gb4, (SSEA-4) to +, - cat production) A and Yangnu At least one of the specific embryonic antigens _4 -4 (SSEA-4) is "a specific part of the embryonic antigen-like part of the G-stage buttf, the thief is composed of GiGbGH or its immunogenic knot; and an adjuvant, It comprises/comprising a content of conjugated to the carrier protein by a linker of =1 (DT-CRM197), and the adjuvant is in the amount of the cancer (2) mouth = disease, wherein the effective cancer, the vaccine Injecting α can reduce the growth of the second f in the long part of the ί: - real J, the Lai system, the group of the following: breast cancer, lung cancer, '201100098 ^ cancer, cheek cancer, lunar cancer, colon cancer, nasopharyngeal Cancer, skin cancer, kidney cancer, brain tumor, prostate cancer, (four) cancer, cervical cancer, intestinal cancer, and bladder cancer. The present invention relates to an immunogenic composition comprising: (a) poly riding, Essentially consisting of a Globo H-related glycan or an immunogenic fragment thereof, wherein the glycan is co-linked with a carrier protein via a linker; and (b) Zozib CDld can bind the glycolipid molecules on the dendritic cells, wherein the

The Globo H-related glycan is selected from the group consisting of *SSEA-3 and SSEA-4, and the immunogenic composition induces an immune response which induces an isoform compared to the IgM isoform A relatively high amount of IgG isotype antibody of the antibody. In a partial aspect of the immunogenic composition, the carrier protein is diphtheria, cross-hybrid (tetra) 197 (DT-CRM197), and the synthesis of the α-galactosyl group of the lion is carried out by guanamine (α-GalCer) Analogs, and the linker is a p-nitrophenyl linker. In a specific example, the adjuvant is C34. The present invention relates to a therapeutic agent for anti-milk scale, which comprises: a Gk conjugated to a diphtheria toxin cross-reactive material [97 (DT-CRMI97) carrier protein via a p-decylphenyl linker; b〇H; and an adjuvant comprising a glycolipid that binds to CDld molecules on dendritic cells. In this treatment example, the adjuvant is C34. The invention relates to a therapeutic agent for age-transfer anti-milk age cells, which comprises: SSEA_3 co-linked with diphtheria toxin cross-reactive material 97 (DT-CRM197) carrier protein via a p-nitrophenyl linker: And an adjuvant comprising a glycolipid C34 which binds to a CDld molecule on dendritic cells. The present invention relates to a therapeutic agent for combating breast cancer stem cells, which comprises conjugated SSEA 4 with a diphtheria toxin cross-reactive material 'Material I' (DT-CRM197) carrier protein via a p-nitrostyl linker. In some embodiments, the therapeutic agent further comprises an adjuvant comprising a glycolipid which binds to a CDld molecule on the dendritic cell. The therapeutic agent of the present invention is administered to a subject to induce antibody production, The antibodies recognize an antigen expressed on breast cancer stem cells (BCSC), wherein the antigen is selected from the group consisting of Globo®, SSEA-3, and SSEA-4. The present invention relates to a method of treating breast cancer, comprising The present invention is directed to the surprising discovery that DT_CRM197 can be used as an effective carrier protein for Globo(R) and SSEA-4, not only because it has been widely used against Diphtheria human vaccination for decades, also because of its high degree of immunity, originality, and most importantly, has been approved by the FDA for various sugar & yoke conjugate vaccines. Diphtheria toxin cross-reactive material 1 97 (DT_CRM197) is a non-toxic wing of DT (G52E), which has immunogenicity and ability to bind heparin-binding epidermal growth factor (HB_EGF), and specificity of heparin-binding epidermal growth factor DT. Sex cell membrane receptors, often in cancer; ^ overexpression (Buzzi S. ❹ /., Cancer Immunology, Immun〇therapy (2004), 53 (11): 1041-1048). Py: using C34 as an adjuvant, GH- Both DT and SSEA-4-DT showed the most effective immune response, and the anti-tumor antigen was induced in comparison with IgM antibody. GH-DT binds to C34 to induce sputum antibody, which not only neutralizes GloboH but also It can neutralize SSEA_3 (Gb5) and SSEA_4, which is specific to breast cancer cells and cancer stem cells. Furthermore, the invention provides (4) micro-funding materials to provide antibody = strong platform 'can be lining Test and test the immune response after immunization. In the following detailed description, 'can be used as a pattern, scales _ form the invention 201100098 invention, and the details '俾 熟 熟 熟 技 技 范围 范围 范围 熟The structure is carried out without departing from the invention of the invention. Explain the preferred method test. The recognition: ==f capacity is regarded as the specific method of s, _%« 〇3 is the instance of Wei, and '申ί=,; The scope of the appendix will only be defined by the definition of f, which is the text and in the scope of the attached patent application, the singular is not L: in addition, 4 is plural, unless the above is touched by the TM; = note that it is ^At least heart 14 201100098

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. These techniques are fully described in the literature. See, for example, Molecular Colonization: Laboratory Manual, 2nd ed. (Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook ed., Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989); DNA selection, I and II (DNA Cloning, Volumes I and II, DN Glover ed) 1985); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 〇 1987); Immobilized Cells and Immobilized Cells And Enzymes, IRL

Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning, 1984; the treatise, Methods In Enzymology, Academic Press, Inc. NY); for mammalian cells Gene Transfer Vectors For Mammalian Cells, JH Miller and Μ. P. Calos eds., 1987, Cold Spring Harbor Laboratory; Methods in Enzymology, Vol. 154 and 155 (Methods In Enzymology, Vols. 154 and 155) , Wu et al. eds·); Immunochemical Methods in Cell and Molecular Biology, Mayer and Walker, eds., Academic Press, 〇London, 1987; Antibody: Laboratory Manual ( 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 liposoluble (lipophilic) molecule involved in the cellular signaling pathway. The term "glycolipid" as used herein refers to a lipid attached to a sugar which serves as a marker for cell recognition. 15 201100098 The term "α-galactosylglycine" or "a_GalCer" as used herein refers to a glycosity that stimulates natural killer T cells to produce tau helper cells, both TH1 and TH2 cytokines. The glycolipid derivative C34 used herein has the following structure:

The α-GalCer analogs of the present disclosure include a_GalCer analogs of bacterial origin (Group I: C2, C3, and C14), a_GalCer analogs modified by lithification (Groups II: C4, C5, and C9) a phenyl-alkyl chain a-GalCer analog (Group III: C6-C8, C10-C11, C15-C16, C18-C34, C8·5, and C8-6), and phytol ( Phyt〇Sphing〇sine) truncated α-GalCer analogs (Group IV: C12, C13, and C17). The structure of C34 and other α-galactosyl-based neuroinhibitor analogs and their use as adjuvants are disclosed in detail in PCT Patent Application No. PCT/US2008/060275 (filed Apr. 14, 2008).

Synthetic alpha-GalCer analogs (including C34) form a co-linkage with CDld molecules. Synthetic α-GalCer analogs can be recognized by NKTs T cell receptors. The synthetic α-GalCer analog can excite TH1-type, TH2-type, or TH1-type, and TH2-type reactions. The α-GalCer analog activates NKTs in test tubes. The a-GalCer analog activates NKTs in vivo. The term "glycan" as used herein refers to a polysaccharide or sugar collection. As used herein, a glycan is also used to refer to a sugar moiety of a sugar conjugate linker, such as a glycoprotein, a glycoprotein, a pickled peptide, a vinegar proteome, a peptide polyester, a lipopolysaccharide, or a proteoglycan. Glycans are usually composed only of a single sputum-glycosidic bond. For example, cellulose is a 16 098 Ο 201 201100098 poly _ (or more specifically dextran), composed of sugar, and chitin is composed of β·Μ _ linkage: sex d-grape The polyphonic listener can be the same or a heteropolymer of a monosaccharide residue and is composed of glucosamine. : It was found to be attached to the protein, such as in sugar eggs. The glycan is found on the outer surface of the cell. 0_ and N_ far from the glycan. In general, cells can also be found in prokaryotic cells (although less common glycans are very common in the eukaryotic sequ〇n), the aspartic acid methionine -Ser or Asn-X-Thr sequence, any amino acid other than ruthenium (Ν). In addition to the guanamine, the term "sounding I white r., t white. Protein: 〗) ^ Linked day ^^ Jia's egg vinegar protein (mucin), 3) Glucosamine polyzyme (GAGs, - linked glycans), 4) GPI-anchored protein. Daegu VIII is also known as protein Sex (a variety of attachments at the same glycosylation site; / structural micro-heterogeneous heterogeneity and structure of the giant body of the text to make the shape of the "antigen" (10) any saki immune response, the term "free silk" A substance or a substance, such as a DNA vaccine. The term "factory immunogenicity" as used herein refers to the ability of a vaccine to stimulate an immune response. ', a few', or a plague, the use of the technique "Immunotherapy" refers to the "---------------------------------------------------------- Dld" Touch-age is now =, cell surface (4) (differentiated group _ _ protein family members. ^ The current 曰 抗原 antigen can activate natural killer τ cells. CDld a deep antigen-binding groove 'for enzyme lipids Antigen binding. It is expressed in dendrimers [cell 17 201100098 including oc-GalCer analogs, CDld molecules can bind and present glycolipids such as C34. - 夕-ίΐΐ! Adaptive immune system" refers to the exclusion of pathogens If the systemic cells and responses. The cell line of the adaptive immune system - the type of white blood cells 'is called lymphocytes. The b cells and tau cells are the main types of lymphocytes. 〇 The term "sputum cells" and rTs are used herein. Refers to a group of white blood cells called lymphocytes that play a central role in cellular regulatory immunity. By the special receptor present on the cell surface, called the T cell receptor (TCR), tau cells can interact with other lymphocytes. Cell type segments, such as sputum cells and NKs. Several different subpopulations of T cells are known, each with unique functions. The helper τ (TH) cell line is the "individual" of the adaptive immune system. Once activated, it rapidly divides and secretes small molecules called cytokines, which regulate or "help" the immune response. These cells differentiate into TH1, TH2 based on the received cytokine signal. One of TH17, or one of the other subpopulations, which secretes different cytokines.

The term "antigen-presenting cell" (APC) as used herein refers to a cell which exhibits a foreign antigen (complexed with a major histocompatibility complex (MHC)) on its surface. T cells can recognize such complexes using their TCR. APCs can be divided into full-time and non-professional. Dendritic cells (DCs) are of a professional type and they can present antigen to T cells by CD1. In an exemplary embodiment, the DCs used in the methods of the invention can be any of a number of DC subpopulations, in one embodiment, produced by lymphoid differentiation' or in another embodiment, It is produced by differentiation of myeloid progenitor cells. The term "naive cell" as used herein refers to an undifferentiated immune system cell, e.g., a CD4 T cell, which has not been specialized to identify a particular pathogen. 18 201100098 Hemiplegia used in this article 1 "Natural killer cells" and "NKs" refer to a lymphocyte that is activated by interferon to participate in the congenital host defense against viruses and other intracellular pathogens. a The term "natural killer tau cells" (NKTs) as used herein refers to - Ο

Ϊΐ 群 Λ Λ Λ 共有 共有 共有 习 习 共有 共有 共有 共有 共有 共有 共有 共有 共有 共有 共有 共有 共有 共有These cells of Zhu Xi can recognize non-polymorphic cmd molecules that can bind to the antigens of the autologous and exogenous lipids f and f. The TCR of NKTs recognizes the enzymatic lipid molecules present (protected) by CDld molecules. The main response of NKTs is to rapidly secrete cells after stimulation; alkaloids, including IL-4, IFN-γ, and IL-10' and thus affect a variety of immune responses and pathogenic effects. NKTs can be homogenous or heterogeneous. In an exemplary embodiment, the population may be "non-invariant NKTs", which may comprise human and mouse bone marrow and human liver T cell populations, for example, non-CDld reactivity indicative of various TCRs. Invariant tau cells, and which can also produce large amounts of IL-4 and IFN-γ. The most well-known CDld-dependent νκβ subpopulation is a constant TCR-α chain. This is specifically referred to as Type I or invariant nkts (iNKTs). These cells are conserved between human (Va24iNKTs) and mouse (Val4iNKTs) and are involved in a variety of immune functions. 〃 The term “cytokine” as used herein refers to any of a number of small, secreted proteins that can affect the differentiation of immune cells (usually involving changes in gene expression, whereby precursor cells can become different The cell type II regulates the intensity and duration of the immune response. Cytokines have been named lymphosteroids, interleukins, and chemotactic hormones based on their putative functions, secretory cells, or targets. For example, & 'partially common Interleukins include, but are not limited to, IL I, IL-18, IL-2, IFN-γ, TNF, IL-4, IL-10, IL_13, IL_21, and TGF-β 〇19 201100098 "chemokine" refers to any of the fine-released substances released at the site of infection, which provides a protective device for moving and neutralizing lymphocytes. Chemokines can attract white blood cells to the infection site. a cysteine residue, which can be made into a region; for tetra?2?^, etc., representative chemotactic hormones are c_c chemotactic hormones (RAN S MCP-di-ΜΐΡ1α, and ΜΙρ_1β), cxc Chemotactic 1 CXXXC tit ^ 0 ^ ^ L^Ptoactin) > and cxxxc tendencies Hormone (fractal hormone, Fractalkine). The production of "Th2 type reaction" refers to a mode in which a cytokine produces a second low, a cytokine, an interferon, or a chemokine. Blood type TH2 cytokines include, but are not limited to, IL_4, IL_5, IL_6, and no. In order to produce a th1 type reaction, it refers to a model in which a cytokine is produced, such as a class i, a phytohormone, an interferon, or a chemokine. Blood group cytokines include, but are not limited to, IL_2, secret gamma, .gmcsf, and middle-counter% bias. This refers to an immunogen reaction. The degree of increase in the production is the term "antigenic" system used in this article. Defined as the green binding site contact portion of an anti-antibody or T cell f body. ', /, anti-shoulder "vaccine" refers to a kind of preparation 'containing antigen, the substance ====== 20 201100098 α-GalCer* class _ is used as an example of the anti-f The term "alum adjuvant" as used in the analog c34 is used to mean an immunosuppressive agent. The salt of the activity of the agent. Such a test shot absorbs the eggs of the scallops from the antigen and allows the sputum to improve the immune compatibility of the vaccine by assisting the slow release of the antigen from the ageing and gradual release. The text is the collection, &

The term "anti-tumor immunotherapeutic active agent" as used herein refers to an antibody produced by a vaccine which inhibits, reduces, or eliminates tumors. As used herein, the term "antigen-specific" refers to the nature of a cell population that allows it to cause proliferation of a particular cell by providing a particular antigen or antigenic fragment. The term "flow cytometry" or rFACS as used herein means a technique for detecting the physical or chemical properties of particles or cells suspended in a liquid stream via optical and electronic detection means. The amino acid residue in the peptide will be abbreviated as follows: phenylalanine phe or F, leucine Leu or L; isoleucine iie or I; thiol amide Met or hydrazine; guanamine Acidic Val or V; serine acid Ser or S; proline acid Pro or P; hydroxybutyric acid Thr or T; alanine Ala or A; tyrosine Tyr or Y; histidine His or Η; glutamic acid Gin or Q; aspartic acid is Asn or N; lysine Lys or K; aspartic acid Asp or D; face acid Glu or E; Amine acid Cys or C; tryptophan Trp or W; arginine Arg or R; and glycine Gly or G. Further explanation of amino acids is described in 'Proteins: Structure and Molecular Properties, Creighton, T. E., W. H. Freeman & Co., New Yorkl 983. 21 201100098 The compositions disclosed herein can be included in a medicinal or nutmceutical composition, in combination with other active agents, carriers, carriers, and forms that are recognized by those skilled in the art upon reading the second volume of the present disclosure. Agent' or adjuvant. Preferably, the pharmaceutical or nutraceutical composition comprises at least one pharmaceutically acceptable carrier. In such pharmaceutical compositions, the compositions disclosed herein form "active compounds", also known as "active agents." The term "pharmaceutically acceptable carrier" as used herein includes solvents, dispersion bases, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. . Supplementary active compounds can also be included in the compositions. The pharmaceutical composition is formulated to be compatible with the route of administration. The route of administration includes (e.g., 'intravenous, intradermal, subcutaneous), oral (e.g., inhalation), perforation, transmucosal, and rectal administration. For parenteral, intradermal, or subcutaneous administration, the solution or suspension may include a lower component, such as no water, a saline solution, a non-volatile oil, polyethylene glycol, glycerin, or propylene glycol. Solvent; antibacterial agent, such as benzoquinone or p-hydroxybenzoic acid, deuterated agent, such as ascorbic acid or sodium bisulfite; chelating agent, such as ethylenediaminetetraacetic acid; buffering agent 'such as acetate, Citrate, or both, and osmotic agents such as 'chlorinated sodium or glucose. Pf can be made of acid glass or plastic, disposable, or more _^ to lie in the glass, the human and the non-human difficult (such as, fine stars, (eg, dogs, such as 'Mianping , cattle, horses, baboons, and sputum), pets ii / each ί,), laboratory test animals (such as, petty, rabbit, rat,, captive wild animals (such as, fox, two reagents obtained An organism can be obtained without limitation to the agent of the present invention. Whether it is a human or a non-human organism, the subject can be referred to as a patient, an individual, a host, or a recipient. 22 201100098, with the doctor of Newtonit The ageing compound includes g-free water (sexual green liquor) and an innocent powder for preparing (4) a sterile injectable solution = ▲ liquid. In the case of intravenous administration, a suitable carrier is water-incorporated, and the brine is inhibited. Cremophor ELTM (BASiI7 TWc. χτ dish; rf, (PBS) is a kind of miscellaneous miscellaneous. It is made in the manufacture of miscellaneous pieces and has antiseptic properties against microorganisms (such as bacteria and cockroaches; a solvent or dispersion matrix containing, for example, water, = erythritol (eg, 'glycerin, propylene glycol, and liquid polyethylene An alcohol, and a suitable mixture thereof. Suitable fluidity can be, for example, a coating agent = phospholipid), the particle size of the dispersion is maintained in the case of a dispersion, and the surfactant is used for transfer. Microorganisms _ B Water, i, and the like. In many cases, an isotonic agent, for example, a saccharide, a polyhydric alcohol (sorbitol), or a sodium chloride may be included. The extended absorption of the injectable composition may be The drug can be delayed in the absorption test, for example, the single hard acid can be used to incorporate the desired amount of the active compound into a suitable solvent, together with the aforementioned ingredients or Combine, and then make a transition to prepare... Generally, the dispersion is prepared by using the active compound in a sterile carrier containing the other components required for g, and is used for the preparation of a non-brittle solution. Finally, the method of preparation can produce a powder of the active ingredient plus any other desired ingredient from its previously innocent filtered > trough solution. 'Λ二ϊίΐϊ: generally includes pure diluent or edible Carrier. For the purpose of oral b treatment The active compound can be mixed with excipients and used in the form of 23 201100098, tablets, or capsules (eg, gelatin capsules). Oral compositions can also be prepared using liquid carriers as a mouthwash. Compatible adhesives or adjuvant materials include as part of the composition. Tablets, pills, capsules, tablets, and the like may contain any of the following ingredients, or compounds having similar properties, a binder, For example, microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as 'alginic acid, Primggd corn starch; lubricants such as magnesium stearate or Ster Glutes; glidants, bismuth dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or citrus flavoring agents. In the case of 吸入京 Inhalation, the compound is in the form of an aerosol spray from a pressurized container or dispenser containing a suitable S propellant (such as a 'gas' such as carbon dioxide) or a sprayer. transfer. Systemic administration can also be done by wearing a mucous membrane or wearing a skin. In the case of transmucosal or transdermal administration, Y uses a penetrating agent suitable for the barrier to be penetrated in the formulation. Such penetrants are generally known in the art and include, for example, in the case of transmucosal administration, a detergent, a bile salt, and a fusidic acid derivative. The administration of the mucosa can be accomplished by using a nasal spray or a plug. For transdermal administration, the active compound may be formulated as an ointment, a peat, a gel, or a cream, as generally known in the art. The compounds (4) may also be prepared in the form of (iv) (e.g., a plug of L, such as cocoa butter or other glycerides) or an enema to retain the rectum. According to the embodiments, the active compound can be prepared together with carriers which can protect the compound from rapid elimination from the body, such as a controlled release formulation, including, and a microcapsule delivery system. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyortive vinegar, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and N〇va Pharmaceuticais, Inc. It is also possible to use liposomes 24 201100098 as directed by the skilled artisan, as described in the accompanying claims, which is incorporated herein by reference. Ruguan special shot 4,522,811

Early, aliquots are formulated with oral or parenteral compositions for ease of administration. 'Dose unit form refers to physical discontinuity ΐ' which is suitable as a single-dose for the subject to be treated;篁 杂 化合物 ( ( ( ( ( ( 杂 杂 杂 杂 杂 杂 杂 杂 έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ έ (10) 5. (It can make 5% of iif) to encourage 5g (the y in the treatment of the scale of the sputum). The dose ratio between f sex and therapeutic efficacy is the therapeutic index, and it can be expressed as the ratio LD5〇/ED5〇. Compounds having a high therapeutic index are preferred. While toxic by-products can be used, care should be taken to design a delivery system that targets these compounds to the affected site to minimize potential damage to the unsensed cells and thereby reduce side effects. Dosage ranges for use in humans can be formulated using data from cell culture assays and animal experiments. Dosages of such compounds are preferably within a range of circulating concentrations' which range includes ED5 oxime with little or no toxicity. The dosage ^ varies within the range of the dosage form used and the route of administration employed. For any of the compounds 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 the animal mode to achieve a certain range of plasma concentrations. This range includes 1 〇 50 as determined in cell culture (ie, the concentration of test compound that achieves half of the maximum symptom suppression). ). This information can be used to more accurately determine the available dose in the human body. The amount in plasma can be measured using, for example, high liquid phase chromatography. ° 25 201100098 - The therapeutically effective amount (i.e., effective dose) of the active compound, as defined herein, may range from about 0.001 to 100 g/kg body weight, or is apparent to those skilled in the art without undue experimentation. And other areas that can be understood. & artisans will be able to understand the 'dose factors that can affect the effective treatment of the subject's time machine' including, but not limited to, the severity of the disease or condition, previous treatment, the general Health conditions or age, and other existing diseases. σ

According to another aspect, one skilled in the art can envision one or more component kits that can perform at least one of the methods disclosed herein, the kit kits g two or more compositions 'these compositions are included An effective amount of the composition of the invention (alone or in combination) of at least one of the above methods. Such kits may also include compositions comprising an active agent, a biological event agent, or other compound recognized by those skilled in the art upon reading this disclosure. The kit may also comprise at least one composition comprising a combination of the inventions of the invention. The compositions and cells of the components can be implemented according to at least one of the methods disclosed herein by the skilled artisan.

The term "polypeptide" as used herein refers to a polymer or polymer of any amino acid residue. Purely composed of two or more polypeptides. Polypeptides include proteins, oligopeptides. The polypeptide may be a straight chain or a branch. The polypeptide may comprise a modified amino acid analog, or a non-naturally occurring seric acid residue, and may be naturally or by intervening, eg, forming a disulfide bond, a hydrazine, a group, a Deuterated, scalded, or manipulated. For example, a total of the link mark components. , , and "specific binding" refers to the binding pair (eg, antibody seedling 7 side. In each case, the specific binding may be about 10-6 material / liter, side .8 her liter, or below 26 201100098癌症 癌症 cancer lean seedlings 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) Sexual embryonic antigen _3 (SSEA_3, also known as Gb5) or SSEA-4) and adjuvants. The types of target cancer include 'but not limited to, breast cancer (including stage 1-4), lung cancer (eg, small cell lung cancer), Liver cancer (eg, hepatocellular carcinoma), oral cancer, gastric cancer (including T1_T4), colon cancer, nasopharyngeal cancer, skin cancer, kidney cancer, brain tumor (eg, astrocytoma, glioblastoma multiforme, and Cerebral meningioma), prostate cancer, ovarian cancer, cervical cancer, bladder cancer, and endometriosis, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, and gastrointestinal stromal tumors. Cancer classified by site includes oral cavity and pharynx Cancer (insult, tongue, salivary gland) , mouth, gums, and other mouth, nasopharynx, tonsils, oropharynx, hypopharynx, other mouth/pharynx); cancer of the digestive system (esophagus; stomach; small intestine; colon and rectum; anal, anal canal, And anorectal; peritoneum, omentum, and mesentery; other digestive, clever); cancer of the respiratory system (nasal, middle ear, and sinus; throat; lung and branch trachea; pleura; trachea, mediastinum, and other respiratory systems); Skin tumors; bones and joints; and cancers of soft tissues (including the heart); skin cancers, including melanoma and other non-epithelial skin cancers; Kaposi's sarcoma and breast cancer; cancer of the female reproductive system (cervix; uterus Uterus, unspecified (N〇s); ovary; vagina; vulva; and other female reproductive systems); cancer of the male reproductive system (g gland; pill; penis; and other male reproductive systems); cancer of the urinary system ( Bladder, kidney and renal pelvis; ureter; and other urinary systems; cancer of the eye and eyelid; cancer of the brain and nervous system (brain; and other cancers of the nervous system endocrine system) Glandular and other endocrine systems, including the thymus; lymphoma (Hodgkin's disease and non-Hodgkin's lymphoma), multiple 27 201100098 myeloma, and leukemia (lymphocytic leukemia; myeloid leukemia) ; cytocellular leukemia; and other leukemias. ' Other cancers classified by histological type that may be appropriate targets for the cancer vaccine of the invention include, but are not limited to, tumors, malignancy; carcinoma, NOS; carcinoma, undifferentiated 'NOS , giant cell and spine cell carcinoma; small cell carcinoma, papillary carcinoma, NOS; squamous cell carcinoma, NOS; lymphoid epithelial carcinoma, N〇s; basal cell carcinoma, NOS; hairy mammary carcinoma; Cancer, N0S; papillary metastatic epithelial cell carcinoma; adenocarcinoma, NOS; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma, NOS; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenoma; Cystic cancer; adenocarcinoma adenocarcinoma; adenocarcinoma, familial colon polyposis; solid cancer, N〇S; carcinoid, malignant; bronchiole-alveolar adenocarcinoma; papillary adenocarcinoma, NOS; Erotic cell carcinoma, eosinophilic carcinoma, eosinophilic adenocarcinoma Hemorrhagic cell carcinoma; clear cell adenocarcinoma, NOS; granulosa cell carcinoma; follicular adenocarcinoma, NOS; papillary and vesicular adenocarcinoma, NOS; non-encapsulated sclerosing carcinoma; adrenal cortical carcinoma; Endometrial cancer; skin appendage cancer; apocrine adenocarcinoma; sebaceous gland cancer; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma, NOS; papillary cystadenocarcinoma, NOS; papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma, NOS; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma NOS; breast cancer; inflammatory cancer; berth (paget) disease, breast; acinar cell carcinoma Glandular squamous cell carcinoma; adenocarcinoma with squamous cell metaplasia; thymoma 'malignant' impression of benignoma, malignant; benign cell tumor, malignant; nest granulosa cell tumor 'malignant; male blastoma 'Malignant; Sertoli's cell tumor; Leydig cell tumor, malignant; lipoma, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; chromaffin Cell tumor; glomus sarcoma; malignant melanoma, NOS; no melanoma melanoma; superficial disseminated black Tumor; NOS; malignant melanoma in giant pigmented nevus; epithelial melanoma; blue sputum malignant; sarcoma, NOS; fibrosarcoma, NOS; fibroblastoma, malignant; mucinous sarcoma; liposarcoma, N 〇s; uterine muscle tumor, 28 201100098 NOS; rhabdomyosarcoma, NOS; embryonal rhabdomyosarcoma; acinar rhabdomyosarcoma; interstitial sarcoma NOS; mixed tumor, malignant, N〇s; Mullerian mixed tumor Wilms tumor; hepatoblastoma; carcinosarcoma, NOS; mesenchymal cell tumor 'malignant; Brenner's face tumor, malignant; phyllodes tumor, malignant; synovial sarcoma, NOS; mesothelioma Malignant; malignant embryonal tumor; embryonic carcinoma 'NOS; teratoma, malignant, NOS; goiter-like ovarian tumor malignant; choriocarcinoma; middle renal tumor, malignant; hemangioma; vascular endothelial cell tumor, malignant; Xi's sarcoma; perivascular cell tumor, malignant; lymphatic carcinoma 'malignant; osteosarcoma, NOS; paracortical osteosarcoma; chondrosarcoma, N〇s; chondroblastoma, malignant; mesenchymal chondrosarcoma; Giant cell tumor Ewing sarcoma 'malignant; odontogenic tumor, malignant; ameloblastic sarcoma; ameloblastoma 'malignant; ameloblastic fibrosarcoma; pineal tumor, malignant; chordoma; glioma' malignant Ependymoma, NOS; astrocytoma, NOS; protoplasmic astrocytoma; fibroblastic astrocytoma; astroblastoma; glioblastoma 'NOS; oligodendroglioma, NOS; oligodendrocyte glioblastoma; primitive neuroectodermal tumor; cerebellar sarcoma, NOS; ganglion neuroblastoma; neuroblastoma, NOS; retinoblastoma, NOS; olfactory neurogenic tumor; meningioma Malignant; neurofibrosarcoma; schwannomas, malignant; granulosa cell tumor 'malignant; malignant lymphoma, NOS; Hodgkin's disease, NOS; Hodgkin's paragranulomatosis, NOS; malignant lymphoma, small lymph Cellular; malignant lymphoma 'large cell' diffuse; malignant lymphoma, follicular, NOS; verrucous granuloma; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; Mast cell meat Immunoproliferative small bowel disease; leukemia 'NOS; lymphocytic leukemia, NOS; plasma cell leukemia; red and platinum disease; lymphosarcoma leukemia; myeloid leukemia, N〇s; basophilic white disease; Leukemia; monocytic leukemia, N〇s; fat 29 201100098 large cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. The term "treating" as used herein refers to the administration or administration of 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 administration and administration. Heal, treat, alleviate, alleviate, alter, remedy, improve, improve, or affect the cancer, the symptoms of cancer, or the predisposition to cancer. As used herein, "effective amount" refers to the amount of each active agent required to produce a therapeutic effect on a subject, either alone or in combination with one or more other active agents. As is known to those skilled in the art, the effective amount will vary depending on the route of administration, the use of excipients, and the total use of other active agents. The immunological composition used in the above method may contain a glycan (i.e., a molecule containing a sugar molecule moiety) which is a Globo® or a fragment thereof, and an adjuvant. G1〇b〇 is a glycan containing a hexasaccharide epitope (匕(10)丨^ Galpi-3 GalNAcpi->3 Galal-4 Gaipi-4 Glc)' and optionally containing a non-sugar moiety. A fragment thereof is a glycan containing a hexose antigen epitope and, if any, a fragment of a non-sugar molecule moiety. Such oligosaccharides can be prepared by conventional methods (see 'Huang et al., 5W. USA 103: 15-20 (2006)). If necessary, it can be linked to a non-sugar molecule. The original application US Patent Application No. 12/485,546 reveals unforeseen findings: (1) SSEA_3, a direct precursor of Globo, has a high level of performance in breast cancer stem cells and can therefore be used as an appropriate target for breast cancer treatment. The target, and (2) 1 galactosyl-neuramide (α-GalCer) is an effective adjuvant that promotes the production of anti-G1〇b〇Η and anti-SSEA-3 antibodies. U.S. Patent Application Serial No. 12/485,546, the disclosure of which is incorporated herein by reference to the entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire all KLH) Conjugated link. 30 201100098 Such an immunological composition elicits an immune response (eg, antibody production) that targets GloboH or a fragment thereof when administered to a subject (eg, a human), and thus is effective in treating cancer (eg, breast cancer, Prostate cancer, ovarian cancer, and lung cancer). U.S. Patent Application Serial No. 12/485,546 is directed to a method of preparing an antibody specific for Globo(R) or a fragment thereof, which is directed against a non-human mammal (e.g., mouse, rabbit, goat, sheep, or horse). Administration of the above-described immunological composition, and isolation of the anti-sputum which binds G1〇b〇Η or a fragment thereof from the mammal, reveals that the Globo(R) or other glycan system described in the inner trough is conjugated to the protein carrier, such as , DT-CRM197. It can then be k-conjugated with an adjuvant (e.g., c34) acceptable carrier (e.g., 'buffered saline' or cesium carbonate) to form an immunological composition (e.g., vaccine) via conventional methods. See, for example, U.S. Patents 4,6,1,9,3; 4,599,231; 4,599,23; and ^4'596,792. The conjugate can be prepared as an injectable substance, a liquid ‘liquid, according to the method of administration and the way of health according to the standard i medical practice. n drug carrier and diluent, and (Remington, s PhaJac ^ 1 composition preferably contains a_GalCer as an adjuvant. Adjuvant ^ Other: Examples include, but are not limited to, scorpion toxin, field heat, Immune irritating co-arms (isc〇m), or a group of people who can assist in the delivery of the body.

The composition in 21:1250-5, 2003^\ D et aL stimulates the challenge against 201100098 intramuscular injection). Or, it may be formulated. In the case of suppositories, the viscous, including suppositories and oral diol or triglyceride furnaces and carriers may include, for example, polyolefin based such as, for example, 11 acaricidal formulations may include excipients for normal use, These group two: ^ Lai, and the like. Formulations, or powder forms, pills, capsules, sustained release compositions. Gas, and contains 1〇-95. /. The immunological group described herein is administered to a subject to be treated in a manner compatible with the dosage formulation, including, for example, the immune activity of the individual (if necessary) to produce a cellular regulatory immune response. The accuracy of the 4 series is determined by the judgment of the medical practitioner. 11 The appropriate dose range can be easily determined by a skilled artisan. The initial treatment and the appropriate therapeutic aspect of the sputum agent 4 are variable and may include initial administration followed by administration. The dosage of the vaccine may also depend on the route of administration and will vary depending on the size of the host. "The immunological compositions of the present invention can also be used to produce antibodies in animals for the preparation of antibodies which are useful in the treatment and diagnosis of cancer. Methods for preparing single and multiple antibodies and fragments thereof in vivo = (e.g., 'mouse, rabbit, goat, sheep, or horse) are well known in the art. See, for example, 'Antibody: Experiment to Handbook (Harlow and Lane, (1988) Oil-cutting Oil Ms (10) βΐ, Cold Spring Harbor Laboratory, New York). The term "antibody" includes intact immunoglobulin molecules as well as fragments thereof, such as Fab, F(ab')2, Fv, scFv (single-chain antibody), and dAb (regional antibody); Ward, et. al. (1989) ) Nature, 341, 544) °

Globo H-DT-CRM197 and related vaccines 32 201100098 Globo(R) (1) and its fragment 2_1〇 were synthesized as described herein. The purified Globo simmered half vinegar 12 was incubated with the individual carrier white for the purpose of performing protein-co-linking as shown in Fig. 14. The characteristics of these Globo Η-proteins were analyzed by MALDI-TOF to determine the number of Globo Η molecules on each carrier protein. The average number of Globo 纳入 is listed in Table 1. Table 1. MALDI-TOF analysis of Globo® inclusion

Ref protein MW after glycosylation a average inclusion (η) GH-BSA 66431 66449 76029 8 GH-DT 58472 58326 62138 2~4 GH-TT GH-KLH* GH-Bamb 〇〇150682 8.6 x 10s 25kDxl600 155609 162902 ND 6 ~700 percentage of sugar Ϊ1Τ?% ~~ 6.8% 4.5% 14.7% a peak in MALDI-TOF m/z; ND: not determined; *GH-KLH is provided by 〇ptimer inc•

The GH-KLH conjugated linker showed the greatest number of G1〇b〇 纳入, mainly due to the large size of KLH and the presence of more Lys residues. The same procedure for the _ nitro-based linker will also be applied to the bamboo mosaic virus, which contains more than 100,000 lysine residues on the viral coat. However, the instability of the virus when reacted in sodium sulphate buffer (pH = 7.2) at 4 °C is a major consideration for further development. Furthermore, GH-BaMV 16 is limited by its large size for speculation through MALDI-TOF analysis. The synthesized Globo(R) and the fragment (Fig. 丨) were attached to the reducing end with a pentylamine linker and covalently immobilized on an NHS coated glass slide. Nine embossings were selected on the microarray in ^* an oligosaccharide. Nine Globo Η analogs (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, ΒΒ4, ΒΒ3, and ΒΒ2) were spotted on each microarray slide with 50 μΜ (12 replicates). 33 201100098 To validate the carbohydrates on the microarray, use mouse monoclonal antibodies (for Globo Η VO ^Mbd ' and anti-SSEA_3) and use the corresponding secondary antibody C Shanzao anti-small IgG and IgM) to test The binding specificity is shown in Figures 2A-2C. The data shows that both VK9 and Mbrl can recognize & 〇b〇 H' and the outer tetrasaccharide BB4, but MBrl can also slightly recognize BB3. In addition, the anti-SSEA-3 antibody specifically recognizes the SSEA_3 antigen (Gb5) without any cross-reactivity. These results indicate that Gl〇b〇 Η microarrays can be used to describe the specificity and potency of multiple antibodies taken from immunized mice. As reported by the uranium, 'immunized mice with fully synthesized Globo sputum vaccine and co-administered QS-21 can produce antibodies against human breast cancer cells, but 'although there are several booster immunizations, These mouse antibodies are still predominantly IgM (Ragupathi G, et al. (1997) Angew Chem Int Ed 36: 125-128). A group of mice were immunized subcutaneously with 1 Pg of synthetic Globo H co-linker with or without the lipid adjuvant a-GalCer (C1). GH-KLH, GH-DT, and GH-BV were found to be the most potent immunogens for inducing IgM, followed by GH-TT and GH-BSA' as described in Figure 3A, while ct-GalCer stimulates immune responses to induce high amounts. IgM antibody. A similar tendency was also observed on mouse IgG antibodies (Fig. 3B), and the relative IgG content was higher than the IgM content. Briefly, although the synthetic sugar conjugate linker has a lower sugar density, GH-DT has immunogenicity similar to GH-KLH, while the α-GalCer adjuvant ij shows increased immune response. Since α-GalCer has been shown to be an effective adjuvant for GH-DT, it was tested against other glycolipids having better adjuvant activity than C1, as shown in Fig. 4. The mice were immunized with GH-DT and GH-BV with or without lipids. Serum was taken and introduced into the glycan microarray analysis. In general, the anti-Globo H IgG titer of mice increased with the progress of the immunization process, but the IgM content of 34 201100098 was almost independent of the number of immunological weights (Figure 5). In the GH-BV immunization group, there was no significant difference in the IgM content between the glycolipid vaccine treatment and the single vaccine treatment. Although these results suggest that GH-BV binding to lipids is not an effective immunological regimen, this poor immunogenicity may be due to the instability of BaMV.

k into. However, the 'α-GalCer analog', particularly 7DW8-5', cooperates well with GH-DT to induce a mouse immune response.

Interestingly, mice with multiple IgG antibodies produced by GH-DT and various glycolipid adjuvants can not only neutralize Globo® but also cross-react with Gb5, SSEA-4, and Gb4, while C34 seems to be most effective. Glycolipid adjuvant (Figure 6). To find novel vaccine compositions that can induce IgG that is much higher than IgM, test Globo®-DT conjugates with glycoforms C1 or C34 or the available adjuvant AIPO4 (salmonic acid) or MF59. · People think outside the field, after the second vaccination, υ 1〇Ε) 〇 H_DT and sugar! Quality C34 can almost exclusively and specifically induce IgG antibodies (Figure 7). In short, the glycoside side 7DW8_5 binds to the GH_DT sister linkage to enhance both the Globo H IgG and IgM antibodies, while the glycolipid adjuvant c34 砝Gh induces a much higher IgG antibody titer than IgM; ; I1; (Gb5) and SSEA-4 antigens also have different degrees of binding affinity, and the antigens are specifically expressed on the surface of breast cancer stem cells. To further compare the different glycolipid adjuvants for the G1〇b() H epidemic, seven groups of mice were immunized with GH-KLH. These results show that mice with the same vaccination can induce higher levels of anti-GbbG, =8). Although MF59 is an adjuvant, it cannot be combined with = to induce antibodies against Globo. a1Pq4 (phosphorus) also showed childhood resistance and induced a duck-free effect. On the other hand, gh_klh combines (3) with the second immune link to read the good immune nucleus, but after the third ugly reading ci there is no difference. Overall, novel glycolipid derivatives such as 2011 201198 have been found to have potential as adjuvants for sugar-based vaccines.

The nature of cellular and humoral immune responses is not only affected by the combination of antigen and adjuvant, but also by the vector and the immune pathway. As described by Sesardic and colleagues, DT-CRM197, a non-toxic mutant toxin, induces antigen-specific tau cell proliferation and increases IL-2, IFN-γ, and IL-6 production in splenocytes, thus Implying that it plays a role in the pathway driven by TM (Miyaji EN ei α/. (2001) Infect Immun 69:869-874; Godefroy S, et al. (2005) Infect Immun 73:4803-4809; Stickings P, et al. (2008) Infect /mm(10)76:1766-1773). Although the cytokine content is mainly TM, the subgroup of anti-CRM197 antibodies is igG1 and has no detectable igG2a, thus indicating a mixed Th1/Th2 response. These results prompted an assessment of the antibody isotype content of the Globo® vaccine, and this study showed that GH_DT or gh_klh combined with a glycolipid adjuvant can primarily induce IgG1 antibodies and trace amounts of igG2a (Figure 9). Although glycolipid adjuvant enhanced Th1 biased cytokine secretion when administered intravenously (i.v.) alone, no antibody type switching (IgG2a) was observed. In general, 'glycolipids play a key role in enhancing both cellular and humoral immune responses.

Globo Η, SSEA-3, and SSEA-4 cancer vaccines were synthesized with DT conjugated SSEA_3 (Gb5) and SSEA_4. Comparison of antibody titers of IgM and IgG after the third vaccination. SSEA-3-DT and SSEA-4-DT were also found to be much higher than igM.

IgG titer (Figure 10). - Since GH-DT and C34 can induce Globo Η, Gb5, and SSEA-4, the array of 24 glycans is used in the presence of adjuvant for 36 201100098 SSEA-3-DT and SSEA-4- The specificity of the DT vaccine was tested and the focus was on IgG studies (Figure 11). As shown in Figure 12, mice immunized with Globo®-DT and C34 adjuvants were able to induce South-selective identification of antibodies against Globo®, SSEA-3 (Gb5), and SSEA-4, while vaccine SSEA-3-DT and Adjuvant MF59 induces a highly selective immune response with low selectivity. On the other hand, SSEA-3-DT combined with adjuvant C34 only induced antibodies against globo Η, SSEA-3, and SSEA-4. Interestingly, SSEA_4_DT (sialic acid-Gb5) induces IgG and IgM antibodies that specifically recognize SSEA-4 and its cleavage structure (SSEA-4 with a lactose-deficient deletion) with or without adjuvant. Without being bound by theory, it is hypothesized that sialic acid is highly immunogenic and can elicit a highly specific immune response. Immunization of mice with SSEA-3-DT-C34 induces antibodies reactive with Gl〇b〇H, SSEA-3, and SSEA-4, suggesting that Globo®-based vaccines can be used to express Globo®, SSEA -3, and SSEA-4 tumor cells and breast cancer stem cells. Immunization of mice with Globo H-DT-C34 induced antibodies with Globo η, SSEA-3, and SSEA-4 reactivity, suggesting that the vaccine based on Gi〇b〇η can target Globo Η, SSEA -3, and SSEA-4 tumor cells and breast cancer stem cells. Immunization of mice with SSEA-4-DT induced an antibody with SSEA-4 reactivity. This suggests that SSEA-4-DT-based vaccines can target tumor cells and breast cancer stem cells that express SSEA-4. The size of the sputum caused by the tumor vaccine was reduced to directly evaluate the efficacy of the synthetic conjugated conjugate vaccine, and the tumor size was measured 3 times per week, as shown in FIG. In general, tumors were grown for 2 weeks after injection of 4T1 (a 37 201100098 breast cancer cell line with Globo®). On day 24, all vaccinated groups that bind glycolipid adjuvants still showed smaller tumor progression compared to the GH-DT and PBS control groups alone. This data suggests that vaccination with GH-DT and glycolipid adjuvant can delay partial tumor progression in vivo. The performance of SSEA-3 and SSEA-4 in breast cancer and BCSCs has confirmed that Globo Η is expressed in BCSCs, but its frequency is lower than that of non-BCSCs'. In breast cancer and BCSCs, SSEA-3 is higher than Globo ❹. Performance (Chang WW. et al., (2008) / Voc " 105(33): 11667-11672, which is incorporated herein by reference in its entirety). Table 2 summarizes the clinical features of 35 breast cancer patients in which SSEA-3 or SSEA-4 performance was measured in vivo. The median age is 48 years old (between 31 and 82 years old). The patient included a phase 0, 10 phase I, 19 π phase, and 5 m phase. Most of the tumor samples had pathological phenomena of invasive ductal carcinoma (80.0%), of which 51.4〇/〇 were positive for £^, and 657% were positive for lymph node metastasis. In Table 2, the range of performance of SSEA-3 or SSEA-4 is expressed as the percentage of positive cells in cancer cells. use? The trial was performed for statistical analysis of SSEA-3 or SSEA-4 performance relative to HER_2 or lymph node metastasis status. 〇 HER_2 expression was determined by immunohistochemistry. There was no significant correlation between the amount of SSEA-3 or SSEA-4 on the tumor and various clinical pathological factors, such as 'segmentation (SSEA-4: P = 0.3498; SSEA-3:, P = 0.9311), or HER_2 (SSEA-4: Ρ = 0.0142; SSEA-3:, P = 0.0128) (Table 2): 38 201100098

Table 2. Bfe Qing Borrowing Characteristics of Breast Cancer Patients %" SSF S SSEA4 has performance & cell percentage Ο

Age of participation, age range 35 48 31- 82 100 Tumor type invasive ductal carcinoma 28 80.0 Invasive breast cancer 1 2.8 In situ ductal carcinoma 1 2.8 Zhao-like carcinoma 1 2.8 Atypical medullary carcinoma 1 2.8 Cancer 2 6.0 Inflammatory cancer 1 2.8 Stage 0 I II III SSEA3 Percentage of cells with expression __Medium (range) Household value ο Negative positive for lymph node metastasis "ER negative positive 1 2.9 33.1 (33.1) 10 28.6 41.4 (0.5 -69.1) 19 54.3 39.3 (0.0-77.1) 5 14.2 49.8 (7.7-70.7)

0.9311 23 65.7 37.8 (0.0-69.1) 12 34.3 49.1 (17.4-77.1) 1.4(1.4) 36.4 (0.0-55.9) 30.9 (0.0-66.4) 32.3 (0.0-36.1) 0.4925 30.9 (0.0-66.4) 35.8 (0.0- 60.7) 18 51·4 36.2 (0.5-60.3) 17 48.6 48.5 (0.0-77.1) 0.0128 29.7 (0.0-38.6) 40.0 (0.0-66.4) CD45 was isolated from the participating patients by enzymatic digestion; CD24'CD44 CD45+ cells To remove white blood cells. To compare the performance of 39 201100098 SSEA-3 or SSEA-4 between BCSCs and non-bcscs, cD45-tumor cells were further separated into BCSCs and non-BCsCs based on their surface markers. b ^fine (four) foxes and other parts of the second use of this method, SSEA_3 or SSEA_4 in BCSCs in 35 tumor samples. Overall, SSEA_4 was detected in m (97.1%) tumors at 27/35 (77, f=_ to SSEA-3 (Table 3). Ssea_3 = SSEA-4 performance was determined by flow cytometry. BCSCs were defined as CD45-CD24_CD44+ cells and BCSCS was defined as the face of the CD45-cell population. The range was calculated as the percentage of positive cells in the total cells. As summarized in Table 3, 27/35 (77.1 The sample of %) showed ssea_3, and the percentage of pot = cell percentage ranged from 14% to 66 4%. The range of positive cells was 2% from the 2nd to the 4th of the tumor / medium ί knife 4^ non-BCSCS. 70.4%. In comparison, it was taken from the illusion of 35 tumors (65 7〇/ j jSCs showed positive staining of SSEA-3, and the percentage of positive cells was from 5.0% to 58.4%. Among the 34/35 (97.1%) samples showing SSEA-4, the positive cells ranged from 〇.5% to 77.1%. Non-Cs isolated from 32/35 tumors showed SSEA-4' percentage of positive cells. The range is from 24% to 1%. In comparison, BCSCs from 3 out of 35 tumors (88.6%) have SSEA slave positive staining's positive cell W score Fan Peripheral range from 5.6% to 83.6%. 40 201100098 Table 3. BCSCs 舆 non-BCSCs jggA4 and SSEA3 performance comparison number of glycan group patients positive number of cells with percentage of performance (range) % of totals SSEA-4 - total 35 34 41.4 (0.5-77.1) 97.1 Non-BCSCs 35 32 43.7 (4.0-78.1) 91.4 BCSCs 35 31 37.1 (5.6-83.6) 88.6 SSEA-3 Total 35 27 36.4 (1.4-66.4) 77.1 Non-BCSCs 35 25 40.5 (24.3 -70.4) 71.4 BCSCs 35 23 24.3 (5.0-58.4) 65.7 The performance of SSEA-4 in BCSCs is to compare the performance of SSEA-4 between BCSCs and non-BCSCs, and to further classify CD45-tumor cells into BCSCs based on the performance of their surface markers. Unlike non-BCSCs, BCSCs are identified by CD45-/CD24-/CD44+ cells, while other parts of the CD45-group are considered non-BCSCs. The SSEA-4 expression of each of the two differentiated populations varies with the tumor sample. Different, as shown in Figure 15. For example, patients with BC0264 of BC0264 accounted for 5.7% of total isolated tumor cells, negative for SSEA-4, and 60.3% of non-BCSCs showed SSEA-4. For the disease BC0266, SSEA-4 performance was detected only in 59.4% of non-BCSCs and 55.7% of BCSCs. For patients with BC〇313, SSEA-4 performance was measured in 32.4% of non-BCSCs and 83.6% of BCSCs. A total of 34/35 (97.1%) of the test samples were SSEA_4, and the percentage of positive cells ranged from 0.5% to 77.1% (Table 32). The performance of SSEA-3 and SSEA-4 in normal woven fabrics 41 201100098 Different, connective group using tissue microarray, immunohistochemical staining, analysis of SSEA-4 performance in 2 〇 organs, as shown in Table 4 ( E, epithelial.p). Normal Tissue Antigen---- SSEA4 Brain0/5 Bone0/5 Lymph node-- 0/5 --- EC Breast 1/5 0/5 Colon Wood 2/4 0/4 Esophagus 0/5 0/5 Intestine 5 /5 0/5 Kidney 2/5 0/5 Liver 0/5 0/5 Lung 1/5 0/5 1/5 0/5 Ovary 1/5 0/5 Pancreas 0/5 0/5 Prostate 5/5 0/5 Rectal 0/5 0/5 Skin 0/5 0/5 Spleen 4/5 0/5 Stomach 4/5 0/5 睪 pill 1/5 0/5 1/5 0/5 Thymus _ Cervical τ咏The clothes are now on several kinds of urea-like woven * epithelial cells, purely. House, colon, gastrointestinal tract, kidney, lung, ovary, pancreas, rectum, stomach, pen, gland, and uterus (Table 4). In addition, the performance of SSEA-4 is similar to that of G1〇b and SSEA_3 (Chang W-W. et al., (2008) /W

42 201100098 (iv) 105(33): 11667-11672) 'Mainly limited to the cytoplasm or top surface of epithelial cells, which is essentially inaccessible to the immune system, as shown in Figure 16. In contrast, Globo tethers are expressed on several epithelial cells of the glandular tissue such as the breast, gastrointestinal tract, pancreas, prostate, and cervix. The distribution of SSEA3 is similar to GloboΗ, except that it does not exist in normal breast tissue, but is present in the kidney, rectum, testis, and thymus. These tissues are G1〇b〇 Η negative (chang

W-W. et al., (2008) proc Acad Sci USA 105(33): 11667-11672). ® EXAMPLE The following examples are illustrative of preferred embodiments of the invention. It will be apparent to those skilled in the art that the technology disclosed in these examples represents the technology discovered by the inventors and can be used to implement the present invention and thus can be considered as a preferred mode of construction. However, it will be apparent to those skilled in the <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; General Methods, Materials, and Instruments 0 Materials Commercially available, the granules and deductants are used at the time of obtaining, and no further purification is purchased from Sigma-Aldrich, Acros, Merck, Echo Chemical, and

Senn Chemical. The monoclonal antibody Mbrl was purchased from ALEXIS biochemicals, C^y3-conjugated anti-mouse IgG (IgG, IgG and IgG2a) and the IgM anti-system was purchased from Jacfoon. DT-CRM197 protein and broken &quot;W wind-like steroids were purchased from Merck and Adimmune, respectively. Acid-filled gel adjuvant (AIPO4) was purchased from Brenntag Bi〇sector. Bamboo virus and VK9 monoclonal antibody 43 201100098 were prepared from the laboratory of Dr. Lin and Yu (YU) respectively. The glycolipid derivative system was synthesized and supplied by Dr. Wang (Wong)'s laboratory. General Method The molecular sieve (Ms, aw_300) used for glycosylation was ground and activated prior to use. Analytical TLC disk (PLC Silicone-60, F254, 2 mm,

Merck) monitors the reaction and visualizes it under UV (254 nm) or with a bacteriophage. Flash column chromatography was performed on gel (40-63 μπι) or LiChroprep RP18 (40-63 μπι). Wash the dialysis membrane 以 (Cellulose Ester, MCCO = 1〇, 〇〇〇) with ddH20 before use. The instrument was recorded on a Bruker Advance 600 (600 MHz / 150 MHz) NMR spectrometer for proton nuclear magnetic resonance (W NMR) spectroscopy and nuclear magnetic resonance (13⁄4 NMRa) spectroscopy. The chemical shifts of protons are reported in ppm (δ grade) and refer to tetramethylnonane (δ = 0). Chemical shifts in carbon are also reported in parts per million (ppm units, δ levels). Multiplicity was determined using DEPT 135 (No Distortion Polarization Transfer Enhancement). The data are expressed as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad peak), enthalpy integral and coupling coefficient ( J) is the Hz unit. A high resolution mass spectrum was obtained with BioTOF III and MALDI-TOF MS was used with Ultraflex II TOF/TOF200. Example 1 Synthesis of GloboH Co-Linked with Different Carrier Proteins GloboH (l; see Figure 11) and its fragment 2-l〇 were synthesized using a programmable one-pot strategy. Huang CY, β α/. (2006) Proc 103:15-20 ). The reaction of 1 is carried out with sufficient homobiifUnctional linker in the absence of 44 201100098 water DMF solution at room temperature (Wu X, ❹/· (2004) Ο 6.4 6.4407-4410; Wu X , Bundle DR (2005) J Org Chem 7〇'l-7388). The reaction can be easily monitored by TLC. Once the free amine disappears, the larger Ry product is now evaporated, i.e., the reaction mixture is evaporated to remove DMF, and washed with dimethyl hydrazine and water to remove * excess linker. Finally, the product was purified by reverse phase (ci8) I column chromatography and eluted with water containing 1% acetic acid to 40% methanol in water. This solution is then carried out; it is dried to produce a pale yellow product 12. Wood ^ for protein co-linking, the purified guanidine ester (9) _4 〇Ζ and individual carrier protein, in phosphate buffer (10 mM, pH 7 2): production = down; co-located for 24 hours (Figure 14 ). Importantly, the protein must be h h 'to make the amino acid residue and G1〇b〇 H half vinegar even. After 24 hours, the sugar was co-diluted and the excess phenyl group was donated. The solution was then dried to a white powder to produce 13, 14, and 15. Each 彳 analysis GlQb was determined. The characteristics of the Η_protein hetero-linker are dissolved in fine ruthenium with pmHiS 13, 14, and 15 to produce about 1 nitrile and deionized hydrazine. The mustard seeds were aged and the mixture was prepared to produce a 10 mg/mL spectrum containing 0.1% TFA. In the mode _ each sample to obtain - show the heterogeneity, the molecular weight of the two. The sugar conjugated linker 14 has a larger ulnar core than the ί ft part of the leg and is caused by the human temple. The same even ε of the p-nitrophenyl linker will also be used, which contains more than 10 〇, _ soil... on the outer shell of the virus, and the disease I is in sodium phosphate buffer at 4 〇C (ρΗ = 45 201100098 =) The instability in the reaction is the main consideration for further development. In addition, and whoever has a huge size, it can be restricted to the 1st test by saying 031-1'017. Finally, the silk candy co-linkage is stored at _3. . . Reconstituted with sterile water using beta. Example 2: The manufacture and verification of a glycan microarray into if°bC &gt;H and a truncated fragment (Fig. 连 attached to the original 钿 with a pentylamine linker and covalently immobilized on an NHS coated glass slide. Of the eleven oligosaccharides, nine were imprinted on the microarray. Experiments were performed on a series of vinegar concentrations U, 5, 10, 20, 40, 50, 80, ΙΟΟμΜ) to allow binding affinity and firefly The light intensity is optimized. Each microarray slide was spotted with 9 GμΜ of nine G1〇b〇Η analogues (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, ΒΒ4, ΒΒ3, and BB2) (12 repeats) ). After the reaction in an atmosphere of 8 % humidity, the slides were stored in a desiccator at room temperature before use. 〃 To verify the sugars on the microarray, mouse monoclonal antibodies (for VK9 and Mbrl ' and anti-SSEA-3 for G1〇b〇Η) and corresponding secondary antibodies (goat anti-mouse IgG and IgM) were used. The binding specificity was examined and the results are shown in Figures 2A-2C. The data suggest that both VK9 and Mbrl can recognize Gl〇b〇Η and the lateral tetrasaccharide 4' but MBrl can also slightly recognize BB3 (Gilewski T e/ al (200\) Proc Natl Acad Sci USA 98:3270-3275; Huang CY, et a/· (2006) /Voc JcW as t/like 103:15-20). In addition, the anti-SSEA-3 antibody specifically recognizes the SSEA-3 antigen (Gb5) without any cross-reactivity. These results indicate that GloboH microarrays can be used to describe the specificity and potency of multiple antibodies taken from immunized mice. 46 201100098 Example 3: Mouse Immunization

In this experiment, a group of mice were immunized subcutaneously with 1 Mg of synthetic GloboH (GH) co-linker with or without the glycolipid adjuvant α-GalCer (C1). After 10 days of immunization three times per week, small air serum was collected and then introduced into the glycan microarray to evaluate the antibody content. GH-KLH, GH-DT, and GH-BV were found to be the most potent immunogens for inducing IgM, followed by GH-TT and GH-BSA' as described in Figure 3A, while α-GalCer stimulated the immune response to induce high Amount of IgM antibody. A similar tendency was observed on mouse lgG antibodies (Fig. 3B), and the relative lgG content was higher than the lgM content. Briefly, although synthetic sugar co-linkers have a lower sugar density, GH-DT has immunogenicity similar to GH-KLH, and a_GalCer adjuvant has been shown to enhance this immune response. Since C1 has been shown to be an effective adjuvant for GH-DT, it has been tested for other glycolipids with better adjuvant activity above C1, as shown in Figure 4 (Fujio M, et al. (2006) *Sbc 128 :9022-9023). Mice were immunized intramuscularly twice a week with 1.6 pg of GH-DT and GH-BV with or without 2 pg of glycoprotein. Serum was taken two weeks after the third vaccination and introduced into the glycan microarray analysis. In general, mouse resistance

The 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 immunization group, the seedling treatment and the single-touch seedling _ IgM content were finely differentiated. $ This special, suggesting that GH_BV combined with _f is not a valid immune ν, poor immunogenicity may be caused by the instability of BaMV 'a_GalCer analogues, especially 7DW8_5, can be used with GH-DT D Good and induced mouse immune response. 47 201100098 Interestingly, mouse 1gG antibodies produced by GH-DT and various glycolipid adjuvants not only neutralize Globo Η but also cross-react with Gb5, SSEA-4, and Gb4' while C34 appears to be The most effective (Figure 6). To find novel vaccine compositions that can induce igG with much higher potency than IgM, test Globo Η-DT conjugated linkers with glycolipids C1 or C34 or commercially available adjuvants Α1Ρ〇4 (scales) Acid Ming) or MF59. Surprisingly, in the second vaccination gas, Globo(R)-DT and glycolipid CM can almost induce IgG antibodies (7). Jane Lu's 'new bile lipid adjuvant 7DW8-5 binds to GH-DT co-light linker to enhance both anti-Globo H IgG and IgM antibodies, while glycolipid adjuvant 0 C34 binds GH-DT to induce higher than IgM Many 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. To further compare the effects of different glycolipid adjuvants on the G1〇b〇 H vaccine, seven groups of mice were immunized with GH-KLH. These results suggest that mice vaccinated with glycolipids can induce higher levels of anti-G1〇b〇/Η antibodies (Figure 8). Although MF59 is a potent adjuvant, it is unable to cooperate with gh-KT Η to induce antibodies against Globo. Α1Ρ〇4 (aluminum phosphate) also showed no significant effect on antibody induction. On the other hand, GH-KLH combines C34 in the first

One and the first immunization showed better immunogenicity, but in the third immunization (J

After inoculation, there was no significant difference from C1. X DT-CRM197 (a heterozygous mutant toxin) can antigen-specific cell proliferation and increase the production of IL-2, IFN_Y, and IL_6 in splenocytes, suggesting that it plays a role in the pathway driven by Thl Role (Miyaji EN et al. (2001) Infect Immun 69: 869-874; Godefroy S, et al. (2005) / ci / w dirty 73: 4803-4809; Stickings P, ed (2008) / (10) 76:1766-1773). Although the cytokine content is predominantly Th1, the subgroup of anti-CRM197 antibodies is igGi and has no detectable IgG2a, suggesting that it is a 48 201100098 mixed Th1/Th2 response. These results contributed to the s-estimation of the antibody isoforms in the G1〇b〇Η vaccine, and this study showed that GH_DT or the human lipid lipid adjuvant can mainly induce _ antibodies and trace amounts of IgG2a (Fig. The agent can enhance the secretion of Th1 cytokines when administered intravenously (iv) alone, but no antibody type conversion (purchasing &amp;) is observed. The sputum of the sputum is enhanced in both cellular and humoral immune responses. Play a key role. ❹ 合成 Also synthesize Gb5 and SSEA_4 linked to DT in the same strategy. After fj vaccination, compare the antibody titers of IgM and IgG, and find that Gb5_DT and SSEA-4-DT can also induce higher than IgM. A lot of IgG titers (Fig. 1〇). Example 4: Specificity of antibodies elicited by different vaccine compositions. GH-DT and C34 can induce antibody recognition G1〇b〇η, Gb5 S^A- 3), and SSEA-4, therefore using the array of 24 glycans, in the next 'special test for SSEA_3-DT and SSEA_4_DT vaccine, focus on the igG study (Figure η). Lu Zheng ίf i2 shown to Gbb. Mice immunized with H_DT and C34 adjuvants can induce the selective identification of Gi〇bo η, SSEA-3 (Gb5), and _Α·4 = body' while the vaccine SSEA-3-DT and adjuvant MF59 A low selective surface immune response can be induced. On the other hand, SSEA_3_DT binds to adjuvant C34 and only antibodies against Globo®, SSEA-3, and SSEA-4. ^ Interestingly, SSEA_4_DT differentially recognizes SSEA_4 and its _ structure (with anterior lactose deletion ^ why-4) with or without adjuvants, IgG and igM antibodies. However, the source of this selectivity is not known. 49 201100098 To directly assess the efficacy of a synthetic sugar conjugated linker vaccine, the tumor was measured weekly = 3 times, as shown in Figure 13. In general, tumors were allowed to grow for 2 weeks after injection of 4T1 (a breast cancer cell line with Globo®). On day 24, all vaccinated groups that bind the glycolipid adjuvant still showed a smaller tumor progression (compared to the GH-DT group and the PBS control group alone). This preliminary data suggests that vaccination with GH-DT and glycolipid adjuvant can delay some of the tumor progression in vivo. Example 5: Preparation of Globo oxime half ester Globo Η half vinegar was prepared as follows:

Globo Η half ester (12) Globo guanamine 1 (5 mg '4.54 μηιοί) was dissolved in anhydrous DMF solution. Next, a p-decylphenyl ether linker (8.8 mg, 22 7 μπκ) 1) was added, and stirred at room temperature for 1 to 3 hours. The reaction was monitored by TLC (1% AcOH in methanol) and Ninhydrin test. The disappearance of free amines and the appearance of larger R/products indicate that the reaction is complete. The reaction mixture was evaporated under reduced pressure without heating to remove DMF, followed by extraction twice with CH.sub.2Cl.sub.2 and 1% EtOAc. The aqueous solution was concentrated and purified by reverse phase (C18) column chromatography eluting with 1% acetic acid 0 to MeOH: H20 = 4: 6. The solution was dried to a pale yellow solid product (5.4 mg, yield 88%) ln NMR (600 MHz, D20) δ 8.25 (d, 2 Η, J = 9.0 Hz), 7.28 (d, 2H, 50 201100098 J= 9.0 Hz), 5.12 (d, IH, J= 3.9 Hz), 4.79 (d, 1H, J= 3.7 Hz), 4·51 (d, 1H, J= 7.7 Hz), 4.44 (d, 1H, J= 7.7 Hz), 4.39 (d, 1H, J = 7·7 Hz), 4.31-4.28 (t, 2H, J= 7.7 Hz), 4.15-4.11 (m, 2H), 3.99 (d, 1H , 2.0 Hz), 3.92 (d, 1H, /= 2.8 Hz), 3.89-3.44 (m, 33H), 3·16 (t, 1H, 8.6 Hz), 3.10 (t, 2H, J= 6.7 Hz), 2.62 (t, 2U, J = 6 9 Hz), 2.20 (t, m, J = 6.6 Hz), 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 = 6.5 Hz) 13C NMR (150 MHz, D20) δ 178.0, 176.1, 176A 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, 70.1, 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 : C55H87N3035Na [M+N a]+ Calculated value: 1372.5018; Experimental value: 1372.5016. Example 6: General procedure for producing a sugar conjugated linker A sugar conjugated linker was prepared as follows:

GfoboΗ

13, n=8 (BSA) 14, n=2-4 (CRM197) 15,n=4 (tetanus bismuth)&gt; 16. (Bamboo disease 軎&gt; BSA, DT-CRM197, and tetanus toxoid ( Adimmune, Taiwan) is dissolved in 100 mM phosphate buffer 7.2) (~5 mg/ml), and 30 to 40 equivalents of Globo oxime half ester 35 are added to the solution. The mixture is gently stirred at room temperature for 24 hours. The mixed 51 201100098 was then diluted with deionized water and dialyzed for 5 times with deionized water. The solution was then lyophilized to a white powder. Globo H-protein conjugates qualitatively obtained by MALDI-TOF analysis. Linkage to determine the sugar inclusion 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) was not determined. Example 7: Recombination of sugar co-linkers 41, 42, 43 and original carrier protein (~1μδ/μΙ〇 with ddH2〇. Freshly prepared with acetonitrile and deionized water: 丨Matrix erucic acid to produce a final matrix concentration of 10 mg/mL including 0.1% TFA. Mild addition and mixing of the matrix solution and sugar conjugated link followed by air drying of the test disk. The amount must be corrected by using bovine serum albumin. The individual sugar conjugated linkers and the original carrier protein samples were detected in linear positive ion mode. The average molecular weight was used to calculate the average number of glycosides on the carrier protein. Example 8: Glycans Preparation of microarray using machine needle (SMP3, TeleChem International Inc. USA), dissolving ~〇·7 nL in imprint buffer (3 mM phosphate buffer containing 5% 5% Tween 2 ,, pH 8.5 Various concentrations of aminoglycans in the deposition from 96-well onto NHS-coated glass slides to print microarrays (Bi〇D〇t, Cartesian)

Technologies, USA). Each of the microarray slides was spotted (12 replicates) with nine Globo(R) analogs (SSEA-4, GH, Gb5, Gb4, Gb3, Gb2, ΒΒ4, ΒΒ3, and ΒΒ2) of 50 μM, respectively. The printed slides were allowed to react in an atmosphere of 80%^ for one hour and then dried overnight. The slides were stored in a desiccator at room temperature prior to use. 52 201100098 Example 9: Serological analysis (polyfluorene microarray) dilution of small ^ I^SA/PBS buffer (pH 7.4) * 〇 〇 5% Tween 20 B 1: 1 for preliminary screening. The cells were blocked with 50 mM and PBS for 1 hour, and were introduced into the serum dilutions such as the 〇2〇 microarray f^ before use, and Α was placed under the dish for 1 hour. PBST (PBS buffer Ο)

G Γ ° Tween_20) and PBS buffer to further clean the microarrays ^ ΐ 2: human. Next-step 'Cy3_affmiPUre-small milk 1gG (H + L), IgCH, IgG2a or anti-mouse 1gM was added to the microarray slides, and the samples were read at room temperature for 1 hour. Finally, the slides were washed three times in the order of PBST, PBS, and fjdH2. Dry the microarray slides, connect the f=microarray fluorescent wafer reader (Genepix 4000B), and analyze the data at 532 mn with the software GenePix Pro 6.0 (Axon Instruments, Union = ty, CA, USA). . To achieve accurate measurements, adjust the photomultiplier tube gain (PMT Gain) to 400 to avoid fluorescence saturation. The local background value is subtracted from the b number of each glycan point. The point with obvious defects or no detectable signals is omitted. The final fluorescence intensity is defined as the average of the "F532 nm - B532nm middle number" of the repeated experimental points. Example 10: Serological analysis (Melkin immunosorbent assay) 〇 2 Globo-H ceramide in 1 μl of sodium bicarbonate buffer (pH 10) was applied to a 96-well plate at 4 °C (NUNC) in the middle of the night. Wash with PBS' and block with 3% fetal bovine serum albumin for 3 minutes at room temperature. Serial dilutions of mouse serum were added to each well and allowed to stand at room temperature for 1 hour, followed by washing with DPBST (Dulbecco's phosphate buffered saline, 0.05% Tween 20). Add goat anti-mouse IgG-AP (1:200, Southern Biotech" 53 201100098 US3) 'And set a total of 45 minutes at room temperature. Wash the test plate five times with PBST, then with alkaline phosphoric acid at 37 ° C The enzyme substrate was co-localized for 8 minutes with Sigma-based stupid acid (Sigma). After co-location, 3 μL of NaOH solution was added to stop the reaction, and 405 was taken on ELISA (SpectraMax, Molecular Devices) to 405. The nm assay assay disk. The titer is defined as the highest dilution that produces an optical density greater than 。 1. Example 11: Dosage and immunization (1) for a group of three mice (6-week-old mother C57BL/6 mice, BioLASCO 'Taiwan', subcutaneously administered GH-KLH (Optimer Inc.), GH-BSA, GH_TT, GH-CRM197, and GH-BaMV, respectively, with or without lipid lipid adjuvant C1 or 7DW8_5 To the abdomen area, three times at weekly intervals. Each vaccination contained 1 Globo sputum with or without 2 pg of glycolipid adjuvant. Control group mice were injected with phosphate buffered saline (PBS) only. The mice were given blood after sub-immunization (before immunization) and after 1 day of the third immunization. (2) Needle Group of three mice (8-week-old female Balb/c mice, BioLASCO, Taiwan), with or without a, C23, or 7DW8-5, respectively, GH-BaMV or GH-CRM197 Two weeks interval was intramuscularly immunized three times. Each vaccination contained 1.6 pg of Globo sputum with or without 2 pg of adjuvant. The control group was injected with phosphate buffered saline (pbs) before and after immunization. Mice were bled 2 weeks later. (3) Groups of three mice (8-week old female Balb/c mice, BioLASCO, Taiwan) with or without adjuvants a, C17, 7DW8-5 In the case of C30, AlP〇4, MF59 (1:1 mixture), immunize as described in (2). Serum was obtained by centrifugation at 4000 g for 10 minutes. Serological reactions were analyzed by glycan microarray or with conventional ELISA analysis comparison 54 201100098 Example 12: Xenograft mode wrong pair of five groups of immunized female secret/G mice (divine PBS, chlorinated C, C23, and 7DW8_5 GH_CRM197), in the final free seed 8 After the week, 2 χ (8) metastatic mouse breast tumors were injected subcutaneously, 4 1 1 (in sterile PBS). (2) Seven groups of immunized mothers TU-small (GH-KLH alone or in combination with Cl, C17, 8-5, C30, AlP〇4, and] VIF59) 'After 6 weeks of final immunization, 2 〇 1〇5 by subcutaneous injection Metastatic mouse mammary tumor cell line 4T1 (in sterile pbs). Mouse anti-Glob(R) sputum serum was monitored before and after tumor xenografts. Weekly

The Vernier caliper measures the tumor size of the mouse three times and defines it as (length X height X width) / 2 (mm3). Example 13: Isolation of primitive swollen cells from human breast cancer samples Human breast cancer samples were obtained from patients who had undergone initial surgery at the Three Military General Hospital (Taipei, Taiwan). The samples were fully coded to protect the patient's privacy and were used in the 〇 operational procedures approved by the Academia Sinica Medical Research Ethics Committee (Taipei, Taiwan). The tumor sample was sectioned into a 1 mm square piece and placed in RPMI1640 medium containing collagenase (1,000 U/ml), hyaluronan (300 U/ml), and DNase I (100 pg/ml). They were co-digested at 37 ° C for 2 hours. The original breast cancer cells were collected by filtration through a 100-μηη cell strainer (BD Biosciences) and resuspended in RPMI1640 medium supplemented with 5% FBS. Example 14: Flow cytometric analysis 55 201100098 The original breast cancer cells were made into 1 x 10 cells in PBS containing 2% FBS and 1% NaN3. Cells were labeled with anti-CD24-PE, anti-CD44-APC, and anti-CD45-PerCP-Cy5.5 antibody mix (1 μl each). A single anti-Globo(R) antibody (VK-9) conjugated to Alexa488 was stained to detect the performance of Globo(R). Analysis was performed on a FACSCanto flow cytometer (Becton Dickinson). BCSCs are defined as CD45-/CD24-/CD44+ cells, whereas BCSCs are defined as the remaining population of CD45-cells. Further analysis of Globo's performance in the interval. Example 15: Cell sorting Cells obtained from human breast cancer transplanted into mice were stained with anti-CD24-PE, anti-CD44-APC, and anti-H2Kd-FITC antibody mixture (BD Biosciences). Fluorescent activated cell sorting of antibody-labeled cells was performed on a FACSAria cell sorter (Becton Dickinson). H2Kd/CD24/CD44+ cells were sorted into BCSCs; the other H2Kd· populations were sorted as non-BCSCs. The typical purity of BCSCs and non-BCSCs is &gt;85% and &gt;90%, respectively. Example 16: Immunohistochemistry. For the performance of SSEA 4 on normal tissues, tissue microarray slides (Biomax) containing 2 different organs were used, each of which was derived from five individuals. The slides were dried at 56 ° C, rehydrated according to standard histopathological procedures, and then subjected to antigen retrieval with AR_1() solution pH 9 (Bi〇Ge Laboratories). Use anti-ship eight_4 anti-〇 performance. The anti-money 1 café drug antibody fine 4 stained 'Asian DAB was color-developed. Using hematoxylin on the slide for 56 Ο ο 201100098 5 transfer dyeing λ T (ttTGTTV / ^ SuperFr〇st ^us to cut the same, ah t toluene in the wood miscellaneous (=) = The sheets were placed side by side in 1 〇mm〇1/L citric acid buffer _·〇. The high-sensitivity polymer-biliary IHC system (Bi〇Genex) has a ;f and patent documents for all purposes and test the dry circumference as each publication or patent file is exhaustive, the previous invention has been The manner in which the description and examples are presented is to be understood as a Variations and modifications without departing from the scope of the appended claims: 37 C.F.R. Valley 1,72 (b) provides an abstract of the invention to enable the reading of the nature and gist of the invention. The Abstract of the Invention is not intended to explain or limit the scope or meaning of the scope of the patent application. [Simple description of the schema] Ming Taixuan forms part of this specification' and it is a detailed description of the specific examples presented in this article, ', ', patent or patent application file containing at least one color Manufactured in the 2011 201100098 patent, the photocopy of the open case (including the color drawing) will be provided by the Intellectual Property Office upon request and payment of the necessary _. Figure 1 shows the structure of Globo(R) and the truncated derivative. 2A-2C show the binding specificity of monoclonal antibodies VK9 and Mbd (for G〗 〇b〇 and anti-SSEA-3, respectively.

—, 3A-3B shows the seroconversion of various G1〇b〇H conjugated linkers and a_Gal(:er mice inoculated with the field. For the group of three C57BL/6 mice, with or without 2 In the case of glycolipids, vaccination was carried out by sc with a 1 Mg sugar conjugate, and mouse serum was diluted 1:6 〇 and i side to perform IgM (Fig. 3A) and IgG (Fig. 3B) antibodies. Analysis. Cy3-anti-mouse igG or IgM secondary antibodies were detected by fluorescence at 532 nm, pMT 5 。. The data were expressed as SEM of the average fluorescence intensity of three mice. Structure of a-GalCer and the like Figure 5 shows the IgM content of mice inoculated with Globo(R) conjugated linker and a_GalCer derivative. Mouse serum was collected and after the second and second vaccination Analysis was performed as indicated. Cy3 secondary anti-mouse IgM was used for detection at 532 (10), pMT 4 。. Results were expressed as mean fluorescence intensity + SEM of three mice.

Figure ό shows the good specificity of multiple antibodies (anti-G1〇b〇 η, anti-Gb5, anti-SSEA-4, and anti-Gb4) in mice after vaccination. Mouse sera (mother's Balb/c' i.m.) were obtained two weeks after the third vaccination with 1.6 pg of GH-DT with or without 2 tons of adjuvant. The igG titer was analyzed by a glycan microarray and was defined as producing a MFI greater than 1000 (1 fold times the background value), the highest dilution of PMT 400. Each point represents individual mouse titers. Figure 7 shows the IgM versus IgG antibody titers produced by Globo®-DT with different adjuvants. 58 201100098 Figure 8 shows that for adjuvant gh_klh % Balb/ca -X 1.6 μ§ GH-KLH ^ 2 μ§ ^ ^;Γ 〇# vaccination' and introduce it into the microarray analysis in the vaccine . Persimmon release blood, and bribe 300). Data system

, 10 shows the IgM versus IgG antibody titer induced by SSEA-3-DT or SSEA. Lunar Figure 11 shows the structure of 24 glycans on the cell surface. The cross-reaction of IgG induced by Sex Research vaccine ΐ Anti-coffee induced by α adjuvant. f '. Figure B. Anti-Gb5 IgG induced by Gb5-DT and Cl adjuvant; Figure 12C. Anti-SSEA-4 IgG induced by SSEA-4-DT and Cl adjuvant Figure 13 shows mouse xenogeneic Migration mode. 2 X(7)5, /11 small metastatic mammary gland tumor cells were prepared in sterile pBS and subcutaneously injected to vaccinate Balb/c t. The mouse tumor size was measured with a ν_Γ caliper and was expressed as (length X height X width)/2 (mm3). Figure 14 shows the flow of synthesizing Globo oxime and sugar conjugated linkers. Figure 15 shows the flow fine, count analysis for SSEA_4 expression in the original breast cancer stem cells. The four-color immunofluorescence color and subsequent flow cytometry were used to analyze the SSEA-4 expression on the surface of BCSCs and non-BCSCs. BCSCs (4) CD45 / CD24 / OM4 + _ ─ BCSCs limbs are the rest of the CD45 - as shown in the left column. The expressions of BcsCs and non-6 (:3(;^ surface) are shown in the middle and right barrier respectively. The dotted line represents the isotype control group, and the number represents the percentage of positive cells. 59 201100098 Figure 16 shows SSEA-4 in normal tissues. Limitations of performance. SSEA-4 expression in the breast, small intestine, and rectum was examined using immunohistochemical staining of normal tissue arrays. The positive staining of SSEA-4 was localized to the top surface of epithelial cells.

Claims (1)

201100098 VII. Patent Application Range: 1. An immunogenic composition comprising: (a) poly-, which consists essentially of G1〇b〇H or an immunogenic fragment thereof, wherein the glycan is via a linker And conjugated to a carrier protein; and an adjuvant comprising a glycolipid that binds to a CDld molecule on a dendritic cell, wherein the immunogenic composition induces an immune response that induces an isoform compared to the IgM isoform A relatively high amount of IgG isotype antibody of the antibody. Q. The immunogenic composition according to claim 1, wherein the carrier protein is a diphtheria toxin cross-reactive material 197 (DT-CRM197). 3. The immunogenic composition according to s. 1, wherein the adjuvant is a synthetic analog of α-galacto-chidamine (α-GalCer). 4. The immunogenic composition according to claim 3, wherein the adjuvant is C34, wherein C34 comprises a structure: 5. The immunogenic composition according to claim 1, wherein the linker is a phenyl linker. 6. The immunogenic composition according to claim 1, wherein the linker is p-phenyl, the carrier protein is diphtheria toxin cross-reactive material 197 (DT-CRM197), and the adjuvant is C34. 7. The immunogenic composition of claim 1 wherein the immune response is directed to the production of an IgG isotype antibody. &quot;^ 61 201100098 8. The immunogenic composition according to claim 7, wherein the carrier protein is a diphtheria toxin cross-reactive material 197 (DT-CRM197), and the adjuvant is C34. 9. The immunogenic composition according to claim 1, which comprises at least one adjuvant which induces body fluids and cellular immune responses. 10. The immunogenic composition according to claim 1, wherein the antibody produced by the immune reaction neutralizes an antigen expressed on a cancer cell or a cancer stem cell. 11. The immunogenic composition according to claim 10, wherein the antibody produced by the immune reaction neutralizes antigen Gb4, stage-specific embryonic antigen-3 (SSEA-3), and stage-specific embryonic antigen-4 ( At least one of SSEA-4). 12. The immunogenic composition according to claim 11, wherein the neutralizable antigen Gb4, stage-specific embryonic antigen_3 (SSEA-3), and stage-specific embryonic antigen-4 (SSEA-4) are at least One antibody comprises a higher relative amount of an IgG isotype antibody compared to an igM isotype antibody. 13. The immunogenic composition according to claim 11, wherein the carrier protein is a diphtheria toxin cross-reactive material 197 (DT-CRM197), and the adjuvant is C34. 14. The immunogenic composition of claim 1 further comprising a cancer vaccine composition that elicits an anti-cancer immune response in the subject. 15. The cancer vaccine according to claim 14, wherein the cancer vaccine is suitable for treating cancer selected from the group consisting of breast cancer, lung cancer, liver cancer, buccal cancer, lunar cancer, colon cancer, nasopharyngeal cancer, skin cancer, Kidney cancer, brain tumor, prostate cancer, ovarian cancer, cervical cancer, intestinal cancer, and bladder cancer. 16. The cancer vaccine according to claim 15, wherein the cancer tissue exhibits a GloboH antigen on the cell surface. 17. The cancer vaccine according to claim 15, wherein the Globo(R) antigen system is expressed on an epithelial cell of a breast cancer tumor of 62 201100098. The cancer vaccine of the antibody of claim 14, wherein at least one of the original Pei h, gm, the stage-specific embryonic antigen-3 ·), and the stage-specific embryonic antigen-4 (SSEA-4) produced by the immune reaction 0 4 main s ^• According to the request M, the cancer vaccine, wherein the antigen Globo Η, stage Ο Embryo, at least one of antigen_3 (SSEA_3;&gt;, and stage-specific embryonic antigen-4 CSSEA-4) is expressed on breast cancer stem cells. 20. A method of treatment comprising inhibiting tumor growth, the method comprising: administering to a subject in need thereof an immunogenic composition comprising: a glycan comprising: consisting essentially of Globo(R) or an immunogenic fragment thereof Wherein the glycan is conjugated to the carrier protein via a linker; and an adjuvant comprising a glycolipid capable of binding to a CDld molecule on the dendritic cell; and ^ '° (b) inducing an immune response, the immune response Higher relative amounts of IgG isotype antibodies can be induced compared to IgM isotype antibodies. 21. The method of claim 20, wherein in the immunogenic composition, the linker is p-nitrophenol, the carrier protein is a diphtheria toxin cross-reactive material 197 (DT-CRM197), and the adjuvant A synthetic analog of α-galactosyl-neuracilide (α-GalCer). 22. The method of claim 21, wherein the adjuvant is C34. 23. The method of claim 20, wherein the immunogenic composition further comprises a cancer vaccine&apos; and wherein treatment with one or more of the effective amount of the cancer vaccine inhibits tumor growth. The method according to claim 23, wherein the administration of the cancer vaccine reduces the tumor size. The method of claim 23, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, liver cancer, frequency cancer, stomach cancer, colon cancer, nasopharyngeal cancer, skin cancer, kidney cancer, brain tumor. , prostate cancer, ovarian cancer, cervical cancer, intestinal cancer, and bladder cancer. 26. The method of claim 21, wherein the immune response is preferably directed to the production of a 1 § isotype antibody. 27. The method according to claim 20, wherein the antibody produced by the immune reaction neutralizes the antigen Globo(R), Gb4, stage-specific embryonic antigen_3 (SSEA-3), and stage-specific embryonic antigen-4 (SSEA) At least one of -4). 28. The method according to claim 27, wherein at least one of the antigens G1〇b〇H, stage-specific embryonic antigen-3 (SSEA-3), and stage-specific embryonic antigen_4 (SSEA_4) is expressed in On breast cancer stem cells. 29. The method of claim 27, wherein the Globeh H antigen system is expressed on a breast cancer tumor epithelial cell. 30. The method according to claim 27, wherein the antibody capable of neutralizing at least one of antigen GM, stage ϋ biogenesis embryo f antigen _3 (SSEA_3), and stage-specific embryo antigen-4-A-4) A relatively southerly relative amount of an IgG isotype antibody compared to an IgM isotype antibody. 31. A cancer vaccine comprising: a composition comprising: a polymerase substantially in the form of a coffee. Ϋϋΐ The (4) susceptibility to the immunoreactant by the linker and the carrier egg; and the correct relative amount of the IgG isotype of the hard body (b) pharmaceutically acceptable excipient. The invention relates to a cancer vaccine according to claim 31, wherein in the immunogenic composition, the linker is p-nitrophenol, and the carrier protein is a diphtheria toxin cross-reactive material 197 (DT-CRM197), and The adjuvant is a synthetic analog of alpha-galactosyl-ceramide (oc-GalCer). 33. The cancer vaccine of claim 32, wherein the adjuvant is C34. 34. The cancer vaccine according to claim 32, wherein the cancer is breast cancer, and wherein the Globo(R) antigen system is expressed on a breast cancer tumor epithelial cell. The cancer vaccine according to claim 32, wherein the antibody produced by the immune reaction neutralizes the antigen Globo(R), Gb4, stage-specific embryonic antigen_3 (SSEA-3), and stage-specific embryonic antigen_4 At least one of (SSEA-4). 36. The cancer vaccine according to claim 32, wherein the cancer is breast cancer, and wherein the antigens are Globo(R), stage-specific embryonic antigen _3 (SSEA_3), and stage-specific embryonic antigen-4 (SSEA-4) At least one of the lines is expressed on breast cancer stem cells. 37. An immunogenic composition comprising: (a) a glycan consisting essentially of a Globo H-related glycan or an immunogenic sheet U segment thereof, wherein the glycan is via a linker a carrier protein co-linking; and (b) an adjuvant comprising a glycolipid that binds to CDld* on the dendritic cell, wherein the Globo H-related glycan is selected from the group consisting of SSEA_3 and SSEA_4, and Wherein the immunogenic composition elicits an immune response that elicits a relatively high amount of an IgG isotype antibody compared to an IgM isotype antibody. 38. The immunogenic composition according to claim 37, wherein the carrier protein is a diphtheria toxin cross-reactive material 197 (DT-CRM197). 65. The immunogenic composition according to claim 37, wherein the adjuvant is a synthetic analog of α-galactoside-neuracine (α-GalCer). 40. The immunogenic composition according to claim 39, wherein the adjuvant is C34, wherein C34 comprises the structure: C34 41. The immunogenic composition according to claim 37, wherein the linker is a p-nitrophenyl linker. 42. The immunogenic composition according to claim 37, wherein the linker is _ succinyl phenyl, the carrier protein is a diphtheria toxin cross-reactive material 197 (DT-CRM197), and the adjuvant is C34. 43. A therapeutic agent for combating breast cancer stem cells, the therapeutic agent comprising: GloboH conjugated to a diphtheria toxin cross-reactive material i 9 7 (DT-CRM197) carrier protein via a p-nitrophenyl linker; An adjuvant comprising a glycolipid that binds to a CDld molecule on a dendritic cell. 44. The therapeutic agent according to claim 43, wherein the adjuvant is C34. 45. The therapeutic agent according to claim 43, wherein the therapeutic agent is administered to a subject to elicit antibody production&apos; such antibodies recognize antigen present on breast cancer stem cells (BCSC). 46. The therapeutic agent according to claim 44, wherein the at least one antigen is selected from the group consisting of Globo®, SSEA-3, and SSEA-4. 47. A therapeutic agent against breast cancer stem cells, the therapeutic agent comprising: 66 201100098 SSEA_3 co-linked with a diphtheria toxin cross-reactive material 97 (DT-CRM197) carrier protein via a p-nitrophenyl linker; The adjuvant 'which contains a glycoprotein C34 that binds to CDld molecules on dendritic cells. 48. The therapeutic agent according to claim 47, wherein the therapeutic agent is administered to a subject to elicit antibody production, the antibodies recognizing an antigen expressed on breast cancer stem cells (BCsc:). 49. The therapeutic of claim 48, wherein the at least one antigen is selected from the group consisting of GloboH, SSEA-3, and SSEA-4. 50. A therapeutic agent against breast cancer stem cells, the therapeutic agent comprising: SSEA-4 conjugated to a diphtheria toxin cross-reactive material (DT-CRM197) carrier protein via a p-nitrophenyl linker. 51. The therapeutic of claim 50, which further comprises an adjuvant comprising a bile lipid that binds to a CD Id molecule on the dendritic cell. The therapeutic agent according to claim 51, wherein the adjuvant is C34. 53. The therapeutic agent according to claim 50, wherein the therapeutic agent is administered to a subject to elicit antibody production, the antibodies being identifiable on breast cancer stem cells (BCSC), antigen SSEA-4 or its cut-out structure. 54. A method of treating breast cancer, the method comprising administering to a subject in need thereof a therapeutic composition according to claim 43. 55. A method of treating breast cancer, the method comprising administering to a subject in need thereof a therapeutic composition according to claim 47. 56. A method of treating breast cancer, the method comprising administering a therapeutic composition according to claim 50 to a subject in need thereof. 57. A method of treating breast cancer, the method comprising administering to a subject in need thereof a therapeutic composition according to any one of claims 43-52. 67